The cytoprotective effects of 7,8-dihydroxyflavone against oxidative stress are mediated by the upregulation of Nrf2-dependent HO-1 expression through the activation of the PI3K/Akt and ERK pathways in C2C12 myoblasts

Recent studies have demonstrated that 7,8-dihydroxyflavone (7,8-DHF), a newly identified tyrosine kinase receptor B agonist, is a potent antioxidant agent. The present study was designed to confirm the cytoprotective effects of 7,8-DHF against oxidative stress-induced cellular damage and to further elucidate the underlying mechanisms in C2C12 myoblasts. We found that 7,8-DHF attenuated hydrogen peroxide (H2O2) -induced growth inhibition and exhibited scavenging activity against intracellular reactive oxygen species (ROS) that were induced by H2O2. We also observed that 7,8-DHF significantly attenuated H2O2-induced comet tail formation, and decreased the phosphorylation levels of the histone, H2AX, as well as the number of Annexin V-positive cells, suggesting that 7,8-DHF prevents H2O2-induced DNA damage and cell apoptosis. Furthermore, 7,8-DHF increased the levels of heme oxygenase-1 (HO-1), which is a potent antioxidant enzyme associated with the induction and phosphorylation of nuclear factor-erythroid 2-related factor 2 (Nrf2), as well as the translocation of Nrf2 from the cytosol to the nucleus. However, the protective effects of 7,8-DHF against H2O2-induced ROS generation and growth inhibition were significantly diminished by zinc protoporphyrin IX, an HO-1 competitive inhibitor. Moreover, the potential of 7,8-DHF to mediate HO-1 induction and protect the cells against H2O2-mediated growth inhibition was abrogated by transient transfection with Nrf2-specific small interfering RNA (siRNA). In addition, 7,8-DHF induced the activation of Akt, a downstream target of phosphatidylinositol 3-kinase (PI3K), and also that of extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK), while specific inhibitors of PI3K and ERK, but not a p38 MAPK inhibitor, abolished the 7,8-DHF induced HO-1 upregulation and Nrf2 induction and phosphorylation. Collectively, these results demonstrate that 7,8-DHF augments the cellular antioxidant defense capacity through activation of the Nrf2/HO-1 pathway, which also involves the activation of the PI3K/Akt and ERK pathways, thereby protecting C2C12 myoblasts from H2O2-induced oxidative cytotoxicity.