JP7497974B2 - Method for activating skin stem cells by suppressing MPC1 and skin stem cell activator - Google Patents

Description

The present invention relates to a method for activating skin stem cells by suppressing MPC1.

Various methods are being 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.

Differentiated cells produce ATP using the electron transport chain in addition to glycolysis, so reactive oxygen species accumulate within the cells. On the other hand, stem cells produce ATP only through glycolysis, so no reactive oxygen species are generated and the cells are protected (Non-Patent Document 1). It has been reported that in mouse epidermis, the number of stem cells in the S phase increases at night when the NADH/NAD+ ratio is high and the glycolysis is dominant, but decreases during the day when the NADH/NAD+ ratio is low and the electron transport chain is dominant, repeating this cycle (Non-Patent Document 2). In addition, it has been shown that hair follicle cells genetically engineered to lack the function of the mitochondrial pyruvate carrier (MPC1) and block the pathway to the electron transport chain have activated stem cell proliferation and promoted the hair cycle (Non-Patent Document 3).

Patent document 3 discloses a cosmetic composition containing a mitochondrial transfer promoter, and describes the use of such a composition in skin fibroblasts, epidermal keratinocytes, adipose-derived mesenchymal stem cells, epidermal stem cells, and dermal stem cells. Patent document 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, respectively, and providing a respective response to the stressor. Patent document 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, respectively, and providing a metabolic pathway-related response to the test substance.

JP 2018-52879 A JP 2019-26617 A JP 2018-123130 A Patent Publication No. 2015-534644 Patent Publication No. 2015-531881

Journal of Cell Science 125(23), 5597-5608 Stringari et al., 2015, Cell Reports 10 , 1-7, January 6, 2015, http://dx.doi.org/10.1016/j.celrep.2014.12.007 Nat Cell Biol. 2017 September ; 19(9): 1017-1026. doi:10.1038/ncb3575.

The objective of the present invention is to provide a method for activating skin stem cells, a skin stem cell activator, and a method for screening skin stem cell activators.

As a result of extensive research, the inventors came up with the idea of activating skin stem cells by inhibiting the pathway to the electron transport chain through MPC1 inhibition, and thus completed the present invention.

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 comprising as an active ingredient at least any one of Akebia extract, black bean extract, peony extract, tea extract, jojoba leaf extract, and ergothioneine.
(4) A skin stem cell activator comprising 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 skin samples contacted with the candidate agent;
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:

The present invention provides an agent and method that are effective in activating skin stem cells by suppressing MPC1. Activation of skin stem cells is effective in inhibiting skin aging.

FIG. 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), and echinomycin (10 nM Echinomycin, 20 nM Echinomycin) were added, with the result for the control (Cont) set at 1 as a relative value. FIG. 2 shows the results of Experiment 1-2, and is a set of photographs showing the expression of MCSP and DAPI when a control (Cont) and UK5099 (20 μM UK5099) were added, respectively. FIG. 3 shows the results of Experiment 1-2, and is a set of photographs showing the expression of Integrin β1 and DAPI when a control (Cont) and UK5099 (20 μM UK5099) were added. 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). FIG. 5 shows the results of Experiment 1-4 and is a set of photographs showing the expression of Integrin α6, MCSP, and DAPI in tissue sections of three-dimensional cultured skin models cultured for 4 days with the addition of control (Cont) or UK5099 (1 μM UK5099, 10 μM UK5099). FIG. 6 shows the results of Experiment 1-5, and is a set of photographs showing the expression of Integrin α6, MCSP, and DAPI in tissue sections of a human skin organ culture model cultured for 5 days after the addition of a control (Cont) or UK5099 (10 μM UK5099). Figure 7 shows the results of the primary screening in Experiment 2-2, and shows the MPC1/B2M values when a control (DMSO; Control) and each evaluation sample (Drug No. 1 to 124) was added, expressed as a relative value with the control result set at 1.0. Figure 8 shows the results of the secondary screening of Experiment 2-2, and shows the MPC1/B2M values when each evaluation sample selected in the primary screening (37 of Drug Nos. 1 to 124) was added, expressed as a percentage (% of control) of the control (DMSO) result, which is taken as 100.0. 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) with the control result taken as 100.0. FIG. 10 shows a graph in which the results of FIG. 9 were statistically processed 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).

The present inventors have discovered that skin stem cells are activated by suppressing MPC1, and have discovered new substances that have MPC1 inhibitory effects. In particular, they have discovered that Akebia extract, black bean extract, peony extract, tea extract, jojoba leaf extract, and ergothioneine have high MPC1 inhibitory effects.

Based on such 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 thereof, and a method for activating skin stem cells by applying them. The methods of the present invention may be methods for cosmetic purposes and may not be treatments administered by a doctor or medical professional.

MPC1 is a protein that forms a heterodimer with MPC2 to form the mitochondrial pyruvate carrier, a transporter that delivers pyruvate to the inside of mitochondria. MPC1 suppression 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 the function of MPC1 include the function of MPC1 to deliver pyruvate to the inside of mitochondria. In one embodiment, MPC1 suppression can mean that the activity, amount, and/or expression level of MPC1 is reduced with a statistically significant difference (e.g., Dunnett's test) with a significance level of 5%, for example, when the agent of the present invention is administered compared to a state (control) in which the agent of the present invention is not administered, or that the activity, amount, and/or expression level of MPC1 is reduced by, 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%.

The expression level of MPC1 can be measured, 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 method described in Patent Document 3 and other documents, but is not limited to these, and any known technique can be used.

As used herein, skin stem cell activation refers to promoting the 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 levels of stem cell markers such as integrin β1, etc. 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.

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

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.

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

Akebia quinata is a deciduous shrub belonging to the genus Akebia in the family Akebiaceae. Akebia has been reported to have hyaluronic acid production promoting effects, collagen production promoting effects, and MMP inhibitory effects, as well as wrinkle prevention and improvement effects, lipase inhibitory activity, blood neutral fat concentration increase suppression activity, and anti-obesity activity (JP Patent Publication No. 2015-17048, JP Patent Publication No. 2010-265182, etc.). As the Akebia extract used in the present invention, an extract of the stem of Akebia is preferable, but since the fruit, peel, seeds, leaves, flowers, roots, etc. of Akebia also contain active ingredients, extracts of any one or more of these can also be used.

Black soybeans (Glycine max 'Kuromame') are a variety of soybean, an annual plant of the legume family, and varieties such as 'Tambaguro' and 'Wachiguro' can be used. They are rich in polyphenols such as anthocyanins, and it has been reported that ingredients derived from black beans have anti-obesity, anti-inflammatory, antioxidant, and glucose tolerance improving effects (JP Patent Publication No. 2015-140298, etc.). The black soybean extract is preferably an extract of the legume of black soybeans, but since active ingredients are also contained in the pods, leaves, stems, flowers, roots, etc. of black soybeans, extracts of one or more of these can also be used.

Peony (Paeonia lactiflora) is a perennial plant of the Peonies 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 (JP Patent Publication No. 2019-11252, JP Patent Publication No. 2012-121856, etc.). Peony extract is preferably an extract of peony root, but peony leaves, stems, flowers, fruits, peels, seeds, etc. also contain active ingredients, so extracts of one or more of these can also be used.

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 catechin, and to have lipid metabolism improving effects, antioxidant effects, anticancer effects, and blood pressure suppression effects. As the tea extract, an extract of tea leaves or stems is preferable, but since tea fruits, flowers, roots, seeds, etc. also contain active ingredients, extracts of one or more of these can also be used.

Jojoba (Simmondsia chinensis) is an evergreen shrub belonging to the family Jojobaceae in the order Caryophyllaceae. Jojoba is known to have moisturizing, emollient, and anti-inflammatory effects, as well as collagenase inhibitory, elastase inhibitory, and hyaluronidase inhibitory effects (JP Patent Publication Nos. 2003-48846, 2003-048812, and 2003-034644, etc.). Jojoba leaf extract is an extract of jojoba leaves, but since active ingredients are also contained in jojoba seeds, fruits, flowers, roots, etc., extracts of one or more of these can also be used.

Ergothioneine (L-Ergothioneine) is an amino acid with a molecular weight of 229.3 and has the following structure, and is known to have antioxidant, anti-inflammatory, elastase inhibitory, and tyrosinase inhibitory effects (JP Patent Publication No. 2019-149972, etc.). Ergothioneine may be chemically synthesized, or may be used in the form of an extract of such natural products, since it is contained in microorganisms such as basidiomycetes, animals, plants, etc.

The above-mentioned various extracts may be commercially available as cosmetic raw materials or health food materials, or may be obtained by a conventional method. The extraction method is not particularly limited, but examples include extraction using a solvent and extraction by hydrolysis. When performing extraction using a solvent, the raw material is immersed or heated under reflux with the extraction solvent at room temperature or with heat, and when performing extraction by hydrolysis, any hydrolysis treatment, such as chemical treatment with alkali or acid, physical treatment with heat or pressure, or biological treatment with enzymes, is performed, and then the extract is filtered and concentrated. The raw material can be used as is, but it is better to crush it into granules or powder and then subject it to extraction, because it is possible to extract the active ingredient with high extraction efficiency under mild conditions in a short time. The extraction temperature is not particularly limited, and may be set appropriately depending on the particle size of the crushed 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 may be set appropriately depending on the particle size of the crushed material, the type of solvent, the extraction temperature, etc. Furthermore, during extraction, stirring may be performed, the mixture may be left to stand without stirring, or ultrasonic waves may be added.

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.

By such an extraction operation, 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 used after undergoing conventional purification treatments such as sterilization, washing, filtration, decolorization, and deodorization. If necessary, it may be concentrated by freeze-drying or diluted with any solvent before use. Furthermore, the solvent or hydrolyzate may be completely volatilized to form a solid (dried product) before use, or the dried product may be redissolved in any solvent before use.

In addition, the squeezed liquid obtained by squeezing the raw materials contains the same active ingredients as the extract, so the squeezed liquid can be used instead of the extract.

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 via MPC1 inhibitory activity.

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 wishes to activate skin stem cells preventively.

The agent or composition of the present invention can be applied by any route, such as external administration or oral administration, but is preferably incorporated into an external skin agent that can be applied directly to the skin. The form of external administration can be selected from any of the following forms: liquid, emulsion, cream, solid, sheet, spray, gel, foam, powder, etc. The agent or composition may be a cosmetic composition such as emulsion, cream, beauty essence, lotion, pack, or face wash. The form of oral administration can be selected from any of the following forms: tablet, supplement, drink, powder, etc. The agent or composition of the present invention can be appropriately incorporated with optional ingredients used in compositions such as cosmetics and pharmaceuticals, as necessary, within a range that does not impair the effects of the agent or composition. Examples of the optional ingredients include excipients, carriers, diluents, oils, surfactants, powders, coloring materials, water, alcohols, thickeners, chelating agents, silicones, antioxidants, ultraviolet absorbers, moisturizers, fragrances, various medicinal ingredients, preservatives, pH adjusters, and neutralizers. For example, other medicinal ingredients that promote the activation of skin stem cells may be included.

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 as needed, but is not limited to these.

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.

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% by weight, 0.0001 to 90% by weight, 0.001 to 50% by weight, 0.01 to 5% by weight, 0.01 to 1% by weight, 0.1 to 0.5% by weight, 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.

The present application also provides a screening method for skin stem cell activators using MPC1 inhibitory activity as an index. 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 whether or not a candidate drug has skin stem cell activation activity, enabling product development and the proposal of new skin care methods.

The skin sample may be a skin sample after collection, for example, a skin sample in an ex vivo state after collection from an animal such as a human, or may be in an in vitro state such as cultured skin cells, for example, cultured keratinocytes or cultured fibroblasts. Alternatively, it 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 effect.

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 inhibition of MPC1.

The present invention will now be described in more detail with reference to examples. Note that the present invention is not limited to these examples.

Experiment 1: Effect of MPC1 inhibition on stem cells Experiment 1-1: Gene expression analysis of MCSP by PCR method
To examine 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.

Cell culture Normal human fetal epidermal keratinocytes (ScienCell, Cat No. 2120) were cultured in epidermal keratinocyte culture medium (Kurabo, KK-2150S). After passage, the cells were seeded in a 6-well plate at 2.5 × 10 ⁵ cells/well/2 mL, and 24 hours later, UK5099, control, and echinomycin were added. Echinomycin is an inhibitor of HIF-1α, which controls mitochondrial activity, like MPC1, but differs in that it inhibits the conversion of pyruvate to acetyl-CoA in mitochondria. Echinomycin was used to examine whether the pathway that inhibits pyruvate delivery into mitochondria or the pathway that inhibits the conversion of pyruvate to acetyl-CoA in mitochondria activates epidermal keratinocyte stem cells. UK5099 (Merck: Catalog No. 504817) was diluted with DMSO to a concentration of 10 mM and 20 mM to prepare a stock solution, which was then added to the medium at a 1000-fold dilution to give a final concentration of 10 μM and 20 μM. DMSO was added at a 1000-fold dilution as a control. Echinomycin (Abcam: Product No. ab144247) was diluted with DMSO to a concentration of 10 μM and 20 μM to prepare a stock solution, which was then added to the medium at a 1000-fold dilution to give a final concentration of 10 nM and 20 nM. mRNA was collected 24 hours after the addition of these substances.

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). The gene expression levels of MCSP and B2M were measured by the Syber Green method using 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)

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

Experiment 1-2: Protein expression analysis of MCSP and Integrin β1 by cell staining Furthermore, in addition to MCSP, we also performed cell staining and analysis of another stem cell marker, Integrin β1.
Epidermal keratinocytes subcultured as in Experiment 1-1 were treated with UK5099 at 20μM. As a control, the same amount of DMSO was added without treatment with UK5099. The cells were seeded on a 4-well chamber slide (Falcon, 354114) at 1.0×10 ⁴ cells/well/0.5mL and cultured for 48 hours. After 48 hours, the cells were reacted with 4% PFA for 10 minutes and fixed. Then, the cells were reacted with 0.01% Triton-X100/PBS for 15 minutes to partially dissolve the cell membrane. After blocking with 12% BSA/PBS, the cells were reacted overnight at 4℃ with anti-MCSP antibody (Millipore, MAB2029, mouse mAb) and anti-β1 integrin antibody (Santa Cruz, sc-13590, mouse mAb) as primary antibodies. The next day, the cells were washed three times for 5 minutes with PBS and reacted with Alexa488-anti-mouse IgG antibody as secondary antibody at room temperature for 1 hour. 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.
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.

Experiment 1-3: FACS analysis of integrin β1 positive cells Next, FACS analysis of the number of integrin β1 positive cells was performed. Epidermal keratinocytes subcultured in the same manner as in Experiment 1-1 were added with UK5099 at 20μM, and as a control, they were not treated with UK5099 and the same amount of DMSO was added and cultured for 48 hours. These cells were detached with trypsin and a cell suspension was prepared at a concentration of 1.0×10 ⁶ cells/500uL. 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 reacted on ice for 1 hour, washed three times by centrifugation with 0.1% BSA/PBS, and FACS analysis was performed using a cell sorter SH800. In addition, no antibody was added to the negative control (Negative cont), and the same treatment was performed.

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

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 medium at 1μM and 5μM, and as a control, the same amount of DMSO was added without treatment with UK5099, and the skin model was cultured at 2.5mL/well. The medium was replaced after 2 days, and 4 days after the start of culture, the skin model was immersed in refrigerated acetone for dehydration and fixation. Then, the solvent was replaced twice with room temperature acetone, twice with room temperature methyl benzoate, and twice with room temperature xylene, and paraffin embedding was performed to create a paraffin block. Sections were made at a thickness of 3μm using a microtome. After deparaffinization using xylene and blocking with 12% BSA/PBS, the skin model was reacted overnight at 4℃ with anti-MCSP antibody (Millipore, MAB2029, mouse mAb) and anti-α6 integrin antibody (Santa Cruz, sc-19622, rat mAb) as primary antibodies. 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.

Figure 5 shows the results of adding 1 μM and 5 μM UK5099 and the control. Addition of UK5099 increased MCSP and integrin β1 expressing cells, and the results were consistent with the results of Experiments 1-1, 1-2, and 1-3, even when using a three-dimensional skin model.

Experiment 1-5: Protein expression analysis of MCSP and integrin β1 by tissue staining. Fresh human abdominal skin purchased from BioPredic was cultured at 3mL/well with 10μM UK5099 added to the medium, and the same amount of DMSO was added to the control without treatment with UK5099. The medium was replaced after 2 and 4 days, and 5 days after the start of culture, the cells were immersed in refrigerated acetone for dehydration and fixation. After that, the solvent was replaced twice with acetone at room temperature, twice with methyl benzoate at room temperature, and twice with xylene at room temperature, and paraffin embedding was performed to create paraffin blocks. Sections were made at a thickness of 3μm using a microtome. After deparaffinization using xylene and blocking with 12% BSA/PBS, the cells were reacted overnight at 4℃ with anti-MCSP antibody (Millipore, MAB2029, mouse mAb) and anti-α6 integrin antibody (Santa Cruz, sc-19622, rat mAb) as primary antibodies. 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.

Figure 6 shows the results of adding 10 μM UK5099 and the control. Addition of UK5099 increased MCSP and integrin β1 expressing cells, and the results were consistent with the results of Experiments 1-1, 1-2, 1-3, and 1-4 when using ex vivo human skin samples.

Experiment 2: Screening for substances with MCP1 inhibitory effects Experiment 1 suggested that inhibiting MCP1 activates stem cells. Therefore, we screened for substances with MCP1 inhibitory effects.
Experiment 2-1: Sample preparation
The following samples were used for evaluation of MPC1 inhibitory activity.

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

Experiment 2-2: Evaluation of MCP1 inhibitory effect Normal human fetal epidermal keratinocytes were cultured in the same manner as in Experiment 1-1, and each drug was added 24 hours after seeding. Candidate samples were diluted to 10% with DMSO in advance and added to the medium at a 1000-fold dilution to a final concentration of 0.01% in the first and second screening, and added to the medium at a 10,000-fold, 1000-fold, and 100-fold dilution to a final concentration of 0.001%, 0.01%, and 0.1% in the third screening. UK5099 was added to the medium at 20 μM as a positive control in the same manner as in Experiment 1. DMSO was added at a 1000-fold dilution as a negative control. mRNA was collected 24 hours after addition of each drug.

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 was measured by the Syber Green method using 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)

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 substances with MPC1 inhibitory activity.
The 37 compounds selected in the primary screening were subjected to secondary screening at a drug concentration of 0.01%, 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 candidates for compounds that have MPC1 inhibitory activity in a concentration-dependent manner.
Tertiary screening was performed on the 15 products selected in the secondary screening at drug concentrations of 0.001%, 0.01%, and 0.1%, N=3 for each. The results of the tertiary screening are shown in Figure 9. Six products (dark grey substances in Figure 9) were selected from the 15 products as candidates for substances that reproducibly inhibit MPC1 in a concentration-dependent manner: Akebia extract, black bean extract, peony extract, tea extract, jojoba leaf extract, and ergothioneine.

Figure 10 shows a graph of the results of Figure 9 for each of the six selected products, which were statistically processed using Dunnett's test. As shown in Figure 10, all substances exerted a concentration-dependent MPC1 inhibitory effect with statistically significant differences.

These results show 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 suppressing skin aging.

Claims (5)

An MPC1 inhibitor containing Akebia extract as an active ingredient. A skin stem cell activator comprising an MPC1 inhibitor,
the skin stem cells are keratinocyte stem cells,
A skin stem cell activator, wherein the MPC1 inhibitor contains Akebia extract as an active ingredient. A method for screening a skin stem cell activator using MPC1 inhibitory activity as an index, comprising:
The screening method, wherein the skin stem cells are keratinocyte or fibroblast stem cells. contacting the keratinocytes with a candidate agent;
measuring the activity, amount, and/or expression level of MPC1 in keratinocytes contacted with the candidate agent;
determining the MPC1 inhibitory effect of the candidate drug from the measured MPC1 activity, amount, and/or expression level;
selecting a keratinocyte stem cell activator based on the determined MPC1 inhibitory activity;
A method for screening for a keratinocyte stem cell activator, comprising: contacting fibroblasts with a candidate agent;
Measuring the activity, amount, and/or expression level of MPC1 in fibroblasts 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 fibroblast stem cell activator based on the determined MPC1 inhibitory activity;
A method for screening a fibroblast stem cell activator, comprising:

Images