Protective Effects of Polyphenol on Reactive Oxygen Species in Cultured Human Endothelial Cells

Tark, Kwan Chul; Seul, Chul Hwan; Kim, Joo Bong; Rah, Dong Kyun

Abstract

Endothelial cells are most sensitively affected by ischemic-reperfusion injury, and also the endothelial cells have very important role in immune reaction of organ transplantation and preservation of the organ. To improve the survival rate of a flap, and also to reduce the possibility of flap necrosis, the protection and preservation of the endothelial cells are very important. Because reactive oxygen species(ROS) are thought to be an important cause of ischemic reperfusion injury, we studied the cytotoxicity of ROS on endothelial cells. We performed an in vitro study to document whether green-tea polyphenol pretreatment play an important role in preventing cytotoxic damage from ROS. Neonatal Dermal Microvascular Endothelial Cells were cultured in EGM-2 MV BulletKit. Endothelial cell suspension in concentration of 4x104, was distributed into the wells of 24-well plate, cultured for 1, 3, 5, 7 days and the growth rates of the cells were measured with ELISA reader. Green-tea polyphenolic compounds(GtPP) were administered on L929 mouse fibroblasts and the possible cytotoxicity was measured with ELISA reader. Cultured endothelial cells in the concentration of 2x105 cells were treated with 0.1mM, 1mM, 10mM H2O2 and also with 0.25mM xanthine and 0.1U/L, 1U/L, 10U/L xanthine oxidase to induce oxidative stresses. Then the morphological findings of the endothelial cells were observed under the light microscope and the growth rate was analyzed with flow cytometry. To evaluate its protective effect, 0.25, 1, 10, 100microgram/ml of GtPP were administered to endothelial cells in the concentration of 2x105cells, one hour before administration of the oxidative agents, and then the cells were cultured for 24 hours. Afterwards, the morphology of endothelial cells were observed under the light microscope and the growth rate was analyzed with flow cytometry. The results are as follows: The growth of human endothelial cells were normal, and polyphenol of each concentration administered in this study did not show cytotoxicity. As a result of oxidative stress induced by H2O2 or xanthine oxidase(+0.25mM xanthine), the endothelial cell viability decreased by more than 25%, thus confirming the effects of ROS to endothelial cells. The GtPP pretreatment before H2O2 or xanthine oxidase(+0.25mM xanthine) administration, resulted significant protective effects for endothelial cells in morphology and growth rate study. Through these studies, the authors confirmed the protective effects of polyphenol against ROS. We also conceived that the polyphenol can possibly be implemented as an agent for organ or tissue preservation.