七氟醚后处理对氧糖剥夺/复氧复糖诱导HT22细胞DNA损伤及SIRT1表达的影响

Effects of sevoflurane postconditioning on DNA damage and SIRT1 expression of HT22 under oxygen and glucose deprivation/reoxygenation

目的探讨七氟醚后处理对HT22细胞氧糖剥夺/复氧复糖后DNA损伤及沉默信息调节因子1(SIRT1)表达的影响。方法随机将对数生长期的HT22细胞分为7组:空白对照组(CON),氧糖剥夺/复氧复糖4 h组(OGD/R 4 h),氧糖剥夺/复氧复糖4 h+1%七氟醚后处理组(OGD/R 4 h+1%SEVO),氧糖剥夺/复氧复糖4 h+2%七氟醚后处理组(OGD/R 4 h+2%SEVO),氧糖剥夺/复氧复糖6 h组(OGD/R 6 h),氧糖剥夺/复氧复糖6 h+1%七氟醚后处理组(OGD/R 6 h+1%SEVO),氧糖剥夺/复氧复糖6 h+2%七氟醚后处理组(OGD/R 6 h+2%SEVO);实验后序部分只取前四组研究。MTT法检测细胞存活率,PI/Hoechst荧光双染检测细胞坏死及凋亡,TUNEL染色检测神经元凋亡,免疫荧光染色测定Cleaved-casepase3表达,免疫荧光染色检测8-OHdG以测定DNA损伤程度,Western blot测定SIRT1、Bcl-2及BAX的蛋白表达。结果前半部分实验结果中,与CON组比较,OGD/R 4 h及OGD/R 6 h组细胞存活率均降低(均P<0.01),凋亡细胞及坏死细胞占比升高;与对应OGD/R 4 h组比较,1%和2%七氟醚后处理的细胞存活率升高(均P<0.01),凋亡及坏死细胞数量占比降低;与对应OGD/R 6 h组比较,1%和2%七氟醚后处理的细胞存活率升高(均P<0.01),凋亡及坏死细胞占比降低。实验后半部分,与CON组比较,OGD/R 4 h组中TUNEL染色阳性细胞数增多,Cleaved-casepase3表达增加(P<0.01),8-OHdG含量增多(P<0.01),BAX蛋白表达上调(P<0.01),SIRT1、Bcl-2蛋白表达下调(均P<0.01);与OGD/R 4 h组比较,1%和2%七氟醚后处理的细胞TUNEL染色阳性细胞数减少,Cleaved-casepase3表达降低(均P<0.01),8-OHdG含量降低(均P<0.01),BAX蛋白表达下调(P<0.05,P<0.01),而SIRT1、Bcl-2蛋白表达上调(均P<0.01;P<0.05,P<0.01)。结论七氟醚后处理改善氧糖剥夺/复氧复糖诱导的HT22海马神经元DNA氧化损伤及凋亡,并上调SIRT1蛋白表达。

Objective To investigate the effect of sevoflurane postconditioning on DNA damage and the expression of silent information regulation(SIRT1) of HT22 cell line following oxygen and glucose deprivation/reoxygenation injury. Methods Firstly, HT22 cells in logarithmic phase were randomly divided into normal control group(CON), oxygen and glucose deprivation/reoxygenation 4 h group(OGD/R 4 h), oxygen and glucose deprivation/reoxygenation 4 h+1% sevoflurane postconditioning group(OGD/R 4 h+1%SEVO), oxygen and glucose deprivation/reoxygenation 4 h+2% sevoflurane postconditioning group(OGD/R 4 h+2%SEVO), oxygen and glucose deprivation/reoxygenation 6 h group(OGD/R 6 h), oxygen and glucose deprivation/reoxygenation 6 h+1% sevoflurane postconditioning group(OGD/R 6 h+1%SEVO), oxygen and glucose deprivation/reoxygenation 6 h+2% sevoflurane postconditioning group(OGD/R 6 h+2%SEVO). Secondly, the former four groups in the first part of this study were further investigated the mechanism of sevoflurane postconditiong in oxygen and glucose deprivation/reoxygenation injury. MTT assay was used to detect cell survival rate, PI/Hoechst fluorescence staining and TUNEL assay were used to detect apoptotic and necrosis rate, immunofluorescence staining was used to detect the expression of 8-OHdG and Cleaved-casepase 3, Western blot analysis was used to detect the expression of SIRT1 and apoptotic protein expression. Results In the first part of the experiment, compared with CON group, the cell survival rates in both OGD/R 4 h and OGD/R 6 h groups reduced(P<0.01, P<0.01), and the number of apoptosis and necrotic cells also increased. Compared with the related OGD/R 4 h group, the cell survival rate in the 1% SEVO and 2% SEVO groups both increased( P < 0. 01,P < 0. 01),and the number of apoptotic cells and necrotic cells also decreased. Moreover,compared with the related OGD/R 6 h group,the cell survival rate in the1% SEVO and 2% SEVO groups increased( P < 0. 01,P < 0. 01),and the number of apoptotic cells and necrotic cells decreased. The second part of the experiment,compared with CON group,there were more TUNEL positive labeled cells,and increased expressions of Cleaved-casepase3( P < 0. 01) and 8-OHdG( P < 0. 01),as well as BAX( P < 0. 01) in the OGD/R 4 h group,however,SIRT1 and Bcl-2 expression reduced( P < 0. 01,P < 0. 01).Compared with the OGD/R 4 h group,there were less TUNEL positive labeled cells,and decreased expressions of Cleaved-casepase3( P < 0. 01,P < 0. 01) and 8-OHdG( P < 0. 01,P < 0. 01),as well as BAX( P < 0. 05,P <0. 01) in the 1% SEVO and 2% SEVO groups,SIRT1 and Bcl-2 expression increased( P < 0. 01,P < 0. 01;P <0. 05,P < 0. 01). Conclusion Sevoflurane postconditioning can alleviate DNA oxidative damage and apoptosis of HT22 hippocampal neurons induced by oxygen and glucose deprivation/reoxygenation,and up-regulate SIRT1 protein expression.