Mechanistic studies of cardio-protection of EGCg by attenuating H2O2-induced oxidative stress in H9c2 cardiomyoblasts

• Main Authors: Chien-Sheng Hsu, 許建生
• Other Authors: Ying-Ming Liou
• Format: Others
• Language: zh-TW
• Published: 2011
• Online Access: http://ndltd.ncl.edu.tw/handle/05668395999954633586

碩士 === 國立中興大學 === 生命科學系所 === 99 === (-)-Epigalocathine-3-gallate (EGCg) is the major polyphenols in green tea, containing many OH- groups for a strong free radical scavenging capacity. When the heart is damaged by ischemia-reperfusion injury, the cells are accompanied by the generation of intracellular hydrogen peroxide, superoxi anions, and hydroxyl radicals and other reactive oxygen species (ROS). ROS would cause oxidative stress and the consequent damage on the heart. The main goal of the present study was to determine the potential mechanism for cardioprotection of EGCg on hydrogen peroxide (H2O2)-induced oxidative stress in H9c2 rat cardiomyoblasts. The results using the MTT assay showed that H9c2 cells exposing to H2O2 suppressed cell viability, while EGCg pretreatment for 30 min prior to oxidative stress effectively improved cell survival of cardiac cells. EGCg prevented ROS formation and attenuated cytosolic Ca2+ overload in H2O2-treated cells. To identify the putative mechanisms underlying the EGCg signaling pathways, EGFP (enhanced green fluorescence protein) was ectopically expressed in H9c2 cells. EGCg exerted a dose-dependent decrease in the EGFP fluorescence. In contrast, H2O2 did not cause significant spectral changes in EGFP fluorescence, but an increase in the sensitivity for the EGCg induced decrease in EGFP fluorescence. Using Triton X-100 to remove plasma membrane of the cells the similar result was obtained for the increased sensitivity of the EGCg induced response. Apparently, both Triton X-100-soluble and -insoluble fractions involved in transmission of the EGCg signals to regulate EGFP fluorescence in H9c2 cells, and the cell membrane damage caused by oxidative stress could enhance the sensitivity of EGCg. Intercellular junctional complexes in the intercalated discs play an important role in modulation of functional recovery of the cardiac injury. To examine whether EGCg could act to maintain the integrity of the intercalated disc and transmitting cell-cell adhesion signals during oxidative stress, immunoblotting with specific antibody were used to analyze the phosphorylated and non-phosphorylated protein levels of the major ventricular gap junction protein connexin 43 (Cx43) and adhesion molecule beta-catenin、N-cadherin and GSK-3beta in cells. Results obtained showed that EGCg could increase cell-cell adhesion by decreasing GSK-3beta dependent pathway to maintain adherens junction, and increase direct communication between adjacent cells by increasing phosphorylated level of Cx43. In summary, EGCg could protect cardiac cells from oxidative stress by preventing ROS formation and cytosolic Ca2+ overload, and by modulating adherens and gap junction proteins on cell membrane.