Combination Of NADPH And NAD~+ Offers Greater Neuroprotection Against Ischemia-reperfusion Injury

Aim:To investigate if combined NADPH and NAD~+could offer better neuroprotective effects on cellular and animal models of ischemic stroke and explore the potential mechanism.Methods:Using oxygen-glucose deprivation/reoxygenation(OGD/R)and transient middle cerebral artery occlusion/reperfusion(tMCAO/R),the cellular and animal models of ischemia/reperfusion(I/R)were established.The level of NAD~+in primary cortical neurons and cerebral cortex was calculated with NAD~+/NADH assay kit.The neuronal morphology in NAD~+-treated neurons was observed under fluorescence microscope.Choose the appropriate concentration and action time of NAD~+by testing the survival rate and the release of LDH in neurons,assessing brain infarct volume,neurological deficit and water content in mice.Intracellular and cortical ROS levels were detected with DHE probe.The levels of rGSH,SOD,MDA,ATP and LD were analyzed by the corresponding detection kits,respectively.Using MTT assay,DHE probe and rGSH,MDA and ATP assay kits to test the effects of NAD~+in combination with NADPH on the survival,oxidative stress and energy metabolism of neuronal cells after OGD/R injury.The brain infarct volume was measured to evaluate the therapeutic effects of the combination drug on I/R-induced damage at different dose and time.The long-term survival rate,shrinkage of the ischemic hemisphere and behavioral deficits were estimated the long-term therapeutic effects of the combination drug in mice suffered I/R injury.To explore DNA damage,comet assay was used.Protein levels related to oxidative damageand apoptosis were measured with Western blot giving a different medication.Results:Exogenous NAD~+could get into neuronal cells and itself did not lead to cell death or change the morphology of neurons in cultured neurons.Results showed that the protective effect of NAD~+on OGD/R-induced injury was concentration-and time-dependent.Basing on this result,we chose to directly add NAD~+at the concentration of 15 mmol/L to neuronal culture at the onset of reoxygenation.Exogenous NAD~+could penetrate the blood brain barrier and be used by the brain neurons.Results showed that the protective effect of NAD~+on ischemic brain damage in a dose-and time-dependent manner.The beneficial effects of 50 mg/kg NAD~+on the infract volume,neurological deficient and water content could be observed only when it was given within 2 h after reperfusion.Neurons and mice treated with NAD~+after I/R had higher levels of rGSH,SOD and ATP and lower levels of ROS,MDA and LD,which suggested that NAD~+could ameliorates metabolic stress in vitro and in vivo models of stroke.In vitro studies,compared with NADPH or NAD~+used alone,the combination drug could significantly increase the survival rate and rGSH,ATP levels of damaged neurons.It also further attenuated the levels of ROS and MDA after OGD/R injury.Similarly,in vivo studies,the combination drug produced greater effect on I/R-induced damage by reducing the infract volume significantly.NAD~+could have better protection when combined with low concentration NADPH,and NADPH could extent the therapeutic window of NAD~+.The combination drug was more effective than NADPH or NAD~+used alone in increasing the long-term survival rate on 28 days in MCAO mice,alleviating the shrinkage of the ischemic hemisphere and improving neurological recovery.Western blot analysis revealed that protein expression related to oxidative damage and apoptosis significantly changed at 16 h after reperfusion in mice subjected to tMCAO.Using NADPH or NAD~+alone could inhibit these changes,and the combination drug could further inhibit the changes of most of them.Conclusion:NAD~+replenishment exhibited neuroprotection against I/R-induced neurons injury both in vivo and in vitro.Combination of NADPH and NAD~+provided better neuroprotective effects both in cellular and animal models of ischemic stroke.Most importantly,NADPH could prolong the therapeutic window of NAD~+,whereas NAD~+could reduce the dosage of NADPH to produce the similar magnitude of protective effects.In addition to improvement of the energy metabolism,the combined medication could effectively inhibit multiple cell death pathways during ischemic damage.The current results suggest that combination of NADPH and NAD~+may provide a novel effective therapy for ischemic stroke.