The Protective Effect Of NADPH On KA-induced Neuronal Excitoxicity And Its Mechanisms

Aim:To investigate the effects and mechanisms of NADPH on KA-induced excitotoxicity in rats striatum and cultured primary cortical neurons.Methods:The in vivo excitotoxic model was produced with stereotaxic injection of Kainic acid(KA)into unilateral striatum.NADPH pretreatment group was established by intravenously injection of NADPH before KA treatment.The in vitro excitoxic model was produced with KA treatment of primary cortical neurons.In vivo,striatal lesion sizes of KA treatment group and NADPH pretreatment group were measured by Nissl staining to assess the protective effect of NADPH on KA-induced excitotoxicity.Western blot was used to detect the changes in TIGAR,LC3-II/LC3-I,p62 and Beclinl of KA treatment group and NADPH pretreatment group.In vitro,the cytotoxicity of KA and neuroprotective effects of NADPH was assessed by CCK-8 to evaluate the damage of KA on primary neurons and the protective effect of NADPH.Intracellular calcium was measured by Fluo-3 AM fluorescent probe.DHE fluorescence probe was used to detect the intracellular ROS levels.Reduced form of glutathione(GSH)in primary neurons was detected by GSH/GSSG assay kit.The changes of protein expression levels of TIGAR,LC3-II/LC3-I,p62,Beclinl,ATG5,active-cathepsin B and active-cathepsin D protein levels were observed by Western blot analysis.Results:In this study,we have successfully established the in vivo and in vitro excitoxic model by KA.In vivo,compared with control group,KA significantly induced wider lesion sizes of striatum,while pretreatment of NADPH markedly ameliorate the injury.Western blot analysis showed that there are decrease in expression of TIGAR and p62 and elevation in the ratio of LC3-Ⅱ/LC3-Ⅰ and Beclinl protein levels in KA treatment group in comparison with control group.Nevertheless,NADPH effectively inhibits these changes induced by KA.In vitro,CCK-8 results showed that KA decreased the cell viability in a time-and dose-dependent manner while NADPH could significantly help neurons to survive.KA increased the intracellular levels of calcium and ROS and decreased the reduced form of GSH,but pretreatment of NADPH effectively reversed these changes.NADPH inhibited KA-induced down-regulation of TIGAR and p62,and up-regulation of ratio of LC3-Ⅱ/LC3-Ⅰ,Beclinl,ATG5,active-cathepsin B and active-cathepsin D in primary neurons by Western blot analysis.Conclusions:Our data provide a possible mechanism that NADPH ameliorated KA-induced excitotoxicity by blocking autophagy/lysosome pathway and up-regulating TIGAR besides its antioxidant properties.