The Role Of SIRT1 In Repeated Sevoflurane Exposure-induced Cognitive Impairment In Developing Mice And Interventional Study

Part ⅠRepeated sevoflurane exposure induced hippocampal neuroapoptosis by SIRT1-BDNF signaling pathway in developing miceObjective:To detect the effect of repeated sevoflurane exposure on SIRT1-BDNF pathway and neuroapoptosis in the hippocampi of developing mice.Methods:The neonatal mice were assigned randomly to two groups:control group;sevoflurane group.The mice in sevoflurane group received 3%sevoflurane plus 60%oxygen(balanced with nitrogen)2 h daily form P6 to P8 as previous studies described.Mice in the control group were given the control gas(60%oxygen balanced with nitrogen)exposure.One hour after the end of sevoflurane anesthesia on P8,the mice were euthanized and decapitated,their hippocampi were harvested.RT-PCR was used to detect the mRNA expression of SIRT1 and BDNF in two groups.Western Blot was used to measure the protein expression of SIRT1,BDNF,MeCP2,Ac-MeCP2,CREB,p-CREB,Bax,Bcl-2 and cleaved caspase-3.Results:There was no difference in the expression level of SIRT1 mRNA between two groups(P>0.05),but BDNF mRNA expression decreased in sevoflurane group compared with control group(P<0.05).Both SIRT1 and BDNF protein expression in hippocampi were suppressed in sevoflurane group compared to control group(P<0.01,P<0.05,respectively).The ratio of Ac-MeCP2/MeCP2 and the expression of total MeCP2 increased after repeated sevoflurane exposure(P<0.05).There was no significant difference in terms of total CREB expression in hippocampi between the sevoflurane group and control group(P>0.05),while the ratio of p-CREB/CREB declined after repeated sevoflurane exposure(P<0.05)Conclusion:Repeated sevoflurane exposure downregulated SIRT1 expression in the hippocampi of developing mice.SIRT1 deficiency increased MeCP2 acetylation and decreased CREB phosphorylation,ultimately resulting in the downregulation of BDNF in the hippocampi of developing mice.The decreased expression of BDNF induced hippocampal neuroapoptosis revealed by elevating the expression of Bax and cleaved caspase 3 and decreasing the expression of Bcl-2.Part ⅡRepeated sevoflurane exposure induced hippocampal neuroinflammation by SIRT1-NF-κB signaling pathway in developing miceObjective:To detect the effect of repeated sevoflurane exposure on SIRT1-NF-κB pathway and neuroinflammation in the hippocampi of developing miceMethods:The neonatal mice were assigned and received sevoflurane exposure as described in Part I.One hour after the end of sevoflurane anesthesia on P8,the mice were euthanized and decapitated,their hippocampi were harvested.Western Blot was used to measure the protein expression of SIRT1,NF-κB,Ac-NF-κB,nuclear NF-κB,IκBα,p-IκBα,IL-6 and TNF-α.Results:The expression of SIRT1 in hippocampi decreased,but the ratio of Ac-NF-κB/NF-κB and the expression of nuclear NF-κB increased significantly(P<0.01)The expression of total NF-κB and the ratio of p-IκBα/IκBα increased,while the expression of IκBα decreased.The inflammatory response was activated and revealed by the increased expression of IL-6 and TNF-α in sevoflurane group(P<0.05)Conclusion:Repeated sevoflurane exposure downregulated SIRT1 expression in the hippocampi of developing mice.SIRT1 deficiency increased NF-κB acetylation and further facilitated NF-κB transportation to nucleus,consequently resulting in the upregulation of IL-6 and TNF-α in the hippocampi of developing mice.The increased expression of IL-6 and TNF-α activated hippocampal neuroinflammationPart ⅢResveratrol pretreatment mitigated sevoflurane-induced cognitive impairment in developing MiceExperiment 1Objective:To investigate the minimum effective concentration of resveratrol on improving the expression of SIRT1 deficiency after repeated sevoflurane exposureMethods:The neonatal mice were assigned randomly to five groups:control group,sevoflurane group,50 mg/kg resveratrol+sevoflurane group,100 mg/kg resveratrol+sevoflurane group,150 mg/kg resveratrol+sevoflurane group.The mice in each group respectively received one intraperitoneal injection of 3%DMSO,3%DMSO,50 mg/kg resveratrol,100 mg/kg resveratrol,150 mg/kg resveratrol from P3 to P5 and 1 h prior to the exposure to sevoflurane or control gas from P6 to P8.The mice in sevoflurane group and resveratrol+sevoflurane group received 3%sevoflurane plus 60%oxygen(balanced with nitrogen)2 h daily form P6 to P8.Mice in the control group were subject to the control gas(60%oxygen balanced with nitrogen)exposures.One hour after the end of sevoflurane anesthesia on P8,the hippocampi of mice were harvested.Western Blot was used to measure the protein expression of SIRT1 in five groupsResults:When compared with control group,the expression of SIRT1 in hippocampi decreased significantly in sevoflurane group(P<0.01);when compared with sevoflurane group,the expression of SIRT1 was not elevated in 50 mg/kg resveratrol+sevoflurane group(P>0.05),but was elevated in 100 mg/kg resveratrol+sevoflurane group and 150 mg/kg resveratrol+sevoflurane group(P<0.01,P<0.01,respectively),especially in the latter group.Conclusion:100 mg/kg is the minimum effective concentration among these concentrations to mitigate the deficiency of SIRT1 after repeated sevoflurane exposure.Thus,we chose 100 mg/kg resveratrol as the dose for the intervention experimentExperiment 2Objective:To detect the effect of resveratrol on sevoflurane-induced changes of SIRT1-BDNF pathway and neuroapoptosisMethods:The neonatal mice were assigned randomly to four groups:control group,control+resveratrol group,sevoflurane group,sevoflurane+resveratrol group.The mice in each group respectively received one intraperitoneal injection of 3%DMSO,100 mg/kg resveratrol,3%DMSO,100 mg/kg resveratrol from P3 to P5 and 1 h prior to the exposure to sevoflurane or control gas from P6 to P8.The mice in sevoflurane group and resveratrol+sevoflurane group received 3%sevoflurane plus 60%oxygen(balanced with nitrogen)2 h daily form P6 to P8.One hour after the end of sevoflurane anesthesia on P8,the mice were euthanized and decapitated,their hippocampi were harvested.RT-PCR was used to detect the mRNA level of SIRT1 and BDNF in four groups.Western Blot was used to measure the protein expression of SIRT1,BDNF,MeCP2,Ac-MeCP2,CREB,p-CREB,Bax,Bcl-2 and cleaved caspase-3 in four groupsResults:There was no difference in the expression level of SIRT1 mRNA in hippocampi between four groups(P>0.05),but BDNF mRNA expression decreased significantly in sevoflurane group compared with control group(P<0.01),and increased in sevoflurane+resveratrol group compared with sevoflurane group(P<0.05).Both SIRT1 and BDNF protein expression in hippocampi were suppressed in sevoflurane group compared to control group(P<0.05,P<0.01,respectively),and were elevated in sevoflurane+resveratrol group compared with sevoflurane group(P<0.05,P<0.01,respectively).The result of CO-IP indicated that SIRT1 could interact with AcMeCP2 and CREB.The ratio of Ac-MeCP2/MeCP2 increased and the ratio of p-CREB/CREB decreased(P<0.05),the expression of total MeCP2 increased in sevoflurane group compared with control group(P<0.05).The ratio of Ac-MeCP2/MeCP2 decreased and the ratio of p-CREB/CREB increased(P<0.05),the expression of total MeCP2 increased in sevoflurane+resveratrol group compared with sevoflurane group(P<0.05).There was no significant difference in terms of total CREB expression in hippocampi among four groups(P>0.05)Conclusion:Pretreatment with resveratrol restored the expression of SIRT1 and thus decreased MeCP2 acetylation and increased CREB phosphorylation which contributed to upregulated the expression of BDNF.Consequently,resveratrol mitigated neuroapoptosis in the hippocampi of developing miceExperiment 3Objective:To detect the effect of resveratrol on sevoflurane-induced changes of SIRT1-NF-κB pathway and neuroinflammationMethods:The neonatal mice were assigned and received resveratrol pretreatment and sevoflurane exposure as described in Experiment 2.One hour after the end of sevoflurane anesthesia on P8,the hippocampi of mice were harvested.Western Blot was used to measure the protein expression of SIRT1,NF-κB,Ac-NF-κB,nuclear NF-κB,IL-6 and TNF-α in four groupsResults:The expression of SIRT1 in hippocampi decreased significantly,the ratio of Ac-NF-κB/NF-κB and the expression of nuclear NF-κB increased significantly(P<0.01),the expression of total NF-κB increased(P<0.05),the ratio of p-IκBα/IκBα increased significantly(P<0.01),the expression of IκBα decreased(P<0.05),the expression of IL-6 and TNF-α increased(P<0.01,P<0.05,respectively)in sevoflurane group,compared with control group.The expression of SIRT1 in hippocampi increased(P<0.05),the ratio of Ac-NF-κB/NF-κB decreased(P<0.05),and the expression of nuclear NF-κB decreased significantly(P<0.01),the expression of total NF-κB decreased(P<0.05),the ratio of p-IκBα/IκBα decreased significantly(P<0.01),the expression of IκBα increased,the expression of IL-6 and TNF-α increased(P<0.05)in sevoflurane+resveratrol group compared with sevoflurane groupConclusion:Pretreatment with resveratrol decreased NF-κB acetylation and its transport to the nucleus,leading to the downregulated expression of IL-6 and TNF-α,and consequently mitigated neuroinflammation in the hippocampi of developing mice Experiment 4Objective:To investigate the role of resveratrol on sevoflurane-induced cognitive impairment.Methods:The neonatal mice were assigned and received resveratrol pretreatment and sevoflurane exposure as described in Experiment 2.The open field test was used to measure the motor function of young mice on P30;the MWM test was used to detect the spatial memory of the different groups of mice from P31 to P35Results:Open field test showed that the total distance traveled and average speed did not differ among the four groups of mice(P>0.05).MWM test indicated that compared with the mice in control group,the mice in sevoflurane group required longer escape latency and escape path length to locate the position of platform on the fifth day of training trial,they spent less time in the target quadrant and crossed the platform fewer times in probe trial test(P<0.05);compared with sevoflurane group,escape latency and escape path length on the fifth day of training trial decreased,time in the target quadrant and the platform crossing times increased in probe trial test in resveratrol+sevoflurane group(P<0.05)Conclusion:Resveratrol pretreatment ameliorated cognitive impairment induced by repeated sevoflurane exposure.