Application of Flavonifratorplautii combined Danyankang capsule in treatment of non-alcoholic fatty liver disease

Abstract

The invention provides an application of a Flavonifratorplatii combined Danyankang capsule in treatment of a non-alcoholic fatty liver disease, and belongs to the field of new application of a traditional Chinese medicine combined intestinal flora. The invention further provides a preparation method of the Flavonifratorplatii combined Danyankang capsule for treating the non-alcoholic fatty liver disease. The application proves that the weight, the liver weight and the white fat weight of an NAFLD mouse are obviously reduced by using the Flavonifratorplatii and the Danyankang capsule in a combined manner; the levels of ALT, AST, TG, TC, LDL-c, GLU, IL-6, TNF-alpha and MDA in serum of NAFLD mice are remarkably reduced, and the level of HDL-c is remarkably increased; the richness and diversity of intestinal flora of the NAFLD mouse are improved, so that the abundance of gate level, genus level and species level flora is close to that of a normal control group. The discovery provides a new treatment strategy for intervention and treatment of the non-alcoholic fatty liver disease.

Description

Technical Field

[0001] This invention belongs to the field of novel applications of intestinal flora in combination with traditional Chinese medicine, specifically involving Flavonifractor_plautii Application of combined cholecystitis-relieving capsules in the treatment of non-alcoholic fatty liver disease. Background Technology

[0002] Nonalcoholic fatty liver disease (NAFLD) is a clinicopathological syndrome caused by factors other than alcohol and other known liver-damaging agents. It is characterized by diffuse macrovesicular steatosis of hepatocellular carcinoma, lipid metabolism disorders, and inflammation induced by lipid peroxidation. It has now surpassed viral hepatitis to become the leading cause of liver disease worldwide. If left uncontrolled, NAFLD can progress to nonalcoholic steatohepatitis (NASH), fatty liver fibrosis, cirrhosis, and even liver cancer. NAFLD is also a major contributing factor to cardiovascular disease, diabetes, heart disease, and chronic kidney disease. With changing lifestyles and the prevalence of obesity globally, the incidence of NAFLD is rising annually, becoming a significant public health issue. According to statistics from relevant organizations in 2019, the number of people with NAFLD worldwide reached 190 million; the prevalence in my country reached 29%. Due to its high incidence and tendency to develop into other liver diseases, NAFLD has become a significant global public health problem, seriously affecting people's normal lives. Therefore, it has significant social implications and medical research value for the prevention and treatment of non-alcoholic fatty liver disease.

[0003] The pathogenesis and progression of NAFLD are complex and not yet fully elucidated. The mainstream academic view is that the pathogenesis of NAFLD is based on the "two-hit" hypothesis; although the "two-hit" hypothesis can explain part of the pathogenesis, it cannot fully explain the pathophysiology and clinical manifestations of NAFLD. In recent years, increasing evidence suggests that disruption of the gut-hepatic axis and microbial-derived metabolites drives the pathogenesis of NAFL / NASH. Disruptions to the gut-hepatic axis play a crucial role in the pathogenesis of NAFLD, mainly manifested in bacterial translocation, intestinal barrier disruption, changes in gut microbiota structure, and liver inflammatory responses, such as activation of inflammatory signaling pathways and changes in the composition of bacterial metabolites. Research data shows that a high-fat, high-sugar diet can induce a decrease in the variety of gut microbiota and an increase in the abundance of harmful bacteria, leading to gut microbiota dysbiosis. This further weakens the intestinal physical barrier and intestinal vascular barrier, promoting the entry of harmful bacterial metabolites such as lipopolysaccharide (LPS), ethanol, trimethylamine (TMA), and intestinal inflammatory factors (IL-6 and TNF-α) into the liver via the portal vein, exacerbating non-hepatic inflammation and lipid metabolism abnormalities.

[0004] Currently, there is a lack of specific treatments for NAFLD. Existing treatments mostly focus on lifestyle interventions or symptomatic treatments (such as lowering enzymes and lipids), and a combined treatment strategy that "targets the gut-liver axis" has not yet been formed. Summary of the Invention

[0005] The purpose of this invention is to provide Flavonifractor_plautii The application of combined cholecystitis-relieving capsules in the treatment of non-alcoholic fatty liver disease (NAFLD). Overcoming the shortcomings of existing NAFLD treatments, such as single-target therapy and neglect of gut microbiota regulation, this study provides a combined treatment regimen of "traditional Chinese medicine preparation + specific gut bacteria." By synergistically regulating liver inflammation and gut microbiota, it enhances the therapeutic effect of NAFLD.

[0006] This invention provides Flavonifractor_plautii The use of a pharmaceutical composition in the preparation of a medicament for improving non-alcoholic fatty liver disease, said pharmaceutical composition being made from raw materials including Lysimachia christinae, Rheum palmatum, Saxifraga stolonifera, Clematis armandii, Pteris vittata, Scutellaria baicalensis, Phellodendron chinense, and Andrographis paniculata.

[0007] Furthermore, the aforementioned Flavonifractor_plautii Its accession number is ATCC 49531, and it is held at the American Center for Type Culture Collection.

[0008] Furthermore, the aforementioned Flavonifractor_plautii The pharmaceutical composition is a freeze-dried bacterial powder; it is made from the following raw materials in the indicated weight ratios: 15-25 parts of Lysimachia christinae, 16-27 parts of Rheum palmatum, 14-28 parts of Saxifraga stolonifera, 15-26 parts of Clematis armandii, 16-25 parts of Pteris multifida, 17-28 parts of Scutellaria baicalensis, 15-26 parts of Phellodendron chinense, and 15-26 parts of Andrographis paniculata.

[0009] Furthermore, the pharmaceutical composition is in the form of capsules.

[0010] Furthermore, the drug is a drug for the prevention, treatment, or relief of non-alcoholic fatty liver disease.

[0011] Furthermore, the non-alcoholic fatty liver disease refers to non-alcoholic steatohepatitis, fatty liver fibrosis, cirrhosis, or liver cancer.

[0012] Furthermore, the drug is a drug that clears the accumulation of reactive oxygen species in hepatocytes, reduces lipid accumulation in the liver, inhibits the inflammatory response in the liver, and reduces the production of malondialdehyde.

[0013] Furthermore, the drug is a drug that improves the richness and diversity of the intestinal flora in mice with non-alcoholic fatty liver disease, and keeps the ratio of the number or abundance of Firmicutes to Bacteroidetes in the intestinal flora within a balanced and stable fluctuation range.

[0014] The present invention also provides a combination drug for the prevention and / or treatment of non-alcoholic fatty liver disease, said combination drug containing ingredients for simultaneous or separate administration. Flavonifractor_plautii And a pharmaceutical composition; said pharmaceutical composition is made from raw materials including Lysimachia christinae, Rheum palmatum, Saxifraga stolonifera, Clematis chinensis, Pteris vittata, Scutellaria baicalensis, Phellodendron chinense, and Andrographis paniculata; the combined drug contains Flavonifractor_plautii The proportion of the active pharmaceutical ingredient in the drug composition is (0.5~2)×10 9 CFU: (450~500) mg / kg.

[0015] Furthermore, in the combined medication Flavonifractor_plautii The proportion of active pharmaceutical ingredient in the pharmaceutical composition is 1×10 9 CFU: 480 mg / kg.

[0016] Furthermore, the pharmaceutical composition is in the form of capsules.

[0017] Furthermore, the pharmaceutical composition is Cholecystitis Relief Capsules.

[0018] Furthermore, in the combined medication Flavonifractor_plautii The ratio of the cholecystitis-relieving capsules to the total cholesterol-lowering capsules is (0.5~2)×10. 9 CFU: (100~200) mg / kg.

[0019] More preferably, in the combined medication Flavonifractor_plautii The ratio of cholecystitis-relieving capsules is 1×10 9 CFU: 150 mg / kg.

[0020] Furthermore, the combined medication is an oral formulation.

[0021] Furthermore, the aforementioned Flavonifractor_plautii The product is a freeze-dried bacterial powder; the Cholecystitis Capsules are made from raw materials including Lysimachia christinae, Rheum palmatum, Saxifraga stolonifera, Clematis armandii, Pteris vittata, Scutellaria baicalensis, Phellodendron chinense, and Andrographis paniculata.

[0022] Furthermore, the Cholecystitis Capsules are made from the following raw materials in the indicated weight ratios: 15-25 parts of Lysimachia christinae, 16-27 parts of Rheum palmatum, 14-28 parts of Saxifraga stolonifera, 15-26 parts of Clematis armandii, 16-25 parts of Pteris multifida, 17-28 parts of Scutellaria baicalensis, 15-26 parts of Phellodendron chinense, and 15-26 parts of Andrographis paniculata.

[0023] Furthermore, the drug is a drug for the prevention, treatment, or relief of non-alcoholic fatty liver disease.

[0024] This invention Flavonifractor_plautiiIn the combined treatment of NAFLD with Cholecystitis Relief Capsules, the non-alcoholic fatty liver disease includes non-alcoholic steatohepatitis, fatty liver fibrosis, cirrhosis, and even liver cancer, covering the entire course of NAFLD from early to late stages, and has a wide range of applications.

[0025] This invention Flavonifractor_plautii In the combined treatment of NAFLD with Cholecystitis Relief Capsules, the aforementioned Flavonifractor_plautii When combined with Cholecystitis Capsules, the treatment of NAFLD alleviates pathological liver damage (reduces hepatocellular steatosis and inflammatory infiltration), reduces liver-related indicators in serological tests: alanine aminotransferase (ALT), aspartate aminotransferase (AST), triglycerides (TG), total cholesterol (TC), malondialdehyde (MDA), serum glucose (GLU), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and low-density lipoprotein (LDL-C) levels, and increases high-density lipoprotein (HDL-C) levels.

[0026] This invention Flavonifractor_plautii In the combined treatment of NAFLD with Cholecystitis Relief Capsules, the aforementioned Flavonifractor_plautii In improving non-alcoholic fatty liver disease, the combined Cholecystitis Capsules clear the accumulation of reactive oxygen species in hepatocytes and reduce the accumulation of lipid peroxide products (MDA), thus exhibiting antioxidant capabilities.

[0027] This invention Flavonifractor_plautii In the combined treatment of NAFLD with Cholecystitis Relief Capsules, the aforementioned Flavonifractor_plautii Combined with Cholecystitis Capsules, the expression of lipid metabolism-related proteins in the liver of NAFLD mice induced by a high-fat, high-sugar diet can be regulated (the expression levels of lipid synthesis-related proteins such as fatty acid synthase-FASN, acetyl-CoA carboxylase α-ACACA, and stearoyl-CoA desaturase 1-SCD-1 are regulated), thereby improving liver lipid metabolism disorders and reducing liver fat accumulation at the molecular level.

[0028] This invention Flavonifractor_plautii In the combined treatment of NAFLD with Cholecystitis Relief Capsules, the aforementioned Flavonifractor_plautii Combined with Cholecystitis Relief Capsules, this formula can increase the richness and diversity of the intestinal flora in NAFLD mice, and increase the number of Firmicutes (Clostridium perfringens) in the intestinal flora. Firmicutes ) and Bacteroidetes ( Bacteroidetes The quantity or abundance ratio of ) remains within a balanced and stable fluctuation range.

[0029] Compared to existing treatment methods, the beneficial effects of this invention are as follows: 1) Therapeutic advantages: Compared with using Cholecystitis Capsules alone or Flavonifractor_plautiiThe combined regimen can significantly reduce liver fat content (TC, TG, LDL-c), liver function indicators (ALT, AST), serum inflammatory factors (IL-6, TNF-α), lipid peroxide accumulation (MDA), and alter the abundance and structure of gut microbiota (regulating F / B ratio) in NAFLD patients. 2) Safety advantages: Cholecystitis Relief Capsules are a marketed drug. Flavonifractor_plautii These are normal gut bacteria in humans, and the combined administration of these two has no obvious toxic side effects, making them suitable for a wide range of people. 3) Mechanism advantages: It covers multiple dimensions such as improving liver pathology, regulating lipid metabolism, anti-oxidation, and repairing gut microbiota. It can intervene in NAFLD from etiology to symptoms in an all-round way. It is the first to combine "traditional Chinese medicine for lowering lipids, lowering blood sugar, anti-inflammation, and anti-oxidation" with "gut microbiota regulation". It addresses the limitations of existing solutions by targeting the "multi-target and multi-mechanism" pathological characteristics of NAFLD.

[0030] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions or alterations can be made without departing from the basic technical concept of the present invention.

[0031] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following embodiments. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Attached Figure Description

[0032] Figure 1 Effects of FP bacteria and DYK alone or in combination on the general condition of mice. (A) Weekly weight monitoring results curve; (B) Observation of mouse body shape; (C) Observation of mouse liver.

[0033] Figure 2 Effects of FP bacteria and DYK alone or in combination on liver and liver function in NAFLD mice. (A) H&E staining of mouse liver (magnification, ×200); (B) Serum AST and ALT levels.

[0034] Figure 3 Effects of FP bacteria and DYK alone or in combination on inflammation and oxidative stress in NAFLD mice. (A) Serum IL-6 level analysis; (B) Serum TNF-α level; (C) Serum MDA level; (D) Immunohistochemical analysis of TNF-α in mouse liver.

[0035] Figure 4 Effects of FP bacteria and DYK alone or in combination on hepatic fat accumulation in NAFLD mice (magnification, ×200).

[0036] Figure 5 Effects of FP bacteria and DYK alone or in combination on serum biochemical parameters in NAFLD mice. (A) Statistical analysis of TG levels; (B) Statistical analysis of TC levels; (C) Statistical analysis of LDL-c levels; (D) Statistical analysis of HDL-c levels; (E) Statistical analysis of GLU levels.

[0037] Figure 6 Effects of FP bacteria and DYK alone or in combination on the expression of key proteins in the liver of NAFLD mice. (A) Western blot analysis of expression levels of AMPK, p-AMPK, ACACA, CPT-1, PGC-1, SCD-1, CD36, FASN and SCD-1; (B) and (C) statistical analysis of protein expression levels, with β-actin as an internal control.

[0038] Figure 7 Abundance ranking curve analysis of mouse gut microbiota.

[0039] Figure 8 Species accumulation curve.

[0040] Figure 9 Alpha diversity analysis of mouse gut microbiota. (A) Sobs index analysis; (B) Chao index analysis; (C) ace index analysis.

[0041] Figure 10 : Analysis of β-diversity of mouse gut microbiota. (A) PCA analysis; (B) PLS-DA analysis; (C) NMDS analysis.

[0042] Figure 11 : Phylogenetic composition of mouse gut microbiota. (A) Bar chart of species distribution; (B) Heatmap of species composition.

[0043] Figure 12 : The genus-level composition of the intestinal flora in mice. Detailed Implementation

[0044] The raw materials and equipment used in this invention are all known products, obtained by purchasing commercially available products.

[0045] The embodiments of the present invention employ Flavonifractor_plautii The freeze-dried bacterial powder purchased from Beijing Bio-Bio Biotechnology Co., Ltd. has the accession number ATCC 49531 and is deposited at the American Center for Type Culture Collection (ATCC).

[0046] The embodiments of this invention use commercially available Cholecystitis Capsules, which can be directly used to treat non-alcoholic fatty liver disease.

[0047] Example 1 Flavonifractor_plautiiExperimental validation of combined cholecystitis-relieving capsules in the treatment of NAFLD animal models 1. Experimental Materials 1.1 Materials Fifty SPF-grade C57BL / 6J male mice, 4 weeks old, weighing 20±2g, were purchased from the Model Animal Institute of Nanjing Medical College. The mice were fed a high-fat diet produced by Jiangsu Xiehe Pharmaceutical Biotechnology Co., Ltd., and sterilized tap water was provided for drinking. During the experiment, the animals had free access to food and water. The laboratory temperature was 21±2℃, and the humidity was 40%-70%. Cholecystitis Relief Capsules were purchased from the market and produced by Guizhou Baili Pharmaceutical Group Co., Ltd. The formula consisted of: 15-25 parts of Lysimachia christinae, 16-27 parts of Rheum palmatum, 14-28 parts of Saxifraga stolonifera, 15-26 parts of Clematis armandii, 16-25 parts of Pteris multifida, 17-28 parts of Scutellaria baicalensis, 15-26 parts of Phellodendron chinense, and 15-26 parts of Andrographis paniculata; each capsule contained 0.5g (equivalent to 1.6g of medicinal slices). Flavonifractor_plautii Freeze-dried bacterial powder purchased from Beijing Bio-Bio Biotechnology Co., Ltd.

[0048] 1.2 Instruments Benchtop centrifuge (Beckman), biosafety cabinet (ThermoScientific), constant temperature water bath (Grant), autoclave (Shenan), Milli-Q water purifier (BioGen), gel electrophoresis apparatus (BIO-RAD), Odyssey & CLX dual-color infrared imaging system (LI-COR Odyssey), multi-functional microplate reader (Biotek).

[0049] 2. Experimental Methods 2.1 Establishing the NAFLD model Four-week-old male C57BL / 6J mice were randomly divided into the CN group (normal diet) and the HFD group (high sugar and high fat diet) and fed for 8 weeks to establish a NAFLD mouse model. The weight of the mice was measured weekly.

[0050] 2.2 Grouped administration (1) Grouping: The HFD mice were randomly divided into the model group (M) and the cholecystitis capsule group (DYK) using a random number table method. Flavonifractor_plautii Group (FP), Cholecystitis Relief Capsules + Flavonifractor_plautii Group (DYK+FP).

[0051] (2) Administration: The dosage for each group was as follows: DYK group: 150 mg / kg / d; FP group: 1×10 9 CFU / animal / day; DYK+FP group: the dosage for each group in the DYK and FP groups. The intervention was followed by 12 weeks of drug administration, with fasting for 12 hours after the last dose.

[0052] 2.3 Handling of laboratory animals At the end of week 12, after the administration of the drug, the animals were fasted for 12 hours but allowed free access to water, and were weighed. Mice in each group were anesthetized with 10% chloral hydrate, euthanized by dislocation, and blood was collected from the orbital cavity. The serum was placed on ice for 2 hours, centrifuged at 3000 rpm for 15 minutes, and stored at -80°C for later use. Simultaneously, the livers of the mice were harvested, and the colon tissue and its contents were collected and immediately placed in a liquid nitrogen container for flash freezing and storage at -80°C.

[0053] 2.4 Detection Indicators 2.4.1 Liver tissue samples Partial liver samples from mice stored at -80℃ were sent to BGI Genomics for transcriptome sequencing analysis; partial liver samples from mice fixed with 4% paraformaldehyde were sent to Wuhan Saive Biotechnology Co., Ltd. for liver tissue pathological analysis (H&E staining and Oil Red O staining).

[0054] 2.4.2 Gut diversity analysis The collected cecal contents of each group of mice were sent to BGI Genomics Co., Ltd. in Shenzhen for sequencing.

[0055] 2.4.3 Western Blotting Experiment Mouse liver tissue homogenate was lysed using RIPA lysis buffer containing 1% PMSF and 1% phosphatase inhibitor, and protein content was determined using a BCA kit. Extracted proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to a membrane with 5% skim milk or 3% BSA for 2 h, and incubated overnight at 4°C with primary antibodies β-actin (AF7018, Affinity), Cpt-1 (ab234111, Abcam), Acaca (ab45174, Abcam), Fasn (DF6106, Affinity), ATP citrate lyase ([Acly]AF7832, Affinity), stearoyl-CoA desaturase-1 ([SCD-1]#2438, CST), CD36 (ab133625, Abcam), Ampkα (AF6423, Affinity), and p-Ampk (AF3423, Affinity). The secondary antibody was incubated at room temperature for 1 hour, then developed using an Odyssey imager, and the images were saved.

[0056] 2.4.4 Statistical Analysis Statistical analysis was performed using SPSS 20.0. Data are presented as mean ± standard deviation (Mean ± SD). Independent samples t-tests were used to compare two groups, and one-way ANOVA and Wilcoxon signed-rank tests were used to compare multiple groups. A p-value ≤ 0.05 was considered statistically significant. GraphPad Prism 8.0 and PowerPoint were used for plotting.

[0057] 3. Experimental Results 3.1. Effects of FP bacteria and DYK alone or in combination on body weight, liver weight, and fat weight in NAFLD mice NAFLD mice were treated with FP bacteria and DYK alone or in combination (FP+DYK). During treatment, the body weight of mice in each group was monitored, and the results are as follows: Figure 1 A showed that all treatment groups could inhibit the rate of weight gain in NAFLD mice, with the FP+DYK group showing the most significant inhibitory effect. Figure 1 Results B showed that mice in group CN had smooth, shiny fur with no sebum secretion, while mice in group M were significantly obese with sebum secretion at the hair roots. After treatment with FP bacteria and DYK alone or in combination, no obvious sebum was observed at the hair roots of the mice, and the obesity was alleviated.

[0058] The results of observation of mouse livers showed that ( Figure 1 In group C and CN, the livers of mice were reddish-brown, normal in size and shape, soft in texture, and smooth on the surface. In group M, the livers were significantly enlarged, yellowish-brown, with thickened, blunt edges, a tense capsule, and an oily, granular texture. The livers of the treatment groups were nearly identical in size and shape to those of the normal groups, reddish-brown in color, and smooth on the surface without any granular texture. The statistical results of the final body weight, liver weight, and white fat weight (Table 1) showed that, compared with group M, the treatment groups (DYK, FP, and FP+DYK) significantly reduced the body weight, liver weight, and white fat weight of NAFLD mice.

[0059] Table 1: Flavonifractor_plautii The effects of combined treatment with Cholecystitis Relief Capsules on body weight, liver weight, and white fat mass in patients with non-alcoholic fatty liver disease (NAFLD). ) Note: Compared with group CN, ### P < 0.001; compared with group M, ** P < 0.01, *** P < 0.001 3.2. Effects of FP bacteria and DYK alone or in combination on liver pathological damage and liver function in NAFLD mice H&E staining was performed on the liver tissue of mice, and the results are as follows: Figure 2As shown in Figure A, compared with group M, the hepatocytes in group DYK were round and neatly arranged, with almost no fat vacuoles and inflammatory infiltration, close to the normal group; the hepatocytes in group FP were swollen, but significantly reduced compared with group M, and fat vacuoles were present, but their number was also significantly reduced, and the damage to the liver tissue structure was improved; the cells in group FP+DYK had a very small number of vacuoles, but the number of vacuoles was significantly reduced compared with groups M and FP, the inflammatory infiltration and lipid accumulation of hepatocytes were improved, and the cells were evenly arranged with round nuclei located in the center of the cell.

[0060] Serum marker test results showed ( Figure 2 (B) Compared with the CN group, the serum ALT and AST levels in the M group were significantly increased. Compared with the M group, the ALT and AST levels in the treatment groups (DYK, FP, and FP+DYK) were significantly decreased, with the FP+DYK group showing a decrease close to that of the normal group.

[0061] 3.3. Effects of FP bacteria and DYK alone or in combination on inflammation and oxidative stress in NAFLD mice To investigate the effects of DYK, FP, and FP+DYK treatments on inflammatory factors and oxidative stress levels in NAFLD mice, serum levels of IL-6, TNF-α, and MDA were measured. Results are as follows: Figure 3 As shown in AC, compared with the CN group, the levels of IL-6, TNF-α, and MDA in the M group mice were significantly increased; compared with the M group, the above indicators were significantly downregulated in NAFLD mice after treatment with DYK and FP alone or in combination, indicating that the inflammatory and oxidative stress status of mice fed a high-sugar, high-fat diet was improved. Furthermore, liver immunohistochemical analysis ( Figure 3 D) showed that the expression level of TNF-α in the liver of mice in group M was significantly higher than that in group CN, while DYK, FP and FP+DYK treatments all significantly reduced the expression level of TNF-α in the liver of mice, with FP+DYK treatment being significantly better than DYK and FP.

[0062] 3.4. Effects of FP bacteria and DYK alone or in combination on blood lipids and liver lipid levels in NAFLD mice The defining characteristic of NAFLD is excessive fat accumulation within hepatocytes. This invention uses Oil Red O staining to observe fat accumulation in hepatocytes of different groups of mice. The results are as follows... Figure 4As shown, in the CN group, hepatocytes showed no obvious lipid droplet aggregation and appeared pale blue. In the M group, numerous Oil Red O-positive fat vacuoles were clearly observed in hepatocytes, appearing bright red. Compared with the M group, the DYK group showed a significant reduction in fat vacuoles, and the staining appeared pale blue. The staining of the FP group was similar to that of the M group, with more lipid droplets in hepatocytes. Compared with the M and FP groups, the FP+DYK group showed a significant reduction in lipid droplets, and the degree of hepatocyte steatosis and lesions was reduced. To detect the effects of FP bacteria and DYK alone or in combination on blood lipids and blood glucose in NAFLD mice, serum TC, TG, HDL-C, LDL-C, and GLU were measured in mice of different treatment groups. The results are as follows: Figure 5 As shown in the AE, compared with group M, serum TC, TG, LDL-C, and GLU levels were significantly decreased and HDL-C levels were significantly increased in NAFLD mice induced by a high-sugar, high-fat diet after treatment with FP bacteria and DYK alone or in combination. Figure 5 The results showed that, compared with the FP and DYK monotherapy groups, the M group, after treatment with the FP+DYK group, showed a significant increase in the serum HDL-C level in NAFLD mice induced by a high-sugar, high-fat diet, achieving a synergistic effect (1+1>2) and obtaining unexpected technical results.

[0063] 3.5. Key Protein Expression Analysis To investigate the effects of FP bacteria and DYK alone or in combination on the expression of key proteins in NAFLD mice, this invention used Western blotting to detect the expression levels of proteins related to fatty acid synthesis, uptake, and oxidation in hepatocytes of different groups of mice. The results are as follows: Figure 6 As shown in AC, compared with group M, the expression of ACACA, carnitine palmitoyltransferase 1 (CPT-1), SCD-1, FASN, and fatty acid transporter (CD36) proteins in mouse hepatocytes were significantly decreased in the DYK, FP, and FP+DYK groups, while the expression of phosphorylated adenosine monophosphate activated protein kinase / adenosine monophosphate activated protein kinase (p-AMPK / AMPK) and peroxisome proliferator-activated receptor gamma coactivator 1α (PGC-1α) proteins were increased. However, although the p-AMPK / AMPK ratio in the FP+DYK group was higher than that in the M group, there was no significant difference between the two groups. Furthermore, compared with the drug monotherapy groups, the FP+DYK group did not show a significant synergistic effect in regulating the expression of key proteins.

[0064] 3.6.16S rRNA sequencing analysis of gut microbiota information 3.6.1.OUT Rank-Abundance Curve To further investigate the effects of FP bacteria and DYK alone or in combination on the gut microbiota of NAFLD mice, cecal contents were collected for 16S rRNA sequencing analysis of gut microbiota information. OUTRank-Abundance curves were used to classify and quantify the microbial community, analyzing species richness and evenness. Results are as follows: Figure 7 As shown, comparing the trends of the curves in each group, the CN group curve is at the top of all curves, with the gentlest downward trend; the M group curve is at the bottom and shows the most dramatic decline; in contrast, the curves of the DYK, FP, and FP+DYK groups are between the CN and M groups, with the FP+DYK group curve trend and height being very similar to the CN group. This indicates that the M group mice had the lowest abundance and evenness of gut microbiota, and that drug intervention could increase the abundance of gut microbiota in NAFLD mice, with FP+DYK intervention showing a more significant increase in microbiota abundance.

[0065] 3.6.2. Species Accumulation Curve Species accumulation curves are used to describe the trend of the number of species observed in different samples increasing with sample size, and are thus used to estimate species diversity. The results are as follows: Figure 8 As shown, the species accumulation curve exhibits an initial sharp rise followed by a gradual flattening, indicating that the number of newly observed species gradually decreases as the sample size increases. This demonstrates the high level of diversity in the sample and that the sampling was sufficiently comprehensive.

[0066] 3.6.3. α-Diversity Analysis Next, the Sobs index, reflecting the number of different species OTUs observed in the sample, is used to describe the species richness of the sample. The Chao and Ace indices are then used to estimate the number of unobserved species, i.e., latent species richness, thus providing a more comprehensive assessment of biodiversity. Figure 9 AC analysis showed that, compared with the M group, the DYK group, FP group, and FP+DYK group exhibited significantly increased sobs, chao, and ace indices, with the FP+DYK group showing a more significant increase. This indicates that treatment with FP bacteria and DYK alone or in combination increased the richness and diversity of the gut microbiota in NAFLD mice, with the combination treatment group showing a more significant effect.

[0067] 3.6.4. β-diversity analysis First, principal component analysis (PCA) was used to observe the clustering and dispersion of samples to assess the variability within each group and the differences in microbial community changes between groups. Then, partial least squares discriminant analysis (PLS-DA) was used to further understand the distribution characteristics of different microbial communities in the samples. Finally, nonmetric multidimensional scaling (NMDS) was used to visualize the dissimilarity between samples to better understand the differences and similarities between different samples. Figure 10As shown in Figure A, the samples within the CN group were relatively dispersed, indicating high variability in the CN group. In contrast, the samples within the M, DYK, FP, and FP+DYK groups showed high clustering, indicating low variability and high intra-group reproducibility in the mouse gut microbiota. The distances between the M, DYK, FP, and FP+DYK groups and the CN group were large, while the distance between the M group and the FP group was small, but the distances between the M group and the DYK and FP+DYK groups were large, suggesting that DYK alone or in combination with FP is more effective than FP alone in changing the gut microbiota in NAFLD mice. The PLS-DA analysis results are as follows. Figure 10 B shows that the samples within groups are highly clustered, while the samples between groups are completely discrete. NMDS analysis results show ( Figure 10 (C) The high dispersion of samples within the CN group indicates significant differences and low similarity among samples within the CN group. Samples within the M, DYK, FP, and FP+DYK groups showed high clustering, indicating small differences and high similarity among samples within each group. Furthermore, the DYK and FP+DYK groups were completely discrete from the M group, indicating large differences and low similarity between them. These results suggest that treatment with FP or DYK alone or in combination improves gut microbiota structure in NAFLD mice, with the combination group showing better results.

[0068] 3.6.5. Colony Composition Analysis Differences in gut microbiota composition were analyzed at the phylum and genus levels. The phylum-level distribution of mouse gut microbiota was as follows: Figure 11 As shown in Figure A, at the level of the door... Firmicutes and Bacteroidetes All of these were the dominant bacterial groups in their respective groups. Compared with the CN group, the F / B ratio of the M group was higher; compared with the M group, the F / B ratio of the DYK group and the FP+DYK group was lower, while that of the FP group was higher. Figure 11 B is a heatmap showing the differences in species composition among samples. At the genus level, the dominant genera in the FP+DYK group accounted for a larger proportion. Figure 12 The above results indicate that after treatment with FP bacteria and DYK alone or in combination, the structure and composition of the intestinal flora in mice were closer to those in the CN group, suggesting that treatment with FP, DYK alone or in combination with DYK can restore the phylum-level composition and structure of the intestinal flora in NAFLD mice.

[0069] In summary, this invention provides Flavonifractor_plautii The application of combined cholecystitis-relieving capsules in the treatment of non-alcoholic fatty liver disease. This invention demonstrates... Flavonifractor_plautiiThe combined use of Cholecystitis Relief Capsules significantly reduced body weight, liver weight, and white fat weight in NAFLD mice; significantly decreased serum levels of ALT, AST, TG, TC, LDL-c, GLU, IL-6, TNF-α, and MDA in NAFLD mice, and significantly increased HDL-c levels; and improved the richness and diversity of gut microbiota in NAFLD mice, bringing the abundance of microbiota at the phylum, genus, and species levels close to that of the normal control group. These findings provide a new treatment strategy for the intervention and treatment of non-alcoholic fatty liver disease.

Claims

1. Flavonifractor_plautii The use of the combined pharmaceutical composition in the preparation of a medicament for improving non-alcoholic fatty liver disease is characterized by, The pharmaceutical composition is made from raw materials including Lysimachia christinae, Rheum palmatum, Saxifraga stolonifera, Clematis armandii, Pteris vittata, Scutellaria baicalensis, Phellodendron chinense, and Andrographis paniculata.

2. The application according to claim 1, characterized in that, The Flavonifractor_plautii Its accession number is ATCC 49531, and it is held at the American Center for Type Culture Collection.

3. The application according to claim 1, characterized in that, The Flavonifractor_plautii The pharmaceutical composition is a freeze-dried bacterial powder; it is made from the following raw materials in the indicated weight ratios: 15-25 parts of Lysimachia christinae, 16-27 parts of Rheum palmatum, 14-28 parts of Saxifraga stolonifera, 15-26 parts of Clematis armandii, 16-25 parts of Pteris multifida, 17-28 parts of Scutellaria baicalensis, 15-26 parts of Phellodendron chinense, and 15-26 parts of Andrographis paniculata.

4. The application according to claim 3, characterized in that, The pharmaceutical composition is in the form of capsules.

5. The application according to claim 1, characterized in that, The drug is used to prevent, treat, or alleviate non-alcoholic fatty liver disease.

6. The application according to claim 5, characterized in that, The non-alcoholic fatty liver disease refers to non-alcoholic steatohepatitis, fatty liver fibrosis, cirrhosis, or liver cancer.

7. The application according to claim 1, characterized in that, The drug is used to clear the accumulation of reactive oxygen species in hepatocytes, reduce lipid accumulation in the liver, inhibit liver inflammation, and reduce malondialdehyde production.

8. The application according to claim 1, characterized in that, The drug is designed to increase the richness and diversity of the gut microbiota in mice with non-alcoholic fatty liver disease, and to maintain the ratio of Firmicutes to Bacteroidetes in the gut microbiota within a stable and balanced range.

9. A combination drug for the prevention and / or treatment of non-alcoholic fatty liver disease, characterized in that, The combined medication contains ingredients for simultaneous or separate administration. Flavonifractor_plautii And a pharmaceutical composition; said pharmaceutical composition is made from raw materials including Lysimachia christinae, Rheum palmatum, Saxifraga stolonifera, Clematis chinensis, Pteris vittata, Scutellaria baicalensis, Phellodendron chinense, and Andrographis paniculata; the combined drug contains Flavonifractor_plautii The proportion of the active pharmaceutical ingredient in the drug composition is (0.5~2)×10 9 CFU: (450~500) mg / kg.

10. The combination drug according to claim 9, characterized in that, The combined medications are oral formulations.

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