Method for screening agent for preventing or improving skin trouble and method for identifying gene associated with skin trouble

Abstract

To highly accurately determine whether a test substance has a skin trouble preventing or improving action.SOLUTION: The method for preventing or ameliorating skin troubles comprises a step of measuring the expression level of genes whose expression level by long-wave UVA (370-400 nm) irradiation is different from that by short-wave UVA (320-370 nm) irradiation in the presence of a test material, and selecting a skin trouble-preventing or ameliorating agent by using the expression level of the genes in the presence of the test material as an indicator.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present disclosure relates to a method for screening for an agent for preventing or improving skin troubles and a method for identifying genes associated with skin troubles. [Background technology]

[0002] It is generally known that ultraviolet rays deteriorate skin conditions, and ultraviolet A rays (UVA, wavelength range: 320 nm to 400 nm) in particular are believed to penetrate deep into the dermis layer, causing wrinkles, sagging, and the like. Non-Patent Document 1 discloses that the expression level of the GDF-15 gene is increased in UV-irradiated aging fibroblasts and photoaged hyperpigmented skin. Non-Patent Document 1 also discloses that when aging-induced fibroblasts are irradiated with UVA in the wavelength range of 320 nm to 400 nm, with a maximum peak at 350 nm, the expression level of GDF15 increases in a time-dependent manner. Furthermore, Non-Patent Document 2 discloses that repeated irradiation with UVA in the wavelength range of 315 nm to 400 nm reduces the expression of the FN1 gene in fibroblasts.

[0003] Furthermore, Patent Document 1 discloses a method for evaluating the effectiveness of an ingredient with a whitening effect by irradiating an epidermis equivalent using a reconstructed epidermis containing melanocytes and keratinocytes as the main components with a mixed light consisting of UVA and ultraviolet B rays (UVB, wavelength range: 280 nm to 320 nm). In Patent Document 1, the skin equivalent is irradiated with ultraviolet light and the ingredient is applied, and pigmentation is visually observed from the stratum corneum side of the skin equivalent.

[0004] Furthermore, Patent Document 2 discloses a method for screening drugs that inhibit long-wavelength UV-induced melanization by irradiating a solution containing melanin monomers and a test sample with long-wavelength UV light, and using the melanin monomer residual rate after UV irradiation, which is obtained from the fluorescence intensity when the solution is irradiated with excitation light. In Patent Document 2, the solution is irradiated with long-wavelength UV light in the wavelength range of 320 nm to 400 nm. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Japanese Patent Application Laid-Open No. 2014-149152 [Patent Document 2] Japanese Patent Application Laid-Open No. 2005-274308 [Non-patent literature]

[0006] [Non-Patent Document 1] Yeongeun Kim et al., Journal of Investigative Dermatology (2020) 140, 2478~2486 [Non-patent document 2] Wongnapa Nakyai et al.,Photochemistry and Photobiology,2017,93,1462-1471 Summary of the Invention [Problem to be solved by the invention]

[0007] However, there is a need for a method for determining with higher accuracy whether a test substance has a skin trouble preventing or improving effect. Therefore, the present disclosure aims to provide, based on completely new findings, a method for screening a skin trouble preventing or improving agent and a method for identifying a skin trouble-related gene, which can determine with higher accuracy whether a test substance has a skin trouble preventing or improving effect. [Means for solving the problem]

[0008] In order to achieve the above-mentioned object, the present inventors have conducted extensive research and found that there are factors whose expression levels differ when exposed to UVA, particularly long-wave UVA, and when exposed to short-wave UVA, and have thus completed the present disclosure.

[0009] <1> measuring the expression level of a gene whose expression level is different when irradiated with long-wave UVA (370 nm to 400 nm) compared to when irradiated with short-wave UVA (320 nm to 370 nm) in the presence of a test substance; A method for screening agents for preventing or improving skin troubles, which selects agents for preventing or improving skin troubles using the expression level of a gene in the presence of a test substance as an indicator. <2> The gene expression level is higher with long-wave UVA irradiation than with short-wave UVA irradiation. <1> A method for screening an agent for preventing or improving skin troubles according to the above item 1. <3> The gene is the GDF-15 gene. <2> A method for screening an agent for preventing or improving skin troubles according to the above item 1. <4> The gene expression level due to long-wave UVA irradiation is lower than that due to short-wave UVA irradiation. <1> A method for screening an agent for preventing or improving skin troubles according to the above item 1. <5> The gene is the FN1 gene <4> A method for screening an agent for preventing or improving skin troubles according to the above item 1. <6> In the step of measuring the expression level of a gene, the mRNA of the gene is measured. <1> ~ <5> 10. A method for screening an agent for preventing or improving skin troubles according to any one of the above. <7> The agent for preventing or improving skin troubles improves the deterioration of skin conditions caused by long-wave UVA. <1> ~ <6> 10. A method for screening an agent for preventing or improving skin troubles according to any one of the above. <8> The agent for preventing or improving skin troubles is at least one agent for preventing or improving wrinkles, blemishes, and dullness. <1> ~ <7> 10. A method for screening an agent for preventing or improving skin troubles according to any one of the above. <9> In the step of measuring the expression level of a gene, a test substance is brought into contact with skin cells or skin tissue, and then the expression level of the gene in the skin cells or skin tissue is measured. <1> ~ <8> 10. A method for screening an agent for preventing or improving skin troubles according to any one of the above. <10> In the step of measuring the expression level of the gene, after irradiating skin cells or skin tissue with long-wave UVA, a test substance is brought into contact with the skin cells or skin tissue, and the expression level of the gene in the skin cells or skin tissue is measured. <1> ~ <9> 10. A method for screening an agent for preventing or improving skin troubles according to any one of the above. <11> In the step of measuring the expression level of the gene, a test substance is brought into contact with the skin cells or skin tissue while irradiating the skin cells or skin tissue with long-wave UVA, and the expression level of the gene in the skin cells or skin tissue is measured. <1> ~ <9> 10. A method for screening an agent for preventing or improving skin troubles according to any one of the above. <12> In the step of measuring the expression level of the gene, a test substance is brought into contact with the skin cells or skin tissue, and then the skin cells or skin tissue are irradiated with long-wave UVA, and the expression level of the gene in the skin cells or skin tissue is measured. <1> ~ <9> 10. A method for screening an agent for preventing or improving skin troubles according to any one of the above. <13> The skin cells are human dermal fibroblasts or human epidermal cells. <9> ~ <12> 10. A method for screening an agent for preventing or improving skin troubles according to any one of the above.

[0010] <14> A step of irradiating skin cells or skin tissue with long-wave UVA (370 nm to 400 nm) or short-wave UVA (320 nm to 370 nm) and measuring gene expression upon irradiation with long-wave UVA and gene expression upon irradiation with short-wave UVA; A step of extracting genes whose expression levels are different when irradiated with long-wave UVA compared to when irradiated with short-wave UVA; The method for identifying genes related to skin troubles comprises identifying the extracted genes as genes that serve as indicators of the effect of preventing or improving skin troubles. <15> In the extraction step, genes whose expression level is greater when irradiated with long-wave UVA than when irradiated with short-wave UVA are extracted. <14> A method for identifying a gene related to skin troubles described above. <16> In the extraction step, genes whose expression level due to long-wave UVA irradiation is lower than that due to short-wave UVA irradiation are extracted. <14> A method for identifying a gene related to skin troubles described above. <17> In the step of measuring gene expression, the amount of mRNA of the gene is measured. <14> ~ <16> A method for identifying a gene related to skin troubles according to any one of the above. <18> The effect of preventing or improving skin problems is the effect of preventing or improving skin problems caused by long-wave UVA. <14> ~ <17> A method for identifying a gene related to skin troubles according to any one of the above. <19> The skin trouble prevention or improvement effect is at least one or more prevention or improvement effects selected from the group consisting of wrinkle prevention or improvement effect, blemish prevention or improvement effect, and dullness prevention or improvement effect. <14> ~ <18> A method for identifying a gene related to skin troubles according to any one of the above. <20> The skin cells or skin tissue are human skin cells or human skin tissue. <14> ~ <19> A method for identifying a gene related to skin troubles according to any one of the above. <21> The human skin cells are human dermal fibroblasts or human epidermal cells. <20> A method for identifying a gene related to skin troubles described above. [Effects of the Invention]

[0011] According to the present disclosure, based on the novel finding that a factor whose expression level changes when irradiated with long-wave UVA and short-wave UVA is used, it is possible to provide a screening method for agents for preventing or improving skin troubles and a method for identifying genes related to skin troubles, which can determine with high accuracy whether a test substance has the effect of preventing or improving skin troubles. [Brief explanation of the drawings]

[0012] [Figure 1] FIG. 1 is a characteristic diagram showing the results of a comparison of the expression levels of the GDF-15 gene in dermal fibroblasts irradiated with short-wave UVA and long-wave UVA. [Figure 2]FIG. 1 is a characteristic diagram showing the results of comparing the expression levels of the GDF-15 gene in epidermal cells irradiated with short-wave UVA and long-wave UVA. [Figure 3] FIG. 1 is a characteristic diagram showing the results of comparing the expression levels of the FN1 gene in dermal fibroblasts irradiated with short-wave UVA and long-wave UVA. [Figure 4] FIG. 1 is a characteristic diagram showing the expression level of the GDF-15 gene in dermal fibroblasts treated with Lagerstroemia indica leaf extract. [Figure 5] FIG. 1 is a characteristic diagram showing the expression level of the FN1 gene in dermal fibroblasts treated with Lagerstroemia indica leaf extract, Hydrangea tea extract, Rehmannia root extract, or Fucus extract. [Figure 6] FIG. 1 is a characteristic diagram showing the expression level of the FN1 gene when dermal fibroblasts irradiated with long-wave UVA were treated with hydrangea extract. [Figure 7] FIG. 1 is a characteristic diagram showing the expression level of the FN1 gene when dermal fibroblasts treated with Hydrangea extract are irradiated with long-wave UVA. [Figure 8] FIG. 1 is a characteristic diagram showing the results of comparing the expression levels of the TM4SF1 gene in a system in which dermal fibroblasts were irradiated with short-wave UVA and a system in which dermal fibroblasts were irradiated with long-wave UVA. [Figure 9] FIG. 1 is a characteristic diagram showing the expression level of the TM4SF1 gene in dermal fibroblasts treated with Lagerstroemia indica leaf extract, Rehmannia root extract, or Fucus vesiculosus extract. DETAILED DESCRIPTION OF THE INVENTION

[0013] Hereinafter, embodiments of the present disclosure will be described. The description is for illustrating the embodiments and is not intended to limit the scope of the present disclosure.

[0014] In the following embodiments, the components (including element steps, etc.) are not essential unless otherwise specified. The same applies to numerical values ​​and their ranges, and do not limit the present disclosure. For example, the present disclosure allows addition, omission, substitution, modification, etc. of the number, amount, position, ratio, material, configuration, type, order, etc., within the scope of the spirit of the present disclosure.

[0015] In the present disclosure, numerical ranges indicated using "to" include the numerical values ​​before and after "to" as the minimum and maximum values, respectively. In the numerical ranges described in stages in this disclosure, the upper or lower limit value described in one numerical range may be replaced with the upper or lower limit value of another numerical range described in stages. Furthermore, in the numerical ranges described in this disclosure, the upper or lower limit value of that numerical range may be replaced with a value shown in the examples. In the present disclosure, each component may contain multiple types of corresponding substances. When multiple types of substances corresponding to each component are present in a composition, the content or amount of each component means the total content or amount of the multiple substances present in the composition, unless otherwise specified.

[0016] In the present disclosure, the term "gene" is not limited to a region that encodes a protein, but also includes, for example, an expression control region, an intron region, and non-coding RNA (miRNA) of unknown function.

[0017] The screening method for skin trouble prevention or amelioration agents disclosed herein includes a step of measuring, in the presence of a test substance, the expression level of a gene whose expression level differs when irradiated with long-wave UVA (wavelength range: 370 nm to 400 nm) compared to when irradiated with short-wave UVA (wavelength range: 320 nm to 370 nm), and selecting a skin trouble prevention or amelioration agent using the gene expression level in the presence of the test substance as an indicator. The screening method disclosed herein is based on the novel finding that there are genes whose expression patterns differ when irradiated with long-wave UVA and when irradiated with short-wave UVA. According to the screening method disclosed herein, genes whose expression level changes when irradiated with long-wave UVA are used as indicators, and therefore candidate substances that primarily prevent or ameliorate skin trouble caused by long-wave UVA can be selected from among the test substances.

[0018] In the present disclosure, the term "skin trouble preventive or ameliorating agent" refers to either or both of a preventive agent that makes skin trouble less likely to occur and an ameliorating agent that cures existing skin trouble. In other words, the screening method of the present disclosure makes it possible to select, from among the test substances tested, a substance that has a preventive effect against skin trouble, a substance that has an ameliorating effect against skin trouble, or a substance that has a preventive effect against skin trouble and an ameliorating effect against skin trouble.

[0019] Skin trouble refers to changes in skin condition caused by ultraviolet radiation, particularly UVA radiation (wavelength range: 320 nm to 400 nm), especially long-wave UVA radiation (wavelength range: 370 nm to 400 nm). Therefore, the skin trouble preventing or ameliorating agent according to the present disclosure can prevent or ameliorate changes in skin condition caused by ultraviolet radiation, especially UVA radiation (wavelength range: 320 nm to 400 nm), especially long-wave UVA radiation (wavelength range: 370 nm to 400 nm). In particular, it is more preferable that the skin trouble preventing or ameliorating agent according to the present disclosure prevents or ameliorate skin trouble caused by long-wave UVA.

[0020] In this disclosure, long-wave UVA refers to ultraviolet light with a wavelength range of 370 nm to 400 nm, preferably ultraviolet light with a wavelength greater than 370 nm to 400 nm, more preferably ultraviolet light with a wavelength of 375 nm to 400 nm, and even more preferably ultraviolet light with a wavelength of 380 nm to 400 nm. Furthermore, in this disclosure, short-wave UVA refers to ultraviolet light with a wavelength range of 320 nm to 370 nm, preferably ultraviolet light with a wavelength of 320 nm or greater but less than 370 nm, more preferably ultraviolet light with a wavelength of 320 nm to 365 nm, and even more preferably ultraviolet light with a wavelength of 320 nm to 360 nm. Furthermore, in the present disclosure, when referring to "a gene whose expression level differs when irradiated with long-wave UVA (wavelength range: 370 nm to 400 nm) compared to when irradiated with short-wave UVA (wavelength range: 320 nm to 370 nm)," the difference in wavelength between long-wave UVA and short-wave UVA (wavelength of long-wave UVA minus wavelength of short-wave UVA) is preferably at least 10 nm or more, more preferably 20 nm or more, even more preferably 30 nm or more, and even more preferably 40 nm or more. By definition, the maximum difference in wavelength between long-wave UVA and short-wave UVA is 80 nm.

[0021] Specifically, skin troubles include wrinkles, age spots, and dullness. Therefore, the skin trouble preventing or improving agent can mean, for example, at least one preventing or improving agent selected from the group consisting of wrinkle preventing or improving agents, age spots preventing or improving agents, and dullness preventing or improving agents. In particular, it is known that irradiation with long-wave UVA can cause at least one skin trouble selected from the group consisting of wrinkles, age spots, and dullness. Therefore, the skin trouble preventing or improving agent according to the present disclosure can have a preventive effect or an improving effect on at least one skin trouble selected from the group consisting of wrinkles, age spots, and dullness caused by irradiation with long-wave UVA.

[0022] [Skin trouble-related genes] In the screening method for skin trouble prevention or amelioration agents disclosed herein, genes serving as indicators (hereinafter referred to as skin trouble-related genes) can be identified, for example, by the following method. Specifically, the disclosed method for identifying skin trouble-related genes includes the steps of irradiating skin cells or skin tissue with long-wave UVA (370 nm to 400 nm) or short-wave UVA (320 nm to 370 nm), measuring gene expression upon long-wave UVA irradiation and short-wave UVA irradiation, and extracting genes whose expression levels differ with long-wave UVA irradiation compared to short-wave UVA irradiation, and identifying the extracted genes as skin trouble-related genes. According to the disclosed method for identifying skin trouble-related genes, genes whose expression levels change specifically in response to UVA, particularly long-wave UVA, can be extracted from the skin. Here, "extracting genes" refers to selecting genes from a group of tested genes whose expression levels differ with long-wave UVA irradiation compared to short-wave UVA irradiation.

[0023] In particular, the above-mentioned method can extract skin trouble-related genes whose expression level is higher when exposed to long-wave UVA than when exposed to short-wave UVA. That is, it can extract skin trouble-related genes whose expression is specifically enhanced in skin by long-wave UVA among UVA. Here, the extracted skin trouble-related genes can be genes whose expression level when exposed to long-wave UVA is statistically significantly higher than that when exposed to short-wave UVA. Statistical significance can be analyzed using known testing methods such as chi-square test, generalized Wilcoxon test, Wilcoxon signed-rank test, Mann-Whitney test, log-rank test, and Cox proportional hazards.

[0024] In addition, it is preferable that the difference between the expression level by long-wave UVA irradiation and the expression level by short-wave UVA irradiation is large. For example, when the expression level by short-wave UVA irradiation is 1, the expression level by long-wave UVA irradiation can be 1.1 or more, preferably 1.2 or more, more preferably 1.3 or more, even more preferably 1.4 or more, even more preferably 1.5 or more, even more preferably 1.7 or more, even more preferably 2.0 or more, and even more preferably 2.5 or more.

[0025] An example of a skin trouble-related gene whose expression level is higher with long-wave UVA irradiation than with short-wave UVA irradiation is the GDF-15 gene. The GDF-15 gene encodes growth differentiation factor 15, and its expression pattern in skin cells or skin tissues upon long-wave UVA irradiation is unknown. In addition to the GDF-15 gene, other skin trouble-related genes whose expression level is higher with long-wave UVA irradiation than with short-wave UVA irradiation include the SCF (Stem Cell Factor) gene and the bFGF (Basic Fibroblast Growth Factor) gene.

[0026] Alternatively, the above-mentioned method can extract skin trouble-related genes whose expression level is lower when exposed to long-wave UVA compared to when exposed to short-wave UVA. That is, it can extract skin trouble-related genes whose expression is specifically suppressed in skin by long-wave UVA among UVA. Here, the skin trouble-related genes to be extracted can be genes whose expression level when exposed to long-wave UVA is statistically significantly lower than that when exposed to short-wave UVA. Statistical significance can be analyzed using known testing methods such as chi-square test, generalized Wilcoxon test, Wilcoxon signed-rank test, Mann-Whitney test, log-rank test, and Cox proportional hazards.

[0027] In addition, it is preferable that the difference between the expression level by long-wave UVA irradiation and the expression level by short-wave UVA irradiation is large. For example, when the expression level by short-wave UVA irradiation is 1, the expression level by long-wave UVA irradiation can be 0.9 or less, preferably 0.8 or less, more preferably 0.7 or less, even more preferably 0.6 or less, even more preferably 0.5 or less, even more preferably 0.4 or less, even more preferably 0.3 or less, and even more preferably 0.2 or less.

[0028] An example of a skin trouble-related gene whose expression level is lower with long-wave UVA irradiation than with short-wave UVA irradiation is the FN1 gene. The FN1 gene encodes fibronectin 1, and its expression pattern in skin cells or skin tissues upon long-wave UVA irradiation is unknown. In addition to the FN1 gene, other skin trouble-related genes whose expression level is lower with long-wave UVA irradiation than with short-wave UVA irradiation include the IVL (involucrin) gene, the TGM1 (transglutaminase 1) gene, and the COL1 (collagen type 1) gene.

[0029] Here, the expression level of a skin trouble-related gene can be measured by detecting the mRNA of the skin trouble-related gene. For example, mRNA in skin cells or skin tissues irradiated with long-wave UVA and mRNA in skin cells or skin tissues irradiated with short-wave UVA can be detected, and the expression level of a skin trouble-related gene irradiated with long-wave UVA and the expression level of a skin trouble-related gene irradiated with short-wave UVA can be compared based on the detected mRNA amount.

[0030] Here, it is particularly preferable to use human skin cells or human skin tissue as the skin cells or skin tissue. The human skin cells are not particularly limited, but fibroblasts present in the dermis layer are preferred, although epidermal cells present in the epidermis layer may also be used. Examples of fibroblasts include human-derived dermal fibroblasts and immortalized human fibroblasts. Dermal fibroblasts and immortalized fibroblasts derived from mammals other than humans, such as mouse dermal fibroblasts and immortalized mouse fibroblasts, can also be used. Among these, it is most preferable to use primary human-derived dermal fibroblasts, from the viewpoints of similarity to human skin tissue and stimuli responsiveness. The irradiance when fibroblasts are irradiated with long-wave UVA or short-wave UVA is 0.1 mW / cm. 2 ~15mW / cm 2 It is preferable to set the light intensity to 0.1 mW / cm 2 ~10mW / cm 2 It is more preferable that the cumulative light dose when irradiating fibroblasts with long-wave UVA or short-wave UVA is 0.5 J / cm. 2 ~100J / cm 2 It is preferable to set the value to 0.5 J / cm 2 ~50J / cm 2 It is more preferable to set the value to 1 J / cm 2 ~30J / cm 2 It is even more preferable to set the irradiance and cumulative light dose within this range when irradiating fibroblasts with long-wave UVA or short-wave UVA. By setting the irradiance and cumulative light dose within this range when irradiating fibroblasts with long-wave UVA or short-wave UVA, it is possible to highly simulate the effects of long-wave UVA and short-wave UVA on actual skin, and to create conditions that enable fibroblast culture. Furthermore, when irradiating fibroblasts with long-wave UVA or short-wave UVA, a non-irradiation period may be set between irradiations while keeping the cumulative light dose within the above range in order to prevent the death of fibroblasts. The non-irradiation period can be the fibroblast culture period. The irradiation period can be 1 minute to 8 hours, preferably 30 minutes to 6 hours, and more preferably 1 hour to 3 hours. Furthermore, the non-irradiation period can be 1 minute to 48 hours, preferably 30 minutes to 24 hours, more preferably 1 hour to 6 hours, and even more preferably 1 hour to 3 hours.

[0031] The expression analysis of mRNA in skin cells or skin tissues can be performed using a so-called transcriptome analysis method. Transcriptome analysis involves analyzing all mRNA (or primary transcription products, transcripts) present in specific cells, tissues, etc. Specific examples of transcriptome analysis include methods using microarrays and next-generation sequencers. The analysis method using next-generation sequencers, also known as RNA-seq, is a technology that comprehensively sequences cDNA synthesized from total RNA or mRNA extracted from cells, tissues, etc.

[0032] Total RNA or mRNA can be extracted from skin cells or skin tissues using, for example, the phenol / chloroform method, the AGPC (acid guanidinium thiocyanate-phenol-chloroform extraction) method, a method using columns such as TRIzol, RNeasy, or QIAzol, a method using special silica-coated magnetic particles, a method using Solid Phase Reversible Immobilization magnetic particles, or extraction using a commercially available RNA extraction reagent such as ISOGEN.

[0033] According to the method described above, it is possible to identify skin trouble-related genes that serve as indicators of the preventive or ameliorative effects on skin troubles caused by long-wave UVA. In particular, the skin trouble-related genes identified as described above serve as indicators of the preventive or ameliorative effects on skin troubles such as wrinkles, age spots, and dullness.

[0034] [Test substance] The test substance is not particularly limited, but examples include plant extracts, natural compounds, synthetic compounds, proteins, peptides, microbial cultures, cell extracts, and animal tissue extracts. Among these, plant extracts, natural compounds, and synthetic compounds are preferred. The test substance may be either a novel substance or a known substance. Furthermore, substances contained in any library of cosmetics or pharmaceuticals may also be used as the test substance. The library may be a compound library or an extract library.

[0035] The test substance is added for a period of preferably 1 minute to 1 week, more preferably 1 hour to 48 hours, and even more preferably 4 hours to 32 hours, from the viewpoint of its action on the skin and culturing conditions.

[0036] The concentration of the test substance is preferably 0.01 μg / mL to 1000 μg / mL, more preferably 0.1 μg / mL to 100 μg / mL, and even more preferably 1 μg / mL to 10 μg / mL, from the viewpoint of possible skin effects and preparation conditions.

[0037] The test substance can be selected from the following, for example: Lagerstroemia indica leaf extract, burnet extract, tea catechin, tea leaf extract, pomegranate extract, pomegranate seed oil, olive leaf extract, tripeptide-1, Ascophyllum nodosum extract, hydrolyzed white lupin protein, loquat leaf extract, rehmannia root extract, marjoram leaf extract, licorice leaf extract, angelica tree leaf / stem extract, oligopeptide-6, cacao extract, perilla leaf extract, royal jelly extract, licorice root extract, hydrangea extract, hollyhock leaf / stem extract, St. John's wort flower / leaf / stem extract, moss bean seed extract, horse chestnut seed extract, emblica fruit extract, Tilia cordata flower extract, comfrey leaf extract, peony extract, and allo Evergreen bark juice, mussel glycogen, pyrrolidone carboxylic acid (PCA), soy milk fermented liquid, eelgrass extract, Salicornia herb extract, Ascaea officinalis extract, arnica flower extract, artemisia capillaris flower extract, fennel fruit extract, tea leaf extract, turmeric rhizome extract, Scutellaria baicalensis root extract, olive leaf extract, fucus extract, artemisia princeps leaf extract, sage leaf extract, birch bark extract, yarrow extract, centella asiatica extract, morus alba root bark extract, clove extract, calendula officinalis flower extract, honeysuckle leaf extract, loquat leaf extract, peppermint leaf extract, hop flower extract, eucalyptus leaf extract, lavender flower extract, and wild thyme extract.

[0038] [Expression analysis of genes related to skin problems] In the screening method of the present disclosure, a test substance is applied to skin cells or skin tissue, and then the expression level of a skin trouble-related gene is measured. Here, the skin cells or skin tissue may be the skin cells or skin tissue used in identifying the above-mentioned skin trouble-related gene, or may be different skin cells or skin tissue, or skin organoids or artificial skin. In the screening method of the present disclosure, the skin cells to which the test substance is applied are not particularly limited, but are preferably fibroblasts present in the dermis layer, but may also be epidermal cells present in the epidermis layer. Examples of fibroblasts include mouse dermal fibroblasts, human-derived dermal fibroblasts, immortalized human fibroblasts, and immortalized mouse fibroblasts. Among these, it is most preferable to use primary human-derived dermal fibroblasts, which are similar to human skin tissue and responsive to stimuli.

[0039] The method for measuring the expression levels of the skin trouble-related genes identified as described above is not particularly limited, and conventionally known methods can be used. For example, various hybridization methods can be used to measure the expression levels of skin trouble-related genes. Examples of such hybridization methods include Northern blotting, Southern blotting, DNA chip analysis, in situ hybridization, Northern hybridization, and Southern hybridization. In addition, in combination with or as an alternative to hybridization methods, PCR methods such as quantitative RT-PCR, Invader, loop-mediated amplification (LAMP), nucleic acid sequence-based amplification (NASBA), strand displacement amplification (SDA), rolling circle amplification (RCA), signal-mediated amplification of RNA technology (SMART), helicase-based nucleic acid detection methods, and next-generation sequencing can be used.

[0040] When using the Northern blot method, the presence or absence of gene expression and the expression level can be detected and measured by using a nucleic acid probe designed based on the base sequence of a skin trouble-related gene. Specifically, the nucleic acid probe (complementary strand) is ionized with a radioisotope (32 P, 33 P, 35 For example, the DNA / RNA duplex is labeled with a radioisotope (e.g., S) or a fluorescent substance, transferred to a nylon membrane or the like in the usual manner, hybridized with RNA derived from skin cells or skin tissue samples treated with the test substance, and then a signal derived from the label (radioisotope or fluorescent substance) in the formed DNA / RNA duplex is detected.

[0041] When using quantitative RT-PCR, the presence or absence of gene expression and its expression level can be detected and measured by using a pair of primers designed based on the base sequence of a skin trouble-related gene. For example, RNA is collected from skin cells or skin tissue treated with a test substance, the 3' end is polyadenylated, and cDNA is prepared from the polyadenylated RNA according to standard methods. Then, using the obtained cDNA as a template, a nucleic acid amplification reaction is performed using the above pair of primers, and the resulting amplified nucleic acid fragment is detected. Examples of methods for detecting amplified nucleic acid fragments include a nucleic acid amplification reaction using primers previously labeled with a radioisotope or fluorescent substance, a method in which the amplified nucleic acid fragment is electrophoresed on an agarose gel and stained with ethidium bromide or the like for detection, and a method in which the resulting amplified nucleic acid fragment is transferred to a nylon membrane or the like and hybridized with a labeled nucleic acid probe for detection.

[0042] When using a microarray, a microarray (RNA chip or DNA chip) is prepared in which nucleic acid probes (single-stranded or double-stranded) designed based on the base sequence of a skin trouble-related gene are immobilized on a substrate (solid phase). In this method, cDNA is prepared using a pair of primers designed based on the base sequence of a skin trouble-related gene, and the amplified nucleic acid fragment is hybridized with the nucleic acid probe immobilized on the substrate under highly stringent conditions. "Highly stringent conditions" refer to conditions defined for hybridization and subsequent washing. "Highly stringent conditions" are described, for example, in Sambrook, J. & Russell, D., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, January 15, 2001, Vol. 1, pp. 7.42-7.45 and Vol. 2, pp. 8.9-8.17.

[0043] The expression level of skin trouble-related genes may also be measured using a sequencer. When using a sequencer, so-called next-generation sequencers can be used. Examples of next-generation sequencers include NovaSeq, HiSeq, NextSeq, and MiSeq (Illumina Inc.); Ion Proton, Ion PGM, Ion S5 / S5 XL (Thermo Fisher Scientific Co., Ltd.); PacBio RS II, and Sequel (Pacific Biosciences Inc.); and when using a nanopore sequencer, for example, MinION (Oxford Nanopore Technologies Inc.) can be used.

[0044] As described above, nucleic acid probes and primers designed to measure the expression levels of skin trouble-related genes specifically recognize the mRNA or cDNA of the skin trouble-related genes. Here, "specifically recognize" means that, for example, in Northern blotting, substantially only the skin trouble-related genes disclosed herein or nucleic acids derived therefrom can be detected, and, for example, in RT-PCR, substantially only the nucleic acids are amplified. Specifically, DNA consisting of the base sequence constituting the skin trouble-related genes disclosed herein or oligonucleotides containing a certain number of nucleotides complementary to its complementary strand can be used. Here, "complementary" does not necessarily mean a perfectly complementary sequence over a certain number of consecutive nucleotides, but preferably has an identity of 80% or more, more preferably 90% or more, and even more preferably 95% or more. The identity of the base sequences can be determined using algorithms such as BLAST.

[0045] In the present disclosure, the nucleic acid probe may be designed to have a chain length of, for example, 8 or more bases, preferably 10 or more bases, more preferably 15 or more bases, and for example, 100 or less bases, preferably 50 or less bases, more preferably 25 or less bases, as long as it is capable of specific hybridization, as described above. Furthermore, in the present disclosure, the primer may be designed to have a chain length of, for example, 10 or more bases, preferably 15 or more bases, more preferably 20 or more bases, and for example, 100 or less bases, preferably 50 or less bases, more preferably 35 or less bases, as long as it is capable of specific annealing, as described above.

[0046] In the screening method of the present disclosure, the expression level of a skin trouble-related gene can be measured by measuring the translation product (protein) of the skin trouble-related gene. For example, a molecule that interacts with the translation product of the skin trouble-related gene can be used to measure the translation product by protein chip analysis, immunoassays (e.g., ELISA), mass spectrometry (e.g., LC-MS / MS, MALDI-TOF / MS), etc. An antibody against the translation product can be used as the molecule that interacts with the translation product. For example, the expression level of a skin trouble-related gene can be measured by contacting the antibody with skin cells or skin tissue treated with a test substance, detecting the protein bound to the antibody, and measuring its level. For example, in the Western blot method, the above-mentioned antibody is used as the primary antibody, and then a secondary antibody labeled with a radioisotope, fluorescent substance, enzyme, or the like is bound to the primary antibody. The signal derived from the labeled substance is measured using a radiation meter, fluorescence detector, or the like, to measure the expression level of the skin trouble-related gene.

[0047] In the screening method of the present disclosure, the expression level of a skin trouble-related gene is measured as described above, and then the test substance's ability to prevent or improve skin trouble is determined using the expression level as an indicator. For a skin trouble-related gene (e.g., GDF-15 gene, etc.) whose expression level is increased upon long-wave UVA irradiation compared to short-wave UVA irradiation, if the expression level of the skin trouble-related gene is reduced in skin cells or skin tissues upon application of the test substance, the test substance can be determined to be a candidate substance with skin trouble-preventing or improving effects. Conversely, for a skin trouble-related gene (e.g., FN1 gene, etc.) whose expression level is reduced upon long-wave UVA irradiation compared to short-wave UVA irradiation, if the expression level of the skin trouble-related gene is increased in skin cells or skin tissues upon application of the test substance, the test substance can be determined to be a candidate substance with skin trouble-preventing or improving effects. Here, the high or low expression level of a skin trouble-related gene is determined by comparing it to a reference value (also referred to as a cutoff value).

[0048] The reference value for comparing the expression levels can be determined in advance by measuring the expression levels of the skin trouble-related genes in skin cells or skin tissues that have not been irradiated with either long-wave UVA or short-wave UVA, and appropriately based on statistical values ​​such as the average expression levels and standard deviations based thereon. Alternatively, the reference value for comparing the expression levels can be determined in advance by measuring the expression levels of the skin trouble-related genes in skin cells or skin tissues that have been irradiated with long-wave UVA, and appropriately based on statistical values ​​such as the average expression levels and standard deviations based thereon.

[0049] If the expression level of a skin trouble-related gene whose expression is increased by long-wave UVA irradiation in skin cells or skin tissue is less than a reference value when the test substance is applied, the test substance is judged to be a candidate substance with the effect of preventing or improving skin trouble. Furthermore, if the expression level of a skin trouble-related gene whose expression is decreased by long-wave UVA irradiation in skin cells or skin tissue is greater than a reference value when the test substance is applied, the test substance is judged to be a candidate substance with the effect of preventing or improving skin trouble. Here, a candidate substance means a substance that may have the effect of preventing or improving skin trouble.

[0050] Furthermore, in the step of measuring the expression level of a skin trouble-related gene in the screening method of the present disclosure, the skin cells or skin tissues can be irradiated with long-wave UVA, followed by contacting a test substance with the skin cells or skin tissues, and measuring the expression level of the skin trouble-related gene in the skin cells or skin tissues. For a skin trouble-related gene whose expression level increases with long-wave UVA irradiation (e.g., the GDF-15 gene), if the expression level of the skin trouble-related gene in the skin cells or skin tissues is reduced when the test substance is applied, the test substance can be determined to be a candidate substance with the effect of preventing or improving skin trouble. Conversely, for a skin trouble-related gene whose expression level decreases with long-wave UVA irradiation (e.g., the FN1 gene), if the expression level of the skin trouble-related gene in the skin cells or skin tissues is increased when the test substance is applied, the test substance can be determined to be a candidate substance with the effect of preventing or improving skin trouble. Here, too, the high or low expression level of a skin trouble-related gene is determined by comparing it with the above-mentioned reference value (also referred to as the cutoff value).

[0051] In this case, since it is presumed that the expression levels of skin trouble-related genes in skin cells or skin tissues are altered by irradiation with long-wave UVA, the candidate substance can be selected as a substance that has a particular effect of improving skin troubles. Note that the candidate substance may also be selected as a substance that has an effect of preventing skin troubles in addition to improving skin troubles.

[0052] Furthermore, in the step of measuring the expression level of a skin trouble-related gene in the screening method of the present disclosure, a test substance can be contacted with the skin cells or skin tissue while irradiating the skin cells or skin tissue with long-wave UVA, and the expression level of the skin trouble-related gene in the skin cells or skin tissue can be measured. In the case of a skin trouble-related gene whose expression level increases with long-wave UVA irradiation (e.g., the GDF-15 gene), if the expression level of the skin trouble-related gene in the skin cells or skin tissue is reduced when the test substance is applied, the test substance can be determined to be a candidate substance with the effect of preventing or improving skin trouble. Conversely, in the case of a skin trouble-related gene whose expression level decreases with long-wave UVA irradiation (e.g., the FN1 gene), if the expression level of the skin trouble-related gene in the skin cells or skin tissue is increased when the test substance is applied, the test substance can be determined to be a candidate substance with the effect of preventing or improving skin trouble. Here, too, the high or low expression level of a skin trouble-related gene is determined by comparing it with the above-mentioned reference value (also referred to as the cutoff value).

[0053] In this case, irradiation with long-wave UVA and contact with the test substance are carried out simultaneously, so candidate substances can be selected as substances that have either or both of the effects of preventing skin troubles and improving skin troubles.

[0054] Furthermore, in the step of measuring the expression level of a skin trouble-related gene in the screening method of the present disclosure, a test substance can be contacted with skin cells or skin tissue, followed by irradiating the skin cells or skin tissue with long-wave UVA radiation, and then measuring the expression level of the skin trouble-related gene in the skin cells or skin tissue. For a skin trouble-related gene whose expression level increases upon long-wave UVA radiation (e.g., the GDF-15 gene), if the expression level of the skin trouble-related gene in the skin cells or skin tissue is reduced upon application of the test substance, the test substance can be determined to be a candidate substance with the effect of preventing or improving skin trouble. Conversely, for a skin trouble-related gene whose expression level decreases upon long-wave UVA radiation (e.g., the FN1 gene), if the expression level of the skin trouble-related gene in the skin cells or skin tissue is increased upon application of the test substance, the test substance can be determined to be a candidate substance with the effect of preventing or improving skin trouble. Here, too, the high or low expression level of a skin trouble-related gene is determined by comparing it with the above-mentioned reference value (also referred to as the cutoff value).

[0055] In this case, since the test substance is irradiated with long-wave UVA after contact, the candidate substance can be selected as a substance having a skin trouble prevention effect. Note that the candidate substance may also be selected as a substance having a skin trouble amelioration effect in addition to the skin trouble prevention effect.

[0056] [Screened uses for preventing or improving skin problems] The skin trouble preventing or ameliorating agent selected by the screening method of the present disclosure can be blended into cosmetics as a cosmetic ingredient, for example. Cosmetics blended with the skin trouble preventing or ameliorating agent of the present disclosure can exert the effect of preventing or ameliorating skin trouble caused by long-wave UVA.

[0057] Cosmetics may be used in a variety of applications, including, but not limited to, serums, lotions, emulsions, creams, body lotions, bath additives, sunscreens, makeup bases, makeup products, lotions, and aftershave creams. The skin trouble prevention or amelioration agent according to the present disclosure can also be incorporated into pharmaceuticals or quasi-drugs. Pharmaceuticals or quasi-drugs can be administered via any route, such as transdermal, intramuscular, oral, or intravenous routes, but transdermal administration is preferred from the perspective of direct action on the skin. Preferred dosage forms for transdermal administration include topical skin preparations and skin patches. The above-mentioned cosmetics, pharmaceuticals, and quasi-drugs can be incorporated with, for example, moisturizers, whitening agents, antioxidants, oily ingredients, UV absorbers, surfactants, thickeners, alcohols, colorants, fragrances, water, solvents, preservatives, pH adjusters, gelling agents, and other active ingredients. [Example]

[0058] The present disclosure will be described in more detail below using examples, but the technical scope of the present disclosure is not limited to the following examples.

[0059] [Example 1] In this example, we searched for genes whose expression levels change with irradiation of long-wave UVA (skin trouble-related genes). First, human dermal fibroblasts (Thermo Fisher) were cultured according to a standard method. 24 hours after the start of culture, the cells were exposed to long-wave UVA (390 nm) or short-wave UVA (340 nm) at 16.7 J / cm. 2 The cells were then irradiated with UVA at an intensity of 1000 kJ / cm and cultured for a further 24 hours. As a control, human dermal fibroblasts were cultured under the same conditions without irradiating with either long-wave or short-wave UVA.

[0060] After the culture was completed, the cells of each test level were collected using the RNeasy Plus Mini Kit (QIAGEN) to obtain a precipitate of total RNA. The collected total RNA was purified using a kit (One Step SYBR PrimeScript TMUsing RT-PCR Kit II (Takara Bio), QuantStudio3 (Applied Biosystems), and One Step TB Green (Takara Bio), the expression of various genes and the expression of the housekeeping gene GAPDH gene as an internal standard were detected. The expression levels of various genes were normalized based on the expression level of the GAPDH gene. In this test system, the expression level of the gene in each group was calculated relative to the expression level of the control gene, which was set at 100%.

[0061] Through the above tests, the GDF-15 gene was identified as a gene whose expression was enhanced when exposed to long-wave UVA compared to when exposed to short-wave UVA. The results of comparing the expression levels of the GDF-15 gene in systems irradiated with short-wave UVA and long-wave UVA are shown in Figure 1. This example enabled the identification of genes whose expression levels differed when exposed to long-wave UVA compared to when exposed to short-wave UVA.

[0062] Furthermore, in this example, the expression level of the GDF-15 gene was measured when human epidermal cells (Thermo Fisher) were irradiated with long-wave UVA (390 nm) or short-wave UVA (340 nm). In the experiment using human epidermal cells, the irradiation intensity of long-wave UVA (390 nm) and short-wave UVA (340 nm) was 8.35 J / cm. 2 The experiment was conducted in the same manner as the experiment using human fibroblasts described above, except that the expression level of the GDF-15 gene was measured. The results are shown in Figure 2. As shown in Figure 2, it was found that the expression level of the GDF-15 gene was higher in human epidermal cells after irradiation with long-wave UVA than after irradiation with short-wave UVA.

[0063] [Example 2] Long-wave UVA (390 nm) or short-wave UVA (340 nm) at 6.4 J / cm 2In the same manner as in Example 1, except that irradiation was performed at an intensity of 1000 nm, genes whose expression levels differed when irradiated with long-wave UVA compared to short-wave UVA were identified in human fibroblasts. In this example, the FN1 gene was identified as a gene whose expression level decreased when irradiated with long-wave UVA compared to short-wave UVA. The results of comparing the expression levels of the FN1 gene between a system irradiated with short-wave UVA and a system irradiated with long-wave UVA are shown in Figure 3.

[0064] [Preventive Improvement Verification Experiment 1] The expression level of the GDF-15 gene identified in Example 1 was examined to determine whether it could be used as an indicator of the preventive or ameliorative effect on skin troubles. First, human dermal fibroblasts (Thermo Fisher) were cultured according to standard methods. 24 hours after the start of culture, the medium was replaced with one containing 10 ppm of Lagerstroemia indica leaf extract, and cultured for another 24 hours. For comparison, a control group was cultured with human dermal fibroblasts under the same conditions except for the medium not containing Lagerstroemia indica leaf extract. After culture was completed, cells from each test level were recovered using an RNeasy Plus Mini Kit (QIAGEN) to obtain a total RNA precipitate. The expression level of the GDF-15 gene contained in the recovered total RNA was measured as in Example 1.

[0065] The results are shown in Figure 4. The Lagerstroemia Intica leaf extract used in this verification experiment is known as a skin conditioning agent. As shown in Figure 4, the expression level of the GDF-15 gene was reduced in human dermal fibroblasts treated with Lagerstroemia Intica leaf extract. When considered together with the results of Example 1, it was shown that substances that reduce the expression level of the GDF-15 gene are highly likely to have the effect of preventing or improving skin troubles. Therefore, it was suggested that the GDF-15 gene identified in Example 1 can serve as an indicator of the preventive or improving effect of skin troubles.

[0066] [Preventive Improvement Verification Experiment 2] The FN1 gene identified in Example 2 was examined to determine whether it could be used as an indicator of the preventive or ameliorative effects of skin troubles. The expression level of the FN1 gene contained in total RNA was measured in the same manner as in Verification Experiment 1, except that in addition to Lagerstroemia indica leaf extract, Hydrangea tea extract, Rehmannia root extract, and Fucus vesiculosus extract were used.

[0067] The results are shown in Figure 5. The Hydrangea extract used in this verification experiment is known to have a whitening effect. Rehmannia root extract is known as a skin conditioning agent. Fucus extract is known to have moisturizing and skin regenerating effects. As shown in Figure 5, the expression level of the FN1 gene was increased in human dermal fibroblasts treated with Lagerstroemia indicum leaf extract, Hydrangea extract, Rehmannia root extract, or Fucus extract. Considering the results of Example 2 comprehensively, it was shown that substances that increase the expression level of the FN1 gene are highly likely to have an effect of preventing or improving skin problems. Therefore, it was suggested that the FN1 gene identified in Example 2 can serve as an indicator of the preventive or improving effect of skin problems.

[0068] [Preventive Improvement Verification Experiment 3] The change in expression level of the FN1 gene identified in Example 2 was measured when hydrangea extract was applied after irradiation with long-wave UVA. First, human dermal fibroblasts (Thermo Fisher) were cultured according to a conventional method. 24 hours after the start of culture, the human dermal fibroblasts were exposed to long-wave UVA (390 nm) at 1.9 J / cm. 2The cells were irradiated at an intensity of 10 ppm and cultured for another 24 hours. The medium was then replaced with one containing 10 ppm of Hydrangea extract, and cultured for another hour. For comparison, a group of human dermal fibroblasts was cultured under the same conditions except for using a medium without Hydrangea extract and not irradiating with long-wave UVA. This group was designated Control 1. For comparison, a group of human dermal fibroblasts was cultured under the same conditions except for using a medium without Hydrangea extract. This group was designated Control 2. After the culture was completed, the cells from each test level were recovered using an RNeasy Plus Mini Kit (QIAGEN) to obtain a total RNA precipitate. The expression level of the FN1 gene contained in the recovered total RNA was measured in the same manner as in Example 1.

[0069] The results are shown in Figure 6. As shown in Figure 6, in human dermal fibroblasts exposed to long-wave UVA and then treated with Hydrangea extract, the expression level of the FN1 gene was increased compared to Control 1 and Control 2. The results of this verification experiment showed that the effect of Hydrangea extract in improving skin problems caused by long-wave UVA irradiation can be monitored using the expression level of the FN1 gene as an indicator. This verification experiment also showed that substances that increase the expression level of the FN1 gene are highly likely to have the effect of preventing or improving skin problems.

[0070] [Preventive Improvement Verification Experiment 4] The change in expression level of the FN1 gene identified in Example 2 was measured when it was treated with Hydrangea extract and then irradiated with long-wave UVA. First, human dermal fibroblasts (Thermo Fisher) were cultured according to a conventional method. 24 hours after the start of culture, Hydrangea extract was added to the cells to a concentration of 10 ppm, and the cells were cultured for another 24 hours. Then, the human dermal fibroblasts were exposed to long-wave UVA (390 nm) at 1.9 J / cm. 2The cells were then irradiated at an intensity of 1000 kJ / ml and cultured for a further 24 hours. For comparison, a group of human dermal fibroblasts was cultured under the same conditions except that a medium containing no hydrangea extract was used and long-wave UVA was not irradiated, and designated Control 1. For comparison, a group of human dermal fibroblasts was cultured under the same conditions except that a medium containing no hydrangea extract was used, and designated Control 2. After the culture was completed, cells from each test level were recovered using an RNeasy Plus Mini Kit (manufactured by QIAGEN) to obtain a total RNA precipitate. The expression level of the FN1 gene contained in the recovered total RNA was measured in the same manner as in Example 1.

[0071] The results are shown in Figure 7. As shown in Figure 7, in human dermal fibroblasts that were treated with Hydrangea extract and then irradiated with long-wave UVA, the expression level of the FN1 gene was similar compared to Control 1, and the expression level of the FN1 gene was increased compared to Control 2. The results of this verification experiment showed that the effect of Hydrangea extract in preventing skin problems caused by long-wave UVA irradiation can be monitored using the expression level of the FN1 gene as an indicator. This verification experiment also showed that substances that increase the expression level of the FN1 gene are highly likely to have the effect of preventing or improving skin problems.

[0072] [Preventive Improvement Verification Experiment 5] For genes whose expression levels did not differ between long-wave UVA and short-wave UVA irradiation, we examined the effect of treating human dermal fibroblasts with Lagerstroemia speciosa leaf extract, Rehmannia root extract, or Fucus extract. The expression level of the TM4SF1 gene contained in total RNA was measured in the same manner as in Experiment 1, except that Lagerstroemia speciosa leaf extract, Rehmannia root extract, and Fucus extract were used.

[0073] Figure 8 shows the results of comparing the expression levels of the TM4SF1 gene between a system irradiated with short-wave UVA and a system irradiated with long-wave UVA. As shown in Figure 8, the expression level of the TM4SF1 gene was found to be increased by both short-wave UVA and long-wave UVA. Furthermore, Figure 9 shows the results of measuring the expression level of the TM4SF1 gene when human dermal fibroblasts were treated with Lagerstroemia speciosa leaf extract, Rehmannia root extract, or Fucus extract. As shown in Figure 9, unlike the results of Verification Experiments 1 and 2, the expression level of the TM4SF1 gene did not change with the treatment with Lagerstroemia speciosa leaf extract, Rehmannia root extract, or Fucus extract. The results of Verification Experiment 5 indicated that genes whose expression levels are increased by both short-wave UVA and long-wave UVA cannot be used as indicators of the preventive or ameliorative effects of skin problems.

Claims

1. measuring, in the presence of a test substance, the expression level of a gene whose expression level is different when irradiated with long-wave UVA in the wavelength range of 370 nm to 400 nm compared to the expression level when irradiated with short-wave UVA in the wavelength range of 320 nm to 370 nm; A method for screening agents for preventing or improving skin troubles, which selects an agent for preventing or improving skin troubles using the expression level of the gene in the presence of the test substance as an index.

2. 2. The method for screening an agent for preventing or improving skin trouble according to claim 1, wherein the gene is expressed in a greater amount by the long-wave UVA irradiation than by the short-wave UVA irradiation.

3. 3. The method for screening an agent for preventing or improving skin trouble according to claim 2, wherein the gene is a GDF-15 gene.

4. 2. The method for screening an agent for preventing or improving skin trouble according to claim 1, wherein the gene is expressed in a smaller amount by the long-wave UVA irradiation than by the short-wave UVA irradiation.

5. The method for screening an agent for preventing or improving skin trouble according to claim 4, wherein the gene is the FN1 gene.

6. 2. The method for screening an agent for preventing or improving skin trouble according to claim 1, wherein the step of measuring the expression level of the gene comprises measuring mRNA of the gene.

7. The method for screening an agent for preventing or improving skin trouble according to claim 1, wherein the agent for preventing or improving skin trouble prevents or improves skin trouble caused by long-wave UVA.

8. The method for screening a skin trouble preventing or improving agent according to claim 1, wherein the skin trouble preventing or improving agent is at least one or more preventing or improving agents selected from the group consisting of wrinkle preventing or improving agents, blemish preventing or improving agents, and dullness preventing or improving agents.

9. 2. The method for screening an agent for preventing or improving skin problems according to claim 1, wherein the step of measuring the expression level of the gene comprises contacting the test substance with skin cells or skin tissue and then measuring the expression level of the gene in the skin cells or skin tissue.

10. 2. The method for screening an agent for preventing or improving skin troubles according to claim 1, wherein in the step of measuring the expression level of the gene, the test substance is brought into contact with the skin cells or skin tissue after irradiating the skin cells or skin tissue with long-wave UVA, and the expression level of the gene in the skin cells or skin tissue is measured.

11. 2. The method for screening an agent for preventing or improving skin troubles according to claim 1, wherein in the step of measuring the expression level of the gene, the test substance is brought into contact with the skin cells or skin tissue while irradiating the skin cells or skin tissue with long-wave UVA, and the expression level of the gene in the skin cells or skin tissue is measured.

12. 2. The method for screening an agent for preventing or improving skin troubles according to claim 1, wherein the step of measuring the expression level of the gene comprises contacting the test substance with skin cells or skin tissue, irradiating the skin cells or skin tissue with long-wave UVA, and measuring the expression level of the gene in the skin cells or skin tissue.

13. The method for screening an agent for preventing or improving skin trouble according to any one of claims 9 to 12, wherein the skin cells are human dermal fibroblasts or human epidermal cells.

14. irradiating skin cells or skin tissue with long-wave UVA in the wavelength range of 370 nm to 400 nm or short-wave UVA in the wavelength range of 320 nm to 370 nm, and measuring gene expression upon irradiation with long-wave UVA and gene expression upon irradiation with short-wave UVA; extracting genes whose expression levels are different when irradiated with long-wave UVA compared to when irradiated with short-wave UVA; and identifying the extracted gene as a gene that serves as an indicator of an effect of preventing or improving skin trouble.

15. The method for identifying skin trouble-related genes according to claim 14, wherein the extraction step extracts genes whose expression levels due to the long-wave UVA irradiation are greater than those due to the short-wave UVA irradiation.

16. The method for identifying skin trouble-related genes according to claim 14, wherein the extraction step extracts genes whose expression levels due to the long-wave UVA irradiation are lower than those due to the short-wave UVA irradiation.

17. The method for identifying a skin trouble-related gene according to claim 14, wherein the step of measuring gene expression comprises measuring an amount of mRNA of the gene.

18. The method for identifying a gene related to skin trouble according to claim 14, wherein the effect of preventing or improving skin trouble is the effect of preventing or improving skin trouble caused by long-wave UVA.

19. The method for identifying genes related to skin troubles described in claim 14, wherein the skin trouble prevention or improvement effect is at least one or more prevention or improvement effects selected from the group consisting of wrinkle prevention or improvement effect, age spot prevention or improvement effect, and dullness prevention or improvement effect.

20. The method for identifying a gene related to skin trouble according to claim 14, wherein the skin cells or skin tissue are human skin cells or human skin tissue.

21. The method for identifying a skin trouble-related gene according to claim 20, wherein the human skin cells are human dermal fibroblasts or human epidermal cells.

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