Use of Nrf2-ARE / hMAO-B / QR2 modulator 1 in the preparation of a medicament for treating non-alcoholic fatty liver

Abstract

The application belongs to the field of new use of medicines, and more particularly relates to application of Nrf2-ARE / hMAO-B / QR2 modulator 1 in preparation of medicines for treating non-alcoholic fatty liver. The compound is a multi-target ligand based on resveratrol, and can act on Nrf2-ARE, hMAO-B and QR2 signal pathways. Experimental results show that the compound can significantly reduce the body weight of NAFLD mice induced by high-fat diet, reduce liver lipid accumulation, improve insulin resistance, reduce blood sugar and liver enzyme levels, and relieve liver steatosis and vacuolization pathological changes. In addition, the mechanism of action is related to regulating Aldh3a2 gene expression and downstream target protein GPCPD1. The application first discloses application value of the compound in prevention and treatment of non-alcoholic fatty liver, and provides a new strategy for developing multi-target anti-non-alcoholic fatty liver medicines.

Description

Technical Field

[0001] This invention belongs to the field of novel uses of pharmaceuticals, and more specifically, relates to the use of Nrf2-ARE / hMAO-B / QR2 modulator 1 in the preparation of a drug for treating non-alcoholic fatty liver disease. Background Technology

[0002] Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disease worldwide, characterized by excessive fat accumulation in hepatocytes, unrelated to excessive alcohol intake. NAFLD has a broad spectrum, progressing from simple hepatic steatosis to nonalcoholic steatohepatitis (NASH), liver fibrosis, cirrhosis, and even hepatocellular carcinoma. Its pathogenesis is complex, closely related to multiple factors including insulin resistance, lipid metabolism disorders, oxidative stress, and genetic susceptibility. Currently, there are no approved drugs for the treatment of NAFLD globally. Clinical treatment mainly relies on lifestyle interventions (such as dietary control and increased physical activity) and the management of metabolic comorbidities (such as diabetes and hyperlipidemia), with limited efficacy for advanced stages. Summary of the Invention

[0003] The purpose of this invention is to provide the application of Nrf2-ARE / hMAO-B / QR2 modulator 1 in the preparation of drugs for treating non-alcoholic fatty liver disease.

[0004] This invention provides the application of Nrf2-ARE / hMAO-B / QR2 modulator 1 in the preparation of drugs for treating non-alcoholic fatty liver disease.

[0005] Nrf2-ARE / hMAO-B / QR2 modulator 1 is a resveratrol-based multi-target ligand that simultaneously acts on three signaling targets closely related to oxidative stress and neurodegenerative diseases: hMAO-B, NRF2, and QR2, thus exhibiting neuroprotective effects. The invention originated from the discovery that Aldh3a2 expression is decreased in NAFLD patients, and animal experiments confirmed that overexpression of Aldh3a2 can improve NAFLD symptoms and reduce hepatic lipid deposition and metabolic disorders. Further proteomic analysis indicated that GPCPD1 is a key downstream effector molecule of Aldh3a2. Based on molecular docking simulations, Nrf2-ARE / hMAO-B / QR2 modulator 1 was found to bind to the active site of the GPCPD1 protein, suggesting that this compound may exert its therapeutic effect by regulating the Aldh3a2-GPCPD1 axis. Subsequent animal experiments verified that the compound has multiple therapeutic effects in reducing hepatic steatosis, improving glucose and lipid metabolism and liver function in a high-fat diet-induced NAFLD model, ultimately establishing its novel use in the treatment of NAFLD.

[0006] Furthermore, the non-alcoholic fatty liver disease mentioned above refers to non-alcoholic fatty liver disease caused by a high-fat diet.

[0007] Furthermore, the Nrf2-ARE / hMAO-B / QR2 modulator 1 reduces the weight of patients with non-alcoholic fatty liver disease.

[0008] Furthermore, the Nrf2-ARE / hMAO-B / QR2 modulator 1 reduces abnormal lipid accumulation in the liver of patients with non-alcoholic fatty liver disease.

[0009] Furthermore, the Nrf2-ARE / hMAO-B / QR2 modulator 1 improves insulin resistance in patients with non-alcoholic fatty liver disease.

[0010] Furthermore, the Nrf2-ARE / hMAO-B / QR2 modulator 1 improves glucose tolerance in patients with non-alcoholic fatty liver disease.

[0011] Furthermore, the Nrf2-ARE / hMAO-B / QR2 modulator 1 reduces blood glucose levels in patients with non-alcoholic fatty liver disease.

[0012] The present invention also provides the use of the composition containing the above-described Nrf2-ARE / hMAO-B / QR2 modulator 1 in the preparation of a medicament for the prevention and / or treatment of non-alcoholic fatty liver disease.

[0013] Furthermore, the composition is a drug made with Nrf2-ARE / hMAO-B / QR2 modulator 1 as the active ingredient and pharmaceutically acceptable excipients.

[0014] The beneficial effects of this invention are as follows: Originality: This invention reveals for the first time a novel pharmaceutical use of compound Nrf2-ARE / hMAO-B / QR2 modulator 1 in the prevention and treatment of NAFLD, expanding the application field of this compound and providing new candidate molecules and therapeutic targets for drug development of NAFLD.

[0015] Synergistic effects across multiple targets: This compound works synergistically across multiple levels, including antioxidant, anti-inflammatory, and energy metabolism improvement, by simultaneously regulating three signaling axes: Nrf2-ARE, hMAO-B, and QR2. Targeting the complex pathogenesis of NAFLD, it is expected to produce therapeutic effects superior to single-target drugs.

[0016] The efficacy is clear: In vivo animal experiments have fully demonstrated that this compound can significantly reduce weight gain, liver weight and lipid accumulation in NAFLD mice induced by a high-fat diet, improve glucose tolerance and insulin resistance, alleviate hepatic steatosis and vacuolation, and effectively reduce hepatic triglyceride and total cholesterol levels, with significant pathological improvement effects. Attached Figure Description

[0017] Figure 1 The figures show the bioinformatics analysis of Aldh3a2. A represents the expression of Aldh3a2 in the population cohort gene expression profiling dataset GSE33814; B represents the expression of Aldh3a2 in the population cohort gene expression profiling dataset GSE15653; C represents the expression of Aldh3a2 in the population cohort gene expression profiling dataset GSE49541; D represents the expression of Aldh3a2 in the population cohort gene expression profiling dataset GSE126848; E represents the Western blot analysis of Aldh3a2 in the serum of patients with metabolic-associated fatty liver disease (NAFLD) and healthy subjects; and F represents the statistical graph of the Western blot analysis.

[0018] Figure 2 A schematic diagram of the experimental strategy for treating NAFLD model mice with AAV-Aldh3a2 (adeno-associated virus aldehyde dehydrogenase 3 family member A2).

[0019] Figure 3 This is a comparison chart of the body weights of mice in each group.

[0020] Figure 4The images show the appearance of mouse tissues. In A, the liver and adipose tissue of each group of mice are stained with hematoxylin and eosin (HE). In B, the weight of each tissue of each group of mice is shown.

[0021] Figure 5 Fasting plasma glucose (FPG) was compared among the groups of mice.

[0022] Figure 6 The graphs are statistical charts for mouse tolerance tests. A is the statistical chart of insulin tolerance test (ITT) for each group of mice, B is the statistical chart of area under the curve of insulin tolerance test, C is the statistical chart of glucose tolerance test (GTT) for each group of mice, and D is the statistical chart of area under the curve of glucose tolerance test.

[0023] Figure 7 The graphs show the serum triglyceride (TG) and total cholesterol (TC) levels of mice in each group. In the graph, A shows the serum triglyceride levels of mice in each group, and B shows the total cholesterol levels of mice in each group.

[0024] Figure 8 The graphs show the serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels in mice of each group. In the graphs, A represents the serum alanine aminotransferase levels in each group of mice, and B represents the serum aspartate aminotransferase levels.

[0025] Figure 9 Images of HE staining and Oil Red O staining of mouse livers in each group.

[0026] Figure 10 The graphs show the liver triglyceride (TG) and total cholesterol (TC) levels of mice in each group. In the graphs, A shows the liver triglyceride levels of mice in each group, and B shows the liver total cholesterol levels of mice in each group.

[0027] Figure 11 Heatmap of differentially expressed proteins in each group of mice for proteomics analysis.

[0028] Figure 12 This is a metabolic pathway diagram of GPCPD1's involvement in lipid synthesis.

[0029] Figure 13 The images show the Western blot images of GPCPD1 and Aldh3a2. In the image, A is the Western blot image of GPCPD1 in the tissues of each group of mice, and B is the grayscale analysis result of GPCPD1 in the tissues of each group of mice.

[0030] Figure 14 This is a three-dimensional structural analysis diagram of the GPCPD1 protein, where A is the three-dimensional structure diagram of the protein and B is a schematic diagram of the protein's amino acid pocket.

[0031] Figure 15This is a schematic diagram of the experimental strategy for treating NAFLD model mice with AAV-Nrf2-ARE / hMAO-B / QR2 modulator 1.

[0032] Figure 16 This is a comparison chart of the body weights of mice in each group.

[0033] Figure 17 The images show the tissue appearance of mice in each group. A shows the liver appearance and HE staining of adipose tissue in each group of mice, and B shows the comparison of total fat in each group of mice.

[0034] Figure 18 A graph showing the comparison of fasting plasma glucose (FPG) in mice from different groups.

[0035] Figure 19 The graphs show the results of insulin tolerance test and glucose tolerance test for each group of mice. In the graphs, A is the glucose tolerance test (GTT) graph for each group of mice, B is the area under the curve (AUC) graph for the glucose tolerance test, C is the insulin tolerance test (ITT) graph for each group of mice, and D is the area under the curve (AUC) graph for the insulin tolerance test.

[0036] Figure 20 The graphs show the serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels in mice of each group. In the graphs, A is the statistical graph of serum aspartate aminotransferase (AST) in mice of each group, and B is the statistical graph of serum alanine aminotransferase in mice of each group.

[0037] Figure 21 These are graphs showing the serum triglyceride (TG) and total cholesterol (TC) levels in each group of mice. Graph A shows the serum triglyceride levels in each group of mice, and graph B shows the serum total cholesterol levels in each group of mice.

[0038] Figure 22 Images of HE staining and Oil Red O staining of the livers of mice in each group.

[0039] Figure 23 The graphs show the liver triglyceride (TG) and total cholesterol (TC) levels of mice in each group. In the graph, A shows the liver triglyceride levels of mice in each group, and B shows the liver total cholesterol levels of mice in each group. Detailed Implementation

[0040] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments, but this should not be construed as limiting the invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following embodiments are commercially available unless otherwise specified.

[0041] Example 1: The expression level of Aldh3a2 is decreased in NAFLD patients.

[0042] Different datasets (GSE33814, GSE15653, GSE49541, and GSE126848) of NAFLD-related population cohorts were retrieved and analyzed from the database. Figure 1 As shown in Figures A-D, Aldh3a2 expression levels were decreased in patients with non-alcoholic steatohepatitis (NASH) and NAFLD compared to healthy individuals, and even lower in patients with advanced fibrosis compared to those with mild liver fibrosis. These results suggest that Aldh3a2 may be involved in the pathological process of NAFLD.

[0043] To further investigate the role of Aldh3a2 in NAFLD, blood samples were collected from a number of clinical samples, including healthy controls and NAFLD patients. Results were obtained using Western blotting. Figure 1 As shown in Figures E to F, serum Aldh3a2 levels in NAFLD patients were significantly lower than those in healthy subjects. These results suggest that decreased Aldh3a2 levels may be associated with the pathological progression of NAFLD.

[0044] Example 2: The effect of Aldh3a2 overexpression on NAFLD mice.

[0045] I. Experimental Materials Laboratory animals: Adult male C57BL / 6J mice, 5-6 weeks old, SPF grade, were housed at the Experimental Animal Center of Wenzhou Medical University at a room temperature of (22±1)℃ and a humidity of (50±10)℃, with free access to food and water. All experimental procedures were strictly in accordance with the "Guidelines for the Care and Use of Laboratory Animals" and approved by the Animal Ethics Committee of the Experimental Animal Center of Wenzhou Medical University.

[0046] Laboratory animal feed: 60% fat high-fat purified feed (purchased from Medison, catalog number MD12033).

[0047] II. Experimental Methods Mice were randomly divided into three groups: a normal diet group and a control group. Mice were fed a normal diet and received an injection of EGFP empty vector virus (1×10⁻⁶) via tail vein injection. 11 PFU / mouse (Cyagen Biotech, Inc.), labeled as AAV-GFP+ND group; high-fat diet feeding group, EGFP empty vector virus (1×10⁻⁶) was injected via tail vein injection. 11PFU / mouse (Cyagen Biotech, Inc.), labeled as the AAV-GFP+HFD group; the high-fat diet plus Aldh3a2 virus overexpression group, administered via tail vein injection of Aldh3a2 virus (1×10⁻⁶). 11 PFU / mouse (Cyagen Biosciences Co., Ltd.), labeled as the AAV-Aldh3a2+HFD group. After 3 weeks of viral adaptation, at 8 weeks of age, the AAV-GFP+ND group was fed a normal diet, while the other two groups were fed a high-fat diet. Mice were harvested at 22 weeks of age. Figure 2 As shown.

[0048] Before collection, mice were starved for 8 hours and weighed before anesthesia. Serum was collected from each mouse via orbital blood sampling. After standing at room temperature for 30 minutes, the serum was centrifuged at 3000 rpm for 10 minutes at 4°C, and the supernatant was collected, aliquoted, and stored at -80°C for subsequent detection of serum TG, TC, ALT, and AST levels. Other organs were removed, collected in cryovials, flash-frozen in liquid nitrogen, and stored at -80°C for later use. Liver sections were paraffin-embedded for subsequent HE staining. Frozen sections were embedded in OCT for Oil Red O staining, and Western blotting was used to detect the protein expression of GPCPD1 in the liver.

[0049] GTT and ITT assays in mice: In the GTT, mice were fasted for 12 h but allowed free water. Blood was collected from the tail tip, and fasting blood glucose concentration was measured using a Roche glucometer and glucose test strips via the glucose dehydrogenase method. Then, a glucose solution (2 g / kg body weight) was injected intraperitoneally. Blood was collected from the tail tip at 0 min, 30 min, 60 min, 90 min, and 120 min after injection to measure blood glucose concentration, and the area under the curve (AUC) was calculated. In the ITT, mice were fasted for 4 h but allowed free water. Blood was collected from the tail tip to measure blood glucose concentration. Then, insulin (0.75 U / kg body weight) was injected intraperitoneally. Blood was collected from the tail tip at 0 min, 15 min, 30 min, 60 min, and 90 min after injection to measure blood glucose concentration, and the AUC value was calculated.

[0050] Liver triglyceride (TG) and total liver cholesterol (TC) were measured according to the kit instructions. Serum triglyceride (TG), total serum cholesterol (TC), serum alanine aminotransferase (ALT), and serum aspartate aminotransferase (AST) were measured according to the kit instructions.

[0051] Western blot (WB) experiment: (1) Prepare tissue protein extraction solution: Mix protein tissue lysis buffer, PMSF, and phosphatase inhibitor in a ratio of 100:1:1; (2) Separate a small piece of tissue with scissors and place it in a 1.5 mL EP tube, add an appropriate amount of the prepared protein extraction solution, add 2 magnetic beads, and homogenize in a high-throughput homogenizer. Homogenize several times until there are no obvious tissue blocks. Lyse on ice for 30 min; (3) Centrifuge at 4℃, 12000 rpm for 15 min, and aspirate the supernatant into a new EP tube; (4) Use a BCA kit to determine the protein concentration; (5) Based on the determined protein concentration, take 30 μg of protein sample, add ddH2O to make up to 16 μL, add 4 μL of 5X loading, and boil in a 100℃ metal bath for 10 min to denature the protein. (6) Gel preparation: Place two clean glass plates in the gel preparation tank, check for leaks, add the prepared 10% separating gel, seal the liquid surface with ddH2O, let stand for 30 minutes, and after the separating gel solidifies, pour off the upper layer of ddH2O. Quickly add the prepared 5% stacking gel, insert the comb, pay attention to air bubbles, and let stand at room temperature for about 15 minutes; (7) Electrophoresis: Place the prepared gel in the electrophoresis tank, pour in the electrophoresis solution to cover the sample well, remove the comb, add the prepared sample and marker, maintain a constant voltage of 80V, and after the marker separates, increase the voltage to 120V for constant voltage electrophoresis; (8) Electrospinning: Prepare a PVDF membrane of the same size as the target gel and soak it in methanol for later use. After SDS-PAGE electrophoresis, remove the gel, cut the target gel, and then place it between the black and white plates in the order of sponge pad, filter paper, gel, membrane, filter paper, and sponge pad. After fixing, place it in the electrophoresis tank and perform membrane transfer under low temperature conditions. The membrane transfer conditions are constant current 280mA for 80min. (9) Blocking: After the membrane transfer is completed, place the membrane in 5% skim milk powder at room temperature for blocking and place it on a shaker for 90min. (10) Antibody incubation: Cut the band, add the corresponding primary antibody, and incubate overnight at 4℃ on a shaker. After recovering the primary antibody the next day, add TBST to wash 3 times, 5min each time, and then add rabbit secondary antibody. Incubate at room temperature for 60min, and wash 3 times again with TBST, 5min each time. (11) Exposure: Wipe the membrane slightly dry with paper, add exposure solution and incubate for about 1min, and expose with an exposure machine. The primary antibodies include: Rabbit Polyclonal ALDH3A2 Antibody (Thermo Fisher Scientific) and Rabbit Polyclonal GPCPD1 Antibody (ORIGENE).

[0052] Statistical analysis: SPSS version 24.0 (IBM, NY, USA) was used for statistical analysis, and GraphPadPrism version 8.0 (San Diego, California, USA) was used for statistical graphing. All statistical tests were two-tailed, and a p-value of 0.05 or less was considered statistically significant. Independent samples t-tests were used for comparisons between two groups at the same time point. One-way ANOVA was used for comparisons between groups.

[0053] III. Experimental Results From week 9 to week 22 (a total of 13 weeks) after intervention, mouse weight was measured weekly, and the weight of mice at 22 weeks of age was recorded. Figure 3 As shown, it can be observed that the AAV-GFP+HFD group had a faster rate of weight gain than the AAV-GFP+ND group, and the final weight was higher than that of the AAV-GFP+ND group. Compared with the AAV-GFP+HFD group, the AAV-Aldh3a2+HFD group had a slower rate of weight gain and a lower final weight.

[0054] The appearance of the livers of the mice in each group was then compared, such as... Figure 4 As shown, compared with the AAV-GFP+ND group, the liver of mice in the AAV-GFP+HFD group was significantly larger, but this increase was alleviated in the AAV-Aldh3a2+HFD group. Furthermore, observation of tissue weight revealed that the liver, white adipose tissue (WAT), subcutaneous adipose tissue (SAT), and brown adipose tissue (BAT) in the AAV-GFP+HFD group were significantly heavier than those in the AAV-GFP+ND group. Conversely, compared with the AAV-GFP+HFD group, the liver, WAT, and SAT were lighter in the AAV-Aldh3a2+HFD group. Additionally, HE staining of adipose tissue showed that the degree of vacuolation in the adipose tissue of the AAV-Aldh3a2+HFD group was significantly reduced compared to the AAV-GFP+HFD group. In conclusion, overexpression of Aldh3a2 can reduce NAFLD-induced weight gain, liver weight increase, and lipid accumulation.

[0055] Fasting blood glucose levels were measured in each group of mice, and it was found that, Figure 5 As shown, the blood glucose level of mice in the AAV-GFP+HFD group was higher than that in the AAV-GFP+ND group, but the blood glucose level of the AAV-Aldh3a2+HFD group was lower than that in the AAV-GFP+HFD group.

[0056] Subsequently, ITT and GTT were performed on mice in each group, and the area under the curve (AUC) was calculated. Figure 6As shown, insulin resistance was alleviated and glucose tolerance was improved in the AAV-Aldh3a2+HFD group.

[0057] Serum TG, TC, ALT, and AST levels in mice were detected using a kit. Compared to the AAV-GFP+ND group fed with a normal diet, the AAV-GFP+HFD group showed significantly elevated serum TG, TC, ALT, and AST levels. However, after Aldh3a2 overexpression, the increase in serum TC and ALT was alleviated, while TG and AST levels remained largely unchanged. Figure 7 and Figure 8 As shown.

[0058] The pathological changes in liver tissue observed under a microscope after HE staining and Oil Red O staining in each group of mice are as follows: Figure 9 As shown in the figure. HE staining results showed that, compared with the AAV-GFP+ND group, the liver tissue of mice in the AAV-GFP+HFD group exhibited extensive fatty degeneration, with round vacuoles of varying sizes visible in the cytoplasm and nuclear displacement. However, compared with the AAV-GFP+HFD group, lipid accumulation and fatty degeneration in the liver tissue of mice in the AAV-Aldh3a2+HFD group were significantly improved. Oil Red O staining results showed that diffuse orange-red lipid droplets were visible in the AAV-GFP+HFD group, while the area of ​​red lipid droplets was significantly reduced in the AAV-Aldh3a2+HFD group.

[0059] Further testing was conducted on the TG and TC levels in the livers of mice in each group, such as... Figure 10 As shown in the figure. The results showed that the liver TG level in the AAV-GFP+HFD group was significantly higher than that in the AAV-GFP+ND group, but the TC level did not change significantly. In contrast, the liver TC level in the AAV-Aldh3a2+HFD group was lower than that in the AAV-GFP+HFD group, while the TG level did not change significantly.

[0060] In summary, overexpression of Aldh3a2 can improve the hyperglycemia caused by NAFLD, alleviate insulin resistance, improve glucose tolerance, improve the degree of fat deposition and vacuolation in liver tissue, and alleviate abnormal liver function.

[0061] Proteomics analysis was performed on mice in each group, such as... Figure 11 As shown, the differentially expressed protein GPCPD1 was found. Figure 12 As shown, GPCPD1 participates in the hydrolysis of sn-glycerol-3-phosphocholine (GPC), and its products can enter the lipid synthesis pathway. Western blot analysis revealed that... Figure 13 As shown, the GPCPD1 protein level decreased in the AAV-GFP+HFD group, but significantly increased in the AAV-Aldh3a2+HFD group.

[0062] In summary, GPCPD1 is one of the key downstream effector molecules of Aldh3a2 and may mediate the ameliorative effect of Aldh3a2 on NAFLD.

[0063] Example 3: The effect of Nrf2-ARE / hMAO-B / QR2 modulator 1 on NAFLD mice.

[0064] Molecular docking simulation analysis of the three-dimensional structure of the GPCPD1 protein revealed that, for example... Figure 14 As shown, the compound Nrf2-ARE / hMAO-B / QR2 modulator 1 can effectively bind to a specific amino acid pocket in the GPCPD1 protein, i.e., a potential active site. This suggests that GPCPD1 may be a potential target for this compound.

[0065] I. Experimental Design Six-week-old male, wild-type C57BL / 6J mice were randomly divided into three groups: a control group fed a normal diet (WT), a high-fat diet-fed NAFLD group (WT+HFD), and a high-fat diet-fed group treated with Nrf2-ARE / hMAO-B / QR2 modulator 1 (WT+HFD+Nrf2-ARE / hMAO-B / QR2 modulator 1). The experimental period was 26 weeks. The compound was administered via gavage at a dose of 30 mg / kg / day, which was increased to 50 mg / kg / day from week 20. ITT and GTT were measured at weeks 25 and 26. Figure 15 As shown.

[0066] II. Experimental Detection Methods The method is the same as in Example 2. III. Experimental Results Measuring the weight of mice, such as Figure 16 As shown, it can be observed that the WT+HFD group had a faster rate of weight gain than the WT group, and the final weight was higher than that of the WT group. Compared with the WT+HFD group, the weight gain rate of the WT+HFD+Nrf2-ARE / hMAO-B / QR2 modulator 1 group was slowed down, and the final weight was reduced.

[0067] The appearance of the livers of the mice in each group was then compared, such as... Figure 17The results showed that, compared with the WT group, the liver of mice in the WT+HFD group was significantly enlarged. However, this enlargement trend was alleviated in the WT+HFD+Nrf2-ARE / hMAO-B / QR2 modulator 1 group. Furthermore, observations of adipose tissue weight at weeks 14 and 19 revealed that the adipose tissue weight in the WT+HFD group was significantly greater than that in the WT group. Compared with the WT+HFD group, the adipose tissue weight in the WT+HFD+Nrf2-ARE / hMAO-B / QR2 modulator 1 group was reduced, with this trend becoming more pronounced at week 19. Additionally, HE staining of adipose tissue revealed that the degree of vacuolation in the adipose tissue of the WT+HFD+Nrf2-ARE / hMAO-B / QR2 modulator 1 group was significantly reduced compared to the WT+HFD group. In conclusion, this compound can reduce NAFLD-induced weight gain, liver enlargement, and lipid accumulation.

[0068] Fasting blood glucose levels were measured in each group of mice, and it was found that, Figure 18 As shown, the blood glucose level of mice in the WT+HFD group was higher than that in the WT group, but the blood glucose level of the WT+HFD+Nrf2-ARE / hMAO-B / QR2 modulator 1 group was lower than that in the WT+HFD group.

[0069] Subsequently, ITT and GTT were performed on mice in each group, such as Figure 19 As shown, insulin resistance was alleviated and glucose tolerance was improved in the WT+HFD+Nrf2-ARE / hMAO-B / QR2modulator group 1.

[0070] The levels of TG, TC, ALT, and AST in mouse serum were detected using a kit. It was found that compared with the WT group, the serum levels of TG, TC, ALT, and AST in the WT+HFD group were significantly increased. However, the increase in serum TG, TC, ALT, and AST was alleviated after intervention with the compound. Figure 20 and 21 As shown.

[0071] The pathological changes in liver tissue observed under a microscope after HE staining and Oil Red O staining in each group of mice are as follows: Figure 22 As shown in the figure. The results indicate that lipid droplet deposition and fat vacuolation degeneration occurred in the liver tissue of mice in the WT+HFD group. Compared with the WT+HFD group, the liver lesions in mice in the WT+HFD+Nrf2-ARE / hMAO-B / QR2 modulator 1 group were significantly improved.

[0072] Further testing was conducted on the TG and TC levels in the livers of mice in each group, such as... Figure 23As shown in the figure. The results showed that the liver TG and TC levels in the WT+HFD group were significantly higher than those in the WT group, while the liver TG and TC levels in the WT+HFD+Nrf2-ARE / hMAO-B / QR2 modulator 1 group were lower than those in the WT+HFD group.

[0073] In summary, Nrf2-ARE / hMAO-B / QR2 modulator 1 significantly improves the core liver pathological features of NAFLD, including reducing hepatocyte lipid deposition and vacuolar degeneration, and lowering serum transaminase levels, thereby effectively restoring liver function. Metabolically, this compound also significantly alleviates insulin resistance and improves glucose tolerance, correcting NAFLD-related glucose homeostasis imbalances.

[0074] Based on the experimental results of Examples 1 to 3, it can be concluded that low expression of Aldh3a2 is associated with the pathological progression of NAFLD, and its overexpression can exert a hepatoprotective effect by upregulating the downstream target protein GPCPD1. This invention is the first to discover that the multi-target small molecule compound Nrf2-ARE / hMAO-B / QR2 modulator 1, which can bind to the GPCPD1 protein, exhibits comprehensive therapeutic effects in an animal model of NAFLD, effectively reducing body weight, improving glucose and lipid metabolism disorders, and alleviating hepatic steatosis and liver function damage. Therefore, this compound has important application value in the preparation of drugs for the prevention and treatment of NAFLD.

[0075] It should be noted that when numerical ranges are mentioned in the claims of this invention, it should be understood that the two endpoints of each numerical range and any value between the two endpoints can be selected. To avoid redundancy, the present invention describes preferred embodiments.

[0076] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including both the preferred embodiments and all changes and modifications falling within the scope of the invention.

[0077] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. The application of Nrf2-ARE / hMAO-B / QR2 modulator 1 in the preparation of drugs for treating non-alcoholic fatty liver disease, characterized in that, The drug improves non-alcoholic fatty liver disease through one or more of the following pathways: (a) Reduce weight in patients with non-alcoholic fatty liver disease; (b) Reduce abnormal lipid accumulation in the liver of patients with non-alcoholic fatty liver disease; (c) Improves insulin resistance in patients with non-alcoholic fatty liver disease; (d) Improve glucose tolerance in patients with non-alcoholic fatty liver disease; (e) Lowering blood glucose levels in patients with non-alcoholic fatty liver disease; (f) Reduce serum triglyceride and / or total cholesterol levels in patients with non-alcoholic fatty liver disease; (g) Reduce serum alanine aminotransferase and / or aspartate aminotransferase levels in patients with non-alcoholic fatty liver disease; (h) Reduce hepatic steatosis and / or vacuolation in patients with non-alcoholic fatty liver disease.

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