Effection Of Up-regulated NAD On Infarct Evolution And Relevant Mechanism During Acute Ischemia In Mice

Objective: Nicotinamide adenine dinucleotide(NAD)is a vital coenzyme in tricarboxylic acid cycle,which plays an important role in myocardial infarction,aging and circadian rhythm.However,the role and mechanism of NAD in cerebral infarction remains unclear.This study was carried out to explore the effects of NAD with different treatments and the underlying mechanism on acute cerebral infarction.Methods: Middle cerebral artery occlusion(MCAO)model was established in C57BL/6J mice,and NAD precursor(either nicotinamide or nicotinamide riboside)was administered as preconditioning(qd×3d,the last injection was given 1 hour before the surgery)and posttreatment(one injection 20 min after the reperfusion of MCAO).Brain infarct volume was determined by TTC staining,and the hypoxia tolerance was observed by monitoring blood flow.Neurobehavioral function was evaluated with renewed Comprehensive Behavioral function scale(r CBS),and the body weight was measured,so as to observe the effects of different treatments on cerebral infarction in mice.As well,the oxygen-glucose deprivation/ reoxygenation(OGD/R)model was induced on primary neurons or astrocytes.Then,either in normal mice(baseline)or in MCAO mice,the contents of NAD and ATP in the infarcted cerebral cortex were measured by reversed-phase high performance liquid chromatography(RP-HPLC),and the contents of MDA,ROS and SOD were determined by biochemical method.Finally,the protein expression and activity of Sirt1,AMPK,PGC1α and PARP1 were detected by Sirt1 activity kit and Western blot.Results: Up-regulation of NAD by post-treatment reduced the volume of cerebral infarction,so did the r CBS score and body weight loss.However,elevating NAD by preconditioning revealed contrary results,and impaired the ischemic tolerance of brain tissue.The in vitro study suggested that post-treatment of NAD precursors during hypoxia elevated the neuronal viability,while the preconditioning made it worse.The results in normal brain tissue showed that long-term intervention by NAD precursors significantly decreased the content of ATP and increased the production of MDA and ROS,while short-term intervention significantly increased the content of ATP and significantly enhanced the production of SOD.By testing the NAD-dependent signaling pathways,it demonstrated that short-term intervention increased the activity of Sirt1,and significantly increased the expression of Sirt1,AMPK,p AMPK and PGC1α.After ischemia,it showed that preconditioning significantly decreased the level of ATP and significantly increased the content of MDA in cerebral cortex,but the post-treatment significantly increased the level of ATP and the content SOD in cerebral cortex.The activation of Sirt1 was significantly inhibited by NAD preconditioning,while the activity of Sirt1 and the expression of Sirt1,p AMPK and PGC1α were significantly increased via up-regulated NAD post-treatment.Conclusion: Although the content of NAD in brain tissue could be increased by either preconditioning or post-treatment,opposite outcomes appeared in early ischemic brains.Long-term activation or pre-ischemic activation of NAD aggravates the ischemic damages during the early stage,while short-time activation or post-ischemic activation of NAD takes a protective effect.The differences of energy metabolism and oxidative stress contribute to these changes,which may relate to the regulation of Sirt1-AMPK-ATP and PGC1α-oxidative stress pathways.