KR102119466B1 - Screening method for material for restoring inhibition of differentiation of keratinocytes by fine particulate matter and composition for restoring inhibition of differentiation of keratinocytes by fine particulate matter - Google Patents

Abstract

The present invention relates to a method for screening a substance capable of restoring the suppression of the differentiation of keratinocytes by fine dust, comprising the step of confirming the relative formation degree of primary cilia of keratinocytes. Through one aspect of the invention, effective screening and excavation of a substance capable of restoring the suppression of differentiation of skin keratinocytes caused by fine dust is possible, and through this, development of a skin health related business by fine dust can be promoted.

Description

Screening method of a substance capable of restoring the suppression of differentiation of skin keratinocytes caused by fine dust, and a composition for restoring the suppression of skin keratinocytes differentiation caused by fine dust {SCREENING METHOD FOR MATERIAL FOR RESTORING INHIBITION OF DIFFERENTIATION OF KERATINOCYTES BY FINE PARTICULATE MATTER AND COMPOSITION FOR RESTORING INHIBITION OF DIFFERENTIATION OF KERATINOCYTES BY FINE PARTICULATE MATTER}

The present invention relates to a method for screening a substance capable of restoring the suppression of differentiation of skin keratinocytes caused by fine dust, and specifically, controlling the length and number of primary cilia, the organelles of cells, to form keratinocytes caused by fine dust It relates to a method for screening a substance capable of restoring the differentiation inhibition of and a composition for restoring the suppression of cell differentiation of skin keratinocytes by fine dust.

The skin plays a very important role as a barrier function that protects the individual from the outside. Barrier function is a function to protect against various stimuli from outside (chemicals, air pollutants, dry environment, ultraviolet rays, etc.) and to prevent excessive dissipation of moisture in the body through the skin, which is a stratum corneum composed of keratinocytes. Its function can be maintained only when it is normally formed.

Among the epidermis, the outermost stratum corneum (horney layer) is formed from keratinocytes and is composed of keratinocytes that have been differentiated and the lipid layer surrounding them (Marcelo CL et al., J. Invest. Dermatol. , 80, pp37-44, 1983).

Keratinocytes are characteristic cells in which basal cells, which proliferate continuously in the lowermost layer of the epidermis, undergo step-by-step morphological and functional changes and rise to the surface of the skin. Dropped and new keratinocytes take over their function, and this repetitive sequence of changes is called "differentiation of epidermal cells" or "ekratinization".

Also, during the differentiation process, keratinocytes form a natural moisturizing factor (NMF) and intercellular lipids (ceramides, cholesterol, fatty acids), forming a stratum corneum to make the stratum corneum firm and flexible, thus preventing the skin barrier. It retains its function as a (skin barrier).

Such a stratum corneum can easily lose its function due to excessive face washing, lifestyle factors such as bathing, or endogenous diseases such as atopic skin or senile skin.

On the other hand, fine dust is dust that is fine enough to be invisible and refers to dust having a diameter of 10 µm or less, and PM10 (fine dust) and PM2.5 with a diameter of 10 µm or less, 2.5 µm or less, and 1.0 µm or less, respectively, depending on the particle size. It is divided into (ultrafine dust) and PM1.0 (ultrafine dust). Here, PM (particulate matter) means'particulate matter (solid or liquid fine particles floating in the air)'.

Fine dust, a harmful substance, causes respiratory and lung diseases, skin and eye diseases, and, unlike ordinary dust, accumulates in the body without being filtered from the nose, mouth, and bronchi. Particularly, in modern times, symptoms of dry skin due to damage to the skin barrier function caused by such fine dust are gradually increasing.

On the other hand, cytokalasin D is known as a type of mycotoxin known as cytokalasin, and the relationship between the damage to the skin barrier function due to fine dust and cytochalasin D is not known.

EZRATTY, E. J. et al., A Role for the Primary Cilium in Notch Signaling and Epidermal Differentiation during Skin Development (Cell, April 24, 2011, 145, pp. 1129-1141)

The present invention is a new method for effectively screening a substance capable of restoring the suppression of the differentiation of skin keratinocytes by fine dust by controlling the number and length of primary cilia of the keratinocytes, and by using fine dust. An object of the present invention is to provide a composition for restoring the suppression of keratinocyte-forming cell differentiation.

In order to achieve the above object, an embodiment of the present invention comprises the steps of treating a test substance on keratinocytes;

Confirming a relative degree of formation of primary cilia before and after treatment with the test substance in the keratinocytes treated with the test substance; And

If the relative degree of formation of primary cilia after treatment with the test substance is greater than before treatment, determining the test substance as a material for restoring cell keratin formation and cell differentiation inhibition by fine dust, forming skin keratin by micro dust Provided is a method for screening a substance capable of restoring the suppression of cell differentiation.

Another embodiment of the present invention provides a composition for restoring the suppression of cell differentiation of keratinocytes formed by fine dust, comprising cytochalasin D as an active ingredient.

The screening method of a material capable of restoring the suppression of differentiation of keratinocytes caused by fine dust according to the present invention includes the step of confirming the relative formation degree of primary cilia in the keratinocytes, thereby more effectively fine It is possible to screen for substances capable of restoring the suppression of the differentiation of skin keratinocytes caused by dust, and identification of substances capable of restoring the suppression of the differentiation of skin keratinocytes caused by fine dust that has not been found by conventional methods As it is possible, it can greatly contribute to the industrial development in related fields.

In addition, the composition for restoring skin keratinocytes differentiation inhibition by fine dust according to the present invention restores the suppression of primary cilia production by fine dust in the skin keratinocytes, and as a result, the differentiation of skin keratinocytes caused by fine dust. Inhibition can be effectively restored.

Figure 1 is observed by confocal microscopy that the length and number of primary cilia increases as human normal cutaneous keratinocytes differentiate (scale bar is 10 μm).
FIG. 2 shows the changes in the length and number of primary cilia when observed by treating fine dust (PM10) and/or cytocalacin D in human normal cutaneous keratinocytes with a confocal microscope (scale bar is 10 μm).
Figure 3a is a human normal cutaneous keratinocytes in the treatment of fine dust (PM10) and / or cytocalin D involucrin (involucrin) expression changes through RT-PCR confirmed.
Figure 3b is a human normal cutaneous keratinocytes treated with fine dust (PM10) and / or cytocalin D when the expression change of lorricrin (loricrin) was confirmed through RT-PCR.
Figure 3c is a human normal cutaneous keratinocytes in the treatment of fine dust (PM10) and / or cytocalin D, the expression change of the pilar green (filaggrin) was confirmed through RT-PCR.
Figure 3d shows the expression change of keratin 1 (keratin 1) when human fine skin keratinocytes are treated with fine dust (PM10) and/or cytocalin D through RT-PCR.
Figure 3e shows the expression change of keratin 10 (keratin 10) through the treatment of fine dust (PM10) and/or cytocalin D in human normal keratinocytes through RT-PCR.

Hereinafter, the present invention will be described in detail.

As used herein, "fine dust" refers to dust that is fine enough to be invisible and refers to dust having a diameter of 10 µm or less, and PM10 (fine dust) having a diameter of 10 µm or less, 2.5 µm or less, 1.0 µm or less, depending on the particle size, It is classified into PM2.5 (ultrafine dust) and PM1.0 (ultrafine dust), and is a broad concept that includes all of them. Here, PM (particulate matter) means'particulate matter (solid or liquid fine particles floating in the air)'.

In the present specification, the term “skin” refers to a tissue that covers the body surface of an animal, and is the broadest concept including scalp and hair, as well as tissue that covers a body surface such as a face or body. Meanwhile, in the present specification, the skin may include not only biological skin, but also artificial skin or a skin mimetic that embodies the state of biological skin.

In the present specification, “skin cell differentiation” may mean a process in which keratinocytes functionally differentiate from the base layer, which is the lowest layer of the skin, to gradually form a polar layer, a granular layer, and a stratum corneum.

As used herein, "primary cilia", also referred to as primary cilium (primary cilia in the case of multiples), is an organelle protruding from the cell body, including various external stimuli including chemical stimulation, physical stimulation, light, osmotic pressure, fluid flow, and gravity signals. It refers to the organelles that sense stimuli.

"Relative degree of formation" herein refers to the formation, expression, number, quantity, quality of formation or expression when comparing the degree of formation or expression of primary cilia in cells with or without any conditions. It may be a degree indicating the difference. In addition, the degree of formation may include, for example, the number or amount of cells forming or expressing primary cilia, or the length of the primary cilia.

In this specification, "cytocalcin D" may be expressed as cytochalasin D or cyto D.

The present invention, in one aspect, the step of treating the test substance on the keratinocytes (keratinocyte); Confirming a relative degree of formation of primary cilia before and after treatment with the test substance in the keratinocytes treated with the test substance; And if the relative degree of formation of primary cilia after treatment with the test substance is greater than before treatment, determining the test substance as a material for restoring cell keratin formation and cell differentiation inhibition by fine dust, and skin keratin caused by fine dust. A method for screening a substance capable of restoring the suppression of differentiation of forming cells is provided.

In an exemplary embodiment of the present invention, the relative formation degree of the primary cilia may be determined through one or more of comparison of the number of skin keratinocytes with primary cilia expressed and comparison of the length of the primary cilia.

In an exemplary embodiment of the present invention, the relative degree of formation of the primary cilia is confirmed by at least one expression level selected from involucrin, loricrin, pilarggrin, and keratin. It may be. For example, by comparing the number of keratinocytes expressing involucrin, loricrin, pilarin or keratin, or by comparing the amount of involukrin, loricrin, pilarrin or keratin expressed in one cell. The number of involucrin, loricrin, pilargreen, or keratin-expressing keratinocytes after the test substance treatment, or the amount of involucrin, loricrin, pilargreen, or keratin expressed in one cell is treated with the test substance If more than before, it can be seen that the expression amount of primary cilia formed or the length thereof is increased, and the test substance can be judged as a substance for restoring the suppression of keratinocyte-forming cell differentiation by fine dust.

In one embodiment of the present invention, the keratin may include at least one selected from keratin 1 (keratin 1) and keratin 10 (keratin 10).

On the other hand, the method of measuring the relative formation degree of the primary cilia, known techniques, such as immunofluorescence analysis, western blot (western blot), dot blot (dot blot), enzyme-linked immunosorbent assay (ELISA) ), Northern blot (northern blot), PCR, RT-qPCR, GC-MS, LC-MS, NMR, etc. can be appropriately selected by those skilled in the art, but is not limited thereto.

In another aspect, the present invention provides a composition for inhibiting and restoring the differentiation of keratinocytes formed by fine dust, comprising cytochalasin D as an active ingredient.

In an exemplary embodiment of the present invention, the cytoklasin D may increase the number of keratinocytes that have primary cilia or the length of the primary cilia.

In an exemplary embodiment of the present invention, the cytokalasin D may enhance expression of at least one selected from involucrine, loricrin, pilargreen and keratin, and the keratin is keratin 1 and keratin 10. It may include at least any one selected from.

That is, the cytocalin D may increase the expression of the primary cilia of the keratinocytes, thereby restoring the suppression of the differentiation of the keratinocytes by the fine dust.

In an exemplary embodiment of the present invention, the composition may be a composition for moisturizing the skin or enhancing the function of the skin barrier by restoring the suppression of cell differentiation formation due to fine dust. If the skin cells are not differentiated well, the stratum corneum of the skin cannot function normally, so the water retention capacity of the skin decreases and the skin barrier function decreases. Therefore, a substance that restores the suppression of skin differentiation-forming cell differentiation by fine dust may exhibit a skin moisturizing and skin barrier strengthening effect against fine dust.

In an exemplary embodiment of the present invention, the content of the cytokalasin D can be easily selected by those skilled in the art within a range not impairing the objects and effects of the present invention, for example, 0.0001 to 30% by weight based on the total weight of the composition. Can be. In addition, the content is 0.0001 wt% or more, 0.0005 wt% or more, 0.001 wt% or more, 0.005 wt% or more, 0.01 wt% or more, 0.05 wt% or more, 0.1 wt% or more, 0.3 wt% or more, based on the total weight of the composition, 0.5% or more, 0.8% or more, 1% or more, 3% or more, 5% or more, 8% or more, 10% or more, 12% or more, 15% or more, or 18% or more Can be. In addition, 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% or less, 8% or less, 5% or less, 3% or less, 1% or less, 0.8% or less, 0.5% or less, 0.3% or less, 0.1% or less, 0.05% or less, It may be 0.01% by weight or less, 0.005% by weight or less, 0.001% by weight or less, or 0.0005% by weight or less.

In one embodiment of the present invention, the composition may be a cosmetic composition.

The cosmetic composition may be provided in any formulation suitable for topical application. For example, it can 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 oil 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 these formulations can be prepared according to conventional methods in the art.

The cosmetic composition may contain other ingredients that can give a synergistic effect to the main effect, within a range that does not impair the main effect, in addition to the above-mentioned substances. The cosmetic composition may include substances selected from the group consisting of vitamins, polymer peptides, polymer polysaccharides, and sphingoli lipids. In addition, the cosmetic composition may include a moisturizer, emollient, surfactant, ultraviolet absorber, preservative, disinfectant, antioxidant, pH adjuster, organic and inorganic pigments, fragrances, coolants or limiting agents. The compounding amount of the components can be easily selected by those skilled in the art within a range that does not impair the object and effect of the present invention, the blending amount can be 0.01 to 5% by weight, specifically 0.01 to 3% by weight based on the total weight of the composition have.

In one embodiment of the present invention, the composition may be a health food composition.

The health food is manufactured using raw materials or ingredients (functional ingredients) having nutrients or functions useful for the human body that are easily deficient in daily meals, and maintains and improves health through maintaining normal functions of the human body or activating physiological functions. It may mean food, but is not limited thereto. The health food may be manufactured and processed in the form of tablets, capsules, powders, granules, liquids, pills, etc., but is not limited thereto, and may be manufactured and processed in any form according to law.

The health food composition of each formulation can be formulated by appropriately selecting the ingredients commonly used in the field other than the active ingredient according to the formulation or purpose of use without difficulty, and synergistic effects may occur when applied simultaneously with other raw materials. .

There are no particular limitations on the liquid components that may be contained in addition to the active ingredients disclosed in the present specification, and may include various flavoring agents, natural carbohydrates, and the like as additional components, such as conventional beverages. Examples of the natural carbohydrate include monosaccharides, disaccharides such as glucose and fructose, polysaccharides such as maltose and sucrose, sugars such as xylitol, sorbitol, and erythritol, such as polysaccharides such as dextrin and cyclodextrin. And so on. As the above-mentioned flavoring agent, natural flavoring agents (taumatine, stevia extract (for example, rebaudioside A, glycyrrhizine, etc.) and synthetic flavoring agents (for example, saccharin, aspartame, etc.) can be advantageously used. The ratio of the natural carbohydrate may be generally about 1 to 20 g per 100 ml of the composition disclosed herein, and about 5 to 12 g in one aspect.

The food composition is, in one aspect, various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic and natural flavoring agents, coloring agents and neutralizing agents (cheese, chocolate, etc.), pectic acid and salts thereof, alginic acid and salts thereof , Organic acids, protective colloidal thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohol, carbonic acid used in carbonated beverages, and the like. In other aspects it may include flesh for the manufacture 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 the range of 0.001 to about 20 parts by weight per 100 parts by weight of the composition disclosed herein.

Hereinafter, the contents of the present invention will be described in more detail through examples and test examples. However, these examples and test examples are only presented to understand the contents of the present invention, the scope of the present invention is not limited to these examples and test examples, and modifications and substitutions commonly known in the art And insertion, etc., which are also included in the scope of the present invention.

[Example 1]

Human epidermal normal keratinocyte preparation

Neonatal Normal Human Epidermal Keratinocytes (NHEK-Neo) from newborns were obtained from LONZA, bovine pituitary extract (BPE), insulin, human epidermal growth factor, gentamicin/amphotericin B (gentamicine/amphotericin B), 36°C according to LONZA's instructions using KGM-Gold medium (LONZA) with supplements containing supplements containing epinephrine, transferrin, and hydrocortisone Cultured in 5% CO ₂ incubator. When the test material was treated, the supplement was treated with KGM-Gold medium without addition.

Immunofluorescence analysis using confocal microscopy

After transferring the human epidermal normal keratinocytes to a Lab-TexTM 2 well glass chamber slide (Tnermo Scienticific, Waltham, MA, USA), the number of cells per well was moved to 0.5X10 ^5, and the time when the cells grew to about 90-100%. 0 day, from this time, the test substance cytochalasin D 0.2μM, ciliobrevin A1 5μM, fine dust PM10 100μg/ml was added to the medium for 2, 4, 6 days, and then fixed and primary cilia Was dyed. DMSO in which the test substance was dissolved was used as a control. cytochalasin D and cilibrevin A1 were purchased from sigma, and fine dust PM10 (ERM-CZ100, Sample No: 0504) was purchased from ERM (European Reference Materials). Primary cilia staining is specifically, 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). Incubated with green) and anti-E-cadherin antibody (1/1000 dilution, Invitrogen, red) was incubated overnight at 4°C. Then, Invitorgen's Alexa Fluor 594 goat anti-mouse IgG and Alexa Fluor 488 goat anti-rabbit IgG were diluted to 1/1000 using PBS as a secondary antibody and incubated at room temperature for 2 hours. Was stained with 2 μg/ml DAPI (blue) for 10 minutes. As a confocal laser microscope, LSM510 (Carl Zeiss Microimaging Inc., Thornwood, NY) was used.

Q-RT-PCR (Quantitative real-time reverse transcriptional polymerase chain reaction) analysis

All RNA is Trizol? (Invitrogen, Carlsbad, CA, USA). The concentration of RNA was measured by a spectrophotometer and the integrity of RNA was evaluated using Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA, USA). 2 μg of RNA was reverse transcribed into cDNA using SuperScript® III reverse transcriptase (Invitrogen, Carlsbad, CA, USA) and aliquots were stored at -20°C. Quantitative measurements of markers associated with keratinocyte differentiation in each cDNA sample were performed using the Assays-on-DemandTM Gene Expression Kit (Applied Biosystems, Foster City, CA, USA). Synthesis of cDNA samples and changes in mRNA amount using respective primers for involucrin, loricrin, pilarggrin, keratin 1 and keratin 10 Was analyzed. In addition, to average mRNA expression levels, RPL13A primers were used to analyze the relative expression of RPL13A. In order to determine the expression level of the selected target gene, Q-RT-PCR (Quantitative real-time TaqMan RT-PCR technology) (Applied Biosystems, Foster City, CA, USA) technology was used. Cycling conditions include denaturation steps 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 an 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, as follows: Involucrine (Product No.: Hs00846307_s1), Loriclean (Product No.: Hs01894962_s1), Filaggrin (Product No.: Hs00856927_g1), Keratin 1 (Product No.: Hs00196158_m1) ), keratin 10 (product number: Hs00166289_m1), RPL13A (product number: Hs04194366_g1).

Statistical analysis

Each data was obtained after at least three independent experiments, and was expressed as mean±SE (standard error). Statistical evaluation of the results was performed using one-way ANOVA (* p<0.05, **p<0.01).

[Experimental Example 1] Recovery of suppression of cell differentiation cell differentiation caused by fine dust by primary cilia formation

Primary cilia are intracellular organs with a polarized structure, in which cilia binding (cilia formation) and degradation are interconnected by a variety of causes, including cell cycle, polarity, spinal development, and genetic disease. Through the immunofluorescence analysis of Example 1, it was confirmed that the length and number of primary cilia (indicated by arrows) increased as human normal cutaneous keratinocytes differentiated (FIG. 1).

In order to confirm the effect of fine dust on the primary cilia of the keratinocytes, the treatment of fine dust (PM10) on the keratinocytes and observed four days later revealed that ciliobrevin A1 is known as a substance that inhibits the production of primary cilia. It was confirmed that primary cilia were not generated, as in the case of processing, and when cytochalasin D was treated with fine dust, the inhibition of the formation of primary cilia was restored again, and only cytochalasin D was treated or nothing was treated. It was confirmed that the number or length of primary cilia (indicated by arrows) increased to a level equal to or higher than that of the control group (FIG. 2).

In addition, when treating fine dust with keratinocytes, involucrin (FIG. 3A) and loricrin (FIG. 3B), which are markers of differentiation of keratinocytes at a level equal to or more severe than that of ciliobrevin A1 treatment, Although the expression levels of pilarin (FIG. 3C), keratin 1 (FIG. 3D) and keratin 10 (FIG. 3E) were significantly reduced, when cytochalasin D was treated with fine dust, expression of differentiation markers of each keratinocytes was decreased. It was confirmed by the Q-RT-PCR analysis of Example 1 that the recovery to a significant level again (Fig. 3a to 3e).

Taken together, it was confirmed that fine dust affects the primary cilia and differentiation processes of keratinocytes. That is, when the fine dust is treated with keratinocytes, the number and length of primary cilia, which play an important role in the differentiation process of keratinocytes, decreases, and the expression of moisturizing factors, which are differentiation markers, decreases. In this regard, skin keratin is increased when the expression of primary cilia is increased through the treatment of cytocalacin D, a substance that increases the expression of primary cilia, to skin keratinocytes whose differentiation is suppressed by fine dust. It was confirmed that the expression of the differentiation markers of the forming cells increased and, as a result, the suppression of the differentiation of the skin keratinocytes differentiation by fine dust was restored. By utilizing this point, it is possible to screen a substance capable of restoring the suppression of cell differentiation of keratinocytes formed by fine dust.

Claims (10)

delete delete delete delete A composition for restoring the suppression of cell differentiation of skin keratinocytes formed by fine dust, comprising cytochalasin D (cytochalasin D) as an active ingredient. The method of claim 5,
Said cytocalin D is a composition for restoring the suppression of differentiation of keratinocytes caused by fine dust, which increases the number of keratinocytes or primary cilia expressing primary cilia. The method of claim 5,
Said cytokalasin D enhances the expression of at least one selected from involucrin, loricrin, filaggrin and keratin, and exfoliation of the skin due to fine dust. A composition for restoring formation cell differentiation inhibition. The method of claim 7,
The keratin, keratin 1 (keratin 1) and keratin 10 (keratin 10) containing at least one selected from the composition for the recovery of skin keratinocytes cell differentiation inhibition by fine dust. The method of claim 5,
The composition is a composition for moisturizing skin or enhancing skin barrier function by restoring the suppression of differentiation of skin keratinocytes caused by fine dust, and a composition for restoring the suppression of skin keratinocytes differentiation by fine dust. The method according to any one of claims 5 to 9,
The composition is a cosmetic composition or a health food composition, a composition for restoring skin keratinocytes cell differentiation inhibition by fine dust.

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