JP2022068272A - Cytokine production suppressor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for evaluating a test sample, the method making it easy to evaluate whether a test sample has the effect of suppressing cytokine production in skin cells, and a cytokine production suppressor capable of effectively suppressing cytokine production in skin cells.

SOLUTION: The present invention pertains to a cytokine production suppressor to be used for suppressing cytokine production in skin cells by activating TRPM4, the suppressor containing an aluminum compound as an active ingredient for activating TRPM4.

SELECTED DRAWING: None

COPYRIGHT: (C)2022,JPO&INPIT

Description

The present invention relates to a method for evaluating a test sample. More specifically, the present invention relates to a method for evaluating a test sample for evaluating whether or not a test substance has a cytokine production inhibitory effect, and a cytokine production inhibitor.

Inflammation on the skin causes acne scars, age 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 calms the inflammation that has already occurred on the skin, it suppresses the onset of inflammation by suppressing the production of cytokines, which are one of the causative factors of inflammation. It is desired to develop anti-inflammatory cosmetics, anti-inflammatory agents, etc. that can be used.

Japanese Unexamined Patent Publication No. 2016-196418

The present invention has been made in view of the above-mentioned prior art, and is an evaluation method of a test sample capable of easily evaluating whether or not a test sample has an action of suppressing cytokine production in skin cells, and a cytokine in skin cells. It is an object of the present invention to provide a cytokine production inhibitor capable of effectively suppressing production.

The present invention is a cytokine production inhibitor used for the purpose of activating TRPM4 and suppressing cytokine production in skin cells, and is characterized by containing an aluminum compound as an active ingredient for activating TRPM4. Regarding production inhibitors.

According to the present invention, it is possible to easily evaluate whether or not a test sample has an action of suppressing cytokine production in skin cells, a method for evaluating a test sample, and effective suppression of cytokine production in skin cells. Cytokine production inhibitors are provided.

In Reference Example 1, it is a drawing substitute photograph which shows the result of the Western blot analysis. It is a graph which shows the result of having examined the expression level of the IL-1α gene in each cell obtained in Examples 1 and 2 and Comparative Examples 1 and 2 in Test Example 1. It is a graph which shows the result of having examined the expression level of the IL-8 gene in each cell obtained in Examples 1 and 2 and Comparative Examples 1 and 2 in Test Example 1. It is a graph which shows the result of having examined the expression level of the TNF gene in each cell obtained in Examples 1 and 2 and Comparative Examples 1 and 2 in Test Example 1. In Test Example 2, it is a graph which shows the result of having examined the expression level of IL-8 gene in each cell obtained in Comparative Examples 3-6. In 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 the relative fluorescence intensity. 3 is a graph showing the results of investigating the relationship between the type of test sample and the relative fluorescence intensity in Example 3. It is a graph which shows the result of having examined the IL-1α expression level in each epidermal keratinized cell obtained in Example 4 and Comparative Example 7 in Test Example 3. 3 is a graph showing the results of examining the IL-6 expression level in each epidermal keratinized cell obtained in Example 4 and Comparative Example 7 in Test Example 3.

1. 1. Evaluation method of test sample The evaluation method of the test sample of the present invention is a method of evaluating the test sample for evaluating the cytokine production inhibitory effect of the test sample in the skin cells, and the TRPM4-expressing cells and the test sample are brought into contact with each other for the test. The sample is characterized by measuring a TRPM4-mediated physiological event in TRPM4-expressing cells and evaluating the cytokine production inhibitory effect of the test sample based on the physiological event.

According to the method for evaluating a test sample of the present invention, a physiological event caused by the test sample in TRPM4 expressing cells via TRPM4 is measured, 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 or not the test sample suppresses the production of cytokines due to the activation of TRPM4 in skin cells. Therefore, according to the method for evaluating a test sample of the present invention, it is possible to easily evaluate whether or not the test sample has an effect of suppressing cytokine production in skin cells. Further, according to the evaluation method of the test sample of the present invention, since TRPM4 expressing cells are used, the evaluation of the cytokine production inhibitory effect of the test sample can be performed many times at the same time under the same conditions with good reproducibility. Can be done.

The test sample is a sample to be evaluated whether or not 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 these examples. When the test sample is a liquid, it may be used as it is, or it may be diluted with a solvent and used if necessary. When the test sample is a solid, it can be used by dissolving it in a solvent.

Since the solvent varies depending on the type of the test sample, the type of the physiological event to be measured, etc., it cannot be unconditionally determined. Therefore, the solvent is determined according to the type of the test sample, the type of the physiological event to be measured, and the like. Is preferable. Examples of the solvent include ethanol, physiological saline, phosphate buffered physiological saline, purified water, medium, and 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) hydrochloride 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 media components suitable for the growth of TRPM4-expressing cells. Examples of the medium component include glucose, amino acids, peptones, vitamins, cell proliferation promoting factors, serum, calcium chloride, magnesium chloride and the like, but the present invention is not limited to these examples. The medium may be a medium produced by supplementing the basal medium with a medium component, or may be a commercially available medium. The basic medium is not particularly limited, and examples thereof include Eagle's minimum medium (hereinafter referred to as "MEM"), Dulbecco's modified Eagle's medium (hereinafter referred to as "DMEM"), RPMI-1640 medium, and the like. It is not limited to the examples. Since the medium differs depending on the type of TRPM4-expressing cells and the like, it cannot be unconditionally determined. Therefore, it is preferable to determine the medium according to the type of TRPM4-expressing cells and the like.

TRPM4-expressing cells are cells that express physiological functions equivalent to those expressing wild-type TRPM4. Physiological functions equivalent to those of cells expressing wild-type TRPM4 include, for example, permeation of potassium and sodium ions from the outside to the inside of the cell by stimulation such as chemical stimulation with a TRPM4 agonist. , Not limited to such examples. The TRPM4 expressing cell may be an endogenous TRPM4 expressing cell or an exogenous TRPM4 expressing cell.

Examples of endogenous TRPM4-expressing cells include normal human epidermal keratinized cells, HaCaT cells, keratinized cells such as NCTC2544; T such as human myocardial cells, human T cells, Jurkat cells, MALT-4 cells, and U-937 cells. Cells; obese cells such as human obese cells, HMC-1; human monocytic cells, monocytic cells such as THP-1 and the like, but the present invention is not limited to such embodiments.

The extrinsic TRPM4 expressing cell may be any cell that overexpresses extrinsic 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 transferred to the chromosome of the host cell. It may be an integrated cell. The exogenous TRPM4 expressing cells may express endogenous TRPM4 as long as the object of the present invention is not impaired. Exogenous TRPM4 expressing cells can be obtained, for example, by introducing a TRPM4 expression vector carrying a nucleic acid encoding TRPM4 into a host cell.

Examples of the nucleic acid encoding TRPM4 include, but are not limited to, the mRNA shown in GenBank accession number: NM_017636, the cDNA obtained from the mRNA, and the like. 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 nucleic acids consisting of sequences and encoding mutant polypeptides exhibiting TRPM4 activity, but the present invention is not limited to such examples. For "sequence identity", PROTEIN BLAST based on the BLAST algorithm is used as a default value, and the amino acid sequence (query sequence) to be evaluated is aligned with the amino acid sequence (reference sequence) shown in SEQ ID NO: 1. It means the value calculated by. The sequence identity is 85% or more, preferably 90% or more, more preferably 95% or more, still more preferably 98% or more, and the upper limit is 100%, from the viewpoint of expressing TRPM4 activity.

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

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

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

Physiological events caused by TRPM4 include, for example, a decrease in the expression level of a cytokine gene due to the activation of TRPM4, a decrease in the expression level of a cytokine due to 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 caused by activation of TRPM4 is used as a physiological event caused by TRPM4, the following evaluation method 1 or the like is a specific example of the evaluation method of the test sample of the present invention. However, the present invention is not limited to such examples.

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

Since TRPM4-deficient cells are used in steps (A3) and (A4) in evaluation method 1, is the physiological event caused by TRPM4-expressing cells by the test sample a physiological event caused via TRPM4? You can check if. From this, the TRPM4 expressing cells used in the evaluation method 1 are preferably endogenous TRPM4 expressing cells from the viewpoint of confirming the cells under the same conditions as when the TRPM4 deficient cells are used, except that they express TRPM4. Further, the TRPM4 expressing cell is preferably a TRPM4 cell having a cytokine expressing ability from the viewpoint of evaluating with high accuracy whether or not the test sample has a cytokine production inhibitory action. Examples of endogenous TRPM4-expressing cells having a cytokine-expressing ability include HaCaT cells, but the present invention is not limited to these examples.

In step (A1), a TRPM4 expressing cell having a cytokine expressing ability is brought into contact with a test sample and a cytokine production promoting substance, 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”), and interleukin. Examples thereof include 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. The cytokine genes are not limited to these examples. 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, but the present invention is not limited to such examples. Among these cytokine genes, the IL-1α gene and the IL-8 gene are preferable from the viewpoint of accurately evaluating the action of suppressing the onset of inflammation in the skin.

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

The order of contacting the TRPM4-expressing cell with the test sample and the cytokine production-promoting substance is, for example, (a1) the order of contacting the TRPM4-expressing cell with the test sample and then contacting the TRPM4-expressing cell with the cytokine production-promoting substance. , (A2) The order in which the TRPM4 expressing cell is brought into contact with the test sample and the cytokine production promoting substance at the same time, (a3) The TRPM4 expressing cell and the cytokine production promoting substance are brought into contact with each other, and then the TRPM4 expressing cell is brought into contact with the test sample. The present invention is not limited to such an example, although the order and the like may be mentioned.

When the order of (a1) is adopted in step (A1), as a method of contacting TRPM4 expressing cells with a test sample, for example, a method of incubating TRPM4 expressing cells in a test sample can be mentioned. The invention is not limited to such embodiments. Examples of the contact method between the TRPM4 expressing cells and the cytokine production promoting substance include a method in which the cytokine production promoting substance is added to the mixture of the test sample and the TRPM4 expressing cells after incubation, and the obtained mixture is incubated. However, the present invention is not limited to such examples. At the time of contact between the TRPM4-expressing cell and the test sample and the contact between the TRPM4-expressing cell and the cytokine production-promoting substance, the reagent used for measuring the 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 differ depending on the type of the TRPM4 expressing cell and the like, they cannot be unconditionally determined. It is preferable to determine according to such factors. 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 35 from the viewpoint of ensuring temperature conditions suitable for the expression of TRPM4 activity in the TRPM4 expressing cell. It is 40 ° C. Further, since the contact time between the TRPM4 expressing cell and the test sample differs depending on the type of the TRPM4 expressing cell, the type of the test sample, etc., it cannot be unconditionally determined. Therefore, the type of the TRPM4 expressing cell, the type of the test sample, etc. It is preferable to determine 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 effect of the test sample on the TRPM4 activity. 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 the cytokine production-promoting substance, the type of the substance whose expression level is to be measured, and the like, and therefore cannot be unconditionally determined. It is preferable to determine according to the type of TRPM4-expressing cell, the type of the cytokine production promoting substance, the type of the substance whose expression level is to be measured, and the like. The contact time between TRPM4-expressing cells and a cytokine production-promoting substance is usually preferably 6 to 72 hours when evaluating the expression level of cytokines from the viewpoint of accurately evaluating the effect of the test sample on TRPM4 activity. When assessing the expression level of a cytokine gene, it is usually preferably 1 to 12 hours.

Upon contact between TRPM4 expressing cells and the test sample, the number of TRPM4 expressing cells per 1 mL of the test sample varies depending on the type of TRPM4 expressing cells, the type of the test sample, etc., and therefore cannot be unconditionally determined. 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 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 TRPM4-mediated physiological events. From the viewpoint of accurately causing TRPM4-mediated physiological events in TRPM4 cells, the number is preferably 1 × 10 ⁹ or less, more preferably 1 × 10 ⁸ or less.

Upon contact between the TRPM4-expressing cells and the test sample, the amount of the cytokine production-promoting substance per 1 mL of the test sample varies depending on the type of the cytokine production-promoting substance, the type of the TRPM4-expressing cell, etc., and therefore cannot be unconditionally determined. , It is preferable to determine according to the type of cytokine production promoting substance, the type 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 effect of the test sample on TRPM4 activity.

When the order of (a2) or (a3) is adopted in step (A1), the number of TRPM4-expressing cells per 1 mL of the test sample and the amount of the cytokine production-promoting substance per 1 mL of the test sample are the order of (a1). The number of TRPM4-expressing cells per 1 mL of the test sample and the amount of the cytokine production-promoting substance per 1 mL of the test sample at the time of adoption are the same.

Since the contact temperature between the TRPM4-expressing cell, the test sample, and the cytokine production-promoting substance differs depending on the type of TRPM4-expressing cell and the like, it cannot be unconditionally determined. Therefore, it can be determined according to the type of TRPM4-expressing cell and the like. 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 ensuring temperature conditions suitable for expressing TRPM4 activity in TRPM4-expressing cells.

The contact time between the TRPM4-expressing cell, the test sample, and the cytokine production-promoting substance varies depending on the type of TRPM4-expressing cell, the type of the test sample, the type of the cytokine production-promoting substance, the type of the substance whose expression level is to be measured, and the like. Since it cannot be determined, it is preferable to determine it according to the type of TRPM4-expressing cell, the type of test sample, the type of the cytokine production promoting substance, the type of the substance whose expression level is to be measured, and the like. The contact time between TRPM4-expressing cells, the test sample, and the cytokine production-promoting substance is usually preferably 6 to 6 when evaluating the expression level of cytokines from the viewpoint of accurately evaluating the effect of the test sample on TRPM4 activity. It is 72 hours, and when evaluating the expression level of the cytokine gene, it is usually preferably 1 to 12 hours.

Examples of the method for measuring the expression level of cytokines include Western blotting, enzyme-linked immunosorbent assay (ELISA), and the like, but the present invention is not limited to these examples. Examples of the method for measuring the expression level of the cytokine gene include Northern blotting method and quantitative RT-PCR method, but the present invention is not limited to these examples.

Examples of the antibody used in the Western blotting method and ELISA include, for example, an anti-cytokine antibody or an antibody fragment thereof, but the present invention is not limited to such examples. The anti-cytokine antibody may be a monoclonal antibody or a polyclonal antibody. Monoclonal antibodies are preferred from the viewpoint of improving the quantification of cytokine expression. For the monoclonal antibody, for example, a hybrid doma that produces a monoclonal antibody that is reactive to a cytokine or a part thereof is cultured to obtain a culture supernatant, and the obtained culture supernatant is subjected to a sulfur fractionation method as necessary. , Obtained by purification by ion exchange chromatography, gel filtration chromatography, affinity chromatography and the like. The hybrid domoma immunizes the animal by, for example, administering the cytokine or a part thereof intravenously, subcutaneously or intraperitoneally to obtain antibody-producing cells, and the antibody-producing cells and myeloma cells are fused. It is obtained by culturing the obtained fusion cells in a HAT medium or the like. In addition, the polyclonal antibody immunizes the animal by administering the cytokine or a part thereof intravenously, subcutaneously or intraperitoneally to obtain an antiserum, and the obtained antiserum is subjected to sulfurization as necessary. It can be obtained by purifying by a painting method, ion exchange chromatography, gel filtration chromatography, affinity chromatography or the like. The antibody fragment can be obtained, for example, by degrading an anti-cytokine antibody with a digestive enzyme and purifying the obtained degradation product as needed. Further, as the anti-cytokine antibody and the antibody fragment thereof, a commercially available anti-cytokine antibody and the antibody fragment thereof can be used.

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 the 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 the cytokine gene and the like, so it can be unconditionally determined. Since it cannot be determined, it is preferable to determine it according to the type of cytokine gene and the like. The length of the probe is usually preferably 20 to 500 nucleotides in length 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 consisting of a part of the base sequence of the nucleic acid encoding the cytokine gene and a primer consisting of a part of the base sequence of the antisense strand of the nucleic acid. Primers and the like can be mentioned, but the present invention is not limited to these examples. Since the length, GC content and Tm value of each primer constituting the primer pair and the distance between the primers on the nucleic acid encoding the cytokine gene differ depending on the type of the cytokine gene and the like, they cannot be unconditionally determined. It is preferable to determine according to the type of cytokine gene and the like. The length of the primer is usually preferably 12 to 30 nucleotides in length 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, each primer constituting the primer pair contains a sequence in which thymine residues or cytosine residues are continuous (polypyrimidine sequence) and an adenine residue or guanine residue from the viewpoint of improving the measurement accuracy of the expression level of the cytokine gene. It is preferable to have a sequence that does not contain both consecutive sequences (polypurine sequences). Each primer constituting the primer pair consists of a part of the base 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, and may be a sequence of other genes or the like. Is preferably composed of sequences that are 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), TRPM4 expressing cells capable of expressing cytokines are brought into contact with a cytokine production promoting substance, and the expression level of cytokines and / or their genes in TRPM4 expressing cells is measured.

The contact method between the TRPM4-expressing cells and the cytokine production-promoting substance used in step (A2), the contact temperature between the TRPM4-deficient cells and the cytokine production-promoting substance, the contact time between the TRPM4-deficient cells and the cytokine production-promoting substance, and the cytokine gene and cytokine. The method for measuring each expression level is 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. It is the same as the method for measuring the contact time with and the expression level 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 in the contact between the TRPM4-expressing cell, the test substance and the cytokine production-promoting substance in step (A1).

In step (A3), TRPM4-deficient cells lacking TRPM4 function in TRPM4-deficient cells are brought into contact with a test sample and a cytokine production-promoting substance, and the expression level of cytokines and / or their genes in the TRPM4-deficient cells is measured.

TRPM4-deficient cells are similar to the TRPM4-expressing cells used in steps (A1) and (A2), except that they lack TRPM4 function. TRPM4-deficient cells can be produced by disrupting the TRPM4 gene in TRPM4-expressing cells by a gene knockout method, deleting the function of TRPM4 in TRPM4-expressing cells, and the like. Examples of the gene knockout method include a method of disrupting the TRPM4 gene by a homologous recombination method, a method of disrupting the TRPM4 gene by a genome editing technique, and the like, but the present invention is not limited to these examples. .. As a method for deleting the function of TRPM4, a method of inhibiting the expression of TRPM4 gene by RNA silencing method using siRNA or shRNA, and a method of expressing a dominant negative mutant in TRPM4 expressing cells are used to obtain normal TRPM4. Examples thereof include methods for inhibiting the function, but the present invention is not limited to such examples.

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

In step (A4), TRPM4-deficient cells lacking TRPM4 function in TRPM4-deficient cells are brought into contact with a cytokine production-promoting substance, and the expression level of cytokines and / or their genes in the TRPM4-deficient cells is measured.

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

In the method for evaluating a test sample of the present invention, it is preferable to use a control sample and calibrate the expression level when the test sample is used from the viewpoint of improving the accuracy of the evaluation. The control sample may be a liquid that does not contain the test sample, and examples thereof include a solvent used for diluting the test sample, a solvent used for dissolving the test sample, and the like, but the present invention is limited to such examples. It is not something that will be done. When a control sample is used, the same operation as in steps (A1) to (A4) is performed in steps (A1) to (A4), except that the control sample is used instead of the test sample, and the cytokine and / or the same are performed. Measure the expression level of the gene.

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

The expression level measured in step (A1) is smaller 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). If so, it can be evaluated that the test sample is a substance having an action of activating TRPM4 and suppressing the production of cytokines in skin cells.

When the expression of the activity of TRPM4 itself is used as a physiological event caused by TRPM4, specific examples of the evaluation method of the test sample of the present invention include the following evaluation method 2 and the like. , Not limited to such examples.

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

The TRPM4 expressing cells used in the evaluation method 2 are preferably exogenous TRPM4 expressing cells, and more preferably cells overexpressing exogenous TRPM4 from the viewpoint of facilitating observation of changes in TRPM4 activity.

Changes in TRPM4 activity are due, for example, to changes in the amount of sodium ions taken up 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. Examples thereof include an increase in current, but the present invention is not limited to such an example.

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

When the intracellular calcium ion concentration is used as an index for measuring the amount of sodium ion taken up into cells by TRPM4, the measurement of the intracellular calcium ion concentration is carried out, for example, by using a calcium indicator that specifically binds to calcium ions as a TRPM4 expressing cell. It can be carried out by binding the calcium indicator to the calcium ion in the TRPM4 expressing cell and measuring the amount of the calcium indicator bound to the calcium ion in the TRPM4 expressing cell.

In TRPM4-activated TRPM4-expressing cells, the influx of sodium ions from TRPM4-mediated extracellular TRPM4-expressing cells into TRPM4-expressing cells is increased as compared with TRPM4-expressing cells in which TRPM4 is not activated. Increases intracellular sodium ion concentration. As the intracellular sodium ion concentration of TRPM4-expressing cells increases, the intracellular calcium ion concentration of the TRPM4-expressing cells decreases. Further, as shown in Examples described later, the activation of TRPM4 and the suppression of cytokine production in skin cells are correlated. Therefore, when the intracellular calcium concentration of TRPM4-expressing cells in contact with the test sample is lower than the intracellular calcium concentration of TRPM4-expressing cells not in contact with the test sample, the test sample activates TRPM4 and cytokines in skin cells. It can be evaluated as a substance having an action of suppressing production. On the other hand, when the intracellular calcium concentration of the TRPM4-expressing cells in contact with the test sample is higher than or equal to the intracellular calcium concentration of the TRPM4-expressing cells not in contact with the test sample, the test sample is It can be evaluated as a substance that does not have an action of suppressing the production of cytokines in skin cells.

When the intracellular calcium ion concentration is used as an index for measuring the amount of sodium ion taken up into cells by TRPM4, the TRPM4 activating action is, for example,
(I) The step of measuring the intracellular calcium ion concentration A of TRPM4-expressing cells using a calcium indicator,
(II) The step of contacting the TRPM4-expressing cell with the 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) can be compared and evaluated by a method including a step for evaluating the TRPM4 activating effect of the test sample.

In step (I), the intracellular calcium ion concentration A of TRPM4-expressing cells is measured using a calcium indicator. As the TRPM4 expressing cells, it is preferable to use HEK293 cells or the like expressing exogenous TRPM4.

A calcium indicator is a reagent that can detect changes before and after binding to calcium ions by changes in optical properties, etc., because the amount of calcium indicators bound to calcium ions can be measured by a simple operation. Is preferable. Examples of changes in optical properties include changes in fluorescence intensity, changes in absorbance, and the like, but the present invention is not limited to these examples. Examples of the calcium indicator include a fluorescent calcium indicator whose fluorescence intensity changes before and after binding to calcium ions, but the present invention is not limited to these examples. Specific examples of the calcium indicator include 1- [6-amino-2- (5-carboxy-2-oxazolyl) -5-benzofuranyloxy] -2- (2-amino-5-methylphenoxy) ester-N. , N, N', N'-pentaacetoxymethyl ester tetraacetate (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'-tetraacetoxymethyl ester tetraacetate (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'-tetraacetoxy tetraacetate Fluorescent calcium indicators such as methyl ester (Fluo 4-AM) and the like can be mentioned, but the present invention is not limited to such examples. Among calcium indicators, fluorescent calcium indicators whose fluorescence intensity changes before and after binding to calcium ions from the viewpoint of easy measurement and easy differentiation from the dynamics of contaminants present in TRPM4-expressing cells. Is preferable.

The fluorescent calcium indicator may have one type of excitation wavelength or may have two or more types of excitation wavelengths. When 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 fluorescent calcium indicator having one kind of excitation wavelength is used for measuring the change in intracellular calcium concentration, the change in intracellular calcium concentration can be measured based on the fluorescence intensity at the excitation wavelength. When a fluorescent calcium indicator having two or more kinds of excitation wavelengths is used in measuring changes in intracellular calcium concentration, the two or more kinds of excitation wavelengths are used from the viewpoint of improving the accuracy of evaluation of the cytokine production inhibitory effect of the test sample. Two types of excitation wavelengths (first excitation wavelength and second excitation wavelength) selected from are selected, and intracellularly based on the fluorescence intensity ratio calculated from the fluorescence intensity at each of the first excitation wavelength and the second excitation wavelength. Changes in calcium concentration can be measured. When measuring the intracellular calcium concentration, for example, when FURA 2-AM, which is a fluorescent calcium indicator having two kinds 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 calculated based on.

Examples of the method for measuring the intracellular calcium ion concentration A using the fluorescent calcium indicator include the following measuring method 1, but the present invention is not limited to such an example.

<Measurement method 1>
Steps of introducing a fluorescent calcium indicator into TRPM4-expressing cells to obtain indicator-introduced cells,
The step of contacting the indicator-introduced cell with calcium ion to bind calcium ion to the fluorescent calcium indicator in the indicator-introduced cell, and the step of measuring the fluorescence intensity of the fluorescent calcium indicator bound to calcium ion in the indicator-introduced cell. How to include.

Examples of the 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. Not limited to just. Examples of the buffer solution include HEPES buffer solution, but the present invention is not limited to such an example.

As a method of contacting the TRPM4 expressing cells into which the fluorescent calcium indicator has been introduced with calcium ions, for example, a buffer solution containing calcium ions is circulated in a recirculation chamber containing the TRPM4 expressing cells into which the fluorescent calcium indicator has been introduced. Methods and the like can be mentioned, but the present invention is not limited to such examples. Examples of the buffer solution include HEPES buffer solution, but the present invention is not limited to such an example.

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

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

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

Examples of the method for measuring the intracellular calcium ion concentration B using the fluorescent calcium indicator include the following measuring method 2, but the present invention is not limited to such an example.

<Measurement method 2>
Steps of introducing a fluorescent calcium indicator into TRPM4-expressing cells to obtain indicator-introduced cells,
The step of contacting the indicator-introduced cell with the test sample,
It includes a step of contacting a cell after contact with a test sample with a calcium ion and binding the calcium ion to the intracellular fluorescent calcium indicator, and a step of measuring the fluorescence intensity of the fluorescent calcium indicator bound to the calcium ion in the cell. Method.

As a method of contacting the TRPM4 expressing cells into which the fluorescent calcium indicator has been introduced with the test sample, a method of circulating a buffer solution containing the fluorescent calcium indicator in a reflux chamber containing the TRPM4 expressing cells into which the fluorescent calcium indicator has been introduced is circulated. However, the present invention is not limited to such an example. Examples of the method of contacting the cells after contact with the test sample with calcium ions include a method of circulating a buffer solution containing calcium ions and not containing the test sample. Not limited. In the present invention, the buffer solution containing the test sample is circulated in the reflux chamber containing the TRPM4 expressing cells into which the fluorescent calcium indicator has been introduced, and then the buffer solution containing the test sample and calcium ions is circulated. You may.

The method for measuring the intracellular calcium ion concentration B used in step (II), the contact temperature and the number of TRPM4 expressing cells are the method for measuring the intracellular calcium ion concentration B used in step (I), the contact temperature and the number of TRPM4 expressing cells. Similar to the number of.

In step (III), the intracellular calcium ion concentration A obtained in step (I) and the intracellular calcium ion concentration B obtained in step (II) are compared, and the TRPM4 activating effect of the test sample is evaluated. do. In step (III), when the calcium ion concentration B is reduced as compared with the calcium ion concentration A, the test sample can be evaluated as having a TRPM4 activating effect, and further, the calcium ion concentration A and calcium. The larger the difference from the ion concentration B, the higher the TRPM4 activating effect of the test sample can be evaluated.

When using the increase in current resulting from the activation of TRPM4 in TRPM4-expressing cells before and after contact with the test sample as the change in TRPM4 activity, the activating effect of TRPM4 is, for example,
(I) The step of measuring the current A in TRPM4-expressing cells under a constant potential,
(Ii) The step of contacting the TRPM4 expressing cell with the test sample and measuring the current B in the TRPM4 expressing cell under the same potential as the potential in step (i), and (iii) obtained in step (i). The current A can be compared with the current B obtained in step (ii) and evaluated by a method including a step for evaluating the TRPM4 activating effect of the test sample.

In step (i), the current A caused by the activation of TRPM4 in TRPM4-expressing cells is measured under a constant potential. Examples of the method for measuring the current A include the patch clamp method, but the present invention is not limited to such an example. According to the patch clamp method, the current caused by the activation of one or more TRPM4s present on the cell membrane of TRPM4 expressing cells can be measured. Examples of the patch clamp method include a whole cell method and a cell attach method, but the present invention is not limited to these examples.

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

In step (iii), the current A obtained in step (i) and the current B obtained in step (ii) are compared to evaluate the TRPM4 activating effect of the test sample. In step (iii), if the current A is smaller than the current B, the test sample can be evaluated as having a TRPM4 activating effect. Further, it can be evaluated that the larger the difference between the current A and the current B, the higher the TRPM4 activating effect of the test sample.

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

2. 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 is 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, it can effectively suppress cytokine production in skin cells. Therefore, the cytokine production inhibitor of the present invention can be suitably used for suppressing cytokine production in skin cells and the like.

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 unconditionally determined. It is preferable to set it 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 usually preferably 0.0001% by mass or more, more preferably 0.0001% by mass or more, from the viewpoint of sufficiently activating TRPM4 and sufficiently expressing the action of suppressing cytokine production in skin cells. Is 0.005% by mass or more, more preferably 0.008% by mass or more, particularly preferably 0.01% by mass or more, and preferably 100% by 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, as long as the object of the present invention is not impaired. When the cytokine production inhibitor of the present invention contains other components, the aluminum compound and the other components form a complex in the cytokine production inhibitor of the present invention to the extent that the object of the present invention is not impaired. It may be formed.

As described above, since the cytokine production inhibitor of the present invention can effectively suppress 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 for applications such as skin inflammation preventive cosmetics and skin inflammation preventive agents.

When the cytokine production inhibitor of the present invention is used as an inflammation preventive cosmetic in the skin or an inflammation preventive agent in the skin, the content of the cytokine production inhibitor of the present invention in the inflammation preventive cosmetic or the anti-inflammatory agent is the inflammation prevention. Uses of anti-inflammatory cosmetics or anti-inflammatory agents because they cannot be unconditionally determined because they differ depending on the use of cosmetics or anti-inflammatory agents, the type of aluminum compound contained in the cytokine production inhibitor of the present invention, and the like. , It is preferable to determine according to the type of the aluminum compound contained in the cytokine production inhibitor of the present invention. The content of the cytokine production inhibitor of the present invention in an inflammation-preventing cosmetic or an inflammation-preventing agent is usually an inflammation-preventing cosmetic or an inflammation-preventing cosmetic or an anti-inflammatory agent from the viewpoint of sufficiently activating TRPM4 to sufficiently exert an action of suppressing inflammation in the skin. The content of the aluminum compound in the anti-inflammatory agent is preferably 0.0001% by mass or more, more preferably 0.0005% by mass or more, still more 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 desirable that the content is preferably adjusted to 20% by mass or less, more preferably 10% by mass or less. Anti-inflammatory cosmetics or anti-inflammatory agents include, for example, solvents, surfactants, moisturizers, thickeners, preservatives, and oxidations to the extent that TRPM4 is not activated and the action of suppressing inflammation in the skin is not hindered. Other ingredients such as an inhibitor and a pH adjuster may be blended. Since the dose and frequency of application of anti-inflammatory cosmetics 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., they cannot be unconditionally determined. 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 by way of examples, but 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. (Volume%) is shown.

Production Example 1
HEK293 of 5 × 10 ⁶ cells in 10% (v / v)% fetal bovine serum-containing DMEM on a petri dish with a diameter of 35 mm maintained at 37 ° C. in an atmosphere of 5% (v / v) carbon dioxide-containing air. The 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 gene transfer reagent [ThermoFisher SCIENTIC] Name: Lipofectamine Transfection Reaction] is used to transiently introduce a nucleic acid encoding human TRPM4 and a nucleic acid encoding a red fluorescent protein into HEK293 cells according to the protocol attached to the gene transfer reagent, and transfect. I got a tant. In the following, transfectants that emit 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. Dissolution buffer for 1 x 10 ⁶ cells after washing [Composition: 25 mM Trishydroxymethylaminomethane (hereinafter referred to as "Tris"), 1 mM ethylenediaminetetraacetic acid, 0.1 mM glycol etherdiaminetetraacetic acid, 5 mM magnesium chloride , 100 mM sodium chloride, 10% (v / v) glycerol and 1% (v / v) polyethylene glycol-tert-octylphenyl ether (Triton X-100)] 100 μL. The resulting mixture was stirred and then incubated on ice for 10 minutes to give cytolysates. The cytolysate 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 / hydrochloric acid 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 cell (Experiment No. 1) obtained in Production Example 1, a human epidermal keratinized cell establishment strain (HaCaT cell) (Experiment No. 2) or a normal human epidermal keratinized cell (Experiment). Except for the fact that No. 3) was used, the same operation as in Experiment No. 1 was carried out to obtain each electrophoretic sample 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 a migration gel [manufactured by Invitrogen, trade name: NuPAGE 4-12% Bis Tris Gel], and electrophoresed at 200 V for 45 minutes. Next, the protein on the gel after electrophoresis was transferred to the membrane using a transfer kit [Invitrogen, trade name: iBlot gel transfer stocks mini] and a transfer device [Invitrogen, trade name: iBlot].

Post-transfer membrane and mouse anti-TRPM4 antibody [manufactured by OriGene, trade name: Anti-TRPM4, Mouse-Mono (10H5) TrueMAB, product number: TA500381] and secondary antibody [HRP-binding anti-mouse IgG antibody [C.] Western blotting analysis was performed using ST (CST) Japan Co., Ltd., trade 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 the result of Western blot analysis. The result of the Western blot analysis of the sample for electrophoresis 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 keratinized cells obtained in Production Example 1 were obtained. It can be seen that all of them express the TRPM4 protein.

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

BTP2 and HaCaT cells were brought into contact with the cell culture by adding BTP2, which is a TRPM4 agonist, so that the concentration thereof was 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 post-incubation mixture to a concentration of 20 ng / mL, and then the obtained mixture was incubated at 37 ° C. for 1 hour to bring TNFα into contact with HaCaT cells. Then, 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 on a 96-well plate maintained at 37 ° C. in an atmosphere of 5% (v / v) carbon dioxide-containing air, and 6.4 × 10 ³ HaCaT cells were cultured. Cell cultures containing were obtained. The cell culture was 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 on a 96-well plate maintained at 37 ° C. in an atmosphere of 5% (v / v) carbon dioxide-containing air, and 6.4 × 10 ³ HaCaT cells were cultured. Cell cultures containing were obtained. After adding TNFα to the cell culture to a concentration of 20 ng / mL, the obtained mixture was incubated at 37 ° C. for 1 hour to bring TNFα into contact with HaCaT cells. The cells were then harvested from the mixture.

Test Example 1
RNA-containing samples were obtained using the cells obtained in Examples 1 and 2 and Comparative Examples 1 and 2 and a cell lysis reagent [manufactured by Roche, trade name: Realtime ReadyCell Lysis Kit]. Using the obtained RNA-containing sample and an RT-PCR kit [manufactured by Takara Bio Co., Ltd., trade name: One step PrimeScript RT-PCR kit], qRT-PCR was performed to compare with Examples 1 and 2. The expression levels of the IL-1α gene, IL-8 gene, and TNF gene in each cell obtained in Examples 1 and 2 were measured. In addition, 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 the cells obtained in Examples 1 and 2 and Comparative Examples 1 and 2 are shown in FIG. 2, Examples 1 and 2 and Comparative Examples 1 and 2. The results of examining the expression level of the IL-8 gene in each of the obtained cells are shown in FIG. 3, and the results of examining the expression level of the TNF gene in each of the cells obtained in Examples 1 and 2 and Comparative Examples 1 and 2 are shown in FIG. Shown in 4. In FIG. 2, lane 1 is the expression level of the IL-1α gene in the cells obtained in Comparative Example 1, lane 2 is the expression level of the IL-1α gene in the cells obtained in Comparative Example 2, and lane 3 is the expression level of the IL-1α gene in Example 1. The expression level of the IL-1α gene in the cells obtained in 1 and lane 4 shows the expression level of the IL-1α gene in the cells obtained in Example 2. In FIG. 3, lane 1 is the expression level of the IL-8 gene in the cells obtained in Comparative Example 1, lane 2 is the expression level of the IL-8 gene in the cells obtained in Comparative Example 2, and lane 3 is the expression level of the IL-8 gene in Example 1. The expression level of the IL-8 gene in the cells obtained in Example 2 and lane 4 indicate the expression level of the IL-8 gene in the cells obtained in Example 2. In FIG. 4, lane 1 is the expression level of the TNF gene in the cells obtained in Comparative Example 1, lane 2 is the expression level of the 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.

From the results shown in FIGS. 2 to 4, it can be seen that the expression levels of the IL-1α gene, the IL-8 gene, and the TNF gene decrease as the concentration of BTP2 increases. These results suggest that TRPM4 activation correlates with suppression of the expression of inflammatory cytokine genes such as the IL-1α gene, IL-8 gene, and TNF gene. In addition, by contacting a substance having an action of activating TRPM4 with skin cells in advance, the expression of inflammatory cytokines induced by TNFα at the gene level can be suppressed, so that the action of activating TRPM4 can be suppressed. It is suggested that the substance possessed can prevent inflammation.

Manufacturing example 2
By disrupting the TRPM4 gene in HaCaT cells by gene knockout technique, knockout HaCaT cells in which the TRPM4 gene was knocked out were obtained.

Comparative Example 3
In Example 1, the same operation as in Example 1 was carried out except that the knockout HaCaT cells obtained in Production Example 2 were used instead of HaCaT cells to obtain cells.

Comparative Example 4
In Example 2, the same operation as in Example 2 was carried out except that the knockout HaCaT cells obtained in Production Example 2 were used instead of HaCaT cells to obtain cells.

Comparative Example 5
In Comparative Example 1, cells were obtained by performing the same operation 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 by performing the same operation 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
In Test Example 1, the same as in Test Example 1 except that the cells obtained in Comparative Examples 3 to 6 were used instead of the cells obtained in Examples 1 and 2 and Comparative Examples 1 and 2. The operation was performed, and the expression levels of the IL-1α gene, the IL-8 gene, and the TNF gene in each cell obtained in Comparative Examples 3 to 6 were measured.

FIG. 5 shows the results of examining the expression level of the IL-8 gene in each cell obtained in Comparative Examples 3 to 6 in Test Example 2. In FIG. 5, lane 1 is the expression level of the IL-8 gene in the cells obtained in Comparative Example 5, lane 2 is the expression level of the IL-8 gene in the cells obtained in Comparative Example 6, and lane 3 is the expression level of the IL-8 gene in Comparative Example 3. The expression level of the IL-8 gene in the cells obtained in Comparative Example 4 and lane 4 indicate the expression level of the IL-8 gene 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 cells were contacted with BTP2 and then contacted with TNFα was such that the knockout HaCaT cells were contacted with TNFα without contacting BTP2. It can be seen that the expression level of the IL-8 gene at that time is about the same. Inflammatory cytokine genes other than the IL-8 gene, such as the IL-1α gene and the TNF gene, also showed the same tendency as the IL-8 gene. From these results, it can be seen that the decrease in the expression level of the inflammatory cytokine gene in TRPM4-expressing cells when contacted with BTP2 is due to the activation of TRPM4.

As described above, by contacting a substance having an action of activating TRPM4 with a skin cell in advance, the expression of an inflammatory cytokine gene in the skin cell is suppressed, so that the substance having an action of activating TRPM4 According to the report, it can be seen that the expression of inflammatory cytokines can be suppressed and inflammation can be prevented.

Production example 3
Calcium-free solution (hereinafter referred to as "Sodium B") so that the concentration of tapcigargin is 1 μM [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)] was added to obtain a tapcigargin-containing bath solution.

Production example 4
Calcium-containing solution (hereinafter referred to as "Sodium A") containing ionomycin so that its concentration becomes 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 HEEPS buffer solution (hereinafter referred to as “solution A”). It was added to pH 7.4)] to obtain an ionomycin-containing solution A.

Production Example 5
U-73122, a TRPM4 agonist, was added to Solution B to a concentration of 10 μM to give a U-73122-containing bath solution.

Production Example 6
U-73122, a TRPM4 agonist, was added to Solution A so that its concentration was 10 μM to obtain U-73122-containing solution A.

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

(2) Measurement of intracellular calcium ion concentration in the absence of TRPM4 agonist The FURA 2-AM-introduced cells obtained in Reference Example 2 (1) were placed in the recirculation chamber of an inverted microscope equipped with a recirculation chamber. In the following, using a CCD camera, fluorescence at an excitation wavelength of 340 nm based on FURA 2-AM in FURA 2-AM-introduced cells and fluorescence at an excitation wavelength of 380 nm based on FURA 2-AM in FURA 2-AM-introduced cells are continuously performed. Fluorescence intensity at an excitation wavelength of 340 nm based on FURA 2-AM in FURA 2-AM-introduced cells using recording and image analysis software [manufactured by Solution Systems Co., Ltd., trade name: IPLab] (hereinafter, "fluorescence"). Fluorescence intensity at an excitation wavelength of 380 nm based on FURA 2-AM in FURA 2-AM-introduced cells (hereinafter referred to as “fluorescence intensity _{380 nm ”} ) was determined.

Solution B was refluxed in a reflux chamber containing FURA 2-AM-introduced cells. After 50 seconds have passed from the start of reflux of Solution B, calcium ions are released from the endoplasmic reticulum by refluxing the tapcigargin-containing bath solution obtained in Production Example 3 in a reflux chamber containing FURA 2-AM-introduced cells. I let you. After 240 seconds from the start of reflux of the tapcigargin-containing bath solution, solution A was refluxed in a reflux chamber containing FURA 2-AM-introduced cells. After 150 seconds had elapsed from the start of reflux of Solution A, 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 up into the cell via TRPM4, and the amount of calcium ions flowing into the cell decreases as the amount of the taken up sodium ions increases. Therefore, TRPM4 activity can be indirectly measured by measuring the change over time in the intracellular calcium ion concentration.

(3) Measurement of intracellular calcium ion concentration in the presence of TRPM4 agonist The FURA 2-AM-introduced cells obtained in Reference Example 2 (1) were placed in the recirculation chamber of an inverted microscope equipped with a recirculation chamber. In the following, the FURA 2-AM-based fluorescence intensity _{340 nm} in the FURA 2-AM-introduced cells and the FURA 2-AM-based fluorescence intensity _{380 nm} 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 _340nm / Fluorescence intensity _380nm (Ia)
The change over time in the fluorescence intensity ratio was determined based on the above.

Solution B was refluxed in a reflux chamber containing FURA 2-AM-introduced cells. After 50 seconds have passed from the start of reflux of Solution B, calcium ions are released from the endoplasmic reticulum by refluxing the tapcigargin-containing bath solution obtained in Production Example 3 in a reflux chamber containing FURA 2-AM-introduced cells. I let you. Tapcigargin-containing bath solution 120 seconds after the start of reflux, the U-73122-containing bath solution obtained in Production Example 5 was refluxed in a reflux chamber containing FURA 2-AM-introduced cells. U-73122-containing bath solution 120 seconds after the start of reflux, 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 had elapsed from the start of reflux of U-73122-containing solution A, 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.

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

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

FIG. 6 shows the results of investigating the relationship between the presence or absence of a 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, since 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, it flows into the HEK293 cells with the activation of TRPM4. It can be seen that the intracellular calcium ion concentration decreased as the sodium ion amount increased. From these results, test samples were examined by investigating the effect of the test sample on the intracellular calcium ion concentration using cells overexpressing extrinsic TRPM4, such as the TRPM4 overexpressing cells obtained in Production Example 1. It can be seen that it is possible to evaluate whether or not TRPM4 has an action of activating TRPM4 and suppressing the production of cytokines in skin cells.

Production example 7
Aluminum chloride as a test sample was added to Solution B so that its concentration was 1 mM to obtain a test sample-containing bath solution.

Production Example 8
As a test sample, aluminum chloride was added to the solution A so that its concentration was 1 mM to obtain a test sample-containing solution A.

Production Example 9
As a test sample, potassium aluminum sulfate was added to Solution B so that its concentration was 1 mM to obtain a test sample-containing bath solution.

Production Example 10
As a test sample, potassium aluminum sulfate was added to the solution A so that its concentration was 1 mM to obtain a test sample-containing solution A.

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

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

FIG. 7 shows the results of investigating 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.

From the results shown in FIG. 7, it can be seen 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 potassium aluminum sulfate have an action of activating TRPM4 and suppressing cytokine production.

Example 4
Epidermal keratinocytes were cultured in DMEM 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 and adjusting 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 potassium aluminum sulfate (potassium aluminum sulfate concentration: 2 mM), and then the mixture was 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).

After culturing the obtained culture solution A at 37 ° C. for 48 hours, the culture supernatant was placed in a centrifuge tube and centrifuged at 14000 × g for 5 minutes to recover the culture supernatant A. Meanwhile, the cells remaining in each hole of the 12-well plate were washed with ice-cold PBS. 200 μL of a protein extraction buffer [manufactured by Santa Cruz, trade name: RIPA buffer] was added to the cells, and the cells were peeled off from the wall surface of each hole using a cell scraper to obtain a cell suspension. The cell suspension was centrifuged at 14000 × g for 5 minutes and the supernatant was collected to obtain a cell extract A.

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 and adjusting 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 the mixture was 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).

After culturing the obtained culture solution B at 37 ° C. for 48 hours, the culture supernatant was placed in a centrifuge tube and centrifuged at 14000 × g for 5 minutes to recover the culture supernatant B. Meanwhile, the cells remaining in each hole of the 12-well plate were washed with ice-cold PBS. 200 μL of a protein extraction buffer [manufactured by Santa Cruz, trade name: RIPA buffer] was added to the cells, and the cells were peeled off from the wall surface of each hole using a cell scraper to obtain a cell suspension. The cell suspension was centrifuged at 14000 × g for 5 minutes and the supernatant was collected to obtain a cell extract B.

Test Example 3
Cell extract A obtained in Example 4 or cell extract B obtained in Comparative Example 7 and an IL-1α quantitative reagent [manufactured by R & D, trade name: Human IL-1 alpha / IL-1F1 DuoSet ELISA DY200] The IL-1α expression level in each cultured cell (epidermal keratinized cell) obtained in Example 4 and Comparative Example 7 was measured using and.

FIG. 8 shows the results of examining the IL-1a expression level in each epidermal keratinized cell obtained in Example 4 and Comparative Example 7 in Test Example 3. 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 keratinized cells obtained in Example 4 is smaller than the IL-1α expression level in the epidermal keratinized cells obtained in Comparative Example 7. You can see that. From this result, it can be seen 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 an IL-6 quantitative reagent [manufactured by R & D, trade name: Human IL-6 DuoSet ELISA DY206] were used. , IL-6 expression level in each cultured cell (epidermal keratinized cell) obtained in Example 4 and Comparative Example 7.

FIG. 9 shows the results of examining the IL-6 expression level in each epidermal keratinized cell obtained in Example 4 and Comparative Example 7 in Test Example 3. 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 IL-6 expression level in the epidermal keratinocytes obtained in Example 4 is lower than the IL-6 expression level in the epidermal keratinocytes obtained in Comparative Example 7. Understand. From this result, it can be seen that potassium aluminum sulfate suppresses the expression of IL-6 induced by TNFα.

From these results, it can be seen that by using the physiological events caused by TRPM4 expressing cells via TRPM4 by the test sample, it is possible to evaluate a substance that activates TRPM4 and suppresses the expression of inflammatory cytokines. Further, it can be seen that the substance that activates TRPM4 can suppress the expression of inflammatory cytokines such as IL-1α and IL-6, and can prevent inflammation.

As described above, TRPM4 agonists such as BTP2; inflammatory cytokines in skin cells and inflammatory cytokines by contacting skin cells with a substance having an action of activating TRPM4 such as aluminum chloride and aluminum compounds such as potassium aluminum sulfate in advance. Since the expression of the gene is suppressed, it can be seen that the substance having an action of activating TRPM4 can suppress the expression of inflammatory cytokines and prevent inflammation. Therefore, it is expected that the present invention will be suitably used for the development of anti-inflammatory cosmetics, anti-inflammatory agents and the like.

In addition, by using TRPM4 expressing cells to measure physiological events caused by TRPM4 expressing cells via TRPM4 and using the physiological events in the evaluation of the test sample, cytokines in the skin cells of the test sample are present. It can be seen that the production inhibitory effect can be evaluated.

Claims (1)

A cytokine production inhibitor used for the purpose of activating TRPM4 and suppressing cytokine production in skin cells, which comprises an aluminum compound as an active ingredient for activating TRPM4.

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