Application of integrin beta3 inhibitor RGDfK in preparation of medicine for treating fatty liver disease related to metabolic dysfunction

Abstract

The invention relates to the technical field of medicines, in particular to application of an integrin beta3 inhibitor RGDfK in preparation of a medicine for treating metabolic dysfunction related fatty liver diseases. The RGDfK provided by the invention can inhibit palmitoylation and membrane localization of CD36 in liver tissues of mice with fatty liver diseases related to metabolic dysfunction induced by high fat diet feeding, so that lipid uptake of the liver is inhibited. Experimental results show that RGDfK can improve obesity and insulin resistance of mice with metabolic dysfunction-related fatty liver diseases, can improve fat accumulation, fibrosis and inflammatory infiltration of liver tissues of the mice with fatty liver diseases, and is expected to become a new medicine for treating the metabolic dysfunction-related fatty liver diseases.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and in particular to the use of the integrin β3 inhibitor RGDfK in the preparation of a medicament for the treatment of fatty liver disease associated with metabolic dysfunction. Background Technology

[0002] Metabolic dysfunction-associated fatty liver disease (MASLD) has become a major global health problem, affecting more than 30% of the adult population and causing serious morbidity and mortality. Its progressive inflammatory subtype—metabolic dysfunction-associated steatohepatitis (MASH)—pathologically manifests as hepatocellular steatosis, ballooning degeneration, lobular inflammation, and varying degrees of fibrosis. Approximately 20% to 30% of MASLD patients will develop MASH, a transformation that significantly increases the risk of cirrhosis, hepatocellular carcinoma (HCC), and liver failure, placing a heavy burden on healthcare systems worldwide. Despite this, drug treatment options remain limited. Recently, the U.S. Food and Drug Administration approved the thyroid hormone receptor-β agonist retemiromeuron, a significant milestone; however, in pivotal trials, only 25% to 30% of treated patients achieved a histological response. This underscores the urgent need for further research into disease-specific pathways related to the pathogenesis of MASH and for identifying potential therapeutic targets.

[0003] Mounting evidence suggests that components of the extracellular matrix (ECM), in addition to their supportive role, are involved in liver pathological changes. Excessive ECM deposition and associated transmembrane signaling abnormalities can trigger liver fibrosis, carcinogenesis, and metabolic reprogramming. ECM remodeling regulates hepatocyte function through mechanotransduction and receptor-mediated signaling, thereby affecting metabolic homeostasis. Specific ECM components have been shown to alter lipid accumulation in hepatocytes, while matrix stiffness activates YAP-dependent adipogenic transcriptional programs. These findings indicate that the role of ECM components in regulating hepatic lipid metabolism in the pathogenesis of MASH has been underappreciated. Integrins are a class of heterodimeric transmembrane receptors that play a crucial role in regulating bidirectional signal transduction between cells and the extracellular matrix. Among integrins, integrin β3 (ITGB3) has been extensively studied for its typical roles in cellular processes including adhesion, endocytosis, survival signaling, angiogenesis, and proliferation; targeted therapies against it have been applied to cardiovascular, oncology, and ophthalmic diseases. Notably, we recently discovered that ITGB3 plays a role in the temporal regulation of RhoA activation in renal epithelial cells. Although ITGB3 has been shown to regulate endothelial cell damage and local lipid accumulation in the vascular wall during atherosclerosis by mediating cell adhesion and survival signaling pathways, current research remains limited to these classic mechanistic models. Crucially, whether extracellular matrix remodeling regulates hepatocyte lipid metabolism through ITGB3, and whether this regulation is achieved through its unconventional membrane scaffold function, remains an unexplored area in the pathogenesis of MASH. Currently, there are no reports on the use of the ITGB3 inhibitor RGDfK in the preparation of drugs for the treatment of MASLD. Summary of the Invention

[0004] The purpose of this invention is to provide the application of the integrin β3 inhibitor RGD in the preparation of a medicament for treating metabolic dysfunction-related fatty liver disease.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides the use of the integrin β3 inhibitor RGDfK in the preparation of a medicament for treating metabolic dysfunction-related fatty liver disease.

[0006] Preferably, the molecular formula of the integrin β3 inhibitor RGDfK is shown in Formula 1:

[0007] Formula 1.

[0008] Preferably, the drug inhibits fatty acid uptake in the liver by inhibiting palmitoylation and membrane localization of CD36.

[0009] This invention provides the use of the integrin β3 inhibitor RGDfK in the preparation of a medicament for treating obesity and / or insulin resistance.

[0010] This invention provides the use of the integrin β3 inhibitor RGDfK in the preparation of a medicament for inhibiting lipid uptake in metabolic dysfunction-related fatty liver disease.

[0011] Preferably, the drug is administered via intraperitoneal injection.

[0012] Preferably, the dosage of the drug is 4-10 mg / kg.

[0013] Preferably, the drug further includes a pharmaceutically acceptable carrier or excipient.

[0014] Compared with the prior art, the present invention has the following beneficial effects: The integrin β3 inhibitor RGDfK of this invention effectively inhibits hepatic lipid uptake by suppressing palmitoylation and membrane localization of CD36 in the liver tissue of mice with high-fat diet-induced metabolic dysfunction-associated fatty liver disease (MASLD). Treatment results showed that RGDfK significantly improved the obesity phenotype and insulin resistance in MASLD mice and effectively reduced fat accumulation, fibrosis, and inflammatory infiltration in the liver tissue. These findings not only indicate that RGDfK holds promise as a novel drug for treating MASLD but also provide important evidence for exploring therapeutic targets for this disease.

[0015] This invention specifically provides the application of the integrin β3 inhibitor RGDfK in the preparation of a drug for inhibiting lipid uptake in MASLD. Its mechanism of action is that RGDfK improves hepatic steatosis by inhibiting hepatic lipid uptake. Compared with untreated fatty liver model mice, mice in the RGDfK treatment group showed significant weight loss, decreased plasma triglyceride levels, improved insulin resistance, and a significantly reduced degree of hepatic steatosis. Further research revealed reduced membrane localization of CD36 in the liver of the treatment group mice, indicating weakened fatty acid uptake capacity. In vitro experiments also confirmed that RGDfK can effectively inhibit the uptake of medium- and long-chain fatty acids by hepatocytes. Attached Figure Description

[0016] Figure 1 The effect of RGDfk on the obesity phenotype in non-alcoholic fatty liver disease (where A represents a schematic diagram of the fatty liver model construction, B represents mouse body weight, C represents mouse liver weight ratio, D represents mouse epididymal fat statistics, and E represents mouse subcutaneous fat statistics. Vehicle represents the fatty liver group, and RGDfk represents the fatty liver + RGDfk treatment group).

[0017] Figure 2The effects of RGDfk on insulin resistance and blood lipids in fatty liver (A represents the results of glucose tolerance in mice, B represents the statistical graph of glucose tolerance in mice, C represents the graph of triglycerides in venous blood in mice, D represents the graph of cholesterol in venous blood in mice, and E represents the graph of triglycerides in liver tissue in mice. Vehicle represents the fatty liver group, and RGDfk represents the fatty liver + RGDfk treatment group).

[0018] Figure 3 The effect of RGDfk on fatty liver (A represents HE staining and ORO staining of mouse liver, B represents the statistical graph of liver NAS score, and C represents the statistical graph of liver lipid droplets. Vehicle represents the fatty liver group, and RGDfk represents the fatty liver + RGDfk treatment group).

[0019] Figure 4 The effect of RGDfk on lipid uptake in fatty liver mice (A represents the expression level of total CD36 protein in each group of mice, B represents the expression level of CD36 membrane protein in each group of mice, and C represents the level of CD36 palmitoylation in each group of mice. Vehicle represents the control group, and RGDfk represents the fatty liver + RGDfk treatment group).

[0020] Figure 5 The effect of RGDfk on fatty acid uptake in hepatocytes (A represents the Bodipy staining fluorescence image of hepatocytes, B represents the C16-BODIPY detection results by flow cytometry. Vehicle represents the PA group, and RGDfk represents the PA+RGDfk treatment group). Detailed Implementation

[0021] The male C57BL / 6J described in this embodiment of the invention was purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.

[0022] All experimental data in the examples are presented as mean ± standard error. All graphs and statistical analyses were performed using GraphPadPrism8 software. Differences between two groups were compared using the unpaired t-test, and differences between multiple groups were analyzed using one-way ANOVA. A p-value less than 0.05 was considered statistically significant.

[0023] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0024] Example 1: Effects of RGDfk on obesity and insulin resistance in fatty liver mice

[0025] according to Figure 1Method A uses a high-fat diet (HFD) to induce MASLD (metabolic dysfunction-associated fatty liver disease), followed by intraperitoneal injection of the drug after 12 weeks of continuous feeding.

[0026] The experiment was divided into two groups: a fatty liver + placebo group and a fatty liver + RGDfk group. Mice in each group received a high-fat diet for 12 weeks, followed by intraperitoneal injection of the drug once daily for 4 weeks. RGDfk was dissolved in physiological saline to a final concentration of 5 mg / kg (e.g., ...). Figure 1 A). Record the weight of the mice during the feeding process and conduct a glucose tolerance test in the last week.

[0027] After euthanizing the mice, epididymal and subcutaneous adipose tissue were extracted and weighed. Blood was collected from the carotid-orbital artery, perfused with PBS, and tissue samples such as the liver were collected for subsequent experiments. Figure 1 and Figure 2 As shown, the drug treatment group significantly improved body weight, fat mass, glucose intolerance, and dyslipidemia in hyperlipidemia-induced mice. Therefore, RGDfk can improve systemic metabolic conditions such as obesity, dyslipidemia, and glucose intolerance.

[0028] Example 2: Effects of RGDfk on the liver of fatty liver mice

[0029] The liver tissues of mice in each group in Example 1 were stained using HE staining and ORO staining methods, and slides were prepared to observe the effects of RGDfk on the degree of liver fibrosis and fat accumulation in fatty liver mice. The results are as follows: Figure 3 As shown, HE staining can show improvement in fibrosis in mice, and ORO staining can show improvement in fat accumulation in mice.

[0030] The above results indicate that RGDfk treatment can significantly improve lipid accumulation and fibrosis in the liver of fatty liver mice.

[0031] Example 3: Effect of RGDfk on lipid uptake in fatty liver mice

[0032] Liver tissue from the mice in Example 1 was collected, and total protein was extracted from the liver tissue. Membrane proteins were extracted from the liver tissue using a cell membrane separation kit. The level of CD36 palmitoylation in the liver tissue was detected using the IP-ABE method.

[0033] Figure 4 The results showed that the total CD36 protein level remained unchanged in the liver tissue of mice in the fatty liver + RGDfk group, while the membrane protein level and palmitoylation level decreased. This indicates that RGDfk significantly inhibited the membrane localization and palmitoylation of CD36 in fatty liver mice.

[0034] Example 4: Effect of RGDfk on fatty acid uptake in hepatocytes

[0035] To verify the effect of RGDfk on hepatic lipid uptake, HepG2 cells were stimulated with palmitic acid (PA, 0.2 μM) for 24 h, followed by intervention with RGDfk (40 μM). Bodipy staining was then performed to assess lipid droplet accumulation. To further verify the effect of RGDfk on the uptake of medium- and long-chain fatty acids, HepG2 cells were stimulated with palmitic acid, followed by intervention with RGDfk. After 1 h of intervention, cells were incubated with C16-BODIPY for half an hour, and then fatty acid uptake was detected by flow cytometry (C16-BODIPY detection can indicate the uptake of medium- and long-chain fatty acids). Figure 5 As shown, RGDfk inhibits the uptake of long-chain fatty acids in hepatocytes.

[0036] Experimental results confirm that RGDfK involved in this invention has a therapeutic effect on fatty liver disease related to metabolic dysfunction.

[0037] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Application of integrin β3 inhibitor RGDfK in the preparation of drugs for the treatment of metabolic dysfunction-related fatty liver disease.

2. The application according to claim 1, characterized in that, The molecular formula of the integrin β3 inhibitor RGDfK is shown in Formula 1: Formula 1.

3. The application according to claim 1 or 2, characterized in that, The drug inhibits fatty acid uptake in the liver by suppressing palmitoylation and membrane localization of CD36.

4. The use of an integrin β3 inhibitor RGDfK in the preparation of a medicament for the treatment of obesity and / or insulin resistance.

5. The use of an integrin β3 inhibitor RGDfK in the preparation of a medicament for inhibiting lipid uptake in metabolic dysfunction-related fatty liver disease.

6. The application according to claim 5, characterized in that, The drug is administered via intraperitoneal injection.

7. The application according to claim 6, characterized in that, The dosage of the drug is 4-10 mg / kg.

8. The application according to claim 5, characterized in that, The drug also includes pharmaceutically acceptable carriers or excipients.

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