JP7423250B2 - Evaluation method, screening method and evaluation kit - Google Patents

Description

The present invention relates to an evaluation method, a screening method, and an evaluation kit.

As a method for evaluating the skin irritation of external skin preparations, methods using transient receptor potential channels (also referred to as TRP channels) are known (Patent Documents 1 to 3). TRP channels are ion channels that function as stimulus receptors.

Conventional methods for evaluating skin irritation using TRP channels measure activation of TPR channels using changes in membrane potential (patch clamp method) or calcium ions flowing into cells as indicators (calcium imaging method). There is. According to these evaluation methods, activation of TRP channels can be directly detected by contacting cells with TRP channel activity with a test substance and then measuring changes in membrane potential or calcium ions flowing into the cells. can be evaluated. Therefore, these evaluation methods can be applied to the evaluation of ingredients that cause acute irritation reactions, such as parabens, alkalis, and alcohol.

JP2008-079528A JP2009-082053A Japanese Patent Application Publication No. 2011-209123

Conventional evaluation methods are suitable for detecting acute stimulus responses, ie, transient responses due to activation of TRP channels. However, skin irritation caused by external skin preparations is often felt not only immediately after application, but also after a certain period of time. Such a delayed stimulus response with a time lag is caused by a reaction mediated by the activation of TRP channels (for example, TRPA1 or TRPV1) that occurs at the nerve terminals in the skin, and is transmitted to the central nervous system through crosstalk between cells. It is transmitted to the nerves and is caused by a reaction that causes pain and irritation. Therefore, it is difficult to detect such a delayed stimulus response using conventional methods.

The present invention was made in view of the above-mentioned problems, and aims to provide a method for evaluating skin irritation based on a new index, and an evaluation kit for evaluating skin irritation. shall be.

Another object of the present invention is to provide a method for screening skin irritants based on a new index, which can screen for skin irritants.

Furthermore, it is an object of the present invention to provide a method for screening anti-skin irritants based on a new index, which can screen for anti-skin irritants that suppress the irritation reaction caused by skin irritants. shall be.

The present inventors have diligently investigated the above-mentioned issues and searched for evaluation factors different from membrane potential or calcium ions. As a result, the present inventors have determined that acute and delayed It was discovered that both stimulus responses can be detected, and the present invention was completed.

That is, the present invention includes the following inventions.

[1] An exposure step of exposing cells having TRPV1 activity and/or TRPA1 activity to a test substance, and at least one indicator selected from the group consisting of the following (a) to (n) in the cells. A method for evaluating skin irritation of a test substance, which comprises a step of measuring gene expression level and/or protein production level:
(a) CXC chemokine (b) CXC chemokine receptor (c) CC chemokine (d) CC chemokine receptor (e) C chemokine receptor (f) CX3C chemokine (g) CX3C chemokine receptor (h) Atypical chemokine receptor Body (i) EGR
(j)FOS
(k) MIF
(l)IL-6
(m)M-CSF
(n) SCF.

[2] An exposure step of exposing cells having TRPV1 activity and/or TRPA1 activity to a candidate skin irritant substance, and at least one selected from the group consisting of the following (a) to (n) in the cells: A method for screening skin irritants, comprising a step of measuring the gene expression level and/or protein production level of one type of indicator:
(a) CXC chemokine (b) CXC chemokine receptor (c) CC chemokine (d) CC chemokine receptor (e) C chemokine receptor (f) CX3C chemokine (g) CX3C chemokine receptor (h) Atypical chemokine receptor Body (i) EGR
(j)FOS
(k) MIF
(l)IL-6
(m)M-CSF
(n) SCF.

[3] An exposure step of exposing cells having TRPV1 activity and/or TRPA1 activity to a candidate substance of a non-skin irritating substance; A method for screening non-skin irritating substances, comprising a step of measuring the gene expression level and/or protein production level of at least one indicator:
(a) CXC chemokine (b) CXC chemokine receptor (c) CC chemokine (d) CC chemokine receptor (e) C chemokine receptor (f) CX3C chemokine (g) CX3C chemokine receptor (h) Atypical chemokine receptor Body (i) EGR
(j)FOS
(k) MIF
(l)IL-6
(m)M-CSF
(n) SCF.

[4] An exposure step in which cells having TRPV1 activity and/or TRPA1 activity are exposed to a candidate substance of a low skin irritation substance, and the above cell is selected from the group consisting of (a) to (n) below. A method for screening low skin irritation substances, comprising a step of measuring the gene expression level and/or protein production level of at least one indicator:
(a) CXC chemokine (b) CXC chemokine receptor (c) CC chemokine (d) CC chemokine receptor (e) C chemokine receptor (f) CX3C chemokine (g) CX3C chemokine receptor (h) Atypical chemokine receptor Body (i) EGR
(j)FOS
(k) MIF
(l)IL-6
(m)M-CSF
(n) SCF.

[5] An exposure step of exposing cells having TRPV1 activity and/or TRPA1 activity to a candidate substance for an anti-skin irritation substance and a skin irritation substance, and the following (a) to (n) in the cells: A method for screening anti-skin irritation substances, comprising a step of measuring the gene expression level and/or protein production level of at least one indicator selected from the group:
(a) CXC chemokine (b) CXC chemokine receptor (c) CC chemokine (d) CC chemokine receptor (e) C chemokine receptor (f) CX3C chemokine (g) CX3C chemokine receptor (h) Atypical chemokine receptor Body (i) EGR
(j)FOS
(k) MIF
(l) IL-6
(m)M-CSF
(n) SCF.

[6] Evaluation of skin irritation of a test substance, which is equipped with a probe that specifically binds to at least one indicator gene or protein selected from the group consisting of (a) to (n) below. Evaluation kit for:
(a) CXC chemokine (b) CXC chemokine receptor (c) CC chemokine (d) CC chemokine receptor (e) C chemokine receptor (f) CX3C chemokine (g) CX3C chemokine receptor (h) Atypical chemokine receptor Body (i) EGR
(j)FOS
(k) MIF
(l) IL-6
(m)M-CSF
(n) SCF.

According to the present invention, it is possible to provide a skin irritation evaluation method and an evaluation kit for evaluating skin irritation based on a new index.

Further, according to the present invention, it is possible to provide a method for screening skin irritants based on a new index, which can screen for skin irritants that cause irritation reactions.

Further, according to the present invention, there is provided a method for screening anti-skin irritants based on a new index, which is capable of screening for anti-skin irritants that suppress irritation reactions caused by skin irritants. Can be done.

It is a graph showing the results of quantifying CCL2 released extracellularly after exposing cells expressing exogenous TRPV1 to capsaicin for 6 hours or 24 hours. It is a graph showing the results of quantifying CCL7 released extracellularly after exposing exogenous TRPV1-expressing cells to capsaicin for 6 hours or 24 hours. It is a graph showing the results of quantifying CXCL8 released extracellularly after exposing cells expressing exogenous TRPV1 to capsaicin for 6 hours or 24 hours. FIG. 2 is a graph showing the results of quantifying M-CSF released extracellularly after exposing cells expressing exogenous TRPV1 to capsaicin for 6 hours or 24 hours. FIG. It is a graph showing the results of quantifying SCF released extracellularly after exposing cells expressing exogenous TRPV1 to capsaicin for 6 hours or 24 hours. FIG. 2 is a graph showing the results of quantifying IL-6 released extracellularly after exposing exogenous TRPV1-expressing cells to capsaicin for 6 or 24 hours. FIG. It is a graph showing the results of quantifying MIF released extracellularly after exposing exogenous TRPV1-expressing cells to capsaicin for 6 hours or 24 hours. FIG. 2 is a graph showing the results of quantifying CXCL8 released extracellularly after exposing exogenous TRPA1-expressing cells to allyl thioisocyanate (AITC) for 6 hours or 24 hours. FIG. It is a graph showing the results of quantifying CCL7 released extracellularly after exposing exogenous TRPA1-expressing cells to AITC for 6 hours or 24 hours. FIG. 2 is a graph showing the results of quantifying IL-6 released extracellularly after exposing exogenous TRPA1-expressing cells to AITC for 6 hours or 24 hours. FIG. It is a graph showing the results of quantifying MIF released extracellularly after exposing exogenous TRPA1-expressing cells to AITC for 6 hours or 24 hours. FIG. 2 is a graph showing the results of quantifying M-CSF released extracellularly after exposing exogenous TRPA1-expressing cells to AITC for 6 hours or 24 hours. FIG. It is a graph showing the results of quantifying SCF released extracellularly after exposing exogenous TRPA1-expressing cells to AITC for 6 hours or 24 hours. It is a graph showing the results of quantifying CCL2 released extracellularly after exposing exogenous TRPA1-expressing cells to AITC for 6 hours or 24 hours.

Hereinafter, an example of an embodiment of the present invention will be described in detail, but the present invention is not limited thereto. The present invention can be modified in various ways within the scope of the claims. Further, embodiments or examples obtained by appropriately combining technical means disclosed in different embodiments or examples are also included in the technical scope of the present invention. Furthermore, new technical features can be formed by combining the technical means disclosed in each embodiment. Note that all academic literature and patent literature described in this specification are incorporated as references in this specification. In this specification, unless otherwise specified, the numerical range "X to Y" is intended to be "more than or equal to X and less than or equal to Y."

[1. Method for evaluating the skin irritation of a test substance]
The method for evaluating a test substance according to the present invention includes an exposure step of exposing cells having TRPV1 activity and/or TRPA1 activity to a test substance, and measuring the amount of gene expression and/or protein production of a specific indicator in the cells. and a measuring step of measuring. With the test substance evaluation method according to the present invention, not only acute stimulation reactions but also delayed stimulation reactions can be evaluated.

[1-1. Exposure process]
The exposure step is a step of exposing cells having TRPV1 activity and/or TRPA1 activity to a test substance. Note that the exposure time is not limited and can be set as appropriate.

(cell)
The cells used in the present invention are cells that have TRPV1 activity and/or TRPA1 activity. The cells may be, for example, wild-type cells collected from a living body, cells established from cells collected from a living body, or transformed cells thereof.

The cells having TRPV1 activity used in the present invention are cells that express the physiological functions of TRPV1. The physiological functions of TRPV1 include, for example, chemical stimulation by capsaicin, camphor, protons, etc., thermal stimulation (for example, stimulation at around 43°C), pain stimulation, mechanical stimulation, etc. from the outside of the cell to the inside of the cell. Examples include permeation of cations such as sodium ions and calcium ions.

Examples of the cells having TRPV1 activity include wild-type cells expressing endogenous TRPV1, cells into which a nucleic acid encoding TRPV1 has been introduced into a host cell so as to be able to express it, and the like. Among the cells having TRPV1 activity, from the viewpoint of measuring physiological events caused through TRPV1 with simple operations, cells in which a nucleic acid encoding TRPV1 has been introduced into a host cell in a manner capable of expressing it (hereinafter referred to as " (also referred to as "exogenous TRPV1-expressing cells") are preferred.

The wild-type cells expressing endogenous TRPV1 are not particularly limited, and include, for example, sensory nerve cells, brain cells, bladder epithelial cells, and the like.

The cells having TRPA1 activity used in the present invention are cells that express the physiological function of TRPA1. The physiological functions of TRPA1 include, for example, chemical stimulation by AITC, cinnamon aldehyde, carvacrol, allicin, paraoxybenzoic acid ester, menthol, nicotine, flufenamic acid, etc., and cold stimulation (for example, stimulation at around 17°C). , the permeation of cations such as sodium ions and calcium ions from the outside of the cell into the cell due to stimulation such as pain stimulation and mechanical stimulation.

Examples of the cells having TRPA1 activity include wild-type cells expressing endogenous TRPA1, cells into which a nucleic acid encoding TRPA1 is introduced into a host cell so as to be able to express it, and the like. Among the above-mentioned cells having TRPA1 activity, from the viewpoint of measuring physiological events caused through TRPA1 with a simple operation, cells in which a nucleic acid encoding TRPA1 has been introduced into a host cell in a manner capable of expressing it (hereinafter referred to as " (also referred to as "exogenous TRPA1-expressing cells") are preferred.

The wild-type cells expressing endogenous TRPA1 are not particularly limited, and include, for example, sensory nerve cells, inner ear cells, and the like.

The cells having TRPV1 activity and TRPA1 activity used in the present invention are cells that express both the physiological functions of TRPV1 and TRPA1.

The cells having TRPV1 activity and TRPA1 activity include wild-type cells expressing both endogenous TRPV1 and endogenous TRPA1, and host cells capable of expressing both the TRPV1-encoding nucleic acid and the TRPA1-encoding nucleic acid. Examples include TRPA1-TRPV1 co-expressing cells introduced into the cell line. In addition, if the host cell is a cell that originally expresses either the physiological function of TRPV1 or the physiological function of TRPA1, either the nucleic acid encoding TRPV1 or the nucleic acid encoding TRPA1 can be expressed. It is also possible to use TRPA1-TRPV1 co-expressing cells introduced into host cells.

In the above-mentioned TRPA1-TRPV1 co-expressing cells, the nucleic acid encoding TRPV1 and the nucleic acid encoding TRPA1 may be under the control of the same expression promoter or may be under the control of different expression promoters. From the viewpoint of separately measuring physiological events caused by a test substance via TRPV1 and physiological events caused by a test substance via TRPA1, the nucleic acid encoding TRPV1 and the nucleic acid encoding TRPA1 are Preferably, each is under the control of separate expression promoters. The expression promoter can be appropriately selected depending on the type of host cell used.

The exogenous TRPV1-expressing cells, exogenous TRPA1-expressing cells, and TRPA1-TRPV1 co-expressing cells may be cells in which the nucleic acid is present as an extrachromosomal element, or cells in which the nucleic acid is integrated into the chromosome. There may be.

The above-mentioned exogenous TRPA1-expressing cells can be obtained, for example, by transforming a host cell with a recombinant vector carrying a nucleic acid encoding TRPA1. Moreover, the above-mentioned exogenous TRPV1-expressing cells can be obtained by transforming host cells with a recombinant vector carrying a nucleic acid encoding TRPV1. The TRPA1-TRPV1 co-expressing cell can be obtained, for example, by transforming a host cell with a recombinant vector carrying a TRPA1-encoding nucleic acid and a TRPV1-encoding nucleic acid.

The above-mentioned nucleic acid encoding TRPA1 may be a nucleic acid encoding human TRPA1 or a nucleic acid encoding TRPA1 of another animal. Moreover, the nucleic acid encoding TRPV1 may be a nucleic acid encoding human TRPV1 or a nucleic acid encoding TRPV1 of another animal.

The nucleic acid encoding TRPV1 is preferably a nucleic acid encoding human TRPV1 from the viewpoint of accurately evaluating the irritation of a test substance to human skin. Examples of the nucleic acid encoding human TRPV1 include the nucleic acid registered in GenBank under the accession number NM_080704.3.

The nucleic acid encoding TRPV1 may be a splicing variant of the nucleic acid encoding human TRPV1. Examples of the splicing variants include nucleic acids registered in GenBank under accession numbers NM_018727.5, NM_080705.3, NM_080706.3, and the like.

The nucleic acid encoding TRPV1 may be any polypeptide encoded by the nucleic acid as long as it expresses the above-mentioned physiological function, and may be a mutant nucleic acid of the nucleic acid registered in GenBank with the above accession number. Alternatively, it may be a splicing variant of a nucleic acid registered in GenBank under the above accession number.

The nucleic acid encoding TRPA1 is preferably a nucleic acid encoding human TRPA1 from the viewpoint of accurately evaluating the irritation of a test substance to human skin. Examples of the nucleic acid encoding human TRPA1 include the nucleic acid registered in GenBank under the accession number NM_007332.

The nucleic acid encoding TRPA1 may be a splicing variant of the nucleic acid encoding human TRPA1.

The nucleic acid encoding TRPA1 may be any polypeptide encoded by the nucleic acid as long as it expresses the above-mentioned physiological function, and may be a mutant nucleic acid of the nucleic acid registered in GenBank with the above accession number. Alternatively, it may be a splicing variant of a nucleic acid registered in GenBank under the above accession number.

Nucleic acids encoding TRPV1 include, for example,
(i) Nucleic acid consisting of the base sequence shown in accession number NM_080704.3, NM_018727.5, NM_080705.3, or NM_080706.3,
(II) Accession Number NM_080704.3, NM_018727.5, NM_080705.3, or NM_080706.3, COST TO OPEN GAP 11, COST TO EXTEN by Blast algorithm. D GAP 1, EXPECT VALUE 10. Consists of a base sequence whose sequence identity value calculated by alignment under the conditions of word size 11 is preferably 90% or more, more preferably 95% or more, and even more preferably 98% or more, and is encoded. A nucleic acid whose polypeptide is a polypeptide expressing at least the above-mentioned physiological function;
(iii) Hybridizes under stringent conditions with a nucleic acid consisting of the base sequence shown in accession number NM_080704.3, NM_018727.5, NM_080705.3, or NM_080706.3 and is encoded by Examples include nucleic acids in which the polypeptide is a polypeptide that expresses the above-mentioned physiological function.

In addition, examples of nucleic acids encoding TRPA1 include:
(iv) a nucleic acid consisting of the base sequence shown in accession number NM_007332,
(v) Sequence identity calculated by aligning the base sequence shown in accession number NM_007332 using the BLAST algorithm under the following conditions: Cost to open gap 11, Cost to extend gap 1, expect value 10, word size 11. is preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and the encoded polypeptide is a polypeptide that expresses at least the above-mentioned physiological function. ,
(vi) A polypeptide that hybridizes under stringent conditions with a nucleic acid that is complementary to the nucleic acid consisting of the base sequence shown in accession number NM_007332, and the encoded polypeptide expresses the function of permeating cations. Examples include certain nucleic acids.

In this specification, the above-mentioned stringent conditions include, for example, a desired nucleic acid and a nucleic acid to be hybridized corresponding to the nucleic acid in a hybridization solution [composition: 6×SSC (composition: 0.9M sodium chloride, 0.09 M sodium citrate, adjusted to pH 7.0), 0.5 mass% sodium dodecyl sulfate, 5x Denhardt's solution, 100 μg/mL denatured salmon sperm DNA, 50 volume% formamide] at room temperature or above. Incubation for 10 hours at a temperature of 42°C or higher for more stringent conditions or 60°C or higher for even more stringent conditions, followed by, for example, 2x SSC or 0.1x for more stringent conditions. Conditions include washing under SSC ionic strength conditions and at a temperature of room temperature or higher, more stringent conditions at a temperature of 42° C. or higher, and even more stringent conditions at a temperature of 60° C. or higher.

The host cell is not particularly limited as long as it can efficiently express the nucleic acid encoding TRPV1 and/or the nucleic acid encoding TRPA1 and can be easily cultured. Examples of the host cells include animal cells, bacterial cells, plant cells, and insect cells. Among these, animal cells are preferred from the viewpoint of fully reproducing the physiological functions of TRPV1 and/or TRPA1 in humans. Examples of animal cells include human cells, monkey cells, hamster cells, and mouse cells. Examples of human cells include, but are not limited to, HepG2 cells, HEK293 cells, HeLa cells, and the like. Examples of monkey cells include, but are not limited to, COS-7 cells. Examples of hamster cells include, but are not limited to, CHO cells. Examples of mouse cells include, but are not limited to, NIH3T3 cells. Among the above host cells, HepG2 cells, HEK293 cells, CHO cells, COS-7 cells and NIH3T3 cells are preferred because they are easy to handle. Among these, HEK293 cells are preferred because they hardly express TRP channels and activation of exogenous TRPV1 and/or TRPA1 can be easily and selectively measured.

The above recombinant vector is a vector obtained by ligating a nucleic acid encoding TRPV1 and/or a nucleic acid encoding TRPA1 with a conventional vector. The above vector may be any vector as long as it is easy to prepare, can be efficiently introduced into host cells, and can efficiently express TRPV1 and/or TRPA1 in host cells. The above-mentioned vector is preferably a vector having a selection marker gene from the viewpoint of easily selecting cells carrying the recombinant vector after transformation. Examples of vectors include plasmid vectors and virus vectors. These vectors can be appropriately selected depending on the host cell used. Note that the vector used as the recombinant vector for producing the exogenous TRPV1-expressing cells and the exogenous TRPA1-expressing cells may have an expression promoter.

Transformation using the above-mentioned recombinant vector can be carried out by transformation methods such as electroporation, lipofection, transfection, and particle gun methods, depending on the type of host cell used. These transformation methods are described, for example, in Molecular Cloning: A Laboratory Manual (Sambrook et al., Cold Spring Harbor Press, 1989), etc. According to the description of can be done. For example, when the recombinant vector used has a selection marker gene, the selection of exogenous TRPV1-expressing cells, exogenous TRPA1-expressing cells, and TRPV1-TRPA1 co-expressing cells from the transformed cells can be carried out depending on the selection marker gene. This can be done by culturing in a selective medium.

To confirm that the obtained cells are cells expressing the physiological function of TRPV1, for example, the cells are brought into contact with 100 nM to 10 μM capsaicin, and the intracellular calcium ion concentration is measured by the method described below. This can be done by measuring the intracellular calcium ion concentration of the cells. If the cells express the physiological function of TRPV1, the intracellular calcium ion concentration of the cells after contact will be higher than the intracellular calcium ion concentration of the cells not contacted with capsaicin. In addition, confirmation that the obtained cells are cells expressing the physiological function of TRPA1 can be made by, for example, contacting the cells with 1 to 10 mM AITC and measuring the intracellular calcium ion concentration as described below. This can be done by measuring the intracellular calcium ion concentration of the cells after contact. If the cells express the physiological function of TRPA1, the intracellular calcium ion concentration of the cells after contact will be higher than the intracellular calcium ion concentration of the cells that have not been contacted with AITC.

In the exposure step, the cells exposed to the test substance may consist of only one type of cell having TRPV1 activity and/or TRPA1 activity, or a combination of two or more types of cells having TRPV1 activity and/or TRPA1 activity. It may be. Furthermore, cells exposed to the test substance may include cells that do not have TRPV1 activity or TRPA1 activity.

In one embodiment of the invention, the cells exposed to the test substance consist of only one type of cell having TRPV1 activity and/or TRPA1 activity. Since the cells are composed of one type of cell having TRPV1 activity and/or TRPA1 activity, a culture system can be easily constructed, and the amount of gene expression and/or protein production per unit cell number can be easily increased. can be measured.

In one embodiment of the present invention, the cells exposed to the test substance are a combination of two or more types of cells having TRPV1 activity and/or TRPA1 activity. Since the cells are a combination of two or more types of cells having TRPV1 activity and/or TRPA1 activity, it is possible to evaluate crosstalk between different types of cells. That is, it is possible to evaluate comprehensive biological reactions that are thought to occur between different types of cells or tissues. For example, by using a combination of cells that have TRPV1 activity and/or TRPA1 activity and cells that do not have TRPV1 activity and TRPA1 activity, the skin irritation of the test substance can be reduced under conditions closer to the in vivo environment. can be evaluated.

(Test substance)
The test substance used in the present invention is not particularly limited, and may be a compound or a composition. Examples of the composition include cosmetics, quasi-drugs, and external skin preparations such as pharmaceuticals. In addition, examples of the compound include each component contained in the above-mentioned skin external preparation.

(Exposure method)
The method of exposing the cells to the test substance is not particularly limited, and may be any known method. Examples include a method in which a test substance is added to a medium for culturing cells and incubation, or a method in which a test substance is directly added to the cells.

The method of adding the test substance to the medium and incubating it can be selected as appropriate depending on the type of cells used. Such methods include monolayer static culture, suspension culture, rotational culture, and tertiary culture. Examples include the original carrier culture method.

The above-mentioned medium contains ingredients suitable for the growth of cells having TRPV1 activity and/or TRPA1 activity, such as glucose, amino acids, peptone, vitamins, cell growth promoting factors (for example, cell growth factors, hormones, binding proteins, A medium containing cell adhesion factors, lipids), serum (eg, FBS, FCS, etc.), calcium chloride, magnesium chloride, etc. can be used. The medium may be a commercially available medium. The medium used for culturing cells having TRPV1 activity and/or TRPA1 activity may be any medium suitable for such cells, and includes, but is not particularly limited to, MEM medium, DMEM medium, RPMI 1640 medium, and the like. For example, when the host cells used are HEK293 cells, a DMEM medium containing 10% by mass FBS or the like may be used.

Furthermore, conditions such as incubation temperature and carbon dioxide concentration are appropriately set depending on the cells used. For example, when using cells obtained from HEK293 cells as cells having TRPV1 activity and/or TRPA1 activity, such cells are usually grown at 36-38°C, preferably at 36°C, in an atmosphere containing 5% carbon dioxide by volume. Incubation may be performed at .5 to 37.5°C.

In the evaluation method according to the present invention, the cell used is a cell into which a nucleic acid encoding TRPV1 and/or TRPA1 has been introduced into the host cell so as to be able to express it, and transiently expresses TRPV1 and/or TRPA1. In the case of cells, from the viewpoint of sufficiently evaluating physiological events mediated by TRPV1 and/or TRPA1 caused by the test substance, preferably 24 hours to 48 hours after introduction of the nucleic acid encoding TRPV1 and/or TRPA1. It is desirable to carry out the exposure step in time.

The amount of cells to be brought into contact with the test substance is preferably 1 x 10 ¹ cells or more, more preferably 1 x 10 2 cells or more, and more preferably 1 x 10 ² cells or more per 100 μL of the test substance, from the viewpoint of reliability of test data. From the viewpoint of preventing the cells from becoming too dense, the number of cells is preferably 3×10 ² cells or less, and more preferably 2×10 ² cells or less.

The method of directly adding the test substance to cells is not particularly limited, and may be coating, patching, dropping, spraying, or the like. The amount of the test substance that is brought into contact with the cells can be appropriately set depending on the type of test substance.

Note that cells having TRPV1 activity and/or TRPA1 activity are incubated in advance under conditions suitable for the cells in order to maintain the cells in a state suitable for measuring the above-mentioned physiological events. Good too.

(Exposure time)
When evaluating the delayed stimulus response of a test substance using the evaluation method according to the present invention, the exposure time for exposing cells having TRPV1 activity and/or TRPA1 activity to the test substance takes into account the time required for intracellular signal transduction. Therefore, it is preferably 1 hour or more, more preferably 6 hours or more, and in consideration of cytotoxic effects, it is preferably 72 hours or less, and more preferably 24 hours or less.

When evaluating the acute stimulus response of a test substance by the evaluation method according to the present invention, the exposure time for exposing cells having TRPV1 activity and/or TRPA1 activity to the test substance is the time required for transient intracellular signal transduction. Taking into account, it is preferably 1 second or more, more preferably 1 minute or more, and considering the timing of gene expression of the indicator, it is preferably within 24 hours, and more preferably within 1 hour. be.

[1-2. Culture process]
The evaluation method according to the present invention may further include, after the exposure step, a culturing step of culturing the cells exposed to the test substance in a medium that does not contain the test substance for a predetermined period of time, if necessary.

Exposure of cells to the test substance during the exposure process causes changes in indicator gene expression and/or protein production, and it may take a certain amount of time for the changes to reach measurable levels. . In this case, it is preferable to perform a culturing step after the exposure step.

The culture method for culturing cells is not particularly limited and can be appropriately selected depending on the type of cells used. Such methods include monolayer static culture, suspension culture, rotation culture, tertiary culture, Examples include the original carrier culture method.

As the medium, the same medium as described above for the exposure step can be used. Furthermore, conditions such as incubation temperature and carbon dioxide concentration are appropriately set depending on the cells used. For example, when cells obtained from HEK293 cells are used as exogenous TRP-expressing cells, such cells are usually grown at 36-38°C, preferably at 36.5-37°C, in an atmosphere containing 5% carbon dioxide by volume. It may be incubated at 5°C.

The culture time is appropriately set depending on the cells used and the index to be measured, but for example, taking into account the time required to start transcription, it is 1 hour or more, more preferably 6 hours or more, and In consideration of toxic effects, it is preferably within 72 hours, more preferably within 24 hours.

[1-3. Measurement process]
The measurement step is a step of measuring the amount of specific gene expression and/or protein production in cells having TRPV1 activity and/or TRPA1 activity exposed to the test substance in the exposure step.

(index)
The indicator used in the present invention is at least one selected from the group consisting of (a) to (n) below, and in the measurement step, the gene expression level and/or protein production amount of the indicator is measured. .

(a) CXC chemokine (α-chemokine)
(b) CXC chemokine receptor (α chemokine receptor)
(c) CC chemokine (β chemokine)
(d) CC chemokine receptor (β-chemokine receptor)
(e) C chemokine receptor (γ chemokine receptor)
(f) CX3C chemokine (δ chemokine)
(g) CX3C chemokine receptor (δ chemokine receptor)
(h) Atypical chemokine receptor (i) EGR (Early growth response)
(j)FOS
(k) MIF (Macrophage migration inhibitory factor)
(l) IL-6 (Interleukin-6)
(m) M-CSF (Macrophage colony stimulating factor)
(n) SCF (skip1-cul1-F-box-protein)
CXC chemokines refer to the CXC chemokine family, also referred to as alpha chemokines (or family thereof). Examples of CXC chemokines include CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL16, CXCL17, etc., and preferably CXCL1, CXCL5, and CX It is CL8.

CXC chemokine receptor is intended to be the CXC chemokine receptor family, including, for example, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCL6, etc., preferably CXCR2 and CXCR4.

CC chemokines are intended for the CC chemokine family, also referred to as beta chemokines (or family thereof). CC chemokines include CCL1, CCL2, CCL3, CCL3L3, CCL3L1, CCL4L2, CCL4, CCL4L1, CCL5, CCL7, CCL8, CCL11, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL 20, CCL21, CCL22, CCL23 , CCL24, CCL25, CCL26, CCL27, CCL28, etc., preferably CCL2, CCL3, CCL4L2, CCL4L1, CCL5, CCL7, CCL8, CCL11, CCL16, CCL18, CCL19, and CCL22, more preferably CCL2 , CCL7, and CCL8.

CC chemokine receptor is intended to be the CC chemokine receptor family, and includes, for example, CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CCRL2, NCCRP1, etc., preferably: It is CCR6.

C-chemokines are also called gamma-chemokines. Examples of C chemokine receptors include XCR1 and the like.

CX3C chemokines are also referred to as delta chemokines. Examples of CX3C chemokines include CX3CL1 and the like. CX3C chemokine receptors include CX3CR1 and the like.

Examples of atypical chemokine receptors include atypical chemokine family receptors, such as ACKR1, ACKR2, ACKR3, ACKR4, etc., with ACKR1 and ACKR3 being preferred.

EGR includes EGR1, EGR2, EGR3, EGR4, etc., and preferably EGR1, EGR2, and EGR3.

Examples of FOS include FOSB, FOS, FOSL1, FOSL2, etc., and preferably FOSB, FOS, and FOSL1.

The accession numbers of the proteins and genes that serve as indicators are shown in Tables 1 to 6 below.

When evaluating the delayed stimulus response of a test substance by the evaluation method according to the present invention, the above-mentioned indicators include CXC chemokine, CXC chemokine receptor, CC chemokine, CC chemokine receptor, C chemokine receptor, CX3C chemokine, CX3C chemokine receptor, atypical chemokine receptor, MIF, IL-6, M-CSF and SCF can be preferably used.

Among the above indicators, CXCL8, CCL2, CCL7, MIF, IL-6, M-CSF, and SCF proteins are particularly preferred because they have advantages such as being secreted outside the cells and being easy to measure.

When evaluating an acute stimulus response using the evaluation method according to the present invention, EGR, FOS, and the genes encoding them can be suitably used as the above-mentioned indicators.

Among the above indicators, CXCL1, CXCL5, CXCL8, CXCR2, CXCR4, CCL2, CCL5, CCL7, CCL8, CCL11, CCL18, CCL22, CCR6, ACKR1, IL-6, M-CSF, SCF, EGR1, EGR2, EGR3, FOSB , FOS, and FOSL1 can be preferably used as indicators in evaluation methods using cells having TRPV1 activity. Among these, CXCL1, CXCL5, CXCR2, CXCR4, CCL2, CCL5, CCL8, CCL11, CCL18, CCR6, ACKR1, EGR2, EGR3, and FOS are indicators specific to cells with TRPV1 activity.

Among the above indicators, CXCL8, CCL4L1, CCL4L2, CCL3, CCL7, CCL16, CCL19, CCL22, It can be preferably used as an indicator in an evaluation method using cells having the following. Among these, CCL4L1, CCL4L2, CCL3, CCL16, CCL19, XCR1, CX3CR1, ACKR3 and MIF are indicators specific to cells having TRPA1 activity.

Among the above indicators, CXCL8, CCL7, CCL22, IL-6, M-CSF, SCF, EGR1, FOSB and FOSL1 can be suitably used in both cells having TRPV1 activity and TRPA1 activity. .

(Measuring method)
The method for measuring the gene expression level and/or protein production level of the indicator is not particularly limited, and may be any known method for measuring the production of the indicator protein or gene expression. Examples of the measurement of gene expression level include a method of measuring mRNA expression.

Methods for measuring mRNA expression include, for example, extracting total RNA from cells and using real-time RT-PCR, RNA degrading enzyme protection assay, Northern blot analysis, dot blotting, RNA-sequence analysis, microarray, Examples include a measurement method such as FISH (fluorescence in situ hybridization).

Examples of methods for measuring protein expression include Western blotting, immunoprecipitation, ELISA, RIA, EIA, and bioassay.

The measurement method described above can be performed in vitro or ex vivo, or can be performed in vivo. Furthermore, when measuring the expression levels of a plurality of genes, proteins, etc., the expression levels may be measured separately, for example, or may be measured all at the same time.

[1-4. Evaluation process]
The evaluation method according to the present invention may further include an evaluation step of evaluating the skin irritation of the test substance based on the gene expression level and/or protein production amount measured in the above measurement step.

In the evaluation step, the amount of gene expression and/or protein production of the indicator in cells having TRPV1 activity and/or TRPA1 activity after exposure to the test substance is compared with the expression amount and/or production amount of the control. The skin irritation of substances can be evaluated.

As controls, cells with TRPV1 activity and/or TRPA1 activity before exposure to the test substance (Control A), cells with TRPV1 activity and/or TRPA1 activity after exposure to a known non-skin irritating substance (Control B) , control cells without TRPV1 activity and TRPA1 activity after exposure to a test substance (control C), cells with TRPV1 activity and/or TRPA1 activity after exposure to a known skin irritant (control D), known skin Cells that do not have TRPV1 activity and/or TRPA1 activity after exposure to a stimulating substance (control E), etc. can be used.

A known non-skin irritating substance is a substance that is already known to be non-irritating to the skin. Furthermore, a known skin irritant substance is a substance that is already known to have skin irritation properties. As known skin irritants, known agonists for TRPV1 and/or TRPA1 can be used. Known agonists for TRPV1 include, for example, capsaicin, camphor, and the like. Further, known agonists for TRPA1 include, for example, AITC, mustard, cinnamon aldehyde, carvacrol, allicin, paraoxybenzoic acid ester, menthol, nicotine, flufenamic acid, and the like.

For evaluation of irritation, for example, when using Control A, Control B, or Control C as a control, the amount of gene expression or protein production in cells having TRPV1 activity and/or TRPA1 activity after exposure to the test substance. is equivalent to or lower than the expression level or production level in these controls (Controls A to C), the test substance can be evaluated as not irritating to the skin, or as irritating to the skin but only to a small extent. can. On the other hand, if the amount of gene expression or protein production in cells with TRPV1 activity and/or TRPA1 activity after exposure to the test substance is higher than the expression amount or protein production amount in these controls (Controls A to C), the test substance The substance can be assessed as having skin irritation properties.

For example, when Control D or Control E is used as a control, the amount of gene expression or protein production in cells having TRPV1 activity and/or TRPA1 activity after exposure to the test substance is -E) If the expression level or production level is equal to or greater than the expression level or production level in E), it can be evaluated that the test substance is irritating to the skin. On the other hand, if the amount of gene expression or protein production in cells with TRPV1 activity and/or TRPA1 activity after exposure to the test substance is lower than the expression amount or protein production amount in these controls (Controls D to E), the test substance The substance can be evaluated as having no or only low irritation to the skin.

In addition, in the present specification, the expression level or production level is low when the expression level of the indicator gene and/or protein production level in cells having TRPV1 activity and/or TRPA1 activity after exposure to the test substance is lower than that in the control. It refers to less than 1.0 times, preferably 1/2 or less, more preferably 1/5 or less, and most preferably 1/10 or less compared to the amount or production amount.

In addition, in the present specification, the term "high expression level or production level" means that the expression level of an indicator gene and/or protein production level in cells having TRPV1 activity and/or TRPA1 activity after exposure to a test substance is higher than that in the control. amount or production amount, it is more than 1.0 times, preferably 1.1 times or more, more preferably 1.3 times or more, more preferably 1.5 times or more, and more It is preferably 2.0 times or more, more preferably 5.0 times or more, and most preferably 10 times or more.

[2. Screening method for skin irritants
The invention also provides a method of screening for skin irritants. The method for screening a skin irritant substance according to the present invention includes an exposure step of exposing cells having TRPV1 activity and/or TRPA1 activity to a candidate substance for a skin irritant substance, and the above-mentioned (a) to ( n) measuring the gene expression level and/or protein production level of at least one indicator selected from the group consisting of: According to the method for screening skin irritants of the present invention, skin irritants that cause acute and/or delayed irritation reactions can be screened.

Note that the term "skin irritating substance" as used herein refers to a substance that causes an acute and/or delayed irritation reaction in the skin.

The cells, exposure method, exposure time, index, and measurement method used in the screening method are the same as those used in the method for evaluating the skin irritation of the test substance described above.

Further, the candidate substance for the skin irritant substance is not particularly limited, and the same test substances as those mentioned in the above-mentioned method for evaluating the skin irritation of a test substance can be used.

This screening method is similar to the above-mentioned method for evaluating the skin irritation of a test substance, and if necessary, after the exposure step, the cells exposed to the candidate skin irritant substance are treated with the candidate substance. The method may further include a culturing step of culturing the cells in a medium that does not contain the cells.

Further, the screening method may further include a screening step of screening for skin irritating substances based on the gene expression level and/or protein production amount measured in the measurement step.

In the screening step, the amount of gene expression and/or protein production of the indicator in cells having TRPV1 activity and/or TRPA1 activity after exposure to the candidate substance is compared with the expression amount or production amount in the control, and the above If the amount of expression or production is high, the candidate substance can be screened as a skin irritating substance.

As controls, cells with TRPV1 activity and/or TRPA1 activity before exposure to the test substance (Control A), cells with TRPV1 activity and/or TRPA1 activity after exposure to a known non-skin irritating substance (Control B) , control cells that do not have TRPV1 activity and TRPA1 activity after exposure to the test substance (control C), etc. can be used. From the viewpoint of evaluating stimulation through TRPV1 activity and/or TRPA1 activity, it is preferable to use Control C, and it is more preferable to use Control C in addition to Control A or Control B.

[3. Screening method for non-skin irritating substances]
The present invention also provides a method of screening for non-skin irritating substances. The screening method for a non-skin irritating substance according to the present invention includes an exposure step of exposing cells having TRPV1 activity and/or TRPA1 activity to a candidate substance for a non-skin irritating substance, and the above-mentioned (a) in the cell. -(n) measuring the gene expression level and/or protein production level of at least one indicator selected from the group consisting of (n). According to the method of screening for non-skin irritating substances according to the present invention, it is possible to screen for non-skin irritating substances that do not cause acute and/or delayed irritative reactions.

As used herein, the term "non-skin irritating substance" refers to a substance that does not cause acute and/or delayed irritating reactions on the skin.

The cells, exposure method, exposure time, index, and measurement method used in the screening method are the same as those used in the method for evaluating the skin irritation of the test substance described above.

In addition, the candidate substance for the non-skin irritating substance is not particularly limited, and the same test substances as mentioned in the above-mentioned method for evaluating the skin irritation of a test substance can be used.

This screening method is similar to the above-mentioned method for evaluating skin irritation of a test substance, and if necessary, after the exposure step, cells exposed to a non-skin irritating candidate substance are treated with the candidate substance. The method may further include a culturing step of culturing the cells in a medium that does not contain.

Further, the screening method may further include a screening step of screening for non-skin irritating substances based on the gene expression level and/or protein production level measured in the measurement step.

In the screening step, the amount of gene expression and/or protein production of the indicator in cells having TRPV1 activity and/or TRPA1 activity after exposure to the candidate substance is compared with the expression amount or production amount in the control, and the above If the expression level or production level is the same or lower, the candidate substance can be screened as a non-skin irritating substance.

As controls, cells with TRPV1 activity and/or TRPA1 activity before exposure to the test substance (Control A), cells with TRPV1 activity and/or TRPA1 activity after exposure to a known non-skin irritating substance (Control B) , control cells that do not have TRPV1 activity and TRPA1 activity after exposure to the test substance (control C), etc. can be used. From the viewpoint of evaluating stimulation through TRPV1 activity and/or TRPA1 activity, it is preferable to use Control C, and it is more preferable to use Control C in addition to Control A or Control B.

[4. Screening method for low skin irritation substances]
The present invention also provides a method of screening for low skin irritation substances. The method of screening for a low skin irritation substance according to the present invention includes an exposure step of exposing cells having TRPV1 activity and/or TRPA1 activity to a candidate substance for a low skin irritation substance, and the above-mentioned (a) in the cell. -(n) measuring the gene expression level and/or protein production level of at least one indicator selected from the group consisting of (n). According to the method of screening for low skin irritation substances according to the present invention, it is possible to screen for low skin irritation substances that have lower acute and/or delayed irritation reactions than a control.

In addition, as used herein, the term "substance with low skin irritation" refers to a substance that causes less acute and/or delayed irritation response on the skin than a control.

The cells, exposure method, exposure time, index, and measurement method used in the screening method are the same as those used in the method for evaluating the skin irritation of the test substance described above.

In addition, the candidate substance for the low skin irritation substance is not particularly limited, and the same test substances as mentioned in the above-mentioned method for evaluating the skin irritation of a test substance can be used.

This screening method is similar to the above-mentioned method for evaluating skin irritation of a test substance, and if necessary, after the exposure step, cells exposed to a candidate substance of a low skin irritation substance are treated with the candidate substance. The method may further include a culturing step of culturing the cells in a medium that does not contain.

Further, the screening method may further include a screening step of screening for a low skin irritation substance based on the gene expression level and/or protein production amount measured in the measurement step.

In the screening step, the candidate substance can be determined by comparing the expression level and/or protein production level of the indicator in cells having TRPV1 activity and/or TRPA1 activity after exposure to the candidate substance with the expression level or production level in a control. can be screened as low skin irritation substances.

As controls, cells with TRPV1 activity and/or TRPA1 activity before exposure to the test substance (Control A), cells with TRPV1 activity and/or TRPA1 activity after exposure to a known non-skin irritating substance (Control B) , control cells without TRPV1 activity and TRPA1 activity after exposure to a test substance (control C), cells with TRPV1 activity and/or TRPA1 activity after exposure to a known skin irritant (control D), known skin Cells that do not have TRPV1 activity and/or TRPA1 activity after exposure to a stimulating substance (control E), etc. can be used. From the viewpoint of evaluating stimulation through TRPV1 activity and/or TRPA1 activity, it is preferable to use Control C and Control E, and it is preferable to use Control C and Control E in addition to Control A, Control B, or Subject D. More preferred.

For example, when Control A, Control B, or Control C is used as a control, the amount of gene expression or protein production in cells having TRPV1 activity and/or TRPA1 activity after exposure to a candidate substance is If the expression level or production level is the same as or lower than those in A to C), the candidate substance can be screened as a low skin irritation substance.

For example, when Control D or Control E is used as a control, the amount of gene expression or protein production in cells having TRPV1 activity and/or TRPA1 activity after exposure to a candidate substance is different from that of these controls (Control D). If the expression level or production level is lower than those in ~E), the candidate substance can be screened as a low skin irritation substance.

[5. Screening method for anti-skin irritation substances]
The present invention also provides a method of screening for anti-skin irritation substances. The method for screening an anti-skin irritant substance according to the present invention includes an exposure step of exposing cells having TRPV1 activity and/or TRPA1 activity to a candidate substance for an anti-skin irritant substance and a skin irritant substance; and a measuring step of measuring the gene expression level and/or protein production level of at least one indicator selected from the group consisting of (a) to (n) described above. According to the method for screening anti-skin irritants of the present invention, it is possible to screen for anti-skin irritants that suppress acute and/or delayed irritation reactions caused by skin irritants.

In this specification, the term "anti-skin irritation substance" refers to a substance that suppresses acute and/or delayed irritation reactions in the skin caused by skin irritation substances.

In the above exposure step, cells having TRPV1 activity and/or TRPA1 activity are exposed to (i) a candidate substance for an anti-skin irritant substance, and then to a known skin irritant substance; or (iii) simultaneous exposure to a candidate anti-skin irritant and a known skin irritant; This can be done by

The candidate substance for the anti-skin irritation substance is not particularly limited, and those similar to the test substances mentioned in the above-mentioned method for evaluating the skin irritation of a test substance can be used.

The amount of the anti-skin irritation candidate substance added to the cells is not particularly limited as long as the amount of gene expression and/or protein production of the indicator changes measurably. Further, the amount of the known skin irritating substance added to the cells is not particularly limited as long as the gene expression level and/or protein production level of the indicator changes measurably.

When screening for anti-skin irritants that suppress the delayed irritation response of skin irritants, the exposure time during which cells with TRPV1 activity and/or TRPA1 activity are exposed to known skin irritants is determined by the intracellular signal. Considering the time required for transmission, it is preferably 1 hour or more, more preferably 6 hours or more, and considering the influence of cytotoxicity, it is preferably within 72 hours, more preferably 24 hours. within

When screening for anti-skin irritants that suppress the acute irritation response of skin irritants, the exposure time during which cells with TRPV1 activity and/or TRPA1 activity are exposed to a known skin irritant is important to detect transient signals. Considering the time required for transmission, preferably 1 second or more, more preferably 5 minutes or more, and considering the timing of gene expression of the indicator, preferably within 24 hours, more preferably Within 1 hour.

The exposure time for exposing cells having TRPV1 activity and/or TRPA1 activity to a candidate substance for an anti-skin irritation substance is particularly within a range where the indicator gene expression level and/or protein production level changes measurably. It is not limited.

The cells used in the screening method, other configurations of the exposure method, exposure time, indicators, and measurement method are the same as those used in the method for evaluating the skin irritation of the test substance described above.

The screening method is similar to the method for evaluating the skin irritation of the test substance described above, and if necessary, after the exposure step, the test substance is exposed to the anti-skin irritation candidate substance and the skin irritation substance. The method may further include a culturing step of culturing the cells in a medium that does not contain the candidate substance and the skin irritating substance.

Further, the screening method may further include a screening step of screening for anti-skin irritation substances based on the gene expression level and/or protein production level measured in the measurement step.

In the screening step, the amount of gene expression and/or protein production of the indicator in cells having TRPV1 activity and/or TRPA1 activity after exposure to the candidate substance is compared with the expression amount or production amount in the control, and the above If the amount of expression or production is low, the candidate substance can be screened as an anti-skin irritation substance.

As a control, cells having TRPV1 activity and/or TRPA1 activity exposed only to known skin irritants can be used.

[6. Evaluation kit for evaluating the skin irritation of test substances]
One embodiment of the present invention provides an evaluation kit for evaluating skin irritation of a test substance (for example, a microarray kit, a PCR kit). According to the evaluation kit for evaluating the skin irritation of a test substance according to the present invention, not only acute irritation reactions but also delayed irritation reactions can be evaluated. Further, this evaluation kit can be used in the above-mentioned screening method for skin irritating substances, non-skin irritating substances, low skin irritating substances, or anti-skin irritating substances.

Examples of probes that specifically bind to mRNA include polynucleotides that specifically hybridize to the mRNA. In addition, examples of probes that specifically bind to proteins include antibodies that specifically bind to the proteins.

The above probe can also be used by being immobilized on any solid phase. Therefore, this evaluation kit may be provided as a microarray chip, a DNA chip, a protein chip, etc., on which the above probes are immobilized. The solid phase is not particularly limited as long as it can immobilize probes, and examples include substrates such as glass plates, nylon membranes, microbeads, silicon chips, and capillaries.

The evaluation kit according to the present invention further includes a labeled second probe that specifically binds to the mRNA or protein captured by the probe, in order to quantify the mRNA or protein captured by the probe. It may also include a probe. Examples of the second probe include a labeled polynucleotide that specifically hybridizes to mRNA and a labeled antibody that specifically binds to protein. Note that the method of labeling is not particularly limited, and for example, labeling can be performed using a fluorescent dye or the like.

According to the evaluation method and evaluation kit according to the present invention, it is possible to evaluate a test substance that causes an acute irritant reaction and a test substance that causes a delayed irritant reaction.

Furthermore, according to the method for screening skin irritating substances according to the present invention, it is possible to screen for substances that cause inflammation due to acute and/or delayed irritative reactions, for example.

Furthermore, according to the method for screening non-skin irritating substances according to the present invention, it is possible to screen for substances that do not cause acute and/or delayed irritative reactions, for example.

Furthermore, according to the method of screening for low skin irritation substances according to the present invention, it is possible to screen for substances that have a lower acute and/or delayed irritation response than a control, for example.

Furthermore, according to the method for screening anti-skin irritation substances according to the present invention, it is possible to screen for substances that suppress inflammation through acute and/or delayed irritation reactions, for example.

EXAMPLES The present invention will be explained in more detail with reference to Examples below, but the present invention is not limited only to these Examples.

(Manufacturing example 1)
The cDNA encoding human TRPV1 [polynucleotide from positions 276 to 2795 of the base sequence shown in GenBank accession number: MN_080704.3] was transformed into a vector for mammalian cells [manufactured by Invitrogen, product name: pcDNA3.1 (+ )] to obtain a human TRPV1 expression vector. 5 μg of the obtained human TRPV1 expression vector was suspended in 10 μL of sterile water to obtain a mixture I.

In addition, HEK293 cells were cultured to 70% confluency in a DMEM medium containing 10 mass% FBS on a 100 mm diameter Petri dish maintained at 37°C in an atmosphere of 5 volume% carbon dioxide, and then electroporated. Cell suspension I was obtained by suspending the cells in 100 μL of the attached R buffer in a gene transfer reagent [manufactured by Thermo Fisher, trade name: Neon Transfection System 100 μL kit, catalog number: MPK10025].

Mixture I and cell suspension I were mixed, and gene transfer was performed using Neon Transfection System [manufactured by Thermo Fisher, catalog number: MPK5000] with Plus Voltage 1100V, Plus Width 20ms, and Plus Number 2. I did. The day after gene transfer, the medium was changed to 10 mass% FBS-containing DMEM medium supplemented with 1 mg/mL G418 sulfate, and cultured for 3 weeks in an atmosphere of 5 volume% carbon dioxide with continued medium exchange every 2 days. By continuing to do so, exogenous TRPV1-expressing cells in which the human TRPV1 expression vector described above was introduced into HEK293 cells were obtained.

(Manufacturing example 2)
In Production Example 1, the cDNA encoding human TRPA1 [polynucleotide from position 63 to position 3888 of the base sequence shown in GenBank accession number: NM_007332] was used instead of the cDNA encoding human TRPV1. Exogenous TRPA1-expressing cells were obtained in the same manner as in Example 1.

(Manufacturing example 3)
In Production Example 1, an empty vector for mammalian cells that does not contain cDNA encoding human TRPV1 [manufactured by Invitrogen, trade name: pcDNA3.1(+)] was used instead of the human TRPV1 expression vector. MOCK cells (HEK293_MOCK) were obtained in the same manner as in Production Example 1.

[1: Search and quantification of intracellular variable proteins in cells with TRPV1 activity by proteome analysis]
The exogenous TRPV1-expressing cells (HEK293_TPRV1) obtained in Production Example 1 were incubated 5x in a DMEM medium containing 10 mass% FBS on a 35 mm diameter Petri dish maintained at 37°C in an atmosphere of 5 volume% carbon dioxide. 10 ⁵ cells expressing exogenous TRPV1 were cultured to 70% confluency.

Next, capsaicin, which is a known agonist for TRPV1, was added to the above DMEM medium containing 10% by mass FBS at a concentration shown in Table 7 below to obtain Samples 1 to 5.

For samples 1 to 3 obtained above, 10 minutes after capsaicin was added to the medium, they were washed with a capsaicin-free DMEM medium containing 10% FBS, and then cultured in the capsaicin-free medium for 6 hours. continued. Next, exogenous TRPV1-expressing cells were collected and proteome analysis was performed.

For Samples 4 and 5 obtained above, exogenous TRPV1-expressing cells were collected 6 hours after capsaicin was added to the medium, and proteome analysis was performed.

As a control (NT), exogenous TRPV1-expressing cells (comparative sample 1) cultured for 6 hours in a capsaicin-free DMEM medium containing 10 mass% FBS were used.

In the proteome analysis, mass spectrometry of peptides was performed using a mass spectrometer (Q-Exactive HF-X, manufactured by Thermo Fisher Scientific).

As a result of proteome analysis, EGR1 and FOSB were found to be proteins whose expression levels were significantly higher than those in the control.

The ratios of the protein expression levels of EGR1 and FOSB measured for samples 1 to 5 to the expression levels in the control are shown in Table 8 below.

As shown in Table 8, a significant increase in the expression level of EGR1 protein was observed in Samples 1 to 5. Furthermore, in Samples 1 to 4, a significant increase in the expression level of FOSB protein was observed.

[2: Quantification of extracellularly released proteins in cells with TRPV1 activity]
In the same manner as in [1] above, exogenous TRPV1-expressing cells (HEK293_TPRV1) were cultured, and capsaicin was added to the medium at the concentrations shown in Table 9 below to obtain samples 6 to 9.

For samples 6 to 8 obtained above, the medium supernatant was collected 6 hours after capsaicin was added to the medium, and the amount of various cytokines in the medium supernatant was measured using the Bio-Plex Human Cytokine Assay Kit. It was measured using Regarding Sample 9, the medium supernatant was collected 24 hours after capsaicin was added to the medium, and the amounts of various cytokines were similarly measured.

As a control, MOCK cells (HEK293_MOCK) (Comparative Samples 2 to 7) and exogenous TRPV1-expressing cells cultured for 6 hours or 24 hours in a DMEM medium containing 10 mass% FBS without capsaicin (Comparative Samples 8 and 9) It was used.

As a result of the measurement, CC chemokines (CCL2, CCL7), CXC chemokines (CXCL8), M-CSF, SCF, and IL-6 were found to be proteins whose expression levels were significantly higher than those in the control. The quantitative results of each protein are shown in Figures 1 to 6. Moreover, the quantitative results of MIF are shown in FIG.

As shown in FIGS. 1 to 6, a significant increase in the expression levels of CC chemokine, CXC chemokine, IL-6, M-CSF, and SCF proteins was observed. On the other hand, regarding MIF, no significant increase was observed compared to the expression level in the control.

[3: Search and quantification of intracellular variable proteins in cells with TRPA1 activity by proteome analysis]
Exogenous TRPA1 was expressed in the same manner as in [1] above, except that the exogenous TRPA1-expressing cells obtained in Production Example 2 (HEK293_TPRA1) were used instead of the exogenous TRPV1-expressing cells obtained in Production Example 1. Cells were cultured.

Next, AITC, a known agonist for TRPA1, was added to the DMEM medium containing 10% by mass FBS at a concentration shown in Table 10 below to obtain samples 10 to 12.

For sample 10 obtained above, 10 minutes after adding AITC to the medium, the culture was continued for 6 hours in the AITC-free medium after washing with DMEM medium containing 10 mass% FBS. Thereafter, exogenous TRPA1-expressing cells were collected and proteome analysis was performed.

For Samples 11 and 12 obtained above, exogenous TRPA1-expressing cells were collected 6 hours after AITC was added to the medium, and proteome analysis was performed.

As a control (NT), exogenous TRPA1-expressing cells (comparative sample 10) cultured for 6 hours in a DMEM medium containing 10 mass % FBS without AITC were used.

Proteome analysis was performed in the same manner as in [1] above.

As a result of proteome analysis, EGR1 and FOSB were found to be proteins whose expression levels were significantly higher than those in the control.

The ratios of the EGR1 and FOSB protein expression levels measured for samples 10 to 12 to the expression levels in the control are shown in Table 11 below.

As shown in Table 11, a significant increase in the expression levels of EGR1 and FOSB proteins was observed in samples 10 to 12.

[4: Quantification of extracellularly released proteins in cells with TRPA1 activity]
In the same manner as in [2] above, exogenous TRPA1-expressing cells (HEK293_TPRA1) were cultured, and AITC was added to the medium at the concentrations shown in Table 12 below to obtain samples 13 to 16.

For samples 13 to 15 obtained above, the medium supernatant was collected 6 hours after AITC was added to the medium, and the amount of various cytokines in the medium supernatant was measured using the Bio-Plex Human Cytokine Assay Kit. It was measured using For sample 16, the medium supernatant was collected 24 hours after AITC was added to the medium, and the amounts of various cytokines were measured in the same manner.

As controls, MOCK cells (HEK293_MOCK) (comparative samples 11 to 16) and exogenous TRPA1-expressing cells cultured for 6 hours or 24 hours in DMEM medium containing 10 mass% FBS without AITC (comparative samples 17 and 18) It was used.

As a result of the measurement, CXC chemokine (CXCL8), CC chemokine (CCL7), IL-6, MIF, M-CSF, and SCF were found to be proteins whose expression levels were significantly higher than those in the control. The quantitative results of each protein are shown in Figures 8 to 13. Further, the quantitative results of CCL2 are shown in FIG. 14.

As shown in FIGS. 8 to 13, a significant increase in the expression levels of CXC chemokine, CC chemokine, IL-6, MIF, M-CSF, and SCF proteins was observed.

[5: Analysis of variation in mRNA expression level due to delayed stimulus response in cells with TRPV1 activity or TRPA1 activity]
In the same manner as in [1] above, exogenous TRPV1-expressing cells were cultured, and capsaicin was added to the medium to a concentration of 10 nM to obtain sample 17. In addition, comparative sample 19 was obtained by culturing exogenous TRPV1-expressing cells in the same manner and adding a capsaicin-free medium to the culture medium.

For sample 17 obtained above, exogenous TRPV1-expressing cells were collected 6 hours after adding capsaicin to the medium, and for comparative sample 19, 6 hours after adding capsaicin-free medium, Changes in mRNA expression levels due to exposure to capsaicin were analyzed using Clariom ^™ S assay (manufactured by Thermo Fisher Scientific).

Similarly, in the same manner as in [3] above, cells expressing exogenous TRPA1 were cultured, and AITC was added to the medium at a concentration of 10 μM to obtain sample 18. In addition, cells expressing exogenous TRPA1 were similarly cultured, and an AITC-free medium was added to the medium to obtain comparative sample 20.

For sample 18 obtained above, 6 hours after adding AITC to the medium, and for comparative sample 20, 6 hours after adding AITC-free medium, exogenous TRPA1-expressing cells were collected, Changes in mRNA expression levels due to exposure to AITC were analyzed using Clariom ^™ S assay (manufactured by Thermo Fisher Scientific).

As a control, MOCK cells (HEK293_MOCK) were cultured, and capsaicin was added to the medium to a concentration of 10 nM to obtain Comparative Sample 21. In addition, MOCK cells were cultured in the same manner, and a capsaicin-free medium was added to the medium to obtain comparative sample 22. Similarly, the above-mentioned MOCK cells were cultured, and AITC was added to the medium at a concentration of 10 μM to obtain comparative sample 23. In addition, MOCK cells were cultured in the same manner, and an AITC-free medium was added to the medium to obtain comparative sample 24.

Regarding Samples 17 and 18 and Comparative Samples 19 to 24, the cell lines used, whether or not capsaicin or AITC was added, the added concentration, exposure time, and cell viability are summarized in Tables 13 and 14 below.

The analysis results for CXC chemokines, CXC chemokine receptors, CC chemokines, CC chemokine receptors, C chemokine receptors, CX3C chemokine receptors, and atypical chemokine receptors are shown in Table 15 below. The mRNA values in the table are expressed as a ratio to the mRNA expression level in exogenous TRPV1-expressing cells, exogenous TRPA1-expressing cells, or MOCK cells (NT) cultured for 6 hours in a DMEM medium containing 10% by mass FBS.

As shown in Table 15, in exogenous TRPV1-expressing cells and/or exogenous TRPA1-expressing cells, delayed stimulation response with capsaicin or AITC induces CXC chemokines, CXC chemokine receptors, CC chemokines, CC chemokine receptors, C A significant increase in the expression levels of chemokine receptor, CX3C chemokine receptor, and atypical chemokine receptor mRNA was observed.

[6: Analysis of changes in mRNA expression level due to acute stimulus response in cells with TRPV1 activity or TRPA1 activity]
Exogenous TRPV1-expressing cells and exogenous TRPA1-expressing cells were cultured in the same manner as in [5] above, and capsaicin or AITC was added to the medium.

10 minutes after capsaicin or AITC was added to the medium, the cells were washed with a DMEM medium containing 10 mass% FBS, continued in the medium for 6 hours, and then capsaicin or AITC was added in the same manner as in [5] above. Changes in mRNA expression levels due to exposure to AITC were analyzed.

MOCK cells (HEK293_MOCK) were used as a control.

The analysis results for EGR and FOS are shown in Table 16 below. The mRNA values in the table are expressed as a ratio to the mRNA expression level in exogenous TRPV1-expressing cells, exogenous TRPA1-expressing cells, or MOCK cells (NT) cultured for 6 hours in a DMEM medium containing 10% by mass FBS.

As shown in Table 16, in exogenous TRPV1-expressing cells and/or exogenous TRPA1-expressing cells, a significant increase in the expression levels of EGR and FOS mRNA was observed upon acute stimulation with capsaicin or AITC.

According to the evaluation method and evaluation kit of the present invention, it is possible to evaluate a test substance that causes an acute irritant reaction and a test substance that causes a delayed irritant reaction.

Claims (6)

an exposure step of exposing cells collected from a living body having TRPV1 activity and/or TRPA1 activity to a test substance;
A measuring step of measuring the gene expression level and/or protein production level of the following indicators (a) and/or (b) in cells collected from the living body ;
An evaluation step of evaluating the skin irritation of the test substance by comparing the gene expression amount and/or protein production amount measured in the measurement step with the gene expression amount and/or protein production amount in a control. and ,
The controls in the evaluation step include cells that have TRPV1 activity and/or TRPA1 activity before exposure to the test substance, cells that have TRPV1 activity and/or TRPA1 activity after exposure to a known non-skin irritating substance, and cells that have TRPV1 activity and/or TRPA1 activity after exposure to the test substance. Control cells without TRPV1 activity and TRPA1 activity after exposure, cells with TRPV1 activity and/or TRPA1 activity after exposure to a known skin irritant, or TRPV1 activity and/or TRPA1 activity after exposure to a known skin irritant. /or cells that do not have TRPA1 activity,
Method for evaluating skin irritation of test substances:
(a) When using cells collected from an organism having TRPV1 activity, CXCL (CXC motif chemokine ligand) 1, CXCL (CXC motif chemokine ligand) 5, CXCR (CXC chemokine receptor) 2, CXCR (CXC chemokine ligand) receptor) 4, CCL (CC motif chemokine ligand) 2, CCL (CC motif chemokine ligand) 5, CCL (CC motif chemokine ligand) 7, CCL (CC motif chemokine ligand) 8, CCL (CC motif chemokine ligand) 11, CCL (CC motif chemokine ligand) 18, CCL (CC motif chemokine ligand) 22, CCR (CC chemokine receptor) 6, ACKR (Atypical chemokine receptor) 1, M-CSF (Macrophage colony stimulating factor), SCF (skip1-cul1-F -box-protein), EGR (Early growth response) 1, EGR (Early growth response) 2, EGR (Early growth response) 3, FOSB (FosB proto-oncogene, AP-1 transcription factor subunit), FOS (Fos proto- at least one indicator selected from the group consisting of oncogene, AP-1 transcription factor subunit), and FOSL1 (FOS like 1, AP-1 transcription factor subunit);
(b) When using cells collected from an organism having TRPA1 activity, CCL (CC motif chemokine ligand) 4L1, CCL (CC motif chemokine ligand) 4L2, CCL (CC motif chemokine ligand) 3, CCL (CC motif chemokine ligand) 7, CCL (CC motif chemokine ligand) 16, CCL (CC motif chemokine ligand) 19, CCL (CC motif chemokine ligand) 22, XCR (XC chemokine receptor) 1, CX3CR (C-X3-C chemokine receptor) ) 1, ACKR (Atypical chemokine receptor) 3, MIF (Macrophage migration inhibitory factor), M-CSF (Macrophage colony stimulating factor), SCF (skip1-cul1-F-box-protein), EGR (Early growth response) 1, At least one indicator selected from the group consisting of FOSB (FosB proto-oncogene, AP-1 transcription factor subunit) and FOSL1 (FOS like 1, AP-1 transcription factor subunit). an exposure step of exposing cells collected from a living body having TRPV1 activity and/or TRPA1 activity to a candidate skin irritant substance;
A measuring step of measuring the gene expression level and/or protein production level of the following indicators (a) and/or (b) in cells collected from the living body ;
The gene expression level and/or protein production level measured in the measurement step is compared with the gene expression level or protein production level in a control, and if the gene expression level or protein production level is higher than the control, the a screening step of screening the candidate substance as a skin irritating substance;
The control in the screening step is a cell having TRPV1 activity and/or TRPA1 activity before exposure to the test substance, a cell having TRPV1 activity and/or TRPA1 activity after exposure to a known non-skin irritating substance, or a cell having TRPV1 activity and/or TRPA1 activity after exposure to a known non-skin irritating substance, or control cells that do not have TRPV1 activity and TRPA1 activity after exposure to the substance,
How to screen for skin irritants:
(a) When using cells collected from an organism having TRPV1 activity, CXCL (CXC motif chemokine ligand) 1, CXCL (CXC motif chemokine ligand) 5, CXCR (CXC chemokine receptor) 2, CXCR (CXC chemokine ligand) receptor) 4, CCL (CC motif chemokine ligand) 2, CCL (CC motif chemokine ligand) 5, CCL (CC motif chemokine ligand) 7, CCL (CC motif chemokine ligand) 8, CCL (CC motif chemokine ligand) 11, CCL (CC motif chemokine ligand) 18, CCL (CC motif chemokine ligand) 22, CCR (CC chemokine receptor) 6, ACKR (Atypical chemokine receptor) 1, M-CSF (Macrophage colony stimulating factor), SCF (skip1-cul1-F -box-protein), EGR (Early growth response) 1, EGR (Early growth response) 2, EGR (Early growth response) 3, FOSB (FosB proto-oncogene, AP-1 transcription factor subunit), FOS (Fos proto- at least one indicator selected from the group consisting of oncogene, AP-1 transcription factor subunit), and FOSL1 (FOS like 1, AP-1 transcription factor subunit);
(b) When using cells collected from an organism having TRPA1 activity, CCL (CC motif chemokine ligand) 4L1, CCL (CC motif chemokine ligand) 4L2, CCL (CC motif chemokine ligand) 3, CCL (CC motif chemokine ligand) 7, CCL (CC motif chemokine ligand) 16, CCL (CC motif chemokine ligand) 19, CCL (CC motif chemokine ligand) 22, XCR (XC chemokine receptor) 1, CX3CR (C-X3-C chemokine receptor) ) 1, ACKR (Atypical chemokine receptor) 3, MIF (Macrophage migration inhibitory factor), M-CSF (Macrophage colony stimulating factor), SCF (skip1-cul1-F-box-protein), EGR (Early growth response) 1, At least one indicator selected from the group consisting of FOSB (FosB proto-oncogene, AP-1 transcription factor subunit) and FOSL1 (FOS like 1, AP-1 transcription factor subunit). an exposure step of exposing cells collected from a living body having TRPV1 activity and/or TRPA1 activity to a candidate substance of a non-skin irritating substance;
A measuring step of measuring the gene expression level and/or protein production level of the following indicators (a) and/or (b) in cells collected from the living body ;
The gene expression level and/or protein production level measured in the measurement step is compared with the gene expression level or protein production level in a control, and if the gene expression level or protein production level is the same or lower than the control. , a screening step of screening the candidate substance as a non-skin irritating substance,
The control in the screening step is a cell having TRPV1 activity and/or TRPA1 activity before exposure to the test substance, a cell having TRPV1 activity and/or TRPA1 activity after exposure to a known non-skin irritating substance, or a cell having TRPV1 activity and/or TRPA1 activity after exposure to a known non-skin irritating substance, or control cells that do not have TRPV1 activity and TRPA1 activity after exposure to the substance,
Screening method for non-skin irritants:
(a) When using cells collected from an organism having TRPV1 activity, CXCL (CXC motif chemokine ligand) 1, CXCL (CXC motif chemokine ligand) 5, CXCR (CXC chemokine receptor) 2, CXCR (CXC chemokine ligand) receptor) 4, CCL (CC motif chemokine ligand) 2, CCL (CC motif chemokine ligand) 5, CCL (CC motif chemokine ligand) 7, CCL (CC motif chemokine ligand) 8, CCL (CC motif chemokine ligand) 11, CCL (CC motif chemokine ligand) 18, CCL (CC motif chemokine ligand) 22, CCR (CC chemokine receptor) 6, ACKR (Atypical chemokine receptor) 1, M-CSF (Macrophage colony stimulating factor), SCF (skip1-cul1-F -box-protein), EGR (Early growth response) 1, EGR (Early growth response) 2, EGR (Early growth response) 3, FOSB (FosB proto-oncogene, AP-1 transcription factor subunit), FOS (Fos proto- at least one indicator selected from the group consisting of oncogene, AP-1 transcription factor subunit), and FOSL1 (FOS like 1, AP-1 transcription factor subunit);
(b) When using cells collected from an organism having TRPA1 activity, CCL (CC motif chemokine ligand) 4L1, CCL (CC motif chemokine ligand) 4L2, CCL (CC motif chemokine ligand) 3, CCL (CC motif chemokine ligand) 7, CCL (CC motif chemokine ligand) 16, CCL (CC motif chemokine ligand) 19, CCL (CC motif chemokine ligand) 22, XCR (XC chemokine receptor) 1, CX3CR (C-X3-C chemokine receptor) ) 1, ACKR (Atypical chemokine receptor) 3, MIF (Macrophage migration inhibitory factor), M-CSF (Macrophage colony stimulating factor), SCF (skip1-cul1-F-box-protein), EGR (Early growth response) 1, At least one indicator selected from the group consisting of FOSB (FosB proto-oncogene, AP-1 transcription factor subunit) and FOSL1 (FOS like 1, AP-1 transcription factor subunit). An exposure step in which cells collected from a living body having TRPV1 activity and/or TRPA1 activity are exposed to a candidate substance for a low skin irritation substance, which is a substance that causes an acute and/or delayed irritation response in the skin compared to a control. and,
A measuring step of measuring the gene expression level and/or protein production level of the following indicators (a) and/or (b) in cells collected from the living body ;
The gene expression level and/or protein production level measured in the measurement step is compared with the gene expression level or protein production level in a control, and if the gene expression level or protein production level is the same or lower than the control. , a screening step of screening the candidate substance as a low skin irritation substance,
The control in the screening step includes: (i) comparing the gene expression amount and/or protein production amount measured in the measurement step with the gene expression amount or protein production amount in the control; If the amount or protein production is equivalent, cells that have TRPV1 activity and/or TRPA1 activity before exposure to the test substance, and cells that have TRPV1 activity and/or TRPA1 activity after exposure to a known non-skin irritating substance cells, control cells that do not have TRPV1 activity and TRPA1 activity after exposure to a test substance, or cells that do not have TRPV1 activity and/or TRPA1 activity after exposure to a known skin irritating substance, (ii) the measurement The gene expression level and/or protein production level measured in the process are compared with the gene expression level or protein production level in a control, and if the gene expression level or protein production level is lower than the control, the test substance cells with TRPV1 activity and/or TRPA1 activity before exposure to a known non-skin irritating substance, cells with TRPV1 activity and/or TRPA1 activity after exposure to a test substance, and cells with TRPV1 activity and/or TRPA1 activity after exposure to a test substance. Control cells without, cells with TRPV1 activity and/or TRPA1 activity after exposure to known skin irritants, or cells without TRPV1 activity and/or TRPA1 activity after exposure to known skin irritants. be,
Screening method for low skin irritation substances:
(a) When using cells collected from an organism having TRPV1 activity, CXCL (CXC motif chemokine ligand) 1, CXCL (CXC motif chemokine ligand) 5, CXCR (CXC chemokine receptor) 2, CXCR (CXC chemokine ligand) receptor) 4, CCL (CC motif chemokine ligand) 2, CCL (CC motif chemokine ligand) 5, CCL (CC motif chemokine ligand) 7, CCL (CC motif chemokine ligand) 8, CCL (CC motif chemokine ligand) 11, CCL (CC motif chemokine ligand) 18, CCL (CC motif chemokine ligand) 22, CCR (CC chemokine receptor) 6, ACKR (Atypical chemokine receptor) 1, M-CSF (Macrophage colony stimulating factor), SCF (skip1-cul1-F -box-protein), EGR (Early growth response) 1, EGR (Early growth response) 2, EGR (Early growth response) 3, FOSB (FosB proto-oncogene, AP-1 transcription factor subunit), FOS (Fos proto- at least one indicator selected from the group consisting of oncogene, AP-1 transcription factor subunit), and FOSL1 (FOS like 1, AP-1 transcription factor subunit);
(b) When using cells collected from an organism having TRPA1 activity, CCL (CC motif chemokine ligand) 4L1, CCL (CC motif chemokine ligand) 4L2, CCL (CC motif chemokine ligand) 3, CCL (CC motif chemokine ligand) 7, CCL (CC motif chemokine ligand) 16, CCL (CC motif chemokine ligand) 19, CCL (CC motif chemokine ligand) 22, XCR (XC chemokine receptor) 1, CX3CR (C-X3-C chemokine receptor) ) 1, ACKR (Atypical chemokine receptor) 3, MIF (Macrophage migration inhibitory factor), M-CSF (Macrophage colony stimulating factor), SCF (skip1-cul1-F-box-protein), EGR (Early growth response) 1, At least one indicator selected from the group consisting of FOSB (FosB proto-oncogene, AP-1 transcription factor subunit) and FOSL1 (FOS like 1, AP-1 transcription factor subunit). an exposure step of exposing cells collected from a living body having TRPV1 activity and/or TRPA1 activity to a candidate substance for an anti-skin irritation substance and a skin irritation substance;
A measuring step of measuring the gene expression level and/or protein production level of the following indicators (a) and/or (b) in cells collected from the living body ;
The gene expression level and/or protein production level measured in the measurement step are compared with the gene expression level or protein production level in a control, and if the gene expression level or protein production level is lower than the control, the a screening step of screening the candidate substance as an anti-skin irritation substance;
The control in the screening step is cells having TRPV1 activity and/or TRPA1 activity exposed only to known skin irritants.
Screening method for anti-skin irritation substances:
(a) When using cells collected from an organism having TRPV1 activity, CXCL (CXC motif chemokine ligand) 1, CXCL (CXC motif chemokine ligand) 5, CXCR (CXC chemokine receptor) 2, CXCR (CXC chemokine ligand) receptor) 4, CCL (CC motif chemokine ligand) 2, CCL (CC motif chemokine ligand) 5, CCL (CC motif chemokine ligand) 7, CCL (CC motif chemokine ligand) 8, CCL (CC motif chemokine ligand) 11, CCL (CC motif chemokine ligand) 18, CCL (CC motif chemokine ligand) 22, CCR (CC chemokine receptor) 6, ACKR (Atypical chemokine receptor) 1, M-CSF (Macrophage colony stimulating factor), SCF (skip1-cul1-F -box-protein), EGR (Early growth response) 1, EGR (Early growth response) 2, EGR (Early growth response) 3, FOSB (FosB proto-oncogene, AP-1 transcription factor subunit), FOS (Fos proto- at least one indicator selected from the group consisting of oncogene, AP-1 transcription factor subunit), and FOSL1 (FOS like 1, AP-1 transcription factor subunit);
(b) When using cells collected from an organism having TRPA1 activity, CCL (CC motif chemokine ligand) 4L1, CCL (CC motif chemokine ligand) 4L2, CCL (CC motif chemokine ligand) 3, CCL (CC motif chemokine ligand) 7, CCL (CC motif chemokine ligand) 16, CCL (CC motif chemokine ligand) 19, CCL (CC motif chemokine ligand) 22, XCR (XC chemokine receptor) 1, CX3CR (C-X3-C chemokine receptor) ) 1, ACKR (Atypical chemokine receptor) 3, MIF (Macrophage migration inhibitory factor), M-CSF (Macrophage colony stimulating factor), SCF (skip1-cul1-F-box-protein), EGR (Early growth response) 1, At least one indicator selected from the group consisting of FOSB (FosB proto-oncogene, AP-1 transcription factor subunit) and FOSL1 (FOS like 1, AP-1 transcription factor subunit). An evaluation kit for evaluating skin irritation of a test substance, comprising a probe that specifically binds to the indicator gene or protein of the following (a) and/or (b) ,
In the evaluation, cells collected from a living body having TRPV1 activity and/or TRPA1 activity are used, and an evaluation kit :
(a) When using cells collected from a living body having TRPV1 activity, CXCL (CXC motif chemokine ligand) 1, CXCL (CXC motif chemokine ligand) 5, CXCR (CXC chemokine receptor) 2, CXCR (CXC chemokine receptor) ) 4, CCL (CC motif chemokine ligand) 2, CCL (CC motif chemokine ligand) 5, CCL (CC motif chemokine ligand) 7, CCL (CC motif chemokine ligand) 8, CCL (CC motif chemokine ligand) 11, CCL ( CC motif chemokine ligand) 18, CCL (CC motif chemokine ligand) 22, CCR (CC chemokine receptor) 6, ACKR (Atypical chemokine receptor) 1, M-CSF (Macrophage colony stimulating factor), SCF (skip1-cul1-F- box-protein), EGR (Early growth response) 1, EGR (Early growth response) 2, EGR (Early growth response) 3, FOSB (FosB proto-oncogene, AP-1 transcription factor subunit), FOS (Fos proto-oncogene , AP-1 transcription factor subunit), and FOSL1 (FOS like 1, AP-1 transcription factor subunit);
(b) When using cells collected from a living body having TRPA1 activity, CCL (CC motif chemokine ligand) 4L1, CCL (CC motif chemokine ligand) 4L2, CCL (CC motif chemokine ligand) 3, CCL (CC motif chemokine ligand) chemokine ligand) 7, CCL (CC motif chemokine ligand) 16, CCL (CC motif chemokine ligand) 19, CCL (CC motif chemokine ligand) 22, XCR (XC chemokine receptor) 1, CX3CR (C-X3-C chemokine receptor) 1. ACKR (Atypical chemokine receptor) 3, MIF (Macrophage migration inhibitory factor), M-CSF (Macrophage colony stimulating factor), SCF (skip1-cul1-F-box-protein), EGR (Early growth response) 1, FOSB (FosB proto-oncogene, AP-1 transcription factor subunit), and at least one indicator selected from the group consisting of FOSL1 (FOS like 1, AP-1 transcription factor subunit).

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