Diabetes and Metabolism Journal

Korean Diabetes Association (대한당뇨병학회)
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The Protective Effect of EGCG on INS-1 Cell in the Oxidative Stress and Mechanism

산화스트레스에 대한 EGCG (Epigallocatechin Gallate)의 INS-1 세포 보호효과와 기전

Kim, Mi-Kyung;Jung, Hye-Sook;Yoon, Chang-Shin;Kwon, Min-Jeong;Koh, Kyung-Soo;Rhee, Byung-Doo;Park, Jeong-Hyun
김미경;정혜숙;윤창신;권민정;고경수;이병두;박정현

  • Published : 2008.04.01
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Abstract

Background: Oxidative stress is important in both diabetic complications and the development and the progression of type 2 diabetes via the effects on the pancreatic β-cells. EGCG (epigallocatechin galleate), a major constituent of green tea, has been known to have beneficial effects on various diseases through the mechanisms of antioxidant and cell signaling modulation. But, very small numbers of studies were published about the direct effects of EGCG on the pancreatic β cell lines. We performed this study to see the protective effect of EGCG on pancreatic β cell line under H2O2 and the mechanisms of this phenomenon.Methods: We used INS-1 cells and hydrogen peroxide as an oxidative stressor. Their viabilities were verified by MTT assay and FACS. The activity of glutathione peroxidase was assessed by total glutathione quantification kit. Western blot and semi-quantitative RT-PCR for the catalase, SOD (superoxide dismutase), PI3K and Akt were performed. Functional status of INS-1 cells was tested by GSIS (glucose stimulated insulin secretion).Results: The biological effects of EGCG were different according to its concentrations. 10 μM EGCG effectively protected hydrogen peroxide induced damage in INS-1 cells. The expression and the activity of SOD, catalase and the glutathione peroxidase were significantly increased by EGCG. EGCG significantly increased PI3K and Akt activity and its effect was inhibited partially by wortmannin. GSIS was well preserved by EGCG.Conclusion: EGCG in low concentration effectively protected INS-1 cells from the oxidative stress through the activation of both antioxidant systems and anti-apoptosis signaling. Further studies will be necessary for the more detailed mechanisms and the clinical implications. (KOREAN DIABETES J 32:121~130, 2008)

연구배경: 산화스트레스는 만성 당뇨병 합병증의 발생에도 중요할 뿐 아니라 췌장 베타세포에 대한 직접적이거나 간접적인 영향으로 제2형 당뇨병의 발생과 진행에도 중요한 역할을 하는 것으로 밝혀지고 있다. 녹차의 주요 추출물 중의 하나인 EGCG (epigallocatechin galleate)는 각종 암 치료를 비롯하여, 심혈관계질환 및 뇌변성질환에 좋은 영향을 미친다고 알려져 왔으며 임상 연구에서도 당뇨병의 발생을 감소시키는 것으로 알려져 왔다. 그 기전은 전통적으로 잘 알려진 항산화제로서의 역할 뿐 아니라 세포 전달 체계의 변화를 통해서 이루어지는 것으로 보고되고 있다. 그러나 췌장 베타세포에서 EGCG가 직접적으로 미치는 영향에 대해서는 많이 알려져 있지 않다. 본 연구는 과산화수소를 이용한 산화스트레스하에서 EGCG가 INS-1 세포를 보호할 수 있는지를 살펴보고 그 기전을 보고자 하였다.방법: INS-1 세포는 RPMI 1640배지에서 배양하였고, 산화스트레스는 과산화수소수를 이용한 모델을 사용하였다. 세포의 생존능은 MTT assay와 annexin V와 porpium iodide (PI)를 사용한 FACS로 확인하였다. glutathione peroxidase 활성도는 total glutathione quantification kit로 측정하였고 catalase, SOD (superoxide dismutase), PI3K와 Akt의 mRNA와 활성도는 Western blot과 semi-quantitative RT-PCR를 이용하여 측정되었다. PI3K 억제제인 wortmannin을 전처리한 후 세포의 생존 정도를 FACS로 측정하였다. INS-1 세포의 기능적인 측면은 포도당 자극에 의한 인슐린분비(GSIS)로 평가하였다 결과: 산화스트레스하에서의 EGCG의 효과는 농도에 따라 차이가 나서, 10 μM의 EGCG에서는 세포 생존도의 증가가 관찰되었으나, 50 μM 이상의 농도에서는 세포 생존율이 감소하였으며, 이는 농도가 증가됨에 따라 더 감소하였다. SOD, catalase와 glutathione peroxidase의 mRNA 발현과 단백질의 양은 EGCG 처리에 의해 유의하게 증가되었다. EGCG는 PI3K와 Akt 활성도를 증가시켰고, PI3K 억제제에 의해 EGCG의 효과는 부분적으로 감소되었다. 포도당 자극에 의한 인슐린분비는 EGCG에 의해 잘 보존되었다. 결론: EGCG는 낮은 농도에서 INS-1 세포를 산화스트레스로부터 효과적으로 보호할 수 있었고, 항산화효과와 세포자연사(apoptosis)방지에 관여하는 세포 신호 전달을 기전으로 하고 있었다. 더 명확한 기전과 임상적 적용을 위해서는 더 많은 연구가 있어야 할 것이다.

Keywords

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