Antioxidants ameliorates glucose/glucose oxidase-induced myocardial damage through mitochondrial and MAPK pathway.

Diabetes is characterized by high blood glucose concentration that leads to the generation of elevated levels of free radicals (oxidative stress) via auto-oxidation. Oxidative stress plays a key role in diabetes-associated progressive pathologies including myocardial complications. The aim of the present study is to investigate the protective effects of antioxidants in glucose/glucose oxidase (G/GO)-dependent oxidative stress-induced cardiac cell damage. We found that exposure of G (33mM)/GO (1.6 milliunits) to cardiac muscle H9c2 cells resulted in a significant increase in apoptosis as indicated by accumulation of membrane phospholipid phosphatidylserine, DNA damage, and intracellular esterase activity. Confocal microscopy and FACS analysis further showed that G/GO induced the production of reactive oxygen and reactive nitrogen species which led to the loss of mitochondrial membrane potential and release of cytochrome c in H9c2 cells. Treatment of H9c2 cells with antioxidants like N-Acetyl Cysteine, catalase or glutathione abolished the G/GO-induced free radicals, perturbed the mitochondrial membrane potential, and induced cytochrome c release. These antioxidants also inhibited G/GO-induced cell death, caspases, and cleavage of PARP. In addition, antioxidants restored G/GO-induced suppression of antiapoptotic proteins, Bcl-2, Bcl-xL, cFLIP, XIAP, and survivin. Furthermore, G/GO impacted the MAPK pathway via activation of Raf1, MEK1 and ERK1/2 in oxidative stress-dependent manner. Pharmacologic inhibition of Raf1 also abolished G/GO-induced apoptosis. Thus, our data suggest that antioxidants have a strong protective efficacy against G/GO-induced oxidative stress through inhibition of mitochondrial and MAPK-mediated pathways in cardiac cells.