Receptors for hydrolyzable tannins and their use

Abstract

This technology identifies cell membrane molecules essential for the expression of tannin bioactivity and provides a method for screening bioactive agents. [Solution] A screening method for a physiologically active agent, comprising the step of evaluating whether a test substance has binding affinity to the Leucine Rich Repeats And Calponin Homology Domain Containing 2 (LRCH2) protein, wherein the test substance having binding affinity to the LRCH2 protein indicates that the test substance is the physiologically active agent, and the physiological activity is neurogenesis promotion, nerve activation, cognitive function promotion, antidiarrheal, intestinal health improvement, treatment of Crohn's disease, treatment of chronic colitis, miR-191-5p expression regulation, Let7a-5p expression regulation, anti-inflammatory, anti-cancer, inhibition of cell migration, anti-allergic, treatment of pulmonary hypertension, treatment of cerebrovascular disease, or treatment of neurodegenerative disease.

Description

[Technical Field]

[0001] The present invention relates to a receptor for hydrolyzable tannins and its use. More specifically, the present invention relates to a screening method for physiologically active agents, a screening kit for physiologically active agents, non-human animals or cells in which Leucine Rich Repeats And Calponin Homology Domain Containing 2 (LRCH2) has been knocked down or knocked out, and physiologically active agents having a binding substance to the LRCH2 protein as an active ingredient. [Background technology]

[0002] Tannins are water-soluble compounds found in plants that react with proteins, alkaloids, and metal ions, forming strongly bound, sparingly soluble salts. They are a general term for components found in polyphenols. More than 4,000 different molecular species of tannins are known to exist, and various biological regulatory effects have been reported. For example, the inventors previously demonstrated that oral administration of hydrolyzable tannins can promote the proliferation of nerve cells and increase brain mass in both young and aged mice. They also demonstrated that learning and memory abilities, as well as spatial memory abilities, are improved in both young and aged mice (see, for example, Patent Document 1). [Prior art documents] [Patent Documents]

[0003] [Patent Document 1] International Publication No. 2023 / 068176 [Overview of the project] [Problems that the invention aims to solve]

[0004] However, the mechanism of action of tannins remains unclear. The present invention aims to identify the cell membrane molecules essential for the expression of tannin physiological activity and to provide a technology for screening physiologically active agents. [Means for solving the problem]

[0005] The present invention includes the following embodiments. [1] A screening method for a physiologically active agent, comprising the step of evaluating whether a test substance has binding affinity to the Leucine Rich Repeats And Calponin Homology Domain Containing 2 (LRCH2) protein, wherein the test substance having binding affinity to the LRCH2 protein indicates that the test substance is the physiologically active agent, and the physiological activity is neurogenesis promotion, nerve activation, cognitive function promotion, antidiarrheal, intestinal health improvement, treatment of Crohn's disease, treatment of chronic colitis, miR-191-5p expression regulation, Let7a-5p expression regulation, anti-inflammatory, anti-cancer, inhibition of cell migration, anti-allergic, treatment of pulmonary hypertension, treatment of cerebrovascular disease, or treatment of neurodegenerative disease. [2] The screening method according to [1], further comprising the steps of administering the test substance to a non-human animal or cell in which LRCH2 has been knocked down or knocked out, and evaluating the physiological activity that the test substance imparts to the non-human animal or cell, wherein the test substance is a physiological agent if it has binding affinity to the LRCH2 protein and does not exhibit significant physiological activity to the non-human animal or cell. [3] The screening method according to [1] or [2], further comprising the steps of administering the test substance to a wild-type non-human animal or wild-type cells, and evaluating the physiological activity that the test substance provides to the wild-type non-human animal or wild-type cells, wherein the test substance is a physiological agent if it has binding affinity to the LRCH2 protein and exhibits significant physiological activity in the wild-type non-human animal or wild-type cells. [4] A screening method for a physiologically active agent, comprising the steps of: administering a test substance to a non-human animal or cell in which LRCH2 has been knocked down or knocked out; and evaluating the physiological activity that the test substance imparts to the non-human animal or cell, wherein the test substance does not exhibit significant physiological activity to the non-human animal or cell, indicating that the test substance is a physiologically active agent, and the physiological activity is neurogenesis promotion, neuronal activation, cognitive function promotion, antidiarrheal, intestinal health improvement, treatment of Crohn's disease, treatment of chronic colitis, miR-191-5p expression regulation, Let7a-5p expression regulation, anti-inflammatory, anti-cancer, inhibition of cell migration, anti-allergic, treatment of pulmonary hypertension, treatment of cerebrovascular disease, or treatment of neurodegenerative disease. [5] The screening method according to [4], further comprising the steps of administering the test substance to a wild-type non-human animal or wild-type cells, and evaluating the physiological activity that the test substance imparts to the wild-type non-human animal or wild-type cells, wherein the test substance is the physiological agent if it does not exhibit significant physiological activity in a non-human animal or cells in which LRCH2 has been knocked down or knocked out, and exhibits significant physiological activity in the wild-type non-human animal or wild-type cells. [6] A screening kit for bioactive agents, comprising the LRCH2 protein, wherein the test substance exhibiting binding affinity to the LRCH2 protein indicates that the test substance is the bioactive agent, and the bioactivity is neurogenesis promotion, nerve activation, cognitive function promotion, antidiarrheal, intestinal health improvement, treatment of Crohn's disease, treatment of chronic colitis, regulation of miR-191-5p expression, regulation of Let7a-5p expression, anti-inflammatory, anti-cancer, inhibition of cell migration, anti-allergic, treatment of pulmonary hypertension, treatment of cerebrovascular disease or treatment of neurodegenerative disease. [7] The kit according to [6], further comprising a non-human animal or cell in which LRCH2 has been knocked down or knocked out. [8] Non-human animals or cells in which LRCH2 has been knocked down or knocked out. [9] A physiologically active agent comprising a binding substance to the LRCH2 protein as an active ingredient, wherein the physiological activity is to promote neurogenesis, activate nerves, enhance cognitive function, stop diarrhea, improve intestinal health, treat Crohn's disease, treat chronic colitis, regulate miR-191-5p expression, regulate Let7a-5p expression, anti-inflammatory, anti-cancer, inhibit cell migration, anti-allergic, treat pulmonary hypertension, treat cerebrovascular disease, or treat neurodegenerative disease.

[0006] The present invention can also be said to include the following embodiments. A physiologically active agent comprising a binding substance to the [P1]LRCH2 protein as an active ingredient, wherein the physiological activity is neurogenesis promotion, nerve activation, cognitive function enhancement, antidiarrheal, intestinal health improvement, treatment of Crohn's disease, treatment of chronic colitis, regulation of miR-191-5p expression, regulation of Let7a-5p expression, anti-inflammatory, anti-cancer, inhibition of cell migration, or anti-allergic. [P2] A screening method for a physiologically active agent as described in P1, comprising the step of evaluating whether or not the test substance has binding affinity to the LRCH2 protein, wherein the test substance having binding affinity to the LRCH2 protein indicates that the test substance is the physiologically active agent. [P3] The screening method according to P2, further comprising the steps of: administering the test substance to an LRCH2 knockout non-human animal or LRCH2 knockout cells if the test substance has binding affinity to the LRCH2 protein; and evaluating the physiological activity that the test substance exerts on the LRCH2 knockout non-human animal or LRCH2 knockout cells, wherein the test substance does not exhibit significant physiological activity on the LRCH2 knockout non-human animal or LRCH2 knockout cells, indicating that the test substance is the physiologically active agent. [P4] The screening method according to P2 or P3, further comprising the steps of administering the test substance to a wild-type non-human animal or wild-type cells, and evaluating the physiological activity that the test substance provides to the wild-type non-human animal or wild-type cells, wherein the test substance exhibiting significant physiological activity to the wild-type non-human animal or wild-type cells indicates that the test substance is the physiological agent. [P5] LRCH2 knockout non-human animals or LRCH2 knockout cells. [P6] A screening kit for the bioactive agent described in P1, comprising the LRCH2 protein. [P7] The kit described in P6, further comprising LRCH2 knockout non-human animals or LRCH2 knockout cells. [Effects of the Invention]

[0007] According to the present invention, a technique for screening bioactive agents based on cell membrane molecules essential for the expression of tannin's bioactivity can be provided. [Brief explanation of the drawing]

[0008] [Figure 1] Figure 1 shows the chemical formula of gallotannin used in Experimental Example 1. [Figure 2] Figure 2 shows the chemical formula of ellagutannin used in Experimental Example 1. [Figure 3] Figure 3 shows the chemical formulas of food polyphenols other than gallotannin and ellagutannin used in Experimental Example 1. [Figure 4] Figure 4 is a graph showing the results of the quartz crystal microbalance (QCM) analysis in Experimental Example 1. [Figure 5] Figure 5 is a graph showing the results of the QCM analysis in Experimental Example 1. [Figure 6] Figure 6 is a graph showing the results of the QCM analysis in Experimental Example 1. [Figure 7] Figure 7 is a graph showing the results of measuring cell proliferation of Caco-2 cells in Experimental Example 2. [Figure 8] Figure 8 is a graph showing the results of the Y-maze test in Experimental Example 3. [Figure 9] The upper part of Figure 9 is a schematic diagram explaining the T-maze test performed in Experimental Example 3. The lower part of Figure 9 is a graph showing the results of the T-maze test in Experimental Example 3. [Figure 10] Figure 10 is a graph showing the results of the novel object recognition test in Experimental Example 3. [Figure 11] Figure 11 is a graph showing the results of measuring the expression levels of phosphorylated CREB, BDNF, ARC, and EGR1 in the hippocampus of mice in Experimental Example 4. [Figure 12] Figure 12 is a graph showing the results of measuring the expression levels of GFAP and Iba-1 in the hippocampus of mice in Experimental Example 4. [Figure 13] Figure 13 is a graph showing the results of measuring the expression level of miR-191-5p in the plasma of mice in Experimental Example 5. [Figure 14] Figure 14 is a graph showing the results of measuring the expression levels of SOX2, DCX, and NeuN in the hippocampus of mice in Experimental Example 6. [Figure 15] Figure 15 is a graph showing the results of immunostaining thin sections of the hippocampus of mice in each group in Experimental Example 6 and quantifying newborn neurons (Ki67+, DAPI+) and immature neurons (NeuroD1+, DAPI+). [Figure 16] Figure 16 is a graph showing the results of the novel object recognition test in Experimental Example 7. [Figure 17] Figure 17 is a graph showing the results of measuring the abundance of phosphorylated CREB in the hippocampus of mice in Experimental Example 7. [Figure 18] Figure 18 is a graph showing the results of measuring the expression levels of SOX2, DCX, and NeuN in the hippocampus of mice in Experimental Example 8. [Figure 19] Figure 19 is a graph showing the measurement results of the expression level of let-7a-5p in the plasma of mice in Experimental Example 9. [Figure 20]Figure 20 is a graph showing the results of measuring the expression levels of ZO-1 and Occludin in the large intestine of mice in Experimental Example 10. [Figure 21] Figure 21 is a graph showing the results of measuring the expression levels of ZO-1 and Occludin in the large intestine of mice in Experimental Example 11. [Figure 22] Figure 22 shows the evaluation criteria for the Disease Activity Index Score (DAI score) used in Experimental Example 12. [Figure 23] Figure 23 is a graph showing the evaluation results for DAI score, stool condition, and bloody stool in Experimental Example 12. [Figure 24] Figure 24 is a graph showing the results of measuring PAK1 phosphorylation in the large intestine of mice in Experimental Example 13. [Figure 25] Figure 24 is a graph showing the results of measuring PAK1 phosphorylation in Caco-2 cells in Experimental Example 13. [Figure 26] Figure 26 is a graph showing the results of the novel object recognition test in Experiment Example 14. [Figure 27] Figure 27 shows images and graphs of the results of measuring the amount of phosphorylated CREB in the mouse hippocampus using the Western blotting method in Experimental Example 14. [Figure 28] Figure 28 is a graph showing the time course of body weight of mice in each group in Experiment Example 15. [Figure 29] Figure 29 shows graphs of DAI scores for each group of mice in Experimental Example 15, as well as representative stool images. [Figure 30] Figure 30 is a graph showing the results of the novel object recognition test and the T-maze test in Experiment Example 15. [Figure 31] Figure 31 shows images and graphs illustrating the results of measuring the amount of phosphorylated CREB in the mouse hippocampus using the Western blotting method in Experimental Example 15. [Modes for carrying out the invention]

[0009] [Notation of gene names and protein names] In this specification, human genes and human proteins shall be represented by uppercase letters. Mouse genes shall be represented by a capital letter for the first letter followed by lowercase letters. Mouse proteins shall be represented by uppercase letters. However, in some cases, human genes, mouse genes, genes of other species, human proteins, mouse proteins, and proteins of other species may not be strictly distinguished.

[0010] [Screening methods for bioactive agents] In one embodiment, the present invention provides a screening method for a physiologically active agent, comprising the step of evaluating whether a test substance has binding affinity to the LRCH2 protein, wherein the test substance having binding affinity to the LRCH2 protein indicates that the test substance is the physiologically active agent, and the physiological activity is neurogenesis promotion, nerve activation, cognitive function promotion, antidiarrheal, intestinal health improvement, treatment of Crohn's disease, treatment of chronic colitis, regulation of miR-191-5p expression, regulation of Let7a-5p expression, anti-inflammatory, anti-cancer, inhibition of cell migration, anti-allergic, treatment of pulmonary hypertension, treatment of cerebrovascular disease, or treatment of neurodegenerative disease.

[0011] As described later in the examples, the inventors identified LRCH2 as a receptor protein essential for the cognitive function-enhancing and hippocampal neurogenesis-promoting effects of hydrolyzable tannins. The inventors further revealed that hydrolyzable tannins (gallotannins and ellagutannins) with glucose as a core structure and three or more esterified phenol groups exhibit binding affinity to LRCH2.

[0012] The NCBI accession number for the human LRCH2 protein is NP_001230892.1, etc. The NCBI accession numbers for the human LRCH2 gene cDNA are NM_001243963.2, NM_020871.4, etc. The NCBI accession number for the mouse LRCH2 protein is NP_001074642.1, etc. The NCBI accession number for the mouse LRCH2 gene cDNA is NM_001081173.1, etc.

[0013] The inventors also succeeded in creating LRCH2 knockout mice. As described later in the examples, they revealed that the effects of hydrolyzable tannins, such as inhibitory action on cancer cell proliferation, cognitive enhancement, neuroactivation, neurogenesis promotion, miRNA expression regulation, tight junction-related protein expression promotion, suppression of diarrhea and bloody stools in inflammatory bowel disease, and anti-inflammatory effects in the large intestine, are all mediated by LRCH2.

[0014] In other words, LRCH2 is a receptor for hydrolyzable tannins, and substances that bind to LRCH2 exhibit physiological activities such as promoting neurogenesis, activating nerves, enhancing cognitive function, stopping diarrhea, improving intestinal health, treating Crohn's disease, treating chronic colitis, regulating miR-191-5p expression, regulating Let7a-5p expression, anti-inflammatory, anti-cancer, inhibiting cell migration, anti-allergic, treating pulmonary hypertension, treating cerebrovascular disease, and treating neurodegenerative diseases.

[0015] Therefore, LRCH2 is useful as a drug target. The screening method of this embodiment can be used to screen for bioactive agents that mimic the physiological effects of hydrolyzable tannins.

[0016] The test substances are not particularly limited and can include, for example, a library of natural compounds, a library of synthetic compounds, a library of existing drugs, a library of metabolites, etc. It is preferable that the test substances are diverse and not limited to hydrolyzable tannins.

[0017] The method for evaluating whether or not a test substance has binding affinity to the LRCH2 protein is not particularly limited; for example, the quartz crystal microbalance (QCM) analysis used in the examples, Biacore TM This can be performed using surface plasmon resonance (SPR) analysis with a device such as Cytiva. Alternatively, the LRCH2 protein may be modified to emit light when the test substance binds to it, and the ability of the test substance to bind to the LRCH2 protein may be evaluated by detecting this emission. Alternatively, the LRCH2 protein may be modified to emit fluorescence upon irradiation with excitation light when the test substance binds to it, and the ability of the test substance to bind to the LRCH2 protein may be evaluated by detecting this fluorescence.

[0018] Hydrolyzable tannins are a general term for compounds that produce gallic acid or ellagic acid upon treatment with tannase or acid hydrolysis, and thousands of types exist in nature. Hydrolyzable tannins that produce gallic acid through the above treatment usually have a galloyl group. Hydrolyzable tannins that produce ellagic acid through the above treatment usually have gallic acid oligomer residues such as a hexahydroxydiphenoyl (HHDP) group.

[0019] Hydrolyzable tannins are classified into gallotannins and ellagtannins. Gallotannins are compounds in which one or more gallic acid groups are bonded to glucose. More specifically, gallotannins are compounds in which one or more hydroxyl groups of glucose are ester-bonded to the carboxyl group of gallic acid. Gallotannins have one or more galloyl groups and produce gallic acid by tannase treatment or acid hydrolysis treatment.

[0020] Examples of gallotannins include galloyl glucose or tannic acid. Examples of galloyl glucose include monogalloyl glucose, digalloyl glucose, trigaloyl glucose, tetragalloyl glucose, pentagalloyl glucose, hexagalloyl glucose, heptagalloyl glucose, octagalloyl glucose, nonagalloyl glucose, decagalloyl glucose, undecagalloyl glucose, dodecagalloyl glucose, etc. More specifically, examples include 1,4,6-tri-O-galloyl-β-D-glucose, 1,2,4,6-tetra-O-galloyl-β-D-glucose, 1,2,3,6-tetra-O-galloyl-β-D-glucose, 2,3,4,6-tetra-O-galloyl-β-D-glucose, and 1,2,3,4,6-penta-O-galloyl-β-D-glucose.

[0021] Ellagtannins are compounds that produce ellagic acid through hydrolysis. Examples of ellagtannins include strictinin, casaulicin, castalagin, becaragin, castalin, casalicin, casaallin, casaallin, kebradic acid, kevric acid, corinusin E, epipunicacortein A, furosin B, gemin D, granatin A, granatin B, grandinin, lagerstroemine, lambertianin C, peduncladin, punicacortein A, punicacortein B, punicacortein C, punicacortein D, punicaphorin, and punica Examples include lagin, punicalin, punigluconin, robulin A, robulin B, robulin C, robulin D, robulin E, rubusaviin C, sanguine H-4, sanguine H-5, sanguine H-6, sanguine H-10, stachiurin, terimiglandin I, terimiglandin II, terkebrin, terflavin A, terflavin B, tergalagin, 2-O-galloyl-punicalin, plecoxin A, geraniin, stenophyllanin A, stenophyllanin B, eugeniflorin D2, etc.

[0022] Hydrolyzable tannins can be obtained, for example, by extracting a raw material containing hydrolyzable tannins with water or alcohol, and then purifying it by column. Plants containing hydrolyzable tannins can be used as raw materials for hydrolyzable tannins. Examples of plants containing hydrolyzable tannins include plants of the Fagaceae, Lythraceae, Myrtaceae, and Rosaceae families.

[0023] The screening method of this embodiment may further include the steps of administering a test substance to a non-human animal or cell in which LRCH2 has been knocked down, or to a non-human animal or cell in which LRCH2 has been knocked out, and evaluating the physiological activity that the test substance provides to a non-human animal or cell in which LRCH2 has been knocked down or knocked out. In this case, the test substance is a physiological agent if it has binding affinity to the LRCH2 protein and does not show significant physiological activity to a non-human animal or cell in which LRCH2 has been knocked down or knocked out.

[0024] The fact that the test substance does not exhibit significant physiological activity in non-human animals or cells in which LRCH2 has been knocked down, or in non-human animals or cells in which LRCH2 has been knocked out, means that the test substance exhibits the above-mentioned physiological activity via LRCH2.

[0025] The step of evaluating the physiological activity of a test substance in non-human animals or cells in which LRCH2 has been knocked down, or in non-human animals or cells in which LRCH2 has been knocked out, can be carried out, for example, as described later in the examples, by administering the test substance to non-human animals or cells in which LRCH2 has been knocked down, or in non-human animals or cells in which LRCH2 has been knocked out, and measuring the physiological activity such as neurogenesis promotion, neuronal activation, cognitive function enhancement, antidiarrheal, intestinal health improvement, treatment of Crohn's disease, treatment of chronic colitis, regulation of miR-191-5p expression, regulation of Let7a-5p expression, anti-inflammatory, anti-cancer, inhibition of cell migration, anti-allergic, treatment of pulmonary hypertension, treatment of cerebrovascular disease, treatment of neurodegenerative disease, etc.

[0026] The promotion of neurogenesis can be evaluated by measuring the expression levels of neurogenesis-related proteins in brain tissue or cells. Examples of neurogenesis-related proteins include, but are not limited to, SOX2, DCX, and NeuN. An increase in the expression level of neurogenesis-related proteins indicates that neurogenesis has been promoted.

[0027] Neuronal activation can be evaluated by measuring the expression levels of neuronal activation-related proteins in brain tissue or cells. Examples of neuronal activation-related proteins include, but are not limited to, phosphorylated CREB. An increase in the expression level of neuronal activation-related proteins indicates that nerves have been activated.

[0028] Cognitive function enhancement can be evaluated using mice by conducting novel object recognition tests, Y-maze tests, T-maze tests, etc., but is not limited to these.

[0029] Treatment for diarrhea, bowel health improvement, Crohn's disease, and chronic colitis can be evaluated, for example, using a DSS-induced inflammatory bowel disease model, by assessing the Disease Activity Index Score (DAI score), stool condition, bloody stools, and immunochemical analysis of intestinal tissue, but are not limited to these methods.

[0030] The regulation of miR-191-5p expression and Let7a-5p expression can be evaluated by administering a test substance to non-human animals or cells in which LRCH2 is knocked out and measuring the changes in the expression of these miRNAs, but is not limited to this method.

[0031] Anti-inflammatory effects can be evaluated, but are not limited to, by administering a test substance to non-human animals or cells in which LRCH2 has been knocked out and measuring the expression levels of inflammatory marker genes or proteins.

[0032] Anticancer effects can be evaluated by, but are not limited to, assessing the proliferation of cancer cells in the presence and absence of the test substance. If the proliferation of cancer cells is suppressed, the test substance demonstrates anticancer activity.

[0033] The suppression of cell migration can be evaluated by, but is not limited to, measuring cell migration in the presence and absence of the test substance.

[0034] Antiallergies can be evaluated, but are not limited to, by administering a test substance to non-human animals or cells in which LRCH2 has been knocked out and measuring the expression levels of allergy-related marker genes or proteins.

[0035] Examples of non-human animals include mammals, such as rodents like mice, rats, hamsters, and guinea pigs; ungulates like pigs, cows, goats, horses, and sheep; carnivores like dogs and cats; and primates like rhesus macaques, crab-eating macaques, marmosets, orangutans, and chimpanzees. The cells themselves may be derived from the non-human animals mentioned above, or they may be derived from humans.

[0036] LRCH2 knockdown can be performed, for example, by introducing siRNA or shRNA targeting LRCH2 into cells. LRCH2 knockout can be performed by genome editing, introduction of targeting vectors, etc. LRCH2 knockout cells may be cells obtained from LRCH2 knockout non-human animals, or cells in which the LRCH2 gene has been disrupted by genome editing or the like.

[0037] miR-191-5p and miR-191-3p are sometimes referred to simply as miR-191. While miR-191 is significantly downregulated in end-stage erythrocyte differentiation, its overexpression has been shown to inhibit erythrocyte nuclear removal and chromatin condensation, suggesting that miR-191 plays an important role in erythrocyte production. Furthermore, it has been reported that miR-191 exhibits different expression patterns in monocytes and monocyte-derived dendritic cells.

[0038] Changes in miR-191 expression have been reported in various malignancies, including breast cancer, colorectal cancer, lung cancer, liver cancer, prostate cancer, pancreatic cancer, gastric cancer, ovarian cancer, pituitary adenoma, esophageal squamous cell carcinoma, oral squamous cell carcinoma, osteosarcoma, bladder cancer, anaplastic large cell lymphoma, acute myeloid leukemia, severe medulloblastoma, retinoblastoma, thyroid follicular tumor, male breast cancer, and malignant melanoma.

[0039] Abnormal expression and dysfunction of miR-191 have been reported in various diseases other than cancer. For example, miR-191 has been suggested to be a potentially useful diagnostic biomarker for differentiating Crohn's disease from ulcerative colitis. Furthermore, miR-191 has been shown to be a potential biomarker for pulmonary hypertension, enabling not only early detection of pulmonary hypertension but also indicating the severity of the disease. In addition, miR-191 has been found to be induced in nephrotic syndrome in children, making it a potential diagnostic marker for this disease.

[0040] miR-let-7a-5p and miR-let-7a-3p are sometimes referred to simply as miR-let-7a. Similarly, miR-let-7a-5p is sometimes referred to as let-7a-5p or Let7a-5p. let-7a-5p is known to be a microRNA that targets multiple oncogenes, inhibiting cell migration, invasion, and epithelial-mesenchymal transition. Furthermore, it has been reported that inducing overexpression of let-7a in breast cancer cells reduces cell proliferation, colony formation, migration, and invasion. Decreased expression of miR-let-7a-5p has been reported to be associated with neoplastic diseases. In other words, miR-let-7a functions as a tumor suppressor, and its expression is decreased in many types of cancer. Decreased expression of miR-let-7a-5p has been reported in many diseases, including lung squamous cell carcinoma, colorectal cancer, urothelial tumors, lymphoma, and breast cancer.

[0041] In addition to its role in tumor suppression, miRNA-let-7a has been reported to be involved in cell proliferation pathways in human cells and has been linked to cerebrovascular and neurodegenerative diseases. miRNA-let-7a has been reported to directly alter cell cycle progression and inflammatory cytokine production in the brain. In inflammatory responses, miRNA-let-7a is involved in suppressing nitrite production, inducible nitric oxide synthase (iNOS), and interleukin-6 expression, and is involved in increasing the expression of brain-derived inflammatory cytokines in microglia. Therefore, miRNA-let-7a is thought to function as a microglial regulatory factor in inflammation.

[0042] The screening method of this embodiment may further include the steps of administering a test substance to a wild-type non-human animal or wild-type cells, and evaluating the physiological activity that the test substance exerts on the wild-type non-human animal or wild-type cells. In this case, the test substance being a physiological agent is indicated by its binding affinity to the LRCH2 protein and its significant physiological activity in wild-type non-human animals or wild-type cells.

[0043] The fact that the test substance does not show significant physiological activity in non-human animals or cells in which LRCH2 has been knocked down, or in non-human animals or cells in which LRCH2 has been knocked out, but shows significant physiological activity in wild-type non-human animals or wild-type cells, means that the test substance exhibits the above-mentioned physiological activity via LRCH2.

[0044] The process of evaluating the physiological activity of the test substance on wild-type non-human animals or wild-type cells can be carried out, for example, by administering the test substance to wild-type non-human animals or wild-type cells, as described later in the examples, and measuring the physiological activity such as neurogenesis promotion, neuronal activation, cognitive function enhancement, antidiarrheal effect, intestinal health improvement, treatment of Crohn's disease, treatment of chronic colitis, regulation of miR-191-5p expression, regulation of Let7a-5p expression, anti-inflammatory effect, anti-cancer effect, inhibition of cell migration, anti-allergic effect, treatment of pulmonary hypertension, treatment of cerebrovascular disease, and treatment of neurodegenerative disease.

[0045] In one embodiment, the present invention provides a screening method for a physiologically active agent, comprising the steps of: administering a test substance to a non-human animal or cell in which LRCH2 has been knocked down or knocked out; and evaluating the physiological activity that the test substance exerts on the non-human animal or cell, wherein the absence of significant physiological activity in the non-human animal or cell indicates that the test substance is the physiologically active agent, and the physiological activity is neurogenesis promotion, neuronal activation, cognitive function promotion, antidiarrheal, intestinal health improvement, treatment of Crohn's disease, treatment of chronic colitis, miR-191-5p expression regulation, Let7a-5p expression regulation, anti-inflammatory, anti-cancer, inhibition of cell migration, anti-allergic, treatment of pulmonary hypertension, treatment of cerebrovascular disease, or treatment of neurodegenerative disease.

[0046] The screening method of this embodiment differs from the screening method described above in that it does not include a step of evaluating whether or not the test substance has binding affinity to the LRCH2 protein.

[0047] The absence of significant physiological activity of the test substance in non-human animals or cells in which LRCH2 has been knocked down, or in non-human animals or cells in which LRCH2 has been knocked out, means that the test substance exhibits the above-mentioned physiological activity via LRCH2.

[0048] The screening method of this embodiment further comprises the steps of administering the test substance to a wild-type non-human animal or wild-type cell, and evaluating the physiological activity that the test substance exerts on the wild-type non-human animal or wild-type cell, wherein the test substance does not show significant physiological activity in a non-human animal or cell in which LRCH2 has been knocked down or knocked out, and shows significant physiological activity in the wild-type non-human animal or wild-type cell, thereby indicating that the test substance is the physiological activator.

[0049] Even without evaluating whether the test substance has binding affinity to the LRCH2 protein, the fact that the test substance does not show significant physiological activity in non-human animals or cells with LRCH2 knocked down, or in non-human animals or cells with LRCH2 knocked out, but shows significant physiological activity in wild-type non-human animals or wild-type cells, means that the test substance exhibits the above-mentioned physiological activity via LRCH2.

[0050] [Screening kit for bioactive agents] In one embodiment, the present invention provides a screening kit for bioactive agents, comprising an LRCH2 protein, wherein the test substance exhibiting binding affinity to the LRCH2 protein indicates that the test substance is the bioactive agent, and the bioactivity is neurogenesis promotion, nerve activation, cognitive function enhancement, antidiarrheal, intestinal health improvement, treatment of Crohn's disease, treatment of chronic colitis, miR-191-5p expression regulation, Let7a-5p expression regulation, anti-inflammatory, anti-cancer, inhibition of cell migration, anti-allergic, treatment of pulmonary hypertension, treatment of cerebrovascular disease, or treatment of neurodegenerative disease. The screening method described above can be suitably carried out using the kit of this embodiment.

[0051] In the kit of this embodiment, the LRCH2 protein, test substance, binding affinity to the LRCH2 protein, physiological activity, etc., are the same as those described above.

[0052] The kit of this embodiment may further include a non-human animal or cell in which LRCH2 has been knocked down, or a non-human animal or cell in which LRCH2 has been knocked out.

[0053] [Non-human animals or cells] In one embodiment, the present invention provides a non-human animal or cell in which LRCH2 is knocked down, or a non-human animal or cell in which LRCH2 is knocked out. The non-human animal or cell of this embodiment can be used as a model for analyzing the mechanism by which the above-mentioned physiological activity is expressed via LRCH2.

[0054] In this embodiment, the LRCH2, non-human animal, cell, etc., are the same as those described above.

[0055] [Physiologically active agents] In one embodiment, the present invention provides a physiologically active agent comprising a binding substance to the LRCH2 protein as an active ingredient, wherein the physiological activity is to promote neurogenesis, activate nerves, enhance cognitive function, stop diarrhea, improve intestinal health, treat Crohn's disease, treat chronic colitis, regulate miR-191-5p expression, regulate Let7a-5p expression, have anti-inflammatory and anti-cancer effects, inhibit cell migration, have anti-allergic effects, treat pulmonary hypertension, treat cerebrovascular disease, or treat neurodegenerative disease.

[0056] The physiologically active agent of this embodiment binds to the LRCH2 receptor and exhibits physiological activity. Preferably, the physiologically active agent of this embodiment is a variety of substances, not limited to hydrolyzable tannins. When the physiologically active agent of this embodiment is a hydrolyzable tannin, the hydrolyzable tannin is a gallotannin or ellagtannin having glucose as its core structure and three or more esterified phenol groups. [Examples]

[0057] The present invention will now be described in more detail with reference to examples, but the present invention is not limited to the following examples.

[0058] [Materials and Methods] (Mice and diet) During the pre-training period, all mice were acclimatized to the environment by being fed MF diet (KBT Oriental). Male C57BL / 6J mice were obtained from Kyudo Co., Ltd. After a one-week acclimatization period with MF diet, 6-8 mice were maintained as groups without isolation and fed standard diet. Aged mice (60 and 64 weeks old) or young mice (6 weeks old) were randomly divided into two groups of 7-8 mice each. The control group (Cont. group) was given water and orally administered PGG (Matrix Scientific, 098343) or strictinin (Nagara Science, NH026102). Each mouse was given 10g of diet per day, and water was freely available. All mice were housed under controlled conditions of 22±1℃ temperature, 50±10% relative humidity, and a 12-hour light-dark cycle (lights on from 8am to 8pm).

[0059] All animal experiments were conducted in accordance with the Act on Welfare of Laboratory Animals (Laboratory Animal Welfare Act No. 105) and its notification (Laboratory Animal Welfare Act No. 6). All research protocols were approved by the Kyushu University Animal Experiment Committee and followed its guidelines.

[0060] (Lrch2 - / - (Creation of mice) C57BL / 6J-Lrch2 em1cyagen Knockout (KO) mice (female) were established using Cyagen Biosciences' CRISPR / Cas9 technology (project number: KOAI-201016-CEA-01). C57BL / 6J Lrch2 KO mice (male) were established using C57BL / 6J-Lrch2. em1cyagenIt was generated by mating knockout mice (female) with C57BL / 6J wild-type mice (male). All in vivo experiments used male Lrch2 KO mice at 6 weeks of age.

[0061] (Mouse behavioral experiments) <Novel object recognition test> To evaluate the recognition memory of mice, a novel object recognition test was conducted. Prior to training, each mouse was acclimated in the test area for 1 hour. In the training phase, the mice were exposed to two known objects for 5 minutes, during which the recognition behavior was measured by recording the time of approaching, contacting, and sniffing the objects. After 24 hours, to evaluate the memory retention of each mouse, one of the original blocks was replaced with a novel object, and the recognition behavior during the 5-minute test period was recorded.

[0062] <Y-maze test> To examine spontaneous alternation behavior and recognition memory, a Y-maze test was performed. The Y-maze was composed of three arms in a Y-shape with a length of 45 cm, a height of 10 cm, and a width of 10 cm, and the angle between the arms was 120°. Each arm of the Y-maze was randomly designated as area A, B, or C. Rodents tend to explore new arms of the maze rather than return to previously entered arms. When a mouse entered three different arms consecutively, it was regarded as one alternation behavior (e.g., A, B, C, or B, C, A). The percentage of spontaneous alternation behavior was calculated using the following formula: (number of spontaneous alternation behaviors) / (total number of entries into the arms - 2) × 100. An entry or exit into an arm was recorded when the mouse's hind foot passed the boundary of the arm.

[0063] <T-maze test> The T-maze test evaluated the spatial learning and memory of mice. The T-maze consisted of a 30 cm long starting arm and two selection arms. The experiment began with placing the mice on the starting arm and allowing them to move freely for 5 minutes. The right arm of the T-maze was blocked. 24 hours after the initial trial, the right arm was unblocked, and the mice were again placed at the bottom of the T-maze and allowed to explore for 5 minutes. The number of times each arm was entered and the number of times the walls were touched were recorded. Spatial perception ability (%) was calculated using the following formula. Spatial perception ability (%) = (Number of times the left and right arms touch the wall) / (Number of times the left and right arms enter the wall) × 100

[0064] (Creation of genetically modified cells) LRCH2 knockout Caco-2 cells were generated using the CRISPR / Cas9 LRCH2 human gene knockout kit (Origene, KN413512D). (5.0 × 10⁶ cells) 4 Caco-2 cells were seeded in 12-well plates and co-transfected with 1 μg of LRCH2 gRNA vector from a pCas-Guide CRISPR vector and 1 μg of linear donor DNA containing LoxP-EF1A-tGFP-P2A-Puro-LoxP, which allows insertion of reporter genes such as fluorescent protein (GFP) and antibiotic resistance marker (puromycin), using the Lipofectamine 3000 transfection kit (Thermo Fisher Scientific, L3000001) according to the manufacturer's protocol. Two days after transfection, cells were selected with puromycin (10 μg / mL) for 5 days, and individual colonies were isolated. Subsequently, Western blotting and GFP fluorescence observation were performed to confirm knockout of the target gene.

[0065] (Quartz Oscillator Microbalance (QCM) Analysis) ≪Purification of LRCH2 protein≫ Escherichia coli BL21 transformed with the pET30a-hLRCH2 vector was cultured with shaking at 37°C in LB medium containing kanamycin (final concentration 100 μg / mL). LRCH2 expression was induced by adding 0.5 mM IPTG and culturing with shaking at 37°C for 2 hours. The E. coli were centrifuged at 6,000 × g for 10 minutes, pelletized, and washed twice with phosphate-buffered saline (PBS). Cell lysis was performed by sonication in buffer A (20 mM Tris-HCl (Nacalai Tesque); 300 mM NaCl (Fujifilm Wako Pure Chemical Industries), 5 mM imidazole (Nacalai Tesque), 0.5 mg / mL lysozyme (Fujifilm Wako Pure Chemical Industries), 1× protease inhibitor cocktail (Sigmar-Aldrich), 100 mM phenylmethylsulfonyl fluoride (Fujifilm Wako Pure Chemical Industries), and 2 μg / mL aprotinin). Subsequently, an equal volume of binding buffer (198 mL of buffer A and 2 mL of 2 M imidazole (Nacalai Tesque)) was added to the lysis solution. The lysis solution was clarified by centrifugation (6,000 × g, 4°C for 10 minutes), and the supernatant was collected as a sample. Samples were subjected to a HiTrap Chelating HP column (GE Healthcare Life Sciences, 17040903), and LRCH2 was eluted with imidazole gradient buffers (100 mM, 300 mM, 500 mM). Elution was performed using a similar gradient with buffer B (buffer A with 500 mM imidazole added). Protein concentration was measured using a BCA kit, and the fraction with the highest protein content was pooled. The sample with the highest protein content was analyzed by SDS-PAGE, and BioDesign Dialysis Tubing was performed. TM The LRCH2 protein was dialyzed overnight at 4°C in buffer A using a cellulose membrane (molecular weight cutoff: 3,500) (BioDesign, D302-50). Finally, the LRCH2 protein was stored at -80°C.

[0066] <<Evaluation of the binding affinity between polyphenols and LRCH2 protein>> QCM analysis (Scinics, single Q0500) was performed by immobilizing LRCH2 protein (10 μg / mL) on a QCM sensor chip by air-drying it for more than 4 hours. Subsequently, PBS (Fujifilm Wako Pure Chemical Industries, 045-29795) was added for 30 minutes and held until a steady state was reached by the change in resonant frequency. Frequency shifts were observed with the injection of each polyphenol, suggesting interaction between LRCH2 and polyphenols. During measurement, frequency stabilization was considered appropriate if the frequency fluctuation was within 3 Hz per minute. Each polyphenol was diluted to a final concentration of 50 μM with PBS immediately before testing. The change in frequency was plotted against each polyphenol concentration, and the dissociation constant (Kd) value was determined by nonlinear least-squares fitting using the Michaelis-Menten equation in Q-UP Analysis version 1.02 software (Scinics).

[0067] (Measurement of cell proliferation and cytotoxicity) Cell proliferation and cytotoxicity were evaluated using the ATP lite 1 step (PerkinElmer, 6016731). Caco-2 cells transduced with scramble siRNA or LRCH2 siRNA were seeded in 96-well plates (2 × 10⁶ cells in 200 μL 10% FBS-DMEM). 4 (Cells / mL). After 24 hours, PGG (0 or 5 μM) was added to the cells and incubated for 48 hours. ATP lite solution was added according to the manufacturer's protocol and evaluated using the Envision plate reader system (PerkinElmer).

[0068] (statistical analysis) All data are presented as mean ± standard error (SEM). Various statistical tests were performed using GraphPad 6.0 software to determine statistical significance. These tests included the two-tailed t-test (independent t-test), Bonferroni test, Dunnett's test, and Newman-Keuls test.

[0069] [Experimental Example 1] (Hydrolyzable tannins bind to LRCH2.) To accurately determine whether LRCH2 is a receptor for hydrolyzable tannins, the binding affinity of the LRCH2 protein to various polyphenols was evaluated using quartz crystal microbalance (QCM) analysis.

[0070] As polyphenols, we used gallotannin shown in Figure 1, ellagutannin shown in Figure 2, and other food polyphenols besides gallotannin and ellagutannin shown in Figure 3.

[0071] Figures 4-6 are graphs showing the results of the QCM analysis. As shown in Figures 4 and 5, it was revealed that hydrolyzable tannins (gallotannins and ellagutannins) with glucose as a core structure and three or more esterified phenol groups exhibit binding affinity to LRCH2. On the other hand, as shown in Figure 6, other food polyphenols did not exhibit binding affinity to LRCH2.

[0072] These results support the idea that hydrolyzable tannins are receptors for LRCH2.

[0073] [Experimental Example 2] (PGG inhibits cancer cell proliferation in an LRCH2-dependent manner.) Caco-2 cells, a cell line derived from human colon adenocarcinoma, were introduced with either LRCH2 siRNA (siLRCH2) or scramble siRNA (siControl), and cell proliferation was compared after treatment with hydrolyzable tannins. Pentagalloyl glucose (PGG), a type of gallotannin, was used as the hydrolyzable tannin.

[0074] Figure 7 is a graph showing the results of measuring the cell proliferation of Caco-2 cells in each group. "***" indicates a statistically significant difference (p<0.001), and "ns" indicates no statistically significant difference.

[0075] As a result, Caco-2 cells introduced with scramble siRNA showed a significant decrease in cell number upon PGG treatment. On the other hand, Caco-2 cells introduced with siRNA against LRCH2 did not show suppression of cell proliferation upon PGG treatment. This result indicates that PGG suppresses cancer cell proliferation in an LRCH2-dependent manner.

[0076] [Experimental Example 3] (PGG enhances cognitive function in an LRCH2-dependent manner) To investigate the role of Lrch2, Lrch2 knockout (KO) mice were generated using CRISPR / Cas9 technology. gRNA and Cas9 protein were microinjected into fertilized embryos. Genotyping analysis of the offspring revealed deletions in exons 2-5 of the Lrch2 gene locus. Subsequently, the mice were bred, and 6-week-old Lrch2 KO male mice, confirmed by gel electrophoresis, were orally administered 10 mg / kg body weight of hydrolyzable tannin for 7 days. The effects on brain function were evaluated on days 6 and 7. PGG was used as the hydrolyzable tannin.

[0077] When wild-type (WT) mice and Lrch2 KO mice were compared after matching age, no pathological abnormalities were observed in physiological parameters such as body weight, food intake, and organ weight.

[0078] Figure 8 is a graph showing the results of the Y-maze test. In Figure 8, "*" indicates a statistically significant difference (p<0.05), and "ns" indicates no statistically significant difference.

[0079] The upper part of Figure 9 is a schematic diagram explaining the T-maze test. The lower part of Figure 9 is a graph showing the results of the T-maze test. In Figure 9, "*" indicates a statistically significant difference at p<0.05, "**" indicates a statistically significant difference at p<0.01, "***" indicates a statistically significant difference at p<0.001, and "ns" indicates no statistically significant difference.

[0080] Figure 10 is a graph showing the results of the novel object recognition test. In Figure 10, "F" indicates a known object, "N" indicates a novel object, "*" indicates a statistically significant difference at p<0.05, "***" indicates a statistically significant difference at p<0.001, and "ns" indicates no statistically significant difference.

[0081] The results showed that the cognitive function improvements observed in WT mice following oral administration of PGG in the Y-maze test, T-maze test, and novel object recognition test were abolished in Lrch2 KO mice. This result indicates that PGG enhances cognitive function in an LRCH2-dependent manner.

[0082] [Experimental Example 4] (PGG activates neurons in an LRCH2-dependent manner.) WT mice and Lrch2 KO mice were orally administered 10 mg / kg body weight of hydrolyzable tannin for 7 days, then euthanized, and their hippocampi were removed. PGG was used as the hydrolyzable tannin. Subsequently, the amounts of neuronal activation-related proteins in the hippocampus were measured. For comparison, the amounts of inflammation-related proteins in the hippocampus were also measured. Neuronal activation-related proteins measured included phosphorylated CREB, BDNF, ARC, and EGR1. Inflammation-related proteins measured included GFAP and Iba-1.

[0083] Figure 11 is a graph showing the results of measuring the expression levels of phosphorylated CREB, BDNF, ARC, and EGR1 in the hippocampus. In Figure 11, "t-CREB" represents all CREB, "p-CREB" represents phosphorylated CREB, "*" indicates a statistically significant difference at p<0.05, "**" indicates a statistically significant difference at p<0.01, "***" indicates a statistically significant difference at p<0.001, and "ns" indicates no statistically significant difference.

[0084] Figure 12 is a graph showing the results of measuring the expression levels of GFAP and Iba-1 in the hippocampus. In Figure 12, "ns" indicates no significant difference.

[0085] The results showed that the expression levels of nerve activation-related proteins, which increased with PGG administration, disappeared in Lrch2 KO mice. On the other hand, no difference was observed in the expression levels of inflammation-related proteins between WT mice and Lrch2 KO mice. These results indicate that PGG activates nerves in an LRCH2-dependent manner.

[0086] [Experimental Example 5] (PGG reduces miR-191-5p expression in an LRCH2-dependent manner.) After orally administering 10 mg / kg body weight of hydrolyzable tannins to WT mice and Lrch2 KO mice for 7 days, the expression level of miR-191-5p in plasma was measured.

[0087] Figure 13 is a graph showing the results of measuring the expression level of miR-191-5p in plasma. In Figure 13, "*" indicates a statistically significant difference (p<0.05), and "ns" indicates no statistically significant difference.

[0088] The results showed that the decrease in plasma miR-191-5p expression observed with PGG administration disappeared in Lrch2 KO mice. This indicates that PGG reduces miR-191-5p expression in an LRCH2-dependent manner.

[0089] [Experimental Example 6] (PGG promotes neurogenesis in an LRCH2-dependent manner.) WT mice and Lrch2 KO mice were orally administered 10 mg / kg body weight of hydrolyzable tannin for 7 days, then euthanized, and their hippocampi were removed. PGG was used as the hydrolyzable tannin. Subsequently, the expression levels of neurogenesis-related proteins in the hippocampus were measured. The neurogenesis-related proteins measured were SOX2, DCX, and NeuN.

[0090] Figure 14 is a graph showing the measurement results of SOX2, DCX, and NeuN expression levels. In Figure 14, "*" indicates a statistically significant difference at p<0.05, "**" indicates a statistically significant difference at p<0.01, and "ns" indicates no statistically significant difference.

[0091] As a result, it was shown that the expression levels of neurogenesis-related proteins, which had increased upon PGG administration, disappeared in Lrch2 KO mice.

[0092] Figure 15 is a graph showing the results of immunostaining thin sections of the hippocampus of mice in each group and quantifying newborn neurons (Ki67 + , DAPI + ) and immature neurons (NeuroD1 + , DAPI + ).

[0093] As a result, it was shown that the increase in newborn neurons (DAPI + , Ki67 + ) and immature neurons (NeuroD1 + , DAPI + ) observed upon oral administration of PGG disappeared in Lrch2 KO mice.

[0094] The above results indicate that PGG promotes neurogenesis in a LRCH2-dependent manner.

[0095] [Experimental Example 7] (Stricin enhances cognitive function in a LRCH2-dependent manner) Using Lrch2 KO mice, 10 mg / kg body weight of hydrolyzable tannin was orally administered for 7 days, and the effects on brain function were evaluated on the 6th and 7th days. As the hydrolyzable tannin, stricin, which is a type of ellagitannin, was used.

[0096] Figure 16 is a graph showing the results of the novel object recognition test. In Figure 16, "Stric." indicates stricin, "F" indicates a known object, "N" indicates a novel object, "*" indicates a significant difference at p < 0.05, "***" indicates a significant difference at p < 0.001, and "n.s." indicates no significant difference.

[0097] The results showed that the improvement in cognitive function observed in WT mice after oral administration of strictin in a novel object recognition test was lost in Lrch2 KO mice. This result indicates that strictin enhances cognitive function in an LRCH2-dependent manner.

[0098] WT mice and Lrch2 KO mice were orally administered 10 mg / kg body weight of hydrolyzable tannin for 7 days, then euthanized, and their hippocampi were removed. Strictinin was used as the hydrolyzable tannin. Subsequently, the amount of neuronal activation-related proteins in the hippocampus was measured. Phosphorylated CREB was measured as the neuronal activation-related protein.

[0099] Figure 17 is a graph showing the results of measuring the amount of phosphorylated CREB present in the hippocampus. In Figure 17, "Stric." represents strictinin, "t-CREB" represents total CREB, "p-CREB" represents phosphorylated CREB, "*" indicates a statistically significant difference (p<0.05), and "ns" indicates no statistically significant difference.

[0100] The results showed that the expression levels of neuronal activation-related proteins, which were increased by strictinin administration, were abolished in Lrch2 KO mice.

[0101] These results indicate that strictin activates neurons in an LRCH2-dependent manner.

[0102] [Experimental Example 8] (Strictinin increases the expression of neurogenesis-related proteins in an LRCH2-dependent manner.) Wild-type (WT) mice and Lrch2 knockout (LKO) mice were orally administered 10 mg / kg body weight of hydrolyzable tannin for 7 days, then euthanized, and their hippocampi were removed. Strictinin was used as the hydrolyzable tannin. Subsequently, the expression levels of neurogenesis-related proteins in the hippocampus were measured. The neurogenesis-related proteins measured were SOX2, DCX, and NeuN.

[0103] Figure 18 is a graph showing the measurement results of SOX2, DCX, and NeuN expression levels. In Figure 18, "*" indicates a statistically significant difference at p<0.05, "**" indicates a statistically significant difference at p<0.001, and "ns" indicates no statistically significant difference.

[0104] The results showed that the expression levels of neurogenesis-related proteins, which were increased by strictinin administration, disappeared in Lrch2 KO mice. This result indicates that strictinin increases the expression of neurogenesis-related proteins in an LRCH2-dependent manner.

[0105] [Experimental Example 9] (PGG increases the expression level of let-7a-5p in an LRCH2-dependent manner.) After orally administering 10 mg / kg body weight of hydrolyzable tannins to WT mice and Lrch2 KO mice for 7 days, the expression level of let-7a-5p in plasma was measured.

[0106] Figure 19 is a graph showing the results of measuring plasma let-7a-5p expression levels. In Figure 19, "*" indicates a statistically significant difference (p<0.05), "**" indicates a statistically significant difference (p<0.01), and "ns" indicates no statistically significant difference. "cel-39" refers to cel-miR-39, a synthetic RNA used as an internal standard.

[0107] The results showed that the increase in plasma let-7a-5p expression observed with PGG administration was absent in Lrch2 KO mice. This indicates that PGG increases let-7a-5p expression in an LRCH2-dependent manner.

[0108] [Experimental Example 10] (PGG increases the expression of tight junction-related proteins in an LRCH2-dependent manner.) WT mice and Lrch2 KO mice were orally administered 10 mg / kg body weight of hydrolyzable tannin for 7 days, then euthanized, and their large intestines were removed. PGG was used as the hydrolyzable tannin. Subsequently, the expression levels of tight junction-related proteins in the large intestine were measured. ZO-1 and Occludin were measured as tight junction-related proteins.

[0109] Figure 20 is a graph showing the measurement results of ZO-1 and Occludin expression levels. In Figure 20, "*" indicates a statistically significant difference (p<0.05), and "ns" indicates no statistically significant difference.

[0110] The results showed that the expression levels of tight junction-related proteins, which increased with PGG administration, disappeared in Lrch2 KO mice. This indicates that PGG increases the expression of tight junction-related proteins in an LRCH2-dependent manner.

[0111] [Experimental Example 11] (Strictinin increases the expression of tight junction-related proteins in an LRCH2-dependent manner.) WT mice and Lrch2 KO mice were orally administered 10 mg / kg body weight of hydrolyzable tannin for 7 days, then euthanized, and their large intestines were removed. Strictinin was used as the hydrolyzable tannin. Subsequently, the expression levels of tight junction-related proteins in the large intestine were measured. ZO-1 and Occludin were measured as tight junction-related proteins.

[0112] Figure 21 is a graph showing the measurement results of ZO-1 and Occludin expression levels. In Figure 20, "*" indicates a statistically significant difference at p<0.05, "**" indicates a statistically significant difference at p<0.01, and "ns" indicates no statistically significant difference.

[0113] The results showed that the expression levels of tight junction-related proteins, which were increased by strictinin administration, were abolished in Lrch2 KO mice. This result indicates that strictinin increases the expression of tight junction-related proteins in an LRCH2-dependent manner.

[0114] [Experimental Example 12] (Albumin tannins suppress diarrhea and bloody stools in an LRCH2-dependent manner.) We conducted a study using a DSS-induced inflammatory bowel disease model in which WT mice and Lrch2 KO mice were fed drinking water supplemented with 2% dextran sulfate sodium salt (DSS) for 7 days to induce inflammatory bowel disease.

[0115] A control group (no DSS), a group administered DSS, and a group administered DSS along with 20 mg / kg body weight orally daily were prepared. Weight loss rate, stool condition, and bloody stools were evaluated based on the Disease Activity Index Score (DAI score) shown in Figure 22.

[0116] Albumin tannin (CAS number: 9006-52-4), known as an antidiarrheal agent, was used as the hydrolyzable tannin.

[0117] Figure 23 is a graph showing the evaluation results for DAI score, stool condition, and bloody stool. In Figure 23, "C" indicates the results for the control group, "DSS" indicates the results for the DSS-administered group, and "DSS+AT" indicates the results for the group administered albumin tannin along with DSS. "*" indicates a statistically significant difference at p<0.05, "**" indicates a statistically significant difference at p<0.01, "***" indicates a statistically significant difference at p<0.001, and "ns" indicates no statistically significant difference.

[0118] The results showed that the suppression of diarrhea and bloody stools observed with albumin tannin administration disappeared in Lrch2 KO mice. This result indicates that albumin tannin suppresses diarrhea and bloody stools caused by inflammatory bowel disease in an LRCH2-dependent manner.

[0119] [Experimental Example 13] (PGG inhibits PAK phosphorylation levels in the colon in an LRCH2-dependent manner.) WT mice and Lrch2 KO mice were orally administered 10 mg / kg body weight of hydrolyzable tannin, euthanized 12 hours later, and their large intestines were removed. PGG was used as the hydrolyzable tannin. Subsequently, PAK1 phosphorylation in the large intestine was measured. PAK1 phosphorylation is known to be associated with various pathological conditions, including inflammation.

[0120] Figure 24 is a graph showing the results of measuring PAK1 phosphorylation. In Figure 24, "t-PAK1" represents all PAK1, "p-PAK1" represents phosphorylated PAK1, "*" indicates a statistically significant difference (p<0.05), and "ns" indicates no statistically significant difference.

[0121] The results showed that the suppression of PAK1 phosphorylation by PGG administration was abolished in Lrch2 KO mice. This result indicates that PGG suppresses PAK1 phosphorylation in an LRCH2-dependent manner.

[0122] The LRCH2 gene in Caco-2 cells, a cell line derived from human colon adenocarcinoma, was knocked out by genome editing. Subsequently, wild-type Caco-2 cells and Caco-2 cells with the LRCH2 gene knocked out were treated with hydrolyzable tannin for 3 hours, and then PAK1 phosphorylation was measured. PGG was used as the hydrolyzable tannin at a concentration of 10 μM.

[0123] Figure 25 is a graph showing the results of measuring PAK1 phosphorylation. In Figure 25, "t-PAK1" represents all PAK1, "p-PAK1" represents phosphorylated PAK1, "*" indicates a statistically significant difference (p<0.05), and "ns" indicates no statistically significant difference.

[0124] The results showed that the suppression of PAK1 phosphorylation by PGG administration was abolished in Lrch2 KO mice. This result indicates that PGG also suppresses PAK1 phosphorylation in an LRCH2-dependent manner in Caco-2 cells.

[0125] [Experimental Example 14] (Hydrolyzable tannins enhance cognitive function in mouse models of neurodegenerative diseases.) Alzheimer's disease model mice injected intravenously with amyloid-beta (Aβ)1-42 peptide were orally administered hydrolyzable tannins at a dose of 10 mg / kg body weight for two weeks (Sham group: n=9, Aβ group: n=6, Aβ+PGG group: n=6). PGG was used as the hydrolyzable tannin.

[0126] Next, a novel object recognition test was conducted. Subsequently, the mice in each group were euthanized, and their hippocampi were removed. Then, the amount of neuronal activation-related proteins in the hippocampus was measured. Phosphorylated CREB was measured as the neuronal activation-related protein.

[0127] Figure 26 is a graph showing the results of the novel object recognition test. In Figure 26, "**" indicates a statistically significant difference (p<0.01), and "ns" indicates no statistically significant difference. "Cont." indicates the results for Alzheimer's disease model mice (control) that were not administered PGG. The results showed that cognitive decline in Alzheimer's disease model mice was suppressed by PGG administration in the novel object recognition test.

[0128] Figure 27 shows images and graphs of the results of measuring the amount of phosphorylated CREB in the hippocampus using Western blotting. In Figure 27, "t-CREB" represents total CREB, "p-CREB" represents phosphorylated CREB, and "*" indicates a statistically significant difference at p<0.05. The results showed that the decrease in the proportion of phosphorylated CREB observed in Alzheimer's disease model mice was suppressed by the administration of PGG.

[0129] These results indicate that administration of hydrolyzable tannins increases neuronal activation-related proteins in the hippocampus of a mouse model of neurodegenerative disease, thereby enhancing cognitive function.

[0130] [Experimental Example 15] (Hydrolyzable tannins improve DAI scores and enhance cognitive function in mouse models of inflammatory bowel disease.) We conducted a study using a DSS-induced inflammatory bowel disease model in which mice were given drinking water supplemented with 2% dextran sulfate sodium salt (DSS) to induce inflammatory bowel disease.

[0131] A control group (no DSS), a DSS-administered group, and a group (DSS plus 10 mg / kg body weight orally administered daily) were prepared, and changes in body weight over time and DAI scores were evaluated. PGG was used as the hydrolyzable tannin. DAI scores were evaluated based on the Disease Activity Index Score (DAI score) shown in Figure 22.

[0132] Figure 28 is a graph showing the time course of body weight in mice from each group. In Figure 28, "Cont." indicates the results for control mice that were not administered DSS, "*" indicates a statistically significant difference (p<0.05), and "ns" indicates no statistically significant difference. The results showed that the inflammatory bowel disease mouse model administered PGG did not show a significant difference in body weight compared to the control mice.

[0133] Figure 29 shows graphs of DAI scores for each group of mice and representative stool images. In Figure 29, "***" indicates a statistically significant difference at p<0.001. The results showed that administration of PGG improved the DAI score in the inflammatory bowel disease mouse model.

[0134] Next, novel object recognition tests and T-maze tests were performed. Subsequently, the mice in each group were euthanized, and their hippocampi were removed. Then, the amount of neuronal activation-related proteins in the hippocampus was measured. Phosphorylated CREB was measured as a neuronal activation-related protein.

[0135] Figure 30 shows graphs illustrating the results of the novel object recognition test and the T-maze test. In Figure 30, "*" indicates a statistically significant difference (p<0.05), and "ns" indicates no statistically significant difference. The results showed that the decline in cognitive function in the inflammatory bowel disease mouse model was suppressed by PGG administration in the novel object recognition test. Furthermore, the decline in spatial working memory ability in the inflammatory bowel disease mouse model was suppressed by PGG administration in the T-maze test.

[0136] Figure 31 shows images and graphs of the results of measuring the amount of phosphorylated CREB in the hippocampus using Western blotting. In Figure 31, "t-CREB" represents total CREB, "p-CREB" represents phosphorylated CREB, "*" indicates a statistically significant difference (p<0.05), and "ns" indicates no statistically significant difference. The results showed that the decrease in the proportion of phosphorylated CREB observed in the inflammatory bowel disease mouse model was suppressed by the administration of PGG.

[0137] These results indicate that administration of hydrolyzable tannins increases neuronal activation-related proteins in the hippocampus of a mouse model of inflammatory bowel disease, thereby enhancing cognitive function. [Industrial applicability]

[0138] According to the present invention, a technique for screening bioactive agents based on cell membrane molecules essential for the expression of tannin's bioactivity can be provided.

Claims

1. A screening method for physiologically active agents, The procedure includes a step of evaluating whether the test substance has binding affinity to the Leucine Rich Repeats and Calponin Homology Domain Containering 2 (LRCH2) protein. The fact that the test substance has binding affinity to the LRCH2 protein indicates that the test substance is the physiologically active agent. A screening method in which the aforementioned physiological activity is neurogenesis promotion, nerve activation, cognitive function enhancement, antidiarrheal, intestinal health improvement, treatment of Crohn's disease, treatment of chronic colitis, regulation of miR-191-5p expression, regulation of Let7a-5p expression, anti-inflammatory, anti-cancer, inhibition of cell migration, anti-allergic, treatment of pulmonary hypertension, treatment of cerebrovascular disease, or treatment of neurodegenerative disease.

2. The process involves administering the aforementioned test substance to a non-human animal or cell in which LRCH2 has been knocked down or knocked out. The process further includes a step of evaluating the physiological activity that the test substance imparts to the non-human animal or the cells, The screening method according to claim 1, wherein the test substance has binding affinity to the LRCH2 protein and does not exhibit significant physiological activity in the non-human animal or the cell, thereby indicating that the test substance is the physiologically active agent.

3. The process involves administering the test substance to a wild-type non-human animal or wild-type cells. The process further includes a step of evaluating the physiological activity that the test substance imparts to the wild-type non-human animal or the wild-type cells, The screening method according to claim 1, wherein the test substance has binding affinity to the LRCH2 protein and exhibits significant physiological activity in the wild-type non-human animal or wild-type cells, thereby indicating that the test substance is the physiologically active agent.

4. A screening method for physiologically active agents, The process involves administering the test substance to a non-human animal or cell in which LRCH2 has been knocked down or knocked out, The step includes evaluating the physiological activity that the test substance imparts to the non-human animal or the cells, The fact that the test substance does not exhibit significant physiological activity in the non-human animal or the cell indicates that the test substance is the physiologically active agent. A screening method in which the aforementioned physiological activity is neurogenesis promotion, nerve activation, cognitive function enhancement, antidiarrheal, intestinal health improvement, treatment of Crohn's disease, treatment of chronic colitis, regulation of miR-191-5p expression, regulation of Let7a-5p expression, anti-inflammatory, anti-cancer, inhibition of cell migration, anti-allergic, treatment of pulmonary hypertension, treatment of cerebrovascular disease, or treatment of neurodegenerative disease.

5. The process involves administering the test substance to a wild-type non-human animal or wild-type cells. The process further includes a step of evaluating the physiological activity that the test substance imparts to the wild-type non-human animal or the wild-type cells, The screening method according to claim 4, wherein the test substance does not show significant physiological activity in non-human animals or cells in which LRCH2 has been knocked down or knocked out, and shows significant physiological activity in wild-type non-human animals or wild-type cells, thereby indicating that the test substance is the physiologically active agent.

6. A screening kit for bioactive agents, Contains LRCH2 protein, The fact that the test substance exhibits binding affinity to the LRCH2 protein indicates that the test substance is the physiologically active agent. The aforementioned physiological activity is neurogenesis promotion, nerve activation, cognitive function enhancement, antidiarrheal, intestinal health improvement, treatment of Crohn's disease, treatment of chronic colitis, regulation of miR-191-5p expression, regulation of Let7a-5p expression, anti-inflammatory, anti-cancer, inhibition of cell migration, anti-allergic, treatment of pulmonary hypertension, treatment of cerebrovascular disease, or treatment of neurodegenerative disease.

7. The kit according to claim 6, further comprising a non-human animal or cell in which LRCH2 has been knocked down or knocked out.

8. Non-human animals or cells in which LRCH2 has been knocked down or knocked out.

9. A physiologically active agent having a binding substance for the LRCH2 protein as its active ingredient, A physiologically active agent whose physiological activity is to promote neurogenesis, activate nerves, enhance cognitive function, stop diarrhea, improve intestinal health, treat Crohn's disease, treat chronic colitis, regulate miR-191-5p expression, regulate Let7a-5p expression, have anti-inflammatory and anti-cancer effects, inhibit cell migration, have anti-allergic effects, treat pulmonary hypertension, treat cerebrovascular disease, or treat neurodegenerative disease.

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