KR101861583B1

KR101861583B1 - Composition for promoting secretion of sebum

Info

Publication number: KR101861583B1
Application number: KR1020160083516A
Authority: KR
Inventors: 김성진; 임명; 정유라; 손경철; 김창덕; 이증훈
Original assignee: 전남대학교산학협력단
Priority date: 2016-07-01
Filing date: 2016-07-01
Publication date: 2018-05-28
Also published as: WO2018004068A1; KR20180003850A

Abstract

The present invention relates to a composition for external application for promoting sebum secretion. By including adiponectin, it can promote the differentiation of sebaceous glands and increase sebum secretion, thereby improving or strengthening functions such as skin barrier, To an external composition for external application for skin sebum secretion which is capable of increasing the production of lipids which perform synthesis of proteins and enzymes and function as a skin barrier.

Description

Technical Field [0001] The present invention relates to a composition for promoting sebum secretion,

The present invention relates to a composition for promoting lipid synthesis of skin comprising adiponectin.

Skin plays a very important role as a barrier function to protect individuals from the outside. The barrier function is a protective function for preventing various external stimuli such as chemicals, air pollutants, dry environment, ultraviolet rays and excessive divergence of body water. This protective function is a function of the stratum corneum (keratinocyte) the function can be maintained only when the horney layer is normally formed.

Here, lipid of the skin plays an important role in maintenance of the skin barrier function. The lipid of the skin is composed of interstitial lipid, which is synthesized and secreted in the keratinocyte, and lipid secreted from sebaceous gland cells.

The lipid of keratinocyte is the lipid produced by differentiation of keratinocyte of epidermis. Ceramide, free fatty acid and cholesterol are typical components of this lipid. Unique lipid synthesized by sebaceous gland activity is squalene, wax ester and so on.

In the case of sebaceous glands located adjacent to the hair of the sebaceous glands, lipids are excreted onto the skin surface, but the role of improving the skin barrier function is largely unknown.

On the other hand, adiponectin is a kind of adipokine, a protein hormone secreted specifically in adipocytes (subcutaneous fat), and treats dermatological diseases such as obesity, diabetes, medical diseases of hyperlipemia, hair growth effect and wound regeneration It is known as a substance that protects skin barrier.

The present inventors have found that adiponectin receptors (adiponectin receptors 1 and 2) are present in sebaceous glands and sebocytes and that skin barrier function is improved when an adiponectin receptor-binding substance is induced to increase sebum secretion The present inventors have completed the present invention.

Patent Publication No. 2016-0008862 (Jan. 25, 2016)

It is an object of the present invention to provide a composition which promotes the lipid synthesis of the skin by promoting the differentiation of sebaceous glands.

It is an object of the present invention to provide a composition containing oligomer adiponectin which effectively increases the differentiation of sebaceous glands in the human body.

An object of the present invention is to provide a skin external composition for promoting sebum secretion containing a substance that increases expression of adiponectin.

The object of the present invention is to provide a cosmetic composition or external ointment for enhancing skin barrier function.

1. A skin external composition for promoting sebum secretion comprising adiponectin.

2. The composition for external application for skin according to 1 above, wherein said composition promotes the proliferation of sebaceous gland cells.

3. The composition for external application for skin according to 1 above, wherein said composition increases lipid synthesis.

4. The composition for external application for skin according to 1 above, further comprising at least one selected from the group consisting of squalane, triglyceride and palmitic acid.

5. The composition for external application for skin according to 1 above, further comprising inollucrine or filaggrin.

6. A pharmaceutical composition according to any one of the preceding claims, which comprises at least one compound selected from the group consisting of L-arabinose, sialic acid, chromone, (-) catechin, S-arylcysteine, arginine, L-carnitine, caffeine, And at least one selected from the group consisting of isoflavones.

7. The composition for external application for skin according to 1 above, wherein said adiponectin is an oligomer.

8. The composition for external application for skin according to 7 above, wherein the oligomer is polymerized with 2 to 12 monomers.

9. The composition for external application for skin according to 1 above, wherein said composition comprises said adiponectin at 40 ng / ml to 250 ng / ml.

The composition for external application of skin for promoting sebum secretion according to the present invention can improve or enhance the functions such as skin barrier by increasing sebum secretion.

The composition for external application of skin for promoting sebum secretion according to the present invention can increase the synthesis of proteins and enzymes involved in the lipid synthesis of the skin.

The composition for external application of skin for promoting sebum secretion according to the present invention may increase the production of lipids which perform a skin barrier function.

Figure 1 shows the expression of adiponectin receptors in human sebaceous glands and sebaceous cells.
FIG. 2 shows the results of MTT assay and [3] H-thymidine incorporation measurement of cell proliferation after treatment of recombinant human adiponectin in adipocytes in a concentration-dependent manner.
Figure 3 shows the effect of adiponectin on lipid synthesis in human sebaceous cells.
Figure 4 shows that lipid synthesis by adiponectin is induced by activating AMP-activated protein kinase (AMPK) signaling in human sebaceous cells.
Figure 5 shows the effect of adiponectin in three-dimensional culture of human sebaceous cells.
FIG. 6 shows that the protein involucrin, filaggrin, etc. involved in the skin barrier function and the HMG CoA enzyme, which plays a pivotal role in lipid metabolism, are well formed or induced in keratinocytes treated with palmitic acid and squalene.
FIG. 7 shows that adiponectin in sebaceous glands of young men with vigorous sebaceous secretion does not increase lipid synthesis.

Hereinafter, the present invention will be described in more detail.

The composition for external application for skin for promoting sebum secretion of the present invention includes adiponectin.

The skin barrier exists in the stratum corneum which is located at the outermost part of the skin among the structure constituting the skin and functions to protect the human body by preventing water evaporation and foreign material intrusion, which is called skin barrier function.

Skin lipid plays an important role in the maintenance of skin barrier function, and skin lipid is stimulated by sebum secreted from sebaceous gland cells.

The sebaceous glands secreted from sebaceous glands contain lipids such as wax ester and squalene, which play a role in the synthesis of skin lipids.

Thus, increased sebum secretion can improve skin barrier function. The present inventors have devised adiponectin as a substance capable of increasing secretion of sebum.

Previously, adiponectin was one of the adipokines mainly produced in adipocytes and was known to affect the insulin delivery mechanism, energy metabolism regulation, and inflammation throughout the body.

According to the results of the present inventors, adiponectin receptors Adipo1 and Adipo2 are located in sebaceous gland cells. Adiponectin treatment on sebaceous gland cells can induce differentiation of sebaceous gland cells to increase secretion of sebaceous glands. Secretion leads to the synthesis of skin lipids, which leads to effects such as skin barrier function improvement.

The adiponectin of the present invention exhibits an amino acid homology of 70% or more with respect to the entire amino acid sequence of adiponectin, and preferably 75% or more or 80% or more, 85% or more, 90% or more, 95% or more, 96% And up to 97% sequence homology.

Adiponectin may also include modifications of adiponectin corresponding to either amino acid substitution, insertion or deletion, or a combination thereof.

The adiponectin receptor is a protein present on the cell membrane and is a substance that selectively binds to adiponectin according to morphological specificity.

The composition for external application for skin may be prepared in any form conventionally produced in the art, and examples thereof include solutions, suspensions, emulsions, pastes, gels, creams, lotions, powders, soaps, surfactants -Containing cleansing, oil, powder foundation, emulsion foundation, wax foundation, or spray formulations.

In this case, the composition for external application for skin may be prepared in various formulations. In addition to containing the composite powder as an active ingredient, the skin external composition composition may contain components commonly used in cosmetic compositions such as antioxidants, stabilizers, Vitamins, pigments and flavoring agents, and / or carriers. An available carrier according to each formulation will be described below.

When the formulation is a powder or a spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used as the carrier component, and in particular, in the case of a spray, additionally chlorofluorohydrocarbons, propane / Propellants such as dimethyl ether may be further utilized.

When the formulation is a solution or an emulsion, a solvent, a solubilizing agent or an emulsifying agent may be used as a carrier component. Examples thereof include water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, 1,3-butyl glycol oil, glycerol aliphatic ester, polyethylene glycol or fatty acid esters of sorbitan.

When the formulation is in the form of a suspension, a carrier, such as water, a liquid diluent such as ethanol or propylene glycol, a suspension such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, microcrystalline cellulose, Aluminum metahydroxide, bentonite, agar or tracant may be used.

When the formulation is a paste, a cream or a gel, an animal oil, a vegetable oil, a wax, a paraffin, a starch, a tracer, a cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide may be used as a carrier component.

When the formulation is an interface-active agent-containing cleansing, the carrier component is selected from aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyltaurate, sarcosinate, fatty acid amide ether sulfate, Alkylamido betaines, aliphatic alcohols, fatty acid glycerides, fatty acid diethanolamides, vegetable oils, lanolin derivatives, or ethoxylated glycerol fatty acid esters.

In addition, the composition for external application for skin may be an external ointment, and may further include appropriate carriers, excipients and diluents commonly used in ointments for external use. Examples of such carriers, excipients and diluents include lactose, dextrose, sucrose, But are not limited to, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, Hydroxybenzoate, hydroxybenzoate, talc, magnesium stearate and mineral oil.

The daily dose of the external ointment varies depending on the condition and the weight of the patient, the degree and the duration of the disease, and can be appropriately selected by those skilled in the art.

The composition for external application for skin of the present invention may further comprise at least one selected from the group consisting of squalane, triglyceride and palmitic acid.

When at least one of squalane, triglyceride and palmitic acid is used in combination with adiponectin, it is possible to effectively promote the synthesis of epidermal differentiation proteins such as involucrine, filaggrin, free fatty acids and HMG-CoA reductase So that the synthesis of skin lipids can be more effectively increased.

Squalane has the chemical formula of 2,6,10,15,19,23-hexamethyltetracosane, and it is a high-quality oily raw material having superior usability, safety and moisturizing effect compared to squalene which is an unsaturated hydrocarbon. Also, Has a high affinity for percutaneous absorption.

Triglycerides may be artificially synthesized or natural triglycerides may be used. Caprylic acid and caproic acid triglycerides can play a role in helping moisture bind to the skin and can activate lipid synthesis transcription factors to induce skin lipid synthesis.

According to one embodiment of the present invention, the composition for external application for skin may further comprise inollucrine or filaggrin.

Involucrine and filaggrin are epidermal differentiation proteins that play an important role in skin barrier and are substances that induce the synthesis of interstitial lipids by differentiating keratinocytes. Therefore, when used together with adiponectin, skin lipid synthesis can be effectively induced.

Oligomer Adiponectin refers to a protein complex formed from two to twelve adiponectin monomers and can be formed by the interaction between adiponectin monomers, not covalent bonds.

Adiponectin present in the human body is an oligomer which is not a monomer, and when oligomer adiponectin is used, lipid differentiation effect is superior to lipid differentiation using adiponectin monomer derived from prokaryote such as Escherichia coli, and lipid synthesis can be more effectively promoted.

According to one embodiment of the present invention, the content of adiponectin may be from 40 ng / ml to 250 ng / ml.

When the content of adiponectin is less than 40 ng / ml, there is a problem in that lipid production due to sebum secretion is hardly induced because the promoting effect of sebum secretion is not sufficient.

When the content of adiponectin is more than 250 ng / ml, cytotoxic effect is exerted and excessive amount of sebum is secreted and skin diseases such as acne induction may occur.

The composition for enhancing the skin barrier function of the present invention may contain at least one compound selected from the group consisting of L-arabinose, syringic acid, chromone, (-) catechin, S-arylcysteine, arginine, But may further comprise at least one selected from the group consisting of L-carnitine, caffeine, dehydrocapsaicin and isoflavones.

L-arabinose, sialic acid, chromone, (-) catechin, S-arylcysteine, arginine, L-carnitine, caffeine, dihydro capsaicin and isoflavones are substances that increase the expression of adiponectin, When used in combination with adiponectin, sufficient secretion of sebum to induce lipid production can be achieved, and further, skin barrier function can be improved and enhanced.

L-arabinose is a substance having the chemical formula of (3R, 4S, 5S) -oxane-2,3,4,5-tetrol, which has a taste close to that of sugar, to be. In the natural world, hemicelluloses of higher plants exist as constituents of polysaccharides such as arabinan, arabinosyllain or arabinogalactan.

Sic acid is one of the phenolic compounds and the IUPAC name is 4-hydroxy-3,5-dimethoxybenzoic acid.

Sering acid is a phenolic compound synthesized by the shikimic acid pathway and has a formula of 4-Hydroxy-3,5-dimethoxybenzoic acid. It is a plant of sorghyum bicolor, Euterpe oleracea (E.g., syringic acid from Sigma-aldrich) that is extracted, separated and purified from a species, or chemically synthesized.

Chromones are a specific type of aromatic compounds, dimers, trimers and other polymerized chromons having benzopyran-4-one represented by the following formula (2) as their main skeleton form:

(2)

Wherein R 1, R 2, R 3, R 4, R 5 and R 6 are independently selected from -H, -OH, -CH 3, -SH, alkyl, alkenyl, oxoalkyl, oxoalkenyl, hydroxyalkyl, hydroxyalkenyl, OCH3, -SCH3, -OR, -SR, -NH2, -NRH, -NR2, -NR3 + X-, gallate, acetate, cinnamoyl and hydroxyl-cinnamoyl ester, trihydroxybenzoyl ester and caffeoyl ester An ester selected from the group consisting of: And hexose or pentose, wherein the hexose or pentose is linked to the chromone by carbon, nitrogen, sulfur or oxygen, and the hexose or pentose is independently selected from the group consisting of aldopentose, Pentose, aldohexose, ketohexose, and chemical derivatives thereof;

The alkyl and / or alkenyl groups are straight and / or branched chain having 1 to 20 carbon atoms, with or without a double bond and a substitution group (s) selected from the group consisting of -OH, = 0 and -OR ;

X is selected from the group of pharmaceutically acceptable counter anions including but not limited to hydroxyl, chloride, iodide, sulfate, phosphate, acetate, fluoride, carbonate and the like;

R is an alkyl group having from 1 to 20 carbon atoms.

(-) Catechin is a (2R, 3R) -2- (3,4-dihydroxyphenyl) chroman-3,5,7-triol, which is an IUPAC chemical name, and contains green tea (Camellia sinensis) Agrimonia eupatolia L.).

S-aryl cysteine

Lt; / RTI >

Arginine is a substance having the chemical formula of (S) -2-Amino-5-guanidinopentanoic acid, which is one of the amino acids constituting the protein.

Carnitine is a substance with the formula of 3-hydroxy-4 trimethylammoniobutanoate, a quaternary ammonium compound that is biosynthesized from amino acids lysine and methionine, and L-carnitine is a biologically active form of carnitine to be.

Caffeine is a substance with the chemical formula 1,3,7-Trimethylpurine-2,6-dione.

Dihydro capsaicin is a substance with the chemical formula of N- (4-Hydroxy-3-methoxybenzyl) -8-methylnonanamide.

Isoflavone is a substance with the chemical formula 3-Phenyl-4H-chromen-4-one.

Hereinafter, the present invention will be described in detail by way of examples, but they should not be construed as limiting the scope of the present invention.

DETAILED DESCRIPTION OF THE DRAWINGS

Figure 1 shows the expression of adiponectin receptors in human sebaceous glands and sebocytes. (A) is an analysis of whether the adiponectin receptors AdipoR1 and AdipoR2 are present in human sebaceous glands via immunohistochemistry. Inset, isotype control. (B) shows the immunofluorescence labeling of AdipoR1 and AdipoR2 (green) in human sebaceous cells, showing the nucleus contrasted with DAPI (blue). Inset, isotype control. (C, D) shows RT-PCR and Western blot of sebaceous cell lysates. Human keratinocytes and fibroblasts expressing AdipoR1 and AdipoR2 were used as positive control. Scale bars = 20 μm. Sebo, sebocytes; KC, keratinocytes; FB, fibroblasts.

Figure 2 shows the effect of adiponectin on cell proliferation in human sebaceous cells. (A) shows the effect of adiponectin on the survival of sebaceous cells by MTT assay, and (B) shows the ratio of [3H] -thymidine incorporation in adipocytes treated with various doses of adiponectin to that of adiponectin- Of thymidine incorporation in the sample. (C) shows the effect of dose-dependency on sebaceous cell proliferation in a low cell seeding assay using a microscope. Scale bars = 400 μm. Data represent means ± SEM (n = 6). Data were analyzed using Student's t test (* P <0.05).

Figure 3 shows the effect of adiponectin on lipid production in human sebaceous cells. (A) shows that adipocyte treated adipocytes were stained with Oil Red O and Nile red and intracellular lipids were formed through a microscope. Scale bars = 20 μm. (B) shows lipid levels produced in adipocyte cells treated with varying doses of adiponectin as a percentage of lipid levels produced in unadopted cells. (C) shows that the relative amount of the major lipid layer was determined by thin-layer chromatography, and that adiponectin was added to cholesterol, triglyceride, and wax in human sebaceous cells grown in the presence of [14C] Changes in specific lipid components such as ester and squalene were analyzed. Data mean mean SEM (N = 8). Data were analyzed using Student's t test (* P <0.05, ** P <0.01).

FIG. 4 shows that the APPL1-AMP electron transport system activating the signal of protein kinase (AMPK) is activated by treatment with adiponectin to induce lipid synthesis in human sebaceous cells. (A) shows that the sebaceous cells were treated with adiponectin, and then the whole cell lysate was prepared and analyzed by Western blot. The blot was incubated with the specific antibody. (B) the concentration of this signal protein was normalized to the concentration of the actin control. (C) shows Western blot analysis of sebaceous cells transduced with adenovirus expressing adiponectin (AD / Adipo) or LacZ (control) and related genes. Data mean mean SEM (N = 5). Data were analyzed by Student's t test (* P <0.05, ** P <0.01, *** P <0.001).

Figure 5 shows the effect of adiponectin on three-dimensional (3D) culture of human sebaceous cells. The sebaceous cells were seeded in the Matrigel layer and treated with adiponectin (200 ng / ml) for 7 days. 3D organelles of sebaceous cells were stained with hematoxylin and eosin (H & E), Nile red, SREPB and epithelial membrane antigen (EMA). Scale bars = 20 μm.

Fig. 6 shows that sebaceous gland cells induce the expression of genes related to epidermal barrier. (A) shows a quantitative RT-PCR analysis performed on various genes expressed in sebaceous cells co-cultured with keratinocytes. (B) shows the change in gene expression of keratinocytes after treatment of palmitic acid and squalene, which are major components of sebum, in human sebaceous glands. The data show mean ± SEM (N = 6). Data were analyzed by Student's t test (* P <0.05, ** P <0.01, *** P <0.001). KC = keratinocytes; Sebo = sebocytes; FAS = fatty acid synthase; HMGCR = HMG-CoA reductase.

FIG. 7 shows that adiponectin did not affect well-differentiated confluent sebocytes. (A) After the staining with Oil Red O, the intercellular lipids were detected in adipocyte-treated sebaceous cells using a microscope, and untreated cells Value as a percentage. Scale bars = 20 μm. (B) shows the analysis of the relative amount of change in the lipid layer by thin-layer chromatography.

Materials and methods

reagent

Full-length adiponectin from recombinant human was purchased from Biobud (Sungnam, Korea). (SREBP) -1, actin (Santa Cruz Biotechnologies, Santa Cruz, CA, USA), AdipoR2 (Phoenix, Pharmaceuticals, Belmont, CA, USA) for Western blotting and immunohistochemical analysis. Antibodies that specifically bind to APPL, phospho-AMP activated protein kinase (AMPK), AMPK, phospho-Akt (Cell Signaling, Danvers, MA, USA) and adiponectin (Abcam, Cambridge, UK) were used.

Palmitic acid and squalene were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Cell culture

Primary cultures of sebocytes are separated from epidermal tissue of human sebaceous glands and transferred to cell culture dishes. (Biochrom, Berlin, Germany) containing 10% fetal bovine serum (Gibco BRL, Rockville, MD, USA) and 5 ng / mL recombinant human epithelial cell growth factor (Invitrogen, Carlsbad, The cells were incubated at 37 [deg.] C in humidified air containing CO ₂ . Cells were subconfluent and harvested by subculture with 0.05% trypsin EDTA (Gibco BRL). A sample for sebaceous cell culture was obtained on the face of the operated patient. All experiments were carried out in accordance with the approval procedure of the Ethics Committee of Chungnam National University Hospital Clinical Trial Audit Committee.

Co-culture of sebaceous and keratinocytes

For the cultivation of primary keratinocytes, human skin samples were treated with dispase overnight at 4 ° C. The epidermis was removed from the treated samples and the detached epidermis was placed in a solution containing 0.05% trypsin and 0.025% EDTA at 37 占 폚 for 15 minutes. After vigorous pipetting, the cells were pelleted and resuspended in keratinocyte growth medium (KGM). Early sebocytes in primary embryonic cells (primary sebocytes) in inserts (5 × 105 per 0.4 μm pore inserts [Nunc A / S, Roskilde, Denmark] were cultured in DMEM / Ham's F12 medium (Invitrogen) 2 x 105 per well in six-well culture plates). Under the same conditions, early keratinocytes were cultured in sham inserts and used as a control. To evaluate epidermal barrier markers, sebaceous cells and control sham inserts were removed, and trypsin treatment was performed to remove keratinocytes from the wells.

Immunohistochemistry

Tissue samples were fixed with 10% formaldehyde, stored in paraffin, and cut to a thickness of 4 ㎛. Paraffin was dissolved in each part with xylene and rehydrated with an alcohol series. Each portion was pressure-cooked for 4 minutes in citrate buffer (10 mM citric acid, pH 6.0) to detect antigens. Each portion was incubated with 2% hydrogen peroxide for 30 minutes at room temperature to block endogenous peroxidase activity. The primary antibody was diluted 1: 200 and then incubated with secondary antibody for 30 minutes at room temperature. Each portion was incubated with diaminobenzidine tetrachloride solution for 5 minutes at room temperature and compared with Mayer's hematoxylin.

Immunocytofluorescence

Primary sebaceous cells were fixed in 4% paraformaldehyde for 20 min, grown on coverslips, and permeabilized with 0.1% Triton X-100 in PBS for 10 min at room temperature. Then, the cells were incubated with the primary antibody overnight at 4 ° C, and then the resulting solution was stirred at room temperature for 1 hour with a secondary antibody conjugated with FITC. Finally, the cells were allowed to be viewed under a fluorescence microscope (Olympus Corporation, Tokyo, Japan).

Western blotting

Cells were lysed with Proprep solution (intron, Daejeon, Korea). Samples were digested with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to nitrocellulose membranes and incubated with appropriate antibodies. blot was visualized by chemiluminescence (intron) after incubation with peroxidase conjugated secondary antibody. The intensity of the protein was quantified using IMT I-Solution software (IMT, Daejeon, Korea) and the obtained values were normalized to the actin signal.

Real-time PCR

RT aliquots were analyzed by quantitative real-time reverse transcription polymerase chain reaction (RTPCR) using specific primer sets. In this study, primers of the following sequences were used.

involucrin: forward 5'-CCACTGGCTCCACTTATTTCG-3 'and reverse 5'-GGACAGAGTCAAGTTCACAGA-3';

filaggrin: forward 5'-CGAAGGAGCCAAAAATATAAA-3 'and reverse 5'-GATGTGCTAGCCCTGATGTTG-3';

fatty acid synthase: forward 5'-AGTACACACCCAAGGCCAAG-3 'and reverse 5'-GGATACTTTCCCGTCGCATA-3';

HMG-CoA reductase: forward 5'-GTCATTCCAGCCAAGGTTGT-3 'and reverse 5'-CTTTGCATGCTCCTTGAACA-3'.

Quantitative real-time RT-PCR was performed according to the protocol of the SYBR Green real-time PCR master mix (Invitrogen).

Reverse transcription Polymerase chain reaction (RT- PCR )

To assess gene expression, total RNA was extracted using the easy-BLUE RNA extraction kit (Intron). Total RNA (2 μg) was reverse transcribed using the reverse transcriptase M-MLV (ELPIS Biotechnology, Taejon, Korea). The aliquots of the reverse transcription mixture were AdipoR1 forward 5'-CTTCTACTGCTCCCCACAGC-3 'and reverse 5'-ACAAAGCCCTCAGCGATAG-3';

AdipoR2 forward 5'-CTTCTACTGCTCCCCACAGC-3 'and reverse 5'-ATGGCCAGCCTCTACATCAC-3'; And

PCR was performed using a specific primer set of GAPDH (control), forward 5'-CGCTCTCTGCTCCTCCTGTT-3 'and reverse 5'-CCATGGTGTCTGAGCGATGT-3'.

Assessment of cell viability

Human mammary gland cells (2 x 105 cells per well) were inoculated into a six-well plate. The following day, the cells were treated with various doses of adiponectin and then cultured for 24 hours. The rate of cytotoxicity was assessed using a 5 mg / ml MTT reagent (3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide) for 2 hours, After formazan was dissolved in dimethyl sulfoxide, the optical density (OD) was measured at 540 nm using a spectrophotometer.

Cell proliferation assay

[3H] -Thymidine uptake assay was performed to confirm the effect of adiponectin on human sebaceous cell proliferation. Cells were cultured in fresh media containing 1 μCi of [3H] -Thymidine (Amersham, Buckinghamshire, UK), and adiponectin in various doses for 6 days was treated every 2 days on sebaceous cells in a 12-well culture plate After incubation for a period of time, the cells were washed twice with PBS and incubated with 0.1 N NaOH at room temperature. After lysing the cells, the concentration of radioactivity was measured using a liquid scintillation counter as a measure of cell proliferation. We also performed low-density seeding assays. Cells were seeded at a density of 102 cells per well in a six-well plate and grown for 6 days. Microscopic photographs were taken daily to confirm relative cell growth rates.

Lipid detection

In order to detect sebaceous lipid, primary sebocytes were inoculated at a density of 3 × 104 cells per well on a 12-well culture plate and cultured overnight and treated with various doses of adiponectin every 2 days for a total of 6 days. The cells were washed with PBS and fixed with 10% formaldehyde at room temperature for 60 minutes. (0.5% Oil Red O in 99% isopropanol) and a distilled H ₂ O mixture of 6: 4 was used to stain cells fixed with filtered Oil Red O (Sigma) working solution for 60 minutes. The stained cells were washed with distilled H ₂ O, counterstained with hematoxylin and then observed under a microscope. For quantitative detection of lipids in cells, Oil Red O was removed by culturing cells with 100% isopropanol, and the concentration of Oil Red O in the supernatant was determined by measuring the OD at 500 nm. To calculate the amount of lipid per cell, OD values were normalized to the number of cells and determined using FACSCanto II ™ Flow cytometer® and FACSDiva software (BD Biosciences, San Jose, CA, USA). For nile red staining, the stock solution of Nile red (Sigma-Aldrich; 1 mg / mL in acetone) was diluted in PBS to a final concentration of 1 μg / mL. The cells were fixed with 4% formaldehyde for 5 minutes at room temperature, stained with Nile red solution for 15 minutes at 37 ° C, and washed with PBS. The stained cells were observed under a fluorescence microscope using 485 nm excitation and a 565 nm emission filter.

Thin layer chromatography (TLC)

The composition of sebocytes containing major neutral lipids was examined by isolating lipid extracts which were not purified by TLC. [14C] -acetic acid (Perkin-Elmer Corp., Norwalk, CT, USA) was added to the medium for a total of 6 hours. After removing the medium, the cells were separated and the separated cells were transferred to a 1.5 mL capacity tube containing 200 μl of a chloroform / methanol (2: 1) mixture. Cell debris was centrifuged at 13,000 rpm for 5 minutes and the tubes were then dried using a vacuum evaporator. The lipid components extracted from sebocytes were analyzed by chromatography on 20 cm silica gel TLC plates previously charged with chloroform. After lipid (40 μL) dissolved in chloroform was spotted, the plate was developed three times as follows. (70: 30: 1) hexane / ether / acetic acid (1) top of hexane, (2) top of toluene, and (3) 10 cm away from the top. To ensure complete evaporation of the solvent, it was dried between each mobile phase for 30 minutes in a standard fume hood.

Adenovirus produce

The aliquot of the reverse transcription (RT) mixture was subjected to PCR cycles with the adiponectin primer set (forward 5'-TTGGGATCCATGCTGTTGCTGGGAGCTG-3 'and reverse 5'-TGCCTCGAGTCAGTTGGTGTCATGGTAG-3'). The amplified full-length adiponectin cDNA was subcloned into a pENT / CMVGFP vector with attL positions for site-specific recombination with a Gateway destination vector (Invitrogen). The Virapower adenovirus expression system (Invitrogen) was used to generate adenoviruses without replication competence. Positive specific recombination between entry vector and adenovirus target vector was performed with LR clonase (Invitrogen). 293A cells were infected with the adenovirus destination vector obtained using Lipofectamine 2000 (Invitrogen). Cells were grown until 80% cytopathic effect was obtained and then denatured recombinant adenovirus was obtained.

3D culture

Lago F, Dieguez C, Gomez-Reino J, Gualillo O. Adipokines as emerging mediators of immune response and inflammation. Nat Clin Pract Rheumatol. Three-dimensional culture of human sebaceous gland cells was performed using Matrigel (BD Technology, Durham, NC, USA) according to the method described in J. Med. 2007; 3: 716-724. Matrigel (50-100 μL) was added to a glass-bottom dish (1 cm in diameter) and settable during incubation on ice for 20 minutes. Next, 400 μL of medium and primary sebocytes cultured in 2% Matrigel were added to the Matrigel layer. The overlay medium was replaced every 2 days for 7 days.

Statistical analysis

All experiments were repeated at least 3 times with different batches of cells. Data were analyzed statistically using student's t test. P <0.05 showed statistical significance in all analyzes.

Result

Adiponectin receptor expression in human sebaceous glands and primary sebocytes

AdipoR1 and AdipoR2 receptors were examined in human sebaceous glands by preliminary studies. Immunohistochemical analysis in preliminary studies confirmed the presence of AdipoR1 and AdipoR2 in human sebaceous glands (FIG. 1A). The presence of adiponectin receptors was then assessed in human sebaceous cells. The results of immunocytochemical, Western blotting and RT-PCR analyzes indicated that human sebaceous cells express AdipoR1 and AdipoR2 at the protein and mRNA levels (Fig. IB-D). As a positive control, adiponectin receptors were detected in human keratinocytes and fibroblasts.

Adiponectin induces proliferation of human sebocytes

To investigate the potential role of adiponectin in vitro, primary adipocyte cells were treated with adiponectin. We first examined the cytotoxicity of adiponectin in sebaceous cells by MTT assay. There was no cytotoxic effect when adiponectin was added to cultured sebaceous cells at various doses up to 200 ng / mL (Fig. 2A). Then, the effect of adiponectin on the growth of sebaceous gland cells was examined by [3H] -thymidine incorporation assay. As a result, adipocyte proliferation was significantly induced by administration of 200 ng / ml of adiponectin (FIG. 2B). In low cell seeding assays, adiponectin increased the rate of growth of human sebaceous cells at 200 ng / ml (Fig. 2C). This suggests that adiponectin effectively induces the proliferation of human sebaceous cells in vitro.

Adiponectin enhances lipid synthesis in human sebocytes

Previous studies have shown that confluent sebocytes contain more intracellular lipid droplets than differentiated sebaceous cells. Thus, the inventors used pre-confluent undifferentiated sebaceous cells to explore the role of adiponectin when sebaceous gland activity decreased. When adiponectin was treated with 200 ng / ml of sebaceous cells, increased lipid accumulation in the cytoplasm was confirmed by Oil Red O or Nile red (Fig. 3A) staining. In addition, OD was measured by measuring the intracellular lipid level as the dose of adiponectin was increased (FIG. 3B). Next, TLC analysis was used to investigate the relative amounts of major neutral lipids. Adiponectin significantly increased all types of lipids, including cholesterol, triglycerides, wax esters and squalene (FIG. 3C).

Adiponectin increases lipid synthesis in human sebocytes via APPL1 - AMPK signaling

Based on the above results, we investigated the molecular mechanism to enhance adiponetin lipid synthesis. Numerous studies have shown that binding of adiponectin to AdipoR1 and AdipoR2 promotes APPL1-AMPK signaling in a variety of cell types and signals intracellular targets from the cell surface. The concentration of APPL1 in human sebaceous cells was increased after adiponectin treatment and the phosphorylated AMPK expression also increased in proportion to the concentration of adiponectin. Further studies have shown that the level of phosphorylated Akt is strongly influenced by adiponectin as a result of a study of the intracellular molecule Akt, which is affected by AMPK signaling. In addition, the level of SREBP-1, which induced Akt to induce the promotion of fat biosynthesis in human sebaceous cells, increased after treatment with adiponectin (Figs. 4A and B). To confirm this molecular signaling pathway, recombinant adenovirus expressing adiponectin was created and introduced into human sebaceous cells. When adiponectin was overexpressed, expression of the APPL1-AMPK signaling molecule was significantly increased (FIG. 4C). These results indicate that adiponectin increases APPL1-AMPK signaling and that expression of signal molecules such as Akt / SREBP, which are involved in APPL1-AMPK signaling, may increase the lipogenesis in human undifferentiated sebaceous cells.

Adiponectin induce lipid production in 3D culture of human sebocytes

To reproduce in vivo sebaceous tissue, we developed a 3D culture system using Matrigel. Human fibroblast cells were cultured in a 3D culture system to form organoids mimicking the sebaceous glands in vivo. Organoids formed in adipocyte cells exposed to adiponectin showed larger and more complex structure, and Nile red staining showed an increase in lipid expression. In addition, expression of SREBP1 and epithelial membrane antigen (EMA), which are potential markers for lipid production in human sebaceous cells, was also increased by treatment with adiponetin (FIG. 5).

Claims

Adiponectin,
Squalane and palmitic acid. &Lt; RTI ID = 0.0 > 11. < / RTI >

The composition for external application for skin according to claim 1, wherein the composition promotes the proliferation of sebaceous gland cells.

The composition according to claim 1, wherein the composition increases lipid synthesis.

The composition for external application for skin according to claim 1, further comprising a triglyceride.

The composition for external application for skin according to claim 1, further comprising an involucrine or filaggrin.

The composition according to claim 1, which is selected from the group consisting of L-arabinose, sialic acid, chromone, (-) catechin, S-arylcysteine, arginine, L- carnitine, caffeine, Wherein the composition further comprises at least one selected from the group consisting of flavonoids and flavones.

The composition for external application for skin according to claim 1, wherein the adiponectin is an oligomer.

[Claim 7] The composition for external application for skin according to claim 7, wherein the oligomer is polymerized with 2 to 12 monomers.

The composition for external application for skin according to claim 1, wherein said composition comprises said adiponectin at 40 ng / ml to 250 ng / ml.