Study On The Effect Of NAD~+ On The Injury Of Neuron By Oxygen-glucose Deprivation

Oxidative DNA damage is a prominent phenomenon occurring during early stages of reperfusion after cerebral ischemia.Without being rapidly repaired,DNA damage will be accumulated in neurons and activate different cell signal pathways to result in cell death.In the brain,oxidative DNA lesions are repaired mainly via the base-excision repair(BER) pathway.Given that the neuronal genome encounters enormous oxidative damage,the functional integrity of the BER pathway is essential for neurons to survive.Our lab's studies have found that BER in the brain is a highly regulated process after ischemia-precondition.It was reported that the exogenous administration of NAD~+ can prevent cell death caused by DNA damage in other types of cells.So the exogenous administration of NAD~+ may prevent ischemia-induced neuronal injuries in the primary neuron OGD model.It is unclear that if its processes is related to modulation to DNA damage and repair systems or not.Basing on the oxygen-glucose deprivation(OGD) model,we observed the effect of the administration of NAD~+ on the OGD-induced neuronal injuries and the changes of BER enzymes' activity and expressing level,and farther investigated the inherent mechanism of changes of BER enzymes' activities caused by ischemia reperfusion and the indirect effect of NAD~+ on BER enzymes' activity.The results are showed as follow:1.The neuroprotection of NAD~+ is a NAD~+ dose-dependent manner in the OGD model of primary neuron culture,and the neuroprotection increases following the higher NAD~+ concentration used.Neuron viability increase from 24.0±2.2%without NAD~+ treatment to 85.1±2.3%with NAD~+(15mM) treatment in the OGD model.The time windows efficacy of NAD~+ administration was showed in before 1 hour after OGD.The strongest neuroprotection is achieved by NAD~+ administration before OGD.There was no significant neuorprotection in administration of NAD~+ at 2 hour after OGD.2.We investigated the effects of NAD~+ on the DNA damage in the OGD model. The results were showed that NAD~+ markedly suppress the increase of AP site and SSB by OGD injury.With measurement of AP sites,PANT assay and PARS immunocytochemistry staining,compared to the PARP-1 inhibitor,extrinsic NAD~+ can prevent simultaneously DNA damage and PARP-1 activation,but 3-AB,a PARP-1 inhibitor,inhibit PARP-1 activation only.These results imply that the resistance of NAD~+ to the neuronal injury induced by oxygen-glucose deprivation(OGD) is mainly achieved by inhibiting DNA damage of neuron, but not by inhibiting the activation of PARP-1.3.In order to investigate if it is via enhancing base-excise repair ability that administration of NAD~+ reduces the neuronal DNA damage caused by OGD,we mensurated the change of enzymes activities in BER pathway.The results indicate that the activity of two key enzymes,AP endonuclease and DNA polymerase-beta,and the total activity of BER are inhibited early after OGD in neurons suffering from oxygen-glucose deprivation,and haven't been reconverted during the following experiment period.Administration of NAD~+ recovered the activity of APE andβ-pol and the total activity of BER.These results imply that extrinsic NAD~+ prevented effectively the neuronal injuries caused by OGD treatment through enhancing the ability of repairing endogenous oxidative DNA damage.4.In order to elucidate the exact reason for the changes of BER enzymes' activities,we observed the changes of expression levels of BER enzymes and the changes of their phosphoralytion in the OGD model at the present or absent NAD~+ treatment.The expression levels of the key BER enzymes haven't remarkable changes from 0 hour to 6 hours after OGD in the nuclear extract by western blotting.The results by immunoprecipitation assay revealed that the activity of BER system in the neuron was modulated in the OGD model via affecting the phosphoralytion levels of APE andβ-pol,which engendered further DNA damage and cell injuries.NAD~+ prevent inactivation of BER enzymes induced by OGD via inhibiting the phosphorylation of BER enzymes.In summary,the disfunction of BER is a reversible process at the early stage after OGD,and extrinsic NAD~+ supplement can reverse the forfeiture of BER activity,whose mechanism bases on inhibiting the phosphorylation of BER enzymes. Our results imply that NAD~+ may be used as an excellent clinic medicine for brain ischemia therapy,meanwhile the highly reversible modulation of BER enzymes and their important effects on injuries outcome may provide a novel therapeutic target to designing the medicine for brain ischemia injury.