Application of intestinal flora composition in preparation of medicine for treating Alzheimer's disease

Abstract

The invention discloses application of an intestinal flora composition in preparation of a medicine for treating Alzheimer's disease. The invention provides an application of an intestinal flora composition in preparation of medicines for preventing and treating brain-intestinal axis related diseases of animals and in prevention and treatment of the brain-intestinal axis related diseases of the animals. In the experiment process, it is found that the intestinal flora composition can achieve a certain treatment effect on AD and other diseases related to the brain-intestinal axis.

Description

Technical Field

[0001] The present application relates to the field of microbial technology, and in particular to the use of an intestinal flora composition in the preparation of a drug for treating Alzheimer's disease. Background Art

[0002] Microbes are an important part of human life. The co-evolution between human hosts and microbes has established a mutually beneficial symbiotic relationship, in which the host provides a habitable environment and nutrients for the microbiota, while the microbiota has a significant impact on the host during homeostasis and disease. Most of these microbes (including bacteria, archaea, fungi and viruses) are found in the human gastrointestinal tract and are collectively referred to as the gut microbiota. This co-evolution of non-human genomes has led to the establishment of a two-way relationship between the host and the gut microbiota, especially the two-way communication between the gastrointestinal tract, the enteric nervous system and the central nervous system. This complex and sophisticated system that connects gastrointestinal function and the central nervous system is called the Brain-Gut Axis.

[0003] The brain-gut axis communication refers to a connection network involving multiple biological systems that enables two-way communication between the intestinal flora and the brain. The brain-gut axis is mainly mediated directly or indirectly through neural pathways, immune pathways, and microbial metabolites to maintain the equilibrium of the microbial system. When the equilibrium state is disrupted, it will lead to intestinal flora imbalance. Intestinal flora imbalance is characterized by reduced microbial richness and abundance, loss of beneficial bacteria, and increase in pathogenic bacteria. This imbalance is associated with a variety of diseases, such as neurodegenerative diseases, psychiatric diseases, metabolic syndrome, obesity, and intestinal-related inflammatory diseases. [1] .

[0004] Alzheimer's disease (AD) is a chronic neurodegenerative disease and the most common and typical form of dementia. Intestinal flora imbalance plays a vital role in the pathogenesis of AD. [2] Therefore, the study of intestinal microbiota provides a new direction for the treatment of AD.

[0005] The purpose of the present invention is to provide a method of using a combination of microbiota to improve the symptoms of diseases such as AD related to the brain-gut axis through FMT technology. Summary of the invention

[0006] The present application proposes the use of an intestinal flora composition in the preparation of a drug for treating Alzheimer's disease.

[0007] In a first aspect of the present application, a use of an intestinal flora composition in the following A1) to A4) is provided:

[0008] A1) preparing a medicament for preventing brain-gut axis related diseases in animals;

[0009] A2) preparing a medicament for treating a brain-gut axis-related disease in an animal;

[0010] A3) Prevent brain-gut axis-related diseases in animals;

[0011] A4) Treating brain-gut axis related diseases in animals.

[0012] In some embodiments of the present application, the brain-gut axis related disease includes at least one of a neurodegenerative disease, an intestinal permeability disease, and an inflammatory disease.

[0013] In some embodiments of the present application, the neurodegenerative disease includes at least one of Alzheimer's disease, Parkinson's disease, and multiple sclerosis.

[0014] In some embodiments of the present application, the intestinal permeability disease includes but is not limited to at least one of inflammatory bowel disease, irritable bowel syndrome, obesity, non-alcoholic fatty liver disease, type 2 diabetes, cardiovascular disease, and cancer.

[0015] In some embodiments of the present application, the inflammatory disease includes at least one of neuroinflammation, inflammatory arthritis, allergy, anxiety, and depression.

[0016] In some embodiments of the present application, the inflammatory disease comprises neurological inflammation in the brain.

[0017] In some embodiments of the present application, the intestinal flora composition is derived from healthy adults.

[0018] In some embodiments of the present application, the intestinal flora composition is derived from feces of healthy adults.

[0019] In some embodiments of the present application, the intestinal flora composition is prepared by the following method:

[0020] Feces of healthy adults are obtained, homogenized in a phosphate buffer, and filtered through a filter device with a pore size of 60 to 80 μm. The obtained filtrate is prepared into an intestinal flora composition.

[0021] In some embodiments of the present application, the phosphate buffer contains 10-30% glycerol by mass volume.

[0022] In some embodiments of the present application, the intestinal flora composition includes the following species:

[0023] Bacteroides, Faecalibacterium, Megasphaera, Bifidobacterium, unclassified_Muribaculaceae, Megamonas, Dialister, Coprococcus, Agathobacter, Prevotella_9, unclassified_Lachnospiraceae, Ruminococcus, Phascolarctobacterium, Lachnoclostridium, Parabacteroides eroides, Roseburia, Lachnospiraceae_NK4A136_group, Blautia, Lachnospiraceae_UCG_010, Odoribacter, unclassified_Christensenellaceae, Collinsella, Alloprevotella, uncultured_Bacteroidales_bacterium, unclassified_Clostridia_vadinBB60_group, Ligilactobacillus, Enterorhabdus.

[0024] In some embodiments of the present application, the abundance of Bacteroides in the bacterial composition is 10-15%.

[0025] In some embodiments of the present application, the abundance of Faecalibacterium in the bacterial composition is 10-15%.

[0026] In some embodiments of the present application, the abundance of Megamonas in the bacterial composition is 5-10%.

[0027] In some embodiments of the present application, the abundance of Bifidobacterium in the bacterial composition is 3-8%.

[0028] In some embodiments of the present application, the abundance of Megasphaera in the bacterial composition is 3-8%.

[0029] In some embodiments of the present application, the abundance of the genus Dialisterae in the bacterial composition is 3 to 8%.

[0030] In some embodiments of the present application, the abundance of unclassified_Lachnospiraceae in the bacterial composition is 2-5%.

[0031] In some embodiments of the present application, the abundance of Coprococcus in the bacterial composition is 1 to 4%.

[0032] In some embodiments of the present application, the abundance of Agarobacterium in the bacterial composition is 1-4%.

[0033] In some embodiments of the present application, the abundance of Prevotella-9 in the bacterial composition is 1-4%.

[0034] In some embodiments of the present application, the abundance of Ruminococcus in the bacterial composition is 1-4%.

[0035] In some embodiments of the present application, the abundance of Phascolarctobacterium in the bacterial composition is 1 to 4%.

[0036] In some embodiments of the present application, the abundance of the genus Lachnoclostridium in the bacterial composition is 1 to 4%.

[0037] In some embodiments of the present application, the abundance of Parabacteroides in the bacterial composition is 0.5-2%.

[0038] In some embodiments of the present application, treating brain-gut axis related diseases includes treating Alzheimer's disease, and treating Alzheimer's disease includes at least one of improving behavioral abnormalities, reducing Aβ levels, reducing p-Tau protein levels, improving neuroinflammation, and improving intestinal permeability.

[0039] In some embodiments of the present application, the bacterial flora composition is administered in the form of a pharmaceutical composition.

[0040] In some embodiments of the present application, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

[0041] In some embodiments of the present application, the pharmaceutically acceptable excipients include but are not limited to at least one of a carrier, a diluent, a stabilizer, a filler solvent, a preservative, a pH adjuster, and the like.

[0042] In some embodiments of the present application, the pharmaceutical composition is any one of a liquid preparation, a solid preparation, and the like.

[0043] In some embodiments of the present application, the administration method includes but is not limited to at least one of oral administration, colon catheterization, and transanal retention enema.

[0044] In some embodiments of the present application, the animal includes a mammal.

[0045] In some embodiments of the present application, the animal includes a primate.

[0046] In some embodiments of the present application, the animal includes a human.

[0047] In a second aspect of the present application, a method for preventing or treating a brain-gut axis-related disease in an animal is provided, the method comprising administering an effective dose of an intestinal flora composition to the animal.

[0048] In some embodiments of the present application, the intestinal flora composition is derived from healthy adults.

[0049] In some embodiments of the present application, the intestinal flora composition is derived from feces of healthy adults.

[0050] In some embodiments of the present application, the intestinal flora composition is prepared by the following method:

[0051] Feces of healthy adults are obtained, homogenized in a phosphate buffer, and filtered through a filter device with a pore size of 60 to 80 μm, and the obtained filtrate is prepared into the intestinal flora composition.

[0052] In some embodiments of the present application, the phosphate buffer contains 10-30% glycerol by mass volume.

[0053] In some embodiments of the present application, the administration method includes but is not limited to at least one of oral administration, colon catheterization, anal retention enema, etc.

[0054] In some embodiments of the present application, the animal includes a mammal.

[0055] In some embodiments of the present application, the animal includes a primate.

[0056] In some embodiments of the present application, the animal includes a human.

[0057] In some embodiments of the present application, the brain-gut axis related disease includes at least one of a neurodegenerative disease, an intestinal permeability disease, and inflammation.

[0058] In some embodiments of the present application, the neurodegenerative disease includes at least one of Alzheimer's disease, Parkinson's disease, Huntington's disease, and multiple sclerosis.

[0059] In some embodiments of the present application, the intestinal permeability disease comprises at least one of inflammatory bowel disease, irritable bowel syndrome, obesity, non-alcoholic fatty liver disease, type 2 diabetes, cardiovascular disease, and cancer.

[0060] In some embodiments of the present application, the inflammatory disease includes at least one of neuroinflammation, inflammatory arthritis, allergy, anxiety, and depression.

[0061] In some embodiments of the present application, the inflammatory disease comprises neurological inflammation in the brain.

[0062] The beneficial effects of the embodiments of the present application are:

[0063] During the experiment of this application, it was found that the intestinal flora composition can play a certain therapeutic role in diseases related to the brain-gut axis such as AD, and can improve behavioral abnormalities, reduce Aβ levels, reduce p-Tau protein levels, improve neuroinflammation, and improve intestinal permeability.

[0064] Additional aspects and advantages of the present application will be given in part in the description below, and in part will become apparent from the description below, or will be learned through the practice of the present application. BRIEF DESCRIPTION OF THE DRAWINGS

[0065] Figure 1 : Schematic diagram of experimental mouse grouping.

[0066] Figure 2 :Statistical graph of mouse behavioral experiment. A and B are the spontaneous alternation rate (% of Alternation) and total number of entries (Total entries) of mice; C is the time required for mice to find the platform (Escape latency) when there is a platform; D is the number of times the mouse crosses the original platform position (Platform crossings) when there is no platform.

[0067] Figure 3 : Immunohistochemistry of Aβ42 (A) and p-Tau181 (C) in the hippocampus of mice, scale bar is 250μm. B and D are the statistical analysis results of A and C respectively.

[0068] Figure 4 :IBA-1 (A) and GFAP (C) immunohistochemistry images of mouse hippocampus, scale bar is 250μm. B and D are the statistical analysis results of A and C respectively.

[0069] Figure 5 : HE staining of mouse colon tissue (A, scale bar: 250 μm), B is the comparison result of histological score in A.

[0070] Figure 6 :Microbial α-diversity analysis diagram.

[0071] Note: Each point in the statistical graph represents a mouse. Statistical method used t test, means ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001, ns means no significant difference (no significant). DETAILED DESCRIPTION

[0072] Exemplary embodiments of the present invention are set forth below. It should be appreciated that such description is not intended as a limitation on the scope of the present invention, but is provided as a description of exemplary embodiments.

[0073] In the description of this application, "several" means more than one, "more" means more than two, "greater than", "less than", "exceed", etc. are understood to exclude the number itself, and "above", "below", "within", etc. are understood to include the number itself. If there is a description of "first" or "second", it is only used for the purpose of distinguishing technical features, and cannot be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features or implicitly indicating the order of the indicated technical features.

[0074] Unless otherwise defined, all technical and scientific terms used in this application have the same meaning as those commonly understood by those skilled in the art to which this application belongs. The terms used herein are only for the purpose of describing the embodiments of this application and are not intended to limit this application.

[0075] In the description of the present application, the description with reference to the terms "one embodiment", "some embodiments", "illustrative embodiments", "examples", "specific examples" or "some examples" means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present application. In this specification, the schematic representation of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner.

[0076] The brain-gut axis involves multiple systems such as the nervous, immune, and endocrine systems. It is mainly mediated directly or indirectly through neural pathways, immune pathways, and microbial metabolites to maintain the homeostasis of the host's gastrointestinal tract, central nervous system, and microbial system. Intestinal microorganisms participate in the functional response of the brain-gut axis and play an important role in it. Therefore, the microbial-brain-gut axis was proposed, reflecting the interactive relationship between the intestinal flora in the gastrointestinal tract and the central nervous system. Dysbiosis of the intestinal flora is associated with a variety of diseases, such as neurodegenerative diseases, psychiatric diseases, metabolic syndrome, obesity, and intestinal-related inflammatory diseases. Fecal Microbiota Transplantation (FMT) is a therapeutic approach that transplants the flora of a healthy donor to an unhealthy recipient. Its purpose is to rebuild the intestinal flora in the patient's body. It has been proven to have beneficial therapeutic effects. [3] However, there is still a lack of relevant research in diseases related to the brain-gut axis.

[0077] The present application proposes the first aspect of the present application based on relevant experimental results, which relates to the use of an intestinal flora composition in the preparation of drugs for preventing and / or treating brain-gut axis related diseases in animals, and in the prevention and / or treatment of brain-gut axis related diseases in animals.

[0078] In some embodiments, the animal includes a mammal, such as a primate, and specifically may be a human, including but not limited to human individuals of different ages such as children and adults.

[0079] The term "brain-gut axis-related diseases" refers to a series of diseases that involve changes in brain-gut axis function during the course of the disease, including but not limited to gastrointestinal tract-related diseases, nervous system-related diseases, and inflammation-related diseases. Related studies have shown that the brain-gut axis is the genetic basis of some gastrointestinal diseases, such as inflammatory bowel disease (IBD), irritable bowel syndrome (IBS), peptic ulcer disease (PUD), and gastroesophageal reflux disease (GERD). Disruption of the bidirectional interaction in the brain-gut axis is also associated with acute or chronic gastrointestinal disease states. [4,5] In addition, a large amount of clinical evidence also shows that intestinal microorganisms affect the development and aging of healthy nerves and are a key susceptibility factor for neurological diseases (such as Alzheimer's disease, autism, multiple sclerosis, Parkinson's disease, stroke, etc.) [6] In addition, inflammatory signals can also be transmitted bidirectionally through the brain-gut axis, thereby regulating the physiological behaviors and pathological changes of inflammatory-related diseases. [7] wait.

[0080] The term "neurodegenerative disease" refers to diseases caused by the gradual degeneration of spinal cord and brain neurons, including acute neurodegenerative diseases and chronic neurodegenerative diseases. The former include cerebral ischemia (CI), brain injury (BI), epilepsy, etc.; the latter include Alzheimer's disease (AD), Parkinson's disease (PD), multiple sclerosis (MS), etc.

[0081] The term "intestinal permeability" is a metric characteristic of the intestinal barrier. Intestinal permeability refers to the process by which molecules pass through the intestinal epithelial cell layer by non-carrier-dependent diffusion. It is a barrier function closely related to the intestinal commensal flora and the mucosal immune system. Changes in intestinal permeability can cause intestinal antigens to disproportionately penetrate the intestinal mucosa and cause immune disorders, leading to gastrointestinal diseases or immune system diseases, such as inflammatory bowel disease, irritable bowel syndrome, obesity, non-alcoholic fatty liver disease, type 2 diabetes, cardiovascular disease, cancer, etc. Changes in intestinal permeability caused by intestinal mucosal damage are a characteristic of inflammatory bowel diseases including Crohn's disease and ulcerative colitis. [8,9] , while the increased intestinal permeability in patients with irritable bowel syndrome is related to immune activation, inflammatory factors, etc., and reversely increases intestinal permeability

[10] The pathophysiological changes of obesity and related metabolic diseases, such as non-alcoholic fatty liver disease, type 2 diabetes, cardiovascular disease, etc., are associated with changes in intestinal permeability.

[11] Other cancers, such as colon cancer, have also been shown to be closely related to changes in intestinal permeability. Therefore, by increasing or decreasing intestinal permeability, the occurrence and development of the above-mentioned intestinal permeability diseases can be promoted or alleviated.

[0082] The term "inflammation" refers to the vascular-centered defensive response of vascularized tissues to the stimulation of inflammatory factors, including biological factors, physicochemical factors, abnormal immune responses, necrotic tissue, foreign bodies, etc. Correspondingly, inflammatory diseases refer to diseases that contain inflammation in the pathogenic factors and disease processes, including but not limited to neuroinflammation, inflammatory arthritis, allergies (such as food allergies), anxiety, depression, etc.

[0083] The term "treatment" includes, but is not limited to, preventing the recurrence of a disease, alleviating one or more symptoms of a disease, changing any one or several pathological manifestations of a disease, reducing or avoiding the progression and metastasis of a disease, improving or alleviating the state of a disease, optimizing the prognosis of a disease, etc. Correspondingly, the term "prevention" refers to preventing the occurrence of a disease.

[0084] In some embodiments, the intestinal flora composition is derived from a healthy adult, such as an adult male, an adult female, etc., and the age of the healthy adult can be, for example, 18 to 80 years old. In some embodiments, the intestinal flora composition is derived from feces of a healthy adult.

[0085] In some embodiments, the method of obtaining the intestinal flora composition through feces includes but is not limited to the following steps: taking feces from healthy adults, homogenizing and filtering, and the obtained filtrate is made into an intestinal flora composition. In some embodiments, the homogenization method includes mixing the feces with a solution so that the feces is fully dispersed in the solution to form a suspension. In some embodiments, the solution used for homogenization is a common physiological solution, for example, it can be physiological saline, physiological buffer (such as phosphate buffer), etc. In some embodiments, filtering includes filtering with a filter device with a pore size of 60 to 80 μm, and the filter device can be any filter device known in the art that meets the pore size requirements, such as a filter screen, a filter, etc. In some embodiments, after filtering through a filter device with a pore size of 60 to 80 μm, it also includes filtering other substances with smaller particles through a filter device with a smaller pore size, for example, it can be a filter device with a pore size of 10 to 50 μm for secondary or more filtration. In some embodiments, the filtrate is used as an intestinal flora composition, or the intestinal flora composition is made by any one or more of the following methods, such as concentration, dilution, drying, and redissolution. In some embodiments, the intestinal flora composition can be stored for different time periods under different temperature conditions within a temperature range of -196° C. to 4° C. In some embodiments, when the intestinal flora composition is stored in liquid form, a certain amount of protective agent, such as glycerol, is also added.

[0086] In some embodiments, the homogenized feces solution contains glycerol, for example, it may contain 10 to 30% glycerol by mass volume.

[0087] In some embodiments, the intestinal flora composition includes at least one of the following species:

[0088] Bacteroides, Faecalibacterium, Megasphaera, Bifidobacterium, unclassified_Muribaculaceae, Megamonas, Dialister, Coprococcus, Agathobacter, Prevotella_9, unclassified_Lachnospiraceae, Ruminococcus, Phascolarctobacterium, Lachnoclostridium, Parabacteroides, Roseburia Roseburia, Lachnospiraceae_NK4A136_group, Blautia, Lachnospiraceae_UCG_010, Odoribacter, unclassified_Christensenellaceae, Collinsella, Alloprevotella, uncultured_Bacteroidales_bacterium, unclassified_Clostridia_vadinBB60_group, Ligilactobacillus, Enterorhabdus.

[0089] In some embodiments, the intestinal flora composition includes bacterial species of at least two of the above-mentioned genera. In some embodiments, different bacterial species have different abundances among all bacterial species in the intestinal flora composition.

[0090] In some embodiments, the abundance of Bacteroides and Faecalibacterium in the bacterial composition is 10-15% respectively;

[0091] The abundance of Megamonas in the bacterial community composition ranged from 5 to 10%;

[0092] The abundance of Bifidobacterium, Megasphaera, and Dialisterella in the bacterial composition was 3 to 8%, respectively;

[0093] The abundance of the genera Coprococcus, Agarobacterium, Prevotella-9, Ruminococcus, Phascolarctobacterium, and Lachnoclostridium in the bacterial community composition ranged from 1 to 4%, respectively;

[0094] The abundance of Parabacteroides in the bacterial composition is 0.5-2%.

[0095] The "abundance" of a bacterial species or a certain genus refers to the ratio of the number of organisms belonging to the bacterial species or a certain genus sequenced in the sequencing library obtained from the fecal sample to the total number of organisms of all microorganisms in the sample. The specific sequencing type may be, for example, 16S rDNA sequencing.

[0096] In some embodiments, the microbiota composition is administered in the form of a pharmaceutical composition. The pharmaceutical composition includes at least one of pharmaceutically acceptable excipients including but not limited to carriers, diluents, stabilizers, filler solvents, preservatives, pH regulators, and the like.

[0097] In some embodiments, the dosage form of the composition includes but is not limited to liquid preparations, solid preparations, etc. The specific dosage form of the liquid preparation can be an injection, a solution, etc. The specific dosage form of the solid preparation can be a capsule, a tablet, etc. In some embodiments, the use of the composition includes but is not limited to medicines, health products, etc.

[0098] In some embodiments, the intestinal flora composition can be used in food, fermentation agent, food additive, dietary supplement, etc., so that the intake of these intestinal flora compositions can play a certain preventive role on brain-gut axis related diseases of animals.

[0099] In some embodiments, the administration method of the intestinal flora composition or pharmaceutical composition includes but is not limited to the upper gastrointestinal route and the lower gastrointestinal route. The upper gastrointestinal route includes but is not limited to oral administration; the lower gastrointestinal route includes but is not limited to colon catheterization, anal retention enema, etc. Relevant evidence shows that there is no statistically significant difference between the two routes in terms of the effectiveness and safety of the intestinal flora composition in colonization of the user's intestines. [12,13] Therefore, the acceptable pharmaceutical practice can be used according to different administration methods. [14,15] The pharmaceutical composition is prepared as a basis.

[0100] The above dosage regimen will vary depending on known factors such as the pharmacokinetic properties of the specific agent and its mode and route of administration, the species, age, sex, health status, medical status and weight of the subject, the nature and extent of the symptoms, the type of concurrent treatment, the frequency of administration, the route of administration, the patient's renal and liver function, and the desired therapeutic effect. A physician or veterinarian can prescribe an effective amount of the drug as needed to prevent, treat or inhibit the progression of a disease state.

[0101] In these pharmaceutical compositions, the active ingredient will usually be present in an amount of about 0.01 to 95% by weight based on the total weight of the composition.

[0102] In some embodiments, the intestinal flora composition is generally prepared by selecting suitable excipients and mixing them evenly according to different dosage forms and in accordance with conventional pharmaceutical operating procedures. For example, in capsules, the active ingredient can be combined with one or more pharmaceutically acceptable excipients such as but not limited to starch, sucrose, methylcellulose, etc. In liquid preparations for oral administration, the excipient components can be combined with any pharmaceutically acceptable excipients or carriers such as but not limited to ethylene glycol, water, etc. One or more combinations. If necessary, suitable excipients such as suitable flavoring agents can also be added to improve medication compliance. Suitable flavoring agents are such as but not limited to glucose, corn sweeteners, etc.

[0103] Suitable pharmaceutical excipients are listed in the book

[15] This reference is a standard reference text recognized by everyone in the field and is well known to professionals in pharmacy or related fields.

[0104] The present application also relates to a method for preventing or treating a brain-gut axis-related disease in an animal, comprising administering an effective dose of the aforementioned intestinal flora composition to the animal. The present application is described below in conjunction with specific examples.

[0105] Abbreviations

[0106] abbreviation Abbreviation abbreviation Abbreviation AD Alzheimer's disease HE staining Hematoxylin-eosin staining Aβ Beta-amyloid μm Micrometer IBA-1 Ionized calcium binding adaptor molecule 1 FMT Fecal microbiota transplantation GFAP glial fibrillary acid protein IHC Immunohistochemistry mg mg mL ml h Hour ℃ Celsius

[0107] References

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[0111] [4]Gong W,Guo P,Li Y,et al.Role of the Gut-Brain Axis in the SharedGenetic Etiology Between Gastrointestinal Tract Diseases and PsychiatricDisorders:A Genome-Wide Pleiotropic Analysis[J].JAMA Psychiatry,2023,80(4):360-370.

[0112] [5]Rhee S H,Pothoulakis C,Mayer E A.Principles and clinicalimplications of the brain-gut-enteric microbiota axis[J].Nat RevGastroenterol Hepatol,2009,6(5):306-14.

[0113] [6]Cryan J F,O'riordan K J,Sandhu K,et al.The gut microbiome inneurological disorders[J].Lancet Neurol,2020,19(2):179-194.

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[0123] Example 1: Microbial Extraction

[0124] After informed consent, fresh feces from healthy adults with no history of illness in the past three years were collected and immediately transferred to sterile phosphate-buffered saline (PBS) containing 20% ​​glycerol (w / v) for homogenization and filtered through a 70 μm filter to remove large particles to prepare the experimental bacterial solution. The prepared bacterial solution was sampled for pathogen detection, cell count and bacterial flora sequencing. The pathogen detection of Clostridium difficile, Campylobacter, Salmonella, Shigella, Shiga toxin-producing Escherichia coli, and eggs, vesicles, parasites, spores, norovirus, rotavirus and COVID-19 in the bacterial solution was negative, and the multidrug resistance gene extended-spectrum β-lactamase and carbapenemase were negative. The number of viable cells in 100 mL of bacterial solution should be no less than 4.5×10 12 After sampling, the bacterial solution was aliquoted and frozen at -80℃.

[0125] The prepared bacterial liquid was subjected to microbial 16S rDNA sequencing. Through analysis of the sequencing results, the distribution of bacterial community combinations is shown in Table 1 below. The total abundance of bacterial community combinations accounts for more than 90%.

[0126] Table 1. Results of 16S rDNA sequencing of bacterial culture

[0127]

[0128]

[0129] Example 2: Mouse behavioral experiment

[0130] According to the standard operating procedures of intestinal flora transplantation experiment, the experimental design grouping is as follows Figure 1 As shown, 4-week-old 3×Tg mice (AD mice) were divided into a control group (3×Tg, n=30) and an experimental group (Y-FMT, n=30). The mice were subjected to FMT from 4 weeks after birth. In the first week of transplantation, the bacterial solution in Example 1 was given to 3×Tg mice by oral gavage for 6 consecutive days, and FMT was performed 4 times a week starting from the second week. The control group mice were given the same volume of sterile PBS. FMT was performed for a total of 8 weeks.

[0131] Compared with the control group, the spontaneous alternation rate of mice in the experimental group ( Figure 2 A) and total arm entry times increased significantly, about 20% to 31% and 25% to 33% ( Figure 2 B in the figure); the time spent on finding the platform is shorter, about 21% to 39% ( Figure 2 C); and after the platform is removed, the number of times the original platform is crossed also increases significantly ( Figure 2 D), about 95% to 98%. This indicates that FMT treatment can alleviate the impairment of spatial learning ability in 3×Tg mice.

[0132] Example 3: Sample collection from mouse dissection in Example 2

[0133] According to the standard operating procedure for mouse brain dissection, the mice in the control group and experimental group described in Example 2 were killed and their brain tissues and colon tissues were collected and stored at -80°C.

[0134] Example 4: Pathological detection of mouse brain

[0135] The brain tissues of the mice described in Example 3 were used to detect AD-related biochemical indicators and glial cells using IHC technology.

[0136] Compared with the control group, the area of ​​Aβ in the hippocampus of the experimental group mice ( Figure 3 A and B) and the area of ​​p-Tau181 ( Figure 3 C and D) were significantly reduced to about 79% to 87% and 71.4% to 76.2%, indicating that FMT treatment can reduce Aβ deposition in 3×Tg mice and inhibit Tau hyperphosphorylation; similarly, IBA-1 positive cells ( Figure 4 A and B) and GFAP-positive cells ( Figure 4 The numbers of C and D in the figure were significantly reduced, by about 63% to 71% and 42% to 52%, respectively, indicating that FMT treatment can also improve neuroinflammation in the brain of 3×Tg mice.

[0137] Example 5: Colon tissue detection in mice

[0138] The colon tissues of the mice described in Example 3 were used to detect colon epithelial tissue pathology using HE staining technology.

[0139] Compared with the control group, the experimental group showed significant improvement in colonic inflammatory infiltration, decreased and absent crypts, goblet cell loss, and epithelial damage, and the H&E histological score was significantly lower than that of the control group, about 79% to 87% ( Figure 5 In conclusion, FMT treatment improved colon damage in 3×Tg mice.

[0140] Example 6: Effect of microbiota transplantation on the composition of intestinal microbiota in 3×Tg mice

[0141] Referring to the flora collection method of Example 1, the intestinal flora of the mice described in Example 2 was collected.

[0142] After extracting the total DNA of the sample, primers are designed according to the conserved region, and sequencing adapters are added to the ends of the primers for PCR amplification. The products are purified, quantified and normalized to form a sequencing library. The constructed library is first subjected to library quality inspection, and the library that passes the quality inspection is sequenced. The original image data file obtained by high-throughput sequencing is converted into the original sequencing sequence through base recognition analysis. The test results at least include the sequence information of the sequencing sequence and its corresponding sequencing quality information.

[0143] Microbiota analysis was performed for the experimental and control groups. Using discriminant analysis of LEfSe, FMT treatment increased the microbial α diversity of 3×Tg mice compared with the control group ( Figure 6 ).

[0144] Example 7: Capsule Preparation

[0145] 90 mL of preparation auxiliary liquid was added to the intestinal flora composition prepared in Example 1, and each 10 mL of preparation auxiliary liquid contained the following components in mass percentage: 3-5% glucose, 2-5% vitamin C, 10-13% skimmed milk powder, and the balance was normal saline; a magnetic stirrer was used to stir and mix, and after mixing, freeze-dried using a two-step method, in which the first step was freezing at a rate of 6-12°C / h, from 25°C to -80°C, and the total pre-freezing time was 24h, and the second step was heating at rates of 12°C / h, 10°C / h, and 8°C / h, and the temperature was increased and dried for 24h, 10h, and 2h, respectively. According to the method, 480 mg of intestinal flora freeze-dried powder was prepared.

[0146] 480 mg of the freeze-dried powder of intestinal flora prepared above was evenly divided into 1000 inner capsules, and the inner capsules were respectively filled into No. 00 capsules and sealed to obtain 1000 double-layer intestinal flora capsules.

[0147] The present application is described in detail above in conjunction with the embodiments, but the present application is not limited to the above embodiments, and various changes can be made within the knowledge of ordinary technicians in the relevant technical field without departing from the purpose of the present application. In addition, the embodiments of the present application and the features in the embodiments can be combined with each other without conflict.

Claims

1. Application of the intestinal flora composition in the following A1) to A4): A1) preparing a medicament for preventing brain-gut axis related diseases in animals; A2) preparing a medicament for treating a brain-gut axis-related disease in an animal; A3) Prevent brain-gut axis-related diseases in animals; A4) Treating brain-gut axis related diseases in animals.

2. The use according to claim 1, characterized in that: The brain-gut axis related diseases include at least one of neurodegenerative diseases, intestinal permeability diseases, and inflammatory diseases; Optionally, the neurodegenerative disease includes at least one of Alzheimer's disease, Parkinson's disease, and multiple sclerosis; Optionally, the intestinal permeability disease includes but is not limited to at least one of inflammatory bowel disease, irritable bowel syndrome, obesity, non-alcoholic fatty liver disease, type 2 diabetes, cardiovascular disease, and cancer; Optionally, the inflammatory disease includes at least one of neurological inflammation, inflammatory arthritis, allergy, anxiety, and depression.

3. The use according to claim 1, characterized in that: The intestinal flora composition is derived from healthy adults; Optionally, the intestinal flora composition is derived from feces of healthy adults; Optionally, the intestinal flora composition is prepared by the following method: Taking feces from healthy adults, homogenizing it in a phosphate buffer, and filtering it through a filter device with a pore size of 60 to 80 μm, and the obtained filtrate is used to prepare the intestinal flora composition; Optionally, the phosphate buffer contains 10-30% glycerol by mass volume.

4. The use according to claim 1, characterized in that: The intestinal flora composition includes the following species: Bacteroides, Faecalibacterium, Megasphaera, Bifidobacterium, unclassified_Muribaculaceae, Megamonas, Dialister, Coprococcus, Agathobacter, Prevotella_9, unclassified_Lachnospiraceae, Ruminococcus, Phascolarctobacterium, Lachnoclostridium, Parabacteroides, Roseburia Roseburia, Lachnospiraceae_NK4A136_group, Blautia, Lachnospiraceae_UCG_010, Odoribacter, unclassified_Christensenellaceae, Collinsella, Alloprevotella, uncultured_Bacteroidales_bacterium, unclassified_Clostridia_vadinBB60_group, Ligilactobacillus, Enterorhabdus; Optionally, the abundance of the genus Bacteroides in the bacterial composition is 10-15%; Optionally, the abundance of Faecalibacterium in the bacterial composition is 10-15%; Optionally, the abundance of the genus Megamonas in the bacterial composition is 5-10%; Optionally, the abundance of the genus Bifidobacterium in the bacterial composition is 3-8%; Optionally, the abundance of the genus Megasphaera in the bacterial composition is 3-8%; Optionally, the abundance of the genus Dialister in the bacterial composition is 3-8%; Optionally, the abundance of the genus Coprococcus in the bacterial composition is 1-4%; Optionally, the abundance of the genus Agarobacter in the bacterial composition is 1-4%; Optionally, the abundance of the genus Prevotella-9 in the bacterial composition is 1-4%; Optionally, the abundance of the Ruminococcus genus in the bacterial composition is 1-4%; Optionally, the abundance of Phascolarctobacterium in the bacterial composition is 1-4%; Optionally, the abundance of the genus Lachnoclostridium in the bacterial composition is 1-4%; Optionally, the abundance of the genus Parabacteroides in the bacterial composition is 0.5-2%.

5. The use according to claim 1, characterized in that: The treatment of brain-gut axis related diseases includes the treatment of Alzheimer's disease, and the treatment of Alzheimer's disease includes at least one of improving behavioral abnormalities, reducing Aβ levels, reducing p-Tau protein levels, improving neuroinflammation, and improving intestinal permeability.

6. The use according to claim 1, characterized in that: The bacterial flora composition is administered in the form of a pharmaceutical composition, and the pharmaceutical composition further comprises a pharmaceutically acceptable excipient; Optionally, the pharmaceutically acceptable excipients include at least one of a carrier, a diluent, a stabilizer, a filler solvent, a preservative, and a pH adjuster; Optionally, the pharmaceutical composition is any one of a liquid preparation and a solid preparation; Optionally, the administration method includes at least one of oral administration, colon catheterization, and transanal retention enema.

7. The use according to claim 1, characterized in that: The animals include mammals; Optionally, the animal comprises a primate; Optionally, the animal comprises a human.

8. A method for preventing or treating a brain-gut axis-related disease in an animal, characterized in that: The method comprises administering an effective dose of an intestinal flora composition to the animal; Optionally, the intestinal flora composition is derived from healthy adults; Optionally, the intestinal flora composition is derived from feces of healthy adults; Optionally, the intestinal flora composition is prepared by the following method: Taking feces from healthy adults, homogenizing it in a phosphate buffer, and filtering it through a filter device with a pore size of 60 to 80 μm, and the obtained filtrate is used to prepare the intestinal flora composition; Optionally, the phosphate buffer contains 10 to 30% glycerol by mass volume; Optionally, the administration method includes but is not limited to at least one of oral administration, colon catheterization, and transanal retention enema; Optionally, the animal comprises a mammal; Optionally, the animal comprises a primate; Optionally, the animal includes a human; Optionally, the brain-gut axis related disease includes at least one of a neurodegenerative disease, an intestinal permeability disease, and inflammation; Optionally, the neurodegenerative disease includes at least one of Alzheimer's disease, Parkinson's disease, and multiple sclerosis; Optionally, the intestinal permeability disease includes but is not limited to at least one of inflammatory bowel disease, irritable bowel syndrome, obesity, non-alcoholic fatty liver disease, type 2 diabetes, cardiovascular disease, and cancer; Optionally, the inflammatory disease includes at least one of neurological inflammation, inflammatory arthritis, allergy, anxiety, and depression.

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