Medical application of UBC9 in preventing or treating non-alcoholic fatty liver disease

Abstract

The invention provides medical application of UBC9 protein in preventing or treating non-alcoholic fatty liver disease, and relates to the technical field of biological medicine. According to the present invention, the results of a large number of experiments prove that the expression of the UBC9 in the liver tissue of the patient with the non-alcoholic steatohepatitis (NASH) is reduced, such that the UBC9 expression can be reduced, and the application of the UBC9 in the liver tissue of the patient with the non-alcoholic steatohepatitis (NASH) can be provided; the UBC9 liver specific knock mouse aggravates the liver lipid aggregation, inflammation and fibrosis of the NAFLD; on the contrary, the liver lipid aggregation, inflammation and fibrosis of the NAFLD are improved by the UBC9 overexpression mouse. The UBC9 is expected to become a new action target spot for treating the non-alcoholic fatty liver disease. The invention can be used in preparation of products for diagnosis and treatment related to the non-alcoholic fatty liver disease, and has certain clinical transformation prospects.

Description

Technical Field

[0001] The present invention relates to the field of biomedical applications, and specifically relates to the use of ubiquitin conjugating enzyme E2I (UBC9) protein, mainly to the medical use of UBC9 protein for preventing or treating non-alcoholic fatty liver disease, and specifically to the use of UBC9 protein or its active fragments in the preparation of drugs for preventing or treating non-alcoholic fatty liver disease. Background Art

[0002] Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease in the world and one of the main causes of end-stage liver disease. The current prevalence of NAFLD in China is 29.2%, and it is expected to become the country with the highest number of NAFLD patients and liver-related deaths in the world, posing a serious threat to national health and social development. As the prevalence of NAFLD increases, the medical and economic burden it imposes on society also increases. The primary goal of treating NAFLD is to lose weight and improve insulin resistance, prevent and treat metabolic syndrome and other complications, thereby reducing the burden of the disease, improving the quality of life of patients and prolonging life; the secondary goal is to reduce liver fat deposition, prevent the progression of liver disease, and reduce the occurrence of cirrhosis, hepatocellular carcinoma and their complications. At present, the drug treatment strategy for NAFLD is mainly aimed at symptomatic treatment of diseases such as obesity, hypertension, type 2 diabetes, dyslipidemia, gout, etc., but there is currently no recognized liver-protecting drug that can be recommended for the routine treatment of NAFLD. Therefore, it is urgent to find new mechanisms and new targets to prevent the progression of NAFLD and develop safe and effective treatment options.

[0003] Ubiquitin conjugating enzyme E2I (UBC9) is a small molecule protein that is mainly localized in the cytoplasm, nucleus and mitochondria in cells. The known functions of UBC9 are mainly in two aspects. One is that it is the only E2 conjugating enzyme in the SUMO modification process, responsible for transferring SUMO protein to the substrate; the other is that it participates in the ubiquitin-dependent proteasome degradation pathway to maintain protein stability. SUMO modification is widely present in a variety of tissues and the protein sequence is highly conserved. It has a clear regulatory effect on cell differentiation and proliferation, transcriptional regulation, protein stability, etc. UBC9 fat-specific knockout can cause adipose tissue inflammation and abnormal lipid accumulation in the liver, leading to hyperinsulinemia and hepatic steatosis; UBC9-specific knockout in pancreatic β cells can affect glucose and lipid metabolism through oxidative stress, ultimately leading to diabetes. Previous studies of our research group also found that the transcription of UBC9 was inhibited by the activation of AMPKα2, resulting in the inhibition of SUMO modification of the fatty acid receptor GPR120, which in turn affects the inflammatory response and lipid deposition in the arteries. The above research results show that UBC9 plays an important role in regulating glucose and lipid metabolism, suggesting that UBC9 may be closely related to the occurrence and development of NAFLD. Summary of the invention

[0004] In view of the deficiencies in the prior art, the object of the present invention is to provide a method for preventing or treating non-alcoholic fatty liver disease using UBC9 protein.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions.

[0006] The invention discloses application of a reagent for overexpressing UBC9 in preparing a medicine for non-alcoholic fatty liver disease.

[0007] Furthermore, the reagent includes a vector overexpressing UBC9, nanoparticles carrying UBC9, protein microspheres encapsulating UBC9, liposomes encapsulating UBC9, or any combination thereof.

[0008] Furthermore, the vector includes a DNA plasmid vector, a lentiviral vector, a retroviral vector, a poxvirus vector, a herpes simplex virus vector, an adenovirus vector, an adeno-associated virus vector, a liposome bound to a DNA plasmid, a molecular couple bound to a DNA plasmid and / or a polymer bound to a DNA plasmid.

[0009] Furthermore, the drug also includes a pharmaceutically acceptable carrier.

[0010] The present invention also discloses the use of UBC9 protein in preparing a drug for treating non-alcoholic fatty liver disease.

[0011] Furthermore, the drug contains the UBC9 gene or the protein encoded by the UBC9 gene.

[0012] Furthermore, the drug also includes a pharmaceutically acceptable carrier.

[0013] The present invention also discloses the use of a detection reagent for the expression level of the BC9 gene or its encoded protein in the preparation of an auxiliary diagnosis product for non-alcoholic fatty liver disease.

[0014] Furthermore, the product detects the expression level of the UBC9 gene or its encoded protein in a sample by reverse transcription PCR, real-time quantitative PCR, chip, high-throughput sequencing platform, immunohistochemical staining or enzyme-linked immunosorbent assay.

[0015] Furthermore, the product is a chip, a preparation or a kit; and the sample is a tissue, serum or a cell.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows: The inventors have proved through a large number of experiments that UBC9 expression is decreased in the liver tissue of patients with nonalcoholic steatohepatitis (NASH). In the mouse NAFLD model established by the method of methionine-choline deficiency diet (MCD), UBC9 expression in liver tissue was significantly downregulated. UBC9 liver knockout mice aggravate liver lipid aggregation, inflammation and fibrosis of NAFLD; conversely, UBC9 overexpression mice improve liver lipid aggregation, inflammation and fibrosis of NAFLD. At the cytological level, it was found that reduced UBC9 expression can aggravate lipid aggregation, inflammation and fibrosis of mouse liver cells caused by palmitic acid (PA); conversely, overexpression of UBC9 inhibits lipid aggregation, inflammation and fibrosis of mouse liver cells caused by PA. The above results show that UBC9 plays an important regulatory role in nonalcoholic fatty liver disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1UBC9 expression is downregulated in the non-alcoholic fatty liver disease model. Figure A is the immunohistochemical staining of the liver of the control group and the nonalcoholic steatohepatitis (NASH) patient group (scale bar = 20 μm); Figure B is the immunohistochemical staining of the liver of the control group and the high-fat diet (HFD) group mice (scale bar = 20 μm); Figure C is the immunohistochemical staining of the liver of the control group and the MCD group mice (scale bar = 20 μm); Figure D is the Western blot result of the liver of the control group and the HFD group mice; Figure E is the Real-time PCR result of the liver of the control group and the HFD group mice; Figure F is the Western blot result of the liver of the control group and the MCD group mice; Figure G is the Real-time PCR result of the liver of the control group and the MCD group mice; Figure H is the Western blot result of the liver cells of the control group and the oleic acid (OA) group; Figure I is the Real-time PCR result of the liver cells of the control group and the OA group; Figure J is the Western blot result of the liver cells of the control group and the palmitic acid (PA) group; Figure K is the Real-time PCR results; (n=3, *p<0.05, **p<0.01 vs.con / ND group).

[0018] Figure 2 Liver-specific knockout of UBC9 exacerbates MCD diet-induced non-alcoholic fatty liver disease. Figures A and B are the liver weight and liver weight to body weight ratio of UBC9 liver-specific knockout mice (UBC9-LKO) and their littermate control mice (UBC9-flox); Figures C and D are statistical graphs of triglyceride and total cholesterol levels in the plasma of UBC9-LKO mice and their littermate control mice; Figure E is a statistical graph of aspartate aminotransferase and alanine aminotransferase levels in the plasma of UBC9-LKO mice and their littermate control mice; Figure F is HE, oil red staining and NAS scores of the liver of UBC9-LKO mice and their littermate control mice (scale bar=20μm); Figures G and H are Western blot results and statistical graphs of UBC9-LKO mice and their littermate control mice; Figures I and J are immunohistochemistry and picrosirius red staining of the liver of UBC9-LKO mice and their littermate control mice (scale bar=20μm); Figures K and L are Real-time chromatographic analysis of liver tissues of UBC9-LKO mice and their littermate control mice PCR results (n=3-6, *p<0.05, **p<0.01 vs. UBC9-flox ND group; # p<0.05, ##p<0.01 vs. UBC9-flox MCD group; UBC9-flox: littermate control mice; UBC9-LKO: UBC9 liver-specific knockout mice).

[0019] Figure 3 UBC9 liver overexpression alleviates non-alcoholic fatty liver disease caused by MCD diet. Figures A and B are liver weight and liver weight to body weight ratio of UBC9 overexpressing mice (AAV-UBC9) and their littermates (AAV-con) mice; Figures C and D are statistical graphs of triglyceride and total cholesterol levels in plasma of AAV-UBC9 mice and AAV-con mice; Figure E is a statistical graph of aspartate aminotransferase and alanine aminotransferase levels in plasma of AAV-UBC9 mice and AAV-con mice; Figure F is HE, oil red staining and NAS scores of livers of AAV-UBC9 mice and AAV-con mice (scale bar = 20μm); Figures G and H are Western Blot results and statistical graphs; Figures I and J are immunohistochemistry and picrosirius red staining of liver tissues of AAV-UBC9 mice and AAV-con mice (scalebar=20μm); Figures K and L are Real-time PCR results of liver tissues of AAV-UBC9 mice and AAV-con mice (n=3-6, *p<0.05, **p<0.01 vs. AAV-con ND group; # p<0.05, ## p<0.01 vs. AAV-conMCD group; AAV-con: littermate control mice; AAV-UBC9: UBC9 liver overexpressing mice).

[0020] Figure 4 Knockdown of UBC9 aggravates lipid accumulation, inflammation and fibrosis in liver cells caused by PA. Figure A is oil red staining of liver cells (scale bar=20μm); Figures B and C are Western blot results and statistical graphs; Figures D and E are Real-time PCR test results (n=3, *p<0.05, **p<0.01 vs. si-con group; # p<0.05, ## p<0.01 vs. si-con+PA, si-con: controlsi-RNA; si-UBC9: UBC9 si-RNA).

[0021] Figure 5Overexpression of UBC9 alleviates lipid accumulation, inflammation and fibrosis in liver cells caused by PA. Figure A is oil red staining of liver cells (scale bar=20μm); Figures B and C are Western blot results and statistical graphs; Figures D and E are Real-time PCR test results (n=3, *p<0.05, **p<0.01 vs. adcon group; # p<0.05, ## p<0.01 vs. adcon+PA, adcon: empty control adenovirus; adUBC9: UBC9 overexpression adenovirus). DETAILED DESCRIPTION

[0022] The embodiments of the present invention will be described in detail below in conjunction with the examples, but those skilled in the art will appreciate that the following examples are only used to illustrate the present invention and should not be considered to limit the scope of the present invention. If the specific conditions are not specified in the examples, they are carried out according to conventional conditions or the conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be purchased commercially. The experimental data of the present invention are all percentages, and the comparison of the two sample rates is applied with the chi-square test, and the statistical processing is all processed using the GraphPad Prism8.0 software package, with P<0.05 being considered statistically significant.

[0023] Example 1: UBC9 expression is downregulated in a non-alcoholic fatty liver disease model.

[0024] 1. Experimental animals and their husbandry.

[0025] Wild-type C57BL / 6J mice (8 weeks old, male) were purchased from Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd., China. The mice were randomly divided into groups, and all mice were housed in a specific pathogen free (SPF) environment with free access to food and water, humidity between 45% and 70%, temperature set between 20 and 24°C, and light cycled every 12 h.

[0026] 2. Establishment of non-alcoholic fatty liver disease model in mice.

[0027] C57BL / 6J mice were randomly divided into a control group and an experimental group. The mice in the experimental group were fed with an HFD diet (specific ingredients: protein, 20%; fat, 60%; carbohydrates, 20%) for 28 weeks to establish a mouse NAFLD model; and the mice in the experimental group were fed with an MCD diet (specific ingredients: protein, 14%; fat, 42%; carbohydrates, 44%; but lacking methionine and choline) for 8 weeks to establish a mouse non-alcoholic steatohepatitis (NASH) model. The mice in the control group were fed with a normal (ND) diet (specific ingredients: protein, 18.3%; fat, 10.2%; carbohydrates, 71.5%) for 8 weeks. HFD, MCD and ND were purchased from Jiangsu Collaborative Pharmaceutical Bioengineering Co., Ltd.

[0028] 3. Immunohistochemical staining of liver.

[0029] The liver tissues of the experimental group and the control group were collected for immunohistochemical staining. The specific operation steps are as follows.

[0030] (1) Fixation: Place a piece of cleaned liver tissue into a 15 mL centrifuge tube containing paraformaldehyde and fix for 24 hours.

[0031] (2) Dehydration: Place the fixed liver in an embedding box and then place it in different concentrations of alcohol for gradient dehydration (70% alcohol for 1 hour, 80% alcohol for 1 hour, 95% alcohol for 1 hour, and 100% alcohol for 1 hour × 3 times).

[0032] (3) Embedding: Place the liver in an embedding box and add an appropriate amount of liquid paraffin. Place the embedding box on a cooling table to allow the paraffin to gradually cool into blocks.

[0033] (4) Sectioning: Place the embedding box into a paraffin slicer and continuously cut paraffin sections of the aorta cross section at a thickness of 4 μm. Carefully attach the cut paraffin sections to a glass slide and place them in a baking oven overnight.

[0034] (5) Dewaxing: Place the sections in xylene I for 10 min, xylene II for 10 min, anhydrous ethanol for 10 min, 95% ethanol for 10 min, 90% ethanol for 5 min, and 85% ethanol for 5 min, dewaxing step by step.

[0035] (6) Antigen repair: Soak the slices in antigen repair solution and boil at 100°C for 40 minutes, then cool to room temperature.

[0036] (7) Place the slices in a humidified chamber with the tissue side facing up. Add one drop or 50 μL of peroxidase blocker to each slice. Incubate at room temperature for 10 min, and then wash three times with PBS, each time for 10 min.

[0037] (8) Remove PBS and add one drop or 50 μL of goat serum to each section and incubate at room temperature for 30 min.

[0038] (9) Remove serum and add 50 μL UBC9 (1:100 dilution in PBS) to each section and incubate at 4°C overnight.

[0039] (10) Rewarm at room temperature for 30 min and wash three times with PBS, each time for 10 min.

[0040] (11) Add 50 μL of reagent C solution (secondary antibody) to the paraffin sections (diluted 1:100 with PBS) and incubate at room temperature for 10 min. Rinse with PBS three times, 5 min each time.

[0041] (12) Add 50 μL of Reagent D solution onto the paraffin sections and incubate at room temperature for 10 min. Rinse with PBS three times, 5 min each time.

[0042] (13) Remove PBS and add 50 μL DAB colorimetric solution to each section. DAB colorimetric solution was diluted 20 times.

[0043] (14) Counterstain the cell nuclei with hematoxylin solution.

[0044] (15) Place the paraffin sections in 1% hydrochloric acid for 30 seconds for differentiation and then rinse with running water.

[0045] (16) Place the paraffin sections in ammonia water for 30 seconds to allow the cell nuclei to turn blue, and then rinse with running water.

[0046] (17) Dehydrate, transparentize with xylene, and seal with neutral gum.

[0047] (18) Observe the staining results under an upright microscope and take photos to save the images.

[0048] The results showed that compared with the con group, the expression of UBC9 in the liver of the NASH patient group was significantly decreased ( Figure 1 A); Compared with the ND group mice, the expression of UBC9 in the liver tissue of the HFD group mice was significantly decreased ( Figure 1 B); Compared with the ND group mice, the expression of UBC9 in the liver tissue of the MCD group mice was significantly decreased ( Figure 1 C).

[0049] 4. The Western blot method was used to detect the expression of UBC9 in liver tissue in the non-alcoholic fatty liver disease model.

[0050] In order to clarify the role of UBC9 in non-alcoholic fatty liver disease, the mouse non-alcoholic fatty liver disease model was established by HFD or MCD feeding, and the cellular model of non-alcoholic fatty liver disease was established by PA and OA stimulation of liver cells. The expression of UBC9 in liver tissue and liver cells was detected by western blot. Tissues and cells of the experimental group and the control group were collected respectively, and appropriate amounts of protein lysate were added. Lysis was carried out on ice for 30 minutes, and the mixture was mixed every 5 minutes. Centrifugation was performed at 4℃, 12000g for 20 minutes, and the supernatant was collected as the total cell protein. The protein concentration in the lysate was determined by BCA colorimetric kit. After sufficient mixing, it was boiled for 5 minutes in a 100℃ water bath, and centrifuged at 12000g for 30 seconds at room temperature. 20μg of protein sample was added to each sample well, the power was turned on, and electrophoresis was started. The following voltage and time were used: 100V voltage, 30min, 120V voltage, 60min, and the power was turned off after bromophenol blue electrophoresis reached the bottom of the glass plate. The samples were transferred to PVDF membrane at 90V for 2h. The PVDF membrane was placed in 5% milk blocking solution for 1h and then incubated overnight at 4℃ with primary antibody. 1:1000 anti-UBC9 (Abcam, USA) antibody, 1:1000 anti-CD36 (CST, USA) antibody, 1:1000 anti-PPARγ (CST, USA) antibody, 1:1000 anti-FASN (CST, USA) antibody, 1:1000 anti-GAPDH (CST, USA) antibody were used as primary antibodies, and horseradish peroxidase-labeled goat anti-mouse (or anti-rabbit) antibody (CST, USA) was used as secondary antibody for Western blot detection, and ECL kit (Amersham, USA) was used for luminescence development. ImageJ 1.51 software was used to measure the gray value of the bands and perform statistical analysis.

[0051] The results showed that in the HFD-induced non-alcoholic fatty liver disease model of mice, the expression of fatty acid metabolism indicators CD36, PPARγ, and FASN increased significantly; the expression of UBC9 protein decreased. Figure 1 D. In the mouse model of non-alcoholic fatty liver disease established by MCD, the expression of fatty acid metabolism indicators CD36, PPARγ, and FASN increased significantly; the expression of UBC9 protein decreased. The results are shown in Figure 1 F. In the liver cell non-alcoholic fatty liver disease model established by OA, the expression of fatty acid metabolism indicators CD36, PPARγ, and FASN increased significantly; the expression of UBC9 protein decreased. The results are shown in Figure 1 H. In the PA-established liver cell non-alcoholic fatty liver disease model, the expression of fatty acid metabolism indicators CD36, PPARγ, and FASN increased significantly; the expression of UBC9 protein decreased. The results are shown in Figure 1 J.

[0052] 5. The fluorescence real-time quantitative PCR (Real-time PCR) method was used to detect the expression of UBC9 in the non-alcoholic fatty liver disease model.

[0053] (1) RNA extraction.

[0054] TRIzol was used to extract total RNA from tissues and cells. 1 mL TRIzol reagent was added to every 100 mg of fresh tissue. 7 Add 1 mL of TRIzol reagent per cell.

[0055] A. Pre-cool centrifuge at 4℃.

[0056] B. Place tissue or cell lysate in 1.5 mL of RNase-free medium and place on ice for 5 minutes.

[0057] C. Add 200 μL of chloroform to the EP tube containing the cells or tissue homogenate, mix thoroughly by inverting, and place on ice for 10 min. Centrifuge at 12,000 rpm for 15 min at 4°C.

[0058] D. Take a new EP tube and add 500μL of pre-cooled isopropanol. Transfer the upper aqueous phase (about 500μL) to the new EP tube. Mix thoroughly by inverting the tube and place on ice for 10 minutes.

[0059] E. Centrifuge at 12000 rpm for 10 min at 4°C.

[0060] F. Remove the supernatant. Add 1 mL of pre-cooled 75% ethanol to wash the RNA precipitate once. Centrifuge at 12,000 rpm for 5 minutes at 4°C.

[0061] G. Discard the supernatant and let it air dry.

[0062] H. Add 20 μL of enzyme-free water to dissolve the precipitate, and then test the RNA concentration and purity before conducting subsequent experiments.

[0063] (2) Reverse transcription reaction: Use Takara reverse transcription kit.

[0064] A. Removal of genomic DNA .

[0065] B. Reverse transcription reaction .

[0066] Reaction conditions: 37°C, 15 min; 85°C, 5 s.

[0067] (3) Primer sequence .

[0068] (4) Fluorescence quantitative PCR .

[0069] According to the volume in the table above, prepare 20 μL qPCR reaction system, and set up 3 replicate wells for each group of samples. Reaction conditions: 95℃5min, (95℃5s, 60℃30s, 72℃30s)×40 cycles, 72℃5s, 95℃15s. －△△Ct The expression of each sample was analyzed.

[0070] The results showed that in the HFD-induced nonalcoholic fatty liver disease model in mice, the transcriptional expression of UBC9 was reduced. Figure 1 E. In the mouse model of non-alcoholic fatty liver disease established by MCD, the expression of UBC9 transcription level was reduced, as shown in Figure 1 G. In the OA-induced nonalcoholic fatty liver disease model, the expression of UBC9 transcription level was reduced. Figure 1 I. In the PA-established liver cell model of non-alcoholic fatty liver disease, the expression of UBC9 transcription level was reduced, as shown in Figure 1 K.

[0071] The above results all show that UBC9 expression is decreased in the mouse non-alcoholic fatty liver disease model, suggesting that UBC9 may be involved in the occurrence and development of non-alcoholic fatty liver disease.

[0072] Example 2: Liver-specific knockout of UBC9 aggravates MCD diet-induced non-alcoholic fatty liver disease.

[0073] 1.UBC9 liver-specific knockout mice (UBC9-LKO) and their corresponding littermate control mice (UBC9-flox) were provided by Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd., and the breeding conditions were the same as those in Example 1.

[0074] 2. Establishment of the non-alcoholic fatty liver disease model in UBC9-LKO mice and their corresponding littermate control mice.

[0075] UBC9-LKO mice and their corresponding littermate control mice were randomly divided into a control group and an experimental group, and fed with MCD feed for 8 weeks to construct a mouse non-alcoholic fatty liver disease model, and the specific method was the same as Example 1. The following indicators were measured at 8 weeks of MCD feeding to evaluate the degree of liver damage in non-alcoholic fatty liver disease.

[0076] (1) Mouse liver weight: At 8 weeks after modeling, the mice were killed and their livers were removed. The liver weight was measured and recorded using an electronic scale.

[0077] (2) Liver weight to body weight ratio: At 8 weeks of modeling, use an electronic scale to measure the weight of the mice, calculate the liver weight to body weight ratio and record it.

[0078] (3) Serum total triglycerides: Serum total triglycerides (TG) were measured using a full-wavelength enzyme-labeled chromatograph (Bio-Rad, USA) using a triglyceride (TG) assay kit (A110-1-1, Nanjing Jiancheng, China).

[0079] (4) Serum total cholesterol: Serum total cholesterol (TC) was measured using a full-wavelength enzyme-labeled chromatograph (Bio-Rad, USA) using a total cholesterol (TC) assay kit (A111-1-1, Nanjing Jiancheng, China).

[0080] (5) Aspartate aminotransferase: Serum aspartate aminotransferase (AST) levels were measured using a full-wavelength ELISA chromatograph (Bio-Rad, USA) using an aspartate aminotransferase (AST) test kit (C010-2-1, Nanjing Jiancheng, China).

[0081] (6) Alanine aminotransferase: Serum alanine aminotransferase (ALT) levels were measured using a full-wavelength ELISA chromatograph (Bio-Rad, USA) and an alanine aminotransferase (ALT) test kit (C009-2-1, Nanjing Jiancheng, China).

[0082] The results showed that compared with the UBC9-flox+ND group, the liver weight and liver-to-body weight ratio of the UBC9-flox+MCD group were increased. Compared with the UBC9-flox+MCD group, the liver-specific knockout of UBC9 in the UBC9-LKO+MCD group aggravated the MCD diet-induced liver weight increase and liver-to-body weight ratio. Figure 2 A, B; Compared with the UBC9-flox+ND group, the serum TC and TG levels in the UBC9-flox+MCD group increased. Compared with the UBC9-flox+MCD group, the serum TC and TG levels in the UBC9-LKO+MCD group increased. Figure 2 C, D; Compared with the UBC9-flox+ND group, the serum AST and ALT levels in the UBC9-flox+MCD group increased. Compared with the UBC9-flox+MCD group, the serum AST and ALT levels in the UBC9-LKO+MCD group increased. Figure 2 E.

[0083] 3. HE staining and oil red staining were used to detect the severity of liver fat accumulation.

[0084] The non-alcoholic fatty liver disease model of mice was established by continuous feeding of MCD diet for 8 weeks, and the specific method was the same as that in Example 1. The livers of the mice at 8 weeks of modeling were collected and HE staining was performed, and the specific steps were as follows.

[0085] (1) Dewaxing: Soak in xylene I, xylene II, anhydrous ethanol I, anhydrous ethanol II, 95% ethanol, and 80% ethanol for 15 min, 15 min, 10 min, 10 min, 5 min, and 5 min, respectively. Finally, wash with water for 1 to 2 min.

[0086] (2) Nuclear staining: Place the sections in a hematoxylin aqueous solution, then stain the nuclear tissue for 10 minutes and wash with water for 1 to 2 minutes.

[0087] (3) Differentiation: Soak in 1% hydrochloric acid ethanol solution for 30-60 seconds.

[0088] (4) Blueing: Place the paraffin sections in ammonia water for 30-60 seconds.

[0089] (5) Cytoplasmic staining: Soak in eosin staining solution for 10 minutes and wash with water for 1 to 2 minutes.

[0090] (6) Transparency: Soak in 80% alcohol, 90% alcohol, 100% alcohol I, and 100% alcohol II for 5 minutes each, and in xylene I and xylene II for 10 minutes each.

[0091] (7) Sealing: After the slices are air-dried, seal them with neutral resin.

[0092] The livers of mice at 8 weeks of modeling were collected and stained with oil red. The specific steps are as follows: (1) Take 5 μm frozen sections and dry them at room temperature overnight; (2) Oil Red O staining for 10 min; (3) Hematoxylin stains the nucleus for 2 minutes; (4) Differentiate with 1% hydrochloric acid and alcohol for 10 seconds, make the slide transparent and seal the slide.

[0093] The results showed that compared with the UBC9-flox+ND group, the UBC9-flox+MCD group had increased lipid accumulation in the liver. Compared with the UBC9-flox+MCD group, the UBC9-LKO+MCD group had more obvious lipid accumulation. Figure 2 F.

[0094] 4. Western blot and Real-time PCR methods were used to detect the expression of lipid metabolism indicators in the non-alcoholic fatty liver disease model of UBC9-LKO group mice and their littermates in the control group. The specific steps were the same as those in Example 1.

[0095] Primer sequences: .

[0096] The results showed that compared with the UBC9-flox+ND group, the protein and transcription levels of lipid metabolism indicators CD36, PPARγ and FASN in the UBC9-flox+MCD group were significantly increased. Compared with the UBC9-flox+MCD group, the protein and transcription levels of CD36, PPARγ and FASN in the UBC9-LKO+MCD group were significantly increased. Figure 2 G, H.

[0097] 5. Immunohistochemical staining was used to detect the degree of liver inflammatory infiltration.

[0098] The non-alcoholic fatty liver disease model of mice was established by continuous feeding of MCD diet for 8 weeks, and the specific method was the same as in Example 1. The livers of the mice at 8 weeks of modeling were collected and immunohistochemical staining was performed, and the specific steps were the same as in Example 1.

[0099] The results showed that compared with the UBC9-flox+ND group, the expression level of F4 / 80, an inflammatory infiltration index, was significantly increased in the UBC9-flox+MCD group; compared with the UBC9-flox+MCD group, the expression level of F4 / 80 in the UBC9-LKO+MCD group was more significantly increased. Figure 2 I.

[0100] 6. Sirius red staining was used to detect the degree of liver fibrosis.

[0101] The non-alcoholic fatty liver disease model of mice was established by continuous feeding of MCD diet for 8 weeks, and the specific method was the same as that in Example 1. The livers of the mice at 8 weeks of modeling were collected and stained with picrosirius red, and the specific steps were as follows.

[0102] (1) Dewaxing: The steps are the same as HE staining.

[0103] (2) Stain with iron hematoxylin for 5-10 minutes, then rinse with distilled water for 10-20 seconds.

[0104] (3) Rinse with running water for 5 minutes.

[0105] (4) Sirius red staining solution for 15-30 minutes.

[0106] (5) Rinse with running water.

[0107] (6) Transparency: The steps are the same as HE staining.

[0108] (7) Sealing: After the slices are air-dried, seal them with neutral resin.

[0109] The results showed that compared with the UBC9-flox+ND group, the liver fibrosis in the UBC9-flox+MCD group was increased; compared with the UBC9-flox+MCD group, the liver fibrosis level in the UBC9-LKO+MCD group was significantly increased. Figure 2 J.

[0110] 7. Real-time PCR method was used to detect the expression of inflammation and fibrosis indicators in the non-alcoholic fatty liver disease model of UBC9-LKO group mice and their littermate control group. The specific steps were the same as those in Example 1.

[0111] Primer sequences: .

[0112] The results showed that compared with the UBC9-flox+ND group, the liver inflammation indicators (IL-1β, IL-6, TNF-α) and fibrosis indicators (Col1a1, Col3a1, α-SMA were significantly increased) in the UBC9-flox+MCD group; compared with the UBC9-flox+MCD group, the liver inflammation indicators IL-1β, IL-6, TNF-α were significantly increased in the UBC9-LKO+MCD group; compared with the UBC9-flox+MCD group, the liver fibrosis indicators Col1a1, Col3a1, α-SMA were significantly increased in the UBC9-LKO+MCD group, the results are shown in Figure 2 K, L.

[0113] Example 3: UBC9 hepatic overexpression alleviates MCD diet-induced non-alcoholic fatty liver disease.

[0114] 1. UBC9 overexpression adeno-associated virus and control virus (OBiO Technology, Shanghai, China) were constructed. UBC9 overexpression virus (AAV-UBC9) and control virus (AAV-con) were injected into C57BL / 6J mice by tail vein injection to establish UBC9 overexpression mice and control mice. The injection volume of virus per mouse was 2×10 11 The breeding conditions were the same as those in Example 1.

[0115] 2. Establishment of non-alcoholic fatty liver disease model in AAV-UBC9 mice and their corresponding littermate control mice.

[0116] AAV-UBC9 mice and their corresponding littermate control group mice were randomly divided into a control group and an experimental group, and fed with MCD feed for 8 weeks to construct a mouse non-alcoholic fatty liver disease model. The specific method was the same as in Example 1.

[0117] The following indices were measured at 8 weeks of MCD feeding: mouse liver weight, liver weight to body weight ratio, serum total triglyceride, serum total cholesterol, aspartate aminotransferase, and alanine aminotransferase. The specific method was the same as in Example 2.

[0118] The results showed that liver-specific overexpression of UBC9 attenuated the increase in liver weight induced by MCD diet and increased the liver-to-body weight ratio. Figure 3 A, B; UBC9 liver-specific overexpression alleviated the increase in serum TC and TG levels induced by MCD diet. Figure 3 C, D; Liver-specific overexpression of UBC9 alleviated the increase in AST and ALT levels induced by MCD diet. Figure 3 E.

[0119] 3. HE staining and oil red staining were used to detect the severity of liver fat accumulation.

[0120] The non-alcoholic fatty liver disease model of mice was established by continuous feeding of MCD diet for 8 weeks, and the specific method was the same as in Example 1. The livers of the mice at 8 weeks of modeling were collected and HE staining and oil red staining were performed, and the specific method was the same as in Example 2.

[0121] The results showed that liver-specific overexpression of UBC9 alleviated MCD diet-induced lipid accumulation. Figure 3 F.

[0122] 4. Western blot and Real-time PCR methods were used to detect the expression of lipid metabolism indicators in the non-alcoholic fatty liver disease model of AAV-UBC9 group mice and their littermates in the control group. The specific steps were the same as those in Example 2.

[0123] The results showed that compared with the AAV-con+MCD group, the lipid metabolism indexes CD36, PPARγ and FASN protein and transcription levels in the AAV-UBC9+MCD group were significantly reduced. Figure 3 G, H.

[0124] 5. Immunohistochemical staining was used to detect the degree of liver inflammatory infiltration.

[0125] The non-alcoholic fatty liver disease model of mice was established by continuous feeding of MCD diet for 8 weeks, and the specific method was the same as in Example 1. The livers of the mice at 8 weeks of modeling were collected and immunohistochemical staining was performed, and the specific steps were the same as in Example 1.

[0126] The results showed that compared with the mice in the AAV-con+MCD group, the expression level of F4 / 80, an inflammatory infiltration index, in the AAV-UBC9+MCD group was significantly reduced. Figure 3 I.

[0127] 6. Sirius red staining was used to detect the degree of liver fibrosis.

[0128] The non-alcoholic fatty liver disease model of mice was established by continuous feeding of MCD diet for 8 weeks, and the specific method was the same as that in Example 1. The livers of the mice at 8 weeks of modeling were collected and stained with picrosirius red, and the specific steps were the same as those in Example 2.

[0129] The results showed that compared with the AAV-con+MCD group, the liver fibrosis level in the AAV-UBC9+MCD group was significantly reduced. Figure 3 J.

[0130] 7. Real-time PCR method was used to detect the expression of inflammation and fibrosis indicators in the non-alcoholic fatty liver disease model of AAV-UBC9 group mice and their littermate control group. The specific steps were the same as those in Example 2.

[0131] The results showed that compared with the AAV-con+MCD group, the liver inflammation indicators IL-1β, IL-6, and TNF-α in the AAV-UBC9+MCD group were significantly reduced. Figure 3 K; Compared with the mice in the AAV-con+MCD group, the liver fibrosis indicators Col1a1, Col3a1, and α-SMA in the AAV-UBC9+MCD group were significantly reduced. Figure 3 L.

[0132] Example 4: Knockdown of UBC9 aggravates PA-induced lipid accumulation, inflammation and fibrosis in liver cells.

[0133] 1. Isolation and culture of primary mouse liver cells.

[0134] Primary hepatocytes were isolated from 8-week-old male C57BL / 6J mice using collagenase perfusion and gradient centrifugation. The mice were anesthetized and perfused twice with liver perfusion medium (17701-038, Thermo Fisher Scientific, Waltham, MA, USA) through the portal vein, followed by liver digestion medium (17701-034, Thermo Fisher Scientific, Waltham, MA, USA). Then, the liver of each mouse was excised and filtered through a 70 μm cell strainer, and the hepatocytes were separated by centrifugation at 50 × g twice and cultured at 37 °C with DMEM medium containing 20% ​​fetal bovine serum (growth medium).

[0135] 2. Construction of non-alcoholic fatty liver disease cell model using PA stimulation.

[0136] 300 μL of 2 mM PA stock solution was added to 1.7 mL of normal culture medium and primary liver cells were stimulated for 24 h to construct a non-alcoholic fatty liver disease cell model. The working concentration of PA was 0.4 mM. The control group was given an equal volume of PBS.

[0137] 3. Establishment of UBC9 knockdown mouse primary liver cell model.

[0138] (1) Primary liver cells were evenly seeded in a sterile culture dish.

[0139] (2) When the density reached 80%, primary liver cells were transfected with siRNA targeting UBC9 (Ribobio Biotechnology, China) and cultured in a 37°C constant temperature incubator. After 6 h of transfection, the culture medium was replaced with 20% serum and cultured for 24 h before proceeding to the next step of the experiment.

[0140] 4. Oil red staining was used to detect the severity of lipid aggregation in primary liver cells.

[0141] The primary liver cell non-alcoholic fatty liver disease cell model was established by using PA stimulation (0.4 mM) for 24 hours, and oil red staining was performed after 24 hours. The specific steps were the same as in Example 2.

[0142] The results showed that compared with the sicon group, lipid aggregation occurred in the liver cells of the sicon+PA group; compared with the sicon+PA group, lipid aggregation in the liver cells of the siUBC9+PA group was more obvious. Figure 4 A.

[0143] 5. Western blot was used to detect the effect of knocking down UBC9 on the expression of PA-induced hepatocyte lipid metabolism markers CD36, PPARγ and FASN. The specific method was the same as Example 1.

[0144] The results showed that compared with the sicon group, the expression of CD36, PPARγ and FASN in the sicon+PA group increased; compared with the sicon+PA group, the expression of CD36, PPARγ and FASN in the siUBC9+PA group increased more significantly. Figure 4 B.

[0145] 6. Real-time PCR was used to detect the effect of knocking down UBC9 on the expression of PA-induced hepatocyte lipid aggregation, inflammation and fibrosis indicators. The specific method was the same as Example 2.

[0146] The results showed that compared with the sicion group, the transcriptional levels of lipid metabolism indicators (CD36, PPARγ and FASN), inflammatory indicators (IL-1β, IL-6 and TNF-α) and fibrosis indicators (Col1a1, Col3a1 and α-SMA) in the sicon+PA group were increased. Compared with the sicon+PA group, the transcriptional levels of CD36, PPARγ and FASN in the siUBC9+PA group were higher; compared with the sicon+PA group, the transcriptional levels of inflammatory indicators IL-1β, IL-6 and TNF-α in the siUBC9+PA group were significantly increased; compared with the sicon+PA group, the transcriptional levels of fibrosis indicators Col1a1, Col3a1 and α-SMA in the siUBC9+PA group were significantly increased. Results are shown in Figure 4 CE.

[0147] Example 5: Overexpression of UBC9 alleviates PA-induced lipid accumulation, inflammation and fibrosis in liver cells.

[0148] 1. Establishment of a primary liver cell model of mice overexpressing UBC9.

[0149] (1) Primary liver cells were evenly seeded in a sterile culture dish.

[0150] (2) When the density reached 80%, the UBC9 adenovirus (Shanghai Heyuan Biotechnology Co., Ltd.) was used to infect primary liver cells (adUBC9). At the same time, the empty vector (adcon) adenovirus (Shanghai Heyuan Biotechnology Co., Ltd.) was used to infect primary liver cells as a control group.

[0151] 2. Oil red staining was used to detect the severity of lipid aggregation in primary liver cells.

[0152] The primary liver cell non-alcoholic fatty liver disease cell model was established by using PA stimulation (0.4 mM) for 24 hours, and oil red staining was performed after 24 hours. The specific steps were the same as in Example 2.

[0153] The results showed that overexpression of UBC9 alleviated PA-induced lipid aggregation in primary liver cells. Figure 5 A.

[0154] 3. Western blot was used to detect the effect of overexpression of UBC9 on the expression of lipid metabolism markers CD36, PPARγ and FASN in primary mouse liver cells. The specific method was the same as Example 1.

[0155] The results showed that compared with the adcon+PA group, the expression of CD36, PPARγ and FASN in the adUBC9+PA group was significantly decreased. Figure 5 B.

[0156] 4. Real-time PCR was used to detect the effect of overexpression of UBC9 on lipid aggregation, inflammation and fibrosis indices in the non-alcoholic fatty liver disease cell model. The specific method was the same as that in Example 2.

[0157] The results showed that compared with the adcon+PA group, the transcription levels of lipid metabolism indicators CD36, PPARγ and FASN in the adUBC9+PA group were significantly reduced; compared with the adcon+PA group, the transcription levels of inflammatory indicators IL-1β, IL-6 and TNF-α in the adUBC9+PA group were significantly reduced; compared with the adcon+PA group, the transcription levels of fibrosis indicators Col1a1, Col3a1 and α-SMA in the adUBC9+PA group were significantly reduced. Figure 5 CE.

[0158] The above description is only a preferred embodiment of the present invention and is not intended to limit the scope of the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. Use of reagents that overexpress UBC9 in the preparation of drugs for non-alcoholic fatty liver disease.

2. The use according to claim 1, characterized in that: The reagent includes a vector overexpressing UBC9, nanoparticles carrying UBC9, protein microspheres encapsulating UBC9, liposomes encapsulating UBC9, or any combination thereof.

3. The use according to claim 2, characterized in that: The vectors include DNA plasmid vectors, lentiviral vectors, retroviral vectors, poxvirus vectors, herpes simplex virus vectors, adenovirus vectors, adeno-associated virus vectors, liposomes combined with DNA plasmids, molecular couples combined with DNA plasmids and / or polymers combined with DNA plasmids.

4. The use according to claim 1, characterized in that: The medicament further includes a pharmaceutically acceptable carrier.

5. Application of UBC9 protein in the preparation of drugs for the treatment of non-alcoholic fatty liver disease.

6. The use according to claim 5, characterized in that: The drug contains the UBC9 gene or the protein encoded by the UBC9 gene.

7. The use according to claim 6, characterized in that: The medicament further includes a pharmaceutically acceptable carrier.

8. Use of a detection reagent for the expression level of the UBC9 gene or its encoded protein in the preparation of an auxiliary diagnosis product for non-alcoholic fatty liver disease.

9. The use according to claim 8, characterized in that: The product detects the expression level of the UBC9 gene or its encoded protein in a sample by reverse transcription PCR, real-time quantitative PCR, chip, high-throughput sequencing platform, immunohistochemical staining or enzyme-linked immunosorbent assay.

10. The application according to claim 8, characterized in that: The product is a chip, a preparation or a kit; the sample is a tissue, serum or a cell.

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