Screening method for substances that reduce oxidative stress

Abstract

To provide a screening method for an oxidative stress-reducing substance.SOLUTION: The present invention discloses a screening method for an oxidative stress-reducing substance by using, as an index, a reduction in the expression level of at least one of genes to be expressed immediately in response to oxidative stress, such as HSPA1A, IL-8, and PTGS2.SELECTED DRAWING: Figure 7

Description

[Technical Field]

[0001] The present invention relates to a method for screening for substances that reduce oxidative stress. [Background technology]

[0002] Living organisms are equipped with an oxidative system necessary for energy production and an antioxidant system to prevent the adverse effects of excessive oxidation by the oxidative system, and their homeostasis maintains the health of the living organism. When the balance between oxidation and antioxidant is disrupted and oxidation becomes excessive, this condition is called oxidative stress.

[0003] Skin is the outermost organ of the body and is constantly exposed to various stimuli, including ultraviolet light and air pollutants. UV exposure, one of these stimuli, is known to cause morphological changes in the skin, and oxidative stress has been shown to play an important role in these morphological changes. Oxidative stress is known to be induced by reactive oxygen species (ROS), which have been reported to induce DNA damage and cell death in cells, as well as promote melanin production and degeneration of the dermal matrix, thereby accelerating skin aging (Non-Patent Document 1).

[0004] In developing topical skin preparations for the purposes of skin protection and anti-aging, the changes that occur within living epidermal cells as a result of signals transmitted from stimuli occurring on the stratum corneum surface to living epidermal cells are an important issue, but there is little knowledge about this issue and many aspects remain unclear. Therefore, the present inventors focused on squalene, a component of sebum, in order to clarify the changes that occur within living epidermal cells.

[0005] Squalene is a substance that is thought to function as a radical scavenger and protect the skin from oxidative stress (Non-Patent Document 2). However, it is known that squalene is easily oxidized by exposure to ultraviolet light, etc., to produce the peroxide squalene peroxide. It has been suggested that squalene peroxide induces a radical chain reaction in cell membranes, ultimately causing the formation of age spots and wrinkles (Non-Patent Documents 3-5). However, the changes that occur within living epidermal cells as a result of squalene peroxide contacting the stratum corneum and transmitting signals into the cells have not been clarified. [Prior art documents] [Patent documents]

[0006] [Non-Patent Document 1] J. Kruk, E. Duchnik, Asian Pac. J. Cancer Prev., 15, 561-568(2014) [Non-patent document 2] G.Kelly, Altern. Med.Rev.4,1,29-36(1999) [Non-patent document 3] T.Uchino, H.Tokunaga, H.Onodera, M.Ando, ​​Biol.Pharm.Bull., 25,605-610(2002) [Non-patent document 4] K.Chiba, K.Kawakami, T.Sone, M.Onoue, Skin Pharmacol.Appl.Skin Physiol.,16.242-251(2003) [Non-patent document 5] A.Ryu, K.Arakane, C.Koide, H.Arai, T.Nagano, Biol.Pharm.Bull.,32,1504-1509(2009) Summary of the Invention [Problem to be solved by the invention]

[0007] In light of the above circumstances, the present inventors have conducted extensive research and have demonstrated that application of squalene peroxide to the stratum corneum results in an immediate increase in the expression of specific genes in living epidermal cells, indicating that oxidative stress induced by squalene peroxide immediately increases the expression of specific genes.

[0008] Specifically, the inventors demonstrated that when a sample prepared by irradiating squalene with ultraviolet light to generate squalene peroxide was applied to the stratum corneum side of a three-dimensional cultured epidermal model (hereinafter referred to as RHE), the expression level of a specific gene immediately increased in living epidermal cells. Based on this finding, the inventors have invented a method for screening substances that reduce oxidative stress by using changes in the expression of the specific gene as an indicator.

[0009] An object of the present invention is to provide a screening method for substances that reduce oxidative stress, which was discovered in the process of clarifying the changes that occur in living epidermal cells. [Means for solving the problem]

[0010] That is, the present invention is as follows. (1) the gene whose expression level immediately increases in response to squalene peroxide is a gene expressed in living epidermal cells and is at least one of HSPA1A, IL-8, and PTGS2; In a cultured epidermal model, Using the reduction in expression level as an indicator, For squalene peroxide A method for screening substances that reduce oxidative stress. (2) (1) In the expression of the gene, The expression level of the gene in a cultured epidermal model to which a candidate substance for an oxidative stress-reducing substance has been applied is When the expression level of the gene is reduced compared to that in a cultured epidermal model to which the candidate substance is not applied, determining that the candidate substance has an oxidative stress-reducing effect; (1) for the squalene peroxide A method for screening substances that reduce oxidative stress. [Effects of the Invention]

[0011] The present invention can provide a method for screening for substances that reduce oxidative stress. [Brief explanation of the drawings]

[0012] [Figure 1] This figure shows the results of measuring the POV (meq / kg) of squalene and UV-irradiated squalene (1 h, 3 h, 6 h). Sq: squalene. UV-Sq (1 h, 3 h, 6 h): squalene irradiated with UV for a specified time (1 h, 3 h, 6 h). [Figure 2] FIG. 1 shows the expression levels of each mRNA in living epidermal cells measured 4 hours after application of squalene and UV-irradiated squalene (1 h, 3 h, 6 h) to RHE. [Figure 3] FIG. 1 shows the results of measuring the viability of living epidermal cells 4 hours after applying squalene and UV-irradiated squalene (1 h, 3 h, 6 h) to RHE. [Figure 4] This figure shows the time course of changes in the expression levels of each mRNA related to oxidative stress and inflammation after applying UV-irradiated squalene (3 h) to RHE. [Figure 5] This figure shows the time course of changes in the expression level of each mRNA related to keratinization after applying UV-irradiated squalene (3 h) to RHE. [Figure 6] This figure shows the results of measuring POV (meq / kg) after mixing hydroxyapatite with squalene and UV-irradiated squalene (3h). Sq+(1%, 5%) HAP: Samples in which hydroxyapatite was added to squalene at a specified concentration (1%, 5%). UV-Sq(3h)+(1%, 5%) HAP: Samples in which hydroxyapatite was added to squalene after 3 hours of UV irradiation. [Figure 7] This figure shows the results of measuring the expression level of each mRNA in living epidermal cells 4 hours after applying the supernatant (reaction product) obtained by mixing hydroxyapatite with squalene and UV-irradiated squalene (3 h) to RHE. DETAILED DESCRIPTION OF THE INVENTION

[0013] An embodiment of the present invention will be described below.

[0014] "Elucidation of genes that respond immediately to oxidative stress"

[0015] In the present invention, oxidative stress refers to a state in which the balance between oxidation and antioxidant activity is disrupted, resulting in excessive oxidation (hereinafter referred to as an "overoxidation state"). An overoxidation state is caused by an increase in the amount of intracellular reactive oxygen species (hereinafter referred to as "ROS"). When the amount of ROS increases, the balance between oxidation and antioxidant activity is disrupted, resulting in excessive oxidation, which can cause cell damage, such as the oxidation of membrane lipids. Examples of ROS include superoxide anions, hydroxyl radicals, and peroxides.

[0016] Cells in an over-oxidized state due to ROS and other factors induce radical chain reactions, resulting in cell damage such as DNA damage, enzyme inactivation, and oxidation of membrane lipids. When cell damage occurs, various proteins are produced within the cell to repair damaged proteins and promote inflammation.

[0017] Previous reports have suggested that squalene peroxide, a peroxide of squalene, oxidizes membrane lipids and causes the formation of age spots and wrinkles. However, it was not clear which genes are expressed when squalene peroxide comes into contact with the skin.

[0018] Therefore, the inventors applied squalene peroxide to RHE to generate oxidative stress and measured the expression levels of genes expressed in living epidermal cells. As a result, they found that the expression levels of the genes encoding heat shock protein 70, interleukin 8, cyclooxygenase-2, heme oxygenase 1, and γ-glutamylcysteine ​​synthetase were immediately increased.

[0019] Heat shock protein 70 acts as a molecular chaperone, enabling other proteins to fold correctly and acquire their functions, and is involved in the repair of damaged cells. The gene encoding heat shock protein 70 is called heat shock protein family A (Hsp70) member 1A and its symbol is HSPA1A. Here, "the expression level of the gene encoding heat shock protein 70 increases immediately" means that the amount of mRNA transcribed from DNA encoding heat shock protein 70 in the cell increases immediately and significantly compared to the amount present in the absence of squalene peroxide.

[0020] Interleukin 8 is a low molecular weight protein that transmits signals that cause inflammation. It is known that the secretion of interleukin 8 increases due to oxidative stress, and it functions to recruit inflammatory cells and promote further inflammation. The name of the gene that encodes interleukin 8 is interleukin 8, and the gene symbol is IL-8. Here, "the expression level of the gene encoding interleukin 8 increases immediately" means that the amount of mRNA present in the cell, transcribed from the DNA encoding interleukin 8, immediately increases significantly compared to the amount present in the absence of squalene peroxide.

[0021] Cyclooxygenase-2 is an enzyme involved in the process of metabolizing arachidonic acid into a group of physiologically active substances called prostanoids. Cyclooxygenase-2 expression is induced during inflammation, and the production of PGE2 and PGI2 is enhanced via cyclooxygenase-2. PGE2 is involved in increased vascular permeability, vasodilation, and pain, while PGI2 is involved in vasodilation, pain, and the progression of the inflammatory response. The gene encoding cyclooxygenase-2 is called prostaglandin-endoperoxide synthase 2 and its symbol is PTGS2. Here, "an immediate increase in the expression level of the gene encoding cyclooxygenase-2" refers to an immediate and significant increase in the abundance of mRNA transcribed from DNA encoding cyclooxygenase-2 in cells compared to the abundance in the absence of squalene peroxide.

[0022] Heme oxygenase 1 is an enzyme that breaks down heme into bilverdin, carbon monoxide, and free iron. Bilverdin is further broken down into bilirubin. Bilverdin and bilirubin, produced by heme breakdown, have strong antioxidant properties and suppress cell damage caused by oxidative stress. Carbon monoxide also regulates the perfusion pressure of the peripheral circulation. The gene encoding heme oxygenase 1 is called heme oxygenase 1 and its symbol is HMOX1. Here, "an immediate increase in the expression level of the gene encoding heme oxygenase 1" means that the amount of mRNA transcribed from the DNA encoding heme oxygenase 1 in the cell immediately increases significantly compared to the amount present in the absence of squalene peroxide.

[0023] γ-Glutamylcysteine ​​synthetase is an enzyme that catalyzes the rate-limiting reaction in glutathione synthesis. Glutathione is an antioxidant present in high concentrations within cells and plays a role in protecting cells from reactive oxygen species and free radicals. The gene encoding γ-glutamylcysteine ​​synthetase is called glutamate-cysteine ​​ligase catalytic subunit and its symbol is GCLC. Here, "the expression level of the gene encoding γ-glutamylcysteine ​​synthetase increases immediately and significantly" means that the amount of mRNA present in the cell, transcribed from the DNA encoding γ-glutamylcysteine ​​synthetase, increases immediately compared to the amount present in the absence of squalene peroxide.

[0024] "Method for screening substances that reduce oxidative stress" As described above, the present inventors have identified genes that are immediately expressed in response to oxidative stress. The present invention applies this finding to a screening method for selecting substances that reduce oxidative stress, using as an index the reduction in the expression level of genes whose expression level is immediately increased in response to oxidative stress.

[0025] Specifically, this is a screening method for oxidative stress-reducing substances, in which, in the expression of a gene whose expression level immediately increases in response to oxidative stress, if the expression level of the gene in a cultured epidermal model to which a candidate substance for an oxidative stress-reducing substance has been applied is reduced compared to the expression level of the gene in a cultured epidermal model to which the candidate substance has not been applied, the candidate substance is determined to have an oxidative stress-reducing effect.

[0026] More specifically, this is a method for screening for oxidative stress-reducing substances, in which the expression level of a gene (at least one of HSPA1A, IL-8, PTGS2, HMOX1, and GCLC) that is immediately expressed when a candidate substance for an oxidative stress-reducing substance is applied to a cultured epidermal model together with ROS is reduced compared to the expression level of a gene (at least one of HSPA1A, IL-8, PTGS2, HMOX1, and GCLC) that is immediately expressed when ROS is applied to the cultured epidermal model, and the candidate substance is determined to have an oxidative stress-reducing effect.

[0027] In the present invention, there is no particular limitation on the substance that causes oxidative stress in living epidermal cells, and examples of such substances that can be used include ROS, preferably peroxides, more preferably lipid peroxides, and even more preferably squalene peroxide, which is a peroxide of squalene.

[0028] The cultured epidermal model used in the screening method of the present invention is not particularly limited as long as it produces similar effects. For example, RHE, in which normal human epidermal cells are layer-cultured, can be used, and LabCyte EPI-MODEL (Japan Tissue Engineering), EPI-200 (Kurabo), EPISKIN (SkinEthic), etc. can be used. Regarding cell culture conditions, in addition to conventional culture conditions, any culture conditions that do not interfere with the implementation of the screening method of the present invention can be used without any particular limitation.

[0029] The method for evaluating the expression level of a gene in the present invention is not particularly limited as long as it can determine that the expression level of the gene is fluctuating. For example, in addition to a method of measuring the expression level of mRNA, evaluation may also be performed using the amount of protein, which is the gene expression product, as an indicator.

[0030] Genes and proteins can be extracted from the cultured epidermal model using known methods. For example, RNeasy Mini Kit (Qiagen) can be used for gene extraction, and the extracted total RNA can be further purified to mRNA only, if necessary.

[0031] In the present invention, the gene expression level can be confirmed by a known method. For example, when the gene expression level is measured as the amount of mRNA as a transcription product, any analytical method known in the art for detecting and measuring the expression of a specific gene can be used according to known genetic engineering and molecular biology techniques, such as in situ hybridization, Northern blotting, dot blotting, RNase protection assay, RT-PCR, qRT-PCR, etc.

[0032] Alternatively, when quantifying the amount of protein, which is the expression product of a gene, as another method for determining changes in gene expression, any analytical method known in the art, such as a method known in the art for detecting the amount of a specific protein, for example, Western blotting or various immunohistological methods, can be used in accordance with known genetic engineering and molecular biology techniques.

[0033] Here, "applying a candidate substance for an oxidative stress-reducing substance together with ROS to a cultured epidermal model" does not only mean "applying the candidate substance and ROS together to a cultured epidermal model," but also includes "applying a mixture of the candidate substance and ROS to a cultured epidermal model," "applying a reaction product of the candidate substance and ROS to a cultured epidermal model," "applying the candidate substance to a cultured epidermal model, followed by further application of ROS," and "applying ROS to a cultured epidermal model, followed by further application of the candidate substance." Furthermore, methods are not limited to these, and any method that can be used as a screening method for oxidative stress-reducing substances can be used.

[0034] Here, "instantaneously" means that the reaction occurs within 6 hours after contact with peroxide. "Instantly expressed gene" refers to a gene that is expressed within 6 hours after ROS is applied to a cultured epidermal model. The "instantly expressed gene" is not particularly limited as long as it is an instantly expressed gene, but is preferably at least one of HSPA1A, IL-8, PTGS2, HMOX1, and GCLC, and more preferably at least one of HSPA1A, IL-8, and PTGS2. Because the screening method of the present invention targets instantly expressed genes, it can be carried out quickly from the start of the test to the end of the determination, thereby advantageously shortening the time required to select candidate substances.

[0035] Furthermore, "reducing the expression level of a gene" means that the expression level of the gene is reduced when the candidate substance is applied to a cultured epidermal model together with ROS and when ROS is applied to the cultured epidermal model. The degree of reduction can be set appropriately depending on the degree of effect desired. Furthermore, "reducing the expression level of a gene" is not particularly limited as long as it is reduced as a measured value, but it is preferably reduced statistically significantly, taking into account general variability in tests. The same can be said when measuring "protein expression level."

[0036] In the present invention, "using as an index" means that the amount of change in real-time gene expression is used as an index, and the "change" in "amount of change" includes an increase, decrease, or no change in the amount of expression. The same can be said when "the amount of protein expression" is used as an index.

[0037] "Oxidative stress reducing substances" In the present invention, the term "oxidative stress-reducing substance" refers to a substance that prevents hyperoxidation or reduces or diminishes hyperoxidation. The term "candidate substance for oxidative stress-reducing substance" refers to a substance that is considered to have an oxidative stress-reducing effect and is a candidate for an oxidative stress-reducing substance. The oxidative stress-reducing substance and candidate substance for oxidative stress-reducing substance are not particularly limited. Examples of candidate substances include animal and plant extracts, fungal cultures or their enzyme-treated products, compounds, or derivatives thereof. These substances may be in liquid, powder, gel, or other forms. Furthermore, if a substance is insoluble in the culture medium, it can be dissolved by appropriately using a solubilizing agent such as a surfactant and used as a candidate substance. Furthermore, the extraction method is not particularly limited. Any concentration of the candidate substance can be added as long as no obvious cell death occurs 24 hours after addition of the candidate substance, such as an extract. Methods for coexisting the candidate substance with the cultured epidermal model include adding the candidate substance to the culture medium side or the stratum corneum side of the cultured epidermal model.

[0038] The oxidative stress-reducing substances selected by the screening method of the present invention have the effect of preventing or reducing hyperoxidation. More specifically, they have the effect of preventing, reducing, or diminishing cellular damage such as DNA damage, enzyme inactivation, and membrane lipid oxidation. Prevention of cellular damage, etc., has the effect of suppressing the expression of various proteins that repair damaged proteins or promote inflammation. Furthermore, they have the effect of suppressing the expression of at least one of the genes HSPA1A, IL-8, PTGS2, HMOX1, and GCLC.

[0039] In short, by applying the oxidative stress-reducing substance of the present invention to the skin, it is possible to prevent, reduce, or diminish the hyperoxidative state at the administration site, and to prevent the associated cell damage, thereby suppressing the expression level of proteins that repair proteins and promote inflammation, and further suppressing the expression level of at least one of the genes HSPA1A, IL-8, PTGS2, HMOX1, and GCLC.

[0040] Furthermore, the oxidative stress-reducing substance of the present invention not only has the effect of improving an overoxidation state and returning it to a normal state, but also has the effect of improving it from the normal state to an even better state.Furthermore, the oxidative stress-reducing substance of the present invention also has the effect of preventing an overoxidation state, so that if the overoxidation state is not yet reached but there is a possibility that it may occur, applying the substance in advance can prevent the overoxidation state from occurring.

[0041] Furthermore, the oxidative stress-reducing substance of the present invention may be formulated so as to be applicable to the skin, as long as the effects of the present invention are not impaired.

[0042] "External skin preparations" The external skin preparation in the present invention refers to a formulation prepared by blending an oxidative stress-reducing substance with a drug, quasi-drug, cosmetic, or the like.

[0043] The dosage form of the topical skin preparation is not particularly limited as long as it does not impair the effect of the oxidative stress-reducing substance, and any dosage form can be used. The type of dosage form is not particularly limited, and examples include semi-solids, liquids, and solids such as ointments, gels, creams, packs, patches, emulsions, lotions, skin lotions, and powders.

[0044] The topical skin preparation of the present invention is not particularly limited as long as it does not impair the effect of the oxidative stress-reducing substance, and additives commonly used in the fields of pharmaceuticals, quasi-drugs, cosmetics, etc. can be appropriately blended. The additives are not particularly limited, and examples thereof include purified water, oily components, surfactants, emulsifiers, lower alcohols, higher alcohols, polyhydric alcohols, water-soluble polymers, viscosity modifiers, gelling agents, moisturizers, disinfectants, anti-inflammatory agents, analgesics, antifungal agents, keratin softening and exfoliating agents, skin colorants, hormones, ultraviolet absorbers, sweat deodorants, vitamins, blood flow promoters (vasodilators, blood circulation promoters), herbal medicines, pH adjusters, sequestering agents, pearlizing agents, natural fragrances, synthetic fragrances, colorants, pigments, antioxidants, preservatives, and perfume oils. [Example]

[0045] The present invention will be explained in more detail below by showing examples, but the present invention is not limited to these examples.

[0046] "Method for screening substances that reduce oxidative stress" The screening method for substances that reduce oxidative stress was carried out as follows.

[0047] 1. Method 1-1. Test sample As an example of a candidate substance for reducing oxidative stress, "plate-shaped HAP" from Taihei Chemical Industry Co., Ltd. was used. The squalene used was "Squalene" manufactured by Fujifilm Wako Pure Chemical Industries, Ltd. UV irradiation Squalene is exposed to UV (285-400 nm, 60 W / m 2 ) was obtained by irradiating squalene for a given period of time. Hydroxyapatite-treated UV-irradiated squalene was obtained by mixing hydroxyapatite and UV-irradiated squalene, reacting them for a predetermined time, separating them using a centrifuge, and then collecting the supernatant (reaction product).

[0048] 1-2.Peroxide Value (POV) Measurement The test was conducted in accordance with "Standards and Criteria for Foods, Food Additives, etc. (Ministry of Health and Welfare Notification No. 370, 1959) - Section 1: Foods, Instant Noodles, 4. Measurement of Peroxides." Specifically, the sample was dissolved in an acetic acid-chloroform mixture, and saturated potassium iodide was added. Starch solution was then added, and the POV was measured by titration with sodium thiosulfate solution.

[0049] 1-3. Culture and cell viability of the 3D cultured human epidermal model (RHE) RHE was prepared using a LabCyte EPI-MODEL 24 manufactured by Japan Tissue Engineering Co., Ltd. The test was performed according to the manufacturer's instructions. RHE obtained from the manufacturer were transferred to culture medium and cultured for 24 hours at 37°C and 5% CO2. Next, the sample was applied to the stratum corneum side of the RHE and cultured for 1 hour at 37°C and 5% CO2. The sample on the stratum corneum was then removed and cultured for an additional 3 hours before use in subsequent analysis. Cell viability was determined by MTT assay.

[0050] 1-4. Quantitative RT-PCR The RHE tissue was transferred from the cell culture insert to Buffer RLT Plus (Qiagen) supplemented with mercaptoethanol. The RHE tissue was pulverized, and the supernatant was purified with a phenol-chloroform mixture. Total RNA was then purified using the RNeasy Plus Mini Kit (Qiagen). cDNA was synthesized using the High-Capacity cDNA Reverse Transcription Kit with RNase Inhibitor (Thermo Fisher Scientific). Quantitative PCR was performed using TB Green Premix EX Taq II (Tli RNaseH Plus) (Takara Bio). Relative expression levels were determined using the △△Ct method and normalized using GAPDH.

[0051] 2.Results 2-1. Induction of oxidative stress by UV-irradiated squalene To confirm that squalene peroxide was produced by UV irradiation of squalene, we measured the POV after UV irradiation for 1, 3, and 6 hours (Figure 1). As a result, the POV increased with increasing UV irradiation time. This indicates that the increase in squalene peroxide is dependent on the UV irradiation time.

[0052] Furthermore, samples exposed to UV for 1, 3, and 6 hours were applied to the RHE, and mRNA expression levels were measured 4 hours later (Figure 2). The results showed that the mRNA expression levels of HSPA1A, IL-8, and PTGS2 increased with increasing UV exposure time. Furthermore, the mRNA expression levels of HMOX1 and GCLC also increased with increasing UV exposure time.

[0053] Next, the samples were irradiated with UV for 1 hour, 3 hours, and 6 hours, and then applied to the RHE. The cell viability after 4 hours was calculated (Figure 3). Samples of squalene exposed to UV for 1 hour, 3 hours, and 6 hours were applied to the stratum corneum side of RHE. After 1 hour, the sample on the stratum corneum was removed, and the cells were cultured for another 3 hours. When the cell viability was measured by MTT assay, there was almost no difference in cell viability.

[0054] 2-2. Changes in mRNA expression over time after application of UV-irradiated squalene After applying UV-irradiated squalene (3 hours of exposure) to RHE, we examined the expression levels of mRNA related to oxidative stress and inflammation (Figure 4). Results showed a significant increase in the expression levels of HSPA1A, IL-8, and PTGS2 mRNA. In particular, the expression levels of HSPA1A and IL-8 mRNA were highest 2 hours after application of UV-irradiated squalene (3 hours of exposure). Furthermore, the expression levels of PTGS2 mRNA continued to increase over time. Furthermore, the expression levels of HMOX1 and GCLC mRNA were also elevated even 4 hours after application.

[0055] We examined the expression of mRNA related to keratinization after applying UV-irradiated squalene (3 hours of exposure) to the RHE (Figure 5). The expression of genes related to keratinization (gene symbols: ABCA12, GBA, OCLN, SPINK5, KRT5, and KRT14) tended to decrease after applying squalene peroxide to the stratum corneum.

[0056] 2-3. Hydroxyapatite reduces oxidative stress caused by UV-irradiated squalene Next, we used a group of genes (HSPA1A, IL-8, and PTGS2) that had previously been shown to fluctuate significantly with oxidative stress as indicators of oxidative stress (hereafter referred to as the indicator gene group) to examine whether hydroxyapatite reduces oxidative stress caused by squalene peroxide. First, we investigated POV, and found that hydroxyapatite-treated, UV-irradiated squalene had a significantly lower POV than UV-irradiated squalene (Figure 6). Furthermore, the mRNA expression levels of HSPA1A, IL-8, and PTGS2 were also reduced in hydroxyapatite-treated, UV-irradiated squalene compared to UV-irradiated squalene (Figure 7).

[0057] 3. Discussion First, UV (285-400 nm, 60 W / m 2 When squalene was irradiated with UV light, it was confirmed that squalene peroxide was produced. It was confirmed that the amount of squalene peroxide produced in squalene increased with increasing UV irradiation time.

[0058] Next, we analyzed RHE treated with UV-irradiated squalene (3 hours of exposure) applied to the stratum corneum side, and found that the mRNA expression levels of HSPA1A, IL-8, PTGS2, HMOX1, and GCLC increased.

[0059] HSPA1A, HMOX1, and GCLC are genes whose expression is known to be induced by oxidative stress. The fact that the induction of these genes was confirmed in this study suggests that when squalene peroxide comes into contact with the stratum corneum, a signal is transmitted through the stratum corneum, causing oxidative stress in living epidermal cells, resulting in cell damage, and that these genes increase in order to repair the cell damage.

[0060] Next, IL-8 and PTGS2 are genes involved in inflammation. The fact that the induction of these genes was confirmed in this study suggests that when squalene peroxide comes into contact with the stratum corneum, signals are transmitted through the stratum corneum, and that metabolites within living epidermal cells may cause inflammation in the skin.

[0061] PGE2 is known to induce the formation of melanocyte dendrites and promote melanin synthesis. Interleukin-8 has also been suggested to promote dermal elastase activity, denaturing elastin fibers and contributing to wrinkle formation. It has been shown that applying squalene peroxide to the skin can cause changes such as pigmentation and wrinkle formation, suggesting that factors derived from epidermal cells induced by squalene peroxide are involved in these morphological changes.

[0062] Furthermore, the expression of genes involved in keratinization (ABCA12, GBA, OCLN, SPINK5, KRT5, and KRT14) tended to decrease after application of squalene peroxide to the stratum corneum. This trend suggests that squalene peroxide disrupts the metabolic balance of the skin, causing abnormal keratinization and reducing barrier function. This finding indicates the importance of reducing oxidative stress induced by squalene peroxide, particularly in skin care.

[0063] Based on the above findings, the inventors conducted further studies to determine whether selective adsorption of squalene peroxide generated on the skin could reduce oxidative stress. Using a set of indicator genes, they investigated whether hydroxyapatite, a substrate that selectively adsorbs peroxides, could reduce oxidative stress induced by UV-irradiated squalene. The results showed that the POV was reduced in hydroxyapatite-treated UV-irradiated squalene. This indicates that squalene peroxide in UV-irradiated squalene was selectively adsorbed by hydroxyapatite. Hydroxyapatite is widely used in cosmetics and has been known to selectively adsorb lipid peroxides. The inventors' experiments yielded results similar to those reported previously. Furthermore, the mRNA expression levels of the indicator genes when this hydroxyapatite-treated UV-irradiated squalene was applied to RHE were suppressed compared to the mRNA expression levels of the indicator genes when UV-irradiated squalene was applied to RHE. These findings indicate that selective adsorption of squalene peroxide by hydroxyapatite can effectively reduce oxidative stress induced by squalene peroxide.

[0064] In this study, we focused on squalene, a component of sebum, and examined changes in mRNA levels expressed in living epidermal cells by applying squalene peroxide to the stratum corneum. Changes in genes related to oxidative stress and inflammation were observed. This suggests that squalene peroxide may be a factor in oxidative stress in the skin. Furthermore, these results may be applicable as a model test system for oxidative stress transmitted to living epidermal cells as a result of signals transmitted from the stratum corneum, such as for screening materials that reduce oxidative stress.

[0065] Below are examples of formulations in which the oxidative stress-reducing substances discovered by the screening method of the present invention are applied to external skin preparations. Hydroxyapatite was used as an example of the oxidative stress-reducing substance. Each formulation example was prepared by a conventional method. <Formulation example 1> Lotion (Component name) (mass%) Hydroxyapatite 0.1 Glycerin 5.0 1,3 Butylene Glycol 5.0 Trimethylglycine 3.0 Polyoxyethylene hydrogenated castor oil (60E.0) 0.5 Ethanol 2.0 Citric acid 0.1 Sodium citrate 0.1 Preservatives (appropriate amount)

[0066] <Formulation example 2> Cosmetic cream (Component name) (mass%) Hydroxyapatite 1.0 Shea Butter 2.0 Jojoba oil 1.0 Stearyl Alcohol 5.0 Squalane 10.0 Self-emulsifying glyceryl monostearate 3.0 Polyoxyethylene cetyl ether (20E.0) 1.0 1,3 Butylene Glycol 5.0 Glycerin 5.0 Sodium hydroxide 0.3 Fragrance 0.1 Preservatives and antioxidants (appropriate amount) Purified water remainder

[0067] <Formulation Example 3> Emulsion (Component name) (mass%) Hydroxyapatite 5.0 Squalane 5.0 Jojoba oil 0.3 Self-emulsifying glyceryl monostearate 1.0 Stearic acid 0.1 Carboxyvinyl polymer 0.1 Glycerin 1.5 Sodium hydroxide (appropriate amount) Ethanol 7.0 Fragrance 0.1 Preservatives and antioxidants (appropriate amount) Purified water remainder

[0068] <Formulation example 4> Foundation (Component name) (mass%) Hydroxyapatite 10.0 Glyceryl stearate 1.0 Stearic Acid 4.0 Behenyl Alcohol 1.0 Cetyl alcohol 0.5 Liquid Lanolin 2.0 Squalane 4.0 Hydrogenated Polydecene 4.0 Titanium dioxide 8.0 Talc 4.0 Bengala 1.0 Yellow iron oxide 0.5 Black iron oxide 0.2 Fragrance 0.1 Preservatives and antioxidants (appropriate amount) Purified water remainder

[0069] Although several embodiments of the present invention have been described above, these embodiments are presented as examples and are not intended to limit the scope of the invention. These embodiments can be implemented in various other forms, and various omissions, substitutions, and modifications can be made without departing from the spirit of the invention. These embodiments and their omissions, substitutions, modifications, etc. are included within the scope and spirit of the invention, as well as within the scope of the invention described in the claims and their equivalents.

Claims

1. The gene whose expression level immediately increases in response to squalene peroxide is a gene expressed in living epidermal cells, and is at least one of HSPA1A, IL-8, and PTGS2, A method for screening for a substance that reduces oxidative stress caused by squalene peroxide, using a reduction in the expression level of the gene in a cultured epidermal model as an indicator.

2. In the expression of the gene according to claim 1, The expression level of the gene in a cultured epidermal model to which a candidate substance for an oxidative stress-reducing substance has been applied is When the expression level of the gene is reduced compared to that in a cultured epidermal model to which the candidate substance is not applied, determining that the candidate substance has an oxidative stress-reducing effect; The method for screening for a substance that reduces oxidative stress caused by squalene peroxide according to claim 1.

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