Application of milk thistle medicinal composition in preparing medicine for treating fatty liver disease

Abstract

The present invention relates to the application of a silymarin pharmaceutical composition in the preparation of a drug for treating fatty liver disease, and belongs to the field of medical technology. The present invention provides the application of a pharmaceutical composition in the preparation of a drug for preventing and / or treating fatty liver disease, wherein the active ingredient of the pharmaceutical composition comprises a 7-KDCA regulator; the 7-KDCA regulator comprises silymarin, or the 7-KDCA regulator comprises silybin, silybin, silybinin and / or isosilybin; the silybin comprises silybin A and / or silybin B; the isosilybin comprises isosilybin A and / or isosilybin B. Studies have shown that the pharmaceutical composition can effectively and safely treat fatty liver disease at a low dose, has anti-inflammatory activity, and has a mimetic nerve growth factor activity, and has great application prospects in the preparation of drugs for preventing and / or treating fatty liver disease, anti-inflammatory, and preventing and / or treating Alzheimer's disease.

Description

Technical Field

[0001] The invention relates to application of a silymarin medicinal composition in preparing a medicament for treating fatty liver disease, and belongs to the technical field of medicines. Background Art

[0002] Metabolic dysfunction associated fatty liver disease (MASLD), formerly known as non-alcoholic fatty liver disease (NAFLD), is a global liver disease that affects at least 25% of the world's population. MASLD can progress from simple steatosis (lipid accumulation) to metabolic dysfunction associated steatohepatitis (MASH), and further to liver fibrosis, cirrhosis and liver cancer. The pathogenesis of MASLD is multifactorial, involving genetic, metabolic and environmental factors. A key feature of MASLD is chronic liver inflammation, which plays a crucial role in the progression of the disease from simple steatosis to MASH and beyond. The production of proinflammatory cytokines, such as tumor necrosis factor (TNF) and interleukin-6 (IL-6), can promote the inflammation of MASLD. Another important factor affecting MASLD is the gut-liver axis, which mediates a bidirectional cycle between the gut microbiota and the liver. Alterations in the gut microbiota, increased intestinal permeability, and the resulting excessive influx of microbial metabolites (such as lipopolysaccharide (LPS)) into the liver contribute to the production of TNF and interleukin-1β (IL-1β), leading to liver inflammation and further progression of MASLD.

[0003] Bile acid (BA) metabolism regulated by intestinal microbiota is also closely related to MASLD. Microorganisms containing bile salt hydrolase (BSH) can dissociate conjugated bile acids into free bile acids, and some primary bile acids can also be converted into secondary bile acids by intestinal flora. Therefore, the composition of BA is affected by changes in intestinal microbiota. As the endogenous ligand of farnesoid X receptor (FXR), bile acid not only affects lipid absorption, but also regulates glucose metabolism, lipid metabolism and energy metabolism by regulating the FXR signaling pathway, playing a role in MASLD. Therefore, many drugs used clinically to treat MASLD are FXR receptor agonists, such as obeticholic acid, Vonafexor, PX-104 and TERN-101. However, at present, these drugs still have some problems. For example, drugs related to FXR receptor agonists are in the clinical verification stage and have not passed clinical trials. As the world's first FXR receptor agonist-related drug to enter phase III clinical trials, obeticholic acid still has the risk of causing drug-induced liver injury. Therefore, there is an urgent need to find therapeutic drugs for MASLD with good therapeutic effects, clear mechanisms of action and few side effects. Summary of the invention

[0004] In order to solve the above problems, the present invention provides the use of a pharmaceutical composition in preparing a drug, wherein the drug has any of the following functions:

[0005] (a) prevention and / or treatment of fatty liver disease;

[0006] (b) anti-inflammatory;

[0007] (c) prevention and / or treatment of neurodegenerative diseases;

[0008] The active ingredient of the pharmaceutical composition comprises a 7-KDCA regulator; the 7-KDCA regulator includes silymarin, or the 7-KDCA regulator comprises silybin, silybintin, silybinin and / or isosilybin; the silybin comprises silybin A and / or silybin B; the isosilybin comprises isosilybin A and / or isosilybin B. Silymarin is a natural active substance extracted from the seeds of milk thistle (Silybummarianum (L.) Gaertn.), mainly containing flavonolignans (silybin, silybintin, silybinin and isosilybin) and dihydroquercetin. Silybin (silybin A and silybin B) is the main active ingredient of silymarin, accounting for 50-70% of silymarin.

[0009] In one embodiment of the present invention, the 7-KDCA regulator is silymarin, or the 7-KDCA regulator consists of silybin and isosilybin A; the silybin consists of silybin A and silybin B.

[0010] In one embodiment of the present invention, in the drug, the mass ratio of silybin to isosilybin A is 7 to 8:1.

[0011] In one embodiment of the present invention, the fatty liver disease includes metabolic dysfunction associated fatty liver disease (MAFLD).

[0012] In one embodiment of the present invention, the treatment of fatty liver disease comprises treating fatty liver disease by regulating bile acid metabolism; the regulation of bile acid metabolism comprises reducing the level of 7-KDCA and thereby reducing the binding of 7-KDCA to farnesoid X receptor (FXR), thereby affecting the stability of farnesoid X receptor.

[0013] In one embodiment of the present invention, the treatment of fatty liver disease includes reducing inflammation in patients with fatty liver disease, reducing liver weight in patients with fatty liver disease, reducing fat weight in patients with fatty liver disease, reducing the liver weight / body weight ratio in patients with fatty liver disease, improving liver fatty degeneration in patients with fatty liver disease, reducing lipid droplet accumulation in patients with fatty liver disease, improving serum indicators in patients with fatty liver disease, improving liver function indicators in patients with fatty liver disease and / or improving intestinal flora in patients with fatty liver disease.

[0014] In one embodiment of the present invention, the reducing inflammation in patients with fatty liver disease includes reducing inflammation in patients with fatty liver disease by regulating the TLR4 / NF-κB signaling pathway; the improving the intestinal flora in patients with fatty liver disease includes regulating the relative abundance of intestinal flora that produces short-chain fatty acids in the intestine of patients with fatty liver disease, regulating the relative abundance of intestinal flora that produces secondary bile acids (secondary bile acids) in the intestine of patients with fatty liver disease and / or regulating the relative abundance of intestinal flora that contains bile salt hydrolase activity in the intestine of patients with fatty liver disease.

[0015] In one embodiment of the present invention, the anti-inflammatory treatment comprises reducing inflammation in patients with inflammation by regulating the TLR4 / NF-κB signaling pathway.

[0016] In one embodiment of the present invention, the treatment of neurodegenerative diseases includes treating neurodegenerative diseases as a nerve growth factor-mimetic active substance.

[0017] In one embodiment of the present invention, the neurodegenerative disease includes Alzheimer's disease.

[0018] In one embodiment of the present invention, the drug further contains pharmaceutical excipients.

[0019] In one embodiment of the present invention, the pharmaceutical excipients include stabilizers, preservatives, disintegrants, sweeteners, glidants and / or diluents.

[0020] In one embodiment of the present invention, the dosage form of the drug is capsule, injection, powder, granule, paste and / or tablet.

[0021] The present invention also provides a nerve growth factor-mimicking active substance, the components of which include a 7-KDCA regulator; the 7-KDCA regulator includes silymarin, or the 7-KDCA regulator includes silybin, silybin, silybinin and / or isosilybin; the silybin includes silybin A and / or silybin B; the isosilybin includes isosilybin A and / or isosilybin B.

[0022] In one embodiment of the present invention, the 7-KDCA regulator is silymarin, or the 7-KDCA regulator consists of silybin and isosilybin A; the silybin consists of silybin A and silybin B.

[0023] In one embodiment of the present invention, in the drug, the mass ratio of silybin to isosilybin A is 7 to 8:1.

[0024] The present invention also provides the use of a pharmaceutical composition in the preparation of a nerve growth factor-mimicking active substance, wherein the components of the nerve growth factor-mimicking active substance include a 7-KDCA regulator; the 7-KDCA regulator includes silymarin, or the 7-KDCA regulator includes silybin, silybin, silybinin and / or isosilybin; the silybin includes silybin A and / or silybin B; the isosilybin includes isosilybin A and / or isosilybin B.

[0025] In one embodiment of the present invention, the 7-KDCA regulator is silymarin, or the 7-KDCA regulator consists of silybin and isosilybin A; the silybin consists of silybin A and silybin B.

[0026] In one embodiment of the present invention, in the drug, the mass ratio of silybin to isosilybin A is 7 to 8:1.

[0027] The technical solution of the present invention has the following advantages:

[0028] 1. The present invention provides an application of a pharmaceutical composition in the preparation of a drug for preventing and / or treating fatty liver disease, wherein the active ingredient of the pharmaceutical composition comprises a 7-KDCA regulator; the 7-KDCA regulator includes silymarin, or the 7-KDCA regulator comprises silybin, silybinin, silybinin and / or isosilybin; the silybin comprises silybin A and / or silybin B; the isosilybin comprises isosilybin A and / or isosilybin B.

[0029] Animal experiments have shown that the pharmaceutical composition provided by the present invention can reduce inflammation in MASLD model mice, reduce liver weight in MASLD model mice, reduce fat weight in MASLD model mice, reduce liver weight / body weight ratio in MASLD model mice, improve liver fatty degeneration in MASLD model mice, reduce lipid droplet accumulation in MASLD model mice, improve serum indexes in MASLD model mice, improve liver function indexes in MASLD model mice, and improve intestinal flora in MASLD model mice (including regulating the relative abundance of intestinal flora producing short-chain fatty acids in the intestine of MASLD model mice). degree, regulating the relative abundance of intestinal flora that produces secondary bile acid in the intestine of MASLD model mice, and regulating the relative abundance of intestinal flora that contains bile salt hydrolase activity in the intestine of MASLD model mice, etc.), which has a good treatment for MAFLD. In addition, animal experiments have shown that the pharmaceutical composition provided by the present invention can significantly improve the liver function indicators of MASLD model mice (including reducing the levels of triglycerides, total cholesterol, aminotransferase, aspartate aminotransferase and total bile acid in the serum of MASLD model mice), has a certain liver protection effect, and will not cause liver damage when used for the treatment of MAFLD;

[0030] Mechanism experiments have shown that the pharmaceutical composition provided by the present invention can reduce inflammation in MASLD model mice by regulating the TLR4 / NF-κB signaling pathway, and the pharmaceutical composition provided by the present invention can reduce the level of 7-KDCA in MASLD model mice and thus reduce the binding of 7-KDCA to farnesoid X receptor in MASLD model mice (negative feedback regulation), thereby affecting the stability of farnesoid X receptor in MASLD model mice, so as to achieve the purpose of treating MASLD.

[0031] Therefore, the pharmaceutical composition provided by the present invention can effectively and safely treat fatty liver disease (especially MAFLD), and has great application prospects in the preparation of drugs for preventing and / or treating fatty liver disease (especially MAFLD).

[0032] Further, the 7-KDCA regulator is composed of silybin and isosilybin A; the silybin is composed of silybin A and silybin B; in the drug, the mass ratio of silybin and isosilybin A is 7 to 8:1. Animal experiments have shown that compared with high-dose silybin (80 mg / kg / day silybin), lower doses of silybin and isosilybin A (44.87 mg / kg / day silybin + 6.09 mg / kg / day isosilybin A) can effectively treat MASLD, and the effect is close to that of high-dose silybin (80 mg / kg / day silybin). Therefore, the pharmaceutical composition provided by the present invention can effectively and safely treat fatty liver disease (especially MAFLD), and has great application prospects in the preparation of drugs for preventing and / or treating fatty liver disease (especially MAFLD).

[0033] 2. The present invention provides an application of a pharmaceutical composition in the preparation of an anti-inflammatory drug, wherein the active ingredient of the pharmaceutical composition comprises a 7-KDCA regulator; the 7-KDCA regulator comprises silymarin, or the 7-KDCA regulator comprises silybin, silybin, silybinin and / or isosilybin; the silybin comprises silybin A and / or silybin B; the isosilybin comprises isosilybin A and / or isosilybin B. Cell experiments have shown that the pharmaceutical composition provided by the present invention can exhibit excellent anti-inflammatory activity at the cellular level, animal experiments have shown that the pharmaceutical composition provided by the present invention can reduce inflammation in MASLD model mice, and mechanism experiments have shown that the pharmaceutical composition provided by the present invention can reduce inflammation in MASLD model mice by regulating the TLR4 / NF-κB signaling pathway. Therefore, the pharmaceutical composition provided by the present invention has great application prospects in the preparation of anti-inflammatory drugs.

[0034] 3. The present invention provides the use of a pharmaceutical composition in the preparation of a drug for preventing and / or treating a neurodegenerative disease, wherein the active ingredient of the pharmaceutical composition comprises a 7-KDCA regulator; the 7-KDCA regulator comprises silymarin, or the 7-KDCA regulator comprises silybin, silybin, silybinin and / or isosilybin; the silybin comprises silybin A and / or silybin B; the isosilybin comprises isosilybin A and / or isosilybin B. Alzheimer's disease is a stubborn disease that seriously threatens the health of the elderly. The drugs currently on the market for treating neurodegenerative diseases such as Alzheimer's disease only play a role in alleviating symptoms, and it is still an incurable disease. It is reported that the onset of neurodegenerative diseases such as Alzheimer's disease is related to nerve cell damage and death, and nerve growth factor (NGF) has an important effect on the growth, survival and functional maintenance of neurons. Since NGF is highly hydrophilic and has a large molecular weight, it cannot pass through the blood-brain barrier, which limits its application in the treatment of neurodegenerative diseases such as Alzheimer's disease. Therefore, it has become a research hotspot to find small molecule compounds with NGF-like activity that can pass through the blood-brain barrier smoothly and have the activity of NGF. Cell experiments have shown that the pharmaceutical composition provided by the present invention has the activity of NGF-like activity, and each component in the pharmaceutical composition is a small molecule compound. Therefore, the pharmaceutical composition provided by the present invention, as a NGF-like active substance, has great application prospects in the preparation of drugs for preventing and / or treating neurodegenerative diseases such as Alzheimer's disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 : HPLC analysis results of silymarin.

[0036] Figure 2 : Chemical structure of the main components of silymarin.

[0037] Figure 3 : Effects of silymarin and its main components on PC12 cells. Figure 3 (A) Pictures of PC12 cells after sample treatment, (A), (a) negative control, (b) silybin (4.8 μg / mL), (c) silybin (4.8 μg / mL), (d) silybin (4.8 μg / mL), (e) silymarin (4.8 μg / mL); (B) quantification results of Figure A.

[0038] Figure 4 :Body weight changes of mice during high-fat diet modeling.

[0039] Figure 5 : Effects of silymarin and its major components on body weight, liver weight, and fat weight in HFD-fed mice. Figure 5In the figure, (A) design of mouse dosing regimen; (B) changes in mouse body weight before dosing; (C) changes in mouse body weight during dosing; (D) mouse body weight after dosing; (E) mouse liver weight after dosing; (F) mouse epididymal fat weight after dosing; (G) mouse liver weight to body weight ratio after dosing.

[0040] Figure 6 : Effects of silymarin on liver steatosis, serum biochemical indexes and liver function indexes in HFD mice. Figure 6 In the figure, (A) histological staining of mouse viscera; (B) quantification result of Oil Red staining of mouse liver; (C) serum triglyceride level of mouse; (D) serum total cholesterol level of mouse; (E) serum ALT level of mouse; (F) serum AST level of mouse; (G) serum total bile acid level of mouse.

[0041] Figure 7 : Effects of silymarin on inflammation in HFD mice. Figure 7 (A) LPS level in mouse feces; (B) LPS level in mouse serum; (C) TNF-α level in mouse serum; (D) IL-6 level in mouse serum; (E) LPS level in mouse liver; (F) TNF-α level in mouse liver; (G) IL-6 level in mouse liver; (H) WB results of TLR4, p-IKKβ, IKKβ, p-IκBα, IκBα, and βactin in mouse liver; (I) quantification results of TLR4 / βactin; (J) quantification results of p-IKKβ / IKKβ; (K) quantification results of p-IκBα / IκBα.

[0042] Figure 8 : Effects of silymarin on intestinal flora in HFD mice. Figure 8 (A) α diversity analysis-Chao1 index; (B) α diversity analysis-Simpson index; (C) phylum level clustering analysis; (D) Firmicutes / Bacteroidota (F / B); (E) Desulfobacterota relative abundance.

[0043] Fig. 9 : Effects of silymarin on the relative abundance of intestinal flora at the genus level in HFD mice. Fig. 9 In the figure, (A) clustering diagram (heat map) of intestinal flora at the genus level; (B) abundance of intestinal flora positively correlated with NASH; (C) relative abundance of intestinal flora producing SCFA; (D) relative abundance of intestinal flora producing secondary bile acid; (E) relative abundance of intestinal flora containing BSH.

[0044] Fig.10 : Effects of silymarin on bile acid metabolism in HFD mice. Fig.10(A) Heat map of targeted metabolic analysis of bile acid in mouse feces; (B) composition and content ratio of bile acid in mice; (C) 7-KDCA level.

[0045] Fig.11 :FXR expression level and the effect of 7-KDCA on FXR stability. Fig.11 In the figure, (A) WB analysis results of mouse liver FXR; (B) FXR / βactin quantification results; (C) CETSA results of 7-KDCA and FXR; (D) quantification analysis results of Figure A; (E) DARTS results of 7-KDCA and FXR; (F) quantification analysis results of Figure C. DETAILED DESCRIPTION

[0046] The following examples are provided for a better understanding of the present invention, but are not intended to limit the best mode of implementation, nor to limit the content and protection scope of the present invention. Any product identical or similar to the present invention obtained by anyone under the inspiration of the present invention or by combining the features of the present invention with other prior arts shall fall within the protection scope of the present invention.

[0047] If no specific experimental steps or conditions are specified in the following examples, the conventional experimental steps or conditions described in the literature in the field can be used. If no manufacturer is specified for the reagents or instruments used, they are all conventional reagent products that can be purchased commercially.

[0048] Experimental Example 1: Analysis of the main chemical components of silymarin

[0049] The experimental process is as follows:

[0050] In order to clarify the main chemical components of silymarin, silymarin samples (purchased from Zhejiang Huisong Pharmaceutical Co., Ltd.) were studied. Silymarin and silybin, silybin, silybinin and silybinin were analyzed by HPLC (Cosmosil 5C18-MS-IIPacked Column (Ф4.6 / 250mm), flow rate 1mL / min, detection wavelength: 210nm, 50% methanol, 60min), and area normalization calculation was performed to determine the main components and content proportions of silymarin: silybin (A, B, 56.09%, t R =27.30min,31.57min), silymarin (24.06%,t R =9.99min), Silybin (6.85%, t R =12.03min), isosilybin A (7.61%, t R =45.70min), isosilybin B (3.25%, t R=50.63min) and dihydroquercetin (2.14%, t R =6.95min), and its retention time was basically consistent with that of the standard. Figure 1 As shown in the figure, the main components of silymarin are structured as follows Figure 2 shown.

[0051] Experimental Example 2: Experiment on the NGF-like Activity of Silymarin and Its Main Components

[0052] The experimental process is as follows:

[0053] Press 2×10 5 Addition amount of CFU / well PC12 cells (purchased from the cell bank of the Chinese Academy of Sciences) were added to each well of a 24-well plate with 1 mL of DMEM medium (Dublecco's modified Eagle medium, purchased from Thermo Scientific) containing 10% (v / v) fetal bovine serum, 5% (v / v) horse serum and 1% (w / v, g / 100 mL) premixed antibiotics (purchased from Invitrogen), and then the 24-well plate was placed in a cell culture incubator at 37°C and 5% (v / v) CO2 for 24 h; after the culture, fresh DMEM medium was replaced, and the 24-well plate was divided into a negative control group (Control), a silybin group (Silybin), a silydianin group (Silydianin), a silychristin group (Silychristin) and a silymarin group (Silymarin), with 3 replicates in each group; after grouping, silybin was added to the silybin group, silydianin group, silychristin group and silymarin group, respectively. Silybin, silybin, silybin and silymarin were added to a final concentration of 4.8 μg / mL, and no substance was added to the negative control group. Silybin was purchased from Chengdu Zhibiao Chemical Purification, silybin and silybin were purchased from Chengdu Aifa Biological, and silymarin was purchased from Zhejiang Huisong Pharmaceutical Co., Ltd.; after the addition of samples, the 24-well plate was placed in a cell culture incubator at 37°C and 5% (v / v) CO2 for 24 hours; after the incubation, the morphological changes of the cell morphology were observed under a microscope and the cell neurite differentiation rate was recorded (cell neurite differentiation rate = the number of cells with neurites longer than one times the longest diameter of the cell body / the total number of cells in the selected field of view × 100%). The results showed that compared with the control group, silymarin and its main components significantly increased the proportion of PC12 cells with neurites ( Figure 3 A and Figure 3 B) in the figure indicates that silymarin and its main components have nerve growth factor-like activity.

[0054] Experimental Example 3: Effects of Silymarin and Its Main Components on MASLD Model Mice

[0055] The experimental process is as follows:

[0056] 1. Evaluation of the efficacy of silymarin and its main components

[0057] 1.1. Establishment of MASLD mouse model using high-fat diet

[0058] Fifty ICR male mice (four weeks old) were purchased from the Hangzhou Medical Academy and fed in the Experimental Animal Center of Zhejiang University (the feeding conditions were room temperature 23°C, 55% humidity, and 12h light / dark cycle). After one week of adaptive feeding of the mice, the mice were divided into a normal feed group (ND group, 10 mice) and a high-fat feed group (HFD group, 40 mice). The normal feed group was fed with normal feed, and the high-fat feed group was fed with high-fat feed for MASLD modeling, and the weight changes of the mice were recorded weekly. The normal feed was provided by the Experimental Animal Center of Zhejiang University, and the high-fat feed was purchased from Nantong Trofi Feed Technology Co., Ltd. (feed code TP23300, containing 60% fat, 20.6% carbohydrates and 19.4% protein). After 18 weeks of modeling, there was a significant difference in the body weight of mice in the ND group and the HFD group. The average body weight gain of mice in the ND group was 25.6g, while the average body weight gain of mice in the HFD group was 39.3g ( Figure 4 ).

[0059] 1.2 Effects of silymarin and its main components on liver weight, fat weight and liver weight / body weight ratio in HFD-induced MASLD model mice

[0060] After 18 weeks of modeling, the MASLD modeling mice were randomly divided into four groups: HFD group (given high-fat diet and water), Silymarin30 group (given high-fat diet and 30 mg / kg / day silymarin), Silymarin80 group (given high-fat diet and 80 mg / kg / day silymarin), and Silybin+Iso group (given high-fat diet and 44.87 mg / kg / day silybin + 6.09 mg / kg / day isosilybin A) (e.g. Figure 5 The dosage of silybin and isosilybin A was obtained by multiplying 80 mg / kg by the proportion of silybin and isosilybin A in silymarin (56.09%, 7.61%). Silybin and isosilybin A were purchased from Chengdu Zhibiao Chemical, and silymarin was purchased from Zhejiang Huisong Pharmaceutical Co., Ltd. During the treatment period, the weight changes of the ND and HFD groups were as follows: Figure 5 After 8 weeks of administration, the body weight of the mice was recorded and then dissected, and the weights of the liver and epididymal fat were measured. The body weight, liver weight, and epididymal fat weight of the HFD group were significantly higher than those of the ND group ( Figure 5 D to Figure 5F, p<0.001, p<0.01, p<0.05). HFD-induced weight gain was reduced among treatment groups, but the differences were not significant ( Figure 5 However, liver weights of the Silymarin30, Silymarin80, and Silybin+Iso treated groups were significantly reduced compared with the HFD group ( Figure 5 E in, p<0.01, p<0.001, p<0.05), epididymal fat weight was also significantly reduced ( Figure 5 F, p<0.01, p<0.05, p<0.05). The liver weight to body weight ratios did not differ between the ND and HFD groups, but were significantly lower in the three treatment groups compared with the HFD group ( Figure 5 G, p<0.05, p<0.001, p<0.05). These results indicate that silymarin or the combination of silybin and isosilybin white A significantly reduced the increase in liver and fat weight and the ratio of liver to body weight induced by HFD in mice, and the effect of silymarin80 group was the best.

[0061] 1.3 Effects of silymarin and its main components on hepatic steatosis, lipid droplet accumulation, and serum liver function indices in HFD-induced MASLD mice

[0062] After 8 weeks of oral administration, 3 liver samples were randomly taken from each group of mice for histological staining and observation under a microscope. Hematoxylin and eosin (H&E) staining was used to observe the morphology of liver cells. HE staining results showed that the HFD group had significant hepatic steatosis and liver damage, which could be improved by Silymarin30, Silymarin80 and Silybin+Iso ( Figure 6 Oil red O staining was then used to observe lipid droplet formation. The results showed that a large amount of lipid droplets accumulated in the HFD group ( Figure 6 A and Figure 6 B, p<0.001), while the lipid droplet accumulation was reduced in the Silymarin30, Silymarin80 and Silybin+Iso groups ( Figure 6 A and Figure 6 B, p<0.05, p<0.01, p<0.01). Sirius red and its contrast dye are both strong acidic dyes that easily bind to the alkaline groups in collagen molecules and appear red under an optical microscope. Therefore, they can be used to observe liver fibrosis. The results showed that a small amount of collagen fibers was observed in the HFD group, but the collagen fibers were reduced in the ND group and the treatment group ( Figure 6A in the figure indicates that liver fibrosis was alleviated after silymarin treatment. In addition, 8 weeks after oral administration, mouse plasma was collected by orbital bleeding, and serum was obtained by centrifugation (12000rpm for 30min) after standing for half an hour. Serum samples (200μL) of 8 mice were randomly selected from each group to evaluate the levels of triglyceride (TG), total cholesterol (TC), aminotransferase (ALT), aspartate aminotransferase (AST) and total bile acid (TBA) (detection was completed by Hangzhou Haoke Biological). Compared with the ND group, the levels of triglyceride (TG) and total cholesterol (TC) in the HFD group were significantly increased ( Figure 6 C and Figure 6 D, p<0.001, p<0.001), the TG and TC levels in each treatment group were significantly decreased ( Figure 6 C, p<0.001, p<0.001, <0.01; Figure 6 D in, p<0.01, p<0.001, p<0.05). The levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST), which reflect liver cell damage, were significantly increased in the HFD group, but significantly decreased in the Silymarin80 and Silybin+Iso groups ( Figure 6 E in, p<0.01, p<0.01; Figure 6 F, p<0.001, p<0.05, p<0.05). Serum total bile acid (TBA) can reflect the level of hepatocyte damage and hepatic excretion dysfunction. The results showed that the HFD-induced increase in serum TBA levels was reduced in each treatment group ( Figure 6 These results suggest that silymarin or the combination of silybin and isosilybin white A can not only alleviate HFD-induced hepatic steatosis and reduce serum TG and TC levels, but also improve liver function indicators.

[0063] 1.4 Effects of silymarin and its main components on serum liver function indices in HFD-induced MASLD model mice

[0064] In addition, 50 ICR male mice (four weeks old) were purchased from Hangzhou Medical College and fed in the Experimental Animal Center of Zhejiang University (the feeding conditions were room temperature 23°C, 55% humidity, and 12h light / dark cycle). After one week of adaptive feeding of mice, the mice were divided into a normal feed group (ND group, 5 mice) and a high-fat feed group (HFD group, 45 mice). The normal feed group was fed with normal feed, and the high-fat feed group was fed with high-fat feed for MASLD modeling, and the weight changes of mice were recorded weekly. The normal feed was provided by the Experimental Animal Center of Zhejiang University, and the high-fat feed was purchased from Nantong Trophy Feed Technology Co., Ltd. (feed code TP23300, containing 60% fat, 20.6% carbohydrates and 19.4% protein).

[0065] After 18 weeks of modeling, the MASLD modeling mice were randomly divided into nine groups: HFD group (given high-fat diet and water), silymarin group (given high-fat diet and 80 mg / kg / day silymarin), silybin 7 & isosilybin A1 group (given high-fat diet and 44.87 mg / kg / day silybin + 6.09 mg / kg / day isosilybin A), silybin 5 & isosilybin A5 group (given high-fat diet and 25.48 mg / kg / day silybin + 25.48 mg / kg / day isosilybin A), silybin group (given high-fat diet and 44.87 mg / kg / day silybin), isosilybin group (given high-fat diet and 6.09 mg / kg / day isosilybin), silybin group (given high-fat diet and 5.48 mg / kg / day silybin), silybin group (given high-fat diet and 19.25 mg / kg / day silybin) and dihydroquercetin group (given high-fat diet and 1.71 mg / kg / day dihydroquercetin) (as shown in Table 1), 5 mice in each group, and the drug was administered by gavage continuously for 8 weeks. Among them, the dose of silybin 7 & isosilybin A1 group was obtained by the ratio of silybin to isosilybin A in silymarin, and the dose of silybin 5 & isosilybin A5 group was obtained by the total weight of silybin 7 & isosilybin A1 group. Silybin and isosilybin A were purchased from Chengdu Zhibiao Chemical Pure, silybin, silybinin and dihydroquercetin were purchased from Chengdu Aifa Biological, and silymarin was purchased from Zhejiang Huisong Pharmaceutical Co., Ltd.

[0066] After 8 weeks of oral administration, mouse plasma was collected by orbital blood sampling, and serum was obtained by centrifugation (12000rpm centrifugation for 30min) after standing for half an hour. Serum samples (200μL) of 5 mice in each group were taken to evaluate the levels of triglyceride (TG), total cholesterol (TC), aminotransferase (ALT) and aspartate aminotransferase (AST) (detection was completed by Hangzhou Haoke Biological). The results further showed that only silymarin or a specific ratio combination of silybin and isosilybin white A could reduce serum TG and TC levels and improve liver function indicators (Table 1, * is p<0.05, ** is p<0.01, *** is p<0.01).

[0067] Table 1 Levels of triglyceride, total cholesterol, aminotransferase and aspartate aminotransferase in serum of mice in different groups

[0068]

[0069] 2. Research on the mechanism of action of silymarin and its main components

[0070] 2.1 Effects of silymarin and its main components on reducing inflammation in HFD-fed mice by regulating the TLR4 / NF-κB signaling pathway

[0071] In order to explore whether the effects of silymarin and its main components are related to regulating inflammation, ELISA kits were used to detect the levels of pro-inflammatory cytokines in the feces, serum and liver of mice. For fecal samples, based on Part 1.2, 30 mg of mouse feces collected after 8 weeks of oral administration were taken, and the LPS level was measured using an Elisa kit (purchased from Hefei Laier Biological). For liver and serum samples, based on Part 1.2, 30 mg of mouse liver collected after dissection of mice that had been administered oral administration for 8 weeks was taken, and the levels of LPS (lipopolysaccharide), TNF-α (tumor necrosis factor) and IL-6 (interleukin-6) in the liver and serum of mice were measured using an Elisa kit (purchased from Suzhou Yikesai Biological). The results of mouse fecal LPS levels showed that silymarin and Silybin+Iso reversed the increase in LPS levels in mouse feces caused by HFD ( Figure 7 A, p<0.05, p<0.05, p<0.01, p<0.01). HFD also led to increased levels of serum LPS, TNF-α, and IL-6 ( Figure 7 B~ Figure 7 D in, p<0.05, p>0.05, p<0.05). Among them, the serum LPS levels of all treatment groups were significantly reduced ( Figure 7 B, p<0.05, p<0.05, p<0.05), but only Silymarin80 and Silybin+Iso reduced serum TNF-α levels ( Figure 7C, p<0.05, p<0.01), only Silymarin80 reduced serum IL-6 levels ( Figure 7 D in the figure, p<0.05). The results of the pro-inflammatory cytokine levels in the liver showed that all treatment groups had reduced HFD-induced liver LPS ( Figure 7 E in, p<0.05, p<0.05, p<0.01, p<0.01), TNF-α( Figure 7 F in , p<0.05, p<0.05, p<0.05, p<0.01) and IL-6 ( Figure 7 G, p<0.05, p<0.05, p<0.05, p<0.05). The TLR4 / NF-κB signaling pathway can be activated by LPS and proinflammatory cytokines. Therefore, protein immunoblotting (WB) analysis was used to evaluate the expression levels of related proteins in the mouse liver. Based on part 1.2, 8 weeks after oral administration, liver samples of 3 mice were randomly selected from each group and mixed. 30 mg of mixed liver samples were taken, and the protein concentration of the mouse liver protein solution was measured using a BCA kit (purchased from Kangwei Century). The test samples were subjected to immunoblotting analysis (Western blot). The results showed that the expression levels of TLR4 and phosphorylated IκBα were significantly increased in the HFD group ( Figure 7 H in Figure 7 I and Figure 7 K in , p<0.05, p<0.01), and was significantly reduced by Silymarin80 ( Figure 7 H in Figure 7 I and Figure 7 K in, p<0.01, p<0.05). The Silybin+Iso group significantly increased the phosphorylation level of IKKβ ( Figure 7 H and Figure 7 J, p<0.05), but reduced the increase in IκBα phosphorylation level caused by HFD ( Figure 7 H and Figure 7 K in (p<0.01, p<0.05). These results suggest that silymarin reduces inflammatory signaling pathways by regulating TLR4 / NF-κB, and it is more effective than the combination of silybin and isosilybin A.

[0072] 2.2 Silymarin regulates the intestinal flora composition of HFD-fed mice

[0073] The gut-liver axis plays an important role in the development of MASLD. To investigate whether silymarin changes the intestinal microbiota, based on section 1.2, 8 weeks after oral administration, the mouse cages were cleaned and disinfected with alcohol, and fecal samples of the mice were collected. Fecal samples (2 pellets) of 8 mice were randomly selected from each group to extract DNA. According to the designated sequencing region, the 16S rDNA V4 region of the samples was sequenced by PE250 using the HiSeq 2500 instrument. The results of the α-diversity analysis of the mouse intestinal microbiota are shown in Figure 2. Figure 8 A and Figure 8 As shown in B. HFD reduced the total number of microorganisms in mouse feces, and silymarin or Silybin+Iso did not restore the total number of microorganisms ( Figure 8 A in Figure 2, p<0.01). HFD did not change the richness of microbial species in mouse feces, but the species richness was significantly increased in the Silymarin80 group ( Figure 8 In order to investigate the differences and similarities among the different groups in more detail, cluster analysis was performed on each group at the phylum level ( Figure 8 The results showed that the microbial composition of the Silymarin80 group and the ND group was the most similar, followed by the Silybin+Iso group and the Silymarin30 group. The composition difference between the HFD group and the ND group was the largest ( Figure 8 C in the figure). These results suggest that HFD altered the composition of the intestinal microbiota in mice, while 80 mg / kg silymarin partially restored it to the level of the ND group. An increase or decrease in the ratio of Firmicutes to Bacteroidota (F / B) is generally considered to be intestinal dysbiosis, where an increase in F / B is often associated with obesity and metabolic disorders, which may be related to increased caloric extraction from food, fat deposition and fat production, and impaired insulin sensitivity. In this experiment, the F / B of the HFD group increased significantly ( Figure 8 D in , p<0.001), while the F / B of Silymarin80 and Silybin+Iso groups decreased ( Figure 8 D in, p<0.001, p<0.05). In addition, Desulfovibrio, which is usually negatively correlated with body mass index and triglyceride levels and improves MASLD by producing acetate, was reduced in the HFD group in this experiment, but did not reach a significant level ( Figure 8 However, the relative abundance of Desulfovibrio was significantly increased in each treatment group ( Figure 8E in (p<0.01, p<0.05, p<0.01). These results suggest that silymarin can modulate the composition of the gut microbiota in HFD-fed mice.

[0074] 2.3 Silymarin regulates the relative abundance of microbial flora producing SCFA or secondary bile acids and intestinal flora with BSH activity

[0075] The results of the relative abundance of intestinal flora at the genus level showed that the changes in relative abundance at the genus level caused by HFD were partially restored to the ND level by each treatment group (especially the Silymarin80 group) ( Fig. 9 In addition, 80 mg / kg of silymarin significantly increased the relative abundance of short-chain fatty acid (SCFA)-producing bacteria (i.e., Allobaculum, Bifidobacterium, and Dubosiella), thereby exerting anti-inflammatory, anti-cancer, and intestinal permeability-reducing effects and maintaining homeostasis ( Fig. 9 Bacteria with the ability to produce secondary bile acids (i.e., Clostridium and Eubacterium, both of which belong to the phylum Firmicutes) also increased significantly in the Silymarin80 group ( Fig. 9 D), however, the relative abundance of bacteria with bile salt hydrolase (BSH) activity showed inconsistent trends among the groups, suggesting that only certain bile acids, but not all bile acids, may play a role in the treatment of MASLD by silymarin. These results suggest that silymarin can regulate the abundance of intestinal microbiota at the genus level to improve HFD-induced hepatic steatosis.

[0076] 2.4 Silymarin regulates bile acid metabolism and reduces the increase in 7-KDCA levels caused by HFD

[0077] To further investigate which bile acids play an important role in improving liver steatosis, based on Section 1.2, bile acid targeted metabolomics analysis was performed on the fecal samples of mice in the ND, HFD, and Silymain80 groups collected 8 weeks after oral administration. The number of samples in each group was 4, and each sample was a mixture of feces from 2 mice in the group, with a sample size of 30 mg (fecal samples were sent to Shanghai Zhongke New Life for bile acid targeted metabolomics analysis). The results showed that HFD changed the levels of different types of bile acids in mouse feces, while Silymarin80 restored the concentrations of some bile acids to ND levels ( Fig.10 In the ND group, the highest concentration of bile acid was 12-KLCA, followed by DCA, ApoCA, HDCA, α-MCA, LCA, 7-KDCA ( Fig.10 B). HFD significantly increased the concentrations of 7-KDCA and α-MCA ( Fig.10 A, p<0.001, p<0.05), and decreased the concentration of TCDCA ( Fig.10 A in Figure 2, p<0.05). After treatment with 80 mg / kg silymarin, the concentration of 7-KDCA decreased to the ND level ( Fig.10 A and Fig.10 C, p<0.001), but the concentrations of α-MCA and TCDCA did not return to ND levels. These results suggest that silymarin may exert its effects by regulating 7-KDCA in bile acids.

[0078] 2.5. FXR is a potential target of 7-KDCA

[0079] Farnesoid X receptor (FXR) is an endogenous ligand of bile acid. Therefore, based on Section 1.2, the expression level of FXR in the liver of mice given oral administration for 8 weeks was detected by Western blot. The results showed that the expression levels of FXR in the Silymarin30, Silymarin 80 and Silybin+Iso groups were significantly increased ( Fig.11 A and Fig.11In the results of bile acid targeted metabolic analysis, the concentration of 7-KDCA increased in the HFD group and decreased to the ND level in the Silymarin80 group. Therefore, it is speculated that 7-KDCA can negatively feedback regulate FXR, thereby regulating its downstream signaling pathways and improving MASLD. To verify this hypothesis, based on Section 1.2, proteins were extracted from the liver samples of mice in the ND group (see Section 2.1 for the extraction method) to obtain a mouse liver protein solution; after measuring the protein concentration of the mouse liver protein solution with a BCA kit (purchased from Kangwei Century), the mouse liver protein solution was diluted with PBS buffer to a protein concentration of 2 μg / μL to obtain a dilution; the dilution was dispensed into Ep tubes (100 μL per tube) and different concentrations (0 μM, 0.001 μM, 0.003 μM, 0.01 μM, 0.03 μM, 0.1 μM, 0.3 μM) of 7-KDCA (purchased from Sigma-Aldrich) were added to the dilution, and then incubated in a 37°C incubator for 3 h (setting a blank control) for subsequent CETSA and DARTS experiments. For CETSA, the incubated protein solution was heated at 66°C for 3 min and then centrifuged (4°C, 12000 rpm, 20 min). The supernatant was collected and 5X SDS-PAGE loading buffer was added to the supernatant at a volume ratio of supernatant: buffer = 4:1. The supernatant was heated and denatured at 100°C for 10 min to obtain the test sample. The test sample was subjected to Western blot analysis to detect the expression of FXR and βactin. For DARTS, 1.5% (w / w) pronase (purchased from MCE) was added to the incubated protein solution (TNC buffer was added to the blank control group), and the solution was incubated at room temperature (25°C) in the dark for 25 minutes to obtain an incubation solution; 5XSDS-PAGE loading buffer was added to the incubation solution at a volume ratio of incubation solution: buffer = 4:1, and the solution was heated at 100°C for 10 minutes to obtain the sample to be tested; the sample to be tested was subjected to Western blot analysis to detect the expression of FXR and βactin. The results showed that 7-KDCA could enhance the thermal stability of FXR in a dose-dependent manner when heated at 66°C ( Fig.11 C and Fig.11 D in (p<0.05, p<0.01, p<0.01). Meanwhile, 1.5% (w / w) pronase can degrade FXR and βactin, while 7-KDCA can inhibit the degradation of FXR by pronase in a dose-dependent manner ( Fig.11 E and Fig.11F in (p<0.01, p<0.001, p<0.01). These results indicate that 7-KDCA can bind to FXR and affect the stability of FXR, indicating that FXR is a potential target of 7-KDCA.

[0080] In summary, the above experimental results show that silymarin mainly treats MASLD by targeting and regulating the intestinal microbial metabolites LPS and 7-KDCA to bind to TLR4 and FXR receptors and regulate downstream signaling pathways. The above research results provide new insights into the mechanism of action of silymarin in the treatment of MASLD and clarify the potential role of 7-KDCA in MASLD.

[0081] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. Use of a pharmaceutical composition in the preparation of a drug for preventing and / or treating fatty liver disease, characterized in that: The active ingredients of the pharmaceutical composition are composed of silybin and isosilybin A; in the pharmaceutical composition, the mass ratio of silybin to isosilybin A is 7 to 8:1; The treating fatty liver disease comprises treating fatty liver disease by regulating bile acid metabolism; the regulating bile acid metabolism comprises reducing the level of 7-KDCA and thereby reducing the binding of 7-KDCA to farnesoid X receptor, thereby affecting the stability of farnesoid X receptor; The treatment of fatty liver disease includes reducing inflammation in patients with fatty liver disease, reducing liver weight in patients with fatty liver disease, reducing fat weight in patients with fatty liver disease, reducing liver weight / body weight ratio in patients with fatty liver disease, improving liver steatosis in patients with fatty liver disease, reducing lipid droplet accumulation in patients with fatty liver disease, improving serum indicators in patients with fatty liver disease, improving liver function indicators in patients with fatty liver disease, and improving intestinal flora in patients with fatty liver disease; The fatty liver disease is fatty liver disease associated with metabolic dysfunction.

2. The use according to claim 1, characterized in that The reducing inflammation of patients with fatty liver disease includes reducing inflammation of patients with fatty liver disease by regulating the TLR4 / NF-κB signaling pathway; the improving the intestinal flora of patients with fatty liver disease includes regulating the relative abundance of intestinal flora that produces short-chain fatty acids in the intestine of patients with fatty liver disease, regulating the relative abundance of intestinal flora that produces secondary bile acids in the intestine of patients with fatty liver disease and / or regulating the relative abundance of intestinal flora that contains bile salt hydrolase activity in the intestine of patients with fatty liver disease.

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