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WO2022213507A1 - 一种细菌菌株及组合物、联用药物和用途 - Google Patents

一种细菌菌株及组合物、联用药物和用途 Download PDF

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Publication number
WO2022213507A1
WO2022213507A1 PCT/CN2021/106579 CN2021106579W WO2022213507A1 WO 2022213507 A1 WO2022213507 A1 WO 2022213507A1 CN 2021106579 W CN2021106579 W CN 2021106579W WO 2022213507 A1 WO2022213507 A1 WO 2022213507A1
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Prior art keywords
obesity
diabetes
disease
diseases
strain
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PCT/CN2021/106579
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English (en)
French (fr)
Inventor
林诠盛
蒋先芝
贤一博
邝祖鹏
黄宝家
孔萍
邓茜莹
赵莹莹
肖晨
张腾勋
邝茜雯
泰利宏
Original Assignee
慕恩（广州）生物科技有限公司
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Priority claimed from CN202110369840.5A external-priority patent/CN113069475B/zh
Application filed by 慕恩（广州）生物科技有限公司 filed Critical 慕恩（广州）生物科技有限公司
Priority to EP21935727.4A priority Critical patent/EP4349350A4/en
Priority to CA3214687A priority patent/CA3214687A1/en
Priority to KR1020237038125A priority patent/KR20230167089A/ko
Priority to US18/285,759 priority patent/US20240366686A1/en
Priority to AU2021439233A priority patent/AU2021439233A1/en
Priority to JP2023561061A priority patent/JP2024513071A/ja
Publication of WO2022213507A1 publication Critical patent/WO2022213507A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22—Hormones
    • A61K38/26—Glucagons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/04—Anorexiants; Antiobesity agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2300/00—Mixtures or combinations of active ingredients, wherein at least one active ingredient is fully defined in groups A61K31/00 - A61K41/00
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales

Definitions

  • the present disclosure relates to the technical field of bacterial strain isolation and application, and in particular, to a bacterial strain and composition, a combined drug and use, the combined drug comprising the bacterial strain and a hypoglycemic and lipid-lowering drug.
  • liver disease is mainly prevented and treated by vaccination against hepatitis, reducing alcohol consumption, improving dietary structure and doing physical exercise.
  • these strategies often only play a preventive role, and the benefits are very small, and the effects vary according to individual physiques. Once faced with liver disease that has already occurred, it seems helpless. Therefore, there is an urgent need for countermeasures that can effectively prevent and treat liver diseases, and it is necessary to develop an effective method or drug that can prevent and treat liver diseases with less side effects.
  • the current drug treatment of diabetes includes oral drug treatment, such as sulfonylurea drugs, biguanide hypoglycemic drugs, ⁇ -glucosidase inhibitors, insulin sensitizers, etc., and insulin injection therapy.
  • T2D type 2 diabetes
  • the efficacy of the drugs varies from person to person, and there are also worrying potential side effects, including: (1) Causes gastrointestinal adverse effects, including nausea, Vomiting and diarrhea; (2) increasing the burden on pancreatic islets, which may cause pancreatitis; (3) possibly causing goiter and thyroid cancer; (4) other side effects such as intestinal, renal function, and hypoglycemia; and, (5) having Tendency to cause depression. Therefore, there is an urgent need to develop effective methods or drugs for treating diabetes with less side effects.
  • Obesity drug treatment has a long history, and modern commonly used weight loss drugs include liraglutide, orlistat, sibutramine and rimonabant. But many weight-loss drugs have been restricted from the market or withdrawn from clinical use because some of them did not work as expected or because some of them caused serious adverse reactions in patients. Therefore, there is an urgent need to develop effective methods or drugs for the treatment of obesity and related diseases with less side effects.
  • the objectives of the present disclosure include, for example, to provide a bacterial strain and composition, a combined drug and use to solve the above technical problems, the combined drug comprising microorganisms and a hypoglycemic and lipid-lowering drug.
  • gut microbes Due to the emerging role of gut microbes in obesity and diabetes, the use of gut microbes themselves to improve diabetes, the interaction of gut microbes with antidiabetic drugs and their effects on drug function have become current research hotspots.
  • gut microbes can affect the host's metabolism, immune function, and brain function through the secretion of short-chain fatty acids, which are indispensable for human health.
  • the metabolic activities of gut microbes and their metabolites can affect the metabolism and efficacy of drugs, and drugs can also manipulate the composition of gut microbes and their metabolic capabilities.
  • the present disclosure provides a Christensenella sp., which can treat initial steatohepatitis lesions, slow down liver fat accumulation, and alleviate liver lesions, thereby effectively preventing and treating liver function damage and related diseases; the strain also has the ability to repair and digest It also has four functions such as reducing the body's fasting blood sugar, regulating insulin levels, reducing body weight, and regulating blood lipids, thereby preventing and treating gastrointestinal mucosal damage and related diseases, diabetes, obesity and obesity-related diseases. role of disease.
  • the present disclosure provides the use of a bacterial strain of Christensenella sp. in the manufacture of a medicament for the treatment or prevention of a disease or condition selected from at least one of liver function impairment and liver function impairment-related diseases, Digestive tract mucosal injury and gastrointestinal mucosal injury-related diseases, diabetes, obesity and obesity-related diseases.
  • Liver function impairment-related diseases include at least one of the following diseases: fatty liver, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, liver fibrosis, liver cirrhosis and liver cancer;
  • Digestive tract mucosal damage refers to increased gastrointestinal mucosal permeability and impaired mucosal barrier function.
  • Diseases related to gastrointestinal mucosal damage include at least one of the following diseases: leaky gut, peptic ulcer, gastroenteritis, inflammatory enteropathy; and
  • Obesity-related diseases include at least one of the following diseases: obesity, metabolic syndrome, cardiovascular disease, hyperlipidemia, hypercholesterolemia, hypertension, insulin resistance syndrome, obesity-related gastroesophageal reflux disease and steatohepatitis.
  • the bacterial strain has a 16s rRNA sequence that is at least 98.65% identical to SEQ ID NO. 1.
  • the bacterial strain has a 16s rRNA sequence that is at least 99% identical to SEQ ID NO. 1.
  • the bacterial strain has a 16s rRNA sequence that is 99%, 99.5%, 99.9%, or 100% identical to SEQ ID NO. 1.
  • the aforementioned medicament is lyophilized.
  • the aforementioned medicament further comprises one or more pharmaceutically acceptable excipients or carriers.
  • the aforementioned medicament is a vaccine composition.
  • the aforementioned medicaments are formulated for oral administration, injection administration, or gavage administration.
  • diabetes includes at least one of the following diseases: type 1 diabetes, type 2 diabetes, insulin resistance syndrome, glucose intolerance, hyperlipidemia, diabetic nephropathy Complications, diabetic neuropathy, diabetic eye disease, cardiovascular disease, diabetic foot and gestational diabetes.
  • the dosage forms of the above-mentioned drugs include tablets, pills, powders, suspensions, gels, emulsions, creams, granules, nanoparticles, capsules, suppositories, injections, sprays and injections.
  • the present disclosure provides a cell of the Christensenella strain or its progeny strain or subcloned strain deposited with the accession number GDMCC No: 61117.
  • the present disclosure provides a composition comprising the Christensenella strain described above and/or a metabolite of the strain.
  • the above-described composition further includes a pharmaceutically acceptable excipient or carrier.
  • the present disclosure provides the use of a combined drug comprising microorganisms and a hypoglycemic and lipid-lowering drug in the preparation of a drug for the treatment or prevention of at least one disease or symptom selected from the group consisting of liver function damage and liver function damage-related diseases , diabetes, obesity and obesity-related diseases; the microorganisms are bacteria of the species Christensenella sp.; the hypoglycemic and lipid-lowering drugs can improve glucagon-like peptide-1 (ie GLP-1 ) pathway sensitizing, supplementing and/or promoting GLP-1 action of one or more of the drugs.
  • glucagon-like peptide-1 ie GLP-1
  • the above-mentioned hypoglycemic and lipid-lowering drugs are GLP-1 receptor agonists (ie GLP-1RA) or GLP-1 mimics, GIP receptor agonists (ie glucose-dependent At least one of an insulin-stimulating polypeptide receptor agonist (also known as a gasprotin receptor agonist) and a dipeptidyl peptidase-4 (ie, DPP-4) inhibitor.
  • GLP-1 receptor agonists ie GLP-1RA
  • GIP receptor agonists ie glucose-dependent At least one of an insulin-stimulating polypeptide receptor agonist (also known as a gasprotin receptor agonist) and a dipeptidyl peptidase-4 (ie, DPP-4) inhibitor.
  • the GLP-1 receptor agonist or GLP-1 mimetic is selected from exenatide, liraglutide, semaglutide, oral dosage forms of semaglutide, benaglutide, lixisenatide and exenatide At least one of the peripeptide preparations.
  • the present disclosure provides a combined drug comprising a hypoglycemic and lipid-lowering drug and a microorganism; the microorganism is a bacterium of the species Christensenella sp.
  • a hypoglycemic and lipid-lowering drug is one or more drugs that can improve the sensitivity of the glucagon-like peptide-1 pathway, supplement and/or promote the action of GLP-1.
  • compositions as above or a combination drug as described above in the preparation of a medicament or a preparation for use in at least one selected from the following:
  • the present disclosure provides a composition
  • a composition comprising a bacterial strain of the above-mentioned Christensenella sp. species or the above-mentioned Christensenella strain or a progeny or subcloned strain thereof and/or its metabolites,
  • a disease or condition selected from the group consisting of liver function damage and liver function damage-related diseases, gastrointestinal mucosal damage and diseases related to gastrointestinal mucosal damage, diabetes, obesity and obesity-related diseases.
  • the above-mentioned excipients include antioxidants, chelating agents, emulsifiers, solvents.
  • the present disclosure provides a combination drug, which includes a microorganism and a hypoglycemic and lipid-lowering drug
  • the microorganism is the above-mentioned bacterial strain of Christensenella sp. species or the above-mentioned Christensenella strain or its Progeny strains or subcloned strains and/or their metabolites
  • the hypoglycemic and lipid-lowering drug is one of the drugs that can improve the sensitivity of the glucagon-like peptide-1 pathway, supplement and/or promote the action of GLP-1 or more, for the treatment or prevention of at least one disease or condition selected from the group consisting of liver function impairment and liver function impairment-related diseases, diabetes, obesity and obesity-related diseases.
  • the present disclosure provides a method for treating or preventing a disease or condition, comprising administering the above-mentioned composition or the above-mentioned combined drug to a subject in need, the disease or condition being selected from at least one of the following: liver function impairment and Liver function impairment-related diseases, diabetes, obesity and obesity-related diseases.
  • the present disclosure provides a kit comprising the above-mentioned combined drug.
  • the Christensen bacteria provided by the present disclosure can be applied to the treatment or prevention of liver function damage and liver function damage-related diseases, gastrointestinal mucosal damage and diseases related to gastrointestinal mucosal damage, diabetes, obesity and obesity-related diseases. It has been verified by the applicant that the Christensen bacteria provided by the present disclosure has no toxic and side effects on the kidney, and includes but is not limited to the following: can reduce liver weight; treat initial steatohepatitis lesions; slow down fat accumulation in liver cells; reduce serum AST and ALT; Reduce abdominal white fat inflammatory lesions. Christensen can also reduce the body's fasting blood sugar, and significantly improve the body's level of insulin resistance, which has the effect of preventing and treating diabetes. In addition, Christensen can reduce body fat in mammals and improve metabolic function in obese patients. Christensen also has the function of repairing damaged digestive tract mucosa and preventing and treating mucosal damage-related diseases.
  • the combination of the hypoglycemic and lipid-lowering drugs provided by the present disclosure and microorganisms can be used for the treatment or prevention of liver function damage and liver function damage-related diseases, diabetes, obesity and obesity-related diseases. It has been verified by the applicant that the combined drug of GLP-1 receptor agonist or GLP-1 mimetic provided by the present disclosure and microorganism has a technical effect of synergism, and the combined drug has a synergistic effect than that of single administration of microorganism or single administration of GLP- 1 receptor agonists or GLP-1 mimics have better therapeutic effects, and microorganisms can enhance the weight loss effect of GLP-1 receptor agonists or GLP-1 mimics, improve glucose tolerance, and reduce fasting blood sugar. In addition, the above-mentioned combined drugs have no toxic and side effects on the kidneys and can reduce the weight of the liver. Combination drugs help to enhance the sensitivity of GLP-1; avoid GLP-1RA resistance and related side effects caused by intestinal flocculation.
  • Fig. 1 is the macroscopic morphological diagram of the isolated strain
  • Figure 2 is a microscopic morphological diagram of the isolated strain
  • Fig. 3 is the macroscopic plate picture after single colony anaerobic cultivation
  • Figure 4 is a phylogenetic evolutionary tree
  • Figure 5 is the NASH liver injury scoring standard
  • Figure 6 shows the effect of MNO-863 on the liver weight of obese model mice
  • Fig. 7 is the result of HE staining of liver tissue
  • Figure 8 is a graph showing the results of oil red staining of liver tissue
  • Figure 9 is the NAFLD/NASH liver pathology score
  • Figure 10 is a graph showing the statistical results of the degree of hepatic steatosis
  • Figure 11 is a graph showing the statistical results of liver lobular inflammation score
  • Fig. 12 is a graph showing the statistical result of liver ballooning degeneration score
  • Figure 13 is AST (aspartate aminotransferase), ALT (alanine aminotransferase) levels in serum;
  • Figure 14 is a micrograph and a total area statistic of inflammatory lesions of white fat in the abdomen of mice;
  • Figure 15 is a graph showing the detection results of the content of serum creatinine (CREA), blood urea (UREA) and blood uric acid (UA) in mice;
  • Figure 16 shows the effect of MNO-863 on oral glucose tolerance in high-fat diet-induced obese mice
  • Figure 17 is the effect of MNO-863 on fasting blood glucose (mmol/L) in high-fat diet-induced obese mice;
  • Figure 18 shows the effect of MNO-863 on the HOMA-IR index of high-fat diet-induced obese mice
  • Figure 19 is the effect of MNO-863 on the body weight (g) of high-fat diet-induced obese mice
  • Figure 20 is the effect of MNO-863 on the body weight (%) of high-fat diet-induced obese mice
  • Figure 21 is the effect of MNO-863 on the food intake (g) of high-fat diet-induced obese mice
  • Figure 22 shows the effect of MNO-863 on TC, TG, LDL and HDL-C in high-fat diet-induced obese mice
  • Figure 23 shows the effect of MNO-863 on inguinal fat, subcutaneous fat and epididymal fat in high-fat diet-induced obese mice
  • Figure 24 is a micrograph of mouse colon tissue in the HFD control group
  • Figure 25 is a micrograph of mouse ileum tissue in the HFD control group
  • Figure 26 is a micrograph of the colon tissue of mice in the MNO-863 treatment group.
  • Figure 27 is a micrograph of mouse ileum tissue in the MNO-863 treatment group
  • Figure 28 is a micrograph of the colon tissue of mice in the NCD control group
  • Figure 29 is a micrograph of mouse ileal tissue in the NCD control group
  • Figure 30 shows the effect of MNO-863 in combination with Liraglutide on the absolute body weight of obese mice during the four-week intervention period and the percentage change in body weight
  • Figure 31 shows body weight and percentage change in body weight after four weeks of intervention
  • Figure 32 is the body weight and the percentage change in body weight after four weeks of intervention and four weeks of drug withdrawal;
  • Figure 33 is the weight of inguinal fat after 4 weeks of drug recovery
  • Figure 34 is the single use of MNO-863 strain and the effect of strain combined with Liraglutide on glucose tolerance in obese mice;
  • Figure 35 is the single use of MNO-863 strain and the effect of strain combined with Liraglutide on glucose hyperglycemia in obese mice;
  • Figure 36 shows the effect of MNO-863 strain on hyperglycemia in obese mice after drug withdrawal for 4 weeks
  • Figure 37 is a graph showing the effect of MNO-863 strain alone and the strain combined with Liraglutide on liver weight after 4 weeks of drug withdrawal.
  • a bacterial strain of Christensenella sp. species in the preparation of a medicament for the treatment or prevention of a disease or condition selected from at least one of the following: liver function damage and liver function damage-related diseases, digestive tract mucosa Injury and gastrointestinal mucosal injury-related diseases, diabetes, obesity and obesity-related diseases.
  • a combined drug comprising a microorganism and a hypoglycemic and lipid-lowering drug in the preparation of a drug for the treatment or prevention of at least one disease or symptom selected from the group consisting of liver function damage and liver function damage-related diseases, diabetes, obesity and obesity Related diseases;
  • the microorganisms are bacteria of the species Christensenella sp. and/or one or more of the drugs that promote the action of GLP-1.
  • Liver function impairment-related diseases include at least one of the following diseases: fatty liver, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, and liver cirrhosis.
  • liver function impairment-related diseases also include liver fibrosis and liver cancer.
  • Digestive tract mucosal damage refers to increased gastrointestinal mucosal permeability and impaired mucosal barrier function.
  • Diseases related to gastrointestinal mucosal damage include at least one of the following diseases: leaky gut, peptic ulcer, gastroenteritis, inflammatory Enteropathy and other diseases; it should be noted that leaky gut is manifested by increased intestinal permeability.
  • Obesity-related diseases include at least one of the following diseases: cardiovascular disease, hyperlipidemia, insulin resistance syndrome, obesity-related gastroesophageal reflux disease, and steatohepatitis.
  • obesity-related diseases also include obesity, metabolic syndrome, hypercholesterolemia, hypertension.
  • the above-mentioned "obesity-related disease” may be selected from the following diseases: overeating, binge eating, bulimia, hypertension, diabetes, elevated plasma insulin concentration, insulin resistance Sex, hyperlipidemia, metabolic syndrome, insulin resistance syndrome, obesity-related gastroesophageal reflux disease, arteriosclerosis, hypercholesterolemia, hyperuricemia, low back pain, cardiac hypertrophy and left ventricular hypertrophy , lipodystrophy, nonalcoholic steatohepatitis, cardiovascular disease, and polycystic ovary syndrome, as well as those with obesity-related disorders and including those who desire weight loss.
  • diseases overeating, binge eating, bulimia, hypertension, diabetes, elevated plasma insulin concentration, insulin resistance Sex, hyperlipidemia, metabolic syndrome, insulin resistance syndrome, obesity-related gastroesophageal reflux disease, arteriosclerosis, hypercholesterolemia, hyperuricemia, low back pain, cardiac hypertrophy and left ventricular hypertrophy , lipodystrophy, nonalcoholic stea
  • Type 1 diabetes is caused by autoimmune damage or idiopathic causes. It is characterized by absolute destruction of islet function. It mostly occurs in children and adolescents. It must be treated with insulin to obtain satisfactory results, otherwise it will be life-threatening.
  • Type 2 diabetes is a multifactorial syndrome characterized by abnormal carbohydrate/fat metabolism, usually including hyperglycemia, hypertension, and abnormal cholesterol. Type 2 diabetes is caused by the ineffectiveness of insulin (less binding to receptors). Therefore, not only fasting blood sugar should be checked, but also blood sugar 2 hours after meals should be observed, especially pancreatic islet function tests.
  • GDM gestational diabetes mellitus
  • diabetes examples include but are not limited to the treatment or prevention of type 1 diabetes (T1D), type 2 diabetes (T2D) and gestational diabetes (GDM).
  • T1D type 1 diabetes
  • T2D type 2 diabetes
  • GDM gestational diabetes
  • the above-mentioned uses for the treatment of obesity and obesity-related diseases include not only the combination drug of the bacterial strain provided by the present disclosure and the hypoglycemic and lipid-lowering drug, but also other active compounds, and the other active compounds may be two or more. combination of other active compounds.
  • the above-mentioned combination drugs are used in combination with anti-obesity compounds such as fenfluramine, dexfenfluramine, phentermine, sibutramine, orlistat, neuropeptide Y5 inhibitors and ⁇ 3-adrenergic receptor agonists.
  • the combination of the above combination drugs and cholesterol lowering agents such as: (i) HMG-CoA reductase inhibitors (lovastatin, simvastatin, pravastatin, fluvastatin, atorvastatin) , rivastatin, ivastatin, rosuvastatin, and other statins); (ii) chelates (cholestyramine, colestipol, and cross-linked dextran dialkylaminoalkyl derivatives); (iii) nicotinol, niacin or other salts; (iv) PPAR ⁇ agonists such as fenofibric acid derivatives (gefibrozil, clofibrate, fenofibrate and bezafibrate) ); (v) PPAR ⁇ / ⁇ dual agonists such as KRP-297; (vi) cholesterol absorption inhibitors such as ⁇ -sitosterol and ezetimibe; (vii) acetyl CoA
  • it may also be used in combination with other drugs for inflammation, such as aspirin, non-steroidal anti-inflammatory drugs, glucocorticoids, sulfasalazine, and selective inhibitors of cyclooxygenase II.
  • drugs for inflammation such as aspirin, non-steroidal anti-inflammatory drugs, glucocorticoids, sulfasalazine, and selective inhibitors of cyclooxygenase II.
  • the bacterial strain has a 16s rRNA sequence that is at least 98.65% identical to SEQ ID NO. 1. For example, with SEQ ID NO. 198.7%, 98.75%, 98.8%, 98.85%, 98.9%, 98.95%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% identical 16s rRNA sequences.
  • the Christensenella bacterial strain has a 16s rRNA sequence that is at least 99% identical to SEQ ID NO. 1.
  • the Christensenella bacterial strain has a 16s rRNA sequence that is 99%, 99.5%, 99.9%, or 100% identical to SEQ ID NO. 1.
  • the aforementioned medicament is lyophilized. Lyophilization is an effective and convenient technique for preparing stable compositions that allow the delivery of bacteria.
  • the above-mentioned drugs are powdered or tableted by freeze-drying to facilitate coating or transportation.
  • the aforementioned medicament further comprises one or more pharmaceutically acceptable excipients or carriers.
  • compositions can be antioxidants, chelating agents, emulsifiers, solvents, and the like.
  • the dosage forms of the drug include, but are not limited to, tablets, pills, powders, suspensions, gels, emulsions, creams, granules, nanoparticles, capsules, suppositories, injections, sprays and injections.
  • the above-mentioned drugs also include their pharmaceutically acceptable salts, their solvates or their stereoisomers, and the pharmaceutical compositions formed with one or more pharmaceutically acceptable carriers and/or diluents, which can be in a manner known in the art. It is formulated into any clinically or pharmaceutically acceptable dosage form and administered to patients in need of such treatment by oral, injection or gavage administration.
  • conventional solid preparations such as tablets, capsules, pills, granules, etc., can be prepared; oral liquid preparations, such as oral solutions, oral suspensions, syrups, etc., can also be prepared.
  • suitable fillers, binders, disintegrants, lubricants and the like can be added.
  • the aforementioned medicament is a vaccine composition.
  • the aforementioned medicaments are formulated for oral administration, injection administration, or gavage administration.
  • the administration of the bacterial strains of the present disclosure showed a therapeutic effect comparable to that of the diabetes drug Liraglutide through gavage in mice experiments.
  • the above-mentioned medicaments also include pharmaceutically acceptable salts
  • pharmaceutically acceptable salts refer to the following salts, which, within sound medical judgment, are suitable for use with humans and The tissue contact of lower animals does not have excessive toxicity, irritation, allergic reaction, etc., and is commensurate with a reasonable benefit/risk ratio.
  • the Christensenella sp. MNO-863 provided by the present disclosure was isolated from a fecal sample of a healthy male volunteer of Han nationality in Guangzhou City, Guangdong Province. On August 4, 2020, it was deposited in the Guangdong Provincial Microorganism Collection Center.
  • the deposit number is: GDMCC No: 61117; the deposit address is: Guangdong Institute of Microbiology, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou City, the test result is survival, and its taxonomic name is Christensenella sp.
  • Macroscopic morphology anaerobic culture at 37°C for 72h, the colonies are light yellow, round, with moist surface, translucent and neat edges.
  • the bacteria are short rod-shaped, without spores, without flagella, without movement, 0.3-0.4 ⁇ m ⁇ 0.6-1.1 ⁇ m, single or paired, Gram-negative.
  • Colony characteristics MNO-863 was cultured anaerobic at 37°C for 72h on 104 plates, and a single colony was round, slightly convex, transparent, white, and smooth surface, with a colony diameter of about 0.46-0.50mm.
  • the present disclosure also provides a composition comprising the above-described Christensenella strain and/or a metabolite of the strain.
  • the above-mentioned Christensen bacteria strain can be obtained by the direct culture of the bacterial strain deposited by the above-mentioned deposit number GDMCC No: 61117, or it can be a progeny strain (progeny) or a bacterial strain cultivated from an original strain (subcloned strain), such as , to separate cells.
  • the Christensenella strain provided by the present disclosure also includes its derivatives, for example, it can be modified at the gene level without eliminating its biological activity.
  • the above-mentioned derivative strain has therapeutic activity, and has the activity equivalent to the Christensenella strain deposited with the deposit number of GDMCC No: 61117.
  • the above-described composition further includes a pharmaceutically acceptable excipient or carrier.
  • compositions as above for the preparation of a medicament or a preparation for use selected from at least one of the following:
  • Reduce liver weight treat initial steatohepatitis lesions; slow fat accumulation in liver cells; reduce serum AST (aspartate aminotransferase), ALT (alanine aminotransferase); reduce abdominal white fat inflammatory lesions; reduce mammalian body weight; reduce mammalian food intake reduce mammalian body fat; reduce the level of at least one of the following indicators in mammalian serum: total cholesterol level, low density lipoprotein and triglyceride level; increase mammalian serum high density lipoprotein level; improve mammalian Impaired oral glucose tolerance; lowers fasting blood glucose in mammals; lowers HOMA-IR index in mammals; and, repairs gastrointestinal mucosal damage.
  • liver damage and related diseases reduce liver weight; treat initial steatohepatitis lesions; slow fat accumulation in liver cells; reduce serum AST (aspartate aminotransferase), ALT (alanine aminotransferase) levels; Fat inflammatory lesions.
  • the following is the use in the treatment or prevention of obesity and related diseases: reduce the body weight of the mammal; reduce the food intake of the mammal; reduce the body fat of the mammal; reduce the level of at least one of the following indicators in the serum of the mammal: total cholesterol level , low-density lipoprotein and triglyceride levels; increase mammalian serum high-density lipoprotein levels.
  • the above-mentioned repairing gastrointestinal mucosal damage refers to repairing gastrointestinal mucosal damage, typically repairing intestinal mucosal damage. Repairing intestinal mucosal damage refers to achieving at least one of the following indicators: restoring the structural integrity of intestinal mucosal tissue, reducing the degree of atrophy of intestinal villi and the number of hyphae.
  • a combined drug comprising microorganisms and a hypoglycemic and lipid-lowering drug; the microorganisms are bacteria of the species Christensenella sp.
  • a hypoglycemic and lipid-lowering drug is one or more drugs that can improve the sensitivity of the glucagon-like peptide-1 pathway, supplement and/or promote the action of GLP-1.
  • the above-mentioned hypoglycemic and lipid-lowering drugs are GLP-1 receptor agonists (ie GLP-1RA) or GLP-1 mimics, GIP receptor agonists (ie glucose-dependent insulin secretion-stimulating polypeptide receptor agonists, also known as gastric at least one of aprotinin receptor agonist), dipeptidyl peptidase-4 (ie DPP-4) inhibitor.
  • GLP-1 receptor agonist or GLP-1 mimetic is selected from exenatide, liraglutide, semaglutide, oral dosage forms of semaglutide, benaglutide, lixisenatide and exenatide At least one of the peripeptide preparations.
  • Intestinal flora disturbance can easily lead to the ineffectiveness and resistance of GLP-1 drugs, and the inventors propose that the combination of probiotics and GLP-1 can enhance GLP-1 sensitivity and avoid GLP-1RA caused by intestinal disturbance. resistance and related side effects.
  • This example provides the isolation and identification of Christensenella sp. MNO-863.
  • the Christensenella sp. MNO-863 of the present disclosure was isolated from a fecal sample of a healthy male volunteer of Han nationality in Guangzhou City, Guangdong province, who had not used antibiotics for the first three months when the sample was collected.
  • anaerobic blood plate Joangmen Kailin anaerobic blood agar medium, Guangdong Machinery Zhuzhun 20172400940
  • MNO-863 was subjected to solid plate coating and liquid culture to observe the microbiological characteristics.
  • Morphological characteristics Referring to the macroscopic morphology shown in Figure 1, anaerobic culture at 37°C for 72h, the colonies were light yellow, round, with moist surface, translucent, and neat edges.
  • MNO-863 was cultured anaerobically at 37°C for 72 hours on a plate of 104 medium, and MNO-863 was subjected to Gram staining (the upper image in Figure 2) and spore staining for microscopy (the lower image in Figure 2). ).
  • the cells are short rod-shaped, without spores, without flagella, without movement, 0.3-0.4 ⁇ m ⁇ 0.6-1.1 ⁇ m, single or paired, and Gram-negative.
  • MNO-863 was cultured anaerobic at 37°C for 72 hours on a plate of 104 medium, and a single colony was round, slightly convex, transparent, white, and smooth surface, with a colony diameter of about 0.46-0.50mm (refer to Figure 3). ).
  • MNO-863 does not grow under aerobic conditions, but grows well under anaerobic conditions, and the optimum growth temperature is 37°C.
  • DSM 22607 standard strain Christensenella minuta
  • Test results are shown with reference to Table 2, as can be seen from Table 2, the physiological and biochemical characteristics of the isolated MNO-863 and standard strain DSM 22607 are basically consistent, and exist when the substrates are glycerol, gelatin hydrolysis, mannose, mannitol and salicyl alcohol. difference.
  • the sequence of MNO-863 strain was determined by 16S sequence fragment (amplification primer and sequencing primer are 27F: 5'-AGAGTTTGATCCTGGCTCAG-3' and 1492R: 5'-GGTTACCTTGTTACGACTT-3'), and the 16s rRNA determination results are shown in the sequence SEQ ID NO.1:
  • MNO-863 The whole genome of MNO-863 was sequenced, and MEGA5.0 software was used.
  • the 16S rDNA sequence phylogenetic tree of "MNO-863" and related species was displayed by the ortho-ligation method, and the similarity calculation was repeated 1000 times.
  • the genome sequences of standard strains of the Christensenellaceae family were aligned with each other.
  • the phylogenetic tree shows (see Figure 4) that MNO-863 is on the same clade with three standard strains Christensenella minuta (DSM 22607), Christensenella timonensis (Marseille-P2437) and Christensenella massiliensis (Marseille-P2438), indicating that MNO-863 belongs to A species within the range of Christensenella sp.
  • MNO-863 belongs to a species within the genus Christensenella from a taxonomic perspective, and is named Christensenella sp. MNO-863.
  • the deposit number is: GDMCC No: 61117; the deposit address is: Guangdong Institute of Microbiology, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou City, and the test result is alive.
  • the taxonomic name is Christensenella sp.
  • the in vivo test of MNO-863 in a high-fat diet-induced obesity mouse model was carried out to verify its application in the treatment or prevention of liver function damage and related diseases.
  • mice 32 C57BL/6J male mice (purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.) were purchased, which were normally raised mice, 5 weeks old. The mice were in the same environment during the growth process, 8 of them were given maintenance feed for SPF rats and mice (Guangzhou Hancheng Experimental Equipment Co., Ltd.), and the remaining 24 were given D12492 high-fat feed (Parker Bio), after about 8-10 weeks of feeding , Weighing, the body weight of the diet-induced obesity model was 38.00 ⁇ 2.00g.
  • Test strain MNO-863 cultivated anaerobic, the medium is the 104 liquid medium in Example 1, cultivated under anaerobic conditions at 37°C for 48h, until the bacterial concentration is about the order of 10 11 CFU/mL, then the As the experimental group gavage. Bacteria were stored anaerobic at 4°C.
  • PBS phosphate buffer solution It is a mixed solution composed of weak acid and its salt, weak base and its salt, which can offset and reduce the influence of external strong acid or strong base on the pH of the solution to a certain extent, so as to maintain the pH of the solution.
  • the pH value is relatively stable.
  • the formulation of PBS phosphate buffered solution is shown in Table 4 below:
  • mice that were given maintenance diet for SPF rats were randomly divided into 2 cages, 4 mice/cage, as the first group. From 24 obese mice, 16 mice with a body weight of 38.00g ⁇ 2.00g were selected and divided into 2 groups (as the second and third groups), 8 mice in each group, 4 mice/cage.
  • the first group was the control group fed with normal feed (NCD-control group)
  • the second group was the high-fat diet-induced obesity mouse model group (HFD-control group)
  • the third group was the bacterial treatment group (MNO-863 )
  • the second and third groups were fed with high-fat diet, and the groupings are shown in Table 5.
  • the virtual administration was started and then the administration was started.
  • the first group and the second group were given the same amount of PBS phosphate buffer solution by gavage, and the third group was given the MNO-863 test strain for gavage intervention, and the intervention lasted for 4 weeks. .
  • the amount of intragastric bacterial solution was 0.2 mL/10 g mouse body weight. Data such as body weight, state, and food intake of the mice were recorded before and after modeling, and every 3 days before and after the intervention. After dosing, the animals were dissected and tissues were harvested.
  • the use of experimental animals pays attention to animal welfare, follows the principle of "reduction, replacement and optimization", and is approved by the laboratory animal ethics committee of this unit. During the experiment, they were supervised and inspected by the experimental animal ethics committee.
  • the effect of MNO-863 on the liver weight of obese model mice is shown in Figure 6.
  • the MNO-863 treatment group can significantly reduce the liver weight of the obese mice and return to the normal level of the liver weight of the NCD control group.
  • the data of the effect on the liver weight of the obese model mice are shown in Table 6.
  • Results are presented as mean, *p ⁇ 0.05 compared to HFD-control group.
  • MNO-863 can treat the initial steatohepatitis lesions and slow down fat accumulation in obese mice under the condition of high-fat diet.
  • NAFLD/NASH liver pathology score is shown in Figure 9. According to the scores, MNO-863 can treat the initial steatohepatitis lesions and slow down fat accumulation in obese mice.
  • Figure 10 shows the degree of hepatic steatosis, and it can be seen from Figure 10 that MNO-863 can effectively alleviate the hepatic steatosis.
  • Figure 11 shows the hepatic lobular inflammation score. It can be seen from Figure 11 that MNO-863 can effectively inhibit the occurrence of hepatic lobular inflammation compared with the HFD control group.
  • the liver ballooning degeneration score is shown in FIG. 12 , and it can be seen from FIG. 12 that the liver ballooning degeneration score of MNO-863 was significantly decreased compared with the HFD control group.
  • AST aspartate aminotransferase
  • ALT alanine aminotransferase
  • Figure 14 shows the micrographs and total area statistics of inflammatory lesions of white fat in the abdomen of mice.
  • the detection of serum creatinine (CREA) in mice is based on the principle of enzymatic detection, through the end-point method (using a creatinine determination kit, Lei She, S03076), and using an automatic biochemical analyzer for detection.
  • the detection of blood urea (UREA) is based on the detection principle of the urease-glutamate dehydrogenase method, through a two-point method (using a urea determination kit, Leishe Life Science Co., Ltd., S03036), and using a fully automatic biochemical method analyzer to detect.
  • the detection of serum uric acid (UA) content is based on the detection principle of the uricase method, through the end-point method (using a uric acid determination kit, Leishe Life Science Co., Ltd., S03035), and using an automatic biochemical analyzer for detection.
  • CREA mouse serum creatinine
  • IUA blood urea
  • UUA blood uric acid
  • mice 40 C57BL/6J male mice (purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.) were purchased, which were normally raised mice, 5 weeks old. The growth process of the mice was in the same environment. Among them, 8 mice were given maintenance feed for SPF rats and mice (Guangzhou Hancheng Experimental Equipment Co., Ltd.), and 32 mice were given D12492 high-fat feed (Parker Biotechnology). After feeding for about 8-10 weeks, they said The body weight of the diet-induced obesity model was 38.00 ⁇ 2.00g.
  • Test strain MNO-863 cultured anaerobic, the medium is 104 liquid medium, cultured under anaerobic conditions at 37°C for 48h, until the bacterial concentration is about the order of 10 11 CFU/mL, it can be used as the experimental group.
  • Stomach Bacteria were stored anaerobic at 4°C.
  • PBS phosphate buffer solution It is a mixed solution composed of weak acid and its salt, weak base and its salt, which can offset and reduce the influence of external strong acid or strong base on the pH of the solution to a certain extent, so as to maintain the pH of the solution.
  • the pH value is relatively stable.
  • the formulation of PBS phosphate buffer solution is the same as Table 4 in Example 2.
  • Liraglutide is a human glucagon-like peptide-1 (GLP-1) analog for the treatment of diabetes. Available from Novo Nordisk under the trade name -Novo Nordisk, administered subcutaneously at 15 ⁇ g/kg/d.
  • GLP-1 human glucagon-like peptide-1
  • mice that were given maintenance diet for SPF rats were randomly divided into 2 cages, 4 mice/cage, as the first group.
  • 24 mice with a weight of 38.00g ⁇ 2.00g were selected from 32 obese mice and divided into 3 groups (respectively as the second group, the third group and the fourth group), 8 mice in each group, 4 mice/ cage.
  • the first group was the control group fed with normal feed (NCD-control group)
  • the second group was the high-fat diet-induced obesity mouse model group (HFD-control group)
  • the third group was the bacterial treatment group (MNO-863 )
  • the fourth group was the liraglutide positive control group
  • the second, third and fourth groups were fed with high-fat diet, and the groupings are shown in Table 7.
  • the virtual administration was started and then the administration was started.
  • the first group and the second group were given the same amount of PBS phosphate buffer solution by gavage, and the third group was given the MNO-863 test strain for gavage intervention, and the intervention lasted for 4 weeks. .
  • the amount of intragastric bacterial solution was 0.2 mL/10 g mouse body weight. Data such as body weight and state of the mice were recorded before and after modeling, and every 3 days before and after the intervention.
  • the use of experimental animals pays attention to animal welfare, follows the principle of "reduction, replacement and optimization", and is approved by the laboratory animal ethics committee of this unit. During the experiment, they were supervised and inspected by the experimental animal ethics committee.
  • Oral glucose tolerance test On the 28th day after the animals were administered, the OGTT of fasting for 12 h was determined (eg, fasting from 20:30:00 in the evening to 08:30:00 the next day). The fasting body weight of the mice was weighed, and glucose was administered to the stomach according to the fasting body weight of the mice. The dose of glucose administered to the stomach was 2g/kg (glucose g/mice fasting body weight kg), and the fasting blood glucose and 15min and 30min after sugar administration were measured respectively. , 60min, 90min, 120min blood sugar value. Each mouse was strictly timed, and the blood glucose level was measured accurately at 6 time points. Oral glucose tolerance test is a glucose load test, which is used to understand the function of islet beta cells and the body's ability to regulate blood sugar, and to observe the ability of patients to tolerate glucose. It is currently recognized as the gold standard for the diagnosis of diabetes.
  • mice were fasted overnight for 10-12 hours, and the fasting body weight was weighed the next day.
  • anesthesia with isoflurane Reward Life Technology Co., Ltd.
  • blood was collected from the eyes of the mice, and a blood glucose meter (ACCU-CHEK) was used.
  • ACCU-CHEK blood glucose meter
  • Type, Roche to detect the blood sugar level of fasting blood sugar, and place the blood in a refrigerator at 4 °C for 3-4 hours.
  • After the blood clots shrink centrifuge at 4500 r/min for 15 min at 4 °C, collect the upper serum, and use mouse insulin (INS).
  • enzyme-linked immunosorbent assay kit (Wuhan Huamei Biological Engineering Co., Ltd.) to detect the content of insulin in serum.
  • HOMA-IR was calculated from fasting blood glucose levels and insulin levels in serum.
  • HOMA-IR is an index used to evaluate the level of insulin resistance of an individual. It has become a common index widely used in clinical evaluation of insulin sensitivity, insulin resistance level and islet ⁇ -cell function in diabetic patients.
  • the calculation method is: fasting blood glucose level. (FPG, mmol/L) ⁇ fasting insulin level (FINS, ⁇ U/mL)/22.5, the HOMA-IR index of normal individuals is 1.
  • FPG fasting blood glucose level.
  • FGS fasting insulin level
  • ⁇ U/mL fasting insulin level
  • Insulin resistance refers to the decrease in the efficiency of insulin to promote glucose uptake and utilization due to various reasons.
  • the body compensatory secretion of excessive insulin produces hyperinsulinemia to maintain the stability of blood sugar. Insulin resistance can easily lead to metabolic syndrome and type 2 diabetes. .
  • Results are expressed as mean ⁇ standard deviation (SD), *p ⁇ 0.05, **p ⁇ 0.01, ***p ⁇ 0.001, ****p ⁇ 0.0001 compared to HFD-control group.
  • Impaired glucose tolerance indicates a decline in the body's ability to metabolize glucose and is commonly seen in type 2 diabetes and obesity.
  • the high-fat diet-induced obese mice in the MNO-863 treatment group had significantly lower blood sugar levels after 15 min of glucose gavage than in the HFD control group.
  • the blood glucose level of the mice in the MNO-863 treatment group gradually decreased, and after 120 minutes, the blood glucose level recovered to close to the NCD control group, much lower than the HFD control group, and there was a significant difference.
  • MNO-863 showed a therapeutic effect comparable to that of the diabetes drug liraglutide.
  • Results are expressed as mean, *p ⁇ 0.05, ****p ⁇ 0.0001 compared to HFD-control.
  • the MNO-863 treatment group can significantly reduce the blood glucose level of the high-fat diet-induced obese mice, and a significant difference is achieved compared with the HFD control group.
  • MNO-863 has more obvious control of blood sugar. It shows that MNO-863 has obvious hypoglycemic effect and can improve the symptoms of diabetes.
  • Insulin resistance is the main cause of type 2 diabetes and can promote the occurrence and progression of complications in patients with type 2 diabetes.
  • Biochemical indicators related to HOMA-IR can effectively reveal the cause of IR.
  • the HOMA-IR of diabetic patients is significantly higher than that of the normal population.
  • the intervention of MNO-863 can significantly reduce the HOMA-IR of high-fat diet-induced obese mice. It shows that MNO-863 can improve insulin resistance and islet ⁇ cell function, so as to achieve the purpose of preventing and treating diabetes.
  • Test process (1) Test grouping
  • mice that were given maintenance diet for SPF rats were randomly divided into 2 cages, 4 mice/cage, as the first group.
  • 24 mice with a weight of 38.00g ⁇ 2.00g were selected from 32 obese mice and divided into 3 groups (respectively as the second group, the third group and the fourth group), 8 mice in each group, 4 mice/ cage.
  • the first group was the control group (control group) fed with ordinary feed
  • the second group was the high-fat diet-induced obesity mouse model group (model group)
  • the third group was the bacterial treatment group
  • the fourth group was liralu
  • the peptide positive control group, the second, third and fourth groups were fed with high-fat diet, and the groupings are shown in Table 11.
  • the virtual administration was started and then the administration was started.
  • the first group and the second group were given the same amount of PBS phosphate buffer solution by gavage, and the third group was given the MNO-863 test strain for gavage intervention, and the intervention lasted for 4 weeks. .
  • the amount of intragastric bacterial solution was 0.2 mL/10 g mouse body weight. Data such as body weight, state, and food intake of the mice were recorded before and after modeling, and every 3 days before and after the intervention. After dosing, the animals were dissected and tissues were harvested.
  • the use of experimental animals pays attention to animal welfare, follows the principle of "reduction, replacement and optimization", and is approved by the laboratory animal ethics committee of this unit. During the experiment, they were supervised and inspected by the experimental animal ethics committee.
  • mice were sacrificed, the fat content was recorded, blood was collected, and the serum was collected by centrifugation at 4500 r/min for 15 min at 4 °C. Serum lipid levels were detected with density lipoprotein (LDLC) assay kit (Nanjing Jiancheng Institute of Bioengineering).
  • LDLC density lipoprotein
  • Results are presented as mean ⁇ SD, ****p ⁇ 0.0001 compared to HFD-control group.
  • the effect of MNO-863 on blood lipids in high-fat diet-induced obesity model mice has a significant effect on the blood lipid levels of mice under continuous high lipid intake.
  • the control effect can reduce the indicators related to cardiovascular diseases such as primary hyperlipidemia: total cholesterol (TC), triglyceride (TG) and low-density lipoprotein (LDLC), and increase high-density lipoprotein (HDL- C) levels, while HDL was inversely correlated with the incidence and severity of cardiovascular disease, with significant differences in total cholesterol (TC) and triglyceride (TG) results.
  • TC total cholesterol
  • TG triglyceride
  • LDLC low-density lipoprotein
  • HDL- C high-density lipoprotein
  • Results are presented as means, *p ⁇ 0.05, **p ⁇ 0.01 compared to HFD-control group.
  • Results are expressed in Mean, *p ⁇ 0.05, **p ⁇ 0.01, ****p ⁇ 0.0001 compared to HFD-control.
  • mice that were given maintenance diet for SPF rats were randomly divided into 2 cages, 4 mice/cage, as the first group. From 24 obese mice, 16 mice with a body weight of 38.00g ⁇ 2.00g were selected and divided into 2 groups (as the second and third groups), 8 mice in each group, 4 mice/cage.
  • the first group was the control group fed with normal feed (NCD-control group)
  • the second group was the high-fat diet-induced obesity mouse model group (HFD-control group)
  • the third group was the bacterial treatment group (MNO-863 )
  • the second and third groups were fed with high-fat feed
  • the grouping method was the same as in Table 5 of Example 2.
  • One week after the animals were grouped the virtual administration was started and then the administration was started.
  • the first group and the second group were given the same amount of PBS phosphate buffer solution by gavage, and the third group was given the MNO-863 test strain for gavage intervention, and the intervention lasted for 4 weeks. .
  • the amount of intragastric bacterial solution was 0.2 mL/10 g mouse body weight. Data such as body weight, state, and food intake of the mice were recorded before and after modeling, and every 3 days before and after the intervention. After dosing, the animals were dissected and tissues were harvested.
  • the use of experimental animals pays attention to animal welfare, follows the principle of "reduction, replacement and optimization", and is approved by the laboratory animal ethics committee of this unit. During the experiment, they were supervised and inspected by the experimental animal ethics committee.
  • mice All animals, including animals that died during the experiment, were euthanized and sacrificed at the end of the experiment, were subjected to gross autopsy examination. The ileum and colon of mice were excised and stored in formalin solution. It was sent to Wuhan Sevier Biotechnology Co., Ltd. to be made into pathological sections, and photographed for observation.
  • the layer structure is clear, the mucosal epithelium is intact, the number of intestinal glands is abundant, and the arrangement is tight, and no obvious abnormality is found (refer to Figure 26).
  • the structure of each layer of the mouse ileum tissue is clear, the number of intestinal villi is abundant, the mucosal epithelium is intact, the number of intestinal glands is abundant, and the arrangement is tight, and no other obvious abnormalities are found (refer to Figure 27).
  • the micrographs of the HFD-control group showed that multiple mucosal damages were seen in the colon tissue of the mice, the mucosal epithelial cells were shed, a small amount of intestinal gland structure was destroyed, and a large number of basophilic hyphae were seen in the intestinal lumen (refer to Figure 24). ; The mucosal layer of the ileum tissue of mice was damaged, local intestinal villi and mucosal epithelium were lost, the intestinal gland structure disappeared, a small number of epithelial cells were swollen, the cytoplasm was loose and lightly stained, and a large number of basophilic hyphae were seen in the intestinal lumen (refer to Figure 25) .
  • the micrograph of the NCD-control group showed that the structure of each layer of the mouse colon tissue was clear, the mucosal epithelium was intact, the number of intestinal glands was abundant, the arrangement was tight, and no obvious abnormality was found (refer to Figure 28); the structure of each layer of the mouse ileum tissue Clear, abundant intestinal villi, intact mucosal epithelium, abundant intestinal glands, closely arranged, no obvious abnormality (refer to Figure 29).
  • MNO-863 has the function of repairing the ileum and colon mucosa, and its administration can effectively repair the mucosa of the digestive tract and has a positive effect on the prevention and treatment of diseases related to the damage of the digestive tract mucosa.
  • an in vivo test of the use of MNO-863 strain and/or Liraglutide drug in a high-fat diet-induced obesity mouse model is carried out to verify its efficacy in the treatment or prevention of obesity, diabetes and liver disease. use.
  • mice 50 C57BL/6J male mice (purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.) were purchased, all of which were normally reared mice, 5 weeks old.
  • the growth process of mice was in the same environment, of which 8 mice were given maintenance feed (purchased from Guangzhou Hancheng Experimental Equipment Co., Ltd.) and 42 mice were given D12492 high-fat feed (purchased from Parker Bio) for about 8-10 weeks
  • the standard of diet-induced obesity model is that the weight reaches 38.00 ⁇ 2.00g.
  • Test strain MNO-863 cultured anaerobic, the medium is 104 liquid medium (formula as shown in Table 1 above), cultured under anaerobic conditions at 37°C for 48h, to a bacterial concentration of about 10 9 CFU/mL order of magnitude, can be used as the experimental group by gavage.
  • the bacterial solution was stored anaerobically at 4°C.
  • PBS phosphate buffer solution It is a mixed solution composed of weak acid and its salt, weak base and its salt, which can offset and reduce the influence of external strong acid or strong base on the pH of the solution to a certain extent, so as to maintain the pH of the solution.
  • the pH value is relatively stable.
  • the formulation of PBS phosphate buffered solution is shown in Table 4 above.
  • the test process is as follows:
  • test groups are as follows:
  • 32 of 42 obese mice with a body weight of 38.00g ⁇ 2.00g were selected and divided into 4 groups, 8 mice in each group, 4 mice/cage.
  • the first group was the control group fed with normal feed (NCD control group)
  • the second group was the high-fat diet-induced obesity mouse model group (HFD control group)
  • the third group was the MNO-863 bacterial treatment group
  • the fourth group was the MNO-863 bacterial treatment group.
  • the group is the Liraglutide drug treatment group (Lira group)
  • the fifth group is the MNO-863 bacterial agent combined with the Liraglutide drug treatment group (Lira+MNO-863).
  • the second, third, fourth and fifth groups were fed with high-fat diet, and the groupings are shown in Table 18. Dosing was started one week after the animals were grouped, and the intervention lasted for 4 weeks.
  • the amount of intragastric bacterial solution was 0.2 mL/10 g mouse body weight.
  • mice Data such as body weight, state, and food intake of the mice were recorded before and after modeling, and every 3 days before and after the intervention. Tissues were collected by dissection of animals at the end of dosing. The use of experimental animals pays attention to animal welfare, follows the principle of "reduction, replacement and optimization", and is approved by the laboratory animal ethics committee of this unit. During the experiment, they were supervised and inspected by the experimental animal ethics committee.
  • Impaired glucose tolerance indicates a decline in the body's ability to metabolize glucose and is commonly seen in type 2 diabetes and obesity.
  • the therapeutic effect of drugs and bacterial agents on diabetes can be evaluated by glucose metabolism test.
  • both MNO-863 bacterial agent and Liraglutide drug can improve the abnormal glucose tolerance in obese mice, and can reduce fasting blood sugar, and the combined use group of bacterial agent and drug is more effective than bacteria. It is more significant when the agent or drug is used alone, indicating that the microecological bacteria agent can play a role in enhancing the effect of drug treatment on diabetes.
  • Figure 37 shows the effect of MNO-863 strain alone and the combination of strain and Liraglutide on liver weight after 4 weeks of drug withdrawal. It can be seen from Figure 37 that both MNO-863 and Liraglutide drugs can reduce obesity in mice. The effect of the combination group of bacterial agents and drugs was more significant than that of bacterial agents or drugs alone, indicating that microecological bacteria agents can play a role in enhancing the effect of drugs on liver diseases.
  • the Christensen bacteria provided by the present disclosure can be industrially cultured in large quantities, and the Christensen bacteria provided by the present disclosure can be applied to the treatment or prevention of liver function damage and liver function damage-related diseases, gastrointestinal mucosal damage and digestive tract mucosal damage. Injury-related diseases, diabetes, obesity, and obesity-related diseases.
  • the Christensenella provided by the present disclosure also has no toxic and side effects on the kidneys, and can reduce liver weight; treat initial steatohepatitis lesions; slow down fat accumulation in liver cells; reduce serum AST and ALT; and reduce abdominal white fat inflammatory lesions.
  • Christensen can also repair the mucosa of the digestive tract, restore the mucosal barrier function, and prevent and treat diseases such as intestinal leakage and peptic ulcer caused by impaired barrier function.
  • Christensen also has the functions of reducing fasting blood sugar, regulating insulin levels, reducing body fat in mammals, preventing and treating diabetes, and improving metabolic function of obese patients.
  • Christensen also has the function of repairing damaged digestive tract mucosa and preventing and treating mucosal damage-related diseases.
  • the combined drug containing Christensenella and the hypoglycemic and lipid-lowering drug provided by the present disclosure can be mass-produced in industry, and the combined drug can be used for the treatment or prevention of liver function damage and liver function damage-related diseases, diabetes, obesity and obesity-related diseases.
  • the combined drug provided by the present disclosure has the technical effect of synergy, that is, the combined drug has a better therapeutic effect than the single administration of Christensenella or the single administration of the hypoglycemic and lipid-lowering drug, and the combined drug is effective on the kidneys. No toxic side effects, can reduce liver weight.

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Abstract

提供了一种克里斯滕森菌及组合物、以及其制药用途。该克里斯滕森菌可用于治疗或预防肝脏功能损伤及相关疾病、消化道黏膜损伤及相关疾病、糖尿病、肥胖症及相关疾病。还可与降糖降脂药物联用，对肝脏功能损伤及相关疾病、糖尿病、肥胖及相关疾病具有协同增效的技术效果。

Description

一种细菌菌株及组合物、联用药物和用途
相关申请的交叉引用
本公开要求申请号为202110369840.5(申请日为2021年04月06日，发明名称为“一种细菌菌株及组合物和用途”)和202110370249.1(申请日为2021年04月06日，发明名称为“包含微生物和降糖降脂药物的联用药物”)的中国专利申请的优先权，其全部内容通过引用结合在本公开中。
技术领域
本公开涉及菌株分离和应用技术领域，具体而言，涉及一种细菌菌株及组合物、联用药物和用途，该联用药物包含该细菌菌株和降糖降脂药物。
背景技术
目前，肝脏疾病主要通过接种肝炎疫苗、减少饮酒、改善膳食结构和进行身体锻炼来进行预防治疗。但这些策略往往只能起到预防作用，且收益甚少，效果也因个人体质而异，一旦面临已发生的肝脏疾病则显得束手无策。因此，目前急需能够有效预防治疗肝脏疾病的应对措施，需要开发一种有效且副作用小的并能够预防治疗肝脏疾病的方法或药物。
消化道黏膜损伤尤其是肠道黏膜损伤不仅会影响营养物质的消化和吸收，还会对黏膜屏障功能和机体免疫功能造成极其不利的影响。对于消化道黏膜损伤常用的中西结合药物修复方法常伴有中药药效慢、难根治、易复发以及西药易对消化系统产生刺激作用和不良反应等缺点。亟需开发对修复消化道黏膜损伤具有药效快、药力持久且无毒副作用的药物。
关于糖尿病，目前尚无根治方法，主要还是通过药物治疗的手段控制糖尿病。而当前糖尿病的药物治疗包括口服药物治疗，诸如磺脲类药物、双胍类降糖药、α葡萄糖苷酶抑制剂、胰岛素增敏剂等，以及胰岛素注射治疗。虽然有几种药物可用于治疗T2D(2型糖尿病)，但药物的疗效因人而异，而且还存在令人担忧的潜在副作用，副作用包括：(1)引起胃肠道不良反应，包括恶心、呕吐和腹泻；(2)加重胰岛负担，可能引起胰腺炎；(3)可能引起甲状腺肿大与甲状腺癌；(4)其他一些肠道、肾功能、低血糖等副作用；以及，(5)有引起抑郁的倾向。因此迫切需要开发有效且副作用小的治疗糖尿病的方法或药物。
肥胖症的药物治疗已有很长的历史，现代常用的减肥药物包括有利拉鲁肽、奥利司他、西布曲明和利莫那班等。但许多减肥药物被市场限制或退出临床应用，因为其中一些药物不能达到预期效果，或是因为其中一些药物让患者产生严重的不良反应。因此，迫切需要开发有效且副作用小的治疗肥胖症及其相关疾病的方法或药物。
发明内容
本公开的目的包括，例如，提供一种细菌菌株及组合物、联用药物和用途以解决上述技术问题，该联用药物包含微生物和降糖降脂药物。
由于肠道微生物在肥胖症和糖尿病中的新兴作用，利用肠道微生物自身改善糖尿病、肠道微生物与抗糖尿病药物的相互作用及其对药物功能的影响等，成为了当前的研究热点。一方面，肠道微生物可以通过分泌短链脂肪酸等途径影响宿主的代谢、免疫功能和脑功能，而这些对人体健康具有不可缺少的作用。另一方面，肠道微生物及其代谢物的代谢活动会影响药物的代谢和药效，且药物也可以操纵肠道微生物的组成及其代谢能力。
本公开提供了一种克里斯滕森菌(Christensenella sp.)，能够治疗初期脂肪肝炎病灶、减缓肝脏脂肪堆积、缓解肝脏病变，从而有效防治肝脏功能损伤及其相关疾病；该菌株同时具有修复消化道黏膜屏障的功能，还具有降低机体的空腹血糖、调节胰岛素水平、降低体重、调节血脂四项等功能，从而具有预防和治疗消化道黏膜损伤及其相关疾病、糖尿病、肥胖症及肥胖症相关疾病的作用。
本公开提供克里斯滕森菌(Christensenella sp.)物种的细菌菌株在制备用于治疗或预防选自以下至少一种的疾病或病症的药物中的用途：肝脏功能损伤及肝脏功能损伤相关疾病、消化道黏膜损伤及消化道黏膜损伤相关疾病、糖尿病、肥胖及肥胖相关疾病。
肝脏功能损伤相关疾病包括如下疾病中的至少一种：脂肪肝、非酒精性脂肪性肝病、非酒精性脂肪性肝炎、肝纤维化、肝硬化和肝癌；
消化道黏膜损伤是指消化道黏膜通透性升高、黏膜屏障作用受损，消化道黏膜损伤相关疾病包括如下疾病中的至少一种：肠漏病症、消化性溃疡、胃肠炎、炎性肠病；以及
肥胖症相关疾病包括如下疾病中的至少一种：肥胖症、代谢综合症、心血管疾病、高脂血症、高胆固醇血症、高血压、胰岛素抗性综合征、肥胖相关的胃食管返流症和脂肪性肝炎。
在本公开的一种或多种实施方式中，细菌菌株具有与SEQ ID NO.1至少98.65％一致的16s rRNA序列。
在本公开的一种或多种实施方式中，细菌菌株具有与SEQ ID NO.1至少99％一致的16s rRNA序列。
在本公开的一种或多种实施方式中，细菌菌株具有与SEQ ID NO.1 99％、99.5％、99.9％或100％一致的16s rRNA序列。
在本公开的一种或多种实施方式中，上述药物是冻干的。
在本公开的一种或多种实施方式中，上述药物还包括一种或多种药学上可接受的赋形剂或载剂。
在本公开的一种或多种实施方式中，上述药物是疫苗组合物。
在本公开的一种或多种实施方式中，上述药物被配制用于经口施用、注射施用或灌胃施用。
在本公开的一种或多种实施方式中，糖尿病包括如下疾病中的至少一种：1型糖尿病、2型糖尿病、胰岛素抗性综合征、葡萄糖不耐症、高血脂絮乱、糖尿病肾病变并发症、糖尿病神经病变、糖尿病眼睛病变、心血管疾病、糖尿病足和妊娠期糖尿病。
在本公开的一种或多种实施方式中，上述药物的剂型包括片剂、丸剂、粉剂、混悬剂、凝胶、乳液、乳膏、颗粒剂、纳米颗粒、胶囊、栓剂、注射剂、喷雾和针剂。
本公开提供了一种以保藏号为GDMCC No：61117保藏的克里斯滕森菌菌株或其子代菌株或亚克隆菌株的细胞。
本公开提供了一种组合物，其包括上述克里斯滕森菌菌株和/或该菌株的代谢产物。
在本公开的一种或多种实施方式中，上述组合物还包括药学上可接受的赋形剂或载剂。
本公开提供了一种包含微生物和降糖降脂药物的联用药物在制备用于治疗或预防选自以下至少一种的疾病或症状的药物中的用途：肝脏功能损伤及肝脏功能损伤相关疾病、糖尿病、肥胖及肥胖相关疾病；所述微生物为克里斯滕森菌(Christensenella sp.)物种的细菌；所述降糖降脂药物为能改善胰高血糖素样肽-1(即GLP-1)通路敏感性、补充和/或促进GLP-1作用的药物中的一种或多种。
在本公开的一种或多种实施方式中，上述降糖降脂药物为GLP-1受体激动剂(即GLP-1RA)或GLP-1模拟物、GIP受体激动剂(即葡萄糖依赖性促胰岛素分泌多肽受体激动剂，也称为胃抑肽受体激动剂)、二肽基肽酶-4(即DPP-4)抑制剂中的至少一种。
GLP-1受体激动剂或GLP-1模拟物选自艾塞那肽、利拉鲁肽、索马鲁肽、口服剂型索马鲁肽、贝那鲁肽、利司那肽和艾塞那肽周制剂中的至少一种。
本公开提供了一种联用药物，其包括降糖降脂药物与微生物；微生物为克里斯滕森菌(Christensenella sp.)物种的细菌。降糖降脂药物为能改善胰高血糖素样肽-1通路敏感性、补充和/或促进GLP-1作用的药物中的一种或多种。
本公开提供了一种如上述组合物或一种如上述联用药物在用于制备药物或制剂中的用途，药物或制剂用于选自如下至少一者的用途：
减少肝脏重量；
治疗初期脂肪肝炎病灶；
减缓肝脏细胞脂肪堆积；
降低血清AST、ALT；
减少腹部白色脂肪炎症性病变；
减轻哺乳动物的体重；
减少哺乳动物的摄食量；
减缓停药后的复胖幅度；
降低哺乳动物的体脂；
降低哺乳动物血清中如下至少一种指标的水平：总胆固醇水平、低密度脂蛋白和甘油三酯水平；
提高哺乳动物血清高密度脂蛋白的水平；
改善哺乳动物口服葡萄糖耐量受损；
降低哺乳动物的空腹血糖；
降低哺乳动物HOMA-IR指标；
增强GLP-1敏感性；
增强胰岛素敏感性；
避免肠道絮乱所造成的GLP-1RA的抗性及相关副作用；以及
修复消化道黏膜损伤。
本公开提供了一种组合物，其包括上述克里斯滕森菌(Christensenella sp.)物种的细菌菌株或上述克里斯滕森菌菌株或其子代菌株或亚克隆菌株和/或其代谢产物，用于治疗或预防选自以下至少一种的疾病或病症：肝脏功能损伤及肝脏功能损伤相关疾病、消化道黏膜损伤及消化道黏膜损伤相关疾病、糖尿病、肥胖及肥胖相关疾病。
在本公开的一种或多种实施方式中，上述赋形剂包括抗氧化剂、螯合剂、乳化剂、溶剂。
本公开提供了一种联用药物，其包括微生物和降糖降脂药物，该微生物为上述的克里斯滕森菌(Christensenella sp.)物种的细菌菌株或上述的克里斯滕森菌菌株或其子代菌株或亚克隆菌株和/或其代谢产物，该降糖降脂药物为能改善胰高血糖素样肽-1通路敏感性、补充和/或促进GLP-1作用的药物中的一种或多种，用于治疗或预防选自以下至少一种的疾病或病症：肝脏功能损伤及肝脏功能损伤相关疾病、糖尿病、肥胖及肥胖相关疾病。
本公开提供了一种治疗或预防疾病或病症的方法，包括向有需要的受试者给药上述组合物或者上述的联用药物，该疾病或病症选自以下至少一种：肝脏功能损伤及肝脏功能损伤相关疾病、糖尿病、肥胖及肥胖相关疾病。
本公开提供了一种试剂盒，其包括上述的联用药物。
本公开具有以下有益效果：
本公开提供的克里斯滕森菌可以应用于治疗或预防肝脏功能损伤及肝脏功能损伤相关疾病、消化道黏膜损伤及消化道黏膜损伤相关疾病、糖尿病、肥胖症及肥胖症相关疾病。经申请人验证，本公开提供的克里斯滕森菌对肾脏无毒副作用，且包括但不限于以下：可减少肝脏重量；治疗初期脂肪肝炎病灶；减缓肝脏细胞脂肪堆积；降低血清AST、ALT；减少腹部白色脂肪炎症性病变。克里斯滕森菌还能够降低机体的空腹血糖，并且明显改善机体得到胰岛素抵抗水平，具有预防和治疗糖尿病的作用。此外，克里斯滕森菌还可以降低哺乳动物的体脂，提高肥胖症患者代谢功能。克里斯滕森菌还具有修复受损的消化道黏膜和防治黏膜损伤相关疾病的功能。
本公开提供的降糖降脂药物与微生物的联用药物可以应用于治疗或预防肝脏功能损伤及肝脏功能损伤相关疾病、糖尿病、肥胖及肥胖相关疾病。经申请人验证，本公开提供的GLP-1受体激动剂或GLP-1模拟物与微生物的联用药物具有协同增效的技术效果，该联用药物具有比单独施用微生物或者单独施用GLP-1受体激动剂或GLP-1模拟物更优的治疗效果，微生物能增强GLP-1受体激动剂或GLP-1模拟物的减肥效果、改善糖耐量异常、降低空腹血糖。此外，上述联用药物对肾脏无毒副作用，可减少肝脏重量。联用药物有助于增强GLP-1的敏感性；避免肠道絮乱所造成的GLP-1RA的抗性及相关副作用。
附图说明
为了更清楚地说明本公开实施方式的技术方案，下面将对实施方式中所需要使用的附图作简单地介绍，应当理解，以下附图仅示出了本公开的某些实施例，因此不应被看作是对范围的限定，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他相关的附图。
图1为分离出的菌株的宏观形态图；
图2为分离出的菌株的微观形态图；
图3为单菌落厌氧培养后的宏观平板图；
图4为系统发育进化树；
图5为NASH肝损伤评分标准；
图6为MNO-863对肥胖模型小鼠肝脏重量的影响；
图7为肝脏组织HE染色结果图；
图8为肝脏组织油红染色结果图；
图9为NAFLD/NASH肝脏病理评分；
图10为肝脏脂肪变性程度统计结果图；
图11为肝小叶炎症评分统计结果图；
图12为肝脏气球样变性评分统计结果图；
图13为血清中的AST(谷草转氨酶)、ALT(谷丙转氨酶)水平；
图14为小鼠腹部白色脂肪炎症性病变的显微图以及总面积统计图；
图15为小鼠血肌酐(CREA)、血尿素(UREA)及血尿酸(UA)的含量检测结果图；
图16为MNO-863对高脂饮食诱导肥胖小鼠口服葡萄糖耐量的影响；
图17为MNO-863对高脂饮食诱导肥胖小鼠空腹血糖(mmol/L)的影响；
图18为MNO-863对高脂饮食诱导肥胖小鼠HOMA-IR指数的影响；
图19为MNO-863对高脂饮食诱导肥胖小鼠体重(g)的影响；
图20为MNO-863对高脂饮食诱导肥胖小鼠体重(％)的影响；
图21为MNO-863对高脂饮食诱导肥胖小鼠摄食量(g)的影响；
图22为MNO-863对高脂饮食诱导肥胖小鼠TC、TG、LDL、HDL-C的影响；
图23为MNO-863对高脂饮食诱导肥胖小鼠腹股沟脂肪、皮下脂肪、附睾脂肪的影响；
图24为HFD对照组的小鼠结肠组织的显微图；
图25为HFD对照组的小鼠回肠组织的显微图；
图26为MNO-863处理组的小鼠结肠组织的显微图；
图27为MNO-863处理组的小鼠回肠组织的显微图；
图28为NCD对照组的小鼠结肠组织的显微图；
图29为NCD对照组的小鼠回肠组织的显微图；
图30为MNO-863与同Liraglutide联用对于肥胖小鼠四周干预期间的绝对体重的影响以及与体重变化百分比；
图31为干预四周后的体重以及体重变化百分比；
图32为干预四周与停药四周后的体重以及体重变化百分比；
图33为药4周复胖后的腹股沟脂肪重量；
图34为MNO-863菌株的单独使用以及菌株与Liraglutide联用对于肥胖小鼠葡萄糖耐受性的影响；
图35为MNO-863菌株的单独使用以及菌株与Liraglutide联用对于肥胖小鼠葡萄糖高血糖的影响；
图36为MNO-863菌株对于肥胖小鼠停药4周复胖后的高血糖影响作用；
图37为MNO-863菌株的单独使用以及菌株与Liraglutide联用对于停药4周复胖后的肝脏重量影响结果图。
具体实施方式
现将详细地提供本公开实施方式的参考，其一个或多个实例描述于下文。提供每一实例作为解释而非限制本公开。实际上，对本领域技术人员而言，显而易见的是，可以对本公开进行多种修改和变化而不背离本公开的范围或精神。例如，作为一个实施方式的部分而说明或描述的特征可以用于另一实施方式中，来产生又一实施方式。
克里斯滕森菌(Christensenella sp.)物种的细菌菌株在制备用于治疗或预防选自以下至少一种的疾病或病症的药物中的用途：肝脏功能损伤及肝脏功能损伤相关疾病、消化道黏膜损伤及消化道黏膜损伤相关疾病、糖尿病、肥胖症及肥胖症相关疾病。
包含微生物和降糖降脂药物的联用药物在制备用于治疗或预防选自以下至少一种的疾病或症状的药物中的用途：肝脏功能损伤及肝脏功能损伤相关疾病、糖尿病、肥胖及肥胖相关疾病；所述微生物为克里斯滕森菌(Christensenella sp.)物种的细菌；所述降糖降脂药物为能改善胰高血糖素样肽-1(即GLP-1)通路敏感性、补充和/或促进GLP-1作用的药物中的一种或多种。
肝脏功能损伤相关疾病包括如下疾病中的至少一种：脂肪肝、非酒精性脂肪性肝病、非酒精性脂肪性肝炎和肝硬化。
在其他实施方式中，肝脏功能损伤相关疾病还包括肝纤维化和肝癌。
消化道黏膜损伤是指消化道黏膜通透性升高、黏膜屏障作用受损，消化道黏膜损伤相关疾病包括如下疾病中的至少一种：肠漏病症、消化性溃疡、胃肠炎、炎性肠病等疾病；需要说明的是，肠漏病症表现为肠道的通透性增加。
肥胖症相关疾病包括如下疾病中的至少一种：心血管疾病、高脂血症、胰岛素抗性综合征、肥胖相关的胃食管返流症和脂肪性肝炎。
在其他实施方式中，肥胖症相关疾病还包括肥胖症、代谢综合症、高胆固醇血症、高血压。
在其他实施方式中，上述“肥胖症相关疾病”可选自如下疾病：过食症(overeating)、暴食症(binge eating)、易饥症、高血压、糖尿病、血浆胰岛素浓度升高、胰岛素抗性、高脂血症、代谢综合征、胰岛素抗性综合征、肥胖相关的胃食管返流症、动脉硬化症、高胆固醇血症、高尿酸血症、下背痛、心脏肥大和左心室肥大、脂肪代谢障碍、非酒精性脂肪性肝炎、心血管疾病和多囊性卵巢综合征，以及具有与肥胖相关的疾病并包括希望减轻体重的那些对象。
糖尿病的三种主要类型是1型糖尿病(T1D)、2型糖尿病(T2D)和妊娠期糖尿病(GDM)。1型糖尿病是由于自身免疫损害或特发性原因引起的，是以胰岛功能绝对破坏为特点的糖尿病，多发生在儿童和青少年，必须用胰岛素治疗才能获得满意疗效，否则将危及生命。2型糖尿病是一种以碳水化合物/脂肪代谢异常为特征的多因子综合征，通常包括高血糖、高血压和胆固醇异常。2型糖尿病是胰岛素不能有效发挥作用(与受体结合含量少)所致，因此不仅要检查空腹血糖，而且要观察餐后2小时血糖，特别应做胰岛功能检查。妊娠期间的糖尿病有两种情况：一种为妊娠前已确诊患有糖尿病，称“糖尿病合并妊娠”；以及，另一种为妊娠前糖代谢正常或有潜在糖耐量减退、妊娠期才出现或确诊的糖尿病，又称为“妊娠期糖尿病(GDM)”，糖尿病孕妇中80％以上为GDM。
需要说明的是，上述制药用途针对糖尿病的应用包括但不限于1型糖尿病(T1D)、2型糖尿病(T2D)和妊娠期糖尿病(GDM)的治疗或预防。
还需要说明的是，上述治疗肥胖以及肥胖相关疾病的用途，不仅包括本公开提供的细菌菌株与降糖降脂药物的联用药物，还包括其他活性化合物，其他活性化合物可以是两种或多种其他活性化合物的联合。例如上述联用药物与抗肥胖症化合物的联用，抗肥胖症化合物如芬氟拉明、右芬氟拉明、芬特明、西布曲明、奥利司他、神经肽Y5抑制剂和β3肾上腺素能受体激动剂。
此外，上述联用药物与胆固醇降低剂的联用，胆固醇降低剂如：(i)HMG-CoA还原酶抑制剂(洛伐他汀、辛伐他汀、普伐他汀、氟伐他汀、阿妥伐他汀、利伐他汀(rivastatin)、伊伐他汀、罗苏伐他汀和其他他汀类药物)；(ii)螯合物(考来烯胺、考来替泊和交联右旋糖酐的二烷基氨基烷基衍生物)；(iii)烟醇、烟酸或其他盐；(iv)PPARα激动剂，如非诺贝酸衍生物(吉非贝齐、氯贝丁酯、非诺贝特和苯扎贝特)；(v)PPARα/γ双效激动剂，如KRP-297；(vi)胆固醇吸收抑制剂，如β-谷甾醇和依泽替米贝；(vii)乙酰基CoA，胆固醇酰基转移酶抑制剂，如阿伐麦布；以及，(viii)抗氧化剂，如普罗布考。
在其他实施方式中，也可与其他的用于炎症的药物联用，例如阿司匹林、非类固醇消炎药、糖皮质类固醇、硫氮磺吡啶和环氧酶II选择抑制剂。
在本公开的一种或多种实施方式中，细菌菌株具有与SEQ ID NO.1至少98.65％一致的16s rRNA序列。例如，具有与SEQ ID NO.198.7％、98.75％、98.8％、98.85％、98.9％、98.95％、99％、99.1％、99.2％、99.3％、99.4％、99.5％、99.6％、99.7％、99.8％、99.9％或100％一致的16s rRNA序列。
在本公开的一种或多种实施方式中，克里斯滕森菌细菌菌株具有与SEQ ID NO.1至少99％一致的16s rRNA序列。
在本公开的一种或多种实施方式中，克里斯滕森菌细菌菌株具有与SEQ ID NO.1 99％、99.5％、99.9％或100％一致的16s rRNA序列。
在本公开的一种或多种实施方式中，上述药物是冻干的。冻干是一项制备允许传递细菌的稳定组合物的有效和便利的技术。上述药物通过冻干制粉或制片便于包衣或者运输。
在本公开的一种或多种实施方式中，上述药物还包括一种或多种药学上可接受的赋形剂或载剂。
药学上可接受的赋形剂可以是抗氧化剂、螯合剂、乳化剂、溶剂等。
药物的剂型包括但不限于片剂、丸剂、粉剂、混悬剂、凝胶、乳液、乳膏、颗粒剂、纳米颗粒、胶囊、栓剂、注射剂、喷雾和针剂。
上述药物还包括其药学上可接受的盐、其溶剂化合物或其立体异构体，与一种或多种药用载体和/或稀释剂形成的药物组合物，可以用本领域已知的方式配制成临床上或药学上可接受的任一剂型，以口服、注射或灌胃给药等方式施用于需要这种治疗的患者。用于口服给药时，可制成常规的固体制剂，如片剂、胶囊剂、丸剂、颗粒剂等；也可制成口服液体制剂，如口服溶液剂、口服混悬剂、糖浆剂等。制成口服制剂时，可以加入适宜的填充剂、粘合剂、崩解剂、润滑剂等。
在本公开的一种或多种实施方式中，上述药物是疫苗组合物。
在本公开的一种或多种实施方式中，上述药物被配制用于经口施用、注射施用或灌胃施用。经过灌胃小鼠实验，施用本公开中的细菌菌株表现出与治疗糖尿病药物利拉鲁肽(Liraglutide)相当的治疗效果。
在其他的一种或多种实施方式中，上述药物还包括药学上可接受的盐，“药学上可接受的盐”是指以下盐，其在正确的医疗判断内，适合用于与人和低等动物的组织接触，并不具有过度毒性、刺激性、过敏反应等，并与合理的利益/风险比相称。
一种以保藏号为GDMCC No：61117保藏的克里斯滕森菌菌株或其子代菌株或亚克隆菌株的细胞。
本公开提供的克里斯滕森菌Christensenella sp.MNO-863分离自广东省广州市的一位汉族健康男性志愿者的粪便样品。于2020年8月4日保藏于广东省微生物保藏中心。保藏编号为：GDMCC No：61117；保藏地址为：广州市先烈中路100号大院59号楼5楼，广东省微生物研究所，检测结果为存活，其分类学名称为Christensenella sp.。
宏观形态：37℃厌氧培养72h，菌落浅黄色，圆形，表面湿润，半透明，边缘整齐。菌体呈短杆状，无芽孢，无鞭毛，不运动，0.3-0.4μm×0.6-1.1μm，单个或成对排列，革兰氏阴性。菌落特征：MNO-863在104平板上，37℃厌氧培养72h，单个菌落呈现圆形微凸、透明、白色、表面光滑，菌落直径约在0.46-0.50mm。
本公开还提供了一种组合物，其包括上述克里斯滕森菌菌株和/或该菌株的代谢产物。
上述的克里斯滕森菌菌株可以是由上述的保藏号为GDMCC No：61117保藏的菌株直接培养获得，也可以是子代菌株(后代)或从原始菌株培养(亚克隆菌株)的菌株，例如，分离细胞。
需要说明的是，本公开提供的克里斯滕森菌菌株还包括其衍生物，例如可在基因水平下将其进行修饰而不消除其生物活性。上述的衍生物菌株具有治疗活性，且具有与保藏号为GDMCC No：61117保藏的克里斯滕森菌菌株相当的活性。
在本公开的一种或多种实施方式中，上述组合物还包括药学上可接受的赋形剂或载剂。
一种如上述的组合物在用于制备药物或制剂中的用途，该药物或制剂用于选自如下至少一者的用途：
减少肝脏重量；治疗初期脂肪肝炎病灶；减缓肝脏细胞脂肪堆积；降低血清AST(谷草转氨酶)、ALT(谷丙转氨酶)；减少腹部白色脂肪炎症性病变；减轻哺乳动物的体重；减少哺乳动物的摄食量；降低哺乳动物的体脂；降低哺乳动物血清中如下至少一种指标的水平：总胆固醇水平、低密度脂蛋白和甘油三酯水平；提高哺乳动物血清高密度脂蛋白的水平；改善哺乳动物口服葡萄糖耐量受损；降低哺乳动物的空腹血糖；降低哺乳动物HOMA-IR指标；以及，修复消化道黏膜损伤。
以下为治疗肝脏损伤及其相关疾病中的用途：减少肝脏重量；治疗初期脂肪肝炎病灶；减缓肝脏细胞脂肪堆积；降低血清AST(谷草转氨酶)、ALT(谷丙转氨酶)水平；以及，减少腹部白色脂肪炎症性病变。
以下为治疗或预防肥胖及其相关疾病中的用途：减轻哺乳动物的体重；减少哺乳动物的摄食量；降低哺乳动物的体脂；降低哺乳动物血清中如下至少一种指标的水平：总胆固醇水平、低密度脂蛋白和甘油三酯水平；提高哺乳动物血清高密度脂蛋白的水平。
以下为治疗或预防糖尿病中的应用或用途：改善哺乳动物口服葡萄糖耐量受损、降低哺乳动物的空腹血糖和降低哺乳动物HOMA-IR指标。
上述的修复消化道黏膜损伤是指修复胃肠道黏膜损伤，典型为修复肠道黏膜损伤。修复肠道黏膜损伤是指达到如下指标中的至少一种：恢复肠道黏膜组织的结构完整性、降低肠绒毛萎缩程度和菌丝数量。
一种联用药物，其包括微生物和降糖降脂药物；微生物为克里斯滕森菌(Christensenella sp.)物种的细菌。降糖降脂药物为能改善胰高血糖素样肽-1通路敏感性、补充和/或促进GLP-1作用的药物中的一种或多种。
上述降糖降脂药物为GLP-1受体激动剂(即GLP-1RA)或GLP-1模拟物、GIP受体激动剂(即葡萄糖依赖性促胰岛素分泌多肽受体激动剂，也称为胃抑肽受体激动剂)、二肽基肽酶-4(即DPP-4)抑制剂中的至少一种。GLP-1受体激动剂或GLP-1模拟物选自艾塞那肽、利拉鲁肽、索马鲁肽、口服剂型索马鲁肽、贝那鲁肽、利司那肽和艾塞那肽周制剂中的至少一种。
上述的联用药物在用于制备药物或制剂中的用途，药物或制剂用于选自如下至少一者的用途：
减少肝脏重量；
改善哺乳动物口服葡萄糖耐量受损；
降低哺乳动物的空腹血糖；
减轻哺乳动物的体重；减少哺乳动物的摄食量；
减缓停药后的复胖幅度；
降低哺乳动物血清中如下至少一种指标的水平：总胆固醇水平、低密度脂蛋白胆固醇水平和甘油三酯水平；
增强GLP-1敏感性；避免肠道絮乱所造成的GLP-1RA的抗性及相关副作用。
肠道菌群絮乱容易导致GLP-1药物无效与抗性，而发明人提出将益生菌与GLP-1联用，能够增强GLP-1敏感性与避免肠道絮乱所造成的GLP-1RA的抗性及相关副作用。
为使本公开实施例的目的、技术方案和优点更加清楚，下面将对本公开实施例中的技术方案进行清楚、完整地描述。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市售购买获得的常规产品。
以下结合实施例对本公开的特征和性能作进一步的详细描述。
实施例1
本实施例提供了克里斯滕森菌Christensenella sp.MNO-863的分离与鉴定。
(1)MNO-863的分离
本公开的克里斯滕森菌Christensenella sp.MNO-863分离自广东省广州市的一位汉族健康男性志愿者的粪便样品，采集样品时该志愿者前三个月没有使用抗生素。
在生物安全柜中分装生理盐水于无菌的10ml离心管中；提前24h将厌氧血平板(江门凯林厌氧血琼脂培养基，粤械注准20172400940)和无菌生理盐水转移入厌氧工作台中，将5-7颗无菌的玻璃珠倒入已经凝固的厌氧血平板中。
取志愿者新鲜粪便样本适量置于含有无菌保存液(3％PEG溶液，即称取30g聚乙二醇3350溶于1000mL生理盐水中，于121℃，15min条件下高压灭菌)的样品保存管中，用涡旋振荡器震荡10分钟混匀，随后于厌氧工作站中吸取1mL溶液，用无菌生理盐水稀释至10 -6稀释度，吸取0.1mL稀释菌液涂布于厌氧血琼脂平板上，在厌氧工作站中于37℃培养72小时。采用分区划线法，用无菌牙签挑取不同形态的单菌落在厌氧血琼脂平板上进行划线分离培养。经72小时培养后，在分区平板上提取分离效果好的菌落进行传代培养。
(2)MNO-863的鉴定
①MNO-863的微生物学特征：
对MNO-863进行固体平板涂布以及液体培养以观察微生物学特征，菌株涂布平板和液体培养溶液使用104培养基，该培养基的1L的配方如下表1所示：
表1 104培养基的配方表。
试剂名称 每L培养基称取量
胰化酪蛋白胨 5g
胨 5g
酵母提取物 10g
牛肉膏 5g
葡萄糖 5g
K 2HPO 4 2g
吐温80 1g
半胱氨酸-HCl×H 2O 0.5g
乙酸钠 2g
5×盐溶液母液 8mL
20×CaCl 2溶液母液 2mL
5×氯化血红素母液 2mL
维生素K 1母液 0.2mL
形态学特征：参照图1所示的宏观形态，在37℃厌氧培养72h，菌落为浅黄色、圆形、表面湿润、半透明、边缘整齐。
微观形态：MNO-863在104培养基的平板上，37℃厌氧培养72h，对MNO-863进行革兰氏染色(图2中的上图)和芽孢染色镜检(图2中的下图)。参照图2所示，菌体呈短杆状，无芽孢，无鞭毛，不运动，0.3-0.4μm×0.6-1.1μm，单个或成对排列，革兰氏阴性。
菌落特征：MNO-863在104培养基的平板上，37℃厌氧培养72h，单个菌落呈现圆形微凸、透明、白色、表面光滑，菌落直径约在0.46-0.50mm(参照图3所示)。
继续进行分离出的菌株的生理生化特征鉴定。MNO-863在有氧条件下不生长，厌氧条件下生长良好，最适生长温度为37℃。使用API 20A反应试剂盒测定MNO-863及标准菌株Christensenella minuta(DSM 22607)对底物的利用情况进行比较分析。
试验结果参照表2所示，由表2可知，分离出的MNO-863与标准菌株DSM 22607的生理生化特征基本保持一致，在底物为甘油、明胶水解、甘露糖、甘露醇和柳醇时存在差异。
表2 MNO-863及标准菌株DSM 22607对底物的利用情况对比表。
Figure PCTCN2021106579-appb-000001
上表中的符号说明：“+”，阳性；“+w”，弱阳性；“-”，阴性。
进行细胞脂肪酸分析：采用气相色谱法对培养后的MNO-863及标准菌株Christensenella minuta(DSM 22607)的磷脂脂肪酸组成和含量分别进行比对分析。比对分析结果参照表3所示，由表3可知，与标准菌株相比，本公开分离出的MNO-863的细胞脂肪酸组成差异明显。
表3 细胞脂肪酸分析结果表。
脂肪酸 MNO-863 DSM22607
C9：0 FAME 0.48 0
C11：0 ISO FAME 27.50 2.89
C11：0 FAME 0.33 1.31
C10：0 2OH FAME 1.33 8.57
C12：0 FAME 4.58 1.13
C13：0 ISO FAME 1.58 0
C13：0 ANTEISO FAME 3.30 2.35
C14：0 FAME 25.01 13.03
C15：0 ISO FAME 20.10 27.40
C15：0 ANTEISO FAME 1.53 3.19
C15：0 ISO DMA 0.60 0
C16：0 FAME 9.34 21.14
C17：0 ISO FAME 0.74 1.68
C18：0 FAME 2.48 3.73
C19：0 CYC 9，10 DMA 1.11 0
②核酸分析鉴定
16srRNA测序：
对MNO-863菌株的序列进行了16S序列片段(扩增引物与测序引物为27F：5'-AGAGTTTGATCCTGGCTCAG-3'与1492R：5'-GGTTACCTTGTTACGACTT-3')测定，16s rRNA测定结果见序列SEQ ID NO.1：
Figure PCTCN2021106579-appb-000002
进化分析：
对MNO-863进行全基因组测序，采用MEGA5.0软件，邻位连接法显示“MNO-863”与相关物种的16S rDNA序列系统发育树，进行1000次的相似度重复计算，结果与NCBI中克里斯滕森菌科族(Christensenellaceae family)的标准菌株基因组序列相互比对。系统发育树显示(参见图4)，MNO-863与三个标准菌株Christensenella minuta(DSM 22607)，Christensenella timonensis(Marseille-P2437)以及Christensenella massiliensis(Marseille-P2438)在同一分支上，表明MNO-863隶属克里斯滕森菌属(Christensenella sp.)范围内的一个物种。
经以上传统微生物形态分析和核酸分析的方式鉴定，以及与标准菌株的比对结果，可在分类学角度认为MNO-863是隶属于Christensenella属内的一个物种，命名为Christensenella sp.MNO-863。于2020年8月4日保藏于广东省微生物保藏中心。保藏编号为：GDMCC No：61117；保藏地址为：广州市先烈中路100号大院59号楼5楼，广东省微生物研究所，检测结果为存活。分类学名称为Christensenella sp。
实施例2
本实施例进行MNO-863在高脂饮食诱导肥胖小鼠模型的体内试验以验证其在治疗或预防肝脏功能损伤及其相关疾病中的用途。
实验材料：
(1)实验动物：购买C57BL/6J雄性小鼠(购自江苏集萃药康生物科技有限公司)32只，为正常饲养小鼠，5周龄。小鼠生长过程处于同一环境，其中8只给予SPF级大小鼠维持饲料(广州市瀚程实验器材有限公司)，剩下24只给予D12492高脂饲料(派克生物)，喂养约8-10周后，称重，饮食诱导肥胖模型成模标准的体重达到38.00±2.00g。
(2)供试菌株：厌氧培养的MNO-863，培养基为实施例1中的104液体培养基，37℃厌氧条件下培养48h，至菌浓约10 11CFU/mL数量级，方可做为实验组灌胃。菌液于4℃条件下厌氧储存。
(3)PBS磷酸盐缓冲溶液：是由弱酸及其盐、弱碱及其盐组成的混合溶液，能在一定程度上抵消、减轻外加强酸或强碱对溶液酸碱度的影响，从而保持溶液的pH值相对稳定。PBS磷酸盐缓冲溶液的配方如下表4所示：
表4 PBS磷酸盐缓冲溶液配方
试剂名称 每升缓冲溶液称取量(g)
KH 2PO 4 0.24
Na 2HPO 4 1.44
NaCl 8.00
KCl 0.20
半胱氨酸-HCl 0.50
试验过程：
(1)试验分组
将8只给予SPF级大小鼠维持饲料的小鼠完全随机分成2笼，4只/笼，作为第一组。从24只肥胖小鼠中挑选体重达到38.00g±2.00g的小鼠16只，分为2组(作为第二组和第三组)，每组8只，4只/笼。第一组为饲喂普通饲料的对照组(NCD-对照组)，第二组为高脂饮食诱导肥胖小鼠模型组(HFD-对照组)，第三组为菌剂治疗组(MNO-863)，第二组和第三组饲喂高脂饲料，分组情况见表5。动物分组后开始虚拟给药一周后再开始给药，第一组和第二组灌胃等量PBS磷酸盐缓冲液，第三组使用MNO-863供试菌株进行灌胃干预，干预持续4周。灌胃菌液的量为0.2mL/10g小鼠体重。分别在造模前后，干预前后每3天记录小鼠的体重、状态、摄食量等数据。给药结束后，将动物解剖并采集组织。实验动物的使用关注动物福利，遵循“减少、替代和优化”的原则，并经本单位实验动物伦理委员会批准。试验过程中接受实验动物伦理委员会的监督检查。
表5 试验分组
序号 组别 受试物/对照品 药物浓度 给药频率 动物数量 饲料
1 NCD-对照 PBS / 1次/天 8 正常饮食
2 HFD-对照 PBS / 1次/天 8 D12492
3 MNO-863 MNO-863 1×10 11CFU/mL 1次/天 8 D12492
所有的动物包括试验期间死亡、安乐死和试验结束处死的动物都要进行大体解剖检查，记录每只动物的大体病理学变化。对肝脏进行称重；剪取大叶肝脏的中间一条置于福尔马林溶液中，剩余组织经液氮速冻后，保存于-80℃。随后将肝脏制作成石蜡包埋的肝脏病理学切片并进行HE染色、Masson染色以及油红染色，通过对原始放大时捕获的显微照片进行评分来评估肝脏组织和NAS病理学判读。根据NASH肝损伤评分系统(Kleiner DE,Brunt EM,Van NM,Behling C,Contos MJ,Cummings OW,等人Design and validation of a histological scoring system for nonalcoholic fatty liver disease.Hepatology 2005；41:1313–21.)(Brunt EM.Histopathology of non-alcoholic fatty liver disease.Clin Liver Dis 2009；13:533–44.)评分，评分标准参照图5所示(0–2分:非NASH；3–4分:所属不清；5–8:NASH)。
实验结果：
MNO-863对肥胖模型小鼠肝脏重量的影响参照图6所示，相比HFD对照组，MNO-863处理组能显著减少肥胖小鼠的肝脏重量，恢复至正常NCD对照组小鼠肝脏重量水平。对肥胖模型小鼠肝脏重量的影响数据参照表6所示。
表6 不同处理组的小鼠肝脏重量
分组 肝脏重量(g)
NCD-对照 0.9875
HFD-对照 1.125
MNO-863 0.9325*
注：结果以平均值表示，*p<0.05与HFD-对照组相比。
(2)进一步通过病理学判读肥胖模型小鼠肝脏病变：结果表明MNO-863可治疗初期脂肪肝炎病灶，减缓肝脏细胞脂肪堆积，并缓解肝脏病变。
具体地，对上述各处理组的小鼠肝脏组织分别进行HE染色和油红染色。实验结果分别参照图7和图8所示。在高脂饮食条件饲养下，MNO-863可治疗肥胖小鼠初期脂肪肝炎病灶与减缓脂肪堆积。
NAFLD/NASH肝脏病理评分参照图9所示。通过评分可知，MNO-863可治疗肥胖小鼠初期脂肪肝炎病灶与减缓脂肪堆积。
图10中示出了肝脏脂肪变性程度，由图10可知，MNO-863可有效缓解肝脏脂肪的变性。图11中示出了肝小叶炎症评分，由图11可知，与HFD对照组相比，MNO-863可有效抑制肝小叶炎症的发生。图12中示出了肝脏气球样变性评分，由图12可知，与HFD对照组相比，MNO-863肝脏气球样变性评分明显下降。
(3)申请人还探究了MNO-863对肥胖模型小鼠血清ALT和AST的影响，结果表明MNO-863能显著降低肥胖小鼠的血清ALT及AST指标。
血清中的AST(谷草转氨酶)、ALT(谷丙转氨酶)水平参照图13所示。
(4)申请人还探究了MNO-863对高脂饮食诱导肥胖模型小鼠腹部白色脂肪炎症性病变的影响，结果表明MNO-863能显著减少肥胖模型小鼠腹部白色脂肪炎症性病变。
小鼠腹部白色脂肪炎症性病变的显微图以及总面积统计图参照图14所示。
(5)申请人还探究了MNO-863对高脂饮食诱导肥胖模型小鼠血肌酐(CREA)、血尿素(UREA)及血尿酸(UA)的影响，结果表明MNO-863对肾脏无毒性。
小鼠血肌酐(CREA)的含量检测依据酶法的检测原理，通过终点法(采用肌酐测定试剂盒，雷社，S03076)，并利用全自动生化分析仪进行检测。血尿素(UREA)的含量检测依据尿素酶-谷氨酸脱氢酶法的检测原理，通过两点法(采用尿素测定试剂盒，雷社生命科学股份有限公司，S03036)，并利用全自动生化分析仪进行检测。血尿酸(UA)的含量检测依据尿酸酶法的检测原理，通过终点法(采用尿酸测定试剂盒，雷社生命科学股份有限公司，S03035)，并利用全自动生化分析仪进行检测。
小鼠血肌酐(CREA)、血尿素(UREA)及血尿酸(UA)的测定结果图参照图15所示。
实施例3
本实施例进行MNO-863在高脂饮食诱导肥胖小鼠模型的体内试验以验证其在治疗或预防糖尿病中的用途。实验材料：
(1)实验动物：购买C57BL/6J雄性小鼠(购自江苏集萃药康生物科技有限公司)40只，为正常饲养小鼠，5周龄。小鼠生长过程处于同一环境，其中8只给予SPF级大小鼠维持饲料(广州市瀚程实验器材有限公司)，32只给予D12492高脂饲料(派克生物)，喂养约8-10周后，称重，饮食诱导肥胖模型成模标准的体重达到38.00±2.00g。
(2)供试菌株：厌氧培养的MNO-863，培养基是104液体培养基，37℃厌氧条件下培养48h，至菌浓约10 11CFU/mL数量级，方可做为实验组灌胃。菌液于4℃条件下厌氧储存。
(3)PBS磷酸盐缓冲溶液：是由弱酸及其盐、弱碱及其盐组成的混合溶液，能在一定程度上抵消、减轻外加强酸或强碱对溶液酸碱度的影响，从而保持溶液的pH值相对稳定。PBS磷酸盐缓冲溶液的配方同实施例2中的表4。
(4)利拉鲁肽(Liraglutide)(阳性对照)：利拉鲁肽是一种人胰高糖素样肽-1(GLP-1)类似物，用于治疗糖尿病。购自诺和诺德，商品名为
Figure PCTCN2021106579-appb-000003
-Novo Nordisk，使用时按照15μg/kg/d皮下注射。
试验过程：
(1)试验分组
将8只给予SPF级大小鼠维持饲料的小鼠完全随机分成2笼，4只/笼，作为第一组。从32只肥胖小鼠中挑选体重达到38.00g±2.00g的小鼠24只，共分为3组(分别作为第二组、第三组、第四组)，每组8只，4只/笼。第一组为饲喂普通饲料的对照组(NCD-对照组)，第二组为高脂饮食诱导肥胖小鼠模型组(HFD-对照组)，第三组为菌剂治疗组(MNO-863)，第四组为利拉鲁肽阳性对照组，第二组、第三组和第四组饲喂高脂饲料，分组情况见表7。动物分组后开始虚拟给药一周后再开始给药，第一组和第二组灌胃等量PBS磷酸盐缓冲液，第三组使用MNO-863供试菌株进行灌胃干预，干预持续4周。灌胃菌液的量为0.2mL/10g小鼠体重。分别在造模前后，干预前后每3天记录小鼠的体重和状态等数据。实验动物的使用关注动物福利，遵循“减少、替代和优化”的原则，并经本单位实验动物伦理委员会批准。试验过程中接受实验动物伦理委员会的监督检查。
表7 试验分组
序号 组别 受试物/对照品 药物浓度 给药频率 动物数量 饲料
1 NCD-对照 PBS / 2次/天 8 D12450B
2 HFD-对照 PBS / 2次/天 8 D12492
3 MNO-863 MNO-863 5×10 11CFU/mL 2次/天 8 D12492
4 利拉鲁肽 利拉鲁肽 40μg/ml 1次/天 8 D12492
口服葡萄糖耐量检查(OGTT)：动物给药后的第28天，测定禁食12h的OGTT(如晚上20:30:00禁食至次日08:30:00)。称量小鼠禁食体重，按照小鼠禁食体重值灌胃葡萄糖，灌胃葡萄糖剂量为2g/kg(葡萄糖g/小鼠禁食体重kg)，分别测定空腹血糖以及给糖后15min、30min、60min、90min、120min的血糖值。每只小鼠严格计时，准确按照6个时间点测定血糖值。口服葡萄糖耐量试验是一种葡萄糖负荷试验，用以了解胰岛β细胞功能和机体对血糖的调节能力，观察病人耐受葡萄糖的能力，是目前公认的诊断糖尿病的金标准。
干预实验结束后对小鼠过夜禁食10-12h，次日称量禁食体重，异氟烷(瑞沃德生命科技有限公司)麻醉后对小鼠进行眼球采血，使用血糖仪(ACCU-CHEK型，Roche)检测空腹血糖的血糖值，并将血液放置在4℃冰箱3-4h后，待血液凝固血块收缩后，4℃下4500r/min离心15min，收集上层血清，使用小鼠胰岛素(INS)酶联免疫试剂盒(武汉华美生物工程有限公司)检测血清中胰岛素的含量。根据血清中的空腹血糖水平和胰岛素水平计算出HOMA-IR。HOMA-IR是用于评价个体的胰岛素抵抗水平的指标，目前已成为广泛应用于临床的评价糖尿病人胰岛素敏感性，胰岛素抵抗水平与胰岛β细胞功能的常用指标，其计算方法为：空腹血糖水平(FPG，mmol/L)×空腹胰岛素水平(FINS，μU/mL)/22.5，正常个体的HOMA-IR指数为1。随着胰岛素抵抗水平升高的升高，HOMA-IR指数将高于1。胰岛素抵抗是指各种原因使胰岛素促进葡萄糖摄取和利用的效率下降，机体代偿性的分泌过多胰岛素产生高胰岛素血症，以维持血糖的稳定，胰岛素抵抗易导致代谢综合征和2型糖尿病。
实验结果：
(1)MNO-863对肥胖模型小鼠口服葡萄糖耐量的影响：MNO-863对高脂饮食诱导肥胖小鼠口服葡萄糖耐量的影响参照表8和图16所示。
表8 MNO-863对高脂饮食诱导肥胖小鼠口服葡萄糖耐量的影响
Figure PCTCN2021106579-appb-000004
注：结果以平均值±标准差(SD)表示，*p<0.05，**p<0.01,***p<0.001,****p<0.0001与HFD-对照组相比。
当糖代谢紊乱时，口服一定量的葡萄糖后血糖急剧升高，或升高不明显，但短时间内不能降至空腹水平(或原来水平)，此为糖耐量异常(IGT)或糖耐量降低。糖耐量异常(IGT)表明机体对葡萄糖的代谢能力下降，常见于2型糖尿病和肥胖症等。
根据表8和图16结果可知，在MNO-863干预4周后，MNO-863处理组的高脂饮食诱导肥胖小鼠在灌胃葡萄糖15min后血糖升高程度显著低于HFD对照组。在后续的检测中，MNO-863处理组小鼠血糖的血糖值逐渐降低，并且在120min后血糖值恢复至接近NCD对照组，远低于HFD对照组，且具有显著性差异。同时，MNO-863表现出与治疗糖尿病药物利拉鲁肽相当的治疗效果。
(2)MNO-863对肥胖模型小鼠空腹血糖的影响：MNO-863对高脂饮食诱导肥胖小鼠空腹血糖的影响参照表9和图17所示。
表9 MNO-863对高脂饮食诱导肥胖小鼠空腹血糖(mmol/L)的影响
分组 空腹血糖(mmol/L)
NCD 9.74±1.53*
HFD 11.81±2.82
MNO-863 6.74±1.28****
利拉鲁肽 7.02±0.93****
注：结果以平均值表示，*p<0.05，****p<0.0001与HFD-对照组相比。
根据表9和图17结果可知，与HFD对照组相比，MNO-863处理组能够显著降低高脂饮食诱导肥胖小鼠的血糖值，与HFD对照组相比达到显著差异。并且相比治疗糖尿病药物利拉鲁肽，MNO-863对于血糖的控制更为明显。表明MNO-863具有明显的降血糖的效果，可以改善糖尿病症状。
(3)MNO-863对高脂饮食诱导肥胖模型小鼠HOMA-IR指数的影响：MNO-863对高脂饮食诱导肥胖小鼠HOMA-IR指数的影响参照表10和图18所示。
表10 MNO-863对高脂饮食诱导肥胖小鼠HOMA-IR的影响
分组 HOMA-IR
NCD 1.74
HFD 3.36
MNO-863 1.24
注：结果以平均值表示。
胰岛素抵抗(IR)是2型糖尿病发生的主要原因，可促进2型糖尿病患者并发症的发生及进展。与HOMA-IR相关的生化指标，可有效揭示IR发生的原因。而通常情况下糖尿病患者的HOMA-IR会明显高于正常人群。
根据表10和图18结果可知，与HFD对照组相比，在MNO-863的干预下能够明显降低高脂饮食诱导肥胖小鼠的HOMA-IR。表明MNO-863具有改善机体胰岛素抵抗与胰岛β细胞功能的作用，从而达到预防和治疗糖尿病的目的。
实施例4
本实施例进行MNO-863在高脂饮食诱导肥胖小鼠模型的体内试验以验证MNO-863在治疗和预防肥胖症以及其相关疾病中的应用。实验材料(包括实验动物、供试菌株、PBS磷酸盐缓冲溶液以及阳性对照)同实施例3中的实验材料。
试验过程：(1)试验分组
将8只给予SPF级大小鼠维持饲料的小鼠完全随机分成2笼，4只/笼，作为第一组。从32只肥胖小鼠中挑选体重达到38.00g±2.00g的小鼠24只，共分为3组(分别作为第二组、第三组、第四组)，每组8只，4只/笼。第一组为饲喂普通饲料的对照组(对照组)，第二组为高脂饮食诱导肥胖小鼠模型组(model组)，第三组为菌剂治疗组，第四组为利拉鲁肽阳性对照组，第二组、第三组和第四组饲喂高脂饲料，分组情况见表11。动物分组后开始虚拟给药一周后再开始给药，第一组和第二组灌胃等量PBS磷酸盐缓冲液，第三组使用MNO-863供试菌株进行灌胃干预，干预持续4周。灌胃菌液的量为0.2mL/10g小鼠体重。分别在造模前后，干预前后每3天记录小鼠的体重、状态、摄食量等数据。给药结束后，将动物解剖并采集组织。实验动物的使用关注动物福利，遵循“减少、替代和优化”的原则，并经本单位实验动物伦理委员会批准。试验过程中接受实验动物伦理委员会的监督检查。
表11 试验分组
序号 组别 受试物/对照品 药物浓度 给药频率 动物数量 饲料
1 NCD-对照 PBS / 2次/天 8 D12450B
2 HFD-对照 PBS / 2次/天 8 D12492
3 MNO-863 MNO-863 5×10 11CFU/mL 2次/天 8 D12492
4 利拉鲁肽 利拉鲁肽 40μg/ml 1次/天 8 D12492
实验结束后处死小鼠，记录脂肪含量，采血并于4℃下4500r/min离心15min收集血清，使用总胆固醇(TC)、甘油三酯(TG)、高密度脂蛋白(HDL-C)和低密度脂蛋白(LDLC)测定试剂盒(南京建成生物工程研究所)检测血清中血脂的含量。
实验结果：
(1)MNO-863对肥胖模型小鼠体重的影响：根据表12、表13和图19、图20结果可知，与HFD-对照组相比，MNO-863干预组在3周内能够有效降低高脂饮食诱导肥胖小鼠体重3g以上以及体重百分数约10％，并达到显著性差异，与阳性对照减肥药物利拉鲁肽减重效果相当，表明MNO-863对高脂质摄入下的机体具有减轻体重的效果。
表12 MNO-863对高脂饮食诱导肥胖小鼠体重(g)的影响
Figure PCTCN2021106579-appb-000005
表13 MNO-863对高脂饮食诱导肥胖小鼠体重(g)的影响
Figure PCTCN2021106579-appb-000006
注：结果以平均值±标准差表示，****p<0.0001与HFD-对照组相比。
(2)MNO-863对肥胖模型小鼠摄食量的影响：根据表14和图21结果可知，与HFD-对照组相比，MNO-863干预组在3周内能够降低高脂饮食诱导肥胖小鼠的摄食量。
表14 MNO-863对高脂饮食诱导肥胖小鼠摄食量(g)的影响
Figure PCTCN2021106579-appb-000007
注：结果以平均值表示
(3)MNO-863对高脂饮食诱导肥胖模型小鼠血脂的影响：根据表15和图22结果可知，MNO-863干预组对于持续高脂质摄入下的小鼠的血脂水平具有明显的控制效果，能够降低与原发性高血脂等心血管疾病相关的指标：总胆固醇(TC)、甘油三酯(TG)和低密度脂蛋白(LDLC)，并提高血液中高密度脂蛋白(HDL-C)水平，而HDL与心血管疾病的发病率和病变程度呈负相关，其中总胆固醇(TC)和甘油三酯(TG)结果达到显著差异。
表15 MNO-863对高脂饮食诱导肥胖小鼠血脂四项的影响
Figure PCTCN2021106579-appb-000008
注：结果以平均值表示，*p<0.05，**p<0.01与HFD-对照组相比。
(4)MNO-863菌对高脂饮食诱导肥胖模型小鼠体脂的影响：根据表16和图23结果可知，与HFD-对照组相比，在MNO-863干预下显著降低了高脂饮食诱导肥胖小鼠的腹股沟脂肪、皮下脂肪和附睾脂肪的重量，表明MNO-863具有降低哺乳动物体脂的作用。
表16 MNO-863对高脂饮食诱导肥胖小鼠体脂(g)的影响
Figure PCTCN2021106579-appb-000009
注：结果以Mean表示，*p<0.05，**p<0.01，****p<0.0001与HFD-对照组相比。
实验例5
本实施例进行MNO-863对小鼠模型回肠和结肠组织黏膜修复的体内试验以验证MNO-863在修复消化道黏膜损伤及防治消化道黏膜损伤相关疾病中的应用。
动物实验过程：
将8只给予SPF级大小鼠维持饲料的小鼠完全随机分成2笼，4只/笼，作为第一组。从24只肥胖小鼠中挑选体重达到38.00g±2.00g的小鼠16只，分为2组(作为第二组和第三组)，每组8只，4只/笼。第一组为饲喂普通饲料的对照组(NCD-对照组)，第二组为高脂饮食诱导肥胖小鼠模型组(HFD-对照组)，第三组为菌剂治疗组(MNO-863)，第二组和第三组饲喂高脂饲料，分组方式同实施例2的表5。动物分组后开始虚拟给药一周后再开始给药，第一组和第二组灌胃等量PBS磷酸盐缓冲液，第三组使用MNO-863供试菌株进行灌胃干预，干预持续4周。灌胃菌液的量为0.2mL/10g小鼠体重。分别在造模前后，干预前后每3天记录小鼠的体重、状态、摄食量等数据。给药结束后，将动物解剖并采集组织。实验动物的使用关注动物福利，遵循“减少、替代和优化”的原则，并经本单位实验动物伦理委员会批准。试验过程中接受实验动物伦理委员会的监督检查。
解剖、观察过程：所有的动物包括试验期间死亡、安乐死和试验结束处死的动物都要进行大体解剖检查。剪取小鼠的回肠及结肠置于福尔马林溶液中保存。送到武汉赛维尔生物科技有限公司制作成病理切片，拍照观察。
结果显示：MNO-863供试菌株对小鼠回肠和结肠组织黏膜层损伤具有修复功能(参照表17所示)：与HFD-对照组相比，在MNO-863干预下，小鼠结肠组织各层结构清晰，黏膜上皮完整，肠腺数量丰富，排列紧密，未见明显异常(参照图26所示)。小鼠回肠组织各层结构清晰，肠绒毛数量丰富，黏膜上皮完整，肠腺数量丰富，排列紧密，未见其他明显异常(参照图27所示)。而HFD-对照组的显微图显示，小鼠结肠组织可见多处黏膜层损伤，黏膜上皮细胞脱落，少量肠腺结构破坏，肠腔内可见大量嗜碱性菌丝(参照图24所示)；小鼠回肠组织黏膜层损伤，局部肠绒毛及黏膜上皮缺失，肠腺结构消失，少量上皮细胞肿胀，胞质疏松淡染，肠腔内可见大量嗜碱性菌丝(参照图25所示)。
NCD-对照组的显微图显示，小鼠结肠组织各层结构清晰，黏膜上皮完整，肠腺数量丰富，排列紧密，未见明显异常(参照图28所示)；小鼠回肠组织各层结构清晰，肠绒毛数量丰富，黏膜上皮完整，肠腺数量丰富，排列紧密，未见明显异常(参照图29所示)。
表明MNO-863具有修复回肠和结肠黏膜的功能，其施用能有效修复消化道黏膜并对消化道黏膜损伤相关疾病的防治产生积极效果。
表17 MNO-863对小鼠结肠黏膜损伤和回肠黏膜损伤的病理评分结果
Figure PCTCN2021106579-appb-000010
以下结合实施例对本公开提供的联用药物作进一步的详细描述。
本实施例进行MNO-863菌株和/或Liraglutide(利拉鲁肽)药物的使用在高脂饮食诱导肥胖小鼠模型的体内试验，以验证其在治疗或预防肥胖症、糖尿病及肝脏疾病中的用途。
(1)实验动物：购买C57BL/6J雄性小鼠(购自江苏集萃药康生物科技有限公司)50只，均为正常饲养小鼠，5周龄。小鼠生长过程处于同一环境，其中8只给予SPF级大小鼠维持饲料(购自广州市瀚程实验器材有限公司)，42只给予D12492高脂饲料(购自派克生物)喂养约8-10周后，称重，饮食诱导肥胖模型成模标准为体重达到38.00±2.00g。
(2)供试菌株：厌氧培养的MNO-863，培养基为104液体培养基(配方如上表1所示)，在37℃厌氧条件下培养48h，至菌浓约10 9CFU/mL数量级，方可做为实验组灌胃。菌液存放为4℃条件厌氧储存。
(3)PBS磷酸盐缓冲溶液：是由弱酸及其盐、弱碱及其盐组成的混合溶液，能在一定程度上抵消、减轻外加强酸或强碱对溶液酸碱度的影响，从而保持溶液的pH值相对稳定。PBS磷酸盐缓冲溶液的配方如上表4所示。
试验过程如下：
(1)试验分组如下：
42只肥胖小鼠挑选体重达到38.00g±2.00g的32只，共分为4组，每组8只，4只/笼。第一组为饲喂普通饲料的对照组(NCD对照组)，第二组为高脂饮食诱导肥胖小鼠模型组(HFD对照组)，第三组为MNO-863菌剂治疗组，第四组为Liraglutide药物治疗组(Lira组)，第五组为MNO-863菌剂与Liraglutide药物联合使用治疗组(Lira+MNO-863)。其中第二、第三、第四组和第五组饲喂高脂饲料，分组见表18。动物分组后开始虚拟给药一周后再开始给药，干预持续4周。灌胃菌液的量为0.2mL/10g小鼠体重。
分别在造模前后，干预前后每3天记录小鼠的体重、状态、摄食量等数据。给药结束动物时解剖采集组织。实验动物的使用关注动物福利，遵循“减少、替代和优化”的原则，并经本单位实验动物伦理委员会批准。试验过程中接受实验动物伦理委员会的监督检查。
表18 试验分组
Figure PCTCN2021106579-appb-000011
(2)Liraglutide药物和MNO-863菌剂联合治疗对体重的影响。
MNO-863与Liraglutide联用对于肥胖小鼠四周干预期间的绝对体重的影响以及与体重变化百分比参照图30所示，干预四周后的体重以及体重变化百分比参照图31所示，干预四周与停药四周后的体重以及体重变化百分比参照图32所示，停药4周复胖后的腹股沟脂肪重量参照图33所示。由图30、图31、图32和图33可知，MNO-863菌剂和Liraglutide药物均能降低肥胖小鼠的体重，抑制其体重增长，及降低腹股沟脂肪，而MNO-863菌剂和Liraglutide药物的联用组在降体重、抑制体重增长及降低腹股沟脂肪的效果明显优于MNO-863菌株单独使用或Liraglutide药物单独使用的效果，说明微生态制剂起到增强药物减肥效果的作用。
(3)Liraglutide药物和MNO-863菌剂联合治疗对糖尿病的治疗作用。
当糖代谢紊乱时，口服一定量的葡萄糖后血糖急剧升高，或升高不明显，但短时间内不能降至空腹水平(或原来水平)，此为糖耐量异常(IGT)或糖耐量降低。糖耐量异常(IGT)表明机体对葡萄糖的代谢能力下降，常见于2型糖尿病和肥胖症等。
通过糖代谢试验可对药物和菌剂对糖尿病的治疗作用进行评价。
MNO-863菌株的单独使用以及菌株与Liraglutide联用对于肥胖小鼠葡萄糖耐受性的影响参照图34所示，MNO-863菌株的单独使用以及菌株与Liraglutide联用对于肥胖小鼠葡萄糖高血糖的影响参照图35所示，MNO-863菌株对于肥胖小鼠停药4周复胖后的高血糖影响作用参照图36所示。
由图34、图35和图36可知，MNO-863菌剂和Liraglutide药物均能改善肥胖小鼠的糖耐量异常状况，且能降低空腹血糖，而菌剂与药物的联用组的效果比菌剂或药物单独使用更为显著，说明微生态菌剂能起到增强药物治疗糖尿病效果的作用。
(4)Liraglutide药物和MNO-863菌剂联合治疗对肝脏疾病的治疗作用。
图37为MNO-863菌株的单独使用以及菌株与Liraglutide联用对于停药4周复胖后的肝脏重量影响结果图，由图37可知，MNO-863菌剂和Liraglutide药物均能降低肥胖小鼠的肝脏重量，而菌剂与药物的联用组的效果比菌剂或药物单独使用更为显著，说明微生态菌剂能起到增强药物治疗肝脏疾病效果的作用。
以上所述仅为本公开的典型实施例而已，并不用于限制本公开，对于本领域的技术人员来说，本公开可以有各种更改和变化。凡在本公开的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本公开的保护范围之内。
工业实用性
本公开提供的克里斯滕森菌能够在工业上大量培养，且本公开提供的克里斯滕森菌可以应用于治疗或预防肝脏功能损伤及肝脏功能损伤相关疾病、消化道黏膜损伤及消化道黏膜损伤相关疾病、糖尿病、肥胖症及肥胖症相关疾病。本公开提供的克里斯滕森菌还对肾脏无毒副作用，可减少肝脏重量；治疗初期脂肪肝炎病灶；减缓肝脏细胞脂肪堆积；降低血清AST、ALT；减少腹部白色脂肪炎症性病变。克里斯滕森菌还能修复消化道黏膜、恢复黏膜屏障功能，防治因屏障功能受损引起的肠漏、消化性溃疡等疾病。克里斯滕森菌还具有降低机体空腹血糖、调节胰岛素水平、降低哺乳动物体脂的功效，并具有预防和治疗糖尿病、提高肥胖症患者代谢功能等作用。克里斯滕森菌还具有修复受损的消化道黏膜和防治黏膜损伤相关疾病的功能。本公开提供的包含克里斯滕森菌和降糖降脂药物的联用药物能够在工业上大量生产，且该联用药物可用于治疗或预防肝脏功能损伤及肝脏功能损伤相关疾病、糖尿病、肥胖及肥胖相关疾病。本公开提供的联用药物具有协同增效的技术效果，即该联用药物具有比单独施用克里斯滕森菌或者单独施用降糖降脂药物更优的治疗效果，且该联用药物对肾脏无毒副作用，可减少肝脏重量。

Claims (20)

  1. 克里斯滕森菌(Christensenella sp.)物种的细菌菌株在制备用于治疗或预防选自以下至少一种的疾病或病症的药物中的用途：肝脏功能损伤及肝脏功能损伤相关疾病、消化道黏膜损伤及消化道黏膜损伤相关疾病、糖尿病、肥胖及肥胖相关疾病。
  2. 根据权利要求1所述的用途，其特征在于，所述肝脏功能损伤相关疾病包括如下疾病中的至少一种：脂肪肝、非酒精性脂肪性肝病、非酒精性脂肪性肝炎和肝硬化；
    所述消化道黏膜损伤是指消化道黏膜通透性升高、黏膜屏障作用受损，消化道黏膜损伤相关疾病包括如下疾病中的至少一种：肠漏病症、消化性溃疡、胃肠炎、炎性肠病；
    所述肥胖相关疾病包括如下疾病中的至少一种：心血管疾病、高脂血症、胰岛素抗性综合征、肥胖相关的胃食管返流症和脂肪性肝炎；以及
    所述糖尿病包括如下疾病中的至少一种：1型糖尿病、2型糖尿病、胰岛素抗性综合征、葡萄糖不耐症、高血脂絮乱、糖尿病肾病变并发症、糖尿病神经病变、糖尿病眼睛病变、心血管疾病、糖尿病足和妊娠期糖尿病。
  3. 根据权利要求1或2所述的用途，其特征在于，所述细菌菌株具有与SEQ ID NO.1至少98.65％一致的16s rRNA序列；
    优选地，所述细菌菌株具有与SEQ ID NO.1至少99％一致的16s rRNA序列；以及
    优选地，所述细菌菌株具有与SEQ ID NO.1 99％、99.5％、99.9％或100％一致的16s rRNA序列。
  4. 根据权利要求1至3中任一项所述的用途，其特征在于，所述药物是冻干的；
    优选地，所述药物还包括一种或多种药学上可接受的赋形剂或载剂；以及
    优选地，所述药物是疫苗组合物。
  5. 一种以保藏号为GDMCC No：61117保藏的克里斯滕森菌菌株或其子代菌株或亚克隆菌株的细胞。
  6. 一种组合物，其特征在于，其包括权利要求5所述的克里斯滕森菌菌株和/或其代谢产物；以及
    优选地，所述组合物还包括药学上可接受的赋形剂或载剂。
  7. 根据权利要求6所述的组合物，其特征在于，所述赋形剂包括抗氧化剂、螯合剂、乳化剂、溶剂。
  8. 一种包含微生物和降糖降脂药物的联用药物在制备用于治疗或预防选自以下至少一种的疾病或症状的药物中的用途：肝脏功能损伤及肝脏功能损伤相关疾病、糖尿病、肥胖及肥胖相关疾病；其特征在于，所述微生物为克里斯滕森菌(Christensenella sp.)物种的细菌；所述降糖降脂药物为能改善胰高血糖素样肽-1通路敏感性、补充和/或促进GLP-1作用的药物中的一种或多种。
  9. 根据权利要求8所述的用途，其特征在于，所述降糖降脂药物为GLP-1受体激动剂或GLP-1模拟物、GIP受体激动剂、二肽基肽酶-4抑制剂中的至少一种；
    优选地，所述GLP-1受体激动剂或GLP-1模拟物选自艾塞那肽、利拉鲁肽、索马鲁肽、口服剂型索马鲁肽、贝那鲁肽、利司那肽和艾塞那肽周制剂中的至少一种。
  10. 根据权利要求8或9所述的用途，其特征在于，所述克里斯滕森菌具有与SEQ ID NO.1至少98.65％一致的16s rRNA序列；
    优选地，所述克里斯滕森菌具有与SEQ ID NO.1至少99％一致的16s rRNA序列；以及
    优选地，所述克里斯滕森菌具有与SEQ ID NO.1 99％、99.5％、99.9％或100％一致的16s rRNA序列。
  11. 根据权利要求8至10中任一项所述的用途，其特征在于，所述微生物为保藏号为GDMCC No：61117保藏的克里斯滕森菌菌株或其子代菌株。
  12. 一种联用药物，其特征在于，其包括微生物和降糖降脂药物；所述微生物为克里斯滕森菌(Christensenella sp.)物种的细菌；所述降糖降脂药物为能改善胰高血糖素样肽-1通路敏感性、补充和/或促进GLP-1作用的药物中的一种或多种。
  13. 一种如权利要求6或7所述的组合物或如权利要求12所述的联用药物在用于制备药物或制剂中的用途，其特征在于，所述药物或制剂用于选自如下至少一者的用途：
    减少肝脏重量；
    治疗初期脂肪肝炎病灶；
    减缓肝脏细胞脂肪堆积；
    降低血清AST、ALT；
    减少腹部白色脂肪炎症性病变；
    减轻哺乳动物的体重；
    减少哺乳动物的摄食量；
    减缓停药后的复胖幅度；
    降低哺乳动物的体脂；
    降低哺乳动物血清中如下至少一种指标的水平：总胆固醇水平、低密度脂蛋白和甘油三酯水平；
    提高哺乳动物血清高密度脂蛋白的水平；
    改善哺乳动物口服葡萄糖耐量受损；
    降低哺乳动物的空腹血糖；
    降低哺乳动物HOMA-IR指标；
    增强GLP-1敏感性；
    避免肠道絮乱所造成的GLP-1RA的抗性及相关副作用；以及，
    修复消化道黏膜损伤。
  14. 一种组合物，其包括权利要求1至4中任一项所述的克里斯滕森菌(Christensenella sp.)物种的细菌菌株或权利要求5所述的克里斯滕森菌菌株或其子代菌株或亚克隆菌株和/或其代谢产物，用于治疗或预防选自以下至少一种的疾病或病症：肝脏功能损伤及肝脏功能损伤相关疾病、消化道黏膜损伤及消化道黏膜损伤相关疾病、糖尿病、肥胖及肥胖相关疾病。
  15. 根据权利要求14所述的组合物，其特征在于，所述肝脏功能损伤相关疾病包括如下疾病中的至少一种：脂肪肝、非酒精性脂肪性肝病、非酒精性脂肪性肝炎和肝硬化；
    所述消化道黏膜损伤是指消化道黏膜通透性升高、黏膜屏障作用受损，消化道黏膜损伤相关疾病包括如下疾病中的至少一种：肠漏病症、消化性溃疡、胃肠炎、炎性肠病；
    所述肥胖相关疾病包括如下疾病中的至少一种：心血管疾病、高脂血症、胰岛素抗性综合征、肥胖相关的胃食管返流症和脂肪性肝炎；以及
    所述糖尿病包括如下疾病中的至少一种：1型糖尿病、2型糖尿病、胰岛素抗性综合征、葡萄糖不耐症、高血脂絮乱、糖尿病肾病变并发症、糖尿病神经病变、糖尿病眼睛病变、心血管疾病、糖尿病足和妊娠期糖尿病。
  16. 根据权利要求14或15所述的组合物，其特征在于，所述细菌菌株具有与SEQ ID NO.1至少98.65％一致的16s rRNA序列；
    优选地，所述细菌菌株具有与SEQ ID NO.1至少99％一致的16s rRNA序列；以及
    优选地，所述细菌菌株具有与SEQ ID NO.1 99％、99.5％、99.9％或100％一致的16s rRNA序列。
  17. 一种治疗或预防疾病或病症的方法，包括向有需要的受试者给药权利要求14至16中任一项所述的组合物或者权利要求6或7所述的组合物，所述疾病或病症选自以下至少一种：肝脏功能损伤及肝脏功能损伤相关疾病、消化道黏膜损伤及消化道黏膜损伤相关疾病、糖尿病、肥胖及肥胖相关疾病。
  18. 一种联用药物，其包括微生物和降糖降脂药物，
    所述微生物为权利要求1至4中任一项所述的克里斯滕森菌(Christensenella sp.)物种的细菌菌株或权利要求5所述的克里斯滕森菌菌株或其子代菌株或亚克隆菌株和/或其代谢产物，
    所述降糖降脂药物为能改善胰高血糖素样肽-1通路敏感性、补充和/或促进GLP-1作用的药物中的一种或多种，用于治疗或预防选自以下至少一种的疾病或病症：肝脏功能损伤及肝脏功能损伤相关疾病、糖尿病、肥胖及肥胖相关疾病。
  19. 一种治疗或预防疾病或病症的方法，包括向有需要的受试者给药权利要求14至16中任一项所述的组合物或者权利要求6或7所述的组合物或者权利要求8至11中任一项所述的联用药物或者权利要求12或18所述的联用药物，所述疾病或病症选自以下至少一种：肝脏功能损伤及肝脏功能损伤相关疾病、糖尿病、肥胖及肥胖相关疾病。
  20. 一种试剂盒，其包括权利要求12或18所述的联用药物。
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