CXCL9 expression enhancer

Abstract

To provide a method for maintaining healthy skin and improving skin aging by promoting removal of aged cells on skin.SOLUTION: Inventors paid attention to a system of removing aged cells by a cytotoxic CD4 positive cell, and studies development of a product of improving skin aging. In the study, the inventors paid attention to CXCL9 which plays an important role in inducing a cytotoxic CD4 positive cell, and performed screening of a component promoting expression of CXCL9, and surprisingly found out camellia extract as a component promoting expression of CXCL9 on skin, and thus a CXCL9 expression enhancer containing camellia extract is provided.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to the development of a component that promotes the expression of CXCL9, which attracts cytotoxic CD4-positive T lymphocytes (CD4CTLs) involved in the elimination of senescent cells. [Background technology]

[0002] T cells that control immunity can be broadly divided into CD4+ T cells and CD8+ T cells. CD4+ T cells are called helper T cells, which support the antibody-secreting B cells. CD8+ T cells are called killer T cells, which kill virus-infected cells, cancer cells, and senescent cells with their potent cytotoxic activity. Methods for enhancing these immune cells have been disclosed (Patent Document 1: JP 2021-534829 A). Meanwhile, CD4+ T cells also contain T cells with cytotoxic activity, and these CD4+ T cells are called cytotoxic CD4+ T lymphocytes (CD4CTLs). Cytotoxic CD4+ cells have been reported to express a group of genes expressed by CD8+ T cells. Similar to CD8+ T cells, they have been reported to be able to kill virus-infected cells and tumor cells, exerting a strong antitumor effect.

[0003] Meanwhile, it has been reported that cytotoxic CD4+ T lymphocytes act on senescent skin cells to induce apoptosis. It is believed that tissue homeostasis is maintained by the specific elimination of senescent cells by cytotoxic CD4+ T lymphocytes. It has been reported that latent cytomegalovirus increases in senescent cells, and that cytotoxic CD4+ T lymphocytes recognize senescent cells and induce apoptosis using membrane proteins produced by the increased intracellular cytomegalovirus as a marker (Non-Patent Document 1: Cell 2023). The same document also identifies the importance of CXCL9 expression in attracting cytotoxic CD4+ T lymphocytes to aged skin. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Special Publication No. 2021-534829 [Non-patent literature]

[0005] [Non-Patent Document 1] Cell (2023)186, 1417-1431 Summary of the Invention [Problem to be solved by the invention]

[0006] The object of the present invention is to provide a method for maintaining healthy skin and improving skin aging by promoting the removal of senescent cells in the skin. [Means for solving the problem]

[0007] The present inventors conducted research into the development of products that improve skin aging, focusing on the system by which cytotoxic CD4+ cells remove senescent cells. In the process, they focused on CXCL9, which plays an important role in attracting cytotoxic CD4+ cells, and screened for ingredients that promote CXCL9 expression. Surprisingly, they discovered camellia extract as an ingredient that promotes CXCL9 expression in the skin, leading to the present invention. Thus, the present invention relates to: [1-1] CXCL9 expression promoter containing camellia extract. [1-2] Use of camellia extract for the manufacture of a CXCL9 expression promoter. [1-3] Camellia extract for use in inhibiting aging through promoting CXCL9 expression. [1-4] A method for suppressing CXCL9 expression in a subject suffering from an increased proportion of senescent cells, comprising administering camellia extract. [2] The invention according to any one of Items 1-1 to 1-4, wherein the camellia extract is an extract extracted from a part selected from the group consisting of camellia seeds, flowers, leaves, and roots. [3] The invention according to Item 2, wherein the camellia extract is camellia seed extract or fermented camellia seed extract. [4] The invention described in any one of Items 1-1 to 1-4 and 2 to 3, which is a topical skin preparation. [5] The invention described in any one of items 1-1 to 1-4 and 2 to 4, which promotes CXCL9 expression in keratinocytes. [6] A cosmetic method comprising applying the CXCL9 expression promoter described in any one of items 1-1 to 1-2 and items 2 to 5 subordinate thereto. [Effects of the Invention]

[0008] The camellia extract selected according to the present invention can promote the expression of CXCL9 in skin cells. [Brief explanation of the drawings]

[0009] [Figure 1] Figure 1A shows the results of Western blotting, which indicates the changes in CXCL9 expression in keratinocytes after application of camellia seed extract, fermented camellia seed extract, and their mixture as candidate ingredients. Figure 1B is a graph showing the changes in the relative expression level of CXCL9 to β-actin. DETAILED DESCRIPTION OF THE INVENTION

[0010] One aspect of the present invention relates to a CXCL9 expression promoter containing camellia extract. The CXCL9 expression promoter can be used as a food, cosmetic, or pharmaceutical product and can exert an anti-aging effect through the removal of senescent cells. Furthermore, the CXCL9 expression promoter can exert any effect based on the induction of CXCR3-expressing cells, particularly cytotoxic CD4-positive cells.

[0011] CXCL9 is a chemokine belonging to the CXC chemokine family that induces cell migration. CXCL9 is expressed in monocytes, epithelial cells, and vascular endothelial cells, and is secreted extracellularly. CXCL9 acts as a ligand for CXCR3, a chemokine receptor expressed by T cells, and is involved in the migration of CXCR3-expressing cells. CXCL9 is also known to be induced by interferon-γ, and its expression increases mainly during inflammation, contributing to the attraction of lymphocytes to sites of infection and inflammation. It is also known to have antibacterial properties and to stimulate collagen production in fibroblasts.

[0012] In the present invention, promotion of CXCL9 expression can be determined by measuring the amount of CXCL9 protein and / or mRNA. The amount of intracellular and / or secreted CXCL9 protein can be determined by immunological techniques using antibodies that specifically bind to CXCL9. Alternatively, the amount of mRNA can be measured by PCR, for example. The amount of mRNA can be measured by quantitative PCR or real-time PCR.

[0013] A CXCL9 expression promoter can also be referred to as a CXCL9 secretion promoter. The expression and secretion of CXCL9 can enhance all or part of the effects induced by CXCL9. For example, it can attract CXCR3-expressing cells. Examples of CXCR3-expressing cells include CD4-positive T cells, CD8-positive T cells, NK cells, NKT cells, and some peripheral blood B cells. Among these, cytotoxic CD4-positive T lymphocytes are particularly noteworthy for their ability to damage senescent cells. Therefore, a CXCL9 expression promoter can also be referred to as an agent for attracting and promoting CXCR3-expressing cells, particularly cytotoxic CD4-positive cells, or an agent for promoting the destruction of senescent cells. Furthermore, a CXCL9 expression promoter can partially eliminate virus-infected cells and / or cancer cells via the attraction of cytotoxic CD4-positive cells, and can thus be referred to as a therapeutic or preventive agent for viral infection or cancer.

[0014] CXCL9 expression promoters can act on any cells that express CXCL9. Examples of cells that express CXCL9 include keratinocytes, monocytes, and vascular endothelial cells. However, from the perspective of percutaneous application, it is preferable to act on keratinocytes. When applied percutaneously, CXCL9 expression promoters can penetrate the stratum corneum, granular layer, and spinous layer of the epidermis and act on keratinocytes present in the basal layer. CXCL9 secreted by acting on keratinocytes passes through the basement membrane and penetrates the dermal layer, thereby attracting CXCR3-expressing cells to the dermal layer. Therefore, CXCL9 expression promoters can also be considered as agents that attract CXCR3-expressing cells, particularly cytotoxic CD4-positive cells. By applying CXCL9 expression promoters percutaneously, cytotoxic CD4-positive cells can be attracted to the skin, particularly the dermal layer.

[0015] Among CXCR3-expressing cells, cytotoxic CD4-positive cells in particular can recognize senescent cells and induce apoptosis. Senescent cells include senescent dermal fibroblasts present in the dermis. Senescent dermal fibroblasts cause wrinkles due to their slow division rate and low production capacity of stromal components. Senescent dermal fibroblasts are also known to secrete senescence factors such as IGFBP7, which can adversely affect neighboring cells. Senescent cells highly express HLA-II and HCMV-glycoprotein B upon human cytomegalovirus (HCMV) activation. Cytotoxic CD4-positive cells can specifically eliminate senescent cells through the induction of apoptosis in a manner dependent on the highly expressed HLA-II. Therefore, CXCL9 expression promoters can also be referred to as senescent cell-eliminating agents. CXCL9 expression promoters can improve skin problems by eliminating senescent cells. In particular, aged dermal fibroblasts cause age spots and wrinkles, so removing aged dermal fibroblasts and promoting the proliferation of young dermal fibroblasts can prevent or improve aging. Examples of aging include skin aging, dermal fibroblast aging, and improvement of wrinkles and firmness. Therefore, a CXCL9 expression promoter can also be called an anti-aging agent, particularly an agent for improving skin aging, and an agent for improving age spots, wrinkles, or firmness.

[0016] The route of administration of the CXCL9 expression promoter of the present invention may be any route, including oral, transdermal, subcutaneous, intramuscular, intravenous, intraarterial, oral, and transmucosal administration. From the viewpoint of acting on the skin, transdermal or subcutaneous administration is particularly preferred. The CXCL9 expression promoter administered transdermally or subcutaneously acts on keratinocytes to promote the secretion of CXCL9, and the secreted CXCL9 penetrates the dermal layer and promotes the attraction of cytotoxic CD4+ T lymphocytes to the dermal layer, thereby exerting its effect.

[0017] The subject to which the CXCL9 expression promoter of the present invention is administered is a subject with an increased number of senescent cells or a subject with reduced CXCL9 expression in the skin. Such subjects have symptoms associated with an increase in senescent cells. More specifically, such subjects suffer from symptoms associated with an increase in skin senescent cells, particularly skin aging such as age spots, wrinkles, etc. The amount or proportion of skin senescent cells, particularly senescent dermal fibroblasts, may be identified by methods well known in the art. The increase in skin senescent cells can be determined by determining the proportion of dermal fibroblasts that highly express HLA-II and / or HCMV-glycoprotein B from a skin sample. After identifying a subject with an increase in skin senescent cells, it is desirable to administer the CXCL9 expression promoter of the present invention to such a subject.

[0018] A further aspect of the present invention may relate to a cosmetic method comprising applying a CXCL9 promoter. In such a cosmetic method, the CXCL9 promoter is applied to the skin of a subject suffering from symptoms associated with senescent skin cells, such as skin aging, wrinkles, and loss of elasticity. Application to the skin enables the removal of senescent skin cells, which are replaced with younger cells, thereby improving the symptoms associated with senescent skin cells. The cosmetic method of the present invention is performed by a non-medical professional at a beauty salon or the like, and does not constitute a therapeutic procedure, and can also be considered a non-therapeutic procedure.

[0019] In a further aspect, the present invention relates to a method for screening for a CXCL9 expression promoter or a skin aging inhibitor using the expression of the CXCL9 according to the present invention in keratinocytes as an index. More specifically, the screening method of the present invention comprises the following steps: culturing a culture comprising keratinocytes in a medium containing the candidate component; Measuring the expression of CXCL9 in the cultured keratinocytes; and A step of comparing the measured CXCL9 expression level with that of a control. When the expression level of CXCL9 is increased, the candidate component can be screened as an agent for promoting the expression level of CXCL9. The control CXCL9 expression can be the expression of CXCL9 in keratinocytes cultured in a different process except that it does not contain the candidate component. A threshold may be set based on the CXCL9 expression in a control group obtained by a preliminary experiment, or the culture and measurement process may be performed in parallel. The CXCL9 expression level may be the amount of CXCL9 protein or mRNA in cultured keratinocytes, and can be measured using techniques well known in the art, such as immunological techniques or quantitative PCR.

[0020] The candidate components used in the screening method of the present invention can be any library of cosmetic materials, food materials, pharmaceutical materials, etc. Such libraries may include compound libraries, extract libraries, etc. The compounds and extracts contained in each library may be commercially available compounds and extracts, or may be synthesized compounds and prepared extracts.

[0021] The plant extracts described herein can be obtained by conventional methods, for example, by immersing or refluxing the plant with an extraction solvent at room temperature or elevated temperatures, followed by filtration and concentration. Any solvent commonly used for extraction can be used as the extraction solvent. For example, aqueous solvents such as water, saline, phosphate buffer, borate buffer, or organic solvents such as alcohols (e.g., 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. A mixed solvent of water and an alcohol (e.g., 1,3-butylene glycol) is preferably used. The extract obtained by extraction with the above solvents can be used directly or after concentration by, for example, freeze-drying. If necessary, the extract can be used after removing impurities using adsorption techniques, such as ion exchange resins, or after adsorption on a porous polymer (e.g., Amberlite XAD-2) column, followed by elution with a desired solvent and further concentration. The plant extract may be an extract commercially available as a cosmetic raw material, and the commercially available extract may be blended at a predetermined concentration.

[0022] Camellia extract is an extract obtained from plants belonging to the genus Camellia in the family Theaceae. It is particularly an extract obtained from the seeds of camellia or Camellia japonica. Camellias are native to Japan but are widely distributed throughout the Japanese archipelago, as well as the Korean Peninsula, China, and Taiwan. Any part of the camellia plant, including the tree, leaves, stems, flowers, seeds, and roots, can be used, but camellia seeds are particularly suitable for extraction. Camellia extract can be extracted from the seeds themselves, or by further solvent extraction of the residue after squeezing camellia oil. Extraction can be performed by drying and / or powdering the seeds. Camellia seeds can also be fermented with microorganisms to obtain an extract. Fermented camellia seeds can be obtained by culturing the residue after squeezing camellia oil using, for example, one of four types of koji mold certified by the national government. Examples of such koji mold include Aspergillus oryzae, known in Japanese as yellow koji mold; Aspergillus sojae, classified as yellow koji mold (oryzae group), and a white mutant strain of yellow koji mold; and Aspergillus luchuensis (Aspergillus luchuensis var. awamori), classified as black koji mold, and white koji mold (Aspergillus luchuensis mut. kawachii), which is a white mutant strain of black koji mold. Camellia seed extract and fermented camellia seed extract are listed as follows in the display name and international display name according to the International Nomenclature of Cosmetic Ingredients (INCI) (Japan Cosmetic Industry Association) for use in labeling all ingredients in cosmetics: Display name / INCI name: Camellia seed extract / CAMELLIA JAPONICA SEED EXTRACT, label name / INCI name: ASPERGILLUS / CAMELLIA JAPONICA SEED FERMENT EXTRACT FILTRATE. Solvent extraction can be performed by extracting with water, alcohol, or a mixed solution of these.Examples of alcohols that can be used include ethanol, glycerol, propylene glycol, and butylene glycol. Extraction can be performed using a mixture of water and alcohol at any ratio, for example, a 10:90 to 90:10 mixture, preferably a 30:70 to 70:30 mixture, and even more preferably a 50:50 mixture. More preferably, 1,3-butylene glycol can be used as the alcohol to be mixed with water. Camellia seed extract or fermented camellia seed extract is blended at a concentration of 0.001% to 1%, more preferably 0.01% to 0.1%. Camellia seed extract and fermented camellia seed extract may be blended at any ratio. For example, camellia seed extract and fermented camellia seed extract may be blended at a ratio of 1:99 to 99:1, preferably 1:2 to 2:1, and even more preferably 1:1. Camellia extract promotes CXCL9 expression.

[0023] The concentration and dosage form of the CXCL9 expression promoter of the present invention can be selected as desired to achieve the desired effect, i.e., prevention of skin aging or improvement of wrinkles. Camellia extract, a CXCL9 expression promoter, can be incorporated into foods, cosmetics, pharmaceuticals, or quasi-drugs. When incorporated into foods, it may be incorporated into dietary supplements such as supplements and nutritional drinks, or into functional foods. When incorporated into cosmetics, it may be incorporated into facial or body cosmetics such as lotions, emulsions, serums, creams, lotions, packs, essences, and gels, makeup cosmetics such as foundations, makeup bases, and concealers, and even bath additives. When incorporated into pharmaceuticals, it may be administered orally or parenterally, for example, transdermally. When administered transdermally, it can be formulated into a topical skin preparation. The use of cosmetics, foods, pharmaceuticals, and quasi-drugs containing the CXCL9 expression promoter removes senescent cells through the promotion of CXCL9 expression, thereby exerting an anti-aging effect. The CXCL9 expression promoter can be administered over a long period of time as the food, cosmetic, or pharmaceutical of the present invention. From the viewpoint of exerting an anti-aging effect, the compound may be administered for several days or more, one week or more, two weeks or more, one month or more, three months or more, or six months or more. The upper limit is not particularly limited, but the compound may be administered for several years or less, for example, one year or less.

[0024] When formulated as a topical skin preparation, camellia seed extract or fermented camellia seed extract can be formulated as a CXCL9 expression promoter at a concentration of 0.001% to 1%. To ensure full efficacy, the topical skin preparation can be formulated at a concentration of preferably 0.01% or more, more preferably 0.1% or more. From the perspective of safety, the concentration can be preferably 1% or less, more preferably 0.1% or less.

[0025] The topical skin preparation is not particularly limited as long as it is applicable to the skin, and any dosage form can be used, for example, a solution, emulsion, solid, semi-solid, powder, powder dispersion, water-oil two-layer separation, water-oil-powder three-layer separation, ointment, gel, aerosol, mousse, stick, etc. When formulated into a topical skin preparation, bases and excipients typically used in topical skin preparations, such as preservatives, emulsifiers, and pH adjusters, may be used.

[0026] All documents mentioned herein are incorporated by reference in their entirety.

[0027] The following examples of the present invention are for illustrative purposes only and do not limit the technical scope of the present invention. The technical scope of the present invention is limited only by the claims. The present invention may be modified, for example, by adding, deleting, or substituting components of the present invention, provided that the modifications do not depart from the spirit of the present invention. [Example]

[0028] Example 1: Culture of human skin keratinocytes Human skin keratinocytes (Kurabo) were used at 5 × 10 4 The cells were seeded onto a 6-well plate at a concentration of 1000×1000×1000 and cultured in KGM medium at 37° C. in a 5% CO 2 atmosphere for 2 days. As candidate components, camellia seed extract (Yuka Sangyo) was added to the medium at 0.01% and fermented camellia seed extract at 0.1%, and the medium was cultured at 37°C for 24 hours. A control group, which differed only in that it did not contain the extract, was also cultured in the same way. After culturing, the cells were harvested, washed with PBS, and separated by SDS-PAGE. The gel was transferred to a PVDF membrane and Western blotting was performed using anti-CXCL9 antibody (Cell Signaling Technology) and anti-β-actin antibody (Santa Cruz) as primary antibodies. AP-labeled antibody (Thermo Fisher Scientific) was used as secondary antibody. The results are shown in Figure 1. CXCL9 expression was enhanced when camellia seed extract was added at 0.01% and when fermented camellia seed extract was added at 0.1%.

Claims

1. A CXCL9 expression promoter comprising camellia seed extract, fermented camellia seed extract, or a mixture thereof.

2. The CXCL9 expression promoter according to claim 1, which is an external preparation for skin.

3. The CXCL9 expression promoter according to claim 1 or 2, which promotes CXCL9 expression in keratinocytes.

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