G6PD Plays A Neuroprotective Role In Brain Ischemia Through Promoting Pentose Phosphate Pathway

Aim: To observe the effects of cerebral ischemia on G6 PD expression;the influence of G6 PD on ischemic neuronal damage and preliminary mechanism of the protective effect of G6 PD on ischemia/reperfusion induced brain injury.Methods: Transient middle cerebral artery occlusion(t MCAO)was produced by using an intraluminal filament technique in mice;in vitro the OGD/R model of primary cortical neurons were established by deprivation of oxygen and glucose and reoxygenation.The changes in the expression of G6 PD in cerebral cortex were assessed with RT-PCR and Western blot analysis.Lentivirus-mediated overexpression or knockdown of G6 PD was achieved by lateral ventricular injection of lentivirus,infection rate and protein expression of G6 PD were detected with immunofluorescence and Western blot analysis.The neuroprotective effect of G6 PD was analyzed by assessing brain infarction volume,scoring neurological deficits and determining the brain water content 24 h after reperfusion.The changes in the expression of oxidative damage related proteins:Nitrotyrosine(an indicative of oxidative protein damage),HNE modified protein(an indicative of oxidative lipid damage),and ?-H2AX(an indicative of oxidative DNA damage)after ischemia/reperfusion was detected by Western blot analysisLentivirus-mediated overexpression or knockdown of G6 PD was also performed in primary cortical neurons.The infection rate and protein expressions of G6 PD were detected with immunofluorescence and Western blot analysis.The changes in the expression of oxidative damage related proteins after OGD/R was detected by Western blot analysis.The protection of G6 PD on OGD/R induced neuronal death was detected with CCK-8,LDH,GSH.The protection of NADPH on OGD/R induced neuronal death was detected.DHE probe detects the reactive oxygen species(ROS)in primary cortical neurons.The effects of NADPH were analyzed by assessing brain infarction volume,scoring neurological deficits and determining the brain water content 24 h after reperfusion.Results: The levels of G6 PD m RNA and protein increased after ischemia/reperfusion.In vivo,lentivirus-mediated G6 PD overexpression in mice markedly reduced neuronal damage after ischemia/reperfusion insult,while lentivirus-mediated G6 PD knockdown exacerbated it.In vitro,overexpression of G6 PD in cultured primary neurons decreased neuronal injury under OGD/R condition,whereas knockdown of G6 PD aggravated it.Overexpression of G6 PD increased levels of NADPH and reduced form of glutathione(r GSH),and ameliorated ROS-induced macromolecular damage.On the contrary,knockdown of G6 PD executed the opposite effects in mice and in primary neurons.Supplementation of exogenous NADPH alleviated the detrimental effects of G6 PD knockdown,whereas further enhanced the beneficial effects of G6 PD overexpression in ischemic injury.Conclusion: G6 PD has a protective effect on mouse ischemia/reperfusion injury,its mechanism may be related to increase PPP and enhance endogenous antioxidant NADPH inhibiting ROS,inhibiting neuronal cell death.