A Chinese medicinal probiotic composition for treating fatty liver and optimizing intestinal flora and its application

Abstract

The present invention provides a Chinese medicine probiotic composition for treating fatty liver and optimizing intestinal flora and its application, belonging to the field of biological medicine technology. The present invention provides a Chinese medicine probiotic composition, including a Chinese medicine composition and probiotics; the Chinese medicine composition includes the following components by weight: 28-32 parts of Lysimachia chinensis, 8-10 parts of Scutellaria baicalensis, 8-10 parts of Toosendan fruit, 8-10 parts of Aucklandia lappa and 5-7 parts of Rhubarb; the probiotics include Lactobacillus plantarum. The present invention combines Lactobacillus plantarum with the Chinese medicine composition, which is beneficial to drug absorption and can also enhance the efficacy of treating fatty liver and regulate the structural homeostasis of intestinal flora in patients with fatty liver.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological medicines, and specifically relates to a Chinese medicinal probiotic composition for treating fatty liver and optimizing intestinal flora and an application thereof. Background Art

[0002] With the change of lifestyle and the adjustment of dietary structure, the prevalence of fatty liver has increased year by year. It is reported that the prevalence of fatty liver in the global population is about 25%. Some studies have also pointed out that the increase in liver mortality is related to the increase in the prevalence of non-alcoholic fatty liver disease (NAFLD). Non-alcoholic fatty liver disease (NAFLD) is an acquired metabolic stress liver injury with hepatocyte fatty degeneration and fat accumulation as the main clinical features. Excessive fat deposition in the liver not only affects the normal function of the liver, but may also lead to a series of serious complications, such as cirrhosis and liver cancer. At present, the treatment of fatty liver mainly includes improving diet, increasing exercise, and drug therapy. Although improving diet and increasing exercise have certain effects on patients with mild fatty liver, it is often difficult to achieve ideal therapeutic effects for moderate and severe patients. Drug treatment mainly includes lipid-lowering drugs, antioxidant drugs, anti-inflammatory drugs, etc. These drugs can improve liver function to a certain extent, but long-term use may lead to side effects and drug dependence. Therefore, finding a safer and more effective treatment for fatty liver has become a hot topic in current research.

[0003] In recent years, with the in-depth study of the pathogenesis of fatty liver, more and more treatment methods have been proposed and applied in clinical practice, including gene therapy, stem cell therapy, immunotherapy, etc. Traditional Chinese medicine and probiotics, as two treatment methods with broad application prospects, have received widespread attention in the treatment of fatty liver in recent years. With its multi-component and multi-target characteristics, Chinese medicine has unique advantages in regulating human metabolism and improving liver function, and has also shown significant advantages in the treatment of obesity and fatty liver. Probiotics also show good effects on the treatment of fatty liver by regulating the balance of intestinal flora and affecting the host's metabolism and immune function.

[0004] Traditional Chinese medicine treatment emphasizes the overall concept and syndrome differentiation and treatment. Traditional Chinese medicine theory believes that the pathogenesis of gallstones and fatty liver is the following two points: one is liver qi stagnation and emotional depression; the other is unclean diet, intake of high-calorie, high-cholesterol foods, and both are mainly for treating "dampness". Therefore, the formula used to treat gallstones should also have a good effect in treating fatty liver in theory, that is, the principle of "different diseases treated with the same method" in traditional Chinese medicine theory. In recent years, with the deepening of modern research on traditional Chinese medicine, more and more traditional Chinese medicine ingredients and compound preparations have been found to have anti-obesity and anti-fatty liver effects, providing a scientific basis for the treatment of obesity and fatty liver with traditional Chinese medicine. For example, Lysimachia chinensis has the effect of relaxing gallbladder smooth muscle, promoting cellular cholesterol outflow, and improving intracellular lipid accumulation; Scutellaria baicalensis and others can resist oxidation and scavenge free radicals, chronically treat hepatitis, and resist liver fibrosis. Studies have shown that Chinese medicine can regulate the imbalance of intestinal flora, maintain the homeostasis of intestinal flora, and protect the intestinal mucosa. It can directly or indirectly regulate the imbalance of intestinal flora by supporting the number of intestinal probiotics and causing the intestinal translocation of probiotics, thereby inhibiting the reproduction of pathogenic bacteria. However, whether Chinese medicine regulates the flora to exert its therapeutic effect or the flora absorbs Chinese medicine to make it work still needs further research to prove.

[0005] Studies have shown that intestinal flora plays a key role in obesity and lipid metabolism. For example, both in vitro and in vivo experiments have confirmed that lactobacillus can reduce serum lipids, low-density lipoproteins and triglyceride levels. Studies have found that the intake of lactic acid bacteria can significantly reduce liver fat content and reduce liver inflammation and fibrosis, but its relief mechanism has not yet been elucidated and is strain specific. Bifidobacterium is a common probiotic that has the functions of regulating intestinal flora and enhancing immunity. It can also reduce the production of harmful substances in the intestine and reduce liver fat deposition, thereby improving fatty liver. The intestinal flora can regulate liver metabolism through the gut-liver axis, and there are a large number of studies that have explored the gut-liver axis mechanism in depth from the aspects of enzyme function and transcriptional regulation.

[20] However, due to the complex structure of the compounds in the metabolic process and the long metabolic pathways, there are still many gaps in this field, and the relationship between intestinal flora and liver metabolism still needs further experiments to explore. Summary of the invention

[0006] In view of this, the purpose of the present invention is to provide a Chinese herbal probiotic composition for treating fatty liver and optimizing intestinal flora. The Chinese herbal formula for treating gallstones is combined with Lactobacillus to prepare a Chinese herbal probiotic agent, which can effectively prevent and treat fatty liver and regulate the balance of intestinal flora.

[0007] The present invention provides a Chinese medicine probiotic composition, comprising a Chinese medicine composition and probiotics;

[0008] The Chinese medicine composition comprises the following components in parts by weight: 28-32 parts of Lysimachia chinensis, 8-10 parts of Scutellaria baicalensis, 8-10 parts of Toosendan fruit, 8-10 parts of Aucklandia lappa and 5-7 parts of Rhubarb;

[0009] The preferred selected bacteria include Lactobacillus plantarum (Lacium gold) 30 parts, Scutellaria baicalensis 9 parts, Toosendan fruit 9 parts, Aucklandia lappa 9 parts and Rheum officinale 6 parts.

[0010] Preferably, the Chinese medicine composition comprises a decoction;

[0011] The preparation method of the water decoction comprises the following steps:

[0012] Soak Herba Lysimachiae, Radix Scutellariae, Fructus Toosendan, Radix Aucklandiae and Rhubarb in water, then decoct and separate the decoction.

[0013] Preferably, the soaking time is 50 to 70 minutes;

[0014] During the soaking, the mass ratio of the total mass of the herb Lysimachia scabra, baicalinium bark, Toosendan fruit, costusroot and rhubarb to water is 1:(7-9).

[0015] Preferably, during the decoction, when the mixture of the Chinese medicine and water is boiled, water is added to continue the decoction, and the liquid phase is separated to obtain the Chinese medicine decoction;

[0016] The water supplement mass is 5 to 7 times the total mass of Lysimachia chinensis, Scutellaria baicalensis, Toosendan fruit, Aucklandia lappa and Rhubarb;

[0017] The time for continuing to decoct is 5 to 10 minutes.

[0018] Preferably, it comprises a decoction of traditional Chinese medicine containing Lactobacillus plantarum;

[0019] The live bacterial concentration of the plant lactobacillus is 1×10 5 ~1×10 10 CFU / mL;

[0020] Preferably, the Lactobacillus plantarum comprises Lactobacillus plantarum strain DM9125.

[0021] The present invention provides the use of the Chinese medicinal probiotic composition, the Chinese medicinal composition or Lactobacillus plantarum in the above scheme in preparing a medicine for preventing and / or treating fatty liver.

[0022] Preferably, the treatment of fatty liver is manifested in at least one of the following aspects: alleviating liver inflammation, improving liver status and regulating liver metabolism.

[0023] Preferably, the relief of liver inflammation includes inhibiting the expression of endotoxin LPS and inflammatory cytokines IL-1β, IL-6, and TNF-α;

[0024] The regulating liver metabolism includes regulating the expression levels of lipid synthesis-related molecules SREBP-1c, FAS, LXR, CD36 and PPARγ.

[0025] The present invention provides the use of the Chinese medicinal probiotic composition, the Chinese medicinal composition or Lactobacillus plantarum in the above scheme in preparing a medicine for preventing and / or treating intestinal damage in patients with fatty liver and / or regulating the balance of intestinal flora.

[0026] The present invention provides a Chinese medicine probiotic composition, comprising a Chinese medicine composition and probiotics; the Chinese medicine composition comprises the following components in parts by weight: 28-32 parts of Lysimachia chinensis, 8-10 parts of Scutellaria baicalensis, 8-10 parts of Toosendan fruit, 8-10 parts of Aucklandia lappa and 5-7 parts of Rhubarb; the probiotics include Lactobacillus plantarum. The Chinese medicine composition of the present invention is a stone-removing prescription for treating gallstones, and Lactobacillus plantarum is combined with the Chinese medicine composition, which is beneficial to drug absorption and enhances the efficacy of the Chinese medicine. The present invention uses a fatty liver mouse model as an experimental subject. Compared with the model group, the combined lactobacillus expelling stone prescription treatment group can improve the problem of enlarged liver of the model mouse, yellowish color and greasy texture, and significantly reduce liver weight; HE staining and oil red O staining results show that compared with the model group, the combined lactobacillus expelling stone prescription treatment group can improve the problems of different sizes of liver cells, disordered arrangement, smaller liver sinusoids, a large number of fat vacuoles in liver cell health care, and obvious inflammatory infiltration in the model mouse, and the NAS score is significantly reduced. It can be seen that the combined lactobacillus expelling stone prescription treatment group can improve the liver tissue morphology of the model mouse. At the same time, the combined lactobacillus expelling stone prescription treatment group can significantly reduce the concentration of TG and LDL-C in the liver, significantly reduce the concentration of serum TNF-α, and regulate the expression of genes related to fatty acid metabolism. It can be seen that the lactobacillus combined expelling stone prescription affects the synthesis of fatty acids, the metabolism of cholesterol, and the uptake of lipoproteins by inhibiting the expression levels of these three indicators, thereby improving the liver metabolism abnormalities caused by fatty liver, and then serving the purpose of treating fatty liver. In addition, the combined treatment group of Lactobacillus and Paishi Fang can improve the intestinal tissue morphology of fatty liver model mice to normal morphology, improve the intestinal mucosal damage of fatty liver mice, and significantly change the intestinal flora compared with the fatty liver model group, increasing the total number and abundance of species. It can be seen that Lactobacillus combined with Paishi Fang can also regulate the intestinal flora of patients with fatty liver, improve the intestinal barrier, and have the potential to alleviate fatty liver. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1The results of the changes in the amount of defecation in mice, N: normal control group; M: fatty liver group; C: Paishifang treatment group; P: Lactobacillus treatment group; CP: combined Lactobacillus Paishifang treatment group;

[0028] Figure 2 The changes in food intake of mice (mouse / g / w), N: normal control group; M: fatty liver group; C: Paishifang treatment group; P: Lactobacillus treatment group; CP: combined Lactobacillus Paishifang treatment group;

[0029] Figure 3 The results are the changes in the amount of water consumed by mice (mouse / ml / w), where N: normal control group; M: fatty liver group; C: Paishifang treatment group; P: Lactobacillus treatment group; CP: combined Lactobacillus Paishifang treatment group;

[0030] Figure 4 The results are the changes in the body weight of mice (g), N: normal control group; M: fatty liver group; C: Paishifang treatment group; P: Lactobacillus treatment group; CP: combined Lactobacillus Paishifang treatment group;

[0031] Figure 5 The results of the effects of the three prescriptions on the liver tissue morphology of mice, N: normal control group; M: fatty liver group; C: Paishi prescription treatment group; P: Lactobacillus treatment group; CP: combined Lactobacillus Paishi prescription treatment group;

[0032] Figure 6 The results of the effects of the three prescriptions on mouse liver tissue, A is liver weight; B is liver index; C is NAS score; D is the result of oil red quantitative analysis; N: normal control group; M: fatty liver group; C: Paishi prescription treatment group; P: Lactobacillus treatment group; CP: combined Lactobacillus Paishi prescription treatment group; * indicates P < 0.05; ** indicates P < 0.01; *** indicates P < 0.001; **** indicates P < 0.0001;

[0033] Figure 7 The results of the three prescriptions on the intestinal tissue morphology of mice, A is the HE staining result of the mouse colon and duodenum; B is the score result of the mouse colon injury index; N: normal control group; M: fatty liver group; C: Paishi prescription treatment group; P: Lactobacillus treatment group CP: combined Lactobacillus Paishi prescription treatment group; *** indicates P < 0.001; **** indicates P < 0.0001;

[0034] Figure 8 The results of fasting blood glucose changes in mice, N: normal control group; M: fatty liver group; C: Paishifang treatment group; P: Lactobacillus treatment group; CP: combined Lactobacillus Paishifang treatment group;

[0035] Fig. 9The results of changes in serum TC, liver tissue TC, serum TG, liver tissue TG, serum HDL-C, and serum LDL-C in mice, N: normal control group; M: fatty liver group; C: Paishifang treatment group; P: Lactobacillus treatment group; CP: combined Lactobacillus Paishifang treatment group; * indicates P < 0.05; ** indicates P < 0.01; *** indicates P < 0.001; **** indicates P < 0.0001;

[0036] Fig.10 The results of changes in serum LPS, IL-1β, IL-6, and TNF-α in mice, N: normal control group; M: fatty liver group; C: Paishifang treatment group; P: Lactobacillus treatment group; CP: combined Lactobacillus Paishifang treatment group; * indicates P < 0.05;

[0037] Fig.11 The results of the relative expression of SREBP-1c, FAS and LXR RNA in liver tissue of mice, N: normal control group; M: fatty liver group; C: Paishifang treatment group; P: Lactobacillus treatment group; CP: combined Lactobacillus Paishifang treatment group; * indicates P < 0.05; ** indicates P < 0.01; **** indicates P < 0.0001;

[0038] Fig.12 The results of the effect of relative expression of CD36, PPARα, and PPARγ RNA in mouse liver tissue, where N: normal control group; M: fatty liver group; C: Paishifang treatment group; P: lactobacillus treatment group; CP: combined lactobacillus Paishifang treatment group; ** indicates P < 0.01; **** indicates P < 0.0001;

[0039] Fig.13 The results of PCR-DGGE changes in the intestinal flora of mice in the normal control group (N) before and after modeling;

[0040] Fig.14 The results of PCR-DGGE changes in intestinal flora of mice in the normal control group (N) and fatty liver group (M);

[0041] Fig.15 The results of PCR-DGGE changes in intestinal flora of mice after intervention, N: normal control group; M: fatty liver group; C: Paishifang treatment group; P: Lactobacillus treatment group; CP: combined Lactobacillus Paishifang treatment group;

[0042] Fig.16 The results of OUT cluster analysis of intestinal flora of mice in each group after the three-prescription intervention, N: normal control group; M: fatty liver group; C: Paishi prescription treatment group; P: Lactobacillus treatment group;

[0043] Fig.17The PCoA analysis results of the intestinal flora of mice in each group after the three prescriptions were intervened. N: normal control group; M: fatty liver group; C: Paishi prescription treatment group; P: Lactobacillus treatment group;

[0044] Fig.18 The Venn analysis results of intestinal flora of mice in each group after the three prescriptions were intervened; N: normal control group; M: fatty liver group; C: Paishi prescription treatment group; P: Lactobacillus treatment group;

[0045] Fig.19 The results of the phylum-level analysis of the intestinal flora of mice in each group after the three-prescription intervention, A is the phylum-level community composition analysis; B is Firmicutes; C is Bacteroidota; N: normal control group; M: fatty liver group; C: Paishi prescription treatment group; P: Lactobacillus treatment group; * indicates P < 0.05; ** indicates P < 0.01; *** indicates P < 0.001;

[0046] Fig. 20 The results of the intestinal flora analysis of each group of mice after the three-prescription intervention, where A is the genus level community composition analysis; B is norank_f_Muribacμlaceae; C is Allobacμlum; N: normal control group; M: fatty liver group; C: Paishi prescription treatment group; P: Lactobacillus treatment group; ** indicates P < 0.01; *** indicates P < 0.001;

[0047] Fig.21 These are the results of Lefse analysis of intestinal flora in each group of mice after the three-prescription intervention, where N: normal control group; M: fatty liver group; C: Paishi prescription treatment group; P: Lactobacillus treatment group. DETAILED DESCRIPTION

[0048] The present invention provides a Chinese medicine probiotic composition, comprising a Chinese medicine composition and probiotics;

[0049] The Chinese medicine composition comprises the following components in parts by weight: 28-32 parts of Lysimachia chinensis, 8-10 parts of Scutellaria baicalensis, 8-10 parts of Toosendan fruit, 8-10 parts of Aucklandia lappa and 5-7 parts of Rhubarb;

[0050] The probiotics include Lactobacillus plantarum.

[0051] In the present invention, the Chinese medicine composition preferably includes the following components in parts by mass: 30 parts of Lysimachia chinensis, 9 parts of Scutellaria baicalensis, 9 parts of Toosendan fruit, 9 parts of Aucklandia lappa and 6 parts of Rhubarb.

[0052] In the present invention, the Chinese medicine composition preferably comprises a decoction. The preparation method of the decoction comprises the following steps: soaking Lysimachia chinensis, Scutellaria baicalensis, Toosendan fruit, Aucklandia lappa and Rhubarb in water, then decocting, and separating the decoction.

[0053] In the present invention, the soaking time is preferably 50 to 70 minutes, and more preferably 60 minutes. During the soaking, the mass ratio of the total mass of the Herba Lysimachiae, Baical Scutellariae, Fructus Toosendan, Aucklandiae Radix and Rhubarb to water is preferably 1: (7 to 9), and more preferably 1: 8. The soaking is conducive to the rapid release of the active ingredients in the Chinese medicinal materials. During the decoction, when the mixture of Chinese medicine and water is boiled to boiling, water is added to continue the decoction, and the liquid phase is separated to obtain a Chinese medicine decoction. The added mass of the water is preferably 5 to 7 times the total mass of the Herba Lysimachiae, Baical Scutellariae, Fructus Toosendan, Aucklandiae Radix and Rhubarb, and more preferably 8 times. The continued decoction time is preferably 5 to 10 minutes, and more preferably 8 minutes. The Chinese medicine probiotic composition preferably includes a Chinese medicine decoction containing Lactobacillus plantarum. The live bacteria concentration of Lactobacillus plantarum is 1×10 5 ~1×10 10 CFU / mL, more preferably 1×10 8 CFU / mL. The plant lactobacillus preferably includes the plant lactobacillus strain DM9125. The plant lactobacillus strain DM9125 has been disclosed in the prior art, specifically the prior art (Effects of single, double and triple probiotics on the recovery and immunity of intestinal flora imbalance mice, Gu Jie, Dalian Medical University, 2023, 6, Master's thesis) records plant lactobacillus (DM9125).

[0054] In the present invention, the preparation method of the Chinese medicine probiotic composition preferably comprises the following steps:

[0055] Prepare a Lactobacillus plantarum bacterial liquid, separate the bacterial bodies, and resuspend the bacterial bodies in the Chinese medicine decoction. The method for preparing the Lactobacillus plantarum bacterial liquid preferably inoculates the activated Lactobacillus plantarum in a selective liquid culture medium and cultures it for 14 hours. The selective liquid culture medium is preferably 200 ml / L tomato juice, 15 g / L peptone, 20 g / L glucose, 6 g / L yeast extract powder, 5 g / L sodium chloride, 1 ml / L Tween, and 0.5 g / L soluble starch. The culture temperature is preferably 36 to 38 ° C, more preferably 37 ° C. The culture time is preferably 12 to 15 hours, and most preferably 14 hours.

[0056] In the present invention, Lactobacillus plantarum can improve the intestinal flora and keep it in a steady state. The lipid-lowering effect of lactic acid bacteria alone is not obvious. The Chinese medicine composition is combined with Lactobacillus plantarum strain DM9125 to improve fatty liver while keeping the intestinal flora in a steady state, thereby further enhancing the efficacy of the Chinese medicine composition in preventing and treating fatty liver.

[0057] The present invention provides the use of the Chinese medicinal probiotic composition, the Chinese medicinal composition or Lactobacillus plantarum in the above scheme in preparing a medicine for preventing and / or treating fatty liver.

[0058] In the present invention, the treatment of fatty liver is preferably manifested in at least one of the following aspects: relieving liver inflammation, improving liver status and regulating liver metabolism. The relieving of liver inflammation preferably includes inhibiting the expression of endotoxin LPS and inflammatory cytokines IL-1β, IL-6, and TNF-α. The regulating liver metabolism includes regulating the expression levels of lipid synthesis-related molecules SREBP-1c, FAS, LXR, CD36, and PPARγ.

[0059] In an embodiment of the present invention, the effect on liver morphology, compared with the model group, the Chinese medicine probiotic composition and the Chinese medicine composition can significantly reduce liver weight, the Chinese medicine composition can significantly reduce liver index, plant lactobacillus, Chinese medicine probiotic composition and the Chinese medicine composition can significantly reduce NAS score and ORO value. For the influence of liver biochemical indexes, plant lactobacillus and Chinese medicine probiotic composition can significantly reduce TG concentration in liver, plant lactobacillus, Chinese medicine probiotic composition and the Chinese medicine composition can significantly reduce LDL-C concentration. For the influence of immune indexes, plant lactobacillus and Chinese medicine probiotic composition can significantly reduce the concentration of proinflammatory factor TNF-α. The treatment of fatty liver mice by Paishi Fang and Lactobacillus may be achieved by reducing oxidative stress or inhibiting the expression of inflammatory factors.

[0060] In the present invention, SREBP-1c (sterol regulatory elementbinding protein-1c) is an important target in the treatment of fatty liver. It is a transcription factor that plays a key regulatory role in lipid metabolism. In fatty liver, the expression level of SREBP-1c usually increases, and it mainly exacerbates the development of fatty liver by promoting the synthesis of fatty acids and triglycerides. SREBP-1c can also activate the transcription of enzymes such as acetyl-CoA carboxylase and fatty acid synthase (fatty acid synthase, FAS), thereby increasing the synthesis rate of fatty acids. FAS (fatty acid synthase) is a key enzyme in the fatty acid synthesis process, which catalyzes the continuous condensation reaction from acetyl-CoA to palmitate. Therefore, in the case of fatty liver, due to the activation of transcription factors such as SREBP-1c, the expression and activity of FAS usually increase, resulting in increased synthesis of fatty acids, further exacerbating the condition of fatty liver. LXR (liver X receptor) is a nuclear receptor that can regulate cholesterol and fatty acid metabolism by regulating SREBP-1c. After binding to the ligand, LXR can activate the transcription of a series of target genes, including cholesterol transport proteins ABCA1 and ABCG1, and fatty acid synthase FAS, etc. The present invention uses fluorescent quantitative PCR detection to show that compared with the fatty liver group, the relative expression of SREBP-1c, FAS, and LXR in the three-way treatment group all showed a downward trend, and the effect of the Paishifang treatment group was the most significant. This shows that Paishifang and Lactobacillus may affect the synthesis of fatty acids, the metabolism of cholesterol, and the uptake of lipoproteins by inhibiting the expression levels of these three indicators, thereby improving the liver metabolic abnormalities caused by fatty liver, and then achieving the purpose of treating fatty liver.

[0061] In the present invention, CD36, also known as fatty acid translocase, plays a key role in lipid metabolism and signal transduction. Abnormal increase of CD36 can increase the uptake of fatty acids and lipid accumulation in hepatocytes, thereby promoting the occurrence and development of fatty liver. Studies have shown that rhubarb can reduce insulin resistance and improve diabetes by regulating the PPARγ pathway. PPARγ, i.e. peroxisome proliferator-activated receptor γ, mainly regulates gene expression and glucose metabolism and the storage of fatty acids. Studies have shown that PPARγ-LXR is the core of regulating SREBP gene transcription and SREBP mRNA stability. In fatty liver, the activation of PPARγ may stimulate the differentiation and fat storage of adipocytes, thereby aggravating the condition of fatty liver. Therefore, a lower level of PPARγ expression in the liver may have certain benefits. The results of fluorescence quantitative PCR showed that compared with the fatty liver group, the expression of CD36 and PPARγ in the three-way treatment group showed a downward trend, and the Paishifang treatment group and the lactobacillus treatment group had better effects. This once again proves that Paishifang and lactobacillus can achieve the purpose of improving the metabolism of fatty liver in mice by reducing the uptake and storage of fatty acids.

[0062] The present invention provides the use of the Chinese medicinal probiotic composition, the Chinese medicinal composition or Lactobacillus plantarum in the above scheme in preparing a medicine for preventing and / or treating intestinal damage in patients with fatty liver and / or regulating the balance of intestinal flora.

[0063] In the present embodiment, after HE staining of the intestinal tissue of mice, it was found that the villi structure of the fatty liver group was of different lengths, the internal gaps became larger, and the muscle layer became thinner. This may be due to intestinal flora imbalance and abnormal lipid metabolism, which led to increased levels of inflammatory factors in the body and caused intestinal tissue damage. The intestinal tissue morphology of the three treatment groups was improved to a certain extent. In addition, changes in the intestinal flora structure will promote LPS entry into the blood by changing intestinal permeability, thereby increasing the level of inflammation.

[0064] In the present invention, the fecal flora of each group of mice was first analyzed by PCR-DGGE method, and then two groups with better intervention effects were selected based on the above experiments. The Paishifang group and the lactobacillus group were subjected to 16S rRNA high-throughput sequencing analysis of the fecal specimens of mice. We found that the intestinal flora of the Paishifang group and the lactobacillus group changed significantly compared with the fatty liver group, such as the increase in the total number and richness of species, the bacterial abundance of Firmicutes and Allobaculum decreased, and the bacterial abundance of Bacteroidota, Actinobacteriota, norank_f__Muribaculaceae, Faecalibaculum and Ileibacterium increased. It is pointed out in the literature that some bacteria in Firmicutes are related to energy harvesting and fat storage, and their increase in number may promote excessive absorption of energy and accumulation of fat, thereby aggravating the development of fatty liver. The increase in Allobaculum may be related to the destruction of intestinal barrier function and the release of inflammatory mediators, which may be involved in the pathological process of fatty liver. The metabolism of some bacteria in Bacteroidota is related to fat absorption, and their reduction may affect the host's energy intake and utilization, thereby affecting the synthesis and accumulation of fat. Some bacteria in Bacteroidota have anti-inflammatory effects, and their reduction may lead to a decrease in the anti-inflammatory ability of the intestine and the whole body. Some bacteria in Actinobacteriota also have the ability to regulate immune responses, and some bacteria may be involved in cholesterol metabolism. The reduction in their abundance may affect the balance of cholesterol, which is also an important factor in the development of fatty liver. Similar to the increase in Firmicutes and Allobaculum, the reduction in Bacteroidota and Actinobacteriota may be a sign of the development of fatty liver, and may also be directly involved in the occurrence and development of the disease. The research results of this example show that the Paishi prescription and Lactobacillus intervention treatment can regulate the intestinal flora of mice with fatty liver, thereby improving the intestinal barrier and having the potential effect of alleviating fatty liver.

[0065] The following is a detailed description of a Chinese medicinal probiotic composition for treating fatty liver and optimizing intestinal flora and its application provided by the present invention in conjunction with the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0066] Example 1

[0067] 1. Experimental Materials

[0068] 1.1 Experimental animals, drugs and strains

[0069] Experimental animals: 30 6-week-old male C57BL / 6J mice were purchased from Changsheng Biotechnology Co., Ltd.

[0070] Experimental medication: Modifications to the prescription for expelling stones from the emergency prescription for elbow.

[0071] Experimental strain: Lactobacillus plantarum subspecies (DM9125), a human strain with multiple metabolic activities isolated in 1991 by the laboratory of this example, is the experimental strain

[0072] 1.2 Experimental Reagents

[0073] Table 1 Main reagents

[0074]

[0075] 1.3 Experimental instruments

[0076] Table 2 Main instruments

[0077]

[0078]

[0079] 2. Experimental Methods

[0080] 2.1 Experimental Grouping

[0081] Thirty male C57BL / 6J mice were randomly divided into five groups, with six mice in each group, namely normal control group (N), fatty liver group (M), Paishifang treatment group (C), Lactobacillus treatment group (P), and combined Lactobacillus Paishifang treatment group (CP).

[0082] 2.2 Experimental treatment

[0083] All mice were adaptively fed for 3 days. Except for the normal control group (N), the other mice were fed with high-fat diet for 12 weeks. After the mice were confirmed to have established the model, the high-fat fed mice were randomly divided into fatty liver group (M), stone-removing prescription treatment group (C), lactobacillus treatment group (P) and combined lactobacillus stone-removing prescription treatment group (CP).

[0084] The normal control group (N) and fatty liver group (M) were gavaged with normal saline, the Paishifang treatment group (C) was gavaged with Paishifang decoction, the Lactobacillus treatment group (P) and the combined Lactobacillus Paishifang treatment group (CP) were gavaged with 1×10 9 CFU / head were gavaged with live bacterial suspension of Lactobacillus plantarum (DM9125) and live bacterial suspension of combined Lactobacillus plantarum (DM9125) and decoction of Shi-pai Fang, 0.2 ml per head per day, for 6 weeks.

[0085] 2.3 Preparation of Paishi Fang decoction

[0086] According to the ingredients in the emergency prescription for elbow, 63g of main ingredients such as Lysimachia chinensis and Rhubarb were taken and soaked in 8 times the volume of water for about 1 hour. Boil until boiling and turn off the heat. Add 6 times the volume of water and boil for another 5-10 minutes. Cool to room temperature, filter with sterile gauze, dilute to 80ml, and store in a 4℃ refrigerator for later use. (The Chinese medicinal materials for the Paishi prescription were purchased from Beijing Tongrentang)

[0087] 2.4 Preparation of selective culture medium for Lactobacillus plantarum (DM9125)

[0088] The main components of the selective liquid culture medium for Lactobacillus plantarum (DM9125) are: 200 ml of tomato juice, 15 g of peptone, 20 g of glucose, 6 g of yeast extract powder, 5 g of sodium chloride, 1 ml of Tween, and 0.5 g of soluble starch. Add 1000 ml of triple-distilled water and heat to dissolve. Adjust the pH to 6.0-7.2 and sterilize at 115° C. and 0.07 MPa for 15 minutes.

[0089] The main components of the plant lactobacillus (DM9125) selective solid culture plate are: 200ml tomato juice, 15g peptone, 20g glucose, 6g yeast extract powder, 5g sodium chloride, 1ml Tween, 0.5g soluble starch, 15g agar, add 1000ml triple distilled water and heat dissolve, adjust its pH to 6.0-7.2, autoclave at 115°C, 0.07MPa for 15 minutes, and cool to 50-60°C pouring plate.

[0090] 2.5 Colony count of Lactobacillus plantarum (DM9125)

[0091] Take 1 ml of the thoroughly mixed bacterial solution, dilute it in multiples, take a plant lactobacillus (DM9125) selective solid culture plate, divide each plate into 3 dilution gradients, take 10 μl of the diluted bacterial solution and drop it on the corresponding dilution gradient, repeat the drop plate for three times for each dilution gradient, place it in a 37°C incubator and culture it for 24 hours, then take out the culture plate, calculate the average number of colonies in three drops of the same dilution gradient, and select the dilution gradient with 30 to 300 colonies to calculate the bacterial content per milliliter.

[0092] 2.6 Preparation of live bacterial suspension of Lactobacillus plantarum (DM9125)

[0093] Pick a single colony of Lactobacillus plantarum (DM9125) and place it in a 50 ml system of Lactobacillus plantarum (DM9125) selective liquid culture medium for enrichment culture. After culturing at 37°C for 24 hours, take 1 ml of the enrichment solution and inoculate it in a 50 ml system of selective liquid culture medium. After culturing at 37°C for 14 hours, absorb the corresponding volume of enrichment solution, centrifuge at 6000g for 5 minutes, discard the supernatant and retain the bottom precipitate, wash with sterile saline for 3 times, and then dilute with sterile saline to a volume of 0.2 ml for each mouse. The bacterial solution is prepared on the day of use and stored in a 4°C refrigerator.

[0094] 2.7 Preparation of live bacterial suspension of Lactobacillus paishi prescription

[0095] After Lactobacillus plantarum (DM9125) was cultured in a selective liquid culture medium at 37°C for 14 hours, a corresponding volume of Lactobacillus plantarum (DM9125) enrichment solution was aspirated and centrifuged at 6000g for 5 minutes. The supernatant was discarded and the bottom precipitate was retained. The suspension was washed three times with sterile saline and then diluted with the Paishifang decoction to a volume of 0.2 ml for each mouse. The suspension was prepared on the day of use and stored in a refrigerator at 4°C.

[0096] 2.8 Collection of mouse feces

[0097] Collect the feces of mice once a week. Use sterile tweezers to collect the feces into a 1.5 ml sterile EP tube and place it on ice during collection. Collect at least 100 mg of feces from each mouse and freeze it in a -80℃ refrigerator.

[0098] 2.9 Mouse serum collection

[0099] The mice were killed by drawing blood from the eyeballs, and the whole blood was collected in a 1.5 ml sterile centrifuge tube, left to stand at room temperature for 2 hours, centrifuged at 3000 g for 20 minutes in a 4°C centrifuge, and the supernatant, i.e. serum, was collected and frozen in a -80°C refrigerator.

[0100] 2.10 Collection of mouse liver, ileum and duodenum tissues

[0101] During the dissection, the liver was photographed and weighed under the same light source and height. A small lobule of liver tissue was fixed in tissue fixative and stored in a 4°C refrigerator. The remaining liver tissue was placed in a 2.0 ml EP tube and frozen in a -80°C refrigerator. Clean segments of the colon and duodenum were picked, gently rinsed with pre-cooled PBS buffer, fixed in tissue fixative, and stored in a 4°C refrigerator.

[0102] 2.11 Measurement of mouse body weight, food intake, and water intake

[0103] The weight, food intake, and water intake of mice were observed and recorded every week, and the average weight, food intake, and water intake of each mouse per week were calculated.

[0104] 2.12 Fasting blood glucose test in mice

[0105] On the 7th day of each week, at 9:00 pm, the bedding was changed and the cages were cleaned. The mice were fasted but not watered. At 9:00 am the next day, the tails of the mice were disinfected with 75% alcohol, and the tail tips were cut off with sterile scissors after the alcohol was completely dry. The first drop of blood was discarded, and the whole blood glucose was tested with blood glucose test strips (glucose dehydrogenase method).

[0106] 2.13 HE staining and pathological scoring of mouse liver and intestinal tissues

[0107] 2.13.1 HE staining of mouse liver and intestinal tissues

[0108] The fixed liver and intestinal tissue samples were embedded in paraffin to make 4-6um tissue sections; the paraffin sections were placed in xylene to dissolve the wax, and then dewaxed in ethanol gradient, and the dewaxed sections were placed in a diluted hematoxylin stain solution for 2-10 minutes and then rinsed with distilled water to remove excess dye; the sections were acid-washed and placed in blue agent acid agarose for 1-5 minutes and rinsed with distilled water; the sections were placed in eosin dye solution for 1-5 minutes and then thoroughly rinsed with distilled water; the sections were dehydrated in an ethanol concentration gradient, and then immersed in a transparent agent to make them transparent and then sealed.

[0109] 2.13.2 Pathological scoring of mouse liver and intestinal tissues

[0110] The NAS scoring system is a semi-quantitative scoring system. It is used to perform pathological scoring on photos of mouse liver HE-stained sections taken under a 100x light microscope. The scoring criteria are as follows: (1) Hepatocyte fatty degeneration <5% is scored as 0 points, 5%-33% is scored as 1 point, 34%-66% is scored as 2 points, and >66% is scored as 3 points; (2) No intralobular inflammation is scored as 0 points, intralobular inflammation <2 is scored as 1 point, 2-4 is scored as 2 points, and >4 is scored as 3 points; (3) No hepatocyte ballooning is scored as 0 points, rare is scored as 1 point, and frequent is scored as 2 points. The sum of these three scores is 8 points at most. NAS ≥ 5 points can be diagnosed as non-alcoholic fatty hepatitis.

[0111] According to the Andújar scoring method, the pathological scoring of the HE-stained sections of the mouse colon was performed under a 100x light microscope. The scoring criteria are as follows: (1) no inflammation is scored as 0 points, mild inflammation is scored as 1 point, moderate inflammation is scored as 2 points, and moderate inflammation is scored as 3 points; (2) no lesions are scored as 0 points, lesions to the mucosa are scored as 1 point, lesions to the mucosa and submucosa are scored as 2 points, and lesions that have penetrated the intestinal wall are scored as 3 points; (3) no crypt destruction is scored as 0 points, 1 / 3 of the crypt cells are damaged as 1 point, 2 / 3 of the crypt cells are damaged as 2 points, crypt cells disappear but epithelial cells exist as 3 points, and both crypts and epithelial cells disappear as 4 points. The scores of these three parameters are added together to calculate the score, with a maximum score of 10.

[0112] 2.14 Oil Red O staining and quantitative analysis of mouse liver tissue

[0113] One day after the liver tissue was fixed, a piece of liver tissue was cut and dehydrated in a sucrose solution with a gradient concentration of 20% and 30%, respectively. After dehydration, the liver tissue was embedded in dry ice and sliced ​​using a freezing slicer with a slice thickness of 8 μm. The slices were slightly washed in distilled water and then immersed in 60% isopropanol for 20-30 seconds; the slices were placed in a modified oil red O staining solution and sealed for 10-15 minutes; the slices were slightly washed in 60% isopropanol and then slightly washed in distilled water; the nuclei were counterstained with Mayer's hematoxylin staining solution for 1-2 minutes and then slightly washed in distilled water, and the surrounding water was dried with filter paper and sealed with glycerol gelatin. According to the quantitative analysis method of Mehlem, the slice photos obtained under the 100x optical microscope were quantitatively analyzed.

[0114] 2.15ELISA method to detect the concentrations of LPS, IL-1β, IL-6 and TNF-α in mouse serum

[0115] ELISA kits were used to detect the concentrations of LPS, IL-1β, IL-6, and TNF-α in mouse serum. The specific operation steps were strictly carried out according to the instructions of the kits (the kits were purchased from Jiangsu ELISA Biological Co., Ltd.).

[0116] 2.16 Enzymatic detection of mouse serum TC, TG, HDL-C, and LDL-C concentrations

[0117] The order of adding samples for serum TC and TG measurement is: add 2.5μl dH2O to the blank well, add 2.5μl standard to the standard well, add μl serum sample to the sample well, then add 250μl working solution to each well, mix well, incubate at 37℃ for 10 minutes, and use an enzyme reader to measure the absorbance value of each well at a wavelength of 510nm. The sample content (mmol / L) is calculated according to formula I:

[0118]

[0119] The order of adding samples for serum measurement of HDL-C and LDL-C is as follows: add 2.5 μl dH2O to the blank well, add 2.5 μl standard to the standard well, add 2.5 μl serum sample to the sample well, then add 180 μl working solution 1 to each well, mix well, incubate at 37°C for 5 minutes, and use a microplate reader to measure the absorbance (OD) value of each well at a wavelength of 550 nm A1; add 60 μl working solution 2 to each well, mix well, incubate at 37°C for 5-10 minutes, and use a microplate reader to measure the absorbance (OD) value of each well at a wavelength of 550 nm A2.

[0120] The sample content (mmol / L) is calculated according to Formula II:

[0121]

[0122] Among them, ΔA=A2-A1.

[0123] 2.17 Enzymatic detection of TC and TG concentrations in mouse liver

[0124] Liver tissue TC and TG measurements are performed in advance by preparing liver tissue homogenate. The samples in this embodiment are high-fat samples, so the homogenate medium is uniformly extracted with anhydrous ethanol. Accurately weigh the tissue weight, according to the ratio of 1 (g): 9 (ml) of body weight, mechanically homogenize under ice bath conditions, centrifuge at 2500g for 10 minutes, and take the supernatant for testing. The order of adding samples to measure TC and TG in liver tissue is: add 2.5μl dH2O to the blank well, add 2.5μl of standard, add 2.5μl of liver tissue homogenate supernatant to the sample well, and then add 250μl of working solution to each well, mix well, incubate at 37°C for 10 minutes, and use an enzyme reader to measure the absorbance of each well at a wavelength of 510nm. The sample content (mmol / gprot) is calculated according to formula III:

[0125]

[0126] Among them, TC, TG, HDL-C, and LDL-C biochemical kits were purchased from Nanjing Jiancheng Bioengineering Institute.

[0127] 2.18 Analysis of mRNA levels in mouse liver tissue

[0128] 2.18.1 Extraction of RNA from mouse liver tissue

[0129] (1) After thawing the liver sample on ice, cut about 30 mg of the liver sample and place it in an EP tube. Add 1 ml of PBS, shake and wash the sample, and discard the PBS.

[0130] (2) After mincing the tissue with scissors, add 300 μl Trizol and homogenize it with a homogenizer until it is completely mixed. Then add 700 μl Trizol and mix it by inverting ten times. Let it stand on ice for 10 minutes.

[0131] (3) Add chloroform (0.2 ml / ml Trizol), mix by inversion for 30 seconds, and incubate on ice for 5 minutes.

[0132] (4) Centrifuge at 12000g and 4°C for 10 min and observe whether the liquid is separated into phases.

[0133] (5) Transfer the colorless upper layer to a new 1.5 ml EP tube, add an equal amount of isopropanol, invert and mix 10 times, and incubate on ice for 10 minutes.

[0134] (6) Centrifuge at 12000g and 4°C for 10 min, discard the supernatant and retain the white precipitate at the bottom.

[0135] (7) Add 1 ml of 75% ethanol prepared in sterile enzyme-free water and flick the solution until the precipitate floats.

[0136] (8) Centrifuge at 12000g and 4°C for 10 min to remove all alcohol.

[0137] (9) Dissolve the precipitate in 20 μl of sterile enzyme-free water.

[0138] (10) Determine the RNA concentration, reverse transcribe it into cDNA according to the kit steps, and store it in a -80°C refrigerator.

[0139] 2.18.2 Fluorescence quantitative PCR assay of mouse liver tissue cDNA

[0140] Prepare the PCR system of mouse liver tissue cDNA: SYBR 10μl, front primer 0.4μl, back primer 0.4μl, cDNA 2μl, ddH2O7.8μl. Add samples to the 96-well plate in sequence and set the qPCR reaction program according to Table 3. The primer sequences are shown in Table 4.

[0141] Table 3 qPCR reaction procedure

[0142]

[0143] Table 4 qPCR primer sequences

[0144]

[0145]

[0146] 2.19 Analysis of total DNA of intestinal flora in mouse feces

[0147] 2.19.1 Extraction of total DNA from intestinal flora in mouse feces

[0148] (1) Weigh 100 mg of collected feces and add it to a 2 mL centrifuge tube containing 200 mg of Glass Beads X. This step is performed on ice.

[0149] (2) Add 540 μl SLX-Mlus Buffer and vortex mix at maximum speed for about 10 minutes. Add 60 μl DS Buffer and 20 μl Proteinase K and vortex mix or shake manually.

[0150] (3) Incubate at 70°C for about 10 minutes (mix twice during this period), and incubate at 95°C for 5 minutes. Then add 200 μl of SP2 Buffer and mix for 30 seconds. Place on ice for 5 minutes, centrifuge, and transfer 400 μl of supernatant to a new 1.5 ml EP tube.

[0151] (4) Add 200 μl of cHTR Reagent stored at 4°C, mix for 10 seconds, incubate at room temperature for 2 minutes, centrifuge, and transfer 250 μl of the supernatant to a new 1.5 ml centrifuge tube.

[0152] (5) Add 250 μl BLBuffer and 250 μl anhydrous ethanol and mix well. Transfer all the liquid to the HiBind*DNAMini pink separation column and centrifuge at full speed for 1 minute, then discard the filtrate.

[0153] (6) Transfer the pink column to a new white tube, add 500 μl VHB Buffer and centrifuge at full speed for 30 seconds. Add 700 μl DNA Wash Buffer, centrifuge and discard the filtrate. Repeat this operation twice. Centrifuge at full speed for 2 minutes and transfer the pink column to a new 1.5 ml centrifuge tube again.

[0154] (7) After opening the lid and letting it stand, add 100 μl of Elution Buffer preheated at 65°C. Incubate at room temperature for 2 minutes, then centrifuge at full speed for 1 minute. Discard the powder column, aliquot, and freeze the extracted DNA sample in a -80°C refrigerator.

[0155] 2.19.2 PCR amplification of total DNA of intestinal flora in mouse feces

[0156] The PCR system configuration of the total DNA of the bacterial flora is shown in Table 5, and the primers are 338F (5'-ACTCCTACGGGAGGCAGACG-3', SEQ ID NO: 15) and 518R (5'-CGGACTACHVGGGTATCTTAT-3', SEQ ID NO: 16). The PCR program after the system is prepared is set as shown in Table 6. After the amplification is completed, the product is tested by agarose gel electrophoresis (110V, 20 minutes) to see if it is qualified, and the product is stored in a -20°C refrigerator.

[0157] Table 5 PCR system for total DNA of intestinal flora in feces

[0158]

[0159] Table 6 PCR reaction program

[0160]

[0161] 2.19.3 DGGE of PCR-amplified DNA of intestinal flora in mouse feces

[0162] (1) 0% solution formula: add 10mL 40% denaturing gradient concentration gel, 0.5mL 50×TAE, 1mL glycerol, and quantify to 50mL with deionized water. 100% solution formula: add 21.08g urea, 10mL 40% denaturing gradient concentration gel, 20mL Formid polyacrylamide, 1mL glycerol, 0.5mL 50×TAE, and quantify to 50mL with deionized water.

[0163] (3) Preparation of separation gel: 0% low concentration solution and 100% high concentration solution were used to prepare a denaturing gradient gel with a concentration of 30%-60%, and 6.6 μl TEMED and 33 μl APS were added at the same time.

[0164] (4) Concentrating gel formula: 3000 μL 0% concentration solution, 4.5 μL TEMED, 18 μL APS.

[0165] (5) After pouring the separation gel, continue to pour the concentration gel, insert the comb, and leave it at room temperature for more than 6 hours to allow the gel to solidify.

[0166] (6) Use a micro-spotter to draw up 30 μL of the PCR amplification product for sample loading, then place it in a water bath preheated at 60°C and filled with 1×TAE buffer. Turn on the electrophoresis device and perform electrophoresis at 150 V for 10 minutes, then at 80 V for 6 hours, and then remove the gel.

[0167] (7) EB staining for 20 minutes, rinse twice with deionized water, use UVI automatic gel imaging instrument to detect and photograph, and keep the sample for storage.

[0168] 2.19.4 High-throughput sequencing analysis of 16S rRNA gene of total DNA of intestinal flora in mouse feces

[0169] The 338F and 806R primers were used to amplify the gene fragments, and the products were sequenced by Majorbio. After sequencing, the species community composition of the samples was calculated by comparing with the database.

[0170] 2.20 Statistical analysis

[0171] SPSS27.0 software was used for statistical analysis, GraphPad for statistical drawing, Image J for quantitative analysis, and Quantity One for PCR-DGGE analysis. Statistical data were expressed as mean ± standard deviation. One-way analysis of variance was used for comparison between experimental groups, followed by pairwise comparison. P < 0.05 indicated that the data were statistically significant, and P < 0.01 indicated that the data difference was extremely significant.

[0172] result

[0173] 1. Effects of Paishi Fang, Lactobacillus Fang, and combined Lactobacillus Paishi Fang on the basic indicators of mice in each group

[0174] 1.1 Effects of the three prescriptions on the defecation volume of mice

[0175] At 6 weeks of intervention, one mouse was randomly selected from each group and its feces excreted within 1 hour were collected. Figure 1 As shown, compared with the fatty liver group (M), the stool volume of the Paishifang treatment group (C) increased; there was no significant change in the Lactobacillus treatment group (P); and the stool volume of the combined Lactobacillus Paishifang treatment group (CP) increased.

[0176] 1.2 Effects of the three prescriptions on food intake and water intake of mice

[0177] like Figure 2 As shown in the figure, compared with the fatty liver group (M), the food intake of mice in the Paishifang treatment group (C), the Lactobacillus treatment group (P), and the combined Lactobacillus Paishifang treatment group (CP) was always lower; after 3 weeks of three-way intervention, the food intake of mice in the Paishifang treatment group (C), the Lactobacillus treatment group (P), and the combined Lactobacillus Paishifang treatment group (CP) showed an upward trend, but there was no statistical difference. Compared with the normal control group (N), the fatty liver group (M) and the three-way intervention group were always lower.

[0178] like Figure 3 As shown in the figure, compared with the fatty liver group (M), the water intake of mice in the Paishifang treatment group (C), the lactobacillus treatment group (P), and the combined lactobacillus Paishifang treatment group (CP) was always lower and tended to be stable, with no statistical significance. Compared with the normal control group (N), the fatty liver group (M) and the three-way intervention group were always lower.

[0179] 1.3 Effects of the three prescriptions on the body weight of mice

[0180] like Figure 4 As shown in Table 7, compared with the fatty liver group (M), the body weights of mice in the Paishifang treatment group (C), the Lactobacillus treatment group (P), and the combined Lactobacillus Paishifang treatment group (CP) were all decreased; after 6 weeks of intervention, there was a significant difference between the combined Lactobacillus Paishifang treatment group (CP) and the fatty liver group (M) (P < 0.05); after 1 week of intervention, there was no statistical significance between the combined Lactobacillus Paishifang treatment group (CP) and the normal group (N); after 3 weeks of gavage intervention, the body weights of mice in the Paishifang treatment group (C), the Lactobacillus treatment group (P), and the combined Lactobacillus Paishifang treatment group (CP) were all reduced compared with the normal group (N).

[0181] Table 7 Changes in mouse body weight (g)

[0182]

[0183] * indicates a significant difference compared with group N; # indicates a significant difference compared with group M

[0184] * and # indicate P < 0.05; ** indicates P < 0.01; *** indicates P < 0.001; **** indicates P < 0.0001; N: normal control group; M: fatty liver group; C: Paishifang treatment group; P: Lactobacillus treatment group; CP: combined Lactobacillus Paishifang treatment group.

[0185] 2. Effects of Paishi Fang, Lactobacillus Fang and combined Lactobacillus Paishi Fang on the tissue morphology of mice in each group

[0186] 2.1 Effects of the three prescriptions on the morphology of mouse liver tissue

[0187] like Figure 5 As shown in the figure, the livers of mice in the fatty liver group (M) became significantly larger, yellowish in color, and more greasy in texture; the Paishifang treatment group (C), the Lactobacillus treatment group (P), and the combined Lactobacillus Paishifang treatment group (CP) all showed some improvement. Figure 6 As can be seen from A and B, compared with the fatty liver group (M), the liver weight of the lactobacillus treatment group (P) decreased significantly (P < 0.01); the liver weight of the combined lactobacillus stone-removing prescription treatment group (CP) decreased significantly (P < 0.05); and the liver index of the lactobacillus treatment group (P) also decreased significantly (P < 0.05). Compared with the normal control group (N), the liver weight of the fatty liver group (M) increased significantly (P < 0.001). Therefore, the liver was further observed by HE staining and Oil Red O staining.

[0188] like Figure 5 As shown in the figure, the liver cells of the normal control group (N) mice had clear boundaries and structures, neatly arranged liver cords, and no inflammatory cell infiltration and fat vacuoles; the liver cells of the fatty liver group (M) mice were of different sizes, disordered arrangement of liver cords, smaller liver sinusoids, a large number of fat vacuoles in the cytoplasm of liver cells, and obvious inflammatory infiltration; the Paishifang treatment group (C), the lactobacillus treatment group (P) and the combined lactobacillus Paishifang treatment group (CP) all showed a certain degree of improvement. All pathological sections of each group were scored by NAS, and the results are shown in the figure. Figure 6 As shown in C, compared with the fatty liver group (M), the score of the combined Lactobacillus Paishi Fang treatment group (CP) was significantly reduced (P < 0.001); the scores of the Paishi Fang treatment group (C) and the Lactobacillus treatment group (P) were significantly reduced (P < 0.05). Compared with the normal control group (N), the score of the fatty liver group (M) was significantly increased (P < 0.0001).

[0189] like Figure 5As shown in the figure, the normal control group (N) had almost no lipid droplet accumulation, the nucleus and cytoplasm were clear and tightly arranged, while the fatty liver group (M) had a large number of lipid droplets and a large number of fat vacuoles. The Paishifang treatment group (C), the lactobacillus treatment group (P) and the combined lactobacillus Paishifang treatment group (CP) all showed some improvement. The results obtained through quantitative analysis of the slices are shown in the figure. Figure 6 As shown in D, compared with the fatty liver group (M), the scores of the Paishifang treatment group (C) and the lactobacillus treatment group (P) were extremely significantly reduced (P < 0.01); the combined lactobacillus Paishifang treatment group (CP) was significantly reduced (P < 0.05). Compared with the normal control group (N), the score of the fatty liver group (M) was significantly increased (P < 0.001).

[0190] 2.2 Effects of the three prescriptions on the histological morphology of the colon and duodenum of mice

[0191] like Figure 7 As shown in A, the colon structure of the normal group (N) was intact and the intestinal villi were normal; the villous structure of the fatty liver group (M) was of varying lengths, the internal spaces of the colon became larger, and the muscular layer became thinner; the colon morphology of the Paishifang treatment group (C), the Lactobacillus treatment group (P) and the combined Lactobacillus Paishifang treatment group (CP) were all improved to a certain extent.

[0192] The duodenal villi of the normal group (N) were relatively intact, with neatly arranged villi; the villi of the fatty liver group (M) were broken and sparsely arranged, with fewer duodenal glands and thinner muscular layer; the submucosal layer and muscular layer of the Paishifang treatment group (C) and the lactobacillus treatment group (P) were restored, and the tips of the villi were only slightly broken; but the combined lactobacillus Paishifang treatment group (CP) was still severely broken.

[0193] The colon was scored for intestinal injury index, such as Figure 7 As shown in B, compared with the fatty liver group (M), the score of the Paishi Fang treatment group (C) was extremely significantly reduced (P < 0.0001), and the scores of the Lactobacillus treatment group (P) and the combined Lactobacillus Paishi Fang treatment group (CP) were significantly reduced (P < 0.001); compared with the normal group (N), the score of the fatty liver group (M) was significantly increased (P < 0.0001).

[0194] 3. Effects of Paishi prescription, Lactobacillus prescription and Paishi prescription combined with Lactobacillus on biochemical and immune indicators of mice in each group

[0195] 3.1 Effects of the three prescriptions on fasting blood glucose in mice

[0196] like Figure 8As shown in the figure, compared with the fatty liver group (M), the fasting blood glucose of the Paishifang treatment group (C) was always lower; the fasting blood glucose of the lactobacillus treatment group (P) was always higher; the fasting blood glucose of the combined lactobacillus Paishifang treatment group (CP) began to be higher than that of the fatty liver group (M) after 3 weeks of intervention; there was no statistical significance. Compared with the normal control group (N), the fasting blood glucose of the fatty liver group (M) was always higher; the fasting blood glucose of the Paishifang treatment group (C) was lower after 6 weeks of intervention, and there was no statistical significance.

[0197] 3.2 Effects of the three prescriptions on TC, TG, HDL-C, and LDL-C in serum and liver of mice

[0198] Depend on Fig. 9 As can be seen from A1, compared with the fatty liver group (M), the TC content in the serum of the Paishifang treatment group (C), the lactobacillus treatment group (P) and the combined lactobacillus Paishifang treatment group (CP) showed a downward trend, but there was no statistical difference; compared with the normal control group (N), the fatty liver group (M) showed an upward trend, but there was no statistical difference. Fig. 9 As can be seen from A2, compared with the fatty liver group (M), the TC concentrations in the livers of the Paishifang treatment group (C), the Lactobacillus treatment group (P) and the combined Lactobacillus Paishifang treatment group (CP) showed a downward trend, but there was no statistical difference; compared with the normal control group (N), the fatty liver group (M) showed a significant upward trend (P < 0.01).

[0199] Depend on Fig. 9 As can be seen from B1, compared with the fatty liver group (M), the TG concentration in the Paishi Fang treatment group (C) showed a significant downward trend (P < 0.05); the Lactobacillus treatment group (P) and the combined Lactobacillus Paishi Fang treatment group (CP) both showed a downward trend, but there was no statistical significance; compared with the normal control group (N), the fatty liver group (M) showed an upward trend, but there was no statistical significance. Fig. 9 As can be seen from B2, compared with the fatty liver group (M), the TG content in the liver of the Paishi Fang treatment group (C) and the combined Lactobacillus Paishi Fang treatment group (CP) showed a significant downward trend (P < 0.05); the Lactobacillus treatment group (P) showed a downward trend, but it was not statistically significant; compared with the normal group (N), the fatty liver group (M) had an extremely significant upward trend (P < 0.0001).

[0200] like Fig. 9 As shown in C, compared with the fatty liver group (M), the serum HDL-C in the Paishifang treatment group (C), the lactobacillus treatment group (P) and the combined lactobacillus treatment group (CP) all showed an upward trend, but there was no statistical significance; compared with the normal group (N), the fatty liver group (M) showed a downward trend, but there was no statistical significance. Fig. 9As shown in D, compared with the fatty liver group (M), the serum LDL-C concentration in the Paishi Fang treatment group (C) showed an extremely significant downward trend (P < 0.01), and the Lactobacillus treatment group (P) and the combined Lactobacillus Paishi Fang treatment group (CP) showed a significant downward trend (P < 0.05); compared with the normal control group (N), the fatty liver group (M) increased significantly (P < 0.001).

[0201] 3.2 Effects of the three prescriptions on serum LPS, IL-1β, IL-6, and TNF-α in mice

[0202] The LPS concentration in mouse serum was Fig.10 As shown in A, compared with the fatty liver group (M), the Paishi Fang treatment group (C) was significantly lower (P < 0.05), the Lactobacillus treatment group (P) and the combined Lactobacillus Paishi Fang treatment group (CP) showed a downward trend, which was not statistically significant; compared with the normal control group (N), the fatty liver group (M) showed an upward trend, which was not statistically significant. Fig.10 As shown in B and C, compared with the fatty liver group (M), the Paishifang treatment group (C), the lactobacillus treatment group (P) and the combined lactobacillus Paishifang treatment group (CP) all showed a downward trend, but there was no statistical significance; compared with the normal control group (N), the fatty liver group (M) showed an upward trend, but there was no statistical significance. Fig.10 As shown in D, compared with the fatty liver group (M), the Paishifang treatment group (C), the Lactobacillus treatment group (P) and the combined Lactobacillus Paishifang treatment group (CP) were significantly reduced (P < 0.05); compared with the normal group (N), the fatty liver group (M) showed an increasing trend, which was not statistically significant.

[0203] 4. Effects of Paishifang, Lactobacillus Fang and Paishifang combined with Lactobacillus on RNA expression in each group of mice

[0204] 4.1 Effects of the three prescriptions on SREBP-1c, FAS, and LXR in mouse liver tissue

[0205] like Fig.11 As shown in Figure A, the relative expression levels of SREBP-1cRNA in mouse liver showed that compared with the fatty liver group (M), the Paishifang treatment group (C), the Lactobacillus treatment group (P) and the combined Lactobacillus Paishifang treatment group (CP) all showed a significant downward trend (P < 0.0001); compared with the normal control group (N), the expression level in the fatty liver group (M) increased extremely significantly (P < 0.0001).

[0206] like Fig.11As shown in B, compared with the fatty liver group (M), the Paishifang treatment group (C), the Lactobacillus treatment group (P) and the combined Lactobacillus Paishifang treatment group (CP) all showed a downward trend, among which the Paishifang treatment group (C) and the Lactobacillus treatment group (P) showed a significant downward trend (P < 0.0001); compared with the normal group (N), the relative expression level of FAS in the liver of mice in the fatty liver group (M) was significantly increased (P < 0.0001).

[0207] like Fig.11 As shown in C, compared with the fatty liver group (M), the Paishifang treatment group (C), the Lactobacillus treatment group (P) and the combined Lactobacillus Paishifang treatment group (CP) all showed a downward trend, and the Paishifang treatment group (C) and the Lactobacillus treatment group (P) showed a significant downward trend (P < 0.05); compared with the normal group (N), the relative expression level of LXR in the liver of mice in the fatty liver group (M) was significantly increased (P < 0.01).

[0208] 4.2 Effects of the three prescriptions on CD36, PPARα, and PPARγ in mouse liver tissue

[0209] like Fig.12 As shown in A, compared with the fatty liver group (M), the relative expression levels of CD36 in the mouse livers of the Paishifang treatment group (C), the Lactobacillus treatment group (P) and the combined Lactobacillus Paishifang treatment group (CP) showed a downward trend, and the differences between the Paishifang treatment group (C) and the Lactobacillus treatment group (P) were extremely significant (P < 0.0001); compared with the normal group (N), the fatty liver group (M) showed an extremely significant upward trend (P < 0.0001).

[0210] like Fig.12 As shown in B, there was no significant trend for PPARα, which may be due to the fact that oral gavage intervention in mice did not affect this target. Fig.12 As shown in C, compared with the fatty liver group (M), the Paishifang treatment group (C), the Lactobacillus treatment group (P) and the combined Lactobacillus Paishifang treatment group (CP) all showed a downward trend, and the Paishifang treatment group (C) was significantly different from the fatty liver group (M) (P < 0.0001), and the Lactobacillus treatment group (P) was significantly different from the fatty liver group (M) (P < 0.01); compared with the normal group (N), the relative expression of PPARγ in the liver of mice in the fatty liver group (M) increased significantly (P < 0.0001).

[0211] 5. Effects of Paishifang, Lactobacillus Fang, and Paishifang combined with Lactobacillus on the intestinal flora of mice in each group

[0212] 5.1 Changes of intestinal flora of mice by PCR-DGGE under the three-way intervention

[0213] Fig.13The PCR-DGGE changes of intestinal flora of normal control group (N) mice before and after modeling. Fig.13 A1 and A2 in the middle are PCR-DGGE band diagrams of intestinal flora DNA of normal control group mice before and after modeling. It can be seen from the figure that the fecal DNA bands of control group mice increased after modeling; Fig.13 B1 and B2 show the results of UPGMA similarity cluster analysis. 1-4 are the control groups before modeling, and 5-8 are the control groups after modeling. The results show that the mice before and after modeling are in one cluster each, indicating that the intestinal flora of the control group mice changed before and after modeling. Fig.13 C1 and C2 show the results of principal component analysis. 1-4 are the control groups before modeling, and 5-8 are the control groups after modeling. It can be seen that the distance between the groups is relatively close. Therefore, the Shannon index and richness of mice were calculated based on the bands for further analysis. Fig.13 D and E in the middle are the Shannon index and richness of the control group mice before and after modeling, respectively. It can be seen that there are significant differences in the Shannon index and richness (P < 0.05).

[0214] Fig.14 The PCR-DGGE changes of intestinal flora in the normal control group (N) and the fatty liver group (M) after successful modeling. Fig.14 A1 and A2 in the middle are PCR-DGGE band diagrams of intestinal flora DNA of mice in the normal control group (N) and fatty liver group (M) after successful modeling. It can be seen from the figure that the fecal DNA bands of mice in the fatty liver group (M) were reduced after successful modeling; Fig.14 B1 and B2 show the results of UPGMA similarity cluster analysis. Fig.14 In B1, 1-4 are the normal control group (N), 5-8 are the fatty liver group (M), Fig.14 In B2, 1-4 are the normal control group (N), and 5-9 are the fatty liver group (M). The results show that the mice in the normal control group (N) and the fatty liver group (M) are in a cluster, indicating that the intestinal flora of mice changed after the model was successfully established; Fig.14 C1 and C2 show the results of principal component analysis. Fig.14 In C1, 1-4 are the normal control group (N), 5-8 are the fatty liver group (M), Fig.14 In C2, 1-4 is the normal control group (N), and 5-9 is the fatty liver group (M). It can be seen that the distance between the groups is relatively close. Therefore, the Shannon index and richness of the mice were calculated based on the bands for further analysis. Fig.14 D and E in the middle are the Shannon index and richness of the normal control group (N) and fatty liver group (M) mice after successful modeling. It can be seen that both the Shannon index and richness of the normal control group (N) are greater than those of the fatty liver group (M), but there is no statistical difference.

[0215] Fig.15 To investigate the changes in intestinal flora of mice in each group after 6 weeks of intervention, Fig.15 Middle A is the PCR-DGGE band diagram of the intestinal flora DNA of each group of mice after oral gavage intervention, from left to right are the normal control group (N), Paishi Fang treatment group (C), Lactobacillus treatment group (P), combined Lactobacillus Paishi Fang treatment group (CP) and fatty liver group (M). It can be seen from the figure that the fecal DNA bands of the experimental group mice were slightly reduced; Fig.15 B shows the results of UPGMA similarity cluster analysis. 1-3 is the normal control group (N), 4-6 is the Paishifang treatment group (C), 7-9 is the lactobacillus treatment group (P), 10-12 is the combined lactobacillus Paishifang treatment group (CP), and 13-15 is the fatty liver group (M). The results show that the normal control group (N) is clustered alone at 0.59, the Paishifang treatment group (C) is clustered alone at 0.81, and the lactobacillus treatment group (P) is clustered alone at 0.74. Although the combined lactobacillus Paishifang treatment group (CP) and the fatty liver group (M) are not clustered alone, they are all around 0.71. Fig.15 Figure C shows the results of principal component analysis. 1-3 are the normal group (N), 4-6 are the group treated with the Stone-Removing Formula (C), 7-9 are the group treated with Lactobacillus (P), 10-12 are the group treated with the combined Lactobacillus Stone-Removing Formula (CP), and 13-15 are the fatty liver group (M). It can be seen that the distances within each group are relatively close, but the distances between the three groups of samples, the Stone-Removing Formula (C), the Lactobacillus (P) and the fatty liver group (M), are also relatively close. Therefore, the Shannon index and richness of mice were calculated according to the bands for further analysis. It can be seen from Table 8 that after 6 weeks of intervention, compared with the normal control group (N), the Shannon index and richness of the fatty liver group (M) showed significant changes (P < 0.01), and the Shannon index and richness of the Lactobacillus treatment group (P) and the combined Lactobacillus stone-removing prescription treatment group (CP) were also significantly changed compared with the normal control group (N) (P < 0.01), but there was no statistical difference between the stone-removing prescription treatment group (C) and the normal group (N).

[0216] Table 8 Changes in Shannon index and richness of intestinal flora in each group of mice after 6 weeks of intervention

[0217]

[0218] *Indicates significant difference compared with group N.

[0219] * indicates P<0.05; ** indicates P<0.01; N: normal control group; M: fatty liver group; C: Paishifang treatment group; P: Lactobacillus treatment group; CP: combined Lactobacillus Paishifang treatment group.

[0220] 5.2 Changes in 16S rRNA gene sequencing results of mouse intestinal flora under the three-party intervention

[0221] 5.2.1 Analysis of Beta Diversity of Intestinal Microbiota

[0222] 5.2.1.1 Cluster analysis of intestinal flora OUT

[0223] like Fig.16 As shown, the normal control group (N) was clustered into one cluster, and the other groups were clustered into one cluster. In this cluster, the high-fat model group (M), the Paishifang treatment group (C), and the lactobacillus treatment group (P) were clustered separately.

[0224] 5.2.1.2 PCoA analysis of intestinal flora

[0225] like Fig.17 As shown, the Paishifang treatment group (C) and the Lactobacillus treatment group (P) were close to the fatty liver group (M), while these three groups were far away from the normal control group (N).

[0226] 5.2.2 Analysis of intestinal flora species composition

[0227] 5.2.2.1 Venn analysis of intestinal flora

[0228] like Fig.18 As shown, the number of OUTs in the fatty liver group (M) was 448, of which 42 were unique; the number of OUTs in the Paishifang treatment group (C) was 535, of which 98 were unique and 353 were overlapped with the fatty liver group (M); the number of OUTs in the Lactobacillus treatment group (P) was 462, of which 42 were unique and 344 were overlapped with the fatty liver group (M); the number of OUTs in the normal control group (N) was 863, of which 511 were unique.

[0229] 5.2.2.2 Changes in intestinal flora at different taxonomic levels

[0230] like Fig.19 As shown in Figure A, from the phylum level results, compared with the fatty liver group (M), the abundance of Firmicutes bacteria in the Paishifang treatment group (C) and the Lactobacillus treatment group (P) was reduced, and Fig.19 As shown in B, the abundance of Firmicutes bacteria in the Paishifang treatment group (C) was significantly reduced (P < 0.05), and Firmicutes was the dominant bacteria in this period; compared with the fatty liver group (M), the abundance of Bacteroidota and Actinobacteriota bacteria in the Paishifang treatment group (C) and the Lactobacillus treatment group (P) increased.

[0231] like Fig. 20As shown in A, the sequencing results at the subordinate level show that compared with the fatty liver group (M), the bacterial abundance of norank_f__Muribaculaceae, Faecalibaculum and Ileibacterium in the Paishifang treatment group (C) and the Lactobacillus treatment group (P) increased; Fig. 20 As can be seen from Figure C, compared with the fatty liver group (M), the abundance of Allobaculum bacteria in the Paishifang treatment group (C) and the Lactobacillus treatment group (P) was significantly reduced (P < 0.001).

[0232] 5.2.3 Lefse analysis of intestinal flora

[0233] The intestinal flora of mice was analyzed by Lefse to find the differential bacteria with LDA score ≥ 4 in each stage. The results are as follows Fig.21As shown: the characteristic differential bacteria in the fatty liver group (M) were p_Firmicutes, c_Bacilli, o_Erysipelotrichales, f_Erysipelotrichaceae, g_Allobaculum, g_Lachnoclostridium, o_Lactobacillales, f_Lactobacillaeae, g_Lactobacillus, g_Dubosiella, and o_Petostreptococcales-Tissierellales; the characteristic differential bacteria in the Paishifang treatment group (C) were g_lleibacterium, c_Coriobacteriia, o_Coriobacteriales, g_norank_f__Desulfovibrionaceae, p__Actinobacteriota, f_Desulfovibrionaceae, p_Desulfobacterota, and p_Proteo bacteria, c_Gammaproteobacteria, o_Desulfovibrionales, c_Desulfovibrionia, g_Coriobacteriaceae_UCG-002, f_Atopobiacea, o_Burkholderiales, g_Parasutterella, f_Sutterellaceae; the characteristic differential bacteria in the Lactobacillus treatment group (P) were g_Faecalibaculum, g_unclassified_f_Lachnospiracea, g_Bacteroides, f_Bacteroidaceae, g_norank_f__Eubacterium_coprostanoligenes_group, f_Eubacterium_coprostanoligenes_group, g_Blautia, g_norank_f__Erysipelotrichaceae. The specific differential bacteria in the normal control group (N) were f_Muribaculaceae, g_norank_f__Muribaculaceae, o_Bacteroidales, c_Bacteroidia, p_Bacteroidota, g_Lachnospiraceae_NK4A136_group, and g_Lachnospiraceae_UCG-010.

[0234] In this example, male C57BL / 6J mice with fatty liver were treated with the stone-removing prescription, lactobacillus intervention, and combined lactobacillus stone-removing prescription for 6 consecutive weeks, and the basic indicators of each group of mice were monitored; tissue sections were used to observe the histomorphological changes of the liver and intestines; enzyme method and ELISA method were used to detect the biochemical and immune indicators of each group of mice; fluorescent quantitative PCR method was used to detect the changes in RNA expression of liver metabolism-related indicators of each group of mice; PCR-DGGE method was used to preliminarily explore the changes in intestinal flora, and 16S rRNA high-throughput sequencing analysis was used to study the specific changes in intestinal flora of each group of mice. Finally, the effects of various intervention methods on liver metabolism and intestinal flora of mice with fatty liver were comprehensively analyzed.

[0235] The weight of mice was monitored weekly, and it was found that during the whole experiment, the weight of mice in each treatment group decreased compared with the fatty liver group. This shows that the three-way intervention treatment of Paishi Fang, Lactobacillus Fang and combined Lactobacillus Paishi Fang can improve the weight of mice, but there is no statistical difference between the Paishi Fang treatment group and the Lactobacillus treatment group and the fatty liver group, probably because the intervention time is slightly short. After Oil Red O staining and HE staining of the liver tissue of mice, it was found that the liver cords in the fatty liver group were disordered, there were a large number of fat vacuoles, and the inflammatory infiltration was obvious. This may be due to the excessive accumulation of fat in the liver cells, which damaged the liver cells and then triggered an inflammatory response, leading to changes in liver tissue morphology. After HE staining of the intestinal tissue of mice, it was found that the length of the villi structure in the fatty liver group was different, the internal gap became larger, and the muscle layer became thinner. This may be due to the imbalance of intestinal flora and abnormal lipid metabolism, which led to the increase of the level of inflammatory factors in the body and caused intestinal tissue damage. The liver and intestinal tissue morphology of the three-way treatment group was improved to a certain extent.

[0236] In this example, the fecal flora of each group of mice was first analyzed by PCR-DGGE method, and then two groups with better intervention effects were selected based on the above experiments, and 16S rRNA high-throughput sequencing analysis was performed on the fecal specimens of mice in the Paishifang group and the Lactobacillus group. We found that the intestinal flora of the Paishifang group and the Lactobacillus group changed significantly compared with the fatty liver group, such as the increase in the total number and richness of species, the bacterial abundance of Firmicutes and Allobaculum decreased, and the bacterial abundance of Bacteroidota, Actinobacteriota, norank_f__Muribaculaceae, Faecalibaculum, and Ileibacterium increased.

[0237] The research results of this example show that the Paishi prescription and Lactobacillus intervention treatment can regulate the intestinal flora of mice with fatty liver, thereby improving the intestinal barrier and having the potential effect of alleviating fatty liver.

[0238] In summary, the present embodiment is through Paishifang, lactobacillus, joint lactobacillus Paishifang to fatty liver mice intervention treatment after 6 weeks, combined with each group of mice basic index, histomorphological changes, immune index, liver tissue RNA relative expression and intestinal flora, the conclusion drawn is: Paishifang, lactobacillus, joint lactobacillus Paishifang treatment can effectively improve fatty liver mice liver, intestinal tissue state, can also be by suppressing the expression of inflammatory cytokines, down-regulate the expression of lipid synthesis related molecules, thereby alleviate fatty liver metabolic abnormalities, and comprehensively Paishifang treatment group effect is better. Paishifang, lactobacillus, joint lactobacillus Paishifang treatment can also regulate mouse intestinal flora, improve intestinal barrier, have the potential effect of alleviating fatty liver. Chinese medicine and probiotics are complex mechanisms in the treatment of fatty liver, and the present embodiment provides part of the theoretical basis and data support, and further experimental verification and clinical research are necessary.

[0239] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A Chinese medicinal probiotic composition, characterized in that: It is composed of a Chinese medicine composition and probiotics; The Chinese medicine composition is prepared from the following raw materials in parts by weight: 28-32 parts of Lysimachia chinensis, 8-10 parts of Scutellaria baicalensis, 8-10 parts of Toosendan fruit, 8-10 parts of Aucklandia lappa and 5-7 parts of Rhubarb; The probiotics are Lactobacillus plantarum ( Lactobacillus plantarum )DM9125.

2. The Chinese medicinal probiotic composition according to claim 1, characterized in that: The traditional Chinese medicine composition is prepared from the following raw materials in parts by weight: 30 parts of Lysimachia chinensis, 9 parts of Scutellaria baicalensis, 9 parts of Toosendan fruit, 9 parts of Aucklandia lappa and 6 parts of Rhubarb.

3. The Chinese medicinal probiotic composition according to claim 1, characterized in that: The Chinese medicine composition comprises a decoction; The preparation method of the water decoction comprises the following steps: Soak Herba Lysimachiae, Radix Scutellariae, Fructus Toosendan, Radix Aucklandiae and Rhubarb in water, then decoct and separate the decoction.

4. The Chinese medicinal probiotic composition according to claim 3, characterized in that: The soaking time is 50 to 70 minutes; During the soaking, the mass ratio of the total mass of the herb Lysimachia scabra, baicalensis scutellaria, Toosendan fruit, costusroot and rhubarb to water is 1:(7-9).

5. The Chinese medicinal probiotic composition according to claim 3 or 4, characterized in that: During the decoction, when the mixture of the Chinese medicine and water is boiled, water is added to continue the decoction, and the liquid phase is separated to obtain a Chinese medicine decoction; The mass of the water supplemented is 5 to 7 times the total mass of Lysimachia chinensis, Scutellaria baicalensis, Toosendan fruit, Aucklandia lappa and Rhubarb; The time for continuing to decoct is 5 to 10 minutes.

6. The Chinese medicinal probiotic composition according to claim 5, characterized in that: It is a decoction of traditional Chinese medicine containing Lactobacillus plantarum; The live bacterial concentration of the plant lactobacillus is 1×10 5 ~1×10 10 CFU / mL.

7. Use of the Chinese medicinal probiotic composition according to any one of claims 1 to 6 in the preparation of a medicament for preventing and / or treating fatty liver.

8. Use of the Chinese medicine probiotic composition according to any one of claims 1 to 5 in the preparation of a medicament for preventing and / or treating fatty liver.

9. The use according to claim 7 or 8, characterized in that: The treatment of fatty liver is manifested in at least one of the following aspects: alleviating liver inflammation, improving liver status and regulating liver metabolism.

10. The use according to claim 9, characterized in that: The relief of liver inflammation includes inhibiting the expression of endotoxin LPS and inflammatory cytokines IL-1β, IL-6, and TNF-α; The regulating liver metabolism includes regulating the expression levels of lipid synthesis-related molecules SREBP-1c, FAS, LXR, CD36, and PPARγ.

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