GSK-3β Regulates Nrf2 In Cultured Cortical Neurons After OGD/R

Background: Oxidative stress is the key of pathological mechanism in brain ischemic and reperfusion injury. Inactivation of antioxidant enzymes in the body and antioxidant can not eliminate excess of reactive oxygen species(ROS). Imbalances of oxidation systems in the body cause oxidative stress damage, eventually lead to the death of neurons. Recent evidence indicates that endogenous antioxidant systems have a close relationship with Nuclear factor(erythroid-derived 2)-like 2(Nrf2)2/antioxidant response element(ARE) pathway. GSK-3β is a serine/threonine protein kinase, recent studies have shown that GSK-3β can regulate Nrf2 in mouse Hepa-1and HEK 293 T cells. However, there have been no studies showing how GSK-3β regulates Nrf2 in brain ischemia and reperfusion injury.Objective:To explore after oxygen–glucose deprivation followed by recovery(OGD/R), GSK-3β regulates Nrf2 in neuron.Methods:(1)Primary cortical neurons were cultured and detected a culture purity.(2)Neuron Biotech(Shanghai, China) constructed foursegments of GSK-3 β siRNA lentivirus and one segments of GSK-3 βoverexpression lentivirus. Neurons were infected with the lentivirus. The infected efficency was estimated by calculating fluorescence expression rate under a fluorescence microscope. GSK-3β overexpression or downregulation efficency were confirmed by qRT-PCR and western blot analysis 72 h after transfection.(3)After 1.5 h of OGD, neurons were reoxygenated for 0.5 h, 1 h, 4 h, or 6 h. Western blot analysis of P-GSK-3β(tyr216), total GSK-3β, β-catenin, and Nrf2. And then chose a optimum reoxygenation time for this study.(4)Neurons were treatment with GSK-3βinhibitors(SB216763 and Licl).(5)Under normal conditions,Western blot analysis of GSK-3β and p-GSK-3β(tyr216), total Nrf2 and nuclear Nrf2.Quantitative RT-PCR analysis of GSK-3 β and Nrf2 mRNA levels in neurons.(6)After OGD/R, Western blot analysis of GSK-3β and p-GSK-3β(tyr216),total Nrf2 and nuclear Nrf2. Quantitative RT-PCR analysis of GSK-3β and Nrf2 mRNA levels in neurons.(7)Electrophoretic Mobility Shift Assay(EMSA) analysis of Nrf2-ARE binding.(8)After OGD/R,Western blot analysis of HO-1 and NQO1. Quantitative RT-PCR analysis of HO-1 and NQO1 levels in neurons.Results:(1)Culture cells were detected a culture purity of about 90%.(2)We have depended on infection of the virus and the change of cell morphology under a fluorescence microscope and chosen MOI of 20.(3)The most effective interference siRNA was GSK-3 β siRNA 969.GSK-3β expresion and mRNA have remarkably increased compare with the normal group after neurons were infected with GSK-3β overexpression lentivirus.(4)The expression of p-GSK-3 β(tyr216) initially reached its highest level after 1 h of reoxygenation. Therefore, we chose 1 h of reoxygenation as the optimum time for this study.(5)The results of Western blot, quantitative RT-PCR and EMSA suggest that GSK-3β does not regulate Nrf2 under normal conditions. But, after OGD/R, GSK-3βdownregulates expression levels of Nrf2, Nrf2-ARE binding activity, and expression levels of genes downstream of Nrf2/ARE, including HO-1 and NQO1 in neurons.Conclusion:After OGD/R, GSK-3β downregulates expression levels of Nrf2, Nrf2-ARE binding activity, and expression of Nrf2/ARE-driven genes, including HO-1 and NQO1 in neurons.