Application of N2L in preparation of medicine for preventing and treating obesity and fatty liver

Abstract

The invention relates to the technical field of biological medicines, and discloses an application of N2L in preparation of medicines for preventing and treating obesity and fatty liver, and the N2L is a dimer compound of alpha-lipoic acid and nicotinic acid. According to the embodiment of the invention, the application value of the GPR109A and GLP-1R double-target collaborative strategy in obesity and fatty liver treatment is proposed and verified for the first time, and the small molecule compound N2L with the GPR109A and GLP-1R double-target collaborative strategy can improve muscle endurance while reducing the weight of obese patients and can improve liver fatty degeneration at the same time. The defect that an existing GLP-1R agonist needs to be injected is overcome, meanwhile, the flushing side effect of an existing GPR109A agonist nicotinic acid medicine is avoided, a larger range is provided for medicine selection in the fields of obesity and fatty liver, and meanwhile, the application range of N2L is widened.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to the application of N2L in the preparation of drugs for the prevention and treatment of obesity and fatty liver. Background Technology

[0002] Obesity is a chronic metabolic disease caused by a combination of genetic and environmental factors (BMI ≥ 30 kg / m²). 2 According to the 2024 edition of the World Obesity Atlas, the overweight and obesity rate among adults in my country has exceeded 50%, and it is showing a trend towards affecting younger people.

[0003] Currently, only two types of weight-loss drugs are approved for long-term use in China: lipase inhibitors and nutrient-stimulating hormone receptor agonists. However, these two types of drugs have several drawbacks: lipase inhibitors can cause gastrointestinal side effects, while nutrient-stimulating hormone receptor agonists require injection and are expensive.

[0004] Furthermore, existing anti-obesity drugs primarily focus on weight control and blood sugar regulation, with limited effectiveness in addressing fatty liver disease, a condition closely related to obesity. As the obese population continues to grow, existing drugs can no longer meet the demands of the current weight-loss market. Therefore, the development of safe and effective weight-loss drugs is urgently needed.

[0005] In view of this, the present invention is proposed. Summary of the Invention

[0006] To address the aforementioned problems, the present invention aims to provide an application of N2L in the preparation of drugs for the prevention and treatment of obesity and fatty liver. The small molecule drug N2L of the present invention possesses synergistic dual-target effects on G protein-coupled receptor 109A (GPR109A) and glucagon-like peptide-1 receptor (GLP-1R), exhibiting good preventive and therapeutic effects against obesity and fatty liver disease. This invention not only expands the drug options for patients with obesity and fatty liver but also further broadens the application scope of N2L.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: The use of N2L in the preparation of drugs for the prevention and treatment of obesity and fatty liver, wherein the N2L is a dimer compound of α-lipoic acid and nicotinic acid, the structural formula of which is shown below: .

[0008] According to an embodiment of the present invention, the drug is a GPR109A agonist.

[0009] According to an embodiment of the present invention, the drug is a GLP-1R agonist.

[0010] According to an embodiment of the present invention, the drug is a dual agonist of GPR109A and GLP-1R.

[0011] According to an embodiment of the present invention, the drug is a hepatoprotective agent that improves liver function.

[0012] According to an embodiment of the present invention, the improvement of liver function is to reduce ALT / AST.

[0013] According to an embodiment of the present invention, the treatment includes reducing the weight of obese patients and improving hepatic steatosis.

[0014] According to an embodiment of the present invention, the treatment includes reducing the accumulation of body fat without reducing lean body mass during the weight loss process.

[0015] According to an embodiment of the present invention, the treatment includes improving muscle endurance during weight loss.

[0016] According to embodiments of the present invention, the treatment includes reducing serum triglyceride (TG), cholesterol (CHO), and low-density lipoprotein (LDL-C) levels.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention presents and verifies for the first time the application value of the dual-target synergistic strategy of GPR109A and GLP-1R in the treatment of obesity and fatty liver. The small molecule compound N2L, which has dual agonist effects of GPR109A and GLP-1R, can improve muscle endurance while reducing the weight of obese patients, and can also improve hepatic steatosis.

[0018] This invention overcomes the drawback of existing GLP-1R agonists requiring injection, while avoiding the hot flash side effect of existing GPR109A agonist nicotinic acid drugs, providing a wider range of drug choices for obesity and fatty liver, and also expanding the scope of N2L use. Attached Figure Description

[0019] Figure 1 A is the structural formula of N2L in this invention; Figure 1 B is a schematic diagram of the results of the FP competitive binding experiment of GLP-1-5-FAM SE (0.04 nM), GLP-1R (10 nM) and different concentrations of GLP-1-31 in Example 1 of the present invention; Figure 1 C is a schematic diagram of the results of the FP competitive binding experiment of GLP-1-5-FAM SE (0.04 nM), GLP-1R (10 nM) and N2L at different concentrations in Example 1 of the present invention; Figure 1D is a schematic diagram of the results of the FP competitive binding test conducted in Example 1 of the present invention on GLP-1-5-FAM SE (0.04 nM), GLP-1R (10 nM), blank group, GLP-1-31 (1 nM), N2L (0.1 μM), and N2L (1 μM); Figure 1 E is a schematic diagram of the results of luciferase reporter gene detection in Example 1 of the present invention; Figure 1 F is a schematic diagram of the cAMP content detection results in Example 1 of the present invention; Figure 2 This is a schematic diagram of the diet (A) and water intake (B) of mice under different administration regimens for 8 weeks in Example 2 of the present invention; Figure 3 A is a representative image of mice in each group after 8 weeks of drug administration in Example 2 of this invention; Figure 3 B is a schematic diagram of the weight changes of mice in each group during the entire experiment in Example 2 of the present invention; Figure 3 C is a schematic diagram showing the percentage change in body weight of mice in each group during the initial (20 days) and final (55 days) stages of the experiment in Example 2 of this invention; Figure 3 D is a schematic diagram of the fat mass of mice in each group after treatment using a mouse magnetic resonance body fat analyzer in Embodiment 2 of the present invention; Figure 4 E is a schematic diagram of the lean body mass of mice in each group after treatment using a mouse MRI body fat analyzer in Embodiment 2 of the present invention. Figure 4 F is a schematic diagram showing the weight of white fat obtained by dissecting and weighing the mouse epididymal adipose tissue (eWAT) after the treatment in Embodiment 2 of the present invention. Figure 4 G is a schematic diagram of the weight of white fat obtained by dissecting and weighing the subcutaneous adipose tissue (scWAT) of a mouse after the treatment in Embodiment 2 of the present invention. Figure 5 A is a representative HE-stained image of the epididymal adipose tissue of each group of mice in Example 2 of this invention; Figure 5 B is Figure 5 The area of ​​adipocytes in each group of mice in each HE-stained image in A; Figure 6 A is a schematic diagram showing the average swimming speed of each group of mice within 1 minute in the swimming experiment of Embodiment 2 of the present invention; Figure 6 B is a schematic diagram of the distance to exhaustion of mice in each group during the mouse running endurance experiment in Example 2 of the present invention; Figure 6C is a schematic diagram of the time to exhaustion of exercise for each group of mice in the mouse running endurance experiment of Embodiment 2 of the present invention; Figure 7 A is a representative image of the liver of each group of mice in Example 2 of the present invention; Figure 7 B is an Oil Red O staining image of the liver of each group of mice in Example 2 of this invention; Figure 8 C is an HE-stained image of the liver of each group of mice in Example 2 of this invention; Figure 8 D is the liver index of each group of mice in Example 2 of this invention; Figure 8 E is Figure 7 The area of ​​red oily lipid droplets in each group of mice in Oil Red O staining in B; Figure 9 This is a schematic diagram showing the levels of AST (A), ALT (B), CHO (C), TG (D), LDL-C (E), and HDL-C (F) in the serum of mice in each group of Example 2 of the present invention; Figure 10 A is a representative Oil Red O staining image of cells in each group in Example 3 of the present invention; Figure 10 B is a statistical diagram of the oil red O lipid droplet area of ​​each group of cells in Example 3 of the present invention. Detailed Implementation

[0020] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0021] This invention provides an application of N2L in the preparation of drugs for the prevention and treatment of obesity and fatty liver, wherein the N2L is a dimer compound of α-lipoic acid and nicotinic acid, and its structural formula is as follows: Figure 1 As shown in Figure A.

[0022] According to an embodiment of the present invention, the drug is a GPR109A agonist.

[0023] According to an embodiment of the present invention, the drug is a GLP-1R agonist.

[0024] According to an embodiment of the present invention, the drug is a dual agonist of GPR109A and GLP-1R.

[0025] According to an embodiment of the present invention, the drug is a hepatoprotective agent that improves liver function.

[0026] According to an embodiment of the present invention, the improvement of liver function is to reduce ALT / AST.

[0027] According to an embodiment of the present invention, the treatment includes reducing the weight of obese patients and improving hepatic steatosis.

[0028] According to an embodiment of the present invention, the treatment includes reducing the accumulation of body fat without reducing lean body mass during the weight loss process.

[0029] According to an embodiment of the present invention, the treatment includes improving muscle endurance during weight loss.

[0030] According to embodiments of the present invention, the treatment includes reducing serum triglyceride (TG), cholesterol (CHO), and low-density lipoprotein (LDL-C) levels.

[0031] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0032] For the synthesis of N2L and the results of the determination of the agonist activity of N2L on GPR109A, please refer to references 1 and 2.

[0033] Document 1: Jiang Y, Jin M, Chen J, et al. Discovery of a novel niacin-lipoic acid dimer N2L attenuating atherosclerosis and dyslipidemia with non-flushing effects. Eur J Pharmacol. 2020;868:172871. doi:10.1016 / j.ejphar.2019.172871 Reference 2: Chinese Patent CN 102532114 A discloses a nicotinic acid derivative, its preparation method, and its pharmaceutical composition.

[0034] Example 1 (N2L stimulating GLP-1R) Reagents: N2L (prepared in Example 1 of Literature 2), GLP-1-31, GLP-1-5-FAM SE, liraglutide, GLP-1R antagonist Exendin (9-39), Tris buffer, GLP-1R, DMSO.

[0035] Experimental methods: (1) Fluorescence polarization competition binding experiment Fluorescence polarimetry was performed using a SpectraMax multimode microplate reader (Molecular Devices) with excitation and emission filters. The microplates used for FP measurements were Corning 384-well plates with a black, non-binding surface. 40 μL of the assay solution (containing 20 μL Tris buffer, 10 μL GLP-1-5-FAM SE (0.04 nM), and 10 μL GLP-1R (10 nM)) was added to each well. Subsequently, serial dilutions of GLP-1-31 and N2L were prepared in 40× DMSO solution. Then, 1 μL of different concentrations of GLP-1-31 or N2L was added, and the mixture was incubated at room temperature for 30 min. FP signals were recorded three times and averaged.

[0036] (2) Luciferase reporter gene detection Luciferase assay of GLP-1 receptor activity in HEK293T-GLP-1R-3C-eGFP cells. 0.1% DMSO was used as a negative control, and liraglutide (200 nM) was used as a positive control. The REPO™ cyclic adenosine monophosphate (cAMP) response element-binding protein (CREB) reporter gene was transfected into HEK293T-GLP-1R-3C-eGFP cells for 20 hours. After stimulation with liraglutide (200 nM) and N2L (10 μM) for 4 hours, luminescence values ​​were detected using a one-step luciferase reporter gene assay kit.

[0037] (3) cAMP content detection GLP-1R-293A cells were seeded in 96-well plates at a density of 8000 cells / 100 μL. After cell adhesion, liraglutide (200 nM), exendin (9-39) (200 nM), and N2L (3, 10, 30 μM) were added to the cells and the cells were treated for 30 min. The cells were then lysed with lysis buffer, and the cAMP content released by the cells in the lysis buffer was detected.

[0038] Data Statistical Analysis Data were analyzed using GraphPad Prism 9.0 data analysis software. Unless otherwise specified, data are expressed as mean ± standard error (mean ± SEM). One-way ANOVA (Dunnet t-tests) was used for comparisons among multiple groups, and independent samples t-tests were used for comparisons between two groups. A statistically significant difference was defined as P < 0.05.

[0039] Experimental results See results Figure 1 Wherein, CT represents the blank control, compared with CT and 0.1% DMSO, #P<0.05, ## P<0.01 and ### P < 0.001. Details are as follows: Figure 1 The results of BD showed that GLP-1-5-FAM SE (0.04 nM), GLP-1R (10 nM), and different concentrations of GLP-1-31 or N2L were subjected to FP competitive binding assays. According to the standard curves prepared based on the fluorescence signal values, the binding inhibition value of N2L against GLP-1-31 and GLP-1R was 29.59 nM.

[0040] Figure 1 The results of E showed that the cAMP response element binding protein (CREB) reporter gene was transfected into HEK293T-GLP-1R-3C-eGFP cells for 20 hours, followed by stimulation with liraglutide (200 nM) and N2L (10 μM) for 4 hours, and the luminescence value was detected by a kit. Figure 1 The results in E show that 10 μM N2L can significantly increase cAMP levels, and its effect is comparable to that of 200 nM liraglutide.

[0041] Figure 1 The results of F showed that GLP-1R-293A cells were treated with liraglutide (200 nM), N2L (3, 10, 30 μM), and N2L (30 μM) in combination with the GLP-1R antagonist Exendin (9-39) (200 nM) for 30 min, and cAMP levels were detected using a kit. Figure 1 The results from F showed that N2L (10 μM) significantly stimulated GLP-1R in a dose-dependent manner. When N2L was used concurrently with a GLP-1R antagonist, its stimulatory effect was inhibited.

[0042] In summary, the above results indicate that N2L is a partial agonist of GLP-1R.

[0043] Experiment Example 2 (Animal Experiment) The grouping and dosing regimens for animal experiments are shown in Table 1: Table 1. Animal experimental grouping and dosing regimen

[0044] in: The CT group consisted of C57BL / KsJ-m / m mice; the other groups consisted of C57BL / KsJ-db / db mice.

[0045] The CT group was the normal control group; the Model group was the blank control group, consisting of model mice that did not receive any treatment.

[0046] Liraglutide was used as a positive control. 9-39 is the GLP-1R antagonist Exendin (9-39). MPN is penembromide, an antagonist of GPR109A.

[0047] (1) Preparation of drugs Preparation of 0.5% sodium carboxymethyl cellulose (CMC-Na): Weigh 0.15 g (calculated based on solution volume 30 mL × solute mass fraction 0.5%) of CMC-Na powder and place it in a 50 mL centrifuge tube. Measure 30 mL of distilled water using a graduated cylinder and slowly pour it along the tube wall while gently shaking the centrifuge tube to initially disperse the powder and prevent agglomeration. Then, place the centrifuge tube on a vortex mixer, adjust the speed appropriately, and vortex for 5 minutes to promote full swelling of the powder until the solution becomes uniform and transparent. Next, place the centrifuge tube in an ultrasonic cleaner, set the ultrasonic power to 250 W, and the ultrasonic time to 6-8 hours to obtain 30 mL of 0.5% CMC-Na solution.

[0048] Preparation of 15 mg / mL N2L solution: Taking 20 mL as an example, dissolve 300 mg N2L powder in 20 mL of 0.5% CMC-Na solution and sonicate until dissolved.

[0049] Preparation of 0.1 mg / mL liraglutide solution: Pipette 6 mL of physiological saline into a 15 mL centrifuge tube, take 0.6 mg of the drug solution from the injection pen and dissolve it in physiological saline. Mix well and store in a refrigerator at 4°C.

[0050] Preparation of 0.1 mg / mL Exendin (9-39) solution: Weigh 2 mg of Exendin (9-39) powder and dissolve it in 40 mL of physiological saline. After it is fully dissolved, dispense it into 5 mL EP tubes and store it in a refrigerator at -20°C.

[0051] Preparation of 2 mg / mL MPN solution: Weigh 10 mg MPN and dissolve it in 5 mL of 0.5% CMC-Na solution. After it is fully dissolved, store it in a refrigerator at 4°C.

[0052] (2) Records of body weight and food and water intake During the experiment, the mice, feed and water bottles of each group were weighed every 3 days using an electronic balance, and the average daily food and water intake and weight gain rate were calculated.

[0053] (3) Body fat analyzer was used to measure the fat content of mice. The instrument was started and warmed up 24 hours before the experiment. After system calibration using standard samples, the testing began. The experimental animals were placed in measuring cages, and their movement was restricted by adjusting the restraint device. The cages were then pushed into the nuclear magnetic resonance body composition analyzer, and body components were determined using a non-invasive method. The instrument automatically output the content values ​​of adipose tissue, free body fluid, and lean body mass.

[0054] (4) Mouse treadmill endurance test At the end of the drug administration period, a treadmill exhaustion test was conducted on mice. Before the formal experiment, all mice underwent a 5-minute acclimatization training session on the treadmill, followed by a 15-minute rest period. For db / db mice, the acclimatization training parameters were set as a 5-minute exercise duration and a 20° track incline. During the formal test, the initial exercise speed was set at 8 m / min, increasing by 2 m / min every 2 minutes thereafter. The maximum exercise speed of db / db mice was limited to the range of 10-12 m / min, and the track incline was maintained at 20°. Mice were considered to have reached exhaustion when they were unable to avoid the electrical stimulation and climbed up the treadmill three times consecutively. The total exercise duration and cumulative distance at exhaustion were recorded as endurance evaluation indicators.

[0055] (5) Swimming experiment Prepare a pool with a diameter of 1 m and a height of 60 cm, and fill it with an appropriate amount of water. Gently and slowly place the mouse into the water along the pool wall, and let it swim freely in the pool for 1 minute. Record the mouse's swimming speed in 1 minute using a simulated device.

[0056] (6) HE staining of tissues After fixation with 4% paraformaldehyde, liver and epididymal adipose tissue were placed in embedding casks for gradient dehydration. Following dehydration, the tissues were embedded in paraffin and then transferred to a cryostat for solidification. The tissue blocks were trimmed to a thickness of 10 μm using a microtome and then serially sectioned at 4 μm thicknesses. The resulting paraffin sections were flattened in a 45°C water bath and, after complete unfolding, transferred to glass slides and dried in a 65°C oven. Dewaxing was then performed, involving immersion in hematoxylin and eosin staining solutions for 5 min each, followed by rinsing with running water and gradient dehydration with ethanol and xylene. Finally, the slides were mounted with glycerol gelatin. The prepared HE-stained sections were observed under an optical microscope to analyze the morphological characteristics of the tissues.

[0057] (7) Oil Red O staining Liver samples were fixed with 4% paraformaldehyde and then dehydrated sequentially in 10%, 20%, and 30% sucrose solutions for 24 h, 48 h, and 48 h, respectively. After dehydration, the tissues were embedded using OCT embedding medium and frozen at -80℃. Tissue sections with a thickness of 4 μm were prepared using a cryostat and then stained. First, the sections were stained with Oil Red O working solution for 8 min, then differentiated with 60% isopropanol, and rinsed three times with distilled water for 30 s each time. After rinsing, the sections were counterstained with hematoxylin for 3 min. After differentiation and blue inversion, the stained sections were mounted with glycerol gelatin. The prepared Oil Red O stained sections were observed under an optical microscope to analyze the distribution characteristics of triglycerides and other neutral lipids in the liver tissue.

[0058] (8) Detection of relevant indicators in mouse serum After the experiment, orbital venous blood was collected from mice. The levels of serum triglycerides (TG), total cholesterol (TC), high-density lipoprotein (HDL-C), low-density lipoprotein (LDL), alanine aminotransferase (ALT), and aspartate aminotransferase (AST) were analyzed using a fully automated biochemical analyzer.

[0059] Data Statistical Analysis Data were analyzed using GraphPad Prism 9.0 data analysis software. Unless otherwise specified, data are expressed as mean ± standard error (mean ± SEM). One-way ANOVA (Dunnet t-tests) was used for comparisons among multiple groups, and independent samples t-tests were used for comparisons between two groups. A statistically significant difference was defined as P < 0.05.

[0060] Experimental results (1) N2L only slightly inhibited the diet and water intake of db / db mice compared to liraglutide. See results Figure 2 Each group contained 7-10 mice, with 2-3 mice per cage. Compared to the Model group, # P<0.05, ## P<0.01 and ### P < 0.001. Details are as follows: Figure 2 Results A and B indicate that db / db mice had significantly higher food and water intake than m / m mice (P < 0.001), consistent with the typical metabolic phenotype of T2DM: polydipsia, polyphagia, and polyuria. Compared to the Model group mice, Liruglutide reduced food intake and significantly suppressed water intake (P < 0.001); however, N2L treatment only slightly suppressed food and water intake in mice, showing no statistically significant difference compared to the Model group.

[0061] (2) N2L administration inhibited the increase in body weight in db / db mice. See results Figure 3-4 Seven groups of mice, with 7-10 mice in each group, are shown in the graph; each dot represents one mouse. Compared to the Model group, # P<0.05, ## P<0.01 and ### P < 0.001. Details are as follows: Figure 3 A represents a representative image of mice in each group after 8 weeks of drug administration.

[0062] Figure 3 B represents the change in body weight of mice in each group throughout the experiment.

[0063] Figure 3 C represents the percentage change in body weight of mice in each group at the beginning (20 days) and end (55 days) of the experiment. The calculation method is: (body weight at 20 / 55 days – body weight at 0 days) / body weight at 0 days.

[0064] Figure 3 D and Figure 4 E represents the fat and lean body mass of mice in each group as measured by a mouse magnetic resonance body fat analyzer after treatment.

[0065] Figure 4 F and Figure 4 The G result represents the weight of white fat obtained by dissecting and weighing the epididymal adipose tissue (eWAT) and subcutaneous adipose tissue (scWAT) of mice at the end of treatment.

[0066] Mice body weight was monitored regularly during the experiment. Results showed that mice in the CT group experienced a slight and stable increase in body weight, while mice in the Model group showed a significant and progressive increase. Compared to the Model group, the weight gain in all treatment groups was inhibited. At the end of treatment, the Model group mice had a significantly higher body weight than the CT group, reaching 2.12 times the CT group weight. All treatment groups showed an inhibitory effect on pathological weight gain, with the N2L(L) and N2L(H) groups showing significant weight loss at the experimental endpoint (P < 0.01 or P < 0.001). Notably, the liraglutide group showed significant weight loss in the early stages of treatment (days 1-20) (P < 0.05), but its long-term intervention effect was not statistically different from that of the Model group. Furthermore, the results indicated that when N2L was combined with 9-39 treatment, weight loss in mice was inhibited; conversely, the combined MPN treatment group showed more significant weight loss. Dynamic body weight change results showed that the N2L group maintained a stable weight loss trend throughout the experimental period, and its weight loss effect was superior to liraglutide.

[0067] After treatment, assessment using a small animal MRI component analysis system revealed a significant increase in fat mass in the Model group mice compared to the CT group. N2L intervention reduced fat mass in db / db mice without decreasing lean body mass, and showed a better fat-reducing effect compared to liraglutide. Dissection of the mice and weighing of white and brown adipose tissue revealed a significant increase in epididymal and subcutaneous fat weight in the Model group mice, while both N2L and liraglutide administration reduced fat weight.

[0068] In summary, the dynamic weight change results indicate that the N2L group maintained a stable weight loss trend throughout the experimental period, and its weight loss effect was superior to liraglutide. Furthermore, body fat analysis showed that N2L reduced fat without affecting lean body mass.

[0069] (3) N2L reduces the volume of epididymal adipocytes in db / db mice See results Figure 5 Compared to the Model group, # P<0.05, ## P<0.01 and ### P < 0.001. Details are as follows: Figure 5 Results A represent representative HE-stained images of epididymal adipose tissue from each group of mice, with a scale bar of 200 μM.

[0070] Figure 5 Results B represent the area of ​​adipocytes in each group of mice in the HE-stained images. Adipose tissue from 3 mice in each group was stained.

[0071] The experimental results showed that adipocytes in the CT group mice were morphologically regular, uniform in size, tightly and orderly arranged, and had clear cell boundaries; in the Model group, the number of cells per unit field of view decreased, while the cell area increased significantly. Compared with the Model group, the N2L treatment group showed an increase in the number of adipocytes per unit field of view, a decrease in cell area (P < 0.001), and clearer cell boundaries, indicating that N2L can effectively inhibit adipocyte hypertrophy. Compared with the N2L group, the liraglutide group showed a decrease in the number of adipocytes per unit field of view, but an increase compared with the Model group; N2L combined with Exendin (9-39) treatment reduced the number of adipocytes in mice, and the cells were larger; while the adipocytes in the combined MPN treatment group were similar to those in the N2L group.

[0072] The above results indicate that N2L can effectively reduce the area of ​​adipocytes in db / db mice.

[0073] (4) N2L can improve muscle strength and motor ability in db / db mice.

[0074] See results Figure 6Compared to the Model group, # P<0.05, ## P<0.01 and ### P < 0.001. Details are as follows: Figure 6 Results A indicate the average swimming speed of mice in each group within 1 minute during the swimming experiment.

[0075] Figure 6 B and Figure 6 Results C represent the distance and time to exhaustion for each group of mice in the mouse running endurance test. Each group contains 7-10 mice, and each dot represents one mouse.

[0076] Swimming speed and running endurance in mice over a certain period of time can reflect their limb muscle strength and exercise endurance. Swimming test results showed that the average swimming speed of the Model group mice within 1 minute was lower than that of the CT group. Compared with the Model group, both N2L and liraglutide treatments improved the average swimming speed of mice, but the results showed only a trend and were not statistically significant. Furthermore, the running endurance of db / db mice was significantly weaker than that of the CT group. Compared with the Model group, N2L significantly improved the time to exhaustion and distance of movement in db / db mice (P < 0.01); however, liraglutide treatment did not improve the exercise endurance of mice. N2L combined with Exendin (9-39) or MPN treatment showed weaker improvements in muscle strength and exercise capacity in mice than N2L treatment alone.

[0077] In summary, the experimental results show that N2L can improve muscle strength and exercise endurance in mice while reducing their body weight.

[0078] (5) N2L alleviates hepatic fat deposition in db / db mice.

[0079] See results Figure 7-8 Compared to the Model group, # P<0.05, ## P<0.01 and ### P < 0.001. Details are as follows: Figure 7 Results A represent representative images of the livers of mice in each group.

[0080] Figure 7 Results B represent Oil Red O staining images of the livers of mice in each group.

[0081] Figure 8 Results C represent HE staining images of the livers of mice in each group. The scale bars in the figures are all 200 μM. Three tissue samples were taken from each group of mice for staining.

[0082] Figure 8 The result D represents the liver index of each group of mice, calculated as: (liver weight / body weight) × 100%.

[0083] Figure 8 E results represent the area of ​​red oily lipid droplets in each group of mice during Oil Red O staining.

[0084] Based on the liver images of the mice in each group, the livers of the CT group mice were dark reddish-brown with a smooth surface and sharp edges; the livers of the Model group mice showed enlarged livers with diffuse yellowish-white greasy discoloration and blunted edges, typical pathological features of fatty liver. Compared with the Model group, both N2L and liraglutide treatments could partially alleviate liver enlargement and greasy discoloration. The livers of the mice were weighed and liver indices were calculated. The results showed that the liver indices of the Model group mice were higher than those of the CT group; compared with the Model group, N2L and liraglutide treatments could reduce the liver indices of the mice.

[0085] HE staining was performed on liver tissue to detect pathological changes after drug administration. Results showed that hepatocytes in the CT group were tightly packed with clear hepatic lamina. In the Model group, hepatocytes showed degeneration, swelling, loose cytoplasmic staining, and ballooning vacuoles. Compared to the Model group, N2L and liraglutide treatment alleviated hepatocyte degeneration. To further assess hepatic steatosis, Oil Red O staining was performed on mouse livers. Results showed that the cytoplasm of hepatocytes in the CT group was pale blue, with no red lipid droplets observed. In the Model group, numerous diffusely distributed red lipid droplets were observed in the liver tissue, often clustered in clumps, indicating significant hepatic lipid accumulation. Compared to the Model group, the N2L and liraglutide treatment groups showed fewer and smaller lipid droplets in the liver (P < 0.001 and P < 0.01), indicating a significant improvement in hepatic lipid accumulation, and N2L showed a superior lipid-lowering effect compared to liraglutide. In mice treated with N2L in combination with Exendin (9-39) or MPN, the degree of lipid accumulation in the liver was aggravated, and the effect of N2L in improving lipid accumulation in hepatocytes was weakened.

[0086] The results suggest that N2L can improve hepatic steatosis in db / db mice and may exert its pharmacological effects by activating the GLP-1R / GPR109A pathway.

[0087] (6) N2L improves liver function and reduces blood lipids in db / db mice.

[0088] See results Figure 9 Compared to the Model group, # P<0.05, ## P<0.01 and ### P < 0.001. Details are as follows: Figure 9This is a schematic diagram showing the levels of AST (A), ALT (B), CHO (C), TG (D), LDL-C (E), and HDL-C (F) in the serum of mice in each group of Example 2 of the present invention.

[0089] ALT and AST are indicators for assessing liver injury. Results showed that serum AST and ALT levels were higher in the Model group than in the CT group, indicating some degree of liver injury in the db / db mice. Compared to the Model group, N2L and liraglutide treatment significantly reduced serum AST and ALT levels in mice (P < 0.05 or P < 0.001). Four lipid profiles were used to assess lipid metabolism. Results showed that serum CHO, TG, and LDL-C levels were significantly higher in the Model group, indicating lipid metabolism disorder. Compared to the Model group, N2L and liraglutide treatment significantly reduced serum CHO and LDL-C levels in mice (P < 0.05 or P < 0.001), and also reduced TG levels, but the difference was not statistically significant. HDL-C levels did not change significantly in any group of mice. Overall, N2L was more effective than liraglutide. Mice treated with N2L in combination with Exendin (9-39) or MPN had higher serum ALT, CHO, TG and LDL-C levels than those treated with N2L alone.

[0090] In summary, N2L can effectively regulate abnormal lipid metabolism in the liver of db / db mice and alleviate liver damage.

[0091] Experimental Example 3 (Effect of N2L on palmitate (PA)-induced lipid accumulation in HepG2 cells) Experimental methods Oil Red O staining: HepG2 cells were stained with oil at a concentration of 1×10⁻⁶ cells / mL. 5Cells were seeded in 24-well plates and grouped and treated as follows: ① CT group received the same volume of control solvent; ② PA group received 500 μM PA; ③ N2L group received 500 μM PA and 10 μM N2L; ④ liraglutide group received 500 μM PA and 200 nM liraglutide; ⑤ niacin (NA) group received 500 μM PA and 100 μM niacin; ⑥ N2L + niacin receptor antagonist penmetabyl bromide (MPN) group received 500 μM PA, 10 μM N2L and 100 nM MPN; ⑦ NA + MPN group received 500 μM PA, 100 μM NA and 100 nM MPN; ④ and ⑤ were positive control groups. Cells were cultured in 10% DMEM medium for 24 h, followed by pre-protection with drugs (N2L: 10 μM, nicotinic acid: 100 μM, liraglutide: 200 nM) for 2 h. PA was then diluted to 500 µM and added to the dish. After 48 h of culture, cells were fixed with 4% paraformaldehyde for 25 min. The paraformaldehyde was discarded, and the cells were washed twice with distilled water. Oil Red O staining solution was added for 30 min. The staining solution was discarded, and the cells were rinsed with 60% isopropanol for 20-30 s. The cells were washed 2-5 times with distilled water until no excess staining solution remained. The cells were then covered with distilled water and observed under a microscope.

[0092] Experimental results: N2L reduced PA-induced lipid accumulation in HepG2 cells.

[0093] See results Figure 10 Compared with the CT group, ## P <0.01; compared to the Model group, * P <0.05, ** P <0.01. Data are expressed as mean ± SEM and analyzed using one-way ANOVA followed by Tukey post-hoc test. Each group was tested in triplicate. Details are as follows: Figure 10 Results A represent representative Oil Red O staining images of cells in each group, with a scale bar of 200 μM.

[0094] Figure 10 Results B represent the statistical graph of the oil red O lipid droplet area in each group of cells.

[0095] Oil Red O staining and quantitative lipid droplet analysis showed that only a small amount of lipids were stained in the cell membranes of the control group, while a large number of lipid droplets were generated in the model group. This indicates that lipid metabolism in hepatocytes was disrupted under 500 μM PA stimulation, and the model was successfully established. Furthermore, the results showed that the positive control drugs liraglutide and norepinephrine (NA) significantly reduced lipid droplet formation; N2L at a concentration of 10 μM significantly reduced the number of lipid droplets, and its lipid-lowering effect was better than the two positive control drugs. When the GPR109A inhibitor MPN was added, the lipid-lowering effects of N2L and NA were inhibited, but fewer lipid droplets were observed compared to the PA group.

[0096] The technical solutions provided by the embodiments of the present invention have been described in detail above. Specific examples have been used to illustrate the principles and implementation methods of the embodiments of the present invention. The descriptions of the embodiments above are only for helping to understand the principles of the embodiments of the present invention. At the same time, for those skilled in the art, there will be changes in the specific implementation methods and application scope based on the embodiments of the present invention. Therefore, the content of this specification should not be construed as a limitation of the present invention.

Claims

1. The use of N2L in the preparation of drugs for the prevention and treatment of obesity and fatty liver, wherein, The N2L is a dimer compound of α-lipoic acid and nicotinic acid, and its structural formula is shown below: 。 2. The application according to claim 1, characterized in that, The drug is a GPR109A agonist.

3. The application according to claim 1, characterized in that, The drug is a GLP-1R agonist.

4. The application according to claim 1, characterized in that, The drug is a dual agonist of GPR109A and GLP-1R.

5. The application according to claim 1, characterized in that, The drug is a liver-protective agent that improves liver function.

6. The application according to claim 5, characterized in that, The improvement in liver function is achieved by reducing ALT / AST.

7. The application according to claim 1, characterized in that, The treatment includes weight loss in obese patients and improvement of hepatic steatosis.

8. The application according to claim 1, characterized in that, The treatment involves reducing the accumulation of body fat without reducing lean body mass during the weight loss process.

9. The application according to claim 1, characterized in that, The treatment includes improving muscle endurance during the weight loss process.

10. The application according to claim 1, characterized in that, The treatment includes lowering serum triglyceride (TG), cholesterol (CHO), and low-density lipoprotein (LDL-C) levels.

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