WO2019177099A1

WO2019177099A1 - Method for evaluating test sample

Info

Publication number: WO2019177099A1
Application number: PCT/JP2019/010547
Authority: WO
Inventors: 香織齋藤; 郁尚藤田; 文裕岡田; 石井　健
Original assignee: 株式会社マンダム; 国立研究開発法人医薬基盤・健康・栄養研究所
Priority date: 2018-03-15
Filing date: 2019-03-14
Publication date: 2019-09-19
Also published as: CN111601898A; JP2022068272A; JP7321662B2; KR20200093030A; JPWO2019177099A1; JP7027521B2

Abstract

Provided is a method for evaluating a test sample, said method making it easy to evaluate whether a test sample has the effect of suppressing cytokine production in skin cells, wherein the cytokine production suppressing effect of the test sample is evaluated on the basis of a physiological event triggered by the test sample through TRPM4 in TRPM4-expressing cells.

Description

Test sample evaluation method

The present invention relates to a method for evaluating a test sample. More specifically, the present invention relates to a test sample evaluation method and a cytokine production inhibitor for evaluating whether a test substance has a cytokine production inhibitory effect.

Inflammation on the skin causes acne scars, spots, wrinkles and the like. Therefore, cosmetics containing an anti-inflammatory agent that suppresses inflammation generated in the skin have been proposed (see, for example, Patent Document 1). However, since the anti-inflammatory agent suppresses inflammation that has already occurred in the skin, it suppresses the onset of inflammation by suppressing the production of cytokine, which is one of the causative factors of inflammation. Development of anti-inflammatory cosmetics and inflammation preventive agents that can be used is desired.

JP-A-2016-196418

The present invention has been made in view of the above prior art, and can be used to easily evaluate whether a test sample has an action of suppressing cytokine production in skin cells and cytokines in skin cells. It aims at providing the cytokine production inhibitor which can suppress production effectively.

The present invention
(1) A test sample evaluation method for evaluating a cytokine production inhibitory action in skin cells of a test sample, wherein the TRPM4 expressing cell is brought into contact with the test sample, and is caused by the test sample via TRPM4 in the TRPM4 expressing cell A method for evaluating a test sample, comprising measuring a physiological event and evaluating a cytokine production inhibitory action of the test sample based on the physiological event,
(2) The physiological event is cytokine production, the TRPM4 expression cell having cytokine expression ability is used as the TRPM4 expression cell, the physiological event is measured, and the cytokine possessed by the test sample based on the physiological event The operation when evaluating the production inhibitory action is
(A1) a step of contacting a TRPM4 expression cell having cytokine expression ability with a test sample and a cytokine production promoting substance, and measuring the expression level of the cytokine and / or its gene in the TRPM4 expression cell;
(A2) a step of contacting a TRPM4 expression cell having cytokine expression ability with a cytokine production promoting substance, and measuring the expression level of the cytokine and / or its gene in the TRPM4 expression cell;
(A3) contacting a TRPM4 deficient cell in which the TRPM4 function in the TRPM4 expressing cell is deficient with a test sample and a cytokine production promoter, and measuring the expression level of the cytokine and / or its gene in the TRPM4 deficient cell;
(A4) a step of contacting a TRPM4-deficient cell deficient in TRPM4 function in the TRPM4-expressing cell with a cytokine production promoting substance, and measuring the expression level of the cytokine and / or its gene in the TRPM4-deficient cell; and (A5) The test sample evaluation method according to (1), comprising the step of evaluating the cytokine production inhibitory action of the test sample based on the expression level measured in steps (A1) to (A4),
(3) The operation in measuring the physiological event and evaluating the cytokine production inhibitory effect of the test sample based on the physiological event,
(B1) contacting the TRPM4-expressing cell with the test sample, measuring a change in TRPM4 activity in the TRPM4-expressing cell before and after contact with the test sample, and (B2) changing the TRPM4 activity measured in step (B1) The method for evaluating a test sample according to (1), including the step of evaluating the cytokine production inhibitory action of the test sample, and (4) the use of activating TRPM4 to suppress cytokine production in skin cells The present invention relates to a cytokine production inhibitor used, comprising an aluminum compound as an active ingredient for activating TRPM4.

ADVANTAGE OF THE INVENTION According to this invention, the test sample evaluation method which can evaluate easily whether a test sample has the effect | action which suppresses the cytokine production in a skin cell, and the cytokine production in a skin cell can be suppressed effectively. Cytokine production inhibitors are provided.

In Reference example 1, it is a drawing substitute photograph which shows the result of a Western blot analysis. 6 is a graph showing the results of examining the expression level of IL-1α gene in each cell obtained in Examples 1-2 and Comparative Examples 1-2 in Test Example 1. 6 is a graph showing the results of examining the expression level of IL-8 gene in each cell obtained in Examples 1-2 and Comparative Examples 1-2 in Test Example 1. 6 is a graph showing the results of examining the expression level of the TNF gene in each cell obtained in Examples 1-2 and Comparative Examples 1-2 in Test Example 1. 6 is a graph showing the results of examining the expression level of IL-8 gene in each cell obtained in Comparative Examples 3 to 6 in Test Example 2. In the reference example 2, it is a graph which shows the result of having investigated the relationship between the presence or absence of a TRPM4 agonist and relative fluorescence intensity. In Example 3, it is a graph which shows the result of having investigated the relationship between the kind of test sample, and relative fluorescence intensity. 6 is a graph showing the results of examining the IL-1α expression level in each epidermal keratinocyte obtained in Example 4 and Comparative Example 7 in Test Example 3. 6 is a graph showing the results of examining the IL-6 expression level in each epidermal keratinocyte obtained in Example 4 and Comparative Example 7 in Test Example 3.

1. Test sample evaluation method The test sample evaluation method of the present invention is a test sample evaluation method for evaluating a cytokine production inhibitory action in skin cells of a test sample, wherein the TRPM4-expressing cells are brought into contact with the test sample, and the test sample is tested. A physiological event caused by TRPM4 in cells expressing TRPM4 is measured by the sample, and the cytokine production inhibitory action of the test sample is evaluated based on the physiological event.

According to the test sample evaluation method of the present invention, a physiological event caused by TRPM4-expressing cells via TRPM4 is measured by the test sample, and the cytokine production inhibitory effect of the test sample is evaluated based on the physiological event. Therefore, it is possible to accurately evaluate whether the test sample suppresses cytokine production due to TRPM4 activation in skin cells. Therefore, according to the test sample evaluation method of the present invention, it can be easily evaluated whether or not the test sample has an action of suppressing cytokine production in skin cells. In addition, according to the test sample evaluation method of the present invention, since TRPM4-expressing cells are used, the evaluation of the cytokine production inhibitory effect of the test sample should be performed with high reproducibility and simultaneously under the same conditions. Can do.

The test sample is a sample to be evaluated as to whether it has a cytokine production inhibitory effect. Examples of the test sample include inorganic compounds, organic compounds, plant extracts, cell cultures, cell extracts and the like, but the present invention is not limited to only such examples. When the test sample is a liquid, it may be used as it is, or may be diluted with a solvent as necessary. Moreover, when a test sample is solid, it can be dissolved in a solvent and used.

Since the solvent differs depending on the type of test sample and the type of physiological event to be measured and cannot be determined unconditionally, it is determined according to the type of test sample and the type of physiological event to be measured. It is preferable. Examples of the solvent include ethanol, physiological saline, phosphate buffered saline, purified water, medium, calcium-containing solution [composition: 140 mM sodium chloride, 5 mM potassium chloride, 2 mM magnesium chloride, 2 mM calcium chloride, 10 mM glucose. And 10 mM 2- [4- (2-hydroxyethyl) piperazin-1-yl] ethanesulfonic acid (HEPES) hydrochloric acid buffer (pH 7.4)], calcium-free solution [composition: 140 mM sodium chloride, 5 mM potassium chloride, 2 mM Magnesium chloride, 5 mM glycol ether diamine tetraacetic acid, 10 mM glucose, and 10 mM HEPES buffer (pH 7.4)], and the like, but the present invention is not limited to such examples. The medium contains medium components suitable for growing TRPM4-expressing cells. Examples of the medium component include glucose, amino acid, peptone, vitamin, cell growth promoting factor, serum, calcium chloride, magnesium chloride, and the like, but the present invention is not limited to such examples. The medium may be a medium produced by supplementing a basic medium with medium components, or a commercially available medium. The basic medium is not particularly limited, and examples thereof include an Eagle's minimum medium (hereinafter referred to as “MEM”), Dulbecco's modified Eagle medium (hereinafter referred to as “DMEM”), RPMI-1640 medium, and the like. It is not limited to illustration only. Since the medium varies depending on the type of TRPM4-expressing cells and the like and cannot be determined unconditionally, it is preferably determined according to the type of TRPM4-expressing cells.

TRPM4-expressing cells are cells that express the same physiological function as cells expressing wild-type TRPM4. Examples of physiological functions equivalent to cells expressing wild-type TRPM4 include permeation of potassium ions and sodium ions from outside the cells by stimulation such as chemical stimulation with a TRPM4 agonist. However, the present invention is not limited to such examples. The TRPM4-expressing cell may be an endogenous TRPM4-expressing cell that expresses endogenous TRPM4, or may be an exogenous TRPM4-expressing cell.

Examples of endogenous TRPM4-expressing cells include keratinocytes such as normal human epidermal keratinocytes, HaCaT cells, NCTC2544; T such as human cardiomyocytes, human T cells, Jurkat cells, MOLT-4 cells, U-937 cells and the like. Examples include cells; human mast cells, mast cells such as HMC-1; human monocyte cells, monocyte cells such as THP-1, and the like, but the present invention is not limited to such examples.

The exogenous TRPM4-expressing cell may be a cell that overexpresses exogenous TRPM4. The exogenous TRPM4 cell may be a cell in which the nucleic acid introduced into the host cell exists as an extrachromosomal element and transiently expresses TRPM4, and the nucleic acid introduced into the host cell is present in the chromosome of the host cell. It may be an integrated cell. In addition, the exogenous TRPM4 expression cell may express endogenous TRPM4 in the range which does not interfere with the objective of this invention. Exogenous TRPM4-expressing cells can be obtained, for example, by introducing a TRPM4 expression vector retaining a nucleic acid encoding TRPM4 into a host cell.

Examples of the nucleic acid encoding TRPM4 include mRNA shown in GenBank accession number: NM — 017636, cDNA obtained from the mRNA, and the like, but the present invention is not limited to such examples. Specific examples of the nucleic acid encoding TRPM4 include, for example, a nucleic acid encoding a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1, and an amino acid having a sequence identity of 80% or more with respect to the sequence shown in SEQ ID NO: 1. Examples thereof include a nucleic acid encoding a mutant polypeptide having a sequence and exhibiting TRPM4 activity, but the present invention is not limited to such examples. “Sequence identity” means that the amino acid sequence (query sequence) to be evaluated is aligned with the amino acid sequence (reference sequence) shown in SEQ ID NO: 1, using PROTEIN BLAST based on the BLAST algorithm as a default value. The value calculated in this way. From the viewpoint of expressing TRPM4 activity, the sequence identity is 85% or more, preferably 90% or more, more preferably 95% or more, still more preferably 98% or more, and its upper limit is 100%.

Examples of TRPM4 activity include regulation activity of potassium ion flux and sodium ion flux in cells, regulation activity of membrane potential in cells, etc., but the present invention is not limited to such examples. These TRPM4 activities are expressed, for example, when TRPM4 is activated by a TRPM4 agonist.

The host cell may be a cell that expresses endogenous TRPM4 or a cell that does not express endogenous TRPM4. Examples of host cells include mammalian cells such as human cells, monkey cells, mouse cells, and Chinese hamster cells, but the present invention is not limited to such examples. Examples of human cells include human kidney cells such as HEK293 cells; human cancer cells such as Hela cells, but the present invention is not limited to such examples. Examples of monkey cells include monkey kidney cells such as COS-7 cells, but the present invention is not limited to such examples. Examples of mouse cells include mouse cells such as mouse fetal skin fibroblasts such as NIH3T3 cells, but the present invention is not limited to such examples. Examples of Chinese hamster cells include Chinese hamster ovary cells such as CHO cells, but the present invention is not limited to such examples. Among these host cells, from the viewpoint of ensuring excellent handleability, ensuring adhesion to culture vessels such as dishes and plates, observation cover glasses, etc., and improving the expression efficiency of exogenous TRPM4 gene, HEK293 cells, CHO cells, COS-7 cells and NIN3T3 cells are preferred, and HEK293 cells are more preferred. A TRPM4 expression vector can be obtained by linking a nucleic acid encoding TRPM4 to a vector. Since the vector differs depending on the type of the host cell and the like and cannot be determined unconditionally, it is preferable to determine the vector according to the type of the host cell. As a method for confirming that the obtained cell is an exogenous TRPM4-expressing cell, for example, the cell is brought into contact with a TRPM4 agonist, and the inflow of sodium ions taken into the cell from the outside using the intracellular calcium ion concentration as an index The method of measuring the amount of decrease in the calcium ion concentration due to the above may be mentioned, but the present invention is not limited to such examples. When the intracellular calcium ion concentration of the cell after contact with the TRPM4 agonist is smaller than the intracellular calcium ion concentration of the cell not contacted with the TRPM4 agonist, it can be determined that the obtained cell is an exogenous TRPM4 expressing cell. it can. Known TRPM4 agonists include, for example, N- [4- [3,5-bis (trifluoromethyl) -1H-pyrazol-1-yl] phenyl] -4-methyl-1,2,3-thiadiazole-5 -Carboxamide) (hereinafter referred to as “BTP2”), 1- (6-[(17β-3-methoxyestradi-1,3,5 (10) -trien-17-yl) amino] hexyl) -1H-pyrrole- Examples include 2,5-dione (hereinafter referred to as “U-73122”), decavanadate, and the like, but the present invention is not particularly limited thereto.

The TRPM4-expressing cells are preferably TRPM4-expressing cells having cytokine expression ability from the viewpoint of evaluating with high accuracy whether or not the test sample has a cytokine production inhibitory effect. Examples of TRPM4-expressing cells having the ability to express cytokines include HaCaT cells, but the present invention is not limited to such examples.

Physiological events triggered through TRPM4 include, for example, a decrease in the expression level of cytokine genes resulting from the activation of TRPM4, a decrease in the expression level of cytokines resulting from the activation of TRPM4, and the expression of the activity of TRPM4 itself However, the present invention is not limited to such examples.

When a decrease in the expression level of a cytokine gene resulting from activation of TRPM4 is used as a physiological event caused by TRPM4, the following evaluation method 1 and the like are specific examples of the test sample evaluation method of the present invention. Although mentioned, this invention is not limited only to this illustration.

<Evaluation method 1>
The physiological event is cytokine production, TRPM4-expressing cells having cytokine expression ability are used as TRPM4-expressing cells, the physiological event is measured, and the cytokine production inhibitory action of the test sample is evaluated based on the physiological event The operation when
(A1) a step of contacting a TRPM4-expressing cell having cytokine expression ability with a test sample and a cytokine production promoting substance, and measuring the expression level of the cytokine and / or its gene in the TRPM4-expressing cell;
(A2) a step of contacting a TRPM4 expression cell having cytokine expression ability with a cytokine production promoting substance, and measuring the expression level of the cytokine and / or its gene in the TRPM4 expression cell;
(A3) a step of contacting a TRPM4 deficient cell in which the TRPM4 function in the TRPM4 expressing cell is deficient with a test sample and a cytokine production promoter, and measuring the expression level of the cytokine and / or its gene in the TRPM4 deficient cell;
(A4) a step of contacting a TRPM4 deficient cell deficient in TRPM4 function in the TRPM4 expressing cell with a cytokine production promoting substance, and measuring the expression level of the cytokine and / or its gene in the TRPM4 deficient cell; and (A5) A method comprising a step of evaluating a cytokine production inhibitory effect of a test sample based on the expression level measured in steps (A1) to (A4).

In Evaluation Method 1, since TRPM4-deficient cells are used in steps (A3) and (A4), whether the physiological event caused by the test sample in the TRPM4-expressing cells is a physiological event caused via TRPM4 You can check whether. From this, the TRPM4-expressing cells used in Evaluation Method 1 are preferably endogenous TRPM4-expressing cells from the viewpoint of performing the confirmation under the same conditions as when TRPM4-deficient cells are used, except that they express TRPM4. The TRPM4-expressing cells are preferably TRPM4 cells having cytokine expression ability from the viewpoint of evaluating with high accuracy whether the test sample has a cytokine production inhibitory effect. Examples of endogenous TRPM4-expressing cells having the ability to express cytokines include HaCaT cells, but the present invention is not limited to such examples.

In step (A1), a TRPM4-expressing cell having cytokine expression ability, a test sample, and a cytokine production promoting substance are brought into contact with each other, and the expression level of the cytokine and / or its gene in the TRPM4-expressing cell is measured.

Examples of cytokines include interleukin-1α (hereinafter referred to as “IL-1α”), interleukin 1β (hereinafter referred to as “IL-1β”), interleukin-2 (hereinafter referred to as “IL-2”), interleukin-1 Leukin-6 (hereinafter referred to as “IL-6”), interleukin-8 (hereinafter referred to as “IL-8”), tumor necrosis factor (hereinafter referred to as “TNF”) and the like. Among these cytokines, IL-1α and IL-8 are preferable from the viewpoint of accurately evaluating the action of suppressing the onset of inflammation in the skin. For example, IL-1α gene, IL-1β gene, IL-2 gene, IL-6 gene, IL-8 gene, TNF gene and the like can be mentioned. However, the present invention is not limited only to such examples, and among these cytokine genes, the IL-1α gene and IL-8 are used from the viewpoint of accurately evaluating the action of suppressing the onset of inflammation in the skin. Genes are preferred.

Examples of cytokine production promoting substances include tumor necrosis factor α (hereinafter referred to as “TNFα”), lipopolysaccharide, phorbol 12-myristate 13-acetate (hereinafter referred to as “PMA”), interferon γ (hereinafter referred to as “IFNγ”). IL-17, IL-22, IL-33, histamine and the like, but the present invention is not limited to such examples.

Examples of the order in which the TRPM4-expressing cell, the test sample, and the cytokine production promoting substance are brought into contact are, for example, (a1) the order in which the TRPM4 expressing cell is brought into contact with the test sample, and then the TRPM4 expressing cell and the cytokine production promoting substance are brought into contact. (A2) Order in which a test sample and a cytokine production promoting substance are contacted simultaneously with a TRPM4 expressing cell, (a3) After contacting the TRPM4 expressing cell and the cytokine production promoting substance, the TRPM4 expressing cell and the test sample are brought into contact with each other The order and the like can be mentioned, but the present invention is not limited to such examples.

In the case of adopting the order of (a1) in step (A1), examples of the method of contacting the TRPM4-expressing cell with the test sample include a method of incubating the TRPM4-expressing cell in the test sample. The invention is not limited to such examples. Examples of the method of contacting a TRPM4-expressing cell with a cytokine production promoting substance include a method in which a cytokine production promoting substance is added to a mixture of a test sample after incubation and a TRPM4-expressing cell, and the resulting mixture is incubated. However, the present invention is not limited to such examples. When the TRPM4-expressing cell is contacted with the test sample and when the TRPM4-expressing cell is contacted with the cytokine production promoting substance, a reagent used for measurement of a physiological event to be measured may be used.

Since the contact temperature between the TRPM4-expressing cell and the test sample and the contact temperature between the TRPM4-expressing cell and the cytokine production promoting substance vary depending on the type of the TRPM4-expressing cell and the like, it cannot be determined unconditionally. It is preferable to determine according to the above. The contact temperature between the TRPM4-expressing cell and the test sample and the contact temperature between the TRPM4-expressing cell and the cytokine production promoting substance are usually preferably 35 to 50 from the viewpoint of securing temperature conditions suitable for the expression of TRPM4 activity in the TRPM4-expressing cell. 40 ° C. In addition, since the contact time between the TRPM4-expressing cell and the test sample varies depending on the type of TRPM4-expressing cell, the type of test sample, and the like and cannot be determined unconditionally, the type of TRPM4-expressing cell, the type of test sample, etc. It is preferable to decide according to. The contact time between the TRPM4-expressing cells and the test sample is usually preferably 1 minute to 24 hours from the viewpoint of accurately evaluating the influence of the test sample on TRPM4 activity. Since the contact time between the TRPM4-expressing cell and the cytokine production-promoting substance varies depending on the type of TRPM4-expressing cell, the type of cytokine production-promoting substance, the type of measurement target substance, etc., it cannot be determined unconditionally. It is preferable to determine according to the type of TRPM4-expressing cell, the type of cytokine production promoting substance, the type of the substance whose expression level is to be measured, and the like. From the viewpoint of accurately evaluating the influence of the test sample on TRPM4 activity, the contact time between the TRPM4-expressing cell and the cytokine production-promoting substance is usually preferably 6 to 72 hours when evaluating the expression level of the cytokine. When evaluating the expression level of a cytokine gene, it is usually preferably 1 to 12 hours.

Since the number of TRPM4-expressing cells per 1 mL of the test sample varies depending on the type of TRPM4-expressing cell, the type of test sample, and the like when the TRPM4-expressing cell is contacted with the test sample, it cannot be determined unconditionally. It is preferable to determine according to the type of cell, the type of test sample, and the like. The number of TRPM4-expressing cells per 1 mL of the test sample is usually preferably 1 × 10 ³ or more, more preferably 1 × 10 ⁴ or more, from the viewpoint of improving the measurement accuracy of physiological events caused via TRPM4. From the viewpoint of accurately producing a physiological event caused by TRPM4 in TRPM4 cells, the number is preferably 1 × 10 ⁹ or less, more preferably 1 × 10 ⁸ or less.

Since the amount of the cytokine production promoting substance per 1 mL of the test sample varies depending on the type of cytokine production promoting substance, the type of TRPM4 expressing cell, and the like when contacting the TRPM4 expressing cell with the test sample, it cannot be determined unconditionally. It is preferable to determine according to the kind of cytokine production promoting substance, the kind of TRPM4-expressing cell, and the like. The amount of the cytokine production promoting substance per 1 mL of the test sample is usually preferably 5 to 500 ng from the viewpoint of accurately evaluating the influence of the test sample on the TRPM4 activity.

When adopting the order of (a2) or (a3) in step (A1), the number of TRPM4-expressing cells per mL of the test sample and the amount of the cytokine production promoting substance per mL of the test sample are the same as the order of (a1). It is the same as the number of TRPM4-expressing cells per mL of the test sample and the amount of the cytokine production promoting substance per mL of the test sample when employed.

Since the contact temperature between the TRPM4-expressing cell, the test sample, and the cytokine production promoting substance varies depending on the type of the TRPM4-expressing cell and the like, it cannot be determined unconditionally. Therefore, it can be determined according to the type of the TRPM4-expressing cell. preferable. The contact temperature between the TRPM4-expressing cell, the test sample and the cytokine production promoting substance is usually preferably 35 to 40 ° C. from the viewpoint of securing a temperature condition suitable for the expression of TRPM4 activity in the TRPM4-expressing cell.

Since the contact time between the TRPM4-expressing cell, the test sample, and the cytokine production promoting substance varies depending on the type of the TRPM4-expressing cell, the type of the test sample, the type of the cytokine production promoting substance, the type of the measurement target substance, etc. Since it cannot be determined, it is preferable to determine according to the type of TRPM4-expressing cells, the type of test sample, the type of cytokine production promoting substance, the type of substance whose expression level is to be measured, and the like. From the viewpoint of accurately evaluating the influence of the test sample on the TRPM4 activity, the contact time between the TRPM4-expressing cell, the test sample and the cytokine production promoter is usually preferably 6 to 6 when evaluating the expression level of the cytokine. 72 hours, and when evaluating the expression level of cytokine genes, it is usually preferably 1 to 12 hours.

Examples of methods for measuring the expression level of cytokines include Western blotting and enzyme-labeled immunoassay (ELISA), but the present invention is not limited to such examples. Examples of methods for measuring the expression level of cytokine genes include Northern blotting and quantitative RT-PCR, but the present invention is not limited to such examples.

Examples of antibodies used in Western blotting and ELISA include anti-cytokine antibodies or antibody fragments thereof, but the present invention is not limited to such examples. The anti-cytokine antibody may be a monoclonal antibody or a polyclonal antibody. From the viewpoint of improving the quantitativeness of the expression level of cytokine, a monoclonal antibody is preferable. The monoclonal antibody is obtained by, for example, culturing a hybridoma that produces a monoclonal antibody having reactivity to a cytokine or a part thereof, obtaining a culture supernatant, and subjecting the obtained culture supernatant to an ammonium sulfate fractionation method as necessary. , Purification by ion exchange chromatography, gel filtration chromatography, affinity chromatography and the like. The hybridoma immunizes the animal by, for example, administering cytokine or a part thereof intravenously, subcutaneously or intraperitoneally to obtain an antibody-producing cell, and the antibody-producing cell and myeloma cell are fused. It can be obtained by culturing the obtained fused cells in a HAT medium. In addition, polyclonal antibodies are used to immunize animals by administering cytokines or a portion thereof intravenously, subcutaneously or intraperitoneally, and to obtain antisera. It can be obtained by purification by fractionation, ion exchange chromatography, gel filtration chromatography, affinity chromatography, or the like. The antibody fragment can be obtained, for example, by decomposing an anti-cytokine antibody with a digestive enzyme and purifying the obtained degradation product as necessary. Further, commercially available anti-cytokine antibodies and antibody fragments thereof can be used as the anti-cytokine antibodies and antibody fragments thereof.

Examples of the probe used in the Northern blotting method include a nucleic acid consisting of all or part of a nucleic acid encoding a cytokine gene, a nucleic acid consisting of all or part of an antisense strand of a nucleic acid encoding a cytokine gene, and the like. The present invention is not limited to such examples. In the case of a nucleic acid consisting of a part of a nucleic acid encoding a cytokine gene or a nucleic acid consisting of a part of the antisense strand of the nucleic acid, the length of the probe varies depending on the type of cytokine gene, etc. Since it cannot be performed, it is preferable to determine according to the type of cytokine gene. The length of the probe is usually preferably 20 to 500 nucleotides from the viewpoint of improving the measurement accuracy of the expression level of the cytokine gene.

The primer pair used in the quantitative RT-PCR method includes, for example, a primer comprising a part of the base sequence of a nucleic acid encoding a cytokine gene and a primer comprising a part of the base sequence of the antisense strand of the nucleic acid. Although a primer etc. are mentioned, this invention is not limited only to this illustration. Since the length of each primer constituting the primer pair, the GC content and the Tm value, and the distance between the primers on the nucleic acid encoding the cytokine gene vary depending on the type of cytokine gene and the like, it cannot be determined unconditionally. It is preferable to determine according to the type of cytokine gene. The length of the primer is usually preferably 12 to 30 nucleotides from the viewpoint of improving the measurement accuracy of the expression level of the cytokine gene. The GC content of the primer is usually preferably 40 to 60% from the viewpoint of improving the measurement accuracy of the expression level of the cytokine gene. The Tm value of the primer is preferably 55 to 80 ° C. from the viewpoint of improving the measurement accuracy of the expression level of the cytokine gene. The distance between the primers on the nucleic acid encoding the cytokine gene is usually preferably 100 to 800 nucleotides in length from the viewpoint of rapidly measuring the expression level of the cytokine gene. In addition, from the viewpoint of improving the measurement accuracy of the expression level of the cytokine gene, each primer constituting the primer pair has a sequence of thymine residues or cytosine residues (polypyrimidine sequence) and an adenine residue or guanine residue. It is preferable to have a sequence that does not include both consecutive sequences (polypurine sequences). Each primer constituting the primer pair consists of a part of the nucleotide sequence of the nucleic acid encoding the cytokine gene or its antisense strand from the viewpoint of improving the measurement accuracy of the expression level of the cytokine gene. Is preferably composed of a sequence not included. The sequence used for the primer can be determined, for example, by using a primer design program such as Primer BLAST. The primer pair may be a commercially available primer pair.

In step (A2), a TRPM4-expressing cell having cytokine expression ability is brought into contact with a cytokine production promoting substance, and the expression level of the cytokine and / or its gene in the TRPM4-expressing cell is measured.

Method for contacting TRPM4-expressing cell and cytokine production promoting substance used in step (A2), contact temperature between TRPM4-deficient cell and cytokine production promoting substance, contact time between TRPM4-deficient cell and cytokine production promoting substance, cytokine gene and cytokine The method for measuring each expression level is as follows: the contact method between the TRPM4-expressing cell and the cytokine production-promoting substance used in step (A1), the contact temperature between the TRPM4-expressing cell and the cytokine production-promoting substance, and the TRPM4-expressing cell and the cytokine production-promoting substance. This is the same as the method for measuring the contact time and the expression levels of cytokines and their genes. The amount of the cytokine production promoting substance to be brought into contact with the TRPM4-expressing cell is the same as the amount of the cytokine production promoting substance used when contacting the TRPM4-expressing cell with the test substance and the cytokine production promoting substance in step (A1).

In step (A3), a TRPM4-deficient cell lacking TRPM4 function in a TRPM4-expressing cell is contacted with a test sample and a cytokine production promoter, and the expression level of the cytokine and / or its gene in the TRPM4-deficient cell is measured.

The TRPM4-deficient cells are the same as the TRPM4-expressing cells used in step (A1) and step (A2) except that the function of TRPM4 is deficient. A TRPM4-deficient cell can be produced by disrupting the TRPM4 gene in a TRPM4-expressing cell by a gene knockout method, or deleting the function of TRPM4 in a TRPM4-expressing cell. Examples of the gene knockout method include a method of destroying the TRPM4 gene by homologous recombination method, a method of destroying the TRPM4 gene by genome editing technology, and the like, but the present invention is not limited only to such examples. . In addition, as a method of deleting the function of TRPM4, a method of inhibiting the expression of the TRPM4 gene by an RNA silencing method using siRNA or shRNA, a method of expressing normal TRPM4 by expressing a dominant negative mutant in a TRPM4-expressing cell, Although the method etc. which inhibit a function are mentioned, this invention is not limited only to this illustration.

In step (A3), by performing the same operation under the same conditions as in step (A1), except that in step (A1), TRPM4-deficient cells are used instead of using TRPM4-expressing cells having cytokine expression ability, The expression level of cytokine and / or its gene is measured.

In step (A4), a TRPM4-deficient cell lacking TRPM4 function in a TRPM4-expressing cell is contacted with a cytokine production promoting substance, and the expression level of the cytokine and / or its gene in the TRPM4-deficient cell is measured.

In step (A4), by performing the same operation under the same conditions as in step (A2), except that in step (A2), TRPM4-deficient cells are used instead of using TRPM4-expressing cells having cytokine expression ability, The expression level of cytokine and / or its gene is measured.

In the test sample evaluation method of the present invention, from the viewpoint of improving the accuracy of the evaluation, it is preferable to use a control sample and calibrate the expression level when the test sample is used. The control sample may be a liquid that does not contain the test sample, and examples include a solvent used for dilution of the test sample, a solvent used for dissolution of the test sample, etc., but the present invention is limited only to such examples. Is not to be done. When using a control sample, in steps (A1) to (A4), the same procedure as in steps (A1) to (A4) is performed except that a control sample is used instead of the test sample. The gene expression level is measured.

In step (A4), the cytokine production inhibitory action of the test sample is evaluated based on the expression level measured in steps (A1) to (A4).

The expression level measured in step (A1) is less than the expression level measured in step (A2), and the expression level measured in step (A3) is equivalent to the expression level measured in step (A4) In this case, it can be evaluated that the test sample is a substance having an action of activating TRPM4 and suppressing cytokine production in skin cells.

When the expression of the activity of TRPM4 itself is used as a physiological event caused through TRPM4, specific examples of the test sample evaluation method of the present invention include the following evaluation method 2, etc. However, the present invention is not limited to such examples.

<Evaluation method 2>
An operation in measuring the physiological event and evaluating the cytokine production inhibitory effect of the test sample based on the physiological event,
(B1) contacting the TRPM4-expressing cell with the test sample, measuring a change in TRPM4 activity in the TRPM4-expressing cell before and after contact with the test sample, and (B2) changing the TRPM4 activity measured in step (B1) And a step of evaluating a cytokine production inhibitory effect of the test sample.

The TRPM4-expressing cells used in Evaluation Method 2 are preferably exogenous TRPM4-expressing cells, and more preferably cells that overexpress exogenous TRPM4 from the viewpoint of easily observing changes in TRPM4 activity.

Examples of changes in TRPM4 activity include changes in the amount of sodium ions taken into cells by TRPM4 in TRPM4-expressing cells before and after contact with the test sample, and activation of TRPM4 in TRPM4-expressing cells before and after contact with the test sample. Although the increase of an electric current etc. are mentioned, this invention is not limited only to this illustration.

When using the change in the amount of sodium ions taken into the cells by TRPM4 in the TRPM4-expressing cells before and after contact with the test sample as the change in TRPM4 activity, as a method for measuring the amount of sodium ions taken into the cells by TRPM4, For example, a method of measuring the amount of sodium ions taken into cells using the intracellular calcium ion concentration of TRPM4-expressing cells as an indicator of the amount of sodium ions taken into the cells can be mentioned. It is not limited to illustration only.

When the intracellular calcium ion concentration is used as an index for measuring the amount of sodium ions taken into cells by TRPM4, the intracellular calcium ion concentration is measured by, for example, using a calcium indicator that specifically binds to calcium ions with TRPM4-expressing cells. The calcium indicator is bound to the calcium ion in the TRPM4-expressing cell, and the amount of the calcium indicator bound to the calcium ion in the TRPM4-expressing cell is measured.

In the TRPM4-expressing cells in which TRPM4 is activated, the inflow of sodium ions from outside the TRPM4-expressing cells via TRPM4 into the TRPM4-expressing cells is increased as compared to TRPM4-expressing cells in which TRPM4 is not activated. Intracellular sodium ion concentration increases. As the intracellular sodium ion concentration of the TRPM4-expressing cell increases, the intracellular calcium ion concentration of the TRPM4-expressing cell decreases. Moreover, as shown in the below-mentioned Example, activation of TRPM4 and the suppression of cytokine production in skin cells are correlated. Therefore, when the intracellular calcium concentration of the TRPM4-expressing cells brought into contact with the test sample is lower than the intracellular calcium concentration of the TRPM4-expressing cells not brought into contact with the test sample, the test sample activates TRPM4 and cytokines in skin cells It can be evaluated that the substance has an action of suppressing production. On the other hand, when the intracellular calcium concentration of the TRPM4-expressing cell brought into contact with the test sample is higher than the intracellular calcium concentration of the TRPM4-expressing cell not brought into contact with the test sample, or when both are equivalent, It can be evaluated that the substance does not have an action of suppressing cytokine production in skin cells.

When the intracellular calcium ion concentration is used as an index for measurement of the amount of sodium ions taken into cells by TRPM4, TRPM4 activation action is, for example,
(I) a step of measuring intracellular calcium ion concentration A of TRPM4-expressing cells using a calcium indicator;
(II) a step of contacting a TRPM4-expressing cell with a test sample, and measuring the intracellular calcium ion concentration B of the TRPM4-expressing cell using a calcium indicator; and (III) the intracellular calcium obtained in step (I) The ion concentration A and the intracellular calcium ion concentration B obtained in step (II) are compared, and evaluation can be performed by a method including a step of evaluating the TRPM4 activation action of the test sample.

In step (I), intracellular calcium ion concentration A of TRPM4-expressing cells is measured using a calcium indicator. As TRPM4-expressing cells, HEK293 cells that express exogenous TRPM4 are preferably used.

Since the calcium indicator can measure the amount of calcium indicator bound to calcium ions with a simple operation, it must be a reagent that can detect changes before and after binding with calcium ions by changes in optical properties, etc. Is preferred. Examples of changes in optical characteristics include changes in fluorescence intensity and changes in absorbance, but the present invention is not limited to such examples. Examples of the calcium indicator include a fluorescent calcium indicator whose fluorescence intensity changes before and after binding with calcium ions, but the present invention is not limited to such examples. Specific examples of calcium indicators include 1- [6-amino-2- (5-carboxy-2-oxazolyl) -5-benzofuranyloxy] -2- (2-amino-5-methylphenoxy) ethane-N , N, N ′, N′-tetraacetic acid pentaacetoxymethyl ester (Fura 2-AM), 1- [2-amino-5- (2,7-dichloro-6-hydroxy-3-oxo-9-xanthenyl) Phenoxy] -2- (2-amino-5-methylphenoxy) ethane-N, N, N ′, N′-tetraacetic acid tetraacetoxymethyl ester (Fluo 3-AM), 1- [2-amino-5- ( 2,7-difluoro-6-acetoxymethoxy-3-oxo-9-xanthenyl) phenoxy] -2- (2-amino-5-methylphenoxy) ethane-N, N, N ′, N′-tetraacetic acid Although fluorescent calcium indicator, such as tiger acetoxymethyl ester (Fluo 4-AM) and the like, and the present invention is not limited only to those exemplified. Among calcium indicators, a fluorescent calcium indicator whose fluorescence intensity changes before and after binding with calcium ions from the viewpoint of easy measurement and easy differentiation from the dynamics of contaminants present in TRPM4-expressing cells. Is preferred.

The fluorescent calcium indicator may have one excitation wavelength or two or more excitation wavelengths. In the case where the calcium indicator is a fluorescent calcium indicator, the fluorescent calcium indicator is preferably a fluorescent calcium indicator having two types of excitation wavelengths because the fluorescence intensity can be easily measured and the detection intensity is high. When a change in intracellular calcium concentration is measured, when a fluorescent calcium indicator having one type of excitation wavelength is used, a change in intracellular calcium concentration can be measured based on the fluorescence intensity at the excitation wavelength. When measuring a change in intracellular calcium concentration, when a fluorescent calcium indicator having two or more types of excitation wavelengths is used, the two or more types of excitation wavelengths are used from the viewpoint of improving the accuracy of the cytokine production inhibitory effect of the test sample. Based on the fluorescence intensity ratio calculated from the fluorescence intensity at each of the first excitation wavelength and the second excitation wavelength, the two excitation wavelengths (first excitation wavelength and second excitation wavelength) selected from Changes in calcium concentration can be measured. When measuring intracellular calcium concentration, for example, when FURA 2-AM, which is a fluorescent calcium indicator having two types of excitation wavelengths, is used, the fluorescence intensity at the first excitation wavelength (hereinafter also referred to as “first fluorescence intensity”) is used. The fluorescence intensity at the excitation wavelength of 380 nm can be used as the fluorescence intensity at the excitation wavelength of 340 nm and the fluorescence intensity at the second excitation wavelength (hereinafter also referred to as “second fluorescence intensity”). The fluorescence intensity ratio is, for example, the formula (I):
[Fluorescence intensity ratio] = [first fluorescence intensity] / [second fluorescence intensity] (I)
Can be determined based on

As a method for measuring intracellular calcium ion concentration A using a fluorescent calcium indicator, for example, the following measurement method 1 can be mentioned, but the present invention is not limited to such examples.

<Measurement method 1>
Introducing a fluorescent calcium indicator into a TRPM4-expressing cell to obtain an indicator-introduced cell;
Bringing the indicator-introduced cell into contact with calcium ions, binding the calcium ion to the fluorescent calcium indicator in the indicator-introduced cell, and measuring the fluorescence intensity of the fluorescent calcium indicator bound to the calcium ion in the indicator-introduced cell. Including methods.

Examples of a method for introducing a fluorescent calcium indicator into TRPM4-expressing cells include a method of circulating a buffer solution containing a fluorescent calcium indicator in a reflux chamber containing TRPM4-expressing cells. It is not limited to only. Examples of the buffer include HEPES buffer, but the present invention is not limited to such examples.

As a method for bringing a TRPM4-expressing cell into which a fluorescent calcium indicator has been introduced into contact with calcium ions, for example, a buffer solution containing calcium ions is circulated in a reflux chamber containing TRPM4-expressing cells into which a fluorescent calcium indicator has been introduced. Although a method etc. are mentioned, this invention is not limited only to this illustration. Examples of the buffer include HEPES buffer, but the present invention is not limited to such examples.

Since the contact temperature between the TRPM4-expressing cell into which the fluorescent calcium indicator has been introduced and the buffer containing calcium ions varies depending on the type of TRPM4-expressing cell and the like, it cannot be determined unconditionally. It is preferable to decide according to. In order to eliminate the influence of temperature on TRPM4 activity, it is preferable that the contact between TRPM4-expressing cells and a buffer containing calcium ions is performed in a constant temperature environment.

Since the number of TRPM4-expressing cells used in step (I) varies depending on the type of measuring means of calcium ion concentration A and cannot be determined unconditionally, it depends on the type of measuring means of calcium ion concentration A. Is preferably determined. For example, when measuring the calcium ion concentration A by observing cells at 100 times magnification using an inverted microscope, the number of TRPM4-expressing cells per visual field (data analysis range) improves the reliability of the measurement results. From the viewpoint, it is preferably 10 or more, more preferably 100 or more, and from the viewpoint of securing the space between cells so that the cells do not become too dense, preferably 300 or less, more preferably 200 or less. is there.

In step (II), the TRPM4-expressing cells are brought into contact with the test sample, and the intracellular calcium ion concentration B of the TRPM4-expressing cells is measured using a calcium indicator.

Examples of a method for measuring intracellular calcium ion concentration B using a fluorescent calcium indicator include measurement method 2 below, but the present invention is not limited to such examples.

<Measurement method 2>
Introducing a fluorescent calcium indicator into a TRPM4-expressing cell to obtain an indicator-introduced cell;
Contacting the indicator-introduced cell with the test sample;
Contacting the cell after contacting the test sample with calcium ion, binding calcium ion to the fluorescent calcium indicator in the cell, and measuring the fluorescence intensity of the fluorescent calcium indicator bound to calcium ion in the cell Method.

As a method of bringing a TRPM4-expressing cell into which a fluorescent calcium indicator has been introduced into contact with a test sample, a method in which a buffer containing the fluorescent calcium indicator is circulated in a reflux chamber containing TRPM4-expressing cells into which a fluorescent calcium indicator has been introduced. However, the present invention is not limited to such examples. Examples of the method for contacting cells after contacting the test sample with calcium ions include a method of circulating a buffer solution containing calcium ions and not containing the test sample. It is not limited. In the present invention, after circulating a buffer solution containing a test sample in a reflux chamber containing TRPM4-expressing cells into which a fluorescent calcium indicator has been introduced, a buffer solution containing the test sample and calcium ions is circulated. May be.

The method of measuring intracellular calcium ion concentration B used in step (II), the contact temperature and the number of TRPM4-expressing cells are the same as the method of measuring intracellular calcium ion concentration B used in step (I), contact temperature and TRPM4-expressing cells. It is the same as the number of.

In step (III), the intracellular calcium ion concentration A obtained in step (I) is compared with the intracellular calcium ion concentration B obtained in step (II), and the TRPM4 activation action of the test sample is evaluated. To do. In step (III), when the calcium ion concentration B is decreased as compared with the calcium ion concentration A, the test sample can be evaluated as having a TRPM4 activation action. Furthermore, the calcium ion concentration A and the calcium ion can be evaluated. It can be evaluated that the said test sample has a high TRPM4 activation effect, so that the difference between ion concentration B is large.

When an increase in current due to TRPM4 activation in TRPM4 expressing cells before and after contact with the test sample is used as a change in TRPM4 activity, the activation effect of TRPM4 is, for example,
(I) measuring a current A in a TRPM4-expressing cell under a constant potential;
(Ii) a step in which a TRPM4-expressing cell is brought into contact with a test sample, and the current B in the TRPM4-expressing cell is measured under the same potential as in step (i); and (iii) obtained in step (i). It can be evaluated by a method including a step of comparing the current A and the current B obtained in step (ii) and evaluating the TRPM4 activation action of the test sample.

In step (i), a current A resulting from activation of TRPM4 in a TRPM4-expressing cell is measured under a constant potential. Examples of the method for measuring the current A include a patch clamp method, but the present invention is not limited to such an example. According to the patch clamp method, it is possible to measure the current resulting from the activation of one or more TRPM4 present in the cell membrane of TRPM4-expressing cells. Examples of the patch clamp method include a whole cell method and a cell attach method, but the present invention is not limited to such examples.

In step (ii), the TRPM4-expressing cell is brought into contact with the test sample, and the current B in the TRPM4-expressing cell is measured under the same potential as that in step (i). The method for measuring current B used in step (ii) is the same as the method for measuring current A used in step (i).

In step (iii), the current A obtained in step (i) is compared with the current B obtained in step (ii), and the TRPM4 activation action of the test sample is evaluated. In step (iii), when the current A is smaller than the current B, the test sample can be evaluated as having a TRPM4 activation action. Moreover, it can be evaluated that the said test sample has a high TRPM4 activation effect, so that the difference between the electric current A and the electric current B is large.

As described above, according to the test sample evaluation method of the present invention, a physiological event caused by TRPM4 in a TRPM4-expressing cell is measured by the test sample, and the cytokine possessed by the test sample is based on the physiological event. Since the operation of evaluating the production inhibitory action is taken, it is possible to easily and accurately evaluate whether or not the test sample has an action of inhibiting cytokine production in skin cells. Therefore, the test sample evaluation method of the present invention is expected to be suitably used for the development of inflammation preventive cosmetics, inflammation preventive agents and the like.

2. Cytokine production inhibitor The cytokine production inhibitor of the present invention is a cytokine production inhibitor used for the purpose of activating TRPM4 to suppress cytokine production in skin cells, and an aluminum compound as an active ingredient for activating TRPM4 It is characterized by containing.

Since the cytokine production inhibitor of the present invention contains an aluminum compound as an active ingredient for activating TRPM4, cytokine production in skin cells can be effectively suppressed. Therefore, the cytokine production inhibitor of the present invention can be suitably used for suppressing cytokine production in skin cells.

Since the content of the aluminum compound in the activity inhibitor of the present invention varies depending on the type of the aluminum compound, the use of the cytokine production inhibitor of the present invention, etc., it cannot be determined unconditionally. It is preferable to set appropriately according to the use of the cytokine production inhibitor. The content of the aluminum compound in the cytokine production inhibitor of the present invention is preferably 0.0001% by mass or more, more preferably from the viewpoint of fully expressing the action of activating TRPM4 and suppressing cytokine production in skin cells. Is 0.005 mass% or more, more preferably 0.008 mass% or more, particularly preferably 0.01 mass% or more, and preferably 100 mass% or less from the viewpoint of suppressing the load on the skin.

The cytokine production inhibitor of the present invention may contain other components such as water, a pH adjuster, and a stabilizer within a range that does not hinder the object of the present invention. In addition, when the cytokine production inhibitor of the present invention contains other components, the aluminum compound and the other components are combined with each other in the cytokine production inhibitor of the present invention as long as the object of the present invention is not hindered. It may be formed.

As described above, since the cytokine production inhibitor of the present invention can effectively inhibit cytokine production in skin cells, it can be suitably used for suppressing the onset of inflammation in the skin. Therefore, the cytokine production inhibitor of the present invention is expected to be used in applications such as cosmetics for preventing inflammation in the skin and inflammation preventing agents in the skin.

When the cytokine production inhibitor of the present invention is used as a skin inflammation preventive cosmetic or skin inflammation preventive agent, the content of the cytokine production suppressor of the present invention in the inflammation preventive cosmetic or inflammation preventive agent is determined to prevent inflammation. Because it varies depending on the use of cosmetic preparations or inflammation preventive agents and the type of aluminum compound contained in the cytokine production inhibitor of the present invention, it cannot be determined unconditionally. It is preferable to determine according to the type of aluminum compound contained in the cytokine production inhibitor of the present invention. The content of the cytokine production inhibitor of the present invention in the inflammation preventing cosmetic or inflammation preventing agent is usually from the viewpoint of activating TRPM4 and sufficiently exhibiting the action of suppressing inflammation in the skin, The content of the aluminum compound in the inflammation preventing agent is preferably 0.0001% by mass or more, more preferably 0.0005% by mass or more, further preferably 0.0008% by mass or more, and particularly preferably 0.01% by mass or more. From the viewpoint of suppressing the load on the skin, it is preferably adjusted to 20% by mass or less, more preferably 10% by mass or less. Cosmetics for inflammation prevention or inflammation prevention agents can be used in the range that does not prevent the action of inhibiting inflammation in the skin by activating TRPM4, for example, solvent, surfactant, moisturizer, thickener, preservative, oxidation Other components such as an inhibitor and a pH adjuster may be blended. Since the dose and the number of applications of cosmetics for preventing inflammation or anti-inflammatory agents vary depending on the type of inflammation, the type of target, the age of the target, the weight of the target, etc. It is preferable to determine according to the type of application, the type of application target, the age of application target, the weight of application target, and the like.

Hereinafter, the present invention will be described in more detail with reference to examples. However, the present invention is not limited to such examples. In the following, “% (m / m)” is mass / mass percent (mass%), “% (m / v)” is mass / volume percent, and “% (v / v)” is volume / volume percent. (% By volume).

Production Example 1
HEK293 of 5 × 10 ⁶ cells in DMEM containing 10% (v / v)% fetal calf serum on a 35 mm diameter petri dish maintained at 37 ° C. in an atmosphere containing 5% (v / v) carbon dioxide Cells were cultured. HEK293 cells after culture and human TRPM4 expression vector (manufactured by OriGene, trade name: TrueClone TRPM4 (NM — 017636) Human cDNA Clone) and a reagent for gene transfer [ThermoFisher Scientific (manufactured by ThermoFisher SCIENTIFIC) Name: Lipofectamine Transfection Reagent], a nucleic acid encoding human TRPM4 and a nucleic acid encoding red fluorescent protein were transiently introduced into HEK293 cells according to the protocol attached to the gene introduction reagent, and transfected. I got the Tanto. In the following, transfectants emitting red fluorescence were used as TRPM4 overexpressing cells.

Reference example 1
The TRPM4 overexpressing cells (Experiment No. 1) obtained in Production Example 1 were washed with phosphate buffered saline. Lysis buffer solution [composition: 25 mM trishydroxymethylaminomethane (hereinafter referred to as “Tris”), 1 mM ethylenediaminetetraacetic acid, 0.1 mM glycol etherdiaminetetraacetic acid, 5 mM magnesium chloride for 1 × 10 ⁶ cells after washing , 100 mM sodium chloride, 10% (v / v) glycerol and 1% (v / v) polyethylene glycol-tert-octylphenyl ether (Triton X-100)] were added at 100 μL. The resulting mixture was stirred and then incubated on ice for 10 minutes to obtain a cell lysate. The cell lysate was subjected to centrifugation (4 ° C.) at 20000 × g for 10 minutes to obtain a supernatant. To 90 μL of the obtained supernatant, a sample buffer [composition: 187 mM Tris / HCl buffer (pH 6.8), 6% (m / v) sodium dodecyl sulfate, 15% (m / v) sucrose, 0.015% ( m / v) Bromophenol blue, 15% (v / v) 2-mercaptoethanol] 45 μL was added to obtain a sample for electrophoresis (Experiment No. 1).

In Experiment No. 1, instead of using the TRPM4 overexpressing cells (Experiment No. 1) obtained in Production Example 1, a human epidermal keratinocyte established strain (HaCaT cell) (Experiment No. 2) or normal human epidermal keratinocytes (Experiment No. 1) Except that No. 3) was used, the same operation as in Experiment No. 1 was performed to obtain electrophoresis samples of Experiment No. 2 and Experiment No. 3.

5 μL of each electrophoresis sample of Experiment Nos. 1 to 3 was injected into a well of an electrophoresis gel (trade name: NuPAGE 4-12% Bis Tris Gel, manufactured by Invitrogen) and electrophoresed at 200 V for 45 minutes. Next, the protein on the gel after the electrophoresis was transferred to the membrane using a transcription kit [manufactured by Invitrogen, product name: iBlot gel transfer stacks mini] and a transfer device [manufactured by Invitrogen, product name: iBlot].

Membrane after transfer and mouse anti-TRPM4 antibody [manufactured by OriGene, trade name: Anti-TRPM4, Mouse-Mono (10H5) TrueMAB, product number: TA500381] and secondary antibody [HRP-conjugated anti-mouse IgG antibody [Sea Western blot analysis was performed using ST (CST) Japan Co., Ltd. product name: Anti-mouse IgG, HRP-linked Antibody, product number: # 7076].

In Reference Example 1, the result of Western blot analysis is shown in FIG. In the figure, Lane M is a marker, Lane 1 is the result of Western blot analysis of the electrophoresis sample of Experiment No. 1, Lane 2 is the result of Western blot analysis of the electrophoresis sample of Experiment No. 2, and Lane 3 is Experiment No. 3 The result of the Western blot analysis of the sample for electrophoresis of is shown. In the figure, arrow A indicates a band corresponding to TRPM4, and arrow B indicates a band corresponding to β-actin.

From the results shown in FIG. 1, since a band was detected at a position corresponding to TRPM4 in lanes 1 to 3, TRPM4 overexpressing cells, HaCaT cells and normal human epidermal keratinocytes obtained in Production Example 1 were obtained. It can be seen that both express the TRPM4 protein.

Examples 1 and 2
96-well plate medium A [10% (v / v) fetal calf serum−100 U / mL penicillin−100 μg / mL streptomycin-1 maintained at 37 ° C. in an atmosphere containing 5% (v / v) carbon dioxide % (V / v) L-alanyl-L-glutamine-containing medium supplement (manufactured by Thermo Fisher Scientific Co., Ltd., trade name: GlutaMAX) -containing DMEM] 0.1 mL of HaCaT cells were cultured for 24 hours. A cell culture containing 6.4 × 10 ³ cells was obtained.

BTP2 and HaCaT cells were brought into contact with each other by adding BTP2, which is a TRPM4 agonist, to the cell culture so as to have a concentration of 1 nM (Example 1) or 10 nM (Example 2). The resulting mixture was incubated at 37 ° C. for 10 minutes. TNFα was added to the mixture after the incubation so that its concentration was 20 ng / mL, and the resulting mixture was incubated at 37 ° C. for 1 hour to contact TNFα and HaCaT cells. Thereafter, HaCaT cells contained in the mixture were collected.

Comparative Example 1
HaCaT cells were cultured for 24 hours in 0.1 mL of medium A in a 96-well plate maintained at 37 ° C. in an atmosphere containing 5% (v / v) carbon dioxide, and 6.4 × 10 ³ HaCaT cells were cultured. A cell culture containing was obtained. Cell cultures were incubated in medium at 37 ° C. for 1 hour and 10 minutes, after which the cells were harvested.

Comparative Example 2
HaCaT cells were cultured for 24 hours in 0.1 mL of medium A in a 96-well plate maintained at 37 ° C. in an atmosphere containing 5% (v / v) carbon dioxide, and 6.4 × 10 ³ HaCaT cells were cultured. A cell culture containing was obtained. TNFα was added to the cell culture so that its concentration was 20 ng / mL, and the resulting mixture was incubated at 37 ° C. for 1 hour to bring TNFα into contact with HaCaT cells. Thereafter, cells were collected from the mixture.

Test example 1
Using each of the cells obtained in Examples 1-2 and Comparative Examples 1-2 and a cell lysis reagent (Roche, trade name: Realtime ReadyCell Lysis Kit), an RNA-containing sample was obtained. Using the obtained RNA-containing sample and RT-PCR kit [manufactured by Takara Bio Inc., trade name: One step PrimeScript RT-PCR kit], qRT-PCR was carried out, so that Examples 1 and 2 were compared. Each expression level of IL-1α gene, IL-8 gene and TNF gene in each cell obtained in Examples 1 and 2 was measured. IL-1α, IL-8 and TNF are inflammatory cytokines induced by TNFα.

In Test Example 1, the results of examining the expression level of the IL-1α gene in each cell obtained in Examples 1-2 and Comparative Examples 1-2 are shown in FIG. 2, Examples 1-2, and Comparative Examples 1-2. FIG. 3 shows the results of examining the expression level of the IL-8 gene in each cell obtained, and FIG. 3 shows the results of examining the expression level of the TNF gene in each cell obtained in Examples 1-2 and Comparative Examples 1-2. 4 shows. In FIG. 2, Lane 1 is the expression level of IL-1α gene in the cells obtained in Comparative Example 1, Lane 2 is the expression level of IL-1α gene in the cells obtained in Comparative Example 2, and Lane 3 is Example 1. IL-1α gene expression level in the cells obtained in (1), lane 4 shows the IL-1α gene expression level in the cells obtained in Example 2. In FIG. 3, Lane 1 is the expression level of IL-8 gene in the cells obtained in Comparative Example 1, Lane 2 is the expression level of IL-8 gene in the cells obtained in Comparative Example 2, and Lane 3 is Example 1. IL-8 gene expression level in the cells obtained in (1), lane 4 shows the IL-8 gene expression level in the cells obtained in Example 2. In FIG. 4, lane 1 is the expression level of TNF gene in the cells obtained in Comparative Example 1, lane 2 is the expression level of TNF gene in the cells obtained in Comparative Example 2, and lane 3 is obtained in Example 1. The expression level of the TNF gene in the cells, lane 4 shows the expression level of the TNF gene in the cells obtained in Example 2.

The results shown in FIGS. 2 to 4 indicate that the expression levels of IL-1α gene, IL-8 gene, and TNF gene decrease as the concentration of BTP2 increases. These results suggest that the activation of TRPM4 is correlated with the suppression of expression of inflammatory cytokine genes such as IL-1α gene, IL-8 gene, and TNF gene. Moreover, since the expression at the gene level of the inflammatory cytokine induced by TNFα can be suppressed by contacting a skin cell with a substance having an action to activate TRPM4 in advance, the action to activate TRPM4 is achieved. According to the substance to have, it is suggested that inflammation can be prevented.

Production Example 2
By destroying the TRPM4 gene in HaCaT cells by gene knockout technology, knockout HaCaT cells in which the TRPM4 gene was knocked out were obtained.

Comparative Example 3
In Example 1, cells were obtained in the same manner as in Example 1 except that the knockout HaCaT cells obtained in Production Example 2 were used instead of HaCaT cells.

Comparative Example 4
In Example 2, cells were obtained in the same manner as in Example 2 except that the knockout HaCaT cells obtained in Production Example 2 were used instead of HaCaT cells.

Comparative Example 5
In Comparative Example 1, cells were obtained in the same manner as in Comparative Example 1, except that the knockout HaCaT cells obtained in Production Example 2 were used instead of HaCaT cells.

Comparative Example 6
In Comparative Example 2, cells were obtained in the same manner as in Comparative Example 2, except that the knockout HaCaT cells obtained in Production Example 2 were used instead of HaCaT cells.

Test example 2
Test Example 1 was the same as Test Example 1 except that each cell obtained in Comparative Examples 3-6 was used instead of each cell obtained in Examples 1-2 and Comparative Examples 1-2. The operation was performed, and the expression levels of IL-1α gene, IL-8 gene, and TNF gene in each cell obtained in Comparative Examples 3 to 6 were measured.

In Test Example 2, the results of examining the expression level of IL-8 gene in each cell obtained in Comparative Examples 3 to 6 are shown in FIG. In FIG. 5, Lane 1 is the expression level of IL-8 gene in the cells obtained in Comparative Example 5, Lane 2 is the expression level of IL-8 gene in the cells obtained in Comparative Example 6, and Lane 3 is Comparative Example 3. IL-8 gene expression level in the cells obtained in (1), lane 4 shows the IL-8 gene expression level in the cells obtained in Comparative Example 4.

From the results shown in FIG. 5, the expression level of the IL-8 gene when the knockout HaCaT cell was brought into contact with BTP2 and then brought into contact with TNFα was determined by bringing the knockout HaCaT cell into contact with TNFα without contacting with BTP2. It can be seen that the expression level of the IL-8 gene is similar to that of Further, inflammatory cytokine genes other than IL-8 gene such as IL-1α gene and TNF gene showed the same tendency as IL-8 gene. From these results, it can be seen that the decrease in the expression level of the inflammatory cytokine gene in the TRPM4-expressing cells when brought into contact with BTP2 is due to the activation of TRPM4.

As described above, since the expression of the inflammatory cytokine gene in skin cells is suppressed by bringing a substance having an action to activate TRPM4 into contact with skin cells in advance, the substance having an action to activate TRPM4 According to the above, it can be seen that the expression of inflammatory cytokines can be suppressed and inflammation can be prevented.

Production Example 3
A solution containing no calcium (hereinafter referred to as “solution B”) so that the concentration of thapsigargin is 1 μM [Composition: 140 mM sodium chloride, 5 mM potassium chloride, 2 mM magnesium chloride, 5 mM glycol etherdiaminetetraacetic acid, 10 mM glucose, and 10 mM glucose HEPES buffer (pH 7.4)] to obtain a thapsigargin-containing bath solution.

Production Example 4
Calcium-containing solution (hereinafter referred to as “solution A”) so that the concentration of ionomycin is 25 μM [Composition: 140 mM sodium chloride, 5 mM potassium chloride, 2 mM magnesium chloride, 2 mM calcium chloride, 10 mM glucose and 10 mM HEPES buffer ( pH 7.4)] to obtain an ionomycin-containing solution A.

Production Example 5
U-73122, which is a TRPM4 agonist, was added to Solution B so that its concentration was 10 μM to obtain a U-73122-containing bath solution.

Production Example 6
U-73122, which is a TRPM4 agonist, was added to solution A so as to have a concentration of 10 μM to obtain U-73122-containing solution A.

Reference example 2
(1) Preparation of Fura 2-AM-introduced cells TRMP4-overexpressing cells expressing exogenous TRPM4 obtained in Production Example 1 as TRPM4-expressing cells were analyzed at room temperature in DMEM containing Fura 2-AM for intracellular calcium ion measurement. Fura 2-AM-introduced cells were obtained by incubation at (25 ° C.) for 60 minutes.

(2) Measurement of intracellular calcium ion concentration in the absence of TRPM4 agonist The FURA 2-AM-introduced cell obtained in Reference Example 2 (1) was placed in the reflux chamber of an inverted microscope equipped with a reflux chamber. In the following, using a CCD camera, fluorescence at an excitation wavelength of 340 nm based on FURA 2-AM in a FURA 2-AM-introduced cell and fluorescence at an excitation wavelength of 380 nm based on FURA 2-AM in a FURA 2-AM-introduced cell are continuously produced. The intensity of fluorescence at an excitation wavelength of 340 nm based on FURA 2-AM in a FURA 2-AM-introduced cell using a software for image analysis (trade name: IPLab, manufactured by Solution Systems Co., Ltd.) (hereinafter “fluorescence”). intensity _340nm "hereinafter) and FURA 2-AM introduced fluorescence intensity at an excitation wavelength of 380nm based on FURA 2-AM in cell (hereinafter, was determined) as" fluorescence intensity _380nm ".

Solution B was refluxed in a reflux chamber containing FURA 2-AM-introduced cells. Calcium ions are released from the endoplasmic reticulum by refluxing the thapsigargin-containing bath solution obtained in Production Example 3 in the reflux chamber containing the FURA 2-AM-introduced cells after 50 seconds from the start of solution B reflux. I let you. After 240 seconds from the start of reflux of the thapsigargin-containing bath solution, solution A was refluxed in a reflux chamber containing FURA 2-AM-introduced cells. After 150 seconds from the beginning of solution A reflux, the ionomycin-containing solution A obtained in Production Example 4 was refluxed for 180 seconds in a reflux chamber containing FURA 2-AM-introduced cells. When TRPM4 is activated, sodium ions are taken into cells via TRPM4, and the amount of calcium ions flowing into the cells decreases with an increase in the amount of taken-in sodium ions. Therefore, the TRPM4 activity can be indirectly measured by measuring the time-dependent change in intracellular calcium ion concentration.

(3) Measurement of intracellular calcium ion concentration in the presence of TRPM4 agonist The FURA 2-AM-introduced cell obtained in Reference Example 2 (1) was placed in the reflux chamber of an inverted microscope with a reflux chamber. Hereinafter, the fluorescence intensity _{340 nm} based on FURA 2-AM in the FURA 2-AM-introduced cells and the fluorescence intensity _{380 nm} based on FURA 2-AM in the FURA 2-AM-introduced cells were measured over time. Using the measured fluorescence intensity of _{340 nm} and fluorescence intensity of _{380 nm} , the formula (Ia):
Fluorescence intensity ratio = fluorescence intensity _{340 nm} / fluorescence intensity _{380 nm} (Ia)
Based on this, the change with time in the fluorescence intensity ratio was determined.

Solution B was refluxed in a reflux chamber containing FURA 2-AM-introduced cells. Calcium ions are released from the endoplasmic reticulum by refluxing the thapsigargin-containing bath solution obtained in Production Example 3 in the reflux chamber containing the FURA 2-AM-introduced cells after 50 seconds from the start of solution B reflux. I let you. After lapse of 120 seconds from the start of reflux of the thapsigargin-containing bath solution, the U-73122-containing bath solution obtained in Production Example 5 was refluxed in a reflux chamber containing FURA 2-AM-introduced cells. After a lapse of 120 seconds from the start of reflux of the U-73122-containing bath solution, the U-73122-containing solution A obtained in Production Example 6 was refluxed in a reflux chamber containing FURA 2-AM-introduced cells. After 150 seconds from the start of reflux of the U-73122-containing solution A, the ionomycin-containing solution A obtained in Production Example 4 was refluxed for 180 seconds in the reflux chamber containing the FURA 2-AM-introduced cells.

(4) Calculation of relative fluorescence intensity Fluorescence intensity ratio A after 40 seconds from the start of solution A reflux in Reference Example 2 (2) and 170 seconds after the start of reflux of ionomycin-containing solution A in Reference Example 2 (2) And the fluorescence intensity ratio B of the formula (II):
[Relative fluorescence intensity] = fluorescence intensity ratio A / fluorescence intensity ratio B (II)
Accordingly, the relative fluorescence intensity in the absence of TRPM4 agonist (control) was calculated.

The fluorescence intensity ratio after 40 seconds from the start of reflux of U-73122-containing solution A in Reference Example 2 (3) and the fluorescence intensity ratio after 170 seconds from the start of reflux of ionomycin-containing solution A in Reference Example 2 (3) Was used to calculate the relative fluorescence intensity in the presence of the TRPM4 agonist according to the formula (II).

FIG. 6 shows the result of examining the relationship between the presence or absence of the TRPM4 agonist and the relative fluorescence intensity in Reference Example 2. In the figure, lane 1 shows the relative fluorescence intensity in the presence of the TRPM4 agonist, and lane 2 shows the relative fluorescence intensity in the absence of the TRPM4 agonist.

From the results shown in FIG. 6, the relative fluorescence intensity in the presence of the TRPM4 agonist is lower than the relative fluorescence intensity in the absence of the TRPM4 agonist, and thus flows into HEK293 cells with the activation of TRPM4. It can be seen that the intracellular calcium ion concentration decreased as the amount of sodium ions increased. From these results, using the cells overexpressing exogenous TRPM4, such as the TRPM4 overexpressing cells obtained in Production Example 1, and examining the effect of the test sample on the intracellular calcium ion concentration, the test sample It can be seen that can activate TRPM4 to suppress cytokine production in skin cells.

Production Example 7
As a test sample, aluminum chloride was added to Solution B so as to have a concentration of 1 mM to obtain a test sample-containing bath solution.

Production Example 8
Aluminum chloride was added as a test sample to solution A so that its concentration was 1 mM to obtain test sample-containing solution A.

Production Example 9
A test sample-containing bath solution was obtained by adding potassium aluminum sulfate as a test sample to Solution B so that its concentration was 1 mM.

Production Example 10
A test sample-containing solution A was obtained by adding potassium aluminum sulfate as a test sample to the solution A so as to have a concentration of 1 mM.

Example 3
In Reference Example 2, the test sample-containing bath solution obtained in Preparation Example 7 was used instead of using the U-73122-containing bath solution, and the test obtained in Preparation Example 8 was used instead of using the U-73122-containing solution A. Except that the sample-containing solution A was used, the same operation as in Reference Example 2 was performed, and the relative fluorescence intensity was calculated.

Further, in the above, the test sample-containing bath solution obtained in Production Example 9 was used instead of the test sample-containing bath solution obtained in Production Example 7, and the test sample-containing solution A obtained in Production Example 8 was used. Relative fluorescence intensity was calculated in the same manner as described above except that the test sample-containing solution A obtained in Production Example 10 was used instead of.

FIG. 7 shows the results of examining the relationship between the type of test sample and the relative fluorescence intensity in Example 3. In the figure, lane 1 is the relative fluorescence intensity when aluminum chloride is used as the test sample, lane 2 is the relative fluorescence intensity when aluminum potassium sulfate is used as the test sample, and lane 3 is the relative fluorescence intensity in the absence of the test sample. Indicates strength.

FIG. 7 shows that the relative fluorescence intensity when aluminum chloride or potassium aluminum sulfate is used as the test sample is lower than the relative fluorescence intensity in the absence of the test sample. From these results, it can be seen that aluminum chloride and aluminum potassium sulfate have the action of activating TRPM4 and suppressing cytokine production.

Example 4
Epidermal keratinocytes were cultured in DMEM for 1 day at 37 ° C. The obtained cultured cells were treated with trypsin. A culture solution was obtained by adding DMEM to the cultured cells after trypsin treatment so as to adjust the concentration of the cultured cells to 1 × 10 ⁵ cells / mL. 1 mL of the obtained culture solution was seeded in each hole of a 12-well plate, and the cultured cells were cultured at 37 ° C. for 24 hours. After culturing, 500 μL of the culture supernatant was replaced with 500 μL of DMEM containing aluminum potassium sulfate (potassium aluminum sulfate concentration: 2 mM), and then allowed to stand at 37 ° C. for 10 minutes. After standing, 100 μL of TNFα-containing DMEM (TNFα concentration: 220 ng / mL) was added to obtain a culture solution A (potassium aluminum sulfate concentration: 1 mM and TNFα concentration: 20 ng / mL).

The obtained culture solution A was cultured at 37 ° C. for 48 hours, and then the culture supernatant was placed in a centrifuge tube and centrifuged at 14000 × g for 5 minutes to recover the culture supernatant A. On the other hand, the cells remaining in each hole of the 12-well plate were washed with ice-cold PBS. 200 μL of a protein extraction buffer (trade name: RIPA buffer, manufactured by Santa Cruz Co., Ltd.) was added to the cells, and the cells were peeled from the wall surfaces of the holes using a cell scraper to obtain a cell suspension. Cell extract A was obtained by centrifuging the cell suspension at 14000 × g for 5 minutes and collecting the supernatant.

Comparative Example 7
Epidermal keratinocytes were cultured in DMEM medium at 37 ° C. for 1 day. The obtained cultured cells were treated with trypsin. A culture solution was obtained by adding DMEM to the cultured cells after trypsin treatment so as to adjust the concentration of the cultured cells to 1 × 10 ⁵ cells / mL. 1 mL of the obtained culture solution was seeded in each hole of a 12-well plate, and the cultured cells were cultured at 37 ° C. for 24 hours. After culturing, 500 μL of the culture supernatant was replaced with 500 μL of DMEM, and then allowed to stand at 37 ° C. for 10 minutes. After standing, 100 μL of TNFα-containing DMEM (TNFα concentration: 220 ng / mL) was added to obtain a culture solution B (TNFα concentration: 20 ng / mL).

The obtained culture broth B was cultured at 37 ° C. for 48 hours, and then the culture supernatant was placed in a centrifuge tube and centrifuged at 14000 × g for 5 minutes to recover the culture supernatant B. On the other hand, the cells remaining in each hole of the 12-well plate were washed with ice-cold PBS. 200 μL of a protein extraction buffer (trade name: RIPA buffer, manufactured by Santa Cruz Co., Ltd.) was added to the cells, and the cells were peeled from the wall surfaces of the holes using a cell scraper to obtain a cell suspension. Cell extract B was obtained by centrifuging the cell suspension at 14000 × g for 5 minutes and collecting the supernatant.

Test example 3
Cell extract A obtained in Example 4 or cell extract B obtained in Comparative Example 7 and IL-1α quantitative reagent [R & D, trade name: Human IL-1 alpha / IL-1F1 DuoSet ELISA DY200] Were used to measure IL-1α expression levels in the cultured cells (epidermal keratinocytes) obtained in Example 4 and Comparative Example 7.

In Test Example 3, the results of examining the expression level of IL-1a in each epidermal keratinocyte obtained in Example 4 and Comparative Example 7 are shown in FIG. In the figure, Lane 1 shows the IL-1α expression level in the epidermal keratinocytes obtained in Example 4, and Lane 2 shows the IL-1α expression level in the epidermal keratinocytes obtained in Comparative Example 7.

From the results shown in FIG. 8, the IL-1α expression level in the epidermal keratinocytes obtained in Example 4 is small compared to the IL-1α expression level in the epidermal keratinocytes obtained in Comparative Example 7. I understand that. This result shows that potassium aluminum sulfate suppresses the expression of IL-1α induced by TNFα.

Further, the culture supernatant A obtained in Example 4 or the culture supernatant B obtained in Comparative Example 7 and IL-6 quantitative reagent [manufactured by R & D, trade name: Human IL-6 DuoSet ELISA DY206] were used. The IL-6 expression level in each cultured cell (epidermal keratinocyte) obtained in Example 4 and Comparative Example 7 was measured.

In Test Example 3, the results of examining the IL-6 expression level in each epidermal keratinocyte obtained in Example 4 and Comparative Example 7 are shown in FIG. In the figure, lane 1 shows the IL-6 expression level in the epidermal keratinocytes obtained in Example 4, and lane 2 shows the IL-6 expression level in the epidermal keratinocytes obtained in Comparative Example 7.

From the results shown in FIG. 9, the expression level of IL-6 in the keratinocytes obtained in Example 4 is less than the expression level of IL-6 in the keratinocytes obtained in Comparative Example 7. Recognize. This result shows that potassium aluminum sulfate suppresses the expression of IL-6 induced by TNFα.

From these results, it can be seen that a substance that activates TRPM4 and suppresses the expression of inflammatory cytokines can be evaluated by using a physiological event caused by TRPM4 of TRPM4-expressing cells by the test sample. It can also be seen that substances that activate TRPM4 can suppress the expression of inflammatory cytokines such as IL-1α and IL-6 and prevent inflammation.

As described above, a TRPM4 agonist such as BTP2; a substance having an action of activating TRPM4 such as an aluminum compound such as aluminum chloride and potassium aluminum sulfate, is brought into contact with the skin cells in advance, thereby causing inflammatory cytokines in the skin cells and Since the expression of the gene is suppressed, it can be seen that according to the substance having an action of activating TRPM4, the expression of inflammatory cytokine can be suppressed and inflammation can be prevented. Therefore, the present invention is expected to be suitably used for the development of inflammation preventing cosmetics, inflammation preventing agents and the like.

In addition, by measuring a physiological event caused by TRPM4-expressing cells via TRPM4 using TRPM4-expressing cells and using the physiological event for evaluation of the test sample, cytokines in skin cells of the test sample It turns out that a production inhibitory effect can be evaluated.

Claims

A test sample evaluation method for evaluating a cytokine production inhibitory action in skin cells of a test sample, comprising contacting a TRPM4-expressing cell with a test sample, and measuring a physiological event caused by the test sample via TRPM4, A test sample evaluation method, comprising: evaluating a cytokine production inhibitory action of the test sample based on the physiological event.
The physiological event is cytokine production, the TRPM4 expression cell having cytokine expression ability is used as the TRPM4 expression cell, the physiological event is measured, and the cytokine production inhibitory action of the test sample is based on the physiological event The operation when evaluating
(A1) a step of contacting a TRPM4 expression cell having cytokine expression ability with a test sample and a cytokine production promoting substance, and measuring the expression level of the cytokine and / or its gene in the TRPM4 expression cell;
(A2) a step of contacting a TRPM4 expression cell having cytokine expression ability with a cytokine production promoting substance, and measuring the expression level of the cytokine and / or its gene in the TRPM4 expression cell;
(A3) contacting a TRPM4 deficient cell in which the TRPM4 function in the TRPM4 expressing cell is deficient with a test sample and a cytokine production promoter, and measuring the expression level of the cytokine and / or its gene in the TRPM4 deficient cell;
(A4) a step of contacting a TRPM4-deficient cell deficient in TRPM4 function in the TRPM4-expressing cell with a cytokine production promoting substance, and measuring the expression level of the cytokine and / or its gene in the TRPM4-deficient cell; and (A5) 2. The test sample evaluation method according to claim 1, comprising a step of evaluating a cytokine production inhibitory action of the test sample based on the expression level measured in steps (A1) to (A4).
An operation in measuring the physiological event and evaluating the cytokine production inhibitory effect of the test sample based on the physiological event,
(B1) contacting the TRPM4-expressing cell with the test sample, measuring a change in TRPM4 activity in the TRPM4-expressing cell before and after contact with the test sample, and (B2) changing the TRPM4 activity measured in step (B1) The method for evaluating a test sample according to claim 1, further comprising a step of evaluating a cytokine production inhibitory effect of the test sample based on the method.
A cytokine production inhibitor used for the purpose of activating TRPM4 and suppressing cytokine production in skin cells, comprising an aluminum compound as an active ingredient for activating TRPM4.