WO2022213664A1 - Application of hdac2 and dnmt1 inhibitors in combined targeted therapy of non-alcoholic steatohepatitis - Google Patents

Application of hdac2 and dnmt1 inhibitors in combined targeted therapy of non-alcoholic steatohepatitis Download PDF

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WO2022213664A1
WO2022213664A1 PCT/CN2021/140096 CN2021140096W WO2022213664A1 WO 2022213664 A1 WO2022213664 A1 WO 2022213664A1 CN 2021140096 W CN2021140096 W CN 2021140096W WO 2022213664 A1 WO2022213664 A1 WO 2022213664A1
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liver
igfbp7
adamts1
hdac2
cells
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French (fr)
Chinese (zh)
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丁楅森
曹中炜
张华�
马永源
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四川大学华西第二医院
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/506—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim not condensed and containing further heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
    • A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/06—Antihyperlipidemics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics

Definitions

  • the invention belongs to the field of biomedicine, and mainly relates to the application of HDAC2 and DNMT1 inhibitors in the treatment of nonalcoholic steatohepatitis.
  • the liver has the regenerative ability to repair itself after injury.
  • NASH nonalcoholic steatohepatitis
  • liver regeneration is inhibited.
  • NASH is on the rise globally.
  • NASH can lead to liver fibrosis, cirrhosis and liver failure.
  • Without effective anti-fibrotic treatment liver fibrosis and liver cirrhosis caused by NASH usually lead to systemic complications and will become a major global health burden.
  • a major obstacle to the clinical development of NASH treatments is the lack of clinical and preclinical studies of cellular and molecular networks that systematically mimic the pathogenesis of NASH.
  • the liver is composed of parenchymal cells (hepatocytes) and non-parenchymal cells (NPCs) such as stellate cells, endothelial cells (ECs), and hematopoietic cells. Liver regeneration relies on the synergy between different cellular components.
  • NASH parenchymal cells
  • ECs endothelial cells
  • hematopoietic cells hematopoietic cells.
  • Liver regeneration relies on the synergy between different cellular components.
  • persistent stress in NASH often causes abnormal cellular interactions (in this context, “interaction”, “crosstalk” can both be understood as “crosstalk”) and leads to dysregulated repair and fibrosis.
  • Activation of stellate cells is a critical step in liver fibrosis, but it remains to be determined how chronic stress in NASH leads to interactions between other hepatic NPCs to facilitate this step.
  • vascular endothelial cells and hematopoietic cells belong to the circulatory system, which can directly transmit systemic stimuli (such as metabolic stress), and can also promote the interaction between parenchymal cells and mesenchymal cells to jointly establish the vascular microenvironment.
  • Vascular endothelial cells are the main component of hepatic NPCs.
  • the blood supply to the liver is accomplished by the sinusoidal vascular system between the hepatic veins, the hepatic artery and the portal vein.
  • the sinusoidal vasculature has a layer of sinusoidal endothelial cells (SECs) expressing CLEC4G and OIT3 and macrovascular endothelial cells (MECs) expressing CD34.
  • SECs sinusoidal endothelial cells
  • MECs macrovascular endothelial cells
  • hepatic ECs from different anatomical sites exhibit specific morphological and phenotypic markers, with "sinusoidal endothelium-macrovascular endothelium” vascular hierarchy and intra-organ classification.
  • vascular ECs produce a large number of regulatory factors to regulate the communication between hematopoietic cells, mesenchymal cells and parenchymal cells (communication, which can also be understood as “information transfer” in this paper).
  • Abnormal changes in sinusoidal endothelial cells (SECs), such as capillary vascularization, are closely related to liver fibrosis.
  • SECs sinusoidal endothelial cells
  • the functional role of hepatic EC subsets on human cirrhosis or NASH pathology has not been systematically elucidated at the single-cell level in current clinical and preclinical models.
  • the present invention provides the application of HDAC2 inhibitor and DNMT1 inhibitor in combined targeted treatment of nonalcoholic steatohepatitis.
  • nonalcoholic steatohepatitis is accompanied by liver cirrhosis or liver fibrosis.
  • the pathological grades of liver fibrosis include F2-F4 grades.
  • the HDAC2 inhibitor is Mocetinostat, and the dosage is 1-20 mg/kg/day or 0.1-2.0 mg/kg/day (preferably 1.1 mg/kg/day).
  • the DNMT1 inhibitor is azacitidine, and the dosage is 0.1-2.0 mg/kg/day or 0.001-0.100 mg/kg/day (preferably 0.055 mg/kg/day).
  • the HDAC2 inhibitor and the DNMT1 inhibitor are administered by injection, and the injection includes one or more of intraperitoneal injection, intramuscular injection, subcutaneous injection or intravenous injection.
  • the specific operation of administration by injection is as follows: injection of DNMT1 inhibitor for the first 5 days of the first week, once a day, and then drug withdrawal for 2 days; injection of HADC2 inhibitor for the first 5 days of the second week, once a day, Then the drug is stopped for 2 days, and the treatment is repeated for 5-10 courses of treatment (preferably 6 courses of treatment).
  • the combined targeted use of the HDAC2 inhibitor and the DNMT1 inhibitor reduces the degree of fibrosis in the liver of nonalcoholic steatohepatitis and promotes liver regeneration; and/or reverses the sinus endothelium-macrovascular endothelial disorder in the liver of cirrhosis; and/or decrease the recruitment of pro-fibrotic Th17 cells in the liver of nonalcoholic steatohepatitis.
  • the combined targeted use of the HDAC2 inhibitor and the DNMT1 inhibitor reduces blood sugar, liver fibrosis index and/or serum liver function index.
  • the combined targeted use of the HDAC2 inhibitor and the DNMT1 inhibitor reduces serum total cholesterol levels.
  • the combined targeted use of the HDAC2 inhibitor and the DNMT1 inhibitor alleviates liver cirrhosis and increases hepatocyte proliferation.
  • the combined targeted use of the HDAC2 inhibitor and DNMT1 inhibitor blocked the increase of IGFBP7 and ADAMTS1 in cirrhotic liver.
  • the present invention provides the application of HDAC2 inhibitor and DNMT1 inhibitor in combined targeted treatment of nonalcoholic steatohepatitis.
  • the technical solution of the present invention reveals through a series of experiments: epigenetic changes in liver endothelial cell subsets, that is, abnormal activation of HDAC2 and DNMT1 (wherein, the expression of HDAC2 and DNMT1 changes significantly in the liver of F2-F4 patients; liver
  • the expression of HDAC2 and DNMT1 is significantly upregulated in endothelial cells of cirrhotic patient liver and cirrhotic minipig liver), which leads to "sinusoidal endothelium-macrovascular endothelial dysregulation", stimulates the production of pro-fibrotic IGFBP7 and ADAMTS1 in extracellular vesicles, and recruits Th17 cells, thereby inhibiting liver regeneration and inducing fibrosis in NASH.
  • HDAC2 and DNMT1 Targeted inhibition of HDAC2 and DNMT1 in minipig and mouse models of NASH normalized epigenetic changes in liver endothelial cells, blocked the expression of IGFBP7 and ADAMTS1, and inhibited the recruitment of Th17 cells to a certain extent.
  • the technical solution of the present invention reduces the fibrosis degree of NASH liver and promotes liver regeneration to a certain extent by using HDAC2 inhibitor and DNMT1 inhibitor to jointly target HDAC2 and DNMT1: for example, in the minipig NASH model in Example 4 , minipigs treated with a combination of an HDAC2 inhibitor (eg, Mocetinostat) and a DNMT1 inhibitor (eg, azacitidine) compared to controls: (i) their blood glucose, liver fibrosis index, Serum liver function index, serum total cholesterol were significantly decreased; (ii) liver cirrhosis was reduced, collagen deposition and lipid accumulation were reduced, and hepatocyte proliferation was increased.
  • HDAC2 inhibitor eg, Mocetinostat
  • a DNMT1 inhibitor eg, azacitidine
  • Example 5 cirrhotic minipigs treated with a combination of an HDAC2 inhibitor (eg, Mocetinostat) and a DNMT1 inhibitor (eg, azacitidine) significantly reversed NASH-induced NASH in the treatment group.
  • HDAC2 inhibitor eg, Mocetinostat
  • DNMT1 inhibitor eg, azacitidine
  • the technical solution of the present invention by using HDAC2 inhibitor and DNMT1 inhibitor to target HDAC2 and DNMT1 in combination, the recruitment of pro-fibrotic Th17 cells in the NASH liver of the treatment group is significantly reduced: for example, in Example 6 and Example 7 , in cirrhotic minipigs treated with a combination of HDAC2 inhibitors (eg, Mocetinostat) and DNMT1 inhibitors (eg, azacitidine), the expression of IGFBP7 and ADAMTS1 in sinus endothelial cells (SECs) of the treatment group The increase in mRNA and protein levels was effectively blocked, and the increase in the concentration of IGFBP7/ADAMTS1 in plasma extracellular vesicles (EVs) was also reduced; In cirrhotic minipig and mouse models of NASH treated with a combination of (eg, Mocetinostat) and a DNMT1 inhibitor (eg, azacitidine), the increase in Th17 cell numbers in the treatment group was effectively blocked.
  • the present invention provides a marker group for evaluating nonalcoholic steatohepatitis, characterized in that, the marker group includes IGFBP7 and ADAMTS1; the expression level of the marker group can be used to evaluate liver Degree of fibrosis and/or impaired liver function.
  • the marker panel can evaluate liver cirrhosis and liver fibrosis without impaired liver function.
  • liver fibrosis includes pathological grades F2-F4.
  • the marker group can also be used to evaluate the response of the pro-fibrotic Th17 signaling pathway.
  • the present invention also provides a kit comprising a marker group for evaluating nonalcoholic steatohepatitis, characterized in that the marker group includes IGFBP7 and ADAMTS1;
  • the expression levels of the above-mentioned marker groups can be used to evaluate the degree of liver fibrosis and/or the impairment of liver function.
  • kit can be used to detect the expression levels of IGFBP7 and ADAMTS1 in plasma.
  • kit can be used to detect the protein expression levels of IGFBP7 and ADAMTS1.
  • the present invention also provides a marker group for distinguishing nonalcoholic steatohepatitis and simple fatty liver, characterized in that, the marker group includes IGFBP7 and ADAMTS1.
  • the present invention provides a kit comprising a marker set for distinguishing between nonalcoholic steatohepatitis and simple fatty liver, characterized in that the marker set includes IGFBP7 and ADAMTS1.
  • kit can be used to detect the expression levels of IGFBP7 and ADAMTS1 in plasma.
  • kit can be used to distinguish the expression levels of IGFBP7 and ADAMTS1 in the plasma of healthy people and patients with liver cirrhosis and liver fibrosis.
  • the present invention provides a marker panel for evaluating nonalcoholic steatohepatitis and a kit comprising the marker panel.
  • the technical solution of the present invention reveals through a series of experiments that epigenetic changes in liver endothelial cell subsets can lead to "sinus endothelium-macrovascular endothelial dysregulation" and stimulate the reprogramming of its related vascular secretion factors; in the vascular secretion factor genes , IGFBP7 and ADAMTS1 are specifically expressed at relatively highest levels in human and minipig ECs and can promote pro-fibrotic Th17 responses to promote liver fibrosis and cirrhosis.
  • the plasma concentrations of IGFBP7 and ADAMTS1 can be used to assess nonalcoholic steatohepatitis (NASH).
  • NASH nonalcoholic steatohepatitis
  • liver biopsy and detection of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels are the main methods for assessing NASH.
  • liver biopsy is invasive, may cause pain, bleeding and other problems, and cannot be applied to all people; and the detection of ALT and AST levels is neither sensitive nor specific, and some patients with liver cirrhosis/NASH may have normal ALT or AST levels .
  • IGFBP7 and ADAMTS1 can be used to evaluate liver cirrhosis and liver fibrosis without impaired liver function.
  • Example 6 the expression of IGFBP7 and ADAMTS1 was gradually increased in the fibrotic liver of F2-F4 patients, and the mRNA and protein levels of IGFBP7 and ADAMTS1 in human and minipig cirrhotic sinusoidal endothelial cells (SECs) Both were significantly elevated, i.e., normal and dysregulated SECs could be effectively distinguished based on the up-regulation of IGFBP7 and ADAMTS1 expression.
  • SECs sinusoidal endothelial cells
  • the plasma concentrations of IGFBP7 and ADAMTS1 were significantly higher in cirrhotic/fibrotic patients with normal ALT or AST than in healthy individuals.
  • the technical solution of the present invention found through a series of experiments that, for example, in Examples 8, 10 and 11, IGFBP7 and ADAMTS1 enhanced the pro-fibrotic Th17 response. Therefore, the marker panel for assessing nonalcoholic steatohepatitis and the kit comprising the marker panel provided by the present invention contribute to a reliable, sensitive, simple and non-invasive method in the absence of impaired liver function. Assess for cirrhosis and fibrosis.
  • the present invention also provides a marker panel for distinguishing nonalcoholic steatohepatitis from simple fatty liver and a kit comprising the marker panel.
  • the technical solution of the present invention reveals through a series of experiments that plasma IGFBP7 and ADAMTS1 can be used to distinguish nonalcoholic steatohepatitis from simple fatty liver: the plasma concentrations of IGFBP7 and ADAMTS1 in patients with cirrhosis/hepatic fibrosis with normal ALT or AST are significantly higher than In healthy human samples, there was no significant increase in plasma ALT or AST concentrations in patients with early stage NASH (F0-F1), while plasma IGFBP7 and ADAMTS1 concentrations in NASH patients were significantly increased, and plasma IGFBP7 and ADAMTS1 concentrations in patients with simple fatty liver were statistically Not elevated.
  • the marker group for distinguishing non-alcoholic steatohepatitis and simple fatty liver and the kit comprising the marker group provided by the present invention are helpful for distinguishing non-alcoholic steatohepatitis reliably, sensitively, easily and non-invasively. steatohepatitis and simple fatty liver.
  • the marker panel for evaluating nonalcoholic steatohepatitis and the kit comprising the marker panel provided by the present invention are helpful for reliable, sensitive, simple and non-invasive evaluation nonalcoholic steatohepatitis.
  • the present invention provides the use of vascular secretion factors in the preparation of biomarkers for the detection of nonalcoholic steatohepatitis, characterized in that, the vascular secretion factors include IGFBP7 and/or ADAMTS1; the IGFBP7 and/or ADAMTS1 is expressed in the HDAC2/DNMT1-IGFBP7/ADAMTS1-Th17 signaling pathway.
  • the mRNA expression and/or protein expression of IGFBP7 and/or ADAMTS1 in the HDAC2/DNMT1-IGFBP7/ADAMTS1-Th17 signaling pathway can be used to evaluate the degree of liver fibrosis and/or liver function impairment.
  • the increase in the expression of IGFBP7 and/or ADAMTS1 is proportional to the degree of liver fibrosis, and the degree of liver fibrosis includes pathological grades F2-F4.
  • IGFBP7 and/or ADAMTS1 are present in plasma.
  • the present invention also provides the application of vascular secretion factors in preparing biomarkers for distinguishing nonalcoholic steatohepatitis and simple fatty liver, characterized in that, the vascular secretion factors include IGFBP7 and/or ADAMTS1; the IGFBP7 and /or ADAMTS1 is expressed in HDAC2/DNMT1-IGFBP7/ADAMTS1-Th17 signaling pathway.
  • the mRNA expression and/or protein expression of IGFBP7 and/or ADAMTS1 in the HDAC2/DNMT1-IGFBP7/ADAMTS1-Th17 signaling pathway can distinguish nonalcoholic steatohepatitis from simple fatty liver.
  • the present invention also provides the use of IGFBP7 and/or ADAMTS1 synergistic effect in preparing a biomarker for detecting nonalcoholic steatohepatitis.
  • nonalcoholic steatohepatitis includes liver cirrhosis or liver fibrosis.
  • non-alcoholic steatohepatitis also includes the case of no liver fibrosis, and the pathological grade of the no fibrosis is F0-F1.
  • the IGFBP7 and/or ADAMTS1 induces a pro-fibrotic Th17 cell response.
  • Th17 cells aggregated in fibrotic livers.
  • the present invention provides the application of vascular secreted factors in the preparation of biomarkers for detecting nonalcoholic steatohepatitis.
  • the technical solution of the present invention reveals through a series of experiments that the HDAC2/DNMT1-IGFBP7/ADAMTS1-Th17 signaling pathway exists in the dysregulated liver endothelial cell subsets, wherein the interactive regulation between HDAC2/DNMT1 will stimulate the pro-fibrotic IGFBP7 and Production of ADAMTS1, thereby recruiting Th17 cells, inhibits liver regeneration and induces fibrosis in nonalcoholic steatohepatitis (NASH).
  • NASH nonalcoholic steatohepatitis
  • IGFBP7 and/or ADAMTS1 are expressed in the HDAC2/DNMT1-IGFBP7/ADAMTS1-Th17 signaling pathway.
  • IGFBP7 and ADAMTS1 are regulators that enhance Th17 responses to promote liver fibrosis: For example, in Example 10, combined targeting of endothelial-derived HDAC2 and DNMT1 reduced IGFBP7 expression in fibrotic liver endothelial cells; gene knockout NASH phenotype mice with IGFBP7 significantly reduced liver fibrotic responses (collagen deposition, serum liver function index, liver hydroxyproline content) and Th17 responses; elevated IGFBP7 levels significantly enhanced hepatic pro-fibrotic responses Th17 response.
  • ADAMTS1 was detected in Hdac2 i ⁇ EC + AZA mice (i.e., NASH phenotype mice with selective knockout of endothelial HDAC2 and treated with a DNMT1 inhibitor to obtain the inhibitory effect of combined targeting of HDAC2 + DNMT1).
  • the technical solution of the present invention has found through a series of experiments that the mRNA expression and/or protein expression of IGFBP7 and/or ADAMTS1 in the HDAC2/DNMT1-IGFBP7/ADAMTS1-Th17 signaling pathway can be used to evaluate the degree of liver fibrosis and the influence of liver function. damage situation.
  • the expressions of IGFBP7 and ADAMTS1 were gradually increased in the fibrotic livers of F2-F4 patients, and the mRNA and protein levels of IGFBP7 and ADAMTS1 in human and minipig cirrhotic sinusoidal endothelial cells were significantly increased.
  • IGFBP7 and ADAMTS1 can be used to assess liver cirrhosis and liver fibrosis without impaired liver function.
  • the present invention also provides the application of the vascular secretory factor in preparing a biomarker for distinguishing between nonalcoholic steatohepatitis and simple fatty liver.
  • the technical solution of the present invention revealed through a series of experiments that IGFBP7 and ADAMTS1 can be used to distinguish nonalcoholic steatohepatitis from simple fatty liver: the plasma ALT or AST concentrations of patients with early stage NASH (F0-F1) did not increase significantly, while patients with NASH Plasma concentrations of IGFBP7 and ADAMTS1 were significantly elevated, and plasma IGFBP7 and ADAMTS1 concentrations were not statistically elevated in patients with simple fatty liver.
  • the present invention also provides the use of IGFBP7 and/or ADAMTS1 synergistic effect in the preparation of biomarkers for detecting nonalcoholic steatohepatitis.
  • Nonalcoholic steatohepatitis is often associated with cirrhosis or fibrosis.
  • IGFBP7 and ADAMTS1 cooperate to detect nonalcoholic steatohepatitis, including but not limited to assessing cirrhosis or fibrosis, distinguishing Nonalcoholic steatohepatitis and simple fatty liver and assessment of Th17 cell accumulation in fibrotic livers.
  • the application of the vascular secretion factor provided in the present invention in preparing a biomarker for detecting non-alcoholic steatohepatitis can reliably, simply and non-invasively evaluate non-alcoholic steatohepatitis.
  • Figure 1 is an experimental image of single-cell RNA sequencing (scRNA-Seq) revealing HDAC2/DNMT1-selectively induced "sinusoidal endothelial-macrovascular endothelial dysregulation" in human cirrhotic liver;
  • Figure 2 is an experimental graph of the combined targeted inhibition of HDAC2 and DNMT1 in hepatic endothelial cells alleviating liver fibrosis in a minipig NASH model;
  • Figure 3 is an experimental diagram of targeting the inhibition of epigenetic dysregulated liver endothelial cells in a minipig NASH model to reverse "sinusoidal endothelium-macrovascular endothelial dysregulation", normalize endothelial classification, and block liver cirrhosis;
  • Figure 4 is an experimental graph of the reprogramming of paracrine/vascular secretory factors in epigenetically dysregulated liver endothelial cells in human patients and minipigs;
  • Figure 5 is an experimental graph of the paracrine/vascular secretory factors IGFBP7 and ADAMTS1 in plasma extracellular vesicles (EVs) as biomarkers for assessing fibrosis progression in human patients and minipig NASH models;
  • Figure 6 is an experimental graph of a dysregulated vascular endothelial microenvironment inducing a pro-fibrotic Th17 response in a human patient and a minipig NASH model;
  • Figure 7 is a graph showing that inhibition of HDAC2/DNMT1 reduces the recruitment of pro-fibrotic Th17 cells in dysregulated liver sinusoidal endothelial cells (SECs) in a mouse model of NASH;
  • Figure 8 is an experimental diagram of IGFBP7 promoting liver fibrosis and pro-fibrotic Th17 responses in a mouse NASH model
  • Figure 9 is an experimental graph showing that inhibition of ADAMTS1 alleviates the pro-fibrotic Th17 cell response in a mouse liver fibrosis model
  • FIG 10 is an experimental diagram of single-cell RNA sequencing (scRNA-Seq) analysis of hepatic non-parenchymal cells (NPCs) from 2 healthy individuals and 2 patients with liver cirrhosis;
  • scRNA-Seq single-cell RNA sequencing
  • Figure 11 is an experimental graph of scRNA-Seq analysis of human liver NPCs from GSE136103 data;
  • Figure 12 is an experimental diagram of endothelial cells (ECs) analysis in human liver scRNA-Seq data
  • Figure 13 is an experimental graph of gene expression analysis of human liver, liver CD45 + NPCs and liver endothelial cells;
  • Figure 14 is an experimental diagram of scRNA-Seq analysis of minipig liver NPCs
  • Figure 15 shows endothelial cells (ECs) analysis in minipig liver scRNA-Seq data
  • Figure 16 is an experimental diagram of differential gene analysis of liver cirrhosis between human and minipigs and expression of paracrine factor genes in liver NPCs;
  • Figure 17 is an experimental graph of Th17 cell analysis in human and minipig livers.
  • the term "about” is typically expressed as +/- 5% of the stated value, more typically +/- 4% of the stated value, and more typically + /-3%, more typically +/-2% of said value, even more typically +/-1% of said value, even more typically +/-0.5% of said value.
  • the "interactive regulation” described in the present invention means that liver fibrosis is regulated by both HDAC2 and DNMT1, and one of HDAC2 or DNMT1 is inhibited alone, and the other (DNMT1 or HDAC2) changes in the direction of pro-fibrosis (increased ).
  • HDAC2 in endothelial cells increases the expression of DNMT1
  • inhibition of DNMT1 increases the expression of HDAC2
  • liver fibrosis is affected by the interactive regulation of HDAC2 and DNMT1.
  • vascular microenvironment in the present invention refers to: vascular endothelial cells actively regulate the function and phenotype of surrounding cells through interaction with surrounding cells, thereby forming a guiding microenvironment.
  • disorder in the present invention refers to changes in the proportion of endothelial cell subsets or changes in gene expression in cirrhotic livers compared to normal livers.
  • the "sinusoidal endothelium-macrovascular endothelium disorder” or “sinusoidal endothelium-macrovascular endothelium classification abnormality" in the present invention refers to: compared with normal livers, the number of sinusoidal endothelial cells in cirrhotic livers decreases, while the number of macrovascular endothelial cells increases , a pathological phenomenon in which sinus endothelial cells express macrovascular endothelial markers.
  • the "epigenetic therapy” in the present invention refers to treating liver cirrhosis or fibrosis miniature pigs and mice with epigenetic inhibitors, thereby reducing liver fibrosis.
  • Figure 1 (A) Method for scRNA-Seq of hepatic non-parenchymal cells (NPCs) from human patients at West China Hospital. (B,C) Cluster analysis of scRNA-Seq data from 2 healthy livers and 2 cirrhotic liver NPCs. (B) Heat map showing different cell lines of NPCs and their marker genes (right). (C) UMAP plot showing different cell lines of NPCs. Endo (EC), endothelial cells; DC, dendritic cells; Neu, neutrophils; Mac, macrophages; EPCAM + , EPCAM + cells and cholangiocytes. (D) Pie chart showing the number of differential genes in different cell lines of NPCs (cirrhotic liver vs. healthy liver).
  • NPCs hepatic non-parenchymal cells
  • HDACs histone deacetylases
  • DNMTs DNA methyltransferases
  • F0 F0
  • M Violin plot showing the expression of DNMTs in healthy and cirrhotic human liver endothelial cells.
  • FIG. 2 (A) Minipig NASH model and treatment regimen.
  • the minipig NASH model was induced by a Western diet (WD: high fat, high cholesterol, high sucrose and fructose) and repeated intraperitoneal injections of hepatotoxic carbon tetrachloride ( CCl4 ).
  • Treatment group Induction of NASH by WD+CCl 4 continued for 5 months, and epigenetic therapy was started after 2 months of induction for 3 months.
  • Cirrhosis group Minipigs were induced with WD+CCl 4 continuously for 5 months.
  • B Dosing regimen for combined inhibition of HDAC2 and DNMT1 in a minipig NASH model.
  • HDAC2i HDAC2 inhibitor
  • HDAC2i DNMT1 inhibitor
  • the minipigs were divided into 3 groups: 1) control group (normal diet + corn oil); 2) liver cirrhosis group (model group) (WD+ CCl4 induced NASH) 3) treatment group (HDAC2i and DNMT1i treatment).
  • D Liver fibrosis index of liver tissue of control, cirrhosis and treatment group minipigs.
  • PC III procollagen type III
  • IV-C collagen type IV
  • HA hyaluronic acid
  • (E) Serum liver function levels of minipigs in the control, cirrhosis and treatment groups. ALP, alkaline phosphatase; ALT, alanine aminotransferase; AST, aspartate aminotransferase; TC, total cholesterol. N 3.
  • (F) Assessment of liver histology, collagen and lipid droplet deposition, and cell proliferation in minipigs by H&E, Sirius Red, Collagen I, Oil Red O, and Ki67 staining. Right: High magnification of the dotted area in the left panel. Scale bar, 200 ⁇ m. N 3.
  • N 3.
  • H qPCR showing the mRNA expression levels of HDAC2 and DNMT1 in liver sinusoidal endothelial cells (SECs) and macrovascular endothelial cells (MECs) of control, cirrhosis, and treatment minipigs.
  • SECs sinusoidal endothelial cells
  • MECs macrovascular endothelial cells
  • N 3.
  • I Schematic illustration of targeted pro-fibrotic epigenetic cross-regulation in a minipig NASH model: combined targeting and inhibition of aberrantly activated HDAC2 and DNMT1 in dysregulated liver endothelial cells attenuates liver fibrosis and promotes hepatic fibrosis in a minipig NASH model Liver regeneration.
  • liver cirrhosis group vs control group #, treatment group vs liver cirrhosis group; *, P ⁇ 0.05; **, P ⁇ 0.01. #, P ⁇ 0.05;##,P ⁇ 0.01.
  • Figure 3 (A) Method for single-cell sequencing of liver non-parenchymal cells (NPCs) in a minipig NASH model.
  • B,C Cluster analysis of non-parenchymal cells (NPCs) in the livers of control, cirrhosis, and treatment minipigs.
  • B Heat map showing different cell lines of NPCs and their marker genes (right).
  • C UMAP plot showing different cell lines of NPCs. Endo (EC), endothelial cells; DC, dendritic cells; Neu, neutrophils; Mac, macrophages; EPCAM + , EPCAM + cells and cholangiocytes.
  • D Pie chart showing the proportion of different cell lines of non-parenchymal cells (NPCs) of minipig liver.
  • E Venn diagram showing the number of differential genes in different cell lines of non-parenchymal cells (NPCs) of minipig livers between cirrhosis and control groups and between treatment and cirrhosis groups, respectively. Numbers in parentheses indicate the number of genes recovered after treatment.
  • F KEGG pathway enrichment analysis in minipig liver endothelial cells. There were 79 KEGG pathways significantly changed between the liver endothelial cells of the cirrhosis group and the control group, and 99 KEGG pathways were significantly changed between the treatment group and the cirrhosis group.
  • HDAC2i+DNMT1i treatment reversed the "sinusoidal-macrovascular endothelial dysregulation" in cirrhotic minipig livers.
  • K Abnormally targeted HDAC2/DNMT1 in cirrhotic minipig liver reverses "sinusoidal endothelium-macrovascular endothelial dysregulation", enhances regeneration, and reduces fibrosis.
  • Figure 4 (A) Differential expression of paracrine/vascular secretory factor genes in cirrhotic human liver endothelial cells. Heat map showing representative vascular secreted factors such as Ephrin/Eph, Notch, insulin growth factor-related protein, ADAM/ADAMTS, and Semaphorin/Plexin families. (B) Paracrine/vascular secretory factor gene expression in human livers with different grades of fibrosis (data from GSE84044). F0-F4, different pathological grades of human liver fibrosis. Expression levels of paracrine/vascular secretory factor genes in F1-F4 grade livers were quantified relative to healthy livers (F0).
  • C Violin plots showing the expression of representative paracrine/vascular secretory factor genes in different cell lines of human and minipig liver non-parenchymal cells (NPCs). IGFBP7 and ADAMTS1 are highly expressed in human and minipig liver endothelial cells.
  • D Schematic representation of the reprogramming of paracrine/vasculature factors associated with "sinus endothelium-macrovascular endothelial dysregulation" including induction of IGFBP7 and ADAMTS1 in endothelial cells.
  • E Expression levels of IGFBP7 in total endothelial cells, different subsets of endothelial cells, and sinusoidal endothelial cells in healthy and cirrhotic human livers.
  • H,I qPCR (H) and ELISA (I) showed the expression of IGFBP7 in liver sinusoidal endothelial cells (SECs) and macrovascular endothelial cells (MECs) of minipigs in control, cirrhosis and treatment groups.
  • N 3.
  • J ADAMTS1 expression in total endothelial cells and different subsets of endothelial cells in control, cirrhosis, and treatment minipig livers.
  • K,L qPCR (K) and ELISA (L) showed ADAMTS1 expression in liver sinusoidal endothelial cells (SECs) and macrovascular endothelial cells (MECs) in control, cirrhosis and treatment groups.
  • N 3.
  • FIG. 5 (A) Plasma concentrations of IGFBP7, ADAMTS1, ALT, AST in healthy and cirrhotic/fibrotic patients. (B,C) Plasma concentrations of IGFBP7 and ADAMTS1 in cirrhosis/fibrosis patients with normal or abnormal liver function. Cirrhosis/fibrosis patients were divided into two groups (B): normal ALT/AST concentration and abnormal ALT/AST concentration. (D) Plasma concentrations of IGFBP7 and ADAMTS in healthy and cirrhotic/fibrotic patients with different predispositions.
  • NASH nonalcoholic steatohepatitis-related cirrhosis/fibrosis
  • HBC hepatitis B-related cirrhosis/fibrosis
  • AIH autoimmune Hepatitis-associated cirrhosis/fibrosis
  • PBC primary biliary cirrhosis/fibrosis
  • CC cryptogenic cirrhosis/fibrosis
  • E ALT and AST plasma concentrations in healthy and different pathological grades of NASH patients.
  • F Plasma concentrations of IGFBP7 and ADAMTS1 in healthy, simple fatty liver and NASH patients with different pathological grades.
  • H IGFBP7/ADAMTS1 concentrations in plasma extracellular vesicles (EVs) of minipigs and humans.
  • I,J IGFBP7/ADAMTS1 concentrations in EVs from cirrhotic patients with normal or abnormal liver function (ALT/AST) (I) or NASH patients with different fibrosis grades (J).
  • FIG. 6 (A) Cellular interaction analysis based on receptor ligand profiles of different cell lines of human and minipig livers (NPCs): Liver endothelial cells of patients with cirrhosis and minipigs interact significantly with T cells. (B) Western blot showing protein levels of phosphorylated Smad2 in healthy and cirrhotic CD45 + liver NPCs. Levels of phosphorylated Smad2 were quantified relative to total Smad2. The results showed TGF- ⁇ 1-Smad2 activation in CD45 + NPCs of cirrhotic human livers. Top: quantification of protein expression; bottom: representative protein bands. Data were analyzed by two-tailed Student's t-test and presented as mean ⁇ SEM.
  • C Cluster analysis of CD4 + T cells in healthy and cirrhotic human livers.
  • D Expression of Th17 + marker genes in T cell clusters in healthy and cirrhotic human livers.
  • E Proportion of Th17 cells in healthy and cirrhotic human liver NPCs. The proportion of Th17 cells in cirrhotic human livers was quantified relative to healthy individuals.
  • F,G Cluster analysis of T cells and CD4 + T cells in the livers of control, cirrhosis, and treatment minipigs.
  • H Expression of Th17 + marker genes in minipig liver CD4 + T cell clusters.
  • FIG. 7 (A) Schematic diagram of NASH model and treatment in Hdac2 endothelial-specific knockout mice.
  • Endothelial-specific Hdac2 knockout mice (Hdac2 i ⁇ EC ) were obtained by crossing endothelial-specific cre mice with Hdac2flox mice.
  • Hdac2 i ⁇ EC mice were also treated with the DNMT1 inhibitor azacitidine (AZA) (Hdac2 i ⁇ EC + AZA). Liver fibrosis, liver function and enrichment of Th17 cells were then analyzed.
  • AZA DNMT1 inhibitor azacitidine
  • (D) Protein levels of HDAC2 and DNMT1 in liver SECs of mice. Top: quantification of protein expression; bottom: representative western blot images. N 3.
  • (E, F) Co-staining of CD34 (green) and desmin (red) (E) or Lyve1 (red) and CD34 (green) (F) of mouse liver sections. The proportion of CD34-positive staining area in panel F was quantified relative to the control group. Scale bar, 20 ⁇ m. N 5.
  • (G) Flow cytometric analysis of Th17 cell numbers in mice. Targeted inhibition of HDAC2 and DNMT1 in endothelial cells blocks Th17 cell responses in a mouse model of NASH. Right: Percentage of Th17 cells. N 5.
  • (E) Flow cytometric analysis of the percentage of Th17+ cells in the liver of Igfbp7 -/- mice. N 6.
  • C Schematic diagram of the method for transplantation of human endothelial cell-derived extracellular vesicles (EVs) into mice. Recipient mice were repeatedly injected with CCl to induce liver fibrosis.
  • EVs extracellular vesicles
  • shADAMTS1-transduced HUVECs were treated with TGF- ⁇ for 2 days.
  • EVs were isolated from culture medium of HUVECs transduced with shADAMTS1 or shNC (control) and transplanted into liver fibrotic mice, respectively. Fibrotic responses were compared between recipient mice transplanted with shADAMTS1 or shNC EVs.
  • FIG. 1 Schematic illustration of the "endothelial HDAC2/DNMT1-IGFBP7/ADAMTS1-Th17" axis promoting liver fibrosis in NASH.
  • Epigenetic reprogramming of hepatic endothelial cells (ECs) leads to "sinusoidal-macrovascular endothelial dysregulation" and promotes the production of profibrotic IGFBP7/ADAMTS1 in EVs.
  • IGFBP7/ADAMTS1 from epigenetically dysregulated SECs stimulates profibrotic Th17 cell responses.
  • data were analyzed by two-tailed Student's t-test and presented as mean ⁇ SEM. *, shADAMTS1 or Hdac2 i ⁇ EC + AZA vs. control. *, P ⁇ 0.05; **, P ⁇ 0.01.
  • Figure 10 (A) Clustering of 22,374 hepatic non-parenchymal cells (NPCs) from 2 healthy and 2 cirrhotic patient livers. Left: total NPCs; right: healthy and cirrhotic NPCs. (B) UMAP plot showing cluster analysis of liver NPCs for each sample, respectively. (C) Proportions of different NPCs cell lines in healthy and cirrhotic human livers. EC, endothelial cell; DC, dendritic cell; Neu, neutrophil; Mac, macrophage; EPCAM + , EPCAM + cell and cholangiocyte. (D) Cluster analysis of liver ECs from 2 healthy and 2 cirrhotic patients. (E) Display of different subpopulations of healthy and cirrhotic ECs.
  • NPCs hepatic non-parenchymal cells
  • Figure 11 (A) Cluster analysis of 42,314 NPCs from 4 healthy and 3 cirrhotic human livers. CD45 + and CD45- NPCs were flow - sorted for scRNA-Seq analysis. Left: total NPCs; right: healthy and cirrhotic NPCs. (B) Heat map showing NPCs clustering marker genes and their marked cell lines (right). (C) UMAP plot showing different cell lines of NPCs. EC, endothelial cell; DC, dendritic cell; Neu, neutrophil; Mac, macrophage; EPCAM + , EPCAM + cell and cholangiocyte. (D) Cluster analysis of 4 healthy and 3 cirrhotic human liver ECs.
  • E UMAP plot showing different subsets of ECs. Left: total ECs; right: healthy and cirrhotic ECs.
  • F UMAP plot showing selected marker gene expression in endothelial cells.
  • G Pie chart showing the proportion of ECs subsets.
  • H Pseudo-chronological analysis of distinct subsets of healthy and cirrhotic human liver ECs.
  • Figure 12 (A,B) Heat maps showing endothelial and mesenchymal cell marker gene expression in different cell lines of human total NPCs (A) and cirrhosis patient NPCs (B). (C) GSEA enrichment analysis of mesenchymal differentiation in healthy and cirrhotic human liver endothelial cells. (D) Schematic illustration of possible "sinusoidal endothelium-macrovascular endothelial dysregulation" in a cirrhotic human liver.
  • Figure 14 (A) Cluster analysis of 40,570 NPCs from 1 control, 2 cirrhotic and 2 treated minipig livers. (B) GO enrichment analysis of minipig liver ECs. Epigenetic treatment of HDAC2i and DNMT2i restored most of the altered functions of liver ECs in a minipig NASH model.
  • Figure 15 (A) Cluster analysis of minipig liver ECs in 1 control group, 2 cirrhosis groups and 2 treatment groups; (B) expression of selected marker genes in ECs. (C) UMAP plot showing different subpopulations of ECs in control, sclerotic, and treated groups. (D,E) Heat maps showing genes associated with epigenetic changes (histone modifications and DNA methylation) in different cell lines (D) and ECs of control, cirrhosis and treated minipig liver NPCs Expression of different subpopulations (E). (F) Venn diagrams showing the number of differential genes in different cell lines or subpopulations (D and E) of minipig liver NPCs between cirrhosis and control groups and between treatment and cirrhosis groups, respectively. Red numbers represent the number of genes recovered.
  • Figure 17 (A) Cluster analysis of T cells in 2 healthy and 2 cirrhotic human livers. (B) UMAP plot showing different cell lineages (CD8 + and CD4 + cells) of human T cells. (CI) scRNA-Seq analysis of human CD45 + cells from GSE136103 data. (C) Cluster analysis of 35,806 CD45 + cells from 5 healthy and 5 cirrhotic patient livers. (D) UMAP plot showing different cell lines of NPCs. EC, endothelial cell; DC, dendritic cell; Neu, neutrophil; Mac, macrophage; EPCAM + , EPCAM + cell and cholangiocyte. (E) UMAP plot showing expression of T cell marker genes in CD45 + cells.
  • G UMAP plot showing different cell lineages (CD8 + and CD4 + cells) of human T cells.
  • FG T cell data derived from GSE136103.
  • H Violin plot showing expression of Th17 + marker genes in different populations of T cells.
  • I Proportion of Th17 cells in CD45 + cells of 5 healthy and 5 cirrhotic patients.
  • HI is Th17 cell data derived from GSE136103.
  • Fibrosis grades in non-NASH livers were determined by Fibrosis-4 Index (Fib-4) and Transient Elastography (TE) using the NAFLD Fibrosis Index (NFS), Fib-4, TE and controlled attenuation parameters (CAP) to determine the grade of fibrosis and steatosis in NASH livers.
  • Fibrosis-4 Index Fibrosis-4 Index
  • TE Transient Elastography
  • NFS NAFLD Fibrosis Index
  • CAP controlled attenuation parameters
  • TBIL total bilirubin
  • DBIL direct bilirubin
  • IBIL indirect bilirubin
  • AST aspartate aminotransferase
  • ALT alanine aminotransferase
  • ALP alkaline phosphatase
  • GGT glutamyl transpeptidase.
  • F0 or F4 different pathological grades of human liver fibrosis. #1 and #2 of healthy and cirrhotic livers were used for scRNA-Seq, and #1, #2 and #3 of healthy and cirrhotic livers were used for liquid chip analysis and gene expression analysis.
  • M male; F, female; NA, not applicable; PBC, primary biliary cirrhosis/fibrosis; AIH, autoimmune hepatitis-related cirrhosis/fibrosis; HBC, hepatitis B-related cirrhosis/ Liver fibrosis; CC, cryptogenic cirrhosis/fibrosis; NASH, nonalcoholic steatohepatitis-associated cirrhosis/fibrosis; F0-F4, different pathological grades of human liver fibrosis; Fib-4, fibrosis Tl-4 index; TE, transient elastography; NFS, NAFLD fibrosis index; CAP, controlled decay parameter; UDCA, ursodeoxycholic acid; MP, methylprednisolone; ETV, entecavir; TAF, replacement Nofovir alafenamide; TDF, tenofovir disoproxil; FNB, fenofibrate; SAMe, S-
  • Miniature pigs Male Bama minipigs were obtained from Chengdu Dossy Biological Technology Co., LTD. Minipigs were housed in individual cages at the Chengdu Dossy Experimental Animals Center and fed a diet containing 2% cholesterol and 30% fat by weight, supplemented with fructose and glucose. The experimental animal ethics committee of the West China Second Hospital of Sichuan University and Chengdu Dashuo Biotechnology Co., Ltd. approved the minipig experiments.
  • mice C57BL/6J mice were obtained from the Institute of Model Animals, Nanjing University.
  • C57BL/6J-Hdac2 em1(flox) Smoc mice were obtained from Shanghai Southern Model Biotechnology Co., Ltd. Igfbp7 -/- mice were described previously (ref. 94).
  • Mice expressing the EC-specific Cdh5-(PAC) -Cre ERT2 were provided by Ralf H. Adam (ref. 95).
  • Cdh5-(PAC) -Cre ERT2 mice were crossed with floxed Hdac2 mice to generate Hdac2 i ⁇ EC/i ⁇ EC mice (Hdac2 i ⁇ EC ).
  • Hdac2 i ⁇ EC mice were treated with tamoxifen (250 mg/kg) i.p. 6 times a day (with a 3-day interruption after the third dose) at two months after birth to induce Hdac2 endothelial-specific deletion.
  • Mice were housed in the SPF-level Experimental Animal Center of West China Second Hospital and fed on a standard 12-hour light/dark cycle. The animal experimental protocol was approved by the Experimental Animal Ethics Committee of the West China Second Hospital of Sichuan University.
  • HUVEC Human umbilical vein endothelial cells
  • SCME002, Millipore EndoGRO-VEGF complete medium
  • HEK293T was obtained from the American Type Culture Collection (ATCC).
  • HEK293T was cultured in DMEM (11965092, Gibco) containing 10% fetal bovine serum (FBS) (1600044, Gibco) at 37°C in a humidified environment with 5% CO2 .
  • NASH model 6-month-old male minipigs were fed a Western diet (Western diet, WD) that is 2% cholesterol, 30% fat and supplemented with high-sugar drinking water: 23.1g/L d-fructose and 18.9g/L d-glucose, The intraperitoneal injection dose was 0.1 ml/kg of carbon tetrachloride (CCl 4 ), twice a week, for 5 months. Control group minipigs were injected with CCl 4 vehicle ( corn oil). Serum samples were collected every 15 days, and plasma samples were collected monthly.
  • Mouse NASH model 8-week-old mice (control C57BL/6J, Hdac2 i ⁇ EC , and IGFBP7 -/- mice) were fed a Western diet of 21.1% fat, 41% sucrose, 1.25% cholesterol and supplemented with high-sugar drinking water: 23.1 g/ L fructose and 18.9 g/L glucose; intraperitoneal injection of CCl 4 at a dose of 0.5 ⁇ l/g (0.8 g/kg), once a week, for 3 months. Wild-type control mice were fed a normal diet and injected with corn oil. Two days after the last injection of CCl4 , all mice were sacrificed and serum and liver samples were collected.
  • Minipigs started treatment two months after CCl 4 injection: 2 weeks as a treatment course, the first week of treatment with the DNMT1 inhibitor Azacitidine (AZA): AZA (dose of 0.1- 2.0 mg/kg/day, but also 0.001-0.100 mg/kg/day, preferably 0.055 mg/kg/day), once a day, then discontinued for 2 days; HADC2 inhibitor Mocetinostat (MGCD0103) in the second week Treatment: Intraperitoneal injection of MGCD0103 (dose of 1-20 mg/kg/day, also 0.1-2.0 mg/kg/day, preferably 1.1 mg/kg/day) for the first 5 days, once a day, then discontinued for 2 days .
  • AZA Azacitidine
  • AZA dose of 0.1- 2.0 mg/kg/day, but also 0.001-0.100 mg/kg/day, preferably 0.055 mg/kg/day
  • MGCD0103 HADC2 inhibitor Mocetinostat
  • Hdac2 i ⁇ EC mice were injected with CCl 4 for one month and then intraperitoneally injected with AZA, once every two days, for two months.
  • Minipig surgical biopsy Minipigs underwent laparotomy for liver biopsy. After an overnight fast, sutai was given into the ear vein The minipigs were anesthetized and all operations were performed under general anesthesia. After laparotomy, a small piece of liver tissue was removed by blunt dissection technique. After hemostasis of the liver incision, a drainage tube was set and the abdominal cavity was sutured. Postoperatively, the minipigs were supplemented with glucose intravenously, and water and diet were allowed one day after the operation.
  • liver tissues were washed twice with cold PBS, minced, and incubated in digestion mix (1 mg/ml type I collagenase and 1 mg/ml type II neutral protease in PBS) at 37°C 30 minutes. After 30 minutes, the liver tissue was suspended to ensure complete dissociation. The digested liver sample is a homogeneous minimal block. Digested tissue was filtered through a cell strainer multiple times and cells were collected by centrifugation at 300 g for 5 minutes.
  • hepatocytes and NPCs were isolated by an additional centrifugation step at 4°C, 50 g for 5 min.
  • the hepatocytes at the bottom were used for LC-chip analysis and DNA methylation analysis, and the NPCs in the supernatant were used for LC-chip, DNA methylation, magnetic bead sorting and western blot.
  • ECs CD45 - CD31 +
  • MECs CD45 - CD34 +
  • SECs CD45 - CD34 - CD31 +
  • wash with 1 ml of pre-chilled MACS wash buffer 2 mM EDTA in DPBS, Dynabeads magnetic beads were washed 3 times with 0.1% BSA, 1% penicillin/streptomycin), and after 4 hours of incubation with CD45, CD34 or CD31 antibodies at 4°C, the beads were washed 3 times with MACS wash buffer.
  • NPCs were resuspended in 300 ⁇ l of MACS wash buffer, and 200 ⁇ l of Dynabeads-CD45 antibody conjugate was added, then incubated at 4° C. on a rotator for 45 minutes. After incubation, the CD45 + cell-bound magnetic beads were detached with a magnet and the supernatant was transferred to a tube containing Dynabeads-CD31 or -CD34 antibody conjugates.
  • Dynabeads-CD31 or -CD34 antibody conjugates were incubated with the collected supernatant for 45 min at 4°C on a rotator, and CD31 + CD45- cells ( ECs ) or CD34 + CD45- cells were isolated using a magnet Magnetic beads for cells (MECs).
  • MECs magnet Magnetic beads for cells
  • SECs isolation negative sorting was performed first using Dynabeads-CD45 and -CD34 antibody conjugates, followed by positive sorting using Dynabeads-CD31 antibody conjugates. Magnetic beads with CD45 + , CD31 + CD45 - , CD34 + CD45 - or CD31 + CD45 - CD34 - cells were washed 5 times with cold MACS wash buffer and used for subsequent experiments.
  • Minipig NPCs were incubated with Percp-Cy5.5-CD45, FITC-CD34 and PE-CD31 antibodies for 30 min, and minipig CD45 + NPCs, MECs (CD45 - CD34 + ) and SECs (CD45 - CD34 + ) were isolated by flow cytometry - CD31 + ). After washing, CD45 + cells, MECs and SECs were isolated by FACSAria TM III flow cytometer (BD Biosciences).
  • the OCT-embedded liver tissue was cut into 6 ⁇ m sections, fixed with 4% paraformaldehyde for 5 min, rinsed with PBS, and blocked with 10% donkey serum for 30 min at room temperature. Then, the membrane was permeabilized with 0.3% Triton X-100 in PBS for 20 minutes, and mixed with anti-Lyve1 (70R-LR003, Fitzgerad), anti-CD34 (ab81289, Abcam), anti-desmin (Ab15200, abcam), anti-type I Collagen (Ab34710, Abcam) or anti-Ki67 (b15580, Abcam) antibodies were incubated overnight at 4°C.
  • the number of positive cells (ki67) and the percentage of positively stained area (CD34) were calculated by comparing with the total number of cells or the area of positive staining in the selected area, respectively.
  • 3 (minipigs) or 5 to 6 (mice) samples per group were used for quantification, and 3 fields were selected from each sample for quantification. The average of 3 fields of view was used as the quantitative value for each sample.
  • the values (positive signal/number of positive cells/percent positive area) for each sample in each group were quantified with those of the wild-type or control group. Results for each group are shown as "fold of control or fold of wild type".
  • HDAC2 Knock down HDAC2 and ADAMTS1 in vitro.
  • the shRNA of human HDAC2 (shHDAC2: 5'-CAGACTGATATGGCTGTTAAT-3') or ADAMTS1 (shADAMTS1: 5'-CAAAAACCACAGGAACTGGAAGCATAA-3') was cloned into pLKO.1 and transfected into HEK293T cells to generate sh-HDAC2 ( Lentiviral particles of shRNA targeting HDAC2) or sh-ADAMTS1 (shRNA targeting ADAMTS1) (a negative control was also obtained as required by the experiment).
  • HUVECs were transduced with lentiviral particles and then individually selected with puromycin (1 ⁇ g/ml) for 48 hours before being used for subsequent experiments.
  • Plasma extracellular vesicles (EVs) isolation Plasma extracellular vesicles (EVs) isolation. Plasma EVs were isolated by ultracentrifugation. Human and minipig plasma were thawed at 4°C and centrifuged at 850 g for 30 minutes at 4°C to remove dead cells and particulates. The supernatant was centrifuged at 12,000 g for 45 minutes at 4°C to remove cell debris. Next, the supernatant was centrifuged at 110,000 g for 2 hours at 4°C. Discard the supernatant. Particles containing EVs were collected, resuspended in cold PBS, filtered through a 0.22 ⁇ m filter, and centrifuged at 110,000 g for 2 h at 4 °C to remove contaminating proteins.
  • HUVEC medium supernatant EVs Discard the supernatant and collect extracellular vesicles for subsequent experiments. Isolation of HUVEC medium supernatant EVs was performed with Total Exosome Isolation Reagent (from cell culture medium) (4478359, Invitrogen) according to the manufacturer's instructions. The collected cell culture supernatant EVs were used for transplantation experiments.
  • Extracellular vesicle (EV) transplantation experiments are shown in Figure 9C. 8-week-old C57BL/6J mice were fed a normal diet and injected intraperitoneally with 1 ml/kg of CCl 4 twice a week for a total of 1 week, followed by intraperitoneal injection of CCl 4 and extracellular vesicles (10 ⁇ g protein/mouse, each The dose is approximately 10 10 extracellular vesicles) 2 times a week for 2 weeks.
  • mice were randomly divided into two groups, and EVs from shNC-infected HUVECs medium (high ADAMTS1) and EVs from shADAMTS1-infected HUVECs medium (low ADAMTS1) were injected via tail vein. Two days after the last injection of CCl and extracellular vesicles, all mice were sacrificed and liver samples were collected.
  • Recombinant IGFBP7 treatment experiments The recombinant IGFBP7 protein treatment experiment is shown in Figure 8F.
  • 8-week-old C57BL/6J mice were fed a normal diet and were injected intraperitoneally with CCl 4 at a dose of 1 ml/kg twice a week for 1 week, followed by intraperitoneal injection of CCl 4 and intravenous injection of exogenous recombinant mouse IGFBP7 protein (20 ⁇ g protein). / mice) once every two days for a total of 2 weeks. Two days after the last injection of IGFBP7, all mice were sacrificed and liver samples were collected.
  • RNA from ECs, SECs, MECs, and CD45 + NPCs in human, minipig, and mouse livers was extracted using the RNeasy Mini Kit (QIAGEN) and performed using the Takara Reverse Transcription Kit (TakaraPrimeScript TM RT Master Mix) (RR036A). Reverse Transcription. All gene expression was measured using the Brilliant III Ultra Fast SYBR Green qPCR Master Mix kit (Agilent Technologies). All samples were replicated in triplicate, data were normalized by GAPDH and analyzed by the ddCt method, with error bars representing Standard Error of Mean (SEM).
  • SEM Standard Error of Mean
  • ELISA assay Liver tissue was weighed and homogenized, centrifuged at 2,000 g for 20 minutes, and the supernatant was collected. Referring to the instructions of the type III procollagen (PC III), hyaluronan (HA) and type IV collagen (IV-C) assay kits, set a blank control and make a standard curve. After incubation, the OD value was measured at 450 nm. The sample concentration was converted according to the standard curve. IGFBP7 and ADAMTS1 levels in human and minipig plasma, extracellular vesicles, SECs, MECs and CD45 + NPCs were measured in the same way. Extracellular vesicles were sonicated (40kHZ) for 3 minutes prior to analysis.
  • PC III procollagen
  • HA hyaluronan
  • IV-C type IV collagen
  • Primary antibodies include rabbit anti-HDAC2 (57156, Cell Signaling Technology) and anti-DNMT1 (5032, Cell Signaling Technology), rabbit anti-GAPDH (GB11002, Servicebio), rabbit anti-CD81 (bs-6954R, Bioss) and anti-GRP94 (bs-0194R) , Bioss) and rabbit anti-Smad2 (5339, Cell Signaling Technology) and anti-Phospho-Smad2 (18338, Cell Signaling Technology).
  • Peroxidase-conjugated goat anti-rabbit secondary antibody (GB23303) was purchased from Wuhan Sevier Biotechnology Co., Ltd. 20 ⁇ g protein was loaded per sample. Three biological samples per group were used for statistical analysis. The optical density of the protein bands was quantified by NIH Image J (http://rsb.info.nih.gov/ij/download.html).
  • biotinylated histone H3 or H4 antibody to form a sandwich complex
  • streptavidin-phycoerythrin (SA-PE) conjugate as a fluorescent indicator to form The final detection complex.
  • SA-PE streptavidin-phycoerythrin
  • Different histone PTMs were detected using Bio-plex200 liquid chip system (171000207, Bio-rad).
  • Different histone H3 and H4 PTMs were normalized to total H3 and H4, respectively.
  • PTM multiplex detection was performed by Hangzhou Jingjie Biotechnology Co., Ltd.
  • Biotinylated H3 antibody was used as the detection antibody for the H3 panel
  • biotinylated H4 antibody was used as the detection antibody for the H4 panel.
  • Other antibodies are coupled to different magnetic beads.
  • scRNA-Seq of human and minipig NPCs were isolated from human and minipig livers, and single cells were obtained and resuspended in PBS. scRNA-Seq was performed by Chromium single cell platform (10X Genomics). Single cells go through the steps of GEM (Gel Bead-in-Emulsion) formation and Barcode serialization, GEM-RT (Gel Bead-in-Emulsion-Reverse Transcription) cleaning, cDNA amplification, library construction and other steps, and finally in Illumina Nova-seq 6000 (Illumina, USA) for sequencing.
  • GEM Gal Bead-in-Emulsion
  • Barcode serialization GEM-RT (Gel Bead-in-Emulsion-Reverse Transcription) cleaning
  • cDNA amplification cDNA amplification
  • library construction and other steps
  • Illumina Nova-seq 6000 Illumina, USA
  • the scRNA-Seq of minipig NPCs was performed by Guangzhou Baseao Biotechnology Co., Ltd., and the scRNA-Seq of human NPCs was performed by Beijing Nuohezhiyuan Technology Co., Ltd.
  • ATAC-seq of minipig ECs were isolated from minipig livers, nuclei were isolated, and the nuclei were isolated by translocation reaction, PCR amplification and purification, library construction and other steps, and finally sequenced using Illumina HiSeqTM 4000. ATAC-seq of minipig ECs was performed by Guangzhou Baseao Biotechnology Co., Ltd., and the data were used for subsequent analysis after quality control and reference sequence alignment.
  • Microarray data (GSE84044) of liver biopsy samples from patients with cirrhosis and scRNA-seq data (GSE136103) of NPCs from patients with cirrhosis were obtained from the GEO database.
  • scRNA-seq data (GSE136103) of NPCs from patients with cirrhosis were obtained from the GEO database.
  • the expression levels of HDACs, DNMTs and IGFBP7 were quantified in fibrotic F1-F4 livers relative to healthy livers (F0).
  • scRNA-Seq data preprocessing The scRNA-seq sequences of human and minipig liver NPCs were compared with the human (Homo sapiens) transcriptome (GRCh38.p13) and pig (Sus scrofa) transcriptome (Sscrofa11.1) using Hisat2v2.0.5, respectively. ) for comparison. Unsupervised clustering and differential gene expression analysis were performed on minipigs, human samples from West China Hospital, and GSE136103 data, respectively, using the Seurat R package v3.1.1.
  • Cell filter Filter low-quality cells expressing less than 200 genes and genes expressing less than 3 cells.
  • Minipig and human liver NPCs were filtered by setting different percentage thresholds for mitochondrial gene content.
  • the experimenters used the cell line marker genes recommended in the database (http://biocc.hrbmu.edu.cn/CellMarker/index.jsp) and the marker genes used in the published articles (references 35 and 36) to define different cell line.
  • T cells CD2 + , KLRB1 + , PTPRC + , CD3E + , TRAC + etc.
  • B cells CD19 + , CD22 + , CD79B + , MS4A1 + , MZB1 + , IGKC + , etc.
  • endothelial cells PECAM1 + , CLECC4G + , FLT1 + , OIT3 + , CLECC4M + , CD34 + , etc.
  • macrophages CD163 + , VSIG4 + , CD68 + , ADGR1 + , MSR1 + , C1QC + , etc.
  • neutrophils S100A8 + , S100A9 + , CXCL8 + , MSRB1 + etc.
  • dendritic cells CLEC9A + , LGALS2 + , IDO1 + , CLORF54 + , CLEC4A + , CD83
  • Endothelial cells were reclustered and defined as sinus endothelial cells (CLEC4G + , OIT3 + , CLEC4M + ), large vessel endothelial cells (CD34 + ) and intermediate endothelial cells (CLEC4G ⁇ , CD34 ⁇ ).
  • T cells were reclustered and defined as CD4 + (CD4 + ) and CD8 + (CD8A + ) T cells.
  • Th17 cells were identified by detecting the expression of KLRB1 + , FOXP3 ⁇ , CCR6 + , CCR4 + , AHR + , IL23R + , IL17A + in CD4 + T cells.
  • Heatmaps showing marker gene expression for different cell lines were generated from the average counts of marker genes for these cell lines. Differentially expressed genes between the two groups of cells were identified using the Wilcoxon Rank Sum test, the differential genes satisfying both expression in more than 10% of cells and a log fold change of at least 0.25 between the two groups of cells. All differentially expressed gene analyses had the same threshold.
  • minipig scRNA-Seq a total of 40,570 cells were obtained from minipig liver NPCs in 1 control group, 2 cirrhosis groups, and 2 treatment groups, representing 28 populations.
  • For West China Hospital human scRNA-Seq a total of 223,74 cells were obtained from 2 healthy and 2 cirrhotic human liver NPCs, showing 25 populations.
  • Different cell lines are identified by cell line marker genes. Methods for integrating multiple sets of samples, clustering, identifying different cell lines, and differentially expressed gene analysis were similar in minipig and human scRNA-Seq data analysis.
  • Cell receptor ligand interaction analysis scRNA-Seq results were uploaded to the Cell Phone DB website for cell receptor ligand interaction analysis.
  • Example 2 scRNA-Seq reveals vascular dysregulation and abnormal endothelial classification in human cirrhotic liver
  • NPCs were isolated from fresh normal and cirrhotic human livers and subjected to scRNA-seq (10x genomics) (Fig. 1A, Table S1). Healthy liver tissue (without fibrosis) was obtained from a patient with hepatic hemangioma in West China Hospital, and cirrhotic liver tissue was obtained from a patient with histologically diagnosed liver cirrhosis. 22,374 NPCs from 2 healthy livers and 2 cirrhotic livers clustered into 25 clusters ( Figure 10A-B).
  • EC endothelial cells
  • Mac macrophages
  • Neu neutrophils
  • DC dendritic cells
  • EPCAM + EPCAM + cells and cholangiocytes
  • vascular endothelial cells can form a vascular microenvironment and regulate liver regeneration and fibrosis through paracrine/vascular secretory factors (ref. 28). Therefore, the present invention further analyzes the subsets of ECs in human liver.
  • Human liver ECs were clustered into 12 clusters (Fig. 10D), defined by the marker genes CLEC4G, OIT3 and CD34 as sinus endothelial cells (SEC) and macrovascular endothelium (MEC) (Fig. 1E, Fig. 10E).
  • SEC sinus endothelial cells
  • MEC macrovascular endothelium
  • Example 3 Epigenetic reprogramming of liver ECs induces pro-fibrotic "sinusoidal endothelial-macrovascular endothelial dysregulation"
  • the experimenters further investigated epigenetic changes in different subsets of NPCs in healthy and cirrhotic human livers.
  • 1,239 genes related to histone modification and DNA methylation were screened by Genome Enrichment Analysis (GSEA) and STRING database, of which 1,008 were found in the scRNA-Seq data of human liver of the present invention.
  • GSEA Genome Enrichment Analysis
  • STRING database of which 1,008 were found in the scRNA-Seq data of human liver of the present invention.
  • histone modification and DNA methylation-related genes had the most changes in vascular ECs compared with the normal group (Fig. 1H).
  • Histone acetylation is regulated by histone deacetylases (HDACs).
  • HDACs histone deacetylases
  • DNMTs DNA methyltransferases
  • HDAC2 and DNMT1 expression levels were significantly up-regulated compared with healthy human livers (Fig. 1O).
  • HDAC2 and DNMT1 expression levels were significantly up-regulated compared with healthy human livers (Fig. 1O).
  • HDAC2 and DNMT1 expression levels were significantly up-regulated compared with healthy human livers (Fig. 1O).
  • shHDAC2 knockdown of HDAC2 by shRNA (shHDAC2) up-regulated the expression of DNMT1
  • shHDAC2 shRNA up-regulated the expression of DNMT1
  • Example 4 Combined targeted inhibition of HDAC2 and DNMT1 attenuates liver fibrosis in a minipig NASH model
  • minipigs The physiological characteristics of minipigs are similar to those of humans, which can better simulate the metabolic disorders of humans. Therefore, we constructed a minipig model of NASH to explore the role of vascular dysregulation on cirrhosis and related mechanisms.
  • Western diet high fat, high cholesterol, high fructose and sucrose diet
  • CCl4 chemical injury induces NASH in mice with rapid fibrosis development. Therefore, the experimenters employed the described Western diet (WD) in combination with repeated CCl4 injections to induce a minipig model of NASH.
  • HDAC2i HDAC2 inhibitor
  • DNMT1i DNMT1 inhibitor
  • the experimenters then isolated minipig SECs (CD45 - CD34 - CD31 + ), MECs (CD45 - CD34 + CD31 + ) and other CD45 + NPCs by flow cytometry.
  • the expressions of HDAC2 and DNMT1 in CD45 + NPCs were not significantly different between groups.
  • the expressions of HDAC2 and DNMT1 were significantly up-regulated in SECs and MECs of cirrhotic minipig livers compared with controls.
  • HDAC2i+DNMT1i treatment blocked the upregulation of HDAC2 and DNMT1 expression in cirrhotic minipig liver SECs and MECs (Fig. 2H).
  • the therapeutic effects of HDAC2i and DNMT1i in the minipig NASH model imply that aberrant induction of HDAC2/DNMT1 plays a pathogenic role in NASH (Fig. 2I).
  • Example 5 Combined Epigenetic Targeting Inhibition Normalizes Dysregulated Liver Endothelial Classification in a Minipig NASH Model
  • Fig. 1P Minipig (normal, cirrhotic, and treated) liver NPCs were isolated and subjected to scRNA-seq to analyze the minipig NASH model at the single-cell level (Fig. 3A). 40,570 NPCs from 1 control group, 2 cirrhosis groups and 2 treatment groups were clustered into 28 clusters ( Figure 14A).
  • ECs Similar to human scRNA-Seq, seven cell lines were identified by the expression of marker genes, including T cells, B cells, EC, Mac, Neu, DC, and EPCAM + cells (Fig. 3B-C). The experimenters also found that the proportion of ECs (9.70% vs. 5.90%) in minipig cirrhotic livers was significantly increased compared with healthy livers, and this increased proportion of ECs was significantly reduced by HDAC2i+DNMT1i treatment (4.55%). % vs. 9.70%) (Fig. 3D). In order to reveal the effect of HDAC2/DNMT1 in different NPCs cell lines, the experimenters treated seven cell lines in cirrhosis group and control group (cirrhosis vs.
  • HDAC2i and DNMT2i restored most of the above-mentioned GO and KEGG changes in liver ECs of the NASH group, indicating normalization of minipig ECs after treatment (Fig. 3F, Fig. 14B).
  • minipig ECs clustered into 15 clusters ( Figure 15), identified by genetic markers as SEC and MEC ( Figure 3G, Figure 15B-C). Similar to the described vascular dysregulation in human cirrhotic livers, the proportion of MEC was increased and the proportion of SEC was decreased in cirrhotic minipig livers compared to control minipig livers. In treated minipig livers, HDAC2i + DNMT1i treatment significantly reversed the NASH-induced "sinusoidal endothelium-macrovascular endothelial dysregulation" (Fig. 3H).
  • the SECs had relatively the highest number of differential genes (sclerosis group vs. control group), and most of the differential genes were recovered after HDAC2i+DNMT1i treatment (treatment group vs. sclerosis group) (Fig. 3I).
  • the temporal analysis of liver EC subsets also showed that there was "sinusoidal endothelial-macrovascular endothelial dysregulation" in cirrhotic minipig livers, which was normalized by HDAC2i+DNMT1i treatment (Fig. 3J).
  • the experimenters also found that in cirrhotic minipigs, histone modification and DNA methylation-related genes changed relatively most in SECs, and most genes were restored after HDAC2i+DNMT1i treatment (Fig.
  • Example 6 Reprogramming of paracrine/vascular secretory factors in epigenetically dysregulated SECs in human patients and minipig NASH models
  • ECs can modulate liver regeneration by interacting with surrounding cells via paracrine/vascular secretory factors.
  • Fig. dysregulation associated with reprogramming of vasosecreted factors
  • Fig. 4A-D the metallopeptidase ADAMTS1, insulin-like growth factor-binding protein 7 (IGFBP7), DLL1, and ADAMTS6 were significantly changed in cirrhotic human liver ECs (Fig. 4A).
  • IGFBP7 insulin-like growth factor-binding protein 7
  • DLL1 ADAMTS6
  • IGFBP7 and ADAMTS1 The expression of IGFBP7 and ADAMTS1 was gradually increased in the fibrotic livers of F2-F4 patients (Fig. 4B).
  • the experimenters found that among the vascular secretory factor genes, IGFBP7 and ADAMTS1 were specifically expressed at relatively highest levels in human and minipig ECs (Fig. 4C, Fig. 16B-C).
  • IGFBP7 and ADAMTS1 were selectively upregulated in human cirrhotic liver ECs compared with healthy ECs, and the mRNA and protein levels of IGFBP7 and ADAMTS1 were significantly increased in cirrhotic SECs (Fig. 4E-F).
  • the mRNA levels of IGFBP7 and ADAMTS1 were significantly increased in cirrhotic SECs and MECs, whereas the protein levels of IGFBP7 and ADAMTS1 were only up-regulated in cirrhotic SECs.
  • HDAC2i and DNMT1i treatment blocked the increase in mRNA and protein levels of IGFBP7 and ADAMTS1 in cirrhotic minipig liver SECs (Fig. 4G-L, Fig. 16D). Analyzing chromatin openness helps reveal epigenetic regulation of gene expression, so we performed assays for chromatin openness (ATAC-seq) in minipig liver ECs from control, cirrhosis, and treatment groups.
  • IGFBP7 and ADAMTS1 promoters Compared with control ECs, the chromatin openness of IGFBP7 and ADAMTS1 promoters was significantly enhanced in cirrhotic ECs, and their chromatin openness was significantly reversed after HDAC2i+DNMT1i treatment (Fig. 4M).
  • IGFBP7 and ADAMTS1 upregulation may discriminate between normal and dysregulated SECs in cirrhotic livers, and epigenetic therapy targeting the profibrotic IGFBP7 + ADAMTS1 + dysregulated EC subset may block liver fibrosis ionization (Fig. 4N).
  • Example 7 IGFBP7 and ADAMTS1 from dysregulated SECs predict progression of liver fibrosis in humans and minipigs
  • Plasma concentrations of IGFBP7 and ADAMTS1 in human cirrhosis/fibrosis or NASH we assessed plasma concentrations of IGFBP7 and ADAMTS1 in human patients (Table S2). Plasma concentrations of IGFBP7, ADAMTS1, alanine aminotransferase (ALT), and aspartate aminotransferase (AST) were significantly higher in cirrhosis/fibrosis patients than in healthy human samples ( Figure 5A). Since some patients with cirrhosis/fibrosis can have normal plasma ALT or AST levels, there is a need to discover sensitive biomarkers for clinical diagnosis of liver cirrhosis/fibrosis.
  • ALT alanine aminotransferase
  • AST aspartate aminotransferase
  • the experimenters assessed the value of plasma IGFBP7 and ADAMTS1 as clinical biomarkers.
  • Patients with cirrhosis/fibrosis were divided into two groups according to their plasma liver function index: normal plasma ALT and AST concentrations and abnormal plasma ALT and AST concentrations (Fig. 5B).
  • cirrhotic/fibrotic patients with normal ALT or AST had significantly higher plasma concentrations of IGFBP7 and ADAMTS1 than healthy subjects (Fig. 5C).
  • the cirrhosis/fibrosis cohort included nonalcoholic steatohepatitis-associated cirrhosis/fibrosis (NASH), hepatitis B-associated cirrhosis/fibrosis (HBC), autoimmune hepatitis-associated cirrhosis/ Liver fibrosis (AIH), primary biliary cirrhosis/fibrosis (PBC) and cryptogenic cirrhosis/fibrosis (CC). Elevated plasma levels of IGFBP7 and ADAMTS1 were observed in all of these patients compared to healthy subjects (Fig. 5D). Therefore, vascular-secreted IGFBP7 and ADAMTS1 may serve as biomarkers for the diagnosis of cirrhosis/fibrosis in the absence of liver dysfunction.
  • NASH nonalcoholic steatohepatitis-associated cirrhosis/fibrosis
  • HBC hepatitis B-associated cirrhosis/fibrosis
  • AIH autoimmune hepatitis-associated
  • NASH patients were divided into different groups according to the stage of liver fibrosis. Plasma ALT or AST concentrations were not significantly increased in patients with early stage NASH (F0-F1) (Fig. 5E). In contrast, plasma concentrations of IGFBP7 and ADAMTS1 were significantly elevated in NASH patients (Fig. 5F). Furthermore, plasma IGFBP7 and ADAMTS1 concentrations were not statistically elevated in patients with simple fatty liver, suggesting that plasma IGFBP7/ADAMTS1 concentrations have important clinical value in distinguishing NASH from simple fatty liver ( Figure 5F).
  • IGFBP7/ADAMTS1 The association of IGFBP7/ADAMTS1 with NASH progression raises the possibility that the profibrotic effects of dysregulated SECs depend on vascularly secreted IGFBP7 and ADAMTS1.
  • the experimenters then evaluated the hypothesis that epigenetically dysregulated SECs release IGFBP7 or ADAMTS1 to contribute to NASH.
  • Extracellular vesicles EVs are unique lipid bilayer particles released by cells. Molecules packaged in EVs may facilitate cellular communication in many biological processes. We therefore analyzed whether endothelial cell-produced IGFBP7 and ADAMTS1 are assembled in EVs and released into the circulation.
  • EVs were extracted from human and minipig plasma by ultracentrifugation and validated by electron microscopy and immunoblot analysis (Fig. 5G).
  • the concentrations of IGFBP7 and ADAMTS1 in minipigs and human EVs in the cirrhosis group were higher than those in the control group.
  • HDAC2i + DNMT1i treatment reduced the elevation of EV IGFBP7/ADAMTS1 concentrations in the liver cirrhosis group (Fig. 5H).
  • IGFBP7/ADAMTS1 concentrations were also significantly higher in EVs from cirrhotic/fibrotic patients or NASH patients with normal ALT/AST than in healthy humans (Figure 5I-J).
  • the present data suggest that IGFBP7 and ADAMTS1 in EVs are useful biomarkers for assessing NASH progression prior to liver dysfunction (Figure 5K).
  • Example 8 IGFBP7 + ADAMTS1 + dysregulated SECs induce pro-fibrotic Th17 cell responses in liver cirrhosis patients and a minipig NASH model
  • Dysregulated ECs can interact with neighboring cells to promote fibrosis by forming a dysregulated vascular microenvironment.
  • the present invention seeks to reveal the cellular mechanism by which IGFBP7 + ADAMTS1 + deregulated SECs enhance liver fibrosis through cellular communication.
  • the receptor and ligand expression profiles of different NPCs cell lines were analyzed based on the CellPhoneDB database.
  • Cell interaction predictions indicated that dysregulated ECs interacted with T cells significantly in cirrhotic patients and NASH minipigs (Fig. 6A).
  • the predicted interactions in the human data were evident between ECs and macrophages, there were less EC-macrophage interactions in minipig NPCs.
  • the present invention mainly analyzes the interaction between ECs and T cells.
  • the experimenters next analyzed cell lines of recruited human and minipig T cells.
  • Human T cells from 2 cirrhotic and 2 healthy livers were clustered and identified into 20 populations (FIG. 17A), defined as CD4 + and CD8 + T cells (FIG. 17B).
  • CD4 + T cells were further clustered (Fig. 6C) and Th17 cells were marked by Th17 + marker genes (Fig. 6D).
  • the number of Th17 cells in the livers of patients with cirrhosis was significantly higher than that in healthy livers (Fig. 6E).
  • the experimenters analyzed Th17 cells in the GSE136103 data (Figure 17C-I).
  • the number of Th17 cells was similarly significantly increased in cirrhotic human livers compared to healthy human livers.
  • T cell clustering of minipig livers in the control, cirrhosis and treatment groups identified 16 populations, defined as CD4 + T cells and CD8 + T cells (Fig. 6F, Fig. 17J).
  • CD4 + T cells Th17 cells were identified by Th17 + marker genes (Fig. 6G-H).
  • the number of Th17 cells was also significantly increased in the cirrhotic minipig livers compared to control minipigs.
  • combined treatment of HDAC2i+DNMT1i blocked the increase in Th17 cell numbers in injured minipig livers (Fig. 6I).
  • Example 9 Epigenetically dysregulated SECs in a mouse model of NASH generate pro-fibrotic Th17 responses
  • Hdac2 i ⁇ EC mice selective knockout of HDAC2 in ECs
  • WD+CCl 4 Induction of a mouse model of NASH The combined targeted inhibition of HDAC2+DNMT1 was obtained by treating Hdac2 i ⁇ EC mice (Hdac2 i ⁇ EC + AZA) with the DNMT1 inhibitor AZA (Fig. 7A).
  • liver fibrosis In Hdac2 i ⁇ EC mice treated with DNMT1i, liver fibrosis, inflammation, collagen deposition, and indices of liver fibrosis and liver function were significantly reduced compared with control-injured mice (Fig. 7B-C).
  • Fig. 7B-C To further investigate the interaction regulation between endothelial-derived HDAC2 and DNMT1 in mouse liver SECs, we isolated liver SECs (CD45 - CD34 - CD31 + ). Western blot revealed that knockout of Hdac2 in mouse liver SECs upregulated the expression of DNMT1 in SECs (Fig. 7D).
  • Example 10 IGFBP7 enhances pro-fibrotic Th17 responses in a mouse NASH model
  • FIG. 8A To determine the role of IGFBP7 in stimulating Th17 responses in liver fibrosis, we analyzed the NASH phenotype of IGFBP7 knockout (IGFBP7 -/- ) mice (Fig. 8B). Knockout of Igfbp7 in mice significantly attenuated liver fibrotic responses, namely collagen deposition (FIG. 8C), serum liver function index, liver hydroxyproline content (FIG. 8D) and Th17 responses (FIG. 8E).
  • IGFBP7 directly affects Th17 biology
  • C57BL/6J mice were tail vein injected with recombinant mouse IGFBP7 protein (Fig. 8F). Elevated IGFBP7 levels significantly enhanced the pro-fibrotic Th17 response in injured mouse livers compared to controls (Fig. 8G). These results suggest that IGFBP7 is a regulator that enhances Th17 responses to promote liver fibrosis.
  • Example 11 Genetic inactivation of ADAMTS1 in a mouse fibrosis model attenuates pro-fibrotic Th17 responses
  • ADAMTS1 expression was significantly reduced in SECs of Hdac2 i ⁇ EC + AZA mice (Fig. 9A). Furthermore, knockdown of ADAMTS1 in human ECs by ADAMTS1 shRNA (shADAMTS1) gene blocked the phosphorylation of Smad2 stimulated by TGF- ⁇ (Fig. 9B). Therefore, we used a "human-to-mouse" extracellular vesicle (EVs) transplantation method (Fig. 9C) to investigate whether endothelial-derived ADAMTS1 regulates Th17 biology.
  • EVs extracellular vesicle
  • EVs were isolated from the culture medium of shADAMTS1 and control (shNC) infected HUVECs and transplanted into mice via the tail vein. Liver fibrosis and Th17 responses were significantly reduced in mice transplanted with ADAMTS1-deficient EVs compared to mice treated with control EVs ( Figure 9D-E). These results implicate a functional role of ADAMTS1 in promoting Th17 responses in the progression of liver fibrosis.
  • the present data reveal an endothelial-derived HDAC2/DNMT1-IGFBP7/ADAMTS1-Th17 axis that promotes liver fibrosis in human patient, minipig, and mouse models of NASH.
  • Epigenetic aberrant interactions in subsets of hepatic ECs lead to "sinusoidal endothelium-macrovascular endothelial dysregulation," characterized by dysregulated and dysregulated SECs that produce profibrotic IGFBP7/ADAMTS1, which recruit Th17 cells through extracellular vesicles, A pro-fibrotic vascular microenvironment was formed (Fig. 9F).
  • the pathogenesis of NASH involves systemic effects including metabolic dysfunction. Endothelial and hematopoietic cells in the circulatory system are directly linked to systemic stimulation, and NASH is similar to many risk factors for circulatory/vascular complications.
  • the present invention utilizes multi-omics analysis to reveal how vessel-specific epigenetic alterations reprogram pro-fibrotic cross-regulation in the hepatic circulatory system (vascular and hematopoietic cells) at the single-cell level.
  • the present invention integrates scRNA-Seq data from human and large animal NASH models to reveal how "sinus endothelium-macrovascular endothelial dysregulation" stimulates pro-fibrotic Th17 cell responses in NASH.
  • the present inventors found that in NASH, abnormal HDAC2-DNMT1 cross-regulation in hepatic ECs leads to abnormal endothelial cell sorting and generation of dysregulated IGFBP + ADAMTS1 + hepatic EC subsets to form pro-fibrotic dysregulated vessels in the circulatory system Microenvironment.
  • the present invention employs a bed to benchside approach to systematically study the cellular phenotypes of human patients and complementary large animal and rodent models of NASH. How aberrant epigenetic cross-regulation leads to pro-fibrotic communication between endothelial and immune cells.
  • the present invention demonstrates that abnormal epigenetic cross-regulation of vascular endothelial cells promotes liver fibrosis in human and large animal NASH models.
  • scRNA-Seq histone modifications, and human patient cohort analysis, the present inventors found that the selective induction of HDAC2 and DNMT1 in specific subsets of hepatic vascular ECs is closely related to the progression of liver fibrosis.
  • HDAC2/DNMT1 reciprocal regulation in dysregulated EC subsets stimulates the activation of profibrotic IGFBP7 and ADAMTS1 in extracellular vesicles produced, thereby recruiting Th17 cells, inhibiting liver regeneration and inducing fibrosis in NASH.
  • scRNA-Seq revealed that in NASH, selective epigenetic alterations in ECs lead to "sinus endothelium-macrovascular endothelial dysregulation" and abnormal endothelial classification, resulting in a pro-fibrotic endothelial microenvironment.
  • the present inventors discovered a pro-fibrotic HDAC2/DNMT1-IGFBP7/ADAMTS1 axis in a subpopulation of dysregulated liver ECs. Previous reports have shown that hepatic fibrosis leads to capillary vascularization of SECs and altered blood flow in hepatic sinusoids (refs 22, 23, 57).
  • liver ECs are the major constituents of NPCs and are reported to account for approximately 5%-15% of NPCs.
  • the proportion of ECs in purified NPCs appears to vary between studies and individuals, depending on individual differences and specific isolation methods.
  • scRNA-Seq analysis of hepatic ECs showed that chronic/metabolic damage to ECs and the resulting abnormal epigenetic modification promotes liver fibrosis in NASH.
  • aberrant histone and DNA modifications in the heterogeneous hepatic vasculature may induce dysregulation of hepatic ECs, and these dysregulated ECs subsets further interact with other circulating cells such as Th17 cells to form microscopic mechanisms that promote liver fibrosis. surroundings.
  • scRNA-Seq analysis can reveal vascular dysregulation in cirrhotic livers at the single-cell level.
  • Analyses of human patients and minipig models of NASH demonstrate that aberrant epigenetic cross-regulation leads to dysregulation from the sinus endothelium to the macrovascular endothelium, resulting in aberrant endothelial classification.
  • the observed vascular dysregulation appears to be predominantly a phenotypic and functional shift within the ECs lineage.
  • the fibrotic livers examined exhibited increased numbers of ECs and a lower enrichment of "mesenchymal differentiation" in the EC population. Thus, this vascular dysregulation process appears to be distinct from endothelial-mesenchymal transition (EndMT).
  • the present study shows that epigenetic-dependent dysregulation of liver ECs in NASH leads to the secretion of pro-fibrotic IGFBP7 and ADAMTS1 into extracellular vesicles, and that, in human and minipig NASH models, plasma levels of IGFBP7 and ADAMTS1 are reduced Elevated prior to detectable parenchymal damage (elevation of plasma ALT and AST), combined targeting to inhibit the aberrant cross-regulation of HDAC2/DNMT1 restored minipig plasma levels of IGFBP7 and ADAMTS1.
  • the clinical findings in human patients are consistent with data that combined targeted inhibition of HDAC2/DNMT1 in ECs normalized plasma AST and ALT levels in a minipig NASH model.
  • the present experimental data demonstrate that chronic stress in NASH stimulates the generation of epigenetically reprogrammed IGFBP7 + ADAMTS1 + SECs and produces profibrotic IGFBP7 and ADAMTS1 to enhance liver parenchymal injury.
  • molecular markers involved in abnormal endothelial classification such as extracellular vesicles IGFBP7 or ADAMTS1 may be used as therapeutic targets or biomarkers to assess the progression of fibrosis in NASH patients, especially to distinguish NASH patients from simple fat liver patients.
  • the present invention reveals a single-cell atlas of pro-fibrotic vascular dysregulation in the circulatory system and identifies key molecules involved in vascular dysregulation through multi-omic analysis of a cohort of patients with cirrhosis, transgenic mice, and minipig and rodent NASH models .
  • vascular dysregulation epigenetically reprogrammed liver SECs produce IGFBP7 or ADAMTS1 into extracellular vesicles to recruit profibrotic Th17 cells.
  • Aberrant recruitment and activation of immune cells can stimulate liver fibrosis.
  • the cellular interaction prediction analysis using scRNA-Seq of NPCs showed the interaction between T cells and reprogrammed ECs in human liver cirrhosis patients and a minipig NASH model. This prediction is supported by data that targeting HDAC2/DNMT1 normalizes epigenetic changes and inhibits Th17 recruitment in hepatic ECs in minipig and mouse models of NASH.
  • scRNA-Seq, Igfbp7 knockout mice, and extracellular vesicle transplantation showed that the EC-Th17 interaction depends at least in part on IGFBP7/ADAMTS1 produced by epigenetically reprogrammed liver SECs.
  • the present invention integrates bioinformatics and experimental approaches to uncover this unique pro-fibrotic endothelial-Th17 cell interaction caused by epigenetic-dependent vascular dysregulation.
  • decoding the molecular and cellular networks involved in circulatory dysregulation may facilitate the systematic identification of therapeutic targets or biomarkers.
  • minipigs and mice Based on clinical findings in human patients, we used complementary preclinical NASH models (minipigs and mice) to determine the role of epigenetic-dependent vascular dysregulation. Feeding a Western diet containing high sucrose and fructose, high cholesterol, and high fat combined with repeated liver injury to induce a model of NASH exhibited clinically relevant NASH phenotypes, including histological and transcriptomic features. Preclinical models of minipigs and mice also contribute to understanding the pathogenesis of NASH. The digestive system of minipigs is very similar to that of humans, and has unique advantages similar to those associated with human metabolic disorders, such as NASH. Liver biopsies can be performed in large animals such as minipigs for multi-omics assessment of treatment effects and underlying mechanisms.
  • transgenic mice such as EC-specific Hdac2 knockout mice and Igfbp7 knockout mice
  • EC-specific Hdac2 knockout mice and Igfbp7 knockout mice provide an efficient tool to study the cellular and molecular mechanisms involved in epigenetic-dependent vascular dysregulation.
  • pharmacological and genetic targeting experiments in minipig and mouse models of NASH demonstrated that aberrant HDAC2/DNMT1 interaction leads to vascular dysregulation and subsequent production of IGFBP7/ADAMTS1 into extracellular vesicles.
  • This data is consistent with the association between HDAC2, DNMT1, IGFBP7 or ADAMTS1 and fibrosis grade identified in the human cirrhosis cohort.
  • Combined targeted inhibition of HDAC2 and DNMT1 showed synergistic anti-fibrotic effects in mouse and minipig NASH models.
  • Experiments injected with exogenous IGFBP7 or ADAMTS1 knockdown endothelial-derived EVs further demonstrated that ADAMTS1/IGFBP7 promotes liver fibrosis by stimulating Th17 responses in the liver.
  • ADAMTS1/IGFBP7 promotes liver fibrosis by stimulating Th17 responses in the liver.
  • vascular dysregulation in which epigenetically reprogrammed EC subsets induce the release of profibrotic factors that stimulate Th17 cell recruitment to collectively promote liver fibrosis in multiple species change.
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Abstract

The present invention belongs to the field of biomedicine, and mainly relates to an application of HDAC2 and DNMT1 inhibitors in the treatment of non-alcoholic steatohepatitis. The provided application of HDAC2 and DNMT1 inhibitors in the combined targeted therapy of non-alcoholic steatohepatitis helps to systematically relieve or treat, to a certain extent, non-alcoholic steatohepatitis and liver cirrhosis and/or liver fibrosis associated therewith.

Description

HDAC2和DNMT1抑制剂在联合靶向治疗非酒精性脂肪性肝炎中的应用Application of HDAC2 and DNMT1 inhibitors in combined targeted therapy of nonalcoholic steatohepatitis 技术领域technical field
本发明属于生物医药领域，主要涉及HDAC2和DNMT1抑制剂在治疗非酒精性脂肪性肝炎中的应用。The invention belongs to the field of biomedicine, and mainly relates to the application of HDAC2 and DNMT1 inhibitors in the treatment of nonalcoholic steatohepatitis.
背景技术Background technique
肝脏在损伤后具有自我修复的再生能力。然而，非酒精性脂肪性肝炎(NASH)中，肝再生受到抑制。NASH同糖尿病和代谢综合征类似，全球发病率呈上升趋势。NASH可导致肝纤维化、肝硬化和肝衰竭，如果没有有效的抗纤维化治疗，NASH导致的肝纤维化和肝硬化通常会导致系统性并发症，将成为全球的主要健康负担。临床上发展NASH治疗的一个主要障碍是缺少系统模拟NASH发病机制的细胞和分子网络的临床及临床前研究。The liver has the regenerative ability to repair itself after injury. However, in nonalcoholic steatohepatitis (NASH), liver regeneration is inhibited. Similar to diabetes and metabolic syndrome, NASH is on the rise globally. NASH can lead to liver fibrosis, cirrhosis and liver failure. Without effective anti-fibrotic treatment, liver fibrosis and liver cirrhosis caused by NASH usually lead to systemic complications and will become a major global health burden. A major obstacle to the clinical development of NASH treatments is the lack of clinical and preclinical studies of cellular and molecular networks that systematically mimic the pathogenesis of NASH.
肝由实质细胞(肝细胞)和非实质细胞(NPCs)如星状细胞、血管内皮细胞(ECs)和造血细胞等组成。肝再生依赖于不同细胞组分间的协同作用。然而，NASH中持续的应激经常引起异常的细胞互作(在本文中，“互作”、“串扰”都可以理解为“交互调控(crosstalk)”)和导致失调的修复和纤维化。星状细胞的激活是肝纤维化的关键步骤，但仍然要确定NASH中的慢性应激是如何导致其他肝脏NPCs之间的互作以促进这一步骤的。The liver is composed of parenchymal cells (hepatocytes) and non-parenchymal cells (NPCs) such as stellate cells, endothelial cells (ECs), and hematopoietic cells. Liver regeneration relies on the synergy between different cellular components. However, persistent stress in NASH often causes abnormal cellular interactions (in this context, "interaction", "crosstalk" can both be understood as "crosstalk") and leads to dysregulated repair and fibrosis. Activation of stellate cells is a critical step in liver fibrosis, but it remains to be determined how chronic stress in NASH leads to interactions between other hepatic NPCs to facilitate this step.
在NPCs中，血管内皮细胞和造血细胞属于循环系统，可以直接传递系统性刺激(如代谢应激)，还可以促进实质细胞与间充质细胞相互作用，共同建立血管微环境。血管内皮细胞是肝脏NPCs的主要组成部分。肝的血液供应由肝静脉、肝动脉和门静脉之间的窦状血管系统完成。窦状血管系统有一层表达CLEC4G和OIT3的窦内皮细胞(SECs)和表达CD34的大血管内皮细胞(MECs)。因此，不同解剖部位的肝ECs表现出特殊的形态和表型标记，具有“窦内皮-大血管内皮”血管层次和器官内分类。在器官修复过程中，血管ECs会产生大量调节因子来调控造血细胞、间充质细胞和实质细胞之间的通讯(communication，在本文中还可以理解为“信息传递”)。窦内皮细胞(SECs)的异常改变(如毛细血管化)与肝纤维化密切有关。然而，目前的临床和临床前模型中，在单细胞水平上还没有系统地阐明肝脏EC亚群对人类肝硬化或NASH病理学的功能作用。In NPCs, vascular endothelial cells and hematopoietic cells belong to the circulatory system, which can directly transmit systemic stimuli (such as metabolic stress), and can also promote the interaction between parenchymal cells and mesenchymal cells to jointly establish the vascular microenvironment. Vascular endothelial cells are the main component of hepatic NPCs. The blood supply to the liver is accomplished by the sinusoidal vascular system between the hepatic veins, the hepatic artery and the portal vein. The sinusoidal vasculature has a layer of sinusoidal endothelial cells (SECs) expressing CLEC4G and OIT3 and macrovascular endothelial cells (MECs) expressing CD34. Thus, hepatic ECs from different anatomical sites exhibit specific morphological and phenotypic markers, with "sinusoidal endothelium-macrovascular endothelium" vascular hierarchy and intra-organ classification. During organ repair, vascular ECs produce a large number of regulatory factors to regulate the communication between hematopoietic cells, mesenchymal cells and parenchymal cells (communication, which can also be understood as "information transfer" in this paper). Abnormal changes in sinusoidal endothelial cells (SECs), such as capillary vascularization, are closely related to liver fibrosis. However, the functional role of hepatic EC subsets on human cirrhosis or NASH pathology has not been systematically elucidated at the single-cell level in current clinical and preclinical models.
发明内容SUMMARY OF THE INVENTION
有鉴于此，本发明提供了HDAC2抑制剂和DNMT1抑制剂在联合靶向治疗非酒精性脂肪性肝炎中的应用。In view of this, the present invention provides the application of HDAC2 inhibitor and DNMT1 inhibitor in combined targeted treatment of nonalcoholic steatohepatitis.
进一步地，所述非酒精性脂肪性肝炎伴有肝硬化或肝纤维化。Further, the nonalcoholic steatohepatitis is accompanied by liver cirrhosis or liver fibrosis.
进一步地，所述肝纤维化的病理等级包括F2-F4级。Further, the pathological grades of liver fibrosis include F2-F4 grades.
进一步地，所述HDAC2抑制剂为莫西司他(Mocetinostat)，剂量为1-20mg/kg/天或0.1-2.0mg/kg/天(优选为1.1mg/kg/天)。Further, the HDAC2 inhibitor is Mocetinostat, and the dosage is 1-20 mg/kg/day or 0.1-2.0 mg/kg/day (preferably 1.1 mg/kg/day).
进一步地，所述DNMT1抑制剂为阿扎胞苷，剂量为0.1-2.0mg/kg/天或0.001-0.100mg/kg/天(优选为0.055mg/kg/天)。Further, the DNMT1 inhibitor is azacitidine, and the dosage is 0.1-2.0 mg/kg/day or 0.001-0.100 mg/kg/day (preferably 0.055 mg/kg/day).
进一步地，所述HDAC2抑制剂和DNMT1抑制剂通过注射方式给药，所述注射方式包括腹腔注射、肌肉注射、皮下注射或静脉注射中的一种或多种。Further, the HDAC2 inhibitor and the DNMT1 inhibitor are administered by injection, and the injection includes one or more of intraperitoneal injection, intramuscular injection, subcutaneous injection or intravenous injection.
进一步地，采用注射方式给药的具体操作为：第一周前5天注射DNMT1抑制剂治疗，每天1次，然后停药2天；第二周前5天注射HADC2抑制剂，每天1次，然后停药2天，重复治疗5-10个疗程(优选为6个疗程)。Further, the specific operation of administration by injection is as follows: injection of DNMT1 inhibitor for the first 5 days of the first week, once a day, and then drug withdrawal for 2 days; injection of HADC2 inhibitor for the first 5 days of the second week, once a day, Then the drug is stopped for 2 days, and the treatment is repeated for 5-10 courses of treatment (preferably 6 courses of treatment).
进一步地，所述HDAC2抑制剂和DNMT1抑制剂联合靶向使用减轻非酒精性脂肪性肝炎肝脏的纤维化程度并促进肝再生；和/或逆转肝硬化肝脏中的窦内皮-大血管内皮失调；和/或减少非酒精性脂肪性肝炎肝脏中促纤维化Th17细胞的募集。Further, the combined targeted use of the HDAC2 inhibitor and the DNMT1 inhibitor reduces the degree of fibrosis in the liver of nonalcoholic steatohepatitis and promotes liver regeneration; and/or reverses the sinus endothelium-macrovascular endothelial disorder in the liver of cirrhosis; and/or decrease the recruitment of pro-fibrotic Th17 cells in the liver of nonalcoholic steatohepatitis.
进一步地，所述HDAC2抑制剂和DNMT1抑制剂联合靶向使用降低血糖、肝纤维 化指数和/或血清肝功能指数。Further, the combined targeted use of the HDAC2 inhibitor and the DNMT1 inhibitor reduces blood sugar, liver fibrosis index and/or serum liver function index.
进一步地，所述HDAC2抑制剂和DNMT1抑制剂联合靶向使用降低血清总胆固醇水平。Further, the combined targeted use of the HDAC2 inhibitor and the DNMT1 inhibitor reduces serum total cholesterol levels.
进一步地，所述HDAC2抑制剂和DNMT1抑制剂联合靶向使用减轻肝硬化并增加肝细胞增殖。Further, the combined targeted use of the HDAC2 inhibitor and the DNMT1 inhibitor alleviates liver cirrhosis and increases hepatocyte proliferation.
进一步地，所述HDAC2抑制剂和DNMT1抑制剂联合靶向使用阻断肝硬化肝脏中IGFBP7和ADAMTS1的增加。Further, the combined targeted use of the HDAC2 inhibitor and DNMT1 inhibitor blocked the increase of IGFBP7 and ADAMTS1 in cirrhotic liver.
有益技术效果：Beneficial technical effects:
本发明提供HDAC2抑制剂和DNMT1抑制剂在联合靶向治疗非酒精性脂肪性肝炎中的应用。本发明技术方案通过一系列实验揭示：肝脏内皮细胞亚群中的表观遗传改变，即HDAC2和DNMT1的异常激活(其中，HDAC2和DNMT1的表达在F2-F4级患者的肝脏中变化显著；肝硬化患者肝脏和肝硬化小型猪肝脏的内皮细胞中HDAC2和DNMT1表达显著上调)，会导致“窦内皮-大血管内皮失调”，刺激细胞外囊泡中促纤维化的IGFBP7和ADAMTS1产生，募集Th17细胞，从而在NASH中抑制肝脏再生和诱导纤维化。而通过靶向抑制小型猪和小鼠NASH模型的HDAC2和DNMT1，在一定程度上正常化肝脏内皮细胞的表观遗传变化、阻断IGFBP7和ADAMTS1的表达并抑制Th17细胞的募集。The present invention provides the application of HDAC2 inhibitor and DNMT1 inhibitor in combined targeted treatment of nonalcoholic steatohepatitis. The technical solution of the present invention reveals through a series of experiments: epigenetic changes in liver endothelial cell subsets, that is, abnormal activation of HDAC2 and DNMT1 (wherein, the expression of HDAC2 and DNMT1 changes significantly in the liver of F2-F4 patients; liver The expression of HDAC2 and DNMT1 is significantly upregulated in endothelial cells of cirrhotic patient liver and cirrhotic minipig liver), which leads to "sinusoidal endothelium-macrovascular endothelial dysregulation", stimulates the production of pro-fibrotic IGFBP7 and ADAMTS1 in extracellular vesicles, and recruits Th17 cells, thereby inhibiting liver regeneration and inducing fibrosis in NASH. Targeted inhibition of HDAC2 and DNMT1 in minipig and mouse models of NASH normalized epigenetic changes in liver endothelial cells, blocked the expression of IGFBP7 and ADAMTS1, and inhibited the recruitment of Th17 cells to a certain extent.
本发明技术方案通过使用HDAC2抑制剂和DNMT1抑制剂以联合靶向HDAC2和DNMT1，在一定程度上减轻NASH肝脏的纤维化程度并促进肝再生：例如，在实施例4中的小型猪NASH模型中，经HDAC2抑制剂(例如，莫西司他(Mocetinostat))和DNMT1抑制剂(例如，阿扎胞苷)联合治疗的小型猪与对照组相比：(i)其血糖、肝纤维化指数、血清肝功能指数、血清总胆固醇都显著降低；(ii)其肝硬化减少、胶原蛋白沉积和脂质积聚减轻且肝细胞增殖增加。并且在实施例5中，经HDAC2抑制剂(例如，莫西司他(Mocetinostat))和DNMT1抑制剂(例如，阿扎胞苷)联合处理的肝硬化小型猪，明显逆转了治疗组中NASH引起的“窦内皮-大血管内皮失调”。The technical solution of the present invention reduces the fibrosis degree of NASH liver and promotes liver regeneration to a certain extent by using HDAC2 inhibitor and DNMT1 inhibitor to jointly target HDAC2 and DNMT1: for example, in the minipig NASH model in Example 4 , minipigs treated with a combination of an HDAC2 inhibitor (eg, Mocetinostat) and a DNMT1 inhibitor (eg, azacitidine) compared to controls: (i) their blood glucose, liver fibrosis index, Serum liver function index, serum total cholesterol were significantly decreased; (ii) liver cirrhosis was reduced, collagen deposition and lipid accumulation were reduced, and hepatocyte proliferation was increased. And in Example 5, cirrhotic minipigs treated with a combination of an HDAC2 inhibitor (eg, Mocetinostat) and a DNMT1 inhibitor (eg, azacitidine) significantly reversed NASH-induced NASH in the treatment group. "Sinus Endothelial-Great Vessel Endothelial Dysregulation".
进一步地，本发明技术方案通过使用HDAC2抑制剂和DNMT1抑制剂以联合靶向HDAC2和DNMT1，治疗组NASH肝脏中促纤维化Th17细胞的募集显著减少：例如，在实施例6和实施例7中，经HDAC2抑制剂(例如，莫西司他(Mocetinostat))和DNMT1抑制剂(例如，阿扎胞苷)联合处理的肝硬化小型猪，治疗组的窦内皮细胞(SECs)中IGFBP7和ADAMTS1的mRNA和蛋白质水平的增加被有效阻断，且其血浆细胞外囊泡(EVs)中IGFBP7/ADAMTS1的浓度的升高也被减少；并且，在实施例8和实施例9中，经HDAC2抑制剂(例如，莫西司他(Mocetinostat))和DNMT1抑制剂(例如，阿扎胞苷)联合处理的肝硬化小型猪和小鼠NASH模型中，治疗组Th17细胞数的增加被有效阻断。Further, the technical solution of the present invention by using HDAC2 inhibitor and DNMT1 inhibitor to target HDAC2 and DNMT1 in combination, the recruitment of pro-fibrotic Th17 cells in the NASH liver of the treatment group is significantly reduced: for example, in Example 6 and Example 7 , in cirrhotic minipigs treated with a combination of HDAC2 inhibitors (eg, Mocetinostat) and DNMT1 inhibitors (eg, azacitidine), the expression of IGFBP7 and ADAMTS1 in sinus endothelial cells (SECs) of the treatment group The increase in mRNA and protein levels was effectively blocked, and the increase in the concentration of IGFBP7/ADAMTS1 in plasma extracellular vesicles (EVs) was also reduced; In cirrhotic minipig and mouse models of NASH treated with a combination of (eg, Mocetinostat) and a DNMT1 inhibitor (eg, azacitidine), the increase in Th17 cell numbers in the treatment group was effectively blocked.
现有技术中，单一的药物治疗可能难以同时缓解或治疗非酒精性脂肪性肝炎和其伴有的肝硬化和肝纤维化。综上所述，本发明技术方案提供的HDAC2和DNMT1抑制剂在治疗非酒精性脂肪性肝炎中的应用，有助于在一定程度上系统地缓解或治疗非酒精性脂肪性肝炎及其伴有的肝硬化和/或肝纤维化。In the prior art, it may be difficult to simultaneously relieve or treat nonalcoholic steatohepatitis and its associated liver cirrhosis and liver fibrosis with a single drug therapy. To sum up, the application of the HDAC2 and DNMT1 inhibitors provided by the technical solution of the present invention in the treatment of non-alcoholic steatohepatitis helps to systematically alleviate or treat non-alcoholic steatohepatitis and its accompanying symptoms to a certain extent. of liver cirrhosis and/or liver fibrosis.
另一方面，本发明提供了一种用于评估非酒精性脂肪性肝炎的标记物组，其特征在于，所述标记物组包括IGFBP7和ADAMTS1；所述标记物组的表达量可用于评估肝纤维化程度和/或肝功能受损情况。In another aspect, the present invention provides a marker group for evaluating nonalcoholic steatohepatitis, characterized in that, the marker group includes IGFBP7 and ADAMTS1; the expression level of the marker group can be used to evaluate liver Degree of fibrosis and/or impaired liver function.
进一步地，所述标记物组可以在没有肝功能受损的情况下在评估肝硬化和肝纤维化情况。Further, the marker panel can evaluate liver cirrhosis and liver fibrosis without impaired liver function.
进一步地，所述肝纤维化程度包括病理等级F2-F4级。Further, the degree of liver fibrosis includes pathological grades F2-F4.
进一步地，所述标记物组还可以用于评估促纤维化的Th17信号通路的应答情况。Further, the marker group can also be used to evaluate the response of the pro-fibrotic Th17 signaling pathway.
另一方面，本发明还提供了一种试剂盒，所述试剂盒包含一种用于评估非酒精性脂肪性肝炎的标记物组，其特征在于，所述标记物组包括IGFBP7和ADAMTS1；所述标 记物组的表达量可用于评估肝纤维化程度和/或肝功能受损情况。In another aspect, the present invention also provides a kit comprising a marker group for evaluating nonalcoholic steatohepatitis, characterized in that the marker group includes IGFBP7 and ADAMTS1; The expression levels of the above-mentioned marker groups can be used to evaluate the degree of liver fibrosis and/or the impairment of liver function.
进一步地，所述试剂盒可用于检测血浆中的IGFBP7和ADAMTS1表达量。Further, the kit can be used to detect the expression levels of IGFBP7 and ADAMTS1 in plasma.
进一步地，所述试剂盒可用于检测IGFBP7和ADAMTS1的蛋白表达量。Further, the kit can be used to detect the protein expression levels of IGFBP7 and ADAMTS1.
本发明还提供了一种用于区分非酒精性脂肪性肝炎和单纯脂肪肝的标记物组，其特征在于，所述标记物组包括IGFBP7和ADAMTS1。The present invention also provides a marker group for distinguishing nonalcoholic steatohepatitis and simple fatty liver, characterized in that, the marker group includes IGFBP7 and ADAMTS1.
另一方面，本发明提供了一种试剂盒，所述试剂盒包含一种用于区分非酒精性脂肪性肝炎和单纯性脂肪肝的标记物组，其特征在于，所述标记物组包括IGFBP7和ADAMTS1。In another aspect, the present invention provides a kit comprising a marker set for distinguishing between nonalcoholic steatohepatitis and simple fatty liver, characterized in that the marker set includes IGFBP7 and ADAMTS1.
进一步地，所述试剂盒可用于检测血浆中的IGFBP7和ADAMTS1表达量。Further, the kit can be used to detect the expression levels of IGFBP7 and ADAMTS1 in plasma.
进一步地，所述试剂盒可用于区分健康人与肝硬化、肝纤维化患者血浆中的IGFBP7和ADAMTS1表达水平。Further, the kit can be used to distinguish the expression levels of IGFBP7 and ADAMTS1 in the plasma of healthy people and patients with liver cirrhosis and liver fibrosis.
有益技术效果：Beneficial technical effects:
本发明提供评估非酒精性脂肪性肝炎的标记物组及包含所述标记物组的试剂盒。本发明技术方案通过一系列实验揭示：肝脏内皮细胞亚群中的表观遗传改变，会导致“窦内皮-大血管内皮失调”并刺激其相关的血管分泌因子重编程；在血管分泌因子基因中，IGFBP7和ADAMTS1在人和小型猪ECs中的特异性表达在相对最高水平，且能促进促纤维化的Th17应答以促进肝纤维化和肝硬化。而血浆IGFBP7和ADAMTS1的浓度可用于评估非酒精性脂肪性肝炎(NASH)。The present invention provides a marker panel for evaluating nonalcoholic steatohepatitis and a kit comprising the marker panel. The technical solution of the present invention reveals through a series of experiments that epigenetic changes in liver endothelial cell subsets can lead to "sinus endothelium-macrovascular endothelial dysregulation" and stimulate the reprogramming of its related vascular secretion factors; in the vascular secretion factor genes , IGFBP7 and ADAMTS1 are specifically expressed at relatively highest levels in human and minipig ECs and can promote pro-fibrotic Th17 responses to promote liver fibrosis and cirrhosis. The plasma concentrations of IGFBP7 and ADAMTS1 can be used to assess nonalcoholic steatohepatitis (NASH).
在现有技术中，肝活检和检测谷丙转氨酶(ALT)和谷草转氨酶(AST)水平是评估NASH的主要方法。其中，肝活检具有侵入性，可能会导致疼痛、出血等问题，无法适用于所有人群；而检测ALT和AST水平法既不敏感也不特异，一些肝硬化/NASH患者可能有正常ALT或AST水平。因此需要可靠、简单和无创的方法来评估非酒精性脂肪性肝炎。In the prior art, liver biopsy and detection of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels are the main methods for assessing NASH. Among them, liver biopsy is invasive, may cause pain, bleeding and other problems, and cannot be applied to all people; and the detection of ALT and AST levels is neither sensitive nor specific, and some patients with liver cirrhosis/NASH may have normal ALT or AST levels . There is therefore a need for reliable, simple and non-invasive methods to assess nonalcoholic steatohepatitis.
本发明技术方案通过一系列实验发现，IGFBP7和ADAMTS1可用于在没有肝功能受损的情况下在评估肝硬化和肝纤维化情况。例如，在实施例6中，F2-F4级患者的纤维化肝脏的IGFBP7和ADAMTS1的表达逐渐升高，且人和小型猪肝硬化窦内皮细胞(SECs)中的IGFBP7和ADAMTS1的mRNA及蛋白水平都显著升高，即可以根据IGFBP7和ADAMTS1的表达上调来有效区分正常和失调的SECs。进一步地，例如，在实施例7中，ALT或AST正常的肝硬化/肝纤维化患者的IGFBP7和ADAMTS1血浆浓度明显高于健康人。并且，本发明技术方案通过一系列实验发现，例如在实施例8、10和11中，IGFBP7和ADAMTS1增强促纤维化的Th17应答。由此，本发明提供的评估非酒精性脂肪性肝炎的标记物组及包含所述标记物组的试剂盒，有助于在没有肝功能受损的情况下，可靠、敏感、简便和无创地评估肝硬化和肝纤维化情况。The technical solution of the present invention found through a series of experiments that IGFBP7 and ADAMTS1 can be used to evaluate liver cirrhosis and liver fibrosis without impaired liver function. For example, in Example 6, the expression of IGFBP7 and ADAMTS1 was gradually increased in the fibrotic liver of F2-F4 patients, and the mRNA and protein levels of IGFBP7 and ADAMTS1 in human and minipig cirrhotic sinusoidal endothelial cells (SECs) Both were significantly elevated, i.e., normal and dysregulated SECs could be effectively distinguished based on the up-regulation of IGFBP7 and ADAMTS1 expression. Further, for example, in Example 7, the plasma concentrations of IGFBP7 and ADAMTS1 were significantly higher in cirrhotic/fibrotic patients with normal ALT or AST than in healthy individuals. Moreover, the technical solution of the present invention found through a series of experiments that, for example, in Examples 8, 10 and 11, IGFBP7 and ADAMTS1 enhanced the pro-fibrotic Th17 response. Therefore, the marker panel for assessing nonalcoholic steatohepatitis and the kit comprising the marker panel provided by the present invention contribute to a reliable, sensitive, simple and non-invasive method in the absence of impaired liver function. Assess for cirrhosis and fibrosis.
本发明还提供用于区分非酒精性脂肪性肝炎和单纯性脂肪肝的标记物组及包含所述标记物组的试剂盒。本发明技术方案通过一系列实验揭示，血浆IGFBP7和ADAMTS1可用于区分非酒精性脂肪性肝炎和单纯性脂肪肝：ALT或AST正常的肝硬化/肝纤维化患者的IGFBP7和ADAMTS1血浆浓度明显高于健康人样本，早期阶段NASH(F0-F1)患者的血浆ALT或AST浓度没有明显增加，而NASH患者血浆IGFBP7和ADAMTS1浓度显著升高，单纯性脂肪肝患者的血浆IGFBP7和ADAMTS1浓度在统计学上没有升高。由此，本发明提供的用于区分非酒精性脂肪性肝炎和单纯性脂肪肝的标记物组及包含所述标记物组的试剂盒，有助于可靠、敏感、简便和无创地区分非酒精性脂肪性肝炎和单纯性脂肪肝。The present invention also provides a marker panel for distinguishing nonalcoholic steatohepatitis from simple fatty liver and a kit comprising the marker panel. The technical solution of the present invention reveals through a series of experiments that plasma IGFBP7 and ADAMTS1 can be used to distinguish nonalcoholic steatohepatitis from simple fatty liver: the plasma concentrations of IGFBP7 and ADAMTS1 in patients with cirrhosis/hepatic fibrosis with normal ALT or AST are significantly higher than In healthy human samples, there was no significant increase in plasma ALT or AST concentrations in patients with early stage NASH (F0-F1), while plasma IGFBP7 and ADAMTS1 concentrations in NASH patients were significantly increased, and plasma IGFBP7 and ADAMTS1 concentrations in patients with simple fatty liver were statistically Not elevated. Therefore, the marker group for distinguishing non-alcoholic steatohepatitis and simple fatty liver and the kit comprising the marker group provided by the present invention are helpful for distinguishing non-alcoholic steatohepatitis reliably, sensitively, easily and non-invasively. steatohepatitis and simple fatty liver.
综上所述，与现有技术相比，本发明提供的评估非酒精性脂肪性肝炎的标记物组及包含所述标记物组的试剂盒，有助于可靠、敏感、简便和无创地评估非酒精性脂肪性肝炎。In conclusion, compared with the prior art, the marker panel for evaluating nonalcoholic steatohepatitis and the kit comprising the marker panel provided by the present invention are helpful for reliable, sensitive, simple and non-invasive evaluation nonalcoholic steatohepatitis.
另一方面，本发明提供了血管分泌因子在制备检测非酒精性脂肪性肝炎的生物标记物中的应用，其特征在于，所述血管分泌因子包括IGFBP7和/或ADAMTS1；所述IGFBP7和/或ADAMTS1表达于HDAC2/DNMT1-IGFBP7/ADAMTS1-Th17信号通路。In another aspect, the present invention provides the use of vascular secretion factors in the preparation of biomarkers for the detection of nonalcoholic steatohepatitis, characterized in that, the vascular secretion factors include IGFBP7 and/or ADAMTS1; the IGFBP7 and/or ADAMTS1 is expressed in the HDAC2/DNMT1-IGFBP7/ADAMTS1-Th17 signaling pathway.
进一步地，所述HDAC2/DNMT1-IGFBP7/ADAMTS1-Th17信号通路中IGFBP7和/或ADAMTS1的mRNA表达量和/或蛋白表达量可用于评估肝纤维化程度和/或肝功能受损情况。Further, the mRNA expression and/or protein expression of IGFBP7 and/or ADAMTS1 in the HDAC2/DNMT1-IGFBP7/ADAMTS1-Th17 signaling pathway can be used to evaluate the degree of liver fibrosis and/or liver function impairment.
进一步地，所述IGFBP7和/或ADAMTS1表达量的升高与肝纤维化程度成正比，所述肝纤维化程度包括病理等级F2-F4级。Further, the increase in the expression of IGFBP7 and/or ADAMTS1 is proportional to the degree of liver fibrosis, and the degree of liver fibrosis includes pathological grades F2-F4.
进一步地，所述IGFBP7和/或ADAMTS1存在于血浆中。Further, the IGFBP7 and/or ADAMTS1 are present in plasma.
本发明还提供了血管分泌因子在制备区分非酒精性脂肪性肝炎和单纯性脂肪肝的生物标记物中的应用，其特征在于，所述血管分泌因子包括IGFBP7和/或ADAMTS1；所述IGFBP7和/或ADAMTS1表达于HDAC2/DNMT1-IGFBP7/ADAMTS1-Th17信号通路。The present invention also provides the application of vascular secretion factors in preparing biomarkers for distinguishing nonalcoholic steatohepatitis and simple fatty liver, characterized in that, the vascular secretion factors include IGFBP7 and/or ADAMTS1; the IGFBP7 and /or ADAMTS1 is expressed in HDAC2/DNMT1-IGFBP7/ADAMTS1-Th17 signaling pathway.
进一步地，所述HDAC2/DNMT1-IGFBP7/ADAMTS1-Th17信号通路中IGFBP7和/或ADAMTS1的mRNA表达量和/或蛋白表达量可区分非酒精性脂肪性肝炎和单纯性脂肪肝。Further, the mRNA expression and/or protein expression of IGFBP7 and/or ADAMTS1 in the HDAC2/DNMT1-IGFBP7/ADAMTS1-Th17 signaling pathway can distinguish nonalcoholic steatohepatitis from simple fatty liver.
本发明还提供了IGFBP7和/或ADAMTS1协同作用在制备检测非酒精性脂肪性肝炎的生物标记物中的用途。The present invention also provides the use of IGFBP7 and/or ADAMTS1 synergistic effect in preparing a biomarker for detecting nonalcoholic steatohepatitis.
进一步地，所述非酒精性脂肪性肝炎包括伴有肝硬化或肝纤维化。Further, the nonalcoholic steatohepatitis includes liver cirrhosis or liver fibrosis.
进一步地，所述非酒精性脂肪性肝炎还包括肝无纤维化的情况，所述无纤维化的病理等级为F0-F1。Further, the non-alcoholic steatohepatitis also includes the case of no liver fibrosis, and the pathological grade of the no fibrosis is F0-F1.
进一步地，所述IGFBP7和/或ADAMTS1诱导促纤维化Th17细胞应答。Further, the IGFBP7 and/or ADAMTS1 induces a pro-fibrotic Th17 cell response.
进一步地，所述Th17细胞在肝纤维化的肝脏中聚集。Further, the Th17 cells aggregated in fibrotic livers.
有益技术效果：Beneficial technical effects:
本发明提供血管分泌因子在制备检测非酒精性脂肪性肝炎的生物标记物中的应用。本发明技术方案通过一系列实验揭示：失调的肝脏内皮细胞亚群中，存在HDAC2/DNMT1-IGFBP7/ADAMTS1-Th17信号通路，其中，HDAC2/DNMT1之间的交互调控会刺激促纤维化的IGFBP7和ADAMTS1的产生，从而募集Th17细胞，在非酒精性脂肪性肝炎(NASH)中抑制肝脏再生和诱导纤维化。The present invention provides the application of vascular secreted factors in the preparation of biomarkers for detecting nonalcoholic steatohepatitis. The technical solution of the present invention reveals through a series of experiments that the HDAC2/DNMT1-IGFBP7/ADAMTS1-Th17 signaling pathway exists in the dysregulated liver endothelial cell subsets, wherein the interactive regulation between HDAC2/DNMT1 will stimulate the pro-fibrotic IGFBP7 and Production of ADAMTS1, thereby recruiting Th17 cells, inhibits liver regeneration and induces fibrosis in nonalcoholic steatohepatitis (NASH).
本发明技术方案通过一系列实验发现，IGFBP7和/或ADAMTS1表达于HDAC2/DNMT1-IGFBP7/ADAMTS1-Th17信号通路。IGFBP7和ADAMTS1是增强Th17应答以促进肝脏纤维化的调节因子：例如，在实施例10中，联合靶向抑制内皮来源的HDAC2和DNMT1，降低了纤维化肝脏内皮细胞中IGFBP7的表达；基因敲除IGFBP7的NASH表型小鼠，明显减轻了肝纤维化反应(胶原沉积、血清肝功指数、肝脏羟脯氨酸含量)和Th17应答；升高的IGFBP7水平，显著增强了肝脏的促纤维化的Th17应答。而在实施例11中，ADAMTS1在Hdac2 iΔEC+AZA小鼠(即选择性敲除内皮细胞HDAC2的NASH表型小鼠，并用DNMT1抑制剂处理，以获得联合靶向HDAC2+DNMT1的抑制效果)的肝脏窦内皮细胞中明显降低；敲低人内皮细胞中的ADAMTS1，阻断了TGF-β刺激下Smad2的磷酸化(Smad2是TGF-β1诱导Th17细胞中重要的下游因子)；移植缺乏ADAMTS1的细胞外囊泡(EVs)的小鼠的肝纤维化和Th17应答显著降低。 According to the technical solution of the present invention, it is found through a series of experiments that IGFBP7 and/or ADAMTS1 are expressed in the HDAC2/DNMT1-IGFBP7/ADAMTS1-Th17 signaling pathway. IGFBP7 and ADAMTS1 are regulators that enhance Th17 responses to promote liver fibrosis: For example, in Example 10, combined targeting of endothelial-derived HDAC2 and DNMT1 reduced IGFBP7 expression in fibrotic liver endothelial cells; gene knockout NASH phenotype mice with IGFBP7 significantly reduced liver fibrotic responses (collagen deposition, serum liver function index, liver hydroxyproline content) and Th17 responses; elevated IGFBP7 levels significantly enhanced hepatic pro-fibrotic responses Th17 response. Whereas, in Example 11, ADAMTS1 was detected in Hdac2 iΔEC + AZA mice (i.e., NASH phenotype mice with selective knockout of endothelial HDAC2 and treated with a DNMT1 inhibitor to obtain the inhibitory effect of combined targeting of HDAC2 + DNMT1). Significantly decreased in liver sinusoidal endothelial cells; knockdown of ADAMTS1 in human endothelial cells blocked TGF-β-stimulated phosphorylation of Smad2 (Smad2 is an important downstream factor in TGF-β1-induced Th17 cells); ADAMTS1-deficient cells were transplanted Liver fibrosis and Th17 responses were significantly reduced in mice with extracellular vesicles (EVs).
本发明技术方案通过一系列实验发现，所述HDAC2/DNMT1-IGFBP7/ADAMTS1-Th17信号通路中IGFBP7和/或ADAMTS1的mRNA表达量和/或蛋白表达量可用于评估肝纤维化程度和肝功能受损情况。例如，在实施例6中，F2-F4级患者的纤维化肝脏的IGFBP7和ADAMTS1的表达逐渐升高，且人和小型猪肝硬化窦内皮细胞中的IGFBP7和ADAMTS1的mRNA及蛋白水平都显著升高，即可以根据IGFBP7和ADAMTS1的表达上调来有效区分正常和失调的SECs。进一步地，例如，在实 施例7中，ALT或AST正常的肝硬化/肝纤维化患者的IGFBP7和ADAMTS1血浆浓度明显高于健康人。也就是说，IGFBP7和ADAMTS1可用于在没有肝功能受损的情况下在评估肝硬化和肝纤维化情况。The technical solution of the present invention has found through a series of experiments that the mRNA expression and/or protein expression of IGFBP7 and/or ADAMTS1 in the HDAC2/DNMT1-IGFBP7/ADAMTS1-Th17 signaling pathway can be used to evaluate the degree of liver fibrosis and the influence of liver function. damage situation. For example, in Example 6, the expressions of IGFBP7 and ADAMTS1 were gradually increased in the fibrotic livers of F2-F4 patients, and the mRNA and protein levels of IGFBP7 and ADAMTS1 in human and minipig cirrhotic sinusoidal endothelial cells were significantly increased. High, that is, normal and dysregulated SECs can be effectively distinguished based on the up-regulation of IGFBP7 and ADAMTS1 expression. Further, for example, in Example 7, the plasma concentrations of IGFBP7 and ADAMTS1 were significantly higher in cirrhotic/fibrotic patients with normal ALT or AST than in healthy individuals. That is, IGFBP7 and ADAMTS1 can be used to assess liver cirrhosis and liver fibrosis without impaired liver function.
本发明还提供血管分泌因子在制备区分非酒精性脂肪性肝炎和单纯性脂肪肝的生物标记物中的应用。本发明技术方案通过一系列实验揭示，IGFBP7和ADAMTS1可用于区分非酒精性脂肪性肝炎和单纯性脂肪肝：早期阶段NASH(F0-F1)患者的血浆ALT或AST浓度没有明显增加，而NASH患者血浆IGFBP7和ADAMTS1浓度显著升高，单纯性脂肪肝患者的血浆IGFBP7和ADAMTS1浓度在统计学上没有升高。The present invention also provides the application of the vascular secretory factor in preparing a biomarker for distinguishing between nonalcoholic steatohepatitis and simple fatty liver. The technical solution of the present invention revealed through a series of experiments that IGFBP7 and ADAMTS1 can be used to distinguish nonalcoholic steatohepatitis from simple fatty liver: the plasma ALT or AST concentrations of patients with early stage NASH (F0-F1) did not increase significantly, while patients with NASH Plasma concentrations of IGFBP7 and ADAMTS1 were significantly elevated, and plasma IGFBP7 and ADAMTS1 concentrations were not statistically elevated in patients with simple fatty liver.
本发明还提供IGFBP7和/或ADAMTS1协同作用在制备检测非酒精性脂肪性肝炎的生物标记物中的用途。非酒精性脂肪性肝炎常伴有肝硬化或肝纤维化，如上所述，IGFBP7和ADAMTS1协同作用有助于检测非酒精性脂肪性肝炎，包括但不限于评估肝硬化或肝纤维化情况、区分非酒精性脂肪性肝炎和单纯性脂肪肝和评估Th17细胞在肝纤维化的肝脏的聚集情况。The present invention also provides the use of IGFBP7 and/or ADAMTS1 synergistic effect in the preparation of biomarkers for detecting nonalcoholic steatohepatitis. Nonalcoholic steatohepatitis is often associated with cirrhosis or fibrosis. As mentioned above, IGFBP7 and ADAMTS1 cooperate to detect nonalcoholic steatohepatitis, including but not limited to assessing cirrhosis or fibrosis, distinguishing Nonalcoholic steatohepatitis and simple fatty liver and assessment of Th17 cell accumulation in fibrotic livers.
与现有技术中相比，本发明提供的血管分泌因子在制备检测非酒精性脂肪性肝炎的生物标记物中的应用，能够可靠、简便且无创地评估非酒精性脂肪性肝炎。Compared with the prior art, the application of the vascular secretion factor provided in the present invention in preparing a biomarker for detecting non-alcoholic steatohepatitis can reliably, simply and non-invasively evaluate non-alcoholic steatohepatitis.
附图说明Description of drawings
图1为单细胞RNA测序(scRNA-Seq)揭示人肝硬化肝脏中HDAC2/DNMT1选择性诱导的“窦内皮-大血管内皮失调”的实验图；Figure 1 is an experimental image of single-cell RNA sequencing (scRNA-Seq) revealing HDAC2/DNMT1-selectively induced "sinusoidal endothelial-macrovascular endothelial dysregulation" in human cirrhotic liver;
图2为肝内皮细胞中HDAC2和DNMT1的联合靶向抑制减轻小型猪NASH模型中肝脏纤维化的实验图；Figure 2 is an experimental graph of the combined targeted inhibition of HDAC2 and DNMT1 in hepatic endothelial cells alleviating liver fibrosis in a minipig NASH model;
图3为小型猪NASH模型中靶向抑制表观遗传失调的肝脏内皮细胞逆转“窦内皮-大血管内皮失调”、使内皮分类正常化并阻断肝硬化的实验图；Figure 3 is an experimental diagram of targeting the inhibition of epigenetic dysregulated liver endothelial cells in a minipig NASH model to reverse "sinusoidal endothelium-macrovascular endothelial dysregulation", normalize endothelial classification, and block liver cirrhosis;
图4为人类患者和小型猪中表观遗传失调的肝脏内皮细胞发生旁分泌/血管分泌因子重编程的实验图；Figure 4 is an experimental graph of the reprogramming of paracrine/vascular secretory factors in epigenetically dysregulated liver endothelial cells in human patients and minipigs;
图5为血浆细胞外囊泡(EVs)中的旁分泌/血管分泌因子IGFBP7和ADAMTS1作为评估人类患者和小型猪NASH模型中纤维化进展的生物标记物的实验图；Figure 5 is an experimental graph of the paracrine/vascular secretory factors IGFBP7 and ADAMTS1 in plasma extracellular vesicles (EVs) as biomarkers for assessing fibrosis progression in human patients and minipig NASH models;
图6为人类患者和小型猪NASH模型中失调的血管内皮微环境诱导促纤维化的Th17应答的实验图；Figure 6 is an experimental graph of a dysregulated vascular endothelial microenvironment inducing a pro-fibrotic Th17 response in a human patient and a minipig NASH model;
图7为失调的肝脏窦内皮细胞(SECs)中抑制HDAC2/DNMT1减少小鼠NASH模型的促纤维化的Th17细胞的募集的实验图；Figure 7 is a graph showing that inhibition of HDAC2/DNMT1 reduces the recruitment of pro-fibrotic Th17 cells in dysregulated liver sinusoidal endothelial cells (SECs) in a mouse model of NASH;
图8为IGFBP7促进小鼠NASH模型的肝脏纤维化和促纤维化的Th17应答的实验图；Figure 8 is an experimental diagram of IGFBP7 promoting liver fibrosis and pro-fibrotic Th17 responses in a mouse NASH model;
图9为ADAMTS1的抑制减轻小鼠肝纤维化模型中促纤维化的Th17细胞应答的实验图；Figure 9 is an experimental graph showing that inhibition of ADAMTS1 alleviates the pro-fibrotic Th17 cell response in a mouse liver fibrosis model;
图10为单细胞RNA测序(scRNA-Seq)分析2个健康人和2个肝硬化患者的肝脏非实质细胞(NPCs)的实验图；Figure 10 is an experimental diagram of single-cell RNA sequencing (scRNA-Seq) analysis of hepatic non-parenchymal cells (NPCs) from 2 healthy individuals and 2 patients with liver cirrhosis;
图11为scRNA-Seq分析来自GSE136103数据的人肝脏NPCs的实验图；Figure 11 is an experimental graph of scRNA-Seq analysis of human liver NPCs from GSE136103 data;
图12为人肝脏scRNA-Seq数据中内皮细胞(ECs)分析的实验图；Figure 12 is an experimental diagram of endothelial cells (ECs) analysis in human liver scRNA-Seq data;
图13为人类肝脏、肝脏CD45 +NPCs和肝脏内皮细胞的基因表达分析的实验图； Figure 13 is an experimental graph of gene expression analysis of human liver, liver CD45 + NPCs and liver endothelial cells;
图14为scRNA-Seq分析小型猪肝脏NPCs的实验图；Figure 14 is an experimental diagram of scRNA-Seq analysis of minipig liver NPCs;
图15为小型猪肝脏scRNA-Seq数据中内皮细胞(ECs)分析；Figure 15 shows endothelial cells (ECs) analysis in minipig liver scRNA-Seq data;
图16为肝硬化人类和小型猪肝脏的差异基因分析及旁分泌因子基因在肝脏NPCs中的表达的实验图；Figure 16 is an experimental diagram of differential gene analysis of liver cirrhosis between human and minipigs and expression of paracrine factor genes in liver NPCs;
图17为人和小型猪肝脏的Th17细胞分析的实验图。Figure 17 is an experimental graph of Th17 cell analysis in human and minipig livers.
具体实施方式Detailed ways
为使本发明实施例的目的、技术方案和优点更加清楚，下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整地描述。显然，所描述的实施例是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be described clearly and completely below with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are some, but not all, embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.
需要说明的是，在本文中，术语“包括”、“包含”或者其任何其他变体意在涵盖非排他性的包含，从而使得包括一系列要素的过程、方法、物品或者装置不仅包括那些要素，而且还包括没有明确列出的其他要素，或者是还包括为这种过程、方法、物品或者装置所固有的要素。在没有更多限制的情况下，由语句“包括一个……”限定的要素，并不排除在包括该要素的过程、方法、物品或者装置中还存在另外的相同要素。It should be noted that, herein, the terms "comprising", "comprising" or any other variation thereof are intended to encompass non-exclusive inclusion, such that a process, method, article or device comprising a series of elements includes not only those elements, It also includes other elements not expressly listed or inherent to such a process, method, article or apparatus. Without further limitation, an element qualified by the phrase "comprising a..." does not preclude the presence of additional identical elements in a process, method, article or apparatus that includes the element.
如在本说明书中使用的，术语“大约”，典型地表示为所述值的+/-5％，更典型的是所述值的+/-4％，更典型的是所述值的+/-3％，更典型的是所述值的+/-2％，甚至更典型的是所述值的+/-1％，甚至更典型的是所述值的+/-0.5％。As used in this specification, the term "about" is typically expressed as +/- 5% of the stated value, more typically +/- 4% of the stated value, and more typically + /-3%, more typically +/-2% of said value, even more typically +/-1% of said value, even more typically +/-0.5% of said value.
在本说明书中，某些实施方式可能以一种处于某个范围的格式公开。应该理解，这种“处于某个范围”的描述仅仅是为了方便和简洁，且不应该被解释为对所公开范围的僵化限制。因此，范围的描述应该被认为是已经具体地公开了所有可能的子范围以及在此范围内的独立数字值。例如，范围
Figure PCTCN2021140096-appb-000001
的描述应该被看作已经具体地公开了子范围如从1到3，从1到4，从1到5，从2到4，从2到6，从3到6等，以及此范围内的单独数字，例如1，2，3，4，5和6。无论该范围的广度如何，均适用以上规则。 In this specification, certain embodiments may be disclosed in a format that is within a range. It should be understood that this description "within a range" is merely for convenience and brevity and should not be construed as an inflexible limitation on the disclosed scope. Accordingly, the description of a range should be considered to have specifically disclosed all possible subranges as well as individual numerical values within that range. For example, the range
Figure PCTCN2021140096-appb-000001
The description should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., and Individual numbers such as 1, 2, 3, 4, 5 and 6. The above rules apply regardless of the breadth of the range.
名词解释Glossary
本发明所述的“交互调控”是指：肝脏纤维化同时受HDAC2和DNMT1调控，单独抑制HDAC2或DNMT1的一种，另一种(DNMT1或HDAC2)发生向着促纤维化的方向变化(升高)。例如，在内皮细胞中抑制HDAC2，DNMT1表达升高，抑制DNMT1，HDAC2表达升高，肝脏纤维化受HDAC2和DNMT1交互调控影响。The "interactive regulation" described in the present invention means that liver fibrosis is regulated by both HDAC2 and DNMT1, and one of HDAC2 or DNMT1 is inhibited alone, and the other (DNMT1 or HDAC2) changes in the direction of pro-fibrosis (increased ). For example, inhibition of HDAC2 in endothelial cells increases the expression of DNMT1, inhibition of DNMT1 increases the expression of HDAC2, and liver fibrosis is affected by the interactive regulation of HDAC2 and DNMT1.
本发明所述的“血管微环境”是指：血管内皮细胞通过与周围细胞的相互作用，主动性调控周围细胞的功能和表型，从而形成引导性微环境。The "vascular microenvironment" in the present invention refers to: vascular endothelial cells actively regulate the function and phenotype of surrounding cells through interaction with surrounding cells, thereby forming a guiding microenvironment.
本发明所述的“失调”是指：与正常肝脏相比，肝硬化肝脏中内皮细胞亚群的比例发生变化或基因表达发生变化。The term "disorder" in the present invention refers to changes in the proportion of endothelial cell subsets or changes in gene expression in cirrhotic livers compared to normal livers.
本发明所述的“窦内皮-大血管内皮失调”或“窦内皮-大血管内皮分类异常”是指：与正常肝脏相比，肝硬化肝脏中窦内皮细胞数量减少，大血管内皮细胞数量增加，窦内皮细胞表达大血管内皮标记物(marker)的一种病理现象。The "sinusoidal endothelium-macrovascular endothelium disorder" or "sinusoidal endothelium-macrovascular endothelium classification abnormality" in the present invention refers to: compared with normal livers, the number of sinusoidal endothelial cells in cirrhotic livers decreases, while the number of macrovascular endothelial cells increases , a pathological phenomenon in which sinus endothelial cells express macrovascular endothelial markers.
本发明所述的“表观遗传治疗”是指：用表观遗传抑制剂对肝硬化或纤维化小型猪及小鼠进行治疗，从而减轻肝脏纤维化。The "epigenetic therapy" in the present invention refers to treating liver cirrhosis or fibrosis miniature pigs and mice with epigenetic inhibitors, thereby reducing liver fibrosis.
附图详细说明Detailed Description of Drawings
图1：(A)华西医院人类患者的肝脏非实质细胞(NPCs)的scRNA-Seq的方法。(B,C)2个健康肝脏和2个肝硬化肝脏NPCs的scRNA-Seq数据的聚类分析。(B)热图显示NPCs的不同细胞系及其标记基因(右)。(C)UMAP图显示NPCs的不同细胞系。Endo(EC)，内皮细胞；DC，树突状细胞；Neu，中性粒细胞；Mac，巨噬细胞；EPCAM +，EPCAM +细胞和胆管细胞。(D)饼图显示NPCs不同细胞系的差异基因数(肝硬化肝脏vs.健康肝脏)。(E)2个健康肝脏和2个肝硬化肝脏的内皮细胞的聚类分析。左上：UMAP图显示内皮细胞的不同细胞系；左下：饼图显示内皮细胞的不同亚群的比例；右：内皮细胞的不同细胞系的标记基因表达。CLCE4G和OIT3标记窦内皮细胞，CD34标记大血管内皮细胞。SEC，窦内皮细胞；MEC，大血管内皮细胞。(F)健康和肝硬化的人肝脏内皮细胞的不同亚群的拟时分析。拟时分析显示，肝硬化的人肝脏发生“窦内皮-大血管内皮失调”。(G)健康和肝硬化的人肝脏的实质细胞(肝细胞)和非实质细胞(NPCs)中组蛋 白修饰的液相芯片分析。右：组蛋白H3和H4不同修饰位点的乙酰化的定量。Hep，肝细胞；NPCs，非实质细胞。N＝3。(H)热图显示与组蛋白修饰和DNA甲基化相关基因在健康和肝硬化的人肝脏的非实质细胞(NPCs)的不同细胞系中的表达。上：与组蛋白修饰和DNA甲基化相关的差异基因数。(I)组蛋白去乙酰化酶(HDACs)在人肝脏纤维化不同病理等级的表达(数据来自GSE84044)。F0-F4，人肝脏纤维化的不同病理等级。F1-F4级肝脏的HDACs的表达水平相对于健康肝脏(F0)进行量化。(J)小提琴图显示HDACs在健康和肝硬化人的肝脏内皮细胞中的表达。(K)定量PCR(qPCR)显示HDAC2在健康和肝硬化人的肝脏内皮细胞中的表达。N＝3。(L)DNA甲基化转移酶(DNMTs)在人肝脏纤维化不同等级的表达(数据来自GSE84044)。F0-F4，人肝纤维化不同病理等级。F1-F4级肝脏的DNMTs的表达水平相对于健康肝脏(F0)进行量化。(M)小提琴图显示DNMTs在健康和肝硬化人肝脏内皮细胞中的表达。(N)qPCR显示DNMT1在健康和肝硬化人肝脏的内皮细胞中的表达。N＝3。(O)qPCR显示HDAC2和DNMT1在健康和肝硬化人肝脏的SECs中表达。N＝3。(P)假说示意图：肝硬化肝脏的窦内皮细胞获得大血管内皮细胞的表型标记。肝脏内皮细胞表观遗传重编程促进“窦内皮-大血管内皮失调”，引起内皮细胞分类异常，并导致肝脏纤维化或肝硬化。在所有统计分析中，2组比较的数据通过双尾学生t检验(two-tailed student’s t-test)进行分析；多于2组比较的数据通过单因素方差分析和随后的Tukey事后检验(one-way ANOVA followed by Tukey’s post-hoc test)进行分析。数据用平均值±SEM表示。*，肝硬化vs.健康或F1-F4vs.F0；*，P<0.05；**，P<0.01；***，P<0.001；****，P<0.0001。 Figure 1: (A) Method for scRNA-Seq of hepatic non-parenchymal cells (NPCs) from human patients at West China Hospital. (B,C) Cluster analysis of scRNA-Seq data from 2 healthy livers and 2 cirrhotic liver NPCs. (B) Heat map showing different cell lines of NPCs and their marker genes (right). (C) UMAP plot showing different cell lines of NPCs. Endo (EC), endothelial cells; DC, dendritic cells; Neu, neutrophils; Mac, macrophages; EPCAM + , EPCAM + cells and cholangiocytes. (D) Pie chart showing the number of differential genes in different cell lines of NPCs (cirrhotic liver vs. healthy liver). (E) Cluster analysis of endothelial cells from 2 healthy livers and 2 cirrhotic livers. Top left: UMAP plot showing different cell lines of endothelial cells; bottom left: Pie chart showing proportions of different subpopulations of endothelial cells; Right: marker gene expression of different cell lines of endothelial cells. CLCE4G and OIT3 marked sinus endothelial cells, and CD34 marked macrovascular endothelial cells. SEC, sinusoidal endothelial cells; MEC, macrovascular endothelial cells. (F) Chronometric analysis of different subsets of healthy and cirrhotic human liver endothelial cells. A chronological analysis showed that "sinusoidal endothelium-macrovascular endothelium dysregulation" occurred in human livers with cirrhosis. (G) Liquid ChIP analysis of histone modifications in parenchymal cells (hepatocytes) and non-parenchymal cells (NPCs) of healthy and cirrhotic human livers. Right: Quantification of acetylation at different modification sites on histones H3 and H4. Hep, hepatocytes; NPCs, non-parenchymal cells. N=3. (H) Heat map showing the expression of genes associated with histone modifications and DNA methylation in different cell lines of non-parenchymal cells (NPCs) of healthy and cirrhotic human livers. Top: number of differential genes associated with histone modifications and DNA methylation. (I) Expression of histone deacetylases (HDACs) in different pathological grades of human liver fibrosis (data from GSE84044). F0-F4, different pathological grades of human liver fibrosis. The expression levels of HDACs in F1-F4 livers were quantified relative to healthy livers (F0). (J) Violin plot showing the expression of HDACs in hepatic endothelial cells of healthy and cirrhotic people. (K) Quantitative PCR (qPCR) showing HDAC2 expression in hepatic endothelial cells of healthy and cirrhotic humans. N=3. (L) Expression of DNA methyltransferases (DNMTs) in different grades of human liver fibrosis (data from GSE84044). F0-F4, different pathological grades of human liver fibrosis. Expression levels of DNMTs in F1-F4 livers were quantified relative to healthy livers (F0). (M) Violin plot showing the expression of DNMTs in healthy and cirrhotic human liver endothelial cells. (N) qPCR showing DNMT1 expression in endothelial cells of healthy and cirrhotic human livers. N=3. (O) qPCR shows that HDAC2 and DNMT1 are expressed in SECs of healthy and cirrhotic human livers. N=3. (P) Schematic of the hypothesis: sinusoidal endothelial cells of cirrhotic livers acquire phenotypic markers of macrovascular endothelial cells. Epigenetic reprogramming of hepatic endothelial cells promotes "sinusoidal-macrovascular endothelial dysregulation," causing endothelial cell sorting abnormalities and leading to liver fibrosis or cirrhosis. In all statistical analyses, data for 2-group comparisons were analyzed by two-tailed student's t-test; data for more than 2-group comparisons were analyzed by one-way ANOVA followed by Tukey's post-hoc test (one- way ANOVA followed by Tukey's post-hoc test) for analysis. Data are presented as mean ± SEM. *, cirrhosis vs. healthy or F1-F4 vs. F0; *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001.
图2：(A)小型猪NASH模型和治疗方案。小型猪NASH模型由西方饮食(WD：高脂肪、高胆固醇、高蔗糖和果糖)和重复腹腔注射肝脏毒性的四氯化碳(CCl 4)诱导。治疗组：通过WD+CCl 4持续诱导NASH 5个月，在诱导2个月后开始进行表观遗传治疗，持续3个月。肝硬化组：用WD+CCl 4持续诱导小型猪5个月。(B)HDAC2和DNMT1联合抑制治疗小型猪NASH模型的给药方案。小型猪用HDAC2抑制剂(HDAC2i)和DNMT1抑制剂(HDAC2i)处理。小型猪共分3组：1)对照组(正常饮食+玉米油)；2)肝硬化组(模型组)(WD+CCl 4诱导NASH)3)治疗组(HDAC2i和DNMT1i治疗)。(C)对照组、肝硬化组和治疗组小型猪的血糖水平。Glu，葡萄糖。N＝3。(D)对照组、肝硬化组和治疗组小型猪的肝脏组织的肝纤维化指数。PC III，III型前胶原；IV-C，IV型胶原；HA，透明质酸；Hyp，羟脯氨酸。N＝3。(E)对照组、肝硬化组和治疗组小型猪血清的肝脏功能水平。ALP，碱性磷酸酶；ALT，谷丙转氨酶；AST，谷草转氨酶；TC，总胆固醇。N＝3。(F)通过H&E、天狼星红、I型胶原、油红O和Ki67染色评估小型猪的肝脏组织学、胶原和脂滴沉积以及细胞增殖情况。右：左图虚线区域的高倍放大图。比例尺，200μm。N＝3。(G)图2F中天狼星红、油红O、胶原I和Ki67染色的定量。阳性染色的比例相对于对照组进行量化。N＝3。(H)qPCR显示对照组、肝硬化组和治疗组小型猪的肝脏窦内皮细胞(SECs)和大血管内皮细胞(MECs)中HDAC2和DNMT1的mRNA表达水平。N＝3。(I)小型猪NASH模型中靶向促纤维化的表观遗传交互调控的示意图：在失调的肝脏内皮细胞中联合靶向抑制异常激活的HDAC2和DNMT1减轻小型猪NASH模型的肝脏纤维化并促进肝再生。所有数据均通过单因素方差分析和随后的Tukey事后检验进行统计分析。数据用平均值±SEM表示。*，肝硬化组vs对照组；#，治疗组vs肝硬化组；*，P<0.05；**，P<0.01。#，P<0.05；##，P<0.01。 Figure 2: (A) Minipig NASH model and treatment regimen. The minipig NASH model was induced by a Western diet (WD: high fat, high cholesterol, high sucrose and fructose) and repeated intraperitoneal injections of hepatotoxic carbon tetrachloride ( CCl4 ). Treatment group: Induction of NASH by WD+CCl 4 continued for 5 months, and epigenetic therapy was started after 2 months of induction for 3 months. Cirrhosis group: Minipigs were induced with WD+CCl 4 continuously for 5 months. (B) Dosing regimen for combined inhibition of HDAC2 and DNMT1 in a minipig NASH model. Minipigs were treated with HDAC2 inhibitor (HDAC2i) and DNMT1 inhibitor (HDAC2i). The minipigs were divided into 3 groups: 1) control group (normal diet + corn oil); 2) liver cirrhosis group (model group) (WD+ CCl4 induced NASH) 3) treatment group (HDAC2i and DNMT1i treatment). (C) Blood glucose levels of the control, cirrhosis and treatment minipigs. Glu, glucose. N=3. (D) Liver fibrosis index of liver tissue of control, cirrhosis and treatment group minipigs. PC III, procollagen type III; IV-C, collagen type IV; HA, hyaluronic acid; Hyp, hydroxyproline. N=3. (E) Serum liver function levels of minipigs in the control, cirrhosis and treatment groups. ALP, alkaline phosphatase; ALT, alanine aminotransferase; AST, aspartate aminotransferase; TC, total cholesterol. N=3. (F) Assessment of liver histology, collagen and lipid droplet deposition, and cell proliferation in minipigs by H&E, Sirius Red, Collagen I, Oil Red O, and Ki67 staining. Right: High magnification of the dotted area in the left panel. Scale bar, 200 μm. N=3. (G) Quantification of Sirius Red, Oil Red O, Collagen I and Ki67 staining in Figure 2F. The proportion of positive staining was quantified relative to the control group. N=3. (H) qPCR showing the mRNA expression levels of HDAC2 and DNMT1 in liver sinusoidal endothelial cells (SECs) and macrovascular endothelial cells (MECs) of control, cirrhosis, and treatment minipigs. N=3. (I) Schematic illustration of targeted pro-fibrotic epigenetic cross-regulation in a minipig NASH model: combined targeting and inhibition of aberrantly activated HDAC2 and DNMT1 in dysregulated liver endothelial cells attenuates liver fibrosis and promotes hepatic fibrosis in a minipig NASH model Liver regeneration. All data were statistically analyzed by one-way ANOVA followed by Tukey's post hoc test. Data are presented as mean ± SEM. *, liver cirrhosis group vs control group; #, treatment group vs liver cirrhosis group; *, P<0.05; **, P<0.01. #, P<0.05;##,P<0.01.
图3：(A)小型猪NASH模型中肝脏的非实质细胞(NPCs)的单细胞测序的方法。(B,C)对照组、肝硬化组和治疗组小型猪肝脏的非实质细胞(NPCs)聚类分析。(B)热图显示NPCs的不同细胞系及其标记基因(右)。(C)UMAP图显示NPCs的不同细胞系。Endo(EC)，内皮细胞；DC，树突状细胞；Neu，中性粒细胞；Mac，巨噬细胞；EPCAM +，EPCAM +细胞和胆管细胞。(D)饼图显示小型猪肝脏的非实质细胞(NPCs) 的不同细胞系的比例。(E)维恩图分别显示肝硬化组和对照组以及治疗组和肝硬化组之间小型猪肝脏的非实质细胞(NPCs)的不同细胞系中的差异基因数。括号中的数字表示治疗后恢复的基因数。(F)小型猪肝脏内皮细胞的KEGG通路富集分析。在肝硬化组与对照组小型猪肝脏内皮细胞之间有79条KEGG通路显著改变，在治疗组与肝硬化组小型猪肝脏内皮细胞之间有99条KEGG通路显著改变。通过HDAC2i和DNMT2i的表观遗传治疗使肝硬化组小型猪肝脏内皮细胞中大部分改变的KEGG通路正常化。白色数字表示校正p值的排序顺序。校正p值<0.05被认为具有统计学意义。(G)对照组、肝硬化组和治疗组小型猪肝脏内皮细胞的聚类分析。(H)饼图显示小型猪肝脏内皮细胞的不同亚群的比例。与对照组相比，肝硬化组小型猪肝脏中大血管内皮细胞(MECs)数量增加和窦内皮细胞(SECs)数量减少，提示发生“窦内皮-大血管内皮失调”。(I)维恩图显示小型猪肝脏内皮细胞的不同亚群中的差异基因数。肝硬化组中肝脏窦内皮细胞(SECs)的差异基因数相对最多，HDAC2i+DNMT1i治疗后大部分差异基因得以恢复。括号中的数字代表恢复的差异基因数。(J)对照组、肝硬化组和治疗组小型猪肝脏内皮细胞的不同亚群的拟时分析：HDAC2i+DNMT1i治疗后逆转了肝硬化小型猪肝脏中的“窦内皮-大血管内皮失调”。(K)肝硬化小型猪肝脏中靶向异常的HDAC2/DNMT1逆转“窦内皮-大血管内皮失调”，增强再生，减轻纤维化。 Figure 3: (A) Method for single-cell sequencing of liver non-parenchymal cells (NPCs) in a minipig NASH model. (B,C) Cluster analysis of non-parenchymal cells (NPCs) in the livers of control, cirrhosis, and treatment minipigs. (B) Heat map showing different cell lines of NPCs and their marker genes (right). (C) UMAP plot showing different cell lines of NPCs. Endo (EC), endothelial cells; DC, dendritic cells; Neu, neutrophils; Mac, macrophages; EPCAM + , EPCAM + cells and cholangiocytes. (D) Pie chart showing the proportion of different cell lines of non-parenchymal cells (NPCs) of minipig liver. (E) Venn diagram showing the number of differential genes in different cell lines of non-parenchymal cells (NPCs) of minipig livers between cirrhosis and control groups and between treatment and cirrhosis groups, respectively. Numbers in parentheses indicate the number of genes recovered after treatment. (F) KEGG pathway enrichment analysis in minipig liver endothelial cells. There were 79 KEGG pathways significantly changed between the liver endothelial cells of the cirrhosis group and the control group, and 99 KEGG pathways were significantly changed between the treatment group and the cirrhosis group. Epigenetic therapy by HDAC2i and DNMT2i normalized most of the altered KEGG pathways in the liver endothelial cells of minipigs in the cirrhosis group. White numbers indicate the sort order of corrected p-values. Adjusted p-values < 0.05 were considered statistically significant. (G) Cluster analysis of liver endothelial cells in control, cirrhosis, and treatment groups of minipigs. (H) Pie chart showing the proportions of different subpopulations of minipig liver endothelial cells. Compared with the control group, the number of macrovascular endothelial cells (MECs) increased and the number of sinusoidal endothelial cells (SECs) decreased in the livers of minipigs in the cirrhosis group, suggesting the occurrence of "sinus-macroendothelial dysregulation". (I) Venn diagram showing differential gene numbers in different subpopulations of minipig liver endothelial cells. The number of differential genes in liver sinusoidal endothelial cells (SECs) was relatively highest in the liver cirrhosis group, and most of the differential genes were recovered after HDAC2i+DNMT1i treatment. The numbers in parentheses represent the number of differential genes recovered. (J) Pseudo-temporal analysis of different subpopulations of liver endothelial cells in control, cirrhotic and treated minipigs: HDAC2i+DNMT1i treatment reversed the "sinusoidal-macrovascular endothelial dysregulation" in cirrhotic minipig livers. (K) Abnormally targeted HDAC2/DNMT1 in cirrhotic minipig liver reverses "sinusoidal endothelium-macrovascular endothelial dysregulation", enhances regeneration, and reduces fibrosis.
图4：(A)肝硬化人肝脏内皮细胞的旁分泌/血管分泌因子基因差异性表达。热图显示Ephrin/Eph、Notch、胰岛素生长因子相关蛋白、ADAM/ADAMTS和Semaphorin/Plexin家族等代表性血管分泌因子。(B)纤维化不同等级的人肝脏中旁分泌/血管分泌因子基因表达(数据来源于GSE84044)。F0-F4，人肝纤维化不同病理等级。F1-F4级肝脏的旁分泌/血管分泌因子基因的表达水平相对于健康肝脏(F0)进行量化。(C)小提琴图显示人和小型猪肝脏的非实质细胞(NPCs)的不同细胞系中代表性旁分泌/血管分泌因子基因的表达。IGFBP7和ADAMTS1在人和小型猪的肝脏内皮细胞中高表达。(D)与“窦内皮-大血管内皮失调”相关的旁分泌/血管分泌因子重编程的示意图，该重编程包括内皮细胞中IGFBP7和ADAMTS1的诱导。(E)健康和肝硬化人肝脏中总内皮细胞、内皮细胞不同亚群和窦内皮细胞中IGFBP7的表达水平。左：小提琴图显示肝脏总内皮细胞IGFBP7的表达；中：小提琴图显示肝脏内皮细胞的不同亚群中IGFBP7的表达；右：qPCR显示肝脏窦内皮细胞中IGFBP7的表达，N＝3。(F)健康和肝硬化人肝脏中总内皮细胞、内皮细胞不同亚群和窦内皮细胞中ADAMTS1的表达水平。左：小提琴图显示肝脏总内皮细胞ADAMTS1的表达；中：小提琴图显示肝脏内皮细胞的不同亚群中ADAMTS1的表达；右：Western blot显示肝脏窦内皮细胞中ADAMTS1的表达，N＝3。(G)对照组、肝硬化组和治疗组小型猪肝脏的总内皮细胞和内皮细胞不同亚群中IGFBP7的表达。(H,I)qPCR(H)和ELISA(I)显示对照组、肝硬化组和治疗组小型猪肝脏窦内皮细胞(SECs)和大血管内皮细胞(MECs)中IGFBP7的表达。N＝3。(J)对照组、肝硬化组和治疗组小型猪肝脏的总内皮细胞和内皮细胞不同亚群中ADAMTS1的表达。(K,L)qPCR(K)和ELISA(L)显示对照组、肝硬化组和治疗组小型猪肝脏窦内皮细胞(SECs)和大血管内皮细胞(MECs)中ADAMTS1的表达。N＝3。(M)ATAC-seq结果显示对照组、肝硬化组和治疗组小型猪肝脏内皮细胞中IGFBP7和ADAMTS1启动子的染色质开放性：肝硬化组小型猪肝脏内皮细胞中HDAC2/DNMT1依赖性地诱导IGFBP7和ADAMTS1。(N)人类患者和小型猪中表观遗传重编程的肝脏窦内皮细胞产生促纤维化IGFBP7和ADAMTS1的示意图。在所有统计分析中，2组比较的数据通过双尾学生t检验进行分析；多于2组比较的数据通过单因素方差分析和随后的Tukey事后检验进行分析。数据用平均值±SEM表示。*，肝硬化vs对照(小型猪)/健康(人类)，或者F1-F4vs.F0；#，治疗组vs.肝硬化组(小型猪)。*，P<0.05；**，P<0.01；***，P<0.001。#，P<0.05；##，P<0.01。Figure 4: (A) Differential expression of paracrine/vascular secretory factor genes in cirrhotic human liver endothelial cells. Heat map showing representative vascular secreted factors such as Ephrin/Eph, Notch, insulin growth factor-related protein, ADAM/ADAMTS, and Semaphorin/Plexin families. (B) Paracrine/vascular secretory factor gene expression in human livers with different grades of fibrosis (data from GSE84044). F0-F4, different pathological grades of human liver fibrosis. Expression levels of paracrine/vascular secretory factor genes in F1-F4 grade livers were quantified relative to healthy livers (F0). (C) Violin plots showing the expression of representative paracrine/vascular secretory factor genes in different cell lines of human and minipig liver non-parenchymal cells (NPCs). IGFBP7 and ADAMTS1 are highly expressed in human and minipig liver endothelial cells. (D) Schematic representation of the reprogramming of paracrine/vasculature factors associated with "sinus endothelium-macrovascular endothelial dysregulation" including induction of IGFBP7 and ADAMTS1 in endothelial cells. (E) Expression levels of IGFBP7 in total endothelial cells, different subsets of endothelial cells, and sinusoidal endothelial cells in healthy and cirrhotic human livers. Left: Violin plot showing IGFBP7 expression in total liver endothelial cells; Middle: Violin plot showing IGFBP7 expression in different subsets of liver endothelial cells; Right: qPCR showing IGFBP7 expression in liver sinusoidal endothelial cells, N=3. (F) ADAMTS1 expression levels in total endothelial cells, different subsets of endothelial cells, and sinusoidal endothelial cells in healthy and cirrhotic human livers. Left: Violin plot showing ADAMTS1 expression in total liver endothelial cells; Middle: Violin plot showing ADAMTS1 expression in different subsets of liver endothelial cells; Right: Western blot showing ADAMTS1 expression in liver sinusoidal endothelial cells, N=3. (G) Expression of IGFBP7 in total endothelial cells and different subsets of endothelial cells in the livers of control, cirrhosis, and treatment groups of minipigs. (H,I) qPCR (H) and ELISA (I) showed the expression of IGFBP7 in liver sinusoidal endothelial cells (SECs) and macrovascular endothelial cells (MECs) of minipigs in control, cirrhosis and treatment groups. N=3. (J) ADAMTS1 expression in total endothelial cells and different subsets of endothelial cells in control, cirrhosis, and treatment minipig livers. (K,L) qPCR (K) and ELISA (L) showed ADAMTS1 expression in liver sinusoidal endothelial cells (SECs) and macrovascular endothelial cells (MECs) in control, cirrhosis and treatment groups. N=3. (M) ATAC-seq results showing chromatin openness of the IGFBP7 and ADAMTS1 promoters in control, cirrhosis, and treatment minipig liver endothelial cells: HDAC2/DNMT1-dependent induction in cirrhosis group minipig liver endothelial cells IGFBP7 and ADAMTS1. (N) Schematic representation of the production of profibrotic IGFBP7 and ADAMTS1 by epigenetically reprogrammed liver sinusoidal endothelial cells in human patients and minipigs. In all statistical analyses, data for 2-group comparisons were analyzed by two-tailed Student's t-test; data for more than 2-group comparisons were analyzed by one-way ANOVA followed by Tukey's post hoc test. Data are presented as mean ± SEM. *, cirrhosis vs control (minipigs)/healthy (humans), or F1-F4 vs. F0;#, treatment group vs. cirrhosis group (minipigs). *, P<0.05; **, P<0.01; ***, P<0.001. #, P<0.05; ##, P<0.01.
图5：(A)健康和肝硬化/肝纤维化患者的IGFBP7、ADAMTS1、ALT、AST血 浆浓度。(B,C)肝脏功能正常或异常的肝硬化/肝纤维化患者IGFBP7和ADAMTS1血浆浓度。肝硬化/肝纤维化患者分为两组(B)：ALT/AST浓度正常和ALT/AST浓度异常。(D)健康和不同诱因的肝硬化/纤维化患者IGFBP7和ADAMTS血浆浓度。肝硬化/肝纤维化患者根据病因分为五类：非酒精性脂肪性肝炎相关的肝硬化/肝纤维化(NASH)、乙型肝炎相关的肝硬化/肝纤维化(HBC)、自身免疫性肝炎相关的肝硬化/肝纤维化(AIH)、原发性胆汁性肝硬化/肝纤维化(PBC)和隐源性肝硬化/肝纤维化(CC)。(E)健康和不同病理等级的NASH患者的ALT和AST血浆浓度。(F)健康、单纯性脂肪肝和不同病理等级的NASH患者的IGFBP7和ADAMTS1血浆浓度。(G)人血浆分离出细胞外囊泡(EVs)。上：分离出的EVs的电子显微镜分析；下：Western blot检测EVs的阳性(CD81)和阴性(GRP94)标记物。(H)小型猪和人的血浆细胞外囊泡(EVs)的IGFBP7/ADAMTS1浓度。(I,J)正常或异常肝脏功能(ALT/AST)的肝硬化患者(I)或不同纤维化等级NASH患者(J)的EVs的IGFBP7/ADAMTS1浓度。(K)假说示意图：表观遗传失调的肝脏窦内皮细胞(SECs)产生促纤维化的IGFBP7和ADAMTS1，并通过细胞外囊泡(EVs)促进肝硬化/纤维化。所有统计分析中，2组比较数据通过双尾学生t检验进行分析；多于2组比较数据通过单因素方差分析和随后的Tukey事后检验进行分析。数据用平均值±SEM表示。*，肝硬化、纤维化或NASH vs.对照组(小型猪)或健康(人类)；#，NASH vs.单纯性脂肪肝(人类)或治疗组vs.肝硬化组(小型猪)。*，P<0.05；**，P<0.01；***，P<0.001。#，P<0.05。Figure 5: (A) Plasma concentrations of IGFBP7, ADAMTS1, ALT, AST in healthy and cirrhotic/fibrotic patients. (B,C) Plasma concentrations of IGFBP7 and ADAMTS1 in cirrhosis/fibrosis patients with normal or abnormal liver function. Cirrhosis/fibrosis patients were divided into two groups (B): normal ALT/AST concentration and abnormal ALT/AST concentration. (D) Plasma concentrations of IGFBP7 and ADAMTS in healthy and cirrhotic/fibrotic patients with different predispositions. Patients with cirrhosis/fibrosis are divided into five categories according to etiology: nonalcoholic steatohepatitis-related cirrhosis/fibrosis (NASH), hepatitis B-related cirrhosis/fibrosis (HBC), autoimmune Hepatitis-associated cirrhosis/fibrosis (AIH), primary biliary cirrhosis/fibrosis (PBC), and cryptogenic cirrhosis/fibrosis (CC). (E) ALT and AST plasma concentrations in healthy and different pathological grades of NASH patients. (F) Plasma concentrations of IGFBP7 and ADAMTS1 in healthy, simple fatty liver and NASH patients with different pathological grades. (G) Extracellular vesicles (EVs) isolated from human plasma. Top: Electron microscopy analysis of isolated EVs; Bottom: Western blot detection of positive (CD81) and negative (GRP94) markers of EVs. (H) IGFBP7/ADAMTS1 concentrations in plasma extracellular vesicles (EVs) of minipigs and humans. (I,J) IGFBP7/ADAMTS1 concentrations in EVs from cirrhotic patients with normal or abnormal liver function (ALT/AST) (I) or NASH patients with different fibrosis grades (J). (K) Schematic of the hypothesis: Epigenetically dysregulated liver sinusoidal endothelial cells (SECs) produce profibrotic IGFBP7 and ADAMTS1 and promote cirrhosis/fibrosis via extracellular vesicles (EVs). In all statistical analyses, 2-group comparison data were analyzed by two-tailed Student's t-test; more than 2-group comparison data were analyzed by one-way ANOVA followed by Tukey's post-hoc test. Data are presented as mean ± SEM. *, cirrhosis, fibrosis or NASH vs. control group (minipigs) or healthy (humans); #, NASH vs. simple fatty liver (humans) or treatment group vs. cirrhosis group (minipigs). *, P<0.05; **, P<0.01; ***, P<0.001. #, P<0.05.
图6：(A)基于人和小型猪肝脏(NPCs)的不同细胞系的受体配体谱的细胞相互作用分析：肝硬化患者和小型猪的肝脏内皮细胞与T细胞相互作用显著。(B)Western blot显示健康和肝硬化CD45 +肝脏NPCs中磷酸化的Smad2的蛋白水平。磷酸化的Smad2的水平相对于总Smad2进行定量。结果显示肝硬化人肝脏的CD45 +NPCs中TGF-β1-Smad2激活。上：蛋白表达的定量；下：代表性的蛋白条带。数据通过双尾学生t检验进行分析，并用平均值±SEM表示。*，肝硬化vs.健康，P<0.05。N＝4。(C)健康和肝硬化人类肝脏的CD4 +T细胞的聚类分析。(D)Th17 +标记基因在健康和肝硬化人类肝脏的T细胞簇中的表达。(E)健康和肝硬化人类肝脏NPCs中Th17细胞的比例。肝硬化人类肝脏Th17细胞的比例相对于健康人进行量化。(F,G)对照组、肝硬化组和治疗组小型猪肝脏的T细胞和CD4 +T细胞的聚类分析。(H)Th17 +标记基因在小型猪肝脏CD4 +T细胞簇(cluster)中的表达。(I)对照组、肝硬化组和治疗组小型猪肝脏NPCs中Th17细胞的比例。肝硬化组和治疗组小型猪Th17细胞的比例相对于对照组进行量化。(J)热图显示肝硬化组和治疗组小型猪肝脏的Th17细胞中纤维化相关差异基因的表达。(K)表观遗传失调的肝脏SECs产生IGFBP7和ADAMTS1，以刺激人和小型猪的促纤维化的Th17细胞应答。这种细胞互作可能依赖于循环系统中内皮来源的IGFBP7/ADAMTS1介导的TGFβ1-Smad2信号增强。 Figure 6: (A) Cellular interaction analysis based on receptor ligand profiles of different cell lines of human and minipig livers (NPCs): Liver endothelial cells of patients with cirrhosis and minipigs interact significantly with T cells. (B) Western blot showing protein levels of phosphorylated Smad2 in healthy and cirrhotic CD45 + liver NPCs. Levels of phosphorylated Smad2 were quantified relative to total Smad2. The results showed TGF-β1-Smad2 activation in CD45 + NPCs of cirrhotic human livers. Top: quantification of protein expression; bottom: representative protein bands. Data were analyzed by two-tailed Student's t-test and presented as mean ± SEM. *, cirrhosis vs. healthy, P<0.05. N=4. (C) Cluster analysis of CD4 + T cells in healthy and cirrhotic human livers. (D) Expression of Th17 + marker genes in T cell clusters in healthy and cirrhotic human livers. (E) Proportion of Th17 cells in healthy and cirrhotic human liver NPCs. The proportion of Th17 cells in cirrhotic human livers was quantified relative to healthy individuals. (F,G) Cluster analysis of T cells and CD4 + T cells in the livers of control, cirrhosis, and treatment minipigs. (H) Expression of Th17 + marker genes in minipig liver CD4 + T cell clusters. (I) Proportion of Th17 cells in NPCs in minipig livers in control, cirrhosis and treatment groups. The proportion of Th17 cells in the cirrhosis and treated minipigs relative to the control group was quantified. (J) Heat map showing the expression of fibrosis-related differential genes in Th17 cells in the livers of minipigs in the cirrhosis group and the treatment group. (K) Epigenetically dysregulated hepatic SECs produce IGFBP7 and ADAMTS1 to stimulate profibrotic Th17 cell responses in humans and minipigs. This cellular interaction may depend on enhanced TGFβ1-Smad2 signaling mediated by endothelial-derived IGFBP7/ADAMTS1 in the circulation.
图7：(A)Hdac2内皮特异性敲除小鼠的NASH模型及治疗示意图。通过内皮特异的cre小鼠与Hdac2flox小鼠杂交获得内皮特异性Hdac2敲除的小鼠(Hdac2 iΔEC)。为了检测HDAC2/DNMT1交互调控的联合靶向抑制的效果，Hdac2 iΔEC小鼠也用DNMT1抑制剂阿扎胞苷(AZA)(Hdac2 iΔEC+AZA)处理。然后分析肝脏纤维化、肝脏功能和Th17细胞的富集。(B)H&E、天狼星红和I型胶原染色分析对照组和Hdac2 iΔEC+AZA小鼠的肝脏组织病理学。Hdac2 iΔEC+AZA小鼠的肝脏纤维化与对照组(Hdac2 +/+)小鼠进行比较。右：天狼星红和I型胶原染色的定量。Hdac2 iΔEC+AZA小鼠的阳性染色的比例相对于对照组小鼠进行量化。比例尺，200μm。N＝6。(C)小鼠的ALT和AST血清浓度及肝脏羟脯氨酸(Hyp)含量。N＝6。(D)小鼠的肝脏SECs的HDAC2和DNMT1的蛋白水平。上：蛋白表达的定量；下：代表性的western blot图像。N＝3。(E，F)小鼠肝脏切片的CD34(绿色)和结蛋白(红色)(E)或Lyve1(红色)和CD34(绿色)(F)的共染色。F 图中CD34阳性染色面积的比例相对于对照组进行量化。比例尺，20μm。N＝5。(G)流式细胞分析小鼠的Th17细胞数。小鼠NASH模型中靶向抑制内皮细胞中的HDAC2和DNMT1阻断了Th17细胞应答。右：Th17细胞的百分比。N＝5。所有统计分析中，2组比较数据通过双尾学生t检验进行分析；多于2组比较数据通过单因素方差分析和随后的Tukey事后检验进行分析。数据用平均值±SEM表示。*，Hdac2 iΔEC或Hdac2 iΔEC+AZA vs.对照组或NASH模型vs.野生型；#，Hdac2 iΔEC+AZA vs.NASH模型(对照)。*，P<0.05；***，P<0.001。##，P<0.01。 Figure 7: (A) Schematic diagram of NASH model and treatment in Hdac2 endothelial-specific knockout mice. Endothelial-specific Hdac2 knockout mice (Hdac2 iΔEC ) were obtained by crossing endothelial-specific cre mice with Hdac2flox mice. To examine the effect of combined targeted inhibition of HDAC2/DNMT1 interaction, Hdac2 iΔEC mice were also treated with the DNMT1 inhibitor azacitidine (AZA) (Hdac2 iΔEC + AZA). Liver fibrosis, liver function and enrichment of Th17 cells were then analyzed. (B) H&E, Sirius red and collagen type I staining analysis of liver histopathology in control and Hdac2 iΔEC + AZA mice. Liver fibrosis in Hdac2 iΔEC + AZA mice was compared to control (Hdac2 +/+ ) mice. Right: Quantification of Sirius Red and Collagen Type I staining. The proportion of positive staining in Hdac2 iΔEC +AZA mice was quantified relative to control mice. Scale bar, 200 μm. N=6. (C) ALT and AST serum concentrations and hepatic hydroxyproline (Hyp) content in mice. N=6. (D) Protein levels of HDAC2 and DNMT1 in liver SECs of mice. Top: quantification of protein expression; bottom: representative western blot images. N=3. (E, F) Co-staining of CD34 (green) and desmin (red) (E) or Lyve1 (red) and CD34 (green) (F) of mouse liver sections. The proportion of CD34-positive staining area in panel F was quantified relative to the control group. Scale bar, 20 μm. N=5. (G) Flow cytometric analysis of Th17 cell numbers in mice. Targeted inhibition of HDAC2 and DNMT1 in endothelial cells blocks Th17 cell responses in a mouse model of NASH. Right: Percentage of Th17 cells. N=5. In all statistical analyses, 2-group comparison data were analyzed by two-tailed Student's t-test; more than 2-group comparison data were analyzed by one-way ANOVA followed by Tukey's post-hoc test. Data are presented as mean ± SEM. *, Hdac2 iΔEC or Hdac2 iΔEC + AZA vs. control or NASH model vs. wild type; #, Hdac2 iΔEC + AZA vs. NASH model (control). *, P<0.05; ***, P<0.001. ##, P<0.01.
图8：(A)IGFBP7在对照组及表观遗传治疗组(Hdac2 iΔEC+AZA)小鼠肝脏内皮细胞(ECs)中的表达。N＝6。(B-E)Igfbp7的基因敲除减轻小鼠NASH模型中促纤维化的Th17应答。(B)诱导Igfbp7敲除(Igfbp7 -/-)小鼠NASH模型的示意图。(C)通过H&E、天狼星红和I型胶原染色评估对照组和Igfbp7 -/-小鼠的肝脏组织病理学。右：天狼星红和I型胶原染色的定量。(D)血清ALP和AST浓度及肝脏羟脯氨酸(Hyp)含量。(E)流式细胞分析Igfbp7 -/-小鼠Th17+细胞在肝脏中的百分比。N＝6。(F)重组IGFBP7蛋白诱导小鼠Th17应答的示意图。C57BL/6J小鼠每周腹腔注射四氯化碳(CCl 4)2次，共注射3周；从第2周开始，每2天尾静脉注射IGFBP7重组蛋白1次。Th17应答在用IGFBP7和对照处理的小鼠之间比较。(G)流式细胞分析IGFBP7蛋白诱导小鼠肝脏NPCs的Th17细胞数。右：Th17细胞的百分比。N＝4。在所有统计分析中，数据通过双尾学生t检验进行分析，用平均值±SEM表示。*，Hdac2 iΔEC+AZA，Igfbp7 -/-或IGFBP7vs.对照组。*，P<0.05；**，P<0.01。 Figure 8: (A) Expression of IGFBP7 in liver endothelial cells (ECs) of control and epigenetically treated (Hdac2 iΔEC + AZA) mice. N=6. (BE) Gene knockout of Igfbp7 attenuates pro-fibrotic Th17 responses in a mouse model of NASH. (B) Schematic representation of the induced Igfbp7 knockout (Igfbp7 -/- ) mouse model of NASH. (C) Liver histopathology in control and Igfbp7 -/- mice was assessed by H&E, Sirius red and collagen type I staining. Right: Quantification of Sirius Red and Collagen Type I staining. (D) Serum ALP and AST concentrations and liver hydroxyproline (Hyp) content. (E) Flow cytometric analysis of the percentage of Th17+ cells in the liver of Igfbp7 -/- mice. N=6. (F) Schematic representation of the induction of Th17 responses in mice by recombinant IGFBP7 protein. C57BL/6J mice were intraperitoneally injected with carbon tetrachloride (CCl 4 ) twice a week for a total of 3 weeks; starting from the 2nd week, IGFBP7 recombinant protein was injected into the tail vein once every 2 days. Th17 responses were compared between mice treated with IGFBP7 and controls. (G) Flow cytometric analysis of Th17 cell numbers in mouse liver NPCs induced by IGFBP7 protein. Right: Percentage of Th17 cells. N=4. In all statistical analyses, data were analyzed by two-tailed Student's t-test and presented as mean ± SEM. *, Hdac2 iΔEC + AZA, Igfbp7 -/- or IGFBP7 vs. control. *, P<0.05; **, P<0.01.
图9：(A)ADAMTS1在NASH及表观遗传治疗(Hdac2 iΔEC+AZA)小鼠肝脏窦内皮细胞(SECs)中的表达。N＝6。(B)Western blot显示用ADAMTS1shRNA(shADAMTS1)或对照shNC转导的人脐静脉内皮细胞(HUVECs)中Smad2的磷酸化(p-Smad2)水平。N＝3。(C)人内皮细胞来源的细胞外囊泡(EV)移植到小鼠的方法示意图。受体小鼠反复注射CCl 4以诱导肝脏纤维化。用TGF-β处理shADAMTS1转导的HUVEC 2天。从shADAMTS1或shNC(对照)转导的HUVECs培养基分离出EVs，并分别移植到肝脏纤维化小鼠体内。纤维化的应答在移植shADAMTS1或shNC EVs的受体小鼠之间比较。(D)注射CCl 4并移植shADAMTS1或shNC EVs后小鼠肝脏的天狼星红染色。N＝5。(E)流式细胞分析EVs移植后小鼠肝脏NPCs中Th17细胞数。右：Th17细胞的百分比。N＝5。(F)NASH中“内皮HDAC2/DNMT1-IGFBP7/ADAMTS1-Th17”轴促进肝脏纤维化示意图。肝脏内皮细胞(ECs)的表观遗传重编程导致“窦内皮-大血管内皮失调”，促进在EVs中促纤维化的IGFBP7/ADAMTS1的产生。来自表观遗传失调的SECs的IGFBP7/ADAMTS1刺激促纤维化的Th17细胞应答。在所有统计分析中，数据通过双尾学生t检验进行分析，用平均值±SEM表示。*，shADAMTS1或Hdac2 iΔEC+AZA vs.对照组。*，P<0.05；**，P<0.01。 Figure 9: (A) ADAMTS1 expression in liver sinusoidal endothelial cells (SECs) of NASH and epigenetically treated (Hdac2 iΔEC + AZA) mice. N=6. (B) Western blot showing phosphorylated (p-Smad2) levels of Smad2 in human umbilical vein endothelial cells (HUVECs) transduced with ADAMTS1 shRNA (shADAMTS1) or control shNCs. N=3. (C) Schematic diagram of the method for transplantation of human endothelial cell-derived extracellular vesicles (EVs) into mice. Recipient mice were repeatedly injected with CCl to induce liver fibrosis. shADAMTS1-transduced HUVECs were treated with TGF-β for 2 days. EVs were isolated from culture medium of HUVECs transduced with shADAMTS1 or shNC (control) and transplanted into liver fibrotic mice, respectively. Fibrotic responses were compared between recipient mice transplanted with shADAMTS1 or shNC EVs. (D) Sirius red staining of mouse liver after CCl 4 injection and transplantation of shADAMTS1 or shNC EVs. N=5. (E) Flow cytometric analysis of Th17 cell numbers in mouse liver NPCs after EVs transplantation. Right: Percentage of Th17 cells. N=5. (F) Schematic illustration of the "endothelial HDAC2/DNMT1-IGFBP7/ADAMTS1-Th17" axis promoting liver fibrosis in NASH. Epigenetic reprogramming of hepatic endothelial cells (ECs) leads to "sinusoidal-macrovascular endothelial dysregulation" and promotes the production of profibrotic IGFBP7/ADAMTS1 in EVs. IGFBP7/ADAMTS1 from epigenetically dysregulated SECs stimulates profibrotic Th17 cell responses. In all statistical analyses, data were analyzed by two-tailed Student's t-test and presented as mean ± SEM. *, shADAMTS1 or Hdac2 iΔEC + AZA vs. control. *, P<0.05; **, P<0.01.
图10：(A)2个健康和2个肝硬化患者肝脏的22,374个肝脏非实质细胞(NPCs)的聚类情况。左：总NPCs；右：健康和肝硬化NPCs。(B)UMAP图分别显示每个样本的肝脏NPCs的聚类分析。(C)健康和肝硬化人类肝脏中不同NPCs细胞系的比例。EC，内皮细胞；DC，树突状细胞；Neu，中性粒细胞；Mac，巨噬细胞；EPCAM +，EPCAM +细胞和胆管细胞。(D)2个健康和2个肝硬化患者的肝脏ECs的聚类分析。(E)健康和肝硬化ECs的不同亚群展示。 Figure 10: (A) Clustering of 22,374 hepatic non-parenchymal cells (NPCs) from 2 healthy and 2 cirrhotic patient livers. Left: total NPCs; right: healthy and cirrhotic NPCs. (B) UMAP plot showing cluster analysis of liver NPCs for each sample, respectively. (C) Proportions of different NPCs cell lines in healthy and cirrhotic human livers. EC, endothelial cell; DC, dendritic cell; Neu, neutrophil; Mac, macrophage; EPCAM + , EPCAM + cell and cholangiocyte. (D) Cluster analysis of liver ECs from 2 healthy and 2 cirrhotic patients. (E) Display of different subpopulations of healthy and cirrhotic ECs.
图11：(A)4个健康和3个肝硬化人类肝脏的42,314个NPCs的聚类分析。流式分选CD45 +和CD45 -NPCs，以用于scRNA-Seq分析。左：总NPCs；右：健康和肝硬化NPCs。(B)热图显示NPCs聚类标记基因及其标记的细胞系(右)。(C)UMAP图显示NPCs的不同细胞系。EC，内皮细胞；DC，树突状细胞；Neu，中性粒细胞；Mac，巨噬细胞；EPCAM +，EPCAM +细胞和胆管细胞。(D)4个健康和3个肝硬化人类肝脏ECs的聚类分析。(E)UMAP图显示ECs的不同亚群。左：总ECs；右：健康和肝硬化ECs。 (F)UMAP图显示内皮细胞中选择的标记基因表达。(G)饼图显示ECs亚群的比例。(H)健康和肝硬化人类肝脏ECs的不同亚群的拟时分析。 Figure 11: (A) Cluster analysis of 42,314 NPCs from 4 healthy and 3 cirrhotic human livers. CD45 + and CD45- NPCs were flow - sorted for scRNA-Seq analysis. Left: total NPCs; right: healthy and cirrhotic NPCs. (B) Heat map showing NPCs clustering marker genes and their marked cell lines (right). (C) UMAP plot showing different cell lines of NPCs. EC, endothelial cell; DC, dendritic cell; Neu, neutrophil; Mac, macrophage; EPCAM + , EPCAM + cell and cholangiocyte. (D) Cluster analysis of 4 healthy and 3 cirrhotic human liver ECs. (E) UMAP plot showing different subsets of ECs. Left: total ECs; right: healthy and cirrhotic ECs. (F) UMAP plot showing selected marker gene expression in endothelial cells. (G) Pie chart showing the proportion of ECs subsets. (H) Pseudo-chronological analysis of distinct subsets of healthy and cirrhotic human liver ECs.
图12：(A,B)热图显示内皮细胞和间充质细胞标记基因在人总NPCs(A)和肝硬化患者NPCs(B)的不同细胞系中的表达。(C)健康和肝硬化人类肝脏内皮细胞中间充质细胞分化的GSEA富集分析。(D)肝硬化人类肝脏中可能存在“窦内皮-大血管内皮失调”的示意图。Figure 12: (A,B) Heat maps showing endothelial and mesenchymal cell marker gene expression in different cell lines of human total NPCs (A) and cirrhosis patient NPCs (B). (C) GSEA enrichment analysis of mesenchymal differentiation in healthy and cirrhotic human liver endothelial cells. (D) Schematic illustration of possible "sinusoidal endothelium-macrovascular endothelial dysregulation" in a cirrhotic human liver.
图13：(A)GSE84044数据中HDACs在人肝纤维化不同等级的表达。F0-F4，人肝脏纤维化的不同病理等级。F1-F4级肝脏HDACs的表达水平相对于健康肝脏(F0)进行量化。(B)qPCR显示HDAC2和DNMT1在健康和肝硬化患者肝脏CD45 +NPCs中的表达。N＝3。(C)Western blot显示HDAC2和DNMT1在培养的人脐静脉内皮细胞中的表达，利用shHDAC2敲低HDAC2上调了DNMT1的表达。上：蛋白表达的定量；下：代表性蛋白条带。数据采用双尾学生t检验进行统计分析，以平均值±SEM表示；*，shHDAC2vs.对照组；*，P<0.05，**，P<0.01。N＝3。 Figure 13: (A) Expression of HDACs in different grades of human liver fibrosis in GSE84044 data. F0-F4, different pathological grades of human liver fibrosis. Expression levels of F1-F4 hepatic HDACs were quantified relative to healthy liver (F0). (B) qPCR showing the expression of HDAC2 and DNMT1 in hepatic CD45 + NPCs from healthy and cirrhotic patients. N=3. (C) Western blot showing the expression of HDAC2 and DNMT1 in cultured human umbilical vein endothelial cells, and knockdown of HDAC2 using shHDAC2 upregulated the expression of DNMT1. Top: quantification of protein expression; bottom: representative protein bands. Data were statistically analyzed by two-tailed Student's t-test and expressed as mean ± SEM; *, shHDAC2 vs. control group; *, P<0.05, **, P<0.01. N=3.
图14：(A)来自1个对照组、2个肝硬化组和2个治疗组小型猪肝脏的40,570个NPCs的聚类分析。(B)小型猪肝脏ECs的GO富集分析。HDAC2i和DNMT2i的表观遗传治疗恢复了小型猪NASH模型中肝脏ECs的大部分改变的功能。Figure 14: (A) Cluster analysis of 40,570 NPCs from 1 control, 2 cirrhotic and 2 treated minipig livers. (B) GO enrichment analysis of minipig liver ECs. Epigenetic treatment of HDAC2i and DNMT2i restored most of the altered functions of liver ECs in a minipig NASH model.
图15：(A)1个对照组、2个肝硬化组和2个治疗组小型猪肝脏ECs的聚类分析；(B)ECs中选择的标记基因表达。(C)UMAP图显示对照组、硬化组和治疗组ECs的不同亚群。(D,E)热图显示与表观遗传变化(组蛋白修饰和DNA甲基化)相关的基因在对照组、肝硬化组和治疗的小型猪肝脏NPCs的不同细胞系(D)和ECs的不同亚群(E)的表达。(F)维恩图分别显示肝硬化组和对照组以及治疗组和肝硬化组之间小型猪肝脏NPCs的不同细胞系或亚群(D和E)中差异基因的数量。红色数字代表恢复的基因数量。Figure 15: (A) Cluster analysis of minipig liver ECs in 1 control group, 2 cirrhosis groups and 2 treatment groups; (B) expression of selected marker genes in ECs. (C) UMAP plot showing different subpopulations of ECs in control, sclerotic, and treated groups. (D,E) Heat maps showing genes associated with epigenetic changes (histone modifications and DNA methylation) in different cell lines (D) and ECs of control, cirrhosis and treated minipig liver NPCs Expression of different subpopulations (E). (F) Venn diagrams showing the number of differential genes in different cell lines or subpopulations (D and E) of minipig liver NPCs between cirrhosis and control groups and between treatment and cirrhosis groups, respectively. Red numbers represent the number of genes recovered.
图16：(A)维恩图显示人类和小型猪肝硬化肝脏的差异基因。括号中的数字表示治疗后小型猪中恢复的基因数。(B,C)小提琴图显示代表性旁分泌因子在人(B)和小型猪(C)肝脏的非实质细胞(NPCs)的不同细胞系中的表达。(D)qPCR和ELISA显示，对照组、肝硬化组和治疗组的小型猪肝脏CD45 +NPCs中IGFBP7和ADAMTS1的表达。左：qPCR；右：ELISA。数据通过单因素方差分析和随后的Tukey事后检验进行分析，并以平均值±SEM表示。*，肝硬化vs.对照组，P＜0.05。N＝3。 Figure 16: (A) Venn diagram showing differential genes in human and minipig cirrhotic livers. Numbers in parentheses indicate the number of genes recovered in minipigs after treatment. (B,C) Violin plots showing the expression of representative paracrine factors in different cell lines of non-parenchymal cells (NPCs) of human (B) and minipig (C) livers. (D) qPCR and ELISA showing the expression of IGFBP7 and ADAMTS1 in CD45 + NPCs in minipig livers of control, cirrhosis and treatment groups. Left: qPCR; right: ELISA. Data were analyzed by one-way ANOVA followed by Tukey's post hoc test and presented as mean ± SEM. *, cirrhosis vs. control group, P<0.05. N=3.
图17：(A)2个健康和2个肝硬化人类肝脏中T细胞的聚类分析。(B)UMAP图显示人类T细胞的不同细胞系(CD8 +和CD4 +细胞)。(C-I)来自GSE136103数据中人类CD45 +细胞的scRNA-Seq分析。(C)5个健康和5个肝硬化患者肝脏的35,806个CD45 +细胞的聚类分析。(D)UMAP图显示NPCs的不同细胞系。EC，内皮细胞；DC，树突状细胞；Neu，中性粒细胞；Mac，巨噬细胞；EPCAM +，EPCAM +细胞和胆管细胞。(E)UMAP图显示T细胞标记基因在CD45 +细胞中的表达。(F)5个健康的和5个肝硬化人类肝脏中的T细胞的聚类分析。(G)UMAP图显示人类T细胞的不同细胞系(CD8 +和CD4 +细胞)。(F-G)为来源于GSE136103的T细胞数据。(H)小提琴图显示Th17 +标记基因在T细胞不同群中的表达。(I)5个健康和5个肝硬化患者的CD45 +细胞中Th17细胞的比例。(H-I)为来源于GSE136103的Th17细胞数据。(J)UMAP图显示小型猪T细胞中T细胞的不同细胞系(CD8 +和CD4 +细胞)。 Figure 17: (A) Cluster analysis of T cells in 2 healthy and 2 cirrhotic human livers. (B) UMAP plot showing different cell lineages (CD8 + and CD4 + cells) of human T cells. (CI) scRNA-Seq analysis of human CD45 + cells from GSE136103 data. (C) Cluster analysis of 35,806 CD45 + cells from 5 healthy and 5 cirrhotic patient livers. (D) UMAP plot showing different cell lines of NPCs. EC, endothelial cell; DC, dendritic cell; Neu, neutrophil; Mac, macrophage; EPCAM + , EPCAM + cell and cholangiocyte. (E) UMAP plot showing expression of T cell marker genes in CD45 + cells. (F) Cluster analysis of T cells in 5 healthy and 5 cirrhotic human livers. (G) UMAP plot showing different cell lineages (CD8 + and CD4 + cells) of human T cells. (FG) T cell data derived from GSE136103. (H) Violin plot showing expression of Th17 + marker genes in different populations of T cells. (I) Proportion of Th17 cells in CD45 + cells of 5 healthy and 5 cirrhotic patients. (HI) is Th17 cell data derived from GSE136103. (J) UMAP plot showing different cell lineages (CD8 + and CD4 + cells) of T cells in minipig T cells.
实施例一：材料和方法Example 1: Materials and Methods
1.患者和临床样本。患者的肝脏和血浆样品在知情同意下在四川大学华西医院收集。健康的肝组织(无纤维化)获取自接受内窥镜肝脏切除术的肝血管瘤患者。肝硬化肝组织获取自经组织学诊断为肝纤维化的患者。表1中显示了用于scRNA-Seq、液相芯片分析和基因表达分析的患者信息。血浆样本获取自健康志愿者(n＝21)、单纯性脂肪肝(n＝16)、早期非酒精性脂肪性肝炎(F0-F1)(n＝12)和不同病理等级的肝硬化/纤维化患者(无癌 症)(n＝75)。通过纤维化-4指数(Fib-4)和瞬态弹性成像(TE)来确定非NASH肝脏的纤维化等级，并使用NAFLD纤维化指数(NFS)、Fib-4、TE和受控衰减参数(CAP)来确定NASH肝脏的纤维化和脂肪变性等级。早期NASH主要通过肝活检标本的组织学鉴定，其他一些标本也通过肝活检标本的组织学鉴定。表2显示了用于血浆分析的样本信息。四川大学华西医院医学伦理委员会批准了本发明的研究。对人体的研究符合《赫尔辛基宣言》的原则。1. Patient and clinical samples. Liver and plasma samples of patients were collected at West China Hospital of Sichuan University with informed consent. Healthy liver tissue (without fibrosis) was obtained from patients with hepatic hemangiomas who underwent endoscopic liver resection. Cirrhotic liver tissue was obtained from patients with histologically diagnosed hepatic fibrosis. Patient information used for scRNA-Seq, liquid microarray analysis, and gene expression analysis is shown in Table 1. Plasma samples were obtained from healthy volunteers (n=21), simple fatty liver (n=16), early nonalcoholic steatohepatitis (F0-F1) (n=12), and cirrhosis/fibrosis of different pathological grades Patients (no cancer) (n=75). Fibrosis grades in non-NASH livers were determined by Fibrosis-4 Index (Fib-4) and Transient Elastography (TE) using the NAFLD Fibrosis Index (NFS), Fib-4, TE and controlled attenuation parameters ( CAP) to determine the grade of fibrosis and steatosis in NASH livers. Early NASH is mainly identified by histology of liver biopsy specimens, and some other specimens are also identified by histology of liver biopsy specimens. Table 2 shows sample information for plasma analysis. The study of the present invention was approved by the Medical Ethics Committee of West China Hospital of Sichuan University. Research on the human body conforms to the principles of the Declaration of Helsinki.
表1患者的基本信息Table 1 Basic information of patients
Figure PCTCN2021140096-appb-000002
Figure PCTCN2021140096-appb-000002
备注：TBIL，总胆红素；DBIL，直接胆红素；IBIL，间接胆红素；AST，谷草转氨酶；ALT，谷丙转氨酶；ALP，碱性磷酸酶；GGT，谷氨酰转肽酶。F0或F4，人类肝纤维化的不同病理等级。健康和肝硬化肝脏的#1和#2用于scRNA-Seq，健康和肝硬化肝脏的#1、#2和#3用于液相芯片分析和基因表达分析。Remarks: TBIL, total bilirubin; DBIL, direct bilirubin; IBIL, indirect bilirubin; AST, aspartate aminotransferase; ALT, alanine aminotransferase; ALP, alkaline phosphatase; GGT, glutamyl transpeptidase. F0 or F4, different pathological grades of human liver fibrosis. #1 and #2 of healthy and cirrhotic livers were used for scRNA-Seq, and #1, #2 and #3 of healthy and cirrhotic livers were used for liquid chip analysis and gene expression analysis.
表2血浆样本临床数据Table 2 Clinical data of plasma samples
Figure PCTCN2021140096-appb-000003
Figure PCTCN2021140096-appb-000003
Figure PCTCN2021140096-appb-000004
Figure PCTCN2021140096-appb-000004
Figure PCTCN2021140096-appb-000005
Figure PCTCN2021140096-appb-000005
Figure PCTCN2021140096-appb-000006
Figure PCTCN2021140096-appb-000006
Figure PCTCN2021140096-appb-000007
Figure PCTCN2021140096-appb-000007
备注：M，男性；F，女性；NA，不适用；PBC，原发性胆汁性肝硬化/肝纤维化；AIH，自身免疫性肝炎相关肝硬化/肝纤维化；HBC，乙肝相关肝硬化/肝纤维化；CC，隐源性肝硬化/肝纤维化；NASH，非酒精性脂肪性肝炎相关肝硬化/肝纤维化；F0-F4，人类肝纤维化的不同病理等级；Fib-4，纤维化-4指数；TE，瞬态弹性成像；NFS，NAFLD纤维化指数；CAP，受控衰减参数；UDCA，熊去氧胆酸；MP，甲基强的松龙；ETV，恩替卡韦；TAF，替诺福韦阿拉芬酰胺；TDF，替诺福韦酯；FNB，非诺贝特；SAMe，S-腺苷甲硫氨酸。Remarks: M, male; F, female; NA, not applicable; PBC, primary biliary cirrhosis/fibrosis; AIH, autoimmune hepatitis-related cirrhosis/fibrosis; HBC, hepatitis B-related cirrhosis/ Liver fibrosis; CC, cryptogenic cirrhosis/fibrosis; NASH, nonalcoholic steatohepatitis-associated cirrhosis/fibrosis; F0-F4, different pathological grades of human liver fibrosis; Fib-4, fibrosis Tl-4 index; TE, transient elastography; NFS, NAFLD fibrosis index; CAP, controlled decay parameter; UDCA, ursodeoxycholic acid; MP, methylprednisolone; ETV, entecavir; TAF, replacement Nofovir alafenamide; TDF, tenofovir disoproxil; FNB, fenofibrate; SAMe, S-adenosylmethionine.
2.小型猪。雄性巴马小型猪获取自成都达硕生物科技有限公司(Dossy Biological Technology Co.,LTD)。小型猪饲养在成都达硕实验动物中心(Dossy Experimental Animals Center)的独立笼子里，喂食含2％胆固醇和30％脂肪(按重量计)的饮食，并补充果糖和葡萄糖。四川大学华西第二医院的实验动物伦理委员会和成都达硕生物科技有限公司批准了小型猪实验。2. Miniature pigs. Male Bama minipigs were obtained from Chengdu Dossy Biological Technology Co., LTD. Minipigs were housed in individual cages at the Chengdu Dossy Experimental Animals Center and fed a diet containing 2% cholesterol and 30% fat by weight, supplemented with fructose and glucose. The experimental animal ethics committee of the West China Second Hospital of Sichuan University and Chengdu Dashuo Biotechnology Co., Ltd. approved the minipig experiments.
3.小鼠。C57BL/6J小鼠获取自南京大学模式动物研究所。C57BL/6J-Hdac2 em1(flox)Smoc小鼠获取自上海南方模式生物科技股份有限公司。Igfbp7 -/-小鼠如前所述(参考文献94)。表达EC特异的Cdh5-(PAC)-Cre ERT2的小鼠由Ralf H.Adam提供(参考文献95)。将Cdh5-(PAC)-Cre ERT2小鼠与floxed Hdac2小鼠杂交产生Hdac2 iΔEC/iΔEC小鼠(Hdac2 iΔEC)。Hdac2 iΔEC小鼠在出生后两个月，用他莫昔芬(250mg/kg)腹腔注射处理6次，每天1次(第3次给药后中断3天)，诱导Hdac2内皮特异性缺失。小鼠饲养在华西第二医院SPF级实验动物中心中，并按标准的12小时明/暗周期喂食。动物实验方案经四川大学华西第二医院实验动物伦理委员会批准。 3. Mice. C57BL/6J mice were obtained from the Institute of Model Animals, Nanjing University. C57BL/6J-Hdac2 em1(flox) Smoc mice were obtained from Shanghai Southern Model Biotechnology Co., Ltd. Igfbp7 -/- mice were described previously (ref. 94). Mice expressing the EC-specific Cdh5-(PAC) -Cre ERT2 were provided by Ralf H. Adam (ref. 95). Cdh5-(PAC) -Cre ERT2 mice were crossed with floxed Hdac2 mice to generate Hdac2 iΔEC/iΔEC mice (Hdac2 iΔEC ). Hdac2 iΔEC mice were treated with tamoxifen (250 mg/kg) i.p. 6 times a day (with a 3-day interruption after the third dose) at two months after birth to induce Hdac2 endothelial-specific deletion. Mice were housed in the SPF-level Experimental Animal Center of West China Second Hospital and fed on a standard 12-hour light/dark cycle. The animal experimental protocol was approved by the Experimental Animal Ethics Committee of the West China Second Hospital of Sichuan University.
4.细胞系。从人脐带中分离人脐静脉内皮细胞(HUVEC)。HUVEC在EndoGRO-VEGF完全培养基(SCME002，Millipore)中，于37℃、5％CO 2的湿润环境中培养。HEK293T获取自美国菌种保藏中心(ATCC)。将HEK293T在含10％胎牛血清(FBS)(1600044，Gibco)的DMEM(11965092，Gibco)中，于37℃、5％CO 2的湿润环境中培养。 4. Cell lines. Human umbilical vein endothelial cells (HUVEC) were isolated from human umbilical cord. HUVECs were cultured in EndoGRO-VEGF complete medium (SCME002, Millipore) at 37°C in a humidified environment with 5% CO 2 . HEK293T was obtained from the American Type Culture Collection (ATCC). HEK293T was cultured in DMEM (11965092, Gibco) containing 10% fetal bovine serum (FBS) (1600044, Gibco) at 37°C in a humidified environment with 5% CO2 .
5.NASH模型。小型猪NASH模型：6月龄雄性小型猪喂养西方饮食(Western diet，WD)即2％胆固醇、30％脂肪并补充高糖饮水：23.1g/L d-果糖和18.9g/L d-葡萄糖，腹腔注射剂量为0.1ml/kg的四氯化碳(CCl 4)，每周2次，连续注射5个月。对照组小型猪注射CCl 4赋形剂(CCl 4vehicle)(玉米油)。每15天收集血清样本1次，并且每月收集血浆样本1次。为了检测肝硬化的进展和相关的分子和细胞改变，每2个月手术活检1次，取下肝组织用于不同的分析。最后一次注射CCl 4的2天后，处死全部小型猪并收集血清、血浆和肝脏样本。小鼠NASH模型：8周龄小鼠(对照C57BL/6J、Hdac2 iΔEC及IGFBP7 -/-小鼠)喂养西方饮食即21.1％脂肪、41％蔗糖、1.25％胆固醇并补充高糖饮水：23.1g/L果糖和18.9g/L葡萄糖；腹腔注射剂量为0.5μl/g(0.8g/kg)的CCl 4，每周1次，连续注射3个月。野生型对照小鼠喂养正常饮食并注射玉米油。最后一次注射CCl 4的2天后，处死全部小鼠并收集血清和肝脏样本。 5. NASH model. Minipig NASH model: 6-month-old male minipigs were fed a Western diet (Western diet, WD) that is 2% cholesterol, 30% fat and supplemented with high-sugar drinking water: 23.1g/L d-fructose and 18.9g/L d-glucose, The intraperitoneal injection dose was 0.1 ml/kg of carbon tetrachloride (CCl 4 ), twice a week, for 5 months. Control group minipigs were injected with CCl 4 vehicle ( corn oil). Serum samples were collected every 15 days, and plasma samples were collected monthly. To detect progression of cirrhosis and associated molecular and cellular changes, surgical biopsies were performed every 2 months, and liver tissue was removed for different analyses. Two days after the last injection of CCl4 , all minipigs were sacrificed and serum, plasma and liver samples were collected. Mouse NASH model: 8-week-old mice (control C57BL/6J, Hdac2 iΔEC , and IGFBP7 -/- mice) were fed a Western diet of 21.1% fat, 41% sucrose, 1.25% cholesterol and supplemented with high-sugar drinking water: 23.1 g/ L fructose and 18.9 g/L glucose; intraperitoneal injection of CCl 4 at a dose of 0.5 μl/g (0.8 g/kg), once a week, for 3 months. Wild-type control mice were fed a normal diet and injected with corn oil. Two days after the last injection of CCl4 , all mice were sacrificed and serum and liver samples were collected.
6.HDAC2和DNMT1的联合靶向抑制。HDAC2和DNMT1的联合靶向抑制小型猪NASH模型的策略如图2B所示。小型猪在注射CCl 4两个月后开始治疗：2周为1个治疗疗程，第一周进行DNMT1抑制剂阿扎胞苷(Azacitidine，AZA)治疗：前5天腹腔注射AZA(剂量为0.1-2.0mg/kg/天，还可以为0.001-0.100mg/kg/天，优选为0.055mg/kg/天)， 每天1次，然后停药2天；第二周进行HADC2抑制剂Mocetinostat(MGCD0103)治疗：前5天腹腔注射MGCD0103(剂量为1-20mg/kg/天，还可以为0.1-2.0mg/kg/天，优选为1.1mg/kg/天)，每天1次，然后停药2天。重复治疗6个疗程(根据实际情况，可调整为5-10个疗程，实验数据未显示)。Hdac2 iΔEC小鼠治疗策略如图7A所示。Hdac2 iΔEC小鼠注射CCl 4一个月后，腹腔注射AZA，每两天1次，治疗两个月。 6. Combined targeted inhibition of HDAC2 and DNMT1. The strategy for combined targeting of HDAC2 and DNMT1 to suppress the minipig model of NASH is shown in Figure 2B. Minipigs started treatment two months after CCl 4 injection: 2 weeks as a treatment course, the first week of treatment with the DNMT1 inhibitor Azacitidine (AZA): AZA (dose of 0.1- 2.0 mg/kg/day, but also 0.001-0.100 mg/kg/day, preferably 0.055 mg/kg/day), once a day, then discontinued for 2 days; HADC2 inhibitor Mocetinostat (MGCD0103) in the second week Treatment: Intraperitoneal injection of MGCD0103 (dose of 1-20 mg/kg/day, also 0.1-2.0 mg/kg/day, preferably 1.1 mg/kg/day) for the first 5 days, once a day, then discontinued for 2 days . Repeat the treatment for 6 courses of treatment (according to the actual situation, it can be adjusted to 5-10 courses of treatment, the experimental data is not shown). The treatment strategy for Hdac2 iΔEC mice is shown in Figure 7A. Hdac2 iΔEC mice were injected with CCl 4 for one month and then intraperitoneally injected with AZA, once every two days, for two months.
7.小型猪手术活检。小型猪经剖腹进行肝脏活检术。禁食一晚后，耳静脉注射舒泰
Figure PCTCN2021140096-appb-000008
麻醉小型猪，所有手术均在全身麻醉下进行。开腹后，用钝性解剖技术取下一小块肝组织，肝切口止血后，设置引流管，缝合腹腔。术后小型猪静脉补充葡萄糖，手术后一天允许饮水和饮食。 7. Minipig surgical biopsy. Minipigs underwent laparotomy for liver biopsy. After an overnight fast, sutai was given into the ear vein
Figure PCTCN2021140096-appb-000008
The minipigs were anesthetized and all operations were performed under general anesthesia. After laparotomy, a small piece of liver tissue was removed by blunt dissection technique. After hemostasis of the liver incision, a drainage tube was set and the abdominal cavity was sutured. Postoperatively, the minipigs were supplemented with glucose intravenously, and water and diet were allowed one day after the operation.
8.肝细胞、NPCs、CD45 +NPCs、ECs、SECs和MECs的分离。人、小型猪和小鼠的肝组织用冷PBS洗涤两次，切碎，并在消化混合物(溶于PBS的1mg/ml I型胶原酶和1mg/ml II型中性蛋白酶)中37℃孵育30分钟。30分钟后，将肝组织悬浮以确保完全分离。消化后的肝脏样本是均匀的最小限度的块。消化后的组织用细胞滤网过滤多次，并且细胞用300g离心5分钟收集。用RBC裂解液(R1010,Solarbio)去除红细胞后，洗涤一次，通过另外的4℃、50g离心5min步骤，分离肝细胞和NPCs。底部的肝细胞用于液相芯片分析和DNA甲基化分析，上清中的NPCs用于液相芯片、DNA甲基化、磁珠分选和western blot。对于CD45 +NPCs、ECs(CD45 -CD31 +)、MECs(CD45 -CD34 +)和SECs(CD45 -CD34 -CD31 +)的分离，用1ml预冷的MACS洗涤缓冲液(溶于DPBS的2mM EDTA、0.1％BSA、1％青霉素/链霉素)洗涤Dynabeads磁珠3次，与CD45、CD34或CD31抗体在4℃下孵育4小时后，用MACS洗涤缓冲液洗涤磁珠3次。将NPCs重悬于300μl MACS洗涤缓冲液中，并添加200μl Dynabeads-CD45抗体偶联物，然后在旋转器上4℃孵育45分钟。孵育后，用磁铁分离CD45 +细胞结合的磁珠，并将上清液转移至含有Dynabeads-CD31或-CD34抗体偶联物的管中。类似于CD45 +细胞收集，将Dynabeads-CD31或-CD34抗体偶联物与收集的上清液在旋转器上4℃孵育45分钟，并用磁铁分离CD31 +CD45 -细胞(ECs)或CD34 +CD45 -细胞(MECs)的磁珠。对于SECs分离，首先使用Dynabeads-CD45和-CD34抗体偶联物进行阴性分选，然后使用Dynabeads-CD31抗体偶联物进行阳性分选。将具有CD45 +、CD31 +CD45 -、CD34 +CD45 -或CD31 +CD45 -CD34 -细胞的磁珠用冷的MACS洗涤缓冲液洗涤5次，然后用于后续实验。小型猪NPCs与Percp-Cy5.5-CD45、FITC-CD34和PE-CD31抗体孵育30分钟，通过流式细胞术分离的小型猪CD45 +NPCs、MECs(CD45 -CD34 +)和SECs(CD45 -CD34 -CD31 +)。洗涤后，通过FACSAria TM III流式细胞仪(BD Biosciences)分离CD45 +细胞、MECs和SECs。 8. Isolation of hepatocytes, NPCs, CD45 + NPCs, ECs, SECs and MECs. Human, minipig and mouse liver tissues were washed twice with cold PBS, minced, and incubated in digestion mix (1 mg/ml type I collagenase and 1 mg/ml type II neutral protease in PBS) at 37°C 30 minutes. After 30 minutes, the liver tissue was suspended to ensure complete dissociation. The digested liver sample is a homogeneous minimal block. Digested tissue was filtered through a cell strainer multiple times and cells were collected by centrifugation at 300 g for 5 minutes. After depletion of erythrocytes with RBC lysis buffer (R1010, Solarbio), washing once, hepatocytes and NPCs were isolated by an additional centrifugation step at 4°C, 50 g for 5 min. The hepatocytes at the bottom were used for LC-chip analysis and DNA methylation analysis, and the NPCs in the supernatant were used for LC-chip, DNA methylation, magnetic bead sorting and western blot. For isolation of CD45 + NPCs, ECs (CD45 - CD31 + ), MECs (CD45 - CD34 + ) and SECs (CD45 - CD34 - CD31 + ), wash with 1 ml of pre-chilled MACS wash buffer (2 mM EDTA in DPBS, Dynabeads magnetic beads were washed 3 times with 0.1% BSA, 1% penicillin/streptomycin), and after 4 hours of incubation with CD45, CD34 or CD31 antibodies at 4°C, the beads were washed 3 times with MACS wash buffer. NPCs were resuspended in 300 μl of MACS wash buffer, and 200 μl of Dynabeads-CD45 antibody conjugate was added, then incubated at 4° C. on a rotator for 45 minutes. After incubation, the CD45 + cell-bound magnetic beads were detached with a magnet and the supernatant was transferred to a tube containing Dynabeads-CD31 or -CD34 antibody conjugates. Similar to CD45 + cell collection, Dynabeads-CD31 or -CD34 antibody conjugates were incubated with the collected supernatant for 45 min at 4°C on a rotator, and CD31 + CD45- cells ( ECs ) or CD34 + CD45- cells were isolated using a magnet Magnetic beads for cells (MECs). For SECs isolation, negative sorting was performed first using Dynabeads-CD45 and -CD34 antibody conjugates, followed by positive sorting using Dynabeads-CD31 antibody conjugates. Magnetic beads with CD45 + , CD31 + CD45 - , CD34 + CD45 - or CD31 + CD45 - CD34 - cells were washed 5 times with cold MACS wash buffer and used for subsequent experiments. Minipig NPCs were incubated with Percp-Cy5.5-CD45, FITC-CD34 and PE-CD31 antibodies for 30 min, and minipig CD45 + NPCs, MECs (CD45 - CD34 + ) and SECs (CD45 - CD34 + ) were isolated by flow cytometry - CD31 + ). After washing, CD45 + cells, MECs and SECs were isolated by FACSAria ™ III flow cytometer (BD Biosciences).
9.流式细胞分析。分离出小鼠NPCs，与BV421标记的大鼠抗小鼠的CD4抗体(BV421-rat anti-mouse CD4)(562891，BD Biosciences)孵育30分钟。洗涤后，用BD Cytofix/Cytoperm溶液(554714，BD Biosciences)固定并透膜30分钟，然后用FITC标记的大鼠抗FOXP3(FITC-rat anti-FOXP3)(11-5773-82，Invitrogen)和PE标记的大鼠抗RORγt(PE-rat anti-RORγt)(12-6988-82，Invitrogen)染色30分钟。细胞固定后，在FACS Calibur(BD Biosciences)上进行流式细胞分析，并用Flow Jo V10进行分析。9. Flow cytometry analysis. Mouse NPCs were isolated and incubated with BV421-labeled rat anti-mouse CD4 antibody (BV421-rat anti-mouse CD4) (562891, BD Biosciences) for 30 minutes. After washing, the membrane was fixed with BD Cytofix/Cytoperm solution (554714, BD Biosciences) and permeabilized for 30 minutes, and then treated with FITC-labeled rat anti-FOXP3 (FITC-rat anti-FOXP3) (11-5773-82, Invitrogen) and PE Labeled rat anti-RORγt (PE-rat anti-RORγt) (12-6988-82, Invitrogen) was stained for 30 minutes. After cell fixation, flow cytometric analysis was performed on a FACS Calibur (BD Biosciences) and analyzed with Flow Jo V10.
10.样本收集和组织学分析。人、小型猪和小鼠的肝脏组织用4％多聚甲醛固定或OCT包埋后切片。小型猪和小鼠的血清样本保存在-20℃，人和小型猪的血浆样本保存在-80℃。石蜡包埋的肝组织用苏木精-伊红(H&E)和天狼星红染色，OCT包埋的肝组织用油红O染色。10. Sample collection and histological analysis. Human, minipig and mouse liver tissues were fixed with 4% paraformaldehyde or embedded with OCT and sectioned. Serum samples from minipigs and mice were stored at -20°C, and human and minipig plasma samples were stored at -80°C. Paraffin-embedded liver tissue was stained with hematoxylin-eosin (H&E) and Sirius Red, and OCT-embedded liver tissue was stained with Oil Red O.
11.免疫荧光分析。将OCT包埋的肝组织切成6μm切片，用4％多聚甲醛固定5min后，用PBS冲洗，室温下用10％驴血清封闭30min。然后，用溶于PBS的0.3％Triton X-100透膜20分钟，并分别与抗Lyve1(70R-LR003，Fitzgerad)、抗CD34(ab81289，Abcam)、抗desmin(Ab15200，abcam)、抗I型胶原(Ab34710，abcam)或抗Ki67(b15580，Abcam) 抗体4℃孵育过夜。清洗后，将切片与Alexa Fluor 488或Alexa Fluor 647标记的二抗(驴抗兔IgG(711-605-152或711-545-152，Jackson ImmunoResearch Labs))孵育1小时。PBS洗涤切片，用4,6-二氨基-2-苯基吲哚(DAPI)(C0065，Solarbio)染核并安上盖玻片。图像由激光共聚焦显微镜(LSM880，Zeiss)捕获并用ZEN(Zeiss)处理。11. Immunofluorescence analysis. The OCT-embedded liver tissue was cut into 6 μm sections, fixed with 4% paraformaldehyde for 5 min, rinsed with PBS, and blocked with 10% donkey serum for 30 min at room temperature. Then, the membrane was permeabilized with 0.3% Triton X-100 in PBS for 20 minutes, and mixed with anti-Lyve1 (70R-LR003, Fitzgerad), anti-CD34 (ab81289, Abcam), anti-desmin (Ab15200, abcam), anti-type I Collagen (Ab34710, Abcam) or anti-Ki67 (b15580, Abcam) antibodies were incubated overnight at 4°C. After washing, sections were incubated with Alexa Fluor 488 or Alexa Fluor 647-labeled secondary antibody (donkey anti-rabbit IgG (711-605-152 or 711-545-152, Jackson ImmunoResearch Labs)) for 1 hour. Sections were washed with PBS, nuclei were stained with 4,6-diamino-2-phenylindole (DAPI) (C0065, Solarbio) and coverslipped. Images were captured by laser confocal microscopy (LSM880, Zeiss) and processed with ZEN (Zeiss).
12.天狼星红、油红O、I型胶原、ki67染色以及CD34-Lyve1和CD34-Desmin共染色的半定量。用Image-Pro Plus 6.0(Media Cybernetics,Rockville,MD)对天狼星红、油红O和I型胶原染色进行半定量分析。通过选定区域中的颜色深度和染色面积来评估阳性染色信号，以阳性染色信号与选定区域总面积之比计算天狼星红、油红O、和I型胶原染色的半定量。用Photoshop CC 2018(Adobe Systems，CA)对ki67和CD34-lyve1染色进行半定量。分别通过与所选区域的总细胞数或阳性染色面积相比，计算出阳性细胞数(ki67)和染色阳性面积(CD34)的百分比。每组3个(小型猪)或5到6个(小鼠)样品用于定量，并从每个样品中选择3个视野用于定量。将3个视野的平均值用作每个样品的定量值。为了量化各个组之间的差异，将每组每个样本的值(阳性信号/阳性细胞数/阳性面积的百分比)与野生型或对照组的值进行量化。每组的结果用“对照的倍数或野生型的倍数”显示。12. Semi-quantification of Sirius Red, Oil Red O, Collagen I, ki67 staining and co-staining of CD34-Lyve1 and CD34-Desmin. Semiquantitative analysis of Sirius Red, Oil Red O and Collagen Type I staining was performed with Image-Pro Plus 6.0 (Media Cybernetics, Rockville, MD). The positive staining signal was assessed by the color depth and staining area in the selected area, and semi-quantification of Sirius Red, Oil Red O, and Type I collagen staining was calculated as the ratio of the positive staining signal to the total area of the selected area. Semi-quantification of ki67 and CD34-lyve1 staining was performed with Photoshop CC 2018 (Adobe Systems, CA). The number of positive cells (ki67) and the percentage of positively stained area (CD34) were calculated by comparing with the total number of cells or the area of positive staining in the selected area, respectively. 3 (minipigs) or 5 to 6 (mice) samples per group were used for quantification, and 3 fields were selected from each sample for quantification. The average of 3 fields of view was used as the quantitative value for each sample. To quantify the differences between the groups, the values (positive signal/number of positive cells/percent positive area) for each sample in each group were quantified with those of the wild-type or control group. Results for each group are shown as "fold of control or fold of wild type".
13.体外敲低HDAC2、ADAMTS1。将人类HDAC2(shHDAC2：5'-CAGACTGATATGGCTGTTAAT-3')或ADAMTS1(shADAMTS1：5'-CAAAAACCACAGGAACTGGAAGCATAA-3')的shRNA克隆到pLKO.1中，并转染到HEK293T细胞中，生成携带sh-HDAC2(即靶向HDAC2的shRNA)或sh-ADAMTS1(即靶向ADAMTS1的shRNA)的慢病毒颗粒(也按照实验需求获得阴性对照)。HUVECs用慢病毒颗粒转导，然后分别用嘌呤霉素(1μg/ml)选择48小时，然后用于后续实验。13. Knock down HDAC2 and ADAMTS1 in vitro. The shRNA of human HDAC2 (shHDAC2: 5'-CAGACTGATATGGCTGTTAAT-3') or ADAMTS1 (shADAMTS1: 5'-CAAAAACCACAGGAACTGGAAGCATAA-3') was cloned into pLKO.1 and transfected into HEK293T cells to generate sh-HDAC2 ( Lentiviral particles of shRNA targeting HDAC2) or sh-ADAMTS1 (shRNA targeting ADAMTS1) (a negative control was also obtained as required by the experiment). HUVECs were transduced with lentiviral particles and then individually selected with puromycin (1 μg/ml) for 48 hours before being used for subsequent experiments.
14.血浆细胞外囊泡(EVs)分离。通过超速离心分离血浆EVs。将人和小型猪的血浆在4℃解冻，并在4℃ 850g离心30分钟，以去除死细胞和颗粒物。将上清液在4℃12,000g离心45分钟以除去细胞碎片。接下来，将上清液在4℃ 110,000g离心2小时。弃去上清液。收集含有EVs的颗粒，将其重悬于冷的PBS中，通过0.22μm的过滤器过滤，并在4℃以110,000g离心2小时以除去污染的蛋白质。弃去上清液，并收集细胞外囊泡用于后续实验。HUVEC培养基上清EVs的分离根据制造商的说明书用总外泌体分离试剂(从细胞培养基)(4478359，Invitrogen)进行。收集的细胞培养基上清EVs用于移植实验。14. Plasma extracellular vesicles (EVs) isolation. Plasma EVs were isolated by ultracentrifugation. Human and minipig plasma were thawed at 4°C and centrifuged at 850 g for 30 minutes at 4°C to remove dead cells and particulates. The supernatant was centrifuged at 12,000 g for 45 minutes at 4°C to remove cell debris. Next, the supernatant was centrifuged at 110,000 g for 2 hours at 4°C. Discard the supernatant. Particles containing EVs were collected, resuspended in cold PBS, filtered through a 0.22 μm filter, and centrifuged at 110,000 g for 2 h at 4 °C to remove contaminating proteins. Discard the supernatant and collect extracellular vesicles for subsequent experiments. Isolation of HUVEC medium supernatant EVs was performed with Total Exosome Isolation Reagent (from cell culture medium) (4478359, Invitrogen) according to the manufacturer's instructions. The collected cell culture supernatant EVs were used for transplantation experiments.
15.细胞外囊泡(EV)移植实验。EV移植实验如图9C所示。8周龄C57BL/6J小鼠喂食正常饮食，每周2次腹腔注射1ml/kg的CCl 4，共1周，然后腹腔注射CCl 4，尾静脉注射细胞外囊泡(10μg蛋白/小鼠，每剂量约10 10个细胞外囊泡)每周2次，共2周。将小鼠随机分为两组，并通过尾静脉注射来自shNC感染的HUVECs培养基(高ADAMTS1)的EVs，和来自shADAMTS1感染的HUVECs培养基(低ADAMTS1)的EVs。最后一次注射CCl 4和细胞外囊泡的两天后，处死所有小鼠，并收集肝脏样品。 15. Extracellular vesicle (EV) transplantation experiments. EV transplantation experiments are shown in Figure 9C. 8-week-old C57BL/6J mice were fed a normal diet and injected intraperitoneally with 1 ml/kg of CCl 4 twice a week for a total of 1 week, followed by intraperitoneal injection of CCl 4 and extracellular vesicles (10 μg protein/mouse, each The dose is approximately 10 10 extracellular vesicles) 2 times a week for 2 weeks. Mice were randomly divided into two groups, and EVs from shNC-infected HUVECs medium (high ADAMTS1) and EVs from shADAMTS1-infected HUVECs medium (low ADAMTS1) were injected via tail vein. Two days after the last injection of CCl and extracellular vesicles, all mice were sacrificed and liver samples were collected.
16.重组IGFBP7治疗实验。重组IGFBP7蛋白处理实验如图8F所示。8周龄C57BL/6J小鼠喂食正常饮食，每周2次腹腔注射剂量为1ml/kg的CCl 4，共1周，然后腹腔注射CCl 4，并静脉注射外源重组小鼠IGFBP7蛋白(20μg蛋白/小鼠)两天1次，共2周。最后一次注射IGFBP7的两天后，处死所有小鼠，并收集肝脏样品。 16. Recombinant IGFBP7 treatment experiments. The recombinant IGFBP7 protein treatment experiment is shown in Figure 8F. 8-week-old C57BL/6J mice were fed a normal diet and were injected intraperitoneally with CCl 4 at a dose of 1 ml/kg twice a week for 1 week, followed by intraperitoneal injection of CCl 4 and intravenous injection of exogenous recombinant mouse IGFBP7 protein (20 μg protein). / mice) once every two days for a total of 2 weeks. Two days after the last injection of IGFBP7, all mice were sacrificed and liver samples were collected.
17.实时定量PCR(qPCR)。使用RNeasy Mini Kit(QIAGEN)提取人、小型猪和小鼠肝脏中的ECs、SECs、MECs和CD45 +NPCs的总RNA，并使用Takara反转录试剂盒(TakaraPrimeScript TM RT Master Mix)(RR036A)进行反转录。使用Brilliant III Ultra Fast SYBR Green qPCR Master Mix试剂盒(Agilent Technologies)测量所有的基因表达。将所有样品重复三次，通过GAPDH对数据归一化，并通过ddCt方法进行分析，误差线表示标准误(Standard Error of Mean，SEM)。 17. Real-time quantitative PCR (qPCR). Total RNA from ECs, SECs, MECs, and CD45 + NPCs in human, minipig, and mouse livers was extracted using the RNeasy Mini Kit (QIAGEN) and performed using the Takara Reverse Transcription Kit (TakaraPrimeScript ™ RT Master Mix) (RR036A). Reverse Transcription. All gene expression was measured using the Brilliant III Ultra Fast SYBR Green qPCR Master Mix kit (Agilent Technologies). All samples were replicated in triplicate, data were normalized by GAPDH and analyzed by the ddCt method, with error bars representing Standard Error of Mean (SEM).
18.ELISA测定。将肝脏组织称重并匀浆，以2,000g离心20分钟，收集上清。参 照III型前胶原(PC III)、透明质(HA)和IV型胶原蛋白(IV-C)测定试剂盒的说明书，设置空白对照并制作标准曲线，经酶标试剂、显色液及终止液孵育后，450nm测OD值。样本浓度根据标准曲线换算获得。人和小型猪血浆、细胞外囊泡、SECs、MECs和CD45 +NPCs的IGFBP7和ADAMTS1水平按照相同方法测量。细胞外囊泡在分析前用超声波(40kHZ)处理3分钟。 18. ELISA assay. Liver tissue was weighed and homogenized, centrifuged at 2,000 g for 20 minutes, and the supernatant was collected. Referring to the instructions of the type III procollagen (PC III), hyaluronan (HA) and type IV collagen (IV-C) assay kits, set a blank control and make a standard curve. After incubation, the OD value was measured at 450 nm. The sample concentration was converted according to the standard curve. IGFBP7 and ADAMTS1 levels in human and minipig plasma, extracellular vesicles, SECs, MECs and CD45 + NPCs were measured in the same way. Extracellular vesicles were sonicated (40kHZ) for 3 minutes prior to analysis.
19.羟脯氨酸分析。将小型猪和小鼠肝脏组织称重，用羟脯氨酸试剂盒(BC0255，Solarbio)提取并测定羟脯氨酸，肝脏中羟脯氨酸水平根据所用的肝脏重量来测定。19. Hydroxyproline analysis. Minipig and mouse liver tissues were weighed, extracted with a hydroxyproline kit (BC0255, Solarbio) and assayed for hydroxyproline, and the level of hydroxyproline in the liver was determined according to the liver weight used.
20.血清和血浆分析。使用多参数分析仪(AU 5400；Olympus，日本)测量小型猪和小鼠血清及人血浆ALT、AST、ALP和总胆固醇水平。使用血糖仪(ACCU-CHEK Performa，Roche，德国)测量小型猪的血糖水平。20. Serum and plasma analysis. Minipig and mouse serum and human plasma ALT, AST, ALP and total cholesterol levels were measured using a multiparameter analyzer (AU 5400; Olympus, Japan). Blood glucose levels in minipigs were measured using a blood glucose meter (ACCU-CHEK Performa, Roche, Germany).
21.蛋白质印迹(WB)分析。用RIPA裂解缓冲液(P0013B,Beyotime Biotechnology)并补充蛋白酶抑制剂和磷酸酶抑制剂，来提取人和小型猪的HUVECs和EVs及人肝脏CD45 +NPCs的总蛋白。一抗包括兔抗HDAC2(57156，Cell Signaling Technology)和抗DNMT1(5032，Cell Signaling Technology)，兔抗GAPDH(GB11002，Servicebio)，兔抗CD81(bs-6954R，Bioss)和抗GRP94(bs-0194R，Bioss)和兔抗Smad2(5339，Cell Signaling Technology)和抗Phospho-Smad2(18338，Cell Signaling Technology)。过氧化物酶偶联的山羊抗兔二抗(GB23303)购买自武汉赛维尔生物科技有限公司。每个样本上样20μg蛋白。每组三个生物样本用于统计分析。通过NIH Image J(http://rsb.info.nih.gov/ij/download.html)对蛋白带的光密度进行定量。 21. Western blot (WB) analysis. RIPA lysis buffer (P0013B, Beyotime Biotechnology) supplemented with protease inhibitors and phosphatase inhibitors was used to extract total protein from human and minipig HUVECs and EVs and human liver CD45 + NPCs. Primary antibodies include rabbit anti-HDAC2 (57156, Cell Signaling Technology) and anti-DNMT1 (5032, Cell Signaling Technology), rabbit anti-GAPDH (GB11002, Servicebio), rabbit anti-CD81 (bs-6954R, Bioss) and anti-GRP94 (bs-0194R) , Bioss) and rabbit anti-Smad2 (5339, Cell Signaling Technology) and anti-Phospho-Smad2 (18338, Cell Signaling Technology). Peroxidase-conjugated goat anti-rabbit secondary antibody (GB23303) was purchased from Wuhan Sevier Biotechnology Co., Ltd. 20 μg protein was loaded per sample. Three biological samples per group were used for statistical analysis. The optical density of the protein bands was quantified by NIH Image J (http://rsb.info.nih.gov/ij/download.html).
22.组蛋白翻译后修饰(PTM)多重检测。从人类肝脏中分离肝细胞和NPCs，使用EpiQuik总组蛋白提取试剂盒提取肝细胞和NPCs中的组蛋白。用不同的位点特异性抗体分别进行组蛋白H3和H4翻译后修饰(Post-translational modification，PTM)多重分析(表3)。首先，针对组蛋白PTM的捕获抗体共价偶联到磁珠上(表4)，偶联珠与含有组蛋白PTM的样品反应后，连续洗涤以去除未结合的蛋白。添加生物素化的组蛋白H3或H4抗体形成夹层复合体(sandwich complex)，最后添加作为荧光指示剂的链霉亲和素-藻红素(Streptavidin-phycoerythrin，SA-PE)偶联物以形成最终的检测复合体。使用Bio-plex200液相芯片系统(171000207,Bio-rad)检测不同的组蛋白PTMs。不同的组蛋白H3和H4PTMs分别用总H3和H4进行归一化处理。PTM多重检测由杭州景杰生物科技有限公司进行。22. Multiplex detection of histone post-translational modifications (PTM). Hepatocytes and NPCs were isolated from human liver, and histones were extracted from hepatocytes and NPCs using the EpiQuik Total Histone Extraction Kit. Multiplex analysis of post-translational modification (PTM) of histone H3 and H4 was performed with different site-specific antibodies (Table 3). First, capture antibodies directed against histone PTMs were covalently coupled to magnetic beads (Table 4), and after the coupled beads were reacted with samples containing histone PTMs, unbound proteins were removed by successive washings. Add biotinylated histone H3 or H4 antibody to form a sandwich complex, and finally add streptavidin-phycoerythrin (SA-PE) conjugate as a fluorescent indicator to form The final detection complex. Different histone PTMs were detected using Bio-plex200 liquid chip system (171000207, Bio-rad). Different histone H3 and H4 PTMs were normalized to total H3 and H4, respectively. PTM multiplex detection was performed by Hangzhou Jingjie Biotechnology Co., Ltd.
表3 PTM多重检测的抗体信息Table 3 Antibody information for PTM multiplex detection
Figure PCTCN2021140096-appb-000009
Figure PCTCN2021140096-appb-000009
Figure PCTCN2021140096-appb-000010
Figure PCTCN2021140096-appb-000010
备注：1.PTM-1002和PTM-1004需要生物素处理。生物素化的H3抗体用作H3面板的检测抗体，生物素化的H4抗体用作H4面板的检测抗体。2.其他抗体与不同的磁珠偶联。Remarks: 1. PTM-1002 and PTM-1004 require biotin treatment. Biotinylated H3 antibody was used as the detection antibody for the H3 panel, and biotinylated H4 antibody was used as the detection antibody for the H4 panel. 2. Other antibodies are coupled to different magnetic beads.
表4 PTM多重检测的磁珠信息Table 4 Magnetic bead information for PTM multiplex detection
Figure PCTCN2021140096-appb-000011
Figure PCTCN2021140096-appb-000011
23.人和小型猪NPCs的scRNA-Seq。从人和小型猪的肝脏中分离NPCs，获得单细胞并重悬于PBS。通过Chromium单细胞平台(10X Genomics)进行scRNA-Seq。单细胞通过GEM(Gel Bead-in-Emulsion)的形成和Barcode序列化、GEM-RT(Gel Bead-in-Emulsion–Reverse Transcription)清理和cDNA扩增、文库构建等步骤，最后在Illumina Nova-seq 6000(Illumina，美国)上进行测序。小型猪NPCs的scRNA-Seq由广州基底奥生物科技有限公司进行，人NPCs的scRNA-Seq由北京诺禾致源科技股份有限公司进行。23. scRNA-Seq of human and minipig NPCs. NPCs were isolated from human and minipig livers, and single cells were obtained and resuspended in PBS. scRNA-Seq was performed by Chromium single cell platform (10X Genomics). Single cells go through the steps of GEM (Gel Bead-in-Emulsion) formation and Barcode serialization, GEM-RT (Gel Bead-in-Emulsion-Reverse Transcription) cleaning, cDNA amplification, library construction and other steps, and finally in Illumina Nova-seq 6000 (Illumina, USA) for sequencing. The scRNA-Seq of minipig NPCs was performed by Guangzhou Baseao Biotechnology Co., Ltd., and the scRNA-Seq of human NPCs was performed by Beijing Nuohezhiyuan Technology Co., Ltd.
24.小型猪ECs的ATAC-seq。从小型猪肝脏中分离ECs(CD45 -CD31 +)，分离细胞核，通过转位反应、PCR扩增和纯化、文库构建等步骤，最后使用Illumina HiSeqTM 4000进行测序。小型猪ECs的ATAC-seq由广州基底奥生物科技有限公司进行，数据经质量控制和参考序列比对后用于后续分析。 24. ATAC-seq of minipig ECs. ECs (CD45 - CD31 + ) were isolated from minipig livers, nuclei were isolated, and the nuclei were isolated by translocation reaction, PCR amplification and purification, library construction and other steps, and finally sequenced using Illumina HiSeqTM 4000. ATAC-seq of minipig ECs was performed by Guangzhou Baseao Biotechnology Co., Ltd., and the data were used for subsequent analysis after quality control and reference sequence alignment.
25.公共数据的再利用。肝硬化患者的肝脏活检样本的微阵列(Microarray)数据(GSE84044)和肝硬化患者NPCs的scRNA-seq数据(GSE136103)来自GEO数据库。微阵列数据中，纤维化F1-F4级的肝脏中HDACs、DNMTs和IGFBP7的表达水平相对于健康肝脏(F0)进行量化。scRNA-seq数据中，CD45 +和CD45 -细胞(GSM4041150、GSM4041151、GSM4041153、GSM4041154、GSM4041155、GSM4041156、GSM4041158、GSM4041159、GSM4041161、GSM4041162、GSM4041164、GSM4041165、GSM4041166和GSM4041167)用于分析ECs图谱；CD45 +细胞(GSM4041150、GSM4041153、GSM4041155、GSM4041158、GSM4041160、GSM4041161、GSM4041164、GSM4041166、GSM4041168和GSM4041169)用于分析T细胞图谱。 25. Reuse of public data. Microarray data (GSE84044) of liver biopsy samples from patients with cirrhosis and scRNA-seq data (GSE136103) of NPCs from patients with cirrhosis were obtained from the GEO database. In the microarray data, the expression levels of HDACs, DNMTs and IGFBP7 were quantified in fibrotic F1-F4 livers relative to healthy livers (F0). scRNA-seq数据中，CD45 +和CD45 -细胞(GSM4041150、GSM4041151、GSM4041153、GSM4041154、GSM4041155、GSM4041156、GSM4041158、GSM4041159、GSM4041161、GSM4041162、GSM4041164、GSM4041165、GSM4041166和GSM4041167)用于分析ECs图谱；CD45 +细胞(GSM4041150, GSM4041153, GSM4041155, GSM4041158, GSM4041160, GSM4041161, GSM4041164, GSM4041166, GSM4041168, and GSM4041169) were used to analyze T-cell profiles.
26.生物信息学分析。scRNA-Seq数据预处理：利用Hisat2v2.0.5将人和小型猪肝脏NPCs的scRNA-seq的序列分别与人(Homo sapiens)转录组(GRCh38.p13)和猪(Sus scrofa)转录组(Sscrofa11.1)进行比对。使用Seurat R包v3.1.1分别对小型猪、来自华西医院的人的样本及GSE136103数据进行无监督聚类和差异基因表达分析。细胞过滤：将表达少于200个基因的低质量细胞和表达少于3个细胞的基因过滤。通过设定不同的线粒体基因含量百分比阈值来过滤小型猪和人肝脏NPCs。对于两种小型猪数据，线粒体基因含 量>15％的细胞被过滤掉，而对于华西医院的人的样本数据，>10％的细胞被过滤掉。实验人员过滤掉了肝细胞标记物(ALB、APOE、APOB等)和间充质细胞标记物(COL1A1、COL3A1等)高表达的群。数据归一化：通过“NormalizeData”函数中的“LogNormalize”全局尺度归一化方法对数据进行归一化处理。样本整合：利用函数“FindIntegrationAnchors”和“IntegrateData”合并数据并消除批次效应(batch effects)。维度1到20用于指定邻居搜索空间(neighbor search space)以查找整合锚点。通过函数“ScaleData”进行标准化(scaled)和中心化(centered)。聚类和可视化：函数“RunPCA”用于主成分分析(Principal component analysis，PCA)，主成分1-15用于函数“FindNeighbors”。用函数“FindClusters”对细胞进行聚类(分辨率：0.77)，并用UMAP方法可视化。UMAP图、小提琴图、热图和点图用SeuratR包(ggplot2、pheatmap和grid)构建。实验人员采用数据库(http://biocc.hrbmu.edu.cn/CellMarker/index.jsp)中推荐的细胞系标记基因及已发表文章(参考文献35、36)中使用的标记基因来定义不同的细胞系。通过标记基因的表达定义了7种细胞系：T细胞(CD2 +、KLRB1 +、PTPRC +、CD3E +、TRAC +等)、B细胞(CD19 +、CD22 +、CD79B +、MS4A1 +、MZB1 +、IGKC +等)、内皮细胞(PECAM1 +、CLECC4G +、FLT1 +、OIT3 +、CLECC4M +、CD34 +等)、巨噬细胞(CD163 +、VSIG4 +、CD68 +、ADGR1 +、MSR1 +、C1QC +等)、中性粒细胞(S100A8 +、S100A9 +、CXCL8 +、MSRB1 +等)、树突细胞(CLEC9A +、LGALS2 +、IDO1 +、CLORF54 +、CLEC4A +、CD83 +、CD40 +、CST3 +、CD74 +等)和EPCAM +细胞和胆管细胞(EPCAM +、KRT7 +、SOX9 +、CFTR +、MMP7 +、KRT19 +等)。内皮细胞被重新聚类和定义为窦内皮细胞(CLEC4G +、OIT3 +、CLEC4M +)、大血管内皮细胞(CD34 +)和中间内皮细胞(CLEC4G -，CD34 -)。T细胞重新聚类并定义为CD4 +(CD4 +)和CD8 +(CD8A +)T细胞。通过检测CD4 +T细胞中KLRB1 +、FOXP3 -、CCR6 +、CCR4 +、AHR +、IL23R +、IL17A +的表达来鉴定Th17细胞。显示不同细胞系的标记基因表达的热图由这些细胞系标记基因的平均计数生成。使用Wilcoxon Rank Sum检验鉴定了两组细胞之间的差异表达基因，差异基因同时满足在超过10％的细胞中表达以及两组细胞之间至少0.25的对数倍数变化。所有差异表达基因分析均具有相同的阈值。对于小型猪scRNA-Seq，1个对照组、2个肝硬化组和2个治疗组的小型猪肝脏NPCs中共获得了40,570个细胞，展示了28个群体。对于华西医院人scRNA-Seq，2个健康和2个肝硬化的人肝脏NPCs中共获得223,74个细胞，展示了25个群。通过细胞系标记基因来确认不同的细胞系。在小型猪和人的scRNA-Seq数据分析中，整合多组样本、聚类、鉴定不同细胞系和差异表达基因分析的方法是相似的。拟时分析：R包-monocle v2.12.0用于构建拟时分析。将标记为内皮细胞的细胞及其亚群的信息输入并构建monocle对象。表达矩阵中的所有数值均进行对数变换。利用函数“differential GeneTest”分析不同组间的差异表达基因(有不同的q值，小型猪<0.045，华西医院人<1e-12)。“DDRTree”用于减少维度(Max_components＝2)。细胞受体配体互作分析：scRNA-Seq结果上传到Cell Phone DB的网站上进行细胞受体配体互作分析。 26. Bioinformatics analysis. scRNA-Seq data preprocessing: The scRNA-seq sequences of human and minipig liver NPCs were compared with the human (Homo sapiens) transcriptome (GRCh38.p13) and pig (Sus scrofa) transcriptome (Sscrofa11.1) using Hisat2v2.0.5, respectively. ) for comparison. Unsupervised clustering and differential gene expression analysis were performed on minipigs, human samples from West China Hospital, and GSE136103 data, respectively, using the Seurat R package v3.1.1. Cell filter: Filter low-quality cells expressing less than 200 genes and genes expressing less than 3 cells. Minipig and human liver NPCs were filtered by setting different percentage thresholds for mitochondrial gene content. For both minipig data, cells with >15% mitochondrial gene content were filtered out, while for the human sample data from West China Hospital, >10% cells were filtered out. The experimenters filtered out groups with high expression of hepatocyte markers (ALB, APOE, APOB, etc.) and mesenchymal cell markers (COL1A1, COL3A1, etc.). Data normalization: The data is normalized by the "LogNormalize" global scale normalization method in the "NormalizeData" function. Sample Integration: Use the functions "FindIntegrationAnchors" and "IntegrateData" to combine data and eliminate batch effects. Dimensions 1 to 20 are used to specify the neighbor search space to find integration anchors. Scaled and centered by the function "ScaleData". Clustering and Visualization: The function "RunPCA" was used for Principal Component Analysis (PCA), and Principal Components 1-15 were used for the function "FindNeighbors". Cells were clustered with the function "FindClusters" (resolution: 0.77) and visualized with the UMAP method. UMAP plots, violin plots, heatmaps and point plots were constructed with the SeuratR package (ggplot2, pheatmap and grid). The experimenters used the cell line marker genes recommended in the database (http://biocc.hrbmu.edu.cn/CellMarker/index.jsp) and the marker genes used in the published articles (references 35 and 36) to define different cell line. Seven cell lines were defined by the expression of marker genes: T cells (CD2 + , KLRB1 + , PTPRC + , CD3E + , TRAC + etc.), B cells (CD19 + , CD22 + , CD79B + , MS4A1 + , MZB1 + , IGKC + , etc.), endothelial cells (PECAM1 + , CLECC4G + , FLT1 + , OIT3 + , CLECC4M + , CD34 + , etc.), macrophages (CD163 + , VSIG4 + , CD68 + , ADGR1 + , MSR1 + , C1QC + , etc.) ), neutrophils (S100A8 + , S100A9 + , CXCL8 + , MSRB1 + etc.), dendritic cells (CLEC9A + , LGALS2 + , IDO1 + , CLORF54 + , CLEC4A + , CD83 + , CD40 + , CST3 + , CD74 + etc.) and EPCAM + cells and cholangiocytes (EPCAM + , KRT7 + , SOX9 + , CFTR + , MMP7 + , KRT19 + etc.). Endothelial cells were reclustered and defined as sinus endothelial cells (CLEC4G + , OIT3 + , CLEC4M + ), large vessel endothelial cells (CD34 + ) and intermediate endothelial cells (CLEC4G − , CD34 − ). T cells were reclustered and defined as CD4 + (CD4 + ) and CD8 + (CD8A + ) T cells. Th17 cells were identified by detecting the expression of KLRB1 + , FOXP3 − , CCR6 + , CCR4 + , AHR + , IL23R + , IL17A + in CD4 + T cells. Heatmaps showing marker gene expression for different cell lines were generated from the average counts of marker genes for these cell lines. Differentially expressed genes between the two groups of cells were identified using the Wilcoxon Rank Sum test, the differential genes satisfying both expression in more than 10% of cells and a log fold change of at least 0.25 between the two groups of cells. All differentially expressed gene analyses had the same threshold. For minipig scRNA-Seq, a total of 40,570 cells were obtained from minipig liver NPCs in 1 control group, 2 cirrhosis groups, and 2 treatment groups, representing 28 populations. For West China Hospital human scRNA-Seq, a total of 223,74 cells were obtained from 2 healthy and 2 cirrhotic human liver NPCs, showing 25 populations. Different cell lines are identified by cell line marker genes. Methods for integrating multiple sets of samples, clustering, identifying different cell lines, and differentially expressed gene analysis were similar in minipig and human scRNA-Seq data analysis. Pseudo-time analysis: The R package -monocle v2.12.0 is used to build pseudo-time analysis. Inputs information on cells labeled as endothelial cells and their subpopulations and constructs a monocle object. All values in the expression matrix are log-transformed. Use the function "differential GeneTest" to analyze the differentially expressed genes between different groups (with different q values, minipig <0.045, West China Hospital human <1e-12). "DDRTree" is used for dimension reduction (Max_components=2). Cell receptor ligand interaction analysis: scRNA-Seq results were uploaded to the Cell Phone DB website for cell receptor ligand interaction analysis.
27.定量与统计分析：所有计算或分析均通过Prism 8软件包(GraphPad)或R进行。实验数据通过双尾学生t检验(2组比较)和单因素方差分析(ANOVA)及Tukey事后检验(2组以上比较)进行统计分析。所有数据均以平均值±SEM表示。P<0.05被认为统计学显著，所有误差线代表SEM。在体内实验中，“n”值表示每组生物样本重复数。*，肝硬化/NASH组vs.健康/对照组；#，治疗组vs.肝硬化/NASH组或肝硬化/NASH组vs.单纯性脂肪肝组。*或#，P<0.05；**或##，P<0.01；***或###，P<0.001；****，P＜0.0001。27. Quantitative and statistical analysis: All calculations or analyses were performed by Prism 8 software package (GraphPad) or R. The experimental data were statistically analyzed by two-tailed Student's t test (comparison of 2 groups), one-way analysis of variance (ANOVA) and Tukey's post-hoc test (comparison of more than 2 groups). All data are presented as mean ± SEM. P<0.05 was considered statistically significant and all error bars represent SEM. In in vivo experiments, the "n" value represents the number of replicates per group of biological samples. *, cirrhosis/NASH group vs. healthy/control group; #, treatment group vs. cirrhosis/NASH group or cirrhosis/NASH group vs. simple fatty liver group. * or #, P<0.05; ** or ##, P<0.01; *** or ###, P<0.001; ****, P<0.0001.
实施例二：scRNA-Seq揭示人肝硬化肝脏中血管失调和内皮分类异常Example 2: scRNA-Seq reveals vascular dysregulation and abnormal endothelial classification in human cirrhotic liver
从新鲜的正常和硬化的人肝脏中分离NPCs，并进行scRNA-seq(10x genomics)(图1A，表S1)。健康的肝脏组织(无纤维化)获得自华西医院的肝血管瘤患者，肝硬化肝脏组织获得自经组织学诊断为肝硬化的患者。2个健康肝脏和2个肝硬化肝脏的22,374 个NPCs聚类成25个群(图10A-B)。通过标记基因的表达定义了7种细胞系：T细胞、B细胞、内皮细胞(EC)、巨噬细胞(Mac)、中性粒细胞(Neu)、树突状细胞(DC)以及EPCAM +细胞和胆管细胞(EPCAM +)(图1B-C)。本发明实验人员发现，在检验的所有细胞类型中，ECs的差异基因最多(图1D)。与健康肝脏相比，肝硬化肝脏ECs的比例显著增加(图10C)。 NPCs were isolated from fresh normal and cirrhotic human livers and subjected to scRNA-seq (10x genomics) (Fig. 1A, Table S1). Healthy liver tissue (without fibrosis) was obtained from a patient with hepatic hemangioma in West China Hospital, and cirrhotic liver tissue was obtained from a patient with histologically diagnosed liver cirrhosis. 22,374 NPCs from 2 healthy livers and 2 cirrhotic livers clustered into 25 clusters (Figure 10A-B). Seven cell lines were defined by expression of marker genes: T cells, B cells, endothelial cells (EC), macrophages (Mac), neutrophils (Neu), dendritic cells (DC), and EPCAM + cells and cholangiocytes (EPCAM + ) (Fig. 1B-C). Experimenters of the present invention found that among all the cell types examined, ECs had the most differential genes (Fig. 1D). The proportion of ECs in cirrhotic livers was significantly increased compared with healthy livers (Fig. 10C).
本发明实验人员的前研究表明，血管内皮细胞能够形成血管微环境，通过旁分泌/血管分泌因子调控肝脏再生和纤维化(参考文献28)。因此，本发明进一步分析了人肝脏中的ECs亚群。人肝脏ECs聚类成12个群(图10D)，由标记基因CLEC4G、OIT3和CD34定义为窦内皮细胞(SEC)和大血管内皮(MEC)(图1E，图10E)。MEC的数量显著增加，SEC的数量显著减少，这意味着肝硬化肝脏中发生一种从SEC到MEC的失调(图1E-F)。为了验证这种“窦内皮-大血管内皮分类异常”，实验人员还分析了人肝脏数据库GSE136103的数据。4个健康肝脏和3个肝硬化肝脏的42,314个NPC聚类成28个群(图11A)，定义了7个细胞系(图11B-C)。肝脏ECs进一步聚类，并定义为成SEC和MEC(图11D-F)。GSE136103数据也显示肝硬化肝脏中MECs显著增加、SECs显著减少(图11G)，并存在“窦内皮-大血管内皮失调”(图11H)。实验人员发现，在总ECs和肝硬化ECs中，间充质细胞标记基因的表达较低，并且在肝脏ECs的所检验的类型中，“间充质细胞分化”富集程度相对较低(图12A-C)。这些数据表明，在纤维化的肝脏中，肝脏SECs可能发生血管失调(图12D)。Previous studies by the present inventors have shown that vascular endothelial cells can form a vascular microenvironment and regulate liver regeneration and fibrosis through paracrine/vascular secretory factors (ref. 28). Therefore, the present invention further analyzes the subsets of ECs in human liver. Human liver ECs were clustered into 12 clusters (Fig. 10D), defined by the marker genes CLEC4G, OIT3 and CD34 as sinus endothelial cells (SEC) and macrovascular endothelium (MEC) (Fig. 1E, Fig. 10E). The number of MECs was significantly increased and the number of SECs was significantly decreased, implying a deregulation of SEC to MECs in cirrhotic livers (Fig. 1E-F). To verify this "sinus endothelium-major vessel endothelium classification abnormality", the experimenters also analyzed data from the human liver database GSE136103. 42,314 NPCs from 4 healthy livers and 3 cirrhotic livers were clustered into 28 clusters (Fig. 11A), and 7 cell lines were defined (Fig. 11B-C). Liver ECs were further clustered and defined as SEC and MEC (Figure 11D-F). The GSE136103 data also showed a significant increase in MECs, a significant decrease in SECs in cirrhotic livers (FIG. 11G), and the presence of "sinusoidal-macrovascular endothelial dysregulation" (FIG. 11H). The experimenters found lower expression of mesenchymal cell marker genes in total ECs and cirrhotic ECs, and a relatively low degree of enrichment for "mesenchymal differentiation" in the examined types of liver ECs (Fig. 12A-C). These data suggest that in fibrotic livers, vascular dysregulation may occur in hepatic SECs (FIG. 12D).
实施例三：肝脏ECs表观遗传重编程诱导促纤维化的“窦内皮-大血管内皮失调”Example 3: Epigenetic reprogramming of liver ECs induces pro-fibrotic "sinusoidal endothelial-macrovascular endothelial dysregulation"
表观遗传调控(组蛋白修饰和DNA甲基化)在肝纤维化进程中起重要作用。然而，组蛋白修饰和DNA甲基化在人肝脏硬化/纤维化的NPCs不同细胞系的功能性作用尚不明确。为此，实验人员首先确定了健康肝脏和肝硬化肝脏中实质细胞和NPCs的组蛋白修饰，组蛋白H3和H4修饰的液相芯片分析显示，与健康肝脏相比，人肝硬化肝脏中NPCs的组蛋白乙酰化显著降低，而实质细胞(肝细胞)没有明显差异(图1G)，这表明人肝硬化肝脏中的NPCs发生表观遗传重编程。Epigenetic regulation (histone modification and DNA methylation) plays an important role in the progression of liver fibrosis. However, the functional roles of histone modifications and DNA methylation in different cell lines of human cirrhotic/fibrotic NPCs remain unclear. To this end, the experimenters first determined the histone modifications of parenchymal cells and NPCs in healthy livers and cirrhotic livers. Liquid-chip analysis of histone H3 and H4 modifications showed that compared with healthy livers, NPCs in human cirrhotic livers were more Histone acetylation was significantly reduced, whereas parenchymal cells (hepatocytes) were not significantly different (Fig. 1G), suggesting epigenetic reprogramming of NPCs in human cirrhotic livers.
实验人员进一步研究了健康和肝硬化的人肝脏中不同NPCs亚群的表观遗传变化。通过基因组富集分析(GSEA)和STRING数据库筛选了1,239个与组蛋白修饰和DNA甲基化相关的基因，其中1,008个在本发明的人肝脏的scRNA-Seq数据中发现。与正常组相比，在检验的的7个NPCs细胞系中，血管ECs中组蛋白修饰和DNA甲基化相关基因变化最多(图1H)。这些发现提示，内皮细胞的表观遗传改变可能通过刺激血管微环境功能的失调来促进纤维化。The experimenters further investigated epigenetic changes in different subsets of NPCs in healthy and cirrhotic human livers. 1,239 genes related to histone modification and DNA methylation were screened by Genome Enrichment Analysis (GSEA) and STRING database, of which 1,008 were found in the scRNA-Seq data of human liver of the present invention. Among the seven NPCs cell lines examined, histone modification and DNA methylation-related genes had the most changes in vascular ECs compared with the normal group (Fig. 1H). These findings suggest that epigenetic alterations in endothelial cells may promote fibrosis by stimulating dysregulation of the vascular microenvironment.
组蛋白乙酰化受组蛋白去乙酰化酶(HDACs)调控。为了确定哪些HDAC(s)在肝硬化肝脏的组蛋白修饰中起重要作用，实验人员分别分析了所有HDACs在纤维化肝脏和内皮细胞中的表达。人纤维化肝脏被分为不同的病理等级，F0-F4级，F4级为最严重的纤维化阶段。在所有HDACs中，HDAC2的表达在F2-F4级患者的肝脏中的持续增加(图1I，图13A，数据来自GSE84044)。此外，与健康人肝脏相比，人肝硬化肝脏的内皮细胞中HDAC2表达上调(图1J)。定量PCR(qPCR)显示，虽然HDAC2和DNMT1也在其他细胞类型(CD45 +NPCs)中表达，但在健康组和肝硬化组之间，肝脏内皮细胞(CD45 -CD31 +)中HDAC2的表达有显著差异(图1K，图13B)。 Histone acetylation is regulated by histone deacetylases (HDACs). To determine which HDAC(s) play an important role in histone modification in cirrhotic livers, we separately analyzed the expression of all HDACs in fibrotic livers and endothelial cells. Human fibrotic livers are classified into different pathological grades, F0-F4, with F4 being the most severe stage of fibrosis. Among all HDACs, HDAC2 expression was consistently increased in the livers of F2-F4 class patients (Fig. 1I, Fig. 13A, data from GSE84044). Furthermore, HDAC2 expression was upregulated in endothelial cells of human cirrhotic livers compared to healthy human livers (Fig. 1J). Quantitative PCR (qPCR) showed that while HDAC2 and DNMT1 were also expressed in other cell types (CD45 + NPCs), there was a significant difference in HDAC2 expression in liver endothelial cells (CD45 - CD31 + ) between healthy and cirrhotic groups difference (Fig. 1K, Fig. 13B).
DNA甲基化是另一种常见的表观遗传修饰形式，通常与组蛋白修饰协同作用。因此，实验人员评估了DNA甲基转移酶(DNMTs)在人纤维化肝脏中的表达。发现DNMT1在F2-F4级患者的纤维化肝脏中变化相对最显著(图1L，来自GSE84044的数据)，并且相对于人类健康肝脏，DNMT1在人类肝硬化肝脏ECs中的表达显著增强。DNA methylation is another common form of epigenetic modification, often in concert with histone modifications. Therefore, we evaluated the expression of DNA methyltransferases (DNMTs) in human fibrotic livers. DNMT1 was found to be relatively most altered in fibrotic livers of F2-F4 patients (Fig. 1L, data from GSE84044), and DNMT1 expression was significantly enhanced in human cirrhotic liver ECs relative to human healthy livers.
在分离出的人肝硬化肝脏CD45 -CD34 -CD31 +SECs中，与健康人肝脏相比，HDAC2和DNMT1表达水平显著上调(图1O)。在人脐静脉内皮细胞(HUVECs)中，通过shRNA (shHDAC2)敲低HDAC2可以上调DNMT1的表达(图13C)。这些数据表明，肝脏ECs中HDAC2和DNMT1的异常激活可能导致“窦内皮-大血管内皮失调”，从而促进肝纤维化和肝硬化。 In isolated human cirrhotic liver CD45 - CD34 - CD31 + SECs, HDAC2 and DNMT1 expression levels were significantly up-regulated compared with healthy human livers (Fig. 1O). In human umbilical vein endothelial cells (HUVECs), knockdown of HDAC2 by shRNA (shHDAC2) up-regulated the expression of DNMT1 (FIG. 13C). These data suggest that aberrant activation of HDAC2 and DNMT1 in hepatic ECs may lead to "sinusoidal endothelial-macrovascular endothelial dysregulation", which promotes liver fibrosis and cirrhosis.
实施例四：联合靶向抑制HDAC2和DNMT1减轻小型猪NASH模型的肝脏纤维化Example 4: Combined targeted inhibition of HDAC2 and DNMT1 attenuates liver fibrosis in a minipig NASH model
小型猪生理学特点与人类相似，能够较好的模拟人的代谢紊乱。因此，实验人员构造了一种小型猪NASH模型，以探讨血管失调对肝硬化和相关机制的作用。文献报道的研究表明(参考文献66)，西方饮食(高脂肪、高胆固醇、高果糖和蔗糖饮食)和化学药品(CCl 4)损伤会诱导快速的纤维化发展的小鼠NASH。因此，实验人员采用所述西方饮食(WD)结合重复的CCl 4注射来诱导小型猪NASH模型。为了描述内皮来源的HDAC2/DNMT1在所述小型猪NASH模型中的病理学作用，实验人员还用HDAC2抑制剂(HDAC2i)和DNMT1抑制剂(DNMT1i)处理了小型猪(图2A-B)。与对照组相比，肝硬化组小型猪的血糖水平更高(图2C)，而且肝硬化组的肝纤维化指数和血清肝功能指数升高，这与人类NASH病理学类似(图2D-E)。与肝硬化小型猪组相比，HDAC2i+DNMT1i联合治疗降低了血糖、肝纤维化指数和血清肝功能指数(图2C-E)。另外，肝硬化组血清总胆固醇升高，而治疗后降低(图2E)。因此，在小型猪NASH模型中，ECs中HDAC2和DNMT1的异常诱导导致肝纤维化和肝功能损伤。 The physiological characteristics of minipigs are similar to those of humans, which can better simulate the metabolic disorders of humans. Therefore, we constructed a minipig model of NASH to explore the role of vascular dysregulation on cirrhosis and related mechanisms. Studies reported in the literature show (ref. 66) that Western diet (high fat, high cholesterol, high fructose and sucrose diet) and chemical ( CCl4 ) injury induces NASH in mice with rapid fibrosis development. Therefore, the experimenters employed the described Western diet (WD) in combination with repeated CCl4 injections to induce a minipig model of NASH. To characterize the pathological role of endothelial-derived HDAC2/DNMT1 in the minipig NASH model, we also treated minipigs with an HDAC2 inhibitor (HDAC2i) and a DNMT1 inhibitor (DNMT1i) (Figure 2A-B). Glucose levels were higher in minipigs in the cirrhosis group compared to the control group (Fig. 2C), and the cirrhosis group had elevated indices of fibrosis and serum liver function, which were similar to human NASH pathology (Fig. 2D-E). ). Compared with the cirrhotic minipig group, HDAC2i+DNMT1i combined treatment reduced blood glucose, liver fibrosis index and serum liver function index (Figure 2C-E). In addition, serum total cholesterol increased in the cirrhosis group, but decreased after treatment (Fig. 2E). Thus, in a minipig NASH model, aberrant induction of HDAC2 and DNMT1 in ECs resulted in liver fibrosis and liver function impairment.
接下来，实验人员通过H&E、天狼星红、I型胶原、油红O和Ki67染色评估了对照组、肝硬化组和治疗组的小型猪肝脏的组织病理学、胶原蛋白沉积、脂质滴沉积和细胞增殖(图2F-G)。与显示出正常的组织学特点的对照组相比，肝硬化动物的肝脏表现出独特的假小叶表型。WD+CCl 4诱导了肝硬化小型猪组的晚期肝硬化、胶原沉积和脂质沉积。HDAC2i+DNMT1i联合治疗改善了组织病理学表型，减少了肝硬化，并减轻了胶原蛋白沉积和脂质积聚。Ki67染色证明，HDAC2i+DNMT1i治疗也增加了肝细胞增殖。 Next, the experimenters assessed the histopathology, collagen deposition, lipid droplet deposition, and micropig livers in the control, cirrhosis, and treatment groups by staining with H&E, Sirius red, collagen type I, Oil Red O, and Ki67. Cell proliferation (Figure 2F-G). The livers of cirrhotic animals exhibited a distinct pseudolobular phenotype compared to controls that displayed normal histology. WD+CCl 4 induced advanced liver cirrhosis, collagen deposition and lipid deposition in the cirrhotic minipig group. Combined HDAC2i+DNMT1i treatment improved the histopathological phenotype, reduced cirrhosis, and attenuated collagen deposition and lipid accumulation. Ki67 staining demonstrated that HDAC2i+DNMT1i treatment also increased hepatocyte proliferation.
然后实验人员通过流式细胞术分离小型猪SECs(CD45 -CD34 -CD31 +)、MECs(CD45 -CD34 +CD31 +)和其他CD45 +NPCs。CD45 +NPCs中HDAC2和DNMT1的表达在不同组间无显著差异。与对照组相比，肝硬化小型猪肝脏的SECs和MECs中HDAC2和DNMT1的表达显著上调。HDAC2i+DNMT1i治疗阻断了肝硬化小型猪肝脏SECs和MECs中HDAC2和DNMT1表达的上调(图2H)。HDAC2i和DNMT1i对小型猪NASH模型的治疗效果意味着HDAC2/DNMT1的异常诱导在NASH中起致病作用(图2I)。 The experimenters then isolated minipig SECs (CD45 - CD34 - CD31 + ), MECs (CD45 - CD34 + CD31 + ) and other CD45 + NPCs by flow cytometry. The expressions of HDAC2 and DNMT1 in CD45 + NPCs were not significantly different between groups. The expressions of HDAC2 and DNMT1 were significantly up-regulated in SECs and MECs of cirrhotic minipig livers compared with controls. HDAC2i+DNMT1i treatment blocked the upregulation of HDAC2 and DNMT1 expression in cirrhotic minipig liver SECs and MECs (Fig. 2H). The therapeutic effects of HDAC2i and DNMT1i in the minipig NASH model imply that aberrant induction of HDAC2/DNMT1 plays a pathogenic role in NASH (Fig. 2I).
实施例五：小型猪NASH模型中联合表观遗传靶向抑制使失调的肝脏内皮分类正常Example 5: Combined Epigenetic Targeting Inhibition Normalizes Dysregulated Liver Endothelial Classification in a Minipig NASH Model
实验人员接下来分析了小型猪中HDAC2/DNMT1的异常诱导是否导致在人类肝硬化肝脏中发现的“窦内皮-大血管内皮失调”(图1P)。分离出小型猪(正常组、硬化组及治疗组)肝脏NPCs，并进行scRNA-seq，在单细胞水平上分析小型猪NASH模型(图3A)。1个对照组、2个肝硬化组和2个治疗组的40,570个NPCs聚类成28个群(图14A)。与人类scRNA-Seq类似，通过标记基因的表达鉴定了7个细胞系，包括T细胞、B细胞、EC、Mac、Neu、DC和EPCAM +细胞(图3B-C)。实验人员还发现，与健康肝脏相比，小型猪肝硬化肝脏中ECs的比例(9.70％对5.90％)明显增加，而这种增加的ECs的比例通过HDAC2i+DNMT1i的治疗而被明显降低(4.55％对9.70％)(图3D)。为了揭示HDAC2/DNMT1在不同NPCs细胞系中的影响，实验人员分别对肝硬化组和对照组(肝硬化vs.对照)以及治疗组和肝硬化组(治疗vs.肝硬化)的7个细胞系中的基因差异情况进行了比较。肝硬化小型猪组的血管内皮细胞的基因差异程度在所有检测的细胞类型中最大，其中大部分在HDAC2i+DNMT1i治疗后得以恢复(图3E)。GO和通路富集显示，参与分子功能(Molecular Function)、生物过程(Biological Process)和细胞组成(Cellular Component)的信号传导发生了显著变化(图14B)，以及Th17细胞分化、非酒精性脂肪 肝、趋化因子、TNF、HIF-1、MAPK、PI3K-Akt等KEGG信号通路也发生了显著变化(图3F)。此外，HDAC2i和DNMT2i的表观遗传治疗恢复了NASH组的肝脏ECs的大部分上述GO及KEGG变化，表明治疗后的小型猪ECs的正常化(图3F，图14B)。 We next analyzed whether aberrant induction of HDAC2/DNMT1 in minipigs led to the "sinusoidal-macrovascular endothelial dysregulation" found in human cirrhotic livers (Fig. 1P). Minipig (normal, cirrhotic, and treated) liver NPCs were isolated and subjected to scRNA-seq to analyze the minipig NASH model at the single-cell level (Fig. 3A). 40,570 NPCs from 1 control group, 2 cirrhosis groups and 2 treatment groups were clustered into 28 clusters (Figure 14A). Similar to human scRNA-Seq, seven cell lines were identified by the expression of marker genes, including T cells, B cells, EC, Mac, Neu, DC, and EPCAM + cells (Fig. 3B-C). The experimenters also found that the proportion of ECs (9.70% vs. 5.90%) in minipig cirrhotic livers was significantly increased compared with healthy livers, and this increased proportion of ECs was significantly reduced by HDAC2i+DNMT1i treatment (4.55%). % vs. 9.70%) (Fig. 3D). In order to reveal the effect of HDAC2/DNMT1 in different NPCs cell lines, the experimenters treated seven cell lines in cirrhosis group and control group (cirrhosis vs. control) and treatment group and cirrhosis group (treatment vs. cirrhosis), respectively. Gene differences were compared. The degree of genetic differentiation of vascular endothelial cells in the cirrhotic minipig group was greatest among all cell types examined, most of which were recovered after HDAC2i+DNMT1i treatment (Fig. 3E). GO and pathway enrichment showed significant changes in signaling involved in Molecular Function, Biological Process, and Cellular Component (Fig. 14B), as well as Th17 cell differentiation, nonalcoholic fatty liver disease , chemokine, TNF, HIF-1, MAPK, PI3K-Akt and other KEGG signaling pathways also changed significantly (Fig. 3F). Furthermore, epigenetic treatment of HDAC2i and DNMT2i restored most of the above-mentioned GO and KEGG changes in liver ECs of the NASH group, indicating normalization of minipig ECs after treatment (Fig. 3F, Fig. 14B).
在小型猪NASH模型中，在ECs的表观遗传变化的独特影响使实验人员探索了HDAC2/DNMT1活性与肝纤维化中“窦内皮-大血管内皮分类异常”之间的联系。小型猪ECs聚类成15个群(图15)，通过基因标记鉴定为SEC和MEC(图3G，图15B-C)。与人肝硬化肝脏的所述血管失调相似，与对照组小型猪肝脏相比，肝硬化小型猪肝脏的MEC比例增加而SEC比例减少。在治疗后的小型猪肝脏中，HDAC2i+DNMT1i治疗后明显逆转了NASH引起的“窦内皮-大血管内皮失调”(图3H)。In a minipig model of NASH, the unique impact of epigenetic changes in ECs led us to explore the link between HDAC2/DNMT1 activity and "abnormal sinus-macrovascular endothelial classification" in liver fibrosis. Minipig ECs clustered into 15 clusters (Figure 15), identified by genetic markers as SEC and MEC (Figure 3G, Figure 15B-C). Similar to the described vascular dysregulation in human cirrhotic livers, the proportion of MEC was increased and the proportion of SEC was decreased in cirrhotic minipig livers compared to control minipig livers. In treated minipig livers, HDAC2i + DNMT1i treatment significantly reversed the NASH-induced "sinusoidal endothelium-macrovascular endothelial dysregulation" (Fig. 3H).
在EC亚群中，SECs的差异基因数量相对最多(硬化组vs.对照组)，HDAC2i+DNMT1i治疗后大部分差异基因得以恢复(治疗组vs.硬化组)(图3I)。肝脏EC亚群拟时分析也表明肝硬化小型猪肝脏存在“窦内皮-大血管内皮失调”，HDAC2i+DNMT1i治疗使其正常化(图3J)。实验人员还发现在肝硬化小型猪中，SECs中组蛋白修饰和DNA甲基化相关基因变化相对最多，HDAC2i+DNMT1i治疗后，大多数基因得以恢复(图15D-F)。因此，小型猪NASH模型的数据表明，异常的表观遗传改变会刺激“窦内皮-大血管内皮失调”、内皮分类异常，并增加肝脏纤维化(图3K)。In the EC subgroup, the SECs had relatively the highest number of differential genes (sclerosis group vs. control group), and most of the differential genes were recovered after HDAC2i+DNMT1i treatment (treatment group vs. sclerosis group) (Fig. 3I). The temporal analysis of liver EC subsets also showed that there was "sinusoidal endothelial-macrovascular endothelial dysregulation" in cirrhotic minipig livers, which was normalized by HDAC2i+DNMT1i treatment (Fig. 3J). The experimenters also found that in cirrhotic minipigs, histone modification and DNA methylation-related genes changed relatively most in SECs, and most genes were restored after HDAC2i+DNMT1i treatment (Fig. 15D-F). Thus, data from the minipig NASH model suggest that aberrant epigenetic alterations stimulate "sinus endothelium-macroendothelial dysregulation," abnormal endothelial classification, and increased liver fibrosis (Fig. 3K).
实施例六：人类患者和小型猪NASH模型中表观遗传失调的SECs发生旁分泌/血管分泌因子重编程Example 6: Reprogramming of paracrine/vascular secretory factors in epigenetically dysregulated SECs in human patients and minipig NASH models
ECs可以通过旁分泌/血管分泌因子与周围细胞相互作用来调节肝脏再生。为了解析表观遗传失调的肝脏ECs促纤维化的机制，实验人员在单细胞水平上分析了肝硬化人和小型猪ECs中差异基因(图16A)，并观察到与“窦内皮-大血管内皮失调”相关的血管分泌因子重编程(图4A-D)。与健康ECs比较，肝硬化人肝脏ECs中金属肽酶ADAMTS1、胰岛素样生长因子结合蛋白7(IGFBP7)、DLL1、ADAMTS6均发生明显变化(图4A)。利用人类公共数据库分析了这些血管分泌因子与人纤维化进展的关系。在F2-F4级患者的纤维化肝脏中，IGFBP7和ADAMTS1的表达逐渐升高(图4B)。实验人员发现，在血管分泌因子基因中，IGFBP7和ADAMTS1在人和小型猪ECs中的特异性表达在相对最高水平(图4C，图16B-C)。此外，与健康ECs相比，IGFBP7和ADAMTS1在人肝硬化肝脏ECs中选择性上调，并且肝硬化SECs中的IGFBP7和ADAMTS1的mRNA及蛋白水平显著升高(图4E-F)。这些结果提示在失调的人肝脏ECs中发生血管分泌因子表观遗传重编程，并且IGFBP7和ADMATS1的内皮生成可能促进肝纤维化和肝硬化。ECs can modulate liver regeneration by interacting with surrounding cells via paracrine/vascular secretory factors. To elucidate the mechanism by which epigenetically dysregulated liver ECs promote fibrosis, we analyzed differential genes in cirrhotic and minipig ECs at the single-cell level (Fig. dysregulation" associated with reprogramming of vasosecreted factors (Fig. 4A-D). Compared with healthy ECs, the metallopeptidase ADAMTS1, insulin-like growth factor-binding protein 7 (IGFBP7), DLL1, and ADAMTS6 were significantly changed in cirrhotic human liver ECs (Fig. 4A). The association of these angiosecreted factors with human fibrosis progression was analyzed using public human databases. The expression of IGFBP7 and ADAMTS1 was gradually increased in the fibrotic livers of F2-F4 patients (Fig. 4B). The experimenters found that among the vascular secretory factor genes, IGFBP7 and ADAMTS1 were specifically expressed at relatively highest levels in human and minipig ECs (Fig. 4C, Fig. 16B-C). Furthermore, IGFBP7 and ADAMTS1 were selectively upregulated in human cirrhotic liver ECs compared with healthy ECs, and the mRNA and protein levels of IGFBP7 and ADAMTS1 were significantly increased in cirrhotic SECs (Fig. 4E-F). These results suggest that epigenetic reprogramming of angiogenic factors occurs in dysregulated human liver ECs, and that endothelial production of IGFBP7 and ADMATS1 may contribute to liver fibrosis and cirrhosis.
在小型猪NASH模型中，肝硬化的SECs和MECs中IGFBP7和ADAMTS1的mRNA水平明显升高，而IGFBP7和ADAMTS1的蛋白水平仅在肝硬化的SECs中上调。此外，HDAC2i和DNMT1i的治疗阻断了肝硬化小型猪肝脏SECs中IGFBP7和ADAMTS1的mRNA和蛋白水平的增加(图4G-L，图16D)。分析染色质开放性有助于揭示基因表达的表观遗传调控，因此实验人员用对照组、肝硬化组和治疗组的小型猪肝脏ECs进行染色质开放性检测(ATAC-seq)。与对照组ECs相比，肝硬化ECs中IGFBP7和ADAMTS1启动子的染色质开放性明显增强，并且HDAC2i+DNMT1i治疗后其染色质开放性明显逆转(图4M)。因此，IGFBP7和ADAMTS1上调可能在肝硬化肝脏中区分正常的SECs和失调的SECs，而表观遗传靶向促纤维化的IGFBP7 +ADAMTS1 +失调的EC亚群的表观遗传治疗可能阻断肝纤维化(图4N)。 In the minipig NASH model, the mRNA levels of IGFBP7 and ADAMTS1 were significantly increased in cirrhotic SECs and MECs, whereas the protein levels of IGFBP7 and ADAMTS1 were only up-regulated in cirrhotic SECs. Furthermore, HDAC2i and DNMT1i treatment blocked the increase in mRNA and protein levels of IGFBP7 and ADAMTS1 in cirrhotic minipig liver SECs (Fig. 4G-L, Fig. 16D). Analyzing chromatin openness helps reveal epigenetic regulation of gene expression, so we performed assays for chromatin openness (ATAC-seq) in minipig liver ECs from control, cirrhosis, and treatment groups. Compared with control ECs, the chromatin openness of IGFBP7 and ADAMTS1 promoters was significantly enhanced in cirrhotic ECs, and their chromatin openness was significantly reversed after HDAC2i+DNMT1i treatment (Fig. 4M). Thus, IGFBP7 and ADAMTS1 upregulation may discriminate between normal and dysregulated SECs in cirrhotic livers, and epigenetic therapy targeting the profibrotic IGFBP7 + ADAMTS1 + dysregulated EC subset may block liver fibrosis ionization (Fig. 4N).
实施例七：来自失调的SECs的IGFBP7和ADAMTS1预测人和小型猪的肝纤维化的进展Example 7: IGFBP7 and ADAMTS1 from dysregulated SECs predict progression of liver fibrosis in humans and minipigs
为了确定循环IGFBP7和ADAMTS1在人肝硬化/肝纤维化或NASH中的临床价值，实验人员评估了人类患者血浆中IGFBP7和ADAMTS1的浓度(表S2)。肝硬化/肝纤维化患者的IGFBP7、ADAMTS1、谷丙转氨酶(ALT)和谷草转氨酶(AST)的血浆浓度明 显高于健康人样本(图5A)。由于一些肝硬化/肝纤维化患者可以有正常的血浆ALT或AST水平，因此需要发现敏感的生物标记物在临床上诊断肝脏肝硬化/肝纤维化。因此，实验人员评估了血浆IGFBP7和ADAMTS1作为临床生物标记物的价值。根据肝硬化/肝纤维化患者的血浆肝功能指数，把他们分为两组：正常血浆ALT和AST浓度组和异常血浆ALT和AST浓度组(图5B)。比较肝功能正常和异常的肝硬化/肝纤维化患者IGFBP7和ADAMTS1的血浆浓度。重要的是，ALT或AST正常的肝硬化/肝纤维化患者的IGFBP7和ADAMTS1血浆浓度明显高于健康人(图5C)。肝硬化/肝纤维化队列包括非酒精性脂肪性肝炎相关的肝硬化/肝纤维化(NASH)，乙型肝炎相关性肝硬化/肝纤维化(HBC)，自身免疫性肝炎相关性肝硬化/肝纤维化(AIH)，原发性胆汁性肝硬化/肝纤维化(PBC)和隐源性肝硬化/肝纤维化(CC)。与健康人相比，所有这些患者的血浆IGFBP7和ADAMTS1水平均观察到升高(图5D)。因此，在没有肝功能障碍的情况下，血管分泌的IGFBP7和ADAMTS1可能作为用于诊断肝硬化/肝纤维化的生物标记物。To determine the clinical value of circulating IGFBP7 and ADAMTS1 in human cirrhosis/fibrosis or NASH, we assessed plasma concentrations of IGFBP7 and ADAMTS1 in human patients (Table S2). Plasma concentrations of IGFBP7, ADAMTS1, alanine aminotransferase (ALT), and aspartate aminotransferase (AST) were significantly higher in cirrhosis/fibrosis patients than in healthy human samples (Figure 5A). Since some patients with cirrhosis/fibrosis can have normal plasma ALT or AST levels, there is a need to discover sensitive biomarkers for clinical diagnosis of liver cirrhosis/fibrosis. Therefore, the experimenters assessed the value of plasma IGFBP7 and ADAMTS1 as clinical biomarkers. Patients with cirrhosis/fibrosis were divided into two groups according to their plasma liver function index: normal plasma ALT and AST concentrations and abnormal plasma ALT and AST concentrations (Fig. 5B). Comparison of plasma concentrations of IGFBP7 and ADAMTS1 in cirrhosis/fibrosis patients with normal and abnormal liver function. Importantly, cirrhotic/fibrotic patients with normal ALT or AST had significantly higher plasma concentrations of IGFBP7 and ADAMTS1 than healthy subjects (Fig. 5C). The cirrhosis/fibrosis cohort included nonalcoholic steatohepatitis-associated cirrhosis/fibrosis (NASH), hepatitis B-associated cirrhosis/fibrosis (HBC), autoimmune hepatitis-associated cirrhosis/ Liver fibrosis (AIH), primary biliary cirrhosis/fibrosis (PBC) and cryptogenic cirrhosis/fibrosis (CC). Elevated plasma levels of IGFBP7 and ADAMTS1 were observed in all of these patients compared to healthy subjects (Fig. 5D). Therefore, vascular-secreted IGFBP7 and ADAMTS1 may serve as biomarkers for the diagnosis of cirrhosis/fibrosis in the absence of liver dysfunction.
接下来，实验人员评估了血浆IGFBP7和ADAMTS1是否可以用作诊断标记物，以预测NASH的严重程度或区分NASH与单纯性脂肪肝(表现为单纯性脂肪变性)。根据肝纤维化阶段将NASH患者分为不同的组。早期阶段NASH(F0-F1)患者的血浆ALT或AST浓度没有明显增加(图5E)。相比之下，NASH患者血浆IGFBP7和ADAMTS1浓度显著升高(图5F)。此外，单纯性脂肪肝患者的血浆IGFBP7和ADAMTS1浓度在统计学上没有升高，这表明血浆IGFBP7/ADAMTS1浓度在区分NASH和单纯性脂肪肝中有重要的临床价值(图5F)。Next, the experimenters assessed whether plasma IGFBP7 and ADAMTS1 could be used as diagnostic markers to predict the severity of NASH or to differentiate NASH from simple fatty liver disease (manifested as simple steatosis). NASH patients were divided into different groups according to the stage of liver fibrosis. Plasma ALT or AST concentrations were not significantly increased in patients with early stage NASH (F0-F1) (Fig. 5E). In contrast, plasma concentrations of IGFBP7 and ADAMTS1 were significantly elevated in NASH patients (Fig. 5F). Furthermore, plasma IGFBP7 and ADAMTS1 concentrations were not statistically elevated in patients with simple fatty liver, suggesting that plasma IGFBP7/ADAMTS1 concentrations have important clinical value in distinguishing NASH from simple fatty liver (Figure 5F).
IGFBP7/ADAMTS1与NASH进展的相关性提出了一种可能性，即失调的SECs的促纤维化作用取决于血管分泌的IGFBP7和ADAMTS1。然后实验人员评估了表观遗传失调的SECs释放IGFBP7或ADAMTS1以有助于NASH的假说。细胞外囊泡(EVs)是细胞释放的独特的脂质双层颗粒。包装在EVs中的分子可能在许多生物过程中促进细胞通讯。因此，实验人员分析了内皮细胞产生的IGFBP7和ADAMTS1是否在EVs中组装并释放到循环中。为此，通过超速离心法从人和小型猪血浆中提取EVs，并通过电子显微镜和免疫印迹分析对其进行验证(图5G)。肝硬化组小型猪和人EVs中的IGFBP7和ADAMTS1的浓度高于对照组。在小型猪NASH模型中，HDAC2i+DNMT1i治疗可降低肝硬化组EV IGFBP7/ADAMTS1的浓度的升高(图5H)。ALT/AST正常的肝硬化/肝纤维化患者或NASH患者EVs中的IGFBP7/ADAMTS1浓度也比健康人类明显升高(图5I-J)。本发明的数据表明EVs中的IGFBP7和ADAMTS1是评估在肝功能障碍之前的NASH进展的有用的生物标记物(图5K)。The association of IGFBP7/ADAMTS1 with NASH progression raises the possibility that the profibrotic effects of dysregulated SECs depend on vascularly secreted IGFBP7 and ADAMTS1. The experimenters then evaluated the hypothesis that epigenetically dysregulated SECs release IGFBP7 or ADAMTS1 to contribute to NASH. Extracellular vesicles (EVs) are unique lipid bilayer particles released by cells. Molecules packaged in EVs may facilitate cellular communication in many biological processes. We therefore analyzed whether endothelial cell-produced IGFBP7 and ADAMTS1 are assembled in EVs and released into the circulation. To this end, EVs were extracted from human and minipig plasma by ultracentrifugation and validated by electron microscopy and immunoblot analysis (Fig. 5G). The concentrations of IGFBP7 and ADAMTS1 in minipigs and human EVs in the cirrhosis group were higher than those in the control group. In the minipig NASH model, HDAC2i + DNMT1i treatment reduced the elevation of EV IGFBP7/ADAMTS1 concentrations in the liver cirrhosis group (Fig. 5H). IGFBP7/ADAMTS1 concentrations were also significantly higher in EVs from cirrhotic/fibrotic patients or NASH patients with normal ALT/AST than in healthy humans (Figure 5I-J). The present data suggest that IGFBP7 and ADAMTS1 in EVs are useful biomarkers for assessing NASH progression prior to liver dysfunction (Figure 5K).
实施例八：肝硬化患者和小型猪NASH模型中IGFBP7 +ADAMTS1 +失调的SECs诱导促纤维化的Th17细胞应答 Example 8: IGFBP7 + ADAMTS1 + dysregulated SECs induce pro-fibrotic Th17 cell responses in liver cirrhosis patients and a minipig NASH model
失调的ECs可以通过形成失调的血管微环境与相邻细胞相互作用以促进纤维化。本发明试图揭示IGFBP7 +ADAMTS1 +失调的SECs通过细胞通讯增强肝纤维化的细胞机制。基于CellPhoneDB数据库分析了不同NPCs细胞系的受体和配体表达谱。细胞相互作用预测表明，在肝硬化患者和NASH小型猪中，失调的EC与T细胞的相互作用明显(图6A)。值得注意的是，尽管在人类数据中预测的相互作用是在ECs和巨噬细胞之间互作明显，但在小型猪NPCs中EC-巨噬细胞的相互作用较少。由于T细胞是相对最丰富的肝脏NPCs类型，因此本发明主要分析ECs和T细胞之间的相互作用。 Dysregulated ECs can interact with neighboring cells to promote fibrosis by forming a dysregulated vascular microenvironment. The present invention seeks to reveal the cellular mechanism by which IGFBP7 + ADAMTS1 + deregulated SECs enhance liver fibrosis through cellular communication. The receptor and ligand expression profiles of different NPCs cell lines were analyzed based on the CellPhoneDB database. Cell interaction predictions indicated that dysregulated ECs interacted with T cells significantly in cirrhotic patients and NASH minipigs (Fig. 6A). Notably, while the predicted interactions in the human data were evident between ECs and macrophages, there were less EC-macrophage interactions in minipig NPCs. Since T cells are the relatively most abundant type of liver NPCs, the present invention mainly analyzes the interaction between ECs and T cells.
已有的研究表明，在IGFBP7、ADAMTS1和转化生长因子-β1(TGF-β1)之间存在协同(synergistic)或补充(complementary)作用(参考文献77、78)，该作用可以募集Th17细胞，其是参与NASH和肝纤维化进展(参考文献80、81)的一个CD4 +T亚群(参考文献79)。由于Smad2是TGF-β1诱导Th17细胞中重要的下游因子，因此实验人员分析了人肝脏CD45 +NPCs中Smad2的磷酸化。与健康NPCs相比，肝硬化人类CD45 +NPCs 中Smad2磷酸化水平显著升高(图6B)。接下来实验人员分析了募集的人类和小型猪T细胞的细胞系。2个肝硬化和2个健康肝脏的人类T细胞聚类并鉴定成20个群(图17A)，定义为CD4 +和CD8 +T细胞(图17B)。CD4 +T细胞进一步聚类(图6C)，Th17细胞被Th17 +标记基因标记出(图6D)。肝硬化患者肝脏中Th17细胞的细胞数明显高于健康肝脏(图6E)。为了验证人类患者的结果，实验人员分析了GSE136103数据中的Th17细胞(图17C-I)。与健康人类肝脏相比，肝硬化人类肝脏中的Th17细胞数量也类似地明显增加。这些数据暗示了Th17细胞在人类肝硬化中的促纤维化作用。 Previous studies have shown that there is a synergistic or complementary effect between IGFBP7, ADAMTS1 and transforming growth factor-β1 (TGF-β1) (refs 77, 78), which can recruit Th17 cells, which is a CD4 + T subset (ref. 79) involved in the progression of NASH and liver fibrosis (refs. 80, 81). Since Smad2 is an important downstream factor in TGF-β1-induced Th17 cells, we analyzed the phosphorylation of Smad2 in human liver CD45 + NPCs. Smad2 phosphorylation levels were significantly elevated in cirrhotic human CD45 + NPCs compared to healthy NPCs (Fig. 6B). The experimenters next analyzed cell lines of recruited human and minipig T cells. Human T cells from 2 cirrhotic and 2 healthy livers were clustered and identified into 20 populations (FIG. 17A), defined as CD4 + and CD8 + T cells (FIG. 17B). CD4 + T cells were further clustered (Fig. 6C) and Th17 cells were marked by Th17 + marker genes (Fig. 6D). The number of Th17 cells in the livers of patients with cirrhosis was significantly higher than that in healthy livers (Fig. 6E). To validate the results in human patients, the experimenters analyzed Th17 cells in the GSE136103 data (Figure 17C-I). The number of Th17 cells was similarly significantly increased in cirrhotic human livers compared to healthy human livers. These data implicate a profibrotic role of Th17 cells in human cirrhosis.
接下来实验人员探讨了小型猪NASH模型中肝ECs和Th17亚群之间的细胞互作。与人类T细胞相似，对照组、肝硬化组和治疗组的小型猪肝脏的T细胞聚类鉴定16个群，定义为CD4 +T细胞和CD8 +T细胞(图6F，图17J)。CD4 +T细胞中，Th17细胞被Th17 +标记基因鉴定出(图6G-H)。与对照组小型猪相对，在硬化小型猪肝脏中Th17细胞数也明显增加。此外，HDAC2i+DNMT1i的联合治疗阻断了在损伤的小型猪肝脏中Th17细胞数的增加(图6I)。实验人员还发现，与肝硬化小型猪相比，治疗组小型猪的Th17细胞中纤维化相关基因的表达显著降低(图6J)。根据本发明的数据，提出假说即表观遗传失调的SECs可能募集并激活人和小型猪肝脏中促纤维化的Th17细胞，其可能由分泌的IGFBP7/ADAMTS1介导(图6K)。 We next explored the cellular interactions between hepatic ECs and Th17 subsets in a minipig NASH model. Similar to human T cells, T cell clustering of minipig livers in the control, cirrhosis and treatment groups identified 16 populations, defined as CD4 + T cells and CD8 + T cells (Fig. 6F, Fig. 17J). Among CD4 + T cells, Th17 cells were identified by Th17 + marker genes (Fig. 6G-H). The number of Th17 cells was also significantly increased in the cirrhotic minipig livers compared to control minipigs. Furthermore, combined treatment of HDAC2i+DNMT1i blocked the increase in Th17 cell numbers in injured minipig livers (Fig. 6I). The experimenters also found that the expression of fibrosis-related genes in Th17 cells of the treated minipigs was significantly reduced compared with that of the cirrhotic minipigs (Fig. 6J). Based on the present data, it was hypothesized that epigenetically dysregulated SECs might recruit and activate pro-fibrotic Th17 cells in human and minipig livers, possibly mediated by secreted IGFBP7/ADAMTS1 (Figure 6K).
实施例九：小鼠NASH模型中表观遗传失调的SECs产生促纤维化的Th17应答Example 9: Epigenetically dysregulated SECs in a mouse model of NASH generate pro-fibrotic Th17 responses
为了确定ECs中HDAC2/DNMT1-IGFBP7/ADAMTS1轴对Th17细胞应答和肝纤维化的功能上的作用，实验人员生成了Hdac2 iΔEC小鼠(选择性敲除ECs中HDAC2)，并通过WD+CCl 4诱导生成小鼠NASH模型。通过用DNMT1抑制剂AZA处理Hdac2 iΔEC小鼠(Hdac2 iΔEC+AZA)获得联合靶向抑制HDAC2+DNMT1的效果(图7A)。DNMT1i治疗后的Hdac2 iΔEC小鼠，与对照组损伤小鼠相比，肝纤维化、炎症、胶原沉积以及肝纤维和肝功能的指数显著降低(图7B-C)。为了进一步研究小鼠肝SECs中内皮来源的HDAC2与DNMT1之间的交互调控，实验人员通过免疫磁珠(Dynabeads)分离出对照组、Hdac2 iΔEC和Hdac2 iΔEC+AZA小鼠的肝SECs(CD45 -CD34 -CD31 +)。Western blot显示，敲除小鼠肝脏SECs中的Hdac2上调了SECs中DNMT1的表达(图7D)。值得注意的是，在这种小鼠NASH模型中，发现CD34在一些窦内皮细胞中表达，并且治疗组中靶向抑制内皮来源的HDAC2和DNMT1降低了CD34的表达(图7E-F)。这一染色结果提示，在这种小鼠NASH模型中存在“窦内皮-大血管内皮失调”。流式分析显示，治疗组小鼠肝脏中靶向抑制内皮来源的HDAC2和DNMT1减少了Th17细胞数(图7G)。因此，在小鼠NASH模型中，表观遗传失调的肝脏ECs可能激活促纤维化的Th17应答。 To determine the functional role of the HDAC2/DNMT1-IGFBP7/ADAMTS1 axis in ECs on Th17 cell responses and hepatic fibrosis, we generated Hdac2 iΔEC mice (selective knockout of HDAC2 in ECs), and treated them with WD+CCl 4 Induction of a mouse model of NASH. The combined targeted inhibition of HDAC2+DNMT1 was obtained by treating Hdac2 iΔEC mice (Hdac2 iΔEC + AZA) with the DNMT1 inhibitor AZA (Fig. 7A). In Hdac2 iΔEC mice treated with DNMT1i, liver fibrosis, inflammation, collagen deposition, and indices of liver fibrosis and liver function were significantly reduced compared with control-injured mice (Fig. 7B-C). To further investigate the interaction regulation between endothelial-derived HDAC2 and DNMT1 in mouse liver SECs, we isolated liver SECs (CD45 - CD34 - CD31 + ). Western blot revealed that knockout of Hdac2 in mouse liver SECs upregulated the expression of DNMT1 in SECs (Fig. 7D). Notably, in this mouse model of NASH, CD34 was found to be expressed in some sinus endothelial cells, and targeted inhibition of endothelial-derived HDAC2 and DNMT1 decreased CD34 expression in the treatment group (Figure 7E-F). This staining result suggests the presence of "sinus endothelium-macrovascular endothelial dysregulation" in this mouse model of NASH. Flow cytometry revealed that targeted inhibition of endothelial-derived HDAC2 and DNMT1 in the livers of treated mice reduced Th17 cell numbers (Fig. 7G). Thus, epigenetically dysregulated liver ECs may activate pro-fibrotic Th17 responses in a mouse model of NASH.
实施例十：小鼠NASH模型中IGFBP7增强促纤维化的Th17应答Example 10: IGFBP7 enhances pro-fibrotic Th17 responses in a mouse NASH model
qPCR显示联合靶向抑制内皮来源的HDAC2和DNMT1降低了纤维化肝脏ECs中IGFBP7的表达(图8A)。为了确定肝纤维化中IGFBP7在刺激Th17应答中的作用，实验人员分析了IGFBP7敲除(IGFBP7 -/-)小鼠的NASH表型(图8B)。小鼠中Igfbp7的基因敲除明显减轻了肝纤维化反应即胶原沉积(图8C)、血清肝功指数、肝脏羟脯氨酸含量(图8D)和Th17应答(图8E)。为了进一步研究IGFBP7是否直接影响Th17生物学，将C57BL/6J小鼠尾静脉注射重组小鼠IGFBP7蛋白(图8F)。与对照组相比，升高的IGFBP7水平显著增强了损伤的小鼠肝脏的促纤维化的Th17应答(图8G)。这些结果表明，IGFBP7是增强Th17应答以促进肝脏纤维化的调节因子。 qPCR showed that combined targeting of endothelial-derived HDAC2 and DNMT1 reduced IGFBP7 expression in fibrotic liver ECs (Figure 8A). To determine the role of IGFBP7 in stimulating Th17 responses in liver fibrosis, we analyzed the NASH phenotype of IGFBP7 knockout (IGFBP7 -/- ) mice (Fig. 8B). Knockout of Igfbp7 in mice significantly attenuated liver fibrotic responses, namely collagen deposition (FIG. 8C), serum liver function index, liver hydroxyproline content (FIG. 8D) and Th17 responses (FIG. 8E). To further investigate whether IGFBP7 directly affects Th17 biology, C57BL/6J mice were tail vein injected with recombinant mouse IGFBP7 protein (Fig. 8F). Elevated IGFBP7 levels significantly enhanced the pro-fibrotic Th17 response in injured mouse livers compared to controls (Fig. 8G). These results suggest that IGFBP7 is a regulator that enhances Th17 responses to promote liver fibrosis.
实施例十一：小鼠纤维化模型中ADAMTS1的遗传失活减轻促纤维化的Th17应答Example 11: Genetic inactivation of ADAMTS1 in a mouse fibrosis model attenuates pro-fibrotic Th17 responses
与IGFBP7表达相似，实验人员发现ADAMTS1表达在Hdac2 iΔEC+AZA小鼠的SECs中明显降低(图9A)。此外，通过ADAMTS1shRNA(shADAMTS1)基因敲低人ECs中的ADAMTS1，阻断了TGF-β刺激下Smad2的磷酸化(图9B)。因此，实验人员利用“人 到小鼠”细胞外囊泡(EVs)移植方法(图9C)来研究内皮来源的ADAMTS1是否调节Th17生物学。从shADAMTS1和对照(shNC)感染的HUVECs的培养基中分离EVs，并通过尾静脉移植到小鼠中。与用对照EVs处理的小鼠相比，移植缺乏ADAMTS1的EVs的小鼠的肝纤维化和Th17应答显著降低(图9D-E)。这些结果暗示，在肝脏纤维化进程中ADAMTS1在促进Th17应答的功能作用。 Similar to IGFBP7 expression, we found that ADAMTS1 expression was significantly reduced in SECs of Hdac2 iΔEC + AZA mice (Fig. 9A). Furthermore, knockdown of ADAMTS1 in human ECs by ADAMTS1 shRNA (shADAMTS1) gene blocked the phosphorylation of Smad2 stimulated by TGF-β (Fig. 9B). Therefore, we used a "human-to-mouse" extracellular vesicle (EVs) transplantation method (Fig. 9C) to investigate whether endothelial-derived ADAMTS1 regulates Th17 biology. EVs were isolated from the culture medium of shADAMTS1 and control (shNC) infected HUVECs and transplanted into mice via the tail vein. Liver fibrosis and Th17 responses were significantly reduced in mice transplanted with ADAMTS1-deficient EVs compared to mice treated with control EVs (Figure 9D-E). These results implicate a functional role of ADAMTS1 in promoting Th17 responses in the progression of liver fibrosis.
本发明数据揭示了人类患者、小型猪和小鼠NASH模型中促进肝纤维化的内皮来源的HDAC2/DNMT1-IGFBP7/ADAMTS1-Th17轴。肝脏ECs亚群的表观遗传异常互作导致“窦内皮-大血管内皮失调”，其特点是内皮分类异常和失调的SECs产生促纤维化的IGFBP7/ADAMTS1，通过细胞外囊泡募集Th17细胞，形成促纤维化的血管微环境(图9F)。The present data reveal an endothelial-derived HDAC2/DNMT1-IGFBP7/ADAMTS1-Th17 axis that promotes liver fibrosis in human patient, minipig, and mouse models of NASH. Epigenetic aberrant interactions in subsets of hepatic ECs lead to "sinusoidal endothelium-macrovascular endothelial dysregulation," characterized by dysregulated and dysregulated SECs that produce profibrotic IGFBP7/ADAMTS1, which recruit Th17 cells through extracellular vesicles, A pro-fibrotic vascular microenvironment was formed (Fig. 9F).
总结Summarize
NASH的发病机制涉及包括代谢功能障碍在内的系统性影响。循环系统中内皮细胞和造血细胞与系统性刺激直接联系，NASH与循环/血管并发症的许多危险因素相似。本发明利用多组学分析，揭示了血管特异性表观遗传改变如何在单细胞水平上对肝脏循环系统(血管和造血细胞)中的促纤维化的交互调控进行重编程。本发明整合了人和大型动物NASH模型的scRNA-Seq数据，揭示“窦内皮-大血管内皮失调”是如何刺激NASH中的促纤维化的Th17细胞应答。此外，本发明发现，NASH中，肝ECs中异常的HDAC2-DNMT1交互调控会导致内皮细胞分类异常并产生失调的IGFBP +ADAMTS1 +肝EC亚群，以在循环系统中形成促纤维化的失调血管微环境。本发明采用从病床到实验室(bed to benchside)的方法，系统地研究了人类患者和补充的大型动物和啮齿动物的NASH模型的细胞表型，这种多物种探究策略阐明了在血管微环境的异常的表观遗传交互调控是如何导致内皮细胞和免疫细胞之间的促纤维化的通讯。 The pathogenesis of NASH involves systemic effects including metabolic dysfunction. Endothelial and hematopoietic cells in the circulatory system are directly linked to systemic stimulation, and NASH is similar to many risk factors for circulatory/vascular complications. The present invention utilizes multi-omics analysis to reveal how vessel-specific epigenetic alterations reprogram pro-fibrotic cross-regulation in the hepatic circulatory system (vascular and hematopoietic cells) at the single-cell level. The present invention integrates scRNA-Seq data from human and large animal NASH models to reveal how "sinus endothelium-macrovascular endothelial dysregulation" stimulates pro-fibrotic Th17 cell responses in NASH. Furthermore, the present inventors found that in NASH, abnormal HDAC2-DNMT1 cross-regulation in hepatic ECs leads to abnormal endothelial cell sorting and generation of dysregulated IGFBP + ADAMTS1 + hepatic EC subsets to form pro-fibrotic dysregulated vessels in the circulatory system Microenvironment. The present invention employs a bed to benchside approach to systematically study the cellular phenotypes of human patients and complementary large animal and rodent models of NASH. How aberrant epigenetic cross-regulation leads to pro-fibrotic communication between endothelial and immune cells.
本发明表明，在人和大型动物NASH模型中，血管内皮细胞的异常的表观遗传交互调控促进肝脏纤维化。整合scRNA-Seq、组蛋白修饰和人类患者队列分析，本发明实验人员发现肝血管ECs的特定亚群中HDAC2和DNMT1的选择性诱导与肝纤维化的进展密切相关。大型动物和啮齿动物NASH模型的HDAC2/DNMT1的药理学和遗传学的靶向进一步揭示，失调的EC亚群中的HDAC2/DNMT1交互调控会刺激细胞外囊泡中促纤维化的IGFBP7和ADAMTS1的产生，从而募集Th17细胞，在NASH中抑制肝脏再生和诱导纤维化。The present invention demonstrates that abnormal epigenetic cross-regulation of vascular endothelial cells promotes liver fibrosis in human and large animal NASH models. By integrating scRNA-Seq, histone modifications, and human patient cohort analysis, the present inventors found that the selective induction of HDAC2 and DNMT1 in specific subsets of hepatic vascular ECs is closely related to the progression of liver fibrosis. Pharmacological and genetic targeting of HDAC2/DNMT1 in large animal and rodent NASH models further revealed that HDAC2/DNMT1 reciprocal regulation in dysregulated EC subsets stimulates the activation of profibrotic IGFBP7 and ADAMTS1 in extracellular vesicles produced, thereby recruiting Th17 cells, inhibiting liver regeneration and inducing fibrosis in NASH.
实验人员首先在单细胞水平上分析了人类患者NPCs中内皮细胞和造血细胞的表型和分子特征。在检验的NPCs中，血管ECs表观遗传相关基因变化最大，这表明，在NASH等慢性疾病中，ECs可能更容易受到表观遗传改变的影响。与造血细胞相比，内皮细胞可能在循环系统中有更长的体内半衰期，从而积累更多的微环境或系统性刺激(如代谢应激)。scRNA-Seq揭示，在NASH中，ECs的选择性表观遗传改变导致“窦内皮-大血管内皮失调”及内皮分类异常，形成促纤维化的内皮微环境。通过多组学和多物种分析，本发明的实验人员在失调的肝脏ECs亚群中发现了促纤维化的HDAC2/DNMT1-IGFBP7/ADAMTS1轴。先前的报道显示，肝纤维化会导致SECs毛细血管化并改变肝窦中的血流(参考文献22、23、57)。应当指出的是，肝脏ECs是NPCs的主要成分，据报道约占NPCs的5％-15％。根据个体差异和特定的分离方法不同，纯化的NPCs中ECs的比例在不同的研究和个体之间似乎有所不同。在本发明中，对肝脏ECs的scRNA-Seq分析表明，在NASH中，对ECs的慢性/代谢性损伤以及由此产生的表观遗传修饰异常促进了肝纤维化。在NASH中，异质的肝血管系统中组蛋白和DNA修饰异常可能诱导肝脏ECs的失调，并且这些失调的ECs亚群进一步与其它循环细胞如Th17细胞相互作用，共同形成促进肝脏纤维化的微环境。We first analyzed the phenotypic and molecular characteristics of endothelial and hematopoietic cells in human patient NPCs at the single-cell level. Among the NPCs examined, vascular ECs showed the greatest changes in epigenetically related genes, suggesting that ECs may be more susceptible to epigenetic alterations in chronic diseases such as NASH. Compared with hematopoietic cells, endothelial cells may have longer in vivo half-lives in the circulatory system, thereby accumulating more microenvironmental or systemic stimuli (eg, metabolic stress). scRNA-Seq revealed that in NASH, selective epigenetic alterations in ECs lead to "sinus endothelium-macrovascular endothelial dysregulation" and abnormal endothelial classification, resulting in a pro-fibrotic endothelial microenvironment. Through multi-omics and multi-species analysis, the present inventors discovered a pro-fibrotic HDAC2/DNMT1-IGFBP7/ADAMTS1 axis in a subpopulation of dysregulated liver ECs. Previous reports have shown that hepatic fibrosis leads to capillary vascularization of SECs and altered blood flow in hepatic sinusoids (refs 22, 23, 57). It should be noted that liver ECs are the major constituents of NPCs and are reported to account for approximately 5%-15% of NPCs. The proportion of ECs in purified NPCs appears to vary between studies and individuals, depending on individual differences and specific isolation methods. In the present invention, scRNA-Seq analysis of hepatic ECs showed that chronic/metabolic damage to ECs and the resulting abnormal epigenetic modification promotes liver fibrosis in NASH. In NASH, aberrant histone and DNA modifications in the heterogeneous hepatic vasculature may induce dysregulation of hepatic ECs, and these dysregulated ECs subsets further interact with other circulating cells such as Th17 cells to form microscopic mechanisms that promote liver fibrosis. surroundings.
scRNA-Seq分析能够在单细胞水平上揭示肝硬化肝脏中的血管失调。人类患者和小型猪NASH模型的分析表明，异常的表观遗传交互调控会导致从窦内皮到大血管内皮的 失调，从而导致内皮分类的异常。在检测的肝脏样本中，观察到的血管失调似乎主要表现为ECs谱系内的表型和功能性转变。所检测的纤维化肝脏表现出ECs数量增加，并且在EC群体中“间质细胞分化”的富集度较低。因此，这种血管失调过程似乎与内皮间质转化(EndMT)不同。从治疗上讲，联合靶向抑制异常的HDAC2/DNMT1交互调控使内皮分类及肝脏功能(AST和ALT血浆水平正常)恢复正常，提示内皮失调可能发生在肝脏损伤之前。器官的再生需要有功能的血管系统，其包括血液供应和促进再生和维持体内平衡的旁分泌因子。因此，肝脏内皮失调导致旁分泌因子异常，从而导致肝脏修复异常。本发明的研究显示，NASH中的表观遗传依赖的肝脏ECs失调导致促纤维化的IGFBP7和ADAMTS1分泌到细胞外囊泡中，而且，在人和小型猪NASH模型中，血浆IGFBP7和ADAMTS1水平的升高在可检测的实质损伤之前(血浆ALT和AST的升高)，联合靶向抑制HDAC2/DNMT1的异常交互调控恢复了小型猪血浆IGFBP7和ADAMTS1的水平。在人类患者上的临床发现与小型猪NASH模型中联合靶向抑制ECs中HDAC2/DNMT1使血浆AST和ALT水平正常化的数据一致。因此，本发明的实验数据表明，NASH中的慢性应激会刺激表观遗传重编程的IGFBP7 +ADAMTS1 +SECs的生成，并产生促纤维化的IGFBP7和ADAMTS1来增强肝脏实质损伤。在此过程中，参与内皮分类异常的分子标记(例如细胞外囊泡IGFBP7或ADAMTS1)可能用作治疗靶点或生物标记物来评估NASH患者的纤维化进展，特别是区分NASH患者与单纯性脂肪肝患者。 scRNA-Seq analysis can reveal vascular dysregulation in cirrhotic livers at the single-cell level. Analyses of human patients and minipig models of NASH demonstrate that aberrant epigenetic cross-regulation leads to dysregulation from the sinus endothelium to the macrovascular endothelium, resulting in aberrant endothelial classification. In the liver samples examined, the observed vascular dysregulation appears to be predominantly a phenotypic and functional shift within the ECs lineage. The fibrotic livers examined exhibited increased numbers of ECs and a lower enrichment of "mesenchymal differentiation" in the EC population. Thus, this vascular dysregulation process appears to be distinct from endothelial-mesenchymal transition (EndMT). From a therapeutic point of view, combined targeted inhibition of the aberrant HDAC2/DNMT1 interaction normalized endothelial classification and liver function (normal plasma levels of AST and ALT), suggesting that endothelial dysregulation may occur before liver injury. The regeneration of organs requires a functional vascular system, which includes blood supply and paracrine factors that promote regeneration and maintain homeostasis. Therefore, dysregulation of the liver endothelium leads to abnormal paracrine factors, which lead to abnormal liver repair. The present study shows that epigenetic-dependent dysregulation of liver ECs in NASH leads to the secretion of pro-fibrotic IGFBP7 and ADAMTS1 into extracellular vesicles, and that, in human and minipig NASH models, plasma levels of IGFBP7 and ADAMTS1 are reduced Elevated prior to detectable parenchymal damage (elevation of plasma ALT and AST), combined targeting to inhibit the aberrant cross-regulation of HDAC2/DNMT1 restored minipig plasma levels of IGFBP7 and ADAMTS1. The clinical findings in human patients are consistent with data that combined targeted inhibition of HDAC2/DNMT1 in ECs normalized plasma AST and ALT levels in a minipig NASH model. Thus, the present experimental data demonstrate that chronic stress in NASH stimulates the generation of epigenetically reprogrammed IGFBP7 + ADAMTS1 + SECs and produces profibrotic IGFBP7 and ADAMTS1 to enhance liver parenchymal injury. In this process, molecular markers involved in abnormal endothelial classification (such as extracellular vesicles IGFBP7 or ADAMTS1) may be used as therapeutic targets or biomarkers to assess the progression of fibrosis in NASH patients, especially to distinguish NASH patients from simple fat liver patients.
NASH中异常的细胞互作能够促进肝纤维化。本发明揭示了循环系统中促纤维化的血管失调的单细胞图谱，通过对肝硬化患者队列、转基因小鼠以及小型猪和啮齿动物NASH模型的多组学分析，确定了参与血管失调的关键分子。在所述血管失调过程中，表观遗传的重编程的肝脏SECs产生IGFBP7或ADAMTS1到细胞外囊泡中以募集促纤维化的Th17细胞。免疫细胞的异常募集和激活可刺激肝纤维化。在本发明中，使用NPCs的scRNA-Seq进行的细胞相互作用预测分析显示，在人肝硬化患者和小型猪NASH模型中，T细胞与重编程的ECs之间的互作。这个预测通过数据证明，小型猪和小鼠NASH模型中，靶向HDAC2/DNMT1治疗后使肝脏ECs的表观遗传变化正常并抑制了Th17的募集。scRNA-Seq、Igfbp7基因敲除小鼠和细胞外囊泡移植表明，所述EC-Th17的互作至少部分取决于表观遗传重编程的肝脏SECs产生的IGFBP7/ADAMTS1。因此，本发明整合了生物信息学和实验方法以揭示这种由表观遗传依赖性血管失调引起的独特的促纤维化的内皮-Th17细胞的相互作用。鉴于血管和造血细胞(循环系统)在多个器官中的系统分布，解码参与循环失调的分子和细胞网络可能有助于系统性鉴定治疗靶点或生物标记物。Abnormal cellular interactions in NASH can promote liver fibrosis. The present invention reveals a single-cell atlas of pro-fibrotic vascular dysregulation in the circulatory system and identifies key molecules involved in vascular dysregulation through multi-omic analysis of a cohort of patients with cirrhosis, transgenic mice, and minipig and rodent NASH models . During such vascular dysregulation, epigenetically reprogrammed liver SECs produce IGFBP7 or ADAMTS1 into extracellular vesicles to recruit profibrotic Th17 cells. Aberrant recruitment and activation of immune cells can stimulate liver fibrosis. In the present invention, the cellular interaction prediction analysis using scRNA-Seq of NPCs showed the interaction between T cells and reprogrammed ECs in human liver cirrhosis patients and a minipig NASH model. This prediction is supported by data that targeting HDAC2/DNMT1 normalizes epigenetic changes and inhibits Th17 recruitment in hepatic ECs in minipig and mouse models of NASH. scRNA-Seq, Igfbp7 knockout mice, and extracellular vesicle transplantation showed that the EC-Th17 interaction depends at least in part on IGFBP7/ADAMTS1 produced by epigenetically reprogrammed liver SECs. Thus, the present invention integrates bioinformatics and experimental approaches to uncover this unique pro-fibrotic endothelial-Th17 cell interaction caused by epigenetic-dependent vascular dysregulation. Given the systematic distribution of blood vessels and hematopoietic cells (circulatory system) in multiple organs, decoding the molecular and cellular networks involved in circulatory dysregulation may facilitate the systematic identification of therapeutic targets or biomarkers.
基于人类患者的临床发现，实验人员利用补充的临床前NASH模型(小型猪及小鼠)来确定了表观遗传依赖的血管失调的作用。采用含有高蔗糖和果糖、高胆固醇和高脂肪的西方饮食喂养与反复的肝脏损伤相结合来诱导NASH模型，表现出临床相关的NASH表型，包括组织学特征和转录组学特征。小型猪和小鼠的临床前模型也有助于了解NASH的发病机制。小型猪的消化系统与人的消化系统非常相似，具有类似人类代谢紊乱相关疾病(如NASH)的独特优势。大型动物如小型猪可以进行肝脏活检，用于多组学评估治疗效果和潜在机制。与小型猪相比，转基因小鼠，如EC特异性Hdac2基因敲除小鼠和Igfbp7基因敲除小鼠，为研究参与表观遗传依赖的血管失调的细胞和分子机制提供了有效工具。的确，在小型猪和小鼠NASH模型中进行的药理和遗传靶向实验表明，异常的HDAC2/DNMT1交互调控会导致血管失调，并随后产生IGFBP7/ADAMTS1到细胞外囊泡中。该数据与人类肝硬化队列中鉴定出的HDAC2、DNMT1、IGFBP7或ADAMTS1与纤维化等级之间的相关性相符。在小鼠和小型猪NASH模型中，联合靶向抑制HDAC2和DNMT1显示协同的抗纤维化效果。注射外源IGFBP7或ADAMTS1敲低的内皮来源的EV的实验进一步表明ADAMTS1/IGFBP7通过刺激肝脏中的Th17应答而促进肝脏纤维化。本发明实验虽然只采用了雄性小型猪来构造NASH模型，但是无论是人类患者样本还是小鼠 模型都不具有性别特异性，因此，本发明的临床前平台可能有助于设计各种纤维化相关疾病(与全球范围内40％的死亡相关)的治疗策略。在本发明中，实验人员阐述了血管失调的单细胞图谱，其中表观遗传重编程的EC亚群诱导了促纤维化因子的释放，刺激了Th17细胞募集，以共同促进多个物种的肝脏纤维化。所述失调的血管微环境的形成，包括内皮分类异常和在EVs中促纤维化因子的产成。阐明这种血管失调下的分子和细胞网络可能有助于探索纤维化疾病的诊断或治疗方案。Based on clinical findings in human patients, we used complementary preclinical NASH models (minipigs and mice) to determine the role of epigenetic-dependent vascular dysregulation. Feeding a Western diet containing high sucrose and fructose, high cholesterol, and high fat combined with repeated liver injury to induce a model of NASH exhibited clinically relevant NASH phenotypes, including histological and transcriptomic features. Preclinical models of minipigs and mice also contribute to understanding the pathogenesis of NASH. The digestive system of minipigs is very similar to that of humans, and has unique advantages similar to those associated with human metabolic disorders, such as NASH. Liver biopsies can be performed in large animals such as minipigs for multi-omics assessment of treatment effects and underlying mechanisms. Compared with minipigs, transgenic mice, such as EC-specific Hdac2 knockout mice and Igfbp7 knockout mice, provide an efficient tool to study the cellular and molecular mechanisms involved in epigenetic-dependent vascular dysregulation. Indeed, pharmacological and genetic targeting experiments in minipig and mouse models of NASH demonstrated that aberrant HDAC2/DNMT1 interaction leads to vascular dysregulation and subsequent production of IGFBP7/ADAMTS1 into extracellular vesicles. This data is consistent with the association between HDAC2, DNMT1, IGFBP7 or ADAMTS1 and fibrosis grade identified in the human cirrhosis cohort. Combined targeted inhibition of HDAC2 and DNMT1 showed synergistic anti-fibrotic effects in mouse and minipig NASH models. Experiments injected with exogenous IGFBP7 or ADAMTS1 knockdown endothelial-derived EVs further demonstrated that ADAMTS1/IGFBP7 promotes liver fibrosis by stimulating Th17 responses in the liver. Although only male miniature pigs were used to construct the NASH model in the experiment of the present invention, neither human patient samples nor mouse models are sex-specific. Therefore, the preclinical platform of the present invention may be helpful for designing various fibrosis-related Treatment strategies for the disease (associated with 40% of deaths worldwide). In the present invention, we illustrate a single-cell map of vascular dysregulation in which epigenetically reprogrammed EC subsets induce the release of profibrotic factors that stimulate Th17 cell recruitment to collectively promote liver fibrosis in multiple species change. The development of the dysregulated vascular microenvironment, including abnormal endothelial classification and production of profibrotic factors in EVs. Elucidating the molecular and cellular networks underlying this vascular dysregulation may help to explore diagnostic or therapeutic options for fibrotic diseases.
上面结合附图对本发明的实施例进行了描述，但是本发明并不局限于上述的具体实施方式，上述的具体实施方式仅仅是示意性的，而不是限制性的，本领域的普通技术人员在本发明的启示下，在不脱离本发明宗旨和权利要求所保护的范围情况下，还可做出很多形式，这些均属于本发明的保护之内。The embodiments of the present invention have been described above in conjunction with the accompanying drawings, but the present invention is not limited to the above-mentioned specific embodiments, which are merely illustrative rather than restrictive. Under the inspiration of the present invention, without departing from the scope of protection of the present invention and the claims, many forms can be made, which all belong to the protection of the present invention.
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Claims (34)

  1. HDAC2抑制剂和DNMT1抑制剂在联合靶向治疗非酒精性脂肪性肝炎中的应用。Application of HDAC2 inhibitor and DNMT1 inhibitor in combined targeted therapy of nonalcoholic steatohepatitis.
  2. 如权利要求1所述的应用，其特征在于，所述非酒精性脂肪性肝炎伴有肝硬化或肝纤维化。The use according to claim 1, wherein the nonalcoholic steatohepatitis is accompanied by liver cirrhosis or liver fibrosis.
  3. 如权利要求2所述的应用，其特征在于，所述肝纤维化的病理等级包括F2-F4级。The application of claim 2, wherein the pathological grades of liver fibrosis include F2-F4 grades.
  4. 如权利要求1所述的应用，其特征在于，所述HDAC2抑制剂为莫西司他(Mocetinostat)，剂量为1-20mg/kg/天或0.1-2.0mg/kg/天。The application according to claim 1, wherein the HDAC2 inhibitor is Mocetinostat, and the dosage is 1-20 mg/kg/day or 0.1-2.0 mg/kg/day.
  5. 如权利要求1所述的应用，其特征在于，所述DNMT1抑制剂为阿扎胞苷，剂量为0.1-2.0mg/kg/天或0.001-0.100mg/kg/天。The application according to claim 1, wherein the DNMT1 inhibitor is azacitidine, and the dosage is 0.1-2.0 mg/kg/day or 0.001-0.100 mg/kg/day.
  6. 如权利要求1所述的应用，其特征在于，所述HDAC2抑制剂和DNMT1抑制剂通过注射方式给药，所述注射方式包括腹腔注射、肌肉注射、皮下注射和静脉注射中的一种或多种。The use according to claim 1, wherein the HDAC2 inhibitor and the DNMT1 inhibitor are administered by injection, and the injection includes one or more of intraperitoneal injection, intramuscular injection, subcutaneous injection and intravenous injection kind.
  7. 如权利要求6所述的应用，其特征在于，采用注射方式给药的具体操作为：第一周前5天注射DNMT1抑制剂治疗，每天1次，然后停药2天；第二周前5天注射HADC2抑制剂，每天1次，然后停药2天，重复治疗5-10个疗程。The application according to claim 6, characterized in that, the specific operation of administering by injection is as follows: injecting DNMT1 inhibitor once a day for the first 5 days of the first week, and then stopping the drug for 2 days; Inject HADC2 inhibitor once a day, then stop the drug for 2 days, and repeat the treatment for 5-10 courses.
  8. 如权利要求1所述的应用，其特征在于，所述HDAC2抑制剂和DNMT1抑制剂联合靶向使用减轻非酒精性脂肪性肝炎肝脏的纤维化程度并促进肝再生；和/或逆转肝硬化肝脏中的窦内皮-大血管内皮失调；和/或减少非酒精性脂肪性肝炎肝脏中促纤维化Th17细胞的募集。The use according to claim 1, wherein the combined targeted use of the HDAC2 inhibitor and the DNMT1 inhibitor reduces the degree of fibrosis in the liver of non-alcoholic steatohepatitis and promotes liver regeneration; and/or reverses liver cirrhosis Sinus-macrovascular endothelial dysregulation in nonalcoholic steatohepatitis; and/or reduced recruitment of pro-fibrotic Th17 cells in the liver of nonalcoholic steatohepatitis.
  9. 如权利要求1所述的应用，其特征在于，所述HDAC2抑制剂和DNMT1抑制剂联合靶向使用降低血糖、肝纤维化指数和/或血清肝功能指数。The application according to claim 1, wherein the HDAC2 inhibitor and the DNMT1 inhibitor are used in combination to target lowering blood sugar, liver fibrosis index and/or serum liver function index.
  10. 如权利要求1所述的应用，其特征在于，所述HDAC2抑制剂和DNMT1抑制剂联合靶向使用降低血清总胆固醇水平。The application of claim 1, wherein the HDAC2 inhibitor and the DNMT1 inhibitor are used in combination to target and reduce serum total cholesterol levels.
  11. 如权利要求1所述的应用，其特征在于，所述HDAC2抑制剂和DNMT1抑制剂联合靶向使用减轻肝硬化并增加肝细胞增殖。The use according to claim 1, wherein the combined targeted use of the HDAC2 inhibitor and the DNMT1 inhibitor reduces liver cirrhosis and increases hepatocyte proliferation.
  12. 如权利要求1所述的应用，其特征在于，所述HDAC2抑制剂和DNMT1抑制剂联合靶向使用阻断肝硬化肝脏中IGFBP7和/或ADAMTS1的增加。The use of claim 1, wherein the HDAC2 inhibitor and the DNMT1 inhibitor are combined for targeted use to block the increase of IGFBP7 and/or ADAMTS1 in cirrhotic liver.
  13. 一种用于评估非酒精性脂肪性肝炎的标记物组，其特征在于，所述标记物组包括IGFBP7和ADAMTS1；所述标记物组的表达量可用于评估肝纤维化程度和/或肝功能受损情况。A marker group for evaluating nonalcoholic steatohepatitis, characterized in that the marker group includes IGFBP7 and ADAMTS1; the expression level of the marker group can be used to evaluate the degree of liver fibrosis and/or liver function damage.
  14. 如权利要求13所述的标记物组，其特征在于，所述标记物组可以在没有肝功能受损的情况下在评估肝硬化和肝纤维化情况。The marker panel of claim 13, wherein the marker panel can evaluate liver cirrhosis and liver fibrosis without impaired liver function.
  15. 如权利要求13所述的标记物组，其特征在于，所述肝纤维化程度包括病理等级F2-F4级。The marker set of claim 13, wherein the degree of liver fibrosis comprises pathological grades F2-F4.
  16. 如权利要求13所述的标记物组，其特征在于，所述标记物组还可以用于评估促纤维化的Th17信号通路的应答情况。The marker panel of claim 13, wherein the marker panel can also be used to assess the response of the pro-fibrotic Th17 signaling pathway.
  17. 包含权利要求13所述标记物组的试剂盒。A kit comprising the set of markers of claim 13.
  18. 如权利要求17所述的试剂盒，其特征在于，所述试剂盒可用于检测血浆中的IGFBP7和ADAMTS1表达量。The kit of claim 17, wherein the kit can be used to detect the expression levels of IGFBP7 and ADAMTS1 in plasma.
  19. 如权利要求17所述的试剂盒，其特征在于，所述试剂盒可用于检测IGFBP7和ADAMTS1的蛋白表达量。The kit of claim 17, wherein the kit can be used to detect the protein expression levels of IGFBP7 and ADAMTS1.
  20. 用于区分非酒精性脂肪性肝炎和单纯性脂肪肝的标记物组，其特征在于，所述标记物组包括IGFBP7和ADAMTS1。A marker group for distinguishing nonalcoholic steatohepatitis and simple fatty liver, characterized in that the marker group includes IGFBP7 and ADAMTS1.
  21. 包含权利要求20所述标记物组的试剂盒。A kit comprising the set of markers of claim 20.
  22. 如权利要求21所述的试剂盒，其特征在于，所述试剂盒可用于检测血浆中的IGFBP7和ADAMTS1表达量。The kit of claim 21, wherein the kit can be used to detect the expression levels of IGFBP7 and ADAMTS1 in plasma.
  23. 如权利要求21所述的试剂盒，其特征在于，所述试剂盒可用于区分健康人与肝硬化、肝纤维化患者血浆中的IGFBP7和ADAMTS1表达水平。The kit of claim 21, wherein the kit can be used to differentiate the expression levels of IGFBP7 and ADAMTS1 in plasma from healthy people and patients with liver cirrhosis and liver fibrosis.
  24. 血管分泌因子在制备检测非酒精性脂肪性肝炎的生物标记物中的应用，其特征在于，所述血管分泌因子包括IGFBP7和/或ADAMTS1；所述IGFBP7和/或ADAMTS1表达于HDAC2/DNMT1-IGFBP7/ADAMTS1-Th17信号通路。The application of vascular secretion factor in the preparation of biomarkers for detecting nonalcoholic steatohepatitis, characterized in that, the vascular secretion factor comprises IGFBP7 and/or ADAMTS1; the IGFBP7 and/or ADAMTS1 are expressed in HDAC2/DNMT1-IGFBP7 /ADAMTS1-Th17 signaling pathway.
  25. 如权利要求24所述的应用，其特征在于，所述HDAC2/DNMT1-IGFBP7/ADAMTS1-Th17信号通路中IGFBP7和/或ADAMTS1的mRNA表达量和/或蛋白表达量可用于评估肝纤维化程度和/或肝功能受损情况。The application according to claim 24, wherein the mRNA expression and/or protein expression of IGFBP7 and/or ADAMTS1 in the HDAC2/DNMT1-IGFBP7/ADAMTS1-Th17 signaling pathway can be used to assess the degree of liver fibrosis and / or impaired liver function.
  26. 如权利要求25所述的应用，其特征在于，所述IGFBP7和/或ADAMTS1表达量的升高与肝纤维化程度成正比，所述肝纤维化程度包括病理等级F2-F4级。The use according to claim 25, wherein the increase in the expression of IGFBP7 and/or ADAMTS1 is proportional to the degree of liver fibrosis, and the degree of liver fibrosis includes pathological grades F2-F4.
  27. 如权利要求24所述的应用，其特征在于，所述IGFBP7和/或ADAMTS1存在于血浆中。The use of claim 24, wherein the IGFBP7 and/or ADAMTS1 are present in plasma.
  28. 血管分泌因子在制备区分非酒精性脂肪性肝炎和单纯性脂肪肝的生物标记物中的应用，其特征在于，所述血管分泌因子包括IGFBP7和/或ADAMTS1；所述IGFBP7和/或ADAMTS1表达于HDAC2/DNMT1-IGFBP7/ADAMTS1-Th17信号通路。The application of vascular secretion factor in the preparation of biomarkers for distinguishing nonalcoholic steatohepatitis and simple fatty liver, characterized in that the vascular secretion factor comprises IGFBP7 and/or ADAMTS1; the IGFBP7 and/or ADAMTS1 are expressed in HDAC2/DNMT1-IGFBP7/ADAMTS1-Th17 signaling pathway.
  29. 如权利要求28所述的应用，其特征在于，所述HDAC2/DNMT1-IGFBP7/ADAMTS1-Th17信号通路中IGFBP7和/或ADAMTS1的mRNA表达量和/或蛋白表达量可区分非酒精性脂肪性肝炎和单纯性脂肪肝。The use according to claim 28, wherein the mRNA expression and/or protein expression of IGFBP7 and/or ADAMTS1 in the HDAC2/DNMT1-IGFBP7/ADAMTS1-Th17 signaling pathway can distinguish nonalcoholic steatohepatitis and simple fatty liver.
  30. IGFBP7和/或ADAMTS1协同作用在制备检测非酒精性脂肪性肝炎的生物标记物中的用途。Use of IGFBP7 and/or ADAMTS1 synergistic action in the preparation of biomarkers for the detection of nonalcoholic steatohepatitis.
  31. 如权利要求30所述的用途，其特征在于，所述非酒精性脂肪性肝炎包括伴有肝硬化或肝纤维化。The use of claim 30, wherein the nonalcoholic steatohepatitis comprises associated liver cirrhosis or liver fibrosis.
  32. 如权利要求30所述的用途，其特征在于，所述非酒精性脂肪性肝炎还包括肝无纤维化的情况，所述无纤维化的病理等级为F0-F1。The use according to claim 30, wherein the nonalcoholic steatohepatitis further includes the condition of no liver fibrosis, and the pathological grade of no fibrosis is F0-F1.
  33. 如权利要求30所述的用途，其特征在于，所述IGFBP7和/或ADAMTS1诱导促纤维化Th17细胞应答。The use of claim 30, wherein the IGFBP7 and/or ADAMTS1 induce a pro-fibrotic Th17 cell response.
  34. 如权利要求33所述的用途，其特征在于，所述Th17细胞在肝纤维化的肝脏中聚集。34. The use of claim 33, wherein the Th17 cells accumulate in fibrotic liver.
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