| Background:The reperfusion injury after cerebral ischemia threat to human life and health seriously. Oxidative stress is very important in the occurrence and development of cerebral ischemia-reperfusion injury. Nrf2, as the key factor of endogenous antioxidant system, can defense against oxidative stress in the process of cerebral ischemia-reperfusion injury effectively.Therefore, finding out the key factor that regulates Nrf2 is vital for defensing against cerebral ischemia reperfusion injury. GSK-3β, as a multi-functional kinase, demonstrate the regulatory of Nrf2 according to the previous studies. It participates in the regulation of Nrf2 both inside and outside the nucleus, and the regulation of its function negatively.Objective:To explore the regulation of GSK-3β to Nrf2 in cerebral ischemia-reperfusion in rats. To enhance endogenous antioxidant effect and neuroprotection by regulating Nrf2.Methods:(1) Experimental subject: SD rat(240-300g).(2) Screening out the time of activated GSK-3β in cerebral ischemia-reperfusion in rats. The rats were subjected by intraluminalmiddle cerebral artery occlusion(MCAO) for 1h, followed by reperfusion for 1h, 6h, 24 h. The expression of GSK-3β, GSK-3β(P-tyr216), β-catenin and Nrf2 were detected by Western Blot at each reperfusion time. The reperfusion time screened out was used as the subsequent experiments.(3) GSK-3β interference/inhibition.(1) : GSK-3β si RNA was injected ipsilaterally into the left lateral cerebral ventricle. Transfection efficiency was confirmed under a fluorescence microscope 48 h after transfection. The protein content of GSK-3β, GSK-3β(P-tyr216), total Nrf2 and nuclear Nrf2 were detected by Western Blot; The quantity of GSK-3β m RNA and Nrf2 m RNA were determined by q RT-PCR. The GSK-3β si RNA resulted in Sustained GSK-3β downregulation was selected out as the subsequent experiments.(2): GSK-3β inhibitors Licl and SB216763 was injected ipsilaterally into the left lateral cerebral ventricle.(4) GSK-3β interference/inhibition without MCAO.(1) : GSK-3β si RNA was injected ipsilaterally into the left lateral cerebral ventricle. The protein content of GSK-3β, GSK-3β(P-tyr216), total Nrf2 and nuclear Nrf2 were determined by Western Blot 55 h after transfection; The quantity of GSK-3β and Nrf2 m RNA was detected by q RT-PCR.(2): GSK-3β inhibitors Licl and SB216763 was injected ipsilaterally into the left lateral cerebral ventricle. The protein content of GSK-3β,GSK-3β(P-tyr216), total Nrf2 and nuclear Nrf2 were determined by Western Blot 6h after inhibition; The quantity of GSK-3β and Nrf2 m RNA was detected by q RT-PCR.(5) GSK-3β interference/inhibition with MCAO.(1) : GSK-3β si RNA was injected ipsilaterally into the left lateral cerebral ventricle. MCAO was performed 48 h after transfection. The protein content of GSK-3β, GSK-3β(P-tyr216), total Nrf2 and nuclear Nrf2 were determined by Western Blot after MCAO; The quantity of GSK-3βand Nrf2 m RNA was detected by q RT-PCR; The binding activity of Nrf2 to ARE was detected by EMSA; The expression of ARE downstream HO-1and NQO1 were detected using Western Blot. The quantity of HO-1 and NQO1 m RNA were detected using q RT-PCR.(2): GSK-3β inhibitors Licl and SB216763 was injected ipsilaterally into the left lateral cerebral ventricle. MCAO was performed 6h after inhibition. The expression of GSK-3β, GSK-3β(P-tyr216), total Nrf2 and nuclear Nrf2 were determined by Western Blot 6h after inhibition; The quantity of GSK-3β and Nrf2 m RNA was detected by q RT-PCR; The binding activity of Nrf2 to ARE was detected by electrophoretic mobility shift assay(EMSA); The expression of ARE downstream HO-1 and NQO1 were detected using Western Blot; The quantity of HO-1 and NQO1 m RNA were detected using qRT-PCR.Results:(1) The expression of GSK-3β, GSK-3β(P-tyr216) decreased 1h of reperfusion after MCAO, but increased both 6h and 24 h of reperfusion after MCAO. The expression of β-catenin and Nrf2 increased 1h of reperfusion after MCAO, but decreased both 6h and 24 h of reperfusion after MCAO. After 6h of reperfusion, GSK-3β was activated.(2) After GSK-3β interference/inhibition without MCAO, The expression of GSK-3β, GSK-3β(P-tyr216) as well as GSK-3β m RNA decreased; No significant changes in the expression of total Nrf2, nuclear Nrf2 as well as Nrf2 m RNA.(3) After GSK-3β interference/inhibition with MCAO for 6h reperfusion, The expression of GSK-3β, GSK-3β(P-tyr216) as well as GSK-3β m RNA decreased; The expression of total Nrf2, nuclear Nrf2 as well as Nrf2 m RNA increased significantly; The binding activity of Nrf2 to ARE increased; The expression of HO-1 and NQO1 as well as HO-1m RNA and NQO1 m RNA increased.Conclusion:Activated GSK-3β downregulates Nrf2 and the Nrf2/ARE pathway in brain ischemia and reperfusion injury。... |
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