Mechanisms And Influences Of Glycogen Synthase Kinase 3β Following Cerebral Ischemia/Reperfusion Injurys In Rats

Background: Oxidative stress is an important part in cerebral ischemia reperfusion injury. NF-E2-related factor 2(Nrf2) participate in the protection of oxidative stress injury. Glycogen synthase kinase 3β(GSK-3β) is verified a significant factor that regulate Nrf2 independent of Keap1. As a result, Nrf2 discharges from nucleus and the injury of Neurons are worse. However, no study has conducted to discuss the effect of GSK-3β on cerebral ischemia-reperfusion injury before.Objective: To explore this problem, we use oxygen-glucose deprivation/reoxygenation(OGD/R) and middle cerebral artery occlusion(MCAO) as the models of ischemia-reperfusion in rats in vitro and in vivo.Methods:(1) Cortical neurons were prepared from newborn Sprague-Dawley rat brain tissue and were cultured. They were conducted OGD/R.(2) Parts of the neurons were transfected with GSK-3β si RNA. They were divide into several groups as follows: Control group, vector group, GSK-3β RNAi group, GSK-3β overexpression group, curcumin-treated group, OGD/R group, vector+OGD/R group, GSK-3β RNAi+OGD/R group, GSK-3β overexpression +OGD/R group, curcumin-treated+OGD/R group, leptomycin B-treated+OGD/R group, GSK-3β overexpression +leptomycin B-treated+OGD/R group.(3) The cellular morphology and transfection efficiency was assessed under a fluorescence microscope. Sustained GSK-3β over-expression or interference were confirmed by q RT-PCR.(4)MTS kit was used to measure neuron viability. LDH kit was used to measure neuron damage. And superoxide dismutase(SOD) kit and Malondialdehycle(MDA) kit were used to detect oxidative stress state of neurons. The relationship between GSK-3β and Nrf2 was further measured by Western Blot and immunofluorescence.(5)Middle cerebral artery occlusion( MCAO) model was established in adult male Sprague-Dawley rats. GSK-3β RNAi fragments were build, and was injected into lateral ventricle 48 h before MCAO.(6)Neurological deficits, infarct proportion and morphological features were performed. SOD kit and MDA kit were used to detect oxidative stress state of cortical tissues. The relationship between GSK-3β and Nrf2 was further measured by Western Blot and immunofluorescence.Results:(1)GSK-3β RNAi improved injury and increased viability of neurons subjected to OGD/R. Levels of total Nrf2, nuclear Nrf2, and Nrf2 downstream proteins sulfiredoxin(Srx1) and thioredoxin(Trx1) increased after transfection with the GSK-3β si RNA virus. GSK-3β si RNA increased SOD activity and decreased MDA levels. Overexpression of GSK-3β with a pc DNA-GSK-3β virus showed opposite results.(2)We also demonstrated that intracerebroventricular injection of GSK-3β si RNA in rats ameliorated neurological deficits, reduced brain infarct volume and water content, and reduced damage to cerebral cortical neurons after MCAO. Changes in total Nrf2, nuclear Nrf2, Srx1, Trx1, SOD, and MDA were similar to those observed in vitro.Conclusion: GSK-3β can influence cerebral ischemia/reperfusion injury. The effects may be due to regulating the distribution of Nrf2, promoting the concentration of Nrf2 in cytoplasm, and reducing the expression of Nrf2, Srx1 and Trx1. These results suggest a potential new drug target for clinical treatment of stroke.