Inhibitor of inflammation in keratinocytes, and method for screening for inhibitor of inflammation in keratinocytes

Abstract

The present invention provides an inhibitor of inflammation in keratinocytes enhanced by a combination of exposure to air pollutants and ultraviolet radiation, the inhibitor comprising one or more substances selected from the group consisting of an Angelica acutiloba extract, a Yoshino cherry extract, and a seaweed extract. The present invention also provides a method for screening for an inhibitor of inflammation in keratinocytes enhanced by a combination of exposure to air pollutants and ultraviolet radiation.

Description

Inhibitor of keratinocyte inflammation and method for screening inhibitor of keratinocyte inflammation

[0001] The present invention relates to an inhibitor of keratinocyte inflammation and a method for screening an inhibitor of keratinocyte inflammation. The present invention also relates to the use of an inhibitor for the manufacture of a composition for inhibiting keratinocyte inflammation.

[0002] Skin aging is a change in the skin mediated by various factors, including internal factors such as aging, hormones, and metabolism, as well as external factors such as ultraviolet light and dryness. Skin aging can sometimes be determined by appearance, such as age spots, wrinkles, sagging, and shrinkage. However, there are also cases where invisible aging progresses, such as changes in the structure and components of internal skin tissue. This type of aging often progresses unnoticed, making it difficult to take preventative measures. It is important to take appropriate measures to prevent and improve skin aging.

[0003] In recent years, attention has been focused on the effects of air pollutants on the skin. Since exposure to air pollutants is mainly outdoors, people are also exposed to ultraviolet rays that can damage the skin.

[0004] Cited Document 1 observes the effects of air pollutants and UVA on keratinocytes. However, this document examines the effects on cytotoxicity using only UVA as the ultraviolet light used, and does not mention the effects on gene expression.

[0005] Cited document 2 describes that PM2.5 alone increases the expression of IL-8, which is known as an inflammatory marker.

[0006] Jeremy Soeur, et al. , Photo-pollution stress in skin: Traces of pollutants (PAH and particulate matter) impair redox homeostasis in keratinocytes exposed to UVA1. J Dermatol Sci. 2017 May; 86(2):162-169. John C. Bierman, et al. , Niacinamide mitigates SASP-related inflammation induced by environmental stressors in human epidermal keratinocytes and skin. Int J Cosmet Sci. 2020 Oct; 42(5):501-511.

[0007] An object of the present invention is to provide an inhibitor of keratinocyte inflammation and a method for screening such an inhibitor.

[0008] As a result of extensive research, the present inventors have discovered a substance capable of suppressing inflammation of keratinocytes that is enhanced by a combination of exposure to air pollutants and ultraviolet irradiation. Based on this discovery, they have also discovered a method for screening for a substance capable of suppressing inflammation of keratinocytes that is enhanced by a combination of exposure to air pollutants and ultraviolet irradiation.

[0009] That is, the present application provides the following inventions: [1] An inhibitor of keratinocyte inflammation enhanced by a combination of exposure to air pollutants and ultraviolet irradiation, comprising one or more substances selected from the group consisting of Angelica acutiloba extract, Somei-Yoshino cherry extract, and seaweed extract. [2] The inhibitor according to item 1, wherein the air pollutants are particles having an average particle size of 10 μm or less in 99% or more and contain aluminum (Al), calcium (Ca), iron (Fe), magnesium (Mg), potassium (K), sodium (Na), titanium (Ti), and / or zinc (Zn) as elemental components. [3] The inhibitor according to item 1 or 2, wherein the air pollutants are NIES No. 28 Urban Aerosols. [4] The inhibitor according to any one of items 1 to 3, wherein the ultraviolet light is UVB. [5] The inhibitor according to any one of items 1 to 4, which suppresses the expression of a factor selected from the group consisting of IL-8 and IL-1β, which is enhanced by a combination of exposure to air pollutants and ultraviolet irradiation.

[0010] [6] A method for screening for an inhibitor of keratinocyte inflammation enhanced by a combination of exposure to an air pollutant and ultraviolet irradiation, comprising: contacting a candidate drug with keratinocytes exposed to an air pollutant and irradiated with ultraviolet irradiation, and culturing the keratinocytes; evaluating the inhibitory effect on keratinocyte inflammation enhanced by a combination of exposure to an air pollutant and ultraviolet irradiation based on an index indicating keratinocyte inflammation; and selecting the candidate drug as the inhibitor if the candidate drug is evaluated to have an inhibitory effect. [7] The method of Item 6, wherein the air pollutant is composed of particles having an average particle size of 10 μm or less at 99% or more, and contains aluminum (Al), calcium (Ca), iron (Fe), magnesium (Mg), potassium (K), sodium (Na), titanium (Ti), and / or zinc (Zn) as elemental components. [8] The method of Item 6 or 7, wherein the air pollutant is NIES No. 28 Urban Aerosols. [9] The method of any one of Items 6 to 8, wherein the ultraviolet light is UVB.

[10] The method according to any one of items 6 to 9, wherein the indicator of inflammation is the expression level of a factor selected from the group consisting of IL-8, IL-1β, and TNF-α.

[11] The method according to item 10, wherein the expression level is the expression level of mRNA.

[0011]

[12] Use of an inhibitor for the manufacture of a composition for suppressing keratinocyte inflammation enhanced by a combination of exposure to air pollutants and ultraviolet irradiation, wherein the inhibitor is one or more selected from the group consisting of Angelica acutiloba extract, Somei-Yoshino cherry extract, and seaweed extract.

[13] The use according to item 12, wherein the air pollutants are particles having an average particle size of 10 μm or less in 99% or more and contain aluminum (Al), calcium (Ca), iron (Fe), magnesium (Mg), potassium (K), sodium (Na), titanium (Ti), and / or zinc (Zn) as elemental components.

[14] The use according to item 12 or 13, wherein the air pollutants are NIES No. 28 Urban Aerosols.

[15] The use according to any one of items 12 to 14, wherein the ultraviolet light is UVB.

[16] The use according to any one of Items 12 to 15, wherein the expression of a factor selected from the group consisting of IL-8 and IL-1β is suppressed by a combination of exposure to air pollutants and ultraviolet irradiation.

[0012] According to the present invention, there is provided a drug for suppressing inflammation of keratinocytes that is synergistically enhanced by a combination of exposure to air pollutants and ultraviolet irradiation.

[0013] Figure 1A shows the results of an investigation into how exposure to air pollutants (AP-, AP+) and UV irradiation (UV-, UV+) affects IL-8 mRNA expression in keratinocytes. The relative IL-8 mRNA expression level is shown, with the IL-8 mRNA expression level in keratinocytes from wells without air pollutant exposure or UV irradiation (AP-, UV-) set to "1." n = 4. **p < 0.01, ***p < 0.001 (Tukey's test). Figure 1B shows the results of an investigation into how exposure to air pollutants (AP-, AP+) and UV irradiation (UV-, UV+) affects IL-1β mRNA expression in keratinocytes. The relative IL-1β mRNA expression level is shown, with the IL-1β mRNA expression level in keratinocytes from wells without air pollutant exposure or UV irradiation (AP-, UV-) set to "1." n=4. *p<0.05, ***p<0.001 (Tukey's test). Figure 1C shows the results of an investigation into how the presence or absence of exposure to air pollutants (AP-, AP+) and the presence or absence of ultraviolet light irradiation (UV-, UV+) affects TNF-α mRNA expression in keratinocytes. The relative TNF-α mRNA expression levels are shown, with the TNF-α mRNA expression levels in keratinocytes in wells without air pollutant exposure or ultraviolet light irradiation (AP-, UV-) set to "1." n=4. ***p<0.001 (Tukey's test). Figure 2 shows the results of an investigation into the level of IL-8 mRNA expression when candidate substances were added to keratinocytes exposed to air pollutants (AP) and exposed to ultraviolet light (UV). The figure shows the relative level of IL-8 mRNA expression, with the level of IL-8 mRNA expression in keratinocytes from wells not exposed to air pollutants or irradiated with UV light (Control) set to "1." n = 3-4. *p<0.05, **p<0.01, ***p<0.001 (Dunnett's test). Figure 3 shows the results of examining the level of IL-1β mRNA expression when candidate substances were added to keratinocytes exposed to air pollutants (AP) or irradiated with UV light (UV). The figure shows the relative level of IL-1β mRNA expression, with the level of IL-1β mRNA expression in keratinocytes from wells not exposed to air pollutants or irradiated with UV light (Control) set to "1." n = 4.**p<0.01, ***p<0.001 (Dunnet's test).

[0014] Unless otherwise defined, all terms (technical and scientific) used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0015] In one embodiment, the present invention provides an agent for suppressing keratinocyte inflammation enhanced by a combination of exposure to air pollutants and ultraviolet irradiation, the agent comprising one or more substances selected from the group consisting of Angelica acutiloba extract, Somei-Yoshino cherry extract, and seaweed extract. The present invention provides a drug for suppressing keratinocyte inflammation synergistically enhanced by a combination of exposure to air pollutants and ultraviolet irradiation. This is expected to improve the skin's moisture retention function and skin barrier function, as well as suppress skin aging and a decline in the stemness of epidermal stem cells, which are caused by keratinocyte inflammation.

[0016] "Air pollution" generally refers to substances emitted into the atmosphere that exceed the natural physical diffusion and deposition functions, chemical removal functions, and biological purification functions, resulting in an increase in the amount of substances emitted into the atmosphere compared to the natural state, which has a direct or indirect impact on ecosystems, including humans, and objects (Reference: Ministry of the Environment website (https: / / www.env.go.jp / earth / coop / coop / materials / 02-apctmj1 / 02-apctmj1-012.pdf)). Among these, representative air pollutants that have an impact on human health include, for example, sulfur dioxide (SO ２ ), nitrogen oxides (NO ｘ ), nitrogen dioxide (NO ２ ), suspended particulate matter (particles suspended in the atmosphere with an average particle size of 10 μm or less), carbon monoxide (CO), photochemical oxidants (O ｘ ), non-methane hydrocarbons (NMHC), etc., and may also include nitrogen monoxide, gaseous nitric acid, gaseous fluorine, hydrogen chloride, ammonia, methyl mercaptan, hydrogen sulfide, methyl sulfide, trimethylamine, methyl disulfide, aldehyde, styrene, etc. In particular, among suspended particulate matter, very small particles with a particle size of 2.5 μm or less are called fine particulate matter (PM).２．５ ) and its components may include carbon components, nitrates, sulfates, ammonium salts, as well as inorganic elements such as silicon, sodium, and aluminum.

[0017] As used herein, the term "air pollutant" may refer to one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 10 or more) of the typical air pollutants described above, or may refer to one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 10 or more) components contained in known standard substances. For example, in one aspect, an air pollutant that can be applied to the present invention or that can be used to evaluate the effects of the present invention may be one in which 99% or more of particles have an average particle size of 10 μm or less, and which contains, for example, aluminum (Al), calcium (Ca), iron (Fe), magnesium (Mg), potassium (K), sodium (Na), titanium (Ti), and / or zinc (Zn) as elemental components. In one embodiment, air pollutants that can be applied to the present invention or that can be used to evaluate the effects of the present invention may further contain, in addition to the above elemental components, arsenic (As), barium (Va), cadmium (Cd), copper (Cu), lead (Pb), manganese (Mn), nickel (Ni), strontium (Sr), uranium (U), and / or vanadium (V). In one embodiment, air pollutants that can be applied to the present invention or that can be used to evaluate the effects of the present invention may be known reference materials, such as urban air dust, and may be, for example, but not limited to, certified reference materials (CRMs) researched and developed by the National Institute for Environmental Studies (NIES), such as NIES CRM No. NIES CRM No. 28 Urban Aerosols (urban airborne particulates) may also be used. For reference, the certified values ​​of the substances contained in NIES CRM No. 28 Urban Aerosols are shown below. Note that 99% or more of the particles contained in NIES CRM No. 28 Urban Aerosols have an average particle size of 10 μm or less.

[0018]

[0019] Ultraviolet rays (ultraviolet light) are light with wavelengths ranging from approximately 100 nm to approximately 400 nm and are classified as UVA (320-400 nm), UVB (280-320 nm), and UVC (100-280 nm). At the Earth's surface, the ozone layer absorbs some of the ultraviolet rays contained in sunlight, particularly UVC and UVB, allowing UVA and UVB to reach the Earth's surface. The present inventors have discovered for the first time that the combination of UV irradiation and exposure to air pollutants can enhance inflammation in keratinocytes, and have further discovered a new drug that can suppress this. In particular, it was previously unknown that the combination of UVB irradiation and exposure to air pollutants can enhance inflammation in keratinocytes.

[0020] As a result of extensive research, the inventors of the present application have discovered that one or more substances selected from the group consisting of Angelica acutiloba extract, Somei-Yoshino cherry extract, and seaweed extract can suppress inflammation of keratinocytes that is enhanced by a combination of exposure to air pollutants and ultraviolet irradiation.

[0021] Although Angelica acutiloba extract, Somei-Yoshino cherry extract, and seaweed extract are used in topical skin preparations, they have not been known to have the effect of suppressing inflammation of keratinocytes, which is enhanced by a combination of exposure to air pollutants and ultraviolet light.

[0022] The extract can be obtained by conventional methods, for example, by immersing or refluxing a part or all of the plant material with an extraction solvent at room temperature or under heat, followed by filtration and concentration. The extracted portion may be dried prior to solvent extraction. Any solvent commonly used in extraction can be used as the extraction solvent. For example, organic solvents such as alcohols (e.g., methanol, ethanol, propylene glycol, 1,3-butylene glycol, glycerin), hydrous alcohols, chloroform, dichloroethane, carbon tetrachloride, acetone, ethyl acetate, hexane, etc., or aqueous solvents (e.g., water, saline, phosphate buffer, borate buffer), etc., can be used alone or in combination. Preferably, one or more solvents selected from water, methanol, ethanol, and 1,3-butylene glycol are used.

[0023] The extract obtained by extraction with the above solvents can be used as is, or the extract can be concentrated by, for example, freeze-drying. If necessary, the extract can be used after removing impurities using an adsorption method, for example, an ion exchange resin, or after adsorption on a column of porous polymer (e.g., Amberlite XAD-2), followed by elution with a desired solvent and further concentration.

[0024] The seaweed extract applicable to the present invention may be an extract extracted from at least one alga selected from the group consisting of brown algae, red algae, and green algae, preferably a mixed extract of brown algae, red algae, and green algae, more preferably an extract of brown algae of the genus Laminaria and the genus Undaria, red algae of the genus Thyme, and green algae of the genus Ulva, and even more preferably Algerex (registered trademark).

[0025] Algerex (registered trademark) is a seaweed extract and a cosmetic ingredient sold by Ichimaru Pharcos Co., Ltd. More specifically, Algerex (registered trademark) is a mixed extract of brown, red, and green algae. It is obtained by soaking whole brown algae in 50% 1,3-butylene glycol for three days and filtering the resulting brown algae extract, and then mixing the resulting brown, red, and green algae extracts with whole brown, red, and green algae in 50% 1,3-butylene glycol for three days and filtering the resulting brown, red, and green algae extracts. Algerex (registered trademark) is believed to improve moisture content and to have an inhibitory effect on rough skin. The brown algae used in Algerex (registered trademark) are algae of the Laminaria and Undaria pinnatifida genera, such as Mitsuishi-kombu and Undaria pinnatifida. The red algae used in Algerex (registered trademark) are algae of the Centipedia genus, such as Eucheuma and Hijirimen. The green algae used in Argerex (registered trademark) are algae of the genus Ulva, an example of which is Ulva gracilis.

[0026] The agent of the present invention can also be a composition in which the above-mentioned active ingredient is combined with other ingredients, such as excipients, carriers, and / or diluents. The composition may have any composition and form, and may be appropriately selected depending on the active ingredient, intended use, and other conditions. The composition can be manufactured using a conventional method in a formulation in which an excipient, carrier, and / or diluent and other ingredients are appropriately combined depending on the dosage form.

[0027] 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 can also be incorporated into various foods, beverages, and feeds for human and animal ingestion.

[0028] When the agent of the present invention is applied to external preparations for skin such as cosmetics, pharmaceuticals, and quasi-drugs, the amount of the agent to be blended can be appropriately determined depending on the type, purpose, form, method of use, etc. The amount of extracts to be blended as the agent, such as Angelica acutiloba extract, Somei-Yoshino cherry extract, and seaweed extract, may be, for example, 0.0001 to 20.0% by mass, preferably 0.0001 to 10.0% by mass, and more preferably 0.001 to 1% by mass, in terms of dry matter, of the total amount of the agent.

[0029] In addition to the above ingredients, if necessary, ingredients typically used in external skin preparations such as cosmetics, pharmaceuticals, and quasi-drugs, such as antioxidants, oils, UV protection agents, surfactants, thickeners, alcohols, powder ingredients, coloring materials, aqueous ingredients, water, various skin nutrients, etc., can be appropriately blended as needed within the range that does not impair the effects of the present invention.

[0030] When the present invention is used as an external preparation for skin, it can be applied as a cosmetic, quasi-drug, or the like to be applied to the outer skin, and is particularly preferably applied as a cosmetic, and the dosage form is not limited as long as it can be applied to the skin, and any dosage form can be applied, such as a solution system, a solubilized system, an emulsion system, a powder dispersion system, a water-oil two-layer system, a water-oil-powder three-layer system, an ointment, a lotion, a gel, or an aerosol.

[0031] When the agent of the present invention is used as a cosmetic, it may be used in the form of a lotion, emulsion, foundation, lipstick, lip balm, cleansing cream, massage cream, pack, hand cream, hand powder, body shampoo, body lotion, body cream, bath cosmetics, etc.

[0032] However, the forms that the agent and composition of the present invention can take are not limited to the dosage forms and shapes described above.

[0033] The agent of the present invention may also be used in combination with other treatments. Examples of other treatments include, but are not limited to, the application of stimuli such as light, electrical, mechanical, and manual stimulation, the administration of skin anti-aging agents, and the application of cosmetics. The skin anti-aging agent may be a natural or chemically synthesized compound, an animal or plant extract, or may be in the form of a single substance, a mixture, or a solution such as an aqueous solution, or in the form of a cosmetic. The route of administration of the skin anti-aging agent may be selected arbitrarily, and examples include oral administration, transdermal administration, subcutaneous administration, transmucosal administration, and intramuscular administration.

[0034] In another aspect, the present invention provides use of an inhibitor for the manufacture of a composition for suppressing inflammation of keratinocytes enhanced by a combination of exposure to air pollutants and ultraviolet irradiation, wherein the inhibitor is one or more selected from the group consisting of Angelica acutiloba extract, Somei-Yoshino cherry extract, and seaweed extract.

[0035] The present invention also provides a method for screening inhibitors of keratinocyte inflammation enhanced by a combination of exposure to air pollutants and ultraviolet irradiation, which may include: contacting a candidate drug with keratinocytes exposed to air pollutants and irradiated with ultraviolet light, and culturing the cells; evaluating the inhibitory effect on keratinocyte inflammation enhanced by a combination of exposure to air pollutants and ultraviolet irradiation based on an index showing the keratinocyte inflammation; and selecting the candidate drug as the inhibitor if it is evaluated to have an inhibitory effect.

[0036] As described above, the present invention is the first to discover that the combination of exposure to air pollutants and ultraviolet irradiation can enhance the inflammatory response in keratinocytes. Based on this discovery, a method for screening inhibitors of inflammation in keratinocytes that is enhanced by the combination of exposure to air pollutants and ultraviolet irradiation has been completed.

[0037] The air pollutant used in the method of the present invention may be any of the above air pollutants, and may, for example, be one in which 99% or more of the particles have an average particle size of 10 μm or less and which contains aluminum (Al), calcium (Ca), iron (Fe), magnesium (Mg), potassium (K), sodium (Na), titanium (Ti), and / or zinc (Zn) as elemental components. Furthermore, for example, the air pollutant may be NIES No. 28 Urban Aerosols.

[0038] The ultraviolet light used in the method of the present invention may be light having a wavelength of about 100 nm to about 400 nm, such as UVA (320-400 nm), UVB (280-320 nm), or UVC (100-280 nm), preferably UVB (280-320 nm). The intensity of the light to be irradiated may be adjusted appropriately depending on the wavelength, irradiation time, cell density, etc., and may be, for example, 0.1 mJ / cm. ２ ~10 J / cm ２ , 1 mJ / cm ２ ~1 J / cm ２ , 10 mJ / cm ２ ~100 mJ / cm ２ , or 20 mJ / cm ２ ~50 mJ / cm ２ may be irradiated.

[0039] The keratinocytes used in the method of the present invention are one of the cells that make up the epidermis. In the epidermal tissue of a living body, they divide in the deepest layer (the basal layer), differentiate toward the upper layers, and then migrate to the surface while differentiating into the spinous layer, the granular layer, and the stratum corneum, and eventually become dandruff and are shed. The keratinocytes used in the method of the present invention may be primary cultured cells collected from living tissue, or may be cells that have been isolated and / or grown in advance and are commercially available or distributed, or may be established cell lines, or may be cells induced to differentiate from pluripotent stem cells such as ES cells, iPS cells, or Muse cells. The keratinocytes may be cultured according to known methods, and the culture medium and culture conditions used may be determined with reference to known methods.

[0040] In the present invention, the indicator of keratinocyte inflammation may be based on the expression level of a known factor, for example, an inflammatory marker. For example, the expression levels of factors such as IL-1α, IL-1β, IL-6, IL-8, TNF-α, and GM-CSF can be determined by measuring, for example, the expression levels of their mRNAs or the expression levels of their produced proteins using known methods (e.g., ELISA, flow cytometry, Western blotting, immunohistochemistry, qPCR, etc.). These expression levels may be compared by normalizing them with the expression level of an internal control protein, such as GAPDH, whose expression level is considered to be constant even between cells. In one aspect, the indicator of keratinocyte inflammation may be the expression level of a factor selected from the group consisting of IL-8, IL-1β, and TNF-α, or the expression level of a factor selected from the group consisting of IL-8 and IL-1β, or the expression level of IL-8.

[0041] In one aspect, the candidate factor may be, for example, a small molecule compound, a peptide, a nucleic acid, a protein, a mammalian cell (e.g., mouse, rat, pig, cow, sheep, monkey, human, etc.), a tissue extract or cell culture supernatant, a plant-derived compound or extract (e.g., herbal extract, a compound derived from a herbal drug), and a microbial compound, extract, or culture product.

[0042] In one aspect, the inhibitory effect of keratinocyte inflammation enhanced by a combination of exposure to air pollutants and ultraviolet irradiation is evaluated based on the index of keratinocyte inflammation. For example, if the value of the index of keratinocyte inflammation when the candidate factor is added is significantly reduced compared to the value of the index of keratinocyte inflammation when the candidate factor is not added, the candidate factor can be evaluated as having an inhibitory effect. In this case, the candidate drug can be selected as the inhibitor.

[0043] All documents cited herein are incorporated by reference in their entirety.

[0044] The present invention will now be described in more detail with reference to examples. The 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 can be modified, for example, by adding, deleting, or substituting constituent elements of the present invention, provided that the modifications do not depart from the spirit of the present invention.

[0045] Example 1 1. Experimental Materials and Methods (1) A 24-well plate (Corning) was filled with 60 μM Ca ２＋ Keratinocytes (product number: KK-4009, manufactured by Kurabo Industries Co., Ltd.) suspended in Epilife medium (MEPICF500, manufactured by Thermo Fisher Scientific) containing 4×10 keratinocytes (product number: KK-4009, manufactured by Kurabo Industries Co., Ltd.) and HuMedia-KG growth additive set (KK-6150, manufactured by Kurabo Industries Co., Ltd.), which is a growth additive for normal human epidermal keratinocytes, were cultured at 4×10 keratinocytes. ５ The cells were seeded at 100 cells / well and incubated in 5% CO ２ (2) 500 μL of Eplife medium and air pollutant (NIES No. 28 Urban Aerosols) (150 μg / mL) suspended in gentamicin / amphotericin B (HK-3510, Kurabo Industries, Ltd.), a growth additive for normal human epidermal keratinocytes, were added to each well (1) ("AP+"), or no air pollutant was added ("AP-"), under the conditions of 5% CO ２ (3) Each well was replaced with a buffer solution (sodium chloride 140 mM, potassium chloride 5.4 mM, magnesium chloride hexahydrate 1 mM, 10 mM HEPES, 10 mM glucose, 0.06 mM calcium carbonate), and the wells were irradiated with 30 mJ / cm using a UV irradiation device (PHILIPS ULTRAVIOLET-B TL 20W / 12RS). ２ (0.3 mW / cm ２ The cells were then exposed to UV-B (at an output of 1 / s for 100 seconds) ("UV+") or not ("UV-"), and the medium was then replaced with Eplife medium and gentamicin / amphotericin B medium. The medium was then incubated in 5% CO ２The plates were incubated at 37°C for 3 hours. (4) After washing each well with PBS, total RNA was collected using an RNeasy mini kit (QIAGEN), and cDNA was synthesized using SuperScript VILO Master Mix (Invitrogen). (5) The mRNA expression levels of IL-8, IL-1β, and TNF-α were quantified by qPCR. As a control, the mRNA level of GAPDH was measured (Figure 1). The primers used are as follows:

[0046] IL-8: 5'-AGACAGCAGAGCACACAAGC-3' (forward primer (SEQ ID NO: 1)), 5'-ATGGTTCCTTCCGGTGGT-3' (reverse primer (SEQ ID NO: 2)); IL-1β: 5'-AAAGCTTGGTGATGTCTGGTC-3' (forward primer (SEQ ID NO: 3)), 5'-GGACATGGAGAACACCACTTG-3' (reverse primer (SEQ ID NO: 4)); TNF-α: 5'-CAGCCTCTTCTCTCCTTCCTGAT-3' (forward primer (SEQ ID NO: 5)), 5'-GCCAGAGGGCTGATTAGAGA-3' (reverse primer (SEQ ID NO: 6)); GAPDH: 5'-GAAGGTGAAGGTCGGAGTC-3' (forward primer (SEQ ID NO: 7)), 5'-GAAGATGGTGATGGGATTTC-3' (reverse primer (SEQ ID NO: 8))

[0047] 2. Results Addition of air pollutants alone did not have any effect on increasing gene expression of the inflammatory cytokines IL-8 and TNF-α, but it was found that combining air pollutants with UV-B irradiation synergistically increased gene expression of the inflammatory cytokines IL-8 and TNF-α (Figures 1A and 1C).

[0048] It was revealed that the combination of air pollutant addition and UV-B irradiation synergistically increased gene expression of the inflammatory cytokine IL-1β compared to either treatment alone (Figure 1B).

[0049] Example 2 1. Experimental Materials and Methods The following (3a) was carried out in parallel with (3) of Example 1. The other steps were basically carried out in accordance with the experimental method described in Example 1.

[0050] (3a) Using a UV irradiation device (PHILIPS ULTRAVIOLET-B TL 20W / 12RS), 30 mJ / cm ２ (0.3 mW / cm ２ The wells that were irradiated with UV-B (at an output of 1 / s for 100 seconds) ("UV+") or not irradiated ("UV-") were replaced with Epilife medium or gentamicin / amphotericin B medium containing a solution containing the following candidate factors diluted to a final concentration of 0.1 wt% or 0.05 wt%, and incubated in 5% CO ２ The mixture was incubated at 37°C for 3 hours.

[0051]

[0052] 2. Results In wells to which Angelica acutiloba extract (0.1 wt%, 0.5 wt%), Somei-Yoshino cherry extract (0.1 wt%), and seaweed extract (0.1 wt%, 0.5 wt%) were added, it was revealed that the gene expression of IL-8, an inflammatory cytokine that was synergistically induced by the combination of air pollutants and UV-B irradiation, was significantly reduced (Figure 2).

[0053] In wells to which Angelica acutiloba extract (0.1 wt%, 0.5 wt%) was added, it was revealed that the gene expression of IL-1β, an inflammatory cytokine that was synergistically induced by the combination of air pollutants and UV-B irradiation, was significantly reduced (Figure 3).

Claims

1. An agent for suppressing inflammation of keratinocytes enhanced by a combination of exposure to air pollutants and ultraviolet light, comprising one or more substances selected from the group consisting of Angelica acutiloba extract, Somei-Yoshino cherry extract, and seaweed extract.

2. The inhibitor according to claim 1, wherein the air pollutant is composed of 99% or more particles having an average particle size of 10 μm or less, and contains aluminum (Al), calcium (Ca), iron (Fe), magnesium (Mg), potassium (K), sodium (Na), titanium (Ti), and / or zinc (Zn) as elemental components.

3. The suppressor of claim 1, wherein said air pollutants are NIES No. 28 Urban Aerosols.

4. The inhibitor of claim 1, wherein the ultraviolet light is UVB.

5. The inhibitor according to claim 1, which suppresses the expression of a factor selected from the group consisting of IL-8 and IL-1β, which is enhanced by a combination of exposure to air pollutants and ultraviolet irradiation.

6. A method for screening for an inhibitor of inflammation of keratinocytes enhanced by a combination of exposure to air pollutants and ultraviolet light irradiation, comprising: contacting a candidate drug with keratinocytes exposed to air pollutants and irradiated with ultraviolet light, and culturing the cells; evaluating the inhibitory effect of the candidate drug on inflammation of keratinocytes enhanced by a combination of exposure to air pollutants and ultraviolet light irradiation based on an indicator of inflammation of the keratinocytes; and selecting the candidate drug as the inhibitor if it is evaluated to have an inhibitory effect.

7. The method of claim 6, wherein the air pollutant comprises 99% or more particles having an average particle size of 10 μm or less and contains aluminum (Al), calcium (Ca), iron (Fe), magnesium (Mg), potassium (K), sodium (Na), titanium (Ti), and / or zinc (Zn) as elemental components.

8. The method of claim 6, wherein said air pollutants are NIES No. 28 Urban Aerosols.

9. The method of claim 6, wherein the ultraviolet light is UVB.

10. The method according to claim 6, wherein the indicator of inflammation is the expression level of a factor selected from the group consisting of IL-8, IL-1β and TNF-α.

11. The method of claim 10, wherein the expression level is an mRNA expression level.

12. Use of an inhibitor for the manufacture of a composition for suppressing inflammation of keratinocytes enhanced by a combination of exposure to air pollutants and ultraviolet irradiation, wherein the inhibitor is one or more selected from the group consisting of Angelica acutiloba extract, Somei-Yoshino cherry extract, and seaweed extract.

13. The use according to claim 12, wherein the air pollutant comprises at least 99% particles with an average particle size of 10 μm or less and contains, as elemental components, aluminum (Al), calcium (Ca), iron (Fe), magnesium (Mg), potassium (K), sodium (Na), titanium (Ti), and / or zinc (Zn).

14. The use according to claim 12, wherein the air pollutants are NIES No. 28 Urban Aerosols.

15. The use according to claim 12, wherein the ultraviolet light is UVB.

16. The use according to claim 12, which suppresses the expression of a factor selected from the group consisting of IL-8 and IL-1β, which is enhanced by a combination of exposure to air pollutants and ultraviolet radiation.

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