산화스트레스가 유도된 망막색소상피세포에서 폴리페놀 화합물이 보이는 세포보호 효과
Cytoprotective Effect of Polyphenolic Compounds against Oxidative Stress in Cultured Retinal Pigment Epithelial Cells
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목적: 포도씨 유래 폴리페놀(Grape seed-derived polyphenols, GSP)의 경우 폴리페놀 함유량이 높은 생화합물로서 본 연구에서는 배양된 인간 망막색소상피세포에서 산화스트레스를 유도한 후 GSP가 보이는 세포보호 효과에 대하여 알아보고자 하였다. 대상과 방법: 배양된 인간 망막색소상피세포인 adult retinal pigment epithelium (ARPE)-19를 GSP에 24시간 동안 다양한 농도로 전처치(0.1, 0.5, 1, 5 and 10 μg/mL, respectively)한 후, 산화스트레스 유도를 위해 과산화수소(H2O2, 0.4 mM)로 24시간 동안 처리하였다. 3-(4, 5-dimethylthiazol-2- yl)-2, 5-diphenyltetrazolium bromide (MTT) assay를 통해 세포의 생존성을 측정하였고, 2′,7′ -dichlorofluorescein diacetate (DCF-DA) assay로 세포 내 반응성산소종의 생성을 측정하였다. 결과: 과산화수소로 산화스트레스가 유도된 망막색소상피세포의 경우, 세포의 생존율이 산화스트레스를 받지 않은 대조군에 비해 유의하게 낮았다. GSP의 전처치 농도에 비례하여 산화스트레스가 유도된 망막색소상피세포의 생존율이 증가하였는데, 0.1, 0.5, 1, 5 and 10 μg/mL 농도에서 생존율이 유의하게 증가하였다. 세포 내 반응성산소종의 발생은 과산화수소로 산화스트레스가 유도된 망막색소상피세포에서 유의하게 증가하였고, GSP로 전처치하였을 경우 농도에 비례하여 유의하게 감소하였다. 결론: 천연 폴리페놀 화합물인 GSP는 산화스트레스가 유도된 망막색소상피세포에서 세포 내 반응성산소종의 발생을 감소시키고, 세포를 보호하는 효과를 보여주었다. 따라서 산화스트레스와 연관이 있는 망막질환을 치료하는 데 폴리페놀 화합물이 도움을 줄 수 있을 것이라 생각된다.
Abstract
Purpose: Grape seed-derived polyphenols (GSPs) provide a concentrated source of polyphenols having antioxidant capacity. In this study we investigated the cytoprotective effect of GSP against oxidative stress-induced cell damage in cultured human retinal pigment epithelial (RPE) cells. Methods: Cultured adult retinal pigment epithelium (ARPE)-19 cells were incubated with GSP from Vitis vinifera (0.1, 0.5, 1, 5 or 10 μg/mL) for 24 hours and treated with hydrogen peroxide (H2O2, 0.4 mM) for 24 hours to induce oxidative stress. Cell viability was measured using 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. Intracellular reactive oxygen species (ROS) was quantified using 2′,7′-dichlorofluorescein diacetate (DCF-DA) fluorescence. Results: The percentage of viable RPE cells was significantly lower in cultures treated with H2O2 0.4 mM than in control cultures. GSP significantly reduced H2O2-induced cell death in a dose dependent manner. GSP at 0.1, 0.5, 1, 5 and 10 μg/mL significantly reduced cell mortality due to the treatment with H2O2. Intracellular ROS production increased significantly in cultures treated with H2O2 0.4 mM compared with control. There was a significant dose-dependent decrease in intracellular ROS levels after treatment of RPE with GSP. Conclusions: GSP, a natural polyphenolic compound, can protect RPE cells from H2O2-induced oxidative stress and reduce intracellular ROS production by scavenging free radicals. This suggests potential effects of polyphenolic compounds against retinal diseases associated with oxidative stress.
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