WO2019199089A1 - 미세먼지에 의한 피부각질 형성 세포의 분화 억제를 회복할 수 있는 물질의 스크리닝 방법 및 미세먼지에 의한 피부각질 형성 세포 분화 억제의 회복용 조성물 - Google Patents

미세먼지에 의한 피부각질 형성 세포의 분화 억제를 회복할 수 있는 물질의 스크리닝 방법 및 미세먼지에 의한 피부각질 형성 세포 분화 억제의 회복용 조성물 Download PDF

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WO2019199089A1
WO2019199089A1 PCT/KR2019/004382 KR2019004382W WO2019199089A1 WO 2019199089 A1 WO2019199089 A1 WO 2019199089A1 KR 2019004382 W KR2019004382 W KR 2019004382W WO 2019199089 A1 WO2019199089 A1 WO 2019199089A1
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fine dust
keratinocytes
differentiation
keratin
composition
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PCT/KR2019/004382
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English (en)
French (fr)
Korean (ko)
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최현정
김형준
이태룡
조동형
조두신
Original Assignee
(주)아모레퍼시픽
경희대학교 산학협력단
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Priority to CN201980039628.3A priority Critical patent/CN112334772B/zh
Publication of WO2019199089A1 publication Critical patent/WO2019199089A1/ko

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/49—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing heterocyclic compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/49—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing heterocyclic compounds
    • A61K8/4906—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing heterocyclic compounds with one nitrogen as the only hetero atom
    • A61K8/4913—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing heterocyclic compounds with one nitrogen as the only hetero atom having five membered rings, e.g. pyrrolidone carboxylic acid
    • A61K8/492—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing heterocyclic compounds with one nitrogen as the only hetero atom having five membered rings, e.g. pyrrolidone carboxylic acid having condensed rings, e.g. indol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5076—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving cell organelles, e.g. Golgi complex, endoplasmic reticulum
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6881—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids from skin
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2200/00—Function of food ingredients
    • A23V2200/30—Foods, ingredients or supplements having a functional effect on health
    • A23V2200/318—Foods, ingredients or supplements having a functional effect on health having an effect on skin health and hair or coat
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
    • G01N2333/4701—Details
    • G01N2333/4742—Keratin; Cytokeratin

Definitions

  • the present invention relates to a method for screening a substance capable of restoring the inhibition of differentiation of keratinocytes by fine dust. Specifically, the present invention relates to skin keratinocytes formed by fine dust by controlling the length and number of primary cilia that are organelles of cells. The present invention relates to a method for screening a substance capable of restoring inhibition of differentiation, and a composition for restoring inhibition of skin keratinocyte differentiation by fine dust.
  • Barrier function is to protect against various stimuli (chemicals, air pollutants, dry environment, ultraviolet rays, etc.) from the outside and to prevent excessive release of moisture through the skin.
  • This protective function is a stratum corneum composed of keratinocytes. The function can be maintained only when is normally formed.
  • the outermost stratum corneum (horney layer) of the epidermis is formed from keratinocytes and consists of complete differentiated keratinocytes and the surrounding lipid layer (Marcelo CL et al., J. Invest. Dermatol. , 80, pp 37-44, 1983).
  • Keratinocytes are characteristic cells whose basal cells, which continuously proliferate in the epidermal layer, undergo morphological and functional changes in stages and rise to the surface of the skin. Exfoliated and new keratinocytes take over their function. This repetitive process of change is called “differentiation” or “ekratinization” of epidermal cells.
  • keratinocytes form natural moisturizing factor (NMF) and intercellular fats (ceramides, cholesterol, fatty acids), forming the stratum corneum, which makes the stratum corneum firm and flexible. It retains its function as a skin barrier.
  • NMF natural moisturizing factor
  • ceramides, cholesterol, fatty acids intercellular fats
  • the stratum corneum can easily lose its function due to excessive facial cleansing, lifestyle factors such as bathing, or endogenous diseases such as atopic or aged skin.
  • fine dust is dust of particles that are invisible to the eye and refers to dust having a diameter of 10 ⁇ m or less, and PM10 (fine dust) and PM2.5 having a diameter of 10 ⁇ m or less, 2.5 ⁇ m or less and 1.0 ⁇ m or less, respectively, depending on the particle size.
  • PM10 fine dust
  • PM2.5 having a diameter of 10 ⁇ m or less, 2.5 ⁇ m or less and 1.0 ⁇ m or less, respectively, depending on the particle size.
  • PM poarticulate matter
  • PM means 'particulate matter' (fine particles in solid or liquid state floating in air).
  • Fine dust a harmful substance, causes respiratory and lung diseases, as well as skin and eye diseases, and unlike ordinary dust, accumulates in the body without being filtered out of the nose, mouth, and bronchus.
  • the symptoms of dry skin due to impaired skin barrier function caused by such fine dust are increasing.
  • cytocalin D is known as a type of mycotoxin known as cytocalin, and there is no known relationship between damage to skin barrier function due to fine dust and cytocalin D.
  • the present invention provides a novel method and effective method for screening a substance capable of restoring the inhibition of differentiation of keratinocytes by microdust by controlling the number and length of primary cilia of keratinocytes.
  • An object of the present invention is to provide a composition for restoring the inhibition of skin keratinocyte differentiation.
  • an embodiment of the present invention comprises the steps of treating the test substance to keratinocyte (keratinocyte);
  • test substance Determining the test substance as a substance for restoring inhibition of skin keratinocyte differentiation by fine dust when the relative degree of formation of primary cilia after the test substance treatment is greater than before treatment.
  • methods for screening materials that can restore inhibition of differentiation of cells.
  • Another embodiment of the present invention provides a composition for restoring the inhibition of skin keratinocyte differentiation by microdust, comprising cytochalasin D as an active ingredient.
  • the screening method of a substance capable of restoring the inhibition of differentiation of keratinocytes by the fine dust according to the present invention further comprises the step of confirming the relative formation of primary cilia in the keratinocytes. It is possible to screen substances that can restore the inhibition of differentiation of keratinocytes by dust, and to identify substances that can restore the inhibition of differentiation of keratinocytes by fine dust, which has not been detected by conventional methods. As a result, it can greatly contribute to industrial development in related fields.
  • composition for restoring the inhibition of differentiation of keratinocytes by the fine dust restores the inhibition of the production of primary cilia by the fine dust in the keratinocytes, and as a result, the differentiation of the keratinocytes by the fine dust. Restraint can be effectively restored.
  • FIG. 1 shows confocal microscopy of the length and number of primary cilia as human normal keratinocytes differentiate (scale bar is 10 ⁇ m).
  • FIG. 2 shows confocal microscopy of changes in the length and number of primary cilia when treated with fine dust (PM10) and / or cytocalin D in human normal keratinocytes (scale bar is 10 ⁇ m).
  • Figure 3a is confirmed by RT-PCR changes in the expression of involucrin when treated with fine dust (PM10) and / or cytocalin D in human normal skin keratinocytes.
  • Figure 3b is confirmed by RT-PCR changes in the expression of loricrin when treated with fine dust (PM10) and / or cytocalin D in human normal skin keratinocytes.
  • Figure 3c is confirmed by RT-PCR changes in the expression of filaggrin when treated with fine dust (PM10) and / or cytocalin D in human normal skin keratinocytes.
  • Figure 3d shows the change in the expression of keratin 1 (keratin 1) through RT-PCR when treated with fine dust (PM10) and / or cytocalin D in human normal skin keratinocytes.
  • Figure 3e shows the change in the expression of keratin 10 (keratin 10) through RT-PCR when treated with fine dust (PM10) and / or cytocalin D in human normal skin keratinocytes.
  • fine dust refers to dust of particles that are minutely invisible and refers to dust having a diameter of 10 ⁇ m or less
  • PM10 fine dust having a diameter of 10 ⁇ m or less, 2.5 ⁇ m or less, or 1.0 ⁇ m or less, respectively, depending on the particle size. It is divided into PM2.5 (ultrafine dust) and PM1.0 (ultrafine dust), and it is a broad concept including all of them.
  • PM partate matter
  • PM means 'particulate matter' (fine particles in solid or liquid state floating in air).
  • the term "skin” refers to a tissue covering the body surface of an animal, and is a broad concept including not only tissues covering the body surface such as the face or body, but also the scalp and hair. Meanwhile, in the present specification, the skin may include not only living skin, but also artificial skin or skin mimetics that implement a state of living skin.
  • skin cell differentiation may mean a process in which keratinocytes are functionally differentiated from the basal layer, which is the lowermost layer of the skin, to gradually form a polar layer, a granular layer, and a stratum corneum.
  • primary cilia also called primary cilium (plural primary cilia) are organelles that protrude from the cell body and include various external sources, including chemical stimuli, physical stimuli, light, osmotic pressure, fluid flow, and gravity signals. Refers to organelles that sense stimuli.
  • the degree of relative formation refers to the formation, expression, number, quantity, and quality of the formation or expression when comparing the degree of formation or expression of primary cilia in cells under or without any conditions. It may be a degree indicating the difference.
  • the degree of formation may also include, for example, the number or amount of cells forming or expressing the primary cilia, or the length of the primary cilia.
  • cytokalacin D may be expressed as cytochalasin D or cyto D.
  • the present invention in one aspect, the step of treating the test substance to keratinocyte (keratinocyte); Confirming the relative degree of formation of primary cilia before and after treatment of the test substance in the keratinocytes treated with the test substance; And judging the test substance as a material for restoring inhibition of skin keratinocyte differentiation caused by fine dust when the relative degree of formation of primary cilia after the test substance treatment is greater than before treatment.
  • keratinocyte keratinocyte
  • Confirming the relative degree of formation of primary cilia before and after treatment of the test substance in the keratinocytes treated with the test substance And judging the test substance as a material for restoring inhibition of skin keratinocyte differentiation caused by fine dust when the relative degree of formation of primary cilia after the test substance treatment is greater than before treatment.
  • the relative degree of formation of the primary cilia may be determined by one or more of comparing the number of keratinocytes expressing the primary cilia and comparing the length of the primary cilia.
  • the relative degree of formation of the primary cilia is confirmed to be at least one expression level selected from involucrin, loricrin, filaggrin and keratin. It may be.
  • the determination is made by comparing the number of keratinocytes expressing involuclin, leucine, filaggrin, or keratin, or by comparing the amount of involuclin, leucine, pilaggrin, or keratin expressed in one cell.
  • the number of involuclin, lorricin, pilagrin or keratin-expressed skin keratinocytes after treatment with the test substance or the amount of involuclin, lorricin, pilaggrin or keratin expressed in one cell is treated with the test substance. If less than before, the amount of expression of the formed primary cilia or its length may be considered to be increased, and the test substance may be determined as a substance for restoring inhibition of skin keratinocyte differentiation by fine dust.
  • the keratin may include at least one selected from keratin 1 (keratin 1) and keratin 10 (keratin 10).
  • the method of measuring the relative degree of formation of the primary cilia known techniques, such as immunofluorescence analysis, Western blot, dot blot, enzyme-linked immunosorbent assay, ELISA ), Northern blot, PCR, RT-qPCR, GC-MS, LC-MS, NMR, etc. may be appropriately selected by those skilled in the art, but is not limited thereto.
  • the present invention in another aspect, provides a composition for restoring the inhibition of differentiation of keratinocytes formed by fine dust, comprising cytochalasin D (cytochalasin D) as an active ingredient.
  • cytochalasin D cytochalasin D
  • the cytocalin D may increase the number of keratinocytes expressing primary cilia or the length of the primary cilia.
  • the cytocalin D may enhance the expression of at least one or more selected from involuclin, lorricin, filaggrin, and keratin, and the keratin is keratin 1 and keratin 10 It may include at least one selected from among.
  • the cytocalin D may increase the expression of primary cilia of the keratinocytes, thereby restoring the inhibition of differentiation of the keratinocytes by the fine dust.
  • the composition may be a composition for skin moisturizing or skin barrier function enhancement by restoring inhibition of skin keratinocyte differentiation by fine dust. If the skin cell differentiation is not well done, the stratum corneum of the skin can not function normally, so the moisture retention of the skin is reduced, the skin barrier function is reduced. Therefore, the material for restoring the inhibition of differentiation of keratinocytes formed by fine dust may exhibit skin moisturizing and skin barrier function enhancement effects on fine dust.
  • the content of the cytocalacin D can be easily selected by those skilled in the art within the range that does not impair the object and effect of the present invention, for example, 0.0001 to 30% by weight relative to the total weight of the composition Can be.
  • the content is at least 0.0001 wt%, at least 0.0005 wt%, at least 0.001 wt%, at least 0.005 wt%, at least 0.01 wt%, at least 0.05 wt%, at least 0.1 wt%, at least 0.3 wt%, based on the total weight of the composition, At least 0.5%, at least 0.8%, at least 1%, at least 3%, at least 5%, at least 8%, at least 10%, at least 12%, at least 15%, or at least 18% Can be.
  • the content is 30 wt% or less, 28 wt% or less, 25 wt% or less, 22 wt% or less, 20 wt% or less, 18 wt% or less, 15 wt% or less, 12 wt% or less, based on the total weight of the composition, 10 wt% or less, 8 wt% or less, 5 wt% or less, 3 wt% or less, 1 wt% or less, 0.8 wt% or less, 0.5 wt% or less, 0.3 wt% or less, 0.1 wt% or less, 0.05 wt% or less, 0.01 wt% or less, 0.005 wt% or less, 0.001 wt% or less, or 0.0005 wt% or less.
  • the composition may be a cosmetic composition.
  • the cosmetic composition may be provided in any formulation suitable for topical application.
  • it may be provided in the form of a solution, an emulsion obtained by dispersing an oil phase in an aqueous phase, an emulsion obtained by dispersing an aqueous phase in an oil phase, a suspension, a cream, a solid, a gel, a powder, a paste, a foam, or an aerosol composition.
  • Compositions of such formulations may be prepared according to conventional methods in the art.
  • the cosmetic composition may contain, in addition to the above-mentioned materials, other ingredients that can give a synergistic effect to the main effect within a range that does not impair the main effect.
  • the cosmetic composition may include a substance selected from the group consisting of vitamins, polymer peptides, polymer polysaccharides, and sphingolipids.
  • the cosmetic composition may include a moisturizer, an emulsifier, a surfactant, a UV absorber, a preservative, a fungicide, an antioxidant, a pH adjuster, organic and inorganic pigments, flavoring, cooling agent or limiting agent.
  • the blending amount of the above components can be easily selected by those skilled in the art within the range that does not impair the object and effect of the present invention, the blending amount may be 0.01 to 5% by weight, specifically 0.01 to 3% by weight based on the total weight of the composition. have.
  • the composition may be a health food composition.
  • the health food is manufactured by using ingredients or ingredients (functional ingredients) having a useful function to the human body or nutrients that are likely to be lacking in daily meals, and maintain and improve the health through maintaining the normal function of the human body or physiological function It may mean a food, but is not limited thereto.
  • the health food may be manufactured and processed in the form of tablets, capsules, powders, granules, liquids, pills, and the like, but is not limited thereto and may be manufactured and processed in any form according to the law.
  • the health food composition of each formulation may be appropriately selected and blended by those skilled in the art according to the formulation or purpose of use, in addition to the active ingredient, and synergistic effects may occur when simultaneously applied with other raw materials. .
  • liquid component that can be contained in addition to the active ingredient disclosed herein, and may include various flavors or natural carbohydrates as additional ingredients, such as ordinary drinks.
  • natural carbohydrates include conventional sugars such as disaccharides such as monosaccharides, glucose and fructose, polysaccharides such as maltose and sucrose, dextrins and cyclodextrins, and sugar alcohols such as xylitol, sorbitol and erythritol. Etc.
  • natural flavoring agents such as, tauumatin, stevia extract (for example, rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (for example, saccharin, aspartame, etc.) can be advantageously used.
  • the proportion of natural carbohydrates may generally be about 1-20 g, in one aspect about 5-12 g, per 100 ml of the composition disclosed herein.
  • the food composition may contain various nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors, flavoring agents such as coloring and neutralizing agents (such as cheese, chocolate), pectic acid and salts thereof, alginic acid and salts thereof.
  • it may include a pulp for the production of natural fruit juices and vegetable drinks.
  • the components can be used independently or in combination.
  • the ratio of the additive may vary, but is generally selected from 0.001 to about 20 parts by weight per 100 parts by weight of the composition disclosed herein.
  • the present invention may be directed to a method for restoring inhibition of cutaneous keratinocyte differentiation by fine dust, comprising administering an effective amount of said cytocalin D to a subject in need thereof.
  • the present invention relates to a method for restoring inhibition of skin keratinocyte differentiation by fine dust, comprising administering to a subject in need thereof a composition comprising the cytocalin D as an active ingredient.
  • a composition comprising the cytocalin D as an active ingredient.
  • the present invention provides a method for enhancing skin moisturization or skin barrier function by restoring inhibition of cutaneous keratinocyte differentiation by fine dust, comprising administering an effective amount of said cytocalin D to a subject in need thereof. It may be about the method.
  • the present invention provides a moisturizing skin by restoring the inhibition of skin keratinocyte differentiation by fine dust, comprising administering to a subject in need thereof a composition comprising the cytocalin D as an active ingredient. Or it may be directed to a method of enhancing skin barrier function.
  • the administration of the method may be carried out according to the administration method and administration dose described herein.
  • the present invention may relate to the use of cytocalasin D for preparing a composition for restoring inhibition of skin keratinocyte differentiation by fine dust.
  • the present invention may relate to the use of cytocalasin D for preparing a composition for moisturizing skin or enhancing skin barrier function by restoring inhibition of skin keratinocyte differentiation by fine dust.
  • the present invention may relate to the use of cytocalacin D for the recovery of inhibition of skin keratinocyte differentiation by microdust.
  • the present invention may relate to the use of cytocalin D for moisturizing skin or enhancing skin barrier function by restoring inhibition of skin keratinocyte differentiation by fine dust.
  • Neonatal Normal Human Epidermal Keratinocytes from newborns were obtained from LONZA, bovine pituitary extract (BPE), insulin, human epidermal growth factor, gentamicin / amphotericin B 36 ° C. in accordance with LONZA's instructions using KGM-Gold medium (LONZA) with supplements including gentamicine / amphotericin B, epinephrine, transferrin, and hydrocortisone Incubated in a 5% CO 2 incubator. The test material was treated in KGM-Gold medium without the supplement.
  • the human epidermal normal keratinocytes were transferred to a cell-well of 0.5 ⁇ 10 5 per well on a Lab-TexTM 2 well glass chamber slide (Tnermo Scienticific, Waltham, MA, USA). 0 day, from which the test substance cytochalasin D 0.2 ⁇ M, ciliobrevin A1 5 ⁇ M, and fine dust PM10 100 ⁇ g / ml were added to the medium for 2 days, 4 days and 6 days, and the cells were fixed and primary cilia. Was stained. As a control, DMSO in which the test substance was dissolved was used.
  • cytochalasin D and cilibrevin A1 were purchased from sigma, and fine dust PM10 (ERM-CZ100, Sample No: 0504) was purchased from European Reference Materials (ERM).
  • Primary cilia staining was specifically performed with anti-ARL13B antibody (1/500 dilution, Proteintech diluted with PBS containing 1% (v / v) BSA and 0.1% (v / v) triton TM X-100 (PBS-T) Inc., stained with green) and anti-E-cadherin antibody (1/1000 dilution, Invitrogen, Inc., red) was incubated overnight at 4 °C.
  • Alexa Fluor 594 goat anti-mouse IgG and Alexa Fluor 488 goat anti-rabbit IgG from Invitorgen were diluted to 1/1000 using PBS as a secondary antibody and incubated at room temperature for 2 hours. Silver was stained for 10 minutes with 2 ⁇ g / ml DAPI (blue). Confocal laser microscope used LSM510 (Carl Zeiss Microimaging Inc., Thornwood, NY).
  • Quantitative real-time reverse transcriptional polymerase chain reaction analysis of Q-RT-PCR
  • cDNA samples were synthesized and mRNA levels changed using primers for involucrin, loricrin, filaggrin, keratin 1 and keratin 10, respectively.
  • RPL13A primers were used to analyze the relative expression of RPL13A.
  • Quantitative real-time TaqMan RT-PCR technology (Applied Biosystems, Foster City, CA, USA) technology was used to determine the expression level of the selected target gene. Cycling conditions include a denaturation step at 95 ° C. for 10 minutes and 50 cycles at 95 ° C. for 15 seconds and 60 ° C. for 1 minute.
  • FAM fluorescence of each PCR cycle was measured with the Applied Biosystems 7500 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). The results of the Q-RT-PCR data are expressed as threshold cycle (Ct) values.
  • the primers were obtained from ThermoFisher Scientific, respectively: Involuclin (product number: Hs00846307_s1), loriclean (product number: Hs01894962_s1), filagrin (product number: Hs00856927_g1), keratin 1 (product number: Hs00196158_m1) ), Keratin 10 (product number: Hs00166289_m1), RPL13A (product number: Hs04194366_g1).
  • Primary cilia are intracellular organs with polarized structures in which the cilia's binding (cilia formation) and degradation are interconnected by various causes, including cell cycle, polarity, spinal development, and genetic diseases.
  • the immunofluorescence analysis of Example 1 confirmed that the length and number of primary cilia (indicated by arrows) increased as human normal dermal keratinocytes were differentiated (FIG. 1).
  • ciliobrevin A1 is known as a substance that inhibits the production of primary cilia. It was confirmed that primary cilia were not produced as in the case of treatment, and when the cytochalasin D was treated with fine dust, the inhibition of the production of the primary cilia was recovered again, and only the cytochalasin D was treated or not treated. It was confirmed that the number or length of primary cilia (indicated by arrows) was increased to the same or higher level as that of the control group (FIG. 2).
  • the fine dust affects the primary cilia and differentiation process of keratinocytes.
  • the number and length of primary cilia which play an important role in the differentiation process of keratinocytes, are reduced, and the expression of moisturizing factors, which are differentiation markers, is also reduced.
  • the expression of primary cilia is increased through the treatment of cytocalin D, a substance that increases the expression of primary cilia in the keratinocytes whose differentiation is inhibited by fine dust, Expression of differentiation markers of the forming cells increases and as a result, it was confirmed that the inhibition of keratinocytes differentiation by fine dust is restored. By utilizing this point, it is possible to screen a substance capable of restoring inhibition of keratinocyte differentiation by fine dust.

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