Neuroprotective Effect And Mechanism Of NADPH And NOX Inhibitors On KA-induced Excitotoxic Injury

Aim:Our previous work has shown that reduced nicotinamide adenine dinucleotide phosphate(NADPH)significantly protects neurons from excitotoxic injury by inhibiting oxidative stress and autophagy pathway.Studies have shown that NADPH can be used as a coenzyme to produce reduced glutathione(GSH)and as a substrate of NADPH oxidase(NOX)to produce reactive oxygen species(ROS).The study was aimed to elucidate the effect of NADPH and NOX inhibitor diphenyleneiodonium(DPI)on the excitotoxicity induced by Kainic acid(KA)and their mechanism.Methods:The neuronal excitotoxicity model was constructed by stereotypically injecting KA into the unilateral striatum of adult ICR mice.NADPH was intravenously injected 30 minutes prior to a single KA administration,and NOX inhibitor DPI was intraperitoneally injected immediately after intracranial injection,dosing once a day for the next week.The lesion of striatum and the survival status of neurons were measured by Nissl staining.The neurobehavioral deficit caused by neural dysfunction were evaluated by the inverted grid test,the adhesive removal test,cylinder test of forelimb asymmetry and their combination scores.Total superoxide dismutase(T-SOD)and malondialdehyde(MDA)kits were used to test SOD activity and MDA concentration to reflect the level of oxidative stress.The mitochondria fraction,nuclear fraction and cytoplasm of the striatum were isolated by differential centrifugation.Western blot was used to detect changes in the expression levels of autophagy-related proteins LC3-II/LC3-I,P62,as well as TIGAR and NOX4 in total homogenate;expression levels of oxidative stress-related protein Nuclear respiration factor2(Nrf2)in nuclear fraction and cytoplasm;expression levels of mitophagy-related proteins PINK1 and COX ? in mitochondria fraction and cytoplasm.Results:In this study,we have successfully established the in vivo excitotoxic model by KA.The Nissl staining results showed that the neurons in KA model group were shrunk,and the healthy neurons were reduced compared with the control group.The SOD activity of KA model group was significantly reduced,and the concentration of lipid peroxides MDA was significantly increased compared with the control group.Western blot results showed that P62 and TIGAR protein expression levels were decreased,while LC3-II/LC3-I and NOX4 protein expression levels were increased;nuclear translocation of Nrf2 was increased in the KA model group,compared with the normal control group;PINK1 protein expression levels was increased in the mitochondrial components.Both NADPH and DPI alone can reduce KA-induced striatum neuronal injury.NADPH reversed the KA-mediated decrease in P62 and TIGAR protein expression,and increased NOX4 expression.DPI also inhibited KA-induced decrease in P62 and TIGAR protein levels.Compared with any single group,the combination of low dose of NADPH and NOX inhibitor DPI showed more significant neuroprotective effect,showing more neurons with normal morphological characteristic in the striatum.The behavioral assessment of neurological deficits showed that compared with the monotherapy group,combination of NADPH and DPI offers better muscular strength,fine motor ability,forelimb symmetry and neurological dysfunction scores.Conclusions:Our data provide a possible mechanism that NADPH ameliorated KA-induced excitotoxicity by blocking autophagy.The combination of NADPH and NOX inhibitor DPI offers better neuroprotection by reducing NADPH as a NOX substrate to generate ROS.