Insulin on hydrogen peroxide-induced oxidative stress involves ROS/Ca2+ and Akt/Bcl-2 signaling pathways

Abstract

Oxidative stress is induced by excess accumulation of reactive oxygen and nitrogen species (RONS). Astrocytes are metabolically active cells in the brain and understanding astrocytic responses to oxidative stress is essential to understand brain pathologies. In addition to direct oxidative stress, exogenous hydrogen peroxide (H₂O₂) can penetrate biological membranes and enhance formation of other RONS. The present study was carried out to examine the role of insulin in H₂O₂-induced oxidative stress in rat astrocytic cells. To measure changes in the viability of astrocytes at different concentrations of H₂O₂ for 3 h, a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT)-based assay was used and 500 μM H₂O₂ was selected to establish a model of H₂O₂-induced oxidative stress. Further assays showed that 3 h of 500 μM H₂O₂-induced significant changes in the levels of lactate dehydrogenase (LDH), reactive oxygen species (ROS) and calcium ion (Ca²⁺) in C6 cells, with insulin able to effectively diminish H₂O₂-induced oxidative damage to C6 cells. Western blotting studies showed that insulin treatment of astrocytes increased the levels of phosphorylated Akt and magnified the decrease in total Bcl-2 protein. The protective effect of insulin treatment on H₂O₂-induced oxidative stress in astrocytes by reducing apoptosis may relate to the PI3K/Akt pathway.