JP6923111B2 - Screening method for skin aging improving agents - Google Patents

Description

The present invention relates to a method for screening a skin aging improving agent.

Since skin aging symptoms such as wrinkles and tarmi have a great influence on the impression of appearance, there is a great deal of interest in improving them. In recent years, anti-aging agents, anti-wrinkle agents, moisturizers and the like have been actively developed for the purpose of improving skin aging symptoms. They mainly target dermal fibroblasts that produce substances that maintain skin tension and elasticity, such as collagen and elastin (Patent Documents 1 and 2 etc.), and epidermal basal cells. (For example, Patent Document 3 etc.).

Japanese Unexamined Patent Publication No. 2015-131977 Japanese Unexamined Patent Publication No. 2015-174857 Japanese Unexamined Patent Publication No. 2003-171225

Although conventional anti-aging agents have some degree of anti-aging effect, it cannot be said that a sufficiently satisfactory effect is obtained. Further elucidation of the mechanism by which skin aging symptoms occur is also required.
In view of such a situation, an object of the present invention is to search for an effective ingredient as a skin aging improving agent by a new approach to skin aging symptoms, and an object of the present invention is to establish a new screening method for that purpose.

As a result of diligent research to solve the above problems, the present inventor focused on the subcutaneous tissue existing under the dermis, and considered that the stimulation received from blood by subcutaneous adipocytes could also cause skin aging symptoms. The effect of subcutaneous adipocytes on adipocytocytosis activity when stimulated in the blood and the effect of adipocytosis activity on dermal fibroblasts were investigated. Then, they have obtained the finding that the skin aging symptom can be improved by activating or inactivating a certain adipocytokine, and have completed the present invention.

That is, the present invention is as follows.
[1] A method for screening a skin aging improving agent using the activity of an aging regulator in subcutaneous adipocytes as an index.
[2] The method according to [1], wherein the aging control factor changes any of the physical quantities involved in the development of skin aging symptoms.
[3] The amount of physical quantity involved in the development of the skin aging symptom is selected from the group consisting of collagen amount, elastin amount, hyaluronic acid amount, versican amount, autophagy activity, and skin support structure, according to [2]. Method.
[4] The method according to any one of [1] to [3], wherein the expression level of the aging regulator varies depending on the stimulation of subcutaneous adipocytes.
[5] The activity of the aging control factor is the expression level of the gene encoding the protein constituting the factor or the protein, and the expression level in the subcutaneous adipocytes to which the stimulus and the test substance are added is the expression level of the stimulus. The method according to any one of [1] to [4], wherein the test substance is determined to have an effect of improving skin aging when the expression level is large or small as compared with the expression level in cells to which the test substance is not added.
[6] The method according to [4] or [5], wherein the stimulus is selected from substances whose amount varies depending on lifestyle and aging.
[7] The substance whose amount varies depending on lifestyle and aging consists of oxidized LDL, glucose, hydrogen peroxide, estradiol, caffeine, acetaldehyde, vitamins, orexin, progesterone, testosterone, DHEA, and methylglyoxal. The method according to [6], which is selected from the group.
[8] The method according to [7], wherein the stimulus is oxidized LDL and the aging regulator is selected from the group consisting of 4-1BB, FasL, IGFBP2, MSPα, IFNγ, and PAI-1.
[9] A group in which the stimulus is glucose and the aging regulator is 4-1BB, CRP, IGFBP2, IL-12, RANTES, SAA, SDF-1, sTNFRI, sTNFRII, VEGF, and IL-6sR. The method according to [7], which is selected from.
[10] The method according to [7], wherein the stimulus is hydrogen peroxide, and the aging regulator is selected from the group consisting of IL-8, leptin, GH1, and OPG.
[11] The stimulus is estradiol, and the aging regulator is selected from the group consisting of ACE2, AgRP, CRP, IGFBP3, LeptinR, sTNFRI, ST2, MCP3, PDGFAB, and VEGF [7]. Method.

INDUSTRIAL APPLICABILITY The present invention provides a screening method capable of searching for an effective ingredient as a skin aging improving agent, which is suitable for being contained in an external preparation for skin or food and drink.

A conceptual diagram showing the effect of stimulated subcutaneous adipocytes on skin aging symptoms.

A new mechanism of skin aging discovered by the present inventors will be described with reference to FIG.
In subcutaneous adipocytes stimulated by factors such as aging, lifestyle, and environment, the expression of certain adipocytokines fluctuates (increases or decreases). The adipocytokine is an "aging regulator" involved in skin aging symptoms. For example, changes in the expression of adipocytocytocytosis enhance or attenuate its activity, and physical quantities involved in the development of skin aging symptoms, such as collagen, elastin, hyaluronic acid, versican, autophagy activity, and skin support structure. Etc. are caused. More specifically, the enzyme that decomposes collagen, elastin, hyaluronic acid, or versican increases to decrease the amount of collagen, elastin, hyaluronic acid, or versican, or the autophagy-deteriorating protein (monodansylcadaverine; MDC) The autophagy activity is increased and attenuated, or the expression level of proteins constituting the network structure of the subcutaneous support zone under the subcutaneous tissue is decreased and the network structure becomes sparse. As a result, skin aging symptoms such as wrinkles and tarmi develop in the dermis. In such a skin aging flow, the activity of the aging regulator in the subcutaneous adipocytes is enhanced or attenuated in the direction opposite to the fluctuation due to the stimulus, whereby the skin aging symptom can be suppressed and improved. In other words, a substance that suppresses fluctuations in aging regulators in stimulated subcutaneous adipocytes can be a skin aging improver.

Therefore, the method for screening a skin aging improving agent of the present invention is characterized by using the activity of an aging regulator in subcutaneous adipocytes as an index.

In the present invention, the aging control factor usually refers to a factor that changes a physical quantity involved in the occurrence of skin aging symptoms such as wrinkles and tarmi. Specifically, adipocytokines involved in changes in collagen amount, elastin amount, hyaluronic acid amount, versican amount, autophagy activity, skin support structure (RC structure) and the like are preferably mentioned.
Table 1 shows an index of skin aging and examples of adipocytokines involved in the fluctuation of the index, but is not limited thereto.

Collagen and elastin are fibrous proteins that make up more than 90% of the dermis, which functions as a support for skin structure and plays a mechanical role. They exist as bundled fiber aggregates and are present in almost all layers of the dermis. They are intertwined to form a mesh-like fibrous structure. In general, aging and ultraviolet rays increase the amount of fibrous protein-degrading enzymes, causing a decrease in the amount of collagen and elastin, resulting in thinning or loosening of the dermis layer. In addition, hyaluronic acid plays a role of storing water inside the skin, and the phenomenon is generally caused by aging.
Versican is a proteoglycan that serves as a scaffold for fibroblasts to work when the dermis is made, and is known to decrease with age, leading to skin aging and decreased elasticity (Fragrance Journal 44 (1)). : 88 -88, 2016). As shown in the examples below, activation of certain aging regulators increases versican-degrading enzymes, causing a decrease in versican content.
Autophagy is one of the mechanisms for degrading intracellular proteins and organelles, and it is known that when autophagy activity is enhanced, anti-aging action works (N. Engl. J. Med. , 2013 Feb 14; 368 (7): 651-62.). As shown in Examples below, activation of certain aging regulators increases autophagy-deteriorating proteins (MDCs) and suppresses autophagy activity.
The skin support structure (RC structure) refers to a network structure called the subcutaneous support band (retinacula cutis; RC) in the lower part of the subcutaneous tissue, and when the structure becomes sparse, the elasticity of the deep part of the skin decreases and tarmi is caused (fragrance). See Journal 44 (2), 23-27, 2016). As shown in Examples described later, when a specific aging regulator is activated, the expression level of proteins constituting the RC structure is reduced, and the RC structure is weakened (the network structure is sparse).

Further, in the present invention, it is preferable that the expression level of the aging regulator varies depending on the stimulation of subcutaneous adipocytes.
In general, the abundance of various components in blood fluctuates due to lifestyle, aging, etc., but the present inventors receive stimulation (blood stimulation) by components that increase or decrease the blood concentration of subcutaneous adipocytes. , Found that the expression level of certain adipocytokines fluctuates. Specifically, blood stimulation increases or decreases the expression level of the gene encoding the adipocytokine protein or the protein.

The blood stimulus is preferably a substance whose amount varies depending on lifestyle or aging, and specifically, but not particularly limited, hydrogen peroxide, glucose, oxidized LDL, estradiol, caffeine, acetaldehyde, vitamins, orexin. , Progesterone, testosterone, DHEA, methylglyoxal and the like are preferable.
It is known that hydrogen peroxide, glucose, and oxidized LDL increase in blood concentration with aging, and estradiol, progesterone, testosterone, and DHEA decrease in blood concentration with aging. In addition, caffeine, acetaldehyde, vitamins, orexin, and methylglyoxal are components whose blood concentrations can fluctuate due to feeding behavior.

In the present invention, examples of combinations of aging regulators whose expression levels vary depending on a specific stimulus include those shown in Table 2.
When hydrogen peroxide is added to subcutaneous adipocytes as a stimulus, the expression of interleukin-8 (IL-8) and leptin increases, and the expression of growth hormone 1 (GH1) and osteoprotegrin (OPG) decreases. do.
When glucose is added as a stimulus to subcutaneous fat cells, 4-1BB, C-receptor protein (CRP), insulin-like growth factor-binding protein 2 (IGFBP2), interleukin-12 (IL-12), activated and regulated expression And secretory normal T cells (RANTES), serum amyloid A protein (SAA), stromal cell-derived factor 1 (SDF-1), TNF superfamily type 1 receptor (sTNFRI), TNF superfamily type 2 receptor (sTNFRII), Expression of vascular endothelial cell growth factor (VEGF) is increased and expression of interleukin-6sR (IL-6sR) is decreased.
Addition of oxidized LDL as a stimulus to subcutaneous adipocytes increases the expression of 4-1BB, Fas ligand (FasL), IGFBP2, MSPα, interferon gamma (IFNγ), and plasminogen activator (PAI-1). Expression is reduced.
When estradiol is added as a stimulus to subcutaneous fat cells, angiotensin converting enzyme 2 (ACE2), agouti-related peptide (AgRP), CRP, insulin-like growth factor binding protein 3 (IGFBP3), leptin receptor (LeptinR), sTNFRI, ST2, The expression of monocytrogenic protein 3 (MCP3), platelet-derived growth factor AB (PDGFAB), and VEGF is reduced.

The activity of the aging regulator, which is an index in the screening method of the present invention, is usually the expression level of the aging regulator. Here, the expression level refers to either the transcription amount of mRNA of the gene or the translation amount of the protein encoded by the gene.

In a preferred embodiment of the screening method of the present invention, the expression level of the aging control factor in the subcutaneous adipocytes to which the stimulus and the test substance were added is compared with the expression level (control) in the cells to which the stimulus was added and the test substance was not added. When it is large or small, it is determined that the test substance has an effect of improving skin aging.

Here, in the case of an aging regulator whose expression level increases due to the addition of a stimulus that increases in blood with aging, when the expression level when the test substance is added is smaller than the control, the test substance improves skin aging. It is determined to have an action. On the contrary, in the case of an aging regulator whose expression level decreases due to the addition of a stimulus that increases in blood with aging, when the expression level when the test substance is added is larger than the control, the test substance improves skin aging. It is determined to have an action.
Further, in the case of an aging regulator whose expression level increases due to the addition of a stimulus that decreases in blood with aging, when the expression level when the test substance is added is larger than the control, the test substance has an effect of improving skin aging. Is determined to have. On the contrary, in the case of an aging regulator whose expression level decreases due to the addition of a stimulus that decreases in blood with aging, when the expression level when the test substance is added is smaller than the control, the test substance improves skin aging. It is determined to have an action.

The expression level of the gene encoding the protein, which is an aging regulator, can be measured by any method. For example, PCR is performed using a DNA fragment having a sequence that specifically binds to the sequence of the gene as a primer to perform quantitative detection. The human gene sequences encoding the above-mentioned adipocytokines have been published, and those skilled in the art can appropriately design primers and use them for PCR.
Further, for example, the amount of the protein on the cell membrane may be measured by a conventional method, for example, by an immunological method using an antibody, and used as the expression level of the gene.

Subcutaneous adipocytes are used as the cells used in the screening method of the present invention.
The cell culture conditions are not particularly limited as long as they do not interfere with the execution of the screening method of the present invention, specifically, the measurement of the expression level of the aging regulator, in addition to the normal culture conditions. Can be applied.

The test substance targeted by the screening method of the present invention may be a pure substance, an extract derived from animals and plants, or a mixture thereof.
The animal or plant-derived extract shall mean not only the animal or plant-derived extract itself, but also the fraction of the extract, the purified fraction, the extract or fraction, and the solvent-removed product of the purified product. Examples of the plant-derived extract include native or grown plants, extracts using those sold as raw materials for Chinese herbal medicine, and commercially available extracts.
In the extraction operation, in addition to using the whole plant part, parts such as the plant body, the above-ground part, the rhizome part, the tree trunk, the leaf part, the stem part, the flower, the flower bud, and the fruit can be used, but these are crushed in advance. Alternatively, it is preferable to cut into small pieces to improve the extraction efficiency. Extraction solvents include water, alcohols such as ethanol, isopropyl alcohol and butanol, polyhydric alcohols such as 1,3-butanediol and polypropylene glycol, ketones such as acetone and methyl ethyl ketone, and ethers such as diethyl ether and tetrahydrofuran. One or more selected from polar solvents such as, etc. can be exemplified as suitable ones. As a specific extraction method, for example, 1 to 30 parts by mass of a solvent is added to 1 part by mass of a part used for extraction of a plant or the like or a dried product thereof, and the solvent is added at room temperature for several days at a temperature near the boiling point. If there is, a method of immersing for several hours, cooling to room temperature, removing insoluble matter and / or solvent if desired, and fractionally purifying by column chromatography or the like can be mentioned.

An example of the procedure in the screening method of the present invention is given below, but the content is not limited to the following as long as the gist of the present invention is not deviated, and the procedure can be appropriately modified.
First, a stimulant is added to subcutaneous adipocytes and incubated for 1 to 2 hours. Then, the test substance is added and incubated for 24 hours. Then, mRNA is extracted from the cells, and the expression level of the gene encoding the aging regulator as an index is quantitatively measured by performing RT-PCR using a primer that specifically detects the gene. The expression level of the gene is also measured in cells to which the stimulant is added as a control and the test substance is not added. When the expression level of the gene in the cells to which the test substance is added fluctuates with respect to the expression level (control) of the gene in the cells to which the test substance is not added, the test substance has an effect of improving skin aging. Determined to have. The determined substance can be a skin aging improving agent that can be suitably contained in a skin external preparation for massage.
As for the degree of variation in the expression level, in the case of an increase in the expression level, 120% or more is preferable, 135% or more is more preferable, and 150% or more is further preferable with respect to the control. In the case of a decrease in the expression level, 80% or less is preferable, 65% or less is more preferable, and 50% or less is further preferable with respect to the control.

Improvements in skin aging symptoms include, for example, subjective evaluation of subjects who applied skin aging improving agents, evaluation of wrinkles and tarmi by image analysis, evaluation of skin viscous properties, evaluation of skin water retention, etc. , Can be confirmed by a well-known method.

A substance (skin aging improving agent) determined to have an action of improving skin aging symptoms by the screening method of the present invention can be contained in a composition by any preparation method, and such a composition includes, for example, skin. External compositions, food and drink compositions and the like are preferably mentioned. Such compositions are preferably applicable for anti-aging applications.
The substance (skin aging improving agent) determined to have an action of improving skin aging symptoms by the screening of the present invention is a new mechanism targeting the occurrence of skin aging caused by blood irritation received by subcutaneous fat cells. In terms of improving skin aging symptoms, it can be a compounding ingredient of an effective anti-aging composition by a new approach.

The content (blending amount) of the skin aging improving agent in the anti-aging composition according to the present invention is usually 0.00001% by mass or more, preferably 0.0001% by mass or more, and more preferably 0.001% by mass. It is usually 15% by mass or less, preferably 10% by mass or less, and more preferably 5% by mass. If the content (blending amount) of the skin aging improving agent is too small, it may be difficult to obtain the desired effect, and if it is too large, the effect may reach a plateau and the degree of freedom in formulating the composition may be impaired. be. Further, the type of the skin aging improving agent contained in the composition may be not only one type but also two or more types.

In the production of the anti-aging composition according to the present invention, it is possible to add an optional ingredient usually used in the formulation of cosmetics, quasi-drugs, pharmaceuticals, etc., and the production of oral compositions such as foods and drinks. can.
For example, in the case of external composition for skin, hydrocarbons such as squalane, vaseline and microcrystalin wax, esters such as jojoba oil, carnauba wax and octyldodecyl oleate, triglycerides such as olive oil, beef fat and coconut oil, Fatty acids such as stearic acid, oleic acid and retinoic acid, higher alcohols such as oleyl alcohol, stearyl alcohol and octyldodecanol, anionic surfactants such as sulfosuccinate and sodium polyoxyethylene alkyl sulfate, and both sexes such as alkyl betaine salts. Surfactants, cationic surfactants such as dialkylammonium salts, sorbitan fatty acid esters, fatty acid monoglycerides, these polyoxyethylene adducts, polyoxyethylene alkyl ethers, nonionic surfactants such as polyoxyethylene fatty acid esters, etc. , Polyethylene glycol, glycerin, polyhydric alcohols such as 1,3-butanediol, thickening / gelling agents, antioxidants, ultraviolet absorbers, coloring agents, preservatives, powders, etc. can be arbitrarily blended. can.

In addition, the anti-aging composition according to the present invention also contains a skin aging improving component other than a substance (skin aging improving agent) determined to have an action of improving skin aging symptoms by the screening of the present invention. May be good.
The anti-aging composition according to the present invention can be produced by treating and blending the above-mentioned components according to a conventional method. The form can be any dosage form such as lotion type, emulsifier type, oil type, etc. in the case of external composition for skin, and tablets, granules, powders, liquids, etc. in the case of oral composition. In addition to the dosage form of, foods and beverages can be used arbitrarily.

Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited to the following Examples as long as the gist thereof is not exceeded.

<Test Example 1> Confirmation of the effect of stimulation on adipocytokine expression The changes in protein secreted from subcutaneous adipocytes when stimulated were evaluated by the following procedure.
^{Normal human subcutaneous adipocytes were seeded on a 24-well plate at 1.0 × 10 5} cells / well using a growth medium (manufactured by CELL) and cultured for 2 days in a 37 ° C., 5% CO _{2 environment.} After culturing, the medium was removed, the cells were washed with PBS, replaced with a differentiation medium (manufactured by CELL), and cultured for 10 days _{in a 37 ° C., 5% CO 2 environment.} After culturing, the medium was removed, the cells were washed with PBS, replaced with a maintenance medium (manufactured by CELL), and after culturing for 6 hours, any of the stimuli shown in Table 3 or a maintenance medium containing a solvent control was added. The cells were cultured at 37 ° C. in a 5% CO ₂ environment for the time shown in Table 3. After culturing, the medium was removed, the cells were washed with PBS, replaced with a maintenance medium (manufactured by CELL), and after culturing for 48 hours, the culture supernatant was collected, and the Human Adipocytokine (Adipokine) Antibody Array kit (manufactured by Ray Biotech) was collected. ) Was detected by an antigen-antibody reaction. When the solvent control group is 1,
The relative expression levels are shown in Table 4.

<Test Example 2> Confirmation of the effect of adipocytokines on skin aging 1
The following procedure was used to evaluate whether adipocytokines affect skin aging.
Using DMEM medium (manufactured by SIGMA) or Tenocyte Culture Medium (manufactured by Ds Pharma Biomedical), normal human dermal fibroblasts or normal human tendon cells were added to a 24-well plate at 3.0 × 10 ⁴ cells / well or 2 Seeded at 0.0 × 10 ⁴ cells / well and cultured for 1 day in a 37 ° C., 5% CO _{2 environment.} After culturing, the medium is removed, the cells are washed with PBS, DMEM medium or Tenocyte Culture Medium containing adipocytokine (recombinant protein shown in Table 5) or solvent control is added, and the temperature is 37 ° C. in a 5% CO ₂ environment. Was cultured for 24 hours or 48 hours.
After extracting mRNA of the above normal human dermal fibroblasts or normal human tendon cells using RNeasy Mini Kit (manufactured by QIAGEN) and synthesizing cDNA using Superscript VILO DNA synthesis Kit (manufactured by Lifetechnologies), versican-versican degrading enzyme Alternatively, the expression level of RC structural protein was measured by the real-time qPCR method. In addition, the mRNA expression level of ACTB, which is an endogenous control gene, was measured in the same manner. For the measurement, the QuantiFact SYBR GREEN PCR kit (manufactured by QIAGEN) and the primers shown in Table 6 were used.
The expression level of each mRNA in the recombinant protein addition group and the solvent control group of the fluctuating protein was corrected by the expression level of ACTB, and when the expression level of each mRNA in the solvent control group was set to 1, the recombinant protein addition of the fluctuating protein was added. The expression level of each mRNA in the group was calculated. Some of the results are shown in Table 7 together with the results of the following Test Example 3.

<Test Example 3> Confirmation of the effect of adipocytokines on skin aging 2
The following procedure was used to evaluate whether the fluctuating protein affects skin aging.
^{Normal human dermal fibroblasts were seeded in 96-well plates at 5.0 × 10 3} cells / well using DMEM medium (manufactured by SIGMA), and cultured for 1 day in a 37 ° C., 5% CO _{2 environment.} After culturing, the medium was removed, the cells were washed with PBS, DMEM medium containing adipocytokine (recombinant protein shown in Table 5) or solvent control was added, and the cells were added at 37 ° C. and 5% CO ₂ environment for 24 hours. Alternatively, the cells were cultured for 48 hours. After culturing, the medium was removed, the cells were washed with PBS, and the amount of MDC in the adipocytokine-added group or the solvent control group was measured using the Autophagy / Cytotoxicity Dual Staining Kit (manufactured by Cayman Chemical). Recombinant protein of fluctuating protein Recombinant protein of fluctuating protein when each MDC amount in the solvent control group and the solvent control group was corrected by each MDC amount in the solvent control group and each MDC amount in the solvent control group was set to 1. The amount of each MDC in the addition group was calculated. Some of the results are shown in Table 7 together with the results of Test Example 2.

<Example 1> Screening of skin aging improving agent A skin aging improving agent was screened using the gene expression of an aging regulator (adipocytokine) as an index according to the following procedure.
^{Normal human subcutaneous adipocytes were seeded on a 24-well plate at 1.0 × 10 5} cells / well using a growth medium (manufactured by CELL), and cultured for 2 days in a 37 ° C., 5% CO _{2 environment.} After culturing, the medium was removed, the cells were washed with PBS, replaced with a differentiation medium (manufactured by CELL), and cultured for 10 days _{in a 37 ° C., 5% CO 2 environment.} After culturing, the medium was removed, the cells were washed with PBS, a maintenance medium (manufactured by CELL) containing the stimulant or solvent control shown in Table 3 was added, and the table _{was prepared in a 37 ° C., 5% CO 2 environment.} The cells were cultured for the time shown in 3. After incubation, the medium was removed and the cells washed, added maintenance medium containing extract or vehicle control, under 37 ℃ · 5% CO ₂ environment at PBS, 2
Incubated for 4 hours. After culturing, the medium is removed, the cells are washed with PBS, the mRNA of the above normal human subcutaneous adipocytes is extracted using QIAzol Lysis Reagent (manufactured by QIAGEN), and the Superscript VILO DNA synthesis Kit (manufactured by Lifetechnologies) is used. After synthesizing the cDNA using the cDNA, the expression level of the protein affecting skin aging was measured by the real-time qPCR method. In addition, the mRNA expression level of ACTB, which is an endogenous control gene, was measured in the same manner. For the measurement, the QuantiFact SYBR GREEN PCR kit (manufactured by QIAGEN) and the primers shown in Table 8 were used.
Each mRNA expression level in the extract-added group and the solvent control group was corrected by the expression level of ACTB, and each mRNA expression level in the extract-added group was calculated when each mRNA expression level in the solvent control group was set to 1. Some of the results are shown in Tables 9-11 together with the results of Example 1.

According to the screening method of the present invention, an effective ingredient as a skin aging improving agent can be searched for. Such a skin aging improving agent is very useful industrially because it can be suitably contained in an external preparation for skin for anti-aging use and foods and drinks.

Claims (9)

It is a method of screening a skin aging improving agent using the activity of an aging regulator in subcutaneous adipocytes as an index.
The expression level of the aging regulator varies depending on the stimulation of subcutaneous adipocytes.
The activity of the aging regulator is the expression level of the gene encoding the protein constituting the factor or the protein, and the expression level in the subcutaneous adipocytes to which the stimulus and the test substance are added is the test to which the stimulus is added. A method for determining that the test substance has an effect of improving skin aging when the expression level is large or small compared to the expression level in cells to which the substance has not been added (except when the stimulus is glucose). The method according to claim 1, wherein the aging control factor changes any of the physical quantities involved in the development of skin aging symptoms. The method according to claim 2, wherein the physical quantity involved in the development of the skin aging symptom is selected from the group consisting of collagen amount, elastin amount, hyaluronic acid amount, versican amount, autophagy activity, and skin supporting structure. The method according to any one of claims 1 to 3, wherein the stimulus is selected from a substance whose amount varies depending on lifestyle and aging. The lifestyle及又are substances that change the amount with aging, is selectively oxidized LDL, hydrogen peroxide, estradiol, caffeine, acetaldehyde, vitamins, orexin, progesterone, testosterone, DHEA, and from the group consisting of methylglyoxal , The method according to claim 4. The stimulus is oxidized LDL and
The method of claim 5 , wherein the aging regulator is selected from the group consisting of 4-1BB, FasL, IGFBP2, MSPα, IFNγ, and PAI-1. The stimulus is hydrogen peroxide
The method of claim 5 , wherein the aging regulator is selected from the group consisting of IL-8, Leptin, GH1, and OPG. The stimulus is estradiol
The method of claim 5 , wherein the aging regulator is selected from the group consisting of ACE2, AgRP, CRP, IGFBP3, LeptinR, sTNFRI, ST2, MCP3, PDGFB, and VEGF. It is a method of screening a skin aging improving agent using the activity of an aging regulator in subcutaneous adipocytes as an index.
The activity of the aging control factor is the expression level of the gene encoding the protein constituting the factor or the protein, and the expression level in the subcutaneous adipocytes to which the stimulus and the test substance are added is the test substance to which the stimulus is added. This is a method for determining that the test substance has an effect of improving skin aging when the expression level is large or small as compared with the expression level in the cells to which the above-mentioned substance is not added.
The stimulus was glucose
A method in which the aging regulator is selected from the group consisting of 4-1BB, CRP, IGFBP2, IL-12, RANTES, SAA, SDF-1, sTNFRI, sTNFRII, VEGF, and IL-6sR.

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