Method for activating skin stem cells by suppressing MPC1 and skin stem cell activator

Abstract

Provided are a method for activating skin stem cells, a skin stem cell activator, and a method for screening for a skin stem cell activator. [Solution] The application of an MPC1 inhibitor is effective in activating skin stem cells. Examples of such MPC1 inhibitors include agents containing at least one of Akebia extract, black soybean extract, peony extract, tea extract, jojoba leaf extract, and ergothioneine as an active ingredient. Furthermore, MPC1 inhibitory activity can be used as an indicator to screen for skin stem cell activators.

Description

[Technical Field]

[0001] The present invention relates to a method for activating skin stem cells by suppressing MPC1. [Background technology]

[0002] Various methods have been investigated for activating skin stem cells. For example, Patent Document 1 discloses an agent for inhibiting cellular aging of stem cells, which contains astaxanthin. Patent Document 2 discloses an agent for maintaining and activating stemness of mesenchymal stem cells, which contains hydroxyproline or a pharmacologically acceptable salt thereof as an active ingredient.

[0003] Differentiated cells produce ATP using both glycolysis and the electron transport chain, leading to the accumulation of reactive oxygen species within the cells. Stem cells, on the other hand, produce ATP solely through glycolysis, preventing the generation of reactive oxygen species and protecting the cells (Non-Patent Document 1). It has been reported that in murine epidermis, stem cells in the S phase increase at night when the NADH / NAD+ ratio is high and glycolysis is dominant, but decrease during the day when the NADH / NAD+ ratio is low and the electron transport chain is dominant, repeating this cycle (Non-Patent Document 2). Furthermore, genetically engineered hair follicle cells lacking the function of the mitochondrial pyruvate carrier (MPC1) to block the pathway to the electron transport chain have been shown to stimulate stem cell proliferation and promote the hair cycle (Non-Patent Document 3).

[0004] Patent Literature 3 discloses a cosmetic composition containing a mitochondrial transfer promoter and describes the use of such a composition on skin fibroblasts, epidermal keratinocytes, adipose-derived mesenchymal stem cells, epidermal stem cells, and dermal stem cells. Patent Literature 4 discloses a method for identifying a test substance as a skin care active agent that improves keratinocyte metabolism, including exposing keratinocytes to a stressor, detecting metabolic indicators associated with glycolysis and oxidative phosphorylation, and providing a response to the stressor. Patent Literature 5 discloses a method for identifying or evaluating synergistic combinations of active ingredients for use in a cosmetic composition, including contacting cells with first and second test substances, non-lethally detecting metabolic indicators associated with glycolysis and oxidative phosphorylation, and providing a response related to the metabolic pathway to the test substance. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Japanese Patent Application Publication No. 2018-52879 [Patent Document 2] Japanese Patent Application Publication No. 2019-26617 [Patent Document 3] Japanese Patent Application Publication No. 2018-123130 [Patent Document 4] Special Publication No. 2015-534644 [Patent Document 5] Special Publication No. 2015-531881 [Non-patent literature]

[0006] [Non-Patent Document 1] Journal of Cell Science 125(23), 5597-5608 [Non-patent document 2] Stringari et al., 2015, Cell Reports 10 , 1-7, January 6, 2015, http: / / dx.doi.org / 10.1016 / j.celrep.2014.12.007 [Non-patent document 3] Nat Cell Biol. 2017 September; 19(9): 1017-1026. doi:10.1038 / ncb3575. Summary of the Invention [Problem to be solved by the invention]

[0007] An object of the present invention is to provide a method for activating skin stem cells, a skin stem cell activator, and a method for screening for a skin stem cell activator. [Means for solving the problem]

[0008] As a result of extensive research, the present inventors came up with the idea that skin stem cells can be activated by inhibiting the pathway to the electron transport chain through MPC1 suppression, and thus completed the present invention.

[0009] This application encompasses the following inventions: (1) A cosmetic method for activating skin stem cells by applying an MPC1 inhibitor. (2) The cosmetic method according to (1), wherein the MPC1 inhibitor contains at least one of Akebia extract, black bean extract, peony extract, tea extract, jojoba leaf extract, and ergothioneine as an active ingredient. (3) An MPC1 inhibitor containing at least one of Akebia extract, black bean extract, peony extract, tea extract, jojoba leaf extract, and ergothioneine as an active ingredient. (4) A skin stem cell activator containing an MPC1 inhibitor. (5) The skin stem cell activator according to (4), wherein the MPC1 inhibitor contains at least one of Akebia extract, black bean extract, peony extract, tea extract, jojoba leaf extract, and ergothioneine as an active ingredient. (6) A method for screening skin stem cell activators using MPC1 inhibitory activity as an indicator. (7) contacting the skin sample with a candidate drug; measuring the activity, amount, and / or expression of MPC1 in the skin sample contacted with the candidate drug; determining the MPC1 inhibitory effect of the candidate drug from the measured MPC1 activity, amount, and / or expression level; selecting a skin stem cell activator based on the determined MPC1 inhibitory activity; The method according to (6), comprising: [Effects of the Invention]

[0010] According to the present invention, an agent and method are provided that are effective in activating skin stem cells by suppressing MPC1. Activation of skin stem cells is effective in inhibiting skin aging. [Brief explanation of the drawings]

[0011] [Figure 1] Figure 1 shows the results of Experiment 1-1, and is a graph showing the MCSP / B2M values ​​when a control (Cont), UK5099 (10 μM UK5099, 20 μM UK5099), or echinomycin (10 nM Echinomycin, 20 nM Echinomycin) was added, expressed as a relative value, with the result for the control (Cont) set at 1. [Figure 2] FIG. 2 shows the results of Experiment 1-2, and is a photograph showing the expression of MCSP and DAPI when a control (Cont) and UK5099 (20 μM UK5099) were added. [Figure 3] FIG. 3 shows the results of Experiment 1-2, and is a photograph showing the expression of integrin β1 and DAPI when a control (Cont) and UK5099 (20 μM UK5099) were added. [Figure 4]FIG. 4 shows the results of Experiment 1-3, showing the number of Integrin β1-positive cells when no antibody was added (Negative cont), when a control was added (Cont), and when UK5099 was added (20 μM UK5099). [Figure 5] Figure 5 shows the results of Experiments 1-4 and is a photograph showing the expression of integrin α6, MCSP, and DAPI in tissue sections of three-dimensional cultured skin models cultured for four days with the addition of control (Cont) or UK5099 (1 μM UK5099, 10 μM UK5099). [Figure 6] FIG. 6 shows the results of Experiments 1-5 and is a photograph showing the expression of integrin α6, MCSP, and DAPI in tissue sections of a human skin organ culture model cultured for 5 days with the addition of a control (Cont) or UK5099 (10 μM UK5099). [Figure 7] Figure 7 shows the results of the primary screening of Experiment 2-2, and shows the MPC1 / B2M values ​​when a control (DMSO; Control) and each evaluation sample (Drug No. 1 to 124) were added, expressed as relative values ​​with the control result set at 1.0. [Figure 8] Figure 8 shows the results of the secondary screening of Experiment 2-2, and shows the MPC1 / B2M values ​​when each of the evaluation samples (37 of Drug Nos. 1 to 124) selected in the primary screening was added, expressed as a percentage (% of control) of the control (DMSO) result, which is set at 100.0. [Figure 9] Figure 9 shows the results of the tertiary screening of Experiment 2-2, and shows the MPC1 / B2M values ​​when a control (DMSO; Cont) and each of the evaluation samples selected in the secondary screening (15 of Drug Nos. 1 to 124) were added, expressed as a percentage (% of control) of the control result, which is 100.0. [Figure 10] FIG. 10 shows a graph in which the results of FIG. 9 were statistically analyzed using Dunnett's test for each of the six products selected in the tertiary screening of Experiment 2-2 (*P<0.05, **P<0.01). DETAILED DESCRIPTION OF THE INVENTION

[0012] The present inventors have discovered that skin stem cells are activated by suppressing MPC1, and have discovered novel substances with MPC1 inhibitory activity. In particular, they have discovered that Akebia extract, black soybean extract, peony extract, tea extract, jojoba leaf extract, and ergothioneine have strong MPC1 inhibitory activity.

[0013] Based on these findings, the present invention provides an MPC1 inhibitor and a skin stem cell activator (hereinafter, these may be collectively referred to as "the agents of the present invention"), a screening method for them, and a method for activating skin stem cells by applying them. The methods of the present invention may be for cosmetic purposes and may not be treatments performed by a doctor or medical professional.

[0014] MPC1 is a protein that forms a heterodimer with MPC2 to form the mitochondrial pyruvate carrier, a transporter that delivers pyruvate into mitochondria. MPC1 inhibition refers to reducing and / or inhibiting the function, production, and / or translation of MPC1, and / or reducing the activity, amount, and / or expression level of MPC1. Examples of MPC1 function include the function of MPC1 to deliver pyruvate into mitochondria. In certain embodiments, MPC1 inhibition can refer to a statistically significant decrease (e.g., Dunnett's test) in MPC1 activity, amount, and / or expression level when the agent of the present invention is administered compared to a state in which the agent of the present invention is not administered (control), for example, with a significance level of 5%. Alternatively, it can refer to a decrease of, for example, 5% or more, 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or 100%.

[0015] The expression level of MPC1 may be determined, for example, by measuring the gene expression level using quantitative PCR as in the Examples, by measuring the protein expression level by staining with an antibody, by measuring using a commercially available kit, or by the methods described in documents such as Patent Document 3, but is not limited to these, and any known technique can be used.

[0016] As used herein, "activation of skin stem cells" refers to the promotion of proliferation of stem cells in skin cells such as keratinocytes and fibroblasts. Stem cell proliferation can be determined by counting the number of stem cells, measuring the expression level of stem cell markers such as integrin β1, or the like. Activation of skin stem cells can be expected to promote cell turnover, rejuvenate the skin, improve skin texture, and speed up recovery from undesirable skin conditions such as acne and pigmentation.

[0017] The MPC1 inhibitor of the present invention may consist of or contain as an active ingredient Akebia extract, black soybean extract, peony extract, tea extract, jojoba leaf extract, and / or ergothioneine, but is not limited to these, as long as it is a substance that can inhibit MPC1.

[0018] The agent of the present invention may contain any one of the above active ingredients alone, or may contain two or more of them in any combination and ratio.

[0019] The agent of the present invention may contain the above-mentioned active ingredient in combination with one or more other ingredients, such as an excipient, a carrier and / or a diluent.

[0020] Akebia quinata is a deciduous shrub belonging to the genus Akebia in the family Akebiaceae. It has been reported that Akebia has the effects of promoting hyaluronic acid production, promoting collagen production, and inhibiting MMP, and has the effects of preventing and improving wrinkle formation, lipase inhibitory activity, suppressing the increase in blood triglyceride concentration, and anti-obesity activity (see, for example, JP 2015-17048 A and JP 2010-265182 A). The Akebia extract used in the present invention is preferably an extract from Akebia stems. However, since active ingredients are also contained in Akebia fruits, peels, seeds, leaves, flowers, roots, etc., extracts from any one or more of these can also be used.

[0021] Black soybeans (Glycine max 'Kuromame') are a variety of soybean, an annual plant in the legume family, and varieties such as "Tamba Kuro" and "Wachi Kuro" can be used. It has been reported that black soybeans are rich in polyphenols such as anthocyanins, and that components derived from black soybeans have anti-obesity, anti-inflammatory, antioxidant, and glucose tolerance-improving effects (see, for example, JP 2015-140298 A). While black soybean extracts are preferably extracts from the legumes of black soybeans, active ingredients are also contained in the pods, leaves, stems, flowers, roots, etc., so extracts from one or more of these can also be used.

[0022] Peony (Paeonia lactiflora) is a perennial plant of the Paeoniaceae family. Peony has been reported to have effects such as inhibiting epidermal thickening, promoting VEGF production, IGF-1 production, HGF production, and BMP-2 production (see, for example, JP 2019-11252 A and JP 2012-121856 A). Peony extract is preferably an extract of peony root, but active ingredients are also contained in peony leaves, stems, flowers, fruits, peels, seeds, etc., so extracts of any one or more of these can also be used.

[0023] Tea (Camellia sinensis) is an evergreen tree of the genus Camellia in the family Theaceae, and various varieties such as tea plant (Camellia sinensis var. sinensis) and Assam tea (Camellia sinensis var. assamica) can be used. For example, Uji tea can be used. Tea is known to be rich in polyphenols such as catechins, and to have lipid metabolism-improving, antioxidant, anti-cancer, and antihypertensive effects. The tea extract is preferably an extract of tea leaves or stems, but since tea fruits, flowers, roots, seeds, etc. also contain active ingredients, extracts of one or more of these can also be used.

[0024] Jojoba (Simmondsia chinensis) is an evergreen shrub belonging to the family Jojobaceae in the order Caryophyllaceae. Jojoba is known to have moisturizing, emollient, anti-inflammatory, collagenase inhibitory, elastase inhibitory, and hyaluronidase inhibitory effects (see, for example, Japanese Patent Application Laid-Open Nos. 2003-48846, 2003-048812, and 2003-034644). Jojoba leaf extract is an extract from jojoba leaves. However, since active ingredients are also contained in jojoba seeds, fruits, flowers, roots, and the like, extracts from any one or more of these may also be used.

[0025] Ergothioneine (L-Ergothioneine) is an amino acid with the following structure and a molecular weight of 229.3, and is known to have antioxidant and anti-inflammatory effects, as well as elastase inhibitory and tyrosinase inhibitory effects (see, for example, JP 2019-149972 A). Ergothioneine may be chemically synthesized, or, since it is contained in microorganisms such as basidiomycetes, plants and animals, it may be used in the form of an extract of such natural products. [ka]

[0026] The various extracts mentioned above may be commercially available as cosmetic raw materials or health food ingredients, or may be obtained by conventional methods. The extraction method is not particularly limited, but examples include solvent extraction and hydrolysis extraction. When performing solvent extraction, the raw material is immersed or heated under reflux with the extraction solvent at room temperature or under heat. When performing hydrolysis extraction, any hydrolysis treatment, such as chemical treatment with alkali or acid, physical treatment with heat or pressure, or biological treatment with enzymes, is performed, followed by filtration and concentration. While the raw material can be used as is, grinding it into granules or powder prior to extraction allows for extraction of active ingredients under mild conditions with high extraction efficiency in a short time. The extraction temperature is not particularly limited and can be set appropriately depending on the particle size of the ground material, the type of solvent, etc. It is usually set within the range from room temperature to the boiling point of the solvent. The extraction time is also not particularly limited and can be set appropriately depending on the particle size of the ground material, the type of solvent, the extraction temperature, etc. Furthermore, during extraction, stirring may be performed, the mixture may be left standing without stirring, or ultrasound may be applied.

[0027] As the extraction solvent, any solvent normally used for extraction can be used, for example, aqueous solvents such as water, physiological saline, phosphate buffer, borate buffer, or organic solvents such as alcohols such as ethanol, propylene glycol, 1,3-butylene glycol, glycerin, hydrous alcohols, chloroform, dichloroethane, carbon tetrachloride, acetone, ethyl acetate, hexane, etc., can be used alone or in combination.

[0028] By such an extraction procedure, the active ingredient is extracted and dissolved in the solvent or hydrolyzed. The solvent or hydrolyzate containing the extract may be used as is, or may be subjected to conventional purification treatments such as sterilization, washing, filtration, bleaching, and deodorization before use. If necessary, it may be concentrated by lyophilization or diluted with an arbitrary solvent before use. Furthermore, the solvent or hydrolyzate may be completely evaporated to form a solid (dried product) before use, or the dried product may be redissolved in an arbitrary solvent before use.

[0029] Furthermore, the squeezed liquid obtained by squeezing the raw material also contains the same active ingredients as the extract, so the squeezed liquid can also be used instead of the extract.

[0030] The present application also provides a composition comprising the agent of the present invention. The composition of the present invention may be a cosmetic composition or a food composition. The composition of the present invention may be, for example, a composition that activates skin stem cells through MPC1 inhibitory activity.

[0031] The subject to which the agent or composition of the present invention is applied may be a subject who needs skin stem cell activation from an objective or subjective standpoint, such as a delay in skin turnover, skin aging, or pigmentation, or a subject who desires preventive skin stem cell activation.

[0032] The agent or composition of the present invention can be administered by any route, such as topical or oral administration, but is preferably incorporated into an external skin preparation that can be applied directly to the skin. The external administration form can be selected from various forms, such as liquid, emulsion, cream, solid, sheet, spray, gel, foam, and powder. It may also be a cosmetic composition, such as an emulsion, cream, serum, lotion, pack, or facial cleanser. Oral administration can be selected from various forms, such as tablets, supplements, beverages, and powders. The agent or composition of the present invention can be appropriately formulated with optional ingredients used in cosmetic and pharmaceutical compositions, as needed, as long as the ingredients do not impair their efficacy. Examples of optional ingredients include excipients, carriers, diluents, oils, surfactants, powders, colorants, water, alcohols, thickeners, chelating agents, silicones, antioxidants, UV absorbers, moisturizers, fragrances, various medicinal ingredients, preservatives, pH adjusters, and neutralizers. For example, other medicinal ingredients that promote skin stem cell activation may also be included.

[0033] The administration frequency can be selected arbitrarily, such as once every 4 weeks, once every 2 weeks, once a week, once every 3 days, once every 2 days, once a day, twice a day, three times a day, four times a day, five times a day, or administration as needed, but is not limited to these.

[0034] However, the forms that the agent or composition of the present invention can take are not limited to the dosage forms and shapes described above.

[0035] The amount of active ingredients, such as Akebia extract, black bean extract, peony extract, tea extract, jojoba leaf extract, and ergothioneine, in the agent or composition of the present invention can be appropriately determined depending on the type, purpose, form, method of use, etc. For example, the amount of Akebia extract, black bean extract, peony extract, tea extract, jojoba leaf extract, and / or ergothioneine can be 0.0001 to 100 wt%, 0.0001 to 90 wt%, 0.001 to 50 wt%, 0.01 to 5 wt%, 0.01 to 1 wt%, 0.1 to 0.5 wt%, etc., based on the total weight of the agent or composition of the present invention, but is not limited thereto as long as the effects of the present invention are exhibited.

[0036] Furthermore, the present application provides a screening method for skin stem cell activators using MPC1 inhibitory activity as an indicator. The screening method of the present invention may include the steps of contacting a skin sample with a candidate drug; measuring the activity, amount, and / or expression level of MPC1 in the skin sample contacted with the candidate drug; determining the MPC1 inhibitory activity of the candidate drug from the measured MPC1 activity, amount, and / or expression level; and selecting a skin stem cell activator based on the determined MPC1 inhibitory activity. The method of the present invention makes it possible to screen candidate drugs for their skin stem cell activating activity, enabling product development and the proposal of new skin care methods.

[0037] The skin sample may be a harvested skin sample, for example, an ex vivo skin sample harvested from an animal such as a human, or an in vitro sample such as cultured skin cells, for example, cultured keratinocytes or cultured fibroblasts. Alternatively, the skin sample may be an artificial skin sample such as a three-dimensional skin model. The skin sample is not limited as long as it allows measurement of the MPC1 inhibitory activity.

[0038] The present application also provides Akebia extract, black bean extract, peony extract, tea extract, jojoba leaf extract, and / or ergothioneine, which activate skin stem cells through MPC1 inhibition. [Example]

[0039] The present invention will now be described in more detail with reference to examples, although the present invention is not limited thereto.

[0040] Experiment 1: Effect of MPC1 inhibition on stem cells Experiment 1-1: Gene expression analysis of MCSP by PCR To investigate the effect of MPC1 inhibition on stem cells, we analyzed the expression of melanoma-associated chondroitin sulfate proteoglycan (MCSP), an epidermal stem cell marker, using UK5099, a known MPC1 inhibitor.

[0041] cell culture Normal human fetal epidermal keratinocytes (ScienCell, Cat No. 2120) were cultured in epidermal keratinocyte culture medium (Kurabo, KK-2150S). After passage, 2.5 × 10 5The cells were seeded into 6-well plates at 2 mL per well, and 24 hours later, UK5099, control, and echinomycin were added. Echinomycin, like MPC1, is an inhibitor of HIF-1α, which regulates mitochondrial activity. However, it differs in that it inhibits the conversion of pyruvate to acetyl-CoA within mitochondria. Echinomycin was used to investigate whether the pathway inhibiting pyruvate delivery to mitochondria or the pathway inhibiting the conversion of pyruvate to acetyl-CoA within mitochondria activates epidermal keratinocyte stem cells. UK5099 (Merck, catalog no. 504817) was diluted in DMSO to 10 mM and 20 mM stock solutions, which were then added to the medium at a 1:1000 dilution to give final concentrations of 10 μM and 20 μM. DMSO was added at a 1:1000 dilution as a control. Echinomycin (Abcam, product number ab144247) was diluted in DMSO to prepare stock solutions of 10 μM and 20 μM, which were then added to the medium at a 1000-fold dilution to give final concentrations of 10 nM and 20 nM. 24 hours after addition of these substances, mRNA was collected.

[0042] Gene expression analysis by quantitative PCR RNA was extracted from the cells 24 hours after drug addition using the Quiagen Rneasy mini Kit (Quiagen), and cDNA was synthesized using the SuperScript VILO cDNA Synthesis Kit (Thermo Fisher Scientific). MCSP and B2M gene expression levels were measured using the Syber Green method with Platinum SYBR Green qPCR superMix-UDG (Invitrogen Japan, Tokyo, Japan). The primers used were as follows: B2M forward: 5'-GTGGGATCGAGACATGTAAGCA-3' (SEQ ID NO: 1) B2M reverse: 5'-CAATCCAAATGCGGCATCT-3' (SEQ ID NO: 2) MCSP forward: 5'-CACGGCTCTGACCGACATAG-3' (SEQ ID NO: 3) MCSP reverse: 5'-CCCAGCCCTCTACGACAGT-3' (SEQ ID NO: 4)

[0043] The results are shown in Figure 1. The addition of UK5099 increased MCSP expression in a concentration-dependent manner. On the other hand, the addition of echinomycin reduced MCSP expression. These results suggest that stem cells are activated by suppressing MCP1.

[0044] Experiment 1-2: Protein expression analysis of MCSP and integrin β1 by cell staining Furthermore, we stained and analyzed cells for not only MCSP but also another stem cell marker, integrin β1. Epidermal keratinocytes subcultured in the same manner as in Experiment 1-1 were added with UK5099 at a concentration of 20 μM. As a control, 1.0 × 10 4 Cells were seeded at 0.5 mL per well onto 4-well chamber slides (Falcon, 354114) and cultured for 48 hours. After 48 hours, cells were fixed with 4% PFA for 10 minutes. Cell membranes were then partially lysed with 0.01% Triton-X100 / PBS for 15 minutes. After blocking with 12% BSA / PBS, cells were incubated overnight at 4°C with primary antibodies: anti-MCSP (Millipore, MAB2029, mouse mAb) and anti-β1 integrin (Santa Cruz, sc-13590, mouse mAb). The next day, the cells were washed three times for 5 minutes with PBS and then incubated with Alexa488-anti-mouse IgG as a secondary antibody at room temperature for 1 hour. After three 5-minute washes with PBS, the cells were mounted in DAPI-containing mounting medium (Vectashield, H-1200) and observed under a microscope. The results when 20 μM UK5099 and the control were added are shown in Figures 2 and 3. The addition of UK5099 increased the expression levels of both MCSP and Integrin β1, which was consistent with the results of Experiment 1-1.

[0045] Experiment 1-3: FACS analysis of integrin β1-positive cells Next, FACS analysis was performed to determine the number of integrin β1-positive cells. Epidermal keratinocytes passaged in the same manner as in Experiment 1-1 were cultured for 48 hours with the addition of 20 μM UK5099. As a control, the cells were not treated with UK5099 and the same amount of DMSO was added. These cells were detached with trypsin and cultured at 1.0 × 10 6 A cell suspension was prepared at a concentration of 1000 cells / 500 μL. After removing the supernatant by centrifugation, the cells were blocked with 0.5% BSA / PBS on ice for 10 minutes. After removing the supernatant by centrifugation, IgG antibody and Pacific blue-labeled anti-β1 integrin antibody (BioLegend, 313620) were added to the cell suspension and incubated on ice for 1 hour. The cells were then washed three times with 0.1% BSA / PBS by centrifugation and subjected to FACS analysis using the SH800 cell sorter. A negative control was prepared in the same manner, but without the addition of antibodies.

[0046] The results are shown in Figure 4. The addition of UK5099 increased the number of integrin β1-positive cells, which was consistent with the results of Experiments 1-1 and 1-2.

[0047] Experiment 1-4: Protein expression analysis of MCSP and integrin β1 by tissue staining Next, using a MatTeK Skin Model EFT-400, UK5099 was added to the culture medium at 1 μM and 5 μM concentrations. For controls, the same amount of DMSO was added without UK5099. The skin models were cultured at 2.5 mL / well. The medium was changed after two days, and four days after the start of culture, the skin models were dehydrated and fixed by immersion in refrigerated acetone. Subsequently, the tissue was subjected to solvent exchange twice with room-temperature acetone, twice with room-temperature methyl benzoate, and twice with room-temperature xylene. Paraffin blocks were then prepared by embedding in paraffin. Sections were cut at 3 μm thickness using a microtome. After deparaffinization using xylene and blocking with 12% BSA / PBS, the tissue was incubated overnight at 4°C with anti-MCSP antibody (Millipore, MAB2029, mouse mAb) and anti-α6 integrin antibody (Santa Cruz, sc-19622, rat mAb). The next day, the sections were washed three times with PBS for 5 minutes each, and then incubated with Alexa488-anti-mouse IgG and Alexa594-anti-rat IgG secondary antibodies for 1 hour at room temperature. After washing three times with PBS for 5 minutes each, the sections were mounted in a mounting medium containing DAPI (Vectashield, H-1200) and observed under a microscope.

[0048] The results of adding 1 μM and 5 μM UK5099 and the control are shown in Figure 5. The addition of UK5099 increased MCSP and integrin β1-expressing cells, a result consistent with the results of Experiments 1-1, 1-2, and 1-3, even when using a three-dimensional skin model.

[0049] Experiment 1-5: Protein expression analysis of MCSP and integrin β1 by tissue staining Next, fresh human abdominal skin samples (purchased from BioPredic) were cultured in medium containing 10 μM UK5099. Controls were cultured without UK5099 and the same volume of DMSO was added at 3 mL / well. The medium was changed after 2 and 4 days. Five days after the start of culture, the cells were dehydrated and fixed by immersion in refrigerated acetone. Subsequently, the cells were solvent-exchanged twice with room-temperature acetone, twice with room-temperature methyl benzoate, and twice with room-temperature xylene. Then, paraffin-embedding was performed to prepare paraffin blocks. Sections were cut at 3 μm thickness using a microtome. After deparaffinization using xylene and blocking with 12% BSA / PBS, the cells were incubated overnight at 4°C with anti-MCSP antibody (Millipore, MAB2029, mouse mAb) and anti-α6 integrin antibody (Santa Cruz, sc-19622, rat mAb). The next day, the sections were washed three times with PBS for 5 minutes each, and then incubated with Alexa488-anti-mouse IgG and Alexa594-anti-rat IgG secondary antibodies for 1 hour at room temperature. After washing three times with PBS for 5 minutes each, the sections were mounted in a mounting medium containing DAPI (Vectashield, H-1200) and observed under a microscope.

[0050] The results for the addition of 10 μM UK5099 and the control are shown in Figure 6. The addition of UK5099 increased MCSP and integrin β1-expressing cells, a result consistent with the results of Experiments 1-1, 1-2, 1-3, and 1-4 when using ex vivo human skin samples.

[0051] Experiment 2: Screening for substances with MCP1 inhibitory activity Experiment 1 suggested that suppressing MCP1 activates stem cells. Therefore, we screened for substances that have MCP1 inhibitory effects. Experiment 2-1: Sample preparation The following samples were used to evaluate the MPC1 inhibitory effect. [Table 1]

[0052] A total of 124 candidate samples were prepared, including natural ingredients such as animal and plant extracts and synthetic ingredients.

[0053] Experiment 2-2: Evaluation of MCP1 inhibitory effect Normal human fetal epidermal keratinocytes were cultured as in Experiment 1-1, and each drug was added 24 hours after seeding. The candidate sample was diluted to 10% with DMSO and added to the medium at a 1000-fold dilution for the primary and secondary screenings to a final concentration of 0.01%. For the tertiary screening, the candidate sample was added at a 10,000-fold, 1000-fold, and 100-fold dilutions for final concentrations of 0.001%, 0.01%, and 0.1%. UK5099 was added to the medium at 20 μM as in Experiment 1 as a positive control. DMSO was added at a 1000-fold dilution for the negative control. mRNA was collected 24 hours after each drug addition.

[0054] Gene expression analysis by quantitative PCR RNA was extracted from the cells 24 hours after drug addition using the Quiagen Rneasy mini Kit (Quiagen), and cDNA was synthesized using the SuperScript VILO cDNA Synthesis Kit (Thermo Fisher Scientific). MPC1 and B2M gene expression levels were measured using the Syber Green method with Platinum SYBR Green qPCR superMix-UDG (Invitrogen Japan, Tokyo, Japan). The primers used were as follows: B2M forward: 5'-GTGGGATCGAGACATGTAAGCA-3' (SEQ ID NO: 1) B2M reverse: 5'-CAATCCAAATGCGGCATCT-3' (SEQ ID NO: 2) MPC1 forward: 5'-GTGCGGAAAGCGGCGGACTA-3' (SEQ ID NO: 5) MPC1 reverse: 5'-GGCAGCAATGGGAAGACCCCA-3' (SEQ ID NO: 6)

[0055] The primary screening was performed with N=1 and a drug concentration of 0.01%. The results of the primary screening are shown in Figure 7. 37 substances (substances below the line shown in Figure 7) were selected from 124 substances as candidates for MPC1 inhibitory activity. The 37 compounds selected in the primary screening were subjected to secondary screening at a drug concentration of 0.01%, with N = 3. The results of the secondary screening are shown in Figure 8. From the 37 compounds, 15 compounds (dark gray compounds in Figure 8) were selected as candidate compounds with concentration-dependent MPC1 inhibitory activity. The 15 products selected in the secondary screening were subjected to tertiary screening at drug concentrations of 0.001%, 0.01%, and 0.1%, with N=3 for each. The results of the tertiary screening are shown in Figure 9. Of the 15 products, six products were selected as candidate substances that reproducibly inhibit MPC1 in a concentration-dependent manner: Akebia extract, black bean extract, peony extract, tea extract, jojoba leaf extract, and ergothioneine (dark gray substances shown in Figure 9).

[0056] For each of the six selected products, a graph obtained by statistically processing the results of Figure 9 using Dunnett's test is shown in Figure 10. As shown in Figure 10, all substances exerted MPC1 inhibitory effects in a concentration-dependent manner with statistically significant differences.

[0057] These results demonstrate that skin stem cells are activated by suppressing MCP1, and that Akebia extract, black bean extract, peony extract, tea extract, jojoba leaf extract, and ergothioneine have MCP1 inhibitory effects. If skin stem cells are activated by the MCP1 inhibitor of the present invention, it will be effective in inhibiting skin aging.

Claims

1. A cosmetic method that activates skin stem cells by applying an MPC1 inhibitor.

2. 2. The cosmetic method according to claim 1, wherein the MPC1 inhibitor comprises at least one of Akebia extract, black bean extract, peony extract, tea extract, jojoba leaf extract, and ergothioneine as an active ingredient.

3. An MPC1 inhibitor comprising at least one of Akebia extract, black bean extract, peony extract, tea extract, jojoba leaf extract, and ergothioneine as an active ingredient.

4. A skin stem cell activator containing an MPC1 inhibitor.

5. The skin stem cell activator according to claim 4, wherein the MPC1 inhibitor comprises at least one of Akebia extract, black bean extract, peony extract, tea extract, jojoba leaf extract, and ergothioneine as an active ingredient.

6. A method for screening skin stem cell activators using MPC1 inhibitory activity as an indicator.

7. contacting skin cells with a candidate drug; measuring the activity, amount, and / or expression level of MPC1 in skin cells contacted with the candidate drug; determining the MPC1 inhibitory effect of the candidate drug from the measured MPC1 activity, amount, and / or expression level; selecting a skin stem cell activator based on the determined MPC1 inhibitory activity; The method of claim 6, comprising:

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