KR102109976B1 - Composition for suppressing Cutaneous Pigmentation Containing Arginase 2 Inhibitors - Google Patents

Abstract

The present invention relates to a composition for inhibiting pigmentation containing an arginase 2 inhibitor as an active ingredient, and the composition promotes skin aging and can suppress the expression of arginase 2, which inhibits melanosome decomposition. It can be usefully used for suppressing formation.

Description

Composition for suppressing pigmentation containing Arginase 2 inhibitor as an active ingredient {Composition for suppressing Cutaneous Pigmentation Containing Arginase 2 Inhibitors}

The present invention relates to a composition for inhibiting pigmentation, which contains an arginase 2 inhibitor as an active ingredient.

MicroRNAs (microRNAs, miRNAs) are small RNA molecules that play a role in regulating the gene expression of an organism and inhibit gene expression by binding to complementary sequences in the 3'-untranslated region of the target gene Or it is known to be involved in skin pigment production by decomposing proteins (Dynoodt et al., 2013; Kim et al., 2014b).

The skin can be roughly divided into epidermis and dermis, and melanocytes present in the epidermis synthesize a pigment called melanin to show the skin's own color. On the other hand, skin pigmentation consists of melanin synthesis in melanocytes, migration of melanosomes to keratinocytes, and decomposition of melanosomes, and decomposition of melanosomes is induced by autophagy. Melasma is one of the most common skin diseases. It is a disease characterized by excessive pigmentation on the skin, as well as decreased collagen and increased abnormal elastic materials.

The present inventors completed the present invention by confirming that arginase2 (arginase2, ARG2) is involved in skin pigmentation by reducing autophagy and increasing the level of proteins involved in pigment synthesis.

1. Republic of Korea Patent Publication No. 10-2013-0001123

An object of the present invention is to provide a composition for inhibiting pigmentation, which contains an arginase 2 (ARG2) inhibitor as an active ingredient.

Another object of the present invention

(a) contacting keratinocytes with a test substance;

(b) culturing keratinocytes in contact with the test substance; And

(c) Arginase 2, tyrosinase, PMEL17, Atg12-5 (autophagy-related protein 12-5), LC3-II (microtubule-associated protein light chain 3-II) in the cultured keratinocytes , phospho-mTOR (mechanistic target of rapamycin), senescence-associated (SA) β-Gal, p53, p21, p16, cyclin A, cyclin B, cyclin D and one or more levels selected from the group consisting of cyclin E It is to provide a method for screening a pigmentation inhibitor comprising the step of determining whether the test substance is effective by measuring.

Another object of the present invention is to overexpress arginase 2 in keratinocytes; And co-culturing the keratinocytes overexpressed with the arginase 2 with melanocytes, to provide a cell model for pigmentation inhibitor screening and a cell model for pigmentation inhibitor screening prepared by the method. will be.

One aspect of the present invention provides a composition for inhibiting pigmentation, which contains an arginase 2 (ARG2) inhibitor as an active ingredient.

As used herein, the term "arginase" is an enzyme that hydrolyzes arginine to ornithine and urea at the final stage of the urea cycle, arginases 1 and 2 Isoforms exist. There have been reports of increased expression in patients with psoriasis, epidermal hyperplasia, or skin wounds in case of arginase 1 (Bruch-Gerharz et al., 2003; Redmond and Rothberg , 1978; Kampfer et al., 2003), there is no known relationship between arginase 2 (hereinafter referred to as ARG2) and pigmentation.

As used herein, the term "arginase 2 inhibitor" includes a substance that inhibits the transcription of the ARG2 gene, thereby inhibiting mRNA production or inhibiting the translation of the mRNA and promoting the degradation of the ARG2 protein. .

According to an embodiment of the present invention, when the expression of ARG2 increases, the protein levels of tyrosinase and premelanosome protein 17 (PMEL17), which are involved in melanin synthesis, increase, so the ARG2 inhibitor can be usefully used to inhibit pigmentation. .
The arginase 2 inhibitor may be an antisense nucleotide that complementarily binds to an arginase mRNA.
The arginase 2 inhibitor may be miRNA, and is preferably miRNA of SEQ ID NO: 1, that is, miRNA-1299.

As used herein, the term "composition for inhibiting pigmentation" refers to a composition capable of suppressing the expression of genes involved in pigmentation or translation of mRNA, or controlling the activity of proteins involved in pigmentation.

According to an embodiment of the present invention, the pigment may be melanin, but is not limited thereto.

In addition, the composition for inhibiting pigmentation may inhibit the expression of the ARG2 gene or the activity of the ARG2 protein to promote melanosomal degradation, and reduce the protein level of tyrosinase or PMEL17 involved in melanin synthesis.

As used herein, the term "melanosome (melanosome)" is a membrane-shaped granule containing melanin and serves to move melanin from melanocytes to keratinocytes. When the melanosomes are decomposed, melanin migrates to the stratum corneum of the skin and causes pigmentation symptoms such as stains.

The composition for inhibiting pigmentation of the present invention may be provided in various forms selected from pharmaceutical compositions or functional cosmetic compositions.

When the composition of the present invention is a pharmaceutical composition, suitable carriers, excipients, disintegrants, sweeteners, coating agents, expanding agents, lubricants, lubricants, flavoring agents, antioxidants, buffers, bacteriostatic agents commonly used in the manufacture of pharmaceutical compositions , One or more additives selected from the group consisting of diluents, dispersants, surfactants, binders and lubricants.

When the composition of the present invention is a cosmetic composition, the formulation of the cosmetic composition may be prepared in any formulation conventionally prepared in the art, for example, solutions, suspensions, emulsions, pastes, gels, creams, lotions, Powder, soap, surfactant-containing cleansing, oil, powder foundation, emulsion foundation, wax foundation, and spray may be formulated, but is not limited thereto. More specifically, it can be prepared in the form of a flexible lotion, nutrition lotion, nutrition cream, massage cream, essence, eye cream, cleansing cream, cleansing foam, cleansing water, pack, spray or powder.

Another aspect of the invention

(a) contacting keratinocytes with a test substance;

(b) culturing keratinocytes in contact with the test substance; And

(c) Arginase 2, tyrosinase, PMEL17, Atg12-5 (autophagy-related protein 12-5), LC3-II (microtubule-associated protein light chain 3-II) in the cultured keratinocytes , phospho-mTOR (mechanistic target of rapamycin), senescence-associated (SA) β-Gal, p53, p21, p16, cyclin A, cyclin B, cyclin D and one or more levels selected from the group consisting of cyclin E It provides a method for screening a pigmentation inhibitor comprising the step of determining whether the test substance is effective by measuring.

According to an embodiment of the present invention, the keratinocytes of (a) may be cells in which arginase 2 is overexpressed. The method for overexpressing Arginase 2 may use a conventional method known in the art, for example, a method including transforming a vector containing a gene encoding Arginase 2, but is not limited thereto.

According to an embodiment of the present invention, the step of culturing the keratinocytes of (b) may be performed by co-culture with melanocytes, but is not limited thereto.

According to an embodiment of the present invention, the step of determining the validity of the test substance of (c) is compared with the test substance before contact with arginase 2, tyrosinase, PMEL17, phospho-mTOR (phosphorylated mTOR) in keratinocytes. , When the level of cyclin A, cyclin B, cyclin D or cyclin E decreases or the level of Atg12-5, LC3-II, senescence-associated β-Gal, p53, p21 or p16 increases, the test substance is pigmented It may be judged to be effective as an inhibitor.

According to an embodiment of the present invention, measuring the 'level' means measuring protein concentration, activity, and expression level of mRNA.

According to an embodiment of the present invention, measuring the protein concentration may be performed using a conventional method known in the art, for example, Western blot, ELISA (enzyme-linked immunospecific assay), or the like.

Another aspect of the present invention is the step of overexpressing arginase 2 in keratinocytes; And co-culturing the keratinocytes over-expressed with the arginase 2 with melanocytes, and a cell model for pigmentation inhibitor screening prepared by the method and a cell model for pigmentation inhibitor screening prepared by the method. .

According to an embodiment of the present invention, keratinocytes overexpressing arginase 2 can be usefully used for screening for pigmentation inhibitors because melanosome degradation is inhibited and pigmentation is promoted.

The composition according to an embodiment of the present invention may be used for the purpose of inhibiting skin pigmentation because it can promote skin aging and suppress the expression of arginase 2, which inhibits melanosomal degradation.

1 is a view showing the results of confirming the expression of tyrosinase, ARG2 (arginase2) and PMEL17 in keratinocytes and melanocytes overexpressing miR-1299.
2 is a view showing the results of measuring the complementary sequence of miR-1299 and ARG2 3'-UTR sites and luciferase activity in keratinocytes.
3 is a view showing the result of measuring the level (level) of miR-1299 and ARG2 in the skin of a spotted patient.
FIG. 4 is a diagram showing the results of confirming the expression level of ARG2 in normal pigmented and hyperpigmented skin tissues from skin tissues isolated from blemish patients.
5 is a diagram showing the results of confirming the expression of tyrosinase and PMEL17 in keratinocytes and melanocytes overexpressing ARG2.
6 is a diagram showing the results of confirming the mRNA levels of tyrosinase and PMEL17 in keratinocytes overexpressing or knocking down ARG2.
7 is a diagram showing the results of comparing the relative expression levels of tyrosinase and PMEL17 in keratinocytes overexpressing or knocking down ARG2.
8 is a diagram showing the results of confirming the degree of melanosome transfer (melanosome transfer) when overexpressing ARG2 in keratinocytes co-cultured with melanocytes.
9 is a diagram showing the results of confirming the expression levels of Atg12-5, Atg 12, Atg5, LC3-II and p62 in keratinocytes overexpressing or knocking down ARG2.
10 is a diagram showing the results of confirming the expression levels of p-AMPK, p-mTOR and Atg13 in keratinocytes overexpressing or knocking down ARG2.
11 is a view showing the results of confirming the expression level of tyrosinase, PMEL17, Atg12-5, LC3-II, p-mTOR in keratinocytes treated with rapamycin.
12 is a diagram showing the results of confirming the protein change according to the presence or absence of rapamycin treatment in keratinocytes overexpressing or knocking down ARG2 by confocal microscopy.
13 is a diagram showing the results of double-staining LC3 and ARG2 in keratinocytes overexpressing or knocking down ARG2.
14 is a diagram showing the results of confirming the protein expression change according to 3-MA (3-methyladenine) treatment and overexpression of ARG2 in keratinocytes co-cultured with melanocytes.
15 is a diagram showing the results of staining LC3 and PMEL17 in the skin tissue of a patient with increased ARG2.
16 is a diagram showing the results of staining SA β-Gal in keratinocytes overexpressing ARG2.
17 is a diagram showing the results of confirming the expression of p53, p16, p21, cyclins (cyclins) A, B, D1 and E1 in keratinocytes overexpressing or knocking down ARG2.
18 is a diagram showing the results of confirming the expression of p53, p16, p21, Atg12-5, LC3-II, p62, p-mTOR and ARG2 in keratinocytes of different passage numbers.
19 is a diagram showing the results of confirming the expression of p16, p21, Atg12-5, LC3-II and p-mTOR in keratinocytes of early or late passage overexpressing ARG2.
20 is a diagram showing the results of staining ARG2 and p16 in hyperpigmentation and normal pigmentation skin tissue of patients with increased ARG2.

Hereinafter, one or more specific examples will be described in more detail through examples. However, these examples are intended to illustrate one or more embodiments by way of example, and the scope of the present invention is not limited to these examples.

Experiment method

1. Skin tissue samples and cell culture

A portion of the skin tissue of the hyperpigmentation and normal pigmentation (hereinafter referred to as the control) site was separated from 6 female patients diagnosed with blemishes (ages 44 to 61, average age 50.3 years) by biopsy.

Adult skin tissues separated in the surgical process were separated into dermis and epidermis to prepare epidermal cells. Keratinocytes among epidermal cells include bovine pituitary extract (hereinafter referred to as BPE), bovine insulin (hereinafter referred to as BI), hydrocortisone, and human epidermal growth factor ( It was cultured in EpiLife Medium (Invitrogen, Carlsbad, CA) containing human epidermal growth factor (hereinafter referred to as hEGF) and bovine transferrin (hereinafter referred to as BT). Melanocytes are BPE, BI, fetal bovine serum (hereinafter referred to as FBS), hydrocortisone, BT, basic fibroblast growth factor (hereinafter referred to as bFGF), heparin And Phobol 12-myristate 13-acetate. Incubated in Medium 254 (Invitrogen). The keratinocytes and melanocytes were used in experiments while passaged up to 3 to 5 and 7 to 20 times, respectively.

2. miRNA Transfection

Human miR-1299 analogues (Dharmacon Research, Lafayette, CO; SEQ ID NO: 1), hairpin inhibitors (negative control) or according to the manufacturer's protocol using TransIT-siQUEST transfection reagent (Mirus, PanVera, Madison, WI) or Each miR-1299 hairpin inhibitor was transfected into cells, and a miRNA analog (miRNA mimics; Dharmacon) was used as a control.

3. ARG2  Overexpression ( overexpression ) And knockdown

For ARG2 overexpression, pCMV-ARG2 was transfected into cells using Lipofectamine 2000 (Invitrogen) according to the manufacturer's protocol. In addition, for ARG2 knockdown, human ARG2 siRNA (100 nM or 50 nM) or negative control (ON-TARGETplus SMARTpool or Non-targeting siRNA; Dharmacon) was transfected into cells according to the manufacturer's protocol using TransIT-siQUEST transfection reagent. Sean. After 24 hours, rapamycin (Sapma-Aldrich, St. Louis, MO, USA) at a concentration of 100 to 200 nM was added to the medium and used for further experiments.

4. Luciferase Assay ( luciferase  activity assay)

In the human ARG2 gene, a 3'-UTR site was amplified by PCR, and the amplification product was inserted after the luciferase gene of pMIR-REPORT luciferase vector (Ambion, Austin, TX) to construct a pMIR-ARG2 vector. The pMIR-ARG2 vector was transfected into keratinocytes and melanocytes.

Primers used for PCR in the construction of the pMIR-ARG2 vector are as follows:

ARG2-3'-UTR-F: 5'-AAAGCTGCGCACTAGTGAGACACTGTGCACTGAC-3 '(SEQ ID NO: 2)

ARG2-3'-UTR-R: 5'-GATATCACGCACGCGTTTTCAGAATCCATTTTTATG-3 '(SEQ ID NO: 3)

Using the QuickChange site-directed mutagenesis kit (Stratagene, Lo Jolla, CA, USA), some base sequences were removed from the 3'-UTR region of the human ARG2 gene according to the manufacturer's protocol, and inserted into the pMIR-REPORT luciferase vector. pMIR-mutant vector was constructed.

Dispense cells into 6-well plates and transfect the pMIR-ARG2 vector or pMIR-mutant vector with pRL-TK vectors (Promega, Madison, WI) containing Renilla luciferase in each well. Specified and transfected with 50 nM miR-1299, 50 nM miR-155, or negative control in each well. As the transfection, Lipofectamine 2000 was used, and after 24 hours, luciferase activity was measured using a Dual-Luciferase Reporter Assay (Promega). All experiments were conducted in triplicate, and a total of three experiments were conducted.

5. Real-time RT- PCR  analysis

Mature miRNA expression levels were measured according to the manufacturer's protocol using human miR-1299 specific TaqMan MicroRNA Assays (Applied Biosystems, Foster, CA). As a housekeeping gene for normalization of experimental results (U18) (Roche, Mannheim, Germany) was used. The relative expression level of mRNA compared to GAPDH was measured using the Light Cycler Real-Time PCR (Roche), and the primers used were as follows:

ARG2-F: 5'-AGCAGAGCTGTGTCAGATGG-3 '(SEQ ID NO: 4)

ARG2-R: 5'-GGCATGGCCACTAATGGTA-3 '(SEQ ID NO: 5)

GAPDH-F: 5'-TCCACTGGCGTCTTCACC-3 '(SEQ ID NO: 6)

GAPDH-R: 5'-GCAGAGATGATGACCCTTT-3 '(SEQ ID NO: 7)

6. Western Blot (Western blot analysis)

20 μg of protein is separated by 10% SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis), the protein is transferred to nitrocellulose membranes (hereinafter referred to as NC membrane), and the NC membrane is reacted with a primary antibody. Ordered. Primary antibodies used were as follows: phospho-mTOR, mTOR, Atg12, Atg12-5, phospho-AMPK, AMPK, cyclin D (rabbit polyclonal; Cell Signaling Technology, Beverly, MA); cyclin B, cyclin E (mouse monoclonal; Cell Signaling Technology); ARG2, p21, cyclin A (rabbit polyclonal; Santa Cruz Biotechnology, Santa Cruz, CA); PMEL17, p53, Atg5 (mouse monoclonal; Santa Cruz Biotechnology); tyrosinase (goat; Santa Cruz Biotechnology); LC3 (rabbit polyclonal; MBL International, Woburn, MA); And Atg13, p16 (rabbit polyclonal; Bethyl Laboratories, Montgomery, TX).

The NC membrane, which has been reacted with the primary antibody, was further reacted with anti-rabbit or anti-mouse secondary antibody (horseradish peroxidase-conjugated antibodies), and developed by treatment with an ELC solution (enhanced chemiluminescence solution; Thermo, Rockford, IL, USA) . The color development signal was confirmed by an image reader (LAS-3000; Fuji Photo Film, Tokyo, Japan), and the protein band was analyzed by densitometry.

7. Immunohistochemistry

Paraffin blocks were made by embedding skin tissues separated from blemish patients in paraffin, and skin tissue slides were prepared from the paraffin blocks. After deparaffinization and rehydration of the slide, the slide was preincubated with 3% bovine serum albumin. Thereafter, an anti-ARG2 or anti-LC3 antibody and a 1: 200 Alexa Fluor-labeled goat anti-rabbit IgG (488; Molecular Probes, Eugene, OR) antibody were sequentially reacted. Next, anti-c-kit, anti-PMEL17, or anti-P16 antibodies were reacted with Alexa Fluor-labeled labeled goat anti-mouse or donkey anti-goat IgG (594; Molecular Probes) antibodies. Fluorescence images were imaged using an image analysis system (Dp Manager 2.1; Olympus Optical Co., Tokyo, Japan) and Wright Cell Imaging Facility (WCIF) ImageJ software (http://www.uhnresearch.ca/facilities/wcif/imagej). Confirmed and evaluated.

8. Confocal microscopy observation

After keratinocytes were cultured with ARG2 overexpressed or knocked down, DMSO or rapamycin was treated, and cells were stained with Cyto-ID (Enzo Life Sciences, Farmingdale, NY, USA) according to the manufacturer's protocol. In addition, the cells were reacted with anti-ARG2 or anti-LC3 antibodies, and then diluted 1: 200 to Alexa Fluor-labeled goat anti-rabbit IgG or Alexa Fluor-labeled goat anti-mouse IgG (Molecular Probes, Eugene, OR) Each was reacted with an antibody. Cell nuclei were cross-stained with Hoechst 33258 (Sigma-Aldrich). Staining images were obtained using EZ-C1 software (EZ-C1; Nikon, Tokyo, Japan), and fluorescence signals were measured using NIS-Elements AR 3.2 software (Nikon Instruments, Melville, NY).

9. β- Galactosidase (β- galactosidase ) dyeing

Cells were fixed with 2% formaldehyde at room temperature for 15 minutes, and the cells were washed with PBS. Thereafter, the cells were reacted with a senescence associated (SA) β-Gal staining solution (Cell Signaling Technology) at 37 ° C., and then observed with an optical microscope (DM LB microscope; Leica Microsystems, Wetzla, Germany). In the aged cells, a blue precipitate is observed around the nucleus.

10. Melanosome  move( Melanosome  transfer)

The amount of melanosomes moving from melanocytes to keratinocytes was measured using FACS (Lin et al., 2008). Cells were first fixed with 2% formaldehyde at room temperature and washed for 10 minutes with PBS containing BD PhosFlow Perm / Wash Buffer I (BD Biosciences, San Jose, CA). Then, the cells were reacted with FITC-conjugated goat anti-TYRP-1 (Santa Cruz Biotechnology) antibody and phycoerythrin-conjugated mouse anti-Cytokeratin 14 (BD Biosciences) antibody at room temperature for 30 minutes. Next, the cells were washed with PBS, and the cells were analyzed using a Cytomics FC500 Flow Cytometer (Beckman Coulter, Hialeah, FL) and CXP software (Beckman Coulter). Melanosome transfer efficiency was calculated by dividing the number of cells stained with cytokeratin 14 (Cytokeratin 14) and TYRP-1 (tyrosinase-related protein 1) by the number of cytokeratin 14 positive cells.

11. Statistical Analysis

Student's t-test was used for statistical analysis of the experimental results, and the analysis results were expressed as mean ± standard deviation (means ± SD). When the p value was less than 0.05, it was judged to be statistically significant.

Experiment result

One. miR -1299 overexpression ( overexpression ) Or by knockdown Tyrosinase (tyrosinase), ARG2  And PMEL17  Confirmation of expression change

Expression changes of tyrosinase and ARG2 by overexpression or knockdown of miR-1299 were confirmed in keratinocytes (KC) and melanocytes (melanocytes, MC).

As a result of over-expressing miR-1299 in primary cultured melanocytes, it was confirmed that the expression of tyrosinase, ARG2, and premelanosome protein 17 (PMEL17) was suppressed in a concentration-dependent manner (50 and 100 nM) of miR-1299. In addition, it was confirmed that miR-1299 was overexpressed in keratinocytes co-cultured with normal melanocytes, and that the same results were observed with melanocytes.

On the other hand, when the expression of miR-1299 was knocked down, it was confirmed that the expression of tyrosinase, ARG2 and PMEL17 was increased in both keratinocytes and melanocytes ( p <0.05).

1 is a result of Western blot expression of tyrosinase, ARG2 and PMEL17 in keratinocytes and melanocytes overexpressing miR-1299: KC- keratinocytes; MC-melanin cells; miR-1299Inh is a miR-1299 inhibitor.

In addition, it was confirmed that ARG2 is a target gene of miR-1299 by confirming that miR-1299 significantly reduces luciferase activity of the ARG2 3'-UTR site.

Figure 2 shows the results of measuring the complementary sequence and luciferase activity of miR-1299 and ARG2 3'-UTR sites, and it can be seen that luciferase activity decreases by miR-1299 treatment.

In addition, as a result of measuring the level of ARG2 in real-time RT-PCR in skin tissues isolated from blemished patients, it was confirmed that the level of ARG2 in hyperpigmented skin increased more than 1.5 times compared to normal pigmented skin. As a result of staining with ARG2 antibody, it was found that ARG2 expression increased in hyperpigmented skin.

Figure 3 shows the results of measuring the levels of miR-1299 and ARG2 in the skin of a spotted patient, and it can be seen that the level of ARG2 was significantly increased.

FIG. 4 shows the result of confirming the expression level of ARG2 in skin tissues isolated from blemish patients, and it can be seen that the expression of ARG2 in hyperpigmented skin (Lesion) is increased compared to normal pigmented skin (Normal).

2. ARG2  By overexpression or knockdown Tyrosinase  And PMEL17  Confirmation of expression change

As a result of overexpression of ARG2 in primary cultured melanocytes, it was found that the expression of tyrosinase and PMEL17 increased, and it was confirmed that overexpression of ARG2 in keratinocytes co-cultured with normal melanocytes showed the same results as melanocytes. . However, as a result of confirming the mRNA level change by ARG2 overexpression, it was confirmed that there was no significant difference in the mRNA levels of both tyrosinase and PMEL17.

On the other hand, when the expression of ARG2 was knocked down, it was confirmed that the expression of tyrosinase and PMEL17 was decreased in both keratinocytes and melanocytes ( p <0.05).

Figure 5 shows the results confirming the expression of tyrosinase and PMEL17 in keratinocytes and melanocytes overexpressing ARG2. KC (ARG2) + MC means co-culture of keratinocytes (KC) and melanocytes (MC) overexpressing ARG2, and KC + MC (ARG2) co-exists melanocytes and keratinocytes overexpressing ARG2. It means cultured (hereinafter the same).

Figure 6 shows the results confirming the mRNA levels of tyrosinase and PMEL17 in keratinocytes overexpressing or knocking down ARG2.

Figure 7 shows the results of comparing the relative expression levels of tyrosinase and PMEL17 in keratinocytes overexpressing or knocking down ARG2.

In addition, as a result of confirming that ARG2 overexpression affects melanosome migration using FACS, it was found that there was no significant difference in the degree of melanosome migration compared to the control group.

Figure 8 shows the results of confirming the degree of melanosome migration when overexpressing ARG2 in keratinocytes co-cultured with melanocytes.

3. ARG2  Expressive Melanosome ( melanosome ) Decomposition reduction effect check

In the case of overexpression of ARG2 in keratinocytes co-cultured with normal melanocytes, it was confirmed that the level of melanosomal decomposition was affected because protein levels of tyrosinase and PMEL17 did not change, but mRNA levels and melanosome migration did not change.

First, since the melanosomes are degraded by autophagy, it was confirmed whether the expression of autophage-related proteins changes due to ARG2 overexpression. As a result, it was found that the protein levels of Atg12-5 (Atg12-Atg5 conjugation) and microtubule-associated protein light chain 3 (LC3) -II decreased by ARG2 overexpression. On the other hand, when ARG2 was knocked down from keratinocytes cultured in a single culture or keratinocytes co-cultured with normal melanocytes, it was confirmed that the opposite result was produced ( P <0.05).

Figure 9 shows the results confirming the expression levels of Atg12-5, Atg 12, Atg5, LC3-II and p62 in keratinocytes overexpressing or knocking down ARG2.

In addition, the protein level of Atg13 was decreased by overexpression of ARG2, and phosphorylation of AMPK (AMP-activated kinase) was also reduced. However, phosphorylation of the mechanistic target of rapamycin (mTOR) C1 increased.

Figure 10 shows the results confirming the expression level of p-AMPK, p-mTOR and Atg13 in keratinocytes overexpressing or knocking down ARG2.

In addition, as a result of inducing macroautophagy by treating rapamycin with ARG2 overexpressed cells, protein levels of tyrosinase, PMEL17 and phospho-mTORC1 did not increase, while protein levels of Atg12-5 and LC3-II increased. I could confirm that.

Figure 11 shows the results confirming the expression level of tyrosinase, PMEL17, Atg12-5, LC3-II, p-mTOR in keratinocytes treated with rapamycin.

In addition, as a result of confirming the degree of autophagy in keratinocytes by confocal microscopy, it was found that overexpression of ARG2 decreases the cytosolic cytotoxicity in the cell and decreases melanosome degradation. On the other hand, in the cells treated with rapamycin, the site of Cyto-ID positive increased.

12 is a diagram showing the results of confirming the protein change according to the presence or absence of rapamycin treatment in keratinocytes overexpressing or knocking down ARG2 by confocal microscopy.

In addition, as a result of observing ARG2 and LC3 by double staining, it was found that overexpressing ARG2 reduced the fluorescence staining signal of LC-3, and knocked down ARG2 to confirm the opposite result.

13 shows the results of double staining of LC3 and ARG2 in keratinocytes overexpressing or knocking down ARG2.

In addition, the treatment of macroautophagy (macroautophagy) inhibitor 3-methyladenine (3-MA) confirmed the protein level. As a result, Atg12-5 and LC3-II decreased, but protein levels of tyrosinase, PMEL17 and phospho-mTOR increased. Was confirmed.

14 is a diagram showing the results of confirming a protein expression change according to 3-MA treatment and overexpression of ARG2 in keratinocytes co-cultured with melanocytes.

In addition, as a result of staining PMEL17 and LC3 in a skin tissue sample isolated from a spotted patient, it was confirmed that the fluorescence signal of LC3 was weak in the hyperpigmented epidermis when ARG2 was overexpressed.

15 shows the results of staining LC3 and PMEL17 in skin tissue of patients with increased ARG2.

4. ARG2  Confirm the effect of promoting aging by expression

Through the results of the above 3, it can be inferred that ARG2 decreases autophagy, and it is already known that autophagy is damaged in aging advanced skin cells (Tashiro et al., 2014; Xiong et al., 2014; Capasso et al., 2015; Ott et al., 2016) It was confirmed whether ARG2 is involved in aging of keratinocytes and whether autophagy is damaged in aged keratinocytes. As the aged keratinocytes, cells with an old passage number were used.

As a result of overexpression of ARG2 in keratinocytes, expression of senescence-associated β-Gal (SA β-Gal) was increased (P <0.05). In addition, the protein levels of p53, p21 and p16 increased, and the protein levels of cyclin A, B, D1 and E1 decreased (P <0.05).

Figure 16 shows the results of staining SA β-Gal in keratinocytes overexpressing ARG2, Figure 17 shows p53, p16, p21, cyclins A, B, in keratinocytes overexpressing or knocking down ARG2. It shows the results confirming the expression of D1 and E1.

In addition, the expression of p53, p21 and p16 increased as the passage number of keratinocytes increased, and the expression of Atg12-5 and LC3-II decreased. On the other hand, mTOR phosphorylation and ARG2 levels increased (P <0.05). In addition, overexpression of ARG2 in the initial passage number of keratinocytes decreased the levels of Atg12-5 and LC3-II, and increased the levels of phospho-mTOR, p21 and p16. This change was remarkable when ARG2 was overexpressed in late passaged keratinocytes.

Figure 18 shows the results confirming the expression of p53, p16, p21, Atg12-5, LC3-II, p62, p-mTOR and ARG2 in keratinocytes of different passage numbers, Figure 19 shows the initial overexpression of ARG2 Or it shows the results confirming the expression of p16, p21, Atg12-5, LC3-II and p-mTOR in keratinocytes of late passage.

In addition, as a result of measuring the p16 level in hyperpigmented epidermis of the patient, it was confirmed that the p16 level increased with the increase of ARG2.

FIG. 20 shows the results of staining ARG2 and p16 in hyperpigmented and normal pigmented skin tissue of patients with increased ARG2.

So far, the present invention has been focused on the preferred embodiments. Those skilled in the art to which the present invention pertains will appreciate that the present invention may be implemented in a modified form without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments should be considered in terms of explanation, not limitation. The scope of the present invention is shown in the claims rather than the foregoing description, and all differences within the equivalent range should be interpreted as being included in the present invention.

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Claims (11)

Arginase 2 (arginase2, ARG2) inhibitor as an active ingredient,
The arginase 2 inhibitor is an antisense nucleotide that complementarily binds to an arginase mRNA,
A composition for inhibiting blemish formation.
The composition of claim 1, wherein the inhibition of blemish formation is caused by inhibition of melanin pigment formation.
The composition of claim 1, wherein the arginase 2 inhibitor is a miRNA of SEQ ID NO: 1.
The composition of claim 1, wherein the composition promotes melanosome degradation.
The composition of claim 1, wherein the composition decreases the protein level of tyrosinase or premelanosome protein 17 (PMEL17).
(a) contacting keratinocytes with a test substance;
(b) culturing keratinocytes in contact with the test substance; And
(c) In the cultured keratinocytes, arginase 2, PMEL17, Atg12-5 (autophagy-related protein 12-5), LC3-II (microtubule-associated protein light chain 3-II), phospho Measure one or more levels selected from the group consisting of -mechanistic target of rapamycin (mTOR), senescence-associated (SA) β-Gal, p53, p21, p16, cyclin A, cyclin B, cyclin D and cyclin E Screening method comprising the step of determining whether the test substance is effective.
The method of claim 6, wherein the keratinocytes of (a) are arginase 2 overexpressed.
The method of claim 6, wherein culturing the keratinocytes of (b) is co-cultured with melanocytes.
The method of claim 6, wherein the step of determining whether or not the test substance in (c) is effective is arginase 2, PMEL17, phospho-mTOR, cyclin A, cyclin B, cyclin D in keratinocytes compared to before contact with the test substance. Or when the level of cyclin E decreases, or the level of Atg12-5, LC3-II, senescence-associated β-Gal, p53, p21 or p16 increases, the test substance is judged to be effective as a mitogenesis inhibitor .
Overexpressing arginase 2 in keratinocytes; And
A method of manufacturing a cell model for screening inhibitors comprising the step of co-culturing keratinocytes overexpressed with arginase 2 with melanocytes.
Cell model for screening inhibitors prepared by the method of claim 10.

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