Neuroprotective Effect And Mechanism Of Mito-apocynin On Kainic Acid-induced Excitotoxicity

Aim: Our previous study found that excessive activation of glutamate receptors upregulated the expression of NADPH oxidase(NOX),which uses NADPH as a substrate to generate reactive oxygen species(ROS).In addition,excessive activation of glutamate receptors also led to mitochondrial damage.Therefore,this study aimed to elucidate the neuroprotective effect and mechanism of the mitochondria-targeted NADPH oxidase inhibitor Mito-apocynin on kainic acid(KA)-induced excitotoxicity.Methods: An in vivo model of excitotoxicity was established by stereotaxic injection of KA into the right striatum of mice,and an in vitro model of excitotoxicity was established by treating primary neurons with KA.The effect of KA on the expression of NOX4,NOX2 and NOX1 in total striatal protein and isolated mitochondrial fractions was determined by immunoblotting.The mitochondrial morphology was observed by transmission electron microscopy.Mitochondrial function was evaluated by detecting ATP levels and the mitochondrial oxygen consumption rate.Western Blot was used to detect the expression levels of DRP1,phosphorylated DRP1(Ser616),Mfn2,PGC-1α,PINK1 and Parkin in the total striatum after KA injection to evaluate the effect of KA on the mitochondrial quality control system.Mice were pre-administered with Mito-apocynin intragastrically before KA injection,and continuously administered Mito-apocynin once a day until they were sacrificed.The protective effect of Mito-apocynin on KA-induced excitotoxic injury was determined by Nissl staining in vivo and a CCK-8 kit in vitro.The ameliorating effect of Mito-apocynin on KA-induced behavioral deficits in mice was evaluated by the cylinder test,adhesive removal test,inverted grid test and neurological dysfunction score.The effects of Mito-apocynin on KA-mediated changes in ATP levels,mitochondrial membrane potential and mitochondrial superoxide levels were determined by an ATP detection kit,JC-1 staining and Mito SOX fluorescent probe,respectively.The effect of Mito-apocynin on KA-induced changes in mitochondrial fraction NOX4 protein expression was determined by immunoblotting.To evaluate the effect of Mito-apocynin on KA-mediated mitochondrial quality control system disorders,Western Blot was used to detect the expression levels of phosphorylated DRP1(Ser616),DRP1,PGC-1α,PINK1 and Parkin in the total protein of the striatum.Results: KA upregulated the expression levels of NOX4 and NOX2 in the total protein of the striatum and significantly upregulated the expression level of NOX4 in the mitochondrial fraction 6 h after injection.Transmission electron microscopy results showed that KA damaged mitochondrial morphology.ATP levels and oxygen consumption rates showed that KA impaired mitochondrial energy metabolism and led to mitochondrial dysfunction.Western Blot results showed that KA disrupted the mitochondrial quality control system,leading to a decrease in the protein expression levels of phosphorylated DRP1(Ser616)/total DRP1 and Mfn2 and an increase in the protein expression levels of PGC-1α,PINK1 and Parkin in the total protein of the striatum.Nissl staining and the CCK-8 kit results revealed that Mito-apocynin significantly improved KA-induced neuronal death both in vivo and in vitro.Behavioral tests showed that Mito-Apocynin ameliorated KA-induced neurobehavioral deficits.Mito-apocynin reversed the KA-induced decrease in ATP levels and mitochondrial membrane potential and increase in mitochondrial superoxide levels in vitro.Western Blot results showed that Mito-apocynin significantly reversed KA-induced upregulation of NOX4 expression in the mitochondrial fraction,Mito-apocynin also reversed the KA-induced decrease in phosphorylated DRP1(Ser616)/total DRP1 protein expression and increase in PGC-1α,PINK1,and Parkin protein expression in the total protein of the striatum.Conclusions: Mito-apocynin alleviated oxidative stress,maintained normal mitochondrial function and energy metabolism levels,and promoted the balance of mitochondrial quality control by regulating the expression of NOX in the mitochondrial fraction,thus reducing KA-induced excitatory toxic damage.