Application of Moronic acid in preparation of medicine for treating non-alcoholic fatty liver disease

Abstract

The invention discloses an application of Moronic acid in preparation of a medicine for treating non-alcoholic fatty liver disease. The Moronic acid is a natural pentacyclic triterpenoid compound. The invention finds that Moronic acid (MA) plays a role in treating the non-alcoholic fatty liver disease by activating a PPAR pathway to relieve the non-alcoholic fatty liver disease and hepatic fibrosis. Therefore, the application range of the Moronic acid (MA) is widened, and a novel safe and reliable medicine is provided for treating the non-alcoholic fatty liver disease.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to the use of Moronic acid in preparing a drug for treating non-alcoholic fatty liver disease. Background Art

[0002] Non-alcoholic fatty liver disease (NAFLD) is becoming a leading cause of chronic liver disease worldwide and has become a major global public health concern. Due to the global obesity epidemic, the prevalence of NAFLD is as high as 24% and continues to rise. Furthermore, NAFLD is closely associated with metabolic syndrome, with a higher incidence in individuals with obesity, hypertension, type 2 diabetes, and dyslipidemia. The initial pathological changes of NAFLD manifest as simple hepatic steatosis, with fat content exceeding 5-10% of liver weight. 47% of patients with NAFLD will progress to non-alcoholic steatohepatitis (NASH). Of these NASH patients, 25%-30% develop irreversible liver fibrosis, which then progresses to cirrhosis and even liver cancer. Given its close relationship with metabolic diseases, current expert consensus is considering renaming it "metabolic-associated fatty liver disease (MAFLD)" to better characterize disease progression. Due to its high prevalence and potentially severe complications, NAFLD has become one of the leading causes of liver disease worldwide and has garnered widespread attention in the medical community. Therefore, finding better drugs to prevent and treat nonalcoholic fatty liver disease has become an urgent issue.

[0003] Pentacyclic triterpenoids are natural organic compounds composed of thirty carbon atoms and are widely found in plants, algae and some animals. Their basic skeleton is a triterpene alkyl skeleton composed of five rings, which can be divided into many types according to their chemical structure, mainly including oleanane, ursane, lupeane, etc. Moronic acid (MA) is a small molecule pentacyclic triterpenoid compound that is mainly found in Piper methysticum. Many pentacyclic triterpenoids have anti-inflammatory and antioxidant effects. However, there is currently no relevant information about Moronic acid (MA) in the treatment of non-alcoholic fatty liver disease (NAFLD). Therefore, the present invention studies the effect and mechanism of Moronic acid (MA) in the treatment of non-alcoholic fatty liver disease (NAFLD). Summary of the Invention

[0004] The purpose of the present invention is to address the deficiencies in the prior art and provide an application of Moronic acid in the preparation of a drug for treating non-alcoholic fatty liver disease.

[0005] To achieve the above object, the technical solution adopted by the present invention is:

[0006] The first aspect is to provide the use of Moronic acid in the preparation of a drug for treating non-alcoholic fatty liver disease. Moronic acid is a natural pentacyclic triterpenoid compound with the chemical formula:

[0007]

[0008] Furthermore, the only active ingredient of the drug for treating non-alcoholic fatty liver disease is Moronic acid.

[0009] Furthermore, the moronic acid alleviates non-alcoholic fatty liver disease and liver fibrosis by activating the PPAR pathway.

[0010] The second aspect is to provide a pharmaceutical preparation for treating non-alcoholic fatty liver disease.

[0011] The moronic acid is used as a single active ingredient or as an active ingredient together with other drugs for treating non-alcoholic fatty liver disease.

[0012] In some embodiments, the pharmaceutical preparation further contains a pharmaceutically acceptable carrier or excipient to prepare a pharmaceutically acceptable dosage form.

[0013] In some embodiments, the pharmaceutically acceptable carrier or excipient comprises one or more solid, semisolid or liquid excipients.

[0014] In some embodiments, the pharmaceutically acceptable dosage forms include but are not limited to injections, emulsions, tablets, capsules, granules, pills, syrups, powders, and ointments.

[0015] The present invention adopts the above technical solution, which has the following technical effects compared with the prior art:

[0016] This invention provides a novel application of moronic acid (MA) in the preparation of a drug for treating nonalcoholic fatty liver disease. The invention discovers that moronic acid (MA) exerts its therapeutic effect on nonalcoholic fatty liver disease by activating the PPAR pathway, alleviating nonalcoholic fatty liver disease and liver fibrosis. Therefore, this invention not only expands the application range of moronic acid (MA) but also provides a new, safe and reliable drug for the treatment of nonalcoholic fatty liver disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The results of serum biochemical analysis of liver and kidney function markers in C57BL / 6J mice after MA treatment are shown; data are expressed as mean ± SD (n = 5), p < 0.05, * p<0.01 indicated a significant difference compared with the control group (one-way analysis of variance).

[0018] Figure 2 Figure 3 shows that MA alleviates hepatic steatosis and fibrosis in C57BL / 6J mice; (A) Schematic diagram of the experimental design: non-alcoholic fatty liver disease (NAFLD) and liver fibrosis models were induced in C57BL / 6J mice, and MA (MA, 5 mg / kg or 10 mg / kg) or resmetirom was subsequently administered by oral gavage every two days; (B) Gross morphology of the livers in different treatment groups: the livers of the model group mice showed an enlarged and greasy appearance, while the appearance of the livers in the MA and resmetirom treatment groups improved; (C) Representative histological sections of the livers in the NAFLD group, stained with hematoxylin-eosin, respectively. (H&E) and Oil Red O staining show the degree of hepatic steatosis and lipid droplet accumulation; (D) Representative sections of the liver in the fibrosis group were stained with H&E, Sirius red (collagen fibers appear red), and Masson's trichrome staining (collagen appears blue), respectively, revealing the severity of fibrosis and the effect of treatment; (E) Serum biochemical parameters including alanine aminotransferase (ALT), aspartate aminotransferase (AST), triglycerides (TG), and total cholesterol (TC), and quantitative analysis of the stained areas is shown in the accompanying figures; data are expressed as mean ± SD (n = 8 per group, n = 5 in the control group), and statistical analysis was performed using one-way analysis of variance. #### p<0.0001 indicates a very significant difference compared with the control group; *** p<0.001, **** p<0.0001 indicated a significant difference compared with the model group.

[0019] Figure 3 The validation results of PPARs in animal liver samples are shown; among them, (A) Western Blotting analysis of PPARG, PPARD and PPARA in liver samples of NAFLD group; (B) Western Blotting analysis of PPARG, PPARD and PPARA in liver samples of fibrosis group; (C) Western Blotting analysis of type III collagen and α-SMA in liver samples of fibrosis group; #, ##, ###, #### respectively indicate p<0.05, p<0.01, p<0.001, p<0.0001 compared with the control group; *, **, *** respectively indicate p<0.05, p<0.01, p<0.001 compared with the model group.

[0020] Figure 4Figure 3 Lipidomics analysis revealed metabolic changes induced by moronic acid in NAFLD and fibrosis models; (AB) principal component analysis (PCA) score plots showed clear separation between the control, model, and MA-treated groups in NAFLD (A) and fibrosis (B) models; (CD) pie charts showed the distribution of eight major lipid classes in liver samples: fatty acids (FA), glycerides (GL), glyceroglycolipids (Gly), glycerophospholipids (GP), phosphatidylethanolamines (PE), prenyl lipids (PR), sphingolipids (SP), and sterol lipids (ST); (E) Venn diagram showing the overlap of significantly upregulated and downregulated lipid metabolites (|log2FC|>1, p≤0.05) in NAFLD and fibrosis models; (F) volcano plot of differential lipid metabolites in NAFLD and fibrosis models; (GH) heat maps of significantly altered lipid species in NAFLD (G) and fibrosis (H) models.

[0021] Figure 5 Figure 3 shows the in vitro validation results of moronic acid (MA) in hepatocytes and hepatic stellate cells; (A) Cell viability assay (CCK-8) shows the effects of different concentrations and exposure times of MA on AML12, BRL, HSC-T6, and JS-1 cells; (B) Oil red O staining of AML12 and BRL cells, showing the reduction of lipid accumulation induced by MA and resmetirom; (C) Western blot analysis of PPARA, PPARD, PPARG, type III collagen, and α-SMA expression in HSC-T6 and JS-1 cells; (D) Western blot analysis of PPARA, PPARD, and PPARG expression in HSC-T6 and JS-1 cells. Blot analysis; (E, G) Quantitative analysis of PPARA, PPARD, PPARG, type III collagen, and α-SMA protein levels in HSC-T6 (E) and JS-1 (G) cells; (F, H) Quantitative analysis of PPARA, PPARD, and PPARG protein levels in AML12 (F) and BRL (H) cells; Data are expressed as mean ± SD (n = 3). # p<0.05, ## p<0.01, ### p<0.001, #### p<0.0001 indicates a significant difference compared with the control group; * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001 indicated a significant difference compared with the model group (one-way analysis of variance).

[0022] Figure 6 Moronic acid alleviates steatosis and fibrosis in a 3D liver model through a PPAR-dependent mechanism; (A) The IC of moronic acid in a 3D liver model was determined using a CCK-8 assay. 50 (B) Representative images of HE staining, Oil Red O staining, and type I collagen immunofluorescence in NAC-Organ-based 3D liver models under different treatment conditions; (CE) Quantitative analysis of triglycerides (C), total collagen levels (D), and IL-6 (E) in 3D culture supernatants; Data are expressed as mean ± SD (n = 3). #### p<0.0001 indicates a very significant difference compared with the control group; * p<0.01, *** p<0.001, **** p<0.0001 indicated a significant difference compared with the model group (one-way analysis of variance). DETAILED DESCRIPTION

[0023] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but they are not intended to limit the present invention. It should be noted that, unless there is a conflict, the embodiments and features in the embodiments of the present invention may be combined with each other.

[0024] Example 1

[0025] In order to verify that MA has a therapeutic effect on non-alcoholic fatty liver disease, the present invention conducted animal experiments and in vitro experiments. The specific experimental methods involved are as follows:

[0026] Sixty-nine 6-week-old male C57BL / 6J mice (15 ± 1 g) were purchased from Zhiyuan (Hangzhou, China) and housed in an SPF-grade environment with a 12-h light-dark cycle, room temperature controlled at (25 ± 2)°C, and relative humidity of (50 ± 10)%. Animal experiments were performed in accordance with the animal care protocol approved by the Animal Care and Use Ethics Committee of Shanghai Tongren Hospital (approval number: A2025-011-01).

[0027] After 5 days of acclimation, 69 male C57BL / 6J mice were randomly divided into two groups: a fatty liver group and a fibrosis group. Each group was further divided into a control group, a model group, a resmetirom (3 mg / kg) group, a MA-L (5 mg / kg) group, and a MA-H (10 mg / kg) group. The fatty liver group was fed a high-fat diet for 12 weeks, with drug administration by gavage every two days starting in week 6. The fibrosis group was fed a high-fat diet with intraperitoneal injections of CCl4 (0.2 μL / g) for 22 weeks, with intraperitoneal injections of CCl4 twice weekly starting in week 6 and drug administration by gavage every two days starting in week 16. The control and model groups received PBS. After a 12-hour fast, mice were anesthetized, and blood was collected from the eyeballs. Serum was separated by centrifugation (3000 g, 10 minutes, 4°C), clarified (6000 g, 3 minutes), and frozen in liquid nitrogen. Mice were euthanized by cervical dislocation, and liver tissues were collected, frozen in liquid nitrogen, and stored at −80°C.

[0028] 2. Rat normal hepatocytes (BRL) and hepatic stellate cells (HSC-T6) were purchased from Pronocell (Wuhan, China), mouse normal hepatocytes (AML12) were purchased from Sewell Biotechnology Co., Ltd. (Wuhan, China), and JS-1 cells were purchased from Chengxi Biotechnology (Shanghai, China). BRL cells, HSC-T6 cells, and JS-1 cells were cultured in high-glucose DMEM supplemented with 10% FBS (fetal bovine serum) and 1% P / S (penicillin and streptomycin), while AML12 cells were cultured in DMEM / F-12 supplemented with 10% FBS (fetal bovine serum), 1% P / S (penicillin and streptomycin), and 40 ng / mL dexamethasone in a humidified incubator at 37°C with 5% CO2. Non-alcoholic fatty liver cell models were successfully established by incubating AML12 and BRL cell lines with 900 μM sodium oleate for 48 hours.

[0029] 3. Based on NAC-Organ technology, a three-dimensional liver physiological model was constructed in a 96-well plate (Pusheng Bio, S-9601) using human primary hepatocytes (LV-BIOTECH, LV-PHH001) and human primary non-hepatocytes (iXCell, 10HU-234), with a cell number of 3000. After successful modeling, a liver fibrosis model was induced using liver fibrosis medium (Puheng Bio, MIF02), and the medium was changed every two days for 5 consecutive days to construct a liver fibrosis model. The fatty liver model was constructed as described above. Except for the control group, DMSO, 100μM resmetirom, 10μM MA and 10μM MA+1μM GW9662 were added to the other four groups according to the model.

[0030] 1) CCK8 experiment

[0031] The day before, AML12, BRL, HSC-T6 and JS-1 cells were seeded into 96-well plates according to the concentration gradient of 0μM, 3μM, 6μM, 9μM, 12μM, 15μM, 18μM and 21μM. After treatment for 24h, 48h and 72h respectively, the original culture medium was removed and fresh culture medium containing 10% CCK-8 solution was added. The cells were incubated at 37℃ for 3 hours and the absorbance was measured at a wavelength of 450nm.

[0032] 2) Lipid levels and liver enzymes

[0033] The concentrations or activities of serum total cholesterol (CHO), triglycerides (TG), aspartate aminotransferase (AST), alanine aminotransferase (ALT), blood urea nitrogen (BUN), creatinine (CREA), uric acid (UA), and blood glucose (GLU) were measured using a fully automatic biochemical analyzer (Chemray 800).

[0034] 3) Dyeing

[0035] The obtained liver tissue was fixed in 4% PFA for more than 24 hours, then dehydrated in alcohol and xylene, immersed in paraffin, embedded, and sectioned. The tissue was then deparaffinized with xylene and hydrated with graded ethanol concentrations according to the instructions of the HE staining kit (C0105S, Beyotime), the Sirius red staining kit (MM1004-A, Shanghai Maokang Biological), and the Masson staining kit (C0189S, Beyotime).

[0036] The obtained liver tissue was embedded in OCT compound (C0171A, Beyotime), and the frozen tissue blocks were cut into 5-10 μm thickness using a freezing microtome (CM1950, Leica) at a constant temperature of -20°C. After slicing, the sliced tissue was directly pasted on a glass slide and air-dried at room temperature. Then, the staining was performed according to the instructions of the Oil Red O Staining Kit (C0157S, Beyotime) (according to the manufacturer's instructions, 4% PFA was first added for fixation for 10 minutes, then washed twice with PBS, and an appropriate amount of staining working solution was added for staining for 30 minutes, and then an appropriate amount of staining washing solution was added for washing 2-3 times, and then an appropriate amount of PBS was added for shooting), and then observed and photographed with an optical microscope. Cell Red O staining was also performed according to the instructions.

[0037] 4) Western Blotting

[0038] Protein was extracted from liver tissue or cell pellets using a protein extraction kit (KeyGEN, BioTECH). Protein concentration was quantified using a BCA protein assay kit (KeyGEN, BioTECH). Samples were mixed with loading buffer (Biyuntian), boiled at 100°C for 10 minutes, and stored at −20°C. Proteins were separated by SDS-PAGE and transferred to a PVDF membrane. The membrane was blocked with 5% skim milk for 2 hours, incubated with the primary antibody in TBST overnight, washed, and then incubated with the secondary antibody in TBST for 1 hour.

[0039] 5) Liver proteomic analysis

[0040] Proteins were extracted using SDT lysis buffer (4% SDS, 100 mM Tris-HCl, pH 7.6), and protein concentrations were determined using the BCA assay. Samples (15 μg) were mixed with loading buffer, boiled for 5 minutes, and then separated by SDS-PAGE (4%-20% gradient gel, 180 V, 45 minutes) and stained with Coomassie. Quality control samples were prepared by pooling protein aliquots. Proteins were digested using the FASP method, desalted using a C18 filter, lyophilized, and resuspended in 0.1% formic acid. Peptide concentrations were measured at OD280. Data-independent acquisition (DIA) mass spectrometry analysis was performed using a Vanquish Neo system and an Astral mass spectrometer in positive ion mode. MS1 resolution was 240,000, and MS2 was run in DIA mode with a scan window of 299 and a collision energy of 25 eV. Data were processed using DIA-NN, with trypsin cleavage and carboxymethylation (C) as fixed modifications and oxidation (M) and acetylation (N-terminus) as dynamic modifications. Protein identification was filtered with an FDR < 1%, ensuring robustness of the proteomic analysis.

[0041] 6) Liver lipidomics analysis

[0042] Lipids were extracted from liver samples using a modified Bligh-Dyer method using chloroform-methanol-water (2:1:0.8, v / v / v). After vortex centrifugation, the organic phase was collected, dried under nitrogen, and reconstituted in isopropanol-acetonitrile (1:1, v / v). Lipid concentrations were quantified using a lipid quantification kit (Sigma-Aldrich). For analysis, 10 μL of each sample was injected onto a Vanquish ultra-high performance liquid chromatography system (Thermo Fisher Scientific) coupled with a Q Exactive HF-X mass spectrometer (Thermo Fisher Scientific). Chromatographic separation was performed using a C18 column (2.1 × 100 mm, 1.7 μm) with a gradient consisting of solvent A (water containing 0.1% formic acid and 10 mM ammonium acetate) and solvent B (acetonitrile-isopropanol, 1:1, v / v, containing 0.1% formic acid). The mass spectrometer was operated in positive and negative ion modes with a resolution of 120,000 (200 m / z), a scan range of 200–2000 m / z, and a maximum ion injection time of 50 ms. Data were acquired in data-dependent acquisition (DDA) mode, and the top 10 most intense ions were selected for MS / MS fragmentation under high-energy collisional dissociation (HCD) conditions at 28 eV. Raw data were processed using LipidSearch 4.2 (Thermo Fisher Scientific) for lipid identification and quantification. Parameters included a precursor ion mass tolerance of 5 ppm, a fragment ion mass tolerance of 10 ppm, and a lipid species identification threshold of FDR < 1%. Data normalization and statistical analysis were performed using MetaboAnalyst 5.0 to ensure robustness and reproducibility of lipid analysis.

[0043] Example 2 Results of Animal Experiments

[0044] 1) Therapeutic doses of MA have no obvious toxicity to mice

[0045] Toxicity evaluation was performed in C57BL / 6J mice, and the results were as follows Figure 1 As shown, MA at a dose of 10 mg / kg did not significantly alter liver (ALT, AST) or kidney (CREA, BUN) function markers compared to the control group. Furthermore, no significant changes were observed in glucose or lipid metabolism. However, a higher dose of 20 mg / kg resulted in significant elevations in transaminase levels, suggesting possible liver stress. Therefore, 10 mg / kg was selected as the maximum safe dose for subsequent animal studies.

[0046] 2) MA can reduce hepatic steatosis and fibrosis in a mouse model of non-alcoholic fatty liver disease

[0047] In order to verify that MA has a therapeutic effect on non-alcoholic fatty liver disease, the present invention established a non-alcoholic fatty liver disease and fibrosis mouse model ( Figure 2 A). The liver of the model mice was enlarged and had a greasy surface ( Figure 2B) confirms successful modeling. Fibrosis is more severe than simple steatosis, indicating progressive pathological changes. MA treatment, particularly at a dose of 10 mg / kg, improves gross liver morphology, with effects comparable to resmetirom, demonstrating a dose-dependent effect of MA.

[0048] HE and Oil Red O staining of NAFLD livers, and Sirius Red and Masson staining of fibrotic livers, showed significant fatty degeneration and structural destruction in the model group. These histological changes were alleviated in the MA and resmetirom groups, with reduced lipid droplets and collagen deposition. Oil Red O more clearly showed lipid droplets, while Sirius Red and Masson staining showed reduced fibrosis and pseudolobule formation. Quantitative analysis confirmed these observations ( Figure 2 CD).

[0049] Because nonalcoholic fatty liver disease reflects systemic lipid metabolism dysfunction, we evaluated serum alanine aminotransferase, aspartate aminotransferase, aspartate aminotransferase, and total cholesterol levels. All of these indicators were significantly elevated in model mice but decreased after treatment with MA or resmetirom, although some differences lacked statistical significance ( Figure 2 E). These results suggest that MA can alleviate liver damage, steatosis, and fibrosis associated with lipid dysregulation.

[0050] 3) MA activates the PPAR pathway in vivo, alleviating liver damage and liver fibrosis

[0051] The present invention confirmed the activation effect of MA on PPARs in treated animal liver samples. In the non-alcoholic fatty liver group, no significant difference was observed in the 5mg / kg MA treatment compared with the model group; however, a statistically significant effect was observed at the 10mg / kg dose ( Figure 3 A). In the liver fibrosis model, it was initially detected that the levels of collagen III and α-SMA proteins were reduced in the resmetirom and MA treatment groups compared with the model group, and the effect of MA was dose-dependent ( Figure 3 C). Similarly, in the liver fibrosis model, MA can significantly upregulate the expression of PPAR. These findings suggest that MA can alleviate non-alcoholic fatty liver disease and liver fibrosis by activating PPAR ( Figure 3 B).

[0052] 4) Lipidomics reveals that MA can regulate lipid metabolism in non-alcoholic fatty liver disease and fibrosis

[0053] To explore the lipid-regulating effects of MA, we performed non-targeted lipidomic analysis of liver tissues. Principal component analysis (PCA) showed that there were significant differences between the MA-treated and model groups in both the non-alcoholic fatty liver disease model and the fibrosis model, indicating that the lipidomic profiles were distinct ( Figure 4 AB). Lipid types were divided into eight categories. Compared with the control group, in both models, the levels of glycerophospholipids (GP) and sphingolipids (SP) in the MA group were increased, while the level of triglycerides (GL) was decreased ( Figure 4 CD). Using a threshold of |log2FC|>1 and p≤0.05, we identified 17 commonly upregulated and 21 downregulated lipid metabolites ( Figure 4 E). Most of the up-regulated metabolites belonged to the GP class, including PS (40:3e)-H, PIP (52:5)-H, and PE (18:0e_20:1)-H. In contrast, most of the down-regulated metabolites belonged to the GL class, such as DG (4:0_16:0)+Na, TG (18:1_10:4_10:4)+H, and TG (8:0_10:2_20:4)+NH4. Differential lipids were analyzed by volcano plots and heat maps ( Figure 4 FH) is further displayed intuitively.

[0054] Example 3 Results of in vitro experiments

[0055] 1) MA inhibits lipid accumulation and hepatic stellate cell activation in vitro

[0056] The present invention uses CCK-8 detection method to evaluate the cytotoxicity of AML12, BRL, HSC-T6 and JS-1 cell lines. The day before, AML12, BRL, HSC-T6 and JS-1 cells were seeded into 96-well plates according to the concentration gradient of 0μm, 3μm, 6μm, 9μm, 12μm, 15μm, 18μm, 21μm. After treatment for 24h, 48h and 72h respectively, the original culture medium was removed and fresh culture medium containing 10% CCK-8 solution was added. After incubation at 37°C for 3 hours, the absorbance was measured at a wavelength of 450nm. At a concentration of 10μM and exposure for 48 hours, MA can maintain the cell viability of all four cell types above 90% ( Figure 5 A), which was selected as the optimal dose and time point for subsequent experiments. To explore the dose-dependency, we treated cells with 5 μM, 10 μM, and 15 μM MA and used 100 μM resmetirom as a positive control because it has been reported to reduce the progression of steatohepatitis. Oil red O staining (same method as above) showed that both MA and resmetirom effectively reduced intracellular lipid accumulation in AML12 and BRL hepatocytes ( Figure 5B). This lipid-lowering effect was accompanied by increased expression of PPARA, PPARD, and PPARG proteins in both cell types ( Figure 5 D, F, H). Given the established role of PPARs in the regulation of hepatic stellate cells (HSCs), we also evaluated the effects of MA on TGF-β-induced activation of HSC-T6 (rat) and JS-1 (mouse) cell lines. In both models, MA treatment significantly reduced the expression of fibrotic markers collagen III and α-SMA, while increasing the expression of PPARA, PPARD, and PPARG ( Figure 5 C, E, G). These results indicate that MA inhibits fibrosis by regulating PPAR signaling in hepatocytes and hepatic stellate cells.

[0057] 2) MA alleviates steatosis and fibrosis in a three-dimensional liver model through PPARs

[0058] To further verify the anti-NAFLD and anti-fibrosis effects of MA, we used NAC-Organ technology to construct a three-dimensional liver pathology model. After the model was constructed in the early stage, MA was added to the professional culture medium in a 96-well plate according to the concentration gradient of 0μM, 5μM, 10μM, 20μM, and 40μM. After 48 hours of treatment, the old culture medium was removed and fresh professional culture medium containing 10% CCK8 solution was added. After incubation for an appropriate time, the absorbance at a wavelength of 450nm was measured. The IC of MA was determined by CCK-8 assay. 50 17.34 μM ( Figure 6 A); therefore, consistent with the two-dimensional cell assay, we chose 10 μM for subsequent experiments. Histological analysis showed that both MA and resmetirom significantly reduced vacuolation (HE staining) and lipid accumulation (Oil Red O staining) compared with the model group. Collagen I immunofluorescence also showed that the degree of fibrosis in the MA and resmetirom groups was reduced. Notably, combined treatment with the PPARα / γ antagonist GW9662 reversed the anti-fibrotic and lipid-lowering effects of MA, as manifested by increased lipid droplets and collagen deposition ( Figure 6 B). Biochemical analysis of culture supernatants confirmed these findings. Triglyceride (TG), total collagen, and IL-6 levels were significantly decreased in the MA and resmetirom groups, but increased again in the GW9662 co-treatment group ( Figure 6 CE). This suggests that the anti-inflammatory and metabolic effects of MA are mediated through PPAR activation.

[0059] The above description is only a preferred embodiment of the present invention and does not limit the implementation mode and protection scope of the present invention. For those skilled in the art, it should be aware that all solutions obtained by equivalent substitutions and obvious changes made using the contents of the description and illustrations of the present invention should be included in the protection scope of the present invention.

Claims

1. Use of Moronic acid in the preparation of a drug for treating non-alcoholic fatty liver disease, characterized in that: Moronicacid is a natural pentacyclic triterpenoid compound with the chemical structural formula:

2. The use according to claim 1, characterized in that The only active ingredient of the drug for treating non-alcoholic fatty liver disease is Moronic acid.

3. The use according to claim 1, characterized in that Moronic acid alleviates non-alcoholic fatty liver disease and liver fibrosis by activating the PPAR pathway.

4. The use according to claim 1, characterized in that The drug for treating non-alcoholic fatty liver disease also includes pharmaceutically acceptable excipients.

Images