Application of UBC9 gene overexpression preparation in preparation of medicine for treating metabolic dysfunction fatty liver disease

Abstract

The research finds that the SUMOylation level of the liver in a clinical specimen and an animal model of the metabolic dysfunction fatty liver disease is obviously reduced. UBC9 is used as the only key E2 ligase for SUMOylation to regulate and control the SUMOylation level. The invention discloses application of a UBC9 gene overexpression preparation in drugs for treating or preventing metabolic dysfunction fatty liver diseases. Through overexpression of liver specificity UBC9 mediated by an AAV adeno-associated virus vector or overexpression of UBC9 in HepG2 cells by a lentiviral vector, lipid deposition of a C57 mouse liver induced by high fat diet and a cell model treated by palmitate can be remarkably improved, and reduction of the SUMOylation level of the mouse liver and the human HepG2 cells under the high fat background can be reversed. Therefore, the UBC9 gene overexpression preparation can be used for developing drugs for treating metabolic dysfunction fatty liver diseases.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of gene therapy and metabolic diseases, in particular to the application of UBC9 gene overexpression preparation in the treatment of metabolic dysfunction associated fatty liver disease (MASLD) and its molecular mechanism for improving lipid metabolism disorder by increasing SUMOylation modification pathway. BACKGROUND

[0002] MASLD is the most common chronic liver disease in the world, and its core pathological feature is liver fat accumulation (hepatic steatosis ≥5%). The prevalence of MASLD is about 25%-30%, and up to 50%-70% in obese or type 2 diabetes populations. According to the global burden of disease research, the mortality rate of MASLD related cirrhosis has increased significantly in the past decade, and has become the second indication for liver transplantation. The prevalence in Asia has increased rapidly, consistent with the trend of metabolic syndrome in China, India and other countries.

[0003] Currently, the treatment of MASLD is still based on lifestyle intervention (diet control, exercise), but the patient compliance is poor and the long-term effect is limited. In terms of drug treatment, only one drug, Resmetirom (a selective agonist of thyroid hormone receptor beta), has been approved by FDA for the treatment of MASH related fibrosis. Other drugs such as vitamin E and pioglitazone are recommended by some guidelines for MASH patients, but there are no significant effects or side effects risks (such as prostate cancer, bone fracture, etc.). In addition, in recent years, although the clinical trials of FXR agonists (obeticholic acid) and GLP-1 receptor agonists (semaglutide) have made progress, there is still a lack of long-term safety and effectiveness evidence. Therefore, the development of new targeted therapies for key regulatory nodes of lipid metabolism has become a research hotspot.

[0004] SUMOylation is a kind of post-translational modification of protein, and UBC9 is the only known E2 conjugase in the SUMOylation pathway, which is responsible for transferring SUMO protein to target protein, and then regulating its stability and function. Knocking out UBC9 can reduce the overall SUMOylation of cells, while overexpressing UBC9 can enhance the SUMOylation of cells. In recent years, studies have found that the SUMOylation pathway plays a key role in the regulation of liver lipid metabolism. ATGL is a key enzyme for lipolysis, and the SUMOylation of its 117th lysine (K117) can enhance its stability and promote the decomposition of liver triglycerides. In the liver of MASLD patients, the decrease of UBC9 expression leads to the decrease of SUMOylation of ATGL, and the decrease of lipolysis capacity, thereby exacerbating lipid deposition; SREBP-1c is a key transcription factor for regulating fatty acid synthesis, and overexpression of UBC9 can inhibit the nuclear translocation of SREBP-1c by enhancing the SUMOylation modification of SREBP-1c, thereby reducing liver lipogenesis; FoxA1 has been proved to protect the liver from steatosis, and its expression is down-regulated in MASLD, and the desumoylation of FoxA1 at K6 promotes the degradation of FoxA1, thereby inhibiting the transcription of Sirt6, and thus inhibiting fatty acid beta oxidation and promoting the occurrence and development of MASLD; Liver kinase B1 (LKB1) is a serine / threonine kinase that controls cell homeostasis, and inhibition of LKB1 sumoylation can disrupt cellular fatty acid oxidation; the desumoylation of PPARa can enhance its ubiquitination and degradation, thereby inhibiting fatty acid oxidation and promoting the progression of MASLD.

[0005] Therefore, there is sufficient research evidence to support the treatment of MASLD by regulating the SUMOylation of cells, so that the overexpression of UBC9 gene preparation has great potential in the application of MASLD disease. The existing treatment method cannot target the SUMOylation regulation network. The present application combines the overexpression of UBC9 and the restoration of SUMOylation modification for the first time, and provides a precise treatment strategy for MASLD. SUMMARY

[0006] The purpose of the present application is to provide the application of UBC9 gene overexpression preparation in the preparation of drugs for treating or preventing MASLD, to improve the abnormal liver lipid metabolism and blood lipid level by targeting the expression of UBC9 gene, and to provide a new gene therapy strategy for the treatment of MASLD.

[0007] The experimental research of the present application found that the SUMOylation in the liver biopsy specimens of MASLD patients was significantly lower than that in the healthy group, and the SUMOylation of human HepG2 cell lines and animal livers was also reduced under the background of high fat;

[0008] Overexpression of UBC9 in HepG2 cells using a lentiviral vector can reduce the lipid deposition in cells after palmitate treatment;

[0009] The C57 mice are injected with the UBC9 overexpression adeno-associated virus vector through the tail vein, so as to reduce the liver lipid deposition and blood lipid level of the C57 mice after being fed with high-fat diet for 12 weeks.

[0010] The UBC9 overexpression lentivirus vector is pLVX-CMV, and the specific vector construction is shown in the following table. Figure 1 The UBC9 overexpression adeno-associated virus AAV vector is constructed as shown in the following table. Figure 2 .

[0011] The UBC9 is overexpressed through a gene delivery system (AAV, lentivirus), the liver SUMO modification network is recovered, so as to inhibit lipid synthesis, promote lipolysis and alleviate inflammatory response.

[0012] The present application focuses on the SUMO network of the whole liver for the first time, and it is found through background research and experimental verification that the overexpression of UBC9 can enhance the overall SUMO level of cells, so as to reverse the reduction of the liver SUMO and UBC9 level in the MASLD model, so that both the human HepG2 cell line and the mouse liver show resistance to excessive lipid deposition, and the development of MASLD is reduced. BRIEF DESCRIPTION OF DRAWINGS

[0013] Figure 1 is a UBC9 overexpression lentivirus vector construction detail diagram;

[0014] Figure 2 is a UBC9 overexpression adeno-associated virus AAV vector construction detail diagram;

[0015] Figure 3 is a Western blot (WB) detection diagram of SUMO1 and SUMO2 / 3 proteins, which are key SUMO molecules of human normal liver specimens and clinical MASLD patient liver specimens;

[0016] Figure 4 is a WB detection diagram of UBC9, SUMO1 and SUMO2 / 3 proteins, which are key SUMO molecules of liver specimens of C57BL / 6 mice fed with control diet and high-fat diet for 12 weeks respectively;

[0017] Figure 5 is an immunohistochemical staining diagram of UBC9, SUMO1 and SUMO2 / 3 proteins, which are key SUMO molecules of liver specimens of C57BL / 6 mice fed with control diet and high-fat diet for 12 weeks respectively;

[0018] Figure 6 is a WB detection diagram of UBC9, SUMO1 and SUMO2 / 3 proteins, which are key SUMO molecules of human HepG2 cell lines treated with palmitate (PA) for 0, 6, 12 and 24 hours respectively;

[0019] Figure 7 Figure 5 is a WB detection chart of SUMOylation key molecules UBC9, SUMOl and SUMO2 / 3 proteins of mouse liver samples after mice were injected with adeno-associated virus AAV8-UBC9 for two weeks;

[0020] Figure 8 Figure 6 is an HE and oil red staining chart of liver samples of control diet group, high-fat diet group and AAV8-UBC9 high-fat diet group after mice were fed for 12 weeks;

[0021] Figure 9 Figure 7 is a detection chart of serum triglyceride and total cholesterol of control diet group, AAV8-UBC9 control diet group, high-fat diet group and AAV8-UBC9 high-fat diet group after mice were fed for 12 weeks;

[0022] Figure 10 Figure 8 is a detection chart of liver triglyceride and total cholesterol of control diet group, AAV8-UBC9 control diet group, high-fat diet group and AAV8-UBC9 high-fat diet group after mice were fed for 12 weeks;

[0023] Figure 11 Figure 9 is a WB detection chart of SUMOylation key molecules UBC9, SUMOl and SUMO2 / 3 proteins of human HepG2 cell line control group and cells after using UBC9 overexpression lentivirus;

[0024] Figure 12 Figure 10 is an oil red staining chart of human HepG2 cell line control group and UBC9 lentivirus overexpression group after 24 hours of PA treatment;

[0025] Figure 13 Figure 11 is a GO analysis chart of human HepG2 cell line control group and UBC9 lentivirus overexpression group after proteomics detection;

[0026] Figure 14 Figure 12 is a KEGG analysis chart of human HepG2 cell line control group and UBC9 lentivirus overexpression group after proteomics detection; DETAILED DESCRIPTION

[0027] The specific embodiments of the present application are further illustrated by the following examples. It should be particularly pointed out that the examples are intended to exemplarily illustrate the technical solutions of the present application, and the specific parameters and operation steps thereof should not be understood as limiting the scope of the claims. Any adaptive adjustment or extension improvement made by the person skilled in the art based on the core idea of the present application shall fall within the protection scope of the present application.

[0028] The chemical reagents involved in the examples are all standard products available on the market and can be directly purchased and used. The experimental methods without specific operation steps are all performed according to the general technical specifications in the field.

[0029] Clinical specimens: The specimens were collected from patients who underwent hepatic hemangioma resection in the First Affiliated Hospital of Xi'an Jiaotong University. Six cases with metabolic-associated steatohepatitis (MASLD) were used as the case group, and six cases without MASLD were used as the control group. The exclusion criteria included severe liver disease, digestive tract disease, tumor, alcoholism, etc. Liver tissue sections were evaluated by blind SAF scoring. This study was approved by the Ethics Committee of the hospital.

[0030] Experimental animals: In this study, 6-8-week-old healthy male C57BL / 6J wild-type mice (18-24 g) were used. All mice were raised in a SPF environment (22℃, 12h light-dark cycle) at the Experimental Animal Center of Xi'an Jiaotong University. The experiments were conducted in accordance with the Chinese experimental animal ethics standards and were approved by the Medical Ethics Committee of the school. All mice were euthanized after isoflurane anesthesia.

[0031] Construction of animal models: The wild-type mice were divided into two groups. At 6 weeks of age, the mice were injected with UBC9 adeno-associated virus (80 μL, 5×10 13 / VG / ml, provided by Shanghai Jikai Company) or control virus via the tail vein. Liver tissue was collected two weeks after injection. After confirming the effect of the virus, the UBC9 overexpression group and the control group were fed a high-fat diet (HFD, 60% kcal) or a control diet (CD) for 12 weeks from 8 weeks of age.

[0032] HE staining of tissues: Mouse liver tissue was fixed with 4% paraformaldehyde for 24 hours (4℃), then dehydrated with gradient alcohol (70%→100%) for 1 hour each, cleared with xylene, and embedded in paraffin. Sections were cut to 4-5 μm and mounted for drying. After dewaxing and rehydrating, the sections were stained with hematoxylin for 3-5 minutes, differentiated with hydrochloric acid alcohol, and returned to blue with running water. Then, the sections were re-stained with eosin for 1-2 minutes, dehydrated with gradient alcohol, cleared with xylene, and mounted with neutral resin for microscopic examination. The cell nucleus was blue, and the cytoplasm was pink.

[0033] Oil red O staining of tissues: Fresh mouse liver tissue was quickly frozen in liquid nitrogen to prepare 5-8 μm frozen sections. After 4% paraformaldehyde fixation for 10 minutes, the sections were washed with running water and 60% isopropanol. Under dark conditions, the sections were stained with freshly prepared oil red O working solution (3:2) at 37℃ for 15-20 minutes, differentiated with 60% isopropanol for 10 seconds, re-stained with hematoxylin for 1 minute and returned to blue with running water. The sections were mounted with glycerol gelatin for microscopic examination. The lipid droplets were bright red, and the cell nucleus was blue.

[0034] Immunohistochemical staining: Paraffin sections were deparaffinated with xylene, rehydrated with gradient alcohol, and then subjected to heat-induced antigen retrieval (sodium citrate buffer, pH 6.0). Endogenous peroxidase was blocked with 3% H2O2. After blocking with 5% BSA for 30 min at room temperature, the sections were incubated with mouse anti-UBC9 (1:50) primary antibody at 4°C overnight, and then incubated with HRP-labeled secondary antibody (1:200) for 1 h at room temperature. The sections were developed with DAB, counterstained with hematoxylin, mounted with neutral resin, and observed under an optical microscope (positive signals were brown, and the cell nuclei were blue).

[0035] HepG2 cell culture and treatment: HepG2 cells were routinely cultured in DMEM high-glucose medium containing 10% fetal bovine serum (FBS) and 1% double-antibiotic (penicillin-streptomycin) in a 37°C, 5% CO2 incubator, and subcultured (1:3-1:5) every 2-3 days by trypsinization at a concentration of 0.25%. The cells were maintained in the logarithmic growth phase. When the cells reached 60% density, 0.25 mM palmitate was added for 24 hours, and the same volume of DMSO was added to the control group.

[0036] HepG2 cell lentivirus transfection: HepG2 cells in the logarithmic growth phase were seeded in culture plates at a density of 60%-70%, and lentivirus containing UBC9 overexpression (MOI=10) and 6-8 μg / ml polybrene were added. After incubation at 37°C for 24 hours, the medium was replaced with fresh medium, and the cells were cultured for another 48 hours. Stable transfectants were obtained by puromycin selection for 7-10 days, and the expression levels of UBC9 protein / mRNA were verified by Western Blot or qPCR.

[0037] Cell oil red O staining: HepG2 cell slides were fixed with 4% paraformaldehyde for 10 minutes, rinsed with 60% isopropanol for 1 minute, and then stained with freshly prepared oil red O working solution (oil red O saturated solution:water=3:2) at 37°C for 15 minutes in the dark. After differentiation with 60% isopropanol for 10 seconds, the nuclei were counterstained with hematoxylin for 1 minute, and then returned to blue by flowing water. The slides were mounted with glycerol gelatin, and observed under a microscope. Lipid droplets were bright red, and cell nuclei were blue.

[0038] Western Blot detection of proteins: Proteins were extracted from liver tissues or cell samples using RIPA lysis buffer and quantified by BCA method. Equal amounts of protein were subjected to SDS-PAGE electrophoresis (80V for concentrated gel and 120V for separation gel), and then wet-transferred to a PVDF membrane. After blocking with 5% skim milk for 1 hour, the membrane was incubated with primary antibody (4°C overnight) and HRP-labeled secondary antibody (room temperature for 1 hour), respectively. The expression levels of target proteins were calibrated by β-actin / GAPDH internal controls after ECL development. Key steps required low-temperature operation throughout, and the antibody concentration and exposure time needed to be controlled.

[0039] qPCR detection: Take the mouse liver tissue or cell samples, extract total RNA with TRIzol and detect purity (A260 / A280≈2.0), after reverse transcription to cDNA, use SYBR Green premix and specific primers (internal control gene uses β-actin) to prepare the reaction system, and perform amplification in the real-time fluorescent quantitative PCR instrument (95°C pre-denaturation for 30 seconds→40 cycles: 95°C denaturation for 5 seconds, 60°C annealing / elongation for 30 seconds), verify the specificity of the primers by melting curve, and calculate the relative expression of the target gene by ΔΔCt method. Cell proteomics detection: Take UBC9 stable overexpression and control HepG2 cells (3 biological replicates), lyse and extract total protein, then quantitate by BCA method, trypsinize into peptides, and use liquid chromatography-mass spectrometry (LC-MS / MS) technology for full spectrum analysis, identify differential proteins by MaxQuant software against UniProt database (screening criteria: Fold Change>1.5 and p<0.05), and perform functional annotation and pathway enrichment by GO, KEGG and other bioinformatics tools, focus on screening SUMOylation modification and lipid metabolism related targets, and verify the expression trend of key differential proteins by Western Blot. Mouse blood lipid detection: After the mouse is anesthetized, blood is collected from the orbital venous plexus, and the blood sample is allowed to stand for 30 minutes before centrifugation (3000xg for 15 minutes at 4°C) to separate the supernatant. It is stored at 4°C for a short period or frozen at -80°C for a long period (avoid repeated freezing and thawing). When detecting, use animal-specific kits, and quantify by colorimetry (500nm for TG, 450nm for TC) and standard comparison

[0040] Experimental results:

[0041] (1) The overall SUMOylation of cells is significantly reduced in fatty liver models.

[0042] As shown in Figure 3 : The SUMO1 and SUMO2 / 3 protein levels of liver samples of clinical MASLD patients are significantly reduced compared with the healthy control group; as shown in Figure 4 : The SUMOylation key enzymes UBC9, SUMO1 and SUMO2 / 3 protein levels of liver samples of HFD high-fat fed mice for 12 weeks are significantly reduced compared with the control diet mice; as shown in Figure 6 : The UBC9, SUMO1 and SUMO2 / 3 protein levels of HepG2 cell lines gradually decrease with the increase of palmitate (PA) treatment time compared with the control (DMSO) group.

[0043] (2) Overexpression of UBC9 in mouse liver using adeno-associated virus can increase the overall SUMOylation level of liver and reduce liver lipid deposition after high-fat feeding.

[0044] As shown inFigure 7 As shown: After AAV2 adeno-associated virus was injected into the tail vein of mice, the UBC9 protein in the mouse liver was significantly overexpressed compared with the control group, and the levels of SUMO1 and SUMO2 / 3 proteins in the liver were significantly increased; Figure 8-10 As shown in the figure, after 12 weeks of HFD feeding, the liver lipid deposition in the UBC9 overexpression group was significantly reduced compared with the control group ( Figure 8 ),serum( Figure 9 ) and liver( Figure 10 )Triglyceride and total cholesterol levels were also significantly reduced.

[0045] (3) Overexpression of UBC9 in human HepG2 cells using lentivirus increased the overall SUMOylation level in the liver and reduced cellular lipid deposition after palmitate treatment.

[0046] like Figure 11 As shown: After HepG2 cell line was transfected with lentivirus, UBC9 protein was significantly overexpressed compared with the control group, and the levels of SUMO1 and SUMO2 / 3 proteins in the cells were significantly increased; Figure 12 As shown in the figure, after 24 hours of palmitate (PA) treatment, the lipid deposition in the HepG2 cells UBC9 overexpression group was significantly reduced compared with the control group.

[0047] (IV) Proteomics showed that UBC9 was significantly associated with cellular lipid metabolism pathways.

[0048] like Figure 13 As shown in &14, HepG2 cell lines were transfected with lentivirus and proteomic analysis was performed, and GO and KEGG analysis were performed on the differential proteins. The results showed that a large number of lipid metabolism-related pathway proteins were significantly different after UBC9 overexpression.

Claims

1. Use of a UBC9 gene overexpression preparation in the preparation of a medicament for treating or preventing metabolic dysfunction-related fatty liver disease.

2. Use according to claim 1, characterized in that: The preparation inhibits liver lipid deposition and blood lipid levels by up-regulating UBC9 expression and enhancing SUMOylation modification levels.

3. The use according to claim 1, wherein The UBC9 gene overexpression preparation comprises a recombinant expression vector carrying a UBC9 gene coding sequence, and the recombinant expression vector is an adeno-associated virus (AAV) or a lentivirus.

4. Use according to claim 3, wherein the compound is ###0002### The adeno-associated virus is an adeno-associated virus type 2 (AAV2), and comprises a liver-specific promoter ApoE / hAAT promoter.

5. The use according to claim 3, wherein the compound is ###0002### The lentivirus vector uses a cytomegalovirus promoter (CMV promoter).

6. The use according to claim 1, characterized in that The medicament is used for reducing liver lipid deposition, reducing blood lipid levels, or improving abnormal liver lipid metabolism.

7. The use according to claim 1, characterized in that The medicament is delivered to the liver by intravenous injection or local targeted delivery.

8. The use according to claim 1, wherein The medicament takes the UBC9 gene overexpression vector as an active ingredient, and is combined with a pharmaceutically acceptable excipient to form an injection, a sustained-release preparation, or a targeted nano-preparation.

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