CN114617970B - Application of HDAC2 and DNMT1 inhibitor in combined targeted therapy of non-alcoholic steatohepatitis - Google Patents

Abstract

The invention belongs to the field of biomedicine, and mainly relates to application of HDAC2 and DNMT1 inhibitors in treating non-alcoholic steatohepatitis. The application of the HDAC2 inhibitor and the DNMT1 inhibitor in the combined targeted therapy of the non-alcoholic steatohepatitis is beneficial to systematically relieving or treating the non-alcoholic steatohepatitis and the cirrhosis and/or the hepatic fibrosis accompanied by the non-alcoholic steatohepatitis to a certain extent.

Description

Application of HDAC2 and DNMT1 inhibitor in combined targeted therapy of non-alcoholic steatohepatitis

Technical Field

The invention belongs to the field of biomedicine, and mainly relates to application of HDAC2 and DNMT1 inhibitors in treating non-alcoholic steatohepatitis.

Background

The liver has a regenerative capacity for self-repair after injury. However, in nonalcoholic steatohepatitis (NASH), liver regeneration is inhibited. NASH, like diabetes and metabolic syndrome, has an increasing global incidence. NASH can lead to liver fibrosis, cirrhosis and liver failure, and without effective anti-fibrotic treatment, NASH-induced liver fibrosis and cirrhosis often lead to systemic complications and will become a major health burden worldwide. One major obstacle to the clinical development of NASH treatment 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, vascular Endothelial Cells (ECs), hematopoietic cells, and the like. Liver regeneration relies on a synergistic effect between different cellular components. However, sustained stress in NASH often causes abnormal cellular interactions (both "interactions" and "crosstalk" are understood herein as "cross regulation") and repair and fibrosis leading to disorders. Activation of stellate cells is a critical step in liver fibrosis, but it is still determined how chronic stress in NASH leads to interaction between other liver NPCs to facilitate this step.

Among NPCs, vascular endothelial cells and hematopoietic cells belong to the circulatory system, can directly transmit systemic stimuli (such as metabolic stress), and can promote interaction between parenchymal cells and mesenchymal cells to jointly establish a vascular microenvironment. Vascular endothelial cells are a major component of NPCs in the liver. The blood supply to the liver is accomplished by the sinusoidal vascular system between the hepatic vein, hepatic artery and portal vein. The sinusoidal vasculature has a layer of Sinus Endothelial Cells (SECs) expressing CLEC4G and OIT3 and large vessel endothelial cells (MECs) expressing CD 34. Thus, hepatic ECs at different anatomical sites exhibit specific morphological and phenotypic markers with "sinoendothelial-macroendothelial" vascular hierarchy and intra-organ classification. During organ repair, vascular ECs produce a number of regulatory factors that regulate communication between hematopoietic, mesenchymal and parenchymal cells (communication, also referred to herein as "message transfer"). Abnormal changes in Sinusoidal Endothelial Cells (SECs), such as capillary vascularization, are closely associated with liver fibrosis. However, the functional role of the hepatic EC subgroup on human cirrhosis or NASH pathology has not been systematically elucidated at the single cell level in current clinical and preclinical models.

Disclosure of Invention

In view of the above, the present invention provides the use of an HDAC2 inhibitor and a DNMT1 inhibitor for the combined targeted treatment of non-alcoholic steatohepatitis.

Further, the non-alcoholic steatohepatitis is accompanied by liver cirrhosis or liver fibrosis.

Further, the pathological grade of liver fibrosis includes F2-F4 grade.

Further, the HDAC2 inhibitor is moxiflostat (Mocetinostat) and the dosage is 1-20 mg/kg/day or 0.1-2.0 mg/kg/day (preferably 1.1 mg/kg/day).

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).

Further, the HDAC2 inhibitor and DNMT1 inhibitor are administered by injection, which includes one or more of intraperitoneal injection, intramuscular injection, subcutaneous injection, or intravenous injection.

Further, the specific operation of administration by injection is as follows: injecting DNMT1 inhibitor 5 days before the first week for 1 time per day, and stopping administration for 2 days; the administration of the HADC2 inhibitor is performed 5 days before the second week, 1 time per day, and then the administration is stopped for 2 days, and the treatment is repeated for 5-10 courses (preferably 6 courses).

Further, the combined targeted use of the HDAC2 inhibitor and the DNMT1 inhibitor reduces the degree of fibrosis of the nonalcoholic steatohepatitis liver and promotes liver regeneration; and/or reversing sinoendothelial-macrovascular endothelial disorders in a liver cirrhosis liver; and/or reducing the recruitment of profibrotic Th17 cells in the non-alcoholic steatohepatitis liver.

Further, the combined targeted use of the HDAC2 inhibitor and the DNMT1 inhibitor reduces blood glucose, an index of liver fibrosis and/or an index of serum liver function.

Further, the HDAC2 inhibitor and the DNMT1 inhibitor are combined for targeted use to reduce serum total cholesterol levels.

Further, the combined targeted use of the HDAC2 inhibitor and the DNMT1 inhibitor alleviates liver cirrhosis and increases hepatocyte proliferation.

Further, the combination of the HDAC2 inhibitor and the DNMT1 inhibitor targeted for use blocks the increase of IGFBP7 and ADAMTS1 in a liver cirrhosis liver.

The beneficial technical effects are as follows:

the invention provides application of an HDAC2 inhibitor and a DNMT1 inhibitor in combined targeted therapy of non-alcoholic steatohepatitis. The technical scheme disclosed by the invention comprises the following steps of: epigenetic changes in the subpopulation of liver endothelial cells, i.e., aberrant activation of HDAC2 and DNMT1 (where the expression of HDAC2 and DNMT1 changes significantly in the liver of patients of class F2-F4; and HDAC2 and DNMT1 expression is significantly upregulated in the liver of cirrhosis patients and endothelial cells of liver of cirrhosis mini-pigs) lead to "sinus endothelium-large vessel endothelium dysregulation", stimulating production of profibrotic IGFBP7 and ADAMTS1 in extracellular vesicles, recruiting Th17 cells, thereby inhibiting liver regeneration and inducing fibrosis in NASH. By targeting inhibition of HDAC2 and DNMT1 of the mini-pig and mouse NASH models, epigenetic changes of liver endothelial cells are normalized to some extent, expression of IGFBP7 and ADAMTS1 is blocked, and recruitment of Th17 cells is inhibited.

The technical scheme of the invention reduces the fibrosis degree of the NASH liver and promotes liver regeneration to a certain extent by using the HDAC2 inhibitor and the DNMT1 inhibitor to jointly target HDAC2 and DNMT 1: for example, in the mini-pig NASH model in example 4, mini-pigs treated with a combination of an HDAC2 inhibitor (e.g., moxiflostat) and a DNMT1 inhibitor (e.g., azacitidine) compared to control groups: (i) The blood sugar, the hepatic fibrosis index, the serum liver function index and the serum total cholesterol of the liver-protecting health-care wine are all obviously reduced; (ii) It has reduced liver cirrhosis, reduced collagen deposition and lipid accumulation and increased hepatocyte proliferation. And in example 5, cirrhosis mini-pigs treated with a combination of an HDAC2 inhibitor (e.g., moxiflostat) and a DNMT1 inhibitor (e.g., azacitidine) significantly reversed the "sinoendothelia-macroendothelia dysregulation" caused by NASH in the treatment group.

Further, the present technical solution by using HDAC2 inhibitor and DNMT1 inhibitor to jointly target HDAC2 and DNMT1, recruitment of profibrotic Th17 cells in the liver of the treatment group NASH was significantly reduced: for example, in examples 6 and 7, in cirrhosis minipigs treated with a combination of an HDAC2 inhibitor (e.g., moxystat) and a DNMT1 inhibitor (e.g., azacitidine), the increase in mRNA and protein levels of IGFBP7 and ADAMTS1 in Sinus Endothelial Cells (SECs) of the treatment group was effectively blocked, and the increase in IGFBP7/ADAMTS1 concentration in plasma Extracellular Vesicles (EVs) thereof was also decreased; also, in examples 8 and 9, the increase in Th17 cell number in the treated group was effectively blocked in the cirrhosis mini-pig and mouse NASH model treated with the combination of the HDAC2 inhibitor (e.g., moxystat) and the DNMT1 inhibitor (e.g., azacitidine).

In the prior art, single drug therapy may be difficult to simultaneously alleviate or treat non-alcoholic steatohepatitis and its accompanying cirrhosis and fibrosis. In summary, the application of the HDAC2 and DNMT1 inhibitor provided in the technical scheme of the present invention in the treatment of non-alcoholic steatohepatitis is helpful for systematically relieving or treating non-alcoholic steatohepatitis and liver cirrhosis and/or liver fibrosis accompanied therewith to a certain extent.

Drawings

FIG. 1 is a diagram of single cell RNA sequencing (scRNA-Seq) experiments revealing HDAC2/DNMT 1-selectively induced "sino-endothelium-large vessel endothelium dysregulation" in human liver cirrhosis;

FIG. 2 is a graph of an experiment in which combined targeted inhibition of HDAC2 and DNMT1 in liver endothelial cells reduces liver fibrosis in a mini-pig NASH model;

FIG. 3 is a graph of an experiment targeting inhibition of epigenetic disorders in the liver endothelial cells to reverse "sinus endothelium-large vessel endothelium disorder", normalize endothelial classifications, and block cirrhosis in a mini-pig NASH model;

FIG. 4 is a graph of an experiment showing the occurrence of paracrine/vaso-secretory factor reprogramming in hepatic endothelial cells with epigenetic disorders in human patients and in miniature pigs;

FIG. 5 is an experimental plot of the paracrine/vascular secretion factors IGFBP7 and ADAMTS1 in plasma Extracellular Vesicles (EVs) as biomarkers for assessing fibrosis progression in human patients and the mini-pig NASH model;

FIG. 6 is an experimental plot of deregulated vascular endothelial microenvironment-induced profibrotic Th17 responses in human patients and mini-pig NASH models;

FIG. 7 is an experimental plot of inhibition of HDAC2/DNMT1 in dysregulated liver Sinusoidal Endothelial Cells (SECs) reducing recruitment of profibrotic Th17 cells in the mouse NASH model;

FIG. 8 is an experimental plot of the Th17 response of IGFBP7 to promote liver fibrosis and profibrosis in the mouse NASH model;

FIG. 9 is an experimental plot of ADAMTS1 inhibition reducing the Th17 cell response to profibrosis in a mouse model of liver fibrosis;

FIG. 10 is an experimental diagram of single cell RNA sequencing (scRNA-Seq) analysis of liver non-parenchymal cells (NPCs) of 2 healthy persons and 2 patients with liver cirrhosis;

FIG. 11 is an experimental plot of scRNA-Seq analysis of human liver NPCs from GSE136103 data;

FIG. 12 is a graph of an experiment showing Endothelial Cell (ECs) analysis in human liver scRNA-Seq data;

FIG. 13 shows human liver, liver CD45 ⁺ Experimental graphs for gene expression analysis of NPCs and liver endothelial cells;

FIG. 14 is an experimental diagram of scRNA-Seq analysis of NPCs in the small pig liver;

FIG. 15 is an analysis of Endothelial Cells (ECs) in the small pig liver scRNA-Seq data;

FIG. 16 is a graph of differential gene analysis of liver cirrhosis of human and mini-pig and experimental results of paracrine factor gene expression in NPCs in liver;

FIG. 17 is an experimental picture of Th17 cell analysis of human and piglet livers.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention. It is to be understood that the embodiments described are only a few embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

It should be noted that, in this document, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. Without further limitation, an element defined by the phrase "comprising a … …" does not exclude the presence of another identical element in a process, method, article, or apparatus that comprises the element.

As used in this specification, the term "about" typically means +/-5% of the stated value, more typically +/-4% of the stated value, more typically +/-3% of the stated value, more typically +/-2% of the stated value, even more typically +/-1% of the stated value, and even more typically +/-0.5% of the stated value.

In this specification, certain embodiments may be disclosed in a range of formats. It should be understood that this description of "within a certain range" is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosure. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range. For example, the range

The description of (a) should be read as having 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., as well as individual numbers within this range, e.g., 1,2,3,4,5 and 6. Regardless of the breadth of the range, the following appliesAnd (4) an upper rule.

Noun interpretation

The invention relates to the following steps: liver fibrosis is simultaneously regulated by HDAC2 and DNMT1, inhibiting one of HDAC2 or DNMT1 alone, the other (DNMT 1 or HDAC 2) undergoes a change (increase) toward profibrosis. For example, inhibition of HDAC2, increased expression of DNMT1, inhibition of DNMT1, increased expression of HDAC2 in endothelial cells, liver fibrosis is affected by HDAC2 and DNMT1 cross-regulation.

The 'blood vessel microenvironment' refers to: vascular endothelial cells actively regulate the function and phenotype of peripheral cells through interaction with the peripheral cells, thereby forming a guided microenvironment.

The "disorder" of the present invention means: the proportion of endothelial cell subpopulations or gene expression in the liver is altered in a cirrhotic liver as compared to normal liver.

The "sinoendothelial-macroendothelial disorder" or "sinoendothelial-macroendothelial disorder" in the present invention refers to: compared with normal liver, the quantity of sinus endothelial cells in the liver cirrhosis liver is reduced, the quantity of large vessel endothelial cells is increased, and sinus endothelial cells express a pathological phenomenon of a large vessel endothelial marker (marker).

The "epigenetic therapy" of the present invention refers to: treatment of liver cirrhosis or fibrosis in miniature pigs and mice with epigenetic inhibitors reduces liver fibrosis.

Detailed description of the drawings

FIG. 1: (A) Method of scRNA-Seq of liver non-parenchymal cells (NPCs) in human patients in Wash's Hospital. (B, C) cluster analysis of scRNA-Seq data for 2 healthy livers and 2 NPCs of liver cirrhosis. (B) The heatmap shows different cell lines of NPCs and their marker genes (right). The (C) UMAP plot shows different cell lines of NPCs. Endo (EC), endothelial cells; DC, dendritic cells; neu, neutrophil; mac, macrophages; EPCAM ⁺ ，EPCAM ⁺ Cells and bile duct cells. (D) The pie charts show the differential gene factors for different cell lines of NPCs (liver cirrhosis vs. healthy liver). (E) Endothelial cells of 2 healthy livers and 2 liver cirrhosisAnd (5) clustering analysis. Upper left: UMAP plots show different cell lines of endothelial cells; left lower: pie charts show the proportion of different subpopulations of endothelial cells; and (3) right: marker gene expression of different cell lines of endothelial cells. CLCE4G and OIT3 marked sinus endothelial cells, and CD34 marked large vessel endothelial cells. SEC, sinus endothelial cells; MEC, large vessel endothelial cells. (F) Pseudo-temporal analysis of different subpopulations of endothelial cells of healthy and cirrhotic human liver. Quasi-temporal analysis showed that human liver with cirrhosis developed "sino-macroendothelial disorder". (G) Liquid chip analysis of histone modifications in parenchymal cells (hepatocytes) and non-parenchymal cells (NPCs) of healthy and cirrhosis human liver. And (3) right: quantification of acetylation of different modification sites of histones H3 and H4. Hep, hepatocytes; NPCs, nonparenchymal cells. N =3. (H) The heat maps show the expression of genes associated with histone modification and DNA methylation in different cell lines of non-parenchymal cells (NPCs) of healthy and cirrhotic human liver. The method comprises the following steps: differential gene factors associated with histone modification and DNA methylation. (I) Expression of Histone Deacetylases (HDACs) at different pathological levels of human liver fibrosis (data from GSE 84044). F0-F4, different pathological grades of human liver fibrosis. The expression levels of HDACs in F1-F4 grade liver were quantified relative to healthy liver (F0). (J) The violin plots show the expression of HDACs in liver endothelial cells of healthy and cirrhosis persons. (K) Quantitative PCR (qPCR) showed the expression of HDAC2 in liver endothelial cells of healthy and cirrhosis people. N =3. (L) expression of DNA methyltransferases (DNMTs) at different levels in human liver fibrosis (data from GSE 84044). F0-F4, different pathological grades of human hepatic fibrosis. Expression levels of DNMTs in F1-F4 grade liver were quantified relative to healthy liver (F0). (M) Violin plot shows the expression of DNMTs in endothelial cells of liver of healthy and cirrhosis human. (N) qPCR showed expression of DNMT1 in endothelial cells of healthy and cirrhosis human liver. N =3. (O) qPCR showed that HDAC2 and DNMT1 were expressed in SECs in the liver of healthy and cirrhosis humans. N =3. (P) schematic hypothesis: sinoendothelial cells of the liver cirrhosis liver acquire phenotypic markers of the large vessel endothelial cells. The apparent genetic reprogramming of the liver endothelial cells promotes the dysfunction of the sinoendothelia-great vessel endothelium to cause the abnormal classification of the endothelial cells and lead toCausing liver fibrosis or cirrhosis. In all statistical analyses, 2 sets of compared data were analyzed by two-tailed student's t-test; more than 2 sets of compared data were analyzed by one-way ANOVA fallen by Tukey's post-hoc test (one-way ANOVA fallen by). Data are presented as mean ± SEM. * Liver cirrhosis vs. healthy or F1-F4 vs. F0; * P is<0.05；**，P<0.01；***，P<0.001；****，P<0.0001。

FIG. 2: (A) a mini-pig NASH model and treatment protocol. The mini-pig NASH model consists of a western diet (WD: high fat, high cholesterol, high sucrose and fructose) and repeated intraperitoneal injections of hepatotoxic carbon tetrachloride (CCl) ₄ ) And (4) inducing. Treatment groups: by WD + CCl ₄ NASH induction was continued for 5 months, and epigenetic treatment was initiated 2 months after induction for 3 months. Group of liver cirrhosis: with WD + CCl ₄ The induction of the piglets was continued for 5 months. (B) A dosing regimen for the combined inhibition therapy of the mini-pig NASH model by HDAC2 and DNMT 1. Piglets were treated with HDAC2 inhibitor (HDAC 2 i) and DNMT1 inhibitor (HDAC 2 i). The miniature pigs are divided into 3 groups: 1) Control group (normal diet + corn oil); 2) Cirrhosis group (model group) (WD + CCl) ₄ Induction of NASH) 3) treatment group (HDAC 2i and DNMT1i treatment). (C) Blood glucose levels in control, cirrhosis and treatment groups of mini-pigs. Glu, glucose. N =3. (D) Liver fibrosis indices of liver tissues of control group, cirrhosis group and treatment group of miniature pigs. PC III, type III procollagen; IV-C, type IV collagen; HA, hyaluronic acid; hyp, hydroxyproline. N =3. (E) Liver function levels of sera of control, cirrhosis and treatment groups of piglets. ALP, alkaline phosphatase; ALT, glutamic-pyruvic transaminase; AST, glutamic-oxaloacetic transaminase; TC, total cholesterol. N =3. (F) By H&E. Sirius red, type I collagen, oil red O and Ki67 staining assessed the liver histology, collagen and lipid droplet deposition and cell proliferation of the minipigs. And (3) right: high magnification of the dashed area of the left image. Scale bar, 200 μm. N =3. (G) Quantitation of sirius red, oil red O, collagen I and Ki67 staining in fig. 2F. The proportion of positive staining was quantified relative to the control group. N =3. (H) qPCR showed livers of control, cirrhosis and treatment groups of mini-pigsHDAC2 and DNMT1 mRNA expression levels in Sinus Endothelial Cells (SECs) and large vessel endothelial cells (MECs). N =3. (I) Schematic representation of epigenetic interactive regulation of targeted profibrosis in the mini-pig NASH model: the combined targeted inhibition of aberrantly activated HDAC2 and DNMT1 in deregulated hepatic endothelial cells reduces hepatic fibrosis and promotes liver regeneration in the mini-pig NASH model. All data were statistically analyzed by one-way anova followed by Tukey post hoc test. Data are presented as mean ± SEM. * A liver cirrhosis group vs control group; #, treatment group vs cirrhosis group; * P is<0.05；**，P<0.01。#，P<0.05；##，P<0.01。

FIG. 3: (A) Method for single cell sequencing of non-parenchymal cells (NPCs) of the liver in the mini-pig NASH model. (B, C) clustering analysis of non-parenchymal cells (NPCs) of the livers of the control group, the cirrhosis group and the treated group of the piglets. (B) The heatmap shows different cell lines of NPCs and their marker genes (right). The (C) UMAP plot shows different cell lines of NPCs. Endo (EC), endothelial cells; DC, dendritic cells; neu, neutrophil; mac, macrophages; EPCAM ⁺ ，EPCAM ⁺ Cells and bile duct cells. (D) The pie chart shows the ratio of different cell lines of parenchymal cells (NPCs) of the small pig liver. (E) Venn plots show the differential gene counts in different cell lines of parenchymal cells (NPCs) of the small pig liver between the cirrhosis group and the control group, and the treated group and the cirrhosis group, respectively. The numbers in parentheses indicate the basis factors recovered after treatment. (F) And (4) carrying out KEGG channel enrichment analysis on the small pig liver endothelial cells. 79 KEGG channels are obviously changed between liver endothelial cells of the piglets in the cirrhosis group and the piglets in the control group, and 99 KEGG channels are obviously changed between the liver endothelial cells of the piglets in the treatment group and the piglets in the cirrhosis group. Epigenetic treatment by HDAC2i and DNMT2i normalized the majority of altered KEGG pathways in liver endothelial cells of piglets in the cirrhosis group. The white numbers indicate the order in which the p-values are corrected. Correcting p-value<0.05 was considered statistically significant. (G) Clustering analysis of the liver endothelial cells of the control group, the cirrhosis group and the treatment group of the miniature pigs. (H) The pie chart shows the proportion of different subpopulations of minicar liver endothelial cells. Compared with the control group, the liver of the small pig in the cirrhosis group has the small blood vessel endotheliumAn increase in the number of cells (MECs) and a decrease in the number of Sinus Endothelial Cells (SECs) suggests the occurrence of a "sinoendothelial-macroendothelial disorder". (I) Venn plots show the differential gene factors in different subpopulations of small pig liver endothelial cells. The differential gene counts of hepatic Sinusoidal Endothelial Cells (SECs) were relatively largest in the cirrhosis group, with most of the differential genes restored after HDAC2i + DNMT1i treatment. The numbers in parentheses represent the recovered difference base factors. (J) Pseudo-temporal analysis of different subpopulations of control, cirrhosis and treatment groups of mini-pig liver endothelial cells: HDAC2i + DNMT1i treatment reversed "sinoendothelia-macroendothelia dysregulation" in the liver of cirrhosis piglets. (K) The abnormal HDAC2/DNMT1 in the liver of the cirrhosis miniature pig reverses the sinus endothelium-large vessel endothelium disorder, enhances regeneration and relieves fibrosis.

FIG. 4: (A) The paracrine/vascular secretion factor gene of liver endothelial cell of liver cirrhosis human is expressed differently. The heatmap shows representative vascular secretion factors such as Ephrin/Eph, notch, insulin growth factor-related proteins, ADAM/ADAMTS, and Semaphorin/Plexin families. (B) Paracrine/vascular secretion factor gene expression in the liver of different grades of fibrosis human (data from GSE 84044). F0-F4, different pathological grades of human hepatic fibrosis. The expression level of paracrine/vaso-secretory factor genes of F1-F4 grade liver was quantified relative to healthy liver (F0). (C) Violin plots show expression of representative paracrine/vaso-secretory factor genes in different cell lines of parenchymal cells (NPCs) of human and miniature pig livers. IGFBP7 and ADAMTS1 were highly expressed in human and miniature pig liver endothelial cells. (D) Schematic representation of the reprogramming of paracrine/vasosecretory factors associated with "sinoendothelial-macroendothelial disorder", including the induction of IGFBP7 and ADAMTS1 in endothelial cells. (E) Expression levels of IGFBP7 in total endothelial cells, different subpopulations of endothelial cells, and sinus endothelial cells in healthy and cirrhosis human liver. Left: violin plots show expression of liver total endothelial cell IGFBP 7; the method comprises the following steps: violin plots show the expression of IGFBP7 in different subpopulations of liver endothelial cells; and (3) right: qPCR showed expression of IGFBP7 in liver sinoendothelial cells, N =3. (F) Expression levels of ADAMTS1 in total endothelial cells, different subpopulations of endothelial cells, and sinus endothelial cells in healthy and cirrhosis human liver. Left: violin diagrams show the expression of liver total endothelial cells ADAMTS 1; the method comprises the following steps: violin plots show the expression of ADAMTS1 in different subpopulations of liver endothelial cells; and (3) right: western blot showed ADAMTS1 expression in liver sinoendothelial cells, N =3. (G) Expression of IGFBP7 in different subsets of total endothelial cells and endothelial cells of the livers of control, cirrhosis and treated piglets. (H, I) qPCR (H) and ELISA (I) showed expression of IGFBP7 in small pig liver Sinusoidal Endothelial Cells (SECs) and large vessel endothelial cells (MECs) in the control, cirrhosis and treated groups. N =3. (J) ADAMTS1 expression in different subsets of total endothelial cells and endothelial cells of the livers of control, cirrhosis and treated piglets. (K, L) qPCR (K) and ELISA (L) showed the expression of ADAMTS1 in small pig liver Sinusoidal Endothelial Cells (SECs) and large vessel endothelial cells (MECs) in the control, cirrhosis and treatment groups. N =3. (M) ATAC-seq results show chromatin opening of IGFBP7 and ADAMTS1 promoters in control, cirrhosis and treatment groups of mini-pig liver endothelial cells: the IGFBP7 and ADAMTS1 were induced dependently by HDAC2/DNMT1 in liver endothelial cells of the small pigs in the cirrhosis group. (N) schematic representation of the production of profibrotic IGFBP7 and ADAMTS1 by epigenetic reprogrammed liver sinusoidal endothelial cells in human patients and mini-pigs. In all statistical analyses, 2 groups of compared data were analyzed by two-tailed student t-test; more than 2 sets of compared data were analyzed by one-way anova followed by Tukey post hoc testing. Data are presented as mean ± SEM. * Cirrhosis vs control (minipig)/healthy (human), or F1-F4 vs.f0; treatment group vs. cirrhosis group (mini-pigs). * P <0.05; * P <0.01; * P <0.001.#, P <0.05; #, P <0.01.

FIG. 5 is a schematic view of: (A) Plasma concentrations of IGFBP7, ADAMTS1, ALT, AST in healthy and cirrhosis/liver fibrosis patients. (B, C) plasma concentrations of IGFBP7 and ADAMTS1 in patients with liver cirrhosis/fibrosis with normal or abnormal liver function. Cirrhosis/liver fibrosis patients are divided into two groups (B): normal ALT/AST concentrations and abnormal ALT/AST concentrations. (D) Plasma concentrations of IGFBP7 and ADAMTS in healthy and differently predisposed cirrhosis/fibrosis patients. Patients with cirrhosis/liver fibrosis are divided into five categories according to etiology: non-alcoholic steatohepatitis-associated cirrhosis/liver fibrosis (NASH), hepatitis b-associated cirrhosis/liver fibrosis (HBC), autoimmune hepatitis-associated cirrhosis/liver fibrosis (AIH), primary biliary cirrhosis/liver fibrosis (PBC), and cryptogenic cirrhosis/liver 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 of different pathological grades. (G) human plasma isolated Extracellular Vesicles (EVs). The method comprises the following steps: electron microscopy analysis of the isolated EVs; the following: western blot detects positive (CD 81) and negative (GRP 94) markers for EVs. (H) IGFBP7/ADAMTS1 concentrations of plasma Extracellular Vesicles (EVs) from minipigs and humans. (I, J) IGFBP7/ADAMTS1 concentration of EVs in patients with cirrhosis of the normal or abnormal liver function (ALT/AST) (I) or in patients with NASH of different fibrosis grade (J). (K) schematic hypothesis: epigenetically dysregulated liver Sinusoidal Endothelial Cells (SECs) produce pro-fibrotic IGFBP7 and ADAMTS1, and promote cirrhosis/fibrosis by Extracellular Vesicles (EVs). Of all statistical analyses, 2 groups of comparison data were analyzed by two-tailed student t-test; more than 2 sets of comparison data were analyzed by one-way anova followed by Tukey post hoc testing. Data are presented as mean ± SEM. * Cirrhosis, fibrosis or NASH vs. control group (mini-pigs) or healthy (human); # NASH vs. simple fatty liver (human) or treatment vs. cirrhosis (mini-pigs). * P <0.05; * P <0.01; * P <0.001. And # P <0.05.

FIG. 6: (A) Cell interaction analysis based on receptor ligand profiles of different cell lines of human and mini-pig liver (NPCs): liver endothelial cells of patients with cirrhosis and minipigs interact significantly with T cells. (B) Western blot showing healthy and liver cirrhosis CD45 ⁺ Protein levels of phosphorylated Smad2 in liver NPCs. The level of phosphorylated Smad2 was quantified relative to total Smad 2. The results show CD45 of liver of a liver-cirrhosis human ⁺ TGF-. Beta.1-Smad 2 activation in NPCs. The method comprises the following steps: quantification of protein expression; the following: representative protein bands. Data were analyzed by two-tailed student's t-test and expressed as mean ± SEM. * Liver cirrhosis vs. health, P<0.05.N =4. (C) CD4 of healthy and liver-hardened human liver ⁺ Clustering analysis of T cells. (D) Th17 ⁺ Expression of marker genes in the T cell cluster of healthy and liver-hardened human liver. (E) Proportion of Th17 cells in healthy and cirrhosis human liver NPCs. The proportion of Th17 cells in a liver of a cirrhosis human is quantified relative to a healthy human. (F, G) T cells and CD4 of liver of miniature pig in control group, liver cirrhosis group and treatment group ⁺ Clustering analysis of T cells. (H) Th17 ⁺ Marker gene in miniature pig liver CD4 ⁺ Expression in T cell clusters (Cluster). (I) The proportion of Th17 cells in the control group, the cirrhosis group and the treatment group of the small pig liver NPCs. The proportion of Th17 cells in the cirrhosis group and the treatment group of the mini-pigs was quantified relative to the control group. (J) The heat maps show the expression of fibrosis-associated differential genes in Th17 cells of the livers of the cirrhosis group and the treated group of piglets. (K) Epigenetically dysregulated liver SECs produce IGFBP7 and ADAMTS1 to stimulate profibrotic Th17 cell responses in humans and small pigs. This cellular interaction may depend on endothelial-derived IGFBP7/ADAMTS 1-mediated enhancement of TGF β 1-Smad2 signaling in the circulatory system.

FIG. 7: (A) NASH model and therapeutic schematic for Hdac2 endothelial specific knockout mice. Endothelial-specific Hdac2 knockout mice (Hdac 2) were obtained by crossing endothelial-specific cre mice with Hdac2 flox mice ^iΔEC ). To test the effect of the combined targeted inhibition of HDAC2/DNMT1 cross-regulation, hdac2 ^iΔEC Mice were also treated with the DNMT1 inhibitor azacitidine (AZA) (Hdac 2) ^iΔEC + AZA) treatment. Liver fibrosis, liver function and enrichment of Th17 cells were then analyzed. (B) H&E. Control group and Hdac2 for analysis of sirius red and type I collagen staining ^iΔEC + liver histopathology in AZA mice. Hdac2 ^iΔEC + AZA mice hepatic fibrosis and control group (Hdac 2) ^+/+ ) Mice were compared. And (3) right: quantification of sirius red and type I collagen staining. Hdac2 ^iΔEC The proportion of positive staining of + 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 of liver SECs in mice. The method comprises the following steps: quantification of protein expression; the following: representative western blot images. N =3. (E, F) co-staining of mouse liver sections for CD34 (green) and desmin (red) (E) or Lyve1 (red) and CD34 (green) (F). The proportion of areas stained positive for CD34 in panel F was quantified relative to the control. Scale bar, 20 μm. N =5. (G) flow cytometry analysis of Th17 cell number in mice. Targeting inhibition of HDAC2 and DNMT1 in endothelial cells blocks Th17 cell responses in the mouse NASH model. And (3) right: percentage of Th17 cells. N =5. In all statistical analyses, 2 groups of comparison data were analyzed by two-tailed student's t-test; more than 2 sets of comparison data were analyzed by one-way anova followed by Tukey post hoc testing. Data are presented as mean ± SEM. * Hdac2 ^iΔEC Or Hdac2 ^iΔEC + AZA vs. control or NASH model vs. wild type; #, hdac2 ^iΔEC Nash model (control). * P is<0.05；***，P<0.001。##，P<0.01。

FIG. 8: (A) IGFBP7 in control group and epigenetic treatment group (Hdac 2) ^iΔEC + AZA) expression in mouse liver Endothelial Cells (ECs). N =6. (B-E) Gene knockout of Igfbp7 attenuated the Th17 response promoting fibrosis in the mouse NASH model. (B) Induction of Igfbp7 knock-out (Igfbp 7) ^-/- ) Schematic representation of the mouse NASH model. (C) By H&E. Control group and Igfbp7 for evaluation of sirius red and type I collagen staining ^-/- Liver histopathology of mice. And (3) right: quantification of sirius red and type I collagen staining. (D) Serum ALP and AST concentrations and hepatic hydroxyproline (Hyp) content. (E) Flow cytometry analysis of Igfbp7 ^-/- Percentage of mouse Th17+ cells in liver. N =6. (F) Schematic representation of the induction of mouse Th17 responses by recombinant IGFBP7 proteins. C57BL/6J mice weekly intraperitoneal injection of carbon tetrachloride (CCl) ₄ ) 2 times, 3 weeks in total; IGFBP7 recombinant protein was injected into the tail vein 1 time every 2 days, starting at week 2. Th17 responses were compared between mice treated with IGFBP7 and controls. (G) Flow cytometry analysis of the number of Th17 cells of mouse liver NPCs induced by IGFBP7 protein. And (3) right: percentage of Th17 cells. N =4. In all statistical analyses, data were analyzed by two-tailed student's t-test, expressed as mean ± SEM. * Hdac2 ^iΔEC +AZA，Igfbp7 ^-/- Or IGFBP7 vs. control. * P is<0.05；**，P<0.01。

FIG. 9: (A) ADAMTS1 in NASH and epigenetic therapy (Hdac 2) ^iΔEC + AZA) expression in liver Sinusoidal Endothelial Cells (SECs) in mice. N =6. (B) Western blot shows phosphorylation (p-Smad 2) levels of Smad2 in Human Umbilical Vein Endothelial Cells (HUVECs) transduced with ADAMTS1 shRNA (shADAMTS 1) or control shNC. N =3. (C) Schematic representation of the method for transplantation of human endothelial cell-derived Extracellular Vesicles (EV) into mice. Recipient mice were repeatedly injected with CCl ₄ To induce liver fibrosis. The shADAMTS 1-transduced HUVEC were treated with TGF-. Beta.for 2 days. EVs were isolated from cultures of huavecs transduced with shADAMTS1 or shNC (control) and transplanted into liver fibrotic mice, respectively. Responses to fibrosis were compared between recipient mice transplanted with shADAMTS1 or shNC EVs. (D) Injection CCl ₄ And sirius red staining of mouse liver after transplantation of shADAMTS1 or shNC EVs. N =5. (E) Flow cytometry analysis Th17 cell counts in mouse liver NPCs after EVs transplantation. And (3) right: percentage of Th17 cells. N =5. (F) Schematic representation of liver fibrosis promotion along the "endothelial HDAC2/DNMT1-IGFBP7/ADAMTS1-Th17" axis in NASH. Epigenetic reprogramming of liver Endothelial Cells (ECs) leads to "sinoendothelial-macroendothelial dysregulation," facilitating the production of profibrotic IGFBP7/ADAMTS1 in EVs. IGFBP7/ADAMTS1 from epigenetically dysregulated SECs stimulates a profibrotic Th17 cell response. In all statistical analyses, data were analyzed by two-tailed student's t-test, expressed as mean ± SEM. * shADAMTS1 or Hdac2 ^iΔEC + AZA vs. control. * P is<0.05；**，P<0.01。

FIG. 10: (A) Clustering of 22,374 liver non-parenchymal cells (NPCs) in the liver of 2 healthy and 2 cirrhosis patients. Left: total NPCs; and (3) right: healthy and cirrhosis NPCs. (B) The UMAP plots show the cluster analysis of liver NPCs for each sample separately. (C) Ratio of different NPCs cell lines in healthy and cirrhosis human liver. EC, endothelial cells; DC, dendritic cells; neu, neutrophil; mac, macrophages; EPCAM ⁺ ，EPCAM ⁺ Cells and bile duct cells. (D) Clustering analysis of liver ECs from 2 healthy and 2 cirrhosis patients. (E) different subpopulations of healthy and cirrhosis ECs.

FIG. 11: (A) 4 healthy and 3 cirrhosisClustering analysis of 42,314 NPCs from human liver. Flow sorting CD45 ⁺ And CD45 ^- NPCs for use in scRNA-Seq assays. Left: total NPCs; and (3) right: healthy and cirrhosis NPCs. (B) The heatmap shows the NPCs cluster marker gene and its tagged cell line (right). The (C) UMAP plot shows different cell lines of NPCs. EC, endothelial cells; DC, dendritic cells; neu, neutrophil; mac, macrophages; EPCAM ⁺ ，EPCAM ⁺ Cells and bile duct cells. (D) Clustering analysis of 4 healthy and 3 cirrhotic human liver ECs. The (E) UMAP plots show different subpopulations of ECs. Left: total ECs; and (3) right: healthy and liver cirrhosis ECs. (F) The UMAP plots show the expression of the selected marker gene in endothelial cells. (G) pie charts show the proportion of the ECs subpopulations. (H) Pseudo-temporal analysis of different subpopulations of healthy and cirrhosis human liver ECs.

FIG. 12: (A, B) heatmap shows the expression of endothelial and mesenchymal marker genes in different cell lines of human total NPCs (A) and liver cirrhosis patients NPCs (B). (C) GSEA enrichment analysis of mesenchymal cell differentiation in endothelial cells of healthy and cirrhosis human liver. (D) Schematic representation of a possible "sinoendothelia-macroendothelia disorder" in the liver of a cirrhosis human.

FIG. 13: (A) The expression of HDACs in different grades of human liver fibrosis in GSE84044 data. F0-F4, different pathological grades of human liver fibrosis. The expression levels of liver HDACs of class F1-F4 were quantified relative to healthy liver (F0). (B) qPCR showed that HDAC2 and DNMT1 are CD45 in liver of healthy and cirrhosis patients ⁺ Expression in NPCs. N =3. (C) Western blot shows the expression of HDAC2 and DNMT1 in cultured human umbilical vein endothelial cells, and the knock-down of HDAC2 by shHDAC2 up-regulates the expression of DNMT 1. The following steps: quantification of protein expression; the following: representative protein bands. The data were statistically analyzed by two-tailed student's t-test and expressed as mean ± SEM; * shHDAC2 vs. control; * P is<0.05，**，P<0.01。N＝3。

FIG. 14: (A) Cluster analysis of 40,570 NPCs from 1 control group, 2 cirrhosis group and 2 treatment groups of miniature pig liver. (B) GO enrichment analysis of miniature pig liver ECs. Epigenetic treatment of HDAC2i and DNMT2i restored most of the altered functions of liver ECs in the mini-pig NASH model.

FIG. 15: (A) Cluster analysis of miniature pig liver ECs in 1 control group, 2 cirrhosis groups and 2 treatment groups; (B) expression of a selectable marker gene in ECs. (C) The UMAP plots show different subpopulations of ECs for control, sclerosing and treatment groups. (D, E) heatmap shows the expression of genes associated with epigenetic changes (histone modification and DNA methylation) in different cell lines (D) of control, cirrhosis and treated mini-pig liver NPCs and different subpopulations (E) of ECs. (F) The venn plot shows the number of distinct genes in different cell lines or subpopulations (D and E) of mini-pig liver NPCs between the cirrhosis and control groups and the treated and cirrhosis groups, respectively. Red numbers represent the number of genes recovered.

FIG. 16: (A) Venn diagram shows the differential genes in human and mini-pig cirrhotic liver. The numbers in parentheses indicate the basis factors recovered in the mini-pigs after treatment. (B, C) Violin plots show the expression of representative paracrine factors in different cell lines of human (B) and miniature pig (C) liver non-parenchymal cells (NPCs). (D) qPCR and ELISA showed that the control, cirrhosis and treated groups of miniature pig liver CD45 ⁺ Expression of IGFBP7 and ADAMTS1 in NPCs. Left: qPCR; and (3) right: and (4) ELISA. Data were analyzed by one-way anova followed by Tukey post hoc testing and are presented as mean ± SEM. * Liver cirrhosis vs. control, P <0.05. N =3.

FIG. 17: (A) Cluster analysis of T cells in 2 healthy and 2 cirrhotic human livers. (B) UMAP picture shows different cell lines of human T cells (CD 8) ⁺ And CD4 ⁺ Cells). (C-I) human CD45 from GSE136103 data ⁺ scRNA-Seq analysis of cells. (C) 35,806 CD45 of the livers of 5 healthy and 5 patients with cirrhosis ⁺ Clustering analysis of cells. (D) UMAP plots show different cell lines of NPCs. EC, endothelial cells; DC, dendritic cells; neu, neutrophil; mac, macrophages; EPCAM ⁺ ，EPCAM ⁺ Cells and bile duct cells. (E) UMAP picture shows T cell marker gene at CD45 ⁺ Expression in a cell. (F) Cluster analysis of T cells in 5 healthy and 5 cirrhosis human livers. (G) UMAP plots showing the differentiation of human T cellsCell line (CD 8) ⁺ And CD4 ⁺ A cell). (F-G) is T cell data derived from GSE 136103. (H) Violin diagram display Th17 ⁺ Expression of marker genes in different populations of T cells. (I) CD45 in 5 healthy and 5 patients with cirrhosis ⁺ Proportion of Th17 cells in the cells. (H-I) is Th17 cell data from GSE 136103. (J) UMAP picture shows different cell lines of T cells in minipig T cells (CD 8) ⁺ And CD4 ⁺ A cell).

The first embodiment is as follows: materials and methods

1. Patient and clinical specimens. Liver and plasma samples of patients were collected with informed consent at the western hospital of the university of Sichuan. Healthy liver tissue (no fibrosis) was obtained from patients with hepatic hemangiomas who underwent endoscopic liver resection. Liver cirrhosis liver tissue is obtained from patients histologically diagnosed as liver fibrosis. Patient information for scRNA-Seq, liquid phase chip 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 stage non-alcoholic steatohepatitis (F0-F1) (n = 12) and cirrhosis/fibrosis patients of different pathological grade (no cancer) (n = 75). The fibrosis grade of non-NASH livers was determined by fibrosis-4 index (Fib-4) and Transient Elastography (TE), and NAFLD fibrosis index (NFS), fib-4, TE and Controlled Attenuation Parameters (CAP) were used to determine the fibrosis and steatosis grade of NASH livers. Early NASH was mainly identified by histology of liver biopsy specimens, and some others were also identified by histology of liver biopsy specimens. Table 2 shows sample information for plasma analysis. The ethical committee of the western hospital, huaxi, university, sichuan, approved the study of the present invention. The study on human body is in accordance with the principles of Helsinki declaration.

TABLE 1 basic information of patients

Remarking: TBIL, total bilirubin; DBIL, direct bilirubin; IBIL, indirect bilirubin; AST, glutamic-oxaloacetic transaminase; ALT, glutamic-pyruvic transaminase; ALP, alkaline phosphatase; GGT, glutamyl transpeptidase. F0 or F4, different pathological grades of human liver fibrosis. Healthy and liver-cirrhosis livers #1 and #2 were used for the scRNA-Seq, and healthy and liver-cirrhosis livers #1, #2 and #3 were used for the liquid-phase chip analysis and the gene expression analysis.

Table 2 clinical data of plasma samples

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Remarking: m, male; f, female; NA, not applicable; PBC, primary biliary cirrhosis/liver fibrosis; AIH, autoimmune hepatitis-related cirrhosis/liver fibrosis; HBC, hepatitis b-related cirrhosis/liver fibrosis; CC, cryptogenic cirrhosis/liver fibrosis; NASH, non-alcoholic steatohepatitis-related cirrhosis/liver fibrosis; F0-F4, different pathological grades of human hepatic fibrosis; fib-4, fiberization-4 index; TE, transient elastography; NFS, NAFLD fibrosis index; CAP, controlled attenuation parameter; UDCA, ursodeoxycholic acid; MP, methylprednisolone; ETV, entecavir; TAF, tenofovir alafenamide; TDF, tenofovir disoproxil fumarate; FNB, fenofibrate; SAMe, S-adenosylmethionine.

2. A miniature pig. Male bama minipigs were obtained from dodsy Biological Technology co. The piglets were kept in individual cages, all reaching the Center of the major laboratory Animals (Dossy Experimental Animals Center), on a diet containing 2% cholesterol and 30% fat (by weight) supplemented with fructose and glucose. Laboratory animal ethics committee and medeto biotechnology limited of the second hospital, western, sichuan university approved the mini-pig experiment.

3. A mouse. C57BL/6J mice were obtained from the university of Nanjing institute for model animals. C57BL/6J-Hdac2 ^{em1(flox)Smoc} Mice were obtained from shanghai square model biotechnology, ltd. Igfbp7 ^-/- Mice were as described previously (reference 94). Expression of EC-specific Cdh5- (PAC) -Cre ^ERT2 Provided by Ralf h.adam (reference 95). Mixing Cdh5- (PAC) -Cre ^ERT2 Hybridization of mice with floxed Hdac2 mice to generate Hdac2 ^iΔEC/iΔEC Mouse (Hdac 2) ^iΔEC )。Hdac2 ^iΔEC Two months after birth, mice were treated with tamoxifen (250 mg/kg) by intraperitoneal injection 6 times, 1 time per day (3 days after 3 rd dose discontinuation), inducing a loss of Hdac2 endothelium specificity. Mice were housed in SPF-grade laboratory animal centers in second hospital, western, and fed on a standard 12 hour light/dark cycle. The animal experimental protocol was approved by the ethical committee on experimental animals in the second hospital, western, sichuan university.

4. A cell line. Human Umbilical Vein Endothelial Cells (HUVEC) were isolated from human umbilical cord. HUVEC in Endogro-VEGF complete medium (SCME 002, millipore) at 37 deg.C, 5% CO ₂ Is cultured in a humid environment. HEK293T was obtained from the American Type Culture Collection (ATCC). HEK293T in DMEM (11965092, gibco) containing 10% Fetal Bovine Serum (FBS) (1600044, gibco) at 37 ℃ 5% CO ₂ Is cultured in a humid environment.

NASH model. Mini-pig NASH model: male mini-pigs of 6 months of age were fed a Western diet (Western diet, WD) of 2% cholesterol, 30% fat and supplemented with high-sugar drinking water: 23.1g/L d-fructose and 18.9g/L d-glucose, intraperitoneal injection of 0.1ml/kg carbon tetrachloride (CCl) ₄ ) Injections were given 2 times weekly for 5 months. Control group of miniature pigs injected with CCl ₄ Excipient (CCl) ₄ vehicle) (corn oil). Each timeSerum samples were collected 1 time 15 days and plasma samples were collected 1 time per month. To detect the progression of cirrhosis and related molecular and cellular changes, surgical biopsies were taken 1 time every 2 months and liver tissue was removed for different analyses. Last injection of CCl ₄ After 2 days, all piglets were sacrificed and serum, plasma and liver samples were collected. Mouse NASH model: 8 weeks 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: 23.1g/L fructose and 18.9g/L glucose; the intraperitoneal injection dose is 0.5 mu l/g (0.8 g/kg) of CCl ₄ Injections were given 1 time per week for 3 months. Wild type control mice were fed normal diet and injected with corn oil. Last injection of CCl ₄ After 2 days, all mice were sacrificed and serum and liver samples were collected.

Combined targeted inhibition of hdac2 and DNMT 1. The strategy for the combined targeted inhibition of the mini-pig NASH model by HDAC2 and DNMT1 is shown in fig. 2B. Injection of CCl for miniature pigs ₄ Treatment was started two months later: 2 weeks were 1 course of treatment, with DNMT1 inhibitor Azacitidine (AZA) treatment in the first week: injecting AZA (dosage of 0.1-2.0 mg/kg/day, or 0.001-0.100 mg/kg/day, preferably 0.055 mg/kg/day) into abdominal cavity in the first 5 days, 1 time per day, and stopping administration for 2 days; therapy with the HADC2 inhibitor Mocetinostat (MGCD 0103) in the second week: MGCD0103 (dosage of 1-20 mg/kg/day, or 0.1-2.0 mg/kg/day, preferably 1.1 mg/kg/day) is intraperitoneally injected in the first 5 days for 1 time per day, and the preparation is stopped for 2 days. The treatment is repeated for 6 courses (5-10 courses can be adjusted according to actual conditions, and experimental data are not shown). Hdac2 ^iΔEC The mouse treatment strategy is shown in figure 7A. Hdac2 ^iΔEC Mice injected with CCl ₄ One month later, the abdomen was injected with AZA 1 time every two days for two months.

7. Small pig surgical biopsy. The small pig underwent liver biopsy through laparotomy. After a night after fasting, the ear was injected intravenously

The mini-pigs were 50 anesthetized and all procedures were performed under general anesthesia. Abdomen openingThen, a small piece of liver tissue is taken down by using a blunt dissection technique, and after hemostasis is performed on the liver incision, a drainage tube is arranged and the abdominal cavity is sutured. The postoperative mini-pigs were intravenously supplied with glucose and allowed to drink and eat one day after the operation. />

8. Liver cells, NPCs, CD45 ⁺ Separation of NPCs, ECs, SECs and MECs. Liver tissue of human, miniature pig and mouse was washed twice with cold PBS, minced, and incubated in digestion mix (1 mg/ml collagenase type I and 1mg/ml neutral protease type II in PBS) for 30 minutes at 37 ℃. After 30 minutes, the liver tissue was suspended to ensure complete separation. The digested liver sample was a uniform minimal block. The digested tissue was filtered multiple times with a cell strainer and cells were collected by centrifugation at 300g for 5 minutes. After removal of erythrocytes by RBC lysis buffer (R1010, solarbio), washed once and the hepatocytes and NPCs were separated by an additional 5min step of centrifugation at 50g at 4 ℃. The hepatocytes at the bottom were used for liquid-phase chip analysis and DNA methylation analysis, and the NPCs in the supernatant were used for liquid-phase chip, DNA methylation, magnetic bead sorting and western blot. For CD45 ⁺ NPCs、ECs(CD45 ^- CD31 ⁺ )、MECs(CD45 ^- CD34 ⁺ ) And SECs (CD 45) ^- CD34 ^- CD31 ⁺ ) The Dynabeads beads were washed 3 times with 1ml of pre-chilled MACS wash buffer (2 mM EDTA, 0.1% BSA, 1% penicillin/streptomycin in DPBS), incubated with CD45, CD34 or CD31 antibodies for 4 hours at 4 ℃ and washed 3 times with MACS wash buffer. The NPCs were resuspended in 300. Mu.l of MACS wash buffer and 200. Mu.l of Dynabeads-CD45 antibody conjugate were added, followed by incubation for 45 minutes at 4 ℃ on a rotator. After incubation, CD45 was separated with a magnet ⁺ Cell-bound magnetic beads and the supernatant was transferred to tubes containing Dynabeads-CD31 or-CD 34 antibody conjugates. Similar to CD45 ⁺ Cells were collected, dynabeads-CD31 or-CD 34 antibody conjugate and the collected supernatant were incubated at 4 ℃ for 45 minutes on a rotator, and CD31 was separated with a magnet ⁺ CD45 ^- Cells (ECs) or CD34 ⁺ CD45 ^- Magnetic beads of cells (MECs). For SECs isolation, negative sorting was first performed using Dynabeads-CD45 and-CD 34 antibody conjugates, followed by positive sorting using Dynabeads-CD31 antibody conjugates. Will have CD45 ⁺ 、CD31 ⁺ CD45 ^- 、CD34 ⁺ CD45 ^- Or CD31 ⁺ CD45 ^- CD34 ^- The magnetic beads of the cells were washed 5 times with cold MACS wash buffer and then used for subsequent experiments. Mini-porcine NPCs incubated with Percp-Cy5.5-CD45, FITC-CD34, and PE-CD31 antibodies for 30min, flow cytometry isolated Mini-porcine CD45 ⁺ NPCs、MECs(CD45 ^- CD34 ⁺ ) And SECs (CD 45) ^- CD34 ^- CD31 ⁺ ). After washing, the mixture is passed through FACSAria ^TM III flow cytometry (BD Biosciences) separation of CD45 ⁺ Cells, MECs and SECs.

9. Flow cytometry analysis. Mouse NPCs were isolated and incubated with BV 421-labeled rat anti-mouse CD4 antibody (BV 421-rat anti-mouse CD 4) (562891, BD Biosciences) for 30min. After washing, it was fixed and permeabilized with BD Cytofix/Cytoperm solution (554714, BD Biosciences) for 30 minutes, and then stained with FITC-labeled rat anti-FOXP3 (FITC-rat anti-FOXP 3) (11-5773-82, invitrogen) and PE-labeled rat anti-ROR γ t (PE-rat anti-ROR γ t) (12-6989-82, invitrogen) for 30 minutes. After cell fixation, flow cytometry analysis was performed on a FACS Calibur (BD Biosciences) and analyzed with Flow Jo V10.

10. Sample collection and histological analysis. Liver tissues of human, mini-pig and mouse were fixed with 4% paraformaldehyde or sectioned after OCT embedding. Serum samples from piglets and mice were stored at-20 ℃ and plasma samples from humans and piglets at-80 ℃. 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. Immunofluorescence analysis. The OCT-embedded liver tissue was cut into 6 μm sections, fixed with 4% paraformaldehyde for 5min, washed with PBS, and blocked with 10% donkey serum at room temperature for 30min. Then, triton X-100 was permeabilized for 20 minutes at 0.3% in PBS and incubated overnight at 4 ℃ with anti-Lyve 1 (70R-LR 003, fitzgerad), anti-CD 34 (Ab 81289, abcam), anti-desmin (Ab15200, abcam), anti-type I collagen (Ab34710, abcam) or anti-Ki 67 (b 15580, abcam) antibodies, respectively. 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, stained with 4,6-diamino-2-phenylindole (DAPI) (C0065, solambio) for nuclei and mounted on coverslips. Images were captured by a confocal laser microscope (LSM 880, zeiss) and processed with ZEN (Zeiss).

12. Sirius red, oil red O, I collagen, ki67 staining and semi-quantification of CD34-Lyve1 and CD34-Desmin co-staining. Semi-quantitative analysis of sirius red, oil red O and type I collagen staining was performed with Image-Pro Plus 6.0 (Media Cybernetics, rockville, MD). Positive staining signals were assessed by color depth and staining area in selected regions, and semi-quantitation of sirius red, oil red O, and type I collagen staining was calculated as the ratio of positive staining signals to the total area of selected regions. Ki67 and CD34-lyve1 staining was semi-quantitated using Photoshop CC 2018 (Adobe Systems, CA). The percentage of positive cell number (ki 67) and staining positive area (CD 34) was calculated by comparison to the total cell number or positive staining area, respectively, of the selected region. Each set of 3 (mini-pigs) or 5 to 6 (mice) samples was used for quantification and 3 fields from each sample were selected for quantification. The average of 3 fields was used as a quantitative value for each sample. To quantify the differences between the groups, the value of each sample (positive signal/number of positive cells/percentage of positive area) for each group was quantified with the value of the wild-type or control group. The results for each group are shown as "fold of control or fold of wild type".

13. HDAC2, ADAMTS1 were knocked down in vitro. Shrnas of human HDAC2 (shHDAC 2: 5'-CAGACTGATATGGCTGTTAAT-3') or ADAMTS1 (shADAMTS 1: 5'-CAAAAACCACAGGAACTGGAAGCATAA-3') were cloned into plko.1 and transfected into HEK293T cells to generate lentiviral particles carrying sh-HDAC2 (i.e., shRNA targeting HDAC 2) or sh-ADAMTS1 (i.e., shRNA targeting ADAMTS 1) (negative controls were also obtained as required in the experiment). HUVECs were transduced with lentiviral particles and then selected with puromycin (1. Mu.g/ml) for 48 hours, respectively, before use in subsequent experiments.

14. Plasma Extracellular Vesicles (EVs) were isolated. Plasma EVs were separated by ultracentrifugation. Human and minipig plasma was thawed at 4 ℃ and centrifuged at 850g for 30 minutes at 4 ℃ to remove dead cells and particulates. The supernatant was centrifuged at 12,000g at 4 ℃ for 45 minutes to remove cell debris. Next, the supernatant was centrifuged at 110,000g at 4 ℃ for 2 hours. The supernatant was discarded. The EVs-containing particles were collected, resuspended in cold PBS, filtered through a 0.22 μm filter, and centrifuged at 110,000g for 2 hours at 4 ℃ to remove contaminating proteins. The supernatant was discarded and extracellular vesicles were collected for subsequent experiments. Isolation of supernatant EVs from HUVEC medium was performed with total exosome-isolating reagents (from cell culture medium) (4478359, invitrogen) according to the manufacturer's instructions. The collected cell culture supernatant EVs were used for transplantation experiments.

15. Extracellular Vesicle (EV) transplantation experiments. The EV transplantation experiment is shown in fig. 9C. 8 week old C57BL/6J mice were fed a normal diet and given 2 intraperitoneal injections of 1ml/kg CCl per week ₄ 1 week in total, then inject CCl intraperitoneally ₄ Tail vein injection of extracellular vesicles (10 μ g protein/mouse, about 10 per dose) ¹⁰ Individual extracellular vesicles) 2 times a week for 2 weeks. Mice were randomized into two groups and injected via tail vein with EVs from shNC-infected HUVECs medium (high ADAMTS 1) and EVs from shADAMTS 1-infected HUVECs medium (low ADAMTS 1). CCl for the last injection ₄ And extracellular vesicles, two days later, all mice were sacrificed and liver samples were collected.

16. Recombinant IGFBP7 therapeutic experiments. The recombinant IGFBP7 protein treatment experiment is shown in FIG. 8F. C57BL/6J mice 8 weeks old were fed a normal diet and were injected intraperitoneally 2 times a week with CCl at a dose of 1ml/kg ₄ 1 week in total, then inject CCl intraperitoneally ₄ And the exogenous recombinant mouse IGFBP7 protein (20. Mu.g protein/mouse) was injected intravenously 1 time two days for 2 weeks. Two days after the last injection of IGFBP7, all mice were sacrificed and liver samples were collected.

17. Quantitative real-time PCR (qPCR). Extraction of ECs, SECs, MECs and CD45 in human, miniature pig and mouse livers Using the RNeasy Mini Kit (QIAGEN) ⁺ Total RNA of NPCs, and use of Takara reverse transcription kit (Takara PrimeScript) ^TM RT Master Mix) (RR 036A) were reverse transcribed. All gene expression was measured using the Brilliant III Ultra Fast SYBR Green qPCR Master Mix kit (Agilent Technologies). Will be provided withAll samples were replicated three times, the data normalized by GAPDH and analyzed by ddCt method, error bars represent Standard Errors (SEM).

ELISA assay. Liver tissue was weighed and homogenized, centrifuged at2,000g for 20 minutes, and the supernatant was collected. Setting a blank control and making a standard curve according to the instruction of the type III procollagen (PC III), hyaluronic Acid (HA) and type IV collagen (IV-C) determination kit, and measuring an OD value at 450nm after incubation by an enzyme labeling reagent, a developing solution and a stop solution. And the sample concentration is obtained by conversion according to a standard curve. Human and minicar plasma, extracellular vesicles, SECs, MECs, and CD45 ⁺ IGFBP7 and ADAMTS1 levels of NPCs were measured in the same manner. Extracellular vesicles were treated with ultrasound (40 kHZ) for 3 minutes prior to analysis.

19. Hydroxyproline analysis. Mini-pig and mouse liver tissues were weighed, hydroxyproline extracted and measured using the hydroxyproline kit (BC 0255, solarbio), and hydroxyproline levels in the liver were determined according to the weight of liver used.

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. The blood glucose levels of the piglets were measured using a blood glucose meter (ACCU-CHEK Performa, roche, germany).

21. Western Blot (WB) analysis. HUVECs and EVs from human and small pigs and human liver CD45 were extracted with RIPA lysis buffer (P0013B, beyotime Biotechnology) supplemented with protease inhibitors and phosphatase inhibitors ⁺ Total protein of NPCs. Primary antibodies include rabbit anti-HDAC 2 (57156, cell Signaling Technology) and anti-DNMT 1 (5032, cell Signaling Technology), rabbit anti-GAPDH (GB 11002, servicobio), rabbit anti-CD 81 (bs-6954R, bioss) and anti-GRP 94 (bs-0194R, bioss) and rabbit anti-Smad 2 (5339, cell Signaling Technology) and anti-Phospho-Smad 2 (18338, cell Signaling Technology). Peroxidase-conjugated goat anti-rabbit secondary antibody (GB 23303) was purchased from Wuhan Severe Biotech, inc. Each sample was loaded with 20. Mu.g of protein. Three biological samples per group were used for statistical analysis. Eggs were incubated with NIH Image J (http:// rsb. Info. NIH. Gov/ij/download. Html)The optical density of the white band was quantified.

22. Multiplex detection of histone post-translational modifications (PTM). Hepatocytes and NPCs were isolated from human livers, and histones were extracted from the hepatocytes and NPCs using an EpiQuik histone extraction kit. Histone H3 and H4 Post-translational modifications (PTM) multiplex analysis was performed with different site-specific antibodies, respectively (table 3). First, a capture antibody against histone PTM was covalently coupled to magnetic beads (table 4), and after the coupled beads were reacted with a sample containing histone PTM, successive washing was performed to remove unbound protein. Biotinylated histone H3 or H4 antibody was added to form a sandwich complex (sandwich complex), and finally Streptavidin-phycoerythrin (SA-PE) conjugate was added as a fluorescent indicator to form the final detection complex. Different histone PTMs were detected using a Bio-plex 200 liquid phase chip system (171000207, bio-rad). Different histone H3 and H4 PTMs were normalized with total H3 and H4, respectively. PTM multiplex assay was performed by hangzhou Jing Jie biotechnology limited.

TABLE 3 antibody information for PTM multiplex assays

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Remarking: 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 were coupled to different magnetic beads.

TABLE 4 bead information for PTM multiplex assays

23. scRNA-Seq of human and miniature porcine NPCs. NPCs were isolated from human and minipig livers, single cells were obtained and resuspended in PBS. scRNA-Seq was performed by a chromosome single cell platform (10X Genomics). Single cells are subjected to the steps of GEM (Gel Bead-in-Emulsion) formation, barcode serialization, GEM-RT (Gel Bead-in-Emulsion-Reverse Transcription) cleaning, cDNA amplification, library construction and the like, and finally, sequencing is carried out on Illumina Nova-seq 6000 (Illumina, USA). The scRNA-Seq of the small pig NPCs was performed by Yokou basic Olympic technologies, inc., guangzhou, and the scRNA-Seq of the human NPCs was performed by Yongyuan technologies, inc., beijing Nuo.

24. ATAC-seq of miniature pig ECs. Isolation of ECs from Mini-pig liver (CD 45) ^- CD31 ⁺ ) Separating cell nucleus, performing transposition reaction, PCR amplification and purification, library construction and other steps, and finally sequencing by using Illumina HiSeqTM 4000. ATAC-seq for Mini-pig ECs was performed by Guangzhou fundamentals Olympic Biotechnology, inc. and the data were quality controlled and aligned to reference sequences for subsequent analysis.

25. Reuse of public data. Microarray (Microarray) data of liver biopsy samples from cirrhosis patients (GSE 84044) and scRNA-seq data of NPCs from cirrhosis patients (GSE 136103) were from the GEO database. In the microarray data, expression levels of HDACs, DNMTs and IGFBP7 were quantified in F1-F4 fibrotic liver relative to healthy liver (F0). In scRNA-seq data, CD45 ⁺ And CD45 ^- Cells (GSM 4041150, GSM4041151, GSM4041153, GSM4041154, GSM4041155, GSM4041156, GSM4041158, GSM4041159, GSM4041161, GSM4041162, GSM4041164, GSM4041165, GSM4041166, and GSM 4041167) were used to analyze ECs profiles; CD45 ⁺ Cells (GSM 4041150, GSM4041153, GSM4041155, GSM4041158, GSM4041160, GSM4041161, GSM4041164, GSM4041166, GSM4041168, and GSM 4041169) were used for analysis of T cell profiles.

26. And (4) bioinformatics analysis. Pretreatment of scRNA-Seq data: the sequences of human and mini-pig liver NPCs scRNA-seq were aligned with human (Homo sapiens) transcriptome (GRCh38. P13) and pig (Sus scrofa) transcriptome (Sscofa 11.1) using Hisat2 v2.0.5, respectively. Samples from minipigs, from people in western hospital, and GSE136103 data were individually blanked using the saurat R package v3.1.1Supervised clustering and differential gene expression analysis. Cell filtration: low quality cells expressing less than 200 genes and genes expressing less than 3 cells were filtered. NPCs in miniature pig and human liver are filtered by setting different threshold values of percentage content of mitochondrial genes. Mitochondrial Gene content for two miniature pig data>15% of the cells were filtered out, and for sample data from people in hospital western china,>10% of the cells were filtered off. The experimenter filtered out the highly expressed populations of hepatocyte markers (ALB, APOE, APOB, etc.) and mesenchymal cell markers (COL 1A1, COL3A1, etc.). Data normalization: the data is normalized by the "Log Normalize" global scale normalization method in the "NormalizeData" function. Sample integration: the functions "FindIntegrationAnchors" and "Integratedata" are used to merge data and eliminate batch effects (batch effects). Dimension 1 to 20 are used to specify a neighbor search space (neighbor search space) to find an integration anchor. Normalization (scaled) and centralization (centered) is performed by the function "ScaleData". Clustering and visualizing: the function "RunPCA" is used for Principal Component Analysis (PCA), and Principal components 1 to 15 are used for functions "findneighborirs". Cells were clustered with the function "FindCusters" (resolution: 0.77) and visualized with the UMAP method. The UMAP, violin, heat, and dot maps were constructed using the saurtat packages (ggplot 2, pheatmap, and grid). The laboratory personnel used the cell line marker genes recommended in the database (http:// bioc. Hrbmu. Edu. Cn/CellMark/index. Jsp) and the marker genes used in the published articles (references 35, 36) to define the different cell lines. 7 cell lines were defined by expression of marker genes: t cells (CD 2) ⁺ 、KLRB1 ⁺ 、PTPRC ⁺ 、CD3E ⁺ 、TRAC ⁺ Etc.), B cells (CD 19) ⁺ 、CD22 ⁺ 、CD79B ⁺ 、MS4A1 ⁺ 、MZB1 ⁺ 、IGKC ⁺ Etc.), endothelial cells (PECAM 1) ⁺ 、CLECC4G ⁺ 、FLT1 ⁺ 、OIT3 ⁺ 、CLECC4M ⁺ 、CD34 ⁺ Etc.), macrophages (CD 163) ⁺ 、VSIG4 ⁺ 、CD68 ⁺ 、ADGR1 ⁺ 、MSR1 ⁺ 、C1QC ⁺ Etc.),Neutrophils (S100A 8) ⁺ 、S100A9 ⁺ 、CXCL8 ⁺ 、MSRB1 ⁺ Etc.), dendritic cells (CLEC 9A) ⁺ 、LGALS2 ⁺ 、IDO1 ⁺ 、CLORF54 ⁺ 、CLEC4A ⁺ 、CD83 ⁺ 、CD40 ⁺ 、CST3 ⁺ 、CD74 ⁺ Etc.) and EPCAM ⁺ Cells and bile duct cells (EPCAM) ⁺ 、KRT7 ⁺ 、SOX9 ⁺ 、CFTR ⁺ 、MMP7 ⁺ 、KRT19 ⁺ Etc.). Endothelial cells were re-clustered and defined as sinus endothelial cells (CLEC 4G) ⁺ 、OIT3 ⁺ 、CLEC4M ⁺ ) Macrovascular endothelial cells (CD 34) ⁺ ) And intermediate endothelial cells (CLEC 4G) ^- ，CD34 ^- ). T cells were re-clustered and defined as CD4 ⁺ (CD4 ⁺ ) And CD8 ⁺ (CD8A ⁺ ) T cells. By detecting CD4 ⁺ KLRB1 in T cells ⁺ 、FOXP3 ^- 、CCR6 ⁺ 、CCR4 ⁺ 、AHR ⁺ 、IL23R ⁺ 、IL17A ⁺ To identify Th17 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, with the differential genes satisfying both expression in more than 10% of the cells and log-fold changes of at least 0.25 between the two groups of cells. All differentially expressed gene analyses had the same threshold. For mini-pig scRNA-Seq, a total of 40,570 cells were obtained from mini-pig liver NPCs in 1 control group, 2 cirrhosis groups and 2 treatment groups, demonstrating 28 populations. 223,74 cells were obtained in total for Wash hospital human scRNA-Seq,2 healthy and 2 cirrhotic human liver NPCs, showing 25 populations. Different cell lines were identified by cell line marker genes. The methods of integrating multiple sets of samples, clustering, identifying different cell lines, and differentially expressed gene analysis are similar in the analysis of scRNA-Seq data in minipigs and humans. And (3) performing time-based analysis: the R packet-monocle v2.12.0 was used to construct a time-mimetic analysis. Information on cells labeled as endothelial cells and their subpopulations was entered and used to construct the monocle subjects. All values in the expression matrix are logarithmically transformed. Using the function "diffExperimental GeneTest "analyses differentially expressed genes between different groups (with different q-values, minipigs)<0.045, people in western China Hospital<1 e-12). "DDRTree" is used to reduce the dimension (Max _ components = 2). Cell receptor ligand interaction assay: the scRNA-Seq results were uploaded to the website of Cell Phone DB for Cell receptor ligand interaction analysis.

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 t-test (2-group comparisons) and one-way analysis of variance (ANOVA) and Tukey post-hoc test (more than 2 groups comparisons). All data are expressed as mean ± SEM. P <0.05 was considered statistically significant and all error bars represent SEM. In vivo experiments, the "n" value indicates the number of replicates per group of biological samples. * Liver 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; * P <0.001, #,; * P < 0.0001.

Example two: scRNA-Seq revealed vascular dysregulation and abnormal endothelial classification in human liver cirrhosis

NPCs were isolated from fresh normal and hardened human liver and scRNA-seq (10 xgenomics) was performed (FIG. 1A, table S1). Healthy liver tissue (without fibrosis) was obtained from patients with hepatic hemangiomas in western, and liver-cirrhosis liver tissue was obtained from patients with a histological diagnosis of cirrhosis. 22,374 NPCs from 2 healthy and 2 hepato-hardened liver were clustered into 25 clusters (FIGS. 10A-B). 7 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 bile duct cells (EPCAM) ⁺ ) (FIGS. 1B-C). The present experimenter found that the most different genes of ECs were found among all cell types examined (fig. 1D). The proportion of hepatosclerotic liver ECs was significantly increased compared to healthy liver (fig. 10C).

Previous studies by the present experimenter have shown that vascular endothelial cells are able to form a vascular microenvironment, regulating liver regeneration and fibrosis by paracrine/vasosecretory factors (ref 28). Thus, the present invention further analyzes subsets of ECs in human liver. Human liver ECs clustered into 12 clusters (fig. 10D), defined by marker genes CLEC4G, OIT and CD34 as Sinus Endothelial Cells (SEC) and large vessel endothelium (MEC) (fig. 1E, fig. 10E). The number of MECs was significantly increased and the number of SEC was significantly decreased, which means that a disorder from SEC to MEC occurred in the liver cirrhosis (fig. 1E-F). To validate this "sinoendothelial-macrovascular endothelial classification abnormality", the experimenter also analyzed the data of the human liver database GSE 136103. 42,314 NPCs of 4 healthy livers and 3 hepato-hardened liver clustered into 28 clusters (FIG. 11A), defining 7 cell lines (FIGS. 11B-C). Liver ECs were further clustered and defined as SEC and MEC (fig. 11D-F). GSE136103 data also showed a significant increase in MECs, a significant decrease in SECs in cirrhosis liver (fig. 11G), and the presence of "sinoendothelial-macrovascular endothelial disorder" (fig. 11H). The experimenter found that expression of mesenchymal marker genes was lower in total ECs and in liver cirrhosis ECs, and that the degree of "mesenchymal differentiation" enrichment was relatively lower in the examined types of liver ECs (fig. 12A-C). These data suggest that hepatic SECs may be vascularly dysregulated in the fibrotic liver (fig. 12D).

Example three: "sinoendothelial-macrovascular endothelial disorders" by epigenetic reprogramming of liver ECs to induce profibrosis "

Epigenetic regulation (histone modification and DNA methylation) plays an important role in the progression of liver fibrosis. However, the functional role of histone modification and DNA methylation in different cell lines of human liver cirrhosis/fibrosis NPCs is not clear. To this end, the experimenter first determined histone modifications of parenchymal cells and NPCs in healthy liver and cirrhosis liver, and liquid chip analysis of histone H3 and H4 modifications showed a significant reduction in histone acetylation of NPCs in human cirrhosis liver compared to healthy liver, without significant difference in parenchymal cells (hepatocytes) (fig. 1G), indicating apparent genetic reprogramming of NPCs in human cirrhosis liver.

The experimenter further investigated the epigenetic changes of different subsets of NPCs in healthy and cirrhotic human liver. 1,239 genes associated with 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. Histone modification and DNA methylation-associated genes were the most varied in vascular ECs among the 7 NPCs cell lines tested compared to the normal group (fig. 1H). These findings suggest that epigenetic changes in endothelial cells may promote fibrosis by stimulating deregulation of vascular microenvironment function.

Histone acetylation is regulated by Histone Deacetylases (HDACs). To determine which HDACs(s) play an important role in histone modification of the liver, the experimenter analyzed the expression of all HDACs in fibrotic liver and endothelial cells, respectively. Human fibrotic livers are classified into different pathological grades, grade F0-F4, with grade F4 being the most severe stage of fibrosis. Of all HDACs, HDAC2 expression was consistently increased in the liver of patients of grade F2-F4 (fig. 1I, fig. 13A, data from GSE 84044). Furthermore, HDAC2 expression was upregulated in endothelial cells of human cirrhotic liver compared to healthy human liver (fig. 1J). Quantitative PCR (qPCR) showed that although HDAC2 and DNMT1 are also in other cell types (CD 45) ⁺ NPCs), but between healthy and cirrhosis groups, liver endothelial cells (CD 45) ^- CD31 ⁺ ) There was a significant difference in HDAC2 expression (fig. 1K, fig. 13B).

DNA methylation is another common form of epigenetic modification, usually synergistic with histone modification. Thus, the experimenter evaluated the expression of DNA methyltransferases (DNMTs) in human fibrotic liver. DNMT1 was found to vary relatively most significantly in fibrotic liver in patients of grade F2-F4 (fig. 1L, data from GSE 84044), and DNMT1 expression in human cirrhotic liver ECs was significantly enhanced relative to human healthy liver.

In the isolated human liver cirrhosis liver CD45 ^- CD34 ^- CD31 ⁺ In SECs, HDAC2 and DNMT1 expression levels were significantly upregulated compared to healthy human liver (fig. 1O). In Human Umbilical Vein Endothelial Cells (HUVECs), knockdown of HDAC2 by shRNA (shHDAC 2) upregulated expression of DNMT1 (fig. 13C). These data suggest that aberrant activation of HDAC2 and DNMT1 in liver ECs may lead to "sinoendothelia-macrovascularisationEndothelial dysfunction ", thereby promoting liver fibrosis and cirrhosis.

Example four: combined targeted inhibition of HDAC2 and DNMT1 in alleviating liver fibrosis in the mini-pig NASH model

The physiological characteristics of the miniature pig are similar to those of human beings, and the miniature pig can better simulate the metabolic disorder of the human beings. Thus, the experimenter constructed a mini-pig NASH model to investigate the effects of vascular disorders on cirrhosis and related mechanisms. Literature reported studies have shown (ref 66), western diets (high fat, high cholesterol, high fructose and sucrose diets) and chemicals (CCl) ₄ ) The injury induces rapid fibrosis development in mouse NASH. Thus, the experimenter used the Western Diet (WD) in combination with repeated CCl ₄ Injection induced the mini-swine NASH model. To describe the pathological role of endothelial-derived HDAC2/DNMT1 in the mini-pig NASH model, the experimenter also treated the mini-pigs with HDAC2 inhibitor (HDAC 2 i) and DNMT1 inhibitor (DNMT 1 i) (fig. 2A-B). The blood glucose levels were higher in the small pigs in the cirrhosis group compared to the control group (fig. 2C), and the liver fibrosis index and serum liver function index were elevated in the cirrhosis group, similar to human NASH pathology (fig. 2D-E). HDAC2i + DNMT1i combination treatment reduced blood glucose, liver fibrosis index, and serum liver function index compared to the cirrhosis mini-pig group (fig. 2C-E). In addition, serum total cholesterol was elevated in the cirrhosis group and decreased after treatment (fig. 2E). Thus, in the mini-pig NASH model, abnormal induction of HDAC2 and DNMT1 in ECs leads to liver fibrosis and liver function impairment.

Next, the experimenter passes through H&E. Sirius red, type I collagen, oil red O and Ki67 staining evaluated the histopathology, collagen deposition, lipid droplet deposition and cell proliferation of the livers of piglets in the control, cirrhosis and treatment groups (fig. 2F-G). The liver of the cirrhosis animals showed a unique pseudosize of She Biaoxing compared to the control group showing normal histological features. WD + CCl ₄ Late stage cirrhosis, collagen deposition and lipid deposition were induced in the cirrhosis mini-pig group. HDAC2i + DNMT1i combination therapy improved the histopathological phenotype, reduced liver cirrhosis, and reduced collagen deposition and lipid accumulation. Ki67 staining patternClearly, HDAC2i + DNMT1i treatment also increased hepatocyte proliferation.

The experimenter then isolated the mini-porcine SECs (CD 45) by flow cytometry ^- CD34 ^- CD31 ⁺ )、MECs(CD45 ^- CD34 ⁺ CD31 ⁺ ) And other CDs 45 ⁺ NPCs。CD45 ⁺ The expression of HDAC2 and DNMT1 in NPCs did not differ significantly between the different groups. Compared to the control group, the expression of HDAC2 and DNMT1 was significantly up-regulated in SECs and MECs of the liver of the cirrhosis mini-pigs. HDAC2i + DNMT1i treatment blocked upregulation of HDAC2 and DNMT1 expression in liver cirrhosis mini-pig liver SECs and MECs (fig. 2H). The therapeutic effect of HDAC2I and DNMT1I on the mini-pig NASH model suggests that aberrant induction of HDAC2/DNMT1 plays a pathogenic role in NASH (fig. 2I).

Example five: combined epigenetic targeted inhibition normalizes dysregulated liver endothelial classifications in the mini-pig NASH model

The experimenter next analyzed whether aberrant induction of HDAC2/DNMT1 in miniature pigs resulted in "sino-endothelium-large vessel endothelium dysregulation" as found in human cirrhotic liver (fig. 1P). The liver NPCs of the mini-pigs (normal, sclerosing and treatment groups) were isolated and subjected to scRNA-seq, and the mini-pig NASH model was analyzed at the single cell level (FIG. 3A). 40,570 NPCs from 1 control, 2 cirrhosis and 2 treatment groups were clustered into 28 clusters (fig. 14A). Similar to human scRNA-Seq, 7 cell lines were identified by expression of marker genes, including T cells, B cells, EC, mac, neu, DC and EPCAM ⁺ Cells (FIGS. 3B-C). The experimenter also found that the proportion of ECs in the small pig cirrhotic liver (9.70% versus 5.90%) was significantly increased compared to healthy liver, while this increased proportion of ECs was significantly reduced by treatment with HDAC2i + DNMT1i (4.55% versus 9.70%) (fig. 3D). To reveal the effect of HDAC2/DNMT1 in different NPCs cell lines, the experimenters compared the gene differences in 7 cell lines of the cirrhosis group and the control group (cirrhosis vs. control) and the treatment group and the cirrhosis group (treatment vs. cirrhosis), respectively. The degree of gene differentiation of vascular endothelial cells in the cirrhosis minipig group was greatest among all cell types tested, most of which were restored following HDAC2i + DNMT1i treatment (fig. 3E)). GO and pathway enrichment shows that the signaling involved in Molecular Function (Molecular Function), biological Process (Biological Process) and Cellular composition (Cellular Component) is significantly changed (FIG. 14B), and the KEGG signaling pathways of Th17 cell differentiation, non-alcoholic fatty liver, chemokine, TNF, HIF-1, MAPK, PI3K-Akt, etc. are also significantly changed (FIG. 3F). Furthermore, epigenetic treatment of HDAC2i and DNMT2i restored most of the above GO and KEGG changes in liver ECs of the NASH group, indicating normalization of minicar ECs after treatment (fig. 3F, fig. 14B).

The unique effect of epigenetic changes in ECs in the mini-pig NASH model led the experimenter to explore the link between HDAC2/DNMT1 activity and "sino-large vessel endothelial classification abnormalities" in liver fibrosis. Minicar ECs were clustered into 15 clusters (fig. 15) and identified as SEC and MEC by gene signature (fig. 3G, fig. 15B-C). Similar to the vascular disorder in human cirrhotic livers, the MEC ratio of cirrhotic mini-pig livers was increased and the SEC ratio decreased compared to control mini-pig livers. In the treated piglet liver, the NASH-induced "sino-endothelium-large vessel endothelium disorder" was significantly reversed after HDAC2i + DNMT1i treatment (fig. 3H).

In the EC subpopulation, the differential genes of SECs were relatively the most abundant (sclerosing vs. control), and most of the differential genes were restored after HDAC2I + DNMT1I treatment (treatment vs. sclerosing) (fig. 3I). The hepatic EC subgroup chronomimetic analysis also showed that liver cirrhosis mini-pigs had "sino-endothelia-great vessel endothelial disorder" that was normalized by HDAC2i + DNMT1i treatment (fig. 3J). The experimenter also found that histone modifications and DNA methylation related genes were relatively most varied among SECs in the cirrhosis mini-pigs, and most genes were restored after HDAC2i + DNMT1i treatment (fig. 15D-F). Thus, data from the mini-pig NASH model indicate that abnormal epigenetic changes stimulate "sino-large vessel endothelial dysfunction", abnormal endothelial classification, and increased liver fibrosis (fig. 3K).

Example six: paracrine/vaso-secretory factor reprogramming of epigenetically dysregulated SECs in human patients and mini-pig NASH models

ECs can regulate liver regeneration by interacting paracrine/vasosecretory factors with surrounding cells. To elucidate the mechanism of hepatic ECs profibrosis in epigenetic disorders, experimenters analyzed the differential genes in cirrhotic human and mini-pig ECs at the single cell level (fig. 16A) and observed reprogramming of the vasosecreted factors associated with "sinoendothelial-macroendothelial disorder" (fig. 4A-D). Compared with healthy ECs, the metallopeptidase ADAMTS1, insulin-like growth factor binding protein 7 (IGFBP 7), DLL1, and ADAMTS6 in liver ECs of liver cirrhosis human were all significantly changed (FIG. 4A). The relationship of these vascular secretion factors to the progression of human fibrosis was analyzed using the human public database. In the fibrotic liver of patients of grade F2-F4, the expression of IGFBP7 and ADAMTS1 was gradually increased (FIG. 4B). The experimenter found that IGFBP7 and ADAMTS1 were specifically expressed at relatively highest levels in human and miniature pig ECs among the genes of the vascular secretion factors (FIG. 4C, FIGS. 16B-C). Furthermore, IGFBP7 and ADAMTS1 were selectively up-regulated in human liver-cirrhosis liver ECs compared to healthy ECs, and mRNA and protein levels of IGFBP7 and ADAMTS1 were significantly elevated in liver-cirrhosis SECs (fig. 4E-F). These results suggest that epigenetic reprogramming of the angiogenic factors occurs in dysregulated human liver ECs and that endotheliogenesis of IGFBP7 and ADMATS1 may contribute to liver fibrosis and cirrhosis.

In the mini-pig NASH model, mRNA levels of IGFBP7 and ADAMTS1 were significantly elevated in liver-cirrhosis SECs and MECs, while protein levels of IGFBP7 and ADAMTS1 were only up-regulated in liver-cirrhosis SECs. In addition, treatment with HDAC2i and DNMT1i blocked increases in mRNA and protein levels of IGFBP7 and ADAMTS1 in liver SECs of cirrhosis piglets (fig. 4G-L, fig. 16D). Analysis of chromatin openness helps to reveal epigenetic regulation of gene expression, so the laboratory personnel performed chromatin openness assays (ATAC-seq) with control, cirrhosis and treatment groups of miniature pig liver ECs. Chromatin openness of IGFBP7 and ADAMTS1 promoters was significantly enhanced in liver cirrhosis ECs compared to control ECs, and was significantly reversed after HDAC2i + DNMT1i treatment (fig. 4M). Thus, IGFBP7 and ADAMTS1 upregulation may differentiate between normal and deregulated SECs in liver cirrhosis, whereas epigenetic targeting of profibrotic IGFBP7 ⁺ ADAMTS1 ⁺ Out of regulationEpigenetic treatment of EC subpopulations may block liver fibrosis (fig. 4N).

Example seven: IGFBP7 and ADAMTS1 from deregulated SECs predict progression of liver fibrosis in humans and mini-pigs

To determine the clinical value of circulating IGFBP7 and ADAMTS1 in human cirrhosis/fibrosis or NASH, the experimenter assessed the concentration of IGFBP7 and ADAMTS1 in the plasma of human patients (table S2). Plasma concentrations of IGFBP7, ADAMTS1, glutamic-pyruvic transaminase (ALT) and glutamic-oxaloacetic transaminase (AST) were significantly higher in patients with cirrhosis/fibrosis than in healthy human samples (fig. 5A). Since some cirrhosis/liver fibrosis patients may have normal plasma ALT or AST levels, there is a need to find sensitive biomarkers to clinically diagnose liver cirrhosis/liver fibrosis. Thus, the experimenter evaluated the value of plasma IGFBP7 and ADAMTS1 as clinical biomarkers. Patients with cirrhosis/liver fibrosis are divided into two groups according to their plasma liver function index: normal plasma ALT and AST concentration panels and abnormal plasma ALT and AST concentration panels (fig. 5B). Plasma concentrations of IGFBP7 and ADAMTS1 were compared in patients with normal and abnormal liver function and cirrhosis/fibrosis. Importantly, plasma concentrations of IGFBP7 and ADAMTS1 were significantly higher in ALT or AST normal cirrhosis/fibrosis patients than in healthy humans (fig. 5C). The cirrhosis/liver fibrosis cohort includes non-alcoholic steatohepatitis-related cirrhosis/liver fibrosis (NASH), hepatitis b-related cirrhosis/liver fibrosis (HBC), autoimmune hepatitis-related cirrhosis/liver fibrosis (AIH), primary biliary cirrhosis/liver fibrosis (PBC) and cryptogenic cirrhosis/liver fibrosis (CC). An increase in plasma IGFBP7 and ADAMTS1 levels was observed in all these patients compared to healthy humans (fig. 5D). Therefore, in the absence of liver dysfunction, IGFBP7 and ADAMTS1 secreted from blood vessels are likely to be biomarkers for diagnosing liver cirrhosis/fibrosis.

Next, the experimenter evaluated 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 (manifested as simple steatosis). NASH patients are 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 IGFBP7 and ADAMTS1 concentrations were significantly elevated in NASH patients (fig. 5F). In addition, plasma IGFBP7 and ADAMTS1 concentrations were not statistically elevated in patients with simple fatty liver, suggesting that plasma IGFBP7/ADAMTS1 concentrations have significant clinical value in differentiating NASH from simple fatty liver (fig. 5F).

The correlation of IGFBP7/ADAMTS1 with NASH progression raises the possibility that the profibrosis of deregulated SECs depends on the vascular secreted IGFBP7 and ADAMTS1. The experimenter then evaluated epigenetically dysregulated SECs to release IGFBP7 or ADAMTS1 to contribute to the NASH hypothesis. Extracellular Vesicles (EVs) are unique lipid bilayer particles released by cells. Molecules packaged in EVs may facilitate cellular communication in many biological processes. Thus, the experimenter analyzed whether endothelial cell-produced IGFBP7 and ADAMTS1 were assembled in EVs and released into the circulation. For this purpose, EVs were extracted from human and piglet plasma by ultracentrifugation and verified by electron microscopy and immunoblot analysis (fig. 5G). The concentration of IGFBP7 and ADAMTS1 was higher in the cirrhosis group mini-pigs and human EVs than in the control group. In the mini-pig NASH model, HDAC2i + DNMT1i treatment reduced the increase in concentration of the cirrhosis group EV IGFBP7/ADAMTS1 (FIG. 5H). IGFBP7/ADAMTS1 concentrations in EVs from ALT/AST normal cirrhosis/fibrosis patients or NASH patients were also significantly elevated compared to healthy humans (FIGS. 5I-J). The data of the present invention indicate that IGFBP7 and ADAMTS1 in EVs are useful biomarkers to assess NASH progression prior to liver dysfunction (fig. 5K).

Example eight: IGFBP7 in patients with cirrhosis and mini-pig NASH model ⁺ ADAMTS1 ⁺ Dysregulated SECs induce a profibrotic Th17 cell response

Dysregulated ECs can interact with neighboring cells by forming a dysregulated vascular microenvironment to promote fibrosis. The present invention seeks to disclose IGFBP7 ⁺ ADAMTS1 ⁺ Deregulated SECs enhance the cellular machinery of liver fibrosis through cellular communication. Receptor and ligand expression profiles of different NPCs cell lines were analyzed based on the CellPhoneDB database. Cellular interaction prediction indicates that, in patients with cirrhosis and in small NASH pigs,the interaction of deregulated EC with T cells was evident (fig. 6A). Notably, although the predicted interaction in human data was significant between ECs and macrophages, there was less EC-macrophage interaction in mini-pig NPCs. Since T cells are the relatively most abundant type of liver NPCs, the present invention primarily analyzes the interaction between ECs and T cells.

Previous studies have shown that there is a synergistic (synergistic) or complementary (complementary) effect between IGFBP7, ADAMTS1 and transforming growth factor-beta 1 (TGF-. Beta.1) (references 77, 78) that can recruit Th17 cells, a CD4 involved in NASH and liver fibrosis progression (references 80, 81) ⁺ Subgroup T (reference 79). Since Smad2 is an important downstream factor in TGF-. Beta.1 induced Th17 cells, the human liver CD45 was analyzed by the experimenter ⁺ Phosphorylation of Smad2 in NPCs. Cirrhosis human CD45 compared to healthy NPCs ⁺ Smad2 phosphorylation levels were significantly elevated in NPCs (fig. 6B). The laboratory personnel were next analyzed cell lines of recruited human and minipig T cells. Human T cells from 2 cirrhosis and 2 healthy livers were clustered and identified as 20 populations (fig. 17A), defined as CD4 ⁺ And CD8 ⁺ T cells (fig. 17B). CD4 ⁺ T cells were further clustered (FIG. 6C), th17 cells by Th17 ⁺ The marker gene was marked out (FIG. 6D). The cell number of Th17 cells in the liver of the cirrhosis patients was significantly higher than that of healthy liver (fig. 6E). To validate the results of human patients, the experimenter analyzed Th17 cells in the GSE136103 data (fig. 17C-I). The number of Th17 cells in liver cirrhosis human liver was also similarly significantly increased compared to healthy human liver. These data suggest a profibrotic role for Th17 cells in human liver cirrhosis.

Next, the experimenter explored the cellular interaction between liver ECs and Th17 subpopulations in the mini-pig NASH model. Similar to human T cells, clustering of T cells from the livers of piglets in the control, cirrhosis and treatment groups identified 16 populations, defined as CD4 ⁺ T cells and CD8 ⁺ T cells (fig. 6F, fig. 17J). CD4 ⁺ Among T cells, th17 cells are replaced by Th17 ⁺ Marker genes were identified (FIGS. 6G-H). Compared with control group miniature pigsThe number of Th17 cells in the hardened piglet liver was also significantly increased. Furthermore, the combination therapy of HDAC2I + DNMT1I blocked the increase in Th17 cell number in the damaged piglet liver (fig. 6I). The experimenter also found a significant reduction in the expression of fibrosis-associated genes in Th17 cells in the treatment group of mini-pigs compared to cirrhosis mini-pigs (fig. 6J). According to the data of the present invention, it was hypothesized that epigenetically dysregulated SECs might recruit and activate profibrotic Th17 cells in human and miniature pig livers, which might be mediated by secreted IGFBP7/ADAMTS1 (FIG. 6K).

Example nine: epigenetic dysregulated SECs generate profibrotic Th17 responses in the mouse NASH model

To determine the effect of HDAC2/DNMT1-IGFBP7/ADAMTS1 axis on Th17 cell response and liver fibrosis function in ECs, experimenters generated Hdac2 ^iΔEC Mice (selective knock-out of HDAC2 in ECs) and by WD + CCl ₄ And (3) inducing to generate a mouse NASH model. By treating Hdac2 with the DNMT1 inhibitor AZA ^iΔEC Mouse (Hdac 2) ^iΔEC + AZA) obtained the effect of combined targeted inhibition of HDAC2+ DNMT1 (fig. 7A). Hdac2 after DNMT1i treatment ^iΔEC Mice, with significantly reduced indices of liver fibrosis, inflammation, collagen deposition, and liver fibrosis and liver function compared to control group injured mice (fig. 7B-C). To further study the interaction between endothelial-derived HDAC2 and DNMT1 in liver SECs of mice, the experimenter isolated the control group, HDAC2, by immunomagnetic beads (Dynabeads) ^iΔEC And Hdac2 ^iΔEC + AZA mice liver SECs (CD 45) ^- CD34 ^- CD31 ⁺ ). Western blot showed that Hdac2 in liver SECs of knockout mice up-regulated the expression of DNMT1 in SECs (FIG. 7D). Notably, in this mouse NASH model, CD34 was found to be expressed in some sinus endothelial cells, and targeted inhibition of endothelial-derived HDAC2 and DNMT1 in the treatment group reduced CD34 expression (fig. 7E-F). This staining result suggests the presence of "sino-macroendothelial dysfunction" in this mouse NASH model. Flow analysis showed that targeted inhibition of endothelial-derived HDAC2 and DNMT1 in the liver of treated mice reduced Th17 cell numbers (fig. 7G). Thus, epigenetic dysregulated liver in the mouse NASH modelDirty ECs may activate a Th17 response that promotes fibrosis.

Example ten: IGFBP7 enhances profibrotic Th17 responses in the mouse NASH model

qPCR showed that combined targeted inhibition of endothelial-derived HDAC2 and DNMT1 reduced IGFBP7 expression in fibrotic liver ECs (fig. 8A). To determine the role of IGFBP7 in stimulating Th17 responses in liver fibrosis, the experimenter analyzed IGFBP7 knockouts (IGFBP 7) ^-/- ) NASH phenotype of mice (fig. 8B). Gene knockout of Igfbp7 in mice significantly reduced the hepatic fibrosis response, i.e., collagen deposition (fig. 8C), serum liver function index, hepatic hydroxyproline content (fig. 8D), and Th17 response (fig. 8E). To further investigate whether IGFBP7 directly affected Th17 biology, C57BL/6J mice were tail-intravenously injected with recombinant mouse IGFBP7 protein (fig. 8F). Elevated IGFBP7 levels significantly enhanced the profibrotic Th17 response of the injured mouse liver compared to the control group (fig. 8G). These results indicate that IGFBP7 is a regulatory factor that enhances Th17 responses to promote liver fibrosis.

Example eleven: genetic inactivation of ADAMTS1 in mouse fibrosis model mitigates profibrotic Th17 responses

Similar to IGFBP7 expression, the experimenter found that ADAMTS1 was expressed in Hdac2 ^iΔEC Significant reduction in SECs in + AZA mice (fig. 9A). In addition, knocking down ADAMTS1 in human ECs by ADAMTS1 shRNA (shADAMTS 1) gene blocked Smad2 phosphorylation under TGF-. Beta.stimulation (FIG. 9B). Thus, the experimenter investigated whether endothelial-derived ADAMTS1 modulates Th17 biology using the "human-to-mouse" Extracellular Vesicle (EVs) transplantation method (fig. 9C). EVs were isolated from culture media of shADAMTS1 and control (shNC) infected HUVECs and transplanted into mice via tail vein. Liver fibrosis and Th17 responses were significantly reduced in mice transplanted with EVs lacking ADAMTS1 compared to mice treated with control EVs (fig. 9D-E). These results suggest a functional role for ADAMTS1 in promoting Th17 responses during liver fibrosis progression.

The present data reveal the endothelial-derived HDAC2/DNMT1-IGFBP7/ADAMTS1-Th17 axis that promotes liver fibrosis in human patients, mini-pig and mouse NASH models. Epigenetic aberrant interactions in liver ECs subsets lead to "sino-large vessel endothelial dysregulation", characterized by aberrant and dysregulated endothelial classification of SECs producing profibrotic IGFBP7/ADAMTS1, recruitment of Th17 cells by extracellular vesicles, forming a profibrotic vascular microenvironment (fig. 9F).

Summary of the invention

The pathogenesis of NASH involves systemic effects including metabolic dysfunction. Endothelial and hematopoietic cells in the circulatory system are directly linked to systemic stimuli, NASH is similar to many risk factors for circulatory/vascular complications. The present invention utilizes multiomic analysis to reveal how vessel-specific epigenetic changes reprogram interactive regulation of profibrosis in the liver circulatory system (blood vessels and hematopoietic cells) at the single cell level. The present invention integrates scRNA-Seq data from human and large animal NASH models and reveals how "sinus endothelium-large vessel endothelium disorders" stimulate a profibrotic Th17 cell response in NASH. In addition, the present invention found that abnormal HDAC2-DNMT1 cross-regulation in liver ECs in NASH leads to abnormal endothelial cell classification and the production of dysregulated IGFBPs ⁺ ADAMTS1 ⁺ Hepatic EC subpopulations to form a profibrotic dysregulated vascular microenvironment in the circulatory system. The present invention systematically studied the cellular phenotypes of NASH models in human patients and supplemented large animals and rodents using a hospital-bed to laboratory (bed to laboratory) approach, and this multi-species exploration strategy explains how aberrant epigenetic interaction regulation in the vascular microenvironment leads to profibrotic communication between endothelial and immune cells.

The present invention shows that abnormal epigenetic cross regulation of vascular endothelial cells promotes liver fibrosis in human and large animal NASH models. Integrating scRNA-Seq, histone modification and human patient cohort analysis, the present experimenter found that selective induction of HDAC2 and DNMT1 in specific subpopulations of hepatic vascular ECs was closely related to progression of liver fibrosis. Pharmacological and genetic targeting of HDAC2/DNMT1 by large animal and rodent NASH models further revealed that HDAC2/DNMT1 cross regulation in deregulated EC subpopulations stimulates production of profibrotic IGFBP7 and ADAMTS1 in extracellular vesicles, thereby recruiting Th17 cells, inhibiting liver regeneration and inducing fibrosis in NASH.

The experimenter first analyzed the phenotypic and molecular characteristics of endothelial and hematopoietic cells in NPCs of human patients at the single cell level. The vascular ECs epigenetic associated genes vary most among the NPCs tested, suggesting that ECs may be more susceptible to epigenetic changes in chronic diseases such as NASH. Endothelial cells may have a longer in vivo half-life in the circulatory system than hematopoietic cells, thereby accumulating more micro-environmental or systemic stimuli (e.g., metabolic stress). scRNA-Seq revealed that, in NASH, selective epigenetic changes in ECs lead to "sino-endothelium-macroendothelial dysregulation" and abnormal endothelial classification, forming a pro-fibrotic endothelial microenvironment. Through multiomic and multi-species analysis, the experimenter of the present invention found the profibrotic HDAC2/DNMT1-IGFBP7/ADAMTS1 axis in the dysregulated liver ECs subpopulation. Previous reports have shown that liver fibrosis can lead to the capillary vascularization of SECs and alter blood flow in the antrum (references 22, 23, 57). It should be noted that liver ECs are the major component of NPCs, and are reported to account for approximately 5% to 15% of NPCs. The proportion of ECs in purified NPCs appears to vary from study to study and from individual to individual, depending on individual differences and the particular isolation method. In the present invention, scRNA-Seq analysis of liver ECs shows that chronic/metabolic damage to ECs and the resulting epigenetic modifications abnormality contribute to liver fibrosis in NASH. In NASH, dysregulation of hepatic ECs may be induced by histone and DNA modification abnormalities in the heterogeneous hepatic vascular system, and these deregulated subsets of ECs further interact with other circulating cells, such as Th17 cells, to form a microenvironment that promotes liver fibrosis.

scRNA-Seq analysis was able to reveal vascular disorders in the liver at the single cell level. Analysis of human patients and the mini-pig NASH model showed that aberrant epigenetic interaction regulation leads to dysregulation from sinus endothelium to large vessel endothelium, resulting in abnormal endothelial classification. The vascular disorders observed in the liver samples examined appear to be predominantly manifested as phenotypic and functional shifts within the ECs lineage. The fibrotic liver tested showed an increase in the number of ECsAnd the enrichment of "stromal cell differentiation" in the EC population is lower. Thus, this vascular dysregulation process appears to be distinct from endothelial mesenchymal transition (EndMT). Therapeutically, the modulation of HDAC2/DNMT1 interaction combined with the targeted inhibition abnormality normalizes endothelial classification and liver function (AST and ALT plasma levels), suggesting that endothelial dysfunction may precede liver injury. Regeneration of organs requires a functional vascular system that includes a blood supply and paracrine factors that promote regeneration and maintain homeostasis. Thus, dysfunction of the liver endothelium leads to abnormalities in paracrine factors, and thus to abnormalities in liver repair. The present study shows that epigenetically dependent liver ECs dysregulation in NASH results in the secretion of profibrotic IGFBP7 and ADAMTS1 into extracellular vesicles, and that, in the human and mini-pig NASH models, elevation of plasma IGFBP7 and ADAMTS1 levels precedes detectable parenchymal damage (elevation of plasma ALT and AST), and aberrant cross-regulation in combination with targeted inhibition of HDAC2/DNMT1 restores mini-pig plasma IGFBP7 and ADAMTS1 levels. Clinical findings in human patients are consistent with data on normalization of plasma AST and ALT levels in a mini-pig NASH model in combination with targeted inhibition of HDAC2/DNMT1 in ECs. Thus, the experimental data of the present invention indicate that chronic stress in NASH stimulates epigenetic reprogrammed IGFBP7 ⁺ ADAMTS1 ⁺ SECs are produced and pro-fibrotic IGFBP7 and ADAMTS1 are produced to enhance liver parenchymal damage. In this process, molecular markers involved in endothelial abnormalities (e.g., extracellular vesicles IGFBP7 or ADAMTS 1) may be used as therapeutic targets or biomarkers to assess fibrosis progression in NASH patients, particularly to differentiate NASH patients from simple fatty liver patients.

Abnormal cellular interactions in NASH can promote liver fibrosis. The invention discloses a single-cell map of a profibrotic vascular disorder in the circulatory system, and key molecules involved in the vascular disorder are determined through multiomic analysis of a liver cirrhosis patient cohort, transgenic mice, and small pig and rodent NASH models. During the vascular disorder, epigenetic reprogrammed liver SECs produce IGFBP7 or ADAMTS1 into extracellular vesicles to recruit profibrotic Th17 cells. Abnormal recruitment and activation of immune cells can stimulate liver fibrosis. In the present invention, predictive analysis of cell interactions using scRNA-Seq of NPCs showed interactions between T cells and reprogrammed ECs in human cirrhosis patients and the mini-pig NASH model. This prediction is demonstrated by data that, in the mini-pig and mouse NASH models, targeting HDAC2/DNMT1 treatment normalizes epigenetic changes in liver ECs and inhibits Th17 recruitment. scRNA-Seq, igfbp7 knock-out mice and extracellular vesicle transplantation demonstrated that the EC-Th17 interaction was at least partially dependent on IGFBP7/ADAMTS1 production by epigenetic reprogrammed liver SECs. Thus, the present invention integrates bioinformatics and experimental approaches to reveal this unique profibrotic endothelial-Th 17 cell interaction resulting from epigenetically dependent vascular disorders. Given the systemic distribution of blood vessels and hematopoietic cells (circulatory system) in multiple organs, decoding of molecules and cellular networks involved in circulatory disorders may help to systematically identify therapeutic targets or biomarkers.

Based on clinical findings of human patients, the role of epigenetic dependent vascular disorders was determined by the experimenter using a complementary preclinical NASH model (minipigs and mice). The NASH model was induced using western diet feeding with high sucrose and fructose, high cholesterol and high fat in combination with repeated liver injury, showing clinically relevant NASH phenotypes including histological and transcriptomic features. Preclinical models of miniature pigs and mice also help to understand the pathogenesis of NASH. The digestive system of miniature pigs is very similar to that of humans, and has unique advantages similar to human metabolic disorder-related diseases (such as NASH). Large animals such as mini-pigs can be biopsied for multiomics assessment of treatment efficacy and underlying mechanisms. Compared with miniature pigs, transgenic mice, such as EC-specific Hdac2 knockout mice and Igfbp7 knockout mice, provide effective tools for studying cellular and molecular mechanisms involved in epigenetic-dependent vascular disorders. Indeed, pharmacological and genetic targeting experiments in mini-pig and mouse NASH models have shown that aberrant HDAC2/DNMT1 cross-regulation leads to vascular dysregulation and subsequent production of IGFBP7/ADAMTS1 into extracellular vesicles. This data is consistent with the correlation between HDAC2, DNMT1, IGFBP7 or ADAMTS1 and fibrosis levels identified in the human cirrhosis cohort. Combined targeted inhibition of HDAC2 and DNMT1 showed synergistic anti-fibrotic effects in mouse and mini-pig NASH models. Experiments with injection of exogenous IGFBP7 or ADAMTS 1-knockdown endothelial-derived EV further demonstrated that ADAMTS1/IGFBP7 promotes liver fibrosis by stimulating Th17 responses in the liver. The experiment of the present invention, although only male minipigs were used to construct the NASH model, neither human patient samples nor mouse models are sex specific, and therefore the preclinical platform of the present invention may be useful in designing therapeutic strategies for a variety of fibrosis-related diseases (associated with 40% mortality worldwide). In the present invention, the experimenter elaborates a single cell map of vascular disorders where a subset of epigenetic reprogrammed ECs induce the release of profibrotic factors, stimulating Th17 cell recruitment to collectively promote liver fibrosis for multiple species. The formation of the dysregulated vascular microenvironment, including endothelial dysclassification and production of profibrotic factors in EVs. Elucidation of molecular and cellular networks under such vascular disorders may be helpful in exploring diagnostic or therapeutic protocols for fibrotic diseases.

While the present invention has been described with reference to the embodiments shown in the drawings, the present invention is not limited to the embodiments, which are illustrative and not restrictive, and it will be apparent to those skilled in the art that various changes and modifications can be made therein without departing from the spirit and scope of the invention as defined in the appended claims.

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Claims (11)

1. Use of an hdac2 inhibitor and a DNMT1 inhibitor for the preparation of a medicament for the combined targeted treatment of non-alcoholic steatohepatitis, wherein said non-alcoholic steatohepatitis is accompanied by cirrhosis and/or liver fibrosis.
2. 2. The use of claim 1, wherein said pathological grade of liver fibrosis comprises grades F2-F4.
3. 3. The use according to claim 1, wherein the HDAC2 inhibitor is moxiflostat (Mocetinostat) in an amount of 1-20 mg/kg/day or 0.1-2.0 mg/kg/day.
4. 4. The use of claim 1, wherein the DNMT1 inhibitor is azacitidine at a dose of 0.1 to 2.0 mg/kg/day or 0.001 to 0.100 mg/kg/day.
5. 5. The use of claim 1, wherein the HDAC2 inhibitor and the DNMT1 inhibitor are administered by injection, which comprises one or more of intraperitoneal injection, intramuscular injection, subcutaneous injection, and intravenous injection.
6. 6. The use of claim 5, wherein the injection is administered according to a regimen comprising: injecting the DNMT1 inhibitor 5 days before the first week, 1 time per day, and then stopping the injection for 2 days; the HADC2 inhibitor was injected 5 days prior to the second week, 1 time per day, followed by 2 days of discontinuation, and the dosing regimen was repeated 5-10 times.
7. 7. The use of claim 1, wherein the HDAC2 inhibitor and DNMT1 inhibitor are used in combination to target the extent of fibrosis in the non-alcoholic steatohepatitis liver and promote liver regeneration; and/or reversing sinoendothelial-macrovascular endothelial disorders in a liver cirrhosis liver; and/or reducing recruitment of profibrotic Th17 cells in non-alcoholic steatohepatitis liver.
8. 8. The use of claim 1, wherein the HDAC2 inhibitor and the DNMT1 inhibitor are used in combination targeted to reduce blood glucose, the liver fibrosis index and/or the serum liver function index.
9. 9. The use according to claim 1, wherein the HDAC2 inhibitor and DNMT1 inhibitor are used in combination to target reduction of serum total cholesterol levels.
10. 10. The use according to claim 1, wherein the combined targeted use of the HDAC2 inhibitor and the DNMT1 inhibitor reduces the cirrhosis and increases hepatocyte proliferation.
11. 11. The use of claim 1, wherein the HDAC2 inhibitor and the DNMT1 inhibitor are used in combination to target block the increase of IGFBP7 and/or ADAMTS1 in a liver cirrhosis liver.

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