Application of auricularia auricula monomer polysaccharide ME-2 in preparation of medicine for preventing and treating fatty liver disease related to metabolic dysfunction

Abstract

The invention discloses application of auricularia auricula monomer polysaccharide ME-2 in preparation of drugs for preventing and treating fatty liver diseases related to metabolic dysfunction. The research of the invention systematically proves that ME-2 has a remarkable intervention effect on the whole course of MASLD for the first time. The invention discloses a breakthrough function of ME-2 as a liver metabolism regulator for the first time: by directly correcting liver lipid metabolism disorder (such as regulating core genes such as Srebp-1c, ACC1, FASN and the like), liver fatty degeneration can be relieved in a dose-dependent manner in the whole course of MASLD, and the serum transaminase level and dyslipidemia can be reduced. Importantly, the curative effect of the traditional Chinese medicine is derived from intervention on a metabolic root source, and is totally different from a known anti-inflammatory application mechanism. Meanwhile, ME-2 has the remarkable advantages of being clear in structure, taking metabolic regulation as a core, and being safe and non-toxic, and a brand new drug candidate is provided for prevention and treatment of MASLD.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and more specifically, to the application of black fungus monomer polysaccharide ME-2 in the preparation of drugs for the prevention and treatment of fatty liver disease related to metabolic dysfunction. Background Technology

[0002] Metabolic dysfunction-associated fatty liver disease (MASLD) is the most common chronic liver disease worldwide, with a global prevalence of approximately 38.8%. Its disease spectrum progresses from simple hepatic steatosis to metabolic dysfunction-associated steatohepatitis (MASH) with inflammation and damage, and even to liver fibrosis, cirrhosis, and liver cancer. Current clinical treatment options are limited: there are no effective drugs to halt disease progression in the early stages; in the MASH stage, only a very few approved drugs (such as retemetine) are available, and these are limited by high cost and a narrow patient population. Other treatments, such as insulin sensitizers, are accompanied by side effects such as weight gain and cardiovascular risks. Therefore, there is an urgent clinical need to develop a novel drug that can safely, effectively, and economically intervene in the entire course of MASLD.

[0003] Black fungus ( Auricularia auricula Auricularia auricula-judae is a fungus used in both medicine and food, rich in polysaccharides and other active ingredients. Its polysaccharide components exhibit broad pharmacological activity in regulating glucose and lipid metabolism and protecting the liver. Chinese patent application CN118599020A discloses a type of Auricularia auricula heteropolysaccharide (AAPs) that can alleviate non-alcoholic steatohepatitis (MASLD) induced by chemical toxins combined with a high-fat diet by regulating intestinal flora. This application provides preliminary clues for the application of Auricularia auricula polysaccharides in the field of liver disease; however, the polysaccharides used are heteropolysaccharides with unclear structures, not single-structure active molecules, and the structure-activity relationship between structure and efficacy cannot be elucidated. Furthermore, the chemical toxin-induced model used cannot simulate the pathological process of MASLD in humans, which is centered on metabolic disorders, making it difficult to confirm its therapeutic value throughout the entire MASLD process. Therefore, it is urgent to identify polysaccharide monomers with well-defined structures and clear activities from Auricularia auricula to directly verify their intervention ability on hepatic lipid metabolism.

[0004] Chinese patent application CN114957508A discloses a structurally well-defined, high-acetyl group of novel black fungus monomeric polysaccharide ME-2 and its application in the prevention and treatment of silicosis. Subsequently, Chinese patent applications CN117752702A and CN118453641A disclosed that this black fungus monomeric polysaccharide ME-2 can be used for tumor immunotherapy and the treatment of pulmonary nodules. However, there are currently no reports on the role of black fungus monomeric polysaccharide ME-2 in metabolic dysfunction-related fatty liver disease. Summary of the Invention

[0005] For ease of description and understanding of the present invention, unless otherwise stated, the terms used below have the following specific meanings: MASLD: refers to the complete spectrum of fatty liver diseases associated with metabolic dysfunction.

[0006] MASL: In this invention, it specifically refers to the early stage of MASLD, whose core pathological feature is simple steatosis of hepatocellular cells. This definition aims to clearly distinguish the different stages of the disease progression.

[0007] MASH: refers to the inflammatory progression stage of MASLD, namely metabolic dysfunction-associated steatohepatitis.

[0008] This invention aims to provide new pharmaceutical uses for the black fungus monomer polysaccharide ME-2; specifically, the purpose of this invention is to provide the use of the novel high-acetyl black fungus monomer polysaccharide ME-2 in the prevention and / or treatment of MASLD or in the preparation of drugs for the prevention and / or treatment of MASLD, so as to overcome the problem in the prior art of lacking novel natural active ingredients with well-defined structure, high safety, and the ability to simultaneously intervene in MASL and MASH, and intervene in the entire course of MASLD.

[0009] The black fungus monomeric polysaccharide ME-2 described in this invention is prepared according to the method of Chinese patent application CN114957508A. The preparation method of the black fungus monomeric polysaccharide ME-2 includes the following steps: S1. Black fungus was extracted using water as the extraction solvent to obtain a black fungus aqueous extract; S2. Macroporous resin was used to adsorb the aqueous extract of black fungus to remove pigments and proteins, followed by elution to obtain a macroporous resin aqueous eluent of black fungus. S3. Retain the portion with a molecular weight ≥3000 Da from the water eluent of macroporous resin of black fungus to obtain refined total polysaccharide of black fungus; S4. The total polysaccharide of black fungus was purified by deep removal of impurities using anion exchange resin, and then purified by charge separation using DEAE ion exchange to obtain monomeric polysaccharide ME-2.

[0010] Furthermore, the macroporous resin mentioned in step S2 is AB-8 type macroporous resin; the eluent is distilled water, and the amount of distilled water used for elution is 5 to 7 times the column volume.

[0011] Further, in step S3, the macroporous resin eluent is treated with a 3kDa ultrafiltration column to obtain refined total polysaccharides from black fungus.

[0012] Furthermore, in step S4, the anion exchange resin is Amber. lite FPA90 Cl Anion exchange resin, wherein the DEAE ion exchange employs DEAE... 650M anion exchange column.

[0013] Preferably, Amber is used. lite FPA90 Cl When using anion exchange resin, elute with distilled water and 0.5–2 mol / L NaCl, respectively, with elution volumes of 3–5 column volumes and 1–3 column volumes, respectively, and collect the distilled water eluent.

[0014] Preferably, DEAE is used. When using a 650M anion exchange column, the eluent is distilled water and 0.3–0.7 mol / L NaCl. Collect the eluted fraction of 0.3–0.7 mol / L NaCl to obtain monomeric polysaccharides.

[0015] This invention established two mouse models, MASL and MASH, in parallel using a high-fat diet (HFD) and a choline-deficient high-fat diet (CDA-HFD), respectively, to simulate the staged progression of metabolic dysfunction-related fatty liver disease in humans. The experimental groups and drug administration regimens were consistent for both groups, specifically: a blank control group (NCD), model groups (MASL model group HFD group and MASH model group CDA-HFD group, respectively), a low-dose ME-2 group (50 mg / kg), a medium-dose ME-2 group (100 mg / kg), a high-dose ME-2 group (200 mg / kg), and a positive control group (PIO, pioglitazone hydrochloride, 5 mg / kg). Each drug administration group received the corresponding dose of ME-2 via gavage daily, while the blank control group and model group received an equal volume of PBS via gavage, with continuous intervention for 10 weeks.

[0016] In the MASL model, ME-2 demonstrated a systemic dose-dependent ameliorative effect. Mice in the model group exhibited significant weight gain, diffuse yellowing of the liver with an oily sheen, and significantly elevated liver weight and organ indices, indicating severe metabolic overload and lipid deposition. After intervention with medium and high doses of ME-2, mouse weight gain was significantly inhibited, liver appearance and color returned to normal, and liver weight decreased in a dose-dependent manner, indicating that ME-2 effectively alleviated the fatty pathological changes in the liver induced by a high-fat diet. Liver histopathology using H&E staining revealed dense, irregularly sized fat vacuoles within hepatocytes in the model group, indicating severe lipid accumulation; all ME-2 dose groups dose-dependently reduced the number of fat vacuoles and alleviated the degree of fatty degeneration, with the high-dose group showing the most significant improvement. Sirius red staining further suggested that ME-2 also alleviated early collagen deposition associated with the model. Regarding metabolic function, the model group exhibited significant impaired glucose tolerance and insulin resistance, with significantly elevated areas under the curve (AUC) for GTT and ITT. Medium and high doses of ME-2 significantly reduced AUC values, with improvement effects comparable to PIO, and also significantly reduced fasting blood glucose levels. Serum biochemical tests showed that ALT, AST, TC, TG, and LDL levels were abnormally elevated in the model group. ME-2 dose-dependently downregulated these indicators, with the high-dose group showing better results than PIO. Molecular mechanism studies revealed that the mRNA expression of lipid synthesis genes (ACC1, Srebp-1c, FASN) and inflammation-related genes (IL-1β, F4 / 80, IL-6, TNF-α) in the liver of the model group was significantly upregulated, while the expression of the key fatty acid oxidation gene CPT1A was inhibited. ME-2 intervention dose-dependently reversed the abnormal expression of the above genes, downregulated lipid synthesis and inflammation genes, and upregulated CPT1A, with the regulatory effect of the high-dose group being similar to that of PIO.

[0017] Building upon this, the present invention further investigated the therapeutic effect of ME-2 on the CDA-HFD-induced MASH model. Experimental results showed that in this MASH animal model, the model group mice not only exhibited severe hepatic steatosis, glucose and lipid metabolism disorders, and insulin resistance consistent with the MASL model, but also displayed typical progressive pathological features: obvious inflammatory cell infiltration and hepatocyte damage were visible in the liver tissue; both Masson staining and Sirius red staining showed a significant increase in collagen fiber deposition area, indicating that the fibrosis process had been activated.

[0018] In the MASH model, ME-2 intervention, while fully preserving and continuing its effects in improving hepatic steatosis, further demonstrated a potent inhibitory effect on characteristic MASH lesions. Specifically, it dose-dependently reduced the degree of liver fibrosis and significantly decreased the area of ​​collagen deposition. At the molecular level, qPCR results showed that ME-2 dose-dependently downregulated the expression of key genes involved in the fibrotic process of the MASH model, including α-smooth muscle actin (α-SMA), transforming growth factor-β1 (TGF-β1), and collagen Iα1 (Col1a1) and collagen IIIα1 (Col3a1). The improvement effect of high-dose ME-2 on these core fibrotic indicators was comparable to that of the positive control drug.

[0019] These results indicate that ME-2 can effectively intervene in early-stage simple fatty liver disease characterized primarily by metabolic disorders, and also has a clear therapeutic effect on the MASH stage, which has progressed to a stage with significant fibrosis. This demonstrates its ability to cover the entire course of MASLD. In summary, ME-2 possesses significant advantages such as a well-defined structure, origin from food-grade medicinal materials, and safety without toxicity. It can serve as a natural candidate drug for the prevention and treatment of MASLD. Compared with existing drugs, it is also inexpensive and suitable for a wide range of patients, providing a novel treatment option for clinical practice and possessing significant application prospects and translational value.

[0020] Furthermore, the metabolic dysfunction-associated fatty liver disease includes the early stage of metabolic dysfunction-associated fatty liver disease (simple hepatocellular steatosis stage, MASL) and the inflammatory progression stage (metabolic dysfunction-associated steatohepatitis, MASH).

[0021] Furthermore, the drug works by reducing liver weight and liver organ index in the subjects.

[0022] Furthermore, the drug works by reducing hepatic steatosis in the subjects.

[0023] Furthermore, the drug exerts its effect by inhibiting or reducing liver fibrosis associated with metabolic dysfunction-related steatohepatitis in the subjects.

[0024] Furthermore, the drug works by reducing early liver collagen deposition associated with MASL in the subjects.

[0025] Furthermore, the drug works by reducing the serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and / or triglycerides (TG) in the subjects.

[0026] Furthermore, the drug exerts its effect by downregulating the mRNA expression of lipid synthesis-related genes sterol regulatory element-binding protein-1c (Srebp-1c), acetyl-CoA carboxylase 1 (ACC1), and fatty acid synthase (FASN) in the liver tissue of the subjects.

[0027] Furthermore, the subjects were selected from mammals.

[0028] Furthermore, the mammals mentioned are selected from rats, cats, dogs, pigs, cattle, horses, sheep, monkeys, and humans.

[0029] Furthermore, the dosage of the drug is 50 to 200 mg per kilogram of body weight per day.

[0030] Furthermore, the dosage form of the drug is a pharmaceutically acceptable oral dosage form.

[0031] Furthermore, the drug also includes other pharmaceutically acceptable excipients.

[0032] Preferably, the excipients are selected from any one or a combination of at least two of the following: fillers, binders, wetting agents, disintegrants, emulsifiers, cosolvents, solubilizers, osmotic pressure regulators, colorants, pH regulators, antioxidants, antibacterial agents, or buffers.

[0033] Based on the known immunomodulatory, anti-infective, and anti-inflammatory activities of the black fungus monomer polysaccharide ME-2, those skilled in the art lack a reasonable motivation to use it to treat MASLD, which is characterized by metabolic disorders. This invention represents an exploration across disease domains, and its results are highly unpredictable. This invention represents a breakthrough discovery achieved over this cognitive barrier. This invention unexpectedly demonstrates that ME-2 exhibits remarkable efficacy throughout the entire course of high-fat diet-induced MASLD. Its effect is not a continuation of known anti-inflammatory or immunomodulatory pathways, but rather through direct downregulation of key genes (Srebp-1c, ACC1, FASN) in pathways such as hepatic lipid synthesis and metabolism. This discovery completely overturns the conventional understanding of ME-2 as merely an immunomodulator or anti-inflammatory agent, redefining it as an unprecedented hepatic metabolic regulator, opening a completely new paradigm for the drug treatment of MASLD.

[0034] Compared with the prior art, the present invention has the following beneficial effects: This invention provides a novel application of the black fungus monomer polysaccharide ME-2 in the preparation of drugs for the prevention and treatment of metabolic dysfunction-related fatty liver disease. This invention evaluated the efficacy of ME-2 throughout the entire course of MASLD by constructing a high-fat diet-induced MASLD mouse model (covering the disease spectrum from simple steatosis to steatohepatitis). The results showed that ME-2 can effectively intervene in early-stage simple fatty liver disease characterized by metabolic disorders, and also has a clear therapeutic effect on the MASH stage, which has progressed to a stage with significant fibrosis. This systematically demonstrates ME-2's coverage of the entire MASLD course and its significant interventional effect throughout the entire course of MASLD. This invention reveals for the first time the breakthrough function of ME-2 as a liver metabolic regulator: by directly correcting hepatic lipid metabolism disorders (such as regulating core genes such as Srebp-1c, ACC1, and FASN), it can dose-dependently reduce hepatic steatosis, decrease serum transaminase levels, and reduce dyslipidemia throughout the entire course of MASLD. Importantly, its efficacy stems from intervention at the metabolic root cause, which is fundamentally different from known anti-inflammatory mechanisms. Meanwhile, ME-2 has significant advantages such as well-defined structure, metabolic regulation as its core function, and safety and non-toxicity, providing a brand-new drug candidate for the prevention and treatment of MASLD. Attached Figure Description

[0035] Figure 1 The study included observations of body weight, organ indices, and liver histology in MASL mice. Figure 1 A shows the change in mouse body weight; B shows a gross anatomical diagram of the liver; C shows gross observation of mice, H&E staining, and Sirius red staining; D shows the change in liver weight of mice in each group; E shows the change in liver organ index of mice in each group. The experimental results were statistically analyzed, with mean ± SEM, n=8, compared with NCD: # P <0.05, ## P <0.01, ### P <0.001; compared to HFD: * P <0.05,** P <0.01, *** P <0.001.

[0036] Figure 2 The results show the effects of ME-2 on insulin resistance and fasting blood glucose in the MASL model. Figure 2Figure A shows the blood glucose level results after intraperitoneal injection of glucose in the glucose tolerance test (GTT); Figure B shows the area under the curve (AUC) calculated from the GTT results; Figure C shows the blood glucose level graph after intraperitoneal injection of insulin in mice and the insulin resistance test (ITT); Figure D shows the area under the curve (AUC) calculated from the ITT results; Figure E shows the effect of fasting blood glucose level. Statistical analysis was performed on the experimental results, where mean ± SEM, n=8, compared with NCD: # P <0.05, ## P <0.01, ### P <0.001; compared to HFD: * P <0.05,** P <0.01, *** P <0.001.

[0037] Figure 3 Liver function, blood lipid levels, and qPCR analysis were performed on the ME-2 intervention MASL model. Figure 3 In the table, A represents alanine aminotransferase (ALT); B represents triglycerides; C represents total cholesterol; D represents aspartate aminotransferase (AST); E represents low-density lipoprotein (LDL); and F represents the mRNA expression of ACC1, Srebp-1c, FASN, CPT1A, IL-1β, F4 / 80, IL-6, and TNF-α in the liver tissue of MASL model mice detected by qPCR. The experimental results were statistically analyzed, with mean ± SEM, n = 8 or 6, compared with NCD: # P <0.05, ## P <0.01, ### P <0.001; compared to HFD: * P <0.05,** P <0.01, *** P <0.001.

[0038] Figure 4 The results show the efficacy of ME-2 treatment in the MASH model. Figure 4 A represents H&E staining, Sirius red staining, and Masson staining; B represents changes in liver weight in each group of mice; C represents changes in liver organ indices in each group of mice; D–G represent analyses of liver function and blood lipid indicators in the ME-2 intervention MASH model; H represents the mRNA expression levels of fibrosis, lipid metabolism, and inflammation-related genes in the liver of ME-2 intervention MASH model mice detected by qPCR. Statistical analysis of the experimental results was performed, with mean ± SEM, n = 8 or 6, compared to NCD: # P <0.05,## P <0.01, ### P <0.001; compared to HFD: * P <0.05,** P <0.01, *** P <0.001. Detailed Implementation

[0039] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0040] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0041] Example 1: Preparation of a novel monomeric polysaccharide ME-2 from black fungus Preparation of black fungus monomeric polysaccharide: A novel black fungus monomeric polysaccharide ME-2 was isolated from black fungus according to the method disclosed in Chinese patent application CN114957508A, specifically including the following steps: Take approximately 5 kg of finely chopped black fungus, and extract 250 g of the chopped fungus each time. Add 8 L of distilled water and extract for 3 hours each time. Filter the residue through gauze. Repeat the extraction twice with the residue and combine the black fungus aqueous extracts. Pass the aqueous extract through AB... Type 8 macroporous resin was used for adsorption for 12 hours, followed by elution with distilled water until the eluent was pale yellow according to the phenol-sulfuric acid method (this method can remove some pigments, proteins, and other substances from the black fungus polysaccharide). The eluent from the macroporous adsorption resin was further purified by passing it through a 3000 Da molecular weight ultrafiltration column to remove oligosaccharides and soluble salts, yielding refined total polysaccharide from the black fungus. The refined total polysaccharide from the black fungus was then processed through an anion exchange resin (Amber). lite FPA90 Cl First, the crude polysaccharide was eluted with distilled water. After determining the elution endpoint using the phenol-sulfuric acid method, it was eluted with 1M NaCl to further decolorize and deproteinize the crude polysaccharide. The distilled water eluate was collected to obtain 500g of refined polysaccharide I from black fungus. The refined polysaccharide I was dissolved in water and purified using DEAE. Further purification was performed using a 650M anion exchange column (mobile phase sequence: distilled water, 0.5 mol / L NaCl). The elution fraction with 0.5 mol / L NaCl was collected to obtain the refined polysaccharide monomer from Auricularia auricula-judae, denoted as ME. 2.

[0042] Example 2: Pharmacological Study on the Prevention and Treatment of MASL by ME-2, a Novel Monomeric Polysaccharide from Black Fungus (1) Modeling and grouping Two batches of healthy SPF-grade male C57BL / 6 mice, 48 mice in each batch, were selected. After 7 days of routine acclimatization feeding, each batch of mice was randomly divided into 6 groups of 8 mice each. A MASL mouse model was established using a high-fat diet (HFD) to simulate simple steatosis of hepatocytes in the early stage of MASLD. The experimental groups and drug regimens were as follows: blank control group (NCD, Control), model group (MASL model group, HFD, Model), low-dose ME-2 group (ME-2-L, 50 mg / kg), medium-dose ME-2 group (ME-2-M, 100 mg / kg), high-dose ME-2 group (ME-2-H, 200 mg / kg), and positive control group (PIO, pioglitazone hydrochloride, 5 mg / kg). Each drug treatment group was administered the corresponding dose of ME-2 by gavage daily, while the blank control group and model group were administered an equal volume of PBS by gavage. The intervention was continued for 10 weeks, and the weight of the mice was recorded weekly. The mice were fasted for 24 hours before the end of the experiment (water was not restricted).

[0043] (2) Monitoring of body weight in MASL model mice after treatment with ME-2, a novel monomeric polysaccharide from black fungus. To investigate the regulatory role of ME-2 on body weight gain in MASL model mice, the body weight of mice in each group was measured and recorded weekly during the experiment. The experimental results are as follows: Figure 1 China A Figure 1 As shown in Figure C, compared with the blank group, the model group mice gained weight rapidly. After administration of low, medium, and high doses of ME-2, weight gain was relatively slow, showing an overall dose-dependent effect. By the end of week 10, the body weight of HFD mice was significantly higher than that of NCD mice (…). ### P <0.001%, significant weight loss after administration of medium and high doses of ME-2 (*) P <0.05、*** P (<0.001, 10w), the positive control drug PIO can improve insulin resistance, but cannot reverse HFD-induced weight gain in mice.

[0044] (3) Observation of liver anatomy and liver index in MASL model mice after treatment with ME-2, a novel monomeric polysaccharide from black fungus. To visually evaluate the effect of ME-2 on liver morphology and lipid deposition in MASL model mice, mice were euthanized by cervical dislocation after anesthesia, and the livers were quickly removed. The surface bloodstains were rinsed with pre-cooled physiological saline, the moisture was absorbed with filter paper, and the appearance of the livers in each group was photographed and recorded. The liver index was also calculated by weighing.

[0045] Experimental results are as follows Figure 1As shown in Figure B, the livers of mice in the HFD model group exhibit typical characteristics of fatty liver degeneration: the surface is noticeably oily and shiny, the liver is diffusely yellow with a pale yellow hue, and the lipid deposition characteristics are significant. In contrast, the livers of mice in the ME-2 dosage groups and the PIO positive drug group are closer to normal reddish-brown in color, and the liver weight and liver index in the ME-2 group decreased in a dose-dependent manner, suggesting that ME-2 can effectively reduce lipid deposition in the liver induced by high lipids and has a significant effect on improving fatty liver. The efficacy of the high-dose group is close to that of the positive drug group.

[0046] Further statistical analysis showed that liver weight and liver index were significantly higher in the HFD group than in the NCD group. ### P<0.001, # P<0.05; After intervention with medium and high doses of ME-2, liver weight and liver index were significantly improved, and the improvement effect of the high-dose ME-2 group was similar to that of the PIO group (P<0.05). Figure 1 D, Figure 1 (E).

[0047] (4) Histopathological examination of liver tissue of MASL model mice treated with ME-2, a novel monomeric polysaccharide from black fungus. MASL is simple hepatic steatosis, caused by factors such as overnutrition and insulin resistance, which leads to the deposition of large amounts of triglycerides in hepatocytes, forming fat vacuoles. It often affects hepatocytes in the central region of the liver lobule, while the liver lobule structure remains basically intact.

[0048] H&E staining results are as follows Figure 1 As shown in Figure C, the hepatocytes in the blank group were arranged radially around the central vein, with regular morphology, a moderate and evenly distributed nucleoplasm ratio, clear sinusoidal structure, and no obvious inflammatory cell infiltration or necrosis, exhibiting normal liver tissue morphology. In contrast, the hepatocytes in the HFD group showed obvious fatty degeneration, with numerous lipid droplets and vacuoles of varying sizes visible in the cytoplasm, disordered arrangement of hepatic cords, and disruption of the normal structural integrity of the liver, indicating successful construction of the MASL model. After intervention, the fatty degeneration of hepatocytes in the positive control group (PIO) and various dose groups of ME-2 was significantly reduced, the pathological vacuoles decreased in a dose-dependent manner, and the pathological state of the liver was significantly improved compared with the model group.

[0049] Sirius red staining results are as follows: Figure 1 As shown in Figure C, this further confirms that in the NCD group, only a small amount of light red, thin collagen fibers were visible around the central vein. Their distribution was limited and sparse, with no obvious tendency to extend towards the liver lobules, suggesting physiological collagen deposition without pathological liver fibrosis. In the HFD group, collagen deposition was increased compared to the NCD group, but no obvious fiber bundles were formed, which is consistent with the pathological progression of MASL. After drug intervention, the proliferation of collagen deposition was alleviated, and showed a dose-dependent improvement trend. The above results are consistent with the morphological manifestations of H&E staining.

[0050] (5) The novel monomeric polysaccharide ME-2 in black fungus improves glucose tolerance, insulin resistance and blood glucose levels. To investigate the effects of ME-2 on glucose metabolism, glucose tolerance tests (GTT) and insulin tolerance tests (ITT) were conducted in weeks 8 and 9, respectively. For the GTT, all mice were fasted for 12 hours before fasting blood glucose measurement. Blood glucose levels were collected from the tail vein and recorded as the 0 min time. Subsequently, D-glucose was injected intraperitoneally at a dose of 1.5 g / kg body weight. Blood glucose levels were measured immediately after injection at 15 min, 30 min, 60 min, 90 min, and 120 min using a Haier (VGM74) blood glucose meter. For the ITT, all mice were fasted for 4 hours before the test. Blood glucose levels were collected from the tail vein before intraperitoneal insulin injection and recorded as the 0 min time. Insulin was then injected intraperitoneally at 0.5 U / kg. Blood glucose levels were measured immediately after injection at 15 min, 30 min, 60 min, 90 min, and 120 min using a Haier (VGM74) blood glucose meter.

[0051] Experimental results are as follows Figure 2 As shown, the AUC in the GTT test of the HFD group was significantly higher than that of the NCD group ( ### P <0.001 indicates impaired glucose tolerance), GTT results showed that the HFD group had severe insulin resistance, while ME-2 at all doses significantly reduced the AUC of the GTT (*** P <0.001)( Figure 2 China A Figure 2 (Medium B). In the ITT trial, ME-2 at all doses showed dose-dependent relief of insulin resistance, and the medium and high doses significantly reduced AUC, with the high dose group showing a more pronounced reduction (***P<0.001), comparable to PIO. Figure 2 C, Figure 2 (Medium D). Furthermore, medium and high doses of ME-2 significantly reduced blood glucose levels in MASL mice (*). P <0.05)( Figure 2 (E), fully demonstrating its medicinal efficacy.

[0052] (6) The novel monomeric polysaccharide ME-2 from black fungus improves serum biochemical index levels To further investigate the regulatory effects of ME-2 on liver function and lipid metabolism in MASL model mice, blood samples from each group of mice were thawed naturally, mixed thoroughly, and analyzed using a fully automated biochemical analyzer (Roche C311). Based on possible changes in MASL indicators, the effects on liver function indicators (ALT, AST), lipid indicators (TC, TG), and low-density lipoprotein (LDL) levels were focused. The test results were used to evaluate and construct the therapeutic effects of different ME-2 doses.

[0053] Experimental results are as follows Figure 3 As shown, compared with the normal diet NCD group, the serum ALT (alanine aminotransferase) level in the HFD group mice was significantly higher. Figure 3 (A) and AST (aspartate aminotransferase, Figure 3 D), TC (total cholesterol), Figure 3 C), TG (triglycerides, Figure 3 B), LDL levels (low-density lipoprotein, Figure 3 Both E and E levels were significantly elevated. ### P <0.001), indicating that a high-fat diet induced hepatocyte damage and lipid metabolism disorders; after intervention with different doses of ME-2, the serum levels of ALT, AST, TG, TC and LDL in mice showed a dose-dependent decrease, and the high-dose ME-2 group had better efficacy than PIO, indicating that ME-2 can effectively improve liver function indicators and lipid metabolism abnormalities caused by a high-fat diet.

[0054] (7) The expression of the novel monomeric polysaccharide ME-2 from black fungus in the liver of MASL mice was detected by qPCR. To clarify the regulatory role of ME-2 on liver gene expression in MASL model mice, qPCR was used to detect the mRNA expression levels of lipid metabolism and inflammation-related genes. Mice were euthanized by cervical dislocation, and the livers were quickly harvested, rinsed with pre-cooled physiological saline, blotted dry with filter paper, ground in liquid nitrogen, and stored at -80℃ for later use. Primers were designed using Oligo7 software and compared with the NCBI database (see Table 1). The internal control gene was β-actin, and the target genes (ACC1, Srebp-1c, FASN, CPT1A, IL-1β, F4 / 80, IL-6, TNF-α) were synthesized by Jilin Kumei Biotechnology Co., Ltd. The working solution concentration was adjusted to 10 μmol / L. After the qPCR reaction, the reliability of the amplification and melting curves was first verified. Then, the relative expression levels of each target gene mRNA were calculated using the 2^(-ΔΔCt) method with β-actin as the internal control, and statistical analysis was performed.

[0055] Table 1 Primer Sequences

[0056] qPCR results as follows Figure 3 As shown in Figure F, ME-2 dose-dependently downregulates the mRNA expression of key liver lipid synthesis genes Srebp-1c, ACC1, and FASN, while upregulating the expression of the fatty acid oxidation gene CPT1A, directly regulating lipid metabolism homeostasis. Simultaneously, the expression of inflammation-related factors (IL-1β, TNF-α, F4 / 80, IL-6, etc.) also decreases accordingly.

[0057] Example 3: Pharmacological Study on the Prevention and Treatment of MASH by ME-2, a Novel Monomer Polysaccharide from Black Fungus Based on the evidence from Example 2 that ME-2 can effectively reverse the core metabolic disorders of MASL, this invention further explores its therapeutic effect on the disease progression stage—metabolic steatohepatitis associated with metabolic dysfunction (MASH). A MASH mouse model was constructed using a cholinergic high-fat diet (CDA-HFD), with experimental grouping and administration regimens consistent with Example 2. It should be clearly noted that the fibrotic phenotype in the MASH models used in this example is a result of severe metabolic disorders induced by cholinergic deficiency combined with a high-fat diet. Therefore, this experiment aims to verify whether ME-2, through its proven core metabolic regulatory function, can intervene at the root of the disease, thereby achieving simultaneous improvement of downstream secondary pathological changes, including fibrosis.

[0058] This embodiment systematically evaluated the therapeutic effect of ME-2 on MASH model mice using the research methods described in Example 2, including liver weight and organ index measurement, H&E, Sirius red, Masson staining pathological analysis, serum biochemical index detection, and liver tissue qPCR gene expression analysis.

[0059] The results showed that ME-2 significantly reduced liver weight and liver organ index in model mice. Figure 4 B, Figure 4 The results (C) indicate that ME-2 can improve liver pathological damage and metabolic disorders in MASH model mice in a dose-dependent manner. H&E, Sirius red, and Masson staining analyses showed that ME-2 can effectively alleviate hepatocyte steatosis and inhibit abnormal collagen fiber deposition (C). Figure 4 (A) indicates that ME-2 has the effect of inhibiting the progression of MASH to liver fibrosis. At the functional level, serum biochemical tests confirmed that ME-2 can significantly reduce liver injury markers such as ALT and AST, as well as TG levels, while increasing HDL levels, systematically improving liver function and lipid metabolism disorders. Figure 4(D~G). Further molecular mechanism studies have shown that ME-2 can significantly regulate the mRNA expression of key lipid metabolism genes (ACC1, Srebp-1c, FASN), fibrosis-related genes (α-SMA, TGF-β1, Col1a1, Col3a1), and inflammation-related genes (IL-1β, TNF-α, IL-6, F4 / 80) in liver tissue. Figure 4 (H). This reveals at the molecular level that ME-2 can simultaneously inhibit lipid accumulation and fibrosis in the MASH process through multiple pathways. In summary, ME-2 exhibits comprehensive preventative and therapeutic effects against MASH, and the high-dose group showed efficacy comparable to the positive control drug PIO, providing systematic experimental evidence for its clinical translation.

[0060] The above results demonstrate that ME-2 can effectively intervene in early-stage simple fatty liver disease characterized primarily by metabolic disorders, and also has a clear therapeutic effect on the MASH stage, which has progressed to a stage with significant fibrosis. This systematically reflects its ability to cover the entire course of MASLD. In summary, ME-2 possesses significant advantages such as a well-defined structure, origin from food-grade medicinal materials, and safety without toxicity. It can serve as a natural candidate drug for the prevention and treatment of MASLD. Compared with existing drugs, it is also inexpensive and suitable for a wide range of patients, providing a novel treatment option for clinical practice and possessing significant application prospects and translational value.

[0061] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit it. Although the present invention has been described in detail in the embodiments through general description, specific implementation and experiments, any modifications or improvements that can be made without departing from the core of the present invention shall fall within the scope of protection of the present invention.

Claims

1. The application of black fungus monomer polysaccharide ME-2 in the preparation of drugs for the prevention and / or treatment of fatty liver disease related to metabolic dysfunction, characterized in that, The preparation method of the black fungus monomer polysaccharide ME-2 includes the following steps: S1. Black fungus was extracted using water as the extraction solvent to obtain a black fungus aqueous extract; S2. Macroporous resin was used to adsorb the aqueous extract of black fungus to remove pigments and proteins, followed by elution to obtain a macroporous resin aqueous eluent of black fungus. S3. Retain the portion with a molecular weight ≥3000 Da from the water eluent of macroporous resin of black fungus to obtain refined total polysaccharide of black fungus; S4. The total polysaccharide of black fungus was purified by deep removal of impurities using anion exchange resin, and then purified by charge separation using DEAE ion exchange to obtain monomeric polysaccharide ME-2.

2. The application according to claim 1, characterized in that, The metabolic dysfunction-related fatty liver disease includes simple steatosis of hepatocytes in the early stage of metabolic dysfunction-related fatty liver disease and metabolic dysfunction-related steatohepatitis in the inflammatory progression stage.

3. The application according to claim 1 or 2, characterized in that, The drug works by reducing liver weight and liver organ indices in the subjects.

4. The application according to claim 1 or 2, characterized in that, The drug works by reducing hepatic steatosis in the subjects.

5. The application according to claim 1 or 2, characterized in that, The drug works by inhibiting or reducing liver fibrosis associated with metabolic dysfunction-related steatohepatitis in the subjects.

6. The application according to claim 1 or 2, characterized in that, The drug works by reducing early liver collagen deposition associated with simple steatosis of hepatocytes in the subjects.

7. The application according to claim 1 or 2, characterized in that, The drug works by lowering serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), and / or triglyceride levels in the subjects.

8. The application according to claim 1 or 2, characterized in that, The drug exerts its effect by downregulating the mRNA expression of lipid synthesis-related genes sterol regulatory element-binding protein-1c, acetyl-CoA carboxylase 1, and fatty acid synthase in the liver tissue of the subjects.

9. The application according to claim 1 or 2, characterized in that, It also includes pharmaceutically acceptable excipients.

10. The application according to claim 9, characterized in that, The excipients are selected from any one or a combination of at least two of the following: fillers, binders, wetting agents, disintegrants, emulsifiers, cosolvents, solubilizers, osmotic pressure regulators, colorants, pH regulators, antioxidants, antibacterial agents, or buffers.

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