Molecular identification for epigallocatechin-3-gallate-mediated antioxidant intervention on the H(2)O(2)-induced oxidative stress in H9c2 rat cardiomyoblasts

BACKGROUND: Epigallocatechin-3-gallate (EGCG) has been documented for its beneficial effects protecting oxidative stress to cardiac cells. Previously, we have shown the EGCG-mediated cardiac protection by attenuating reactive oxygen species and cytosolic Ca(2+) in cardiac cells during oxidative stress and myocardial ischemia. Here, we aimed to seek a deeper elucidation of the molecular anti-oxidative capabilities of EGCG in an H(2)O(2)-induced oxidative stress model of myocardial ischemia injury using H9c2 rat cardiomyoblasts. RESULTS: Proteomics analysis was used to determine the differential expression of proteins in H9c2 cells cultured in the conditions of control, 400 μM H(2)O(2) exposure for 30 min with and/or without 10 to 20 μM EGCG pre-treatment. In this model, eight proteins associated with energy metabolism, mitochondrial electron transfer, redox regulation, signal transduction, and RNA binding were identified to take part in EGCG-ameliorating H(2)O(2)-induced injury in H9c2 cells. H(2)O(2) exposure increased oxidative stress evidenced by increases in reactive oxygen species and cytosolic Ca(2+) overload, increases in glycolytic protein, α-enolase, decreases in antioxidant protein, peroxiredoxin-4, as well as decreases in mitochondrial proteins, including aldehyde dehydrogenase-2, ornithine aminotransferase, and succinate dehydrogenase ubiquinone flavoprotein subunit. All of these effects were reversed by EGCG pre-treatment. In addition, EGCG attenuated the H(2)O(2)-induced increases of Type II inositol 3, 4-bisphosphate 4-phosphatase and relieved its subsequent inhibition of the downstream signalling for Akt and glycogen synthase kinase-3β (GSK-3β)/cyclin D1 in H9c2 cells. Pre-treatment with EGCG or GSK-3β inhibitor (SB 216763) significantly improved the H(2)O(2)-induced suppression on cell viability, phosphorylation of pAkt (S473) and pGSK-3β (S9), and level of cyclin D1 in cells. CONCLUSIONS: Collectively, these findings suggest that EGCG blunts the H(2)O(2)-induced oxidative effect on the Akt activity through the modulation of PIP3 synthesis leading to the subsequent inactivation of GSK-3β mediated cardiac cell injury.