WO2023088086A1 - 组合因子及其应用 - Google Patents

组合因子及其应用 Download PDF

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WO2023088086A1
WO2023088086A1 PCT/CN2022/128995 CN2022128995W WO2023088086A1 WO 2023088086 A1 WO2023088086 A1 WO 2023088086A1 CN 2022128995 W CN2022128995 W CN 2022128995W WO 2023088086 A1 WO2023088086 A1 WO 2023088086A1
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trim31
rhbdf2
mice
liver
expression
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French (fr)
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谭君
徐敏轩
葛晨旭
孙岩
代先玲
况琴
赵俊杰
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成都夸克致远生物科技有限公司
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Publication of WO2023088086A1 publication Critical patent/WO2023088086A1/zh
Priority to US18/663,136 priority Critical patent/US20240293579A1/en

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Definitions

  • the invention belongs to the technical field of cell engineering, and in particular relates to a combined factor pair and its application.
  • NAFLD nonalcoholic fatty liver disease
  • NASH will in turn further promote its complications, for example, the development of insulin resistance, hypertension, chronic kidney disease (CKD) and type 2 diabetes (T2D).
  • CKD chronic kidney disease
  • T2D type 2 diabetes
  • Rhomboid 5Homolog 2 also known as iRhom2
  • Rhomboid 5Homolog 2 is an inactive member of the Rhomboid intramembrane protease family that has been identified as a key pathogenic regulator of inflammation-related diseases such as obesity, arthritis, nephritis, atherosclerosis and fibrosis and other diseases.
  • RHBDF2 significantly increased its phosphorylation level and activated downstream inflammatory signals by recruiting MAP3K7. Activation of the RHBDF2-MAP3K7 signaling pathway contributes to the development of NAFLD, which can lead to liver fibrosis.
  • RHBDF2 has the ability to be regulated by ubiquitination, another important regulatory modification.
  • NAFLD and NASH are entangled progression associated with metabolic syndrome and systemic inflammatory response syndrome (SIRS)
  • SIRS systemic inflammatory response syndrome
  • long-term high-calorie diet therapy can stimulate severe insulin resistance in the liver, inhibiting the normal function of insulin in the liver to regulate glucose metabolism.
  • the adipogenesis-promoting function of insulin was fully preserved.
  • severe insulin resistance leads to abnormal accumulation of excess lipids in the liver.
  • high fat intake also triggers an inflammatory response and synergistically promotes the development and development of insulin resistance.
  • E3 ubiquitin ligase-triple-domain protein 31 has been identified as a "Janus-faced" regulator of the innate immune response, promoting target substrate degradation or signal transduction through ubiquitin modification .
  • Animals with TRIM31 dysregulation usually exhibit severe intestinal inflammation and intestinal flora imbalance, and TRIM31 dysregulation induces endotoxemia and increases the activation of NLRP3 inflammasome.
  • TRIM31 leads to myocardial dysfunction by promoting apoptosis and NF- ⁇ B signaling pathway. Chronic inflammation mediated by NF- ⁇ B signaling can enhance the invasion and metastasis of colorectal cancer.
  • the purpose of the present invention is to provide a combination factor pair, the combination factors are respectively TRIM31 and RHBDF2 derived from hepatocytes.
  • the TRIM31 includes the amino acid sequence shown in sequence NO: 1 or sequence NO: 2; the RHBDF2 includes the amino acid sequence shown in sequence NO: 3 or sequence NO: 4.
  • the nucleic acid sequence encoding TRIM31 includes the sequence shown in sequence NO: 5 or sequence NO: 6; the nucleic acid sequence encoding RHBDF2 includes the sequence shown in sequence NO: 7 or sequence NO: 8.
  • nucleic acid sequence encoding TRIM31 may also be the humanized sequence of the sequence shown in sequence NO: 6; the nucleic acid sequence encoding RHBDF2 may also be the humanized sequence of the sequence shown in sequence NO: 8.
  • TRIM31 if the secretion of TRIM31 is increased, the secretion of RHBDF2 will be inhibited, that is, the secretion of RHBDF2 will be reduced.
  • hepatic TRIM31 as a significant negative regulator of high-fat diet (HFD/HFHF)-induced or genetically induced chronic inflammation, insulin resistance, hepatic steatosis, and NASH.
  • HFD/HFHF high-fat diet
  • RHBDF2-MAP3K7 signaling In the presence of TRIM31 dysfunction, strongly activated RHBDF2-MAP3K7 signaling further mediated increased expression of downstream signaling pathways, including JNK-IRS1-associated insulin signaling, I ⁇ B ⁇ -NF- ⁇ B-c-Jun pathway, AKT-GSK3 ⁇ -Inhibition of phosphorylation levels of FOXO1 and CTGF-TIMP1 pathway, promoting impaired insulin signaling (insulin resistance), chronic inflammatory effects, abnormal glucose metabolism disorders and collagen deposition in the liver, thereby exacerbating abnormal lipid metabolism , and the development of hepatic steatosis and the NASH phenotype.
  • downstream signaling pathways including JNK-IRS1-associated insulin signaling, I ⁇ B ⁇ -NF- ⁇ B-c-Jun pathway, AKT-GSK3 ⁇ -Inhibition of phosphorylation levels of FOXO1 and CTGF-TIMP1 pathway, promoting impaired insulin signaling (insulin resistance), chronic inflammatory effects, abnormal glucose
  • TRIM31 may have a "Janus-face" (double-sided) role in regulating immune-related diseases, which can catalyze the degradation of targeted substrates or degrade substrates through ubiquitination signal transduction. Studies have shown that TRIM31 deficiency loses its inhibitory effect on the inflammatory response of colitis; but on the other hand, TRIM31 was identified as a positive promoter that can exacerbate cardiomyopathy caused by inflammation-related signals. These seemingly contradictory conclusions may indicate that the function of TRIM31 is closely related to organ type and cell type.
  • the purpose of the present invention is also to provide the application of the aforementioned combined factor pair as a marker of NAFLD/NASH and/or metabolic disorder.
  • the TRIM31 includes the amino acid sequence shown in sequence NO: 1 or sequence NO: 2; the RHBDF2 includes the amino acid sequence shown in sequence NO: 3 or sequence NO: 4.
  • the nucleic acid sequence encoding TRIM31 includes the sequence shown in sequence NO: 5 or sequence NO: 6; the nucleic acid sequence encoding RHBDF2 includes the sequence shown in sequence NO: 7 or sequence NO: 8.
  • the purpose of the present invention is to also provide a use of TRIM31 as an inhibitor/inhibitor for the preparation of NAFLD/NASH and metabolic disorders.
  • the TRIM31 includes the amino acid sequence shown in sequenceNO:1 or sequenceNO:2; the nucleic acid sequence encoding TRIM31 includes the sequence shown in sequenceNO:5 or sequenceNO:6.
  • the purpose of the present invention is to also provide a TRIM31 promoter as an application in the preparation of drugs for NAFLD, NASH or metabolic disorders.
  • the TRIM31 includes the amino acid sequence shown in sequenceNO:1 or sequenceNO:2; the nucleic acid sequence encoding TRIM31 includes the sequence shown in sequenceNO:5 or sequenceNO:6.
  • the accelerator includes one or more combinations of the following substances:
  • Alkaloid compounds or compositions including berberine, magnolialine, haematine, serpentine, leonurine, tetrandrine, ethosine, peycinine, colchicine, piperine, Rhynchophylline, camptothecin, tetrahydropalmatine, jatrorrhizine, chelidonine, lycorine, behenicine, matrine, oxymatrine, a compound or a combination of multiple compounds thing;
  • Flavonoid compounds or compositions including quercetin, dihydroquercetin, luteolin, morin, morin, apigenin, diosmin, chrysin, alfalfa, puerarin, and anthocyanins Proanthocyanidins, daidzein, apigenin, baicalein, scutellarein, chrysin, wogonin, cyanidin, silymarin, silybin, daidzein, genistein, galangin, kaempferen Phenol, fisetin, isorhamnetin, myricetin, hesperetin, naringenin, liquiritigenin, catechin, epicatechin, gallocatechin, cyanidin, delphinidin, flower A compound or a combination of compounds of sunflowerin, breviscapine, afotoin, rutin, luteolin, baicalin, apigenin, and juglandin;
  • Polyphenol compounds or compositions including chlorogenic acid, cynaric acid, glycosides, glycosides, tea polyphenols, resveratrol, pomegranate polyphenols, curcumin, ferulic acid A compound or a combination of compounds;
  • Amine compounds or compositions penicillamine, pomalidomide, lenalidomide, a compound or a combination of multiple compounds among thalidomide;
  • Chinese medicine extracts including astragalus, Codonopsis pilosula, Danshen, Angelica, Chinese yam, Panax notoginseng, Schisandra chinensis, Polygonatum, Ganoderma lucidum, Yunzhi, artichoke, thistle, milk thistle, kudzu root, Coptis chinensis, Cortex Phellodendron, Turmeric, honeysuckle, Hovenia dulcis, tangerine peel, mulberry leaf, ginkgo biloba, lily, bear gall, pig gall, capillary, citrus aurantium, licorice, knotweed, pagoda seed, paeonol, weeping grass, gardenia, wolfberry, white peony root, chuanxiong, peach kernel , rhubarb, chrysanthemum, Xihuangcao, Hedyotis diffusa, Fangji, Imperata rh
  • Glycyrrhizic acid glycyrrhizin, adenosylmethionine, ursodeoxycholic acid, tauroursodeoxycholic acid, chenodeoxycholic acid, taurochenodeoxycholic acid, bicyclol, bifendate, One or more of sorafenib, regorafenib, lenvatinib, betalain, metformin, and carminic acid.
  • the medicine also includes some pharmaceutical auxiliary materials.
  • the purpose of the present invention is to also provide an application of TRIM31 as an inhibitor of RHBDF2.
  • the TRIM31 comprises the amino acid sequence shown in sequenceNO:1 or sequenceNO:2; wherein the nucleic acid sequence encoding TRIM31 includes the sequence shown in sequenceNO:5 or sequenceNO:6.
  • the nucleic acid sequence encoding TRIM31 may also be a humanized sequence of the sequence shown in sequence NO:6.
  • the purpose of the present invention is also to provide an application of TRIM31 as an agent for eliminating insulin resistance or hepatic steatosis or inflammation and liver fibrosis.
  • the TRIM31 comprises the amino acid sequence shown in sequenceNO:1 or sequenceNO:2; wherein the nucleic acid sequence encoding TRIM31 includes the sequence shown in sequenceNO:5 or sequenceNO:6.
  • the nucleic acid sequence encoding TRIM31 may also be a humanized sequence of the sequence shown in sequence NO:6.
  • RHBDF2 upregulated RHBDF2 triggered by a high-fat diet significantly drives all three pathological phenotypes in vivo, revealing the potential therapeutic promise of RHBDF2 in metabolic diseases associated with hepatic steatosis.
  • TRIM31 an important member of E3 ubiquitin ligase, is a potential effective inhibitor of RHBDF2 expression.
  • the protective function of TRIM31 in hepatocytes was not limited to the liver, but also affected the entire metabolic system, including reducing body weight gain and visceral fat, improving dyslipidemia, and down-regulating serum inflammatory cytokines and chemokines .
  • TRIM31 regulates the liver and thus the whole human body. Moreover, we demonstrated that TRIM31 dysfunction leads to the subsequent marked upregulation of RHBDF2 levels and is closely related to inflammation-related diseases. In addition, RHBDF2 has been shown to be regulated by ubiquitination modification. Similarly, we determined that TRIM31 levels were significantly reduced in the livers of mice fed a high-fat diet and in patients with hepatic steatosis.
  • the purpose of the present invention is to also provide a method for promoting the degradation of RHBDF2, which is characterized in that the degradation of RHBDF2 is promoted by polyubiquitination of the TRIM31 linked by K48.
  • the purpose of the present invention is to also provide an application of TRIM31 as a diagnostic marker for hepatocyte degeneration.
  • the purpose of the present invention is to also provide a method for judging hepatocyte degeneration, which is characterized in that the expression level of TRIM31 in the isolated sample to be tested is detected, and compared with the normal group, when the TRIM31 expression level in the isolated sample to be tested is When the expression level of is lower than that of the normal group, it is judged as hepatocyte degeneration.
  • the expression level of RHBDF2 in the isolated sample to be tested is detected and compared with the normal group, and when the expression level of RHBDF2 in the isolated sample to be tested is lower than the normal group, it is determined as hepatocyte degeneration.
  • the purpose of the present invention is to also provide a TRIM31 expression regulator as a liver glycogen storage regulator.
  • the expression promoter of TRIM31 is used as an inhibitor of liver glycogen storage.
  • the purpose of the present invention is to also provide an application of TRIM31 as an inhibitor of high-fat diet-induced insulin resistance and glucose metabolism disorder.
  • TRIM31 promotes RHBDF2 degradation through K48-linked ubiquitin modification, further inhibits RHBDF2-MAP3K7 activity and the expression of downstream signaling pathways, and thus has the ability to inhibit hepatic steatosis, impaired insulin signaling and liver inflammation.
  • TRIM31 mainly exerts its biological functions at the molecular level based on different motifs. It has been reported that the C-terminal coiled-coil domain (CC) of TRIM31 is basically responsible for the binding and interaction with the target, while the N-terminal RING domain is responsible for the ubiquitin ligase activity and catalytic function.
  • CC C-terminal coiled-coil domain
  • the present invention found that K48-linked RHBDF2 ubiquitination can be catalyzed in vitro, and the deletion of TRIM31 greatly invalidated this endogenous ubiquitination modification of RHBDF2.
  • TRIM31 directly binds to RHBDF2 through the helical domain (CC) to target RHBDF2 activity, and then the RING finger of TRIM31 mediated RHBDF2 proteasomal ubiquitination degradation through K48 to ubiquitinate RHBDF2.
  • CC helical domain
  • TRIM31 Once the interaction between RHBDF2 and TRIM31 was blocked, the protective effect of TRIM31 against high-fat-induced hepatic steatosis was terminated. Moreover, non-functional TRIM31 (RING domain mutant) lost its E3 ligase activity to continue to reduce the activation of the RHBDF2-MAP3K7 signaling pathway and the downstream JNK1/2-NF-kB pathway and enhance insulin signaling. Therefore, TRIM31 with RING domain mutants also has a considerable effect on PA-induced abnormal lipid metabolism in hepatocytes. Thus, Trim31-RHBDF2 binding and subsequent RHBDF2 ubiquitination modification are required for hepatic RHBDF2-MAP3K7 signaling-induced hepatic steatosis, insulin resistance and relief of liver inflammation.
  • RHBDF2 as an important inactive member of the rhombic intramembrane protease family, has been shown to be involved in the regulation and transmission of inflammatory signals.
  • TNF- ⁇ the pro-inflammatory factor
  • Previous studies have shown that in cells other than immune cells, the production of the pro-inflammatory factor TNF- ⁇ is independent of RHBDF2.
  • Existing literature also shows that the loss of RHBDF2 in non-immune cells does not affect the expression and secretion of inflammatory factors such as TNF- ⁇ , IL-6 and IL- ⁇ .
  • TNF- ⁇ in non-immune cells such as hepatocytes
  • MAP3K7 downstream signaling pathways under stimulating conditions, activate NF- ⁇ B inflammatory signaling and accelerate inflammatory cytokines secretion.
  • TRIM31 significantly promoted the degradation of RHBDF2 through K48 ubiquitin linkage, and then the expressions of TNF- ⁇ and other inflammatory factors were greatly reduced.
  • the present invention establishes that hepatic TRIM31 expression is associated with non-alcoholic fatty liver disease/non-alcoholic steatohepatitis and metabolic disorders, such as insulin resistance and glucose metabolism disorders in humans and mice. Furthermore, mouse liver TRIM31 expression responded to dietary intervention, which significantly attenuated insulin resistance, hepatic steatosis, and inflammatory phenotypes by modulating RHBDF2 proteasomal degradation. These findings advance TRIM31 as a viable therapeutic target for nonalcoholic fatty liver disease/nonalcoholic steatohepatitis (NAFLD/NASH) and related metabolic diseases, and link TRIM31 to diagnostic and therapeutic options for these diseases.
  • NAFLD/NASH nonalcoholic fatty liver disease/nonalcoholic steatohepatitis
  • the combined factor of TRIM31 and RHBDF2 provided by the present invention is used as a marker of metabolic disorder, and can accurately and effectively identify NAFLD/NASH and/or metabolic disorder or hepatocyte degeneration.
  • the promoting factor/promoting agent for the preparation of TRIM31 provided by the present invention can effectively improve the situation of NAFLD/NASH and/or metabolic disorder.
  • the TRIM31 used in the present invention can effectively relieve insulin resistance, hepatic steatosis, inflammation and liver fibrosis as or prepare an eliminating agent for insulin resistance or hepatic steatosis or inflammation and hepatic fibrosis.
  • the method for judging hepatocyte degeneration provided by the invention can accurately identify whether hepatocyte degeneration occurs.
  • Figure 1a shows the expression of TRIM31 in the liver samples of HFD mice and NCD mice.
  • Figure 1b shows the expression of TRIM31 in ob/ob mouse liver samples.
  • Figure 1c shows the expression of TRIM31 in HFD mice at 0-16 weeks.
  • Figure 1d shows the expression of TRIM31 in the liver of NASH patients.
  • Figure 1e shows the expression of TRIM31 after administration of palmitic acid (PA) and TNF- ⁇ .
  • Figure 1f shows the expression of TRIM31 after adding active oxygen scavenger N-Acetyl-L-cysteine (NAC) and antioxidant catalase (CAT).
  • NAC active oxygen scavenger N-Acetyl-L-cysteine
  • CAT antioxidant catalase
  • Figure 1g shows the expression of RHBDF2 after PA+CHX treatment.
  • Figure 1h shows the expression of RHBDF2 after treatment with PA+CHX+chloroquine.
  • Figure 1i shows the expression of RHBDF2 after treatment with PA+CHX+MG132.
  • Figure 1j shows the immunofluorescence analysis of mouse liver.
  • Figure 1k is the immunohistochemical analysis of human liver slices and intracellular triglyceride (TG) in L02 cells transfected with adenovirus-mediated TRIM31 expression (AdTRIM31).
  • Figure 2a shows the expression of RHBDF2 in the liver samples of HFD mice, NCD mice, and ob/ob mice.
  • Figure 2b shows oil red staining and HE staining of liver samples from HFD mice at 0-16 weeks.
  • Figure 2c shows the contents of TC, TG and NEFA in HFD mice and NCD mice.
  • Figure 2d shows the expression of RHBDF2 in the liver of NASH patients.
  • Figure 2e shows the expression of RHBDF2 after administration of palmitic acid (PA) and TNF- ⁇ .
  • Figure 2f shows the ubiquitination of RHBDF2 in vivo.
  • Figure 3a is a schematic diagram of the THKO mouse production process.
  • Figure 3b shows the expression of TRIM31 in various body components of THKO mice.
  • Figure 3c shows the food intake of THKO mice treated with NCD and HFD.
  • Figure 4a is a schematic diagram of the production process of THTG mice.
  • Figure 4b shows the expression of TRIM31 in various body components of THTG mice.
  • Figure 4c shows the food intake of THTG mice treated with NCD and HFD.
  • Figure 4d shows the expression of TRIM 31 in primary hepatocytes treated with PA.
  • Figure 5a shows the body weight of THTG mice from 0 to 16 weeks of long-term HFD.
  • Figure 5b shows the fasting blood glucose of THTG mice on HFD for 0-16 weeks.
  • Figure 5c shows the fasting and fasting insulin levels of THTG mice with long-term HFD from 0 to 16 weeks.
  • Figure 5d shows the insulin resistance of THTG mice on HFD for 0-16 weeks.
  • Fig. 5e shows the blood glucose level of THTG mice tested by the glucose tolerance test (GTT).
  • Fig. 5f shows the blood glucose level of THTG mice tested by insulin tolerance test (ITT).
  • Figure 5g shows the relationship between TRIM31 overexpression and liver glycogen storage.
  • Figure 5h shows the dysregulation of hepatic insulin signaling in THTG mice-HFD diet.
  • Figure 6a shows the body weight of differently treated THKO mice at 0-16 weeks.
  • Figure 6b shows the fasting blood glucose of THKO mice with different treatments from 0 to 16 weeks.
  • Figure 6c shows the fasting insulin levels of THKO mice with different treatments from 0 to 16 weeks.
  • Figure 6d shows the insulin resistance of differently treated THKO mice at 0-16 weeks.
  • Fig. 6e shows the blood glucose level of THKO mice tested by the glucose tolerance test (GTT).
  • Fig. 6f shows the blood glucose level of THKO mice tested by insulin tolerance test (ITT).
  • Figure 6g shows the relationship between TRIM31 deletion and liver glycogen storage.
  • Figure 6h shows the dysregulation of hepatic insulin signaling induced by THKO mice-HFD diet.
  • Figure 7a shows the liver weight and liver weight/body weight of HFD-THKO mice.
  • Figure 7b shows the serum alanine aminotransferase (ALT) of HFD-THKO mice.
  • Figure 7c shows the serum aspartate aminotransferase (AST) status of HFD-THKO mice.
  • Fig. 7d is the serum alkaline phosphatase (AKP) situation of HFD-THKO mice.
  • Figure 7e shows the expression of genes related to fatty acid uptake and synthesis and genes related to fatty acid ⁇ -oxidation in HFD-THKO mice.
  • Figure 7f shows the liver weight and liver/body weight ratio of THTG mice and NTG mice fed a high-fat diet.
  • Figure 7g is a solid map of liver fat in THTG mice fed a high-fat diet.
  • Figure 7h shows the liver function-related indicators of THTG mice fed a high-fat diet.
  • Figure 7i shows oil red staining and HE staining of liver samples from THTG mice fed a high-fat diet.
  • Figure 7j shows the expression of genes related to fatty acid uptake and synthesis and the expression of genes related to fatty acid ⁇ -oxidation in THTG mice fed a high-fat diet.
  • Figure 7k is the solid image and TRIM 31 expression of primary hepatocytes treated with PA.
  • Figure 8a shows the visceral fat of THKO and THTG mice fed a high-fat diet.
  • Figure 8b shows the size of adipocytes in THKO and THTG mice fed a fat diet.
  • Figure 9a shows the expression of factors in the signaling pathways related to inflammation in HFD-THKO mice.
  • Figure 9b shows the expression of factors in the signaling pathways related to inflammation in HFD-THTG mice.
  • Figure 9c shows the secretion of inflammation-related factors in the serum of THKO mice fed a high-fat diet.
  • Fig. 9d shows the inflammatory signal mediated by activation of NF- ⁇ B in primary hepatocytes treated with PA.
  • Fig. 10a is a schematic flow diagram of lentivirus encapsulating the complete mouse TRIM31 gene sequence (LV-TRIM31).
  • Figure 10b shows the weight, body weight, and liver weight/body weight ratio of high-fat diet-THKO mice.
  • Figure 10c is the situation of AST and ALT in high-fat diet-THKO mice.
  • Figure 10d shows the lipid accumulation in the liver of high-fat diet-THKO mice.
  • Figure 10e shows the blood glucose concentration of THKO mice tested by GTT.
  • Figure 10f shows the blood glucose concentration of THKO mice tested by ITT.
  • Figure 10g shows the TG, TC and NEFA conditions in the liver of THKO mice.
  • Figure 10h shows the expression of TRIM31 in hepatocytes and the expression of fatty acid ⁇ -oxidation genes in high-fat diet-THKO mice.
  • Figure 10i shows the impaired insulin signaling and activated inflammation-related signaling in THKO mice.
  • Figure 10j shows impaired insulin signaling and activated inflammation-related signaling in THKO mice.
  • Figure 11a shows the role of TRIM31 in alleviating liver lipid accumulation, islet resistance and inflammation.
  • Figure 11b shows the levels of IL-1 ⁇ /YNF- ⁇ /IL-6 in the blood of lentivirus-mediated Trim31 expression and hyperlipidemia.
  • Fig. 11c shows the mRNA levels of signaling factors in blood with high fat-drinking blood caused by lentivirus-mediated Trim31 expression.
  • Figure 12a is a schematic diagram of the in vitro gene therapy process mediated by LV-hTRIM31 and the expression of TRIM 31.
  • Fig. 12b shows the liver weight and body weight of mice repairing TRIM31 by in vitro gene therapy mediated by LV-hTRIM31.
  • Fig. 12c shows oil red staining and HE staining of mouse liver samples repaired by gene therapy in vitro LV-hTRIM31-mediated TRIM31.
  • Fig. 12d shows the status of serum AST and ALT in mice repairing TRIM31 by in vitro gene therapy mediated by LV-hTRIM31.
  • Fig. 12e is the fasting blood glucose level of mice repairing TRIM31 by in vitro gene therapy mediated by LV-hTRIM31.
  • Fig. 12f is the blood glucose level of mice repairing TRIM31 by in vitro gene therapy mediated by LV-hTRIM31.
  • Fig. 12g shows the insulin resistance of TRIM31 mice repaired by LV-hTRIM31-mediated gene therapy in vitro.
  • Figure 12h shows the TG, TC, and NEFA conditions of mice repairing TRIM31 by in vitro gene therapy mediated by LV-hTRIM31.
  • Figure 12i shows the secretion of factors related to TRIM31 repaired by LV-hTRIM31-mediated gene therapy in vitro.
  • Fig. 12j is LV-hTRIM31-mediated in vitro gene therapy to restore the expression of TRIM31-related proteins in mice.
  • Fig. 12k shows the secretion of serum cytokines in mice that repaired TRIM31 by gene therapy in vitro mediated by LV-hTRIM31.
  • Figure 13a shows the results of immunofluorescence sections of high-fat diet-THKO mice and THTG mice.
  • Figure 13b shows the situation of ADAM17, TNFR1/2, p-MAP3K7 and their downstream components in mice.
  • Figure 13c shows the conditions of ADAM17, TNFR1/2, p-MAP3K7 and their downstream components in mice in vitro.
  • Figure 13d shows the impaired insulin signaling induced by PA in THTG hepatocytes transfected with AdshRHBDF2.
  • Figure 14a shows the impaired insulin signaling induced by PA in THTG hepatocytes transfected with AdRHBDF2.
  • Fig. 14b is the results of the immunofluorescence section of the interaction between RHBDF2 and TRIM31.
  • Figure 14c shows the binding of TRIM31 and RHBDF2 in transfected mouse hepatocytes.
  • Figure 15a is the situation of the in vitro interaction test.
  • FIG. 15b shows the labeling of glutathione sulfur transferase (GST).
  • Fig. 15c is a schematic diagram of the structure of TRIM31.
  • Figure 15d shows the ubiquitination of RHBDF2 in vivo.
  • Figure 15e shows the ubiquitination of RHBDF2 catalyzed by the TRIM31 expression vector with the RING-finger domain excision mutant.
  • Figure 15f shows the ubiquitination of RHBDF2 linked to K63 in TRIM31-deficient L02 cells (THKO-L02).
  • Figure 15g is the in vitro transfection experiment of ubiquitin mutant plasmids.
  • Figure 15h shows the change of intracellular TG concentration stimulated with PA in TRIM31-deficient L02 cells (THKO-L02) transfected with the vector.
  • Figure 16a is a schematic diagram of the process of lentiviral load TRIM 31 (LV-TRIM 31 RING) with RING-finger knockout.
  • Figure 16b shows the liver weight, body weight, and liver weight/body weight ratio of TRIM31 RING-finger knockout-hepatocyte transplantation (THKO) (LV+) mice induced by HFD.
  • THKO RING-finger knockout-hepatocyte transplantation
  • Figure 16c shows the AST and ALT conditions induced by HFD in mice of TRIM31 RING-finger knockout-hepatocyte transplantation (THKO) (LV+).
  • Figure 16d is the liver lipid accumulation induced by HFD in TRIM31 RING-finger knockout-hepatocyte transplantation (THKO) (LV+) mice.
  • Figure 16e is the fasting blood glucose level induced by HFD in mice of TRIM31 RING-finger knockout-hepatocyte transplantation (THKO) (LV+).
  • Figure 16f is the blood glucose level induced by HFD in mice of TRIM31 RING-finger knockout-hepatocyte transplantation (THKO) (LV+).
  • Figure 16g shows the hepatic triglyceride, total cholesterol and NEFA contents induced by HFD in TRIM31 RING-finger knockout-hepatocyte transplantation (THKO) (LV+) mice.
  • Fig. 16h shows the expression of genes related to fatty acid intake and synthesis process and the gene expression related to fatty acid ⁇ -oxidation process in THKO (LV+) mice fed a high-fat diet.
  • Fig. 16i is the western blot of RHBDF2-MAP3K7 signaling pathway, mRNA level and concentration of pro-inflammatory mediators in THKO (LV+) mice fed a high-fat diet.
  • Figure 16j is the immunoblotting of RHBDF2-MAP3K7 downstream pathway in THKO (LV+) mice fed a high-fat diet.
  • Figure 17a is a schematic flow chart 2 of lentiviral load TRIM 31 (LV-TRIM 31 RING) with RING-finger knockout.
  • Figure 17c shows the mRNA levels of THKO (LV+) mice fed a high-fat diet.
  • Fig. 18a is a schematic flow chart of LV-hTRIM31 gene therapy in vitro.
  • Figure 18b shows the liver weight, body weight, and liver weight/body weight ratio of mice obtained by LV-hTRIM31 in vitro gene therapy.
  • Figure 18c shows the oil red staining and HE staining of mouse liver samples obtained by LV-hTRIM31 gene therapy in vitro.
  • Figure 18d shows the serum AST and ALT conditions of mice obtained by LV-hTRIM31 in vitro gene therapy.
  • Fig. 18e is the mouse blood glucose level obtained by LV-hTRIM31 in vitro gene therapy.
  • Figure 18f shows the TG, TC and NEFA conditions of mice obtained by LV-hTRIM31 in vitro gene therapy.
  • Figure 18g shows the insulin resistance of mice obtained by LV-hTRIM31 gene therapy in vitro.
  • Figure 19a shows the expression of Rhbdf2 in AAV-TRIM31 RING WT mice.
  • Figure 19b is the body weight of AAV-TRIM31 RING WT mice.
  • Figure 19c shows the fasting blood glucose level of AAV-TRIM31 RING WT mice.
  • Figure 19d shows the blood glucose level of AAV-TRIM31 RING WT mice.
  • Figure 19e shows the liver weight of AAV-TRIM31 RING WT mice.
  • Figure 19f shows the oil red staining and HE staining of AAV-TRIM31 RING WT mouse liver samples.
  • Figure 19g shows the expression of RHBDF2 in AAV-TRIM31 RING WT mice.
  • Figure 19h shows the downstream inflammatory signaling of RHBDF2 in AAV-TRIM31 RING WT mice.
  • Figure 19i is the mRNA expression level of related signals in AAV-TRIM31 RING WT mice.
  • Figure 20a is the situation of DHKO mouse liver samples analyzed by western blot.
  • Figure 20b shows the body weight, liver weight, and liver weight/body weight ratio of DHKO mice.
  • Figure 20c shows the fasting blood glucose level of DHKO mice.
  • Figure 20d shows the oil red staining and HE staining of liver samples from DHKO mice.
  • Figure 20e shows the TG, TC, and NEFA conditions of DHKO mice.
  • Figure 20f shows the RHBDF2-MAP3K7 axis and downstream signaling cascades in DHKO mice.
  • Figure 20g shows the lipid deposition in the liver of DHKO mice.
  • Figure 21a shows the blood glucose levels of different mice.
  • Figure 21b shows abnormal blood glucose levels in different mice.
  • Figure 21c is a graph of PAS analysis of different mouse livers.
  • Figure 21d shows the expression of pro-inflammatory related genes in different mice.
  • Figure 21e shows the signal expression of RHBDF2-MAP3K7 axis in different mice.
  • Figure 22 shows the transfection efficiency of hepatocytes induced by adenoviral vectors.
  • Figure 23a shows the liver weight and liver weight/body weight ratio of THTG-HFHF mice.
  • Figure 23b shows the body weight of THTG-HFHF mice.
  • Figure 23c shows the concentration of TG, TC and NEFA in the liver of THTG-HFHF mice.
  • Figure 23d shows the oil red staining and HE staining of the liver samples of THTG-HFHF mice.
  • Figure 23e shows the expression of genes related to inflammation and collagen in THTG-HFHF mice.
  • Figure 23f is the liver lipid deposition in THTG-HFHF mice.
  • Figure 23g shows the expression of genes related to fatty acid synthesis in THTG-HFHF mice.
  • Figure 23h shows the Masson staining of liver samples from THTG-HFHF mice.
  • Figure 23i shows the expression of inflammation-related genes in THTG-HFHF mice.
  • Figure 23j shows the expression of genes related to liver fibrosis and collagen synthesis in THTG-HFHF mice.
  • Figure 23k is a schematic diagram of RHBDF2-MAP3K7 signal transduction in THTG-HFHF mice.
  • Fig. 24 is a schematic diagram of the production process of hepatocyte-specific TRIM31 deletion (THKO) mice.
  • Figure 25 is the effect of betalain intervention on the expression of related proteins in THTG mice.
  • Figure 26 is the effect of fisetin intervention on the mRNA levels of related protein genes in THTG mice.
  • Figure 27 shows the effect of carminic acid intervention on the expression of relevant proteins or mRNA levels in THTG mice.
  • the primary antibody used and its dilution ratio include: anti-GAPDH (#2118, dilution 1:10000), p-AKT (#4060, dilution 1:1000), AKT (#4691, dilution 1: 1000), p-GSK3 ⁇ (#9322, dilution 1:1000), GSK3 ⁇ (#12456, dilution 1:1000), FOXO1(#2880, dilution 1:1000), anti-p-JNK(#4668, dilution 1: 1000), anti-JNK(#9258, dilution 1:1000), anti-MAP3K7(#4505, dilution 1:1000), anti-p-MAP3K7(#9339, dilution 1:1000) and p-I ⁇ B ⁇ (#2859 , dilution 1:1000) was purchased from CST Company; anti-TNFR1 (#ab223352, dilution 1:1000), anti-TNFR2 (#ab109322, dil
  • a CRISPR/Cas9-mediated genetic engineering system was used to construct Trim31 flox/flox mice based on a C57BL/6N background. Exons 4 and 5 of Trim31 were selected as the conditional knockout region (CKO). The selected Trim31 exon flanks were used as loxP sites, and then two guide RNAs (gRNA1 and gRNA2) targeting the Trim31 intron were designed. A targeting vector containing Trim31 exons 4 and 5 flanked by two loxP sites and two homology arms was used as template. The targeting vector, guide RNA1 and guide RNA2, and Cas9 mRNA were co-injected into fertilized eggs for CKO mouse production.
  • CKO conditional knockout region
  • mice had loxP sites flanking both alleles of exons 4 and 5, and were then designated as Trim31 flox/flox mice.
  • Offspring mice produced by mating Trim31 flox/flox mice with Alb-Cre (Alb-Cre) mice (Jackson Laboratory, Bar Harbor, Maine, USA), and selected homozygotes as hepatocyte-specific Trim31 deletion ( THKO) mice were used (as shown in Figure 24).
  • THKO hepatocyte-specific Trim31 deletion
  • the CRISPR/Cas9 system was used to create hepatocyte-specific Rhbdf2 gene knockout (RHKO) mice by specifically knocking out the fourth exon of Rhbdf2 in hepatocytes.
  • RHKO hepatocyte-specific Rhbdf2 gene knockout mice by specifically knocking out the fourth exon of Rhbdf2 in hepatocytes.
  • the detailed methods and information of the establishment of these mice and the genotype measurement have been in the xu min-xuan, tan jun, et al., Dysfunctional RHBDF2 of Proopiomelanocortin Mitigates Ambient PARTICULE MATER Exposure-Induced Neur. Ological Injury and Neuron Loss by Antagonizing Oxidative Stress and inflammatory reaction. Journal of Hazardous Materials, 2020, 400:123158 has been made public.
  • DHKO hepatocyte-specific Trim31 and Rhbdf2 double deletion mice were generated by crossing Trim31flox/flox mice with
  • the plasmid CAG-loxP-CAT-loxP-Trim31 was microinjected into fertilized eggs isolated from C57BL/6 mice to construct conditional Trim31 transgenic (TG) (overexpression) mice. The resulting pups were then genotyped by PCR and sequencing analysis. The obtained mice were identified by PCR analysis of tail genomic DNA. The offspring of these TG mice were mated with Alb-Cre mice to establish hepatocyte-specific Trim31 transgenic (THTG) mice. Corresponding littermates without Trim31 overexpression in hepatocytes served as control (NTG).
  • mice were acclimatized for 7 days before all experiments normally started. The mice were housed in a constant temperature, constant humidity and pathogen-free environment (temperature 25°C ⁇ 2°C, ambient humidity 50%-60%) and given a 12-hour light/12-hour dark cycle.
  • the high-fat diet (HFD) feed (#D12492) standard is: 20% kcal protein, 60 kcal% fat and 20% kcal carbohydrate; Available from Research Diets, New Brunswick, NJ, USA.
  • the normal food (#D12450H) standard is: 20% kcal protein, 10 kcal% fat and 70% kcal carbohydrate; purchased from Research Diets, New Brunswick, NJ, USA.
  • ob/ob mice (#N000103) were purchased from Nanjing Institute of Biomedicine, Nanjing University.
  • animal experiment 1# was carried out: 6-8 week-old WT male mice (15 in total) were fed a high-fat diet (HFD) feed for 16 weeks to induce fatty liver. Additional WT mice (15 in total) were fed standard normal chow for 16 weeks as a control group (NCD). 6-8 week old ob/ob mice (10 in total) were fed NCD diet and then served as another fatty liver model. A total of 10 WT mice were included per time point in the time-gradient process experiments. At the end of the experiment, liver tissue samples were collected from the mice to detect changes in the abundance of corresponding signaling pathway proteins.
  • animal experiment 2# was carried out: in order to verify the protective effect of Trim31 on HFD-induced insulin resistance, hepatic steatosis and inflammation, pretreated THKO mice fed with HFD for 8 weeks were injected with lentivirus-loaded The wild-type Trim31 gene sequence (LV-Trim31) or Trim31 carrying a RING domain mutation was transduced and transplanted in vivo for gene therapy (ex vivo), and the ex vivo therapeutic effect of Trim31 was observed. At the end of the ex vivo therapeutic intervention, the mice were fasted for 8 hours and then tested for glucose and insulin tolerance. Eye blood and liver samples were collected, weighed, and stored at -80°C. Another part of the liver tissue was subjected to histological analysis and biochemical analysis. At least 3 independent experiments were performed on all available data.
  • animal experiment 3# is carried out: in vivo experiments, adeno-associated virus serotype 8 (AAV8)-thyroxine-binding globulin (TBG) encodes a full-length Trim31 sequence (AAV-Trim31) and AAV8 encodes a RING Mutant Trim31 of the domain-mutated Trim31 (AAV-Trim31 RING ⁇ ) delivery system achieves specific overexpression of Trim31 in hepatocytes after injection. Briefly, the Trim31 open reading frame (ORF) without a stop codon encoding was cloned into an AAV8 vector to generate AAV-Trim31.
  • ORF Trim31 open reading frame
  • Pretreated WT mice fed a HFD for 6 weeks were injected with AAV-Trim31 or AAV- Trim31 RING ⁇ (100 ⁇ l virus containing 2 ⁇ 10 11 vg), then fed HFD for another 10 weeks.
  • Empty vector (AAV-GFP) was injected into mice as a corresponding control.
  • animal experiment 4# is carried out, and the method for establishing the mouse model of fatty liver is as follows: 6-8 week-old male THKO, RHKO, DHKO, THTG mice and their corresponding littermate control mice are fed with HFD Feed for 16 weeks and observe the pathological changes. In addition, week-aged THKO, RHKO, DHKO, THTG mice and littermates were fed with NCD diet for 16 weeks and served as controls.
  • animal experiment 5# was carried out.
  • a mouse model of non-alcoholic steatohepatitis (14% protein, 42% Fat, 44% carbohydrates, 0.2% cholesterol, and a total of 42 g/L carbohydrates mixed in drinking water at a weight ratio of 55% fructose and 45% sucrose) were fed for 16 weeks to produce a NASH phenotype.
  • NASH mice used in this design served as controls and were synchronously given ad libitum access to this diet for 16 weeks.
  • PA was used to conduct in vitro experiments. Previous studies have shown that increased release of free fatty acids from adipocytes will lead to the accumulation of triglyceride (TG) in liver cells, and promote the development of steatosis to steatohepatitis. Saturated fats including palmitic acid (PA) and stearic acid are more toxic than other fatty acids and promote hepatotoxicity in a steatohepatitis model. The massive deposition of TG in the liver is caused by disturbance of lipid metabolism. Metabolic disturbance not only promotes steatosis and liver damage, but also significantly inhibits insulin signaling and promotes insulin resistance.
  • TG triglyceride
  • the L02 cell line (normal human liver cell line) used was obtained from the Type Culture Collection Center of the Chinese Academy of Sciences, and all the recovered cell lines used in the laboratory of the applicant of the present invention were passaged no more than 30 times.
  • the cell lines involved in the examples of the present invention need to be tested by PCR analysis to exclude mycoplasma contamination.
  • L02 cells were cultured in 10% fetal bovine serum (#16140071, Gibco TM ) and 1% penicillin-streptomycin (#15140-122; Gibco TM ) and in 5% CO 2 , 37°C water-jacketed cell culture incubator ( Thermo Fisher Scientific).
  • the primary hepatocytes used are isolated and collected from corresponding experimental mice by liver perfusion. Briefly, the mouse abdominal cavity was opened under painless anesthesia. Carefully perfuse through the hepatic portal vein with 1X Liver Perfusion Medium (#17701-038, Gibco TM ) and 1X Liver Digestion Medium (#17703-034, Gibco TM ). Then, the digested liver tissue was ground and filtered with a 100 ⁇ m stencil. Primary hepatocytes were collected by centrifuging the filtrate at 800 rpm, 4° C. for 5 min, and further purified with 50% percoll cell separation medium (#17-0891-01, GE Healthcare Life Sciences). The obtained hepatocytes were cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin in a cell culture incubator at 5% CO 2 at 37°C.
  • palmitic acid (dissolved in 0.5% fatty acid-free bovine serum albumin BSA) was prepared and obtained at a corresponding concentration. Then, primary hepatocytes or L02 cells were treated with PA containing cell culture medium for 10 hr or 4 hr. BSA (0.5%) without fatty acid alone was used as vehicle control.
  • the construction and method of the Trim31 knockout cell line used are as follows: construct a Trim31-deleted cell line through the CRISPR/Cas9 gene editing system, produce sgRNA targeting the human Trim31 gene and package it into the lentiviral CRISPR- V2 vector to form Cas9-sgRNA lentivirus.
  • the oligonucleotide sequences used to generate the sgRNA expression vector were: sgRNA-F: CACCCAACTCGCTGTTGCGGAATC; and sgRNA-R: AAACGATTCCGCAACAGCGAGTTG. then use 6 Transfection reagents will be packaged in vectors pSPAX2 and pMD2.
  • G and sgRNA expression vectors were transfected into HEK293T cells for 42 hours. Next, the obtained supernatant containing lentivirus was transferred into L02 cells to construct a gene knockout cell line. Cell clones lacking the target gene were selected by immunoblotting.
  • human or mouse full-length Rhbdf2 and Trim31 expression plasmids were established by PCR-based cDNA amplification, and then cloned into 3 ⁇ Flag-tagged pcDNA3.1 vector or 3 ⁇ HA-tagged pcDNA3.1 vector (Invitrogen )middle.
  • Truncated Rhbdf2 and Trim31 fragment expression vectors were obtained using standard PCR, including TRIM31-Flag RING ⁇ , TRIM31-Flag Box ⁇ , TRIM31-Flag CC ⁇ , RHBDF2-HA TMD ⁇ , RHBDF2-HA IRHD ⁇ and RHBDF2-HA Tail ⁇ , As shown in the legend, clone into the corresponding vector.
  • the Myc-tagged ubiquitin WT expression vector was constructed based on the pcDNA3.1 vector.
  • ubiquitin and corresponding derivatives include ubiquitin-K48O, ubiquitin-K63O, ubiquitin-K33O, ubiquitin-K6O, ubiquitin-K29O, ubiquitin-K27O and controls containing only a single complete amino acid residue vector, and then loaded into Myc-tagged pcDNA3.1 plasmid (Thermo Fisher Scientific). The vector was carefully transfected into L02 cells using Lipofectamine TM 3000 transfection reagent (Invitrogen TM ) according to the instruction manual.
  • the method for constructing an adenovirus-loaded Trim31 expression vector is as follows: separately target the specific short hairpin RNA oligonucleotide sequence (shTRIM31) targeting the human TRIM31 sequence (shRNA sequence RNAi#1: TTCCCGTCAAAGGAAGTTTGG; RNAi #2: TATGATGGACTCATGCCTTGC), loaded into adenovirus (AdTRIM31; AdshTRIM31) by Easy Adenoviral Vector System Kit (#240009, Agilent Technologies).
  • AdshGFP was used as a control for knockout or overexpression, respectively.
  • Recombinant adenovirus was purified and titrated to 5 ⁇ 10 10 (PFU). Afterwards, the hepatocytes were infected with the adenovirus diluted in the culture medium, the number of infections was 50 times, and the infection time was 24 hours.
  • CAG promoter sequence is shown in sequence NO:9
  • CMV promoter sequence is shown in sequence NO:10.
  • the TRIM31 expression gene of the mouse is shown in sequence NO: 6, and the RHBDF2 expression gene of the mouse is shown in sequence NO: 8.
  • the AAV8-TBG vector used is a prepackaged 8 serotype AAV with GFP overexpression, which is used to produce a recombinant AAV8-TBG-target gene-GFP expression vector.
  • the recombinant AAV8-TBG-gene of interest-GFP expression vector contains transcriptional control elements from the thyroxine-binding globulin (TBG) promoter, a cloning site for insertion of complementary DNA and a polyA signal.
  • TBG thyroxine-binding globulin
  • the terminal repeats of AAV serotype 2 flank the expression cassette.
  • the mouse full-length Trim31 sequence or the Trim31 sequence with RING domain deletion were respectively cloned into the vector AAV8-TBG-GFP.
  • the newly created vector AAV-TBG-Trim31-GFP was purified by ViraBind TM AAV Purification Mega Kit (VPK-141/VPK-141-5, Cell Biolabs, VPK-141/VPK-141-5, San Diego, USA).
  • AAV-TBG-Trim31 RING ⁇ -GFP was packaged into AAV8 and titrated accordingly by QuickTiter TM AAV Quantitaiton Kit (Cell Biolabs, VPK-145). Dilute the virus particles to a total volume of 50 ⁇ L with saline immediately before injection.
  • lentivirus-Trim31 lentivirus-Trim31
  • lentivirus-Trim31 RING ⁇ lentivirus-Trim31
  • the full-length Trim31 cDNA sequence was loaded into pLenti-CMV-GFP -Puro (Addgene) to upregulate Trim31 expression (pLenti-CMV-Trim31-GFP-Puro or pLenti-CMV-Trim31 RING ⁇ -GFP-Puro).
  • a commercial Lenti-Pac HIV expression packaging kit (LT002, GeneCopoeia, MD, USA) and the corresponding Lenti-Pac 293Ta cell line were used to prepare LV particles.
  • the newly created vector was concentrated and purified by ViraBind TM PLUS Lentivirus Concentration and Purification Kit (Cell Biolabs, VPK-095), and then titrated by QuickTiter TM Lentivirus Quantification Kit (Cell Biolabs, VPK-112).
  • intracellular triglycerides were detected as follows: according to the kit’s experimental instructions, a commercial triglyceride detection kit (#ab65336, Abcam) was used to quantitatively detect intracellular triglyceride (TG) levels.
  • the detection of metabolic indexes and serum cytokines is as follows: in order to carry out glucose tolerance test (GTT) and insulin tolerance test (ITT), the experimental mice in this part were fasted for 8 hours to ensure that the physiological response was normal, and then gave the mice The mice were intraperitoneally injected with glucose (2g/kg BW) (#158968, Sigma-Aldrich, Shanghai, China). Then, use commercially available blood glucose test strips ( Roche Diabetes Care GmbH, Shanghai, China) were used to detect blood glucose levels in mice. In order to examine the insulin resistance test, a certain amount of insulin (1 U/kg BW, Sigma Aldrich) was intraperitoneally injected into the mice, and then the insulin content in the blood of the mice was measured.
  • GTT glucose tolerance test
  • ITT insulin tolerance test
  • HOMA Homeostasis Model Assessment
  • Cytokine and chemokine levels in mouse sera were assessed using corresponding commercial enzyme-linked immunosorbent assay (ELISA) kits.
  • ELISA enzyme-linked immunosorbent assay kits.
  • TNF- ⁇ (#MTA00B), IL-1 ⁇ (#MLB00C), IL-10(#M1000B), IL-6(#M6000B) and CCL-2(#MJE00B) ELISA kits were purchased from R&D system (Shanghai, China ), and use according to the product instructions. All corresponding sera were guaranteed to be stored in a -80°C freezer until use.
  • the liver function parameters and liver lipid levels are detected as: serum alanine aminotransferase (ALT) (#MAK052, Sigma-Aldrich), aspartate aminotransferase (AST) (#MAK055, Sigma-Aldrich), Alkaline phosphatase (AKP) (#ab83369, Abcam), serum insulin (#ab277390, Abcam) and hepatic triglycerides (TG) (#MAK266, Sigma-Aldrich), total cholesterol (TC) (#ab65359, Abcam) and non-esterified free fatty acids (NEFA) (#E-BC-K014, Elabscience, Inc., Houston, USA) were detected in designated experimental groups using commercially available detection kits.
  • the histopathological examination method is: in order to carry out histological and immunohistochemical analysis, liver samples are correspondingly fixed with 10% neutral formalin tissue fixative (#HT501128, Sigma-Aldrich), paraffin Embedded (#YA0010, Solarbio Life Sciences, Beijing, China), and then thinly cut tissue sections with a microtome. Thin liver sample sections were stained with hematoxylin and eosin (H&E) (#abs9217, hematoxylin and eosin staining kit, Absin, Shanghai, China) to visualize lipid deposition and tissue damage.
  • H&E hematoxylin and eosin stain
  • the immunoprecipitation method is: use Lipofectamine TM 3000 transfection reagent (Invitrogen TM ) to transfect the corresponding vector into L02 cells, and then culture for 36 hours. Subsequently, cells were harvested and homogenized into immunoprecipitation (IP) specific lysis buffer (#87787, Pierce TM IP Lysis Buffer, Thermo Scientific Pierce) at 4°C, and then centrifuged at 13000 rpm in a refrigerated centrifuge for 20 minute.
  • IP immunoprecipitation
  • the collected cell lysates were mixed with Protein A/G Magnetic Agarose Beads (#78609, Thermo Scientific Pierce) and incubated at room temperature for 2 hours, and then mixed with the indicated antibodies overnight at 4°C. Immunized compounds were harvested after washing with immunoprecipitation buffer and subjected to western blot analysis by incubation with the indicated primary and corresponding secondary antibodies.
  • the glutathione s-transferase (GST) pull down assay is as follows: the direct protein interaction between Trim31 and Rhbdf2 is combined with the GST pull down assay.
  • Pierce TM GST protein interaction pull down kit (#21516, Thermo Fisher Scientific) was used for the detection of this part of the experiment. Briefly, Rosetta (DE3) E.
  • coli cells were transformed with plasmid pGEX-4T-1-GST-Trim31 or pGEX-4T-1-GST-Rhbdf2, followed by incubation with 0.5 mM isopropyl ⁇ -D- to induce expression of thio Co-incubation with galactopyranoside (IPTG) (#I5502, Sigma-Aldrich). Then, the lysed E. coli extract was mixed with GST beads at 4°C for 1 hour. GST beads were then incubated with Flag-tagged Trim31 or Flag-tagged Rhbdf2 for 4 hr. Interacting protein complexes were then eluted by elution buffer and analyzed by Western blot using an anti-Flag antibody. A vector constructed in E. coli expressing only the GST tag was used as a negative control.
  • the in vitro binding and ubiquitination experiment method is as follows: use Trim31, Rhbdf2 and Trim31 protein with RING domain deletion Expressed in microtubules in vitro by the Rapid Coupled Transcription/Translation System (Promega). Protein interaction binding assays were performed by mixing the corresponding Flags. Rhbdf2 and Trim31 were tagged together, followed by immunoprecipitation with Flag antibody and immunoblotting with Trim31 antibody. Ubiquitination levels were assayed using the Ubiquitination Kit (Boston Biochem).
  • the Western blot analysis method is as follows: cells or liver tissue are homogenized into RIPA lysate and extraction buffer (#9806, CST) to obtain a single homogenate. Then, the liquid supernatant was concentrated by centrifugation at 13500 rpm, 4°C for 30 minutes. Protein concentration The total protein concentration in the homogenate was determined by Pierce TM Rapid Gold BCA Protein Assay Kit (Thermo Fisher Scientific) using bovine serum albumin as a standard. Total protein extraction samples were then subjected to Western blot analysis.
  • the relevant experiments proposed were carried out independently at least three times, and all the data were analyzed using this statistical method, unless otherwise specified.
  • the statistical method is as follows: the quantitative values of the data are expressed as mean ⁇ standard error of the mean.
  • One-way analysis of variance (ANOVA) was used for comparison, Dunnett's multiple comparison test was used for multiple groups, and two-tailed t-test was used for two groups.
  • Data analysis was performed using GraphPad Prism software (Version 8.2.0 for Mac OS X Snow Leopard; Graph Pad Software, Inc., San Diego, CA) and SPSS statistical software (Version 26.0.0.2 for Mac OS X Snow Leopard; IBM, Inc. , New York, USA).
  • a P value less than 0.05 (P ⁇ 0.05) was considered statistically significant.
  • the collection of animal experiment data is random. No data were excluded in the final statistical analysis.
  • Example 1 In the liver with hepatic steatosis, the activity of TRIM31 is inhibited and verified
  • TRIM31 in order to verify that TRIM31 is involved in hepatic steatosis and metabolism, the expression level of TRIM31 was verified in liver tissues isolated from normal diet mouse models and high-fat diet mouse models.
  • CFD normal normal diet
  • Rhbdf2 Figures 1a, 1b and 2a.
  • the dynamic expression level results of HFD-fed mice from 0 to 16 weeks showed that the expression of TRIM31 was gradually suppressed in fatty liver (Fig. 1c and Fig. 2b, 2c).
  • the embodiment of the present invention further detects its expression pattern in the liver of NAFLD (nonalcoholic fatty liver disease) and NASH (nonalcoholic steatohepatitis) patients.
  • NAFLD nonalcoholic fatty liver disease
  • NASH nonalcoholic steatohepatitis
  • TRIM31 levels were significantly reduced and Rhbdf2 levels were increased in the liver compared with non-steatosis samples.
  • TRIM31 expression levels were significantly reduced in livers of NASH patients compared with those with pure steatosis (Fig. 1d and Fig. 2d).
  • ROS reactive oxygen species
  • NAC active oxygen scavenger N-Acetyl-L-cysteine
  • CAT antioxidant catalase
  • RHBDF2 has also been shown to be regulated by ubiquitination.
  • RHBDF2 levels were significantly elevated in livers of patients with NASH and pure steatosis phenotype compared with non-steatosis samples.
  • the expression of RHBDF2 in the liver of NASH patients was significantly higher than that of patients with pure steatosis (Fig. 2d).
  • the embodiment of the present invention also uses cycloheximide (CHX), a protein synthesis inhibitor in eukaryotes, to detect the expression of RHBDF2 protein after PA treatment.
  • CHX cycloheximide
  • the increase of Rhbdf2 was suppressed at 5h and decreased at 10h compared to the control group (0h).
  • the degradation of RHBDF2 was significantly inhibited when co-treated with the proteasome inhibitor MG132, but the degradation effect of RHBDF2 was not obvious when co-incubated with chloroquine (a lysosomal inhibitor) (Fig. 1g-1i). It was found that Rhbdf2 degradation is through the proteasomal pathway. Virtually all RHBDF2 was ubiquitinated in PA-treated primary hepatocytes.
  • Example 2 TRIM31 can improve insulin resistance and abnormal glucose metabolism verification
  • TRIM31 is closely related to fatty liver
  • a series of mouse models were constructed in the embodiment of the present invention to verify the role of TRIM31 in regulating the hallmark influencing factors of insulin resistance and glucose metabolism disorders, HFD-induced insulin resistance and glucose metabolism disorders It is a common complication and an important cause of fatty liver.
  • Hepatocyte-specific TRIM31 knockout mice (THKO) (Fig. 3a, 3b) and hepatocyte-specific TRIM31 overexpression mice (THTG) (Fig. 4a, 4b) were established to verify the effect of TRIM31 on HFD-induced insulin resistance and glucose Protection from metabolic disorders.
  • the final results showed that long-term HFD led to an increase in body weight, fasting blood glucose and fasting insulin levels in mice, which were significantly increased in TRIM31 knockout, but were alleviated to some extent in TRIM31 overexpression ( Figure 5a-5d ).
  • TRIM31 significantly accelerated the reduction of hepatic glycogen storage (Fig. 6g), while the overexpression of TRIM31 inhibited the reduction of hepatic glycogen storage (Fig. 5g).
  • hepatic TRIM31 upregulation significantly reduced mRNA and protein expression profiles associated with markers of gluconeogenesis including glucose-6-phosphatase (G6Pase) and phosphoenolpyruvate carboxykinase (PEPCK); however, phosphoenolpyruvate carboxykinase (PEPCK) Increases in AKT (p-AKT), glycogen synthase kinase 3 ⁇ (p-GSK3 ⁇ ), and forkhead box O1 (p-FOXO1). HFD diet-induced dysregulation of hepatic insulin signaling was enhanced in THKO mice but significantly restored in THTG mice compared with corresponding controls (Fig. 6h and Fig. 5h). The results showed that in the liver, TRIM31 was an important factor in inhibiting HFD-induced insulin resistance and glucose metabolism disorders.
  • Example 3 TRIM31 can prevent hepatic steatosis and inflammation verification
  • the embodiment of the present invention verifies the effect of TRIM31 on improving the main features of hepatic steatosis and inflammation.
  • liver weight and liver weight/body weight were significantly increased in THKO mice fed a long-term HFD diet compared with controls (Fig. 7a).
  • liver appearance changes were used to measure the liver, and the concentrations of triglyceride (TG), total cholesterol (TC) and non-esterified fatty acid (NEFA) in liver tissue were measured , to determine the deposition of hepatic lipids.
  • TG triglyceride
  • TC total cholesterol
  • NEFA non-esterified fatty acid
  • the final results showed that the liver function-related indicators of THKO mice in the HFD group, serum alanine aminotransferase (ALT), aspartate aminotransferase (AST) and alkaline phosphatase (AKP) were significantly increased (Figure 7b-7d).
  • liver fat content and indicators related to liver function such as TG, TC, NEFA, ALT, AST, and AKP were significantly reduced in THTG mice fed a high-fat diet (Fig. 7g-7i).
  • the expression of genes related to fatty acid uptake and synthesis and the expression of genes related to fatty acid ⁇ oxidation also tended to normal levels in THTG mice compared with their corresponding control groups (Fig. 7j).
  • TRIM 31 participates in the regulation of inflammation in the liver
  • the activity of the inflammatory signaling pathway and the corresponding changes of cytokines and chemokines related to inflammation were studied.
  • the signaling pathways related to inflammation caused by HFD were significantly enhanced in THKO mice and significantly decreased in THTG mice ( Figure 9a, 9b).
  • TRIM 31 is an important inhibitor of HFD-induced inflammation and liver lipid accumulation in mice.
  • Example 4 In vitro gene therapy mediated by Example 4 helps to alleviate fatty liver verification
  • THKO(LV+) mice had lower blood glucose concentration than THKO(LV-) mice (Fig. 10e, 10f).
  • the results further verified the significant downregulation trend of hepatic TG, TC and NEFA in THKO(LV+) mice (Fig. 10g).
  • elevated TRIM31 in hepatocytes significantly decreased the gene expression of fatty acid uptake and synthesis and promoted the gene expression of fatty acid ⁇ -oxidation in THKO (LV+) mice fed a high-fat diet (Fig. 10h).
  • LV-hTRIM31-mediated gene therapy in vitro further demonstrated that restoration of TRIM31 in hepatocytes indeed significantly attenuated high-fat diet-induced insulin resistance, hepatic steatosis, and inflammation (Fig. 12a-12k).
  • Example 5 Verifies that TRIM31 inactivates RHBDF2-MAP3K7 to inhibit the development of non-alcoholic fatty liver
  • TRIM31 has a consistent and significant inhibitory effect on fatty liver and related pathological phenotypes
  • the examples of the present invention continue to verify the molecular mechanism and intrinsic function of TRIM31. Because RHBDF2 is ubiquitinated and plays a key role in the phenotypes of NAFLD and NASH, the effect of TRIM31 on RHBDF2 and its downstream signaling events was further verified.
  • RHBDF2-MAP3K7 signaling including ADAM17, TNFR1/2, MKK7, and c-Jun was also identified to be involved in the development of fatty liver.
  • the examples of the present invention verified that the levels of ADAM17, TNFR1/2, and p-MAP3K7 and its downstream component MKK7 were upregulated in the absence of TRIM31 in vivo and in vitro, but were inhibited in the overexpression of TRIM31 (Fig. 13b, 13c).
  • TRIM31 in order to further verify the role of TRIM31 in the function of RHBDF2, a PA-induced injury model of insulin signaling in primary hepatocytes was constructed.
  • RHBDF2 was overexpressed or inactivated in TRIM31-specific deletion or TRIM31-transgenic hepatocytes using adenovirus-encapsulated full-length RHBDF2 sequence (AdRHBDF2) or shRNA targeting RHBDF2 (AdshRHBDF2).
  • AdRHBDF2 adenovirus-encapsulated full-length RHBDF2 sequence
  • AdshRHBDF2 shRNA targeting RHBDF2
  • THTG hepatocytes transfected with AdshRHBDF2 showed a tendency to effectively alleviate PA-induced impaired insulin signaling (e.g., p-AKT, p-GSK3 ⁇ , p-FOXO1) compared to AdRHBDF2-transfected THTG hepatocytes (Fig. 13d and Fig. 14a). It is further proved that dysfunction of the RHBDF2-MAP3K7 signaling pathway is the key to the protective effect of TRIM31 against hepatic steatosis.
  • PA-induced impaired insulin signaling e.g., p-AKT, p-GSK3 ⁇ , p-FOXO1
  • the embodiment of the present invention uses the immunoprecipitation method to verify that TRIM31 directly interacts with RHBDF2 during the development of non-alcoholic fatty liver disease.
  • TRIM31 is mainly composed of three parts: N-terminal ring finger domain, B-box domain and C-terminal helical domain (Fig. 15c).
  • the embodiment of the present invention chose to generate a series of truncated TRIM31 mutants with marker vectors, including wild type (TRIM31-Flag WT), ring finger domain excision mutants (TRIM 31-Flag RING), B-Box domain deletion mutant (TRIM 31-Flag Box) and helical domain deletion mutant (TRIM 31-FlagCC) vectors for the following binding experiments.
  • the embodiment of the present invention also established three RHBDF2 truncated mutants of plasmids with HA tags, including wild type (RHBDF2-HA WT), transmembrane domain (TMD) deletion mutant (RHBDF2-HA TM), inactive rhomboid homology domain (IRHD) deletion mutant (RHBDF2-HA IRHDD) and N-terminal cytoplasmic tail (tail) domain deletion mutant (RHBDF2-HA Tail) vectors.
  • TMD transmembrane domain
  • IRHD inactive rhomboid homology domain
  • RHBDF2-HA IRHDD N-terminal cytoplasmic tail domain deletion mutant
  • RHBDF2-HA Tail N-terminal cytoplasmic tail domain deletion mutant
  • the embodiment of the present invention examines whether they have similar biological functions in mice. Therefore, the binding domains of mouse TRIM31 and RHBDF2 were also detected with TRIM31-Flag CC ⁇ , RHBDF2-HA Tail ⁇ , and corresponding wild-type vectors. Consistent with the results of protein binding experiments, in the transfected mouse hepatocytes, the CC domain-deleted TRIM31 could not bind to RHBDF2 (Fig. 14c).
  • Example 7 Verifies that TRIM31 promotes the degradation of RHBDF2 through K48-linked polyubiquitination
  • the embodiment of the present invention verifies that TRIM31 can promote the degradation of RHBDF2 through K48-linked polyubiquitination. It was previously speculated that RHBDF2 may have a binding site for K48 ubiquitination on the cytoplasmic domain, thereby degrading the proteasome. Example 6 has confirmed that TRIM31 and RHBDF2 are able to interact (Figure 15c).
  • the embodiment of the present invention verifies whether TRIM31 and its corresponding E3 ubiquitination ligase can regulate the activity of RHBDF2.
  • the experimental results show that the ubiquitination level of RHBDF2 is significantly increased in vivo and in vitro (Fig. 15d and Fig. 2f).
  • RHBDF2, Myc-ubiquitin and marked wild-type TRIM31 were transferred into L02 cells together.
  • the ubiquitination level of RHBDF2 was significantly increased with the presence of TRIM31 carrier.
  • TRIM31 expression vectors with RING-finger domain excision mutants failed to catalyze RHBDF2 ubiquitination, suggesting that functional TRIM31 requires the RING-finger domain to regulate RHBDF2 ubiquitination (Fig. 15e).
  • RHBDF2 can be modified by K63-linked ubiquitination. Consistent with previous studies, endogenous RHBDF2 was nearly ubiquitinated and associated with K48 and K63 in PA-treated L02 cells. In TRIM31-deficient L02 cells (THKO-L02), only the K48-linked polyubiquitination of RHBDF2 was significantly inhibited, but the effect on the K63-linked ubiquitination of RHBDF2 was negligible (Fig. 15f).
  • the examples of the present invention used ubiquitin mutant plasmids K48, K63, K33, K6, K29 and K27 to conduct in vitro transfection experiments.
  • the labeling of "O" in K48O, K63O, K33O, K6O, K29O and K27O revealed that all lysine residues in ubiquitin except themselves were completely mutated.
  • a substantial TRIM31-mediated increase in RHBDF2 polyubiquitination could be observed in the presence of the K48 vector, but not in the other vectors (Fig. 15g).
  • the embodiment of the present invention verifies that the RING-finger domain mutant actually eliminates the E3 ubiquitin ligase activity of TRIM31.
  • deletion of the RING-finger domain of TRIM31 not only abolished the polyubiquitination of RHBDF2, but also prevented TRIM31 from inhibiting the activation of the RHBDF2-MAP3K7 pathway and its downstream signaling cascades. capabilities (e.g., ADAM17, p-NF- ⁇ B, p-I ⁇ B ⁇ and p-JNK1/2) (Fig. 15g).
  • TRIM31-induced degradation of RHBDF2 via K48-linked polyubiquitination is dependent on its E3 ubiquitin ligase activity.
  • the embodiment of the present invention established a lentiviral load TRIM 31 (LV-TRIM 31 RING) with RING-finger knockout, and was used again for further research on the effect of TRIM 31 RING on HFD-induced hepatic steatosis, insulin resistance and inflammation Effect (Fig. 16a and Fig. 17a).
  • mice treated with TRIM31 RING-finger knockout-hepatocyte transplantation (THKO) (LV+) failed to alter the induction of HFD compared to controls (THKO) (LV-).
  • THKO RING-finger knockout-hepatocyte transplantation
  • mice transplanted with human TRIM31 RING-hepatocytes did not significantly alleviate insulin resistance, hepatic steatosis and inflammation induced by high-fat diet (Fig. 18a -18g).
  • Fig. 18a -18g mice transplanted with human TRIM31 RING-hepatocytes did not significantly alleviate insulin resistance, hepatic steatosis and inflammation induced by high-fat diet.
  • adeno-associated virus subtype 8 (AAV8)-thyroxine-binding globulin (TBG) of TRIM31 carrying a RING-finger domain mutant (AAV-TRIM31 RING) was generated accordingly and injected in parallel with AAV-TRIM31 into Among pretreated WT mice fed HFD for 6 weeks, mice injected with AAV-GFP served as controls (Fig. 19a).
  • mice administered AAV-TRIM31 RING domain mutants ameliorated impaired insulin signaling, gene expression changes in fatty acid uptake/synthesis and fatty acid ⁇ -oxidation, and RHBDF2-associated downstream inflammation It did not show any significant changes in signaling cascades (Fig. 19g-19i).
  • the TRIM31-RHBDF2 interaction and the RING-finger domain of TRIM31 are critical for TRIM31-mediated hepatic steatosis and inflammation.
  • Example 9 Verifies that TRIM31 regulates and protects hepatic steatosis by targeting RHBDF2
  • RHBDF2 deletion can alleviate steatohepatitis induced by long-term HFD.
  • RHBDF2 deletion blocked the effect of TRIM31 ablation on HFD-stimulated RHBDF2-MAP3K7 axis and Effects of downstream signaling cascades, body weight, liver weight, liver weight/body weight ratio, impaired insulin signaling, abnormal blood glucose levels, hepatic lipid deposition, and elevated expression of proinflammatory-related genes (Figs. 20a-20f and Figs. 21a-21e)
  • the PA-induced changes in intracellular triglyceride levels in vitro also showed the same trend (Fig. 20g and Fig. 22).
  • Example 10 Feasibility and effect evaluation of non-alcoholic steatohepatitis targeting TRIM31-RHBDF2 signaling therapy
  • the embodiment of the present invention verifies the therapeutic feasibility and effect of targeting the TRIM31-RHBDF2-MAP37K pathway in the development and progression of NASH.
  • THTG mice were then fed a HFHF diet containing 14% protein, 42% fat, 44% carbohydrate, 0.2% cholesterol, and a total of 42 g/L carbohydrate mixed in drinking water at a weight ratio of 55% fructose and 45% sucrose compound for 16 weeks to create mice with a NASH phenotype (THTG-HFHF).
  • NTG mice were used as controls (NTG-HFHF) and fed the same diet ad libitum for 16 weeks.
  • NASH model induced by HFHF, liver weight, liver weight/body weight ratio, and hepatic TG, TC, and NEFA concentrations in the THTG group were significantly lower than those in the NTG control group after 16 weeks of HFHF diet, while THTG-HFHF There was no significant difference in body weight between NTG-HFHF and NTG-HFHF mice (Fig. 23a-23c).
  • genes related to inflammation and collagen in THTG Hepatic lipid deposition e.g., CD36, FASN, and ACACa
  • genes involved in inflammation e.g., TNF- ⁇ , IL-1 ⁇ , IL-6, and CCL-2
  • liver fibrosis i.e., collagen deposition
  • expression of collagen synthesis-related genes e.g., TIMP1, CTGF, COL1A1, and COL3A1 were significantly reduced (Fig. 23d-23i).
  • TRIM31 After 16 weeks of HFHF diet, overexpression of TRIM31 also significantly downregulated mouse serum AST, ALT and AKP levels, concentrations of pro-inflammatory cytokines TNF- ⁇ , IL-6 and IL-1 ⁇ , and TRIM31-regulated RHBDF2-MAP3K7 Signaling pathways and downstream signaling pathways.
  • the data in the examples of the present invention reveal the efficacy of TRIM31 in the treatment of mouse steatohepatitis and related metabolic syndrome.
  • liver TRIM31 is a significant negative regulator of high-fat diet (HFD/HFHF)-induced or genetically induced chronic inflammation, insulin resistance, hepatic steatosis, and NASH.
  • HFD/HFHF high-fat diet
  • RHBDF2-MAP3K7 signaling further mediated increased expression of downstream signaling pathways, including JNK-IRS1-associated insulin signaling, I ⁇ B ⁇ -NF- ⁇ B-c-Jun pathway, AKT-GSK3 ⁇ -Inhibition of phosphorylation levels of FOXO1 and CTGF-TIMP1 pathway, promoting impaired insulin signaling (insulin resistance), chronic inflammatory effects, abnormal glucose metabolism disorders and collagen deposition in the liver, thereby exacerbating abnormal lipid metabolism , and the development of hepatic steatosis and NASH phenotypes (Fig. 23k).
  • JNK-IRS1-associated insulin signaling I ⁇ B ⁇ -NF- ⁇ B-c-Jun pathway
  • AKT-GSK3 ⁇ -Inhibition of phosphorylation levels of FOXO1 and CTGF-TIMP1 pathway promoting impaired insulin signaling (insulin resistance), chronic inflammatory effects, abnormal glucose metabolism disorders and collagen deposition in the liver, thereby exacerbating abnormal lipid metabolism , and
  • Example 11 Betaine (BCN) activates TRIM31 to alleviate liver fibrosis and inflammation in mice
  • mice with NASH phenotype were constructed.
  • a blank group (Ctrl), a NASH group (THTG) and an administration group (BCN) were set up, and the administration group was fed after mixing betalain (BCN) with feed at a ratio of 0.2 g/kg.
  • BCN betalain
  • Fisetin activates TRIM31 to alleviate liver fibrosis and inflammation in mice
  • mice with NASH phenotype were constructed.
  • a blank group (Con) a NASH-THTG group (HFD) and an administration group (FIS) were set up, and the administration group was administered with 20 mg/kg, 40 mg/kg, and 80 mg/kg of fisetin (FIS).
  • FID NASH-THTG group
  • FIS administration group
  • the mRNA levels of ⁇ -SMA, collagen I (Collagen I), TGF- ⁇ 1, Fibronectin and other protein genes in the liver of THTG-HFHF mice significantly increased, and the mRNA levels of TRIM31 protein gene The level was significantly reduced, indicating that high sugar and high fat cause collagen accumulation and oxidative stress leading to non-alcoholic steatohepatitis.
  • ⁇ -SMA Collagen I
  • TGF- ⁇ 1 Fibronectin protein genes in the liver tissues of mice treated with low, medium, and high doses of FIS were significantly reduced, and the mRNA levels of TRIM31 protein genes were significantly increased , especially the high-dose administration group was close to the normal level of the control group, effectively alleviating liver fibrosis and inflammation.
  • Example 12 Carminic acid (CA) activates TRIM31 to alleviate liver fibrosis and inflammation in mice
  • mice with NASH phenotype were constructed.
  • Set blank group Con
  • NASH-THTG group Fru
  • low-dose administration group CAL, 0.5% CA+Fru
  • high-dose administration group CAH, 1%CA+Fru
  • administration group adopts 0.5 % and 1% carminic acid (CA) orally.
  • ⁇ -SMA Collagen I
  • TGF- ⁇ 1 TGF- ⁇ 1
  • MMP-9 MMP-9 protein genes in the liver tissue of mice treated with low and high doses of CA were significantly reduced, the expression level of Keap1 protein was significantly reduced, and the expression level of TRIM31 protein was significantly reduced.
  • the expression was significantly increased, especially in the high-dose administration group approaching the normal level of the control group, effectively alleviating liver fibrosis and inflammation.
  • TRIM31 can activate TRIM31 to relieve liver fibrosis and inflammation in mice, the following substances ((1)-( 6)) One or more combinations can achieve the effect of activating TRIM31 to relieve liver fibrosis and inflammation in mice:
  • Alkaloid compounds or compositions including berberine, magnolialine, haematine, serpentine, leonurine, tetrandrine, ethosine, peycinine, colchicine, piperine, Rhynchophylline, camptothecin, tetrahydropalmatine, jatrorrhizine, chelidonine, lycorine, behenicine, matrine, oxymatrine, a compound or a combination of multiple compounds thing;
  • Flavonoid compounds or compositions including quercetin, dihydroquercetin, luteolin, morin, morin, apigenin, diosmin, chrysin, alfalfa, puerarin, and anthocyanins Proanthocyanidins, daidzein, apigenin, baicalein, scutellarein, chrysin, wogonin, cyanidin, silymarin, silybin, daidzein, genistein, galangin, kaempferen Phenol, isorhamnetin, myricetin, hesperetin, naringenin, glycyrrhizin, catechin, epicatechin, gallocatechin, cyanidin, delphinidin, peladin, breviscapine A compound or a combination of compounds of flower element, afotoin, rutin, luteolin, baicalin, apigenin, juglandin;
  • Polyphenol compounds or compositions including chlorogenic acid, cynaric acid, glycosides, glycosides, tea polyphenols, resveratrol, pomegranate polyphenols, curcumin, ferulic acid A compound or a combination of compounds;
  • Amine compounds or compositions penicillamine, pomalidomide, lenalidomide, a compound or a combination of multiple compounds among thalidomide;
  • Chinese medicine extracts including astragalus, Codonopsis pilosula, Danshen, Angelica, Chinese yam, Panax notoginseng, Schisandra chinensis, Polygonatum, Ganoderma lucidum, Yunzhi, artichoke, thistle, milk thistle, kudzu root, Coptis chinensis, Cortex Phellodendron, Turmeric, honeysuckle, Hovenia dulcis, tangerine peel, mulberry leaf, ginkgo biloba, lily, bear gall, pig gall, capillary, citrus aurantium, licorice, knotweed, pagoda seed, paeonol, weeping grass, gardenia, wolfberry, white peony root, chuanxiong, peach kernel , rhubarb, chrysanthemum, Xihuangcao, Hedyotis diffusa, Fangji, Imperata rh
  • Glycyrrhizic acid glycyrrhizin, adenosylmethionine, ursodeoxycholic acid, tauroursodeoxycholic acid, chenodeoxycholic acid, taurochenodeoxycholic acid, bicyclol, bifendate, One or more of sorafenib, regorafenib, lenvatinib, metformin.

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Abstract

本发明涉及一对组合因子及其应用，该组合因子分别为肝细胞来源的TRIM31、RHBDF2，所述TRIM31与所述RHBDF2存在此消彼长的关系。所述组合因子可作为非酒精性脂肪肝病(NAFLD)、非酒精性脂肪肝炎(NASH)、和/或代谢紊乱的标志物，其可用于有效鉴定NAFLD、NASH、代谢紊乱、和/或肝细胞变性。所述TRIM31的促进剂还可用于有效消除或缓解胰岛素抵抗、肝脂肪变性、肝脂肪炎症和肝纤维化。

Description

组合因子及其应用 技术领域
本发明属于细胞工程技术领域，具体涉及一种组合因子对及其应用。
背景技术
最新的流行病学研究发现，肥胖及其代谢并发症已成为最严重的公共卫生危机之一。随着肥胖高危人群的增加，持续、长时间的高能量饮食摄入增加了发生包括肥胖、高脂血症、高脂血症以及严重的心脑血管疾病等全身性代谢综合征的风险。事实上，非酒精性脂肪性肝病(NAFLD)等与肥胖相关的共病的患病率同步增加，NAFLD已经是最常见的慢性肝病。NAFLD的严重程度从单纯脂肪变性到伴有恶性转化和坏死性炎症改变的肝细胞损伤，以非酒精性脂肪性肝炎(NASH)为特征，揭示了患者更容易发生肝纤维化和肝细胞癌。更严重的是，形成的NASH反过来将进一步促进其并发症,例如，胰岛素抵抗的发展，高血压，慢性肾病(CKD)和2型糖尿病(T2D)。然而不幸的是，到目前为止，世界上还没有经过批准和证明的完全有效的治疗NASH的药物治疗方法，缓解该疾病引起的相关并发症的努力也没有完全达到预期。NAFLD/NASH的有效药物和治疗方案的开发，主要依赖于靶向关键信号调节或与致病机制密切相关的调控过程。
Rhomboid 5Homolog 2(RHBDF2)，也被称为iRhom2，是Rhomboid膜内蛋白酶家族的非活性成员，已被确定为炎症相关疾病的关键致病调节因子，如肥胖、关节炎、肾炎、动脉粥样硬化和纤维化等疾病。而且RHBDF2通过招募MAP3K7显著提高其磷酸化水平并激活下游炎症信号。RHBDF2-MAP3K7信号通路的激活有助于NAFLD的发生，而NAFLD可导致肝纤维化。同时，RHBDF2具有被泛素化(另一个重要的调控修饰)调控的能力。有研究推测，在蛋白酶抑制剂的存在下，RHBDF2的半衰期显著延长，进一步表明在细胞质结构域可能存在K48连接的泛素化位点，从而发生蛋白酶体降解。然而，是否存在某些调控因子调控RHBDF2泛素化和RHBDF2介导的NAFLD/NASH发病机制仍不清楚。
同时考虑到NASH的复杂发病机制，目前尚无临床有效的治疗NASH的药物。因此，全面细致地了解非酒精性脂肪性肝病的发病机制和过程，寻找关键的信号调节因子，已成为NASH治疗药物开发的迫切问题。
发明内容
鉴于NAFLD和NASH的病理过程是一种与代谢综合征和全身炎症反应综合征(SIRS)相关的纠缠性进展，应针对致病信号通路中的关键因素设计完善的靶点。胰岛素抵抗、肝脏脂肪变性、长期炎症、胶原沉积等因素共同促进了代谢综合征和NASH的病理形成。而且，长期高热量饮食治疗能够刺激肝脏产生严重的胰岛素抵抗，使肝脏中胰岛素调节葡萄糖代谢的正常功能被抑制。然而，胰岛素促进脂肪生成的功能被完全保留。因此，严重的胰岛素抵抗导致肝脏中过量脂质的异常积累。此外，高脂的摄入也会引发炎症反应，并协同促进胰岛素抵抗的产生和发展。
具体地，E3泛素连接酶——三结构域蛋白31(TRIM31)的三部分已经被确定为先天免疫应答的“Janus-faced”调控因子，通过泛素修饰促进目标底物降解或信号转导。TRIM31失调的动物通常表现出严重的肠道炎症和肠道菌群失衡，TRIM31失调会诱导内毒素血症的产生，并提高NLRP3炎症小体的活化。另一方面，在败血症中，TRIM31通过促进细胞凋亡和NF-κB信号通路，导致心肌功能障碍。而NF-κB信号介导的慢性炎症可增强结直肠癌的侵袭和转移。这些看似矛盾的发现揭示了TRIM31在不同的病理过程中发挥了完全不同的功能。
所以，本发明目的在于提供一种组合因子对，所述组合因子分别为肝细胞来源的TRIM31、RHBDF2。所述TRIM31包含如sequenceNO：1或sequenceNO：2所示的氨基酸序列；所述RHBDF2包含如sequenceNO：3或sequenceNO：4所示的氨基酸序列。其中编码TRIM31的核酸序列包括如sequenceNO：5或sequenceNO：6所示的序列；编码RHBDF2的核酸序列包括如sequenceNO：7或sequenceNO：8所示的序列。
在某些具体实施例中，编码TRIM31的核酸序列也可以是sequenceNO：6所示的序列的人源化序列；编码RHBDF2的核酸序列也可以是sequenceNO：8所示的序列的人源化序列。
进一步，所述TRIM31与所述RHBDF2存在此消彼长的关系。即若所述TRIM31分泌增加，则会抑制RHBDF2分泌，即RHBDF2分泌减少。
具体地，在某些具体实施例中，使用肝细胞特异性TRIM31敲除的小鼠(THKO)/转基因小鼠(THTG)和慢病毒介导的离体基因治疗(LV-TRIM31)小鼠，本发明确定肝脏TRIM31是高脂饮食(HFD/HFHF)诱导或遗传诱导的慢性炎症、胰岛素抵抗、肝脏脂肪变性和NASH的显著负调节因子。对潜在分子机制的研究发现，在长期的高热量饮食下，体内和体外实验中明显观察到TRIM31的减少，这导致RHBDF2-MA3K7信号通路激活的增加。在TRIM31功能失调的情况下，强烈激活的RHBDF2-MAP3K7信号传导进一步介导下游信号通路表达的增加，包括JNK-IRS1相关的胰岛素信号传导、IκBα-NF-κB-c-Jun通路、AKT-GSK3β-FOXO1磷酸化水平的抑制和CTGF-TIMP1通路，促进受损的胰岛素信号传导(胰岛素抵抗)、慢性炎症作用、异常的葡萄糖代谢紊乱和肝脏中的胶原沉积的发生，从而加重异常的脂质代谢，以及肝脏脂肪变性和NASH表型的产生。
在复杂的肝脏代谢调节的网络中，高脂饮食诱导的肝脏脂肪变性期间RHBDF2-MAP3K7信号通路活性和下游促炎因子的增加被肝脏特异性TRIM31过表达显著抑制，表明TRIM31对RHBDF2-MAP3K7介导的炎症反应、胰岛素抵抗和脂肪变性具有抑制作用。利用阳性离体基因治疗恢复小鼠TRIM31的表达也表明，它能有效抑制RHBDF2的活性，缓解上述三种病理表型。而且，有研究发现TRIM31的功能可能在调节免疫相关疾病中具有“Janus-face”(双面)作用，表现为可催化靶向底物降解或者通过泛素化的信号转导使底物降解。有研究表明，TRIM31缺乏失去了对结肠炎炎症反应的抑制作用；但另一方面，TRIM31被确定为阳性启动子，可加剧炎症相关信号引发的心肌病。这些看似相反的结论可能表明TRIM31的功能与器官类型和细胞类型密切相关。
本发明目的在于还提供一种前述的组合因子对作为NAFLD/NASH和/或代谢紊乱的标志物中的应用。所述TRIM31包含如sequenceNO：1或sequenceNO：2所示的氨基酸序列；所述RHBDF2包含如sequenceNO：3或sequenceNO：4所示的氨基酸序列。其中编码TRIM31的核酸序列包括如sequenceNO：5或sequenceNO：6所示的序列；编码RHBDF2的核酸序列包括如sequenceNO：7或sequenceNO：8所示的序列。
本发明目的在于还提供一种TRIM31作为制备NAFLD/NASH和代谢紊乱的抑制因子/抑制剂中的应用。所述TRIM31包含如sequenceNO：1或sequenceNO：2所示的氨基酸序列；编码TRIM31的核酸序列包括如sequenceNO：5或sequenceNO：6所示的序列。
本发明目的在于还提供一种TRIM31的促进剂作为制备NAFLD、NASH或代谢紊乱的药物中的应用。所述TRIM31包含如sequenceNO：1或sequenceNO：2所示的氨基酸序列；编码TRIM31的核酸序列包括如sequenceNO：5或sequenceNO：6所示的序列。
进一步，所述促进剂包括以下几种物质中的一种或多种组合：
(1)生物碱类化合物或组合物，包括小檗碱，木兰花碱，骆驼蓬碱，蛇根碱，益母草碱，粉防己碱，常山碱，浙贝母碱，秋水仙碱，胡椒碱，钩藤碱，喜树碱，延胡索乙素，药根碱，白屈菜碱，石蒜碱，山油柑碱，苦参碱，氧化苦参碱中的一种化合物或多种化合物组成的组合物；
(2)黄酮类化合物或组合物，包括槲皮素，二氢槲皮素，木犀草素，桑色素，桑皮黄素，芹菜素，香叶木素，白杨素，苜蓿素，葛根素，花青素，原花青素，大豆黄素，芹菜素，黄芩素，野黄芩素，白杨素，汉黄芩素，矢车菊素，水飞蓟素，水飞蓟宾，黄豆苷元，染料木素，高良姜黄素，山奈酚，漆黄素，异鼠李素，杨梅黄酮，橙皮素，柚皮素，甘草素，儿茶素，表儿茶素，没食子儿茶素，矢车菊素，飞燕草素，花葵素，灯盏花素，阿福豆素，芦丁，木犀草苷，黄芩苷，芹菜苷，胡桃苷中的一种化合物或多种化合物组成的组合物；
(3)多酚类化合物或组合物，包括绿原酸，洋蓟酸，菜蓟糖苷，菜蓟苦素，茶多酚，白藜芦醇，石榴多酚，姜黄素，阿魏酸中的一种化合物或多种化合物组成的组合物；
(4)胺类化合物或组合物：青霉胺，泊马度胺，来那度胺，沙利度胺中的一种化合物或多种化合物组成的组合物；
(5)中药提取物，包括黄芪、党参、丹参、当归、山药、三七、五味子、黄精、灵芝、云芝、洋蓟、小蓟、水飞蓟、葛根、黄连、黄柏、姜黄、金银花、枳椇、陈皮、桑叶、银杏叶、百合、熊胆、猪胆、茵陈、枳壳、甘草、虎杖、槐米、丹皮、垂盆草、栀子、枸杞、白芍、川芎、桃仁、大黄、菊花、溪黄草、白花蛇舌草、防己、白茅根、半枝莲、柴胡、茯苓、山药中的一种提取物或多种提取物组成的组合物；
(6)甘草酸，甘草酸苷，腺苷蛋氨酸，熊去氧胆酸，牛磺熊去氧胆酸，鹅去氧胆酸，牛磺鹅去氧胆酸，双环醇，联苯双酯，索拉非尼，瑞戈非尼，乐伐替尼，甜菜红素，二甲双胍，胭脂红酸中的一种或多种。
进一步，所述药物还包括一些可药物的辅料。
本发明目的在于还提供一种TRIM31作为RHBDF2的抑制剂中的应用。所述TRIM31包含如sequenceNO：1或sequenceNO：2所示的氨基酸序列；其中编码TRIM31的核酸序列包括如sequenceNO：5或sequenceNO：6所示的序列。
在某些具体实施例中，编码TRIM31的核酸序列也可以是sequenceNO：6所示的序列的人源化序列。
本发明目的在于还提供一种TRIM31作为胰岛素抵抗或肝脂肪变性或炎症和肝纤维化的消除剂的应用。
所述TRIM31包含如sequenceNO：1或sequenceNO：2所示的氨基酸序列；其中编码TRIM31的核酸序列包括如sequenceNO：5或sequenceNO：6所示的序列。
在某些具体实施例中，编码TRIM31的核酸序列也可以是sequenceNO：6所示的序列的人源化序列。
具体地，由高脂饮食触发的上调的RHBDF2显著驱动体内所有三种病理表型，揭示了RHBDF2在肝脏脂肪变性相关代谢疾病中的潜在治疗前景。通过对RHBDF2的研究，发现E3泛素连接酶的重要成员TRIM31是RHBDF2表达的潜在有效抑制剂。而且，随着研究的进一步深入，发现肝细胞TRIM31的保护功能不仅只局限于肝脏，还影响整个代谢系统，包括减少体重增加和内脏脂肪，改善血脂异常，以及下调血清炎症细胞因子和趋化因子。因为肝脏是人体最大的代谢、免疫器官，这可能是TRIM31调节肝脏进而调节整个人体的原因。而且， 我们证实了TRIM31功能失调，导致随后RHBDF2水平显著上调，且与炎症相关疾病密切相关。此外，RHBDF2已被证明受泛素化修饰调节。同样的，我们确定高脂饮食的小鼠和肝脂肪变性患者的肝脏中TRIM31水平显著降低。
本发明目的在于还提供一种促进RHBDF2降解的方法，其特征在于，将所述TRIM31通过K48连接的多泛素化促进RHBDF2降解。
本发明目的在于还提供一种TRIM31作为肝细胞变性的诊断标志物中的应用。
本发明目的在于还提供一种判断肝细胞变性的方法，其特征在于，检测待测离体样本中的TRIM31的表达水平，并与正常组进行比较，当所述待测离体样本中的TRIM31的表达水平低于正常组时，判定为肝细胞变性。
进一步，检测待测离体样本中的RHBDF2的表达水平，并与正常组进行比较，当所述待测离体样本中的RHBDF2的表达水平低于正常组时，判定为肝细胞变性。
本发明目的在于还提供一种TRIM31的表达量调节剂作为肝糖原储备调节剂中的应用。
进一步，TRIM31的表达量促进剂是作为肝糖原储备抑制剂中的应用。
本发明目的在于还提供一种TRIM31作为高脂饮食诱发胰岛素抵抗和糖代谢紊乱的抑制剂中的应用。
具体地，本发明发现TRIM31通过K48连接的泛素修饰促进RHBDF2降解，进一步抑制RHBDF2-MAP3K7活性和下游信号通路的表达，从而具有抑制肝脏脂肪变性、受损胰岛素信号传导和肝脏炎症的能力。TRIM31主要基于不同的基序在分子水平上发挥其生物学功能。据报道，TRIM31的C端卷曲螺旋结构域(CC)基本上负责与靶标的结合和相互作用，而N端RING结构域则负责泛素连接酶活性和催化功能。
本发明发现K48连接的RHBDF2泛素化可以在体外被催化，并且TRIM31的缺失极大使RHBDF2的这种内源性泛素化修饰无效。分子生物学功能的体外研究表明，TRIM31通过螺旋结构域(CC)直接结合RHBDF2以靶向RHBDF2活性，然后TRIM31的RING finger通过K48介导RHBDF2蛋白酶体泛素化降解而泛素化RHBDF2。然而，内源性RHBDF2和TRIM31在肝细胞中的相互作用仍需进一步研究。一旦RHBDF2和TRIM31之间的相互作用被阻断，TRIM31对高脂诱导的肝脂肪变性的保护作用就终止了。而且，非功能性TRIM31(RING结构域突变体)失去了其E3连接酶活性，以继续降低RHBDF2-MAP3K7信号通路和下游JNK1/2-NF-kB通路的激活，并提高胰岛素信号传导。所以带有RING结构域突变体的TRIM31对PA诱导的肝细胞中异常的脂质代谢也有相当大的影响。因此，Trim31-RHBDF2的结合和随后的RHBDF2泛素化修饰是肝细胞RHBDF2-MAP3K7信号诱导的肝脏脂肪变性、胰岛素抵抗和肝脏炎症缓解所必需的。
而且，RHBDF2作为菱形膜内蛋白酶家族的一个重要的非活性成员，已被证实与炎症信号的调节和传递有关。但是，以前研究表明，在免疫细胞以外的细胞中，促炎因子TNF-α的产生与RHBDF2无关。现有文献也表明，非免疫细胞中RHBDF2的缺失并不影响炎症因子如TNF-α、IL-6和IL-β的表达和分泌。但是，尽管TNF-α在非免疫细胞如肝细胞中的释放并不完全依赖于RHBDF2的转运，但它能够在刺激条件下大量地募集MAP3K7下游信号通路，激活NF-κB炎症信号并加速炎症因子的分泌。本发明中，发现在棕榈酸(PA)诱导的肝细胞中，TRIM31通过K48泛素连接显著促进RHBDF2的降解，随后TNF-α和其他炎症因子的表达大大降低。
本发明确定了肝脏TRIM31表达与非酒精性脂肪性肝病/非酒精性脂肪肝炎和代谢紊乱相关，例如人类和小鼠的胰岛素抵抗和糖代谢紊乱。此外，小鼠肝脏TRIM31表达对饮食干预有反应，饮食干预通过调节RHBDF2蛋白酶体降解显著缓解胰岛素抵抗、肝脏脂肪变性和炎症表型。这些发现促使TRIM31可作为非酒精性脂肪性肝病/非酒精性脂肪性肝炎(NAFLD/NASH)和相关代谢疾病的可行治疗靶点，并使TRIM31与这些疾病的诊断和治疗方案相关联。
本发明有益效果在于
本发明提供的TRIM31、RHBDF2组合因子作为代谢紊乱的标志物，可以精确有效的鉴定NAFLD/NASH和/或代谢紊乱或肝细胞变性。
本发明提供的TRIM31制备的促进因子/促进剂可以有效改善NAFLD/NASH和/或代谢紊乱的情况。
本发明采用的TRIM31作为或者制备胰岛素抵抗或肝脂肪变性或炎症和肝纤维化的消除剂可以有效缓解胰岛素抵抗或肝脂肪变性或炎症和肝纤维化的情况。
本发明提供的判断肝细胞变性的方法可以准确的鉴定出肝细胞是否产生变性。
附图说明
图1a为HFD小鼠和NCD小鼠肝脏样本中TRIM31表达情况。
图1b为ob/ob小鼠肝脏样本中TRIM31表达情况。
图1c为HFD小鼠0-16周TRIM31表达情况。
图1d为NASH患者的肝脏中TRIM31的表达情况。
图1e为给药棕榈酸(PA)和TNF-α后TRIM31表达情况。
图1f为加入活性氧清除剂N-Acetyl-L-cysteine(NAC)和抗氧化过氧化氢酶(CAT)后TRIM31表达情况。
图1g为PA+CHX处理后的RHBDF2的表达情况。
图1h为PA+CHX+氯喹处理后的RHBDF2的表达情况。
图1i为PA+CHX+MG132处理后的RHBDF2的表达情况。
图1j为小鼠肝脏免疫荧光分析情况。
图1k为免疫组化分析人肝脏切片情况及腺病毒介导的TRIM31表达(AdTRIM31)转染的L02细胞中细胞内甘油三酯(TG)情况。
图2a为HFD小鼠、NCD小鼠、ob/ob小鼠肝脏样本中RHBDF2表达情况。
图2b为HFD小鼠在0-16周肝脏样本油红染色和HE染色情况。
图2c为HFD小鼠和NCD小鼠TC、TG、NEFA含量情况。
图2d为NASH患者的肝脏中RHBDF2的表达情况。
图2e为给药棕榈酸(PA)和TNF-α后RHBDF2表达情况。
图2f为体内RHBDF2的泛素化情况。
图3a为THKO小鼠生产流程示意图。
图3b为THKO小鼠身体各组分表达TRIM31情况。
图3c为NCD和HFD处理的THKO小鼠食物摄入量情况。
图4a为THTG小鼠生产流程示意图。
图4b为THTG小鼠身体各组分表达TRIM31情况。
图4c为NCD和HFD处理的THTG小鼠食物摄入量情况。
图4d为PA处理的原代肝细胞的TRIM 31表达情况。
图5a为长时间HFD的THTG小鼠0-16周体重情况。
图5b为长时间HFD的THTG小鼠0-16周空腹血糖情况。
图5c为长时间HFD的THTG小鼠0-16周空腹空腹胰岛素水平情况。
图5d为长时间HFD的THTG小鼠0-16周胰岛素抵抗情况。
图5e为葡萄糖耐量试验(GTT)试验测试THTG小鼠血糖水平情况。
图5f为胰岛素耐量试验(ITT)试验测试THTG小鼠血糖水平情况。
图5g为TRIM31过表达与肝糖原储备情况。
图5h为THTG小鼠-HFD饮食引起的肝胰岛素信号失调情况。
图6a为不同处理的THKO小鼠0-16周体重情况。
图6b为不同处理的THKO小鼠0-16周空腹血糖情况。
图6c为不同处理的THKO小鼠0-16周空腹胰岛素水平情况。
图6d为不同处理的THKO小鼠0-16周胰岛素抵抗情况。
图6e为葡萄糖耐量试验(GTT)试验测试THKO小鼠血糖水平情况。
图6f为胰岛素耐量试验(ITT)试验测试THKO小鼠血糖水平情况。
图6g为TRIM31缺失与肝糖原储备情况。
图6h为THKO小鼠-HFD饮食引起的肝胰岛素信号失调情况。
图7a为HFD-THKO小鼠的肝重和肝重/体重情况。
图7b为HFD-THKO小鼠的血清丙氨酸转氨酶(ALT)情况。
图7c为HFD-THKO小鼠的血清天冬氨酸转氨酶(AST)情况。
图7d为HFD-THKO小鼠的血清碱性磷酸酶(AKP)情况。
图7e为HFD-THKO小鼠的脂肪酸摄取和合成相关基因、脂肪酸β氧化相关基因表达情况。
图7f为高脂饮食的THTG小鼠与NTG小鼠肝重和肝脏/体重比例情况。
图7g为高脂饮食的THTG小鼠肝脏脂肪实图。
图7h为高脂饮食的THTG小鼠的肝脏功能相关指标情况。
图7i为高脂饮食的THTG小鼠肝脏样本油红染色和HE染色情况。
图7j为高脂饮食的THTG小鼠的脂肪酸摄取和合成相关基因的表达和脂肪酸β氧化相关基因表达情况。
图7k为PA处理的原代肝细胞的实图与TRIM 31表达情况。
图8a为高脂饮食的THKO和THTG小鼠内脏脂肪情况。
图8b为脂饮食的THKO和THTG小鼠脂肪细胞的大小情况。
图9a为HFD-THKO小鼠内炎症相关的信号通路中因子表达情况。
图9b为HFD-THTG小鼠内炎症相关的信号通路中因子表达情况。
图9c为高脂饮食的THKO小鼠血清中炎症相关的因子分泌情况。
图9d为PA处理的原代肝细胞中通过活化NF-κB介导的炎症信号情况。
图10a为慢病毒包裹完整的鼠TRIM31基因序列(LV-TRIM31)流程示意图。
图10b为高脂饲料-THKO小鼠重量、体重、肝重/体重比情况。
图10c为高脂饲料-THKO小鼠AST和ALT情况。
图10d为高脂饲料-THKO小鼠肝脏脂质堆积情况。
图10e为GTT测试THKO小鼠血糖浓度情况。
图10f为ITT测试THKO小鼠血糖浓度情况。
图10g为THKO小鼠肝脏TG、TC和NEFA情况。
图10h为肝细胞TRIM31的表达与高脂饮食-THKO小鼠脂肪酸β-氧化基因表达情况。
图10i为THKO小鼠受损的胰岛素信号和激活的炎症相关信号情况。
图10j为THKO小鼠受损的胰岛素信号和激活的炎症相关信号情况。
图11a为TRIM31在缓解肝脏脂质堆积、胰岛抵抗和炎症中的作用。
图11b为慢病毒介导的Trim31表达高饮脂血液中IL-1β/YNF-α/IL-6水平。
图11c为慢病毒介导的Trim31表达高饮脂血液中信号因子的mRNA水平。
图12a为LV-hTRIM31介导的体外基因治疗流程示意图及TRIM 31表达情况。
图12b为LV-hTRIM31介导的体外基因治疗修复TRIM31的小鼠肝脏重量和体重情况。
图12c为LV-hTRIM31介导的体外基因治疗修复TRIM31的小鼠肝脏样本油红染色和HE染色情况。
图12d为LV-hTRIM31介导的体外基因治疗修复TRIM31的小鼠血清AST、ALT情况。
图12e为LV-hTRIM31介导的体外基因治疗修复TRIM31的小鼠空腹血糖水平。
图12f为LV-hTRIM31介导的体外基因治疗修复TRIM31的小鼠血糖水平。
图12g为LV-hTRIM31介导的体外基因治疗修复TRIM31的小鼠胰岛素抵抗情况。
图12h为LV-hTRIM31介导的体外基因治疗修复TRIM31的小鼠TG、TC、NEFA情况。
图12i为LV-hTRIM31介导的体外基因治疗修复TRIM31的小鼠相关的因子分泌情况。
图12j为LV-hTRIM31介导的体外基因治疗修复TRIM31的小鼠相关蛋白表达情况。
图12k为LV-hTRIM31介导的体外基因治疗修复TRIM31的小鼠血清细胞因子分泌情况。
图13a为高脂饮食-THKO小鼠与THTG小鼠免疫荧光切片结果。
图13b为小鼠体内ADAM17,TNFR1/2,和p-MAP3K7及其下游成分情况。
图13c为小鼠体外ADAM17,TNFR1/2,和p-MAP3K7及其下游成分情况。
图13d为转染了AdshRHBDF2的THTG肝细胞对PA诱导受损胰岛素信号情况。
图14a为转染了AdRHBDF2的THTG肝细胞对PA诱导受损胰岛素信号情况。
图14b为RHBDF2与TRIM31的相互结合免疫荧光切片结果。
图14c为转染的鼠肝细胞中的TRIM31和RHBDF2结合情况。
图15a为体外相互作用试验情况。
图15b为谷胱甘肽硫转移酶(GST)标记情况。
图15c为TRIM31的结构示意简图。
图15d为体内RHBDF2的泛素化情况。
图15e为具有RING-finger结构域切除突变体的TRIM31表达载体催化RHBDF2的泛素化情况。
图15f为TRIM31缺陷的L02细胞(THKO-L02)中，RHBDF2与K63连接的泛素化情况。
图15g为泛素突变质粒体外转染实验情况。
图15h为载体转染的TRIM31缺陷型L02细胞(THKO-L02)中，用PA刺激的细胞内TG浓度变化情况。
图16a为带有RING-finger敲除的慢病毒负载TRIM 31(LV-TRIM 31 RING)的流程示 意图。
图16b为TRIM31 RING-finger敲除-肝细胞移植(THKO)(LV+)的小鼠HFD诱导的肝脏重量、体重、肝重/体重比率情况。
图16c为TRIM31 RING-finger敲除-肝细胞移植(THKO)(LV+)的小鼠HFD诱导的AST和ALT情况。
图16d为TRIM31 RING-finger敲除-肝细胞移植(THKO)(LV+)的小鼠HFD诱导的肝脏脂质累计情况。
图16e为TRIM31 RING-finger敲除-肝细胞移植(THKO)(LV+)的小鼠HFD诱导的空腹血糖水平情况。
图16f为TRIM31 RING-finger敲除-肝细胞移植(THKO)(LV+)的小鼠HFD诱导的血糖水平情况。
图16g为TRIM31 RING-finger敲除-肝细胞移植(THKO)(LV+)的小鼠HFD诱导的肝甘油三酯、总胆固醇和NEFA含量情况。
图16h为高脂饮食的THKO(LV+)小鼠与脂肪酸摄取、合成过程有关的基因表达和与脂肪酸β-氧化过程有关的基因表达情况。
图16i为高脂饮食的THKO(LV+)小鼠RHBDF2-MAP3K7信号通路免疫印迹、mRNA水平和促炎介质浓度情况。
图16j为高脂饮食的THKO(LV+)小鼠RHBDF2-MAP3K7下游通路免疫印迹情况。
图17a为带有RING-finger敲除的慢病毒负载TRIM 31(LV-TRIM 31 RING)的流程示意图2。
图17b高脂饮食的THKO(LV+)小鼠促炎介质浓度情况。
图17c为高脂饮食的THKO(LV+)小鼠mRNA水平情况。
图18a为LV-hTRIM31体外基因疗法流程示意图。
图18b为LV-hTRIM31体外基因疗法获得的小鼠肝脏重量、体重、肝重/体重比率情况。
图18c为LV-hTRIM31体外基因疗法获得的小鼠肝脏样本油红染色和HE染色情况。
图18d为LV-hTRIM31体外基因疗法获得的小鼠血清AST、ALT情况。
图18e为LV-hTRIM31体外基因疗法获得的小鼠血糖水平。
图18f为LV-hTRIM31体外基因疗法获得的小鼠TG、TC、NEFA情况。
图18g为LV-hTRIM31体外基因疗法获得的小鼠胰岛素抵抗情况。
图19a为AAV-TRIM31 RING WT小鼠Rhbdf2的表达情况。
图19b为AAV-TRIM31 RING WT小鼠体重情况。
图19c为AAV-TRIM31 RING WT小鼠空腹血糖水平情况。
图19d为AAV-TRIM31 RING WT小鼠血糖水平情况。
图19e为AAV-TRIM31 RING WT小鼠肝脏重量情况。
图19f为AAV-TRIM31 RING WT小鼠肝脏样本油红染色和HE染色情况。
图19g为AAV-TRIM31 RING WT小鼠RHBDF2相关表达情况。
图19h为AAV-TRIM31 RING WT小鼠RHBDF2下游炎症信号情况。
图19i为AAV-TRIM31 RING WT小鼠相关信号的mRNA表达水平。
图20a为DHKO小鼠蛋白质印迹分析肝脏样品情况。
图20b为DHKO小鼠体重、肝重、肝重/体重比情况。
图20c为DHKO小鼠空腹血糖水平情况。
图20d为DHKO小鼠肝脏样本油红染色和HE染色情况。
图20e为DHKO小鼠TG、TC、NEFA情况。
图20f为DHKO小鼠RHBDF2-MAP3K7轴和下游信号级联情况。
图20g为DHKO小鼠肝脂质沉积情况。
图21a为不同小鼠血糖水平情况。
图21b为不同小鼠异常血糖水平情况。
图21c为不同小鼠肝脏PAS分析图。
图21d为不同小鼠促炎症相关基因表达情况。
图21e为不同小鼠RHBDF2-MAP3K7轴信号表达情况。
图22为为腺病毒载体诱导的肝细胞的转染效率。
图23a为THTG-HFHF小鼠脏重量、肝重/体重比情况。
图23b为THTG-HFHF小鼠体重情况。
图23c为THTG-HFHF小鼠肝脏TG、TC和NEFA浓度情况。
图23d为THTG-HFHF小鼠肝脏样本油红染色和HE染色情况。
图23e为THTG-HFHF小鼠炎症和胶原相关基因表达情况。
图23f为THTG-HFHF小鼠肝脂质沉积情况。
图23g为THTG-HFHF小鼠脂肪酸合成相关基因表达情况。
图23h为THTG-HFHF小鼠肝脏样品Masson染色情况。
图23i为THTG-HFHF小鼠炎症相关基因表达情况。
图23j为THTG-HFHF小鼠肝纤维化和胶原蛋白合成相关基因表达情况。
图23k为THTG-HFHF小鼠RHBDF2-MAP3K7信号传导示意图。
图24为肝细胞特异性TRIM31缺失(THKO)小鼠生产流程示意图。
图25为甜菜红素干预对THTG小鼠的相关蛋白表达的影响。
图26为漆黄素干预对THTG小鼠的相关蛋白基因的mRNA水平的影响。
图27为胭脂红酸干预对THTG小鼠的相关蛋白表达或mRNA水平的影响。
具体实施方式
所举实施例是为了更好地对本发明进行说明，但并不是本发明的内容仅局限于所举实施例。所以熟悉本领域的技术人员根据上述发明内容对实施方案进行非本质的改进和调整，仍属于本发明的保护范围。
实施例试剂
本发明实施例中，使用的一抗及其稀释比例包括：anti-GAPDH(#2118,dilution 1:10000),p-AKT(#4060,dilution 1:1000),AKT(#4691,dilution 1:1000),p-GSK3β(#9322,dilution 1:1000),GSK3β(#12456,dilution 1:1000),FOXO1(#2880,dilution 1:1000),anti-p-JNK(#4668,dilution 1:1000),anti-JNK(#9258,dilution 1:1000),anti-MAP3K7(#4505,dilution 1:1000),anti-p-MAP3K7(#9339,dilution 1:1000)and p-IκBα(#2859,dilution 1:1000)购自CST公司；anti-TNFR1(#ab223352,dilution 1:1000),anti-TNFR2(#ab109322,dilution 1:1000),anti-MKK7(#ab52618,dilution 1:1000),anti-p-MKK7(#ab192592,dilution 1:1000),anti-c-Jun(#ab40766,dilution  1:1000),anti-p-c-Jun(#ab32385,dilution 1:1000),anti-IRS-1(#ab52167,dilution 1:1000),anti-NF-κB(#ab16502,dilution 1:1000),anti-p-NF-κB(#ab86299,dilution 1:1000),p-FOXO1(#ab131339,dilution 1:1000),anti-IκBα(#ab32518,dilution 1:1000),anti-IKKβ(#ab124957,dilution 1:1000)and anti-Rhbdf2(#ab116139,dilution 1:1000)购自Abcam公司；anti-p-MAP3K7(#PA5-99340,dilution 1:1000),anti-p-IKKβ(#PA5-36653,dilution 1:1000),anti-Trim31(#PA5-40961,dilution 1:1000),anti-ADAM17(#PA5-27395,dilution 1:1000)and anti-p-IRS-1(Ser 307)(#PA1-1054,dilution 1:1000)购自赛默飞公司。
本发明实施例中，其他抗体购自Santa公司和密理博公司。
实施例动物模型
本发明实施例中，使用CRISPR/Cas9介导的基因工程系统构建基于C57BL/6N背景的Trim31 flox/flox小鼠。选择Trim31的外显子4和5作为条件性敲除区域(CKO)。选定的Trim31外显子两侧作为loxP位点，随后设计两个靶向Trim31内含子的向导RNA(gRNA1和gRNA2)。含有侧翼为两个loxP位点和两个同源臂的Trim31外显子4和5的靶向载体作为模板。将靶向载体、向导RNA1和引导RNA2和Cas9mRNA共同注射到受精卵中，用于CKO小鼠生产。获得的小鼠在外显子4和5两个等位基因两侧都具有loxP位点，然后作为Trim31 flox/flox小鼠。通过将Trim31 flox/flox小鼠与Alb-Cre(Alb-Cre)小鼠(Jackson Laboratory,Bar Harbor,Maine,USA)交配而产生的后代子鼠，并选取纯合子作为肝细胞特异性Trim31缺失(THKO)小鼠使用(如图24所示)。同时使用Trim31 flox/flox小鼠的同窝小鼠作为获得的THKO小鼠的对照。
本发明实施例中，使用CRISPR/Cas9系统通过特异性敲除肝细胞中Rhbdf2的第4个外显子来创建肝细胞特异性Rhbdf2基因敲除(RHKO)小鼠。有关这些小鼠的建立和基因型测定的详细方法和信息已在Xu Min-Xuan,Tan Jun,et al.,Dysfunctional Rhbdf2 of proopiomelanocortin mitigates ambient particulate matter exposure-induced neurological injury and neuron loss by antagonizing oxidative stress and inflammatory reaction.Journal of Hazardous Materials,2020,400:123158已经公开。此外，肝细胞特异性Trim31和Rhbdf2双缺失(DHKO)小鼠是通过Trim31flox/flox小鼠与RHKO小鼠杂交产生的。
本发明实施例中，将质粒CAG-loxP-CAT-loxP-Trim31微注射到从C57BL/6小鼠分离的受精卵中构建条件Trim31转基因(TG)(过表达)小鼠。然后通过PCR和测序分析对获得的幼崽进行基因分型。通过尾部基因组DNA的PCR分析鉴定获得的小鼠。这些TG小鼠的后代与Alb-Cre小鼠交配以建立肝细胞特异性Trim31转基因(THTG)小鼠。在肝细胞中没有Trim31过表达的相应的同窝子鼠作为对照(NTG)。
本发明实施例中，使用的所有正常野生型(WT)C57BL/6N小鼠均购自北京维通利华实验动物科技有限公司(中国北京)。
动物实验
本发明实施例中，所有动物实验过程和实验方案都得到了所有参与单位的动物保护和使用委员会的批准。在所有实验正常开始之前，小鼠要适应生活环境7天。将小鼠圈养在恒温、恒湿和无病原体的环境(温度25℃±2℃，环境湿度50％-60％)并给与12小时光照/12小时黑暗循环。
本发明实施例中，高脂饮食(HFD)饲料(#D12492)标准为：20％kcal蛋白质、60 kcal％脂肪和20％kcal碳水化合物；购自Research Diets,New Brunswick,NJ,USA。
本发明实施例中，正常食物(#D12450H)标准为：20％kcal蛋白质、10kcal％脂肪和70％kcal碳水化合物；购自Research Diets，New Brunswick，NJ，USA。
本发明实施例中，ob/ob小鼠(#N000103)，从南京大学南京生物医学研究所购入。
本发明实施例中，进行动物实验1#：将6-8周龄WT雄性小鼠(共15只)饲喂高脂饮食(HFD)饲料16周诱导脂肪肝。额外的WT小鼠(总共15只)被喂食标准的正常食物持续16周作为对照组(NCD)。6-8周龄ob/ob小鼠(共10只)饲喂NCD饮食，然后作为另一种脂肪肝模型。在时间梯度过程实验中，每个时间点总共包括10只WT小鼠。在实验结束时，从小鼠身上收集肝组织样本以检测相应的信号通路蛋白丰度变化。
本发明实施例中，进行动物实验2#：为了验证Trim31对HFD诱导的胰岛素抵抗、肝脂肪变性和炎症的保护作用，在8周HFD喂养的预处理THKO小鼠中,通过注射慢病毒荷载的野生型Trim31基因序列(LV-Trim31)或者携带RING结构域突变的Trim31进行在体基因治疗(ex vivo)转导和移植,观察Trim31的ex vivo治疗效果。在离体治疗干预结束时，这些小鼠禁食8小时，然后进行葡萄糖和胰岛素耐受性测试。收集眼球血和肝脏样品，称重，并在-80℃条件下储存。另一部分肝组织进行组织学分析和生化分析。对所有现有数据进行了至少3次独立实验。
本发明实施例中，进行动物实验3#：在体内实验中，腺相关病毒血清型8(AAV8)-甲状腺素结合球蛋白(TBG)编码全长Trim31序列(AAV-Trim31)和AAV8编码携带RING结构域突变的Trim31(AAV-Trim31 RING Δ)递送系统的突变体Trim31，注射后实现肝细胞中特异性过表达Trim31。简而言之，将没有终止密码子编码的Trim31开放阅读框(ORF)克隆到AAV8载体中以生成AAV-Trim31。6周HFD喂养的预处理WT小鼠通过尾静脉注射AAV-Trim31或AAV-Trim31 RING Δ(含有2×10 11vg的100μl病毒)，然后用HFD喂养另外10周。将空载体(AAV-GFP)注射到小鼠体内作为相应的对照。
本发明实施例中，进行动物实验4#，建立脂肪肝小鼠模型的方法为：将6-8周龄的雄性THKO、RHKO、DHKO、THTG小鼠及其对应的同窝对照小鼠喂食HFD饲料16周，观察病理变化。此外，周龄相当的THKO、RHKO、DHKO、THTG小鼠和同窝仔鼠分别用NCD饲料喂养16周并作为对照处理。
本发明实施例中，进行动物实验5#，为了进一步验证Trim31对肝脂肪变性的保护作用，先建立非酒精性脂肪性肝炎(NASH)小鼠模型，然后用HFHF饮食(14％蛋白质，42％脂肪、44％碳水化合物、0.2％胆固醇和总共42g/L的碳水化合物以55％果糖和45％蔗糖的重量比混合在饮用水中)饲喂16周，以产生NASH表型。此外，在此设计中使用的NTG小鼠作为对照，并被同步允许随意获取这种饮食16周。
本发明实施例中，使用PA进行于体外实验，以前的研究表明游离脂肪酸从脂肪细胞里释放增加会导致肝细胞中甘油三酯(TG)的累积，促进脂肪变性向脂肪肝炎发展。饱和脂肪包括棕榈酸(PA)和硬脂酸比其他脂肪酸毒性更大，在脂肪肝炎模型中促进肝细胞毒性。在肝脏中TG的大量沉积是由脂质代谢紊乱引起的。代谢紊乱不但促进脂肪变性和肝损伤，而且还显著抑制胰岛素信号，促进胰岛素抵抗。
细胞培养与处理
本发明实施例中，使用的L02细胞系(人类正常肝细胞系)获自中国科学院典型培 养物保藏中心，本发明申请人的实验室使用的所有复苏细胞系均传代不超过30次。参与本发明实施例的细胞系需要通过PCR分析检测以排除支原体污染。L02细胞培养在含有10％胎牛血清(#16140071，Gibco TM)和1％青霉素-链霉素(#15140-122；Gibco TM)和在5％CO 2、37℃水套式细胞培养箱(Thermo Fisher Scientific)中。
本发明实施例中，使用的原代肝细胞通过肝脏灌注方法从相应的实验小鼠中分离和收集。简而言之，在无痛麻醉条件下，打开小鼠腹腔。通过肝脏门静脉用1×肝脏灌注培养基(#17701-038，Gibco TM)和1×肝脏消化培养基(#17703-034，Gibco TM)仔细灌注。然后，用100μm钢网研磨和过滤消化后的肝组织。将滤液以800rpm、4℃5min离心收集原代肝细胞，并用50％percoll细胞分离液(#17-0891-01，GE Healthcare Life Sciences)进一步纯化。获得的肝细胞在含10％胎牛血清和1％青霉素-链霉素的DMEM培养基中培养，在5％CO 2、37℃细胞培养箱中培养。
本发明实施例中，为了构建体外脂质沉积的细胞模型，制备并获得相应浓度的棕榈酸(PA)(溶于0.5％不含脂肪酸的牛血清白蛋白BSA)。然后，原代肝细胞或L02细胞用含有细胞培养基的PA处理10小时或4小时。单独使用不含脂肪酸的BSA(0.5％)作为载体对照。
敲除细胞系的建立
本发明实施例中，使用的Trim31敲除细胞系的构建和方法为：通过CRISPR/Cas9基因编辑系统构建Trim31缺失的细胞系，产生靶向人类Trim31基因的sgRNA并将其包装到慢病毒CRISPR-V2载体中以形成Cas9-sgRNA慢病毒。用于生成sgRNA表达载体的寡核苷酸序列为：sgRNA-F：CACCCAACTCGCTGTTGCGGAATC；和sgRNA-R：AAACGATTCCGCAACAGCGAGTTG。然后使用
Figure PCTCN2022128995-appb-000001
6转染试剂将包装载体pSPAX2和pMD2。G以及sgRNA表达载体分别转染到HEK293T细胞中42小时。接下来，将获得的含有慢病毒的上清液转入L02细胞以构建基因敲除细胞系。通过免疫印迹法选择靶基因缺失的细胞克隆。
质粒构建与转染
本发明实施例中，通过基于PCR的cDNA扩增建立人或小鼠全长Rhbdf2和Trim31表达质粒，然后克隆到3×Flag-tagged pcDNA3.1载体或3×HA-tagged pcDNA3.1载体(Invitrogen)中。使用标准PCR获得截断的Rhbdf2和Trim31片段表达载体，包括TRIM31-Flag RING Δ、TRIM31-Flag Box Δ、TRIM31-Flag CC Δ、RHBDF2-HA TMD Δ、RHBDF2-HA IRHD Δ和RHBDF2-HA Tail Δ，如图例所示方法，然后克隆到相应的载体中。基于pcDNA3.1载体构建Myc-标记的泛素WT表达载体。此外，泛素和相应的衍生物包括仅含有唯一完整的氨基酸残基的泛素-K48O、泛素-K63O、泛素-K33O、泛素-K6O、泛素-K29O、泛素-K27O和对照载体，然后荷载入Myc标记的pcDNA3.1质粒(Thermo Fisher Scientific)。根据使用说明书，使用Lipofectamine TM3000转染试剂(Invitrogen TM)将载体小心地转染到L02细胞中。
本发明实施例中，构建一个负载腺病毒的Trim31表达载体方法为：分别将针对靶向人TRIM31序列的特异性短发夹RNA寡核苷酸序列(shTRIM31)(shRNA序列RNAi#1：TTCCCGTCAAAGGAAGTTTGG；RNAi#2：TATGATGGACTCATGCCTTGC)，通过Easy Adenoviral Vector System Kit试剂盒(#240009，安捷伦科技)荷载入腺病毒(AdTRIM31；AdshTRIM31)中。AdshGFP分别用作敲除或过表达的对照。纯化重组腺病毒并滴定至5×10 10(PFU)。之后用培养基稀释的腺病毒感染肝细胞，感染次数为50次，感染时间为24小时。
腺相关病毒和慢病毒构建与制备
本发明实施例中，CAG启动子序列如sequenceNO：9所示，CMV启动子序列如sequenceNO：10所示。
本发明实施例中，小鼠的TRIM31表达基因如sequenceNO：6所示，小鼠的RHBDF2表达基因如sequenceNO：8所示。
本发明实施例中，使用的AAV8-TBG载体是一种预包装的8血清型AAV，具有GFP过表达，用于生产重组AAV8-TBG-目的基因-GFP表达载体。重组AAV8-TBG-目的基因-GFP表达载体包含来自甲状腺素结合球蛋白(TBG)启动子的转录控制元件、用于插入互补DNA和polyA信号的克隆位点。AAV血清型2的末端重复位于表达框的两侧。
本发明实施例中，将鼠全长Trim31序列或具有RING结构域缺失的Trim31序列分别克隆到载体AAV8-TBG-GFP中。通过ViraBind TMAAV Purification Mega Kit试剂盒(VPK-141/VPK-141-5,Cell Biolabs,VPK-141/VPK-141-5,San Diego,USA)将新创建的载体AAV-TBG-Trim31-GFP或AAV-TBG-Trim31 RING △-GFP被包装到AAV8中，并相应地通过QuickTiter TMAAV Quantitaiton Kit(Cell Biolabs,VPK-145)进行滴定。注射前立即用生理盐水将病毒颗粒稀释至50μL的总体积。
本发明实施例中，为了构建慢病毒-Trim31(LV-Trim31)或具有RING结构域缺失的慢病毒-Trim31(LV-Trim31 RING △)载体，将全长Trim31cDNA序列荷载入pLenti-CMV-GFP-Puro(Addgene)中以进行上调Trim31表达(pLenti-CMV-Trim31-GFP-Puro或pLenti-CMV-Trim31 RING △-GFP-Puro)。通过商业化的Lenti-Pac HIV表达包装试剂盒(LT002，GeneCopoeia，MD，美国)和相应的Lenti-Pac 293Ta细胞系用于制备LV颗粒。接下来，根据产品说明，收集含有病毒颗粒的293T细胞培养基上清液。新创建的载体通过ViraBind TMPLUS慢病毒浓缩和纯化试剂盒(Cell Biolabs,VPK-095)进行浓缩和纯化，然后通过QuickTiter TM慢病毒定量试剂盒(Cell Biolabs,VPK-112)进行滴定。病毒产出量介于10 6TU/ml范围内的功能性LV滴度，并且在浓缩后保证达到10 9TU/ml的滴度产量。
本发明实施例中，细胞内甘油三酯检测的为：根据试剂盒实验使用说明，使用商业化甘油三酯检测试剂盒定量(#ab65336，Abcam)检测细胞内甘油三酯(TG)水平。
本发明实施例中，代谢指标与血清细胞因子的检测为：为进行葡萄糖耐量试验(GTT)和胰岛素耐量试验(ITT)，本部分实验小鼠禁食8h，以确保生理反应正常，而后给小鼠腹腔注射葡萄糖(2g/kg BW)(#158968,Sigma-Aldrich,上海,China)。然后，使用市售血糖试纸(
Figure PCTCN2022128995-appb-000002
Roche Diabetes Care GmbH,上海,中国)进行小鼠血糖含量检测。为了检查胰岛素耐受性测试，给小鼠腹膜内注射定量的胰岛素(1U/kg BW，Sigma Aldrich)，而后测定小鼠血液中的胰岛素含量。在注射后0分钟、15分钟、30分钟、60分钟和120分钟时从尾静脉采集血样，用于检测葡萄糖水平。稳态模型评估(HOMA)-IR指数分别根据空腹血糖和血清胰岛素水平计算。使用相应的商业化酶联免疫吸附测定(ELISA)试剂盒评估小鼠血清中的细胞因子和趋化因子水平。TNF-α(#MTA00B)、IL-1β(#MLB00C)、IL-10(#M1000B)、IL-6(#M6000B)和CCL-2(#MJE00B)ELISA试剂盒购自R&D system(上海,中国)，并按产品使用说明使用。所有相应的血清在使用前均保证储存在-80℃冰箱中。
本发明实施例中，肝功能参数和肝脂水平检测为：血清丙氨酸转氨酶(ALT)(#MAK052,Sigma-Aldrich)、天冬氨酸转氨酶(AST)(#MAK055,Sigma-Aldrich)、碱性磷 酸酶(AKP)(#ab83369,Abcam)、血清胰岛素(#ab277390,Abcam)和肝甘油三酯(TG)(#MAK266，Sigma-Aldrich)、总胆固醇(TC)(#ab65359，Abcam)和非酯化游离脂肪酸(NEFA)(#E-BC-K014，Elabscience,Inc.，休斯顿，美国)均使用市售检测试剂盒在指定的实验组中进行检测。
本发明实施例中，病理组织学检查方法为：为了进行组织学和免疫组织化学分析，肝脏样本相应地用10％中性福尔马林组织固定剂(#HT501128，Sigma-Aldrich)固定，石蜡包埋(#YA0010，Solarbio Life Sciences，Beijing，China)，然后切片机薄切组织切片。薄肝样品切片用苏木精和伊红(H&E)(#abs9217，苏木精和伊红染色试剂盒，Absin，上海，中国)染色，以可视化脂质沉积和组织损伤。为了显示肝脏中的脂质积累，将切片在Tissue-Tek(OCT)(#4583,Tissue-Tek,Sakura Finetek,USA)中冷冻包埋，然后用油红O染色试剂盒(#ab150678,Abcam)染色10分钟，后用60％异丙醇(#I9030,Sigma-Aldrich)冲洗后，将组织切片用苏木精重新染色以显色脂质沉积水平。此外，为了可视化肝脏样品中的糖原水平，切片用高碘酸-希夫染色(PAS)(#G1281，索莱宝生命科学，北京，中国)染色。为了进行免疫组织化学测定，在用包括抗Rhbdf2(#orb386934，Biobyt，稀释1:250)、抗Trim31(#PA5-40961，稀释1:200)或抗p-MAP3K7在内的一抗处理之前，对包埋切片进行脱石蜡处理(#PA5-99340，稀释1:200)后，进行染色。相应的山羊抗小鼠或抗兔IgG抗体(Abcam)用作二抗。
本发明实施例中，免疫沉淀法为：使用Lipofectamine TM3000转染试剂(Invitrogen TM)转染相应载体进入L02细胞，而后再培养36小时。随后，收集细胞并在4℃下将细胞匀浆到免疫沉淀(IP)特异性裂解液(#87787，Pierce TMIP裂解缓冲液，Thermo Scientific Pierce)中，然后在冷冻离心机中以13000rpm离心20分钟。将收集的细胞裂解物与Protein A/G Magnetic Agarose Beads(#78609，Thermo Scientific Pierce)在室温下混合孵育2小时，然后与指定的抗体在4℃下混合过夜。在用免疫沉淀缓冲液洗涤后收获免疫化合物，并通过与指定的一抗和相应的二抗孵育进行蛋白质印迹分析。
本发明实施例中，谷胱甘肽s-转移酶(GST)pull down测定法为：Trim31和Rhbdf2之间的直接蛋白质相互作用结合使用GST pull down测定法进行。Pierce TM GST蛋白质相互作用pull down试剂盒(#21516，Thermo Fisher Scientific)用于本部分实验检测。简言之，用质粒pGEX-4T-1-GST-Trim31或pGEX-4T-1-GST-Rhbdf2转化Rosetta(DE3)大肠杆菌细胞，然后与0.5mM异丙基β-D-孵育诱导表达硫代吡喃半乳糖苷(IPTG)(#I5502，Sigma-Aldrich)共同孵育。而后，将裂解的E.coli提取物与GST珠子在4℃下混合1小时。然后将GST珠子与带Flag标签的Trim31或带Flag标签的Rhbdf2一起孵育4小时。而后通过洗脱缓冲液中洗脱相互作用的蛋白质复合物，并使用抗Flag抗体进行蛋白质印迹分析。仅表达GST标签的大肠杆菌构建的载体用作阴性对照。
本发明实施例中，体外结合与泛素化实验方法为：将Trim31、Rhbdf2和具有RING结构域缺失的Trim31蛋白使用
Figure PCTCN2022128995-appb-000003
快速偶联转录/翻译系统(Promega)在体外微管中表达。通过混合相应的Flag进行蛋白质相互作用结合测定。将Rhbdf2和Trim31标记在一起，然后用Flag抗体进行免疫沉淀并用Trim31抗体进行免疫印迹。泛素化水平使用泛素化试剂盒(Boston Biochem)进行分析测定。
本发明实施例中，免疫印迹分析方法为：将细胞或肝组织匀浆到RIPA裂解液和提取缓冲液(#9806，CST)中以获得单一匀浆。然后，在13500rpm、4℃下离心30分钟浓缩 液体上清液。蛋白质浓度通过Pierce TM Rapid Gold BCA蛋白质测定试剂盒(Thermo Fisher Scientific)以牛血清白蛋白作为标准测定匀浆液中的总蛋白浓度。然后对总蛋白质提取样品进行蛋白质印迹分析。等量的细胞或组织总蛋白上样10％或12％十二烷基硫酸钠聚丙烯酰胺凝胶电泳(SDS-PAGE)系统，然后转移到0.45μM PVDF膜(Millipore Company，USA)，使用指定的免疫印迹一抗进行一抗孵育。接下来，将PVDF膜置于含有0.1％Tween-20(#1247ML100，BioFROXX，德国)的1×TBS缓冲液(#T1080，Solarbio，北京，中国)的5％脱脂牛奶(DifcoTM脱脂牛奶，BD，美国)中一起孵育(TBST)1小时，并在4℃下与一抗混合过夜。随后，膜在1×TBST中冲洗3次，随后与HRP偶联的抗兔或抗小鼠二抗(Abcam)在室温(25℃)下孵育1-2小时。通过Pierce TM ECL Plus Western Blotting Substrate(#32134，Thermo Fisher Scientific)观察免疫印迹条带，并于暗室中曝光于Kodak(Eastman Kodak Company，USA)X射线胶片上。然后将相应的蛋白质表达标定为灰度值(Version 1.52g，Mac OS X Snow Leopard，Image J，美国国立卫生研究院)并标准化为管家基因(GAPDH)，并表示为对照的倍数。
本发明实施例中，提出的相关实验至少独立进行了三次，所有的数据都使用该统计方法进行分析，除另有说明外。统计方法为：数据的定量值均以均数±均数标准误差表示。比较采用单因素方差分析(ANOVA)，多组采用Dunnett多重比较检验，两组采用双尾t检验。数据分析采用GraphPad Prism软件(Version 8.2.0 for Mac OS X Snow Leopard；Graph Pad Software,Inc.，San Diego,CA)和SPSS统计软件(Version 26.0.0.2 for Mac OS X Snow Leopard；IBM,Inc.，New York,USA)。P值小于0.05(P<0.05)被认为有统计学意义。动物实验数据的采集是随机的。在最终的统计分析中没有数据被排除。
实施例1在肝脂肪变性的肝脏中，TRIM31的活性受到抑制验证
本发明实施例为了验证TRIM31参与了肝脏脂肪变性和代谢，从正常饮食小鼠模型和高脂饮食小鼠模型中分离的肝组织中验证TRIM31的表达水平。
结果发现，16周高脂饲料(HFD)和ob/ob小鼠的肝脏样本中TRIM31蛋白的表达显著低于标准正常饲料(NCD)和较瘦的对照组小鼠，同时伴随Rhbdf2的表达增加(图1a,1b和图2a)。此外，HFD饮食小鼠0-16周的动态表达水平结果表明，TRIM31在脂肪肝中的表达逐渐被抑制(图1c和图2b,2c)。
此外，本发明实施例进一步检测了其在NAFLD(非酒精性脂肪性肝病)和NASH(非酒精性脂肪性肝炎)患者肝脏中的表达图谱。在NAFLD或NASH患者中，发现与非脂肪变性样本相比，TRIM31水平显著降低，肝脏中的Rhbdf2水平增加。而且，与单纯脂肪变性患者相比，NASH患者的肝脏中TRIM31的表达水平显著降低(图1d和图2d)。
此外，本发明实施例为了探究TRIM31表达下降的分子机制，进一步验证了其体外表达图谱。在培养的小鼠原代肝细胞中，观察到给药棕榈酸(PA)和TNF-α显著降低了TRIM31蛋白的表达水平，提高了RHBDF2的表达水平(图1e,图2e)。
此外，活性氧(ROS)是一种有效的促脂肪变性诱导剂，已在若干已建立的动物模型中得到证实。本发明实施例在体外实验中加入活性氧清除剂N-Acetyl-L-cysteine(NAC)和抗氧化过氧化氢酶(CAT)。结果表明，抑制ROS显著地改善了PA诱导的TRIM31表达的下降(图1f)，这表明ROS在TRIM31功能中具有拮抗作用。
表明，在炎症相关疾病中明显观察到TRIM31失活，同时RHBDF2活性显著增加。此外，RHBDF2也被证明是受泛素化修饰调控的。与非脂肪变性样本相比，NASH和单纯脂肪变 性表型患者的肝脏中RHBDF2水平明显升高。RHBDF2在NASH患者肝脏中的表达明显高于单纯脂肪变性患者(图2d)。
此外，本发明实施例还使用真核生物中的一种蛋白合成抑制剂环己酰亚胺(CHX)用来检测PA处理后的RHBDF2蛋白表达。与控制组(0h)相比，在5h时Rhbdf2的增加被抑制，在10h时降低。并且，与蛋白酶体抑制剂MG132共处理时，RHBDF2的降解受到显著抑制，而与氯喹(溶酶体抑制剂)共孵育时，RHBDF2的降解效果不明显(图1g-1i)。结果发现Rhbdf2降解是通过蛋白酶体途径。实际上，在PA处理的原代肝细胞中，所有RHBDF2会被泛素化。当活性氧消除后，其泛素化作用也会消除。(图2f)。而小鼠肝脏免疫荧光分析结果进一步证实了RHBDF2和TRIM31在脂肪肝中的表达变化(图1j)，免疫组化分析(图1k)了人肝脏切片和腺病毒介导的TRIM31表达(AdTRIM31)转染的L02细胞中细胞内甘油三酯(TG)水平(图1k)。上述结果表明，RHBDF2和TRIM31在脂肪肝中发挥关联作用的可能分子机制。
实施例2 TRIM31可改善胰岛素抵抗和糖代谢异常验证
因为TRIM31与脂肪肝密切相关，所以本发明实施例构建了一系列小鼠模型，以验证TRIM31在调节胰岛素抵抗和糖代谢紊乱的标志性影响因素中的作用，HFD诱导的胰岛素抵抗和糖代谢紊乱是脂肪肝的常见并发症和重要诱因。
建立肝细胞特异性TRIM31敲除小鼠(THKO)(图3a,3b)和肝细胞特异性TRIM31过表达小鼠(THTG)(图4a,4b)，来验证TRIM31对HFD诱导的胰岛素抵抗和糖代谢紊乱的保护作用。最终结果显示，长时间的HFD导致了小鼠的体重、空腹血糖和空腹胰岛素水平的升高，而且在TRIM31敲除中显著增加，但在TRIM31过表达中则得到一定程度缓解(图5a-5d)。
NCD和HFD处理的THKO小鼠(图3c)和THTG小鼠(图4c)的食物摄入量没有显著差异。从葡萄糖耐量试验(GTT)和胰岛素耐量试验(ITT)结果得出THKO小鼠血糖水平显著高于TRIM31 flox/flox(图6e,6f)，而在所有测试中，THTG小鼠的血糖水平都明显降低(图5e,5f)。
并且，通过使用糖原检测试剂盒检测显示TRIM31的缺失显著加速了肝糖原储备的降低(图6g)，而TRIM31过表达则抑制了肝糖原储备的降低(图5g)。
此外，肝脏TRIM31上调显著降低了mRNA和蛋白表达图谱，这些表达图谱与糖异生相关的指标包括葡萄糖-6-磷酸酶(G6Pase)和磷酸烯醇式丙酮酸羧激酶(PEPCK)有关；但是磷酸化AKT(p-AKT)、糖原合成酶激酶3β(p-GSK3β)和forkhead box O1(p-FOXO1)增加。与相应的对照组相比，HFD饮食引起的肝胰岛素信号失调在THKO小鼠中得到了加强，但在THTG小鼠中得到了显著恢复(图6h和图5h)。结果表明，在肝脏中，TRIM31是抑制HFD诱发胰岛素抵抗和糖代谢紊乱的重要因子。
实施例3 TRIM31可以预防肝脏脂肪变性和炎症验证
由于TRIM31对胰岛素抵抗和糖代谢紊乱具有抑制作用，本发明实施例验证了TRIM31在改善肝脏脂肪变性和炎症的主要特征方面的作用。
通过将长期HFD饮食的THKO小鼠与对照组相比，肝重和肝重/体重显著增加(图7a)。
同时，通过肝脏外观变化、TEM-image分析、H&E染色、油红染色对肝脏进行测定，并对肝组织中甘油三酯(TG)、总胆固醇(TC)、非酯化脂肪酸(NEFA)浓度测定，判断肝脂质的沉积。最终结果表明，HFD组THKO小鼠肝功能相关指标，血清丙氨酸转氨酶(ALT)、天冬氨酸转氨酶(AST)和碱性磷酸酶(AKP)明显升高(图7b-7d)。
在HFD喂养的THKO小鼠中，肝脏中TRIM31的功能缺失也与脂肪酸摄取和合成相关基因的表达显著上调和脂肪酸β氧化相关基因的表达显著下调有关(图7e)。但与之相反的是，在THTG中出现了与之截然相反的现象。
而且，将高脂饮食的THTG小鼠与高脂饮食的非转基因普通小鼠(NTG)相对比，前者的肝重和肝脏/体重比例更低(图7f)。通过使用相同的方法，结果显示，肝脏的脂肪含量和与肝脏功能相关指标例如TG、TC、NEFA、ALT、AST和AKP在高脂饮食的THTG小鼠中显著降低(图7g-7i)。同样的，与其相应的控制组相比，在THTG小鼠中，与脂肪酸摄取和合成相关基因的表达和脂肪酸β氧化相关基因的表达也趋向于正常水平(图7j)。
用PA处理的原代肝细胞也表明了TRIM 31在调控脂肪累计上的作用(图7k和图4d)。脂肪在内脏的累计通常与代谢综合征紧密相关。因此，内脏脂肪的重量、体重和脂肪/体重比在高脂饮食的THKO小鼠中升高，但是这种升高在THTG小鼠中被显著抑制(图8a)。因此，脂肪细胞的大小与高脂饮食和普通饮食高度相关，但是在高脂饮食的THKO和THTG中并没有显著性(图8b)。这个结果进一步揭示了与对照组相对比，脂肪细胞的累计可能与更高水平的高脂饮食小鼠的内脏脂肪含量有关。
本发明实施例为了进一步验证TRIM 31参与了肝脏的炎症调控，研究了炎症信号通路的活性和相应的与炎症相关的细胞因子趋化因子变化情况。通过磷酸化IKKβ、IκBα和NF-κB蛋白表达的变化以及炎症相关指标mRNA表达的变化，可以发现HFD引起的与炎症相关的信号通路在THKO小鼠内显著增强而在THTG小鼠内显著降低(图9a,9b)。
而且与炎症相关的因子包括TNF-α、IL-1β、IL-6和CCL-2在高脂饮食的THKO小鼠血清中比正常饮食更高，而在高脂饮食的THTG小鼠血清中比正常饮食更低(图9c)。与此同时，通过活化NF-κB介导的炎症信号，在PA处理的原代肝细胞中进一步观察到类似的结果(图9d)。
以上实施例的结果表明，TRIM 31是小鼠体内由HFD诱发的炎症和肝脏脂质堆积的重要抑制剂。
实施例4介导的体外基因治疗有助于减轻脂肪肝验证
本实施例更好地验证明TRIM 31对脂质堆积的抑制作用，用慢病毒包裹完整的鼠TRIM31基因序列(LV-TRIM31)或者人TRIM 31基因序列(LV-hTRIM31)进行干预，进一步评估TRIM31在缓解肝脏脂质堆积、胰岛抵抗和炎症中的作用(图10a和图11a)。结果显示，在体外培养和转导的肝细胞中，具有肝细胞特异性TRIM31功能获得(THKO)(LV+)的小鼠与相应的对照组(THKO)(LV-)比较，高脂饲料诱导的肝重量、体重、肝重/体重比、AST和ALT、肝脏脂质堆积显著降低(图10b-10d)。
并且，从GTT和ITT的结果来看，THKO(LV+)小鼠比THKO(LV-)小鼠的血糖浓度更低(图10e,10f)。结果进一步验证了THKO(LV+)小鼠中肝脏TG、TC和NEFA的显著性下调趋势(图10g)。同时，肝细胞TRIM31的升高显著降低了高脂饮食的THKO(LV+)小鼠脂肪酸摄取和合成的基因表达，促进了脂肪酸β-氧化的基因表达(图10h)。
而且，通过对THKO小鼠和对照组进行免疫印迹、mRNA水平和促炎因子的浓度测定，THKO小鼠中与对照组相比，受损的胰岛素信号和激活的炎症相关信号被显著抑制(图10i,10j和10b,10c)。与上述结果一致，LV-hTRIM31介导的体外基因治疗进一步证明肝细胞中TRIM31的修复确实显著减轻了高脂饮食诱导的胰岛素抵抗、肝脂肪变性和炎症(图12a-12k)。
实施例5验证TRIM31使RHBDF2-MAP3K7的失活从而抑制非酒精性脂肪肝的发展
由于TRIM31对脂肪肝和相关病理表型有一致且显著的抑制作用，本发明实施例继续验证TRIM31的作用的分子机制及其内在功能。因为RHBDF2是泛素化形式的，而且在NAFLD和NASH的表型中起关键作用，进一步验证了TRIM31对RHBDF2及其下游信号事件的影响。
如免疫荧光切片结果所示，高脂饮食加速了THKO小鼠中RHBDF2-MAP3K7的活性，但是在THTG小鼠中得到显著抑制(图13a)。同样，RHBDF2-MAP3K7信号包括ADAM17、TNFR1/2、MKK7和c-Jun也被确定参与脂肪肝的发展。
本发明实施例验证了体内和体外ADAM17,TNFR1/2,and p-MAP3K7及其下游成分MKK7的水平在TRIM31缺失的时候得到上调，但在TRIM31过度表达中得到抑制(图13b,13c)。
本发明实施例为了进一步验证TRIM31在RHBDF2功能中的作用，构建了PA诱导的原代肝细胞胰岛素信号传导损伤模型。用腺病毒包裹的全长RHBDF2序列(AdRHBDF2)或靶向RHBDF2的shRNA(AdshRHBDF2)，在TRIM31特异性缺失或TRIM31转基因的肝细胞中过度表达或失活RHBDF2。与对照组相比，在AdRHBDF2转染的THKO肝细胞中，由PA刺激的胰岛素信号传导的损伤显著恶化，但在AdshRHBDF2转染的THKO肝细胞中，该损伤实际上得到缓解。然而，转染了AdshRHBDF2的THTG肝细胞相对于转染了AdRHBDF2的THTG肝细胞，表现出对PA诱导受损胰岛素信号(比如p-AKT,p-GSK3β,p-FOXO1)的有效缓解趋势(图13d和图14a)。进一步证明使RHBDF2-MAP3K7信号通路功能障碍，是TRIM31抗肝脂肪变性，起保护作用的关键。
实施例6验证TRIM31直接与RHBDF2相互作用
本发明实施例使用免疫沉淀的方法验证了在非酒精性脂肪性肝病的发展过程中TRIM31直接与RHBDF2相互作用。
体外相互作用试验表明，TRIM31的外源表达可以直接与RHBDF2结合，反之亦然(图15a)。随后，谷胱甘肽硫转移酶(GST)标记的RHBDF2很明显地与TRIM31结合，GST标记的TRIM31也与RHBDF2结合(图15b)。这些数据揭示了TRIM31与RHBDF2的直接相互作用。
作为E3泛素连接酶的关键成员，TRIM31主要由三部分组成:N端环指结构域、B盒结构域和C端螺旋结构域(图15c)。为了确定TRIM31的哪一个结构域与RHBDF2结合,本发明实施例选择了产生一系列带有标记载体的截短的TRIM31突变体，包括野生型(TRIM31-Flag WT)、环指结构域切除突变体(TRIM 31-Flag RING)、B-Box结构域缺失突变体(TRIM 31-Flag Box)和螺旋结构域缺失突变体(TRIM 31-FlagCC)载体，用于以下结合实验。
免疫共沉淀分析表明RHBDF2与野生型、环指结构域切除突变体和B-Box结构域缺失突变体共沉淀。这些结果表明，卷曲螺旋结构域有助于其结合RHBDF2的能力。
接下来，本发明实施例还建立了3个带有HA标签的质粒的RHBDF2截短突变体，一共包括野生型(RHBDF2-HA WT)、跨膜结构域(TMD)缺失突变体(RHBDF2-HA TM)、非活性菱形同源结构域(IRHD)缺失突变体(RHBDF2-HA IRHDD)和N末端细胞质尾(tail)结构域缺失突变体(RHBDF2-HA Tail)载体。用这些RHBDF2突变体进行的免疫共沉淀结果表明，RHBDF2-HA Tail没有与TRIM31结合的能力。
因此，RHBDF2的氮末端胞质尾区(tail)结构域对于与TRIM31的相互结合作用是必需的(图15c和图14b)。
另外，因为人体内TRIM31的CC结构域和RHBDF2的N端结构域(Tail)对于它们的相互作用是必须的，所以本发明实施例检验了它们是否在老鼠身上也有相似的生物功能。因 此，老鼠TRIM31和RHBDF2的结合域也用TRIM31-Flag CCΔ,RHBDF2-HA Tail Δ,和相应的野生型载体来检测。与蛋白结合实验结果一致，在转染的鼠肝细胞中，CC域缺失的TRIM31不能和RHBDF2结合(图14c)。
实施例7验证TRIM31通过K48连接的多泛素化促进RHBDF2的降解
本发明实施例验证TRIM31通过K48连接的多泛素化能促进RHBDF2的降解，之前推测RHBDF2在细胞质内的结构域上可能会有K48泛素化的结合位点，由此使蛋白酶体降解。实施例6已经确认了TRIM31和RHBDF2能够相互作用(图15c)。
因此，本发明实施例验证通过TRIM31及其相应的E3泛素化连接酶是否能RHBDF2的活性对进行调控，实验结果表明，体内外RHBDF2的泛素化水平显著提高(图15d和图2f)。
此外，本发明实施例将RHBDF2和Myc-ubiquitin和标记的野生型TRIM31一起转入L02细胞。此时RHBDF2泛素化水平伴随着TRIM31载体的存在而显著上升。而且，具有RING-finger结构域切除突变体的TRIM31表达载体不能催化RHBDF2的泛素化，这表明功能性TRIM31需要RING-finger结构域来调节RHBDF2的泛素修饰(图15e)。
此外，之前的研究表明RHBDF2可以通过K63连接的泛素化进行修饰。而与之前的研究一致，在PA处理的L02细胞中内源性RHBDF2几乎是泛素化的，且能和K48和K63连接。在TRIM31缺陷的L02细胞(THKO-L02)中，只有RHBDF2的K48连接的多泛素化被显著抑制，但是对RHBDF2与K63连接的泛素化的影响可以忽略不计(图15f)。
此外，为了准确研究TRIM31调节RHBDF多泛素化的方式，本发明实施例用泛素突变质粒K48、K63、K33、K6、K29和K27进行体外转染实验。K48O、K63O、K33O、K6O、K29O和K27O中“O”的标记揭示了泛素中除了它们本身之外的所有赖氨酸残基都被完全突变。相反，在K48载体存在的情况下，可以观察到大量的TRIM31介导的RHBDF2多泛素化的增加，但在其他载体中没有(图15g)。
所以本发明实施例验证了，RING-finger结构域突变体实际上消除了TRIM31的E3泛素连接酶活性。鉴于RING-finger结构域在TRIM31功能中的重要作用，TRIM31的RING-finger结构域缺失不仅消除了RHBDF2的多泛素化，而且阻碍了TRIM31抑制RHBDF2-MAP3K7通路及其下游信号级联的激活的能力(e.g.,ADAM17,p-NF-κB,p-IκBαand p-JNK1/2)(图15g)。
本发明实施例在载体转染的TRIM31缺陷型L02细胞(THKO-L02)中，用PA刺激的细胞内TG浓度变化进一步证明了RING-finger结构域缺失对TRIM31功能的负面影响(图15h)。
综上所述，TRIM31诱导的RHBDF2通过K48连接的多泛素化降解依赖于其E3泛素连接酶活性。
实施例8验证TRIM31-RHBDF2相互作用是TRIM31调节的肝脂肪变性和炎症所必需的
考虑到RHBDF2活性与TRIM31的E3泛素连接酶活性之间的紧密相关性，为了彻底了解TRIM31-RHBDF2相互作用是否是TRIM31对肝脂肪变性的保护功能所必需的。
本发明实施例建立了一个带有RING-finger敲除的慢病毒负载TRIM 31(LV-TRIM 31 RING)，并再次用于进一步研究TRIM 31 RING对HFD诱导的肝脂肪变性、胰岛素抵抗和炎症的影响(图16a和图17a)。正如本发明在细胞实验中所预期的结果，与对照组(THKO)(LV-)相比，采用TRIM31 RING-finger敲除-肝细胞移植(THKO)(LV+)的小鼠未能改变HFD诱导的肝脏重量、体重、肝重/体重比率、肝功能指标AST和ALT以及肝脏脂质累计(图16b-16d)。
此外，血糖水平和肝甘油三酯、总胆固醇和NEFA含量的分析表明没有显著差异(图16e-16g)。同时，在高脂饮食的THKO(LV+)小鼠中，缺失RING-finger结构域的肝细胞TRIM31不能调节与脂肪酸摄取、合成过程有关的基因表达和与脂肪酸β-氧化过程有关的基因表达(图16h)。更重要的是，与对照组相比，胰岛素信号传导没有任何活性变化，并且可以明显观察到RHBDF2-MAP3K7信号通路和下游通路，如免疫印迹、mRNA水平和促炎介质浓度没有显著性改变(图16i,16j和图17b,17c)。
与这些发现一致，本发明实施例还用LV-hTRIM31体外基因疗法进一步证实了老鼠被移植了人TRIM31 RING-肝细胞也没有明显缓解高脂饮食诱导的胰岛素抵抗，肝脂肪变性和炎症(图18a-18g)。这些结果进一步表明鼠和人的TRIM31的RING结构对E3泛素蛋白连接酶的保护性功能是必须的。
另外，携带有RING-finger结构域突变体(AAV-TRIM31 RING)的TRIM31的腺相关病毒亚型8(AAV8)-甲状腺素结合球蛋白(TBG)被相应地产生并和AAV-TRIM31平行注射到6周HFD喂养的预处理WT小鼠中，注射AAV-GFP的小鼠作为对照(图19a)。毫无疑问，注射AAV-TRIM31 RING结构突变体在体重、血糖水平、血胰岛素水平、肝脏重量、LW/BW比值、肝功能指标AST、ALT和AKP、病理表型改变以及肝脏TG、TC和NEFA水平方面调节功能的能力丧失，而AAV-TRIM31则没有(图19b-19f)。同时，与细胞实验得出的数据一致，给予AAV-TRIM31 RING结构域突变体的小鼠在改善受损的胰岛素信号、脂肪酸摄取/合成和脂肪酸β-氧化的基因表达变化以及RHBDF2相关的下游炎症信号级联方面没有显示任何显著变化(图19g-19i)。总的来说，TRIM31-RHBDF2相互作用和TRIM31的RING-finger结构域对于TRIM31调节的肝脂肪变性和炎症至关重要。
实施例9验证TRIM31通过靶向RHBDF2调节保护肝脏脂肪变性
本发明实施例为了进一步研究抑制RHBDF2信号介导TRIM31对脂肪肝的保护作用，将肝细胞特异性TRIM31缺乏小鼠(THKO)与RHBDF2 flox/flox小鼠(Alb-Cre；如“实施例动物模型”一节所述，使用RHBDF2 flox/flox(以下简称RHKO)产生肝细胞特异性TRIM31和RHBDF2双缺失小鼠(Alb-Cre；RHBDF2 flox/flox，TRIM31flox/flox，以下简称DHKO)。通过蛋白质印迹分析确定肝脏样品中TRIM31和RHBDF2的含量(图20a)。之前的研究已经证实了RHBDF2缺失能缓解长期HFD诱导引起的脂肪性肝炎。RHBDF2缺失阻断了TRIM31消融对HFD刺激的RHBDF2-MAP3K7轴和下游信号级联、体重、肝重、肝重/体重比、受损胰岛素信号、异常血糖水平、肝脂质沉积和促炎症相关基因表达升高的影响(图20a-20f和图21a-21e)。此外，在体外检测PA诱导的细胞内甘油三酯水平的变化也有相同的趋势(图20g和图22)。这些结果都证明了TRIM31对NAFLD的保护作用是通过RHBDF2信号途径进行调控的。
实施例10非酒精性脂肪性肝炎靶向TRIM31-RHBDF2信号治疗的可行性和效果评价
基于TRIM31对非酒精性脂肪性肝病的有效保护作用，本发明实施例验证了靶向TRIM31-RHBDF2-MAP37K通路在NASH发展和进展中的治疗可行性和效果。然后用含14％蛋白质、42％脂肪、44％碳水化合物、0.2％胆固醇的HFHF饮食喂养THTG小鼠，并以55％果糖和45％蔗糖的重量比在饮用水中混合总共42g/L的碳水化合物，持续16周，以构建具有NASH表型的小鼠(THTG-HFHF)。
此外，NTG小鼠被用作对照(NTG-HFHF)，并用相同的饮食随意喂养16周。不出所料，在HFHF诱导产生的NASH模型中，在HFHF饮食16周后，THTG组的肝脏重量、肝重/体重比 以及肝脏TG、TC和NEFA浓度明显低于NTG对照组，同时THTG-HFHF和NTG-HFHF小鼠之间的体重没有显著差异(图23a-23c)。
而且，与NTG小鼠相比，在HFHF饮食处理后，通过H&E染色、油红O染色、F4/80免疫组化分析、天狼星红染色、Masson染色、炎症和胶原相关基因表达水平测定，在THTG小鼠中观察到肝脂质沉积，脂肪酸合成相关基因表达(e.g.,CD36,FASN,and ACACa)，炎症相关基因表达(e.g.,TNF-α,IL-1β,IL-6,and CCL-2)，肝纤维化(i.e.,collagen deposition)和胶原蛋白合成相关基因表达(e.g.,TIMP1,CTGF,COL1A1,and COL3A1)显著减少(图23d-23i)。在16周的HFHF饮食后，TRIM31的过度表达还明显下调了小鼠血清AST、ALT和AKP水平、促炎细胞因子TNF-α、IL-6和IL-1β的浓度以及TRIM31调节的RHBDF2-MAP3K7信号通路和下游信号通路。总的来说，本发明实施例数据揭示了TRIM31在治疗小鼠脂肪性肝炎和相关代谢综合征中的疗效。
在本发明实施例中，使用肝细胞特异性TRIM31敲除的小鼠(THKO)/转基因小鼠(THTG)和慢病毒介导的离体基因治疗(LV-TRIM31)小鼠，本发明确定肝脏TRIM31是高脂饮食(HFD/HFHF)诱导或遗传诱导的慢性炎症、胰岛素抵抗、肝脏脂肪变性和NASH的显著负调节因子。对潜在分子机制的研究发现，在长期的高热量饮食下，体内和体外实验中明显观察到TRIM31的减少，这导致RHBDF2-MA3K7信号通路激活的增加。在TRIM31功能失调的情况下，强烈激活的RHBDF2-MAP3K7信号传导进一步介导下游信号通路表达的增加，包括JNK-IRS1相关的胰岛素信号传导、IκBα-NF-κB-c-Jun通路、AKT-GSK3β-FOXO1磷酸化水平的抑制和CTGF-TIMP1通路，促进受损的胰岛素信号传导(胰岛素抵抗)、慢性炎症作用、异常的葡萄糖代谢紊乱和肝脏中的胶原沉积的发生，从而加重异常的脂质代谢，以及肝脏脂肪变性和NASH表型的产生(图23k)。
实施例11甜菜红素(BCN)激活TRIM31缓解小鼠肝纤维化和炎症
按照实施例10构建具有NASH表型的小鼠(THTG-HFHF)。设空白组(Ctrl)、NASH组(THTG)和给药组(BCN)，给药组采用0.2g/kg比例将甜菜红素(BCN)与饲料混合后喂养。如图25所示，16周后，THTG-HFHF小鼠肝脏中的胶原蛋白I(Collagen I)，TGF-β1，Keap1蛋白表达水平显著升高，TRIM31蛋白表达显著降低，说明高糖高脂引起胶原蛋白积累和氧化应激导致非酒精性脂肪肝炎。用BCN给药干预的小鼠肝组织中胶原蛋白I(Collagen I)，TGF-β1，Keap1蛋白表达水平显著降低，TRIM31蛋白表达显著升高，接近控制组正常水平，有效缓解了肝脏纤维化和炎症。
实施例12漆黄素(FIS)激活TRIM31缓解小鼠肝纤维化和炎症
按照实施例10构建具有NASH表型的小鼠(THTG-HFHF)。设空白组(Con)、NASH-THTG组(HFD)和给药组(FIS)，给药组采用20mg/kg，40mg/kg，80mg/kg剂量的漆黄素(FIS)灌胃。如图26所示，16周后，THTG-HFHF小鼠肝脏中的α-SMA、胶原蛋白I(Collagen I)，TGF-β1，Fibronectin等蛋白基因的mRNA水平显著升高，TRIM31蛋白基因的mRNA水平显著降低，说明高糖高脂引起胶原蛋白积累和氧化应激导致非酒精性脂肪肝炎。用低、中、高剂量的FIS治疗的小鼠肝组织中α-SMA，胶原蛋白I(Collagen I)，TGF-β1，Fibronectin蛋白基因的mRNA水平显著降低，TRIM31蛋白基因的mRNA水平显著升高，特别是高剂量给药组接近控制组正常水平，有效缓解了肝脏纤维化和炎症。
实施例12胭脂红酸(CA)激活TRIM31缓解小鼠肝纤维化和炎症
按照实施例10构建具有NASH表型的小鼠(THTG-HFHF)。设空白组(Con)、NASH-THTG 组(Fru)，低剂量给药组(CAL，0.5％CA+Fru)，高剂量给药组(CAH，1％CA+Fru)，给药组采用0.5％和1％的胭脂红酸(CA)灌胃。如图27所示，16周后，THTG-HFHF小鼠肝脏中的α-SMA、胶原蛋白I(Collagen I)，TGF-β1，MMP-9等蛋白基因的mRNA水平显著升高，Keap1蛋白表达水平显著升高，TRIM31蛋白表达显著降低，说明高糖高脂引起胶原蛋白积累和氧化应激导致非酒精性脂肪肝炎。用低、高剂量的CA治疗的小鼠肝组织中α-SMA，胶原蛋白I(Collagen I)，TGF-β1，MMP-9蛋白基因的mRNA水平显著降低，Keap1蛋白表达水平显著降低，TRIM31蛋白表达显著升高，特别是高剂量给药组接近控制组正常水平，有效缓解了肝脏纤维化和炎症。
需要说明的是，除了本发明实施例11-实施13提供的甜菜红素、漆黄素、胭脂红酸可以激活TRIM31缓解小鼠肝纤维化和炎症外，以下几种物质((1)-(6))的一种或多种组合均可以达到激活TRIM31缓解小鼠肝纤维化和炎症的效果：
(1)生物碱类化合物或组合物，包括小檗碱，木兰花碱，骆驼蓬碱，蛇根碱，益母草碱，粉防己碱，常山碱，浙贝母碱，秋水仙碱，胡椒碱，钩藤碱，喜树碱，延胡索乙素，药根碱，白屈菜碱，石蒜碱，山油柑碱，苦参碱，氧化苦参碱中的一种化合物或多种化合物组成的组合物；
(2)黄酮类化合物或组合物，包括槲皮素，二氢槲皮素，木犀草素，桑色素，桑皮黄素，芹菜素，香叶木素，白杨素，苜蓿素，葛根素，花青素，原花青素，大豆黄素，芹菜素，黄芩素，野黄芩素，白杨素，汉黄芩素，矢车菊素，水飞蓟素，水飞蓟宾，黄豆苷元，染料木素，高良姜黄素，山奈酚，异鼠李素，杨梅黄酮，橙皮素，柚皮素，甘草素，儿茶素，表儿茶素，没食子儿茶素，矢车菊素，飞燕草素，花葵素，灯盏花素，阿福豆素，芦丁，木犀草苷，黄芩苷，芹菜苷，胡桃苷中的一种化合物或多种化合物组成的组合物；
(3)多酚类化合物或组合物，包括绿原酸，洋蓟酸，菜蓟糖苷，菜蓟苦素，茶多酚，白藜芦醇，石榴多酚，姜黄素，阿魏酸中的一种化合物或多种化合物组成的组合物；
(4)胺类化合物或组合物：青霉胺，泊马度胺，来那度胺，沙利度胺中的一种化合物或多种化合物组成的组合物；
(5)中药提取物，包括黄芪、党参、丹参、当归、山药、三七、五味子、黄精、灵芝、云芝、洋蓟、小蓟、水飞蓟、葛根、黄连、黄柏、姜黄、金银花、枳椇、陈皮、桑叶、银杏叶、百合、熊胆、猪胆、茵陈、枳壳、甘草、虎杖、槐米、丹皮、垂盆草、栀子、枸杞、白芍、川芎、桃仁、大黄、菊花、溪黄草、白花蛇舌草、防己、白茅根、半枝莲、柴胡、茯苓、山药中的一种提取物或多种提取物组成的组合物；
(6)甘草酸，甘草酸苷，腺苷蛋氨酸，熊去氧胆酸，牛磺熊去氧胆酸，鹅去氧胆酸，牛磺鹅去氧胆酸，双环醇，联苯双酯，索拉非尼，瑞戈非尼，乐伐替尼，二甲双胍中的一种或多种。
最后说明的是，以上实施例仅用以说明本发明的技术方案而非限制，尽管参照较佳实施例对本发明进行了详细说明，本领域的普通技术人员应当理解，可以对本发明的技术方案进行修改或者等同替换，而不脱离本发明技术方案的宗旨和范围，其均应涵盖在本发明的权利要求范围当中。

Claims (10)

  1. TRIM31在制备NAFLD/NASH中的标志物中的应用，所述TRIM31包含如sequenceNO：1或sequenceNO：2所示的氨基酸序列。
  2. TRIM31联合RHBDF2共同制备NAFLD/NASH中的标志物中的应用，所述TRIM31包含如sequenceNO：1或sequenceNO：2所示的氨基酸序列。
  3. 组合因子对，其特征在于，肝细胞来源的TRIM31和RHBDF2；所述TRIM31包含如sequenceNO：1或sequenceNO：2所示的氨基酸序列；所述RHBDF2包含如sequenceNO：3或sequenceNO：4所示的氨基酸序列。
  4. 根据权利要求3所述的组合因子对，其特征在于，所述TRIM31的与所述RHBDF2在量上存在此消彼长的关系。
  5. 权利要求3所述的组合因子对在制备代谢紊乱的标志物中的应用。
  6. TRIM31的促进剂作为制备NAFLD、NASH或代谢紊乱的药物中的应用。
  7. 根据权利要求6所述的应用，其特征在于，所述促进剂包括基因类促进剂、蛋白类促进剂、化学类促进剂、中药类促进剂。
  8. 根据权利要求6所述的应用，其特征在于，所述促进剂包括以下几种物质中的一种或多种：
    (1)生物碱类化合物或组合物，包括小檗碱，木兰花碱，骆驼蓬碱，蛇根碱，益母草碱，粉防己碱，常山碱，浙贝母碱，秋水仙碱，胡椒碱，钩藤碱，喜树碱，延胡索乙素，药根碱，白屈菜碱，石蒜碱，山油柑碱，苦参碱，氧化苦参碱中的一种化合物或多种化合物组成的组合物；
    (2)黄酮类化合物或组合物，包括槲皮素，二氢槲皮素，木犀草素，桑色素，桑皮黄素，芹菜素，香叶木素，白杨素，苜蓿素，葛根素，花青素，原花青素，大豆黄素，芹菜素，黄芩素，野黄芩素，白杨素，汉黄芩素，矢车菊素，水飞蓟素，水飞蓟宾，黄豆苷元，染料木素，高良姜黄素，山奈酚，漆黄素，异鼠李素，杨梅黄酮，橙皮素，柚皮素，甘草素，儿茶素，表儿茶素，没食子儿茶素，矢车菊素，飞燕草素，花葵素，灯盏花素，阿福豆素，芦丁，木犀草苷，黄芩苷，芹菜苷，胡桃苷中的一种化合物或多种化合物组成的组合物；
    (3)多酚类化合物或组合物，包括绿原酸，洋蓟酸，菜蓟糖苷，菜蓟苦素，茶多酚，白藜芦醇，石榴多酚，姜黄素，阿魏酸中的一种化合物或多种化合物组成的组合物；
    (4)胺类化合物或组合物：青霉胺，泊马度胺，来那度胺，沙利度胺中的一种化合物或多种化合物组成的组合物；
    (5)中药提取物，包括黄芪、党参、丹参、当归、山药、三七、五味子、黄精、灵芝、云芝、洋蓟、小蓟、水飞蓟、葛根、黄连、黄柏、姜黄、金银花、枳椇、陈皮、桑叶、银杏叶、百合、熊胆、猪胆、茵陈、枳壳、甘草、虎杖、槐米、丹皮、垂盆草、栀子、枸杞、白芍、川芎、桃仁、大黄、菊花、溪黄草、白花蛇舌草、防己、白茅根、半枝莲、柴胡、茯苓、山药中的一种提取物或多种提取物组成的组合物；
    (6)甘草酸，甘草酸苷，腺苷蛋氨酸，熊去氧胆酸，牛磺熊去氧胆酸，鹅去氧胆酸，牛磺鹅去氧胆酸，双环醇，联苯双酯，索拉非尼，瑞戈非尼，乐伐替尼，甜菜红素，二甲双胍，胭脂红酸中的一种或多种。
  9. 判断肝细胞变性的方法，其特征在于，检测待测离体样本中的TRIM31的表达水平，并与正常组进行比较，当所述待测离体样本中的TRIM31的表达水平低于正常组时，判定为肝细胞变性。
  10. 根据权利要求9所述的方法，其特征在于，检测待测离体样本中的RHBDF2的表达水平，并与正常组进行比较，当所述待测离体样本中的RHBDF2的表达水平低于正常组时，判定为肝细胞变性。
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