JPWO2020111239A1 - Anti-inflammatory agent - Google Patents

Abstract

The present invention provides means and methods useful for suppressing inflammation (eg, prevention and treatment of inflammatory bowel disease). More specifically, the present invention provides the following inventions.
[I] An anti-inflammatory agent containing 5-hydroxyindoleacetic acid.
[II] Screening method for anti-inflammatory agents, including:
(1) Measure the amount of 5-hydroxyindoleacetic acid in a sample taken from a mammal to which the test substance was administered; and (2) Test substance that increases the amount of 5-hydroxyindoleacetic acid in the sample. , To be selected as an anti-inflammatory agent.
[III] Screening method for anti-inflammatory agents, including:
(1) Culturing mammalian-derived cells capable of producing 5-hydroxyindoleacetic acid in a medium containing a test substance;
(2) Measuring the amount of 5-hydroxyindoleacetic acid in the medium;
(3) Select a test substance that increases the amount of 5-hydroxyindoleacetic acid in the medium as an anti-inflammatory agent.

Description

The present invention relates to anti-inflammatory agents and the like.

In recent years, prevention of lifestyle-related diseases [eg, metabolic syndrome, type 2 diabetes (T2D) and inflammatory bowel disease (IBD)] by nutritional intervention has become common, and consumers are conscious of nutrition intake to maintain their health. doing. Traditional Japanese foods such as natto, miso, and matcha are famous for improving health. They have a positive effect on the intestinal environment and act as prebiotics and / or probiotics by maintaining a healthy host state.

For example, rice bran (RB) contained in brown rice is one of the commonly consumed Japanese foods, and it has been reported that it can suppress colitis (Non-Patent Documents 1 to 4). RB is a rich source of dietary fiber, vitamins, free amino acids, and bioactive ingredients such as antioxidants (γ-oryzanol and ferulic acid) that may promote gastrointestinal health and anti-colitis effects. Yes (Non-Patent Documents 2, 3, 5, 6). However, due to the complexity of the constituents of RB, the molecular mechanism by which RB suppresses colitis has not yet been elucidated.

Al-Fayez M et al. , Cancer Chemother Pharmacol. 2006; 58 (6): 816-25. Islam MS et al. , Br J Pharmacol. 2008; 154 (4): 812-24. Komiyama Y et al. , Scand J Gastroenterol. 2011; 46 (1): 40-52. Shafie NH et al. , Int J Mol Sci. 2013; 14 (12): 23454-58. Choi J et al. , Nutr J. et al. 2014; 13 (1): 35. Islam J et al. , Nutrients. 2017; 9 (7): 747.

An object of the present invention is to provide means and methods useful for suppressing inflammation (eg, prevention / treatment of inflammatory bowel disease).

As a result of diligent studies, the present inventors have found that suppression of colitis by RB can be caused by the production of 5-hydroxyindoleacetic acid (5-HIAA) by intestinal bacteria, and 5-HIAA has an anti-inflammatory effect. Found to have.
We also developed anti-inflammatory agents by screening for test substances that increase the amount of 5-HIAA in mammals or mammalian-derived cells in light of the anti-inflammatory activity of 5-HIAA. I found out what I could do.

That is, the present invention is as follows.
[1] An anti-inflammatory agent containing 5-hydroxyindoleacetic acid.
[2] The anti-inflammatory agent according to [1], wherein the anti-inflammatory agent is an anti-inflammatory agent against inflammation in the intestine.
[3] The anti-inflammatory agent according to [2], wherein the inflammation in the intestine is an inflammatory bowel disease.
[4] The anti-inflammatory agent according to any one of [1] to [3], which is an oral composition or a composition for transrectal administration.
[5] The anti-inflammatory agent according to any one of [1] to [4], which is a medicine or food.
[6] Screening method for anti-inflammatory agents including the following:
(1) Measure the amount of 5-hydroxyindoleacetic acid in a sample taken from a mammal to which the test substance was administered; and (2) Test substance that increases the amount of 5-hydroxyindoleacetic acid in the sample. , To be selected as an anti-inflammatory agent.
[7] The method of [6], wherein the sample is feces.
[8] Screening method for anti-inflammatory agents including the following:
(1) Culturing mammalian-derived cells in a medium containing a test substance;
(2) Measuring the amount of 5-hydroxyindoleacetic acid in the medium;
(3) Select a test substance that increases the amount of 5-hydroxyindoleacetic acid in the medium as an anti-inflammatory agent.
[9] The method of [8], wherein the mammalian-derived cells capable of producing 5-hydroxyindoleacetic acid are enterobacteria.

According to the present invention, 5-HIAA can realize suppression of inflammation (eg, prevention / treatment of inflammatory bowel disease).
Further, according to the present invention, an anti-inflammatory agent can be screened by using the amount of 5-HIAA as an index.

FIG. 1 is a diagram showing an outline of an animal experiment. FIG. 2-1 is a diagram showing the effect of a rice bran (RB) diet on weight loss. In FIGS. 2-1 to 2-3, the significance between the control and each group is shown. ^* P <0.05, ^** P <0.01, ^*** P <0.001. FIG. 2-2 is a diagram showing the effect of the RB diet on the disease activity index (DAI). See Figure 2-1 for significance. FIG. 2-3 is a diagram showing the effect of the RB diet on the length of the large intestine. See Figure 2-1 for significance. FIG. 2-4 is a diagram showing the effect of the RB diet on the morphology of the large intestine. FIG. 2-5 is a diagram showing the effect of the RB diet on colorectal histology. The magnification is 200x and the bar indicates 100 μm. FIG. 2-6 is a diagram showing the effect of the RB diet on the gene expression level. FIG. 3-1 is a diagram showing the effect of the fractionation component of the RB diet on weight loss. In FIGS. 3-1 to 3-3, the significance between the control and each group is shown. ^* P <0.05, ^** P <0.01, ^*** P <0.001. FIG. 3-2 is a diagram showing the effect of the fractionation component of the RB diet on DAI. FIG. 3-3 is a diagram showing the effect of the fractionation component of the RB diet on the length of the large intestine. FIG. 3-4 is a diagram showing the effect of the fractionation component of the RB diet on the morphology of the large intestine. FIG. 3-5 is a diagram showing the effect of the fractionation component of the RB diet on the intestinal bacterial flora. FIG. 4-1 is a diagram showing changes in the composition of the fecal bacterial flora by RB in DSS-induced colitis mice. The relative abundance of Enterobacteriaceae detected in the top 20 with a relative abundance exceeding 1% of the total sequence is represented by a bar graph. FIG. 4-2 is a diagram showing changes in the diversity of Simpson. In FIGS. 4-2 and 4-3, the significance between the control and each group is shown. * P <0.05, ** P <0.01. FIG. 4-3 is a diagram showing a box plot of a part of Enterobacteriaceae that changed significantly on day 0. FIG. 5-1 is a diagram showing principal component analysis (PCA) of fecal metabolite data. FIG. 5-2 is a diagram showing the top 10 and bottom 10 factor loadings of PC2 in the principal component analysis (PCA) of fecal metabolite data. FIG. 5-3 is a diagram showing metabolites related to tryptophan metabolism in feces. FIG. 5-4 is a diagram showing the concentration of 5-HIAA in feces. In FIG. 5-5, the significance between the control and each group is shown. * P <0.05, ** P <0.01. FIG. 6-1 is a diagram showing the relative expression level of Cyp1a1 when AhR forced expression RAW264.7 was treated with 5-HIAA. FIG. 6-2 is a diagram showing the 5-HIAA concentration in feces of GF mice in which each bacterial group has been established.

1. 1. Anti-inflammatory agents The present invention provides anti-inflammatory agents, including 5-HIAA.

5-HIAA is a metabolite of serotonin and is biosynthesized from serotonin in a given cell (eg, a mammalian cell capable of producing serotonin, a resident microorganism such as an intestinal bacterium). Therefore, 5-HIAA can be prepared using serotonin-producing cells (eg, natural cells, or transformed cells incorporating a biosynthetic system as described above). 5-HIAA can also be synthesized by organic chemical methods. Alternatively, as 5-HIAA, a commercially available product may be used.

The inflammation to which the anti-inflammatory agent of the present invention can be applied is not particularly limited as long as it is any inflammation that can occur in mammals. Examples of sites where such inflammation can occur include the intestine (eg, small intestine, large intestine), nasal cavity, bronchi, skin, and oral cavity. As the site, the inside of the intestine is preferable, and the inside of the large intestine is more preferable.

Preferably, the inflammation is inflammation in an inflammatory disease. Examples of such inflammatory diseases include inflammatory bowel diseases (eg, ulcerative colitis, Crohn's disease, Behcet's disease), stomatitis, nephritis, and autoimmune diseases (eg, allergic diseases). As inflammation, inflammatory bowel disease is preferable. Therefore, the anti-inflammatory agent of the present invention can be used as a prophylactic or therapeutic agent for such inflammatory diseases.

The amount of 5-HIAA used as an anti-inflammatory agent is not particularly limited as long as it can exert an anti-inflammatory effect, but is as follows as a guide. In the examples, the concentration of 5-HIAA in feces collected from a mammal (mouse) of an inflammatory bowel disease model was about 100 nmol / g, 5- in feces collected from an RB feeding mammal (mouse). It has been confirmed that the concentration of HIAA is about 800 mM / g. That is, it is considered desirable that the concentration of 5-HIAA in the target site of the mammal is in the range of 300 nmol / g or more (eg, 300 to 1500 nmol / g). For example, in order to achieve a concentration in this range in the intestine of a mammal, the dose or intake of 5-HIAA in the mammal is, for example, 0.01-100 g / kg, preferably 0.5-50 g / kg. It is kg, more preferably 1 to 10 g / kg. Therefore, the anti-inflammatory agent of the present invention may contain such an amount of 5-HIAA. Alternatively, when 5-HIAA is applied to the intestinal or non-intestinal site of a mammal, it is, for example, 0.01 to 1000 mg / ml, preferably 0.1 to 100 mg / ml, and more preferably 1. As 5-HIAA is applied in a concentration range of 10 mg / ml, the anti-inflammatory agent of the present invention may contain 5-HIAA in such a concentration range.

In certain embodiments, the anti-inflammatory agents of the invention may be provided in the form of compositions.

For example, the anti-inflammatory agent of the present invention may include a pharmaceutically acceptable carrier in addition to 5-HIAA when provided in the form of a pharmaceutical composition. Pharmaceutically acceptable carriers include, for example, excipients such as sucrose, starch, mannit, sorbit, lactose, glucose, cellulose, talc, calcium phosphate, calcium carbonate, cellulose, methylcellulose, hydroxypropylcellulose, polypropylpyrrolidone. , Gelatin, gum arabic, polyethylene glycol, sucrose, starch and other binders, starch, carboxymethyl cellulose, hydroxypropyl starch, sodium hydrogen carbonate, calcium phosphate, calcium citrate and other disintegrants, magnesium stearate, aerodyl, talc, lauryl Lubricants such as sodium sulfate, citric acid, menthol, glycyrrhizin / ammonium salt, glycine, fragrances such as orange powder, preservatives such as sodium benzoate, sodium hydrogen sulfite, methylparaben, propylparaben, citric acid, sodium citrate, acetic acid Stabilizers such as, methyl cellulose, polyvinylpyrrolidone, suspending agents such as aluminum stearate, dispersants such as surfactants, diluents such as water, physiological saline, orange juice, cocoa butter, polyethylene glycol, white kerosene, etc. Examples include, but are not limited to, base waxes. Pharmaceutical compositions include solids (eg capsules, tablets, powders, granules), semi-solids (eg gels), liquids (eg oral solutions, or transrectal solutions, eye drops, etc. Parenteral liquids such as ear drops and nasal drops).

In certain preferred embodiments, the anti-inflammatory agent of the invention, which is in the form of a pharmaceutical composition, comprises, in addition to 5-HIAA, other substances having anti-inflammatory activity and / or microorganisms having anti-inflammatory activity. You may. Examples of other substances having an anti-inflammatory effect include drugs such as steroidal anti-inflammatory drugs and non-steroidal anti-inflammatory drugs (NSAIDs); extracts from natural products such as animals and plants; antibody drugs. Examples of microorganisms having an anti-inflammatory effect include Clostridium bacteria, Bacteroides bacteria, Lactobacillus bacteria, Bifidobacterium bacteria and the like.

When the anti-inflammatory agent of the present invention is provided in the form of a food composition, 5-HIAA may be provided in the form of being added to the food, or may be used as a main component such as a supplement. .. Examples of foods include liquids (eg, beverages, alcohols), semi-solids (eg, yogurt, jelly), solids (eg, snacks, chocolate).

In certain preferred embodiments, the anti-inflammatory agent of the invention, which is in the form of a food composition, comprises, in addition to 5-HIAA, other substances having anti-inflammatory activity and / or microorganisms having anti-inflammatory activity. You may. Such other substances and microorganisms are similar to those described above.

In a preferred embodiment, the anti-inflammatory agent of the present invention may be an oral composition or a transrectal administration composition. Such an anti-inflammatory agent of the present invention can exert an anti-inflammatory effect in the intestine of a mammal. The content of 5-HIAA in the oral composition and the transrectal administration composition is similar to the above-mentioned administration or intake of 5-HIAA. Oral compositions can be used as solids, semi-solids, or liquids as described above in pharmaceutical and food compositions. The composition for transrectal administration can be used as a semi-solid or liquid.

The anti-inflammatory agent of the present invention can be applied to any mammal. Such mammals include, for example, primates (eg, humans, monkeys, chimpanzees), rodents (eg, mice, rats, rabbits), livestock and working animals (eg, cows, pigs, horses, goats, etc.). Sheep). As mammals, primates or rodents are preferable, primates are more preferable, and humans are even more preferable from the viewpoint of clinical application.

2. Anti-inflammatory agent screening method The present invention provides an anti-inflammatory agent screening method using a sample collected from a mammal or a mammalian-derived cell. Mammals and anti-inflammatory agents are similar to those described above.

In one embodiment, the screening method of the present invention includes:
(1) Measure the amount of 5-HIAA in a sample taken from a mammal to which the test substance was administered; and (2) The test substance that increases the amount of 5-HIAA in the sample is an anti-inflammatory agent. To select as.

In step (1), the test substance is any substance, including known substances and novel substances. Such test substances include, for example, organic low molecular weight compounds, compound libraries prepared using combinatorial chemistry techniques, nucleic acids (eg, nucleosides, oligonucleotides, polypeptides), sugars (eg, monosaccharides, di). Sugars, oligosaccharides, polysaccharides), lipids (eg, fatty acids containing saturated or unsaturated linear, branched and / or rings), amino acids, proteins (eg, oligopeptides, polypeptides, antibodies or fragments thereof), solids Examples thereof include a random peptide library prepared by phase synthesis or a phage display method, or natural components derived from microorganisms, animals and plants, marine organisms, and the like. Alternatively, as the test substance, a microorganism (eg, a resident microorganism such as an intestinal bacterium) may be used.

The sample is a sample that can be collected non-invasively from a mammal, or a sample that can be collected invasively from a mammal. Samples that can be collected non-invasively from mammals include, for example, excrement samples (eg, feces, urine), liquid samples (eg, saliva, tears, sweat), mucosal samples (eg, intranasal mucosa, etc.) Oral mucosa). Samples that can be invasively collected from mammals (eg, biopsy samples) include, for example, samples that can be collected from the skin, bronchi, blood, small intestine, large intestine, cecum, and kidney. As the sample, a sample that can be collected non-invasively from a mammal is preferable, and feces are more preferable. The sample may be subjected to pretreatment. Examples of such pretreatment include dissolution in a liquid, centrifugation, extraction, heating, freezing, and refrigeration.

The amount of 5-HIAA can be measured by any method. Examples of such a method include high performance liquid chromatography (HPLC), mass spectrometry, immunoassay, and colorimetric method. The amount of 5-HIAA may be measured as the 5-HIAA concentration per unit amount of sample.

The present embodiment can further include administering the test substance to the mammal prior to step (1). The amount of test substance to be administered can be adjusted as appropriate. For example, the amount of test substance to be administered can be set to a low amount (eg, 0.05-10 g / kg) if screening for a potent anti-inflammatory agent is particularly desired. Alternatively, the amount of test substance to be administered may be set to a higher amount (eg, 10-100 g / kg) if screening for a potent anti-inflammatory agent is not particularly desired.

In another embodiment, the screening method of the present invention includes:
(1) Culturing mammalian-derived cells capable of producing 5-HIAA in a medium containing a test substance;
(2) Measuring the amount of 5-HIAA in the medium;
(3) Select a test substance that increases the amount of 5-HIAA in the medium as an anti-inflammatory agent.

In step (1), mammalian-derived cells are cultured in a medium containing the test substance. The test substance is the same as that described above. The concentration of the test substance in the medium can be adjusted as appropriate. For example, the concentration of the test substance in the medium can be set to a low concentration (eg, 1 nM-10 μM) if screening for a highly potent anti-inflammatory agent is particularly desired. Alternatively, the concentration of the test substance in the medium may be set to a higher concentration (eg, 100 nM-10 mM) if screening for a potent anti-inflammatory agent is not particularly desired.

The mammalian-derived cells used in step (1) are not particularly limited as long as they have the ability to produce 5-HIAA. Such mammalian-derived cells are mammalian cells, or microorganisms present in mammals, preferably microorganisms present in mammals. The microorganism present in the mammal is a resident microorganism present in a site where inflammation can occur in the mammal (eg, the above-mentioned site), and is preferably an intestinal bacterium.

Indigenous microorganisms such as Gut microbiota can be cultivated in the form of a mixture with other microorganisms or in the form of a non-mixture with other microorganisms. For example, if certain gut microbiota are cultured in the form of a mixture with other gut microbiota, such a mixture can be obtained by recovering from mammalian feces as described above.

Culturing of indigenous microorganisms such as intestinal bacteria can be carried out under general culture conditions of indigenous microorganisms using the same medium as the general medium used for culturing indigenous microorganisms. For example, common media and culture conditions for gut microbiota are well known (eg, (1) Fukuda S. et al., J Vet Med Sci., 2002 Nov; 64 (11): 987-92, (. 2) Fukuda S. et al., J Gen Appl Microbiol., 2005 Appr; 51 (2): 105-13, (iii) Fukuda S. et al., Sci Rep., 2018 Jan 11; 8 (1) :. 435).

For example, the medium can contain components such as carbon sources, nitrogen sources, organic micronutrient sources, vitamins and inorganic ions. Examples of carbon sources include carbohydrates such as monosaccharides (eg, glucose), disaccharides, oligosaccharides, and polysaccharides; converted sugars obtained by hydrolyzing sucrose; glycerol; methanol, formaldehyde, formates, carbon monoxide, and dioxide. Compounds with 1 carbon number such as carbon; Oils such as corn oil, palm oil, soybean oil; Short-chain fatty acids such as acetic acid, propionic acid, butyric acid; Organic acids such as succinic acid and lactic acid; Animal fats and oils; Animal oils; Saturated Fatty acids such as fatty acids and unsaturated fatty acids; lipids; phospholipids; glycerolipids; glycerin fatty acid esters such as monoglyceride, diglyceride, triglyceride; polypeptides such as microbial proteins and vegetable proteins; regeneration of hydrolyzed biomass carbon sources, etc. Possible sources of carbon; yeast extract; horse serum; fecal extract; meat extract; vegetable extract; stomach content extract; or a combination thereof. Examples of the nitrogen source include inorganic ammonium salts such as ammonium sulfate, ammonium chloride and ammonium phosphate, organic nitrogen such as soybean hydrolyzate, ammonia gas and aqueous ammonia. As the organic micronutrient source, for example, it is desirable to contain an appropriate amount of a required substance such as L-homoserine or yeast extract. Examples of vitamins include vitamins B1, B2, B3, B6, B12, C and K1. Examples of the inorganic ions include potassium phosphate, magnesium sulfate, iron ions, and manganese ions.

Explaining the culture conditions, for example, the intestinal bacterial density in the medium is, for example, 1 × 10 ⁶ to 1 × 10 ¹¹ cells / mL, preferably 1 × 10 ⁷ to 1 × 10 ¹⁰ cells / mL, and more. It is preferably 1 × 10 ⁸ to 1 × 10 ⁹ cells / mL. The culture temperature is, for example, 30-40 ° C. The culture period is, for example, 1 to 7 days. Either anaerobic or aerobic culture conditions can be utilized, but anaerobic culture conditions are preferred. The oxygen concentration under anaerobic culture conditions is, for example, 0 to 5%, preferably 0 to 3%, more preferably 0 to 2%, and even more preferably 0 to 1%.

The screening method of the present invention is useful, for example, for the development of a new drug or food having an anti-inflammatory action, and for the development of a drug or food capable of exerting a complex action having an anti-inflammatory action in addition to the existing action. Is.

Next, the present invention will be described in more detail with reference to Examples, but the present invention is not limited to the following Examples.

(Method)
(1) Mice and feed Five-week-old female C57BL / 6J mice were obtained from Clear Japan, Inc. I bought from. Mice were bred under controlled light conditions (12 hour light / 12 hour dark cycle). Feed tap water or 2.0% DSS (molecular weight 36,000-50,000; MP Bio Japan), AIN-93G (Oriental yeast) or 5% RB, 10% RB, 20% RB, γ-oryzanol, RB Oil and defatted RB (AIN-93G) were given. RB samples are from Richech Inc. Γ-Oryzanol, RB oil and degreased RB provided by Oryzanol & Fat Chemical Inc. Provided by.

(2) Animal experiments In this study, two animal experiments were conducted. In the first experiment, the effect of rice bran (RB) was verified and the RB concentration dependence was investigated. In the second experiment, the components of RB were fractionated to investigate which component contributed to the colitis-suppressing effect of RB. In both experiments, after a 1 week acclimation period, mice were randomly assigned to groups 4 or 6 (n = 5-7 in each group) as follows: Control: AIN-93G diet and 2. Colitis was induced by administration of 0% (w / v) DSS water for 10 weeks. 5% RB, 10% RB, 20% RB, RB, RBG (gamma-oryzanol), RBO (RB oil) and RBD (defatted RB): 5% RB, 10% RB, 20% RB, 20% RB, γ- AIN-93G diet supplemented with oryzanol, RB oil and defatted RB and tap water were administered for 1 week, followed by 2.0% DSS water for 10 days each. Fecal samples from all mice were collected daily for 16S rRNA gene analysis during the experiment. In addition, during the DSS administration period, the severity of colitis was assessed as described in the DAI assessment below. All mice were dissected 10 days after DSS administration. The large intestine and the contents of the large intestine were collected and the length of the large intestine was measured. All samples were stored at −80 ° C.

(3) Fecal collection Fecal samples were collected for capillary electrophoresis electrospray ionization time-of-flight mass spectrometry (CETOFMS) and 16S rRNA gene analysis. All mice were placed in separate cages. Every day, 5 fecal pellets per mouse were collected in tubes with autoclaved tweezers and placed on ice. These samples were stored at -80 ° C until subsequent analysis.

(4) DAI evaluation The disease activity index (DAI) was measured daily to evaluate the severity of colitis. DAI was weight change (0: <1%; 1: 1-5%; 2: 5-10%; 3: 10-15%; 4:> 15%), feces (0: negative, 1: +, It was determined by calculating the average of three parameters: 2: ++, 3: ++++, 4: ++++), and stool hardness (0: normal, 2: soft, 4: diarrhea).

(5) Histopathology For histopathological analysis, a representative sample from the central part of the large intestine was fixed in 10% formalin, embedded in paraffin, sectioned, and with hematoxylin and eosin (H & E). It was stained and examined at a magnification of 200.

(6) DNA extraction Fecal DNA extraction was performed according to a previously described method with some modifications (Furusawa Y et al., Nature. 2013; 504 (7480): 446-50.). Fecal samples were lyophilized for 24 hours using a VD-800R lyophilizer (TAITEC). The lyophilized feces were destroyed by vigorous shaking (1,500 rpm for 10 minutes) with 3.0 mm zirconia beads (Biomedical Science) using Shake Master (Biomedical Science). Fecal sample (10 mg) in 1.0% (w / v) SDS / TE (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) 400 μL and phenol / chloroform / isoamyl alcohol (25: 24: 1) 400 μL. Suspended. 0.1 mm zirconia / silica beads were added to the mixed solution and shaken vigorously using a shake master (1,500 rpm for 5 minutes) to further destroy the mixture. After centrifugation at 17,800 xg for 5 minutes at room temperature, bacterial genomic DNA was purified from the fecal extract by the standard phenol / chloroform / isoamyl alcohol method. RNA was removed from the sample by RNase A treatment and then the DNA sample was purified once more by the standard phenol / chloroform / isoamyl alcohol method.

(7) 16S rRNA gene sequencing The 16S rRNA gene in the fecal DNA sample was analyzed using the MiSeq sequencer (Illumina) according to the method previously described with some modifications (Murakami S et al., Altern). Med. 2015; 2015: 824395.). In summary, the V1-V2 region of the 16S rRNA gene contains the bacterial universal primer set 27Fmod (5'-AGRGTTTGATYMTGGCTCAG-3'(SEQ ID NO: 1)) (Kim SW et al., DNA Res. 2013; 20 (3): 241. -53.) And 338R (5'-TGCTGCCTCCCCGTAGGAGT-3' (SEQ ID NO: 2)) were amplified from DNA isolated from feces. PCR was performed using Tks Gflex DNA Polymerase (manufactured by Takara Bio Inc.), and amplification was performed at 98 ° C. for 1 minute, 98 ° C. for 10 seconds, 55 ° C. for 15 seconds, and 68 ° C. for 30 seconds for 30 cycles. , 68 ° C. for 3 minutes at the end. The amplified product is confirmed by agarose gel electrophoresis, purified using Agencourt AMPure XP (Beckman Controller), and then a forward primer containing the P5 sequence, which is the 8 bp barcode sequence (indicated by N) unique to each sample. A reverse primer (5'-CAAGCAGAA) containing a P7 sequence, including (5'-NNNNNNNNNTATTGGTAAATTGTAGRGTTTGATYMTGGCTCAGT-3'(SEQ ID NO: 3)), Rd1 SP sequence and 27Fmod primer and a unique 8bp barcode sequence (indicated by N). -GACGGCTATACGAGATANNNNNNN-AGTCAGTCAGCCCTGCTGCCTC CCGAGGAGT-3'(SEQ ID NO: 4), Rd2 SP sequence, and 338R primer. Amplification products purified with the Agent AMPure XP were quantified using the Quant-iTPicoGreen DNA assay (Invitrogen) and approximately equal volumes of PCR amplifiers from each sample whose quality was controlled with the Agilent 2100 Bioanalyzer (Agilent Technologies). Mixed samples were prepared by pooling. Finally, MiSeq sequencing was performed according to the protocol. In this study, 2 × 300 bp paired-end sequencing was used.

(8) Metabolome analysis using a capillary electrophoresis electrospray ionization time-of-flight mass analyzer Metabolome analysis was performed according to the method described previously [(1) Murakami S et al. , Altern Med. 2015; 2015: 824395. , And (2) Mishima E et al. , JAm Soc Nephrol. 2015; 26 (8): 1787-94. ]. Briefly, fecal samples were lyophilized and destroyed with 3.0 mm zirconia beads as described in the DNA extraction above. To extract metabolites from the stool sample, 500 μL of methanol containing internal standards (20 μM each of methionine sulfone and D-shonow-10-sulfonic acid (CSA)) was added to the 10 mg stool sample. The mixture was further disrupted with 100 mg of 0.1 mm zirconia / silica beads with vigorous shaking using a shake master (1,500 rpm for 5 minutes). The mixture was then mixed with 200 μL of ultrapure water and 500 μL of chloroform and then shaken vigorously (1500 rpm for 5 minutes). After centrifugation at 4,600 xg at 4 ° C. for 15 minutes, the supernatant was transferred to a centrifugal filter tube (Ultrafree MC-PLHCC 250 / pk for Metabolome extraction of Human Metabolome Technologies) to remove protein and lipid molecules. The filtrate was centrifuged and dissolved in 100 μL of ultrapure water containing reference compounds (3-aminopyrrolidine and 200 μM trimesic acid, respectively) immediately prior to CE-TOFMS analysis.

Ionic metabolites were analyzed using CE-TOFMS in both positive and negative modes. All CE-TOFMS experiments were performed using an Agilent CE capillary electrophoresis system (Agilent Technologies). The annotation table was prepared from measurements of standard compounds and aligned with the dataset according to similar m / z values and standardized travel times. The peak area was then normalized to the peak area of the internal standard methionine sulfone or CSA for cationic and anionic metabolites, respectively. The concentration of each metabolite was calculated based on the relative peak area and concentration of the standard compound. Peak annotations and quantifications were confirmed by in-house software (MasterHands) (Sugimoto M et al., Metabolomics. 2010; 6 (1): 78-95.). Metabolite set enrichment analysis (MSEA) was performed using controls on day 0 of DSS administration and metabolites from 10% RB [(1) Xia J et al. , Nucleic Acids Res. 2010; 38 (SUPPL. 2). , And (2) Xia J et al. , In: Current protocols in bioinformatics. 2016. p. 14.10.1-14.10.91. ].

(9) Statistical analysis Differences between two or more groups were evaluated by Student's t-test or Dunnett's test, respectively. Weight changes, DAI were analyzed by bidirectional repeated measures ANOVA followed by Holm-Bonferroni procedure, which used anovakun version 4.8.0. Principal component analysis (PCA) was performed by SIMCAP 13.0.3 (Umetrics). Hierarchical clustering and heatmaps were drawn using MeV TM4 software 4.7.4.

(10) AhR in vitro test In mouse-derived RAW264.7 cells (RAW / Neo) or RAW cells (RAW / AhR) in which AhR was forcibly expressed, 5-HIAA was added to solvent only (DMSO), 2 μM, 20 μM, and 200 μM medium. The relative expression level of Cyp1a1 when added and cultured for 24 hours was measured.

(11) Gnotobiotic test Sterile mice were orally administered with solvent alone or enterococcus bacteria group, Lactobacillus bacteria group, Bacteroides bacteria group, and Clostridium bacteria group. Feces were collected 1 month after administration, and the concentration of 5-HIAA was measured using CE-TOFMS.

(12) FISH analysis The large intestine was collected and treated by the Methacarn fixation method to prepare paraffin sections. The prepared paraffin sections were deparaffinized with xylene, and then Clostridium, which specifically increases in rice bran ingested mice, was stained on 16S rRNA targets using the Fluorescence in situ hybridization (FISH) method. An Erec482 (5'-GCTTCTTAGTCARCTACCG-3') fluorescently labeled FISH probe that specifically stains Clostridium cluster XIV was used for FISH staining. After FISH staining of Clostridium, DNA and mucin are stained with DAPI and fluorescently labeled Wheat Germ Agglutinin (WGA) lectin, respectively.

(Results and discussion)
(1) Colitis is suppressed by ingestion of RB (Fig. 2)
In RB-fed mice, animal experiments were performed using a DSS-induced colitis mouse model to evaluate the effects of the RB diet on weight loss, DAI, colorectal shortening, and colitis as compared to controls. Weight loss was reduced in the control, but was suppressed in the RB intake group (Fig. 2-1). Similarly, there was a significant difference in DAI between the control group and the RB intake group (Fig. 2-2). A significant difference in colon length between the control group and the RBG intake group was observed (Figs. 2-3, 2-4). Furthermore, there was a difference in the amount of blood in the large intestine (Fig. 2-3). Tissue sections of the large intestine showed RB-fed mouse group with crypt distortion and reduced leukocyte and neutrophil mucosa and infiltration (Fig. 2-5). Taken together, these results suggest that an RB diet can suppress DSS-induced colitis.

(2) The RB degreasing component suppresses colitis (Fig. 3)
Three components, RBG, RBD and RBO, were used to identify the active ingredient that contributes to the colitis-suppressing effect of RB. RBDs consisting of fat-free RBs have a significant colitis-suppressing effect on body weight, DAI, and colorectal length and morphology (FIGS. 3-1 to 3-4). In particular, the observed colitis-suppressing effect was similar to that of whole RB (RB). In contrast, RBG and RBO, which were previously thought to have a colitis-suppressing effect, did not show a significant colitis-suppressing effect. These results suggest that the colitis-suppressing effect of RB may be mainly due to its degreasing component.

(3) Changes in fecal bacterial flora due to RB intake (Fig. 4)
In order to evaluate the dynamics of the composition of fecal microorganisms by RB dietary intervention, 16S rRNA gene analysis was performed using fecal samples collected daily during the experiment. 59,662 OTUs (operational taxonomy units) were constructed from 308 samples (some stool samples were lost during stool collection). Taxonomic profiling at the family level of the fecal flora is shown (Fig. 4-1). It was observed that the composition of the fecal flora of DSS-induced colitis mice was dramatically altered. The α-diversity in the RB-fed mouse group was significantly higher or delayed compared to the control (Fig. 4-2). To examine changes in the fecal flora, the relative abundance of each Bacterial family on day 0 was compared between the control group and the other groups. In the RB intake group, 20 families, especially Bifidobacteriaceae, Lactobacillaceae and Tracybacteraceae, were significantly increased, and Bifidobacteriae, Erycipelotrichaceae and Enterobacteriaceae were significantly decreased compared to the control (Fig. 3).

(4) Changes in fecal metabolites due to RB intake (Fig. 5)
Metabolome analysis was performed using fecal samples collected on days 0, 5, and 10 to assess the effects of RB intake at the molecular level. Metabolome analysis by CE-TOFMS and gas chromatography-mass spectrometer (GC-MS) identified 360 metabolites using feces of at least one mouse per group per time point. Concentrations of these metabolites were compared between the control and other groups (data not shown). The temporal changes in control and fecal metabolite profiles of RB mice in PCA were in different directions (Fig. 5-1). This indicates that the fecal metabolome profile was affected by DSS, but RB intake suppressed the effect. Compared with the control, 41 metabolites containing short-chain fatty acids such as acetate and butyrate were significantly changed in RB-fed mice (Fig. 5-2). SCFA produced by fermentation of dietary fiber by the intestinal microflora induces apoptosis in neutrophils (Maslowski KM et al., GPR43. Nature. 2009; 461 (7268): 1282-6.) And colonic regulation. Suppresses enteritis in mice via T cell (Treg) differentiation (Furusawa Y et al., Nature. 2013; 504 (7480): 446-50.). In addition, increasing concentrations of SCFAs result in lower pH. In the presence of lower pH, the number of pathogenic microorganisms and the solubility of bile acids are reduced, resulting in reduced tumor promoter activity of secondary bile acids [(1) Cook SI et al. , Aliment Pharmacol Ther. 1998; 12 (6): 499-507. , (2) Grubben MJAL et al. , Dig Dis Sci. 2001; 46 (4): 750-6. , And (3) Wong JM et al. , J Clin Gastroenterol. 2006; 40 (3): 235-43. ]. Therefore, an increase in intake of fermentable dietary fiber such as RB may be beneficial in controlling colitis [(1) Daou C et al. , J Food Sci Technology. 2014; 51 (12): 3878-85. , And (2) Park HY et al. , Planta Med. 2016; 82 (07): 606-11. ]. In our study, the concentration of SCFAs was increased by RB consumption. The most prominent SCFA, acetate, is important for lowering the pH of the colon [(1) Duncan SH et al. , Br J Nutr. 2004; 91 (06): 915-23. , And (2) Ye Het al. , Scientific Rep. 2016; 6: 20329. ], Thereby the number of pathogenic microorganisms and the solubility of bile acids have anti-inflammatory properties and promote the integrity of the intestinal epithelium of mice [(1) Fukuda S et al. , Nature. 2011; 469 (7331): 543-7. , And (2) Macia L et al. , Nat Commun. 2015; 6 (August): 6734. ]. On day 0, MSEA was performed using the control and 10% RB metabolome data (Table 1). Vitamin B6 metabolism, tryptophan metabolism and ubiquinone biosynthesis were significantly altered between control and 10% RB. Among the tryptophan metabolism-related metabolites, 5-hydroxyindoleacetic acid (5-HIAA) was increased by RB intake (Fig. 5-3, 5-4).

(5) Suppression of colitis by 5-HIAA (Fig. 6)
Treatment of AhR forced expression RAW264.7 cells with 5-HIAA (concentrations 2, 20, and 200 μM in medium) to determine if 5-HIAA was an AhR ligand increased Cyp1a1 gene expression compared to controls. (Fig. 6-1). In addition, when the Clostridium bacterium group was colonized in GF mice, more 5-HIAA was produced than when sterile mice and other bacteria were colonized (Fig. 6-2). As a result of FISH analysis of the cross section of the large intestine, a group of Clostridium bacteria was localized in the fibrous part of RB. Previous studies have shown that tryptophan metabolism by the intestinal microflora plays a role in AhR-mediated mucosal immune responses by regulating the production of IL-22, a cytokine with a well-known effect on intestinal homeostasis. It has been suggested [(1) Lamas B et al. , Nat Med. 2016; 22 (6): 598-605. , (2) Rutz S et al. , Immunol Rev. 2013; 252 (1): 116-32. , And (3) Zelante T et al. , Immunoty. 2013; 39 (2): 372-85. ]. In addition, impaired microbial production of AhR ligands is observed in IBD patients (Lamas B et al., Nat Med. 2016; 22 (6): 598-605.). In our study, tryptophan metabolism was significantly upregulated by RB consumption. Therefore, tryptophan metabolism from the gut microbiota can be a target for the development of new therapeutic agents for patients with colitis.

The anti-inflammatory agent of the present invention is useful for suppressing inflammation (eg, prevention / treatment of inflammatory bowel disease).
The screening method of the present invention is useful for the development of anti-inflammatory agents.

Claims (9)

An anti-inflammatory agent containing 5-hydroxyindoleacetic acid. The anti-inflammatory agent according to claim 1, wherein the anti-inflammatory agent is an anti-inflammatory agent for inflammation in the intestine. The anti-inflammatory agent according to claim 2, wherein the inflammation in the intestine is an inflammatory bowel disease. The anti-inflammatory agent according to any one of claims 1 to 3, which is an oral composition or a composition for transrectal administration. The anti-inflammatory agent according to any one of claims 1 to 4, which is a pharmaceutical or food product. Anti-inflammatory agent screening methods, including:
(1) Measure the amount of 5-hydroxyindoleacetic acid in a sample taken from a mammal to which the test substance was administered; and (2) Test substance that increases the amount of 5-hydroxyindoleacetic acid in the sample. , To be selected as an anti-inflammatory agent. The method of claim 6, wherein the sample is stool. Anti-inflammatory agent screening methods, including:
(1) Culturing mammalian-derived cells capable of producing 5-hydroxyindoleacetic acid in a medium containing a test substance;
(2) Measuring the amount of 5-hydroxyindoleacetic acid in the medium;
(3) Select a test substance that increases the amount of 5-hydroxyindoleacetic acid in the medium as an anti-inflammatory agent. The method according to claim 8, wherein the mammalian-derived cell capable of producing 5-hydroxyindoleacetic acid is an intestinal bacterium.

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