Drugs and cosmetic methods for anti-photoaging, and screening methods for anti-photoaging agents

Abstract

The present invention provides methods and agents for preventing and / or ameliorating photoaging, as well as methods for evaluating screening methods for anti-photoaging agents. [Solution] Photoaging can be prevented and / or ameliorated by increasing cutaneous IL-34 and / or CSF-1R. By using IL-34 and / or CSF-1R as indicators, it becomes possible to objectively evaluate the degree of photoaging, and based on this method, drugs for preventing and / or ameliorating photoaging can be explored.

Description

[Technical Field]

[0001] The present invention relates to pharmaceuticals and cosmetic methods for anti-photoaging, and to a method for screening anti-photoaging agents. [Background technology]

[0002] The aging phenomenon of human skin can be broadly divided into "natural aging" and "photoaging." Photoaging is a skin-specific phenomenon that occurs in specific areas exposed to light. In photoaging, the effects of UV rays and other factors induce the production of reactive oxygen species and damage to cellular DNA, which is thought to damage skin fibrous tissue and cause phenotypes such as wrinkles and sagging. For example, skin photoaging can result in a decrease in collagen fibers made of collagen and degeneration of elastic fibers made of elastin. Melanocytes are also damaged, resulting in the overproduction of melanin pigment, which can cause age spots and other skin conditions.

[0003] As an anti-photoaging cosmetic method, for example, Patent Document 1 discloses an agent for preventing or suppressing skin photoaging by preventing the inhibition of leukocyte elastase. It has also been suggested that inflammation is a contributing factor to the skin photoaging phenomenon, and many anti-inflammatory agents have been developed. Patent Document 2 discloses an anti-aging cosmetic composition containing a compound that increases adiponectin expression and induces autophagy activation.

[0004] The inventors have also reported that an imbalance between M1 and M2 macrophages is associated with photoaging, and that adjusting the M1 / M2 balance is particularly important in preventing and improving photoaging (Patent Document 3). [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Patent No. 5657723 [Patent Document 2] Japanese Patent Application Publication No. 2018-177805 [Patent Document 3] International Publication No. 2020 / 213743 [Patent Document 4] Japanese Patent Application Publication No. 09-187248 [Patent Document 5] Japanese Patent Application Laid-Open No. 2003-261455 Summary of the Invention [Problem to be solved by the invention]

[0006] An object of the present invention is to provide an anti-photoaging agent and cosmetic method, as well as a screening method for anti-photoaging agents. [Means for solving the problem]

[0007] As a result of further intensive research, the inventors discovered that a decrease in cutaneous IL-34 and / or its receptor, CSF-1R, is involved in photoaging, and further discovered that cutaneous IL-34 is associated with the M1 / M2 balance. The present application provides the following inventions: (1) An anti-photoaging agent for preventing and / or ameliorating photoaging by increasing skin IL-34. (2) An anti-photoaging agent according to (1), which contains thyme extract as an agent for increasing skin IL-34. (3) An anti-photoaging agent according to (1) or (2), which prevents and / or improves photoaging by increasing epidermal IL-34 and thereby adjusting the dermal M1 / M2 balance. (4) A method for screening an anti-photoaging agent, comprising: applying a candidate agent to the skin sample; Measuring IL-34 in skin samples before and after application of the candidate drug; and assessing the drug to have an anti-photoaging effect when IL-34 in the skin sample to which the candidate drug has been applied increases compared to before the drug is applied; The method comprises: (5) A cosmetic method for preventing and / or improving photoaging by increasing skin IL-34. (6) The cosmetic method according to (5), which prevents and / or improves photoaging by adjusting the dermal M1 / M2 balance through increasing epidermal IL-34. (7) The cosmetic method according to (5) or (6), which comprises administering an IL-34 production promoter containing thyme extract. (8) An IL-34 production promoter containing thyme extract. (9) The cosmetic method according to (5) or (6), which comprises administering an IL-34 production promoter containing nicotinamide. (10) An IL-34 production promoter containing nicotinamide. (11) An anti-photoaging agent for preventing and / or ameliorating photoaging by increasing dermal CSF-1R. (12) The anti-photoaging agent according to (11), which prevents and / or improves photoaging by increasing dermal CSF-1R and thereby inducing M2 macrophages. (13) A method for screening an anti-photoaging agent, comprising: applying a candidate agent to the skin sample; Measuring CSF-1R in skin samples before and after application of the candidate drug; and assessing the drug to have an anti-photoaging effect when CSF-1R in the skin sample to which the candidate drug has been applied increases compared to before application of the drug; The method comprises: (14) A cosmetic method for preventing and / or improving photoaging by increasing dermal CSF-1R. (15) The cosmetic method according to (14), which prevents and / or improves photoaging by increasing dermal CSF-1R to induce M2 macrophages. [Effects of the Invention]

[0008] The methods and agents of the present invention can prevent and / or ameliorate photoaging by increasing skin IL-34 and / or dermal CSF-1R. Furthermore, by using skin IL-34 and / or dermal CSF-1R as indicators, it becomes possible to objectively evaluate the degree of photoaging, and based on such methods, it becomes possible to search for agents and cosmetic treatments for preventing and / or ameliorating photoaging. [Brief explanation of the drawings]

[0009] [Figure 1] Figure 1 is a graph showing the number of IL-34+ cells (cells / mm2) in the epidermis and dermis of young and elderly subjects (Mann-Whitney u test, *; P<0.05). [Figure 2] Figure 2 is a graph showing the number of CSF-1R+ CD68+ cells in the dermis of young and elderly subjects (cells / mm2). [Figure 3] Figure 3 is a graph showing the relationship between the number of IL-34+ cells (cells / mm2) in the epidermis and the M1 / M2 balance in the dermis (r = Spearman's rank correlation coefficient). [Figure 4] Figure 4 is a graph showing the relationship between the number of CSF-1R+ CD68+ cells in the dermis and the number of IL-34+ cells in the dermis (left), and the number of CSF-1R+ CD68+ cells in the dermis and the number of CD206+ CD68+ cells (number of M2 macrophages) in the dermis (right) (r = Pearson's correlation coefficient). [Figure 5] Figure 5 is a graph showing the level of IL-34 mRNA expression in epidermal cells without (left: control) or with (right: thyme extract) added, as a percentage (%) of the control (t-test, *; P<0.05). [Figure 6] The left side of Figure 6 is an immunostaining photograph showing IL-34+ cells in human skin without (top: Cont) or with (bottom: NAM) nicotinamide addition. The right side of Figure 6 is a graph showing the relative values ​​of IL-34 protein expression in epidermal cells without (left: Cont) or with (right: NAM) nicotinamide addition, with the control (Cont) result set at 1 (t-test). [Figure 7] Figure 7 is a graph showing the level of IL-34 mRNA expression in epidermal cells without (left: Cont) or with (right: NAM) nicotinamide added, expressed as a relative value with the control (Cont) result set at 1 (t-test, ***; P<0.001). [Figure 8] Figure 8 is a graph showing the mRNA expression levels of CD206 (left) and IL-10 (right) when various concentrations of IL-34 or CSF-1 were added to CD14+ monocytic cells differentiated into macrophages (r is Pearson's correlation coefficient). The mRNA expression levels of CD206 and IL-10 are shown as relative values ​​to the mRNA expression level of GAPDH. DETAILED DESCRIPTION OF THE INVENTION

[0010] The present inventors have shown that adjusting the M1 / M2 balance, which is the ratio of M1 to M2 macrophages, is effective against photoaging (Patent Document 3). Further research by the present inventors revealed that cutaneous IL-34 is involved in photoaging, and that a decrease in cutaneous IL-34, particularly epidermal IL-34, is associated with a worsening of the M1 / M2 balance. Furthermore, the present inventors discovered that dermal CSF-1R is also involved in photoaging, and that dermal IL-34 and dermal CSF-1R are correlated. Furthermore, while it is known that IL-34 induces M2 macrophages, the present inventors discovered that epidermal IL-34 is correlated with the M1 / M2 balance, and that it is correlated with the dermal CSF-1R and the number of dermal M2 macrophages.

[0011] Interleukin 34 (IL-34) is one of the ligands for CSF-1R and is a cytokine that has a signaling pathway different from that of colony-stimulating factor-1 (CSF-1). IL-34 has been suggested to be necessary for maintaining Langerhans cells and to be involved in atopic dermatitis. IL-34 is also known to increase IL-10 expression and induce M2 macrophages.

[0012] CSF-1R (colony-stimulating factor-1 receptor), also known as M-CSFR (macrophage colony-stimulating factor receptor) or CD115 (Cluster of Differentiation 115), is a receptor that binds CSF-1 and IL-34 as ligands. It is specifically expressed in macrophages and controls their differentiation and function. It has been reported that CSF-1R promotes the formation of tumor-associated macrophages, and that inhibition of CSF-1R enhances anti-tumor immune responses.

[0013] However, it was not known that a decrease in skin IL-34 or dermal CSF-1R is associated with photoaging. Surprisingly, the inventors discovered that skin IL-34 and dermal CSF-1R are specifically decreased in skin exposed to light.

[0014] The present invention provides an anti-photoaging agent and a cosmetic method for preventing and / or ameliorating photoaging by increasing skin IL-34 and / or dermal CSF-1R. The method of the present invention is a cosmetic method and may not be a treatment administered by a doctor or medical professional. In one embodiment, photoaging is prevented and / or ameliorated by adjusting the M1 / M2 balance through increasing skin IL-34, particularly epidermal IL-34. In one embodiment, photoaging is prevented and / or ameliorated by inducing M2 macrophages through increasing dermal CSF-1R. In one embodiment, the dermal M1 / M2 balance may be adjusted by increasing skin IL-34 and thereby increasing dermal CSF-1R, thereby inducing M2 macrophages.

[0015] The present invention also provides a method for screening anti-photoaging agents using IL-34 and / or CSF-1R in the skin as an index of skin photoaging. Furthermore, the present invention also provides a method for screening anti-photoaging agents, comprising: applying a candidate drug to a skin sample; measuring IL-34 and / or CSF-1R in the skin sample before and after application of the candidate drug; and evaluating the drug as having anti-photoaging effects if IL-34 and / or CSF-1R in the skin sample to which the candidate drug has been applied increase compared to before application of the drug. The method of the present invention makes it possible to screen candidate drugs for their anti-photoaging effects, enabling product development and the proposal of new skin care methods.

[0016] An increase in skin IL-34 means that the amount of IL-34 protein, the amount of IL-34 gene expression, or the number of IL-34-positive cells in the epidermis and / or dermis increases with a statistically significant difference (e.g., Wilcoxon rank sum test, Student's t-test, etc.) at a significance level of 5%, for example, when contacted with a candidate substance compared to a state where the subject is not contacted with the candidate substance (control), or that the increase is, 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, 100% or more, or more.

[0017] Skin IL-34 can be determined by measuring the amount of IL-34 protein, the amount of IL-34 gene expression, or the number of IL-34-positive cells in skin or a skin sample. A skin sample refers to a harvested skin sample, for example, an ex vivo skin sample harvested from an animal such as a human, an artificial skin sample such as a 3D skin model, or the epidermal or dermal equivalent portion of such a skin sample, or an in vitro sample such as cultured skin cells, for example, monolayer or stratified cultured cells, cultured keratinocytes, or cultured fibroblasts. Epidermal / dermal IL-34 can be determined by measuring the amount of IL-34 protein, the amount of IL-34 gene expression, or the number of IL-34-positive cells in skin or the epidermal / dermal equivalent portion of a skin sample, respectively.

[0018] An increase in dermal CSF-1R can mean that the amount of CSF-1R protein, the amount of CSF-1R gene expression, or the number of CSF-1R-positive cells in the dermis increases with a statistically significant difference (e.g., Wilcoxon rank sum test, Student's t-test, etc.) at a significance level of 5%, for example, when contacted with a candidate substance compared to a state where the subject is not contacted with the candidate substance (control), or that the increase is, 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, 100% or more, or more.

[0019] Dermal CSF-1R can be determined by measuring the amount of CSF-1R protein, the amount of CSF-1R gene expression, or the number of CSF-1R-positive cells in the skin or a portion of the skin sample that corresponds to the dermis.

[0020] As used herein, the M1 / M2 balance may refer to the ratio between the number of M1 macrophages and the number of M2 macrophages, or may refer to the ratio between the amount of mRNA of an M1 macrophage marker (e.g., CD86, CD80, iNOS, etc.) and the amount of mRNA of an M2 macrophage marker (e.g., CD206, CD163, Agr1, IL-10, etc.). Adjusting the M1 / M2 balance may involve decreasing the M1 / M2 ratio (the number of M1 / the number of M2, and / or the amount of mRNA of an M1 marker / the amount of mRNA of an M2 marker). The reduction may be, for example, a reduction that is statistically significant at a significance level of 5% (e.g., Wilcoxon rank sum test, Student's t-test, etc.) and / or may be, for example, a reduction of 1% or more, 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%. In one embodiment, the dermal M1 / M2 balance is adjusted by increasing epidermal IL-34 and / or dermal CSF-1R. In one embodiment, the adjustment of the M1 / M2 balance is achieved by inducing M2 macrophages.

[0021] Induction of M2 macrophages may mean increasing the mRNA level of an M2 macrophage marker or the number of M2 macrophages by increasing skin IL-34 and / or CSF-1R, for example, an increase that is statistically significant at a 5% significance level (e.g., Student's t-test) and / or an increase of, for example, 1% or more, 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% or more. In one embodiment, photoaging is prevented and / or ameliorated through the induction of M2 macrophages by increasing skin IL-34 and / or dermal CSF-1R.

[0022] As a result of screening various substances using the above method, the inventors discovered that thyme extract and nicotinamide function as IL-34 production promoters. Therefore, the present invention also provides IL-34 production promoters, anti-photoaging agents, and compositions containing them, which consist of thyme extract and / or nicotinamide or contain thyme extract and / or nicotinamide as active ingredients. In one aspect, the anti-photoaging agent increases IL-34 in the skin, e.g., the epidermis and / or dermis, thereby adjusting the dermal M1 / M2 balance and thereby preventing and / or ameliorating photoaging.

[0023] Thyme extract has been reported to have anti-allergic, antibacterial, and M1 / M2 balance adjusting effects (Patent Documents 3, 4, and 5). However, the present inventors have now discovered for the first time that thyme extract has the effect of promoting epidermal IL-34 production.

[0024] Thymus is a perennial plant of the genus Thymus in the family Lamiaceae. In the present invention, various types of thyme can be used, such as common thyme (T. vulgaris), citrus thyme (T. x citriodorus), and wild thyme (T. serpyllum). The thyme extract used in the present invention can be an extract of one or more parts of the whole plant, seeds, flowers, roots, etc. Commercially available thyme extracts can also be used.

[0025] The extraction method used for the extract is not particularly limited, but extraction methods using a solvent are preferred. When performing extraction, the plant body can be used as is, but crushing it into granules or powder before subjecting it to extraction allows for extraction of the active ingredients under milder conditions, in a shorter time, and with higher extraction efficiency. The extraction temperature is not particularly limited and can 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 can 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 ultrasound may be applied.

[0026] The type of solvent is not particularly limited, but water, hydrous ethanol, lower alcohols such as ethanol, organic solvents such as hexane, or mixed solvents of these, such as hexane / ethanol, are preferred. Extraction can be performed at room temperature, or under heating (for example, using a heated solvent such as warm water or hot water). Furthermore, the extraction process can be performed by adding an enzyme to the solvent. Adding an enzyme can disrupt the plant's cellular tissue, thereby further increasing the extraction efficiency. It is preferable to use a cellular tissue-disintegrating enzyme as the enzyme. Examples of such enzymes include pectinase, cellulase, hemicellulase, α-amylase, and phytase. One of these enzymes may be used alone, or two or more may be used in combination.

[0027] Through this extraction procedure, the active ingredients are extracted and dissolved in the solvent. The solvent containing the extract may be used as is, or may be subjected to conventional purification processes such as sterilization, washing, filtration, bleaching, and deodorization before use. It may also be concentrated or diluted as necessary before use. Furthermore, the solvent may be completely evaporated to form a solid (dried product) before use, or the dried product may be redissolved in any solvent before use.

[0028] In addition, the squeezed liquid obtained by squeezing the raw plant material also contains the same active ingredients as the extract, so the squeezed liquid can also be used instead of the extract.

[0029] Nicotinamide (CAS No. 98-92-0) is an amide compound of nicotinic acid, and is known to have effects such as promoting blood circulation, improving rough skin, and whitening. However, the present inventors have now discovered for the first time that nicotinamide also has the effect of promoting epidermal IL-34 production. The nicotinamide used in the present invention may be an extract from a natural product or may be synthesized by a known method. For example, specifically, those listed in the 17th edition of the Japanese Pharmacopoeia can be used.

[0030] However, the anti-photoaging agent or composition is not limited to the above substances, and any substance known to increase IL-34 and / or CSF-1R, such as vitamin D, may be used. The route of administration can also be selected arbitrarily, for example, transdermal administration, oral administration, subcutaneous administration, transmucosal administration, intramuscular administration, etc. However, because photoaging is a phenomenon that occurs specifically in areas of the skin exposed to light, transdermal administration, which can administer to specific areas of the skin, is preferred. Furthermore, transdermal administration allows the anti-photoaging agent or composition of the present invention to be applied directly to the epidermis.

[0031] The agent of the present invention can also be a composition in which the active ingredient of the present invention is combined with one or more other ingredients, such as excipients, carriers and / or diluents. The composition may have any composition or form, and may be appropriately selected depending on the conditions such as the active ingredient and intended use. The composition can be manufactured using conventional methods in a formulation in which excipients, carriers and / or diluents and other ingredients are appropriately combined depending on the dosage form.

[0032] The agent of the present invention can be used by humans and animals by being incorporated into cosmetics, etc., or can be administered to humans and animals as a pharmaceutical preparation. It may also be incorporated into various foods, drinks, and feeds for consumption by humans and animals.

[0033] When the present invention is applied to external skin preparations such as cosmetics, pharmaceuticals, and quasi-drugs, the amount (dry mass) of the plant body or its extract can be appropriately determined depending on the type, purpose, form, method of use, etc. For example, thyme extract and / or nicotinamide can be blended in an amount of 0.00001% to 50% (in the case of extracts or herbal medicines, converted into dry mass) of the total amount of the cosmetic.

[0034] In addition to the above ingredients, if necessary, ingredients typically used in skin topicals such as cosmetics, pharmaceuticals, and quasi-drugs, such as antioxidants, oils, UV protection agents, surfactants, thickeners, alcohols, powder ingredients, colorants, aqueous ingredients, water, various skin nutrients, anti-photoaging agents, etc., can be appropriately blended as needed within the scope that does not impair the effects of the present invention.

[0035] The topical skin preparation of the present invention can be applied to the outer skin as a cosmetic, quasi-drug, etc., and is particularly suitable as a cosmetic, and its formulation is not limited as long as it can be applied to the skin, and any formulation can be used, such as a solution system, solubilized system, emulsion system, powder dispersion system, water-oil two-layer system, water-oil-powder three-layer system, ointment, lotion, gel, aerosol, etc.

[0036] When the agent of the present invention is used as a cosmetic, it may be used in the form of lotion, emulsion, foundation, lipstick, lip balm, cleansing cream, massage cream, pack, hand cream, hand powder, body shampoo, body lotion, body cream, bath cosmetics, etc.

[0037] However, the forms that the agent and composition of the present invention can take are not limited to the dosage forms and forms described above. In addition, the agent or composition of the present invention may be used in combination with the device or method of the present invention or other devices or methods.

[0038] The subjects to which the methods, agents, and compositions of the present invention are applied may be subjects in whom skin photoaging is objectively or subjectively recognized, or subjects in whom skin photoaging is desired to be prevented. For example, the subjects may be judged to have low skin IL-34 and / or CSF-1R, an imbalance in the M1 / M2 balance, or a low number or induction of M2 macrophages. In one embodiment, the subjects may be judged to have a high degree of skin photoaging using IL-34 and / or CSF-1R in the skin as an indicator. Alternatively, the subjects may be concerned with phenotypes specific to skin photoaging, such as age spots, wrinkles, and sagging. Age spots, wrinkles, sagging, and the like can be determined by visual assessment or using known indicators.

[0039] In this specification, a candidate drug refers to a drug whose anti-photoaging effect is being investigated, and includes drugs that are already on the market as products as well as drugs in the development stage, and may also be drugs selected for use in cosmetics during the development of cosmetics. [Example]

[0040] The present invention will now be described in more detail with reference to examples, although the present invention is not limited to these examples.

[0041] Experiment 1: Relationship between photoaging and skin IL-34 The epidermis and dermis from the corner of the eye of young (n=10, mean age 34.2 years) and elderly (n=10, mean age 74.5 years) Caucasians with photoaging were used. The dermis was cut into frozen sections and stained with an IL-34 marker (ab101443, manufactured by Abcam). + The cells were counted.

[0042] The graph of the counting results is shown in Figure 1. As shown in Figure 1, elderly people experiencing photoaging showed high levels of IL-34 in both the epidermis and dermis. + The number of cells was significantly reduced compared to younger subjects, indicating that a decrease in skin IL-34 is associated with photoaging.

[0043] Experiment 2: Relationship between photoaging and dermal CSF-1R The same young (n=10, mean age 34.2 years) and elderly (n=10, mean age 74.5 years) Caucasian skin from the corners of the eyes as used in Experiment 1 was prepared as frozen sections, thinly sliced, and double stained with anti-CSF-1R antibody (Abcam ab183316) and anti-CD68 antibody (Abcam). CSF-1R in the dermis from just below the epidermis to 200 μm deep was analyzed. + CD68 + The cells were counted.

[0044] The graph of the counting results is shown in Figure 2. As shown in Figure 2, in elderly people who are experiencing photoaging, CSF-1R is increased in the dermis. + CD68 + The number of cells tended to decrease compared to younger subjects, suggesting that the decrease in dermal CSF-1R is related to photoaging.

[0045] Experiment 3: Relationship between skin IL-34 and M1 / M2 balance Frozen section samples of the dermis of the skin of young and elderly subjects used in Figure 1 were stained with the macrophage markers shown below. (1) M1 macrophage staining: Double staining using goat-derived anti-human CD86 antibody (R&D) and rabbit-derived anti-human CD11b antibody (Abcam). (2) Staining of M2 macrophages: Double staining using mouse anti-human CD206 antibody (BD) or mouse anti-human CD163 antibody (Leica) and rabbit anti-human CD11b antibody (Abcam). (3) Total macrophage staining: Double staining using mouse-derived anti-human CD68 antibody (Abcam) and rabbit-derived anti-human CD11b antibody (Abcam).

[0046] The number of cells double positive for antibodies (1) to (3) was counted as each macrophage, and the number of M1 macrophages / the number of M2 macrophages was calculated as the M1 / M2 balance. + The correlation with cell number was examined.

[0047] The results are shown in Figures 3 and 4. As shown in Figure 3, epidermal IL-34 + There was a negative correlation between the cell number and the M1 / M2 balance in the dermis. + Although there was no correlation between the cell number and the M1 / M2 balance in the dermis, there was also a correlation between the dermal CSF-1R and the number of dermal M2 macrophages (Fig. 4 right). + The cell number correlated with dermal CSF-1R (Fig. 4, left).

[0048] Experiment 4: Screening of anti-photoaging agents that prevent and / or improve photoaging by increasing epidermal IL-34 The results of the above experiments suggest that increasing epidermal IL-34 could prevent and / or improve photoaging. Therefore, we screened for drugs that increase epidermal IL-34. A total of six ingredients, including thyme extract, were examined as screening target drugs. The thyme extract was a butylene glycol extract of the whole wild thyme plant purchased from Koei Kogyo. The thyme extract was dissolved in butylene glycol.

[0049] Epidermal cells obtained from Kurabo Industries, Ltd. were cultured for 24 hours at 37°C in medium (ThermoFishser Scientific, EpiLife Medium, MEPI500CA) supplemented with growth additives (Kurabo, HuMedia-KG Growth Additive Set, KK-6150). Each of the screening target drugs was added to the medium at 0.1%. As a control, butylene glycol was added at the same volume, and the cells were cultured for an additional 24 hours at 37°C. Cells were harvested, and RNA was extracted (RNeasy Plus Mini Kit, QIAGEN, 74136). IL-34 expression was quantified by real-time PCR. Real-time PCR was performed using a reaction kit (ThermoFishser Scientific, TaqMan RNA-to-CT 1-Step Kit, 4392938) and IL-34 and GAPDH-specific probes (ThermoFishser Scientific, IL-34: Hs01050926_m1, GAPDH: Hs02786624_g1). GAPDH was used as an endogenous control. We searched for anti-photoaging agents that reproducibly increase IL-34 expression levels compared to the control (cont).

[0050] The results are shown in Figure 5. It was found that thyme extract had the effect of significantly increasing the amount of IL-34 expressed in epidermal cells.

[0051] Experiment 5: Further exploration of drugs that increase epidermal IL-37 In order to search for further substances, the applicant investigated the effect of nicotinamide on promoting epidermal IL-34 production. Nicotinamide (CAS No. 98-92-0) was purchased from Wako Pure Chemical Industries, Ltd. (now Fujifilm Wako Pure Chemical Co., Ltd.).

[0052] Fresh abdominal skin samples from human subjects (in their 20s and 30s) who had given informed consent were purchased from KAC Co., Ltd. and cultured in an equal mixture of 10% FBS-DMEM (Thermo Fisher Science, 11885084) and Humedia-KG2 (KURABO, KK-2150S) supplemented with nicotinamide (final concentration 15 mM). Controls were cultured in the same medium without nicotinamide. The medium was changed daily, and tissue fragments were collected on the second day.

[0053] The collected fresh human skin was dehydrated and fixed using cold acetone according to the AMeX method, then substituted with acetone, methyl benzoate, and xylene in that order, and embedded in paraffin. Sections were cut at a thickness of 3 μm and used for histological staining.

[0054] Paraffin sections (3 μm thick) were deparaffinized with xylene and hydrated with EtOH. Fluorescent immunostaining was performed using an antibody against IL-34 (Abcam, ab101443).

[0055] Fresh human skin samples were collected and heat-treated on a 60°C hot plate to separate the epidermis. mRNA was extracted using the RNeasy mini kit (QIAGEN), and cDNA was synthesized using SuperScript VILO (Invitrogen). Quantitative PCR analysis was then performed using the synthesized cDNA with platinum SYBER green (Invitrogen). The primers used were the Human IL-34 qPCR Primer Pair (Sinobiological, Cat: HP100825) and the B2M primer set (shown in Table 1 below) as an endogenous control. Anti-photoaging agents were sought that reproducibly increased IL-34 expression levels compared to the control (cont). [Table 1]

[0056] The results are shown in Figures 6 and 7. It was found that nicotinamide has the effect of significantly increasing the amount of IL-34 protein and gene expression in epidermal cells.

[0057] Experiment 6: IL-34 regulates M1 / M2 balance As mentioned above, CSF-1R is a receptor for CSF-1 and IL-34, but the following experiment was conducted to demonstrate that IL-34, rather than CSF-1, is strongly involved in the regulation of the M1 / M2 balance in the present invention. Peripheral blood-derived CD14+ monocytes (Lifeline Cell Technology) were seeded at 300,000 cells per well in RPMI 1640 medium (Nacalai Tesque, Kyoto, Japan) supplemented with 10% fetal bovine serum, 1 mM sodium pyruvate, and 2 mM L-glutamine, and human CSF-1 (R&D) was added at 100 ng / ml. After seeding, the cells were cultured at 37°C for 6 days to differentiate into undifferentiated macrophages (Patent Document 3).

[0058] After differentiation, the culture medium was removed and the cells were washed with PBS. IL-34 or CSF-1 (both from R&D) was added at concentrations of 0, 60, 120, 180, and 240 ng / ml, respectively. After 72 hours of culture, mRNA was extracted using the RNeasy Plus Mini Kit (QIAGEN, 74136).

[0059] The expression levels of M2 macrophage markers CD206 and IL-10 were quantified by real-time PCR using the recovered mRNA. Real-time PCR was performed using a reaction kit (ThermoFishser Scientific, TaqMan RNA-to-CT 1-Step Kit, 4392938) and CD206, IL-10, and GAPDH-specific probes (ThermoFishser Scientific, CD206: Hs07288635_g1, IL-10: Hs00961622_m1, GAPDH: Hs02786624_g1).

[0060] The results are shown in Figure 8. When IL-34 was added, a concentration-dependent increase in the expression levels of CD206 and IL-10 was observed, suggesting the induction of M2 macrophages. On the other hand, CSF-1, which binds to the same receptor, CSF-1R, had no such effect, suggesting that M2 induction is specific to IL-34. These results suggest that the anti-photoaging effect of the present invention induces dermal M2 macrophages and adjusts the dermal M1 / M2 balance by increasing skin IL-34. Furthermore, it is suggested that the induction of dermal M2 macrophages may be mediated by an effect on dermal CSF-1R, such as an increase in dermal CSF-1R due to an increase in skin IL-34.

[0061] Previous studies have shown that light stimulation induces an imbalance in the M1 / M2 balance, leading to photoaging, and that the M1 / M2 balance can be adjusted by applying ingredients such as thyme extract, which have M1 differentiation inhibitory effects, as M1 / M2 balance adjusters. This study further demonstrated that skin IL-34 and dermal CSF-1R are involved in photoaging, and found correlations between epidermal IL-34 and the M1 / M2 balance, between dermal IL-34 and dermal CSF-1R, and between dermal CSF-1R and the number of dermal M2 macrophages. These findings suggest that epidermal IL-34 may induce M2 macrophages in the dermis, that increased dermal IL-34 may increase CSF-1R, which is its recipient, and that increased CSF-1R may increase the amount of IL-34 received.

[0062] Furthermore, it was shown that thyme extract and nicotinamide promote the production of epidermal IL-34 and inhibit differentiation into M1 macrophages while inducing M2 macrophages. Therefore, increasing epidermal IL-34 by applying thyme extract and / or nicotinamide may potentially prevent and / or improve photoaging through an increase in dermal CSF-1R and / or adjustment of the M1 / M2 balance. [Industrial Applicability]

[0063] The present invention makes it possible to prevent and / or improve photoaging, evaluate the degree of photoaging, and search for anti-photoaging agents.

Claims

1. An anti-photoaging agent for preventing and / or ameliorating photoaging by increasing skin IL-34.

2. The anti-photoaging agent according to claim 1, comprising thyme extract as an agent for increasing skin IL-34.

3. The anti-photoaging agent according to claim 1 or 2, which prevents and / or improves photoaging by increasing epidermal IL-34 and thereby adjusting the dermal M1 / M2 balance.

4. A method for screening an anti-photoaging agent, comprising: applying a candidate agent to the skin sample; Measuring IL-34 in skin samples before and after application of the candidate drug; and assessing the drug to have an anti-photoaging effect when IL-34 in the skin sample to which the candidate drug has been applied increases compared to before the drug is applied; The method comprising:

5. A cosmetic method for preventing and / or ameliorating photoaging by increasing skin IL-34.

6. The cosmetic method according to claim 5, wherein photoaging is prevented and / or improved by increasing epidermal IL-34 to regulate the dermal M1 / M2 balance.

7. The cosmetic method according to claim 5 or 6, comprising administering an IL-34 production promoter comprising a thyme extract.

8. An IL-34 production promoter containing thyme extract.

9. The cosmetic method according to claim 5 or 6, comprising administering an IL-34 production promoter comprising nicotinamide.

10. An IL-34 production promoter containing nicotinamide.

11. An anti-photoaging agent for preventing and / or ameliorating photoaging by increasing dermal CSF-1R.

12. The anti-photoaging agent according to claim 11, which prevents and / or improves photoaging by increasing dermal CSF-1R and thereby inducing M2 macrophages.

13. A method for screening an anti-photoaging agent, comprising: applying a candidate agent to the skin sample; Measuring CSF-1R in skin samples before and after application of the candidate drug; and assessing the drug to have an anti-photoaging effect if CSF-1R in the skin sample to which the candidate drug has been applied increases compared to before application of the drug; The method comprising:

14. A cosmetic method for preventing and / or ameliorating photoaging by increasing dermal CSF-1R.

15. The cosmetic method according to claim 14, wherein photoaging is prevented and / or improved by increasing dermal CSF-1R to induce M2 macrophages.

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