Application of Akkermansia muciniphila in preparing a composition for improving alcoholic liver dysfunction, composition, and application thereof

Abstract

The present invention relates to the use of Akkermansia muciniphila in the preparation of a pharmaceutical composition for improving alcoholic liver dysfunction. The Akkermansia muciniphila is Akkermansia muciniphila AM06, Akkermansia muciniphila AM02, or a combination of the two; wherein the deposit number of AM06 is CGMCC No. 22793, and the deposit number of AM02 is CGMCC No. 22794. Improving alcoholic liver dysfunction includes preventing and treating alcoholic fatty liver. In this application, liver function indicators (such as ALT and AST) can be improved by reducing triglyceride levels and total cholesterol in the liver and serum, reducing liver fatty degeneration, etc., thereby effectively preventing the occurrence, development, and deterioration of alcoholic fatty liver. It also relates to the use of a composition containing the Akkermansia muciniphila in the preparation or use of a medicine for preventing and treating alcoholic fatty liver.

Description

Technical Field

[0001] The present invention relates to the technical field of prevention and treatment of alcoholic fatty liver disease, and in particular to the use of Akkermansia muciniphila in preparing a pharmaceutical composition for improving alcoholic liver dysfunction, and further relates to the use of a composition containing Akkermansia muciniphila in preparing or as a medicament for preventing and treating alcoholic fatty liver disease, and further relates to a composition containing Akkermansia muciniphila. Background Art

[0002] Alcoholic fatty liver disease (AFLD) is a liver disease caused by long-term, heavy alcohol consumption. It is a subtype of alcoholic liver disease (ALD) and one of the most common chronic forms of the disease worldwide. AFLD patients have a history of long-term alcohol consumption, generally exceeding five years. It is characterized by lipid accumulation exceeding 50% in hepatocytes, dysbiosis of the intestinal flora, increased permeability, and altered levels of bile acids, ethanol, and choline metabolites. Clinical symptoms are nonspecific and may be asymptomatic or include right upper abdominal distension and pain, loss of appetite, fatigue, and weight loss. While AFLD was long considered a benign condition, increasing evidence suggests that AFLD represents an underlying pathological condition. In the initial stages of the disease, triglycerides accumulate in hepatocytes, leading to the development of fatty liver (steatosis). With continued alcohol consumption and in the presence of other co-morbid factors, such as hepatitis virus infection, diabetes, smoking, or drug exposure, steatosis can progress to steatohepatitis, fibrosis, cirrhosis, and even hepatocellular carcinoma.

[0003] Currently, there are no specific medications for AFLD, and treatment options are not clearly differentiated from other types of ALD. Based on the pathogenesis and clinical pathways of ALD, treatment options include anti-inflammatory cytokines, antioxidants, immune system activation, and alterations in the gut microbiome. Widely used ALD treatment options include alcohol abstinence, glucocorticoids, biologics such as anti-TNF-α drugs, and liver transplantation, but these treatments do not always meet actual needs. Summary of the Invention

[0004] Based on this, the invention objectives of the present application include providing the use of Akkermansia muciniphila in the preparation of a pharmaceutical composition for improving alcoholic liver dysfunction, and also include providing the use of a composition containing Akkermansia muciniphila in the preparation or as a medicine for preventing and treating alcoholic fatty liver disease, wherein the Akkermansia muciniphila is Akkermansia muciniphila AM06 (deposit number CGMCC No. 22793), Akkermansia muciniphila AM02 (deposit number CGMCC No. 22794) or a combination of the two.

[0005] In a first aspect of the present invention, there is provided a use of Akkermansia muciniphila in preparing a pharmaceutical composition for improving alcoholic liver dysfunction, wherein the Akkermansia muciniphila is Akkermansia muciniphila AM06, Akkermansia muciniphila AM02, or a combination thereof; wherein,

[0006] The Akkermansia muciniphila AM06 was deposited in the General Microbiology Center of the China Culture Collection Administration on June 28, 2021, with the deposit number CGMCC No. 22793;

[0007] The Akkermansia muciniphila AM02 was deposited in the General Microbiology Center of the China Culture Collection Administration on June 28, 2021, with the deposit number CGMCC No. 22794.

[0008] In some embodiments, the alcoholic liver dysfunction includes at least one of alcoholic steatosis, alcoholic steatohepatitis, liver fibrosis, and liver cirrhosis.

[0009] In some embodiments, improving alcoholic liver dysfunction includes preventing and / or treating alcoholic fatty liver;

[0010] Preferably, the alcoholic fatty liver includes at least one of alcoholic steatosis, alcoholic steatohepatitis, liver fibrosis and liver cirrhosis.

[0011] In some embodiments, the Akkermansia muciniphila AM06 and the Akkermansia muciniphila AM02 are each independently live bacteria, inactivated bacteria, or a combination thereof.

[0012] In some embodiments, the pharmaceutical composition includes the Akkermansia muciniphila and a pharmaceutically acceptable carrier.

[0013] In some embodiments, the pharmaceutical composition is a medicine, and its dosage form is tablets, capsules, granules, pills, ointments, solutions, suspensions, emulsions, creams, sprays, drops, patches or tube feeding preparations.

[0014] In a second aspect of the present invention, there is provided use of a composition containing Akkermansia muciniphila in the preparation of or as a medicament for preventing and treating alcoholic fatty liver disease, wherein the Akkermansia muciniphila is as defined in the first aspect of the present invention.

[0015] In some embodiments, the composition containing Akkermansia muciniphila is a composite probiotic, and the composite probiotic further contains a probiotic different from the Akkermansia muciniphila;

[0016] Preferably, the probiotic bacteria different from the Akkermansia muciniphila include one or more of Bacteroides fragilis, Christensenella, Saccharomyces boulardii, Enterococcus hilarii, Lactobacillus rhamnosus and Lactobacillus bifidum.

[0017] In some embodiments, the composition containing Akkermansia muciniphila further comprises a second active ingredient, wherein the second active ingredient is a drug different from the Akkermansia muciniphila;

[0018] Preferably, the second active ingredient comprises one or more of simvastatin, diammonium glycyrrhizinate, prototype glutathione, silybin, hepatocyte growth promoting factor and fenofibrate;

[0019] Also preferably, the second active ingredient comprises simvastatin.

[0020] In a third aspect of the present invention, there is provided a composition containing Akkermansia muciniphila, comprising the Akkermansia muciniphila defined in the first aspect of the present invention and a second active ingredient;

[0021] Preferably, the second active ingredient comprises one or more of simvastatin, diammonium glycyrrhizinate, prototype glutathione, silybin, hepatocyte growth promoting factor and fenofibrate;

[0022] Also preferably, the second active ingredient comprises simvastatin.

[0023] The inventors of the present application isolated strains AM06 (deposit number CGMCC No. 22793) and AM02 (deposit number CGMCC No. 22794). Based on comprehensive analyses including 16S rRNA analysis, morphological analysis, metabolite composition analysis, and efficacy analysis (such as tolerance to artificial gastric and artificial intestinal fluids, the ability to inhibit inflammatory factors from destroying tight junction proteins in intestinal cells, and the effect of inhibiting LPS-induced liver slice hepatitis), both strains belong to Akkermansia muciniphila and are identified as new strains of Akkermansia muciniphila that are different from ATCC BAA-835 (standard strain).

[0024] The inventors have discovered that the isolated Akkermansia muciniphila AM06 and / or AM02 can be used to improve alcoholic liver dysfunction, including but not limited to preventing and treating alcoholic fatty liver disease (preventing and / or treating alcoholic fatty liver disease). Therefore, the isolated Akkermansia muciniphila AM06 and / or AM02 can be used to prepare pharmaceutical compositions, and further, pharmaceuticals. The isolated Akkermansia muciniphila AM06 and / or AM02 can effectively prevent the development, progression, and worsening of alcoholic fatty liver disease by reducing triglyceride (TG) levels and total cholesterol in the liver and serum, improving liver function indicators (such as ALT and AST), and reducing hepatic steatosis, thereby improving patients' quality of life. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the technical solutions in the embodiments of the present application and to more fully understand the present application and its beneficial effects, the following is a brief introduction to the drawings required for describing the embodiments. Obviously, the drawings described below are only some embodiments of the present application. Those skilled in the art can also derive other drawings based on these drawings without inventive effort.

[0026] Figure 1 This is a colony characteristic diagram of Akkermansia muciniphila AM02 cultured in one embodiment of the present invention;

[0027] Figure 2 This is a colony characteristic diagram of Akkermansia muciniphila AM06 obtained in one embodiment of the present invention;

[0028] Figure 3 This is a microscopic observation of Akkermansia muciniphila AM02 cultured in one embodiment of the present invention after Gram staining;

[0029] Figure 4 This is a microscopic observation of Akkermansia muciniphila AM06 cultured in one embodiment of the present invention after Gram staining;

[0030] Figure 5 This is a PCA analysis chart of metabolites from the culture supernatant of several Akkermansia muciniphila in one embodiment of the present invention;

[0031] Figure 6 These are fluorescence microscopy images showing the effects of several strains of Akkermansia muciniphila on the reduction of tight junction protein ZO-1 expression in Caco2 cells induced by TNF-α and IFN-γ in one embodiment of the present invention.

[0032] The Akkermansia muciniphila AM06 isolated in the present invention is classified and named Akkermansia muciniphila. It was deposited in the General Microbiology Center of China Culture Collection Administration on June 28, 2021, with the address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with the deposit number CGMCC No. 22793. The strain was received and registered by the collection center on June 28, 2021, and was detected as a viable strain by the collection center on June 28, 2021.

[0033] The Akkermansia muciniphila AM02 isolated in the present invention is classified and named Akkermansia muciniphila. It was deposited in the General Microbiology Center of China Culture Collection Administration on June 28, 2021, with the address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with the deposit number CGMCC No. 22794. The strain was received and registered by the collection center on June 28, 2021, and was detected as a viable strain by the collection center on June 28, 2021. DETAILED DESCRIPTION

[0034] The present invention will be further described in detail below in conjunction with the accompanying drawings, embodiments and examples. It should be understood that these embodiments and examples are only used to illustrate the present invention and are not used to limit the scope of the invention. The purpose of providing these embodiments and examples is to make the understanding of the disclosure of the present invention more thorough and comprehensive. It should also be understood that the present invention can be implemented in many different forms and is not limited to the embodiments and examples described herein. Those skilled in the art can make various changes or modifications without violating the connotation of the present invention, and the equivalent forms obtained also fall within the protection scope of the present invention. In addition, in the description below, a large amount of specific details are given in order to provide a more complete understanding of the present invention. It should be understood that the present invention can be implemented without one or more of these details.

[0035] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention herein are only for the purpose of describing embodiments and examples and are not intended to limit the present invention.

[0036] the term

[0037] Unless otherwise specified or incompatible herewith, the terms and phrases used herein shall have the following meanings:

[0038] The terms "and / or", "or / and", and "and / or" used herein include any one of two or more related listed items, and also include any and all combinations of the related listed items, wherein the arbitrary and all combinations include any combination of two related listed items, any more related listed items, or all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or", "or / and", and "and / or", it should be understood that in this application, the technical solution undoubtedly includes technical solutions connected by "logical and" and also undoubtedly includes technical solutions connected by "logical or". For example, "A and / or B" includes three parallel solutions of A, B and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, the technical solution of all being connected by "logical OR"), and also includes any and all combinations of A, B, C, and D, that is, the combination of any two or any three of A, B, C, and D, and also includes the four-item combination of A, B, C, and D (that is, the technical solution of all being connected by "logical AND").

[0039] In the present invention, "plurality", "multiple", "multiple times", etc., unless otherwise specified, refer to a number greater than or equal to 2. For example, "one or more" means one or more than or equal to two.

[0040] As used herein, "combination thereof", "any combination thereof", "any combination thereof" and the like include all suitable combinations of any two or more of the listed items.

[0041] Herein, the “suitable” mentioned in “suitable combination”, “suitable method”, “any suitable method”, etc. shall be based on the ability to implement the technical solution of the present invention, solve the technical problem of the present invention, and achieve the expected technical effect of the present invention.

[0042] Herein, the terms "preferred," "better," "more preferred," and "suitable" are merely used to describe preferred implementations or examples and should not be construed as limiting the scope of protection of the present invention. If multiple "preferred" terms appear in a technical solution, each "preferred" term is considered independent unless otherwise specified and there are no contradictions or mutual constraints.

[0043] In the present invention, “further”, “further”, “particularly”, etc. are used for descriptive purposes to indicate differences in content, but should not be understood as limiting the scope of protection of the present invention.

[0044] In the present invention, the terms "optionally," "optional," and "optional" mean optional or dispensable, i.e., they refer to either option being selected from two parallel options: "with" or "without." If a technical solution contains multiple "optional" clauses, each "optional" clause is independent unless otherwise specified and there are no conflicts or constraints.

[0045] In the present invention, the terms "first," "second," and "third," etc., in "the first aspect," "the second aspect," "the third aspect," etc., are used for descriptive purposes only and should not be construed as indicating or implying relative importance or quantity, nor as implicitly indicating the importance or quantity of the technical features indicated. Furthermore, "first," "second," and "third," etc., serve only as non-exhaustive enumeration and description and should be understood not to constitute closed-ended limitations on quantity.

[0046] In the present invention, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0047] In the present invention, 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 considered continuous and includes the two numerical endpoints (i.e., the minimum and maximum values) of the numerical interval, and each numerical value between the two numerical endpoints. Unless otherwise specified, when a numerical interval refers only to integers within the numerical interval, including the two endpoint integers of the numerical range, and each integer between the two endpoints, is equivalent to directly listing each integer. When multiple numerical ranges are provided to describe features or characteristics, these numerical ranges can be merged. 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, a percentage, a ratio, etc. "Numerical interval" allows for broadly including quantitative intervals such as percentage intervals, ratio intervals, and ratio intervals.

[0048] 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.

[0049] In the present invention, the term "room temperature" generally refers to 4°C to 35°C, preferably 20°C ± 5°C. In some embodiments of the present invention, room temperature refers to 20°C to 30°C.

[0050] In the present invention, 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. For example, 3-5h means that the units of the left endpoint "3" and the right endpoint "5" are both h (hours).

[0051] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as a reference separately. Unless they conflict with the invention purpose and / or technical solution of this application, the cited documents involved in this invention are cited with all their contents and all their purposes. When the present invention involves cited documents, the definitions of relevant technical features, terms, nouns, phrases, etc. in the cited documents are also cited. When the present invention involves cited documents, the examples and preferred embodiments of the cited relevant technical features may also be incorporated into this application as references, but are limited to the ability to implement the present invention. It should be understood that when the cited content conflicts with the description in this application, the present application shall prevail or be modified adaptively according to the description in this application.

[0052] The pathogenesis of alcoholic fatty liver disease includes the following three aspects:

[0053] (1) Ethanol and nutrition

[0054] Ethanol is a significant source of energy, containing 7.1 kcal (29.7 kcal) per gram, a value that exceeds the energy content of carbohydrates or protein. On average, when alcohol comprises half of total intake, it can displace normal nutrients, leading to malnutrition, including deficiencies in folate, thiamine, and other vitamins. Secondary malnutrition can also occur through malabsorption caused by gastrointestinal complications, such as pancreatic insufficiency and impaired hepatic metabolism of nutrients. Furthermore, alcohol promotes nutrient degradation, as exemplified by its effects on vitamin A. In some experiments, nutrient deficiency has been shown to lead to liver damage, suggesting this is also a mechanism for the development of alcoholic liver disease. The interplay of nutrient deficiencies caused by primary or secondary malnutrition and the direct toxicity of ethanol may affect the function and structure of the liver and gastrointestinal tract. Studies in mice and human volunteers have also demonstrated that alcohol can induce fatty liver disease even under appropriate dietary conditions, and that blood alcohol concentrations do not necessarily result in toxicity. Increased dietary long-chain triglycerides enhance alcohol-induced steatosis, while medium-chain triglycerides attenuate this effect. Reducing dietary fat reduces steatosis; however, this effect is reversed when the fat content of the diet drops below 10%, likely due to the increased carbohydrate content of the diet. Alcohol's effects have been shown to be due to its metabolism in the liver.

[0055] (2) Toxic effects of alcohol oxidation

[0056] Ethanol is oxidized via the ADH (acetaldehyde dehydrogenase) pathway to produce acetaldehyde, which is then converted to acetate. Both reactions reduce nicotinamide adenine dinucleotide (NAD) to NADH. Excessive NADH can lead to numerous metabolic disorders, including inhibition of the Krebs cycle and fatty acid oxidation, which in turn contributes to steatosis and hyperlipidemia.

[0057] (3) Microsomal Ethanol Oxidation System (MEOS)

[0058] Following chronic ethanol consumption, adaptive increases in ethanol metabolism occur, raising the possibility that additional pathways exist. This system, termed MEOS, has been demonstrated in vitro in liver microsomes and found to be induced in vivo by chronic ethanol feeding. The key enzyme in MEOS is the ethanol-inducible cytochrome P450 family member 2E1 (CYP2E1). This enzyme is found to be increased 4- to 10-fold in liver biopsies from alcohol-consuming subjects, with a corresponding increase in mRNA. This induction contributes to metabolic tolerance to ethanol (in addition to central nervous system tolerance). The most common cause of CYP2E1 induction is early alcoholic liver injury, such as alcoholic steatosis and alcoholic steatohepatitis, which can be understood from the physiological role of CYP2E1. CYP2E1 is hypothesized to have a dual role: one of detoxification and the other of nutritional support. CYP2E1 contributes to the body's defense mechanisms against the penetration of toxic foreign substances, as indicated by its location of entry and inducibility, as well as its broad substrate specificity. Induction of CYP2E1 has been shown to play a key role in the pathogenesis of alcoholic liver injury, including alcoholic steatohepatitis, and may be related to the generation of oxidative stress.

[0059] While alcohol abuse and nutrition play a central role in disease pathogenesis, preclinical models support a role for the gut microbiome in ALD. Acute and chronic alcohol consumption increases gastrointestinal permeability, potentially facilitating the translocation of bacterial endotoxins, such as lipopolysaccharide (LPS). Elevated levels of endotoxins have been found in acute alcoholics and patients at any stage of ALD. Compared with controls, the gut microbiome differs after alcohol consumption / feeding, and levels of toxic ethanol metabolites are elevated in the liver. Endotoxemia appears to correlate with disease severity in ALD. Linking the microbiome, intestinal pathology, and liver disease, alcohol consumption or a diet rich in saturated fatty acids, cholesterol, and fructose weakens mucus-associated defenses, thereby disrupting the intestinal barrier. Inflammation mediated by bacterial products is closely linked to activation of the innate immune system through Toll-like receptors (TLRs). Studies in mice have further demonstrated that alcohol consumption reduces intestinal immunoglobulin A (IgA) plasma cells, potentially leading to weakened mucosal defenses. Further studies have demonstrated a close connection between ethanol metabolism, the circadian clock, and intestinal permeability. Circadian rhythms regulate gastrointestinal function, and disruptions in this rhythm contribute to intestinal damage and ALD. Alcohol disrupts circadian rhythms by increasing Per2 protein in the duodenum and colon of rats. This disrupted circadian rhythm leads to increased intestinal permeability and ethanol-related liver damage. Furthermore, circadian rhythms regulate the expression of TLRs, suggesting that dysregulated microbial mucus degradation and host immune dysfunction may enhance epithelial damage. Endotoxins, upon entering the portal vein, mediate liver injury by activating Kupffer cells through pathogen recognition receptors such as TLRs. Ligand binding leads to the release of proinflammatory cytokines such as TNF-α (tumor necrosis factor-α), IL-1β (interleukin-1β), IL-6, IL-12, IL-18, and reactive oxygen species (ROS) from Kupffer cells. TGF-β release activates hepatic stellate cells, increases extracellular matrix deposition, and contributes to liver fibrosis. Pathogen-associated molecular patterns may also directly damage hepatocytes. Data from patients with ALD suggest that alcohol abuse activates intestinal lamina propria macrophages, causing them to exhibit an activated phenotype. Markers of intestinal endothelial permeability are increased in patients with celiac disease and ALD who also have elevated transaminases. This suggests that, in addition to mucus, intestinal epithelial cells, and immune cells, the vascular wall may also control the systemic dissemination of bacteria or their products.

[0060] Early intervention is usually through the use of non-absorbable antibiotics to modify the microbiota, reduce endotoxemia, and improve liver disease.

[0061] The first aspect of the present invention

[0062] In a first aspect of the present invention, there is provided use of Akkermansia muciniphila in preparing a pharmaceutical composition for improving alcoholic liver dysfunction, wherein the Akkermansia muciniphila is Akkermansia muciniphila AM06, Akkermansia muciniphila AM02, or a combination of the two.

[0063] In the present invention, "Akkermansia muciniphila" is consistent with the common definition in the art, including but not limited to Akkermansia muciniphila AM06 and AM02.

[0064] In the present invention, "Akkermansia muciniphila" can be a living bacterium, or it can be an Akkermansia muciniphila that has been inactivated, genetically recombined, transformed or modified, attenuated, chemically treated, or physically treated and retains all or part of its original biological activity. It can also be a lysate of the bacterium, a culture (such as a supernatant), or a component extracted from the culture.

[0065] The inventors of the present application isolated strains AM06 (deposit number CGMCC No. 22793) and AM02 (deposit number CGMCC No. 22794). Based on comprehensive analyses including 16S rRNA analysis, morphological analysis, metabolite composition analysis, and efficacy analysis (such as tolerance to artificial gastric and artificial intestinal fluids, the ability to inhibit inflammatory factors from destroying tight junction proteins in intestinal cells, and the effect of inhibiting LPS-induced liver slice hepatitis), both strains belong to Akkermansia muciniphila and are identified as new strains of Akkermansia muciniphila that are different from ATCC BAA-835 (standard strain).

[0066] Akkermansia muciniphila (A. muciniphila) belongs to the phylum Verucomicrobia, family Akkermansiaceae, genus Akkermansia. A. muciniphila is a mucin-degrading, anaerobic, non-motile, non-spore-forming, oval, Gram-negative bacterium that grows optimally at temperatures between 20°C and 40°C and a pH between 5.5 and 8.0 (optimum temperature at 37°C and pH 6.5). It is ubiquitous in the human intestine. Although an anaerobic bacterium, it can tolerate a certain level of oxygen. A. muciniphila colonizes the mucus layer and colon by degrading and utilizing mucin as an energy source. Furthermore, mucin degradation produces short-chain fatty acids (SCFAs). The production of these SCFAs, including acetate, propionate, and butyrate, plays an important role in human health.

[0067] In the present invention, “Akermansia muciniphila according to the present invention” specifically refers to Akkermansia muciniphila AM06, Akkermansia muciniphila AM02 or a combination of the two, which can be expressed as “AM06 and / or AM02”.

[0068] Akkermansia muciniphila AM06 was deposited in the General Microbiology Center of the China Culture Collection Administration on June 28, 2021, with the deposit number CGMCC No. 22793. It can be isolated by the method of Example 1, and the strain can also be identified by one or more methods including but not limited to Examples 2 to 5.

[0069] In some embodiments, the colony culture characteristics of Akkermansia muciniphila AM06 include: rounded protrusions, neatly edged, opaque, white, and unevenly sized colonies. Furthermore, the colony size is approximately 0.08 to 2.2 mm.

[0070] In some embodiments, Akkermansia muciniphila AM06 is isolated from breast milk.

[0071] Akkermansia muciniphila AM02 was deposited in the General Microbiology Center of the China Culture Collection Administration on June 28, 2021, with the deposit number CGMCC No. 22794. It can be isolated by the method of Example 1, and the strain can also be identified by one or more methods including but not limited to Examples 2 to 5.

[0072] In some embodiments, the colony culture characteristics of Akkermansia muciniphila AM02 of CGMCC No. 22794 include: rounded, convex, neatly edged, opaque, white, and unevenly sized colonies, and further, the colony size is approximately 0.08 to 2.2 mm.

[0073] In some embodiments, the applicants also conducted artificial gastric and artificial intestinal fluid tolerance studies. In the artificial gastric fluid tolerance study, statistical analysis of the viable bacterial counts in different groups of 0.9wt% NaCl solution, pH 3 artificial gastric fluid, and pH 2 artificial gastric fluid groups after anaerobic incubation at 37±2°C for 0, 1.5, and 3 hours was performed. The results showed that the artificial gastric fluid tolerance of the Akkermansia muciniphila strain was in the order of AM02 > AM06 > standard strain ATCC BAA-835. In the artificial intestinal fluid tolerance study, statistical analysis of the viable bacterial counts after anaerobic incubation at 37±2°C for 0, 4, and 8 hours showed that the artificial intestinal fluid tolerance was better than AM06 > AM02 > ATCC BAA-835.

[0074] In some in vitro experiments, Akkermansia muciniphila AM06 and AM02 were better than the standard strain BAA-835 in tolerance to artificial gastric juice and artificial intestinal juice, ability to inhibit inflammatory factors from destroying tight junction proteins of intestinal cells, and ability to inhibit LPS-induced liver slice hepatitis.

[0075] Akkermansia muciniphila AM06 and AM02 can each independently be a living bacterium, or an inactivated Akkermansia muciniphila (i.e., inactivated bacteria, which can be fully or partially inactivated), or a bacterial lysate, culture (such as supernatant) or a component extracted from the culture.

[0076] In some embodiments, Akkermansia muciniphila AM06 and Akkermansia muciniphila AM02 are each independently live bacteria, inactivated bacteria, or a combination thereof (i.e., a combination of live bacteria and inactivated bacteria). The inactivated bacteria can be inactivated bacteria with complete morphology and structure, inactivated bacteria with incomplete morphology and structure, or a combination thereof.

[0077] In some embodiments, Akkermansia muciniphila AM06 and Akkermansia muciniphila AM02 are each independently one or more of live bacteria, inactivated bacteria with intact morphology and structure, and inactivated bacteria with incomplete morphology and structure.

[0078] In some embodiments, the Akkermansia muciniphila of the present invention is selected from live Akkermansia muciniphila cells.

[0079] In some embodiments, the Akkermansia muciniphila of the present invention is selected from inactivated Akkermansia muciniphila bacteria.

[0080] In the present invention, "morphologically intact" may include but is not limited to Akkermansia muciniphila that has been completely or partially inactivated through an inactivation treatment.

[0081] In the present invention, the situation of "incomplete morphological structure" may include but is not limited to bacterial lysate, culture (such as supernatant) or components extracted from the culture.

[0082] Through extensive experiments, the inventors have discovered that the isolated Akkermansia muciniphila AM06 and / or AM02 can be used to improve alcoholic liver dysfunction (including preventing and / or treating alcoholic fatty liver disease). The isolated Akkermansia muciniphila AM06 and / or AM02 can be used to prepare pharmaceutical compositions and, further, medicines. The isolated Akkermansia muciniphila AM06 and / or AM02 can reduce triglyceride (TG) levels and total cholesterol in the liver and serum, improve liver function indicators (ALT and AST), and reduce hepatic steatosis, thereby effectively preventing the development, progression, and worsening of alcoholic fatty liver disease and improving patients' quality of life.

[0083] In the present invention, "alcoholic liver dysfunction" refers to "liver dysfunction caused by dietary alcohol", such as alcoholic fatty liver, further such as one or more of alcoholic steatosis, alcoholic steatohepatitis, liver fibrosis and cirrhosis.

[0084] In the present invention, "improving alcoholic liver dysfunction" refers to providing physiological and / or pharmacological beneficial effects on liver dysfunction caused by dietary alcohol. "Improving alcoholic liver dysfunction" includes but is not limited to preventing and / or treating alcoholic fatty liver.

[0085] In some embodiments, the alcoholic liver dysfunction comprises at least one of alcoholic steatosis, alcoholic steatohepatitis, liver fibrosis, and liver cirrhosis.

[0086] In some embodiments, improving alcoholic liver dysfunction includes preventing and / or treating alcoholic fatty liver.

[0087] In some embodiments, alcoholic fatty liver disease comprises at least one of alcoholic steatosis, alcoholic steatohepatitis, liver fibrosis, and liver cirrhosis.

[0088] For preparing pharmaceutical compositions

[0089] In the present invention, "prevention and treatment" and "prevention and / or treatment" have the same meaning and can be used interchangeably. In the present invention, "prevention and treatment" includes aspects such as prevention, treatment, and adjuvant treatment. As used herein, "prevention and treatment" refers to alleviating, delaying progression, attenuating, preventing, or maintaining an existing disease or condition. "Prevention and treatment" also includes curing one or more symptoms of a disease or condition, preventing its development, or alleviating it to a certain extent. Among them, as used herein, "treatment" refers to alleviating, delaying progression, attenuating, or maintaining an existing disease or condition, and treatment also includes curing one or more symptoms of a disease or condition, preventing its development, or alleviating it to a certain extent.

[0090] In the present invention, a "composition" may be a combination of multiple substances, and further, may be used in combination or may be a mixture of the combination.

[0091] For purposes of this invention, "drug" includes any agent, compound, composition, or mixture that provides a pharmacological effect in vivo or in vitro, often with a beneficial effect. The scope of the pharmacological effect of 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.

[0092] In the present invention, a "pharmaceutical composition" refers to a composition that has the effect of preventing and treating a disease or condition and can be used as a medicine or can be used to prepare a medicine.

[0093] In the present invention, "drug" refers to a pharmaceutical preparation that can be directly administered, which usually has a prescribed usage and dosage.

[0094] In some embodiments, the composition for preventing and treating alcoholic fatty liver disease may be a composition for preventing and treating at least one of alcoholic steatosis, alcoholic steatohepatitis, liver fibrosis, and liver cirrhosis.

[0095] In some embodiments, the Akkermansia muciniphila improves one or more of liver weight, serum ALT (alanine aminotransferase) and AST (aspartate aminotransferase) levels, serum LDL-c (low-density lipoprotein cholesterol) levels, steatosis severity score, serum triglyceride (serum TG) levels, liver triglyceride (liver TG) levels, and serum endotoxin (e.g., lipopolysaccharide (LPS)) levels. In some embodiments, "improvement" is relative to not administering the Akkermansia muciniphila of the present invention. In some embodiments, "improvement" is relative to administering the standard strain ATCC BAA-835. See Examples 6-7.

[0096] In some embodiments, Akkermansia muciniphila AM06 and / or Akkermansia muciniphila AM02 can effectively prevent the occurrence, development and deterioration of alcoholic fatty liver disease by reducing triglyceride (TG) levels and total cholesterol in the liver and serum, improving liver function indicators (ALT and AST), reducing liver fatty degeneration, reducing liver fat mass, and inhibiting the expression of genes related to liver fat synthesis, thereby improving the quality of life of patients.

[0097] In some embodiments, the pharmaceutical composition comprises the Akkermansia muciniphila (AM06 and / or AM02) of the present invention and a pharmaceutically acceptable carrier.

[0098] In the present invention, "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.

[0099] In the present invention, a "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, solvent, or encapsulating material. As used herein, the phrase "pharmaceutically acceptable carrier" includes buffers, sterile water for injection, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, that are compatible with drug administration. Each carrier must be "pharmaceutically acceptable" in the sense of being compatible with the other ingredients in the formulation and not harmful to the patient. Suitable examples include, but are not limited to: (1) sugars such as lactose, glucose, and sucrose; (2) starches such as corn starch, potato starch, and substituted or unsubstituted β-cyclodextrins; (3) cellulose and its derivatives such as sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients such as cocoa butter and suppository waxes; (9) oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn starch, and maltose. Rice oil and soybean oil; (10) glycols such as propylene glycol; (11) polyols such as glycerol, sorbitol, mannitol and polyethylene glycol; (12) esters such as ethyl oleate and ethyl laurate; (13) agar; (14) buffers such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethanol; (20) phosphate buffer; and (21) other nontoxic compatible substances used in pharmaceutical formulations.

[0100] In the present invention, "carrier" includes but is not limited to mannitol, sorbitol, sodium metabisulfite, sodium bisulfite, sodium thiosulfate, cysteine hydrochloride, thioglycolic acid, methionine, vitamin C, disodium ethylenediaminetetraacetic acid (disodium EDTA), sodium calcium EDTA, carbonates, acetates, phosphates of monovalent alkali metals or their aqueous solutions, hydrochloric acid, acetic acid, sulfuric acid, phosphoric acid, amino acids, sodium chloride, potassium chloride, sodium lactate, xylitol, maltose, glucose, fructose, oligofructose, dextran, glycine, starch, sucrose, dextrin (such as maltodextrin), lactose, mannitol, silicon derivatives, cellulose and its derivatives, alginate, gelatin, polyvinyl pyrrolidone, glycerol, Tween 80, agar, calcium carbonate, calcium bicarbonate, surfactants, polyethylene glycol, cyclodextrin, phospholipid materials, kaolin, talc, calcium stearate, and magnesium stearate.

[0101] In some embodiments, the pharmaceutical composition may further include other active pharmaceutical ingredients. The other active pharmaceutical ingredients may be pharmaceutical ingredients having the effect of preventing and treating alcoholic fatty liver disease, or pharmaceutical ingredients having the effect of preventing and treating other diseases.

[0102] In some embodiments, other active pharmaceutical ingredients can be appropriately selected from existing alcoholic fatty liver prevention and treatment drugs.

[0103] Preparation type

[0104] In some embodiments, the pharmaceutical composition is a drug.

[0105] In the present invention, "drug" refers to a pharmaceutical preparation that can be directly administered, which usually has a prescribed usage and dosage, consistent with the above definition.

[0106] In some embodiments, the pharmaceutical preparation can be a liquid preparation or a solid preparation. Liquid preparations refer to preparations containing a liquid phase, and non-limiting examples include solutions, suspensions, emulsions, etc. Non-limiting examples of solid preparations include tablets, capsules, granules, pills, etc.

[0107] In some embodiments, depending on the mode of administration, the pharmaceutical preparation can be an oral preparation, an injection, a drop, a patch, a tube feeding preparation, etc.

[0108] In some embodiments, the dosage form of the drug is a tablet, capsule, granule, pill, ointment, solution, suspension, emulsion, cream, spray, drop, patch or tube feeding preparation.

[0109] It should be understood that the medicament contains a therapeutically effective amount of Akkermansia muciniphila (AM06 and / or AM02) of the present invention.

[0110] In the present invention, "therapeutically effective amount" refers to the amount of the active pharmaceutical ingredient that will elicit a biological or medical response in an individual for a disease, disorder and / or symptom, for example, the amount of Akkermansia muciniphila of the present invention that brings a pharmacologically positive effect to an individual, including but not limited to reducing or inhibiting enzyme or protein activity or improving symptoms, alleviating symptoms, slowing or delaying disease progression, or preventing disease.

[0111] In the present invention, the "subject" refers to a patient who consumes the pharmaceutical composition.

[0112] In the present invention, a "subject" is an animal, preferably a mammal, more preferably a human. The subject includes, but is not limited to, a patient suffering from a disease, condition, and / or symptom. The subject in the present invention is preferably a mammal. The term "mammal" primarily refers to warm-blooded vertebrate mammals, including, but not limited to, cats, dogs, rabbits, bears, foxes, wolves, monkeys, deer, mice (e.g., rats, mice), pigs, cattle, sheep, horses, humans, etc., preferably primates, and more preferably humans.

[0113] In some embodiments, the subject is a mammal.

[0114] In some embodiments, the subject is a human or other mammal. When the subject is other mammals, the drug may also be referred to as an "animal drug."

[0115] In some embodiments, the pharmaceutical composition is suitable for use in humans or other mammals. In the present invention, "other mammals" is not a human. Non-limiting examples of "other mammals" include cats, dogs, rabbits, bears, foxes, wolves, monkeys, deer, mice (e.g., rats, mice), pigs, cattle, sheep, horses, and further examples include mice. Non-limiting examples of "other mammals" include primates.

[0116] In some embodiments, the subject is a human or a mouse.

[0117] In the present invention, "patient" refers to an animal, preferably a mammal, such as a human or a mouse.

[0118] Different subjects may result in different selection ranges of other ingredients in the aforementioned pharmaceutical composition.

[0119] Second aspect of the present invention

[0120] In a second aspect, the present invention provides the use of a composition containing Akkermansia muciniphila in the preparation or use as a medicament for preventing or treating alcoholic fatty liver disease. The "composition containing Akkermansia muciniphila" herein refers to a composition containing "Ackermansia muciniphila (AM06 and / or AM02) described herein." The definition of "Ackermansia muciniphila described herein" can be found in the first aspect of the present invention.

[0121] Furthermore, the Akkermansia muciniphila described in the present invention is Akkermansia muciniphila AM06 (deposit number: CGMCC No. 22793), Akkermansia muciniphila AM02 (deposit number: CGMCC No. 22794), or a combination of the two.

[0122] In some embodiments, the composition containing Akkermansia muciniphila is a pharmaceutical composition.

[0123] In the second aspect of the present invention, the definitions of "composition," "prevention and treatment," "alcoholic liver dysfunction," "improvement of alcoholic liver dysfunction," "alcoholic fatty liver," "pharmaceutical composition," and "drug" may refer to the first aspect of the present invention. The definition of "drug" may also refer to the fourth aspect of the present invention.

[0124] The Akkermansia muciniphila described in the present invention can be a living bacterium, or an inactivated Akkermansia muciniphila (i.e., an inactivated bacterium, which can be completely or partially inactivated), or a lysate or culture (e.g., supernatant) of the bacterium, or a component extracted from the culture.

[0125] In some embodiments, the composition comprising Akkermansia muciniphila further comprises a second active ingredient.

[0126] In the present invention, the "second active ingredient" refers to an active ingredient other than the Akkermansia muciniphila (AM06 and / or AM02) of the present invention that has the effect of improving alcoholic liver dysfunction.

[0127] In some embodiments, the second active ingredient can be a modified strain of the Akkermansia muciniphila (AM06 and / or AM02) described herein. Modification methods include, but are not limited to, gene editing, chemical treatment, and physical treatment. The modified Akkermansia muciniphila forms a new strain distinct from AM06 and AM02. This modified strain can still improve alcoholic liver dysfunction (such as preventing and treating alcoholic fatty liver disease) and can also be endowed with other new pharmacological and / or physiological functions.

[0128] In some embodiments, the composition containing Akkermansia muciniphila is a composite probiotic. In this case, in addition to the Akkermansia muciniphila (AM06 and / or AM02) described in the present invention, the composition also contains other probiotics different from the Akkermansia muciniphila described in the present invention.

[0129] In the present invention, a "composite probiotic" contains at least two probiotics. It should be understood that a "composite probiotic containing Akkermansia muciniphila" includes the Akkermansia muciniphila described in the present invention, as well as other probiotics different from the Akkermansia muciniphila described in the present invention.

[0130] In some embodiments, other probiotics (different from the Akkermansia muciniphila described in the present invention) may include modified strains of the Akkermansia muciniphila described in the present invention and probiotics of other species. Probiotics of other species are probiotics different from Akkermansia muciniphila, and may include one or more of Saccharomyces spp., Lactobacillus spp., and probiotics or microorganisms of normal human intestinal flora, and may also include but are not limited to one or more of Bacteroides fragilis, Christensenella, Saccharomyces boulardii, Enterococcus sibiricum, Lactobacillus rhamnosus, Lactobacillus bifidum, etc.

[0131] In some embodiments, the complex probiotic comprises multiple species of Akkermansia muciniphila.

[0132] In some embodiments, the composite probiotic consists of at least one of Akkermansia muciniphila AM06 and AM02, and at least one of a modified bacterium of AM06 and a modified bacterium of AM02.

[0133] In some embodiments, the composite probiotic includes Akkermansia muciniphila described herein and probiotics of other species. Probiotics of other species are defined above. The composite probiotic further includes modified Akkermansia muciniphila described herein.

[0134] When the composition containing Akkermansia muciniphila is used to prepare medicines, it is a pharmaceutical composition, in which the active pharmaceutical ingredients include at least one of Akkermansia muciniphila AM06 and Akkermansia muciniphila AM02.

[0135] The second active ingredient may be a drug different from the Akkermansia muciniphila.

[0136] In some embodiments, the composition comprising Akkermansia muciniphila further comprises a second pharmaceutically active ingredient.

[0137] In the present invention, a "second active pharmaceutical ingredient" refers to an active pharmaceutical ingredient other than Akkermansia muciniphila (AM06 and / or AM02) described herein that has the potential to prevent and treat alcoholic fatty liver disease. This "second active pharmaceutical ingredient" falls within the scope of the "second active ingredient."

[0138] In some embodiments, the second active ingredient includes but is not limited to one or more of simvastatin, diammonium glycyrrhizinate, prototypic glutathione, silybin, hepatocyte growth factor, fenofibrate, and the like.

[0139] In some embodiments, the second active ingredient comprises simvastatin.

[0140] In some embodiments, the second active ingredient is simvastatin.

[0141] In the present invention, each component of the "composition containing Akkermansia muciniphila" can appear at any appropriate time. For example, it can be directly formulated into a mixture and opened for use, or it can be packaged separately and formulated into a mixture when used, or it can be administered to a subject separately so that it appears simultaneously at a local location in the body and exerts a combined effect. When the "composition containing Akkermansia muciniphila" also contains other active pharmaceutical ingredients, the combination of Akkermansia muciniphila (AM06 and / or AM02) and other active pharmaceutical ingredients described in the present invention can also appear at any appropriate time; the definition of "other active pharmaceutical ingredients" can refer to the first aspect of the present invention. The "composition containing Akkermansia muciniphila" that appears at any appropriate time is within the scope of protection of the present invention.

[0142] The third aspect of the present invention

[0143] In a third aspect of the present invention, a composition comprising Akkermansia muciniphila is provided;

[0144] The definition of "composition containing Akkermansia muciniphila" may refer to the first aspect or the second aspect of the present invention.

[0145] In some embodiments, the composition containing Akkermansia muciniphila of the present invention comprises Akkermansia muciniphila described in the present invention and a second active ingredient. The definition of "second active ingredient" can be found in the second aspect of the present invention.

[0146] In some embodiments, the second active ingredient includes but is not limited to one or more of simvastatin, diammonium glycyrrhizinate, prototypic glutathione, silybin, hepatocyte growth factor, fenofibrate, and the like.

[0147] In some embodiments, the second active ingredient includes simvastatin. In this case, the composition containing Akkermansia muciniphila comprises the Akkermansia muciniphila of the present invention and simvastatin.

[0148] In some embodiments, the second active ingredient is simvastatin.

[0149] The fourth aspect of the present invention

[0150] In a fourth aspect of the present invention, a method for improving alcoholic liver dysfunction (such as a method for preventing and treating alcoholic fatty liver disease) is provided, comprising administering a therapeutically effective amount of Akkermansia muciniphila of the present invention to a subject (i.e., administering a therapeutically effective amount of Akkermansia muciniphila of the present invention to a subject).

[0151] In some embodiments, a method for improving alcoholic liver dysfunction (such as a method for preventing and treating alcoholic fatty liver) is provided, comprising administering to a subject a therapeutically effective amount of a pharmaceutical composition containing the Akkermansia muciniphila of the present invention.

[0152] In some embodiments, a method for improving alcoholic liver dysfunction (such as a method for preventing and treating alcoholic fatty liver) is provided, comprising administering to a subject a therapeutically effective amount of a medicament containing the Akkermansia muciniphila of the present invention.

[0153] In the fourth aspect of the present invention, the definitions of "Akkermansia muciniphila", "Akkermansia muciniphila according to the present invention", "composition", "pharmaceutical composition", "drug", "alcoholic liver dysfunction", "improvement of alcoholic liver dysfunction", "prevention and treatment", "alcoholic fatty liver disease", "prevention and treatment of alcoholic fatty liver disease", and "therapeutically effective amount" can refer to the first aspect or the second aspect of the present invention.

[0154] In a fourth aspect of the present invention, the Akkermansia muciniphila described herein can be used in combination with other active pharmaceutical ingredients to improve alcoholic liver dysfunction (e.g., for preventing and treating alcoholic fatty liver disease). The definition of "other active pharmaceutical ingredients" may refer to the first aspect of the present invention. For example, the other active pharmaceutical ingredients may be pharmaceutical ingredients that improve alcoholic liver dysfunction (e.g., prevent and treat alcoholic fatty liver disease), or may be pharmaceutical ingredients that have other disease-preventing effects. In some embodiments of the present invention, the other active pharmaceutical ingredient is the second active ingredient defined in the second aspect of the present invention.

[0155] In the present invention, the Akkermansia muciniphila of the present invention, the pharmaceutical composition containing the Akkermansia muciniphila of the present invention, and the medicine containing the Akkermansia muciniphila of the present invention all fall within the category of "medicine" defined in the present invention.

[0156] In some embodiments, the administration of the drug includes, but is not limited to, oral, rectal, parenteral (intravenous, intramuscular or subcutaneous) injection, topical administration, and inhalation.

[0157] In some embodiments, the drug can be administered orally, by enema, or parenterally.

[0158] In some embodiments, the drug administration cycle can be intermittent administration, periodic administration, continuous administration or long-term administration.

[0159] Solid dosage forms for oral administration may include capsules, tablets, pills, powders, and granules. In these solid dosage forms, the active ingredient is mixed with at least one conventional inert excipient (or carrier), such as sodium citrate or dicalcium phosphate, or with the following ingredients: (a) fillers or extenders, for example, starches, lactose, sucrose, glucose, mannitol, and silicic acid; (b) binders, for example, hydroxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose, and acacia; (c) humectants, for example, glycerol; (d) disintegrants, for example, agar, calcium carbonate, potato or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate; (e) solubilizers, for example, paraffin; (f) absorption accelerators, for example, quaternary ammonium compounds; (g) wetting agents, for example, cetyl alcohol and glyceryl monostearate; (h) adsorbents, for example, kaolin; and (i) lubricants, for example, talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, or mixtures thereof. In capsules, tablets, and pills, the dosage form may also include a buffer. Solid dosage forms such as tablets, dragees, capsules, pills, and granules may be prepared using coatings and shell materials, such as enteric coatings and other materials known in the art. They may include opacifying agents, and the release of the active ingredient or compound in such compositions may be delayed in a certain portion of the digestive tract. Examples of embedding components that may be used are polymeric substances and waxes. If desired, the active ingredient may also be microencapsulated with one or more of the above-mentioned excipients.

[0160] 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 inert diluents commonly used in the art, such as water or other solvents, solubilizers, and emulsifiers, specifically ethanol, isopropyl alcohol, ethyl carbonate, ethyl acetate, propylene glycol, 1,3-butylene glycol, dimethylformamide, and oils, particularly cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil, and sesame oil, or mixtures thereof. In addition to these inert diluents, the composition may also contain adjuvants, such as wetting agents, emulsifiers and suspending agents, sweeteners, flavoring agents, and fragrances. For example, a suspension may contain a suspending agent, specifically ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methoxide, and agar, or mixtures thereof.

[0161] 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, ethanol, polyols and suitable mixtures thereof.

[0162] Dosage forms for topical administration include ointments, powders, patches, sprays and inhalants, which are prepared by mixing the active ingredient with a pharmaceutically acceptable carrier and any preservatives, buffers, or propellants as necessary under sterile conditions.

[0163] The "active ingredient" in the above-mentioned pharmaceutical preparations of this aspect 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 invention can be added with different pharmaceutically acceptable carriers to prepare suitable clinical dosage forms, which include but are not limited to the dosage forms described above.

[0164] Some specific examples are provided below.

[0165] The embodiments of the present invention will be described in detail below with reference to the examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the invention. For experimental methods in the following examples where specific conditions are not specified, reference is made to the guidance provided in the present invention, and may also be made according to experimental manuals or conventional conditions in the art, or according to conditions recommended by the manufacturer, or with reference to experimental methods known in the art.

[0166] In the following specific 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 operational accuracy are allowed.

[0167] In the following examples, “% (v / v)” and “% (vol / vol)” have the same meaning and can be used interchangeably, both referring to volume percentages.

[0168] Example 1. Isolation and identification of Akkermansia muciniphila

[0169] 1.1. Isolation and identification of strain AM02

[0170] Use a sterile sampling spoon to take soybean-sized feces (samples are from healthy adult men) and place them in a 10 mL centrifuge tube. After sampling, transfer the sample immediately to a 37°C anaerobic workstation (85% N2, 10% H2, 5% CO2, volume percentage), and dilute the sample to 10 by 10-fold gradient. -9 1 mL of each dilution solution was inoculated into 9 mL of basal culture medium with mucin as the sole carbon source and cultured anaerobically for 7 days. -4 Dilution: 1 mL of culture medium was inoculated and diluted to 10 in a 1:10 gradient dilution. -6100 μL of each dilution was plated on mucin agar and incubated anaerobically for 7 days. A single colony was then picked and inoculated into 2 mL of BHI broth (containing N-acetyl-D-glucosamine). 16S rRNA sequencing was performed on the cultured bacterial broth, and the 16S rRNA sequence was aligned to the NCBI database. The isolated strain was identified as Akkermansia muciniphila. The 16S rRNA sequencing result is shown below (SEQ ID No.: 1):

[0171]

[0172] The sequence was aligned with the 16S rRNA sequence of ATCC BAA-835 at NCBI and the Per.Ident value was 99.43%.

[0173] 1.2. Isolation and identification of strain AM06

[0174] Freshly collected breast milk samples (from healthy adult women) were immediately injected into 5 mL anaerobic penicillin bottles for storage. The samples were then transferred to a 37°C anaerobic workstation (85% N2, 10% H2, 5% CO2, volume percentage) and diluted to 10% by 10-fold gradient. -6 , take 1 mL of each dilution solution and inoculate it into 9 mL of basal culture medium with mucin as the sole carbon source, and culture it anaerobically for about 1 month. -1 ~10 -4 Dilution: 1 mL of culture medium was inoculated and diluted to 10 at a rate of 1:10. -6 100 μL of each dilution was plated on mucin agar and incubated anaerobically for 7 days. A single colony was then picked and inoculated into 2 mL of BHI broth (containing N-acetyl-D-glucosamine). The cultured bacteria were identified by 16S rRNA sequencing. The 16S rRNA sequence was aligned with the NCBI database and identified as Akkermansia muciniphila. The 16S rRNA sequencing result is shown below (SEQ ID No.: 2):

[0175]

[0176] The sequence was aligned with the 16S rRNA sequence of ATCC BAA-835 at NCBI and the Per.Ident value was 99.22%.

[0177] 1.3. Strain deposit information

[0178] Akkermansia muciniphila AM02, whose classification name is Akkermansia muciniphila, was deposited in the General Microbiology Center of China Culture Collection Administration on June 28, 2021, at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with the deposit number CGMCC No. 22794; the strain was received and registered by the collection center on June 28, 2021, and was tested as a viable strain by the collection center on June 28, 2021.

[0179] Akkermansia muciniphila AM06, whose classification name is Akkermansia muciniphila, was deposited in the General Microbiology Center of China Culture Collection Administration on June 28, 2021, at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with the deposit number CGMCC No. 22793; the strain was received and registered by the collection center on June 28, 2021, and was tested as a viable strain by the collection center on June 28, 2021.

[0180] Example 2. Cultivation of Akkermansia muciniphila and preparation of inactivated bacteria

[0181] 2.1. Streak Akkermansia muciniphila onto BHA plates and incubate anaerobically for 3 days. Observe colony morphology, staining properties, size, club shape, and distribution.

[0182] Colony characteristics: After 3 days of culture on the above medium, Akkermansia muciniphila AM02 and AM06 both showed round, convex, neatly edged, opaque, white, and unevenly sized colonies, ranging in size from 0.08 to 2.2 mm. Figure 1 (Akkermansia muciniphila AM02) and Figure 2 (Akkermansia muciniphila AM06).

[0183] Microscopic morphology: Gram staining of Akkermansia muciniphila AM02 and AM06 revealed that they are Gram-negative bacteria, oval in shape, arranged singly or in chains. Figure 3 (Akkermansia muciniphila AM02) and Figure 4 (Akkermansia muciniphila AM06).

[0184] The colony characteristics of the standard strain ATCC BAA-835 are: round, raised, opaque, white, and unevenly sized colonies with neat edges. Microscopically, they appear to be Gram-negative bacteria, oval in shape, and arranged singly or in chains.

[0185] 2.2. A single colony was selected and inoculated into BHI broth for 48 hours (at 37°C). The resulting bacterial suspension was centrifuged at 16,000 × g for 30 minutes. The supernatant was removed and the precipitate was collected to obtain Akkermansia muciniphila slurry. Akkermansia muciniphila strains AM02, AM06, and ATCC BAA-835 were obtained.

[0186] 2.3. Preparation of Inactivated Bacteria: Take an appropriate amount of bacterial sludge and add physiological saline at a ratio of 1:10 (m:v). Stir at 1000 rpm using a stirrer for 5-20 minutes to evenly disperse the bacteria. Place 100 mL of the dispersed bacterial solution in a sterile three-necked round-bottom flask (try to ensure that the bacterial solution does not adhere to the inner wall of the flask). Place the three-necked round-bottom flask on the heating plate of a magnetic stirrer, add a sterile stir bar, and insert a temperature probe. Set the speed to 300-500 rpm and the temperature to 70°C. Heat for 30 minutes to obtain inactivated bacteria of Akkermansia muciniphila.

[0187] Example 3. Non-targeted metabolic differential analysis of Akkermansia muciniphila culture supernatants

[0188] Sample preparation

[0189] After completing the culture in Example 2, 1 mL of the culture supernatant from each strain of Akkermansia muciniphila (AM02, AM06, and ATCC BAA-835) was centrifuged at 12,000 rpm for 5 minutes. The supernatant was filtered through a 0.22 μm filter, and the filtrate was used as the test sample for non-targeted metabolomics analysis. Five replicates were prepared for each strain.

[0190] 3.2 Experimental results

[0191] PCA is a data dimensionality reduction method that reduces multiple variables to a new set of composite variables. The principal components that best reflect the original variables are then selected to achieve this dimensionality reduction. PCA plots reflect the true distribution of samples and are primarily used to observe the separation trend between sample groups and the presence of outliers. They also reflect the variability between and within groups in the original data.

[0192] The experimental results can be found in Figure 5 . Figure 5The PCA analysis included quality control samples (QC samples) and all samples. The QC samples clustered together in both principal component analysis plots, indicating instrument stability and good reproducibility of the collected data during testing. The results also showed that the metabolites in the AM06 culture supernatant were similar to those in the BAA-835 culture supernatant, while the metabolites in the AM02 and BAA-835 culture supernatants differed significantly.

[0193] The results of the comparison of the number of differential metabolites between the strains can be found in Table 1. Compared with the standard strain BAA-835, the number of differential metabolites detected by positive ion (pos) mode and negative ion (neg) mode for AM02 was 205 and 135, respectively. Compared with the standard strain BAA-835, the number of differential metabolites detected by positive ion (pos) mode and negative ion (neg) mode for AM06 was 111 and 62, respectively.

[0194] Table 1. Statistics of differential metabolites

[0195]

[0196] Example 4. Efficacy verification of Akkermansia muciniphila

[0197] 4.1. Tolerance of Akkermansia muciniphila to artificial gastric fluid

[0198] 4.1.1. Experimental methods and grouping

[0199] Table 2. Experimental groups

[0200]

[0201] Table 3. Experimental methods

[0202]

[0203] “+” indicates that testing is required.

[0204] 4.1.1.1. Take one tube of Akkermansia muciniphila, remove the label, and disinfect the outer surface of the glycerol cryovial with 75% (v / v) ethanol. Vortex to mix thoroughly, and then open the tube. Pipette 100-500 μL of the bacterial solution into 10 mL / tube of BHI broth and shake well. Prepare three tubes in total. Simultaneously, keep the tube uninoculated as a negative control and incubate anaerobically at 37°C for 2-4 days to obtain the primary seed solution.

[0205] Gram staining microscopic examination of the first-level seed liquid should show G-bacilli, no spores, and no other bacteria.

[0206] 4.1.1.2. Take 10 mL of the above-mentioned first-level seed solution, centrifuge at 12,000×g and 4°C for 10 min, discard the supernatant, add 1 mL of 0.9 wt% NaCl solution and resuspend to prepare bacterial suspension for later use.

[0207] 4.1.1.3. According to Tables 2 and 3, add bacterial suspensions of AM06, AM02, and the standard strain to 0.9 wt% NaCl, pH 3.0, and pH 2.0 artificial gastric fluid, respectively. Mix thoroughly, aliquot into 5 mL tubes, and incubate in an anaerobic glove box at 37°C for 0 h, 1.5 h, and 3 h. Afterward, remove the samples and measure the bacterial concentration. Perform three replicates for each experimental group.

[0208] 4.1.1.4. Determination of viable count:

[0209] Take the experimental sample and dilute it in a 10-fold series gradient to different dilutions. Take 100 μL of the dilution and inoculate it on the BHA plate. Spread it evenly. Make 2 plates for each dilution. Generally, make 2 to 3 dilutions. At the same time, take 100 μL of the dilution on the BHA plate as a negative control. All the coated plates are placed upright and cultured under anaerobic conditions for about 3 to 5 days. Observe the growth of the colonies on the plates and count them.

[0210] The number of viable bacteria was calculated based on the sum of the colony counts on the two plates using the following formula:

[0211] Viable bacteria count (CFU / mL) = sum of colony counts on two plates / 2 × 10 × final dilution

[0212] Survival rate calculation:

[0213]

[0214] 4.1.2 Experimental results

[0215] The experimental results can be found in Table 4. The order of artificial gastric fluid tolerance of different Akkermansia muciniphila strains from high to low is AM02 > AM06 > standard strain BAA-835.

[0216] Table 4. Statistical table of survival rate of Akkermansia muciniphila in artificial gastric fluid tolerance

[0217]

[0218] 4.2. Tolerance of Akkermansia muciniphila to artificial intestinal fluid

[0219] 4.2.1. Experimental methods and grouping

[0220] Table 5. Experimental groups

[0221]

[0222] 4.2.1.1. Preparation of primary seed solution

[0223] Remove the label from one Akkermansia muciniphila tube and disinfect the exterior of the glycerol cryovial with 75% (v / v) ethanol. Vortex to mix thoroughly and then open the tube. Pipette 100 μL of the bacterial suspension into 10 mL / tube of BHI broth and shake well. Prepare three tubes in total. Simultaneously, keep the tubes uninoculated as a negative control and incubate them anaerobically at 37°C for 2–4 days to obtain the primary seed solution.

[0224] Gram staining microscopic examination of the first-level seed liquid should show G-bacilli, no spores, and no other bacteria.

[0225] 4.2.1.2. Mushroom sludge preparation

[0226] The above-mentioned first-level seed liquid was divided into 1.5 mL / tube, centrifuged at 12000×g for 10 min, and the supernatant was discarded to prepare bacterial slurry. 3 tubes of bacterial slurry were prepared for each of AM06, AM02 and standard strains.

[0227] 4.2.1.3. Evaluation of strain tolerance to artificial intestinal fluid

[0228] As shown in Tables 5 and 6, 1.5 mL of artificial intestinal fluid was added to each tube of the prepared bacterial sludge and mixed thoroughly. The solution was then aliquoted into three tubes at a volume of 0.5 mL per tube. The tubes were anaerobically incubated at 37°C for 0, 4, and 8 hours, and samples were collected for viable bacterial counts. Three replicates were performed for each group.

[0229] Table 6. Experimental methods

[0230]

[0231]

[0232] “+” indicates that testing is required.

[0233] 4.2.1.4. Determination of viable bacterial count

[0234] Take the incubated samples separately, dilute them in 10-fold gradient series, take 100 μL of the dilution and inoculate them on BHA plates, spread them evenly, make 2 plates for each dilution, and generally make 2 to 3 dilutions. At the same time, take 100 μL of the dilution on the BHA plate as a negative control. All the coated plates are placed upright and cultured under anaerobic conditions for about 3 to 5 days. Observe the growth of the colonies on the plates and count them.

[0235] Viable bacteria count (CFU / mL) = sum of colony counts on two plates / 2 × 10 × final dilution

[0236] Survival rate calculation:

[0237] Survival rate = number of viable bacteria at each time point / corresponding number of viable bacteria at 0 h × 100%

[0238] 4.2.2 Experimental results

[0239] The results can be seen in Table 7. ATCC BAA-835, AM02, and AM06 strains tolerated artificial intestinal fluid well, and the tolerance order from high to low was AM02 > AM06 > standard strain BAA-835.

[0240] Table 7. Survival rate statistics

[0241]

[0242] 5. Effects of Akkermansia muciniphila on the expression of tight junction protein ZO-1 in Caco2 cells induced by TNF-α and IFN-γ

[0243] 5.1. Experimental methods and grouping

[0244] Caco2 cells were seeded into 96-well plates and cultured to a confluence of 80% to 90%. After induction with 100 ng / mL TNF-α and 100 ng / mL IFN-γ for 24 hours, AM02, AM06, and BAA-835 were added and incubated with the cells for another 24 hours. Experimental groupings are shown in Table 8. Five replicate wells were prepared for each group. Immunofluorescence was used to investigate the effects of BAA-835, AM06, and AM02 on the expression of the tight junction protein ZO-1 in Caco2 cells induced by TNF-α and IFN-γ.

[0245] Table 8. Experimental groups

[0246]

[0247]

[0248] 5.2 Experimental results

[0249] The fluorescence intensity of the captured images was statistically analyzed. The results can be found in Figure 6 and Table 9. Compared with the blank control group, the fluorescence intensity of the intercellular space in the inflammatory model group was significantly reduced after 48 hours of TNF-α and IFN-γ induction, indicating reduced ZO-1 protein expression and disruption of tight junctions between cells. Compared with the inflammatory model group, the fluorescence intensity of the treated groups treated with AM02, AM06, and BAA-835 significantly increased (p < 0.05), and AM02 and AM06 were significantly more effective than BAA-825 in inhibiting the inflammatory factor-induced reduction of ZO-1 protein in Caco2 cells (p < 0.05).

[0250] Table 9. Fluorescence intensity statistics

[0251] serial number Group Fluorescence intensity A Blank control group 36.2647±5.8977** B Inflammation model group 25.6588±4.5303 C AM02 group 41.7059±6.5664**aa D AM06 group 40.2949±4.5437**aa E BAA-835 group 33.6688±4.13336**

[0252] Note: ** indicates significant difference compared with the model group, p < 0.01; aa indicates significant difference compared with the BAA-835 group, p < 0.01.

[0253] Example 6. Experimental study on the efficacy of Akkermansia muciniphila in treating alcoholic fatty liver disease in mice

[0254] Experimental design and methods

[0255] Experimental design: 210 male C57BL / 6 mice (backcrossed for more than 10 generations), 8 to 10 weeks old. After a one-week adaptation period, 20 mice were randomly selected as blank controls and gavaged with normal saline every day; the remaining 190 mice were gavaged with 30% (v / v) ethanol water at 5g / kg.d (5 grams per kilogram of body weight per day), and the other feeding conditions were the same. After 15 days of gavage, 180 mice weighing about 20g were selected from the 190 mice and formed the experimental group with 20 blank control group mice. Experimental groups: blank group, model group, low-dose AM06 (10 6 CFU / mL), medium dose (10 8 CFU / mL), high dose (10 10 CFU / mL) group; AM02 (10 10 CFU / mL) group; ATCC BAA-835 (10 10 CFU / mL) group; AM06 (10 10 CFU / mL) inactivated bacteria group; AM02(10 10 CFU / mL) inactivated bacteria group; ATCC BAA-835 (10 10 CFU / mL) inactivated bacteria group, 20 mice per group. After 6 weeks of gavage, each treatment group began to receive medication, while the blank group and model group were given normal saline. Akkermansia muciniphila was prepared using the method of Example 1.

[0256] After 10 weeks, 10 mice were randomly selected from each group and euthanized with CO₂ after a 6-hour fast. Blood was collected from the eyeballs. Serum was separated and stored at -80°C for insulin and biochemical analyses. Final body weight and liver weight were recorded. The liver was cut into four pieces: one was quick-frozen for biochemical analysis, one was fixed in formalin for histopathological examination, and the remaining two pieces were kept for later use. After 15 weeks, the above procedure was repeated for the remaining mice.

[0257] 6.2 Experimental results

[0258] 6.2.1. At week 10, mice exhibited significant steatosis. Liver weight, serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), low-density lipoprotein cholesterol (LDL-c), and hepatic steatosis score were measured in the index mice. The results of each test indicator can be found in Table 10.

[0259] SPSS 25.0 software was used to process the data, and p < 0.05 was considered to be statistically significant.

[0260] Table 10. Statistics of various test indicators in mice with fatty degeneration (mean ± SD, n = 10)

[0261]

[0262]

[0263] Note: * indicates significant difference compared with the model group (p<0.05).

[0264] 6.2.1.1. Mouse liver weight

[0265] As shown in Table 10 above, compared with the blank group, the liver weight of the model group mice increased to a certain extent; compared with the model group, the strength of the liver of the mice in each drug-treated group decreased; among the Akkermansia muciniphila and the same inactivated bacteria groups, the decrease rate was AM06>AM02>BAA-835.

[0266] 6.2.1.2. Mouse serum liver function test

[0267] According to the kit instructions, the serum alanine aminotransferase (ALT), aspartate aminotransferase (AST) and low-density lipoprotein cholesterol (LDL-c) of mice were detected.

[0268] (1) ALT and AST

[0269] As shown in Table 10 above, ALT and AST levels in the model group were significantly elevated compared to the blank group (p<0.05). ALT and AST levels decreased to some extent in all treatment groups compared to the model group. Among the Akkermansia muciniphila and the same inactivated bacteria groups, the magnitude of decrease was AM06 > AM02 > BAA-835.

[0270] In terms of ALT level, there were significant differences between the high-dose AM06 and inactivated bacteria groups and the model group (p<0.05); in terms of AST level, there were significant differences between the various doses of AM06 groups and the model group (p<0.05).

[0271] (2)LDL-c

[0272] As shown in Table 10, the LDL-c levels of mice in the model group were significantly higher than those in the blank group (p<0.05). Compared with the model group, LDL-c levels decreased in all treatment groups. Among the Akkermansia muciniphila and the same inactivated bacteria groups, the decrease rate was AM06 > AM02 > BAA-835.

[0273] (3) Hepatic steatosis score

[0274] The livers of mice in each group were pathologically stained with HE and the degree of fatty change was scored.

[0275] The scoring criteria are as follows:

[0276] 0 points: hepatocytes containing fat droplets are scattered and sparse; 1 point: hepatocytes containing fat droplets ≤ 1 / 4; 2 points: hepatocytes containing fat droplets ≤ 1 / 2; 3 points: hepatocytes containing fat droplets ≤ 3 / 4; 4 points: liver tissue is almost replaced by fat droplets, and its area is greater than 3 / 4.

[0277] As shown in Table 10, compared with the blank group, the steatosis scores of mice in the model group were significantly increased (p < 0.05). Compared with the model group, the steatosis scores of all treatment groups were significantly decreased (p < 0.05). Among the Akkermansia muciniphila groups and the same inactivated bacteria groups, the decrease in the score was AM06 > AM02 > BAA-835.

[0278] In conclusion, Akkermansia muciniphila can effectively treat alcoholic fatty liver steatosis in mice, with the efficacy of AM06>AM02>BAA-835.

[0279] 6.2.2. At week 16, the primary symptom of the mice was alcoholic steatohepatitis (ASH). Liver weight, serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), triglycerides (TG), total cholesterol (TC), and serum endotoxin (LPS) were measured in the index mice. For details on these parameters, see Table 11.

[0280] SPSS 25.0 software was used to process the data, and p < .05 was considered to be statistically significant.

[0281] Table 11. Statistics of various test indicators in mice with alcoholic fatty liver disease (mean ± SD, n = 10)

[0282]

[0283] Note: * indicates that the difference is significant compared with the model group, p < 0.05; ** indicates that the difference is extremely significant compared with the model group, p < 0.01.

[0284] 6.2.2.1. Mouse liver weight

[0285] As shown in Table 11, compared with the blank group, the liver weight of mice in the model group increased; compared with the model group, the liver weight of mice in each treatment group decreased. Among the Akkermansia muciniphila and the same inactivated bacteria groups, the decrease rate was AM06 > AM02 > BAA-835.

[0286] 6.2.2.2. Mouse serum liver function test

[0287] According to the kit instructions, the mouse serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), triglyceride (TG), liver triglyceride (TG), and endotoxin (LPS) were detected.

[0288] (1) ALT and AST

[0289] As shown in Table 11, compared with the blank group, ALT and AST in the model group were significantly increased (p < 0.01). Compared with the model group, ALT and AST in all treatment groups decreased. Among them, in the Akkermansia muciniphila and inactivated bacteria groups, the decrease was AM06>AM02>BAA-835.

[0290] (2) Serum TG and liver TG

[0291] As shown in Table 11, compared with the blank group, the serum TG and liver TG of the model group mice were significantly increased (p < 0.05). Compared with the model group, the TG of the drug group decreased.

[0292] (3) Serum LPS

[0293] As shown in Table 11, LPS levels in the model group were significantly elevated compared to the blank group (p < 0.05). Compared to the model group, LPS levels decreased in all treatment groups. Among the Akkermansia muciniphila and inactivated bacteria groups, the magnitude of decrease was AM06 > AM02 > BAA-835. Significant differences were observed between the high-dose AM06 group and the inactivated bacteria groups and the model group (p < 0.05).

[0294] In conclusion, Akkermansia muciniphila can effectively treat alcoholic steatohepatitis in mice, with the efficacy of AM06>AM02>BAA-835.

[0295] Example 7. Experimental study on the efficacy of Akkermansia muciniphila in preventing alcoholic fatty liver disease in mice

[0296] Experimental design and methods

[0297] 7.1.1. Induction of simple steatosis in mice: 130 male C57BL / 6 mice aged 8-10 weeks weighing more than 20 g were selected. After a one-week adaptation period, 120 mice of similar weight were divided into the following experimental groups: blank group, model group, positive drug (simvastatin, 10 mg / kg) group, AM06 low-dose (10 6 CFU / mL), medium dose (10 8 CFU / mL), high dose (10 10 CFU / mL) live bacteria group; AM02 (10 10 CFU / mL) live bacteria group; ATCC BAA-835 (10 10 CFU / mL) live bacteria group, simvastatin (10 mg / kg) + AM06 (10 10 CFU / mL) group; AM06 (10 10 CFU / mL) inactivated bacteria group; AM02(10 10 CFU / mL) inactivated bacteria group; ATCC BAA-835 (10 10 The blank group was fed with ordinary feed, the model group was fed with Lieber-DeCarli feed (containing 5% (v / v) ethanol), and the drug-treated groups were fed with Lieber-DeCarli feed (containing 5% (v / v) ethanol) and given corresponding drugs at the same time.

[0298] After 4 weeks, animals were euthanized with CO₂ after a 6-hour fast. Blood samples were collected retroorbitally. Serum was separated and stored at −80°C for insulin and biochemical analyses. Terminal body weight and liver weight were recorded. The liver was cut into four pieces: one was flash-frozen for biochemical analysis, one was fixed in formalin for histopathological examination, and the remaining two pieces were reserved for later use.

[0299] 7.1.2. Induction of alcoholic steatohepatitis in mice: 130 male C57BL / 6 mice aged 8-10 weeks were selected. 120 mice of similar weight were divided into the following experimental groups: blank group, model group, positive drug (simvastatin, 10 mg / kg) group, AM06 low dose (10 6 CFU / mL), medium dose (10 8 CFU / mL), high dose (10 10 CFU / mL) live bacteria group; AM02 (10 10 CFU / mL) live bacteria group; ATCC BAA-835 (10 10 CFU / mL) live bacteria group; simvastatin (10 mg / kg) + AM06 (10 10 CFU / mL) group; AM06 (10 10 CFU / mL) inactivated bacteria group; AM02(1010 CFU / mL) inactivated bacteria group; ATCC BAA-835 (10 10 CFU / mL) inactivated bacteria group, 10 mice per group. The blank group was fed a normal diet; the model group and each drug-treated group were fed a Lieber-DeCarli diet (containing 5% (v / v) ethanol) and the corresponding drugs simultaneously for 10 days. On the morning of day 11, the model group and each drug-treated group were gavaged with a single dose of ethanol (5g / kg, concentration not exceeding 31.5% (vol / vol); the blank group was gavaged with normal saline.

[0300] The Lieber-DeCarli diet formula can be found in Table 12.

[0301] Table 12. Lieber-DeCarli diet formula

[0302]

[0303] Nine hours after gavage on day 11, mice were deeply anesthetized with isoflurane, and the eyeballs were avulsed from the orbits with forceps, and blood was collected from the orbits. Under general anesthesia, mice were killed by cervical dislocation. Serum was isolated and stored at −80°C for insulin and biochemical analyses. Terminal body weight and liver weight were recorded. The liver was cut into four pieces: one piece was flash-frozen for biochemical analysis, one piece was fixed in formalin for histopathological examination, and the remaining two pieces were reserved for later use.

[0304] 7.2 Experimental results

[0305] 7.2.1. The test results of mice with simple fatty degeneration as the main symptom can be found in Table 13.

[0306] SPSS 25.0 software was used to process the data, and p < 0.05 was considered to be statistically significant.

[0307] Table 13. Statistics of various test indicators in mice with simple steatosis (mean ± SD, n = 10)

[0308]

[0309]

[0310] Note: * indicates significant difference compared with the model group, p<0.05.

[0311] 7.2.1.1. Mouse liver wet weight

[0312] As shown in Table 13, compared with the blank group, the liver weight of the mice in the model group increased; compared with the model group, the liver weight of the mice in each drug-treated group decreased.

[0313] 7.2.1.2. Mouse serum liver function test

[0314] According to the kit instructions, the serum alanine aminotransferase (ALT), aspartate aminotransferase (AST) and low-density lipoprotein cholesterol (LDL-c) of mice were detected.

[0315] (1) ALT and AST

[0316] As shown in Table 13, compared with the blank group, ALT and AST levels in the model group were significantly elevated (p < 0.05). Compared with the model group, ALT and AST levels in all treatment groups were significantly decreased. ALT levels were significantly lower in all AM06 dose groups compared with the model group (p < 0.05). AST levels were significantly different between the AM06 dose groups, AM02, and the inactivated bacteria group and the model group (p < 0.05).

[0317] (2)LDL-c

[0318] As shown in Table 13, compared with the blank group, the LDL-c levels of the model group mice were significantly elevated (p < 0.05). Compared with the model group, LDL-c levels decreased in all treatment groups. Significant differences were observed between the AM06 dose groups and the inactivated bacteria group and the model group (p < 0.05).

[0319] (3) Liver fatty degeneration score

[0320] Liver pathology HE staining was performed on mice in each group, and the degree of fatty change was scored. The scoring criteria are as follows:

[0321] 0 points: hepatocytes containing fat droplets are scattered and sparse; 1 point: hepatocytes containing fat droplets ≤ 1 / 4; 2 points: hepatocytes containing fat droplets ≤ 1 / 2; 3 points: hepatocytes containing fat droplets ≤ 3 / 4; 4 points: liver tissue is almost replaced by fat droplets, and its area is greater than 3 / 4.

[0322] As shown in Table 13, compared with the blank group, the steatosis scores of the model group mice were significantly increased. Compared with the model group, the steatosis scores of all treatment groups decreased. Significant differences were observed between the AM06 dose groups, the AM02 group, and the inactivated bacteria group and the model group (P < 0.05).

[0323] In conclusion, Akkermansia muciniphila can effectively prevent simple steatosis of alcoholic fatty liver in mice, with the efficacy of AM06>AM02>BAA-835.

[0324] 7.2.2. Results of various test indicators for inducing alcoholic steatohepatitis in mice (see Table 14).

[0325] SPSS 25.0 software was used to process the data, and p < 0.05 was considered to be statistically significant.

[0326] Table 14. Statistics of various test indicators in mice with alcoholic steatohepatitis (mean ± SD, n = 10)

[0327]

[0328]

[0329] Note: * indicates that the difference is significant compared with the model group, p < 0.05; ** indicates that the difference is extremely significant compared with the model group, p < 0.01.

[0330] 7.2.2.1. Mouse liver wet weight

[0331] As shown in Table 14, compared with the blank group, the liver weight of the mice in the model group increased; compared with the model group, the liver weight of the mice in each drug-treated group decreased.

[0332] 7.2.2.2. Mouse serum liver function test

[0333] According to the kit instructions, serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), triglyceride (TG), endotoxin (LPS) and liver triglyceride (TG) of mice were detected.

[0334] (1) ALT and AST

[0335] As shown in Table 14, compared with the blank group, ALT and AST levels in the model group were significantly elevated (P < 0.01). Compared with the model group, ALT and AST levels decreased in all drug-treated groups, with AM06 > AM02 > BAA-835. The combination of AM06 and simvastatin had the most significant therapeutic effect, significantly outperforming the active drug group and exhibiting a synergistic effect.

[0336] (2) Serum TG and liver TG

[0337] As shown in Table 14, compared with the blank group, the serum TG and liver TG of the model group mice were significantly increased (P < 0.05). Compared with the model group, the TG of each drug group decreased.

[0338] (3) Serum LPS

[0339] As shown in Table 14, compared with the blank group, serum LPS levels in the model group were significantly elevated (P < 0.05). Compared with the model group, LPS levels decreased in all treatment groups. Among the Akkermansia muciniphila and inactivated bacteria groups, the magnitude of decrease was AM06 > AM02 > BAA-835. The combination of AM06 and simvastatin was the most effective, significantly superior to the active drug group and exhibiting a synergistic effect.

[0340] In summary, Akkermansia muciniphila can effectively prevent alcoholic steatohepatitis in mice, with the efficacy of AM06>AM02>BAA-835.

[0341] The technical features of the above-mentioned embodiments and examples can be combined in any appropriate manner. In order to make the description concise, not all possible combinations of the technical features in the above-mentioned embodiments and examples 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.

[0342] The above embodiments only express several implementation methods of the present invention, which are convenient for understanding the technical solutions of the present invention in a specific and detailed manner, but they cannot be understood as limiting the scope of protection of the present invention. It should be pointed out that, for those of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present invention, and these all fall within the scope of protection of the present invention. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and the equivalent forms obtained also fall within the scope of protection of the present invention. It should also be understood that the technical solutions obtained by those skilled in the art through logical analysis, reasoning or limited experiments on the basis of the technical solutions provided by the present invention are all within the scope of protection of the claims attached to the present invention. Therefore, the scope of protection of the patent of the present invention shall be based on the contents of the attached claims, and the description and drawings can be used to interpret the contents of the claims.

Claims

1. Akkermansia muciniphila ( Akkermansia muciniphila ) in the preparation of a pharmaceutical composition for improving alcoholic liver dysfunction, characterized in that: The Akkermansia muciniphila ( Akkermansia muciniphila ) is Akkermansia muciniphila ( Akkermansia muciniphila )AM06, Akkermansia muciniphila ( Akkermansia muciniphila )AM02 or a combination of the two; wherein, The Akkermansia muciniphila ( Akkermansia muciniphila AM06 was deposited in the General Microbiology Center of China Culture Collection Administration on June 28, 2021, with the deposit number CGMCC No. 22793. The Akkermansia muciniphila ( Akkermansia muciniphila ) AM02 was deposited in the General Microbiology Center of China Culture Collection Administration on June 28, 2021, with the deposit number CGMCC No. 22794.

2. The use according to claim 1, characterized in that The alcoholic liver dysfunction includes at least one of alcoholic steatosis, alcoholic steatohepatitis, liver fibrosis and liver cirrhosis.

3. The use according to claim 1 or 2, characterized in that The improvement of alcoholic liver dysfunction includes preventing and / or treating alcoholic fatty liver; The alcoholic fatty liver includes at least one of alcoholic steatosis, alcoholic steatohepatitis, liver fibrosis and liver cirrhosis.

4. The use according to claim 1, wherein The Akkermansia muciniphila ( Akkermansia muciniphila ) AM06 and the Akkermansia muciniphila ( Akkermansia muciniphila ) AM02 are each independently live bacteria, inactivated bacteria or a combination thereof.

5. The use according to claim 1, wherein The pharmaceutical composition comprises the Akkermansia muciniphila ( Akkermansia muciniphila ) and a pharmaceutically acceptable carrier.

6. The use according to claim 5, characterized in that The pharmaceutical composition is a medicine, and its dosage form is tablets, capsules, granules, pills, ointments, suspensions, emulsions, sprays, drops or tube feeding preparations.

7. Contains Akkermansia muciniphila ( Akkermansia muciniphila ) in the preparation of a medicament for preventing and treating alcoholic fatty liver, characterized in that: The Akkermansia muciniphila ( Akkermansia muciniphila ) as defined in claim 1 or 4.

8. The use according to claim 7, wherein The bacteria containing Akkermansia muciniphila ( Akkermansia muciniphila ) is a composite probiotic, wherein the composite probiotic further contains a probiotic different from the Akkermansia muciniphila ( Akkermansia muciniphila ) of probiotics.

9. The use according to claim 8, wherein The present invention is different from the Akkermansia muciniphila ( Akkermansia muciniphila ) includes one or more of Bacteroides fragilis, Christensenella, Saccharomyces boulardii, Enterococcus hilarii, Lactobacillus rhamnosus, Lactobacillus bifidum and Lactobacillus bifidum.

10. The use according to claim 7, wherein The bacteria containing Akkermansia muciniphila ( Akkermansia muciniphila ) further comprises a second active ingredient, wherein the second active ingredient is different from the Akkermansia muciniphila ( Akkermansia muciniphila ) of the drug.

11. The use according to claim 10, characterized in that The second active ingredient comprises one or more of simvastatin, diammonium glycyrrhizinate, prototype glutathione, silybin, hepatocyte growth promoting factor and fenofibrate.

12. The use according to claim 11, characterized in that The second active ingredient comprises simvastatin.

13. Akkermansia muciniphila for improving alcoholic liver dysfunction Akkermansia muciniphila ), characterized in that It comprises the Akkermansia muciniphila defined in claim 1 or 4 ( Akkermansia muciniphila ) and a second active ingredient; The second active ingredient includes one or more of diammonium glycyrrhizinate, prototype glutathione, silybin, and hepatocyte growth promoting factor.

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