Screening method for agent for improving skin condition

Abstract

To provide a novel screening method for an agent for improving skin condition.SOLUTION: The present invention provides a screening method for an agent for improving skin condition, using proteasome activity in epidermal keratinocytes as an indicator.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a method for screening for skin condition improving agents using proteasome activity in epidermal keratinocytes as an indicator. [Background technology]

[0002] In recent years, research has been conducted into ingredients that can improve skin conditions that have deteriorated with age, and various ingredients have been discovered. Proteasomes, present in cells, are proteolytic enzymes distributed in both the cytoplasm and nucleus of eukaryotic cells, and play an important role in cellular function and maintenance. It is known that proteasome activity decreases with age (Non-Patent Document 1).

[0003] An extract from peach (Prunus persica) belonging to the Rosaceae family is known as a naturally occurring functional material that has the effect of enhancing proteasome activity (Patent Document 1). [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Japanese Patent Application Laid-Open No. 2016-124827 [Non-patent literature]

[0005] [Non-Patent Document 1] G. Carrard et al. Int J Biochem Cell Biol. 34 1461-1474 Summary of the Invention [Problem to be solved by the invention]

[0006] In view of the above-mentioned prior art, an object of the present invention is to provide a novel method for screening skin condition improving agents. [Means for solving the problem]

[0007] The present invention, which solves the above-mentioned problems, provides a method for screening for skin condition improving agents using proteasome activity in epidermal keratinocytes as an indicator.

[0008] In a preferred embodiment of the present invention, the test substance is judged to be a more excellent skin condition improving agent as the proteasome activity in epidermal keratinocytes cultured in the presence of the test substance increases.

[0009] In a preferred embodiment of the present invention, the method comprises the steps of culturing epidermal keratinocytes in the presence of a test substance, measuring proteasome activity in the epidermal keratinocytes, and determining that the test substance is the skin condition improving agent if the proteasome activity is higher than the proteasome activity of epidermal keratinocytes cultured in the absence of the test substance.

[0010] In a preferred embodiment of the present invention, the skin condition improving agent is used to inhibit pigmentation and / or inhibit the decline in function of the cell adhesion apparatus.

[0011] In a preferred embodiment of the present invention, the skin condition improving agent is used to inhibit melanocyte activation and / or promote tight junction (TJ) formation.

[0012] In a preferred embodiment of the present invention, the skin condition improving agent is used for suppressing ADM gene expression and / or enhancing ZO-1 gene expression.

[0013] In a preferred embodiment of the present invention, the skin condition is one or more selected from the group consisting of age spots, dullness, freckles, sunburn, darkening due to skin inflammation or irritation, and a decrease in stratum corneum barrier function. [Effects of the Invention]

[0014] According to the present invention, a method for screening for skin condition improving agents using proteasome activity in epidermal keratinocytes as an indicator can be provided. [Brief explanation of the drawings]

[0015] [Figure 1] FIG. 1 shows changes in proteasome activity ratio in epidermal keratinocytes due to the addition of test substances. [Figure 2] FIG. 1 shows changes in the ADM gene in epidermal keratinocytes due to inhibition of proteasome activity. [Figure 3] FIG. 1 shows changes in ZO-1 gene expression levels in epidermal keratinocytes due to inhibition of proteasome activity. DETAILED DESCRIPTION OF THE INVENTION

[0016] Preferred embodiments of the present invention will be described below, but it goes without saying that the technical scope of the present invention is not limited to the following embodiments.

[0017] In the screening method of the present invention, the skin condition improving agent to be screened is not particularly limited, and examples of test substances include commercially available compounds (including peptides), known compounds (including peptides), groups of compounds obtained by combinatorial chemistry techniques, extracts derived from living organisms, and mixtures thereof.

[0018] The term "biological extract" refers to not only the extract itself derived from animals or plants, but also a collective term for fractions of extracts, purified fractions, and solvent-removed extracts, fractions, and purified products. Examples of plant extracts include extracts made from wild or cultivated plants, extracts sold as herbal medicine ingredients, and commercially available extracts.

[0019] The screening method of the present invention uses proteasome activity in epidermal keratinocytes as an index.

[0020] The screening method of the present invention comprises adding a test substance to a culture system of epidermal keratinocytes and measuring proteasome activity in the epidermal keratinocytes.

[0021] Specifically, the screening method of the present invention makes it possible to determine that the test substance is a superior skin condition improving agent as the proteasome activity in epidermal keratinocytes cultured in the presence of the test substance is higher.

[0022] Furthermore, preferably, when the proteasome activity in epidermal keratinocytes cultured with the addition of a test substance is statistically significantly greater than the proteasome activity in epidermal keratinocytes cultured without the addition of the test substance, the test substance can be determined to be a candidate for a skin condition improving agent. More specifically, if the proteasome activity in epidermal keratinocytes cultured with the test substance is greater than one-fold the proteasome activity in epidermal keratinocytes cultured without the test substance, the test substance can be selected as a candidate for a skin condition improving agent.

[0023] Proteasome activity can be measured by measuring the chymotrypsin-like, trypsin-like, or caspase-like protease activity of the proteasome complex present in the cultured cells. Measurement is performed by detecting the fluorescent substance produced when a substrate (SUC-LLVY-AMC) is cleaved by the proteasome. Commercially available measurement kits include Promega's Proteasome-Glo® Assay, Cosmo Bio's Proteasome 20S assay kit, and Funakoshi's Proteasome 20S assay kit.

[0024] The present invention can also be in a form comprising the steps of culturing epidermal keratinocytes in the presence of a test substance, measuring proteasome activity in the epidermal keratinocytes, and determining that the test substance is the skin condition improving agent if the proteasome activity is higher than the proteasome activity of epidermal keratinocytes cultured in the absence of the test substance.

[0025] Each step in a preferred embodiment of the present invention will be described in detail below.

[0026] (1)Culture process The culture step is a step of culturing epidermal keratinocytes in the presence of a test substance. The cell culture method is not particularly limited, and adherent culture or suspension culture can be appropriately selected depending on the properties of the cells used.

[0027] In the present invention, skin collected from a living body or a three-dimensional cultured skin model may also be used. The skin is preferably a skin fragment collected from a living body, with hair removed if desired, and consisting of the granular layer and basal layer of the epidermis.

[0028] A preferred three-dimensional cultured skin model is one in which cells collected from human skin (such as normal human epidermal keratinocytes (NHEK) or normal human dermal fibroblasts (NHDF)) are cultured to create a three-dimensional structure that mimics the structure of skin, and a commercially available product in this form can also be purchased and used. A preferred example of a commercially available product is "EFT-400" sold by Kurabo Industries Co., Ltd.

[0029] The culture medium is not particularly limited, and any known medium can be used, with suitable examples including DMEM (Dulbecco's Modified Eagle Medium).

[0030] The duration of the culture step is not particularly limited, but is preferably 3 hours or more, more preferably 6 hours or more, even more preferably 12 hours or more, and particularly preferably 1 day or more. The upper limit of the culture period is also not particularly limited, but as a guideline, it is preferably 1 month or less, more preferably 3 weeks or less, even more preferably 2 weeks or less, and particularly preferably 1 week or less.

[0031] In the culturing step, culturing is carried out in the presence of a test substance. Specific examples include an embodiment in which the test substance is added to a medium and then cultured. The type of test substance is not limited, and any low molecular weight compound, high molecular weight compound, protein, peptide, nucleic acid, or the like can be applied. The test substance may be a pure compound or a mixture containing two or more components, such as a plant or animal extract.

[0032] In the culturing step, one or more test substances may be used, and when evaluating a synergistic effect due to a combination of test substances, it is preferable to add two or more test substances.

[0033] In the culturing step, cells may be cultured in the absence of the test substance as a control in parallel with the culture in the presence of the test substance. In this case, it is preferable to keep the culture conditions the same except for the presence or absence of the test substance.

[0034] (2) Measurement process The measuring step is a step of measuring proteasome activity in epidermal keratinocytes that have been subjected to cell culture. The method for measuring proteasome activity in epidermal keratinocytes is not particularly limited, and any experimental method can be used as long as it can confirm the degradation of a desired protein within the cells. For example, a method for measuring proteasome activity includes monitoring the hydrolysis of AMC from Suc-LLVY-AMC using a fluorescent peptide. Suc-LLVY-AMC is a 7-amino-4-methylcoumarin (AMC)-labeled fluorescent substrate that can be used to measure the chymotrypsin-like activity of the 20S and 26S proteasomes, as well as calpain and other chymotrypsin-like proteases. Upon enzymatic cleavage of the peptide, the fluorophore AMC is released, resulting in a strong increase in fluorescence.

[0035] (3) Judgment process The determination step is a step of determining that the test substance is a skin condition improving agent based on the proteasome activity measured in the measurement step. Specifically, the test substance is determined to be the skin condition improving agent when the proteasome activity in epidermal keratinocytes cultured in the presence of the test substance is higher than the proteasome activity in epidermal keratinocytes cultured in the absence of the test substance.

[0036] Here, the test substance determined in the determination step may be determined as an active ingredient as is, or may be determined as a candidate active ingredient to be subjected to secondary screening.

[0037] The skin condition improving agent obtained by the screening method of the present invention can be used to inhibit pigmentation and / or inhibit the decline in the function of the cell adhesion apparatus.

[0038] In the present invention, inhibition of pigmentation specifically refers to the prevention and / or improvement of age spots, dullness, freckles, sunburn, darkening due to inflammation or irritation of the skin, and the like. The skin condition improving agent obtained by the screening method of the present invention can be preferably used for preventing and / or improving spots and dullness.

[0039] In the present invention, suppression of functional decline of the cell adhesion apparatus specifically refers to suppression of functional decline of tight junctions and / or adherens junctions. Promoting the formation of tight junctions and / or adherens junctions strengthens the bonds between epithelial cells, improving the skin's defense function. The improved skin defense function improves skin condition. An example of the skin's defense function is the stratum corneum barrier function. The skin condition improving agent obtained by the screening method of the present invention can be preferably used to prevent and / or improve a decrease in barrier function and dryness.

[0040] The skin condition improving agent obtained by the screening method of the present invention can be preferably used to inhibit melanocyte activation and / or promote tight junction (TJ) formation.

[0041] The skin condition improving agent obtained by the screening method of the present invention can be more preferably used to suppress ADM gene expression and / or enhance ZO-1 gene expression.

[0042] Pigmentation can be suppressed by suppressing ADM gene expression.

[0043] Increased expression of the ZO-1 gene can suppress the decline in the function of the cell adhesion apparatus. [Example]

[0044] The present invention will be described in more detail below with reference to examples, but it goes without saying that the present invention is not limited to these examples. In addition, the rice bran extract hydrolysate used in the examples can be, for example, a rice bran extract hydrolyzed solution obtained by the following production example, such as the technique described in JP-A-2022-138725.

[0045] Example of manufacturing rice bran extract hydrolyzed solution 1500 g of 0.1 M lactic acid solution was added to 500 g of rice bran, stirred to thoroughly mix the rice bran and lactic acid solution, and then allowed to stand at room temperature for one day. Insoluble matter was then removed by filtration, and the filtrate was treated with papain. The enzyme treatment was carried out using 1.2 mg of enzyme at the enzyme's optimal pH, by incubating at 80°C for one hour. The resulting insoluble matter was filtered off, and the filtrate was adjusted to pH 6.5 with phytic acid, yielding 731 g of a pale yellow, transparent rice bran extract hydrolyzed solution (solids concentration 3.7%).

[0046] <Test Example 1> Screening of test substances that enhance proteasome activity in human epidermal keratinocytes Human epidermal keratinocytes were cultured in a black 96-well plate at 8.0 × 103 The cells were seeded at 100 cells / well and cultured at 37°C in a 5% CO2 environment for 24 hours. 24 hours after seeding, a hydrolyzate of rice bran extract was added to a final concentration of 0.5%, and the culture was continued for another 24 hours.

[0047] The cell culture supernatant was then removed, and each well was washed with Tris-HCl buffer ((pH 8.0): 12 mM trishydroxymethylaminomethane, 5 mM EDTA, 150 mM NaCl, hydrochloric acid). Then, 100 μL of cell lysis solution (Tris-HCl buffer (pH 8.0): 12 mM trishydroxymethylaminomethane, 5 mM EDTA, 150 mM NaCl, hydrochloric acid containing 1% Triton X-100) was added to the wells used to measure total substrate degradation capacity, and 90 μL was added to the wells used to measure non-proteasome activity. The cells were disrupted and used as a crude enzyme solution. 10 μL of proteasome activity inhibitor solution (cell lysis solution containing 2 mM epigallocatechin gallate) was added to the wells used to measure non-proteasome activity, and the wells were left to stand at room temperature for 10 minutes.

[0048] 10 μL of a buffer containing 65 μM Suc-Leu-Leu-Val-Tyr-AMC as a substrate was added to each plate, and the mixture was allowed to react for 1 hour at 37° C. After the reaction was completed, the fluorescence intensity (excitation wavelength: 355 nm, emission wavelength: 460 nm) was measured using a fluorescent plate reader (ARVO X4, PerkinElmer).

[0049] Similarly, a black plate containing the extract but without cell disruption was prepared, the cell supernatant was removed, and each well was washed with PBS(-). 100 μL of PBS containing Hoechst 33342 was added, and the reaction was allowed to proceed at 37°C for 30 minutes. After the reaction was complete, the fluorescence intensity (excitation wavelength: 355 nm, emission wavelength: 460 nm) was measured using a fluorescent plate reader (ARVO X4, PerkinElmer) to measure the fluorescence intensity due to DNA staining.

[0050] The non-proteasome activity measurement results were subtracted from the total substrate degradation measurement results, and the value obtained was normalized by the fluorescence intensity of DNA staining to determine the proteasome activity. The same procedure as above was also performed for the control (PBS(-) sample), and the relative value of the proteasome activity obtained when each sample was added to the control was calculated, and this was used as the proteasome activity ratio. The results are shown in Table 1 and Figure 1.

[0051] [Table 1]

[0052] As shown in Table 1, it was confirmed that the hydrolysate of rice bran extract has the effect of enhancing proteasome activity in epidermal keratinocytes. Thus, using proteasome activity as an indicator, it is possible to screen for substances involved in skin conditions caused by proteasome activity in skin tissue.

[0053] <Test Example 2> Enhancement of pigmentation-related gene expression by inhibiting proteasome activity Human epidermal keratinocytes were cultured in a collagen I-coated 24-well plate at 3.0 × 10 4 The cells were seeded at 100 cells / well and cultured at 37°C in a 5% CO2 environment for 24 hours. 24 hours after seeding, MG-132 (Carbobenzoxy-L-leucyl-L-leucyl-L-leucinal, a proteasome activity inhibitor) diluted with DMSO was added to a concentration of 100 nM, and the cells were cultured for a further 72 hours, after which RNA was collected from the cells. The control was prepared under the same conditions as above except that MG-132 was not added.

[0054] The collected RNA was analyzed using real-time PCR to evaluate the expression of the ADM gene (a pigmentation-related gene). Specifically, the gene expression level after the addition of MG-132 was calculated based on the gene expression level in cells without MG-132. The results are shown in Table 2 and Figure 2. Table 2 shows the ratio of the ADM gene expression level after the addition of MG-132, assuming that the ADM gene expression level in cells without MG-132 was "1.00."

[0055] [Table 2]

[0056] As shown in Table 2, it was confirmed that inhibition of proteasome activity promoted ADM gene expression in epidermal keratinocytes. This indicates that enhancing proteasome activity can reduce the expression level of melanocyte activating factor in epidermal keratinocytes. Furthermore, enhancing proteasome activity is expected to have an inhibitory effect on pigmentation. In other words, it is possible to screen for substances involved in the inhibition of pigmentation using proteasome activity as an indicator.

[0057] <Test Example 3> Inhibition of cell adhesion apparatus-related gene expression by inhibiting proteasome activity Human epidermal keratinocytes were cultured in a collagen I-coated 24-well plate at 3.0 × 10 4 The cells were seeded at 100 cells / well and cultured at 37°C in a 5% CO2 environment for 24 hours. 24 hours after seeding, MG-132 (Carbobenzoxy-L-leucyl-L-leucyl-L-leucinal, a proteasome activity inhibitor) diluted with DMSO was added to a concentration of 100 nM, and the cells were cultured for a further 72 hours, after which RNA was collected from the cells. The control was prepared under the same conditions as above except that MG-132 was not added.

[0058] The collected RNA was analyzed using real-time PCR to evaluate the expression of the ZO-1 gene (a gene associated with the cell adhesion apparatus). Specifically, the gene expression level after the addition of MG-132 was calculated based on the gene expression level in cells without MG-132. The results are shown in Table 3 and Figure 3. Table 3 shows the ratio of the ZO-1 gene expression level after the addition of MG-132, when the ZO-1 gene expression level in cells without MG-132 was set at 1.00.

[0059] [Table 3]

[0060] As shown in Table 3, it was confirmed that inhibition of proteasome activity inhibited the expression of the ZO-1 gene in epidermal keratinocytes. This demonstrates that enhancing proteasome activity can increase the expression of tight junction (TJ)-related genes in epidermal keratinocytes. Furthermore, enhancing proteasome activity is expected to have an effect of suppressing the decline in the function of the cell adhesion apparatus. In other words, it is possible to screen for substances involved in suppressing the decline in the function of the cell adhesion apparatus using proteasome activity as an indicator. [Industrial Applicability]

[0061] According to the present invention, a method for screening for skin condition improving agents using proteasome activity in epidermal keratinocytes as an indicator can be provided.

Claims

1. A method for screening skin condition improving agents using proteasome activity in epidermal keratinocytes as an indicator.

2. The screening method according to claim 1, wherein the test substance is judged to be a superior skin condition improving agent as the proteasome activity in epidermal keratinocytes cultured in the presence of the test substance increases.

3. Culturing epidermal keratinocytes in the presence of a test substance; measuring proteasome activity in the epidermal keratinocytes; determining that the test substance is the skin condition-improving agent when the proteasome activity is higher than the proteasome activity of epidermal keratinocytes cultured in the absence of the test substance; The screening method according to claim 1, comprising:

4. The screening method according to any one of claims 1 to 3, wherein the skin condition improving agent is used for inhibiting pigmentation and / or inhibiting a decline in the function of the cell adhesion apparatus.

5. The screening method according to any one of claims 1 to 3, wherein the skin condition improving agent is used for inhibiting melanocyte activation and / or promoting tight junction (TJ) formation.

6. The screening method according to any one of claims 1 to 3, wherein the skin condition improving agent is used for suppressing ADM gene expression and / or enhancing ZO-1 gene expression.

7. The screening method according to any one of claims 1 to 3, wherein the skin condition is one or more selected from the group consisting of age spots, dullness, freckles, sunburn, darkening due to skin inflammation or irritation, and a decrease in stratum corneum barrier function.

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