Screening method for laminin 511 production promoter, epidermal basement membrane stabilizer and / or epidermal stem cell decrease inhibitor or increase promoter

Abstract

To provide a method for easily screening an agent that proliferates MCSP-positive epidermal basal stem cells or an agent that suppresses the decrease of MCSP-positive epidermal basal stem cells, thereby suppressing the decrease of epidermal stem cells or increasing the cells.SOLUTION: Provided is a screening method for a laminin 511 expression promoter, using the expression of laminin 511 as an indicator. Also provided is an agent that promotes the expression of laminin 511 containing 1-(2-hydroxyethyl)-2-imidazolidinone as an active ingredient, using the screening method.SELECTED DRAWING: Figure 5

Description

Technical Field

[0001] The present invention relates to the technical field of suppressing the decrease or promoting the increase of epidermal stem cells in the epidermal basement membrane.

Background Art

[0002] The epidermis is a skin tissue present in the outermost layer of the skin. The epidermis is mainly composed of the stratum corneum, stratum granulosum, stratum spinosum, and basal layer. The basal cells present in the basal layer divide and move to the outer layer. During this movement, enucleation occurs in the cells, they flatten, and differentiate into the stratum corneum, and the stratum corneum finally peels off. This turnover period is said to be about 45 days. However, in aging skin, the turnover rate slows down and the entire epidermis becomes thinner. As a result, it is known that skin functions such as a decrease in barrier function and a decrease in water content occur. Basal cells are cells rich in mitotic activity, but they do not repeat proliferation infinitely and stop dividing after a certain number of divisions. Basal cells are newly supplied by the differentiation of a part of the epidermal basal stem cells present on the basement membrane. However, it is known that the number of epidermal basal stem cells decreases with aging, and when the number of epidermal basal stem cells decreases, signs of aging such as thinning of the epidermis, drying of the epidermis, and decrease in barrier function appear.

[0003] In recent years, the MCSP antibody, which is an epidermal basal stem cell marker, has been developed, and by using this antibody, it has become possible to identify epidermal basal stem cells. In the non-exposed skin of men and women of each age group, visualization of epidermal basal cells using the MCSP antibody has been performed, and it has been shown that the number of MCSP-positive epidermal basal stem cells decreases with aging (Non-Patent Document 1). Development of drugs having an action of promoting the increase or suppressing the decrease in the number of MCSP-positive epidermal basal stem cells is required.

Prior Art Documents

Non-Patent Documents

[0004]

Non-Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0005] An object of the present invention is to provide a method for promoting the increase or suppressing the decrease of the number of MCSP-positive epidermal basal stem cells.

Means for Solving the Problems

[0006] The inventors of the present invention conducted intensive research on MCSP-positive epidermal basal stem cells and found the relationship between MCSP-positive epidermal basal stem cells and laminin 511 present near the basement membrane. That is, when the amount of laminin 511 near the basement membrane decreases, the number of MCSP-positive epidermal basal stem cells also decreases accordingly, and it was first found that the number of MCSP-positive epidermal basal stem cells can be increased by increasing the expression level of laminin 511. In addition, it was found that laminin is a complex of laminin α, β, and γ, and the abundance of laminin 511 can be determined by using the expression levels of laminin α5, β1, and γ1 as indicators.

[0007] Based on this finding, the inventors arrived at an invention related to a method for screening a laminin 511 expression promoter using the expression of laminin 511 as an indicator. Since the laminin 511 expression promoter can stabilize the basement membrane, the method for screening a laminin 511 expression promoter can also be referred to as a method for screening a basement membrane stabilizer. In addition, when the abundance of laminin 511 increases and the basement membrane is stabilized, an increasing or decreasing inhibitory effect on epidermal basal stem cells occurs. Therefore, the method for screening a laminin 511 expression promoter can also be referred to as a method for screening an epidermal basal stem cell increasing promoter or a decreasing inhibitor.

[0008] More specifically, it relates to the following invention: [1] A method for screening a laminin 511 expression promoter using the expression level of laminin 511 in epidermal cells as an index. [2] A step of culturing epidermal cells in a culture medium containing a candidate drug, A step of measuring the expression level of laminin 511 in epidermal cells, A step of determining that the candidate drug has a laminin 511 expression promoting effect when the expression of laminin 511 increases as compared with the laminin 511 expression level in the control The method according to item 1, comprising: [3] The method according to item 1 or 2, wherein the expression level of laminin 511 is determined from one or more mRNA levels selected from the group consisting of the protein amount of laminin 511 or the mRNA amounts of laminin α5, laminin β1, and laminin γ1. [4] The method according to any one of items 1 to 3, wherein the expression level of laminin 511 is determined from the total amount of the mRNA amounts of laminin α5, laminin β1, and laminin γ1. [5] The method according to any one of items 1 to 4, wherein the laminin 511 expression promoter is an epidermal basement membrane stabilizer. [6] The method according to any one of items 1 to 5, wherein the epidermal basement membrane stabilizer is an agent for suppressing or increasing the decrease of epidermal basal stem cells. [7] The method according to any one of items 1 to 6, wherein the epidermal cells include epidermal stem cells. [8] The method according to any one of items 1 to 7, wherein the epidermal stem cells include epidermal basal stem cells. [9] The method according to any one of items 1 to 8, wherein the epidermal cells include MCSP-expressing cells.

[10] The method according to any one of items 1 to 9, wherein the epidermal cells further include cells expressing integrin.

[11] The method according to any one of items 1 to 10, wherein the epidermal cells are derived from a fetus.

[12] A laminin 511 expression promoter comprising at least one extract selected from the group consisting of brown algae, red algae, and green algae or 1-(2-hydroxyethyl)-2-imidazolidinone as an active ingredient.

[13] An epidermal basement membrane stabilizer that promotes the expression of laminin 511 and contains at least one extract selected from the group consisting of brown algae, red algae, and green algae or 1-(2-hydroxyethyl)-2-imidazolidinone as an active ingredient.

[14] An agent for suppressing the decrease or increasing the number of epidermal basal stem cells by promoting the expression of laminin 511 and containing at least one extract selected from the group consisting of brown algae, red algae, and green algae or 1-(2-hydroxyethyl)-2-imidazolidinone as an active ingredient.

[15] An inhibitor of the activity of extracellular matrix degrading enzymes containing 1-(2-hydroxyethyl)-2-imidazolidinone.

[16] The inhibitor of the activity of extracellular matrix degrading enzymes according to item 15, wherein the extracellular matrix degrading enzyme is matrix metalloproteinase or heparanase.

[17] The inhibitor of the activity of extracellular matrix degrading enzymes according to item 15, wherein the extracellular matrix degrading enzyme is matrix metalloproteinase 9.

[18] An inhibitor of laminin 511 degradation containing the inhibitor of the activity of extracellular matrix degrading enzymes according to any one of items 15 to 17.

[19] An epidermal basement membrane stabilizer containing the inhibitor of the activity of extracellular matrix degrading enzymes according to any one of items 15 to 17.

[20] An agent for suppressing the decrease or promoting the increase of epidermal basal stem cells containing the inhibitor of the activity of extracellular matrix degrading enzymes according to any one of items 15 to 17.

[21] A method for enhancing the expression of laminin 511 in the epidermis, which includes administering at least one extract selected from the group consisting of brown algae, red algae, and green algae or 1-(2-hydroxyethyl)-2-imidazolidinone.

[22] A method for stabilizing the epidermal basement membrane by administering at least one extract selected from the group consisting of brown algae, red algae, and green algae or 1-(2-hydroxyethyl)-2-imidazolidinone and by the action of promoting the expression of laminin 511.

[23] A method of suppressing the decrease or promoting the increase of epidermal basal stem cells by administering at least one extract selected from the group consisting of brown algae, red algae, and green algae or 1-(2-hydroxyethyl)-2-imidazolidinone, through the action of promoting the expression of laminin 511.

[24] A beauty method of administering at least one extract selected from the group consisting of brown algae, red algae, and green algae or 1-(2-hydroxyethyl)-2-imidazolidinone to enhance the expression of laminin 511 in the epidermis.

[25] A beauty method of administering at least one extract selected from the group consisting of brown algae, red algae, and green algae or 1-(2-hydroxyethyl)-2-imidazolidinone to enhance the expression of laminin 511 in the epidermis and stabilize the epidermal basement membrane through the action of promoting the expression of laminin 511.

[26] A beauty method of administering at least one extract selected from the group consisting of brown algae, red algae, and green algae or 1-(2-hydroxyethyl)-2-imidazolidinone to enhance the expression of laminin 511 in the epidermis and suppress the decrease or promote the increase of epidermal basal stem cells through the action of promoting the expression of laminin 511.

[27] Use of at least one extract selected from the group consisting of brown algae, red algae, and green algae or 1-(2-hydroxyethyl)-2-imidazolidinone for the manufacture of a cosmetic or pharmaceutical for promoting the expression of laminin 511.

[28] Use of at least one extract selected from the group consisting of brown algae, red algae, and green algae or 1-(2-hydroxyethyl)-2-imidazolidinone for the manufacture of a cosmetic or pharmaceutical for stabilizing the epidermal basement membrane.

[29] Use of at least one extract selected from the group consisting of brown algae, red algae, and green algae or 1-(2-hydroxyethyl)-2-imidazolidinone for the manufacture of a cosmetic or pharmaceutical for suppressing the decrease or promoting the increase of epidermal basal stem cells.

[30] At least one extract selected from the group consisting of brown algae, red algae, and green algae or 1-(2-hydroxyethyl)-2-imidazolidinone for use in anti-aging treatment through promoting the expression of laminin 511.

[31] At least one extract or 1-(2-hydroxyethyl)-2-imidazolidinone selected from the group consisting of brown algae, red algae, and green algae for use in anti-aging treatment through epidermal basement membrane stabilization.

[32] At least one extract or 1-(2-hydroxyethyl)-2-imidazolidinone selected from the group consisting of brown algae, red algae, and green algae for use in anti-aging treatment through suppression or increase of epidermal basal stem cells.

[33] 1-(2-hydroxyethyl)-2-imidazolidinone for use in anti-aging treatment through inhibition of the activity of extracellular matrix degrading enzymes.

[34] The 1-(2-hydroxyethyl)-2-imidazolidinone according to item 33, wherein the extracellular matrix degrading enzyme is matrix metalloproteinase or heparanase.

[35] The 1-(2-hydroxyethyl)-2-imidazolidinone according to item 33, wherein the extracellular matrix degrading enzyme is matrix metalloproteinase 9.

[36] An agent for improving skin barrier, firmness, moisture, and inflammation, comprising at least one extract selected from the group consisting of brown algae, red algae, and green algae.

[37] An anti-inflammatory agent, comprising at least one extract selected from the group consisting of brown algae, red algae, and green algae.

[38] Use of at least one extract selected from the group consisting of brown algae, red algae, and green algae for the manufacture of an agent for improving skin barrier, firmness, moisture, and inflammation.

[39] Use of at least one extract selected from the group consisting of brown algae, red algae, and green algae for the manufacture of an anti-inflammatory agent.

[40] A cosmetic method for improving skin barrier, firmness, moisture, and inflammation, comprising applying a cosmetic containing at least one extract selected from the group consisting of brown algae, red algae, and green algae to a subject in need of improving skin barrier, firmness, moisture, and inflammation.

[41] A cosmetic method for suppressing inflammation, comprising applying a cosmetic containing at least one extract selected from the group consisting of brown algae, red algae, and green algae to a subject suffering from inflammation.

Advantages of the Invention

[0009] Screening for a drug that increases MCSP-positive epidermal basal stem cells can be performed in a simpler cell experiment by using laminin 511 as an indicator.

Brief Description of the Drawings

[0010]

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Mode for Carrying Out the Invention

[0011] The present invention relates to a method for screening a laminin 511 expression promoter using the expression of laminin 511 in epidermal cells as an index. More specifically, the method for screening a laminin 511 expression promoter is as follows: a step of culturing epidermal cells in a culture medium containing a candidate drug, a step of measuring the expression of laminin 511 in epidermal cells, a step of determining that the candidate drug has a laminin 511 expression promoting effect when the expression of laminin 511 increases as compared with the laminin 511 expression level in the control It includes. Further, it may include a step of irradiating the cultured epidermal cells with ultraviolet rays. By irradiating with ultraviolet rays, the expression level of laminin 511 decreases. The irradiation with ultraviolet rays, as an example, can be performed by irradiating the cultured cells with 1 mJ / cm 2 ~200 mJ / cm 2 . The irradiation with ultraviolet rays can enhance the activity of matrix metalloproteinase and result in the degradation of laminin. Therefore, under ultraviolet irradiation, when the candidate drug can suppress the decrease in the protein amount of laminin 511, it can be known that the candidate drug has an inhibitory effect on the degradation of laminin 511 and a promoting effect on its expression. By examining the inhibitory effect of the candidate drug on the activity of matrix metalloproteinase, it can be determined that the candidate drug has a promoting effect on the expression of laminin 511.

[0012] The step of culturing epidermal cells in a culture medium containing a candidate drug may be performed in any manner as long as the culture medium containing the candidate drug comes into contact with the cultured epidermal cells. The candidate drug or its dilution may be directly added to the culture medium being cultured, or the culture medium may be replaced with a culture medium containing the candidate drug. The culture is carried out under normal conditions used for culturing human epidermal cells. As an example, it is cultured in an incubator under an atmosphere of 37°C and 5% CO2 humidity. The culture time after the addition of the candidate drug can be arbitrarily set according to the effect of the candidate drug. From the viewpoint of fully exerting the effect of the candidate drug, for example, it can be cultured for 300 minutes or more, preferably 6 hours or more, more preferably 48 hours or more. Also, from the viewpoint of preventing saturation of the effect of the candidate drug, for example, it can be cultured for a period of 7 days or less, preferably 5 days or less, more preferably 72 hours or less.

[0013] The measurement of the expression of laminin 511 in cultured epidermal cells can be measured by any method of molecular biology using an antibody that recognizes laminin 511. For the measurement of the protein amount of laminin 511, for example, Western blot or immunostaining can be used. In addition, the expression of laminin 511 can be measured based on the mRNA transcription levels of the constituent subunits (α5, β1, γ1) of laminin 511. The transcription level of mRNA can be measured, for example, by performing Northern blot or quantitative PCR. The constituent subunits can be measured based on the transcription level of any one, or can be measured as a combination of the transcription levels of two or three. In one aspect, the expression level can be measured by summing the respective expression levels of laminin α5, laminin β1, and laminin γ1. In another aspect, the amount of the constituent subunit with the lowest expression level can also be used as the expression level of laminin 511.

[0014] When the expression of laminin 511 increases in epidermal cells cultured in a culture medium containing a candidate agent as compared to the expression level of laminin 511 in the control, it can be determined that the candidate agent has an effect of promoting laminin 511 expression. In one aspect, when there is a significant difference in the expression level, it can be determined that the candidate agent has an effect of promoting laminin 511 expression. In another aspect, when the expression level increases at a predetermined ratio, it can be determined that the candidate agent has an effect of promoting laminin 511 expression. The predetermined ratio can be arbitrarily determined by those skilled in the art. For example, 10%, more preferably 20%, still more preferably 30%, and even more preferably 50% can be selected as the predetermined ratio compared to the expression level of the control. This determination can be made by the experimenter or by software analysis. From the perspective of performing the screening method of the present invention on a large scale, the determination step is preferably performed by a computer including a device that measures expression or a processor that acquires data from the device. The expression level of laminin 511 in the control refers to the expression level of laminin 511 in epidermal cells that have undergone the same operations except that the candidate agent is not included. Therefore, the aforementioned culture step and expression measurement step may include a step of culturing control epidermal cells in a culture medium not containing the candidate agent and measuring the expression of laminin 511 in the control epidermal cells. The culture step and expression step for the control may be performed simultaneously and in parallel with the culture step and expression step using a culture medium containing the candidate agent, or may be performed in advance.

[0015] In addition to the aforementioned steps, the screening step of the present invention may include an optional pre-culture step performed before the step of culturing epidermal cells in a culture medium containing a candidate agent, a post-culture step of further culturing in a culture medium not containing the candidate agent after the step of culturing epidermal cells in a culture medium containing the candidate agent, a recovery step of recovering cells, and a step of storing the recovered cells, or proteins, mRNAs, or DNAs reverse-transcribed from mRNAs extracted from the recovered cells.

[0016] Laminin is a protein belonging to the laminin family and is one of the proteins that make up the basement membrane. Among the laminin family, in particular, laminin 332 composed of α3β3γ2 subunits and laminin 511 composed of α5β1γ1 subunits are known to be present in the basement membrane. Laminin is recognized by integrins expressed on cells and functions as a cell scaffold. Using the laminin present in the basement membrane as a scaffold, epidermal basal cells form layers.

[0017] Laminin 511 is a protein involved in cell adhesion and proliferation and is mainly present in the epidermal basement membrane. Laminin 511 has binding affinity with β1 integrin, especially α6β1 integrin. In cell experiments, it is involved in the proliferation of stem cells expressing β1 integrin and is used for the culture of iPS cells and ES cells. In vivo, it is known that laminin 511 decreases with aging. The inventors of the present invention have clarified that laminin 511 also decreases due to the influence of ultraviolet rays in addition to aging (Figure 1). Although not intended to be limited by theory, it is considered that ultraviolet rays activate matrix metalloproteinase (MMP), thereby reducing the amount of laminin. However, the inventors of the present invention have shown that laminin 332 is less affected by aging and ultraviolet rays, while laminin 511 decreases with aging and further decreases due to the influence of ultraviolet rays (Figure 2A). This tendency is the same as that of β1 integrin expressed in stem cells, and the same tendency was also observed in MCSP-expressing stem cells (Figure 1A).

[0018] The laminin 511 expression promoter can be interpreted in the broadest sense and can include any agent as long as it can increase the protein amount of laminin 511. Therefore, not only an agent that can simply promote the gene expression of laminin 511, but also an agent that can increase the protein amount of laminin 511 by its heparanase inhibitory action or MMP inhibitory action can be called a laminin 511 expression promoter. The expression of laminin 511 can be determined by the protein amount or the mRNA amount. Since laminin 511 is a complex protein, the laminin 511 expression promoter can promote the expression of each mRNA of the constituent subunits. By increasing the expression of laminin 511 in the epidermal basement membrane, the laminin 511 expression promoter can exert an epidermal basement membrane stabilization action, an epidermal stem cell decrease inhibitory action, and an epidermal stem cell increase promoting action. Therefore, the laminin 511 expression promoter can also be called an epidermal basement membrane stabilizer, an epidermal stem cell decrease inhibitor, or an epidermal stem cell increase promoter.

[0019] The epidermal basement membrane is present in the lowermost layer of the epidermis and constitutes the boundary between the epidermis and the dermis. The epidermal basement membrane is a thin membranous extracellular matrix mainly composed of collagen, proteoglycan, entactin, and laminin. Basal cells are arranged on the basement membrane, and the basement membrane and basal cells together are called the basal layer of the epidermis. The basement membrane mainly contains type I collagen, type IV collagen, and type VII collagen as collagen. The basement membrane mainly contains laminin 511 and laminin 332 as laminin. The basement membrane is partially decomposed and destabilized by the influence of extracellular matrix degrading enzymes such as matrix metalloproteinase and heparanase. In the destabilized basement membrane, laminin decreases, and epidermal cells that grow using laminin as a cell scaffold, such as β1 integrin-expressing cells, can be lost. Among epidermal cells, epidermal stem cells, especially epidermal basal stem cells, are considered to be β1 integrin-expressing cells, and these cells can be lost along with the decrease in laminin.

[0020] The collagen contained in the dermis has different shapes depending on its position in the dermis, and the types of collagen that make it up are also different. The epidermal basement membrane region contains type I collagen, type IV collagen, and type VII collagen. In the region immediately below the basement membrane, there are thin collagen fiber bundles called the fibroreticular lamina. As we go deeper, collagen fibrils are located, and thick collagen fibers are located in the deepest part. The fibroreticular lamina in the region immediately below the basement membrane is mainly composed of type V collagen. The collagen fibrils are composed of type III collagen and type V collagen, and the deepest collagen fibers are composed of type I and type III collagen.

[0021] Type V collagen immediately below the basement membrane is produced by dermal fibroblasts in the dermis layer. However, our research has shown that it decreases with age (Figure 12). Furthermore, surprisingly, we have found that the presence of type V collagen is closely related to the presence of the basement membrane (Figure 13A). On the other hand, the total amount of collagen production in the entire dermis layer was not affected by the addition of MMP inhibitors and heparanase inhibitors that protect the basement membrane (Figure 13B). Based on these results, we have hypothesized that some factor is secreted from the basement membrane side, activating only the dermal fibroblasts immediately below the basement membrane to produce type V collagen. To prove this hypothesis, in a three-dimensional skin model containing the dermis layer, basement membrane, and epidermal layer, the medium of a culture with MMP inhibitors and heparanase inhibitors that protect the basement membrane added to the medium was collected, and this medium was added to a fibroblast culture to examine the expression of type V collagen (Figure 14). We found that the medium obtained from the culture with MMP inhibitors and heparanase inhibitors that protect the basement membrane has an effect of promoting type V collagen expression.

[0022] The epidermis was recovered from a culture of a three-dimensional skin model supplemented with an MMP inhibitor and a heparinase inhibitor that protect the basement membrane, and when attention was paid to growth factors whose expression changed depending on the presence or absence of the addition of the inhibitors, PDGF-BB was identified as a growth factor having an effect of promoting type V collagen expression (FIG. 15). PDGFRβ, which is a receptor for PDGF-BB, was shown to be expressed in dermal fibroblasts immediately beneath the basement membrane. Although not intended to be limited by theory, from these findings, it can be explained that as the basement membrane is damaged with aging, the production amount of PDGF-BB decreases, and accordingly, the expression amount of type V collagen decreases, resulting in loss of skin firmness, an aging model. Referring to this aging model, the laminin 511 expression promoter, epidermal basement membrane stabilizer, epidermal stem cell decrease inhibitor or increase promoter identified by the present invention can also be referred to as a PDGF-BB production promoter, and can also be used as a collagen production promoter, particularly preferably a type V collagen production promoter, and can be formulated in cosmetics as a firmness improver.

[0023] An epidermal basement membrane stabilizer refers to a drug that can stabilize the epidermal basement membrane. The basement membrane stabilizer may be an inhibitor of extracellular matrix degrading enzymes that degrade the basement membrane, or may be an expression promoter of basement membrane constituent molecules. Examples of inhibitors of extracellular matrix degrading enzymes include heparanase inhibitors, and drugs such as mucrodi extract powder, cantharides extract E, citrus peel extract BG, white lily, houttuynia cordata extract, IBR, S-173, and BIBIPU are known. Also, matrix metalloproteinase (MMP) inhibitors are also included as inhibitors of extracellular matrix degrading enzymes, and drugs such as turmeric extract BG, tormentilla extract, mangostin extract BG, and CGS27023A are known. Through the stabilization of the epidermal basement membrane, the decrease in epidermal basal stem cells can be suppressed or their proliferation can be promoted. Therefore, an epidermal basement membrane stabilizer can also be referred to as an inhibitor of the decrease in epidermal basal stem cells or a promoter of their increase. An inhibitor of the decrease in or promoter of the increase in epidermal basal stem cells refers to a drug that can exert an effect of inhibiting the proliferation, decreasing, or maintaining epidermal basal stem cells. Also, through an inhibitor of the decrease in or promoter of the increase in epidermal basal stem cells, an anti-aging effect on the epidermis can be exerted. Therefore, an epidermal basement membrane stabilizer can also be referred to as an anti-skin aging agent.

[0024] The inhibitor of the decrease in or promoter of the increase in epidermal stem cells of the present invention refers to a drug that can inhibit the decrease, maintain, or promote the increase in epidermal stem cells in the skin. The number of epidermal stem cells decreases with aging and ultraviolet irradiation, but by applying the inhibitor of the decrease in or promoter of the increase in epidermal stem cells of the present invention, the decrease in epidermal stem cells can be inhibited or maintained. In another aspect, the number of epidermal stem cells can also be increased.

[0025] As the epidermal cells used in the present invention, cultured epidermal cells can be used. As the cultured epidermal cells, a three-dimensional epidermal model obtained by inducing differentiation can also be used. The epidermal cells can include keratinocytes, Langerhans cells, Merkel cells, melanocytes, and epidermal basal cells, and preferably further include epidermal stem cells. More preferably, such epidermal stem cells are preferably epidermal stem cells expressing MCSP, that is, epidermal basal stem cells. Among epidermal cells, in addition to epidermal basal stem cells, epidermal stem cells are also considered to exist in the bulge region and sebaceous glands. Among these, only epidermal basal stem cells can express MCSP. Therefore, MCSP-expressing epidermal stem cells usually refer to epidermal basal stem cells.

[0026] The epidermal cells preferably express integrin. Examples of the integrin to be expressed include α integrin and β integrin. In particular, epidermal stem cells preferably express β1 integrin. The epidermal cells may be cells derived from any animal species, but for the purpose of eliminating the influence of each species, they are preferably human cells. Human cultured cells may be derived from any source such as adults, children, infants, neonates, and fetuses, but are preferably derived from fetuses from the viewpoint of using cells containing a large number of basal stem cells.

[0027] The integrin expressed in epidermal cells refers to a molecule that exists in the cell membrane and functions as a receptor for the extracellular matrix. Integrin is composed of an α chain and a β chain. About 18 types of α chains have been found, and about 8 types of β chains have been found. β1 integrin refers to integrin in which the β chain is a β1 subunit and the α chain is an arbitrary subunit. Integrin containing the β1 subunit has a high binding affinity for laminin. Without intending to be limited by theory, it is considered that the loss of laminin 511 due to the influence of aging or ultraviolet rays results in the loss of stem cells expressing β1 integrin, and as a result, the number of MCSP-expressing stem cells decreases.

[0028] Candidate agents can be drugs included in any library of cosmetics or pharmaceuticals. As these libraries, any library such as a compound library or an extract library can be used. Among the candidate agents, by selecting an agent that can increase the expression of laminin 511, it can be screened as a laminin 511 expression promoter, an epidermal basement membrane stabilizer, an inhibitor of epidermal basal stem cell decrease or a promoter of epidermal basal stem cell increase.

[0029] The control refers to an experimental group that provides the laminin 511 expression level serving as a reference for comparison in order to determine the laminin 511 expression promoting effect of the candidate agent in the determination step. Therefore, the expression level of laminin 511 determined using cultured cells cultured under the same conditions and for the same period without only the candidate agent is provided for comparison as the expression level of the control. Such an expression level of the control may be determined by culturing the cells in parallel with the step of culturing epidermal cells in a culture medium containing the candidate agent and measuring the expression of laminin 511, or may be determined by separately culturing in advance under the same conditions and for the same period and measuring the expression of laminin 511.

[0030] When the screening method of the present invention was applied to cosmetic raw materials, algelex was selected as an agent having an effect of promoting laminin 511 production. The agent thus selected is an agent that promotes the expression of laminin 511, is also an epidermal basement membrane stabilizer, is also an inhibitor of epidermal basal stem cell decrease, and is also a promoter of epidermal basal stem cell increase.

[0031] Algirex is a cosmetic raw material sold by Ichimaru Pharcos Co., Ltd. and relates to seaweed extracts. More specifically, Algirex relates to a mixed extract of brown algae, red algae, and green algae. The whole brown algae are immersed in 50% 1,3-butylene glycol for 3 days, and the filtered brown algae extract, and the whole brown algae, red algae, and green algae are immersed in 50% 1,3-butylene glycol for 3 days, and the filtered extracts of brown algae, red algae, and green algae are mixed to obtain it. Algirex is considered to improve the moisture content and also exert an inhibitory effect against rough skin. The brown algae used in Algirex are algae of the genera Laminaria and Undaria, such as Mitsui Kombu and Undaria pinnatifida as an example. The red algae used in Algirex are algae of the genus Gracilaria, such as Eucheuma muricatum and Gracilaria verrucosa as an example. The green algae used in Algirex are algae of the genus Ulva, such as Enteromorpha intestinalis as an example.

[0032] In the present invention, the seaweed extract relates to an extract extracted from at least one alga selected from the group consisting of brown algae, red algae, and green algae. In a more preferred embodiment, the seaweed extract relates to extracts of brown algae, red algae, and green algae. Even more preferably, it is an extract of brown algae of the genus Laminaria and Undaria, red algae of the genus Gracilaria, and green algae of the genus Ulva, and most preferably, it is Algelex. The solvent used may be any solvent, and water, alcohol, ether, ester, etc. can be used alone or in combination. As the alcohol used, monohydric alcohols such as methanol, ethanol, propanol, and butanol, dihydric alcohols such as ethylene glycol, propylene glycol, and butylene glycol, and trihydric alcohols such as glycerin may be used. As the ether, dimethyl ether, diethyl ether, ethyl methyl ether, tetrahydrofuran, etc. may be used. As the ester, methyl acetate, ethyl acetate, etc. may be used. When used as a mixture, it can be used in any mixing ratio. For example, a mixture of water and 1,3-butylene glycol can be used in the range of 1:10 to 10:1, and more preferably in the range of 3:10 to 10:3. A 1:1 mixture may also be used. The seaweed extract can be formulated in cosmetics. For example, the seaweed extract can be formulated in cosmetics in an amount of 0.0001% to 10%, preferably 0.001% to 1.0%. As an example, 0.01% of the seaweed extract can be formulated in cosmetics such as skin care lotions, lotions, emulsions, creams, etc.

[0033] As a laminin 511 expression promoter, the seaweed extract of the present invention screened exhibits an effect of promoting laminin 511 expression, a basement membrane stabilization effect, and an effect of promoting epidermal stem cell proliferation or suppressing its decrease. Also, due to or independently of these effects, the seaweed extract suppresses the expression of the heparanase gene. Thereby, it exerts an effect of improving the skin barrier function. Therefore, the laminin 511 expression promoter containing the seaweed extract can also be used as an inhibitor of heparanase gene expression or an agent for improving the skin barrier function. Also, due to or independently of these effects, the seaweed extract increases the expression of the hyaluronic acid synthase 2 gene and suppresses the expression of the hyaluronidase gene. Thereby, the moisture of the skin is improved. Therefore, the laminin 511 expression promoter containing the seaweed extract can also be used as an agent for promoting hyaluronic acid increase or an agent for improving skin moisture. Also, due to or independently of these effects, the seaweed extract increases the expression of PDGF-BB. Since PDGF-BB acts on dermal fibroblasts to promote the production of collagen, especially type V collagen, the laminin 511 expression promoter containing the seaweed extract exerts an effect of improving firmness (Biomedical Engineering (2017) 55(2):97-102). Also, PDGF-BB is known to contribute to the stabilization of mesenchymal stem cells. Mesenchymal stem cells include dermal stem cells, and PDGF-BB acts on dermal stem cells to exert an effect of improving firmness. Therefore, the laminin 511 expression promoter containing the seaweed extract can also be used as an agent for promoting PDGF-BB expression or an agent for improving firmness. Also, due to or independently of these effects, the seaweed extract suppresses the expression of IL-8. Thereby, it exerts an anti-inflammatory effect. Therefore, the laminin 511 expression promoter can also be used as an inhibitor of IL-8 expression and an anti-inflammatory agent. Therefore, the seaweed extract can also be said to be an agent for improving the skin barrier, firmness, moisture, and inflammation, and can be applied to subjects suffering from or in need of improvement of the skin barrier, firmness, moisture, and inflammation.

[0034] In another aspect of the present invention, the present invention relates to a cosmetic method for improving at least one, preferably all, of skin barrier, firmness, moisture, and inflammation. Such a cosmetic method includes applying a cosmetic containing at least one extract obtained from the group consisting of brown algae, red algae, and green algae to a subject suffering from at least one, preferably all, of skin barrier, firmness, moisture, and inflammation or in need of improvement. Such a subject may have at least one, or all, of the following symptoms: 1) decrease in hyaluronic acid in the skin, 2) decrease in skin barrier function, 3) onset of inflammation, 4) decrease in the amount of fibers in the papillary layer, and 5) decrease in PDGF-BB production. In yet another aspect, a subject suffering from at least one, preferably all, of skin barrier, firmness, moisture, and inflammation or in need of improvement is also a subject suffering from symptoms such as decrease in skin moisture content, rough skin, pigmentation and skin darkening, and decrease in skin flexibility. In the cosmetic method of the present invention, an appropriate amount can be applied to the face and body surface at least once a day, preferably at least twice a day, to such a subject. A particularly preferred embodiment can be applied after bathing, before going to bed, and / or after waking up.

[0035] 1-(2-Hydroxyethyl)-2-imidazolidinone (HEI) has the following chemical formula:

Chemical formula

[0036] The promoter for laminin 511 expression, the epidermal basement membrane stabilizer, and the inhibitor of epidermal basal stem cell decrease or promoter of epidermal basal stem cell increase screened in the present invention can each be formulated into cosmetics as cosmetic materials. The cosmetics formulated with this cosmetic material can exert an anti-aging effect or an anti-UV effect on the epidermis through promoting the expression of laminin 511, through stabilizing the epidermal basement membrane, or through inhibiting the decrease or promoting the increase of epidermal basal stem cells. As cosmetics, they can be formulated into any cosmetics, for example, they can be used in skin care lotions, lotions, emulsions, creams, body milks, bath agents, sunscreens, makeup bases, makeup products, lotions, after-shaving creams, etc. They can also be formulated into pharmaceuticals, quasi-drugs, etc. Such pharmaceuticals can be administered via any route such as orally, transdermally, intramuscularly, intravenously, etc., but from the perspective of directly acting on the skin, it is preferable to administer them by transdermal administration. For transdermal administration, it is preferably formulated into a topical skin preparation, a skin patch, etc. In particular, it can be formulated into a topical skin preparation. These cosmetics and pharmaceuticals can contain drugs that can generally be added, for example, moisturizers, whitening agents, antioxidants, oily components, UV absorbers, surfactants, thickeners, alcohols, colorants, fragrances, water, solvents, preservatives, storage agents, pH adjusters, gelling agents, and other active ingredients.

[0037] Administration of HEI increased the gene expression level of filaggrin, and improvement of skin barrier function and water content was confirmed. These effects are considered to be due to the stabilization of the basement membrane by promoting the expression of laminin 511. A decrease in epidermal stem cells may be involved in the aging phenomenon of the epidermis in every aspect. Examples of epidermal aging include a decrease in moisture, rough skin, color unevenness, and a decrease in skin firmness. These aging phenomena are caused by a decrease in hyaluronic acid, a decrease in skin barrier function, inflammation induction, and a decrease in the production of papillary layer fibers and PDGF-BB, respectively. All of these aging phenomena can be solved by suppressing the decrease or promoting the increase in the number of epidermal basal stem cells. Therefore, a laminin 511 expression promoter, an epidermal basement membrane stabilizer, and an agent for suppressing the decrease or promoting the increase in epidermal basal stem cells can also be referred to as anti-aging agents having one or more actions selected from the group consisting of an action of promoting filaggrin gene expression, an action of improving skin barrier function, an action of improving water content, an action of increasing hyaluronic acid, an action of calming inflammation, and an action of promoting the production of papillary layer fibers and PDGF-BB. In addition, a laminin 511 expression promoter, an epidermal basement membrane stabilizer, and an agent for suppressing the decrease or promoting the increase in epidermal basal stem cells can also be referred to as a filaggrin gene expression promoter, a skin barrier function improver, a water content improver, a hyaluronic acid increaser, an anti-inflammatory agent, and a papillary layer fiber and PDGF-BB production promoter, respectively.

Example

[0038] Example 1: Measurement of age-related changes and the effect of ultraviolet rays on MCSP-positive epidermal basal stem cells in human skin Sample Skin samples from the face and abdomen of subjects in their 20s to 70s (20s: 9 subjects, 30s: 10 subjects, 40s: 10 subjects, 50s: 9 subjects, 60s: 10 subjects, and 70s: 9 subjects) who gave informed consent were obtained, fixed using cold acetone according to the AMex method, and embedded in paraffin.

[0039] Embedded tissues were sectioned into 3-μm thick slices, and stained using a melanoma chondroitin sulfate proteoglycan (MCSP) antibody (MAB2029, Chemicon, Billerica, MA) as the primary antibody and an Alexa488-labeled anti-mouse IgG antibody (Life technologies, Carlsbad, CA) as the secondary antibody. Nuclear staining was further performed using DAPI. Additionally, an anti-α6 integrin antibody (GOH3, Santa Cruz, Dallas, TX) was used as the primary antibody, and an Alexa594-labeled anti-rat IgG antibody (Life technologies, Carlsbad, CA) was used as the secondary antibody for staining. Visualization was performed using an Olympus BX51 microscope, and images were acquired using a DP control digital camera. The staining results of the facial and abdominal regions of subjects in their 20s and 60s are shown in Fig. 1A.

[0040] From the results in Fig. 1A, it was found that the number of MCSP-positive basal cells (hereinafter referred to as epidermal basal stem cells) present in the epidermal basal layer decreased with aging in both the facial and abdominal regions of subjects in their 20s and 60s. Also, from a comparison between the facial region (exposed area) and the abdominal region (non-exposed area) of the same subjects, it was found that the number of epidermal basal stem cells was lower in the facial region (exposed area). Additionally, in each subject, the number of epidermal basal stem cells relative to the length of the basement membrane was measured, and the average was graphed and shown (Fig. 1B).

[0041] Next, keratinocytes obtained from subjects of each age from 0 to their 60s were cultured, separated, and recovered, and mRNA was extracted and purified using an RNeasy mini kit (QIAGEN, Tokyo, Japan) according to the product manual. The expression level of the MCSP gene in keratinocytes was determined by quantitative PCR using Platinum SYBR Green qPCR super MIX-UDG (Invitrogen Japan, Tokyo Jaqpan). The primers used were as follows: [Table 1] The changes in the expression levels in each age group are shown in Fig. 1C.

[0042] From the results of Fig. 1A, it was found that aging and ultraviolet light each contributed to the decrease in the number of epidermal basal stem cells. Also, from the results of Fig. 1B and C, it was shown that with aging, the number of MCSP-positive cells increased while the expression level of MCSP decreased.

[0043] The embedded tissue was sectioned into 3-μm thick slices, and anti-laminin 332 antibody (prepared: BM165, mouse monoclonal antibody), anti-laminin 511 antibody (4C7, Abcam, Cambridge, UK), and anti-β1 integrin antibody (vendor: P5D2, Santa Cruz, CA) were used as primary antibodies, respectively, and Alexa488-labeled anti-mouse IgG antibody (vendor: Lifetechnologies, Carlsbad, CA) was used as a secondary antibody for staining. Further, nuclear staining was performed using DAPI. Visualization was carried out using an Olympus BX51 microscope, and images were acquired with a DP control digital camera. The staining results of the facial and abdominal regions of subjects in their 20s and 60s are shown in Fig. 2A.

[0044] In addition, keratinocytes obtained from subjects of each age group were isolated and collected, and mRNA was extracted and purified using an RNeasy mini kit (QIAGEN, Tokyo, Japan) according to the product manual. The expression level of the laminin α5 gene in keratinocytes was measured by quantitative PCR using Platinum SYBR Green qPCR super MIX-UDG (Invitrogen Japan, Tokyo Jaqpan). The primers used were as follows:

Table 2

[0045] Example 2: Association between laminin 511 and stem cell markers Human epidermal keratinocytes were cultured for 6 days in a Primeria flask (Coaster, Tokyo, Japan) coated with iMatrix-511 (Wako Pure Chemical Industries, Ltd.) using Humedia-KG2 medium (Kurabo Col, Ltd, Japan). As a control, cells were cultured in an uncoated flask. Cells that had grown to sub-confluence were detached with Typsin (Nakarai co, ltd, Japan), seeded onto a chamber slide (Thermo Fisher Science, Waltham, MA) coated with iMatrix-511, and cultured. The control group was cultured on an uncoated chamber slide.

[0046] After culturing, the cells were fixed with 4% PFA, stained using the MCSP antibody (MAB2029, Chemicon, Billerica, MA) as the primary antibody and the Alexa488-labeled anti-mouse IgG antibody (Life technologies, Carlsbad, CA) as the secondary antibody. Nuclear staining was further performed using DAPI. Visualization was carried out using an Olympus BX51 microscope, and images were acquired with a DP control digital camera. It was found that more MCSP-positive cells were maintained in the iMatrix511-coated group compared to the uncoated control (Figure 3A).

[0047] Next, after culturing, the medium was discarded and the cells were washed with PBS. Total RNA was extracted and purified from the collected cells using the RNeasy mini kit (QIAGEN, Tokyo, Japan) according to the manufacturer's instructions. To measure the expression levels of the genes of stem cell markers CD46, DLL1, Lrig1, and CD44, quantitative PCR was performed using Platinum SYBR Green qPCR super MIX-UDG (Invitrogen Japan, Tokyo Jaqpan). The primers used were as follows: [Table 3] Compared with the expression levels of the genes for CD46, DLL1, Lrig1, and CD44 in the control, in the laminin 511-coated group, the expression levels of the genes for CD46, DLL1, Lrig1, and CD44 were increased (Figure 3B), indicating that laminin 511 contributes to the maintenance of stem cells.

[0048] Example 3: Maintenance of the number of MCSP-positive epidermal basal stem cells by addition of a laminin degrading inhibitor Skin samples were obtained from the abdomen of subjects (aged in their 20s to 30s) who had given informed consent. The obtained samples were cultured for 4 days in a culture medium containing CGS270234 (10 μM), an MMP9 inhibitor, and BIPBIU (10 μM), a heparanase inhibitor. The culture medium used was William's E medium (Thermo Fisher Science, Waltham, MA). CGS270234 and BIPBIU are compounds represented by the following chemical formulas respectively, and are known to be usable as an MMP9 inhibitor and a heparanase inhibitor (Iriyama S, et al., Exp Dermatol. 2011; 20 (11): 953-5).

Chemical formula

[0049] Embedded tissues were sectioned into 3-μm sections, and anti-laminin 551 antibody (4C7, Abcam, Cambridge, UK) was used as the primary antibody, followed by co-staining with Alexa488-labeled anti-mouse IgG antibody (manufacturer: Life technologies, Carlsbad, CA) as the secondary antibody. Furthermore, nuclear staining was performed using DAPI. The results are shown in Fig. 4(A). Next, the embedded skin samples were co-stained using anti-MCSP antibody (MAB2029, Chemicon, Billerica, MA) as the primary antibody, followed by Alexa488-labeled anti-mouse IgG antibody (Life technologies, Carlsbad, CA) as the secondary antibody. Furthermore, nuclear staining was performed using DAPI. The results are shown in Fig. 4(B).

[0050] From the comparison between the non-cultured group (Day0) and the control drug-free group (Day4: Control), it was found that laminin 551 present in the basement membrane decreased and MCSP-positive cells also decreased after 4 days of tissue culture. On the other hand, when cultured with the addition of inhibitors of matrix metalloproteinase 9 (MMP9) and heparanase, which degrade laminin, laminin 551 was maintained and MCSP-positive cells were also maintained.

[0051] Example 4: Screening for drugs with an effect of promoting laminin 511 production Epidermal cells obtained from the fetus were cultured for 6 days in a Primaria flask (Coaster, Tokyo, Japan) coated with iMatrix-511 (Wako Pure Chemical Industries, Ltd.) using Humedia-KG2 medium (Kurabo Col, Ltd, Japan). When the cells became sub-confluent, the cells were detached with Trypsin, seeded in a 6-well plate, and replaced with a medium in which the candidate drug was dissolved 1 day later, followed by culturing for 48 hours. As candidate drugs, 178 registered raw materials for cosmetics ( breakdown: 135 extracts and 43 single compounds) were used. The medium was discarded, washed with PBS, and mRNA was extracted and purified using an RNeasy mini kit (QIAGEN, Tokyo, Japan) according to the product manual. Quantitative PCR was performed for the expression levels of the laminin α5, laminin β1, and laminin γ1 genes using Platinum SYBR Green qPCRsuper MIX-UDG (Invitrogen Japan, Tokyo Jaqpan). The primers used were as follows:

Table 4

[0052] Regarding the screened Alegerex, the drug concentration was changed to 0.001%, 0.01%, and 0.10% for testing, and the dose-dependent promoting effect on laminin 511 expression was examined. The cells, culture medium, culture conditions, and PCR conditions used were the same as those used in the drug screening method of Example 4. The results are shown in Fig. 5A. Furthermore, under the same conditions, protein quantitative analysis was performed by ELISA using an anti-laminin 511 antibody (vendor: cloud-clone corp). The results are shown in Fig. 5B.

[0053] Example 5: Verification of the skin improvement function by Alegerex Epidermal cells obtained from the fetus were cultured in a Primaria flask (Coaster, Tokyo, Japan) coated with iMatrix-511 (Wako Pure Chemical Industries, Ltd.) using Humedia-KG2 medium (Kurabo Col, Ltd, Japan). When the cells became sub-confluent, the cells were detached with Trypsin, seeded into a 6-well plate, and replaced with a medium containing 0.01% Aljetex after 1 day, followed by culturing for 48 hours. As a control, a medium without Aljetex was used. The medium was discarded, washed with PBS, and mRNA was extracted and purified using the RNeasy mini kit (QIAGEN, Tokyo, Japan) according to the product manual. The expression levels of the heparanase (HPA) gene, PDGF-BB gene, hyaluronan synthase 2 (HAS2) gene, hyaluronidase 1 (HYAL1), and interleukin 8 (IL-8) gene were determined by quantitative PCR using Platinum SYBR Green qPCR super MIX-UDG (Invitrogen Japan, Tokyo Jaqpan). The primers used were as follows: [Table 5] The changes in the expression levels of each are shown in Figures 6A to 6E. Aljetex significantly reduced the expression of the heparanase (HPA) gene (p < 0.01), significantly increased the expression of the PDGF-BB gene (p < 0.001), significantly increased the expression of the hyaluronan synthase 2 (HAS2) gene (p < 0.01), significantly reduced the expression of the hyaluronidase 1 (HYAL1) gene (p < 0.01), and reduced the expression of the interleukin 8 (IL-8) gene (p < 0.05) in cultured epidermal cells.

[0054] Example 6: Inhibitory effect on laminin degradation of HEI and maintenance effect on MCSP-positive epidermal basal stem cells Abdominal skin samples were obtained from subjects (aged in their 20s to 30s) who gave informed consent. The obtained samples were cultured for 5 days in a culture medium containing 0.01% 1-(2-hydroxyethyl)-2-imidazolidinone (HEI, or S-173). William's E medium (Thermo Fisher Science, Waltham, MA) was used as the culture medium. The control was cultured using a culture medium without the addition of HEI. The cultured skin samples were fixed using cold acetone according to the AMex method and then embedded in paraffin.

[0055] The embedded tissue was sectioned into 3-μm sections, and stained using the MCSP antibody (MAB2029, Chemicon, Billerica, MA) as the primary antibody and Alexa488-labeled anti-mouse IgG antibody (Life technologies, Carlsbad, CA) as the secondary antibody. Further nuclear staining was performed using DAPI, and further stained using the anti-laminin 551 antibody (4C7, Abcam, Cambridge, UK) as the primary antibody and Alexa488-labeled anti-mouse IgG antibody (Life technologies, Carlsbad, CA) as the secondary antibody. Visualization was performed using an Olympus BX51 microscope, and images were acquired with a DP control digital camera and shown in Fig. 7A. By performing the culture, in the control, laminin 511 decreased, and furthermore, the number of MCSP-positive cells also decreased. On the other hand, when cultured with the addition of 0.01% HEI, the amount of laminin 511 could be maintained, and the number of MCSP-positive cells on the basement membrane could also be maintained.

[0056] Example 7: Heparanase inhibitory effect of HEI The heparanase assay was performed using a heparan sulfate immobilized plate described in Behzad F, et al., Analytical Biochemistry, 320, pp.207-213, 2003. Specifically, heparan sulfate (Sieikagaku, Tokyo, Japan) was reacted with Photo-biotin to prepare biotinylated heparan sulfate. The biotinylated heparan sulfate was immobilized on a carbohydrate-binding surface plate (Coastar, Tokyo, Japan). 0% HEI, 0.0005% HEI, 0.005% HEI, and 0.05% HEI were added onto the biotinylated heparan sulfate immobilized plate together with A431 cell lysate (50 μg / ml), incubated at 37°C for 3 hours, and washed with PBS-T. Then, it was incubated with peroxidase-avidin (Vector Laboratories, Inc. CA, USA) at 37°C for 1 hour. A 3,3’,5,5’-tetramethylbenzidine (TMB) solution (Bio-Rad, Tokyo, Japan) was added, and the plate was further incubated at room temperature for 30 minutes. The absorbance at 450 nm was measured to determine the inhibitory effect on heparanase. HEI was shown to inhibit the activity of heparanase in a dose-dependent manner (Figure 8).

[0057] Example 8: MMP9 inhibitory effect of HEI The MMP9 assay was performed using a Matrix Metalloproteinase-9 (MMP-9) colorimetric drug discovery kit (Enzo life sience Inc.). Specifically, HEI was added to a solution of recombinant human MMP-9 enzyme to concentrations of 0.0001%, 0.001%, and 0.01%. After mixing the chromogenic MMP-9 substrate, the absorbance at 412 nm was measured every minute. The inhibition rate (%) of MMP-9 by HEI was calculated from the slope of the absorbance (OD / min). HEI was shown to inhibit the activity of MMP9 in a dose-dependent manner (Figure 9).

[0058] Example 9: Epidermal improvement effect of HEI Skin samples were obtained from the abdomen of informed consent subjects (aged in their 20s to 30s). The obtained samples were cultured in a culture medium containing 0.01% HEI for 5 days. William's E medium (Thermo Fisher Science, Waltham, MA) was used as the culture medium. As a control, culture was performed using a culture medium without the addition of HEI. After wiping off the excess moisture adhering to the cultured skin samples with a Kim towel and leaving them to stand for 5 minutes, TEWL was measured using a Vapometer, and the stratum corneum moisture content was measured using a Corneometer. The results are shown in Figures 10B and C. Also, the cultured skin samples were fixed using cold acetone according to the AMex method and then embedded in paraffin.

[0059] The embedded tissue was cut into 3-μm sections, and using a filaggrin antibody (vendor: AKH-1, Santa Cruz Biotechnology, Dallas, TX) as the primary antibody and an Alexa488-labeled anti-mouse IgG antibody (Life technologies, Carlsbad, CA) as the secondary antibody, staining was performed. Further, nuclear staining was performed using DAPI. Visualization was performed using an Olympus BX51 microscope, and the images were acquired with a DP control digital camera and shown in Figure 10A.

[0060] Next, for 20 men aged 20 to 50 years, a 1.5% HEI aqueous solution was applied to one side of the face and water was applied to the other side of the face, twice a day for 4 weeks. At the time points before application, 2 weeks after application, and 4 weeks after application, TEWL was measured using a Vapometer, and the stratum corneum moisture content was measured using a Corneometer. The results are shown in Figures 10D and E.

[0061] In the HEI addition group, in the ex vivo experiment, a tendency for an increase in filaggrin was observed. And in both the ex vivo experiment and the in vivo experiment, since TEWL decreased, the skin barrier function was improved, and the stratum corneum moisture content also increased.

[0062] Example 10: Changes in Collagen Beneath the Basement Membrane by the Addition of an MMP Inhibitor and a Heparanase Inhibitor Skin tissue purchased from Bio Predic was used as skin tissue pieces and cultured in William's E medium supplemented with CGS27023A (final concentration 10 -5 M) and BIPBIPU (final concentration 10 -5 M). In the control, the culture was performed without these inhibitors. The medium was changed daily, and the skin tissue pieces were collected on the 5th day of culture. The collected skin tissue pieces were immersed in Zamboni fixative and post-fixed with 1% osmium. The skin samples were embedded in epoxy resin. Ultrathin sections were prepared and observed with a transmission electron microscope (JEOL JEM1230) (Figure 11A).

[0063] Six skin sections from each group were observed, and 30 photographs were taken from one section. The size and number of collagen fibers present in the dermis of all the photographs were calculated using the image analysis software "win ROOF 2013" to create a histogram (Figure 11B). Similarly, using the image analysis software "win ROOF 2013", the area of the dermis was also extracted, and the density of collagen fibers in the dermis and the thickness per fiber were calculated to derive the average values (Figure 11C).

[0064] Example 11: Expression of Type V Collagen Skin purchased from Bio Predic was subjected to the AMeX method to create paraffin blocks. Sections were prepared at a thickness of 3 μm, and fluorescence immunostaining was performed using an antibody against type V collagen (ORIGENE, Cat#: AM1015PU-N) and an antibody against cytokeratin 14 (K-14) (Fitzgerald, Cat#; 20R-CP002) (Figure 12A). It was shown that the basement membrane region was stained by cytokeratin 14, and type V collagen was present directly beneath it, and its amount decreased with aging.

[0065] Normal human epidermal cells and normal human fibroblasts were purchased from Kurabo and Bio Predic. Six specimens obtained from subjects in their 20s to 30s and six specimens obtained from subjects in their 50s to 60s were purchased respectively. The epidermal cells were cultured in serum-free medium Humedia-KG2, and the fibroblasts were cultured in DMEM medium containing 10% serum. After one passage, each sample was seeded in a 6-well plate. After becoming confluent, each cell was collected, cDNA was prepared, and the gene expression level of the gene COL5A1 of type V collagen was evaluated by real-time PCR using the following primers (Figure 12B). Type V collagen was shown not to be expressed from epidermal cells, but to be expressed from fibroblasts, and its expression level was shown to decrease with aging.

Table 6

[0066] The skin tissue purchased from Bio Predic was made into skin tissue pieces and cultured in William's E medium supplemented with CGS27023A (final concentration 10 -5 M) and BIPBIPU (final concentration 10 -5 M). In the control, the culture was carried out without these inhibitors. The medium was changed daily, and the skin tissue pieces were collected on the 5th day of culture. The collected skin tissue pieces were subjected to the AMeX method to prepare paraffin blocks. Sections were made at a thickness of 3 μm, and fluorescence immunostaining was performed using an antibody against type V collagen (ORIGENE, Cat#: AM1015PU-N) and an antibody against cytokeratin 14 (K-14) (Fitzgerald, Cat#; 20R-CP002) (Figure 13A).

[0067] The skin three-dimensional model (EFT-400) purchased from MatTeK was treated with CGS27023A (final concentration 10 -5 M), BIPBIPU (final concentration 10 -5They were cultured in a dedicated medium (EFT400-ASY) supplemented with -5 M) of CGS27023A and -5 M) of BIPBIPU. As a control, they were cultured without including these inhibitors. The medium was changed once every two days, and on the seventh day, the tissue pieces were collected and subjected to the AMeX method to create paraffin blocks. Sections were made at a thickness of 3 μm, and fluorescence immunostaining was performed using an antibody against type V collagen (ORIGENE, Cat#: AM1015PU-N) and an antibody against cytokeratin 14 (K-14) (Fitzgerald, Cat#; 20R-CP002) (Figure 13A).

[0068] The skin three-dimensional models were cultured for 4 days and 7 days in both the group with the addition of inhibitors and the non-addition (control) group. After culturing, the epidermis was removed, and only the dermis was placed in Trizol to extract mRNA. cDNA was prepared immediately, and using the above-mentioned primers, the gene expression level of the gene COL5A1 of type V collagen was evaluated by real-time PCR (Figure 13B).

[0069] In both the organ culture of skin tissue and the skin three-dimensional model, compared with the control, the expression of type V collagen increased when MMP inhibitors and heparinase inhibitors were added. On the other hand, in the entire dermis layer, no significant change was observed in the expression of type V collagen due to the addition of MMP inhibitors and heparinase inhibitors.

[0070] In the TEM image of Example 10, attention was paid to the fibroblasts present directly under the basement membrane (Figure 13C). In the control (non-inhibitor addition group) of Figure 13C, collagen fibers were not observed much in the fibroblasts, while in the inhibitor addition group, collagen fibers were observed in the vesicles within the fibroblasts. This vesicle is suggested to be the collagen secretion process. This suggests that the fibroblasts directly under the basement membrane actively produce collagen due to the addition of MMP inhibitors and heparinase inhibitors.

[0071] The skin three-dimensional model (EFT-400) purchased from MatTeK Corporation was used with CGS27023A (final concentration 10 -5 M), BIPBIPU (final concentration 10 -5It was cultured in a dedicated medium (EFT400-ASY) supplemented with CGS27023A (final concentration 10

[0072] Example 12: Search for factors that promote the expression of type V collagen M), BIPBIPU (final concentration 10 -5 M). As a control, it was cultured without these inhibitors. Culture supernatants were collected on the 2nd, 4th, and 7th days of culture. Separately, normal human fibroblasts derived from skin collected from a 14-month-old child purchased from Bio Predic were added to a culture that had been cultured in DMEM supplemented with 10% FBS for 24 hours, and the culture supernatant collected from the culture of the skin three-dimensional model was added. The cells were collected 1 day after the addition, and mRNA was extracted using Trizol. cDNA was prepared immediately, and using the above-mentioned primers, the gene expression level of the gene COL5A1 of type V collagen was evaluated by real-time PCR (Figure 14). The addition of the culture supernatant of the skin three-dimensional model significantly increased the expression of type V collagen. This suggested that the culture supernatant of the skin three-dimensional model contains factors that promote the production of type V collagen. -5 The skin three-dimensional model (EFT-400) purchased from MatTeK was cultured in a dedicated medium (EFT400-ASY) supplemented with CGS27023A (final concentration 10

[0073] M), BIPBIPU (final concentration 10

[0073] M). On the 2nd, 4th, and 7th days of culture, the epidermis was collected from the skin model, and a protein extract was prepared using RIPA buffer manufactured by Nacalai. Using a membrane array kit (ab134002) manufactured by Abcam, the amounts of various cytokines in the protein extract were detected using LAS-1000UVmini manufactured by Fujifilm, and were quantified using the attached software to create a graph (data not shown). Cytokines with increased amounts compared to the control were selected as candidate cytokines that promote type V collagen expression. As such candidate cytokines, three cytokines including PDGF-BB were selected.Recombinant cytokine proteins were purchased from R&D. Normal human fibroblasts derived from the skin of a 14-month-old child were cultured in DMEM supplemented with 10% FBS for 24 hours, and then the culture medium was replaced with DMEM medium containing 25% serum so that each cytokine reached a predetermined concentration, and the cells were further cultured for 24 hours. After culturing, the cells were collected. Then, mRNA was extracted using Trizol. cDNA was immediately prepared, and the gene expression level of the gene COL5A1 of type V collagen was evaluated by real-time PCR using the above primers (Figure 15). Among the three cytokines, only PDGF-BB significantly increased the production of type V collagen. Thus, it is considered that PDGF-BB is a cytokine produced from the epidermal side and acts on fibroblasts directly below the basement membrane to promote the expression of type V collagen.

[0074] Example 13: Action site of PDGF-BB and changes in PDGF-BB expression Paraffin blocks obtained in the same manner as in Example 11 were sectioned at a thickness of 3 μm, and fluorescence immunostaining was performed using an antibody against PDGFR-β (R&D Systems, Cat#; MAB1263) and an antibody against cytokeratin 14 (K-14) (Fitzgerald, Cat#; 20R-CP002) (Figure 16A). It was found that there were many cells expressing PDGFR-β directly below the basement membrane regardless of age. This confirmed that PDGF-BB secreted from the basement membrane or the epidermal side can act on fibroblasts directly below the basement membrane.

[0075] Normal human epidermal cells of each age purchased from KAC and Bio Predic were cultured, and mRNA was extracted. cDNA was immediately prepared, and the gene expression level of PDGFB was evaluated by real-time PCR using the following primers (Figure 16B).

Table 7

Claims

1. An inhibitor for suppressing the decrease in the protein amount of laminin 511 in the epidermal basement membrane, which contains 1-(2-hydroxyethyl)-2-imidazolidinone as an active ingredient.

2. An inhibitor for suppressing the decrease in epidermal stem cells or a promoter for promoting the increase thereof, which contains 1-(2-hydroxyethyl)-2-imidazolidinone as an active ingredient and suppresses the decrease in the protein amount of laminin 511.

3. An inhibitor for inhibiting the activity of matrix metalloproteinase, which contains 1-(2-hydroxyethyl)-2-imidazolidinone.

4. The inhibitor for inhibiting the activity according to Claim 3, wherein the matrix metalloproteinase is matrix metalloproteinase 9.

5. An inhibitor for suppressing the decrease in the protein amount of laminin 511 in the epidermal basement membrane, which contains the inhibitor for inhibiting the activity of matrix metalloproteinase according to Claim 3 or 4.

6. An inhibitor for suppressing the decrease in epidermal stem cells or a promoter for promoting the increase thereof, which contains the inhibitor for inhibiting the activity of matrix metalloproteinase according to Claim 3 or 4.

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