Mechanistic Study On The Roles Of NAD~+ And NAD~+-Dependent Deacetylase SIRT2 In The Tissue Injury

NAD~+is a fundamental molecule in cells,which plays critical roles in various biological processes,such as calcium homeostasis,mitochondrial functions and energy metabolism.Multiple studies have indicated that NAD~+treatment could decrease oxidative stress-induced cell death in vitro,and attenuate both ischemic and traumatic brain damage in vivo.These studies suggested that NAD~+could be a potential drug for multiple tissue injuries.To further explore the potential of NAD~+in the treatment of multiple diseases,we investigated the effects and mechanisms of NAD~+and NAD~+-dependent deacetylase SIRT2 in different injury models,including doxorubicin-induced liver damage model,acute myocardial infarction model and neuroinflammation model.One of the major obstacles for cancer treatment is the toxic side effects of anticancer drugs.Doxorubicin(DOX)is one of the most widely used anticancer agents,which produces significant toxic side effects on heart,liver,brain and kidney.It is of great significance to find a novel therapeutic approach to limit the DOX-induced liver injury,thus enhancing the beneficial effects of DOX.In this study we investigated the effects of NAD~+administration on the DOX-induced liver injury by using a mouse model,showing that NAD~+administration can significantly attenuate DOX-induced increase in serum glutamate oxaloacetate transaminase activity and decrease in liver weight and body weight.The NAD~+administration also attenuated the DOX-induced increases in the levels of double-strand DNA damage,TUNEL signals,and active Caspase-3.Furthermore,our data has suggested that the NAD~+administration could produce protective effects at least partially by restoring the antioxidation capacity of the liver,because NAD~+administration can attenuate the decreases in both the GSH levels and the glutathione reductase activity,both of which contributes to the DOX-induced hepatotoxicity.Oxidative stress plays an important role in DOX-induced liver damage and antioxidants can decrease DOX-induced liver injury.However,most antioxidants may protect the cancer cells as well.Because NAD~+can also selectively decrease tumor cell survival,it may have significant merits over antioxidants for applying jointly with DOX to decrease the toxic side effects of DOX.Acute myocardial infarction is one of the leading causes of death around the world.Although percutaneous coronary intervention has been widely applied in clinical settings,myocardial ischemia/reperfusion(I/R)injury remains poorly solved.Therefore,it is critical to establish novel therapeutic strategies for myocardial I/R injury.To test our hypothesis that intravenous administration of NAD~+can attenuate I/R injury by reducing apoptotic damage and enhancing antioxidant capacity,we used a rat model of myocardial I/R.Our study found that NAD~+administration can dose-dependently reduce myocardial infarct induced by I/R,with an approximately 85%reduction of the infarct at the dosage of 20 mg/kg NAD~+.We further found that the injection of NAD~+can significantly decrease I/R-induced apoptotic damage in the heart:NAD~+administration can both decrease the TUNEL signals,Bax,cleaved Caspase-3 levels and increase the Bcl-XL levels in rat heart that are subjected to myocardial I/R injury.NAD~+administration can also significantly attenuate I/R-induced decreases in SOD activity and SOD-2 protein levels in the hearts.NAD~+can profoundly decrease myocardial I/R injury at least partially by attenuating apoptotic damage and enhancing the antioxidant capacity,thus suggesting that NAD~+may become a promising therapeutic agent for myocardial I/R injury.Moreover,we investigated the effects and mechanism of the NAD~+-dependent deacetylase SIRT2 on neuroinflammation.It has been reported that SIRT2 inhibition can decrease the injury in cellular and animal models of PD,HD and ischemia stroke,and SIRT2 is necessary for bacterial infection.And our previous cell culture study has indicated that SIRT2siRNA-produced decrease in SIRT2 levels can lead to significant inhibition of lipopolysaccharides(LPS)-induced activation of BV2 microglia,suggesting that SIRT2 is required for LPS-induced microglial activation.In our current study,we used a mouse model of neuroinflammation to determine the role of SIRT2 in LPS-induced inflammation.We found that administration of SIRT2 inhibitor AGK2 can significantly decrease LPS-induced increases in CD11b signals and the mRNA of TNF-?and IL-6.We further found that AGK2 can block LPS-induced nuclear translocation of NF-?B.In addition,our study has shown that AGK2 can decrease not only LPS-induced increase in TUNEL signals,but also LPS-induced increases in the levels of active Caspase-3 and Bax.Collectively,our current in vivo study,together with our previous cell culture study,has suggested that SIRT2 is required for LPS-induced neuroinflammation and brain injury.And SIRT2 may become a new therapeutic target for decreasing the neuroinflammation in multiple major neurological diseases.In conclusion,in this project we investigated the effects and mechanisms of NAD~+and NAD~+-dependent deacetylase SIRT2 on tissue injury,and provided the theoretical basis for the clinical application of NAD~+-and SIRT2-based therapy.