[논문]인간피부 섬유아세포에서 UVB 유도된 산화 스트레스와 기질금속단백질가수분해효소 발현에 블루베리의 저해능

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doi:10.5352/jls.2019.29.12.1321

인간피부 섬유아세포에서 UVB 유도된 산화 스트레스와 기질금속단백질가수분해효소 발현에 블루베리의 저해능
Inhibitory Activity of Blueberries on UVB-induced Oxidative Stress and Matrix Metalloproteinase Expression in Human Skin Fibroblasts 원문보기

생명과학회지 = Journal of life science, v.29 no.12, 2019년, pp.1321 - 1328

초록
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자외선B(UVB: 290-320 nm) 방사선으로도 알려진 중간 파장 태양광선은 인간에게 조기 노화 및 산화손상에 의존하는 피부암을 유발할 수 있다. UVB가 유발하는 반응성 산소종(ROS)의 형성은 종종 이러한 광선에 과도하게 노출되는 결과로 MMP-1과 MMP-3와 같은 매트릭스 금속단백질(MMP)을 활성화할 수 있다. 이 효소들은 인간의 섬유질에서 type 1형 콜라겐을 분해한다. 본 연구에서는 블루베리(EEB)의 에틸 아세테이트 추출물의 항산화 및 항노화 효과를 평가했다. 블루베리의 항산화 실험은 DPPH 분석과 CCD-986sk 세포를 사용하여 ROS 생성을 평가했다. 블루베리의 주름방지 효능을 평가하기 위해 MMP-1 생성과 type 1형 procollagen 합성을 평가하고 Western Blot과 RT- PCR을 통해 MMP 1, 3의 발현량을 평가하였다. EEB는 2,2-diphenyl-1-picrylhydrazyl (DPPH) 라디칼의 소거능을 보이며 UVB에 유도된 ROS의 생성을 저해하였다. 또한 EEB는 procollagen 생성감소 및 MMP-1 생성량의 증가 등 광노화 및 피부암과 관련된 UVB로 야기되는 과정을 억제하였다. 더 정확히 말하면, EEB (50㎍/ml)는 MMP-1과 -3의 mRNA와 단백질 발현을 현저히 억제하였다. EEB의 항노화 효과는 항산화 작용에 기인한다. 이러한 연구 결과는 EEB가 type 1형 procollagen, MMP-1, MMP-3의 수준을 조절함으로써 인간 섬유아세포에서 UVB가 유발하는 노화로부터 보호 효과를 가지고 있음을 나타낸다.

Abstract ▼ AI-Helper

Intermediate-wavelength solar radiation, also known as ultraviolet B (UVB: 290-320 nm) radiation, may cause premature aging and oxidative damage-dependent skin cancer in humans. UVB-induced formation of reactive oxygen species (ROS)-often a consequence of excessive exposure to these rays-could activate matrix metalloproteinases (MMPs) such as MMP-1 and MMP-3. These enzymes break down type I collagen in human fibroblasts. In this study, we assessed the antioxidant and anti-aging effects of ethyl acetate extract of blueberry (EEB). An antioxidant test in blueberries evaluated ROS production using CCD-986sk cells and DPPH assay. In order to evaluate the anti-wrinkle efficacy of blueberries, the MMP-1 production and type 1 procollagen synthesis evaluated and the expression of MMP 1, 3 were tested through Western blot and RT- PCR. EEB exhibited 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity and reduced the production of UVB-induced ROS. Also, EEB inhibited UVB-induced processes associated with photoaging and skin cancer, such as reduction in procollagen production and increase in MMP-1 production. More precisely, EEB (50 ㎍/ml) markedly suppressed mRNA and protein levels of MMP-1 and -3. The anti-aging effects are attributable to the antioxidant activity of EEB. These findings indicate that EEB has a protective effect against UVB-induced aging in human fibroblast cells by regulating the levels of type-1 procollagen, MMP-1, and MMP-3.

주제어

표/그림 (6)

그림 Fig. 1. DPPH radical-scavenging activity of ethyl acetate extract of blueberries (EEB).
그림 Fig. 2. The viability of CCD-986sk cells after treatment with ethyl acetate extract of blueberries (EEB). MTT assay was performed to measure viabilities of cells.
그림 Fig. 3. Effects of ethyl acetate extract of blueberries (EEB) on the generation of reactive oxygen species (ROS) induced by UVB irradiation of human skin fibroblasts. CCD-986sk cells were grown and exposed to UVB for 1 min and were then treated with indicated concentrations of EEB.
그림 Fig. 5. The expressions of matrix metalloproteinase (MMP)-1 (A) and -3 (B) mRNA were evaluated using realtime PCR. CCD-986sk cells were grown and exposed to UVB for 1 min, after which the cells were treated with 10, 25, or 50 μg/ml of EEB. The levels of mRNA were detected by RT-PCR. β-Actin mRNA was used as an internal control.
그림 Fig. 4. (A) Matrix metalloproteinase (MMP)-1 inhibition and (B) type-1 procollagen synthesis in CCD-986sk cells after treatment with ethyl acetate extract of blueberries (EEB). Fibroblast cells were exposed to UVB for 1 min and treated with EEB (10, 25, or 50 μg/ml). MMP-1 inhibition and Type-1 procollagen synthesis were measured with an assay kit.
그림 Fig. 6. Protein levels of matrix metalloproteinase (MMP)-1 and -3 were investigated after treatment of CCD-986sk fibroblasts with ethyl acetate extract of blueberries (EEB) through western blotting analysis. After 24 hr incubation of CCD-986sk cells, they were exposed to UVB radiation for 1 min and were treated with 10, 25, or 50μg/ml of EEB. Antigen-specific antibodies as primary antibodies and horseradish peroxidase (HRP)-conjugated secondary antibodies were used. Chemiluminescence was detected using enhanced chemiluminescence (ECL) solution. All experiments were performed in triplicate.

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문제 정의

Most of these studies are focused on the antioxidant, anti-diabetic, and anti-cancer properties of blueberry extracts; the anti-aging properties are rarely investigated. In this study, we clarified the roles of MMPs and type I procollagen in the potential anti-aging effects exerted by blueberries on UVB-irradiated human CCD-986sk fibroblasts.
Of all the MMPs, MMP-1 and MMP-3 are the major contributors to UVB-induced photodamage [3]. Research has shown some natural products to be effective in inhibiting the expression of MMPs to delay photoaging.

제안 방법

For example, the anthocyanins in bilberries have been reported to scavenge UVA-stimulated production of ROS [14], and strawberry extracts have been shown to possess strong anti-inflammatory [9] and antioxidant activities [33]. Previous studies have shown that some natural foods and their derived products have an inhibitory effect on MMP expression due to reduced photodamage [15] In the light of this observation, this study demonstrated the antioxidant properties of EEB measured by decreased ROS production and a DPPH free radical-scavenging assay. We showed that EEB treatment decreases mRNA and protein levels of MMP-1 and MMP-3 in UVB-irradiated human skin fibroblast cells in vitro.
This study also investigated the inhibitory effect of EEB on MMP-1 and MMP-3 mRNA expression in UVB-irradiated cells. To verify whether MMP-1 and MMP-3 mediated anti-aging effects of EEB, we quantified MMP-1 and MMP-3 mRNA levels by RT-PCR before and after 48 hr EEB treatment of UVB-irradiated cells by RT-PCR (Fig.
This study also investigated the inhibitory effect of EEB on MMP-1 and MMP-3 mRNA expression in UVB-irradiated cells. To verify whether MMP-1 and MMP-3 mediated anti-aging effects of EEB, we quantified MMP-1 and MMP-3 mRNA levels by RT-PCR before and after 48 hr EEB treatment of UVB-irradiated cells by RT-PCR (Fig. 5). The results showed an increase in both mRNA levels immediately after treatment followed by a decrease over time.

대상 데이터

DPPH radical-scavenging activity of ethyl acetate extract of blueberries (EEB). All experiments were performed in triplicate. The shown values represent mean ± SEM of three different assays.
CCD-986sk human skin fibroblast cells were purchased from American Type Culture Collection (ATCC; Manassas, VA, USA). All media required for cell growth were pur-chased from Gibco BRL (Franklin Lakes, NJ, USA).
The reagent 3-(4,5-Dimethylthiazol-2yl)-2,5-diphenyl-tetrazoliumbromide (MTT) was purchased from Sigma-Aldrich (St.Louis, MO, USA). The MTT cell proliferation assay was performed by modifying a previouslyl published protocol [22].

성능/효과

4B). The results indicated that the expression of MMP-1 was reduced, while the production of procollagen was increased after treating UVB-irradiated cells with EEB. Also, the inhibition of MMP-1 expression and the production of procollagen in UVB-irradiated cells were related to the antioxidant effect of EEB.

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