Application of Eubacterium mucilaginosus in preparation of medicine for preventing and treating non-alcoholic fatty liver disease

Abstract

The invention discloses an application of Eubacterium mucilaginosus in preparation of a medicine for preventing and treating a non-alcoholic fatty liver disease. Wherein the preservation number of the Eubacterium mucilaginosus is JCM 6421. In the application, the Eubacterium mucilaginosus can be used for preventing and treating the non-alcoholic fatty liver disease by inhibiting liver fat accumulation, and the Eubacterium mucilaginosus is from healthy people, is a relatively common bacterium genus, is smaller in possibility of adverse reaction and is wider in applicable crowd range.

Description

Technical Field

[0001] The present application relates to the field of biomedicine, and specifically to the use of Eubacterium muciniphilum in the preparation of drugs for preventing and treating non-alcoholic fatty liver disease. Background Art

[0002] Non-alcoholic fatty liver disease is a chronic disease with multiple complex pathological manifestations, in which the accumulation of fatty acids in the liver triggers inflammation and fibrosis, leading to further liver damage. Studies have found that intestinal flora imbalance is key to the pathogenesis of fatty liver disease, and microbial imbalance is associated with the onset and progression of liver disease. Based on the association between intestinal microbiota and fatty liver disease in the gut-liver axis, microbiome-related interventions may serve as an alternative to conventional treatment options for fatty liver disease. Although researchers have conducted a lot of research on bacterial intestinal microbiota, the number of probiotic preparations that can be used in clinical practice is scarce, and the effects are difficult to provide patients with diversified choices. In addition, the objects for which probiotics and traditional Chinese medicine compositions are applicable are limited.

[0003] Therefore, seeking new treatment options for fatty liver disease is a technical issue that urgently needs to be addressed. Summary of the Invention

[0004] Based on this, it is necessary to provide the use of Eubacterium mucilaginosus in the preparation of drugs for preventing and treating non-alcoholic fatty liver disease.

[0005] A first aspect of the present application provides use of Eubacterium limosum in preparing a medicament for preventing and treating non-alcoholic fatty liver disease. The deposit number of Eubacterium limosum is JCM 6421.

[0006] In some embodiments, the Eubacterium mucronulate comprises live Eubacterium mucronulate bacteria, inactivated Eubacterium mucronulate bacteria, or a combination thereof.

[0007] In some embodiments, the drug further includes a pharmaceutically acceptable excipient; optionally, the pharmaceutically acceptable excipient includes one or more of a diluent, a wetting agent, a binder, a disintegrant, an aqueous solvent, a solubilizer, a pH regulator, and an isotonic or isotonic regulator.

[0008] In some embodiments, the dosage form of the drug is a liquid preparation.

[0009] In some embodiments, the dosage form of the drug includes at least one of a suspension, an oral solution, and an enema.

[0010] In some embodiments, the drug is in a dosage form for oral administration, enema administration, or parenteral administration.

[0011] In some embodiments, Eubacterium mucilaginosus is used as the sole probiotic in the medicament.

[0012] In some embodiments, the subject is a mammal; alternatively, the subject is a human or a mouse.

[0013] In some embodiments, the subject has abnormalities in at least one of the following indicators in the liver:

[0014] Abnormal low-density lipoprotein levels;

[0015] Abnormal proportion of fat droplets in the liver;

[0016] abnormal expression levels of SCD1 or CHREBP;

[0017] The expression level of LCAT was abnormal.

[0018] The second aspect of the present application provides the use of Eubacterium mucilaginosum with the deposit number JCM 6421 in the preparation of a preparation having at least one of the following functions:

[0019] Preparations that suppress blood lipid levels;

[0020] An agent that inhibits fat accumulation in the liver.

[0021] In some embodiments, the formulation is a pharmaceutical formulation.

[0022] This application demonstrates through experiments that Eubacterium limosum, deposited with accession number JCM 6421, can be used to prevent and treat non-alcoholic fatty liver disease by inhibiting hepatic fat accumulation. The provided Eubacterium limosum can be used to prepare a medicament for preventing and treating fatty liver disease. Furthermore, Eubacterium limosum is a relatively common genus of bacteria obtained from healthy individuals, is less likely to cause adverse reactions, and is applicable to a wider range of patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the technical solutions in the embodiments and examples of this application and to provide a more complete understanding of the application and its beneficial effects, the following briefly introduces the drawings required for use in the description of the embodiments or examples. Obviously, the drawings described below are only some embodiments of this application. Those skilled in the art can also derive other drawings based on these drawings without inventive effort.

[0024] Figure 1The colonization of Eubacterium mucilaginosus and Lactobacillus rhamnosus in the intestines of different groups of mice at different feeding times in one embodiment of the present application, wherein A is the colonization of Eubacterium mucilaginosus in the intestines of mice in the high-fat Myxobacterium group at week 3, B is the colonization of Lactobacillus rhamnosus in the intestines of mice in the high-fat Lactobacillus group at week 3, C is the colonization of Eubacterium mucilaginosus in the intestines of mice in the Myxobacterium ordinary feed group at week 3, D is the colonization of Lactobacillus rhamnosus in the intestines of mice in the Lactobacillus ordinary feed group at week 3, E is the colonization of Eubacterium mucilaginosus in the intestines of mice in the high-fat Myxobacterium group at week 9, F is the colonization of Lactobacillus rhamnosus in the intestines of mice in the high-fat Lactobacillus group at week 9, G is the colonization of Eubacterium mucilaginosus in the intestines of mice in the Myxobacterium ordinary feed group at week 9, and H is the colonization of Lactobacillus rhamnosus in the intestines of mice in the Lactobacillus ordinary feed group at week 9;

[0025] Figure 2 The low-density lipoprotein levels in the serum of different groups of mice in one embodiment of the present application;

[0026] Figure 3 The liver fat content of different groups of mice in one embodiment of the present application, wherein A is the HE staining image of the liver of different groups of mice, and B is the percentage of liver fat reduction of different groups of mice;

[0027] Figure 4 The expression levels of liver lipid synthesis-related genes in different groups of mice in one embodiment of the present application, wherein A is the expression level of SCD1 in different groups of mice, B is the expression level of CHREBP in different groups of mice, and C is the expression level of LCAT in different groups of mice;

[0028] Among them, * indicates p < 0.05, ** indicates p < 0.01, and ns indicates no statistical difference. DETAILED DESCRIPTION

[0029] To facilitate understanding of the present application, a more comprehensive description of the present application will be provided below with reference to the accompanying drawings. The accompanying drawings illustrate preferred embodiments of the present application. However, the present application may be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a more thorough and comprehensive understanding of the disclosure of the present application.

[0030] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this application pertains. The terms used herein in the specification of this application are for the purpose of describing specific embodiments only and are not intended to limit this application. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0031] As used herein, the terms "having," "containing," "including," and "comprising" are synonymous and are inclusive or open-ended, not excluding additional, unrecited members or features. Examples of members or features include materials or components, structures, elements, and instruments. Non-limiting examples of members or features include actions, conditions for the occurrence of actions, timing, and states.

[0032] In this application, the technical features or technical solutions described in open language include closed technical features or technical solutions composed of the listed contents, and also include open technical features or technical solutions containing the listed contents.

[0033] In this application, when referring to the unit of a data range, if the unit is only after the right endpoint, it means that the units of the left endpoint and the right endpoint are the same.

[0034] In this application, if a method flow involves multiple steps, unless otherwise specified herein, there is no strict order restriction on the execution of these steps, and they can be executed in an order other than the order described. Moreover, any step can include multiple sub-steps or multiple stages, and these sub-steps or stages do not necessarily need to be completed at the same time, but can be executed at different times, and their execution order is not necessarily sequential, but can be executed in turn, alternating, or simultaneously with other steps or parts of sub-steps or stages of other steps.

[0035] In this application, exemplary descriptions such as "in some embodiments (or examples)" and "in one embodiment (or example)" may include but are not limited to the following meanings: these solutions can be combined with other solutions in a suitable manner to form new technical solutions.

[0036] In this application, the terms "first" and "second" in "the first aspect" and "the second aspect" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or quantity, nor should they be understood as implicitly indicating the importance or quantity of the technical features indicated. Furthermore, "first" and "second" serve only as non-exhaustive enumeration and description and should be understood not to constitute a closed-ended limitation on quantity.

[0037] In this application, when referring to a numerical interval (i.e., a numerical range), unless otherwise specified, the distribution of the optional numerical values ​​within the numerical interval is deemed to be continuous and includes the two numerical endpoints of the numerical interval (i.e., the minimum and maximum values), as well as every numerical value between these two numerical endpoints. Unless otherwise specified, when a numerical interval refers only to integers within the numerical interval, it includes the two endpoint integers of the numerical range, as well as every integer between the two endpoints, which is equivalent to directly listing every integer. When multiple numerical ranges are provided to describe a feature or characteristic, these numerical ranges can be combined. In other words, unless otherwise specified, the numerical ranges disclosed herein should be understood to include any and all subranges included therein. The "numerical value" in the numerical interval can be any quantitative value, such as a number, percentage, ratio, etc. "Numerical interval" allows for a broad range of numerical interval types including percentage intervals, ratio intervals, and ratio intervals.

[0038] The temperature parameters in the present invention, unless otherwise specified, may be either constant temperature or fluctuating within a certain temperature range. It should be understood that the constant temperature treatment allows for temperature fluctuations within the precision range of the instrument control. Fluctuations within ranges such as ±5°C, ±4°C, ±3°C, ±2°C, and ±1°C are permitted.

[0039] At present, for non-alcoholic fatty liver disease, although researchers have conducted a lot of research on bacterial intestinal microbiota, the number of probiotic preparations that can be used in clinical practice is scarce, and the effects are difficult to provide patients with diversified choices. In addition, the objects that probiotics and Chinese medicine compositions are applicable to are limited. Currently, Lactobacillus rhamnosus, as a probiotic, has shown certain potential in auxiliary intervention studies for fatty liver. In addition to Lactobacillus rhamnosus, the inventors of this application discovered for the first time that Eubacterium mucilaginosum can also be used to treat fatty liver disease, and Eubacterium mucilaginosum comes from healthy people and is a relatively common genus of bacteria, with a lower possibility of adverse reactions and a wider range of applicable populations.

[0040] Based on this, the embodiments of the present application at least provide the use of Eubacterium mucilaginosum in the preparation of drugs for preventing and treating non-alcoholic fatty liver disease.

[0041] In a first aspect of the present application, a use of Eubacterium limosum in preparing a medicament for preventing and treating non-alcoholic fatty liver disease is provided. The deposit number of Eubacterium limosum is JCM 6421.

[0042] In this application, "non-alcoholic fatty liver disease (NAFLD)" is a chronic liver disease associated with metabolic dysfunction, characterized by excessive fat deposition (steatosis) in hepatocytes, in the absence of a history of excessive alcohol consumption. The disease spectrum ranges from simple hepatic steatosis (NAFL) to non-alcoholic steatohepatitis (NASH), which can progress to liver fibrosis, cirrhosis, and even hepatocellular carcinoma (HCC).

[0043] In some embodiments, the Eubacterium mucronulate may be a living bacterium, or a Eubacterium mucronulate that has been inactivated, genetically recombined, transformed or modified, attenuated, chemically treated, or physically treated to retain biological activity, or may be a lysate of the bacterium, a culture (e.g., a supernatant), or a component extracted from the culture.

[0044] In some embodiments, the Eubacterium muciniphilum is selected from live bacteria of Eubacterium muciniphilum.

[0045] Through extensive experiments, the inventors of this application have discovered that Eubacterium mucronum, deposited with accession number JCM 6421, can be used to prevent and treat fatty liver disease by inhibiting hepatic fat accumulation. The provided Eubacterium mucronum can be used to prepare a medicament for preventing and treating fatty liver disease. Furthermore, Eubacterium mucronum is a relatively common genus derived from healthy individuals, is less likely to cause adverse reactions, and is applicable to a wider range of patients.

[0046] In this application, "prevention" includes aspects such as prevention, treatment, and adjuvant therapy. As used herein, "prevention" means to alleviate, slow the progression of, attenuate, prevent, or maintain an existing disease or condition. "Prevention" also includes curing, preventing the development of, or alleviating to a certain degree one or more symptoms of a disease or condition.

[0047] As used herein, "drug" includes any agent, compound, composition, or mixture that provides a physiological and / or pharmacological effect in vivo or in vitro, often with a beneficial effect. The scope of the physiological and / or pharmacological effect produced by a "drug" in vivo is not particularly limited and may be systemic or localized. The activity of the "drug" is not particularly limited and may be an active substance that interacts with other substances or an inert substance that does not interact.

[0048] In some embodiments, the drug further comprises a pharmaceutically acceptable excipient.

[0049] As used herein, "pharmaceutically acceptable" refers to those ligands, materials, compositions, and / or dosage forms that are suitable for administration to a patient within the scope of sound medical judgment and commensurate with a reasonable benefit / risk ratio.

[0050] In some embodiments, the excipients are selected from, but are not limited to, one or more of the following excipient categories: diluents, wetting agents, binders, disintegrants, solvents, solubilizers, pH adjusters, and isotonic or isotonic agents. Diluents may include, but are not limited to, starches, sugars, celluloses, and inorganic salts. Wetting agents may include, but are not limited to, water. Binders may include, but are not limited to, starch slurry, dextrin, sugars, cellulose derivatives, and gelatin. Disintegrants may include, but are not limited to, starch. Solvents may include, but are not limited to, water, ethanol, and glycerol. Solubilizers may include, but are not limited to, Tweens. pH adjusters may include, but are not limited to, hydrochloric acid, sodium hydroxide, and sodium bicarbonate. Isotonic or isotonic agents may include, but are not limited to, glucose, sodium chloride, sodium citrate, sorbitol, and xylitol.

[0051] In some embodiments, the drug can be administered using a suitable route of administration according to clinical needs, and the route of administration may include but is not limited to oral administration, enema administration, or parenteral administration.

[0052] In some embodiments, the drug for preventing or treating fatty liver disease contains an effective amount of Eubacterium mucilaginosus.

[0053] In this application, "effective amount" refers to the dosage of the component corresponding to the term to achieve the treatment, prevention, alleviation and / or relief of a specific disease, condition and / or symptom in a subject. In this application, unless otherwise specified, it refers to the dosage to achieve the treatment, prevention, alleviation and / or relief of hypertensive diseases, conditions and / or symptoms.

[0054] In this application, a "subject" is an animal, preferably a mammal, more preferably a human, and includes, but is not limited to, a patient with a disease, condition, and / or symptom. The subject in this application is preferably a mammal. The term "mammal" primarily refers to a warm-blooded vertebrate mammal.

[0055] In some embodiments, the subject is an APOE- / - mouse.

[0056] In the present application, "APOE- / - mice" refer to apolipoprotein E gene knockout mice.

[0057] In some embodiments, the subject has abnormalities in at least one of the following indicators in the liver:

[0058] Abnormal low-density lipoprotein levels;

[0059] Abnormal proportion of fat droplets in the liver;

[0060] abnormal expression levels of SCD1 or CHREBP;

[0061] The expression level of LCAT was abnormal.

[0062] In this application, "abnormal" means that the detection value of an indicator deviates from the normal reference range or the expected value under physiological conditions, which may appear to be increased or decreased.

[0063] In some embodiments, an abnormal low-density lipoprotein level refers to an elevated low-density lipoprotein level. The low-density lipoprotein level can be detected by conventional detection methods in the art. When the detection value is significantly higher than the upper limit of the normal reference range of a healthy control group of the same species and under the same feeding conditions (for example, through statistical analysis, p < 0.05), it is determined to be an abnormal low-density lipoprotein level.

[0064] In some embodiments, an abnormal liver fat droplet ratio refers to an increased liver fat droplet ratio. Under normal physiological conditions, the liver fat droplet content is extremely low. In a pathological examination of liver tissue sections, when the proportion of fatty degenerated hepatocytes significantly exceeds the normal threshold (for example, statistically significantly higher than the healthy control group), it is determined to be an abnormal liver fat droplet ratio.

[0065] In some embodiments, abnormal expression level of SCD1 or CHREBP refers to increased expression level of SCD1 or CHREBP. The expression level of SCD1 or CHREBP can be detected by conventional detection methods in the art. When the detection value is significantly higher than the average level of a healthy control group of the same species and under the same feeding conditions (for example, through statistical analysis, p<0.05), it is determined that the expression level of SCD1 or CHREBP is abnormal.

[0066] In some embodiments, the abnormal expression level of LCAT refers to a decrease in the expression level of LCAT. The expression level of LCAT can be detected by conventional detection methods in the art. When the detection value is significantly lower than the average level of a healthy control group of the same species and under the same feeding conditions (for example, by statistical analysis, p<0.05), it is determined that the expression level of LCAT is abnormal.

[0067] In some embodiments, the drug for preventing or treating fatty liver disease contains a therapeutically effective amount of Eubacterium limosum.

[0068] In this application, "therapeutically effective amount" refers to the amount of the active ingredient of the drug that will cause a biological or medical response in an individual to a disease, disease and / or symptom, for example, the amount of the compound of the present invention that brings about a physiological and / or pharmacologically positive effect to an individual, and the physiological and / or pharmacologically positive effects include but are not limited to reducing or inhibiting enzyme or protein activity or improving symptoms, alleviating symptoms, slowing or delaying disease progression or preventing disease, etc.

[0069] In some embodiments, Eubacterium mucilaginosus is used as the sole probiotic in the medicament.

[0070] In some embodiments, the concentration of Eubacterium mucilaginosum in the medicament is not less than 1×10 8 CFU / mL.

[0071] In a second aspect of the present application, there is provided the use of the above-mentioned Eubacterium mucilaginosum in the preparation of a preparation having at least one of the following functions:

[0072] preparing a preparation for suppressing blood lipid levels;

[0073] Preparation of a preparation for inhibiting hepatic fat accumulation.

[0074] In some embodiments, the formulation is a pharmaceutical formulation.

[0075] In some embodiments, the pharmaceutical preparation is a liquid preparation. Liquid preparations refer to preparations containing a liquid phase, including, but not limited to, solutions, suspensions, emulsions, and the like.

[0076] A third aspect of the present application provides a method for preventing and treating fatty liver disease, comprising administering an effective amount of Eubacterium limosum to a subject.

[0077] In some embodiments, the drug is administered by routes including, but not limited to, oral or rectal administration.

[0078] In some embodiments, the drug may be administered orally, by enema, or parenterally.

[0079] In some embodiments, the drug administration cycle can be intermittent administration, periodic administration, continuous administration, or long-term administration.

[0080] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups or tinctures. In addition to the active ingredient, the liquid dosage form may contain an inert diluent conventionally used in the art, such as water.

[0081] Compositions for parenteral injection may comprise physiologically acceptable sterile aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous or nonaqueous carriers, diluents, solvents or excipients include water.

[0082] The "active ingredient" in the above-mentioned pharmaceutical preparation refers to the component in the pharmaceutical composition that can exert the "drug" effect. It is understood that the drugs of the embodiments of the present application can be added with various pharmaceutically acceptable excipients to prepare suitable clinical dosage forms, which include but are not limited to the dosage forms described above.

[0083] Some examples are provided below.

[0084] The embodiments of the present application will be described in detail below with reference to the examples. It should be understood that these examples are intended to illustrate the present application only and are not intended to limit the scope of the present application. The experimental methods for which the conditions are not specified in the following examples are preferably referred to the guidance provided in the present application, and may also be based on the experimental manuals or conventional conditions in this area, or on the conditions recommended by the manufacturer, or with reference to experimental methods known in the art.

[0085] In the following examples, the measured parameters of raw material components may have slight deviations within the range of weighing accuracy unless otherwise specified. For temperature and time parameters, acceptable deviations caused by instrument testing accuracy or operating accuracy are allowed.

[0086] Example 1

[0087] 1. Preparation of strains and establishment and grouping of animal models of non-alcoholic fatty liver disease

[0088] Lactobacillus rhamnosus was purchased from Guangdong Institute of Microbiology, with the strain number GDMCC 1.2223; Eubacterium limosum was purchased from the JCM strain bank in Japan, with the strain number JCM6421.

[0089] Resuscitation of Myxobacterium: Dissolve the lyophilized powder completely in 1 mL of sterile PBS solution, pipette 500 µL of the bacterial solution into BHI broth (brain heart infusion broth), and then spread 100 µL of the bacterial solution onto a BHI agar plate. Incubate the plate at 37°C in an anaerobic chamber for 72 hours, and store the remaining bacterial solution at -80°C. Subculture: Pipette the revived bacterial solution into fresh BHI broth and subculture. Take 2 mL of the bacterial solution for each generation and freeze it. Subculture the remaining bacterial solution until the third generation, and then expand the culture at the fourth generation. Count the colonies of the cultured bacterial solution, and then resuspend the bacteria in PBS buffer containing 25% glycerol to a concentration of 1x10 8 CFU / mL, and then aliquot and store at -80℃ until use.

[0090] Lactobacillus resuscitation: Take 1mL of sterile PBS solution to completely dissolve the lyophilized powder, draw 500µL of the bacterial solution and add it to MRS broth, then take 100µL of the bacterial solution and spread it on an MRS agar plate. Incubate at 37℃ in an anaerobic chamber for 48h, and store the remaining bacterial solution at -80℃. Passage: Aspirate the revived bacterial solution into fresh MRS broth and subculture. Take 2mL of the bacterial solution for each generation and freeze it. The remaining bacterial solution is subcultured until the third generation, and then expanded in the fourth generation; count the colonies of the cultured bacterial solution, and then resuspend the bacteria in PBS buffer containing 25% glycerol to a bacterial concentration of 1x108 CFU / mL, and then aliquot and store at -80℃ until use.

[0091] A high-fat diet was used to induce non-alcoholic fatty liver disease in ApoE- / - mice (reference: Polysaccharides from Ostrea rivularis rebuild the balance of gut microbiotato ameliorate non-alcoholic fatty liver disease in ApoE- / - mice). After adaptive feeding, 44 7-week-old ApoE- / - mice were randomly divided into 6 groups, half male and half female. The specific group settings were as follows: 6 mice in the high-fat diet group (HFD) were fed a high-fat and high-choline diet (42% fat + 1.25% cholesterol + 1% choline), and 0.2 mL of sterile distilled water was gavaged every day; 8 mice in the high-fat lactobacillus group (HFD+LGG) were fed a high-fat and high-choline diet, and 1×10 8 CFU / mL Lactobacillus rhamnosus (LGG) bacterial solution 0.2 mL; high-fat myxobacterium group (HFD+E.limosum) 8 mice, fed with high-fat and high-choline feed, gavage 1×10 8 CFU / mL E.limosum bacterial solution 0.2mL; Lactobacillus common feed group (Chow+LGG) 8 mice, fed with common feed, gavage 1×10 8 CFU / mL of Lactobacillus rhamnosus bacterial solution 0.2mL; Chow+E.limosum common feed group 8 mice, fed with common feed, gavage 1×10 8 The Chow group consisted of six mice fed a standard chow diet and gavaged with 0.2 mL of sterile distilled water daily for 16 weeks. Feces were collected every two weeks during the experiment and stored at -80°C. Fecal samples were collected one week before the end of the experiment. The day before dissection, mice were deprived of food and water for 12 hours and sacrificed. Blood, small intestine, cecum, liver, and heart tissues were collected. The intact heart and a portion of the liver were fixed with 4% paraformaldehyde, and the remaining tissue samples were stored in cryovials at -80°C.

[0092] 2. qPCR Amplification of Intestinal Bacteria Colonization

[0093] Total DNA was extracted according to the instructions of a fecal genomic DNA extraction kit. The extracted fecal DNA was used as a template for 16sRNA amplification of the V3-V4 variable region (upstream primer: 5'-GTGCCAGCMGCCGCGGTAA-3' (SEQ ID NO: 1), downstream primer: 5'-GGACTACHVGGGTWTCTAAT-3' (SEQ ID NO: 2)). Amplification was also performed using primers specific for Myxobacterium (upstream primer: 5'-GGCTTGCTGGACAAATACTG-3' (SEQ ID NO: 3), downstream primer: 5'-CTAGGCTCGTCAGAAGGATG-3' (SEQ ID NO: 4)) and Lactobacillus (upstream primer: 5'-CCAAATTGGCAACAGACCTT-3' (SEQ ID NO: 5), downstream primer: 5'-GCCATCTGGTGCTTTTGTTT-3' (SEQ ID NO: 6)). The amplification system was 20 µL. See Table 1 for details. The specific PCR amplification procedures are shown in Table 2. After qPCR amplification, the Ct value (threshold cycle) of each group of samples was calculated, and the 16sRNA V3-V4 variable region was used as the internal reference gene. -ΔΔCt The relative expression values ​​were calculated.

[0094] Table 1 PCR reaction system

[0095]

[0096] Table 2 PCR amplification program

[0097]

[0098] The feces of mice at week 3 and week 9 of the experiment were tested. For example, the results were as follows: Figure 1 As shown, Figure 1 AD in the figure represents the colonization of E.limosum and LGG in the intestine of mice in the third week of the experiment. Figure 1 The EH in the figure represents the colonization of E. limosum and LGG in the mouse intestines at week 9 of the experiment. The test results show that E. limosum and LGG can colonize in the mouse intestines, and the number of colonization sites generally accumulates with the increase of gavage time.

[0099] 3. Effects of Eubacterium mucilaginosus on blood lipid levels in mice

[0100] The blood lipid levels in the serum of the above modeling mice were detected, and the results were shown as follows: Figure 2As shown in the results, the probiotic E. limosum supplementation significantly reduced the serum LDL (low-density lipoprotein) level in the high-fat diet group mice compared with that after LGG intervention.

[0101] Effects of Eubacterium muciniphila on liver fat accumulation in mice

[0102] HE staining was performed on the liver of the above modeling mice. For example, the results were as follows: Figure 3 As shown in Figures A and B, the results show that mice fed a high-fat diet (high-fat diet group) all developed fatty liver degeneration, confirming the successful establishment of the aforementioned animal model of non-alcoholic fatty liver disease. Following intervention with the probiotics E. limosum and LGG, the E. limosum-supplemented group (high-fat Myxobacterium group) showed significantly smaller liver lipid droplet areas, demonstrating that E. limosum has a positive effect on reducing fat accumulation in the liver.

[0103] 5. Effects of Eubacterium mucilaginosus on genes related to hepatic lipid synthesis

[0104] The lipid synthesis-related genes in the liver of the above-mentioned modeling mice were detected. For example, the results were as follows: Figure 4 As shown in A, B and C, the results showed that compared with LGG intervention, E. limosum significantly reduced the expression of lipid synthesis-related genes SCD1 and CHREBP, and significantly increased the expression of lipid transfer-related gene LCAT.

[0105] The technical features of the above-mentioned embodiments can be combined arbitrarily. In order to make the description concise, not all possible combinations of the technical features in the above-mentioned embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0106] The above-described embodiments merely represent several implementation methods of the present application. While the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent. It should be noted that a person skilled in the art could make several modifications and improvements without departing from the spirit of the present application, all of which fall within the scope of protection of the present application. Therefore, the scope of protection of the patent in this application shall be based on the appended claims, and the specification and drawings may be used to interpret the claims.

Claims

1. Use of Eubacterium limosum in the preparation of a medicament for preventing and treating non-alcoholic fatty liver disease, characterized in that: The deposit number of the Eubacterium mucilaginosum is JCM 6421.

2. The use according to claim 1, characterized in that The Eubacterium mucilaginosum includes live Eubacterium mucilaginosum bacteria, inactivated Eubacterium mucilaginosum bacteria or a combination thereof.

3. The use according to claim 1, characterized in that The drug also includes pharmaceutically acceptable excipients; Optionally, the pharmaceutically acceptable excipients include one or more of a diluent, a wetting agent, a binder, a disintegrant, an aqueous solvent, a solubilizer, a pH regulator, and an isotonic or isotonic regulator.

4. The use according to claim 3, characterized in that The dosage form of the medicine is a liquid preparation.

5. The use according to claim 4, characterized in that The dosage form of the drug includes at least one of a suspension, an oral solution and an enema.

6. The use according to any one of claims 1 to 5, characterized in that In the medicine, Eubacterium mucilaginosus serves as a single probiotic.

7. The use according to any one of claims 1 to 5, characterized in that The subject is a mammal; optionally, the subject is a human or a mouse.

8. The use according to claim 7, characterized in that The subject's liver shows abnormalities in at least one of the following indicators: Abnormal low-density lipoprotein levels; Abnormal proportion of fat droplets in the liver; abnormal expression levels of SCD1 or CHREBP; The expression level of LCAT was abnormal.

9. Use of Eubacterium mucilaginosum with the accession number JCM 6421 in the preparation of a preparation having at least one of the following functions: Preparations that suppress blood lipid levels; An agent that inhibits fat accumulation in the liver.

10. The use according to claim 9, characterized in that The preparation is a pharmaceutical preparation.

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