Role Of Sirt1 In Cerebral Ischemic Tolerance Induced By Hgyperbaric Oxygen Preconditioning In Rats And Neurons

Background:Clinical treatment of this debilitating disorder is inadequate,especially for neurological impairment,although many advances have been madein the pharmacotherapy of stroke．There is a huge umnet medical need fordeveloping novel and rational strategies aimed at redcing damage after strokeand other neurological conditions．The discovery that ischemic preconditioninginduces ischemic tolerance to brain offers a new idea to the treatment of stroke,but the approach of ischemic preconditioning is difficult to be used in clinic．InOur previous studies,we have demonstrated that hyperb'dTic oxygenpreconditioning(HBO—PC)induces a tolerance to subsequent ischemic injury inthe brain and spinal cord,and perfectly mimics the neuroprotective effect ofischemic preconditioning．Because of the safety and feasibility,HBO—PC is apromising approach for limiting or preventing ischemic injury．Furthermore,aclinical trial has reported the protective effect of HBO—PC on cardiacischemia—reperfusion injury in patients undergoing coronary artery b~ass grafting．Our previous studies showed that the tolerance induced by HBO—PC tocerebral ischemia is mediated by antioxidant enzymes through an appropriateincrease of endogenous ROS in vivo and in vitro．The administration of oxygenfi'ee radical scavengers attenuates the neuroprotective effect of HBO—PC．However,the underlying mechanism is not fully understood and more evidence isneeded for HBO—PC to be accepted clinically．The covalent modification of proteins includes phosphorylation／dephosphorylation, acetylation／deacetylation, methylation／demethylation,adenylation／deadenylation and oxidation-reduction SO on,which can change theactivity of proteins．Previous studies mainly focused on the protein function ofphosphorylation／dephosphorylation．However,it has been demonstrated recentlythat acetylation／deacetylation also play an importmat role in regulation ofprotein．SifTl,served as class III histone deacetylase,activates several key transcriptionfactors which involve in signaling pathways of cell survival．Especially under theoxidative stress,SifTl remaJcably promotes cell survival．Autophagy is a criticalcatabolic process in cells,which is involved in degrading misfolded proteins andclearing excess or damaged organelles．This process aids in the balance betweendegradation,synthesis,and the recycling of cellular components．Autophagy isessential for maintaining cell homeostasis and is induced during the response tostress,including starvation,oxidative stress,and hypoxia．There is evidence tosuggest that SifTl activation involve in the protective effect of ischemicpreconditioning on oxygen glucose deprivation in rat hippocampal slices．Ischemic preconditioning offers a remarkable tolerance to a subsequent fatalischemic insuk through autophagy activation in rats．In addition,SifTl mediatedthe formation ofautophagosomes via regulation ofautophagy associated proteins．Besed on these findings,we phypothesis that HBO—PC upregulates the expression of SirT1 activating downstream signal molecule and then inducesautophagN which provides the neuroprotection for cerebral ischemia injury．Inthe present study,we applied SifTl short interfering RNA fsiRNAl in a rat modelof focal cerebral ischemia induced by middle cerebral artery occlusion,andadenovirus--mediaed SirT1 up--regulation or SifTl down-regulation in primarycortical neuron followed by oxygen-glucose deprivation《OGD)injury toinvestigate the mechanism ofneuroprotection induced by HBO—PC．Experiment I:Effect of SirT1 in the ischemic tolerance induced by HBO—PCin rat brain．Objective:To investigate whether SifTl is involved in the neuroprotectionelicitedbyHBO—PC in a ratbrain．Methods:1)To assess the expression ofSifTl in ischemic penumbra in rat brainwith HBO—PC after cerebral focal ischemia．Twenty-four hours after thecompletion of HBO—PC r2．5 atmospheres absolute in 100％oxygen for 60minutes a day for 5 consecutive days),male Sprague—Dawley rats were subjectedto focal cerebral ischemia elicited by middle cerebral artery occlusion《MCAO)with nylon monofilament for 120 minutes and then reperfusion for twenty-fourhours．The expression of SifTl protein was detecd in the ischemic penumbrabefore the onset ofMCAO and 1,3,6,12 and 24 h after reperfusion．Furthermore,the level of SirT1 mRNA and the localization of SirT1 were examined 3 h afterreperfusion．2)To determine the effect of SifTl on the neuroprotection ofHBO—PC against ischemic injury．SifTl inhibitor EX527 and SifTl siRNA wereused to suppress the increase of SifTl by HBO—PC,and SifTl activatorresveratrol was used to activate SirT1．The neurobehavioral score mad infarctvolume wete evaluated 24 h after reperfusion．The intracerebroventricular (i．C．V) injection of EX527《1,10,30 ug)was administered before HBO—PC for 5 days．Resveratrol(50mg·kg-1)was delivered intragastrically once a day for 1 5 days．The level of SirT1 protein was detected 1,3,5,7 d after an i．C．V．injection ofSirT1 siRNA．Results:HBO—PC improved the neurobehavioral score and reduced the infarctvolume 24 h after reperfusion．Compared with MCAO alone,HBO—PC increasedthe expression ofSirT1 protein and mRNA after focal cerebral ischemic injury．The expression SifTl protein was increased beginning at 3 h after reperfusionand remained at least for 24 h．SifTl was primarily expressed in neuronal nucleiin ischemic penumbra．The ischemic tolerance induced by HBO—PC wasa~enuaed by EX527 but was mimicked by pretreatment with resveratr01．siRNAdelivered by a single i．C．V injection significantly down-regulated SkTl at 1,3,5,7 d after injection．Knockdown ofSkTl by siRNA in vivo reverses theneuroprotective effect ofHBO—PC．Conclusion:These results suggest that HBO—PC up—regulates the level ofSkTl,which induces ischemic tolerance against transient focal cerebral ischemia．Experiment II:Effect of SirT1 in ischemic tolerance induced by HBO—PC inrat cortical neurons．Objective:To explore whether SkTl is involved in the neuroprotection elicitedby HBO—PC in a rat cortical neurons．Methods:1)effect of HBO—PC on neuronal injury by oxygen glucosedeprivation(OGD)．Primary cortical neurons were obtained fcom embryo(17～18d1 SD rats．HBO—PC(3．5 amospheres absolute for 60 min,oxygen>90％)wasperformed at 8 days in culture．Twenty-four hours after HBO—PC,neuronssubjectedtoOGDinjuryf95％N2,5％C02)forl20min andthe reoxygenation for 24 h．The cell viability and lactate dehydrogenase《LDH)leakage ratio wereexamined after reoxygenation．2)The expression of SifTl protein was detected24 h after OGD／reoxygenation．3)Effect of SifTl on neuroprection of HBO—PCagainst OGD injury in neurons．SifTl(Ad—SifTl)and SifTl siRNAAd-siRNA-SirT1)were transferred into neurons mediated by adenovirus carryinggreen fluorescent protein to up—regulate and down-regulate SifTl．Thetransfection efficiency and the expression of SifTl protein were evaluated 48 hafter transfection．HBO—PC was conducted 48 h after adenovirus—mediated SirT1transfection．The cell viability and lactate dehydrogenase《LDH)leakage ratiowere examined 24 h after reoxygenation．Results:After 6 days in culture,immunofluorescent staining showed the percentof 13-tubulin positive neurons was more than 95％．HBO—PC enhanced the cellviability and decreased LDH leakage ratio．Compared with OGD injury alone,HBO—PC increased the expression ofSirT1 protein 24 h after reoxygenation．Thetransfection efficiency of Ad—SifTl was 30％,88％,95％,and the transfectionefficiency of Ad-siRNA-SirT1 was 20％,60％,80％at 10,50,100 MOIrespectiveM Ad-SirT1 up—regulated the expression of SifTl protein,whileAd-siRNA-SirT1 down-regulated SifTl 48 h after transfection．In addition,Ad-SirT1 up—regulated SifTl 24 h after reoxygenation and enhanced the cellviability and decreased LDH leakage ratio．In contrast,Ad—siRNA-SirT1suppressed the increase of SifTl induced by HBO—PC and abolished theprotective effect ofHBO—PC on OGD injury in neurons．Conclusion:HBO—PC protects cortical neurons against OGD／reoxygenationinjurythroughup—regulationofSifTl．Experiment III:Effect of autophayg in ischemic tolerance induced by HBo-PC in rat brain．Objective:To nvestigate the role of autophagy in the neuroprotection elicited byHBO—PC in a rat model oftransient focal cerebral ischemia．Methods:1)To assess the activation of autophagy in ischemic penumbra in ratbrain with HBO—PC after cerebral focal ischemia．Male SD rats subjected toHBO—PC and MCAO which were described in experiment I．The expression ofautophagy associated protein LC3一11 was detected in the ischemic penumbrabefore the onset of MCAO and 1,3,6,12 and 24 h after reperfusion．Theexpression of Beclin 1,the formation of autophagosmes and the localization ofLC3 were examined 6 h after reperfusion．2)To determine effect ofautophagy onneuroprotection of HBO—PC against ischemic injury．The i．C．V injection ofautophagy inhibitor 3-methyladenine(3-MA)(30 ug,10 ul)was administeredbefore HBO—PC for 5 days in order to down-regulate autophagy activiation．Asingle i．C．V injection of rapamycin9(30 ng,10UL) was delivered 24 h beforeischemia．The expression of LC3一II and Beclin 1 was detected 6 h afterreperfusion with treatment 3-MA or rapamycin．The neurobehavioral score andinfarct volume wete evaluated 24 h after reperfusion．Results:HBO--PC increased the expression of LC3--II fcom 3 h until at least 24 hafter reperfusion．The elevation of Beclin 1 and formation of autophagosomes byHBO—PC were detected 6 h after reperfusion．LC3 was primarily expressed inneurons in ischemic penumbra．3-MA treatment suppressed the increase ofLC3一II and Beclin 1 induced by HBO—PC．and attenuated neuroprotection ofHBO--PC against cerebral ischemic injury indicating that HBO--PC improvedneurobehavioral score and reduced infarct volume 24 h after reperfusion．Incontrast,rapamycin pretreatment up—regulated LC3一II and Beclin 1,andmimicked the neuroprotective effect ofHBO—PC． Conclusion:HBO--PC up--regulates autophagy,which elicits the neuroprotectiveeffect in cerebral ischemic injury,suggesting a novel mechanism ofHBO—PC——induced tolerance against transient focal cerebral ischemia．...