A strain of Faecalibacterium prausnitzii capable of improving metabolism-related fatty hepatitis and its application

Abstract

The present application relates to the field of biotechnology, and specifically discloses a strain of Faecalibacterium prausnitzii and its application that can improve metabolism-related fatty hepatitis. The Faecalibacterium prausnitziiGZSY3214 is deposited in the Guangdong Provincial Microbiological Culture Collection Center on August 7, 2024, with a deposit number of GDMCC No: 64992. After treatment with the above-mentioned Faecalibacterium prausnitzii, it has a good therapeutic effect in improving the progression of metabolic-related fatty hepatitis, and has a significant inhibitory effect on the NF‑κB and MAPK signaling pathways involved in the inflammatory response of the liver; among them, the strain can also improve liver function and lipid metabolism, downregulate the levels of ALT, AST, TG and LDL, and inhibit the expression of inflammation-related inflammatory cytokines and chemokines, and significantly inhibit the expression of collagen and transforming growth factor-β related to liver fibrosis.

Description

Technical Field

[0001] The present application relates to the field of biotechnology, and in particular to a strain of Faecalibacterium prausnitzii capable of improving metabolism-related fatty hepatitis and its application. Background Art

[0002] Metabolic-associated fatty liver disease (MAFLD) is a multi-system metabolic dysfunction disease that mainly affects the liver and is closely related to obesity, metabolic disorders, and type 2 diabetes. In recent years, the prevalence of MAFLD has increased year by year due to the trend of fast-food eating habits, the prevalence of high-fat, high-sugar, high-calorie diets, and sedentary lifestyles. MAFLD is mainly characterized by fat deposition in the liver and diffuse macrovesicular fatty degeneration of hepatocytes. Accompanied by inflammatory response and damage repair, it can further develop into metabolic-associated steatohepatitis (MASH), and even cirrhosis and liver cancer. It can also lead to cardiovascular and cerebrovascular diseases, chronic kidney disease, etc. At present, there is still a lack of specific drugs for the treatment of MAFLD in clinical practice.

[0003] Faecalibacterium prausnitzii (F. prausnitzii) is a Gram-negative bacterium of the Faecalibacterium prausnitzii group, belonging to the family Clostridiaceae and the phylum Firmicutes. Faecalibacterium prausnitzii is one of the most important bacteria in the human intestinal flora, accounting for 5-15% of the total bacteria detected in fecal samples of healthy people. Studies have shown that Faecalibacterium prausnitzii is one of the main butyrate producers in the intestine. Butyrate can participate in the enhancement of the colon barrier by increasing the synthesis of tight junction proteins and the production of antimicrobial peptides; butyrate plays an anti-tumor role by reducing the secretion of tumor necrosis factor (TNF) in intestinal epithelial cells and inducing differentiation and apoptosis of tumor cells. However, the therapeutic effect of Faecalibacterium prausnitzii in metabolic-related fatty liver disease or metabolic-related fatty hepatitis is still unclear. Summary of the invention

[0004] The purpose of the present application is to overcome the deficiencies of the above-mentioned prior art and to provide a strain of Faecalibacterium prausnitzii that can improve metabolism-related fatty hepatitis and its application.

[0005] To achieve the above purpose, the technical solution adopted by this application is:

[0006] The present application provides a strain of Faecalibacterium prausnitzii that can improve metabolic-related fatty liver disease or metabolic-related fatty hepatitis. The Faecalibacterium prausnitzii is Faecalibacterium prausnitziiGZSY3214, which has been deposited in the Guangdong Provincial Microbiological Culture Collection Center on August 7, 2024, with a deposit number of GDMCC No: 64992, and a classification name of Faecalibacterium prausnitzii. The deposit address is: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0007] The colony morphology of Faecalibacterium prausnitziiGZSY3214 is: about 1.5 to 3 microns in length, about 0.5 to 0.8 microns in width, arranged in single cells or pairs, non-spore-producing, non-flagellated, non-motile bacilli, and showing convex, translucent, complete-edge and moist colonies in solid culture medium. The morphological characteristics in liquid culture medium show a series of cell morphologies from spherical, pleomorphic spherules to rods.

[0008] The physiological and biochemical characteristics of Faecalibacterium prausnitziiGZSY3214 are: Gram-negative, strictly anaerobic, extremely sensitive to oxygen, and the dominant bacteria of the Clostridium group, belonging to the family Clostridiaceae, the genus Clostridium of the phylum Firmicutes. Faecalibacterium prausnitziiGZSY3214 produces a large amount of formate, butyrate and a small amount of D-lactate through glucose fermentation.

[0009] The Faecalibacterium prausnitziiGZSY3214 is involved in multiple metabolic pathways such as sugar metabolism, amino acid and vitamin metabolism, and the production of short-chain fatty acids. Its metabolites mainly include butyrate and 15KD microbial anti-inflammatory molecule (MAM). The Faecalibacterium prausnitziiGZSY3214 can only survive stably in an anaerobic environment, and the optimal growth pH range is 5.7 to 6.7.

[0010] After extensive research and experiments, the inventors of the present application found that Faecalibacterium prausnitzii GZSY3214 screened in the present application has good therapeutic effects in preventing, alleviating, improving and / or treating metabolic-related fatty liver disease or metabolic-related fatty liver disease, and has a significant inhibitory effect on the signaling pathways of the liver involved in inflammatory response.

[0011] As a preferred embodiment of the Faecalibacterium prausnitzii described in the present application, the sequence of 16s rDNA of Faecalibacterium prausnitzii is shown as SEQ ID NO:1.

[0012] The present application also provides a product containing the Faecalibacterium prausnitzii.

[0013] As a preferred embodiment of the product described in the present application, the product includes a medicine or a microbial agent.

[0014] As a preferred embodiment of the product described in the present application, the product is a microbial agent, and the concentration of Faecalibacterium prausnitzii in the microbial agent is 1~6×10 9 CFU / mL.

[0015] The microbial agent is prepared by the following steps:

[0016] (1) Inoculate the glycerol culture of Clostridium prausnitzii into YCFA liquid culture medium, and culture it statically at 35-37°C for 16-19 hours under anaerobic conditions to obtain a primary seed solution;

[0017] (2) inoculating the first-level seed solution obtained in step (1) into YCFA liquid culture medium, and culturing the culture medium at 35-37° C. under anaerobism for 7-9 hours to obtain a second-level seed solution;

[0018] (3) The secondary seed liquid obtained in step (2) is inoculated into YCFA liquid culture medium, and cultured at 35-37° C. for 16-19 hours under anaerobism to obtain a fermentation liquid of Clostridium prausnitzii.

[0019] (4) centrifuging the obtained F. prausnitzii fermentation broth at 4-8° C. to obtain F. prausnitzii cells;

[0020] (5) resuspending the Clostridium prausnitzii cells obtained in step (4) using a phosphate buffer to obtain Clostridium prausnitzii bacterial slurry;

[0021] (6) Mixing the obtained Clostridium prausnitzii slurry with a cryoprotectant and freezing the mixture at -80°C; the volume ratio of the Clostridium prausnitzii slurry to the cryoprotectant is 1:1, thereby obtaining the microbial agent.

[0022] The microbial agent containing the Faecalibacterium prausnitzii obtained by culture can be directly added to auxiliary materials permitted in the field of microbial preparations to prepare a liquid agent, or the bacteria in the microbial agent can be collected and mixed with auxiliary materials permitted in the field of microbial preparations such as freeze-drying protective agents to prepare the microbial agent.

[0023] As a preferred embodiment of the product described in the present application, the product is a medicine, and the medicine also includes excipients.

[0024] As a preferred embodiment of the product described in the present application, the auxiliary materials include excipients and additives.

[0025] In some specific embodiments, the excipients include anti-adhesive agents, penetration enhancers, buffers, plasticizers, surfactants, defoamers, thickeners, inclusion agents, absorbents, humectants, solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, pH regulators, adhesives, disintegrants, fillers, lubricants, wetting agents, integrators, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, foaming agents, suspending agents, coating materials, fragrances, diluents, flocculants and deflocculants, filter aids and release retardants.

[0026] In some specific embodiments, the additional agent comprises microcrystalline cellulose, hydroxypropyl methylcellulose and refined lecithin.

[0027] As a preferred embodiment of the product described in the present application, the dosage form of the drug includes one of granules, capsules, tablets, pills and oral liquids.

[0028] The present application also provides the use of the Faecalibacterium prausnitzii or the above-mentioned product in the preparation of a drug for preventing, alleviating, improving and / or treating metabolism-related fatty liver disease or metabolism-related fatty hepatitis.

[0029] The application is at least one of the following (1) to (5):

[0030] (1) Reduce body weight and liver weight;

[0031] (2) Improve liver tissue lesions, relieve fatty degeneration of liver cells, and reduce ballooning of liver cells;

[0032] (3) Improve liver function and lipid metabolism, and reduce the levels of ALT, AST, TG, and LDL;

[0033] (4) Inhibit the expression of inflammation-related MAPK, NF-κB, inflammatory cytokines, and chemokines;

[0034] (5) Inhibit the expression of liver fibrosis-related collagen and transforming growth factor-β.

[0035] This application also selected two typical metabolic-associated fatty liver disease (MAFLD) and metabolic-associated steatohepatitis (MASH) animal models for experiments, namely (1) spontaneous type 2 diabetic mice (db / db) given a high-fat, high-sugar, and high-cholesterol Western diet; (2) methionine-choline deficient diet (MCD), which can better simulate the disease progression of metabolic-associated fatty liver disease or metabolic-associated steatohepatitis.

[0036] According to animal experiments, compared with the representative strain A2-165, the body weight and liver weight of mice can be significantly reduced after treatment with FaecalibacteriumprausnitziiGZSY3214 of the present application, and the fatty degeneration of liver cells of mice can be alleviated, liver tissue lesions can be improved, and the ballooning of liver cells is reduced. It shows that Faecalibacterium prausnitziiGZSY3214 of the present application can significantly improve the fatty degeneration and inflammatory response of the liver of MAFLD mice induced by Western diet. Faecalibacterium prausnitziiGZSY3214 of the present application can also effectively inhibit fatty degeneration of liver cells, ballooning of liver cells and fibrosis of mice induced by MCD diet, reduce serum ALT, AST and TNFα levels, have the effect of improving MASH, and GZSY3214 has a better improvement effect on MASH than A2-165 strain.

[0037] Moreover, compared with the representative strain A2-165, the ALT, AST, TG and LDL of mice treated with FaecalibacteriumprausnitziiGZSY3214 of the present application were significantly decreased (P<0.05), and the expression of signaling pathway molecules such as inflammation-related MAPK, NF-KAPPA B (NF-κB), inflammatory cytokines, and chemokines was significantly inhibited, and the expression of collagen and transforming growth factor-β related to liver fibrosis was significantly inhibited. It can also significantly reduce the expression of 1143 WD non-induced genes (Profile 3), which are enriched in signaling pathways such as cytokine-cytokinereceptor interaction, calcium signaling pathway, and antigen processing and presentation. The above results show that treatment with Faecalibacterium prausnitziiGZSY3214 has a good therapeutic effect in improving the progression of metabolic-related fatty liver disease.

[0038] In addition, Faecalibacterium prausnitziiGZSY3214 can also affect the metabolism of the entire pathway through the IKK-dependent NF-κB classical pathway and the NIK-dependent non-classical pathway, thereby affecting the inflammatory response of metabolic-related fatty liver disease.

[0039] In summary, treatment with Faecalibacterium prausnitziiGZSY3214 has a good therapeutic effect in the treatment of the inflammatory response of metabolic-related fatty hepatitis, and has a significant inhibitory effect on the NF-κB and MAPK signaling pathways involved in the inflammatory response of the liver.

[0040] The present application also provides the use of the Faecalibacterium prausnitzii or the above-mentioned product in the preparation of a drug for reducing liver function indicators and blood lipid factor levels.

[0041] As a preferred embodiment of the application described in the present application, the liver function index includes alanine aminotransferase or aspartate aminotransferase; the blood lipid factors include triglycerides or low-density lipoprotein.

[0042] The present application also provides the use of the Faecalibacterium prausnitzii or the above-mentioned product in the preparation of a drug for reducing the expression of molecules in the inflammatory signaling pathway.

[0043] As a preferred embodiment of the application described in the present application, the inflammatory signaling pathway molecules include at least one of MAPK, NF-κB, inflammatory cytokines and chemokines.

[0044] In the technical solution of the present application, after being treated with Faecalibacterium prausnitziiGZSY3214 of the present application, the liver and blood lipid factors such as ALT, AST, TG and LDL of mice were significantly decreased (P<0.05).

[0045] In addition, the strain can significantly inhibit the expression of inflammation-related MAPK, NF-KAPPA B (NF-κB), inflammatory cytokines, and chemokines, and can also significantly inhibit the expression of liver fibrosis-related collagen and transforming growth factor-β, and can also significantly reduce the expression of 1143 WD non-induced genes (Profile 3). This indicates that the treatment with Faecalibacterium prausnitziiGZSY3214 of the present application can significantly inhibit the inflammatory response of metabolism-related fatty hepatitis, which is beneficial for the treatment of metabolism-related fatty hepatitis.

[0046] Compared with the prior art, this application has the following beneficial effects:

[0047] The present application provides a strain of Faecalibacterium prausnitziiGZSY3214 that can improve metabolic-related fatty hepatitis and its application. After being treated with Faecalibacterium prausnitziiGZSY3214 screened by the present application, it has a good therapeutic effect in treating the progression of metabolic-related fatty hepatitis and has a significant inhibitory effect on the NF-κB and MAPK signaling pathways involved in the inflammatory response of the liver; wherein, the use of Faecalibacterium prausnitziiGZSY3214 to treat metabolic-related fatty liver disease or metabolic-related fatty hepatitis animal models can improve liver tissue lesions, alleviate hepatocyte fatty degeneration, and reduce hepatocyte ballooning. In addition, the strain can also improve liver function and lipid metabolism, downregulate the content of ALT, AST, TG and LDL, and inhibit the expression of inflammation-related MAPK, NF-κB, inflammatory cytokines, and chemokines, and significantly inhibit the expression of liver fibrosis-related collagen and transforming growth factor-β. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 This is the morphological diagram of Faecalibacterium prausnitziiGZSY3214;

[0049] Figure 2 This is the evolutionary tree of Faecalibacterium prausnitziiGZSY3214;

[0050] Figure 3 The treatment results of WT mice using a low-fat, low-sugar control diet (CD), db / db mice using a low-fat, low-sugar control diet (CD), a Western diet (WD), and the microbial agent prepared in Example 2 (containing Faecalibacterium prausnitzii GZSY3214) or the representative strain of Faecalibacterium prausnitzii A2-165 ( Figure 3 A in the middle is a graph showing the weight gain of mice in each group; Figure 3 Middle B is a graph showing the liver weight of mice in each group; Figure 3 Middle C shows hematoxylin-eosin and oil red staining of liver tissues of mice in each group; compared with db / db-WD, * P<0.05, ** P<0.01, *** P<0.001, scale bar is 100 μm);

[0051] Figure 4The results of serum ALT, AST, TG, and LDL levels of mice in each group after WT mice were treated with a low-fat, low-sugar control diet (CD), db / db mice were treated with a low-fat, low-sugar control diet (CD), a Western diet (WD), and the microbial agent prepared in Example 2 (containing Faecalibacteriumprausnitzii GZSY3214) or the representative strain of Faecalibacterium prausnitzii A2-165 (* P<0.05, ** P<0.01, *** P<0.001);

[0052] Figure 5 The MASH mouse model was established by feeding WT mice with a control diet (MCS) or a methionine-choline deficient diet (MCD). The treatment results of the microbial agent prepared in Example 2 (containing Faecalibacterium prausnitziiGZSY3214) or the representative strain of Faecalibacterium prausnitzii A2-165 were shown in Figure ( Figure 5 A in the middle shows the hematoxylin-eosin staining, oil red staining and Masson staining of the liver tissue of mice in each group; the scale bar is 100 μm; Figure 5 BD in the middle are the results of serum ALT, AST and TNFα levels of mice in each group; * P<0.05, ** P<0.01);

[0053] Figure 6 The PCA analysis, volcano plot and KEGG pathway results of mice in different treatment groups ( Figure 6 A in the middle shows the intra-group and inter-group differences of samples from mice in different treatment groups through PCA analysis; Figure 6 Middle B is a volcano plot of differentially expressed genes in db / db mice treated with a Western diet and the microbial agent prepared in Example 2 (containing Faecalibacterium prausnitziiGZSY3214, Fp); Figure 6 Middle C shows the upregulated KEGG pathways in db / db mice treated with the microbial agent prepared in Example 2 (containing Faecalibacterium prausnitziiGZSY3214); Figure 6 D in the middle is a diagram of the down-regulated KEGG pathways in db / db mice treated with the microbial agent (containing Faecalibacterium prausnitziiGZSY3214) prepared in Example 2;

[0054] Figure 7The heat map of differential gene expression of db / db mice in three groups: low-fat and low-sugar control diet (CD), Western diet (WD), and the microbial agent prepared in Example 2 (containing Faecalibacterium prausnitziiGZSY3214) ( Figure 7 A in the middle is the expression result diagram of inflammation-related genes; Figure 7 (B) is the expression result of liver fibrosis related genes);

[0055] Figure 8 The treatment results of db / db mice treated with a low-fat and low-sugar control diet (CD), a Western diet (WD), and the microbial agent prepared in Example 2 (containing Faecalibacterium prausnitziiGZSY3214) (wherein, Figure 8 A in the middle is a trend analysis diagram of significantly differentially expressed genes among the three groups of mice; Figure 8 Middle B is the KEGG pathway of Profile 5 gene group; Figure 8 (C) shows the KEGG pathway of Profile 3 gene group);

[0056] Fig. 9 The heat map of differential gene expression of db / db mice in three groups: low-fat and low-sugar control diet (CD), Western diet (WD), and the microbial agent prepared in Example 2 (containing Faecalibacterium prausnitziiGZSY3214) ( Fig. 9 A in the middle is the expression heat map of genes in the NF-κB signaling pathway; Fig. 9 (B) is the expression heat map of genes in the MAPK signaling pathway). DETAILED DESCRIPTION

[0057] In order to better illustrate the purpose, technical solutions and advantages of the present application, the present application will be further described below in conjunction with the accompanying drawings and specific embodiments.

[0058] In the following examples, the experimental methods used are conventional methods unless otherwise specified, and the materials, reagents, etc. used are all commercially available unless otherwise specified, and the components and raw materials used in each parallel experiment are of the same type.

[0059] The following Faecalibacterium prausnitziiGZSY3214 is referred to as GZSY3214 or Fp.

[0060] The YCFA liquid medium mentioned in the following examples includes the following components:

[0061] 10g / L tryptone, 2.5g / L yeast extract, 4.0g / L sodium bicarbonate, 2.0g / L glucose, 2.0g / L maltose, 2.0g / L cellobiose, 1.0g / L cysteine ​​hydrochloride, 150mL / L mineral solution I, 150mL / L mineral solution II, 1mg / L resazurin, 6.2mL / L organic acid mixture, 10.0mL / L heme solution, 1.0mL / L vitamin solution I, 1.0mL / L vitamin solution II, plus 15% agar to make YCFA solid culture medium (YCFA plate culture medium).

[0062] Mineral solution I is a 3.0 g / L dipotassium hydrogen phosphate aqueous solution.

[0063] Mineral solution II includes the following components: 3.0 g / L potassium dihydrogen phosphate, 6.0 g / L ammonium sulfate, 6.0 g / L sodium chloride, 0.6 g / L magnesium sulfate heptahydrate, and 0.6 g / L calcium chloride dihydrate.

[0064] The heme solution includes the following components: 2.8 g / L potassium hydroxide, 250 mL / L anhydrous ethanol and 1.0 g / L hemin chloride.

[0065] The organic acid mixture includes the following components: 17.0 mL acetic acid, 6.0 mL propionic acid, 1.0 mL n-valeric acid, 1.0 mL isovaleric acid and 1.0 mL isobutyric acid.

[0066] Vitamin solution I includes the following components: 0.01 g / L biotin, 0.01 g / L vitamin B12, 0.03 g / L p-aminobenzoic acid, 0.05 g / L folic acid and 0.15 g / L vitamin B6 hydrochloride.

[0067] Vitamin solution II includes the following components: 0.05 g / L thiamine hydrochloride and 0.05 g / L riboflavin.

[0068] Example 1: Isolation and screening of Faecalibacterium prausnitzii GZSY3214

[0069] This example provides the steps for separation and screening of Faecalibacterium prausnitzii GZSY3214:

[0070] Firstly, the freshly collected feces of healthy donors were diluted in multiples and cultured on YCFA plate culture medium. The strictly anaerobic characteristics of Faecalibacterium prausnitzii were used to establish a "negative screening" method to select colonies for 16sRNA identification.

[0071] The specific steps are as follows: take about 1 g of freshly collected feces and add it to 9 mL of sterile saline, shake and mix, and dilute it to 10 by 10-fold gradient. -8 200 μL of the dilution gradient was 10 -6 times, 10 -7 times, 10 -8 The 100-fold dilution was placed in the YCFA plate medium that had been placed in the anaerobic workstation for at least 24 hours, and was evenly spread using a disposable spreader rod, and then placed in the anaerobic workstation for 72 hours at 37°C. After the culture was completed, the colonies were selected according to the morphology, size, and transparency of the colonies and placed on two plates with squares. Plate A was exposed to the air for 30 minutes and then placed back in the anaerobic workstation for culture, while plate B was always placed in the anaerobic workstation. After 72 hours of culture, the strains that did not grow in plate A were picked from plate B for subsequent 16s rDNA sequencing identification: the amplified 16s rDNA sequence data was submitted to GenBank, and homology comparison was performed using the online software Blast on the NCBI website; the phylogenetic tree was constructed using the Maximum likelihood method in the bioinformatics analysis software MEGA-X, and the strain was further identified as Faecalibacterium prausnitzii.

[0072] The Faecalibacterium prausnitzii obtained by the above screening was preserved and named Faecalibacterium prausnitziiGZSY3214. The Faecalibacterium prausnitzii was deposited in the Guangdong Provincial Microbiological Culture Collection Center on August 7, 2024, with a preservation number of GDMCC No: 64992, a classification name of Faecalibacterium prausnitzii, and a preservation address of 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0073] The colony morphology of Faecalibacterium prausnitziiGZSY3214 is: about 1.5 to 3 microns in length, about 0.5 to 0.8 microns in width, arranged in single cells or pairs, non-spore-forming, non-flagellated, non-motile rods, and convex, translucent, intact-edge and moist colonies in YCFA solid culture medium. The morphological characteristics in YCFA liquid culture medium are a series of cell morphologies from spherical, pleomorphic spherules to rods, such as Figure 1 shown.

[0074] The physiology and biochemistry of Faecalibacterium prausnitziiGZSY3214 is: Gram-negative, strictly anaerobic, extremely sensitive to oxygen, and is the dominant bacteria of the Clostridium group, belonging to the family Clostridiaceae, the genus Clostridium of the phylum Firmicutes. Faecalibacterium prausnitziiGZSY3214 produces a large amount of formate, butyrate and a small amount of D-lactate through glucose glycolysis. Faecalibacterium prausnitziiGZSY3214 participates in multiple metabolic pathways such as sugar metabolism, amino acid and vitamin metabolism, and the production of short-chain fatty acids. The metabolites mainly include butyrate and 15KD microbial anti-inflammatory molecules (MAM). FaecalibacteriumprausnitziiGZSY3214 can only survive stably in an anaerobic environment, and the optimal growth pH range is 5.7 to 6.7.

[0075] The sequence of 16s rDNA of Faecalibacterium prausnitziiGZSY3214 is shown in SEQ ID NO: 1. The phylogenetic tree of Faecalibacterium prausnitziiGZSY3214 is shown in Figure 2 shown.

[0076] SEQ ID NO: 1 is:

[0077]

[0078] Example 2: Microbial agent containing Faecalibacterium prausnitziiGZSY3214

[0079] This embodiment provides a microbial agent containing Faecalibacterium prausnitziiGZSY3214 and a preparation method thereof, wherein the preparation method comprises the following steps:

[0080] (1) The glycerol bacteria of Faecalibacterium prausnitziiGZSY3214 in Example 1 was inoculated into YCFA liquid culture medium at an inoculum rate of 10%, and cultured anaerobically and statically for 16-19 hours to obtain a primary seed solution;

[0081] (2) inoculating the first-level seed solution obtained in step (1) into YCFA liquid culture medium at an inoculum rate of 4%, and culturing anaerobically and statically for 7-9 hours to obtain a second-level seed solution;

[0082] (3) The secondary seed liquid obtained in step (2) is inoculated into YCFA liquid culture medium at an inoculum rate of 0.5%, and anaerobically and statically cultured for 16-19 hours to obtain F. prausnitzii fermentation liquid.

[0083] (4) centrifuging the F. prausnitzii fermentation broth obtained in step (3) at 4° C. and 8000 rpm for 7 min to obtain F. prausnitzii cells;

[0084] (5) resuspending the Clostridium prausnitzii cells obtained in step (4) using a phosphate buffer to obtain Clostridium prausnitzii bacterial slurry;

[0085] (6) The Faecalibacterium prausnitzii GZSY3214 slurry obtained in step (5) was mixed with a cryoprotectant (10 wt% maltodextrin, 5 wt% trehalose, 1 wt% arginine and 1 wt% histidine), and the mixture was frozen at -80°C; the volume ratio of the Faecalibacterium prausnitzii slurry to the cryoprotectant was 1:1, and the number of viable bacteria after freezing was calculated. The concentration of Faecalibacterium prausnitzii GZSY3214 in the microbial agent was 5×10 9 CFU / mL.

[0086] Example 3: Experiment on the improvement of metabolism-related fatty liver disease by Faecalibacterium prausnitziiGZSY3214

[0087] 1. Faecalibacterium prausnitziiGZSY3214 can reduce the body weight and liver weight of MAFLD mice, and improve the liver tissue lesions of MAFLD mice.

[0088] In this example, 8-week-old spontaneously diabetic mice (db / db) of the BSK strain were selected and fed with a western diet to establish a metabolic-associated fatty liver disease (MAFLD) model. At the same time, the microbial agent prepared in Example 2 (containing Faecalibacterium prausnitziiGZSY3214, with a dosage of 1×10 8 CFU / unit) or the representative strain of Faecalibacterium prausnitzii A2-165 inoculum (the A2-165 cells were resuspended in PBS buffer to obtain the inoculum, and the dosage of the inoculum was 1×10 8 CFU / mouse), gavage was performed every other day, body weight was measured every 3 days, and the experiment was terminated after 30 days.

[0089] like Figure 3 As shown, WT mice were treated with a low-fat, low-sugar control diet (CD), and db / db mice were treated with a low-fat, low-sugar control diet (CD), a Western diet (WD), the microbial agent prepared in Example 2 (containing Faecalibacterium prausnitzii GZSY3214), and an agent containing the representative strain of Faecalibacterium prausnitzii A2-165, respectively, to form the following groups: WT-CD, db / db-CD, db / db-WD, db / db-WD- GZSY3214, and db / db-WD- A2-165.

[0090] in, Figure 3 A in the middle shows the weight gain of mice in each group; Figure 3 Middle B shows the liver weight of mice in each group; Figure 3 Middle C shows the hematoxylin-eosin and oil red staining of the liver tissues of mice in each group. Compared with db / db-WD, * P<0.05, ** P<0.01, *** P<0.001.

[0091] Figure 3The results showed that compared with the db / db-WD group, the body weight of the mice in the db / db-WD-GZSY3214 group decreased significantly from the 12th day (P<0.05), and the liver weight of the mice decreased significantly (P<0.01). Liver H&E staining and Oil Red O staining showed that compared with the WT-CD group, the mice in the db / db-WD group had severe diffuse fatty degeneration of hepatocytes, with a large number of fat vacuoles, widespread ballooning of hepatocytes, and diffuse red lipid droplets were observed by Oil Red O staining; after administration of Faecalibacterium prausnitzii, the fatty degeneration of hepatocytes in the db / db-WD-GZSY3214 group was alleviated, and the ballooning of hepatocytes was reduced. In addition, compared with the representative strain A2-165, the treatment with Faecalibacterium prausnitziiGZSY3214 of the present application had a better effect on improving the body weight, liver weight and lipid deposition of mice.

[0092] The above results indicate that Faecalibacterium prausnitzii GZSY3214 screened in this application can significantly improve liver fatty degeneration and inflammatory response in MAFLD mice induced by Western diet.

[0093] 2. Faecalibacterium prausnitziiGZSY3214 can improve liver function and lipid metabolism in MAFLD mice

[0094] In order to study the effect of Faecalibacterium prausnitziiGZSY3214 on the liver function of MAFLD mice, the present application detected the indicators of ALT (alanine amiotransferase), AST (aspartate aminotransferase), TG (triglyceride) and LDL (low-density lipoprotein) in the serum of mice. The results are shown in Table 1 and Figure 4 shown.

[0095] like Figure 4As shown, WT mice were treated with a low-fat, low-sugar control diet (CD), and db / db mice were treated with a low-fat, low-sugar control diet (CD), a Western diet (WD), and the microbial agent prepared in Example 2 (containing Faecalibacterium prausnitziiGZSY3214), forming the following groups: WT-CD, db / db-CD, db / db-WD, db / db-GZSY3214, and db / db-WD-A2-165. The serum ALT, AST, TG, and LDL levels of mice in each group were detected. Compared with db / db-WD, * P<0.05, **P<0.01, *** P<0.001.

[0096] like Figure 4 The results showed that compared with the WT-CD group, the ALT, AST, TG and LDL of the db / db-WD group mice were increased, indicating that the db / db-WD group mice had severe liver function and lipid metabolism abnormalities; after administration of Faecalibacterium prausnitziiGZSY3214, compared with the db / db-WD model group mice, the ALT, AST, TG and LDL of the db / db-WD-GZSY3214 treatment group mice were significantly decreased (P<0.05). Moreover, compared with the representative strain A2-165, the liver function indicators ALT, TG and LDL after treatment with Faecalibacterium prausnitziiGZSY3214 of this application decreased more significantly (P<0.05).

[0097] The results (average values) of different groups in the db / db WD model are shown in Table 1:

[0098] Table 1

[0099]

[0100] 3. In order to further demonstrate the effect of Faecalibacterium prausnitziiGZSY3214 in improving MAFLD, the present application selected 8-week-old wild-type C57 mice and fed them with a methionine-choline deficient diet (MCD) to construct a MASH model. After 14 days of modeling, the mice were gavaged with the microbial agent prepared in Example 2 above once every other day, and their body weight was measured every 3 days. The experiment was terminated after 30 days.

[0101] like Figure 5 As shown, WT mice were fed a control diet (MCS) and the microbial agent prepared in Example 2 (containing Faecalibacterium prausnitziiGZSY3214, with a dosage of 1×10 8CFU / ) and a bacterial agent containing the representative strain of Faecalibacterium prausnitzii A2-165 (the bacterial agent was obtained by resuspending A2-165 cells in PBS buffer, and the dosage of the bacterial agent was 1×10 8 CFU / unit) treatment results ( Figure 5 A in the middle shows hematoxylin-eosin staining, oil red staining and Masson staining of liver tissues of mice in each group; Figure 5 BD in the figure are the serum ALT, AST and TNFα (tumor necrosis factor-α) levels of mice in each group (*P<0.05, **P<0.01).

[0102] like Figure 5 As shown in A, WT mice were fed a control diet (MCS), mice were fed a methionine-choline deficient diet (MCD), and mice were fed the microbial agent prepared in Example 2 (containing Faecalibacterium prausnitziiGZSY3214 at a dose of 1×10 8 CFU / ) and a bacterial agent containing the representative strain of Faecalibacterium prausnitzii A2-165 (the bacterial agent was obtained by resuspending A2-165 cells in PBS buffer, and the dosage of the bacterial agent was 1×10 8 CFU / mouse) treatment, forming the following groups: MCS, MCD, MCD-GZSY3214, and MCD-A2-165.

[0103] The results (average values) of different groups in the MASH model are shown in Table 2:

[0104] Table 2

[0105]

[0106] Depend on Figure 5 As shown in the results of Table 2, Faecalibacterium prausnitzii GZSY3214 screened by the present application can effectively inhibit MCD diet-induced fatty degeneration, ballooning degeneration and fibrosis of hepatocytes in mice, reduce serum ALT, AST and TNFα levels, and improve MASH. The improvement effect of GZSY3214 on MASH is better than that of the A2-165 strain.

[0107] 4. Faecalibacterium prausnitziiGZSY3214 can change MAFLD-related metabolic pathways.

[0108] In order to further explore the molecular mechanism of Faecalibacterium prausnitziiGZSY3214 in improving MAFLD, this application performed transcriptome sequencing on the liver tissues of mice in the modeling group and the treatment group.

[0109] like Figure 6 As shown, Figure 6 A in the middle shows the intra-group and inter-group differences of samples from mice in different treatment groups through PCA analysis; Figure 6 Middle B is a volcano plot of differentially expressed genes in db / db mice treated with a Western diet and the microbial agent prepared in Example 2 (containing Faecalibacterium prausnitzii GZSY3214); Figure 6 C is the microbial agent prepared in Example 2 (containing Faecalibacterium prausnitziiGZSY3214, with a dosage of 1×10 8 The up-regulated KEGG pathways in db / db mice treated with 1000 CFU / mouse; Figure 6 D is the microbial agent prepared in Example 2 (containing Faecalibacterium prausnitziiGZSY3214, with a dosage of 1×10 8 The KEGG pathways downregulated in db / db mice treated with 100% CFU / mouse CFU / mouse were analyzed.

[0110] according to Figure 6 The results showed that PCA analysis showed that there were significant differences among the CD group, WD group and WD-Fp group, and the differences between the groups were small.

[0111] The enrichment of differentially expressed genes and KEGG pathways showed that compared with the WD group, the genes related to metabolic pathways in the WD-Fp group were upregulated and significantly enriched, indicating that Faecalibacterium prausnitzii (Fp) treatment can significantly inhibit the expression of inflammation-related signaling pathway molecules such as MAPK, NF-KAPPA B (NF-κB), inflammatory cytokines, and chemokines ( Figure 6 ).

[0112] like Figure 7 As shown, the differential gene expression heat map of db / db mice in three groups: low-fat and low-sugar control diet (CD), Western diet (WD) and the microbial agent prepared in Example 2 (containing Faecalibacterium prausnitziiGZSY3214) ( Figure 7 A in the middle is the expression level of inflammation-related genes; Figure 7 (B in the figure is the expression level of liver fibrosis-related genes).

[0113] The results showed that Faecalibacterium prausnitziiGZSY3214 (Fp) could effectively inhibit the expression of inflammation-related cytokines such as IL1b, IL1a, Tnf, Il15 and chemokines such as Cxcl5, Cxcl9, Ccl6 induced by WD diet; at the same time, the expression of liver fibrosis-related genes such as collagens Col1a1, Col1a2, Col4a5 and transforming growth factor-β such as Tgfb1, Tgfb2, Tgfb3 also decreased significantly.

[0114] like Figure 8 As shown, db / db mice were fed a low-fat and low-sugar control diet (CD), a Western diet (WD), and the microbial agent prepared in Example 2 (containing Faecalibacterium prausnitziiGZSY3214 at a dose of 1×10 8 CFU / mouse) treatment.

[0115] in, Figure 8 A in the middle is a trend analysis diagram of significantly differentially expressed genes among the three groups of mice; Figure 8 Middle B is the KEGG pathway of Profile 5 gene group; Figure 8 Middle C is the KEGG pathway status of Profile 3 gene group.

[0116] according to Figure 8 The results show that the trend analysis results ( Figure 8 Middle A) shows that Faecalibacterium prausnitzii GZSY3214 can significantly reduce the expression of 5916 genes induced by WD (Profile 5); KEGG pathway enrichment of these genes revealed that inflammation-related MAPK, inflammatory cytokines, chemokines and other signaling pathway molecules were significantly enriched ( Figure 8 Middle B).

[0117] At the same time, Faecalibacterium prausnitziiGZSY3214 could significantly reduce the expression of 1143 genes that were not induced by WD (Profile 3), which were enriched in signaling pathways such as cytokine-cytokine receptor interaction, calcium signaling pathway, and antigen processing and presentation ( Figure 8The above results show that Faecalibacterium prausnitziiGZSY3214 can significantly inhibit the inflammatory response of MAFLD.

[0118] like Fig. 9 As shown, the differential gene expression heat map of db / db mice in three groups: low-fat and low-sugar control diet (CD), Western diet (WD) and the microbial agent prepared in Example 2 (containing Faecalibacterium prausnitziiGZSY3214) ( Fig. 9 A in the middle is the expression heat map of genes in the NF-κB signaling pathway; Fig. 9 (B) is the expression heat map of genes in the MAPK signaling pathway).

[0119] The results showed that Faecalibacterium prausnitziiGZSY3214 (Fp) can effectively inhibit the expression of genes such as TLR4, Ikbkb, Nfkb2, Ripk1, Trim25, etc., which are key components of the NF-κB signaling pathway induced by WD diet; at the same time, the expression of kinases such as Map3k8, Map4k1, Map4k4, etc. in the MAPK signaling pathway also decreased significantly, thereby blocking the activation of the MAPK signaling pathway induced by WD diet.

[0120] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present application rather than to limit the scope of protection of the present application. Although the present application has been described in detail with reference to the preferred embodiments, ordinary technicians in this field should understand that the technical solution of the present application can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present application.

Claims

1. A strain of Faecalibacterium prausnitzii capable of improving metabolic-related fatty liver disease or metabolic-related fatty hepatitis, characterized in that: The Faecalibacterium prausnitzii is Faecalibacterium prausnitziiGZSY3214, which has been deposited in Guangdong Provincial Microbiological Culture Collection Center on August 7, 2024, with a deposit number of GDMCC No: 64992, and a deposit address of 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou; The sequence of the 16s rDNA of Faecalibacterium prausnitzii is shown in SEQ ID NO:

1.

2. A product containing Faecalibacterium prausnitzii according to claim 1, characterized in that The products include medicines or microbial agents.

3. The product according to claim 2, characterized in that The product is a microbial agent, and the concentration of Faecalibacterium prausnitzii in the microbial agent is 1~6×10 9 CFU / mL.

4. The product according to claim 2, characterized in that The product is a medicine, and the medicine also includes excipients.

5. The product according to claim 4, characterized in that The auxiliary materials include excipients and additives.

6. The product according to claim 4, characterized in that The dosage form of the medicine includes one of granules, capsules, tablets, pills and oral liquid.

7. Use of the Faecalibacterium prausnitzii as claimed in claim 1 or the product as claimed in claim 2 in the preparation of a medicament for preventing, alleviating, improving and / or treating metabolism-related fatty liver disease or metabolism-related fatty hepatitis.

8. The use according to claim 7, characterized in that The application is at least one of the following (1) to (5): (1) Reduce body weight and liver weight; (2) Improve liver tissue lesions, relieve fatty degeneration of liver cells, and reduce ballooning of liver cells; (3) Improve liver function and lipid metabolism, and reduce the levels of ALT, AST, TG, and LDL; (4) Inhibit the expression of inflammation-related MAPK, NF-κB, inflammatory cytokines, and chemokines; (5) Inhibit the expression of liver fibrosis-related collagen and transforming growth factor-β.

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