Use of ribo-adenosine in the preparation of a medicament for treating fatty liver and reducing obesity

Abstract

The present application belongs to the field of biological medicine, and particularly relates to the use of ribo-adenosine in the preparation of a medicine for treating fatty liver and reducing obesity. The present application finds and confirms, through cell experiments, organoid experiments and animal experiments, that ribo-adenosine can reduce lipid accumulation, reduce body weight and thus reduce obesity, and can prevent and improve metabolic diseases related to obesity; in particular, the present application finds that ribo-adenosine can inhibit the synthesis of fatty acids and cholesterol in liver cells, reduce the levels of fatty acids, triglycerides and free cholesterol, improve liver function, reduce liver damage, and play a preventive and therapeutic role in metabolic fatty liver.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically, it relates to the use of levodopaline in the preparation of drugs for treating fatty liver and reducing obesity. Background Technology

[0002] Lipoadenosine (N) 6 -Isopentenyl adenosine (i6A), with the molecular formula C6 15 H 21 N5O4 is a naturally occurring modified nucleoside widely found in plants and microorganisms. In plant physiology, levofloxacin, as an important growth regulator, not only promotes cell division and growth but also enhances the plant's adaptability to adverse conditions such as drought and salt stress. In medical research, levofloxacin exhibits diverse biological activities and potential therapeutic value. Studies have shown that levofloxacin demonstrates antitumor activity in various models: it can inhibit the proliferation of human breast cancer MDA-MB-231 cells and induce apoptosis. [1] In a melanoma model, lipoadenosine activates adenosine monophosphate-activated protein kinase (AMPK) to induce autophagosome accumulation, thereby exhibiting significant anti-melanoma activity. [2] In gliomas and glioblastomas, levodopa induces necrotizing apoptosis in cancer cells via a proteasome-mediated pathway, thus achieving an anti-tumor effect. [3, 4] The above studies indicate that lipoadenosine has promising applications in disease treatment. However, research on lipoadenosine in the field of lipid metabolism diseases remains limited, especially its effects on metabolic-associated fatty liver disease (MAFLD) and obesity, which have not been systematically reported. MAFLD is fatty liver disease driven primarily by metabolic dysfunction, mainly due to insulin resistance leading to increased hepatic fat synthesis and decreased fat degradation. Simultaneously, abnormal oxidative stress, mitochondrial dysfunction, chronic inflammation, and gut microbiota dysbiosis further exacerbate hepatic lipid metabolism abnormalities. Meanwhile, obesity has become a global epidemic, seriously threatening public health. Currently, there are limited drug options for treating MAFLD and obesity; therefore, developing more effective treatment strategies and drugs for fatty liver and / or obesity and related diseases has become an urgent need to address this major health challenge. Summary of the Invention

[0003] This invention proposes new uses for lipoadenosine through extensive and in-depth research. This invention discovers that lipoadenosine exhibits potent activity in improving metabolic-related fatty liver disease and reducing obesity, thereby completing this invention.

[0004] The technical solution of the present invention is as follows:

[0005] In one aspect, the present invention discloses the use of lipoadenosine in the preparation of a medicament for reducing obesity in subjects.

[0006] In one embodiment, the levoadenosine of the present invention can reduce the accumulation of white fat in the abdomen of the subject, reduce weight, and alleviate obesity.

[0007] One aspect of the present invention discloses the use of levodopaline in the preparation of drugs for the prevention / improvement of obesity-related metabolic diseases in subjects.

[0008] In this invention, obesity-related metabolic diseases include one or more of the following: hyperlipidemia, type 2 diabetes, hyperuricemia, hypertension, coronary heart disease, heart failure, arrhythmia, atrial fibrillation, atherosclerosis, obstructive sleep apnea syndrome, and metabolic fatty liver.

[0009] Obese patients often have dyslipidemia, with elevated triglyceride (TG) levels being particularly prominent and positively correlated with the degree of obesity. In addition, elevated low-density lipoprotein cholesterol and total cholesterol levels and decreased high-density lipoprotein cholesterol levels are also common. The mechanisms include: (1) reduced mobilization and utilization of free fatty acids by body tissues, leading to excessive accumulation of free fatty acids in the blood; (2) obese patients often have hyperinsulinemia, and insulin has the effects of promoting fat synthesis and inhibiting fat breakdown.

[0010] Overweight and obesity are significant causes of prediabetes and type 2 diabetes mellitus (T2DM). The greater the degree of obesity, the greater the risk of developing prediabetes and T2DM.

[0011] Obese patients often have hypertension. The pathophysiological mechanisms of obesity-induced hypertension mainly involve increased cardiac output, plasma volume expansion and sodium retention (salt sensitivity), sympathetic nervous system activation, renin-angiotensin-aldosterone system activation, insulin resistance, abnormal brain-gut axis function, adipokines imbalance, inflammation / oxidative stress, extravascular fat dysfunction, and sleep apnea syndrome. Hypertension in obese patients may manifest as refractory hypertension, often requiring more antihypertensive medications than in non-obese individuals. Obesity is an independent risk factor for cardiovascular disease.

[0012] Obese patients often have co-existing conditions such as atherosclerosis, coronary heart disease, congestive heart failure, arrhythmia, and cardiomyopathy, significantly increasing their risk of cardiovascular events. The underlying mechanisms are related to the fact that obese patients often also have hypertension, dyslipidemia, inflammatory responses, and insulin resistance.

[0013] Obesity is also associated with an increased risk of infertility, gestational diabetes, gestational hypertension, and premature birth in women with polycystic ovary syndrome. Obesity, especially central obesity, is also a significant risk factor for obstructive sleep apnea syndrome.

[0014] In one aspect, the present invention discloses the use of lipoadenosine in the preparation of a medicament for the prevention / treatment of metabolic-associated fatty liver disease (MAFLD) in subjects.

[0015] Metabolic fatty liver disease (MAFLD) is a series of clinicopathological syndromes, including nonalcoholic fatty liver (NAFL), nonalcoholic steatohepatitis (NASH), liver fibrosis, and cirrhosis. Pathological manifestations include hepatocellular steatosis, hepatocellular damage, liver inflammation, and fibrosis. Overweight and obesity are important causes and risk factors for MAFLD.

[0016] In one embodiment, the lipoadenosine of the present invention can reduce lipid molecules such as total cholesterol, triglycerides and fatty acids in the liver, thereby reducing the number and size of lipid droplets in the liver.

[0017] In one embodiment, the lipoadenosine of the present invention, as a drug for the prevention / treatment of metabolic-associated fatty liver disease (MAFLD), can lower blood lipids, reduce hepatic steatosis and blistering degeneration, and reduce liver damage.

[0018] In one embodiment, the lipoadenosine of the present invention, as a drug for the prevention / treatment of metabolic-associated fatty liver disease (MAFLD), can reduce serum total cholesterol, low-density lipoprotein, alanine aminotransferase and aspartate aminotransferase, reduce lipid molecules such as total cholesterol, triglycerides and fatty acids in the liver, and reduce the number and size of lipid droplets in the liver.

[0019] In this invention, levodopa can reduce serum total cholesterol and low-density lipoprotein, reduce abdominal white fat accumulation, reduce weight, alleviate obesity, and thus prevent / improve obesity-related metabolic diseases.

[0020] In this invention, the subject is an animal, preferably a mammal;

[0021] In this invention, the subject is a human.

[0022] In this invention, those skilled in the art will understand that the drug also includes pharmaceutically acceptable excipients. In the pharmaceutical field, excipients refer to all substances in a pharmaceutical preparation other than the active pharmaceutical ingredient. While they generally do not have therapeutic effects themselves, they are crucial for the safety, efficacy, stability, and user compliance of the preparation. Commonly used excipients include: fillers, binders, disintegrants, lubricants, flow aids, coating agents, solubilizers, emulsifiers, suspending agents, preservatives, antioxidants, pH adjusters, buffers, etc.

[0023] In this invention, those skilled in the art will understand that the drug can be prepared into any desired dosage form for convenient use by the subject. Common dosage forms include tablets, capsules, granules, liquid preparations, and gels. Capsules can be soft capsules or hard capsules. Liquid preparations include injections and oral liquid preparations. Granules can be made into large pills or into small powders. Beneficial effects

[0024] This invention provides a new use for lipoadenosine, which can reduce lipid accumulation, lower weight, and alleviate obesity. It not only has preventive and therapeutic effects on obesity, but also prevents and improves obesity-related metabolic diseases. The lipoadenosine of this invention inhibits the synthesis of fatty acids and cholesterol in hepatocytes, improves liver function, reduces liver damage, and plays a role in the prevention and treatment of metabolic-related fatty liver disease. Attached Figure Description

[0025] Figure 1 The preventive effect of lipoadenosine on metabolic-related fatty liver disease. A. Flowchart of the prevention experimental protocol. Eight-week-old male C57BL / 6J mice were divided into four groups (n=8). Mice were fed a high-sugar, high-fat, high-cholesterol diet and simultaneously administered the drug via gavage for 8 weeks. The groupings were as follows: Con diet group; solvent control group (HFHC-con); lovastatin group (HFHC-Lov); lipoadenosine group (HFHC-i6A). B. Lipoadenosine reduced the content of total cholesterol (TC) in the liver. C. Lipoadenosine reduced the content of triglycerides (TG) in the liver, with a better effect than lovastatin. D. Oil Red O staining of liver sections showed that lipoadenosine reduced lipid droplets in the liver, with a better effect than lovastatin. Scale bar: 100 m. E. Liver lipidomics showed that a HFHC diet led to an increase in lipids such as free fatty acids and triglycerides in the liver, which decreased after administration of lipoadenosine or lovastatin. Data are expressed as mean ± standard deviation. p-values ​​were calculated using one-way ANOVA, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns: not significant.

[0026] Figure 2 The therapeutic effects of lipoadenosine on metabolic-associated fatty liver disease (MAF): It reduces lipid levels in serum and liver, alleviating liver damage. A. Treatment experimental protocol flowchart. Eight-week-old male C57BL / 6J mice were fed a high-fat diet (60 kcal%) for 16 weeks to induce MAF, and then administered lipoadenosine via drinking water for 20 weeks. Grouping was as follows: normal diet group (Con diet), solvent control group (HFD-con), and lipoadenosine group (HFD-i6A). B. Lipoadenosine reduces liver / body weight percentage. C. Lipoadenosine reduces serum alanine aminotransferase (ALT) levels. D. Lipoadenosine reduces serum aspartate aminotransferase (AST) levels. E. Lipoadenosine reduces serum total cholesterol (TC) levels. F. Lipoadenosine reduces serum low-density lipoprotein (LDL) levels. G. Lipoadenosine reduces total cholesterol (TC) levels in the liver. H. lipoadenosine reduces triglyceride (TG) levels in the liver. Data are expressed as mean ± standard deviation. p-values ​​were calculated using one-way ANOVA; *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns: not significant.

[0027] Figure 3 The therapeutic effects of lipoadenosine on metabolic-associated fatty liver disease: reducing hepatic steatosis and inflammatory response. Specifically, A. Oil Red O staining of liver sections showed that lipoadenosine reduced lipid droplets in the liver. B. HE staining of liver sections showed that lipoadenosine reduced vesicular lipid droplets and ballooning degeneration of hepatocytes induced by a high-fat diet, and also reduced the aggregation of inflammatory cells. Arrows indicate ballooning degeneration, asterisks indicate vesicular lipid droplets, and blue dashed lines indicate inflammatory cell aggregation. Scale bar: 100 (AB). C. Liver lipidomics results showed that HFD led to an increase in lipids such as free fatty acids and triglycerides in the liver, which decreased after administration of lipoadenosine or lovastatin. Data are expressed as mean ± standard deviation. p-values ​​were calculated using one-way ANOVA, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns: not significant.

[0028] Figure 4Lipoadenosine reduces abdominal white adipose tissue accumulation and lowers body weight, thereby alleviating obesity. Specifically: A. Lipoadenosine does not affect food intake and reduces body weight gain induced by a high-sugar, high-fat, high-cholesterol (HFHC) diet. B. Lipoadenosine reduces the weight of white adipose tissue (WAT) from an HFHC diet. C. HE staining of white adipose tissue sections shows that lipoadenosine reduces adipocyte enlargement and decreases mean adipocyte size induced by an HFHC diet. Scale bar: 300 m, magnified to 50 m. D. Lipoadenosine does not affect food intake and reduces body weight gain induced by a 60 kcal% high-fat diet (HFD). E. Lipoadenosine reduces white adipose tissue (WAT) weight in HFD diets. F. HE staining of white adipose tissue sections shows that lipoadenosine reduces adipocyte enlargement and decreases mean adipocyte size induced by HFD diets. Scale bar: 300 m, magnified to 50 m. Data are expressed as mean ± standard deviation. p-values ​​were calculated using one-way ANOVA, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns: not significant.

[0029] Figure 5 Lipoadenosine reduces free cholesterol levels in human liver organoids. A. Confocal microscopy image of human liver organoids. Scale bar: 100 m. B. Statistical results of staining area of ​​human liver organoids. Lipoadenosine significantly reduced free cholesterol levels. Data are expressed as mean ± standard deviation. p-values ​​were calculated using an unpaired two-tailed t-test. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns: not significant.

[0030] Figure 6Lipoadenosine inhibits the synthesis of fatty acids and cholesterol in hepatocytes, thereby reducing the levels of fatty acids, triglycerides, and free cholesterol. Specifically, AH. Lipoadenosine inhibits the synthesis of fatty acids and cholesterol in hepatocytes. A. Heatmap of differentially expressed genes in the HepG2 fatty acid biosynthesis pathway. B. Heatmap of differentially expressed genes in the HepG2 unsaturated fatty acid biosynthesis pathway. C. Heatmap of differentially expressed genes in the HepG2 terpenoid backbone biosynthesis pathway. D. Heatmap of differentially expressed genes in the HepG2 steroid biosynthesis pathway. E. GSEA analysis of the fatty acid biosynthesis pathway. F. GSEA analysis of the unsaturated fatty acid biosynthesis pathway. G. GSEA analysis of the terpenoid backbone biosynthesis pathway. H. GSEA analysis of the steroid biosynthesis pathway. IJ. Lipoadenosine reduces the levels of fatty acids, triglycerides, and free cholesterol. I. Lipoadenosine reduces the levels of fatty acids (FA) and triglycerides (TG) in hepatocytes. J. Left: High-content imaging results of free cholesterol staining in HepG2 cells before and after lipoadenosine treatment. Right: Dose-response curve of lipoadenosine concentration and average staining area of ​​free cholesterol in HepG2 cells. Lipoadenosine reduces the level of free cholesterol in hepatocytes in a dose-dependent manner. Detailed Implementation

[0031] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. Unless otherwise specified, the equipment and reagents used in the embodiments and experimental examples are commercially available. Unless otherwise stated, all reagents used in this invention are analytical grade reagents. The specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention.

[0032] The chemical structure of the compound lipoadenosine described in this invention is shown in formula (I):

[0033]

[0034] The control drug was lovastatin (CAS No. 75330-75-5; MCE, HY-N0504, purity: 99.75%).

[0035] The test process is illustrated as follows: Figure 1As shown in Figure A. To conduct mouse modeling and drug administration experiments for metabolically related fatty liver disease, 8-week-old male C57BL / 6J mice (Beijing Vital River Laboratory Animal Technology Co., Ltd.) were purchased and fed a high-sugar, high-fat, high-cholesterol diet (Keao Xieli Tianjin Feed Co., Ltd.) containing 15% fat, 20% sucrose, and 1.2% cholesterol, hereinafter referred to as HFHC. The control diet was a basic feed without added lipids (Keao Xieli Tianjin Feed Co., Ltd.), hereinafter referred to as Condiet. Mice were randomly divided into the following 4 groups (n=8 mice / group) and underwent 7 days of acclimatization: normal diet group (Con diet), HFHC solvent control group (HFHC-con, solvent being physiological saline supplemented with 0.5% Tween-80 and 0.5% sodium carboxymethyl cellulose), HFHC diet plus 60 mg / kg lovastatin by gavage group (HFHC-Lov), and HFHC diet plus 60 mg / kg lipoadenosine by gavage group (HFHC-i6A), administered once daily by gavage. After 8 weeks of drug administration, liver tissue samples were collected from the mice for analysis of relevant indicators. Lovastatin is reported to be an inhibitor of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR), thereby reducing cholesterol synthesis in the liver and regulating liver lipid metabolism.

[0036] Determination of total cholesterol and triglycerides in liver. Liver tissue was collected after mouse sacrifice, approximately 50 mg of liver was taken and its weight recorded. 500 L of methanol and one 3 mm steel ball were added to a 1.5 mL centrifuge tube, and the liver tissue was homogenized using a cryo-homogenizer. The tissue fluid was vortexed and mixed thoroughly. 200 μL of the homogenate was then collected. L tissue homogenate, add 100 L frozen methanol and 700 L frozen methyl tert-butyl ether (MTBE), vortexed for 2 min, then added 50 L water. Centrifuge at 7500 rpm for 20 min at 4℃, aspirate the upper MTBE phase into a clean glass bottle, freeze-dry, and then use 200 The dried sample was dissolved in L phosphate-buffered saline (PBS, 1×, pH=7.4). Total cholesterol (TC) in the liver was determined using the Amplex™ Red Cholesterol Assay Kit (Thermo Fisher Scientific), and triglyceride (TG) content was determined using a tissue cell TG enzymatic assay kit (Beijing Pulilai Gene Technology Co., Ltd.), according to the manufacturer's instructions. Results are as follows... Figure 1 As shown in the BC diagram, lipoadenosine significantly reduced the levels of total cholesterol and triglycerides in the liver, with a more pronounced decrease in triglycerides than lovastatin, indicating that lipoadenosine significantly reduced lipid accumulation in hepatocytes.

[0037] Liver oil red O staining analysis. First, frozen liver samples were embedded using OCT embedding medium (Sakura, 4583). Then, the liver samples were cut into 8 sections using a cryostat. The sections were sliced ​​and adhered to a slide to prevent detachment. The Oil Red O staining procedure was as follows: (1) Dilute the saturated Oil Red O solution with distilled water at a ratio of 2:3 to prepare the Oil Red O working solution; (2) Bring the frozen sections to room temperature and incubate with the Oil Red O working solution for 10 min; (3) Wash with 60% isopropanol and distilled water; (4) Incubate with hematoxylin dye for 3 min; (5) Rinse with distilled water; (6) Finally, mount with glycerol. Images were taken at 20x magnification using an Olympus VS200 slide scanner and quantitatively analyzed using ImageJ. Figure 4 As shown in Figure D, Oil Red O is a specific staining for lipid droplets, and a HFHC diet leads to the accumulation of lipid droplets in the liver. Administration of lovastatin and lipoadenosine significantly reduced the formation and size of lipid droplets in the liver, with lipoadenosine showing a more potent effect.

[0038] Liver lipidomics analysis. Lipidomics studies were conducted to further analyze changes in fatty acids, triglycerides, and other lipids in the mouse liver. (Add 100...) L frozen methanol and 700 L MTBE, vortex oscillation for 2 min, then add 50 L water. Centrifuge at 7500 rpm for 20 min at 4℃, aspirate the upper MTBE phase into a clean glass bottle, freeze-dry, and re-adjust the volume of the freeze-dried sample to 200 L by adding a chloroform:methanol:water mixture (1:2:0.8, v:v:v). L. A high-performance liquid chromatography-mass spectrometry (UHPLC-TripleTOF 5600+, AB SCIEX) system was used, equipped with an Acquity UPLC CSHC18 column (100 mm × 2.1 mm, 1.7 m²). (m), using Acquity VanGuard CSH C18 pre-pillars (5 × 2.1mm; 1.7m). The mobile phases used were acetonitrile / water (6:4, v:v) + 10 mM ammonium formate + 0.1% formic acid (aqueous phase, A) and isopropanol / acetonitrile (9:1, v:v) + 10 mM ammonium formate + 0.1% formic acid (organic phase, B). The flow rate was set at 0.4 mL / min, the column temperature at 55 °C, and the injection volume at 5 mL / min. The specific settings for the mobile phase gradient are as follows: the initial mobile phase is 15% B; B increases from 15% to 30% within 0-2 min, to 48% within 2-2.5 min, to 82% within 2.5-11 min, to 99% within 11-11.5 min and maintains this level until 12 min; then, B decreases to 15% within 12-12.1 min and maintains this level until 15 min. The mass spectrometry data acquisition mode is IDA, and the mass spectrometry scan range is set to 50-1700 (m / z). Raw data were systematically processed using MS-DIAL (v5.5). First, metabolic features were extracted using a peak identification algorithm, and secondary mass spectrometry fragment signals of co-eluted lipids were analyzed using deconvolution technology. Then, background noise was filtered by comparing with blank samples, and peak alignment across samples was performed based on a retention time drift correction algorithm. Annotation was performed using the software's built-in lipid database. Statistical analysis and data normalization were performed on the obtained results, as shown below. Figure 1 As shown in Figure E, a high-sugar, high-fat, and high-cholesterol diet leads to a significant upregulation of various lipids in the liver, such as fatty acids (FA 18:1, etc.), triglycerides (TG 50:2, etc.), and bile acids (…). α-mouse bile acid, ceramide (Cer), and various phospholipids (LPE, PI, PC, PE, and PG) were significantly increased, and all of these lipids were significantly downregulated after treatment with lovastatin and lipoadenosine.

[0039] The testing process is as follows Figure 2 As shown in Figure A. Eight-week-old male C57BL / 6J mice (Beijing Vital River Laboratory Animal Technology Co., Ltd.) were purchased and fed a 60 kcal% high-fat diet (Research Diets, D12492, hereinafter referred to as HFD) for 16 weeks to induce the formation of metabolic-related fatty liver disease. A 10 kcal% control diet (Research Diets, D12450B, hereinafter referred to as Con diet) was used. Mice were randomly divided into the following three groups (n=6-8 / group): a normal diet group (Con diet), a control group (HFD-con, with HFD and solvent added to drinking water, solvents being 0.5% Tween-80 and 0.2% DMSO), and a group (HFD-i6A) with HFD and lipoadenosine added to drinking water at a concentration of 1 mg / mL. Mice weighing 50 g drank an average of 5 mL of water per day, and the i6A dosage was 100 mg / kg / day. After 20 weeks of administration, the mice were sacrificed and blood and liver were collected for subsequent analysis.

[0040] Calculation of liver-to-body weight ratio in mice. After drug administration, mouse livers were collected and weighed, and the liver-to-body weight ratio (Liver / body weight %) was calculated by comparing it to the mouse's body weight. Results are as follows: Figure 2In patients with hepatitis B, HFD significantly increased the liver-to-body ratio, with the absolute weight gain of the liver exceeding the weight gain, indicating edema and fatty degeneration within the liver. Following treatment with lipoadenosine, the liver-to-body ratio decreased, suggesting that lipoadenosine reduced hepatomegaly and fatty accumulation.

[0041] Determination of blood biochemical parameters in mice. Blood was collected after sacrifice of mice, allowed to clot at room temperature for 1 hour, and then centrifuged at 2000 g for 10 min at 4°C. The supernatant was collected. All blood biochemical parameters were measured using a blood biochemistry analyzer. The results of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in mouse blood are as follows: Figure 2 As shown in C and D, these are markers of liver damage. Lipoadenosine significantly reduces the levels of ALT and AST in the blood, indicating that lipoadenosine can reduce liver damage. The results for total cholesterol (TC) and low-density lipoprotein (LDL) in mouse blood are shown below. Figure 2 As shown in Figures E and F. High total cholesterol (TC) indicates abnormal blood lipids, while high low blood lipid (LDL) is the most direct risk factor for atherosclerosis and cardiovascular health. Lipoadenosine can significantly reduce the levels of TC and LDL in the blood, indicating that it has a good lipid-lowering effect and is of great significance in protecting the cardiovascular system.

[0042] The methods for determining total cholesterol and triglycerides in mouse liver were the same as in Example 1. The results are as follows: Figure 2 As shown in G and H, lipoadenosine significantly reduced the levels of total cholesterol (TC) and triglycerides (TG) in the liver, indicating that lipoadenosine significantly reduced lipid accumulation in hepatocytes.

[0043] The specific Oil Red O staining analysis of mouse liver lipid droplets was the same as in Example 1. The results are as follows: Figure 3 As shown in Figure A, levodopa significantly reduced the number and size of lipid droplets in hepatocytes.

[0044] Histopathological analysis of mouse liver. Hematoxylin-eosin (HE) staining was performed on paraffin sections of liver tissue. After fixation in 4% paraformaldehyde and paraffin embedding, the tissue was cut into 5-micrometer thick sections. The sections were baked at 60°C for 30 minutes, dewaxed with xylene, and rehydrated with a series of ethanol solutions. Hematoxylin-eosin staining was performed on the sections, followed by dehydration with 80%, 90%, and 100% ethanol sequentially, followed by clearing with xylene and mounting with neutral resin. Images were captured at 20x magnification using an Olympus VS200 slide scanner, and quantitative analysis was performed using ImageJ. The tissues were scored according to hepatic steatosis (0-3), ballooning degeneration (0-2), and intralobular inflammation (0-3). The results are as follows: Figure 3 As shown in Figure B, the liver in HFD exhibits numerous vesicular lipid droplets, ballooning degeneration, and a small number of aggregated inflammatory cells. Lipoadenosine significantly reduces these features of metabolic-related fatty liver disease.

[0045] Liver lipidomics analysis. The analytical method was the same as in Example 1 above, and the results are as follows: Figure 3 As shown in Figure C, HFD significantly upregulated multiple lipids in the liver, such as fatty acids (FA 20:4, etc.), diglycerides (DG 33:1, etc.), triglycerides (TG 38:6, etc.), bile acids (BA 24:1, etc.), cholesterol esters (CE 18:2, etc.), sterol esters (SE), sphingomyelin (SM), carnitine (CAR), ceramide (Cer), and various phospholipids (LPA, LPC, LPE, PA, PC, PE, PG, and PI). Following lipoadenosine administration, the levels of these lipids in the liver were significantly downregulated, reshaping hepatic metabolic homeostasis.

[0046] In summary, Figure 2 and Figure 3 Data indicate that levodopa can reduce lipid accumulation in the liver and has a therapeutic effect on metabolic-associated fatty liver disease.

[0047] Excess weight is a major characteristic of obesity. Of particular concern is central obesity, characterized by the accumulation of white adipose tissue in the abdomen, which poses significant health risks. When adipose tissue stores excess energy, its weight increases, and fat cells enlarge. High-sugar, high-fat, high-cholesterol (HFHC) diets and 60 kcal% high-fat diets (HFD) are also commonly used models of mouse obesity, allowing for the simultaneous study of the effects of levodopaminergic agonism on obesity. The experimental design was the same as in Examples 1 and 2. Mouse body weight and food intake were measured weekly. After drug administration, white adipose tissue from the abdomen was collected, weighed, and subjected to pathological analysis.

[0048] Lipoadenosine reduces body weight gain caused by a high-sugar, high-fat, high-cholesterol (HFHC) diet. Figure 4 In mice on the high-fat, high-cholesterol diet (HFHC), body weight gradually increased. After administration of lipoadenosine, body weight gradually decreased compared to the HFHC group, with the difference appearing at week 7 and persisting into week 8. Food intake was monitored; due to the high energy density of the high-fat diet, food intake decreased in the HFHC group compared to the normal diet, but there was no difference in food intake between the lipoadenosine group and the HFHC group. Lipoadenosine can alleviate obesity induced by a high-sugar, high-fat, and high-cholesterol diet without affecting appetite.

[0049] Lipoadenosine reduces the accumulation of white abdominal fat caused by a high-sugar, high-fat, high-cholesterol (HFHC) diet. Figure 4 As shown in Figure B, the HFHC diet significantly increased the weight of abdominal white adipose tissue (WAT), increasing the accumulation of abdominal fat in mice. After administration of lipoadenosine, the weight of white adipose tissue decreased significantly, indicating that lipoadenosine significantly reduced the accumulation of abdominal white adipose tissue.

[0050] Lipoadenosine inhibited the enlargement of abdominal white fat cells induced by a high-sugar, high-fat, and high-cholesterol (HFHC) diet. Hematoxylin-eosin (HE) staining analysis was performed on paraffin sections of white adipose tissue, following the same procedure as in Example 2. Results are as follows... Figure 4 As shown in Figure C, under the HFHC diet, adipocytes store excess energy, leading to an increase in adipocyte size. Lipoadenosine significantly shrinks adipocytes, inhibiting the cell enlargement process and reducing the accumulation of white fat in the abdomen.

[0051] Lipoadenosine reduces weight gain caused by a high-fat diet (HFD) by 60 kcal. Figure 4 As shown in Figure D, mice treated with lipoadenosine experienced a decrease in body weight compared to the HFD group, with the difference becoming apparent at week 20. Food intake was also monitored, demonstrating that lipoadenosine could alleviate obesity induced by a high-fat diet without affecting appetite.

[0052] Lipoadenosine reduces the fat content of white abdominal fat caused by a high-fat diet (HFD) with a 60 kcal reduction. Figure 4 As shown in Figure E, after administration of lipoadenosine, the weight of white adipose tissue decreased significantly, indicating that lipoadenosine significantly reduced the accumulation of white adipose tissue in the abdomen.

[0053] Lipoadenosine inhibited the enlargement of abdominal white fat cells induced by a 60 kcal% high-fat diet (HFD). Figure 4 As shown in Figure F, levodopa significantly shrinks adipocytes, inhibits cell enlargement, and reduces the accumulation of white fat in the abdomen.

[0054] In summary, Figure 4 Data shows that levodopa can reduce the accumulation of white fat in the abdomen and reduce weight, thereby achieving a significant effect in reducing obesity.

[0055] A mature human liver organoid can realistically simulate the microenvironment of the human liver, providing a predictive in vitro model that closely resembles the human liver in structure and function.

[0056] Human liver organoids were obtained from DaXiang Technology (Liver Organoid Expansion and Differentiation Kit). The resuscitation, passage, cryopreservation, and differentiation of the organoids were all performed according to the DaXiang Technology kit instructions, resulting in mature differentiated liver organoids. Differentiated liver organoids were treated with lipoadenosine M for 72 hours. The culture medium was removed from the plate, and the cells were washed twice with PBS. They were then fixed in 4% tissue cell fixative at 37°C in the dark for 1 hour, followed by washing twice with PBS. Cell staining solution was prepared using PBS, with 7AAD diluted 1:50 for labeling cell nuclei, and Filipin Complex concentration was 200 μL. g / mL was used to label free cholesterol. The staining solution was added to the plate and stained in a 37°C incubator in the dark for 1 hour. After staining, the staining solution was removed from the plate, and the plate was washed twice with PBS.

[0057] Images were acquired using the confocal mode of a Nikon A1RSi+ laser scanning confocal microscope. The excitation wavelength for the detection channel of 7AAD in the cell nucleus was 560.5 nm, and the excitation wavelength for free cholesterol Filipin was 403.4 nm. The interval between scans was set to 5 nm. The number of scan layers varies from 18 to 22, depending on the size of the organoid. The maximum intensity on the z-axis of the scan results is projected to obtain a two-dimensional planar projection image of the organoid. The built-in deconvolution function of the software is used to sharpen and enhance the image, and ImageJ software is used for quantitative analysis.

[0058] The specific calculation process is as follows: a uniform threshold is set based on fluorescence intensity. Positive areas exceeding this threshold are considered the staining area of ​​free cholesterol, and the 7AAD staining range is considered the organoid area. The ratio of these two is the Filipin staining percentage (Filipin stain area%). The results are as follows: Figure 5 As shown in Figures A and B, the level of free cholesterol in organoids was significantly downregulated after treatment with levodopa.

[0059] In summary, Figure 5 Data shows that levodopa can significantly reduce the level of free cholesterol in human liver organoids.

[0060] To further investigate the mechanism of action of lipoadenosine, transcriptome sequencing analysis of lipoadenosine-treated hepatocytes (HepG2) was performed. HepG2 cells were seeded in 6-well plates at 500,000 cells per well and cultured overnight in a cell culture incubator to allow adherence. Lipoadenosine was diluted with complete cell culture medium (EMEM mixture containing 10% bovine serum and 1% penicillin / streptomycin) to a final concentration of 1 mg / well. Cells were cultured in triplet wells with complete medium containing 0.01% DMSO as a control for 48 hours. Total RNA was extracted from the cells using Trizol, and RNA integrity and total amount were accurately detected using an Agilent 2100 bioanalyzer. The starting RNA for library construction was total RNA. Poly(A) tailed mRNA was enriched using Oligo(dT) magnetic beads and randomly fragmented using divalent cations to obtain fragmented mRNA as a template. The first strand of cDNA was synthesized using random oligonucleotides as primers. The RNA strand was degraded, and the second strand of cDNA was synthesized using dNTPs. The purified doublet cDNA underwent repair, screening, amplification, and purification before being quantified using a luorometer and an Agilent 2100 bioanalyzer to ensure library quality. After passing library inspection, sequencing was performed using an Illuminanovaseq 6000 in paired-end mode. To ensure data quality, the raw data was filtered to remove low-quality reads, obtaining clean reads for subsequent analysis. The HISAT2 software was used to perform rapid and accurate alignment of clean reads with the reference genome. The featureCounts tool in the subread software was used to quantitatively analyze gene expression levels in the samples. DESeq2 was used for significance analysis, and KEGG (Kyoto Encyclopedia of Genes and Genomes database) pathway enrichment analysis was performed on differentially expressed genes (p<0.05). A local version of the GSEA analysis tool was used to perform GSEA analysis on the KEGG dataset.

[0061] The results are as follows Figure 6 As shown in the gene heatmap, levodopa significantly downregulated fatty acid biosynthesis in hepatocytes. Figure 6 (A) Biosynthesis of unsaturated fatty acids ( Figure 6 (B) Biosynthesis of Terpenoid Skeletons ( Figure 6 (C) and steroid biosynthesis pathway ( Figure 6 Key genes of D) and corresponding GSEA analysis results ( Figure 6 The EH (Chinese version) also indicates this conclusion.

[0062] Lipomics analysis was performed to analyze changes in fatty acids and triglycerides in hepatocytes after treatment with lipoadenosine. HepG2 cells (purchased from ATCC) were seeded in 10 cm culture dishes and incubated overnight to allow adhesion. Lipoadenosine was serially diluted with complete cell culture medium to a final concentration of 1 g / L in the dishes. M, with complete medium containing 0.01% DMSO as a control, was used in 4 replicates. Cells were cultured in an incubator for 48 hours. The culture medium was removed from the dish, and the cells were quenched with liquid nitrogen. Cells were collected in 1.5 mL centrifuge tubes using 1 mL of ultrapure water and a cell scraper. The samples were then homogenized using a non-contact sonicator (Bioruptor) at 4°C with a program of 10 seconds of sonication followed by a 15-second pause, for a total of 60 cycles. 200 Add 700 to L cell fluid L of frozen MTBE and 50 The sample was treated with ultrapure water (L), sonicated for 30 min, and centrifuged at 7500 rpm for 20 min (both sonication and centrifugation were performed at 4℃). The supernatant was then transferred to a 1.5 mL vial for lyophilization. The lyophilized sample was reconstituted to 200 mL using a chloroform:methanol:water mixture (1:2:0.8, v:v:v). L, and using 0.22 The sample was filtered through a PFTE membrane and then subjected to lipidomics analysis. The analytical method was the same as in Example 1.

[0063] The results are as follows Figure 6 As shown in Figure I, after treatment with levoprine, the levels of various fatty acids (FA) and triglycerides (TG) in HepG2 cells were significantly downregulated.

[0064] Analysis of free cholesterol in hepatocytes. HepG2 cells were seeded in 96-well plates at 30,000 cells per well and incubated overnight in a cell culture incubator to allow adhesion. Lipoadenosine was serially diluted with complete cell culture medium to a final concentration of 1 mg / L in the plate. M, 0.5 Cells were cultured at concentrations of 250 nM, 125 nM, 62.5 nM, and 31.25 nM, with a complete culture medium containing 0.01% DMSO as a control. To ensure accuracy, each concentration was tested in triplicate, and cells were cultured for 48 hours. Cells were fixed in 4% tissue fixative (Solepro, P1110) at room temperature in the dark for 30 minutes, followed by washing twice with PBS. Cell staining solutions were prepared using PBS, with 7AAD (BD Biosciences, 559925) diluted 1:100 for labeling nuclei, and Filipin Complex (MCE, HY-N6716) at a concentration of 100... g / mL was used to label free cholesterol. The staining solution was added to the plate and stained at room temperature in the dark for 1 hour. After staining, the staining solution was removed, the plate was washed twice with PBS, and immediately analyzed using a high-content cell imaging analysis system (ImageXpress). At least 5 fields of view were selected per well, and images were acquired using a 20x lens. The fluorescence image of each field of view was analyzed, and the area of ​​all fluorescently stained cells in the same image was calculated by normalizing to the total cell count, thus obtaining the mean stained area (MSA). Results are as follows: Figure 6 As shown in Figure J, levodopa-adenosine reduces intracellular free cholesterol levels in HepG2 cells in a dose-dependent manner. Figure 6 The lipid molecular changes in I and J in hepatocytes were consistent with the pathway results from the hepatocyte transcriptome analysis.

[0065] In summary, Figure 6 Data shows that levodopa can significantly inhibit the synthesis of fatty acids and cholesterol in hepatocytes, thereby reducing the levels of fatty acids, triglycerides, and free cholesterol in hepatocytes.

[0066] References:

[0067] [1] LAEZZA C, MALFITANO AM, DI MATOLA T, et al. Involvement of Akt / NF‐κB pathway in N6‐isopentenyladenosine‐induced apoptosis in human breastcancer cells [J]. Molecular Carcinogenesis, 2010, 49(10): 892‐901.

[0068] [2] RANIERI R, CIAGLIA E, AMODIO G, et al. N6-isopentenyladenosinedual targeting of AMPK and Rab7 prenylation inhibits melanoma growth through the impairment of autophagic flux [J]. Cell Death Differ, 2018, 25(2): 353-67.

[0069] [3] CIAGLIA E, ABATE M, LAEZZA C, et al. Antiglioma effects of N6-isopentenyladenosine, an endogenous isoprenoid end product, through thedownregulation of epidermal growth factor receptor [J]. International Journal of Cancer, 2016, 140(4): 959-72.

[0070] [4] PAGANO C, NAVARRA G, COPPOLA L, et al. N6-isopentenyladenosineinduces cell death through necroptosis in human glioblastoma cells [J]. CellDeath Discovery, 2022, 8(1).

[0071] The above description, in conjunction with specific embodiments, provides a further detailed explanation of the present invention. It should not be construed that the specific implementation of the present invention is limited to these descriptions. For those skilled in the art, several simple deductions or substitutions can be made without departing from the concept of the present invention, and all such deductions or substitutions should be considered to fall within the scope of protection defined by the claims submitted herein.

Claims

1. The use of lipoadenosine in the preparation of a medicament for reducing obesity in subjects, characterized in that, The drug is used to reduce the accumulation of white fat in the abdomen of the subjects.

2. The use of lipoadenosine in the preparation of a medicament for the prevention or improvement of obesity-related metabolic diseases in subjects, characterized in that, The obesity-related metabolic disease mentioned is metabolic-associated fatty liver disease (MAFLD).

3. The application according to claim 2, characterized in that, The drug reduces total cholesterol, triglycerides, and fatty acids in the liver of the subjects, and reduces the number and size of lipid droplets in the liver.

4. The application according to claim 2, characterized in that, The subjects were mammals.

5. The application according to claim 2, characterized in that, The subjects were humans.

6. The application according to claim 2, characterized in that, The drug also includes pharmaceutically acceptable excipients.

7. The application according to claim 1, characterized in that, The dosage forms of the drug include tablets, capsules, granules, liquid preparations, and gels.

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