Use of hydronidone in treating metabolic dysfunction-associated steatotic liver disease

Abstract

The present invention relates to use of hydronidone or a pharmaceutically acceptable salt thereof in treating metabolic dysfunction-associated steatotic liver disease (MASLD). The present invention can ameliorate liver injury, inflammatory responses, and lipid deposition caused by MASLD. Moreover, hydronidone can alleviate the progression of MASLD by regulating the CD36 / AMPK signaling pathway. The present invention provides a novel approach and strategy for treating MASLD.

Description

Application of hydroxynidone in the treatment of fatty liver disease associated with metabolic dysfunction

[0001] This invention claims priority to the prior application filed by the applicant with the China National Intellectual Property Administration on January 25, 2025, with patent application number 202510120100.6 and invention title "Application of Hydroxynidone in the Treatment of Fatty Liver Disease Related to Metabolic Dysfunction", the contents of which are incorporated herein by reference. Technical Field

[0002] This invention belongs to the field of pharmaceutical technology, specifically relating to the use of hydroxynidone or a pharmaceutically acceptable salt thereof in the treatment of fatty liver disease associated with metabolic dysfunction. Background Technology

[0003] Metabolic dysfunction-associated fatty liver disease (MASLD) is a chronic liver disease characterized by excessive lipid deposition in the liver and various metabolic dysfunctions. Formerly known as non-alcoholic fatty liver disease (NAFLD), MASLD was officially renamed MASLD in 2022. As the disease progresses, the damage to the liver increases. The scope of NAFLD / MASLD includes non-alcoholic metabolic dysfunction-associated fatty liver (NAFL / MAFL) and non-alcoholic steatohepatitis / metabolic dysfunction-associated steatohepatitis (NASH / MASH), both of which can develop into more serious liver diseases such as liver fibrosis, cirrhosis, and even hepatocellular carcinoma.

[0004] Statistics show that the prevalence of MASLD is close to 30% globally, making it a serious public health problem. Increased lipid deposition in the liver leads to continued deterioration of inflammatory responses and liver fibrosis. Currently, only resimeltiro is officially approved by the U.S. Food and Drug Administration (FDA) for the treatment of NASH. Most drugs for treating MASLD are symptomatic treatments. The development of new drugs for MASLD faces significant challenges.

[0005] Hydroxynidone (HDD) is a small molecule drug modified with pirfenidone, approved by the FDA in 2014 for the treatment of idiopathic pulmonary fibrosis. HDD is a pirfenidone-modified product, giving it better affinity for the liver. Our previous studies have shown that HDD in combination with entecavir can effectively reverse the progression of fibrosis in patients with HBV-related liver fibrosis and reduce Ishak liver fibrosis scores.

[0006] CD36 is a classic scavenger class B receptor, first isolated from platelets in 1989. CD36 has been identified as one of the core molecules in the progression of many metabolic diseases, especially lipid metabolism, and therefore the regulation of CD36 expression has become an important therapeutic target for MASLD. Summary of the Invention

[0007] To address the aforementioned technical problems, this invention provides the use of hydroxynidone or a pharmaceutically acceptable salt thereof in the preparation of a medicament for the treatment or adjunctive treatment of metabolic dysfunction-associated fatty liver disease (MASLD).

[0008] The hydroxynidone (HDD), with CAS number 851518-71-3, molecular formula C12H11NO2, and molecular weight 201.22, has the following structural formula:

[0009] According to embodiments of the present invention, pharmaceutically acceptable salts of hydroxynidone include salts that may be present in acidic or basic groups of hydroxynidone. Examples include sodium salts, calcium salts, potassium salts, hydrobromide salts, sulfate salts, hydrogen sulfate salts, phosphate salts, hydrogen phosphate salts, dihydrogen phosphate salts, acetate salts, succinate salts, citrate salts, tartrate salts, lactate salts, mandelate salts, methanesulfonate salts, and p-toluenesulfonate salts.

[0010] According to an embodiment of the present invention, the metabolic dysfunction-associated fatty liver disease (MASLD) is selected from one or more of the group consisting of non-alcoholic metabolic dysfunction-associated fatty liver (NAFL / MAFL), non-alcoholic steatohepatitis / metabolic dysfunction-associated steatohepatitis (NASH / MASH), and simple hepatic steatosis.

[0011] According to an embodiment of the present invention, the metabolic dysfunction-associated fatty liver disease (MASLD) is selected from MASLD liver injury, MASLD inflammatory response, and MASLD lipid deposition.

[0012] According to embodiments of the present invention, in the above applications, the drug can reduce lipid synthesis, increase lipid breakdown in the liver, and alleviate liver inflammation.

[0013] According to an embodiment of the present invention, in the above-described application, the drug can reduce the levels of ALT and AST in the blood.

[0014] According to an embodiment of the present invention, in the above application, the drug can inhibit the expression of lipid synthesis genes Fasn / Scd1 / Acc1 in the liver and increase the expression of lipid breakdown-related genes Cpt1α / Pgc1α / Ppar-α in the liver.

[0015] According to an embodiment of the present invention, in the above-described application, the drug is able to reduce the expression of α-SMA, Col1, and fibronectin in the liver.

[0016] According to an embodiment of the present invention, in the above-described application, the drug can reduce the expression of M1 pro-inflammatory macrophage markers such as IL-1β / IL-6 / Tnf-α in the liver and increase the expression of M2 anti-inflammatory macrophage markers such as ARG1 / Fizz1 / IL-12 in the liver.

[0017] According to an embodiment of the present invention, in the above-described application, the drug is able to regulate the CD36 / AMPK signaling pathway.

[0018] According to an embodiment of the present invention, in the above-described application, the drug is able to negatively regulate the expression of CD36.

[0019] According to embodiments of the present invention, in the above applications, the drug further includes a pharmaceutically acceptable carrier and / or excipients (such as diluents), suitable for preparation into formulations for intravenous infusion, intravenous drip, subcutaneous administration, intradermal administration, intramuscular injection, oral spray, oral administration, etc.

[0020] According to embodiments of the present invention, in the above applications, the drug may be an oral preparation, an injectable preparation, or an enema liquid preparation. Oral preparations include tablets, capsules, pills, powders, granules, suspensions, drops, etc., and injectable preparations include injection solutions, powder injections, etc.; more preferably, the tablets include ordinary tablets, orally disintegrating tablets, dispersible tablets, or sustained-release tablets.

[0021] According to embodiments of the present invention, in the above applications, the drug may be administered alone or in combination with one or more other therapeutic agents.

[0022] According to an embodiment of the present invention, in the above application, the metabolic dysfunction-associated fatty liver disease (MASLD) is CD36 / AMPK-related metabolic dysfunction-associated fatty liver disease.

[0023] The present invention also provides the use of a pharmaceutical composition containing hydroxynidone or a pharmaceutically acceptable salt thereof in the preparation of a medicament for the treatment or adjunctive treatment of metabolic dysfunction-associated fatty liver disease (MASLD).

[0024] According to an embodiment of the present invention, in the above-described application, subjects receiving treatment or adjunctive treatment for metabolic dysfunction-related fatty liver disease did not exhibit symptoms of fibrosis.

[0025] The present invention also provides a medicament for treating or adjuvant treatment of metabolic dysfunction-associated fatty liver disease (MASLD), comprising hydroxynidone or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier or excipient.

[0026] The present invention also provides a method for treating or adjuvant treatment of metabolic dysfunction-associated fatty liver disease (MASLD), the method comprising administering to a subject in need a therapeutically effective amount of hydroxynidone or a pharmaceutically acceptable salt thereof.

[0027] According to an embodiment of the present invention, the metabolic dysfunction-associated fatty liver disease (MASLD) is selected from non-alcoholic metabolic dysfunction-associated fatty liver (NAFL / MAFL), non-alcoholic steatohepatitis / metabolic dysfunction-associated steatohepatitis (NASH / MASH), and simple hepatic steatosis.

[0028] According to an embodiment of the present invention, the metabolic dysfunction-associated fatty liver disease (MASLD) is pre-activated metabolic dysfunction-associated fatty liver disease (MASLD), including pre-activated non-alcoholic metabolic dysfunction-associated fatty liver (NAFL / MAFL) and pre-activated non-alcoholic steatohepatitis / metabolic dysfunction-associated steatohepatitis (NASH / MASH).

[0029] According to an embodiment of the invention, the subject did not exhibit symptoms of fibrosis. The invention also provides a method for reducing hepatic lipid synthesis and / or increasing lipid breakdown in the liver, the method comprising administering a therapeutically effective amount of hydroxynidone or a pharmaceutically acceptable salt thereof to a subject in need.

[0030] According to an embodiment of the invention, the amount of lipid accumulation in the liver of the subject is reduced by at least 30% relative to the pre-treatment baseline, preferably by at least 40%, and more preferably by at least 50%.

[0031] The present invention also provides a method for preventing the development of fibrosis in nonalcoholic steatohepatitis / metabolic dysfunction-associated steatohepatitis (NASH / MASH), the method comprising administering to a subject in need a therapeutically effective amount of hydroxynidone or a pharmaceutically acceptable salt thereof.

[0032] The present invention also provides a method for preventing the progression of metabolic dysfunction-associated fatty liver disease (MASLD) to fibrotic MASLD, the method comprising administering a therapeutically effective amount of hydroxynidone or a pharmaceutically acceptable salt thereof to a subject in need.

[0033] According to an embodiment of the present invention, the daily dose of the hydroxynidone or its pharmaceutically acceptable salt or analogue is 1-15 mg / kg.

[0034] According to an embodiment of the invention, the subject in need may be a mammal, such as a human.

[0035] The present invention also provides hydroxynidone or a pharmaceutically acceptable salt thereof for the treatment or adjunctive treatment of metabolic dysfunction-associated fatty liver disease (MASLD), or a pharmaceutical composition containing hydroxynidone or a pharmaceutically acceptable salt thereof for the treatment or adjunctive treatment of metabolic dysfunction-associated fatty liver disease (MASLD). Beneficial effects

[0036] This invention validates the role of hydroxynidone or a pharmaceutically acceptable salt thereof in metabolic dysfunction-associated fatty liver disease (MASLD), which can improve liver damage, inflammatory response and lipid deposition in MASLD. Furthermore, hydroxynidone can alleviate the progression of MASLD by modulating the CD36 / AMPK signaling pathway, providing a new treatment method and strategy for MASLD. Attached Figure Description

[0037] Figure 1: Histological staining results of each group in Example 1 (A: H&E staining, B: Oil Red staining, C: IHC-F4 / 80, D: Sirius Red staining), scale bar 50 μm;

[0038] Figure 2: Analysis results of histological staining data of each group in Example 1. The data are shown as SEM ± mean; * p < 0.05 compared with NCD group; # p < 0.05 compared with HFHC group;

[0039] Figure 3: Serum biochemical test results of each group in Example 1. The data are shown as SEM ± mean; * Compared with the NCD group, p<0.05; # Compared with the HFHC group, p<0.05;

[0040] Figure 4: qPCR analysis of lipid metabolism markers in each group in Example 1. Data are shown as SEM ± mean; * p < 0.05 compared with the NCD group; # p < 0.05 compared with the HFHC group;

[0041] Figure 5: Effects of each group on the expression levels of marker genes in M1 and M2 macrophages in Example 1. Data are shown as SEM ± mean; * p < 0.05 compared with the NCD group; # p < 0.05 compared with the HFHC group;

[0042] Figure 6: qPCR analysis of fibrosis markers in each group in Example 1. Data are shown as SEM ± mean; * p < 0.05 compared with NCD group; # p < 0.05 compared with HFHC group;

[0043] Figure 7: Western Blot analysis of fibrosis markers in each group of Example 1;

[0044] Figure 8: Histological staining results of each group in Example 2 (A: H&E staining, B: Oil Red staining, C: IHC-F4 / 80, D: Sirius Red staining), scale bar 50 μm;

[0045] Figure 9: Analysis results of histological staining data of each group in Example 2. The data are shown as SEM ± mean; * p < 0.05 compared with the MCS group; # p < 0.05 compared with the MCD group;

[0046] Figure 10: Serum biochemical test results of each group in Example 2. The data are shown as SEM ± mean; *Compared with the MCS group, p<0.05; #Compared with the MCD group, p<0.05;

[0047] Figure 11: qPCR analysis of lipid metabolism markers in each group in Example 2. Data are shown as SEM ± mean; * p < 0.05 compared with the MCS group; # p < 0.05 compared with the MCD group;

[0048] Figure 12: Effects of each group on the expression levels of marker genes in M1 and M2 macrophages in Example 2. Data are shown as SEM ± mean; * p < 0.05 compared with the MCS group; # p < 0.05 compared with the MCD group;

[0049] Figure 13: qPCR analysis of fibrosis markers in each group in Example 2. Data are shown as SEM ± mean; * p < 0.05 compared with the MCS group; # p < 0.05 compared with the MCD group;

[0050] Figure 14: Western Blot analysis of fibrosis markers in each group in Example 2;

[0051] Figure 15: Experimental results of mpHCs cells in each group in Example 3. The data are shown as SEM ± mean; * p < 0.05 compared with CN group; # p < 0.05 compared with PA group;

[0052] Figure 16: Experimental results of AML12 cells in each group of Example 3. Data are shown as SEM ± mean; * p < 0.05 compared with CN group; # p < 0.05 compared with PA group;

[0053] Figure 17: Effect of each group in Example 3 on lipid deposition of mpHCs, scale bar 100 μm;

[0054] Figure 18: Effect of each group in Example 3 on AML12 lipid deposition, scale bar 100 μm;

[0055] Figure 19: Liver transcriptome sequencing heatmaps of the HFHC group, NC group, HFHC group, and HFHC+50mg / kg HDD group in Example 4.

[0056] Figure 20: Transcriptome sequencing volcano plots of the HFHC group, NC group, and HFHC group and HFHC+50mg / kg HDD group in Example 4;

[0057] Figure 21: GO enrichment analysis of differentially expressed genes in the HFHC and NCD groups in Example 4;

[0058] Figure 22: GO enrichment analysis of differentially expressed genes in the HFHC+50mg / kg HDD group and HFHC group in Example 4;

[0059] Figure 23: KEGG enrichment analysis of differentially expressed genes in the HFHC and NCD groups in Example 4;

[0060] Figure 24: KEGG enrichment analysis of differentially expressed genes in the HFHC+50mg / kg HDD group and HFHC group in Example 4;

[0061] Figure 25: GSEA analysis of differentially expressed genes between the HFHC+50mg / kg HDD group and the HFHC group in Example 4;

[0062] Figure 26: Modeling and screening of differentially expressed genes before and after HDD treatment in Example 4;

[0063] Figure 27: Effects of each group on mRNA expression in Example 5. Data are shown as SEM ± mean; * p < 0.05 compared with the NCD group; # p < 0.05 compared with the HFHC group;

[0064] Figure 28: Western Blot analysis of important proteins in each group in Example 5;

[0065] Figure 29: Western Blot analysis of important proteins in each group in Example 6;

[0066] Figure 30: H&E and Oil Red staining results for each group in Example 6, scale bar 50 μm;

[0067] Figure 31: Analysis results of H&E and Oil Red staining data for each group in Example 6. The data are shown as SEM ± mean; * p < 0.05 compared with the HFHC group; # p < 0.05 compared with the HFHC+OE-NC group;

[0068] Figure 32: Serum biochemical test results of each group in Example 6. The data are shown as SEM ± mean; * Compared with the HFHC group, p<0.05; # Compared with the HFHC+OE-NC group, p<0.05;

[0069] Figure 33: qPCR analysis of lipid metabolism markers in each group in Example 6. Data are shown as SEM ± mean; * p < 0.05 compared with the HFHC group; # p < 0.05 compared with the HFHC+OE-NC group;

[0070] Figure 34: IHC-F4 / 80 and Sirius Red staining results for each group in Example 6, scale bar 50 μm;

[0071] Figure 35: Analysis results of IHC-F4 / 80 and Sirius red staining data for each group in Example 6. The data are shown as SEM mean ±; * Compared with the HFHC group, p<0.05; # Compared with the HFHC+OE-NC group, p<0.05;

[0072] Figure 36: qPCR analysis of inflammatory markers in each group in Example 6. Data are shown as SEM mean ±; * p < 0.05 compared with the HFHC group; # p < 0.05 compared with the HFHC+OE-NC group;

[0073] Figure 37: qPCR analysis of fibrosis markers in each group in Example 6. Data are shown as SEM mean ±; * p < 0.05 compared with HFHC group; # p < 0.05 compared with HFHC+OE-NC group;

[0074] Figure 38: Western Blot analysis of important proteins in Example 7;

[0075] Figure 39: Experimental results of each group on AML12 cells in Example 7. Data are shown as SEM ± mean; * p < 0.05 compared with PA group; # p < 0.05 compared with PA+HDD+OE-NC group;

[0076] Figure 40: Effect of each group in Example 7 on AML12 lipid deposition, scale bar 100 μm. Detailed Implementation

[0077] The technical solution of the present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanatory of the present invention, and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are covered within the scope of protection intended by the present invention.

[0078] Unless otherwise stated, the raw materials and reagents used in the following examples are commercially available products or can be prepared by known methods.

[0079] The term “metabolic dysfunction-associated fatty liver disease (MASLD)” in this article refers to a chronic liver disease in which excessive lipids are deposited in the liver and accompanied by a variety of metabolic disorders, including non-alcoholic metabolic dysfunction-associated fatty liver (NAFL / MAFL), non-alcoholic steatohepatitis / metabolic dysfunction-associated steatohepatitis (NASH / MASH), and simple hepatic steatosis.

[0080] The term "adjunctive therapy" in this article refers to additional treatment, usually given after surgery.

[0081] The term "subject" in this article refers to an animal, such as a mammal. Mammals include, for example, mice, rats, dogs, cats, pigs, sheep, horses, cattle, and humans.

[0082] The term "therapeutic effective amount" or "effective amount" herein refers to the amount of a compound disclosed and / or described herein that, when administered to a patient requiring this treatment, is sufficient to achieve the treatment as defined herein. A therapeutically effective amount of a compound may be an amount sufficient to treat ischemia-reperfusion injury. Therapeuticly effective amounts will vary depending on factors such as the subject being treated and their disease condition, the subject's weight and age, the severity of the disease condition, the specific compound, the dosing regimen to be followed, the timing of administration, and the method of administration, all of which can be readily determined by one of ordinary skill in the art.

[0083] The term “treatment” in this article includes one or more of the following: suppressing a disease or condition; slowing or halting the development of clinical symptoms of a disease or condition; and / or alleviating a disease or condition (i.e., causing relief or resolution of clinical symptoms), and both complete or partial reduction of clinical symptoms of a disease or condition.

[0084] The term "therapeutic effect" in this article refers to the effect resulting from treatment, which at the cellular level manifests as an inhibition rate of cell growth or a cell death rate, and at the animal level manifests as an alteration, usually a reduction or improvement of the symptoms of a disease or disease condition, or a cure of a disease or disease condition.

[0085] The term "pharmaceutically acceptable salt" in this article refers to a salt that can be present in the acidic or basic groups of hydroxynidone. Examples include sodium salts, calcium salts, potassium salts, hydrobromide salts, sulfates, hydrogen sulfates, phosphates, hydrogen phosphates, dihydrogen phosphates, acetates, succinates, citrates, tartrates, lactates, mandelates, methanesulfonates, and p-toluenesulfonates.

[0086] The mice used in the following experiments were purchased from Shanghai Slack Laboratory Animal Center and housed at the Laboratory Animal Center of Shanghai People's Hospital. They were males, C57BL / 6, and weighed 22-25g.

[0087] Experimental methods:

[0088] Isolation of primary HC from mice

[0089] Primary mouse hepatocytes (mpHCs) were isolated by in situ perfusion. The liver was perfused via the portal vein with protease and collagenase. MpHCs were isolated and then cultured in Williams E medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. Plates used for inoculation of mpHCs were coated with mouse tail collagen one day prior to inoculation to aid mpHC adhesion.

[0090] Cell processing and transfection

[0091] MpHCs and AML12 cells were used for in vitro studies. Lipotoxic hepatocytes were generated by treating primary HCs with 200 μM PA (Sigma-Aldrich Corporation, St. Louis, MO, USA) for 24 hours. Hydroxynesone (HDD) solution was dissolved in dimethyl sulfoxide (DMSO), and different concentrations of HDD were added to the mixture with PA. Cells were stimulated for 24 hours, and then collected for subsequent experiments.

[0092] CD36 overexpression (OE-CD36) plasmid and blank control (OE-NC) plasmid were used for transfection of AML12 cells. Plasmid transfection was performed using the Lipo3000 transfection kit.

[0093] Histological staining

[0094] Hematoxylin and eosin (H&E) were used to stain paraffin-embedded sections of mouse liver to observe liver damage. Sirius red (SR) staining was used to observe liver fibrosis, and Oil Red staining was used to observe lipid deposition. Images were acquired using the Leica Application Suite X system (Leica Microsystems GmbH, Wetzlar, Germany) and analyzed using ImageJ software.

[0095] Immunohistochemical analysis

[0096] Paraffin-embedded sections of mouse liver were selected for immunohistochemical staining. F4 / 80 antibody was used to stain for mouse liver inflammation, following the instructions of the immunohistochemical staining kit. Images were acquired using an optical microscope and analyzed using ImageJ software.

[0097] qPCR

[0098] RNA was extracted from mouse liver tissue and cells using the Trizol kit. The RNA was reverse transcribed into cDNA using a reverse transcription kit. The relative expression of mRNA in different samples was detected using real-time quantitative PCR (RT-PCR) with the Yeasen PrimeScript Buffer kit.

[0099] Protein blot

[0100] Protein samples were extracted from liver tissue and cells using RIPA (Radioimmunoprecipitation assay lysis buffer) and prepared using SDS-PAGE buffer. Protein quantification was performed using ImageJ software.

[0101] Transcriptome sequencing

[0102] Reverse transcription and complementary cDNA libraries from each sample were used for transcriptome sequencing. Analysis was performed using the Illumina Novaseq 6000 sequencing platform. Subsequent differentially expressed genes and pathway analyses were then conducted using the sequencing data. For differentially expressed data, genes with an absolute logFC value >1 and an adjusted p-value <0.05 were considered differentially expressed genes. GO and KEGG analyses were used to evaluate changes in the biological functions and pathways associated with differentially expressed genes before and after different treatments. GSEA analysis was used to evaluate changes in gene sets for different pathways.

[0103] Biochemical testing

[0104] After modeling in each group of mice, blood was collected and serum samples were separated by centrifugation. The levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in the serum of each group of mice were analyzed using a Chemray 800 fully automated biochemical analyzer.

[0105] Statistical analysis

[0106] All experimental data are expressed as mean ± standard deviation. Student's t-test was used to analyze statistical differences between two groups, and analysis of variance was used to analyze statistical differences among multiple groups. SPSS 19.0 software was used for analysis. A p-value less than 0.05 was considered statistically significant.

[0107] Example 1: Establishment and Experimental Results of the HFHC MASLD Mouse Model

[0108] Mice were randomly divided into 5 groups (n=8 per group): control group (NCD), model group (HFHC), low-dose HDD treatment group (HFHC + 25 mg / kg HDD / HDD-L), medium-dose HDD treatment group (HFHC + 50 mg / kg HDD / HDD-M), and high-dose HDD treatment group (HFHC + 100 mg / kg HDD / HDD-H). The control group (NCD) was fed a normal diet for 24 weeks, the model group (HFHC) was fed a high-fat, high-cholesterol (HFHC) diet for 24 weeks, and the low-dose, medium-dose, and high-dose HDD treatment groups were fed an HFHC diet for 24 weeks. Treatment with different doses of HDD was initiated at week 13.

[0109] Liver tissue and serum samples were collected from the control and model groups. As shown by H&E staining (Figure 1), a large number of lipid droplets were deposited in the liver lobules of mice fed the HFHC diet compared with the control group. Furthermore, more hepatocyte sac degeneration and inflammatory cell infiltration were observed, and the NAS scores of mice fed the HFHC diet were significantly higher than those of the control group (Figure 2). Biochemical tests revealed significantly elevated ALT and AST levels in the serum of mice in the HFHC group compared with the control group (Figure 3). These results indicate that the HFHC diet-induced MASLD mouse model was successfully established.

[0110] In the treatment groups, lipid droplets in the livers of mice in all three groups were significantly reduced, and NAS scores were also significantly lower (Figure 2). According to the H&E staining and Oil Red staining results in Figure 1, the medium-dose group showed the best treatment effect among the three treatment groups, and all three groups of mice showed significant improvement compared to the model group. The reduction in lipid deposition in the livers of mice in the medium-dose treatment group was the most significant, with the deposition pattern changing from large lipid droplets to small lipid droplets, and the NAS score decreasing most significantly (Figure 2). Similar to the H&E staining results, compared with the model group, serum ALT and AST levels in mice in all three treatment groups were significantly reduced, indicating a significant reduction in liver damage (Figure 3). In addition, compared with the model group (HFHC), the expression of lipid synthesis genes Fasn / Scd1 / Acc1 was significantly inhibited in all three treatment groups, while the expression of lipid breakdown-related genes Cpt1α / Pgc1α / Ppar-α was significantly increased (Figure 4). Among the three treatment groups, the medium-dose treatment showed the most significant improvement in biochemical indicators. H&E staining and biochemical marker results showed that HDD treatment could effectively reduce liver damage in HFHC diet-induced MASLD mice.

[0111] Immunohistochemical F4 / 80 staining was used to assess macrophage infiltration in the livers of mice in each group. According to the staining results (Figures 1 and 2), the proportion of F4 / 80-positive cells in the livers of the model group was significantly increased compared to the control group. Compared to the HFHC group, the number of F4 / 80-positive cells in the livers of mice in all treatment groups was reduced, with the most significant reduction observed in the medium-dose treatment group, consistent with previous lipid metabolism findings. Subsequently, the expression levels of M1 and M2 macrophage marker genes in the livers of mice in each group were examined (Figure 5). Compared to the model group, the expression of M1 pro-inflammatory macrophage markers such as IL-1β / IL-6 / Tnf-α was significantly decreased in all treatment groups, while the expression of M2 anti-inflammatory macrophage markers such as ARG1 / Fizz1 / IL-12 was significantly increased. These results indicate that HDD exhibits a significant anti-inflammatory effect on HFHC diet-induced MASLD mice.

[0112] Sirius Red staining was used to assess liver injury in mice. Compared with the model group (HFHC), the proportion of collagen-positive areas and the length of fiber bundles were significantly reduced in the sections of the three treatment groups (Figure 1). The expression of α-SMA, Col1, and fibronectin in the liver of the three treatment groups was significantly reduced (Figures 6 and 7). Based on these findings, the results indicate that HDD can alleviate liver injury induced by the HFHC diet in MASLD mice.

[0113] Example 2: Establishment and Experimental Results of the MCD MASLD Mouse Model

[0114] Mice were randomly divided into 5 groups (n=8 per group): control group (MCS), model group (MCD), low-dose HDD treatment group (MCD+25mg / kg HDD / HDD-L), medium-dose HDD treatment group (MCD+50mg / kg HDD / HDD-M), and high-dose HDD treatment group (MCD+100mg / kg HDD / HDD-H). The control group (MCS) was fed a methionine and cystine supplement diet (MCS) for 8 weeks, the model group (MCD) was fed a methionine and cystine deficient diet for 8 weeks, and the low-dose, medium-dose, and high-dose HDD treatment groups were fed a methionine and cystine deficient diet (MCD) for 8 weeks. Different doses of HDD were administered in week 5.

[0115] H&E staining and Oil Red staining results (Figure 8) showed that, compared with the control group, the model group mice had significant lipid droplet deposition and extensive inflammatory cell infiltration in the liver, exhibiting steatohepatitis. ALT and AST were significantly elevated, indicating the successful establishment of the MCD diet-induced MASLD mouse model. Compared with the MCD group, the liver lipid deposition H&E staining, Oil Red staining, NAS scores (Figures 8 and 9), and biochemical indicators (Figure 10) were significantly reduced in all three treatment groups. Compared with the model group, the expression of lipid synthesis genes Fasn / Scd1 / Acc1 was significantly inhibited in all three treatment groups, while the expression of lipid breakdown-related genes Cpt1α / Pgc1α / Ppar-α was significantly increased (Figure 11). Similar to the HFHC model, the moderate-dose treatment group showed the most significant effect among the three treatment groups in the MCD model.

[0116] Immunohistochemical F4 / 80 staining showed that the proportion of F4 / 80 positive areas was significantly reduced in all three treatment groups compared to the model group (MCD) (Figures 8 and 9). The moderate-dose group showed the most significant effect among the three treatment groups. Compared to the model group, the mRNA expression of the M1 pro-inflammatory macrophage markers Il-1β / Il-6 / Tnf-α was significantly reduced in all treatment groups, while the mRNA expression of the M2 anti-inflammatory macrophage markers Fizz1 / Arg1 / Il-12 was significantly increased in all treatment groups (Figure 12), indicating that HDD has an anti-inflammatory effect on MCD diet-induced MASLD.

[0117] Sirius staining and qPCR results showed that, compared with the model group (MCD), the proportion of collagen-positive staining in the livers of mice in the three treatment groups was significantly reduced (Figures 8, 13, and 14). Based on these findings, the results indicate that HDD can alleviate MCD diet-induced liver damage in MASLD mice.

[0118] Example 3: In vitro experiment of HDD

[0119] Mouse primary hepatocytes (mpHCs) or AML12 cells were stimulated with 200 μM palmitic acid for 24 hours to simulate lipotoxic injury. The PA group was treated with 200 μM palmitic acid for 24 hours, while the CN group was treated with 0.5% BSA solution. Four different treatment groups were PA+50 μM HDD, PA+100 μM HDD, PA+200 μM HDD, and PA+400 μM HDD. Specifically, after preparing 400 μM palmitic acid medium, cells were co-incubated with 50 μM, 100 μM, 200 μM, and 400 μM HDD.

[0120] Changes in the mRNA of lipid metabolism-related genes in each treatment group were detected by qPCR. Compared with the PA group, the expression of Fasn / Scd1 / Acc1 in the four HDD treatment groups of mpHCs was significantly decreased, while the expression of Cpt1α / Pgc1α / Ppar-α was significantly increased, and the expression of AML12 was significantly increased in HDD treatment (Figures 15 and 16). Furthermore, mRNA expression showed a dose-dependent pattern. At the overall level, the expression levels of lipid synthesis genes gradually decreased, while the expression levels of lipolysis genes gradually increased with increasing HDD dose. The changes in the expression of lipid synthesis and degradation genes were most significant at 400 μM HDD. Oil Red staining was then used to observe the effect of HDD on lipid deposition in mpHCs and AML12 (Figures 17 and 18, where the PA+HDD group was PA+400 μM HDD). The results indicate that HDD can significantly reduce lipid deposition in hepatocytes. The above results indicate that HDD can effectively inhibit lipid deposition in the MASLD cell model, with the 400 μM dose group showing the most significant therapeutic effect among all dose groups.

[0121] Example 4:

[0122] Transcriptome sequencing was performed on three groups of mouse liver samples from Example 1 (NCD group, HFHC group, and HFHC+50mg / kg HDD group, respectively). The mouse modeling method was the same as in Example 1, and four samples from each of the three groups of mouse liver samples were sequenced.

[0123] Gene expression changes were compared between the HFHC and NCD groups. The absolute value of Log2FC ≥ 1 and the adjusted p-value < 0.05 were used as criteria for screening differentially expressed gene sets. A total of 1502 genes were found to have significantly different expression in gene set S1, of which 1017 genes were upregulated and 485 genes were downregulated in the HFHC group (Figure 19). Volcano plots and heatmaps of differentially expressed genes are shown in Figure 20. Among the differentially expressed genes, Cd36, Col1a1, Mmp7, Cd68, Ccl6, Ccl9, Nos2, Ppar-a, Trem2, Col3a1, and Col5a1 are known to be closely related to the progression of MASLD. These differentially expressed genes are involved in the three main dimensions of lipid metabolism, inflammatory response, and fibrosis in MASLD.

[0124] Results were analyzed in the HFHC group and the HFHC+50mg / kg HDD group. A total of 847 genes were identified in the differentially expressed gene set S2, with 353 genes upregulated and 494 genes downregulated in the treatment group (Figure 19). Volcano plots and heatmaps of the differentially expressed genes are shown in Figure 20. Among the differentially expressed genes with the highest fold change, those closely associated with the progression of MASLD included Timp1, Cd36, Trem2, Fasn, Scd1, Pnpla3, Col1a1, Col3a1, Col5a1, Hgf, Pdgfra, Cxcl9, Fgf21, Fgfr1, Loxl2, Ccl5, Ccl2, Acta2, and Tlr4. These differentially expressed genes were highly consistent with the phenotype of HDD-treated MASLD mice and were involved in three main dimensions: lipid metabolism, inflammatory response, and fibrosis. In particular, differentially expressed genes related to lipid metabolism ranked relatively high.

[0125] GO and KEGG enrichment analyses were performed to explore the potential roles of differentially expressed genes. According to GO enrichment, differentially expressed genes after HDD treatment played roles in cellular responses to lipid metabolism, extracellular matrix organization, regulation of smooth muscle cell proliferation, antigen processing and presentation, cellular responses to endogenous stimuli, collagen fiber organization, and other aspects of collagen fiber synthesis (Figures 21, 22). KEGG analysis revealed enrichment responses in the extracellular matrix, MAPK signaling pathway, AMPK signaling pathway, PI3K-ATK signaling pathway, and TGF-β signaling pathway (Figures 23, 24). These signaling pathways are closely related to lipid metabolism, fibrosis, and other disease processes. Furthermore, we used GSEA analysis to explore which specific disease processes might be influenced by differentially expressed genes after HDD treatment (Figure 25). GSEA analysis results indicated that these differentially expressed genes may play important roles in triglyceride metabolism, the tricarboxylic acid cycle, cholesterol metabolism, carbohydrate metabolism, and extracellular matrix regulation. Sequencing analysis further confirmed the therapeutic effects of HDD on MASLD mice through lipid metabolism, inflammatory responses, and liver fibrosis.

[0126] After analyzing the differentially expressed gene characteristics before and after modeling and treatment, a total of 159 genes showed significant expression differences. During the gene set screening process, we focused on the top-ranked genes related to lipid metabolism (Figure 26). Among these differentially expressed genes, we found that CD36 was both a top-ranked differentially expressed gene and closely related to lipid metabolism (Figure 26).

[0127] This study analyzed the pathways by which CD36 may participate in the MASLD process. Literature review revealed that CD36 is closely related to TLRs, mTOR, AMPK, and other signaling pathways. Combined with the aforementioned KEGG pathway enrichment analysis results, we found that among the lipid metabolism pathways regulated by CD36, the AMPK pathway showed significant differences in KEGG enrichment analysis.

[0128] Example 5:

[0129] The three groups of mouse liver samples selected in Example 4 were the NCD group, the model group (HFHC group), and the medium-dose HDD treatment group (i.e., HDD group, HFHC + 50mg / kg HDD / HDD-M). The transcriptome sequencing results were verified by qPCR and Western Blot. The mouse modeling method was the same as in Example 4.

[0130] The results showed that, compared with the control group, the expression of CD36 mRNA and protein in the liver of mice in the model group (HFHC) was significantly increased (Figures 27 and 28). Furthermore, compared with the model group (HFHC), the expression of CD36 mRNA and protein in the HDD treatment group was significantly decreased, consistent with the sequencing results. Western blot analysis showed that, compared with the control group, the expression of phosphorylated AMPK protein in the model group (HFHC) was significantly decreased, indicating that this pathway was inhibited. However, compared with the model group (HFHC), the expression of phosphorylated AMPK protein in the HDD treatment group was significantly increased, indicating that this pathway was reactivated (Figure 28). Activation of the AMPK pathway during MASLD significantly inhibited excessive lipid deposition in the liver and exerted a therapeutic effect. The results indicate that after HDD treatment, CD36 expression in MASLD mice decreased, and the AMPK pathway was activated.

[0131] Example 6: Establishment of an overexpression mouse model and experimental results

[0132] Thirty-two male C57BL / 6 mice were randomly divided into four groups of eight mice each. The four groups were further divided into a model group (HFHC), a medium-dose HDD treatment group (i.e., HDD group, HFHC + 50 mg / kg HDD / HFHC + HDD), an overexpression control group (i.e., OE-NC group, HFHC + 50 mg / kg HDD + OE-NC / HFHC + HDD + OE-NC), and a CD36 overexpression group (i.e., OE-CD36 group, HFHC + 50 mg / kg HDD + OE-CD36 / HFHC + HDD + OE-CD36). The model group (HFHC) was induced with a high-fat, high-cholesterol (HFHC) diet for 24 weeks, as was the treatment group. Treatment with 50 mg / kg HDD began at week 13. The overexpression control group and the CD36 overexpression group were also induced with a high-fat, high-cholesterol (HFHC) diet for 24 weeks. Mice were injected with adeno-associated virus via the tail vein at week 10, and treatment with 50 mg / kg HDD began at week 13. After model establishment, mice were sacrificed by cervical dislocation, and liver tissue and serum samples were collected.

[0133] The results showed that, compared with the overexpression control group, the expression of CD36 protein in the liver of the CD36 overexpression group was significantly increased, and the phosphorylated AMPK protein was significantly decreased, indicating that the downstream AMPK pathway was inhibited with the increase of CD36 expression (Figure 29). We then further investigated the effect of CD36 overexpression in HDD-treated MASLD mice. The effect of OE-CD36 on HDD treatment was evaluated by fatty degeneration, inflammation, and fibrosis. H&E staining, as well as NAS scores and Oil Red staining, showed that CD36 overexpression increased lipid deposition in the liver of HDD-treated MSDLD mice (Figures 30, 31). The comparison between the overexpression control group and the CD36 overexpression group showed that ALT and AST levels were also significantly increased in the CD36 overexpression group (Figure 32). In addition, compared with the overexpression control group, the mRNA expression of lipid synthesis-related genes Fasn / Scd1 / Acc1 in the liver of the CD36 overexpression group was significantly increased, while the mRNA expression of lipid breakdown genes Cpt1α / Pgc1α / Ppar-α was significantly decreased (Figure 33). This indicates that HDD treatment of MASLD mice blocked the therapeutic effect of HDD on lipid metabolism disorders, and that CD36 overexpression exacerbated lipid metabolism dysfunction.

[0134] The inflammatory response in four groups of mice was assessed. F4 / 80 staining results showed that, compared with the overexpression control group, the proportion of F4 / 80 positive areas in the liver of the CD36 overexpression group was significantly increased (Figs. 34 and 35). The mRNA expression of the M1 pro-inflammatory macrophage markers Il-1β / Il-6 / Tnf-α in the liver of the treatment group mice was significantly decreased, while the mRNA expression of the M2 anti-inflammatory macrophage markers Fizz1 / Arg1 / Il-12 was significantly increased (Fig. 36). Therefore, the anti-inflammatory effect of HDD on the liver of MASLD mice was inhibited after CD36 overexpression. We further evaluated the changes in liver fibrosis in the four groups of mice. Sirius staining and qPCR results showed that, compared with the overexpression control group, the proportion of α-SMA positive areas and fibrosis markers in the liver of the CD36 overexpression group was significantly increased (Figs. 34 and 37). These evidences suggest that CD36 overexpression in the liver of HDD-treated MASLD mice blocked the therapeutic effect of HDD on MASLD mice.

[0135] Example 7: In vitro overexpression experiment

[0136] AML12 cells were divided into a modeling group (PA), a treatment group (HDD group, PA+400μM HDD), an overexpression control group (OE-NC group, PA+400μM HDD+OE-NC), and an overexpression CD36 group (OE-CD36 group, PA+400μM HDD+OE-CD36). Cells were treated with 200μM PA and 400μM HDD to observe whether CD36 overexpression affected the therapeutic effect of HDD on hepatic lipid deposition.

[0137] Western blot results showed that, compared with the OE-NC group, the expression of CD36 protein in AML12 cells of the OE-CD36 group was significantly increased, while the expression of phosphorylated AMPK protein in AML12 cells was significantly decreased (Figure 38), indicating that the AMPK pathway was inhibited after CD36 overexpression. Furthermore, the mRNA expression of lipid synthesis-related genes in mouse liver showed a significant increase in Fasn / Scd1 / Acc1, while the mRNA expression of lipid degradation genes Cpt1α / Pgc1α / Pparα showed a significant decrease in the OE-CD36 group (Figure 39). We then observed lipid droplet deposition in the four groups using Oil Red staining. The staining results showed that, compared with the OE-NC group, the lipid droplet area in AML12 cells of the OE-CD36 group was significantly increased (Figure 40). The results indicate that the therapeutic effect of HDD on lipotoxic hepatocytes was blocked after CD36 overexpression.

[0138] The exemplary embodiments of the present invention have been described above. However, the scope of protection of this application is not limited to the above embodiments. Any modifications, equivalent substitutions, improvements, etc., made by those skilled in the art within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. Use of a hydroxynidone or a pharmaceutically acceptable salt thereof in the preparation of a medicament for the treatment or adjunctive treatment of metabolic dysfunction-associated fatty liver disease (MASLD).

2. Use according to claim 1, characterized in that, Pharmaceutically acceptable salts of the hydroxynidone include salts that may be present in the acidic or basic groups of the hydroxynidone, such as sodium salts, calcium salts, potassium salts, hydrobromide salts, sulfate salts, hydrogen sulfate salts, phosphate salts, hydrogen phosphate salts, dihydrogen phosphate salts, acetate salts, succinate salts, citrate salts, tartrate salts, lactate salts, mandelate salts, methanesulfonate salts, and p-toluenesulfonate salts.

3. Use according to claim 1, characterized in that, The metabolic dysfunction-associated fatty liver disease (MASLD) is selected from one or more of the following groups: non-alcoholic metabolic dysfunction-associated fatty liver (NAFL / MAFL), non-alcoholic steatohepatitis / metabolic dysfunction-associated steatohepatitis (NASH / MASH), and simple hepatic steatosis. Alternatively, the metabolic dysfunction-associated fatty liver disease (MASLD) is selected from MASLD liver injury, MASLD inflammatory response, and MASLD lipid deposition.

4. Use according to claim 1, characterized in that, The drug can reduce lipid synthesis, increase lipid breakdown in the liver, and alleviate liver inflammation. Alternatively, the drug can lower ALT and AST levels in the blood; Alternatively, the drug can inhibit the expression of lipid synthesis genes Fasn / Scd1 / Acc1 in the liver and increase the expression of lipid breakdown-related genes Cpt1α / Pgc1α / Ppar-α in the liver. Alternatively, the drug can reduce the expression of α-SMA, Col1, and fibronectin in the liver; Alternatively, the drug can reduce the expression of M1 pro-inflammatory macrophage markers such as IL-1β / IL-6 / Tnf-α in the liver and increase the expression of M2 anti-inflammatory macrophage markers such as ARG1 / Fizz1 / IL-12 in the liver.

5. The use according to claim 1, characterized in that, The drug can regulate the CD36 / AMPK signaling pathway; furthermore, the drug can negatively regulate the expression of CD36.

6. Use according to any one of claims 1 to 5, characterized in that, The drug also includes a pharmaceutically acceptable carrier and / or excipients (such as diluents); Furthermore, the drug is in the form of an intravenous infusion, intravenous drip, subcutaneous administration, intradermal administration, intramuscular injection, oral spray, or oral administration.

7. The use according to any one of claims 1 to 6, characterized in that, The drug may be administered alone or in combination with one or more other therapeutic agents.

8. The use according to claim 1, characterized in that, The metabolic dysfunction-associated fatty liver disease (MASLD) mentioned above is a CD36 / AMPK-related metabolic dysfunction-associated fatty liver disease.

9. Use of a pharmaceutical composition containing hydroxynidone or a pharmaceutically acceptable salt thereof in the preparation of a medicament for the treatment or adjunctive treatment of metabolic dysfunction-associated fatty liver disease (MASLD).

10. A medicament for the treatment or adjunctive treatment of metabolic dysfunction-associated fatty liver disease (MASLD), comprising hydroxynidone or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier or excipient.

Images