Use of madecassoside in the preparation of a liver-protecting drug for treating alcoholic liver disease

Abstract

This invention discloses the application of asiaticoside in the preparation of hepatoprotective drugs for treating alcoholic liver disease. Experiments show that oral administration of this compound significantly reduces serum ALT, AST, ALP, TBIL, and inflammatory factors IL-1β and IL-6 levels in mice with alcoholic liver disease, alleviates hepatocellular edema and steatosis, repairs colonic damage, and upregulates the expression of tight junction proteins occludin and claudin1. 16S rRNA sequencing shows that it can increase beneficial bacteria. Duncaniella , Bifidobacterium Abundance, reducing pathogenic bacteria Escherichia , Klebsiella Abundance and improvement of gut microbiota imbalance; Western blot confirmed its inhibition of hepatic PI3K / AKT signaling pathway phosphorylation. This invention reveals for the first time that asiaticoside exerts multi-target hepatoprotective effects through anti-inflammatory activity, gut microbiota regulation, and PI3K / AKT pathway blockade, providing a new strategy for the treatment of alcoholic liver disease.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to the application of asiaticoside in the preparation of hepatoprotective drugs for treating alcoholic liver disease. Background Technology

[0002] Alcoholic liver disease (ALD) refers to a series of liver diseases caused by long-term excessive alcohol consumption, including alcoholic fatty liver, alcoholic hepatitis, liver fibrosis, cirrhosis, and even liver cancer. It has become one of the leading causes of chronic liver disease worldwide. In recent years, with the continuous increase in per capita alcohol consumption in my country, the incidence and mortality rates of ALD have also been rising year by year, placing a heavy burden on the public health system. Currently, the main clinical treatments for ALD include strict abstinence from alcohol, nutritional support, and symptomatic drug treatments such as glucocorticoids. However, these approaches have limitations in efficacy, significant side effects, or poor patient compliance, necessitating the development of safe and effective new therapeutic drugs.

[0003] The pathological mechanisms of alcoholic liver injury are extremely complex, involving multiple aspects such as the direct hepatotoxicity of alcohol and its metabolite acetaldehyde, oxidative stress damage, lipid metabolism disorders, and the cascade amplification of hepatic inflammatory responses. Among these, the inflammatory response plays a central role in the initiation and progression of ALD. Studies have shown that reactive oxygen species (ROS) generated from alcohol metabolism can activate Kupffer cells in the liver, thereby activating inflammation-related signaling pathways such as PI3K / AKT, promoting the release of pro-inflammatory factors such as tumor necrosis factor-α, interleukin-1β, and interleukin-6, and inducing and exacerbating persistent inflammatory damage to the liver. Therefore, inhibiting the hepatic inflammatory response and blocking the abnormal activation of the PI3K / AKT signaling pathway has become one of the important strategies for the development of anti-ALD drugs.

[0004] In recent years, in-depth research on the "gut-liver axis" theory has revealed the crucial regulatory role of gut microbiota in the development and progression of ALD. Long-term alcohol intake can damage the intestinal mucosal mechanical barrier, reduce the expression of tight junction proteins occludin and claudin1, increase intestinal permeability, and lead to a large influx of enterogenic endotoxins (such as lipopolysaccharide) into the portal circulation and reaching the liver, activating hepatic immune cells and thus exacerbating inflammatory responses and hepatocellular damage. Simultaneously, the gut microbiota structure of ALD patients and animal models generally exhibits an ecological imbalance characterized by decreased abundance of Firmicutes and increased abundance of Proteobacteria and opportunistic pathogens (such as Escherichia and Klebsiella). Targeted regulation of gut microbiota balance and repair of intestinal barrier function have gradually become new directions in ALD treatment research.

[0005] Madecassoside (MA) is a pentacyclic triterpenoid active ingredient extracted from the traditional medicinal plant Centella asiatica. Existing literature reports its pharmacological activities, including anti-inflammatory effects, promotion of wound healing, and reduction of high-fat diet-induced hepatic steatosis. However, there are currently no reports, either domestically or internationally, on the effects of madecassoside in treating alcoholic liver disease by inhibiting the PI3K / AKT signaling pathway, reducing inflammatory responses, and regulating intestinal flora imbalance. Its application in the preparation of hepatoprotective drugs for treating ALD is also undisclosed. Based on the shortcomings of the existing technology, this invention proposes for the first time the use of madecassoside in the preparation of hepatoprotective drugs for treating alcoholic liver disease, and provides sufficient experimental evidence for this new indication. Summary of the Invention

[0006] This invention discloses the application of asiaticoside in the preparation of hepatoprotective drugs for treating alcoholic liver disease. Validated in a mouse model of alcoholic liver disease, this compound significantly reduces serum transaminase, bilirubin, and inflammatory factor levels, alleviates hepatocellular steatosis and edema, repairs the intestinal barrier, upregulates tight junction proteins, and increases beneficial bacteria. Duncaniella and Bifidobacterium Abundance, reducing pathogens Escherichia and Klebsiella It increases the abundance of liver cells and achieves multi-target hepatoprotective effects by inhibiting phosphorylation of the hepatic PI3K / AKT signaling pathway.

[0007] The present invention provides the application of asiaticoside in the preparation of a hepatoprotective drug for treating alcoholic liver disease, using the following technical solution: The application of asiaticoside in the preparation of hepatoprotective drugs for the treatment of alcoholic liver disease, wherein the drugs use asiaticoside as the sole active ingredient or contain asiaticoside as one of the active ingredients.

[0008] Preferably, the drug is an oral or injectable preparation, wherein the oral preparation is a tablet, capsule, granule, oral liquid, or pill.

[0009] Preferably, the dosage of the drug is 10–40 mg / kg.

[0010] Preferably, the drug exerts its hepatoprotective effect by inhibiting the phosphorylation activation of the hepatic PI3K / AKT signaling pathway.

[0011] Preferably, the drug improves liver function by reducing serum levels of alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, and total bilirubin.

[0012] Preferably, the drug reduces liver inflammation by lowering the levels of the inflammatory factors interleukin-1β and interleukin-6 in the serum.

[0013] Preferably, the drug exerts its hepatoprotective effect by regulating the balance of intestinal flora, specifically by: increasing the abundance of Firmicutes and decreasing the abundance of Proteobacteria; and increasing beneficial bacteria. Duncaniella , Muribaculum , Kineothrix , Bifidobacterium The abundance of [unclear] reduced the number of opportunistic pathogens. Escherichia , Klebsiella , Prevotella The abundance of.

[0014] Preferably, the drug repairs the intestinal mucosal barrier by upregulating the expression of colonic tight junction proteins occludin and claudin1.

[0015] Preferably, alcoholic liver disease includes alcoholic fatty liver, alcoholic hepatitis, alcoholic liver fibrosis, or alcoholic cirrhosis.

[0016] Preferably, the drug exerts its hepatoprotective effect through a multi-target synergistic mechanism that simultaneously inhibits the PI3K / AKT signaling pathway, reduces inflammatory factor levels, regulates gut microbiota balance, and repairs the intestinal barrier.

[0017] In summary, the beneficial effects of the present invention are as follows: This invention is the first to discover that asiaticoside has a clear protective effect against alcoholic liver disease. Animal experiments have verified that it can significantly reduce serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), and total bilirubin in mice with alcoholic liver disease. Simultaneously, it effectively inhibits the release of pro-inflammatory factors interleukin-1β and interleukin-6, reduces hepatocyte edema and fatty vacuolar degeneration, and improves liver morphology and pathological structure. Compared with existing clinical treatments, asiaticoside, derived from a natural plant extract, has low toxicity and good safety profile, and can simultaneously improve liver function damage and inflammation across multiple indicators, providing a safer and more effective new drug option for patients with alcoholic liver disease.

[0018] Furthermore, this invention reveals the multiple mechanisms by which asiaticoside exerts its hepatoprotective effect: it can upregulate the expression of colonic tight junction proteins occludin and claudin1, repairing alcohol-induced intestinal mucosal barrier damage; simultaneously, it increases Firmicutes and beneficial bacteria by regulating the intestinal flora structure. Duncaniella , Bifidobacterium The relative abundance of Proteobacteria and pathogenic bacteria was reduced. Escherichia , KlebsiellaIt increases the abundance of [specific enzymes / products] and restores the balance of the intestinal microecology; in addition, it can significantly inhibit the phosphorylation activation of the hepatic PI3K / AKT signaling pathway and block the inflammatory cascade response. This invention is the first to demonstrate that hydroxyasiaticoside achieves comprehensive intervention for alcoholic liver disease through multi-target synergistic effects such as "gut-liver axis" regulation, intestinal barrier repair, microbiota balance reconstruction, and inhibition of anti-inflammatory signaling pathways, which has outstanding innovation and clinical application value. Attached Figure Description

[0019] Figure 1 The effect of MA on body weight and liver index in mice with alcoholic liver disease; in, Figure 1 In Figure A, the curves showing the changes in body weight of mice in each group are shown. Figure 1 B represents a comparison of the net weight gain of mice in each group; Figure 1 The middle C represents a comparison of liver indices among the different groups of mice; Figure 2 The effects of MA on the liver epigenesis and histopathology of mice with alcoholic liver disease; in, Figure 2 In section A, the liver appearance and histopathology of the normal group are shown. Figure 2 B represents the liver epithelial and histopathological findings in the model group; Figure 2 C represents the liver epiphysiology and histopathology of the silymarin group; Figure 2 The liver epithelial and histopathological features in the low-dose MA group are shown in Figure D. Figure 2 E represents the liver epithelial and histopathological findings in the high-dose MA group; Figure 3 The effects of MA on colonic tissue pathology and tight junction protein expression in mice with alcoholic liver disease; in, Figure 3 Image A shows representative HE-stained colon tissue images from each group of mice; Figure 3 In the middle B diagram, there are bands representing the expression of occludin and claudin1 proteins in colon tissue. Figure 3 C represents a comparison of the relative expression levels of occludin protein in colon tissue; Figure 3 D represents a comparison of the relative expression levels of claudin1 protein in colon tissue; Figure 4 The effects of MA on serum transaminase, bilirubin and inflammatory factor levels in mice with alcoholic liver disease; in, Figure 4 In Figure A, the serum ALT activity of mice in each group was compared. Figure 4 B represents a comparison of serum AST activity in mice across different groups; Figure 4 C represents a comparison of serum TBIL levels in mice across different groups; Figure 4 D represents a comparison of serum ALP levels in mice across different groups; Figure 4 E represents a comparison of serum IL-1β levels in mice across different groups; Figure 4 F: Comparison of serum IL-6 levels in mice of different groups; Figure 5 The effect of MA on the expression of PI3K / AKT signaling pathway proteins in the liver of mice with alcoholic liver disease; in, Figure 5 Image A shows the protein expression bands of AKT, p-AKT, PI3K, and p-PI3K in liver tissue. Figure 5 B represents a comparison of the relative protein expression levels of p-PI3K / PI3K; Figure 5 C represents a comparison of the relative protein expression levels of p-AKT / AKT; Figure 6 The effects of MA on gut microbiota diversity and composition in mice with alcoholic liver disease; in, Figure 6 In the middle A, the alpha diversity of gut microbiota in each group of mice (Chao1 and Observed species indices) is represented. Figure 6 In the middle, B represents Beta diversity based on principal coordinate analysis (PCoA); Figure 6 In the middle (C), the differences in bacterial community distance between groups are compared. Figure 6 D in the diagram is a Venn diagram based on the ASV / OTU level; Figure 6 The middle section (E) is a bar chart showing the species composition at the phylum level; Figure 6 The F-value represents a comparison of the Firmicutes / Bacteroidetes (F / B) ratio at the phylum level. Figure 6 G is a heatmap of species composition at the genus level; Figure 6 H represents linear discriminant analysis of gut microbiota (LEfSe). Detailed Implementation

[0020] The present invention will be further described in detail below with reference to the embodiments.

[0021] Example Example 1 Observation of drug administration in mice and calculation of liver index, the specific steps are as follows: S1. Experimental animals and feeding SPF-grade male C57BL / 6J mice aged 6-8 weeks with a body mass of 18-22 g were selected, which were purchased from Henan Speks Biotechnology Co., Ltd. (Production License No.: SCXK(Yu)2025-0005). The usage license number of the experimental institution is SCXK(Gui)2020-0004. The mice were housed in an environment with a controlled temperature of (25±2)°C, a relative humidity of 60%-70%, and a 12 h light / dark cycle, with free access to food and water. The experiment was started after 7 days of adaptive feeding.

[0022] S2. Grouping and drug administration 40 mice were randomly divided into 5 groups (8 mice in each group): normal group, model group, silybin group (50 mg / kg), low-dose madecassoside group (10 mg / kg) and high-dose madecassoside group (40 mg / kg). Madecassoside (MA, batch No. MP23, Shanghai Dibai Bio) and silybin (batch No. S817884, Shanghai Macklin) were both prepared with normal saline, and administered by gavage at a volume of 0.1 mL / 10 g body mass; the normal group and the model group were given an equal volume of normal saline. The administration was performed once a day for 14 consecutive days.

[0023] S3. Establishment of alcoholic liver disease model From day 12 to day 14 of administration, except for the normal group, the mice in the other groups were gavaged daily with 50% edible alcohol solution (batch No. 20250311, Chongqing Chuandong Chemical Industry) at 12 mL / kg body mass for 3 consecutive days to establish the ALD model.

[0024] S4. General condition observation and body mass measurement During the experimental period, the basic conditions of the mice such as mental state, activity, and hair were observed daily, and the body mass was measured every 2 days to record the change trend.

[0025] S5. Sample collection and liver index calculation On the next day (day 15) after the last alcohol gavage, all mice were anesthetized by intraperitoneal injection of urethane (batch No. C16702548, Shandong Keyuan), blood was collected from the eyeballs, the blood was placed in a 1.5 mL EP tube, allowed to stand at room temperature for 1 h and then centrifuged (4°C, 3000 r / min, 15 min), the serum was separated, aliquoted and stored at -80°C for later use.

[0026] The liver was rapidly dissected and separated, rinsed thoroughly with pre-cooled normal saline at 4°C, blotted dry with gauze, photographed, and the wet weight of the liver was weighed. The liver index was calculated according to the formula: Liver index = (wet liver weight / final body mass) × 100%.

[0027] At the same time, approximately 1 mm of tissue was cut from the same location in the left lobe of the liver. 3 Liver tissue and approximately 1 cm of colon tissue were fixed in 10% neutral paraformaldehyde for pathological examination; additional portions of liver and colon tissue were flash-frozen in liquid nitrogen and stored at -80°C; cecal contents were collected in sterile 1.5 mL EP tubes and frozen at -80°C for later use.

[0028] Figure 1 The results showed that, regarding changes in body weight, Figure 1 The results showed that the body weight of the normal group mice increased slowly and steadily during the experimental period, while the body weight of the model group and each drug-treated group decreased significantly after alcohol gavage modeling (days 12-14 of the experiment). Figure 1 Further quantification of net body weight gain in the model group showed that the net body weight gain of the model group mice was significantly lower than that of the normal group. p <0.01), while the weight loss in the high-dose MA group was significantly lower than that in the model group, suggesting that MA has an ameliorative effect on the physical weakness of ALD mice. Regarding liver indices, Figure 1 The C-ray discrepancy showed that the liver index of the model group mice was significantly higher than that of the normal group ( p <0.01 indicates that alcohol intake causes liver edema and compensatory enlargement; compared with the model group, the liver index of the silymarin group and the high- and low-dose MA groups was significantly reduced ( p The result of <0.01 further indicates that MA can effectively alleviate alcohol-induced liver edema and lipid accumulation, and its effect shows a dose-dependent trend.

[0029] Example 2 The specific steps for pathological examination of the liver and colon are as follows: S1. Tissue sampling and fixation After dissecting and separating the liver, a 1 mm section was taken from the same location on the left lobe of the liver. 3 Collect liver tissue of various sizes; simultaneously separate the colon and cut a colonic segment of approximately 1 cm in length. Quickly transfer the above tissues into centrifuge tubes containing 10% neutral paraformaldehyde fixative (standard preparation or purchased from a reputable reagent supplier), ensuring that the volume of fixative is more than 10 times the volume of the tissue. Fix at room temperature for 24–48 hours to allow the tissue to fully harden.

[0030] S2, Paraffin Embedding and Sectioning After fixation, the tissue was dehydrated with a gradient of ethanol (70%, 80%, 95%, 100%) and cleared with xylene. It was then immersed in molten paraffin (melting point 56-58℃, commercially available pathological grade paraffin) and embedded in an embedding machine to create paraffin blocks. The paraffin blocks were then serially sectioned using a paraffin microtome (such as a Leica RM2235) to a thickness of 4-5 μm. The sections were flattened in a 40℃ water bath, transferred to glass slides, and baked in a 60℃ oven for 2 hours.

[0031] S3, hematoxylin-eosin (HE) staining After baking, the sections were dewaxed in xylene (twice, 10 minutes each time), rehydrated in a gradient of ethanol (100%→95%→80%→70%, 5 minutes each), then immersed in hematoxylin staining solution (commercially available HE staining kit, Solarbio G1120) for 5-8 minutes, and rinsed with running water; then differentiated in 1% hydrochloric acid ethanol for a few seconds, and then blued with running water; subsequently immersed in eosin staining solution (0.5% water-soluble eosin) for 1-2 minutes, and rinsed with running water. After staining, the sections were dehydrated in a gradient of ethanol, cleared in xylene, and finally mounted with neutral resin.

[0032] S4. Microscopic observation and image acquisition Prepared hematoxylin and eosin (HE) sections were placed in bright-field mode on an upright fluorescence microscope (Olympus, Japan) for observation of pathological changes such as liver lobule structure, hepatic cord arrangement, hepatocyte edema, and fat vacuoles, as well as colonic villi integrity, goblet cell count, and mucosal structure, in both low-power (×100) and high-power (×400) fields of view. Representative fields of view were photographed, and histological scoring or descriptive analysis was performed by pathologists using a double-blind method.

[0033] Figure 2 In the normal group A mice, the liver was reddish-brown, smooth, soft, and had sharp edges. HE staining showed that the liver lobule structure was intact, the liver cords were arranged in a neat radial pattern, the hepatocytes were regular in shape, the cytoplasm was uniform, and no fat vacuoles or inflammatory cell infiltration were observed. Figure 2 In the B model group, the livers appeared whitish, slightly smaller in size, and softer in texture. Fine granules were visible on the surface of some livers. HE staining showed disordered lobular structure, significant edema of hepatocytes, and fat vacuoles of varying sizes. In some areas, the hepatic cords were loosely arranged, suggesting that alcohol gavage successfully induced hepatocyte damage. Figure 2 The liver appearance of the silymarin group was similar to that of the normal group, with a reddish-brown color and uniform luster. HE staining showed that the liver lobule structure was basically intact, and the hepatocyte edema and fat vacuoles were significantly reduced compared with the model group, with only scattered slight changes, indicating that the positive drug has a protective effect against liver damage. Figure 2 The liver appearance of the low-dose MA group was improved compared with that of the model group, with a slightly reddish color. HE staining showed that a small number of fat vacuoles and mild edema were still visible in the hepatocytes, but the degree was significantly less than that of the model group. Figure 2In the high-dose MA group, the liver appearance was reddish-brown with a smooth surface, similar to the normal group and the silymarin group. HE staining showed that the liver lobule structure was intact and clear, the hepatic cords were arranged in a more regular manner, and hepatocyte steatosis and edema had basically disappeared, with only a few minor abnormalities. The high-dose MA group showed the most significant improvement in liver appearance and pathological damage, and the effect was positively correlated with the dose, indicating that MA can effectively reverse the gross morphological and histopathological abnormalities of the liver caused by alcohol, with an effect comparable to silymarin.

[0034] exist Figure 3 In HE staining of the colon of mice with Aldosterone (MA), the intestinal tissue structure of normal mice was clear and intact, with regular and uniform villi arrangement, abundant mucosa and goblet cells, and no obvious pathological changes were observed. The colon structure of the model group was significantly disordered, with shorter and broken villi, a significant reduction in the number of goblet cells, and partial mucosal epithelial shedding, suggesting that alcohol gavage caused severe damage to the intestinal mucosal mechanical barrier. The colon tissue morphology of the silymarin group and the high- and low-dose MA groups was significantly improved compared with the model group, with villi arrangement becoming more regular and the number of goblet cells recovering. Among them, the high-dose MA group showed the most significant improvement, and the tissue structure was close to that of the normal group. Figure 3 The B protein bands showed that the band signal intensity of occludin and claudin1 in the model group was significantly weaker than that in the normal group, while the band signal of both proteins was significantly enhanced after MA intervention. The silymarin group also showed a similar enhancing trend. Figure 3 C and Figure 3 Quantitative analysis of D further confirmed that, compared with the normal group, the relative expression levels of occludin and claudin1 in the model group were significantly reduced. p <0.05 or p <0.01); Compared with the model group, the expression levels of both proteins in the high- and low-dose MA groups and the silymarin group were significantly increased ( p <0.05 or p The MA concentration was <0.01), and the high-dose group showed better efficacy than the low-dose group, exhibiting a dose-dependent effect. Combined with Figures A to D, MA effectively repaired alcohol-induced colonic tissue damage and enhanced intestinal mucosal barrier function by upregulating the expression of tight junction proteins occludin and claudin1. This provides direct morphological and molecular biological evidence for its ability to improve alcoholic liver disease through the gut-liver axis pathway.

[0035] Example 3 The specific steps for detecting serum biochemical indicators and inflammatory factors are as follows: S1. Serum Sample Preparation Take mouse serum samples frozen at -80℃ and thaw them slowly at 4℃, avoiding repeated freeze-thaw cycles. After thawing, centrifuge at 4℃ and 3000 r / min for 5 minutes, and collect the supernatant for later use, ensuring that the test sample is free of precipitation or turbidity.

[0036] S2, Serum Biochemical Indicators Detection Strictly follow the kit instructions to detect the levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), and total bilirubin (TBIL) in serum. The kits used are as follows: AST kit (batch number 20250724), ALT kit (batch number 20250724), ALP kit (batch number 20250725), and TBIL kit (batch number 20250802), all purchased from Nanjing Jiancheng Bioengineering Institute. Specific operating steps (taking ALT as an example): Prepare 96-well plates, including blank wells, standard wells, and sample wells. Add the corresponding reagents and serum samples sequentially, mix well, and incubate at 37℃ for a specific time (each indicator according to the instructions). Stop the reaction by adding stop solution. Measure the absorbance (OD value) of each well using a multi-mode microplate reader (Berten Instruments, Inc., USA) at the corresponding wavelengths (510 nm for ALT / AST, 520 nm for ALP, and 450 nm for TBIL). The actual levels of AST, ALT, ALP, and TBIL in each serum sample were calculated based on the standard curve.

[0037] S3, Detection of serum inflammatory factors IL-1β and IL-6 The levels of interleukin-1β (IL-1β) and interleukin-6 (IL-6) in serum were detected using enzyme-linked immunosorbent assay (ELISA). The kits used were as follows: IL-1β ELISA kit (batch number 090188803D076M) and IL-6 ELISA kit (batch number 090188803D049M), both purchased from Shanghai Ruixin Biotechnology Co., Ltd. Specific operating steps: Remove the pre-coated antibody-labeled ELISA plate, add standards and serum samples (100 μL per well), cover with a membrane, and incubate at 37°C for 90 minutes; discard the liquid, pat dry, add 100 μL of biotinylated antibody working solution to each well, and incubate at 37°C for 60 minutes; wash the plate 3 times with washing buffer, add 100 μL of enzyme conjugate working solution to each well, and incubate at 37°C in the dark for 30 minutes; wash the plate 5 times, add 100 μL of chromogenic substrate (TMB), and develop at 37°C in the dark for 15 minutes; finally, add 50 μL of stop solution to terminate the reaction. Immediately measure the OD value of each well at 450 nm using a multi-functional ELISA reader (Berten Instruments, Inc., USA). Plot a standard curve based on the standard concentrations and corresponding OD values, and calculate the IL-1β and IL-6 content in each serum sample.

[0038] S4. Data Recording and Quality Control All tests were performed in duplicate, and the average value was taken as the final result. Ensure the correlation coefficient R of the standard curve is accurate. 2≥0.99, and the sample coefficient of variation (CV) is controlled within 10% to ensure the reliability of the test results.

[0039] Figure 4 China A and Figure 4 Serum ALT and AST activities in the B model group were significantly higher than those in the normal group. p <0.01 indicates that alcohol gavage caused severe hepatocellular damage; after intervention with low and high doses of MA and silymarin, the levels of ALT and AST were significantly reduced ( p <0.01), with the highest reduction in the high-dose MA group, approaching the level of the silymarin group. Figure 4 C and Figure 4 The levels of TBIL and ALP in the model group of D were significantly higher than those in the normal group. p <0.01, indicating that alcohol causes hepatocellular damage and bile excretion dysfunction; both indicators were significantly reduced after low-dose MA, high-dose MA, and silymarin intervention. p <0.01), and shows a dose-dependent trend. Figure 4 China E and Figure 4 The results of the detection of inflammatory factors in the model group showed that the serum IL-1β and IL-6 levels were significantly higher than those in the normal group. p <0.01 indicates that alcohol induced a systemic inflammatory response; after low and high dose interventions of silymarin and MA, the levels of both inflammatory factors were significantly reduced ( p <0.01), the high-dose group showed particularly outstanding effects, consistent with the improvement trend of biochemical indicators. Combined with Figures A to F, MA dose-dependently reduced serum transaminase, bilirubin, and pro-inflammatory factor levels in mice with alcoholic liver disease, suggesting that it exerts a hepatoprotective effect by reducing hepatocellular damage, improving liver function, and inhibiting inflammatory responses.

[0040] Example 4 Western blot analysis of liver PI3K / AKT pathway and colonic tight junction protein expression was performed as follows: S1, tissue protein extraction Mouse liver tissue (approximately 50 mg) and colon tissue (approximately 30 mg) stored at -80℃ were separately placed on ice and minced. RIPA lysis buffer (high-potency type, purchased from Shanghai Beyotime Biotechnology Co., Ltd.) containing a protease inhibitor (PMSF, final concentration 1 mmol / L) was added, with a tissue-to-lysis buffer ratio of 1:10 (g / mL). The homogenate was homogenized 3-5 times on ice using a handheld homogenizer (Tiangen OSE-Y30), 10 seconds each time, with 10-second intervals, until no obvious tissue fragments remained. The homogenate was centrifuged at 12000 r / min for 15 minutes at 4℃ (Thermo low-temperature high-speed centrifuge), and the supernatant was transferred to a new EP tube, which is the total protein extract.

[0041] S2, Protein Concentration Measurement Quantification was performed using the Thermo Scientific Pierce BCA Protein Assay Kit. An appropriate amount of supernatant was taken and, following the instructions, the BCA working solution (solution A:solution B = 50:1) was mixed with the sample or standard (BSA, concentration gradient 0–2 mg / mL) at an 8:1 ratio in a 96-well plate and incubated at 37°C for 30 minutes. The absorbance was measured at 562 nm using a multi-sensor microplate reader (Berten Instruments, Inc.). The protein concentration of each sample was calculated based on the standard curve, and all samples were diluted to the same concentration (2 μg / μL) with lysis buffer and aliquoted at -80°C for later use.

[0042] S3, SDS-PAGE electrophoresis Prepare SDS-PAGE gels (10% separating gel, 5% stacking gel; reagents such as 30% acrylamide / methylenebisacrylamide, 1.5 mol / L Tris-HCl pH 8.8, 1.0 mol / L Tris-HCl pH 6.8, 10% SDS, 10% ammonium persulfate, and TEMED were all purchased from Bio-Rad). Take equal amounts of protein sample (30 μg per well), add 5×SDS loading buffer (containing β-mercaptoethanol), and denature in a boiling water bath for 5 minutes. Add the protein sample and pre-stained protein molecular weight standards (Marker, range 10–250 kDa, such as Thermo Fisher PageRuler Plus) to the gel wells. During electrophoresis, first concentrate at a constant voltage of 80 V for 30 minutes, then separate at a constant voltage of 120 V for approximately 60 minutes, until the bromophenol blue indicator reaches the bottom of the gel.

[0043] S4, Transfer Film After electrophoresis, the gel containing the target band was cut off, and the transfer sandwich was assembled in the order of "sponge-filter paper-gel-PVDF membrane-filter paper-sponge" (the PVDF membrane (0.45 μm pore size, Millipore, USA) needs to be activated by soaking in methanol for 15 seconds, and then transferred to transfer buffer for equilibration for 5 minutes). The sandwich was placed in the transfer tank (Bio-Rad Mini Trans-Blot), pre-cooled transfer buffer (containing 20% ​​methanol) was added, and transfer was performed at a constant current of 250 mA in an ice bath for 90 minutes to ensure that the protein was transferred to the PVDF membrane.

[0044] S5, Closed After the transfer was complete, the PVDF membrane was removed and rinsed three times for 5 minutes each time with TBST buffer (Tris-HCl 50 mmol / L, NaCl 150 mmol / L, 0.1% Tween-20, pH 7.4). The membrane was then placed in rapid blocking buffer (Shanghai Beyotime) and blocked on a shaker at room temperature for 30 minutes to block non-specific binding sites on the membrane.

[0045] S6, primary antibody incubation After blocking, the PVDF membranes were washed three times with TBST, cut to size according to the molecular weight of the target protein, and then incubated with specific primary antibodies (all antibodies were diluted with blocking buffer at the recommended ratio) overnight (12-16 hours) at 4°C on a shaker. The primary antibody information used is as follows: rabbit anti-p-PI3K polyclonal antibody (batch number T41006F, dilution 1:1000, Shanghai ABMART); mouse anti-PI3K polyclonal antibody (batch number M15DEZC, dilution 1:1000, Chengdu Zhengneng Biotechnology Co., Ltd.); rabbit anti-p-AKT polyclonal antibody (batch number N140C02, dilution 1:1000, Chengdu Zhengneng); mouse anti-AKT polyclonal antibody (batch number N11AP7P, dilution 1:1000, Chengdu Zhengneng); rabbit anti-occl... Rabbit anti-claudin polyclonal antibody (lot number X2896, dilution ratio 1:800, Hangzhou Nature Biosciences); rabbit anti-claudin1 polyclonal antibody (lot number S6050, dilution ratio 1:800, Hangzhou Nature Biosciences); rabbit anti-GAPDH polyclonal antibody (lot number N26AP9P, dilution ratio 1:5000, Chengdu Zhengneng); and rabbit anti-β-actin polyclonal antibody (lot number P4903, dilution ratio 1:5000, Hangzhou Nature Biosciences) were used as internal control controls.

[0046] S7, Second Antibiotic Incubation After primary antibody incubation, the membrane was washed three times with TBST for 10 minutes each time. The membrane was then incubated with the corresponding fluorescently labeled secondary antibody at room temperature in the dark for 1 hour. The secondary antibody information was as follows: fluorescently labeled goat anti-mouse IgG (lot number WK340127, dilution 1:15000, Invitrogen, USA) and fluorescently labeled goat anti-rabbit IgG (lot number WD322231, dilution 1:15000, Invitrogen, USA). After incubation, the membrane was washed three times with TBST for 10 minutes each time in the dark.

[0047] S8. Development and Image Analysis The PVDF membrane incubated with secondary antibody was placed in a near-infrared dual-color fluorescence imaging system (LI-COR Odyssey, USA). The corresponding fluorescence channels (excitation wavelengths of 700 nm and 800 nm) were selected for scanning and development, and protein band images were acquired. ImageJ software (NIH) was used to analyze the grayscale values ​​of the bands. The relative expression level of the target protein was expressed as the ratio of the grayscale value of the target protein band to that of the internal control (GAPDH or β-actin) band. Data from each group are expressed as mean ± standard deviation and statistically compared.

[0048] Figure 5 The analysis of p-PI3K and p-AKT bands in the liver tissue of the model group showed that, compared with the normal group, the band signal intensity of p-PI3K and p-AKT was significantly enhanced, while the band signals of total PI3K and total AKT showed no significant difference among the groups, suggesting that alcohol gavage induced phosphorylation activation of PI3K and AKT proteins. After intervention with MA and silymarin, the band signals of p-PI3K and p-AKT were significantly weakened, indicating that MA can effectively inhibit the phosphorylation level of both proteins. Figure 5 China B and Figure 5 Quantitative analysis of C further confirmed that the relative protein expression levels of p-PI3K / PI3K and p-AKT / AKT in the model group were significantly higher than those in the normal group. p <0.01), while the above ratios in the low-dose MA group, high-dose MA group, and silymarin group were all significantly lower than those in the model group ( p The inhibitory effect of MA at the high-dose group was greater than that at the low-dose group, showing a dose-dependent effect. This indicates that MA can significantly inhibit the abnormal phosphorylation and activation of the PI3K / AKT signaling pathway in the liver of mice with alcoholic liver disease, which may be one of the key molecular mechanisms by which it reduces liver inflammation and exerts a hepatoprotective effect.

[0049] Example 5 The specific steps for high-throughput sequencing analysis of 16S rRNA from intestinal contents are as follows: S1. Sample Collection and Preservation After the last administration and anesthesia, mice were dissected under aseptic conditions. The intact intestines were separated, and the intestinal segments were cut open in a sterile petri dish. Approximately 200 mg of cecal contents were scraped off using sterile ophthalmic forceps and immediately transferred to a sterile 1.5 mL EP tube. After labeling, the tubes were rapidly frozen in liquid nitrogen and then transferred to an ultra-low temperature freezer at -80°C to avoid repeated freeze-thaw cycles. All procedures were performed in a clean bench (such as the SW-CJ-2FD type), and instruments were autoclaved before use.

[0050] S2, Sample delivery and testing entrustment The intestinal contents samples of the frozen normal group, model group and MA high-dose group (8 animals in each group, 24 samples in total) were placed in dry ice incubators and the 16S rRNA gene high-throughput sequencing analysis was performed by Shanghai Paisenno Biotechnology Co., Ltd.

[0051] S3, Alpha diversity analysis The Chao1 index (reflecting species richness) and the Observed species index (number of observed species) were calculated to compare the richness and diversity differences of gut microbiota among the groups. Data are expressed as mean ± standard deviation, and the results were calculated and exported using the diversity plugin of QIIME2.

[0052] S4, Beta Diversity Analysis Principal Coordinates Analysis (PCoA) was used to assess the differences in microbial community composition among samples based on the Bray-Curtis distance algorithm. Two-dimensional scatter plots were drawn to observe the aggregation distribution of samples from the normal group, model group, and high-dose MA group.

[0053] S5, Species Composition Analysis At the phylum level, the relative abundance of major phyla such as Firmicutes and Proteobacteria was compared among the groups, and the F / B ratio (Firmicutes / Bacteroidetes) was calculated. At the genus level, heatmaps were used to display the abundance of each group. Duncaniella , Muribaculum , Kineothrix , Escherichia , Klebsiella , Prevotella Changes in the relative abundance of key bacterial genera.

[0054] S6, LEfSe Difference Analysis: LEfSe software (linear discriminant analysis of effect size) was used to analyze intergroup species differences and screen for biomarkers with statistically significant differences between groups (LDA score > 2). p <0.05).

[0055] S7, Data Statistics and Visualization Sequencing data are expressed as mean ± standard deviation. One-way ANOVA (SPSS 27.0) was used for comparisons between groups. p <0.05 was considered statistically significant. All results charts (PCoA plots, species abundance bar charts, heatmaps, LEfSe branching plots, etc.) were created with the assistance of the client, who provided vector graphics files for use in subsequent paper writing.

[0056] Figure 6In the Alpha diversity analysis of A, the Chao1 and Observed species indices in the model group were significantly higher than those in the normal group, suggesting that alcohol intake led to an abnormal increase in gut microbiota richness. After MA intervention, both indices returned to near the level of the normal group, indicating that MA can restore the homeostasis of gut microbiota diversity. Figure 6 China B and Figure 6 In the Beta diversity analysis of C, principal coordinate analysis (PCoA) showed that the normal group, model group and MA high-dose group samples each clustered together. The normal group and MA high-dose group were closer in distribution and had similar microbiota structure characteristics, while the model group was farther away from the former two, indicating that the overall structure of the gut microbiota of ALD mice tended to normalize after MA intervention. Figure 6 The Venn diagram of the middle D showed that the common ASV / OTU ratio in the normal group and the high-dose MA group was 16.13%, which was higher than the common ratio in the model group and the high-dose MA group (13.77%), further supporting that MA caused the gut microbiota composition of ALD mice to regress to that of the normal group. Figure 6 China E and Figure 6 Analysis at the F phylum level showed that the abundance of Firmicutes in the model group was significantly lower than that in the normal group, while the abundance of Proteobacteria was significantly higher, resulting in a decrease in the F / B ratio. After MA intervention, the above changes were reversed, significantly increasing the abundance of Firmicutes and decreasing the abundance of Proteobacteria, causing the F / B ratio to recover. Figure 6 The horizontal heatmap of the G genus shows that the model group Muribaculum , Dubosiella , Ligilactobacillus When the abundance of beneficial bacteria decreases, Escherichia , Klebsiella , Prevotella Increased abundance of opportunistic pathogens; after MA intervention, Duncaniella , Muribaculum , Kineothrix When the abundance of beneficial bacteria increases, at the same time Escherichia , Klebsiella The abundance of pathogenic bacteria species decreased. Figure 6 LEfSe analysis of H in the middle group screened out differentially expressed microbial biomarkers between groups. In the MA group, Firmicutes and Duncaniella , Bifidobacterium The abundance of the model group is characterized by an increase, while the model group is characterized by an increase in abundance. Escherichia , Klebsiella Characterized by an increase in the abundance of opportunistic pathogens. MA can effectively improve intestinal flora imbalance in ALD mice, increase the abundance of beneficial bacteria, reduce the abundance of pathogenic bacteria, and restore flora diversity and community structure, suggesting that regulating the balance of intestinal microecology is one of the important mechanisms by which MA exerts its hepatoprotective effect.

[0057] The above are all preferred embodiments of the present invention and are not intended to limit the scope of protection of the present invention. Therefore, all equivalent changes made in accordance with the structure, shape and principle of the present invention should be covered within the scope of protection of the present invention.

Claims

1. The application of asiaticoside in the preparation of hepatoprotective drugs for treating alcoholic liver disease, characterized in that, The drug has asiaticoside as its sole active ingredient or contains asiaticoside as one of its active ingredients.

2. The application according to claim 1, characterized in that, The drug is an oral or injectable preparation, and the oral preparation is a tablet, capsule, granule, oral liquid, or pill.

3. The application according to claim 1, characterized in that, The dosage of the drug is 10–40 mg / kg.

4. The application according to claim 1, characterized in that, The drug exerts its hepatoprotective effect by inhibiting the phosphorylation activation of the hepatic PI3K / AKT signaling pathway.

5. The application according to claim 1, characterized in that, The drug improves liver function by lowering serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), and total bilirubin.

6. The application according to claim 1, characterized in that, The drug reduces liver inflammation by lowering serum levels of the inflammatory cytokines interleukin-1β and interleukin-6.

7. The application according to claim 1, characterized in that, The drug exerts its hepatoprotective effect by regulating the balance of intestinal flora, specifically by increasing the abundance of Firmicutes and decreasing the abundance of Proteobacteria; and increasing beneficial bacteria. Duncaniella , Muribaculum , Kineothrix , Bifidobacterium The abundance of [unclear] reduced the number of opportunistic pathogens. Escherichia , Klebsiella , Prevotella The abundance of.

8. The application according to claim 1, characterized in that, The drug repairs the intestinal mucosal barrier by upregulating the expression of colonic tight junction proteins occludin and claudin1.

9. The application according to claim 1, characterized in that, The alcoholic liver disease mentioned refers to alcoholic fatty liver, alcoholic hepatitis, alcoholic liver fibrosis, or alcoholic cirrhosis.

10. The application according to claim 1, characterized in that, The drug exerts its hepatoprotective effect through a multi-target synergistic mechanism that simultaneously inhibits the PI3K / AKT signaling pathway, reduces inflammatory factor levels, regulates gut microbiota balance, and repairs the intestinal barrier.

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