国际麻醉学与复苏杂志   2015, Issue (9): 3-3
    
七氟醚预处理对缺氧/复氧损伤心肌细胞中自噬的影响
王露, 牛力, 许鹏程1()
1.徐州医学院
Effects of sevoflurane preconditioning on autophagy during hypoxia/reoxygenation injury in cardiomyocyte
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摘要：

目的　探讨七氟醚预处理对大鼠心肌细胞缺氧/复氧（hypoxia/reoxygenation， H/R）损伤中自噬变化的影响及机制。　方法　大鼠H9C2心肌细胞采用随机数字表法分为5组（免疫印迹每组4瓶细胞，共20瓶；细胞存活率检测时细胞培养在96孔板中，每组8孔，共40孔）：空白对照组（Con组）、H/R组（缺氧4 h、复氧2 h）、七氟醚预处理组（Sev+H/R组，给予2.5％七氟醚预处理30 min后行H/R）、叔丁基过氧化氢（tert?蛳butylhydroperoxide， TBHP）组（TBHP+H/R组，培养基内加入浓度为25 mmol/L TBHP后行H/R）和七氟醚预处理+叔丁基过氧化氢组（Sev+TBHP+H/R组，2.5％七氟醚预处理后于25 mmol/L TBHP的培养基行H/R）。复氧结束后取心肌细胞，四氮唑蓝（thiazolyl blue tetrazolium bromide， MTT）检测细胞存活率，二氯荧光素法（dichlorofluorescein diacetate， DCFH-DA）检测细胞内活性氧（reactive oxygen species， ROS）含量，免疫印迹法检测Ⅱ型微管相关蛋白1轻链3（microtubule-associated protein 1 light chain 3， LC3-Ⅱ）和Beclin l蛋白的表达。　结果　与Con组（100%）比较，H/R组和Sev+H/R组心肌细胞存活率［（52±14）%，（76±20）%］均降低（P<0.05），ROS的产生量均增加；其余4组Beclin1和LC3?蛳Ⅱ的表达均升高（P<0.05）。与H/R组比较，Sev+H/R组心肌细胞存活率增加（P<0.05），ROS的产生量降低，LC3-Ⅱ和Beclin1的表达均降低（P<0.05）。与Sev+H/R组［（0.75±0.10），（0.65±0.08）］比较，Sev+TBHP+H/R组LC3-Ⅱ和Beclin1的表达［（1.02±0.08），（0.85±0.04）］均升高（P<0.05）。　结论　七氟醚预处理通过降低H/R期间ROS的产生量来降低大鼠心肌细胞H/R损伤中自噬的水平，对大鼠心肌细胞H/R损伤产生保护作用。
关键词： 七氟醚；　预处理；　心肌细胞；　缺氧/复氧损伤；　活性氧；　自噬
Abstract:

Ｏｂｊｅｃｔｉｖｅ　To study the effects of sevoflurane preconditioning on autophagy during hypoxia/reoxygenation（H/R）in rat's cardiomyocytes and its possible mechanism.　Methods　The embryonic cardiomyocytes of rats（H9C2 cells） were randomly divided into 5 group（Western blot 4 bottles of cells in each group， total 20 bottles. Cells were cultured in 96 well plates to detect the cell survival rate， 8 wells in each group， total 40 wells）： control group（Con group）， H/R group （hypoxia for four hours and followed by two hours of reoxygenation）， sevoflurane preconditioning group（Sev+H/R group， preconditioned with 2.5% sevoflurane for 30 min and followed by establishing H/R）， tert-butylhydroperoxide（TBHP） plus H/R group（TBHP+H/R group， H/R with 25 mmol/L TBHP in the medium）， and sevoflurane preconditioning plus TBHP group（Sev+TBHP+H/R group， preconditioned with 2.5% sevoflurane for 30 min and followed by H/R with 25 mmol/L TBHP in the medium）. At the end of reoxygenation， cell viability was measured by the thiazolyl blue tetrazolium bromide（MTT） method. The production of reactive oxygen species（ROS） was detected by dichlorofluorescein diacetate（DCFH?蛳DA） method. The expression of autophagy marker microtubule-associated protein 1 light chain 3（LC3-Ⅱ） and Beclin1 were determined by Western blot.　Results　Compared with Con group（100%）， the viabilities of cells in H/R group（52±14）% and Sev+H/R group（76±20）% both deceased（P<0.05）， and the productions of ROS were enhanced in H/R group and Sev+H/R group. And when the other four groups compared with Con group， the expressions of LC3-Ⅱ and Beclin1 were enhanced（P<0.05）. Compared with H/R group， the viability of cells in Sev+H/R group significantly enhanced（P<0.05）， the production of ROS was decreased and the expression of LC3-Ⅱ and Beclin1 was down-regulated（P<0.05）. Compared with Sev+H/R group［（0.75±0.10），（0.65±0.08）］， the viability of cells significantly decreased and the expression of LC3-Ⅱ（1.02±0.08） and Beclin1（0.85±0.04） was enhanced in Sev+TBHP+H/R group（P<0.05）.　Conclusions　Sevoflurane preconditioning can decrease autophagy during H/R in the rat's cardiomyocytes，and the possible mechanism is through decreasing ROS and Beclin1，thus protecting rat's cardiomyocytes against H/R injury.
Key words: Sevoflurane；　Preconditioning；　Cardiomyocyte；　Hypoxia/reoxygenation injury；　Reactive oxygen species；　Autophagy