JP2015000001A - Method for producing irritable bowel syndrome model animal - Google Patents

Abstract

PROBLEM TO BE SOLVED: To produce a method for producing an irritable bowel syndrome diarrhea model animal in which chronic diarrhea is induced.SOLUTION: The present inventors take aim at the fact that abnormal proliferation of enterobacteria is caused by the reduced immunity and the abnormal proliferation of enterobacteria is related to the onset of an irritable bowel syndrome diarrhea type. From this, the fact that chronic diarrhea is induced by administering an immunosuppressive drug to an animal is found, and the present invention is completed.

Description

  The present invention relates to a method for producing a diarrhea-type irritable bowel syndrome model animal and a propionic acid bacterium effective for the treatment of diarrhea-type irritable bowel syndrome.

Functional gastrointestinal disorders (FGIDs) are a group of gastrointestinal disorders that do not have organic disorders. Abnormal functions such as gastrointestinal motility and perception cause various symptoms such as abdominal pain, nausea, vomiting, abdominal bloating, and defecation abnormalities such as diarrhea and constipation.
Irritable bowel syndrome (IBS), which is included in FGIDs, is caused by a malfunction of the lower gastrointestinal tract, and its pathophysiology is known to be an intestinal motility abnormality. Symptoms include abdominal pain and abdominal discomfort (characterized by relief from defecation), bowel movement abnormalities (diarrhea, constipation, mixed diarrhea / constipation), fecal abnormalities (mixed mucus, abnormal hardness) Abdominal fullness is observed. In the diarrhea type, the intestinal transit time is shortened, and in the constipation type, the intestinal transit time is prolonged.
Conventionally, stress has been a cause of IBS, but in recent years, disturbance of the intestinal bacterial flora has also attracted attention as a cause of IBS (Non-Patent Documents 1 to 6).

  As the IBS diarrhea type disease model animal, fear-conditioned stress-induced defecation enhancement model, restraint stress-induced defecation enhancement model, serotonin-induced diarrhea model (Patent Document 1), castor oil-induced diarrhea and the like are known. However, there is a further need for a disease state animal model that can reproduce chronic diarrhea as observed in actual IBS.

JP 2012-140415 A

DuPont A.W. and DuPont H.L., Nat Rev Gastroenterol Hepatol 8, 523-531,2011 Al-Khatib K. and Lin H.C., Gut and Liver 3 (1), 14-19, 2009 Posserud I. et al, Gut 56, 802-808, 2007 Lee H.R. and Pimentel M., Curr Gastroenterol Rep 8 (4), 305-311, 2006 Van Citters G.W. and Lin H.C., Curr Gastroenterol Rep 7 (4), 317-320,2005 Malinen E., et al, Am J Gastroenterol 100 (2), 373-382, 2005

  An object of the present invention is to provide a method for producing an irritable bowel syndrome diarrhea model animal in which chronic diarrhea is induced.

  The inventors of the present invention focused on the abnormal growth of intestinal bacteria due to decreased immunity, and the abnormal growth of intestinal bacteria leads to the development of irritable bowel syndrome diarrhea type. From this, it was found that chronic diarrhea is induced by administering an immunosuppressive agent to animals, and the present invention was completed.

  Specifically, in an in vivo test using mice, diarrhea is induced after administration of an immunosuppressant, and further a decrease in intestinal barrier function, a decrease or breakdown of immune function, an increase in the total number of enteric bacteria, and Disturbances in the internal bacterial flora were observed. It has been reported that many of these phenomena are characteristically seen in human irritable bowel syndrome diarrhea type patients (Non-Patent Documents 1 to 6). Therefore, an irritable bowel syndrome diarrhea model animal in which chronic diarrhea was induced by the method of the present invention could be produced. Moreover, the material which can improve the pathological condition of irritable bowel syndrome diarrhea type | mold was also able to be found by the method of this invention. Furthermore, the method of the present invention can also produce a model animal of diarrhea symptoms that occur in a state in which immunity is suppressed (such as an immunodeficiency disease).

That is, the present invention
[1] A method for inducing chronic diarrhea, comprising administering an immunosuppressive agent to a non-human mammal,
[2] The method according to [1] above, wherein the immunosuppressive agent is at least one selected from the group consisting of alkylating agents, antimetabolites, and calcineurin inhibitors.
[3] The method according to [2] above, wherein the immunosuppressive agent is a calcineurin inhibitor,
[4] The method according to [3] above, wherein the immunosuppressive agent is tacrolimus,
[5] The method according to any one of [1] to [4] above, wherein the dose of the immunosuppressant per day is 0.6 to 50 mg / kg body weight.
[6] The method according to any one of [1] to [5] above, wherein the administration period of the immunosuppressive agent is continuous 5 to 25 days,
[7] The method according to any one of [1] to [6], wherein the non-human mammal is a rodent or a rabbit.
[8] The method according to [7] above, wherein the non-human mammal is a mouse,
[9] An irritable bowel syndrome diarrhea model animal obtained by the method according to any one of [1] to [8], wherein chronic diarrhea is induced,
[10] Production of irritable bowel syndrome diarrhea model animal comprising obtaining a non-human mammal in which chronic diarrhea has been induced by the method according to any one of [1] to [8] above Method,
[11] An irritable bowel syndrome diarrhea model animal is prepared by the method described in [10], a test substance is administered to the irritable bowel syndrome diarrhea model animal, and diarrhea symptoms, intestinal motility in the animal are further administered. A method for screening a substance that promotes or inhibits irritable bowel syndrome diarrhea, characterized by evaluating at least one selected from the group consisting of sex and visceral hyperalgesia,
[12] The screening method according to [11] above, which is a screening method for a prophylactic and / or therapeutic agent for irritable bowel syndrome diarrhea type,
[13] The screening method according to [11] above, which is a screening method for foods for preventing and / or treating irritable bowel syndrome diarrhea type,
[14] A model animal of diarrhea due to an immunodeficiency disease, which is obtained by the method according to any one of [1] to [8], wherein chronic diarrhea is induced,
[15] A model animal of diarrhea due to an immunodeficiency disease, comprising obtaining a non-human mammal in which chronic diarrhea has been induced, obtained by the method according to any one of [1] to [8] above Production method,
[16] Propionibacterium freudenreichii ET-3 strain (accession number: FERM) that suppresses chronic diarrhea induced by administration of immunosuppressants
BP-8115),
[17] Propionibacterium freudenreichii ET-3 strain (accession number: FERM BP-8115), which suppresses irritable bowel syndrome diarrhea type,
[18] Propionibacterium freudenreichii ET-3 strain (accession number: FERM BP-8115), which suppresses diarrhea caused by an immunodeficiency disease.

  By the method of the present invention, a model animal of IBS diarrhea type in which chronic diarrhea has been induced can be produced. Moreover, an effective material can be screened by prevention / treatment of IBS diarrhea type using the obtained model animal.

In Example 1, the result of having evaluated the property of feces. The graph represents mean ± SE (n = 5) of fecal score. **: p <0.01 vs. control group (Student's t-test). In Example 1, the result of having measured the blood LPS density | concentration and IL-6 density | concentration. The graph represents mean ± SE (n = 5). *: P <0.05 vs. control group (Student's t-test). In Example 2, the result of having evaluated the property of feces. The graph represents mean ± SE (n = 5) of fecal score. **: p <0.01 vs. control group (Student's t-test). In Example 2, the result of having measured the blood cytokine density | concentration is shown. The graph represents mean ± SE (n = 5). #: P <0.1, *: p <0.05 (Student's t-test). In Example 2, the result of having analyzed the immune cell population of the spleen is shown. The graph represents the abundance ratio mean ± SE (n = 5). *: P <0.05 (Student's t-test). In Example 2, the result of having measured the blood LPS density | concentration is shown. The graph represents mean ± SE (n = 5). *: P <0.05 (Student's t-test). In Example 2, the result of having detected Bacterial Translocation is shown. The graph represents the positive rate of Bacterial Translocation. In Example 2, the result of having measured the gene expression level of occludin in an ileal tissue is shown. The graph represents mean ± SE (n = 5) for the expression level of occludin mRNA against GAPDH. *: P <0.05 (Student's t-test). In Example 3, the result of having evaluated the property of feces. The graph represents mean ± SD (n = 7) of fecal score. **: p <0.01 vs. control group (Student's t-test). In Example 3, the result of having analyzed the intestinal microflora with the next-generation sequencer. The graph represents mean ± SD (n = 7). *: P <0.05, **: p <0.01 (Student's t-test). In Example 3, the result of having analyzed the intestinal microflora by q-PCR. The graph represents mean ± SD (n = 7). *: P <0.05, **: p <0.01 (Student's t-test). In Example 3, the result of measuring sIgA of cecal contents. The graph represents mean ± SE (n = 7). *: P <0.05 (Student's t-test). In Example 3, the result of having measured the gene expression level of Reg3 (gamma) in an ileum tissue is shown. The graph represents mean ± SE (n = 7) for the mRNA expression level of Reg3γ with respect to GAPDH. *: P <0.05 (Student's t-test). In Example 3, the result of having measured the amount of occludin protein of an ileal tissue is shown. The graph represents mean ± SE (n = 7) for the amount of occludin protein relative to β-actin. The result of measuring granulysin concentration in blood in Example 3. It represents mean ± SE (n = 7). *: P <0.05, **: p <0.01 (Student's t-test).

  Hereinafter, the present invention will be described in detail. However, the present invention is not limited to the following preferred embodiments, and can be freely changed within the scope of the present invention.

  The present invention relates to a method for inducing chronic diarrhea, which comprises administering an immunosuppressive agent to a non-human mammal. The present invention also relates to an irritable bowel syndrome diarrhea model animal in which chronic diarrhea is induced by the above method. Alternatively, the present invention relates to a method for producing an irritable bowel syndrome diarrhea model animal in which chronic diarrhea is induced by the above method. Then, the present invention creates an irritable bowel syndrome diarrhea model animal in which chronic diarrhea has been induced by the above-described method, and administers a test substance to the irritable bowel syndrome diarrhea model animal and evaluates it. The present invention relates to a screening method for a substance that promotes or suppresses diarrhea type of irritable bowel syndrome.

  Immunosuppressants have the effect of suppressing the function of the immune system, and are used for suppressing rejection of transplanted organs, treating autoimmune diseases, treating inflammatory diseases, and the like. Immunosuppressive agents can be classified into alkylating agents (such as cyclophosphamide), antimetabolites (such as azathioprine), calcineurin inhibitors (such as FK506, cyclosporine) and the like based on their mechanism of action.

FK506 is one of the immunosuppressants, also known as Tacrolimus and chemical names (3S, 4R, 5S, 8R, 9E, 12S, 14S,
15R, 16S, 18R, 19R, 26aS) -5, 19-Dihydroxy-3-{(1E) -2-[(1R, 3R, 4R) -4-hydroxy-3-methoxycyclohexyl] -1-methylethenyl} -14 ,
16-dimethoxy-4, 10, 12,18-tetramethyl-8- (prop-2-en-1-yl) -15, 19-epoxy-5, 6,
8, 11, 12, 13, 14, 15, 16, 17, 18, 19,24, 25, 26, 26a-hexadecahydro-3H-pyrido [2,
1-c] [1, 4] oxaazacyclotricosine-1, 7, 20, 21 (4H, 23H) -tetrone monohydrate. Cas of FK506
No. is 104987-11-3.
FK506 forms a complex with FK506 binding protein in helper T cells and inhibits activation of calcineurin. In this way, the activation of T cells (especially helper T cells) is strongly inhibited to suppress rejection of organ transplants, and cytokine production (IL-12, IFNγ) from T cells is suppressed to treat ulcerative colitis. Has been applied. One of the side effects of FK506 is severe diarrhea.

It is known that intestinal bacteria overgrow when immunity decreases after enteritis. And it has been reported that abnormal growth of intestinal bacteria leads to the onset of irritable bowel syndrome. It is also known that malnutrition and intestinal mucosa damage occur when intestinal bacteria grow abnormally. Furthermore, there is a possibility that the abnormally grown intestinal bacteria may migrate to the small intestine where the intestinal bacteria are not normally present.
When immunity decreases, the bacteria that make up the intestinal flora also change, and the intestinal flora worsens. At this time, if the intestinal barrier function breaks down due to stress or damage to the intestinal mucosa, pathogenic bacteria etc.
Translocation is easier and the risk of infection is higher.

In the method of the present invention, chronic diarrhea can be induced by administering an immunosuppressive agent to a non-human mammal. When the inventors administered FK506 to mice, induction of diarrhea, decreased intestinal barrier function, increased blood IL-6 concentration, decreased blood Th1-type cytokines (IL-12, IFNγ), number of helper T cells Decrease, intestinal total bacteria count, intestinal Lactobacillus occupancy reduction, intestinal Bacteroides
Increased fragilis group, decreased ileal Reg3γ level and decreased blood granulysin level were observed.
This result indicates a decrease in immune function, a decrease in intestinal barrier function, and a deterioration in the intestinal bacterial flora caused by administration of FK506. As a result, it is considered that intestinal motility was enhanced and diarrhea occurred. In addition, it has been reported that many of these phenomena are characteristically seen in human irritable bowel syndrome diarrhea type patients (Non-Patent Documents 1 to 6). Therefore, it can be said that an irritable bowel syndrome diarrhea model animal in which chronic diarrhea was induced by the method of the present invention could be produced.

  The method of the present invention comprises the step of administering an immunosuppressive agent to a non-human mammal. Non-human mammals that can be used in the present invention include those generally used as laboratory animals such as rodents (rats, mice, guinea pigs, etc.), rabbits, dogs, etc. It is not limited.

  The immunosuppressant used in the method of the present invention can be appropriately selected depending on the type of non-human mammal used, the degree of the pathological condition to be evaluated, the administration route, and the like. Specific examples of the immunosuppressant used in the method of the present invention include alkylating agents (such as cyclophosphamide), antimetabolites (such as azathioprine), calcineurin inhibitors (such as FK506, cyclosporine), and the like. However, it is not limited to these examples. Any immunosuppressive agent may be used as long as it suppresses the immune function, and one type or a plurality of types of immunosuppressive agents may be used in combination.

In the method of the present invention, the dose, administration frequency, and administration period of the immunosuppressive agent should be appropriately adjusted depending on the type of immunosuppressive agent, the type of non-human mammal used, the degree of the pathological condition to be evaluated, the administration route, etc. Can do. For example, in the examples described below, using ICR mice, FK506 is 1 mg / kg.
When administered intraperitoneally at a body weight dose of once a day for 14 days, diarrhea symptoms suddenly worsened from the fourth day of administration, and the symptoms worsened as the number of administration days increased. Furthermore, on the 14th day after administration of FK506, the intestinal barrier function decreased, the blood IL-6 concentration increased, the blood Th1-type cytokine (IL-12, IFNγ) decreased, the helper T cell count decreased, the intestinal total Increase in the number of bacteria, decrease in the intestinal Lactobacillus occupancy rate, intestinal Bacteroides
Increased fragilis group, decreased ileal Reg3γ level and decreased blood granulysin level were observed.
In the method of the present invention, the dose of the immunosuppressive agent per day is 0.6 to 50 mg / kg body weight in the case of intraperitoneal administration, preferably 0.8 to 10 mg / kg.
body weight,
More preferably, 0.9-5 mg / kg body weight can be mentioned. Further, examples of the administration period of the immunosuppressive agent include 5 to 25 days, preferably 10 to 21 days. The immunosuppressive agent may be administered continuously or intermittently.
In the method of the present invention, the administration route to be used can be appropriately adjusted depending on the type of non-human mammal used, the degree of the pathological condition to be evaluated, the type of immunosuppressive agent, and the like. For example, any of oral administration, intraperitoneal administration, subcutaneous administration, intramuscular administration, intravenous administration, and the like may be used.

  In the method of the present invention, abnormal growth of intestinal bacteria can also be induced by administering an immunosuppressive agent to a non-human mammal. Examples of the intestine capable of inducing abnormal bacterial growth in the method of the present invention include the small intestine (duodenum, jejunum, ileum) and the large intestine (colon, rectum). Furthermore, by administering an immunosuppressive agent to a non-human mammal, abnormal growth of intestinal bacteria can be induced to produce an irritable bowel syndrome diarrhea model animal.

  In the present invention, chronic diarrhea can be caused by combining other methods in addition to administration of the immunosuppressive agent. For example, combined with known stress loads (fear-conditioned stress-induced defecation model, restraint stress-induced defecation model, etc.), administration of nitric oxide synthase inhibitors (such as L-NAME), administration of dextran sulfate sodium, etc. It is possible.

Furthermore, the present invention produces an irritable bowel syndrome diarrhea model animal in which chronic diarrhea is induced by administering an immunosuppressant to a non-human mammal, and the irritable bowel syndrome diarrhea model animal is tested. The present invention relates to a method for screening a substance that promotes or suppresses irritable bowel syndrome diarrhea, which comprises administering a substance and evaluating the substance.
Irritable bowel syndrome Diarrhea-type pathological conditions include diarrhea symptoms (deterioration of stool properties, increase in stool volume, increase in the number of defecations, etc.), abnormal intestinal motility, and visceral hyperalgesia. In the present invention, examples of items for evaluating substances that promote or inhibit irritable bowel syndrome diarrhea types include diarrhea symptoms, intestinal motility and visceral hyperalgesia, but are not limited to these examples. In the present invention, when at least one of the improvement of diarrhea symptoms, the improvement of intestinal motility and the improvement of visceral hyperalgesia is satisfied by administering the test substance, the test substance has an irritable bowel syndrome diarrhea type. It can be said that it was suppressed.

  It is known that immunity is suppressed by various factors such as stress, inflammatory diseases, and immunodeficiency diseases. Immunodeficiency diseases are congenital and acquired immunodeficiency diseases (due to drug therapy (immunosuppressants, cancer chemotherapeutic agents, radiation therapy, etc.) and chronic diseases (HIV infection, hepatitis, cancer, etc.) Can be classified. Both cause abnormalities in the immune system, so patients are more susceptible to infections and their resistance to cancer is reduced. Severe diarrhea is known as one of the symptoms of immunodeficiency diseases. The model animal of the present invention can also reproduce diarrhea that occurs in a state in which immunity is suppressed. In addition, the model animal of the present invention can be used to screen for substances that promote or suppress diarrhea that occurs in a state in which immunity is suppressed.

Using the screening method of the present invention, the present inventors have found that Propionibacterium freudenreichii ET-3 strain (hereinafter also referred to as ET-3) has an effect of suppressing irritable bowel syndrome diarrhea type. Specifically, diarrhea symptoms caused by administration of FK506 were improved in mice that were orally administered with live cells of ET-3. In addition, administration of live ET-3 cells restored intestinal barrier function, restored blood Th1-type cytokine levels, improved intestinal flora, improved production of endogenous antimicrobial peptides, and restored innate immune function. The effect was obtained.
The inventors deposited the Propionibacterium freudenreichii ET-3 strain at the Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology. The contents specifying the deposit are described below.
(1) Depositary name: National Institute of Advanced Industrial Science and Technology, Patent Biological Depositary Center (2) Contact: Chuo 6th 1-1 1-1 Higashi 1-chome Tsukuba City, Ibaraki Prefecture 305-8566
(3) Accession number: FERM BP-8115
(4) Display for identification: ET-3
(5) Date of original deposit: August 9, 2001 (6) Date of transfer to deposit under the Budapest Treaty: July 11, 2002 The above Propionibacterium freudenreichii ET-3 strain was isolated from Emmental cheese It is a positive bacilli and has the ability to produce DHNA (2,4-dihydroxy-2-naphthoicacid) and ACNQ (2-amino-3-carboxy-1,4-naphthoquinone).

  The daily intake of live ET-3 cells varies depending on age, symptoms, body weight, application, route of administration, etc., but is not particularly limited. In the case of oral administration, 0.1 to 10000 mg can be ingested per day for an adult, preferably 1 to 5000 mg, more preferably 10 to 500 mg.

  The live cells of ET-3 can be used in the form of pharmaceuticals or foods and drinks. For example, chronic diarrhea by directly administering the nutritional composition of the present invention as a pharmaceutical or by directly ingesting as a special-purpose food such as a food for specified health use, a nutritional functional food, a nutritional supplement, a liquid food or a supplement, It is expected to improve diarrhea that occurs when immunity is suppressed, or irritable bowel syndrome.

Hereinafter, although an example is given and explained about the present invention, the present invention is not limited by this.

[Example 1: Induction of diarrhea by immunosuppressant]
(Immunosuppressant) FK506 (Tacrolimus)
FK506 (Astellas Pharma) was suspended in physiological saline to adjust the concentration to 20, 40, 80 μg / ml, and this was administered to animals.

(Animal experimentation)
Twenty 5-week-old male ICR mice (CLEA Japan) were individually housed for 1 week on the CRF-1 diet and acclimated to the environment. After the acclimatization period, mice were grouped into 4 groups (control group, 0.5 mg / kg (low dose) administration group, 1.0 mg / kg (medium dose) administration group, and 2.0 mg / kg (high dose) administration group). Groups were divided into groups n = 5). The grouped mice were bred in cages laid with poly filter paper (Whatman bench coat plus) and stainless steel wire mesh (16 mesh, mesh opening 1.2 mm) without using flooring.
From the day of grouping (day 0), the immunosuppressive agents shown in Table 1 were administered to each group for 14 days. In the control group, physiological saline having the same volume as that of the immunosuppressant was administered.
The stool of each mouse was received by poly filter paper through a stainless steel wire mesh, and all of the stool adhered to the wire mesh and the stool adhered to the filter paper through the wire mesh were visually observed every day.

(Measurement)
-Fecal properties: Feces were visually observed on each day of days 1 to 14, and evaluated with the following four grades of fecal properties.
1. Normal flights Soft stool (stool with a high moisture content)
3. Mud stool (stool that does not maintain the shape of the stool and adheres to the gaps in the wire mesh)
4). Watery stool (nearly liquid and passes through a wire mesh)

・ Blood LPS concentration:
Serum is aseptically prepared from day 14 blood, and using Lipopolysaccharide (Endospecy ES-50M set (Seikagaku Biobusiness) and Toxicolor DIA-MP set (Seikagaku Biobusiness) according to the attached protocol. LPS) concentration was measured.
・ Blood cytokine concentration:
Serum was prepared from day 14 blood, and Bio-plex ^TM (registered trademark) Pro Mouse
IL-6 was measured using Cytokine 23-plex Assay, M60-009RDPD (manufactured by BIO-RAD) according to the attached protocol.

(result)
The result regarding the property of feces is shown in FIG. The administration of 1 mg / kg body weight and 2 mg / kg body weight with FK506 induced significant diarrhea symptoms on day 14.
The results regarding the blood LPS concentration and the blood IL-6 concentration are shown in FIG. The group administered 1 mg / kg body weight with FK506 showed significantly higher LPS concentration than the control group.
In addition, blood IL-6 showed a significantly higher IL-6 concentration in the group administered with 2 mg / kg body weight of FK506 than in the control group.

[Example 2: Effectiveness test of propionic acid bacteria]
(Test substance) Viable cells of Propionibacterium freudenreichii ET-3 strain
ET-3 was statically cultured in an GAM medium at 30 ° C. for 72 hours under anaerobic conditions, and collected by centrifugation (8000 × g, 15 minutes). The viable cells washed twice with physiological saline and once with distilled water were suspended in distilled water, immediately stored at −80 ° C. and freeze-dried, and the dry weight of the viable cells was calculated. The dried live cells were suspended in distilled water to a concentration of 6 mg / ml and administered to animals.
(Immunosuppressant) FK506 (Tacrolimus)
FK506 (Wako Pure Chemical Industries) was suspended in physiological saline to prepare a concentration of 40 μg / ml, and this was administered to animals.

(Animal experimentation)
Fifteen 5-week-old male ICR mice (CLEA Japan) were individually housed for 1 week on CRF-1 feed and acclimated to the environment. After the acclimatization period, the mice were divided into three groups (each group n = 5) of control group, FK506 group and FK506 + ET-3 group using body weight as an index. The grouped mice were bred in cages laid with poly filter paper (Whatman bench coat plus) and stainless steel wire mesh (16 mesh, mesh opening 1.2 mm) without using flooring.
From the day of grouping (day 0), the immunosuppressive agents and test substances shown in Table 2 were administered to each group once a day for 14 days. To the group not administered with the immunosuppressant or test substance, physiological saline or distilled water having the same volume as the dose of the immunosuppressant or test substance was administered.
The stool of each mouse was received by poly filter paper through a stainless steel wire mesh, and all of the stool adhered to the wire mesh and the stool adhered to the filter paper through the wire mesh were visually observed every day.
Two weeks after the start of administration of the immunosuppressant and test substance (day 14), the mice were sacrificed after whole blood collection under isoflurane anesthesia. The spleen, mesenteric lymph nodes, cecum, and ileum were removed by dissection.

(Measurement)
・ Fecal properties:
The stool properties of each day from day 1 to 14 were visually observed and evaluated with the following four stool scores.
1. Normal flights Soft stool (stool with a high moisture content)
3. Mud stool (stool that does not maintain the shape of the stool and adheres to the gaps in the wire mesh)
4). Watery stool (nearly liquid and passes through a wire mesh)
・ Blood cytokine concentration:
Serum was prepared from day 14 blood and Bio-plex ^TM Pro Mouse Cytokine 23-plex Assay, M60-009RDPD
(BIO-RAD) was used, and various cytokine concentrations were measured according to the attached protocol. Measurement items are IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12 (p40), IL-12 (p70), IL-13, IL-17, eotaxin, G-CSF, GM-CSF, IFN-γ, KC, MCP-1, MIP-1α, MIP-1β, RANTES, TNF-α .
・ Blood LPS concentration:
Serum is aseptically prepared from day 14 blood, and using Lipopolysaccharide (Endospecy ES-50M set (Seikagaku Biobusiness) and Toxicolor DIA-MP set (Seikagaku Biobusiness) according to the attached protocol. LPS) concentration was measured.

Spleen immune cell population:
Cells were prepared from the spleen of day 14 and positive for each surface antigen as DX5, CD4, CD8, B220, CD11b, and CD11c markers for NK cells, helper T cells, killer T cells, B cells, macrophages, and dendritic cells, respectively. FACS for cell number
The ratio of each immune cell was detected by counting with a Calibur ^™ flow cytometer (Becton, Dickinson and Company).
・ Bacterial Translocation:
Aseptically collected day 14 mesenteric lymph nodes were homogenized, mixed with BL medium for anaerobic bacteria test, and cultured for 72 hours (BL medium) under anaerobic conditions at 37 ° C. Furthermore, the positive rate of anaerobic bacteria was calculated according to the following formula.
Positive rate (%) = (number of samples in which anaerobic bacteria were detected / number of all samples) × 100

-Gene expression level related to tight junctions in the ileum:
The gene expression level of occludin, which is a membrane protein constituting a tight junction, was measured.
Extract RNA from day 14 ileum according to standard methods, PrimeScript RT reagent Kit, and SYBR Premix Ex Taq,
Using Perfect Real Time (manufactured by Takara Bio Inc.), cDNA synthesis and analysis by q-PCR were performed according to the attached protocol. GAPDH was used as a housekeeping gene.

(result)
In FIG. 3, the result of having evaluated the property of feces is shown. As shown in FIG. 3 (a), diarrhea was induced in the FK506 group, and the fecal score further increased after the fourth day of administration (day 4). As a result, the FK506 group showed a significantly higher stool score than the control group in the average of the whole administration period (FIG. 3 (b)). On the other hand, in the FK506 + ET-3 group, the increase in the stool score was suppressed compared to the FK506 group, and the stool score was significantly lower in the mean of the whole administration period.
From this, it was found that remarkable diarrhea was induced in the group administered with 1 mg / kg body weight of FK506, and that diarrhea induced with FK506 was suppressed by co-administration of 3 mg / body of live ET-3 bacteria. It was.

FIG. 4 shows the measurement results of blood cytokine concentration. The FK506 group showed significantly lower IL-12 (p70) values than the control group, and IL-12 (p40) and IFN-γ also showed lower tendencies than the control group. On the other hand, IL-12 (p40), IL-12 (p70) and IFN-γ were significantly higher in the FK506 + ET-3 group than in the FK506 group.
From this, it was found that in the group administered with FK506, Th1-type cytokines decreased, and by co-administration of live ET-3 bacteria, the Th1-type cytokines decreased with FK506 were recovered.

FIG. 5 shows the analysis results of spleen immune cell population. The number of CD4 + T cells in the FK506 group was significantly lower than that in the control group, but no significant difference was observed in other groups.
From this, it was found that the proportion of helper T cells decreased in the group administered with FK506.

FIG. 6 shows the measurement result of blood LPS concentration. The FK506 group showed significantly higher LPS concentration than the control group, but there was no significant difference in the FK506 + ET-3 group.
From this, it was found that the intestinal barrier function decreased in the group administered with FK506.

FIG. 7 shows the detection result of Bacterial Translocation.
Regarding anaerobic bacteria, FK506 group showed Bacterial Translocation positive rate about 4 times higher than control group.
On the other hand, in the FK506 + ET-3 group, the positive rate decreased to about half that of the FK506 group.
From these results, it was found that in the group administered with FK506, the intestinal barrier function was lowered, and the intestinal barrier function was partially recovered by co-administration with live ET-3 bacteria.

FIG. 8 shows the results of measuring the gene expression level of occludin in the ileal tissue.
occludin is a component of the intestinal tight junction.
The gene expression level in the FK506 group was significantly lower than that in the control group, but no significant difference was observed in the FK506 + ET-3 group.
From this, it was found that in the group administered with FK506, the intestinal barrier function was lowered, and the decrease in the intestinal barrier function was suppressed by co-administration of viable ET-3 bacteria.

[Example 3: Effectiveness test of propionic acid bacteria]
(Test substance) Viable cells of Propionibacterium freudenreichii ET-3 strain
ET-3 was statically cultured in an GAM medium at 30 ° C. for 72 hours under anaerobic conditions, and collected by centrifugation (8000 × g, 15 minutes). The viable cells washed twice with physiological saline and once with distilled water were suspended in distilled water, immediately stored at −80 ° C. and freeze-dried, and the dry weight of the viable cells was calculated. The dried live cells were suspended in distilled water to a concentration of 6 mg / ml and administered to animals.
(Immunosuppressant) FK506 (Tacrolimus)
FK506 (Wako Pure Chemical Industries) was suspended in physiological saline to prepare a concentration of 40 μg / ml, and this was administered to animals.

(Animal experimentation)
Twenty-one 5-week-old male ICR mice (CLEA Japan) were individually housed for 1 week on CRF-1 diet and acclimated to the environment. After the acclimatization period, the mice were divided into 3 groups (each group n = 7) of control group, FK506 group and FK506 + ET-3 group using body weight as an index. The grouped mice were bred in cages laid with poly filter paper (Whatman bench coat plus) and stainless steel wire mesh (16 mesh, mesh opening 1.2 mm) without using flooring.
From the day of grouping (day 0), the immunosuppressive agents and test substances shown in Table 2 were administered to each group once a day for 14 days. To the group not administered with the immunosuppressant or test substance, physiological saline or distilled water having the same volume as the dose of the immunosuppressant or test substance was administered.
The stool of each mouse was received by poly filter paper through a stainless steel wire mesh, and all of the stool adhered to the wire mesh and the stool adhered to the filter paper through the wire mesh were visually observed every day.
About 2 weeks after the start of administration of the immunosuppressant and test substance (day 15), the mice were sacrificed after whole blood collection under pentobarbital anesthesia. The spleen, mesenteric lymph nodes, cecum, ileum and colon were removed by dissection.

(Measurement)
・ Fecal properties:
The stool properties of each day from day 1 to 15 were visually observed and evaluated with the following four stool scores.
1. Normal flights Soft stool (stool with a high moisture content)
3. Mud stool (stool that does not maintain the shape of the stool and adheres to the gaps in the wire mesh)
4). Watery stool (nearly liquid and passes through a wire mesh)
・ Microflora in feces:
Day 15 stool was collected, and the next generation sequencer (Illumina) of the Miseq model used Lactobacillus, Bacteroides
The occupancy rate (%) of the fragilis group was measured, and the number of bacteria shown in Table 3 was measured by q-PCR using each genus-specific primer.

・ Amount of secretory IgA in cecal contents:
Collect the cecal contents of day15,
Maciel et al. (Maciel M. et al, Braz J Med Biol Res
37 (6): 817-826, 2004)
The extraction was performed with reference to FIG.
20 mg of mouse cecal contents were collected in a 1.5 ml tube, 1 ml of PBS was added, mixed and centrifuged (3000 × g 10 min).
2 μl of protease inhibitor cocktail (SIGMA) was added to 800 μl of the supernatant, mixed, and stored at −80 ° C. until measurement.
A dilution of this mixture was used for the Secretory IgA ELISA (Eagle
Biosciences)
Secretory IgA (sIgA) was measured according to the attached protocol.

・ Reg3γ gene expression level in the ileum:
Extract RNA from day 15 ileum according to standard methods, PrimeScript RT reagent Kit, and
Use SYBR Premix Ex Taq, Perfect Real Time (Takara Bio) and follow the attached protocol.
cDNA synthesis and q-PCR analysis were performed. GAPDH was used as a housekeeping gene.

・ Amount of protein related to tight junction in the ileum:
Protein was extracted from each tissue using Allprotect Tissue Reagent (QIAGEN) according to the attached protocol. Pierce
The total protein amount was measured with BCA Protein Assay Kit (Thermo scientific), and SDS-PAGE was performed after aligning the protein amount. Rabbit
Anti-Occludin antibody (Invitrogen) to occludin, Rabbit polyclonal antibody to beta Actin
Β-actin was measured by (IMGENEX) by Western Blotting according to the attached protocol. The amount of Occludin protein was expressed by dividing the measured value of occludin by the measured value of β-actin.

・ The concentration of granulysin in the blood:
Serum was prepared from day 15 blood, and the concentration of granulysin was measured using the Mouse Granulinsin ELISA Kit (TSZ ELISA) according to the attached protocol.
Statistical analysis of the obtained results was performed by Student's t-test.

(result)
FIG. 9 shows the results of evaluating the properties of stool. As shown in FIG. 9 (a), diarrhea was induced in the FK506 group, and the fecal score further increased after the fourth day of administration (day 4). As a result, the FK506 group also showed a significantly higher stool score than the control group in the average over the entire administration period (FIG. 9 (b)). On the other hand, in the FK506 + ET-3 group, the increase in the stool score was suppressed compared to the FK506 group, and the stool score was significantly lower than that in the FK506 group in the average of the whole administration period.
From this, it was found that remarkable diarrhea was induced in the group administered with 1 mg / kg body weight of FK506, and that diarrhea induced with FK506 was suppressed by co-administration of 3 mg / body of live ET-3 bacteria. It was.

FIG. 10 and FIG. 11 show the results of analyzing the flora in the stool of day15. The total number of bacteria in the FK506 group was significantly higher than that in the control group (FIG. 11). Also Bacteroides
The FK506 group also showed significantly higher values in the fragilis group occupancy and the number of bacteria compared to the control group, but no significant difference was observed in the FK506 + ET-3 group (FIGS. 10 and 11). . On the other hand, in the occupation rate of Lactobacillus genus, the FK506 group showed a significantly lower value compared to the control group (FIG. 10). As for the number of bifidobacteria, the FK506 + ET-3 group showed a significantly higher value than the control group (FIG. 11).
From this, it was found that in the group administered with FK506, disturbance of the intestinal flora occurred, and the intestinal flora improved by co-administration of live ET-3 bacteria.

  FIG. 12 shows the measurement results of the secretory IgA content in the cecal contents. The FK506 + ET-3 group showed significantly higher sIgA levels compared to the control group and the FK506 group. From this, it was found that the secretion of sIgA was increased by administering live ET-3 bacteria.

FIG. 13 shows the results of measuring the expression level of Reg3γ gene in ileal tissue. Reg3γ is an antibacterial peptide produced by intestinal epithelial cells. The expression level of Reg3γ gene in the FK506 group was significantly lower than that in the control group. On the other hand, the FK506 + ET-3 group has a significantly higher g than the FK506 group.
Reg3γ gene expression level was shown.
From these results, it was found that the gene expression level of the endogenous antibacterial peptide Reg3γ decreased in the group administered with FK506 and recovered by co-administration with live ET-3 bacteria.
FIG. 14 shows the results of measuring the amount of occludin protein in the ileal tissue. There was no significant difference between any groups.

FIG. 15 shows the results of measuring the concentration of granulysin in serum.
Granulysin is a cytotoxic intragranular protein produced by natural killer (NK) cells and cytotoxic T cells, and is known to correlate strongly with NK activity. The concentration of granulysin was significantly lower in the FK506 group than in the control group. On the other hand, the FK506 + ET-3 group showed a significantly higher granulysin concentration than the FK506 group.
From this, it was found that the innate immunity function decreased in the group administered with FK506, and the innate immunity function was recovered by co-administration with live ET-3 bacteria.

  From the above results, administration of FK506 induced diarrhea symptoms, decreased intestinal barrier function, decreased blood Th1-type cytokine (IL-12, IFNγ) level, decreased helper T cell count, disturbed gut microbiota It has been found that an increase in intestinal bacteria, a decrease in endogenous antimicrobial peptide production and a decrease in innate immune function occur. In addition, co-administration of live ET-3 with FK506 improves diarrhea symptoms, recovery of intestinal barrier function, recovery of blood Th1-type cytokine levels, immune activation, improvement of intestinal microflora, endogenous antimicrobial peptide Effects such as improved production and recovery of innate immune function were obtained.

  Since chronic diarrhea can be induced by the method of the present invention, a substance effective for preventing and treating chronic diarrhea such as irritable bowel syndrome and immunodeficiency syndrome can be screened.

Claims (6)

  A method for inducing chronic diarrhea, comprising administering an immunosuppressive agent to a non-human mammal.   A diarrhea model animal of irritable bowel syndrome, wherein chronic diarrhea is induced, obtained by the method according to claim 1.   A method for producing an irritable bowel syndrome diarrhea model animal, comprising obtaining a non-human mammal in which chronic diarrhea has been induced by the method according to claim 1.   An irritable bowel syndrome diarrhea model animal is prepared by the method according to claim 3, a test substance is administered to the irritable bowel syndrome diarrhea model animal, and diarrhea symptoms, intestinal motility, visceral pain sensation in the animal A screening method for a substance that promotes or inhibits irritable bowel syndrome diarrhea, characterized by evaluating at least one selected from the group consisting of hypersensitivity.   A model animal of diarrhea caused by an immunodeficiency disease, wherein chronic diarrhea is induced, obtained by the method according to claim 1.   A method for producing a model animal of diarrhea due to an immunodeficiency disease, comprising obtaining a non-human mammal in which chronic diarrhea has been induced, obtained by the method according to claim 1.