Application of nicotiflorin in preparation of medicine for treating metabolism-related fatty liver disease / metabolism-related steatohepatitis disease

Abstract

The invention belongs to the field of medicines, and discloses application of nicotiflorin in preparation of medicines for treating metabolism-related fatty liver diseases / metabolism-related steatohepatitis diseases. The invention discloses application of nicotiflorin in preparation of a medicine for treating fatty liver diseases related to metabolic dysfunction and application of nicotiflorin in preparation of a medicine for treating hepatic fibrosis or slowing down the development process of hepatic fibrosis. The invention discloses an application of nicotiflorin in preparation of a health care product with an auxiliary protection effect on chemical liver injury. The nicotiflorin can obviously reduce the lipid content of AML-12 cells induced by palmitic acid and obviously reduce the expression of alpha-SMA and Collagen1 of human hepatic stellate cells, so that the activation of LX-2 cells is inhibited, and the liver fibrosis induced by CCl4 is obviously improved.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of medicine, and relates to application of Nicotiflorin in relieving metabolic dysfunction associated fatty liver disease (MASLD) / metabolic dysfunction associated steatohepatitis (MASH), in particular, to application of Nicotiflorin in preparing a medicament for treating metabolic associated fatty liver disease / metabolic associated steatohepatitis, application of Nicotiflorin in functional food, and application of Nicotiflorin in preparing health care products with auxiliary protection effect on chemical liver injury. BACKGROUND

[0002] Metabolic dysfunction associated fatty liver disease (MASLD), formerly known as non-alcoholic fatty liver disease (NAFLD), is a group of metabolic disorders involving multiple organs, and the characteristic pathological manifestation is excessive deposition of triglyceride in hepatocytes, characterized by central obesity, high blood pressure, hyperglycemia and dyslipidemia. At present, the drug for treating MASLD in clinic has no significant effect, and the process of MASLD is mainly controlled by changing the way of life such as controlling diet and exercising. Therefore, it is of great practical significance to find safe and effective drugs for preventing and treating MASLD.

[0003] Hepatic fibrosis (HF) is a wound repair reaction caused by continuous stimulation of various chronic liver injury factors (such as viral hepatitis, alcohol abuse, metabolic dysfunction associated fatty liver disease, etc.), and its pathological characteristics are excessive deposition and imbalance of degradation of extracellular matrix (especially collagen fibers) in the liver, which leads to destruction of liver structure and impairment of liver function. If not effectively intervened, liver fibrosis can progress to cirrhosis and even liver failure, which is the main pathological basis of end-stage liver disease. At present, the research and development of drugs for treating liver fibrosis is still in the early stage, and there are defects such as limited clinical efficacy, high treatment cost, obvious side effects, high failure rate of clinical trials, complexity of metabolic dysfunction associated steatohepatitis (MASH) treatment, and difficulty in reversing liver fibrosis.

[0004] Hepatic stellate cells (HSCs), also known as perisinusoidal cells, are the core effector cells of liver fibrosis. In physiological state, HSCs are in a resting phenotype, and after liver injury, hepatocytes, immune cells and other cells release various mediators to promote the activation of HSCs. During liver injury, necrotic hepatocytes release TGF-β, which inhibits apoptosis of HSCs and induces HSCs to synthesize excessive matrix proteins, promotes the production of ECM and inhibits its degradation, thereby leading to liver fibrosis. Nicotiflorin has anti-inflammatory and immunoregulatory, antioxidant and neuroprotective, and liver protection activities. Search shows that Nicotiflorin can protect against acute liver injury induced by concanavalin A, galactoside and alcohol-induced liver injury, and no report has been found that Nicotiflorin can relieve MASLD / MASH. SUMMARY

[0005] In view of the deficiencies of the prior art, the present application aims to provide a new medical use of Yinhuaosan, i.e., it can be applied to treat hepatocyte lipid accumulation and liver fibrosis diseases.

[0006] The object of the present application is achieved by the following technical solutions.

[0007] The application of Yinhuaosan, Yinhuaosan derivatives (i.e., Yinhuaosan modified in chemical structure), traditional Chinese medicine containing Yinhuaosan or prescription in the preparation of functional food or medicine, nutrient supplement, dietary supplement for preventing and / or treating metabolic dysfunction related fatty liver disease (MASLD) / metabolic dysfunction related fatty liver hepatitis (MASH).

[0008] The metabolic dysfunction related fatty liver disease / metabolic dysfunction related fatty liver hepatitis is manifested as simple liver steatosis or accompanied by liver inflammation, accompanied by liver fibrosis.

[0009] The metabolic dysfunction related fatty liver disease / metabolic dysfunction related fatty liver hepatitis is manifested as overweight / obesity (BMI≥24.0 kg / m 2 ), persistent increase in serum transaminase level (>6 months), liver steatosis (liver fat content≥5%), dyslipidemia (triglyceride≥1.70 mmol / L, or high-density lipoprotein cholesterol≤1.0 mmol / L (male), ≤1.3 mmol / L (female)), liver lobular inflammation and ballooning degeneration, fibrosis type F0-F3 index characteristics.

[0010] Preferably, Yinhuaosan is applied in the preparation of functional food or medicine, nutrient supplement, dietary supplement for preventing and / or treating metabolic dysfunction related fatty liver disease / metabolic dysfunction related fatty liver hepatitis by exerting at least one of the following effects: sustained weight loss, liver fat content reduction, improvement of serum transaminase level and blood lipid level, and reduction of fibrosis degree.

[0011] As one of the specific technical solutions of the present application, Yinhuaosan is applied in the preparation of functional food or medicine, nutrient supplement, dietary supplement for preventing and / or treating metabolic dysfunction related fatty liver disease (MASLD) / metabolic dysfunction related fatty liver hepatitis (MASH).

[0012] As one of the specific technical solutions of the present application, Yinhuaosan is applied in the preparation of medicine for treating metabolic dysfunction related fatty liver disease (MASLD).

[0013] As one of the specific technical solutions of the present application, Yinhuaosan is applied in the preparation of medicine for treating metabolic dysfunction related fatty liver hepatitis (MASH) and liver fibrosis diseases.

[0014] As one of the specific technical solutions of the present application, the application of Yinhuaoside in the preparation of a drug for treating or slowing the development of liver fibrosis.

[0015] The liver fibrosis is chemical liver fibrosis.

[0016] Preferably, the drug comprises a pharmaceutically acceptable carrier; and the dosage form of the drug is oral preparation, injection preparation, transdermal preparation or mucosal preparation.

[0017] As one of the specific technical solutions of the present application, the application of Yinhuaoside in the preparation of a health care product having an auxiliary protective effect on chemical liver injury.

[0018] A pharmaceutical composition for preventing and / or treating metabolic dysfunction-associated steatohepatitis (MASLD) / metabolic dysfunction-associated steatohepatitis (MASH), which comprises Yinhuaoside, a Yinhuaoside derivative (i.e., Yinhuaoside modified in chemical structure) or a Chinese medicinal material containing Yinhuaoside as an effective component, and further comprises a pharmaceutically acceptable carrier.

[0019] The dosage form of the pharmaceutical composition is oral preparation, injection preparation, transdermal preparation or mucosal preparation.

[0020] As one of the specific technical solutions of the present application, Yinhuaoside can be prepared into an injection preparation for intraperitoneal injection.

[0021] In specific embodiments, the lipid accumulation in hepatocytes is in vitro palmitic acid (PA)-induced AML-12 cells, which is an in vitro pathological model of MASLD; the liver fibrosis is in vitro TGF-β-induced LX-2 cell activation and in vivo CCl4-induced liver fibrosis and diet-induced MASH and liver fibrosis.

[0022] Compared with the prior art, the present application has the following beneficial effects:

[0023] Yinhuaoside can reduce the increase of triglyceride (TG) in AML-12 cells induced by palmitic acid (PA) and reduce the lipid content in hepatocytes; at the same time, Yinhuaoside can inhibit the collagen secretion and fibrosis deposition of hepatic stellate cells and slow the development of liver fibrosis, thereby providing a theoretical research basis for developing Yinhuaoside into a drug for treating MASLD / MASH and fibrosis diseases such as liver fibrosis. Specifically, Yinhuaoside can:

[0024] (1) Under the stimulation of exogenous PA, Yinhuaoside can reduce the TG level and lipid level in AML-12 cells, suggesting that Yinhuaoside can help improve MASLD;

[0025] (2) Under the stimulation of exogenous TGF-β, Yinhuaoside can reduce the α-SMA, collagen I and collagen III levels in LX-2 cells, suggesting that Yinhuaoside can help improve liver fibrosis. The expression level of Collagen 1 is inhibited, and the collagen deposition is inhibited, which can help to inhibit the activation of LX-2 cells and slow down the development process of liver fibrosis.

[0026] (3) In the CCl4-induced liver fibrosis model, intraperitoneal injection of Yanhuangoside can reduce the serum ALT and AST levels and reduce the liver HYP content. The pathological results show that Yanhuangoside can reduce the enrichment of liver fibrosis bridges, inhibit collagen deposition, and improve inflammatory cell infiltration. At the same time, Yanhuangoside can down-regulate the expression levels of fibrosis-related proteins α-SMA and Collagen 1 in the liver, suggesting that Yanhuangoside can help to slow down the occurrence of MASH and liver fibrosis and delay the development process of liver fibrosis, thereby achieving the purpose of improving liver fibrosis. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 The effect of Yanhuangoside on the activity of LX-2 cells.

[0028] Figure 2 The effect of Yanhuangoside on the lipid content change of PA-induced AML-12 cells; wherein A is the change of TG content of hepatocytes in Yanhuangoside-induced high-fat model, and B is the lipid Nile red staining of Yanhuangoside-induced high-fat model (scale is 50 μm).

[0029] Figure 3 The effect of Yanhuangoside on α-SMA and Collagen 1 proteins of LX-2 cells; wherein A is the change of α-SMA and Collagen 1 protein Western Blot content, B is the relative expression change of Collagen 1 protein content, and C is the relative expression change of α-SMA protein content.

[0030] Figure 4 The immunofluorescence staining of α-SMA and Collagen 1 proteins of LX-2 cells (scale is 25 μm).

[0031] Figure 5 The effect of Yanhuangoside on serum ALT, AST and liver HYP of CCl4-induced liver fibrosis mice; wherein A is the change of serum ALT content, C is the change of serum AST content, and C is the change of liver HYP content.

[0032] Figure 6 The effect of Yanhuangoside on α-SMA and Collagen 1 proteins of CCl4-induced liver fibrosis mice, wherein A is the change of α-SMA and Collagen 1 protein Western Blot content, B is the relative expression change of α-SMA protein content, and C is the relative expression change of Collagen 1 protein content.

[0033] Figure 7 H&E staining (scale bar, 200 μm), Masson's trichrome staining (scale bar, 100 μm), and immunofluorescence staining of α-SMA and Collagen 1 (scale bar, 100 μm) of the liver of the mice with liver fibrosis induced by YH and CCl4. DETAILED DESCRIPTION

[0034] In order for those skilled in the relevant art to better understand the content of the present application, the technical solutions of the present application are described in detail below through examples, which are implemented on the premise of the technical solutions of the present application, and detailed implementation modes and specific operation processes are given, but the content of the present application is not limited to the examples described below.

[0035] The reagents or instruments and equipment used are not marked with the manufacturer, and are all regarded as conventional products that can be purchased in the market.

[0036] Palmitic acid (PA) is a free long-chain saturated fatty acid, which can cause lipid droplet accumulation in hepatocytes by promoting intracellular triglyceride synthesis and lipid metabolism disorder. In the present application, PA is used to induce AML-12 hepatocytes to produce lipid accumulation, simulating the pathological environment of metabolic dysfunction-associated steatohepatitis (MASLD).

[0037] TGF-β (Transforming Growth Factor-β) is a multifunctional cytokine belonging to the TGF-β superfamily, which is expressed in almost all tissues and is involved in key physiological processes such as cell proliferation, differentiation, apoptosis, immune regulation, and extracellular matrix (ECM) synthesis. In liver fibrosis, TGF-β drives HSC activation and collagen deposition through Smad-dependent and independent pathways. In the present application, TGF-β is used as an inducer to establish a human hepatic stellate cell fibrosis model.

[0038] CCl4 is a classic inducer of liver fibrosis, which is metabolized by cytochrome P450 enzyme system in the body to generate trichloromethyl free radical (CCl3), which can attack the membrane of hepatocytes to initiate lipid peroxidation, leading to hepatocyte damage and necrosis; at the same time, it activates HSCs, which are transformed into myofibroblast-like cells, which synthesize a large amount of ECM components such as collagen, and ultimately form a fibrosis model. In the present application, 25% CCl4 is used as an inducer of liver fibrosis in mice to establish an in vivo liver fibrosis model.

[0039] Experimental cells:

[0040] Human hepatic stellate cells (LX-2 cell line) were cultured in 1640 complete medium (1640 basal medium + 10% FBS + 1% double antibiotics (streptomycin and penicillin)) at a ratio of 1:3 in a cell culture incubator (37℃, 5% CO2) and passaged.

[0041] AML-12 cells were cultured in DMEM-F12 complete medium (DMEM-F12 basal medium + 10% FBS + 1% double antibiotics (streptomycin and penicillin)) at a ratio of 1:6 in a cell culture incubator (37℃, 5% CO2) and passaged.

[0042] Experimental animals: C57BL / 6J mice.

[0043] All experimental data were analyzed using GraphPad Prim 9.5 statistical software and are expressed as Mean ± SD. One-way ANOVA was used to analyze significant differences between experimental groups. * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001, **** indicates P < 0.0001, and ns indicates no statistically significant difference.

[0044] Example 1

[0045] LX-2 cell Cell Counting Kit-8 (CCK8) experiment

[0046] A 200 mM stock solution of pyroxin was prepared using DMSO. The stock solution was then serially diluted using 1640 complete medium to obtain drug-containing mediums with final concentrations of pyroxin of 400, 200, 100, 50, 25, 12.5, 6.25, and 3.125 μM.

[0047] The effect of pyroxine on LX-2 cell viability was evaluated using the CCK-8 assay. The specific procedure was as follows: LX-2 cells were evenly seeded at 5000 cells / well in 96-well plates and cultured in a cell culture incubator for 24 h. Before drug administration, the old culture medium was discarded, and the experimental groups were replaced with different concentrations of drug-containing medium, with three replicates for each concentration. The control group was replaced with fresh 1640 complete medium, and the 10% DMSO group was replaced with 1640 complete medium containing 10% DMSO. 24 h after drug administration, the medium was discarded, and 10% CCK-8 solution (prepared from 1640 basal medium) was added to each well. The control group contained only 10% CCK-8 solution. The cells were incubated in a cell culture incubator for an appropriate time, and the absorbance (OD) was measured at 450 nm to calculate cell viability.

[0048]

[0049] The results are shown in Table 1. Figure 1 As shown, after treatment with different concentrations of pyroside (3.125-400 μM) for 24 h, cell viability did not change significantly, indicating that pyroside had no significant toxicity to LX-2 cells within this concentration range. This result provides a safe dosage reference for subsequent studies on the anti-fibrotic effect of pyroside on human hepatic stellate cells.

[0050] Table 1. Effect of pyroxine on LX-2 cell viability (Mean±SD)

[0051]

[0052] Note: Compared to the Control group, **** has p<0.0001.

[0053] Example 2

[0054] AML-12 cell TG detection and Nile Red staining experiment

[0055] A 20 mM palmitic acid (PA) stock solution was prepared using 20% ​​BSA (BSA powder dissolved in double-distilled water). Before administration, the PA stock solution was diluted to a 0.2 mM PA solution using DMEM-F12 complete culture medium.

[0056] A 250 mM pyroside stock solution was prepared using DMSO. Before administration, the pyroside stock solution was diluted with DMEM-F12 medium to form drug-containing mediums with pyroside concentrations of 10 μM, 50 μM, and 250 μM.

[0057] Evaluation of the efficacy of NF-κB in improving palmitic acid (PA)-induced lipid accumulation in AML-12 cells. The specific methods are as follows: AML-12 cells were evenly seeded at 10,000 cells / well in 96-well plates (4 replicates per group) and at 30,000 cells / well in 24-well plates (24-well plates pre-placed with cell spreaders), and cultured in a cell culture incubator for 24 h. The next day, the PA-0.2 mM group and the NF-κB group were replaced with 0.2 mM PA solution, and the CON group was replaced with fresh DMEM-F12 complete medium. All were incubated for 24 h, and the medium was discarded. The CON group and the PA-0.2 mM group were replaced with DMEM-F12 complete medium, and the NF-κB groups were replaced with NF-κB media containing 10 μM, 50 μM, and 250 μM NF-κB concentrations (denoted as NF-10 μM group, NF-50 μM group, and NF-250 μM group), and incubated for 24 h.

[0058] After discarding the culture medium, the cells in the 96-well plates were frozen at -80°C to lyse. The absorbance was measured at 550 nm using a triglyceride (TG) assay kit from Nanjing Jiancheng Biotechnology Co., Ltd., and the TG content of the cells was calculated. The results are shown in Table 2. Figure 2 As shown in Figure A.

[0059] After discarding the culture medium in the 24-well plates, cells were fixed with 4% paraformaldehyde for 10 min, washed three times with PBS buffer, incubated with Nile Red staining solution at room temperature for 30 min, washed three times with PBS buffer, and mounted on a glass slide with DAPI-containing anti-fluorescence quenching solution. The slide was then inverted onto the glass slide and observed and photographed using an FV3000 confocal microscope. The results are as follows: Figure 2 As shown in B.

[0060] The results showed that PA significantly increased intracellular lipid content, while pyroside significantly decreased intracellular lipid content, suggesting that pyroside may help improve fatty liver disease associated with metabolic dysfunction.

[0061] Table 2. Changes in TG content in PA-induced AML-12 cells induced by pyroxine (Mean±SD)

[0062]

[0063] Note: Compared with the PA-0.2mM group, **** is p<0.0001.

[0064] Example 3

[0065] LX-2 cell experimental procedures

[0066] A stock solution of TGF-β with a concentration of 40 μg / mL was prepared using PBS, and then diluted with 1640 complete culture medium to a concentration of 0.15 ng / mL before administration.

[0067] A 200 mM solution of pyroside was prepared using DMSO. Before administration, the pyroside solution was diluted with DMEM-F12 medium to form drug-containing medium with concentrations of 8 μM, 40 μM, and 200 μM.

[0068] Efficacy evaluation of pyroxine in treating TGF-β-induced human liver fibrosis cells (LX-2 cell line). The specific methods are as follows: LX-2 cells were uniformly seeded at a concentration of 30,000 cells / well in 24-well plates (the 24-well plates were pre-placed with cell crawling smears) and divided into four groups: Con group, TGF-β group, low-concentration pyroxine group (TGF-β+NL group), medium-concentration pyroxine group (TGF-β+NM group), and high-concentration pyroxine group (TGF-β+NH group). After incubation for 24 h, except for the Con group, all other groups were treated with 0.15 ng / mL TGF-β solution for modeling. After 24 h, the Con group and TGF-β group were replaced with 1640 complete medium, while the low-concentration, medium-concentration, and high-concentration pyroxine groups were treated with pyroxine-containing medium at the corresponding concentrations (8 μM, 40 μM, and 200 μM, respectively). Protein and immunofluorescence samples were collected 24 h after pyroxine administration.

[0069] Western blot and immunofluorescence staining experiments

[0070] Western blot detection of Collagen1 and α-SMA proteins in LX-2 cells: LX-2 cell proteins were collected and electrophoresed at 80V for 30 min, then at 120V for 1 h. After wet transfer and blocking for 2 h, the cells were incubated with primary antibody overnight at 4℃. The next day, after washing the membrane, the cells were incubated with the corresponding secondary antibody at room temperature for 2 h, followed by exposure. The results are shown in Table 3. Figure 3 As shown.

[0071] Table 3. Effects of pyroside on the levels of α-SMA and Collagen1 proteins in LX-2 cells (Mean ± SD)

[0072]

[0073] Note: Compared with the TGF-β group, ** means p<0.01, *** means p<0.001, and **** means p<0.0001.

[0074] Immunofluorescence staining was used to examine the degree of fibrosis in LX-2 cells in each group: LX-2 cell slides were collected from each group. The slides were washed three times with PBS, fixed with 4% paraformaldehyde at room temperature for 15 min, washed three times with PBS, permeabilized with 0.1% Triton X-100 at room temperature for 10 min, washed three times with PBS, blocked with 10% goat serum for 1.5 h, incubated with primary antibody at 4°C overnight, washed three times with PBS, stained with secondary immunofluorescence antibody, incubated at room temperature for 1 hour, washed three times with PBS, and mounted. The slides were observed and photographed under an FV3000 laser confocal microscope. Results are as follows: Figure 4 As shown.

[0075] The results showed that after administration of pyroside, the expression of related fibrosis factors Collagen1 and α-SMA in LX-2 cells was significantly downregulated, indicating that pyroside could inhibit the activation of LX-2 cells and suggesting that pyroside may help delay the development of liver fibrosis.

[0076] Example 4

[0077] Animal experimental procedures

[0078] Thirty male, 6-week-old (approximately 21g) SPF-grade C57BL / 6J mice were randomly divided into five groups: Control group, 25% CCl4 group, low-dose pyroxine (NF-L group, 25mg / kg) group, medium-dose pyroxine (NF-M group, 50mg / kg) group, and high-dose pyroxine (NF-H group, 100mg / kg) group. Mice were provided with sufficient food and water, and kept in a barrier + IVC environment with alternating 12-hour light and 12-hour dark cycles. Except for the Control group, mice in all other groups underwent intraperitoneal injection of 25% CCl4 (prepared with corn oil) at a dose of 2mL / kg, twice weekly for 6 weeks. Mice in the Control group received the same dose of corn oil intraperitoneally. Simultaneously, mice in the three pyroxine administration groups received intraperitoneal injections of 25, 50, and 100mg / kg of pyroxine solution (prepared with 0.5% CMC-Na), respectively, once daily for 6 weeks. At the end of the experiment, blood samples were collected from mice by orbital blood sampling, livers were taken for pathological examination and cryopreservation, and the remaining liver tissue was stored at -80℃ for later use.

[0079] Animal serum ALT, AST and liver HYP detection

[0080] Animal blood samples were incubated at room temperature for 3 hours, then centrifuged at 3500 rpm for 15 minutes at 4°C. The supernatant serum was collected. Using ALT and AST kits from Wittmann Biotechnology (Nanjing) Co., Ltd., and Roche standards, the absorbance of the samples was measured at 340 nm using the rate method. The levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in the samples were calculated. The results are shown in Table 4. Figure 5 As shown in A and 5B.

[0081] Approximately 80 mg of liver sample was weighed and the liver weight was recorded. The HYP content in the liver sample was detected using a hydroxyproline (HYP) assay kit (alkaline hydrolysis method) from Nanjing Jiancheng Biotechnology Co., Ltd. The results are shown in Table 4. Figure 5 As shown in C.

[0082] The results showed that, compared with the control group, mice treated with 25% CCl4 intraperitoneally for 6 weeks exhibited significantly upregulated serum ALT, AST, and liver HYP levels, indicating severe liver inflammation and increased collagen production. Treatment with pyroside reduced liver inflammation and decreased collagen production in mice, suggesting that pyroside may help improve inflammation caused by liver fibrosis.

[0083] Table 4. Effects of pyroside on serum ALT, AST and liver HYP in mice with liver fibrosis (Mean±SD)

[0084]

[0085] Note: Compared with the 25% CCl4 group, * indicates p<0.05, ** indicates p<0.01, *** indicates p<0.001, and **** indicates p<0.0001.

[0086] Animal liver protein Western blot experiment

[0087] Approximately 20 mg of liver tissue was weighed into a 2 mL grinding tube, and 200 μL of SDS lysis buffer and magnetic beads were added. The mixture was then ground in a low-temperature high-speed grinder (60 Hz, 45 s on, 15 s off, 5 times). The homogenate was transferred to a 1.5 mL centrifuge tube, and 400 μL of SDS lysis buffer was added. The mixture was mixed, incubated on ice for 10 min, and centrifuged at 12000 × g for 10 min at 4 °C. The supernatant was collected, and protein quantification was performed using the BCA method. A protein loading buffer containing 1 × Loading buffer was prepared according to the protein concentration. Western blot procedures were the same as in Example 3. The results are shown in Table 5. Figure 6 As shown, after 6 weeks of intraperitoneal injection of 25% CCl4 into mice, the protein expression levels of α-SMA and Collagen1 in the liver were significantly upregulated. After treatment with pyroside, the protein expression levels of α-SMA and Collagen1 in the liver were significantly downregulated, suggesting that pyroside can improve collagen deposition in the liver of fibrotic mice.

[0088] Table 5. Effects of pyroside on α-SMA and Collagen1 proteins in CCl4-induced liver fibrosis mice (Mean ± SD)

[0089]

[0090] Note: Compared with the 25% CCl4 group, * indicates p<0.05, ** indicates p<0.01, and **** indicates p<0.0001.

[0091] Animal liver pathological examination and immunofluorescence staining

[0092] Liver pathology samples were stained with hematoxylin-eosin (H&E) and Masson staining. After fixation with 4% paraformaldehyde, the staining was performed by Saive Biotechnology Co., Ltd. The stained sections were observed and photographed under a BX-53 microscope.

[0093] Frozen liver samples were sectioned using a cryostat, with sections 8 μm thick, and adhered to glass slides. The staining procedure was the same as in Example 3.

[0094] The results are as follows Figure 7 As shown in the results, after 6 weeks of intraperitoneal injection of 25% CCl4 in mice, significant liver fibrosis was observed, with numerous fibrous bridging and inflammatory cell infiltration, and significantly increased protein expression levels of α-SMA and Collagen1. After treatment with pyroside, the number of liver fibrous bridging was significantly reduced, inflammatory cell infiltration was alleviated, protein expression levels of α-SMA and Collagen1 were significantly downregulated, and the degree of fibrosis was alleviated, suggesting that pyroside may help alleviate liver inflammation and fibrosis.

[0095] In summary, this invention investigated the therapeutic effects of flamingoside on lipid accumulation and liver fibrosis using internationally recognized PA-induced AML-12 cell lipid accumulation models, TGF-β-induced LX-12 cell activation models, and CCl4-induced mouse liver fibrosis models. A systematic study of the effects of flamingoside on lipid accumulation and liver fibrosis was conducted from pharmacodynamic and molecular biological perspectives. Results showed that flamingoside could improve lipid accumulation in hepatocytes under high-fat lipid models; in the liver fibrosis model, flamingoside reduced ALT, AST, and HYP levels, downregulated the expression of fibrosis-related proteins Collagen1 and α-SMA, alleviated the accumulation of hepatic collagen fibers, and delayed the progression of liver fibrosis, thereby achieving the goal of improving liver fibrosis. Therefore, this study provides a theoretical basis for the development of flamingoside for the treatment of MASLD / MASH diseases.

[0096] The scope of protection of this invention is not limited to the above embodiments. Variations and advantages that can be conceived by those skilled in the art without departing from the spirit and scope of the inventive concept are included in this invention and are protected by the appended claims.

Claims

1. The use of pyroside or its derivatives, Chinese medicinal materials or prescriptions containing pyroside in the preparation of functional foods or drugs, nutrient supplements, and dietary supplements for the prevention and / or treatment of metabolic dysfunction-related fatty liver disease / metabolic dysfunction-related fatty liver inflammation.

2. The application according to claim 1, characterized in that: Metabolic dysfunction-related fatty liver disease / metabolic dysfunction-related fatty liver disease manifests as simple hepatic steatosis or is accompanied by liver inflammation and liver fibrosis.

3. The application according to claim 2, characterized in that: Metabolic dysfunction-related fatty liver disease / metabolic dysfunction-related steatohepatitis is characterized by overweight / obesity (BMI ≥ 24.0 kg / m²). 2 ), persistently elevated serum transaminase levels (>6 months), hepatic steatosis (liver fat content ≥%), dyslipidemia (triglycerides ≥1.70 mmol / L, or high-density lipoprotein cholesterol ≤1.0 mmol / L (male), ≤1.3 mmol / L (female)), intralobular inflammation and ballooning degeneration of the liver, and fibrosis classification F0-F3 indicators.

4. The application according to claim 1, characterized in that: Application of pyrolysis glycoside in the preparation of functional foods, drugs, nutrient supplements, and dietary supplements that prevent and / or treat metabolic dysfunction-related fatty liver disease / metabolic dysfunction-related steatohepatitis by exerting at least one of the following effects: sustained weight loss, decrease in liver fat content, improvement in serum transaminase and blood lipid levels, and reduction in fibrosis.

5. Application of pyroside in the preparation of drugs for treating fatty liver disease related to metabolic dysfunction.

6. Application of pyroside in the preparation of drugs for treating liver fibrosis or slowing the progression of liver fibrosis.

7. The application according to claim 1, 5, or 6, characterized in that: The drug includes a pharmaceutically acceptable carrier; the dosage form of the drug is an oral preparation, an injectable preparation, a transdermal preparation, or a mucosal preparation.

8. Application of pyrolysis glycoside in the preparation of health products that have an auxiliary protective effect against chemically induced liver damage.

9. A pharmaceutical composition for the prevention and / or treatment of metabolic dysfunction-related fatty liver disease / metabolic dysfunction-related steatohepatitis, characterized in that: The pharmaceutical composition described herein uses pyroside, pyroside derivatives, and traditional Chinese medicinal materials containing pyroside as active ingredients, and includes pharmaceutically acceptable carriers.

10. The pharmaceutical composition according to claim 9, characterized in that: The dosage form of the pharmaceutical composition is an oral preparation, an injectable preparation, a transdermal preparation, or a mucosal preparation.

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