七氟烷预处理对大鼠离体心肌缺血／再灌注损伤线粒体细胞色素C释放的影响

The effect of sevoflurane preconditioning on cytochrome C release from mitochondria during ischemia/reperfusion in isolated rat hearts

目的探讨七氟烷预处理对大鼠离体心肌缺血／再灌注损伤（ischemidreperfusioninjury，I／RI）时心肌细胞凋亡及线粒体细胞色素C释放的影响。方法40只SD大鼠应用随机数字表法随机分为4组（每组10只）：对照组（C组），0．75％、1．5％、3．0％七氟烷预处理组（s1、s2、s3组）。应用Langendofff离体心脏灌注模型，经主动脉用Krebs-Henseleit（K-H）液逆行灌注。各组均缺血30min，再灌注60rain。S1、s2、S3组在缺血前分别以含相应浓度七氟烷的K-H液灌注10rain，再冲洗5min，重复2次。记录平衡灌注末、缺血前即刻、再灌注30、60rain时的心功能指标。再灌注60min时用DNA原位末端缺口标记技术（terminaldeoxynucleotidyltransferase-mediateddUTPnickendlabelling，TUNEL）测定凋亡细胞，提取心肌线粒体，免疫印迹法测定线粒体和胞浆的细胞色素c水平。结果与c组比较，S1、S2、s3组再灌注30、60rain时左室舒张末压（1eftventricularend—diastolicpressure，LVEDP）降低、左室发展压（1eftventriculardevelopedpressure，LVDP）升高（P〈0．05）；与c组比较，s1、s2、s3组凋亡率明显下降（P〈0．05或0．01），再灌注末心肌细胞凋亡率c、s1、s2、s3组分别为（33．10±2．57）％、（28．03±3．11）％、（25．20±3．04）％、（24．06±2．58）％；与c组比较，s1、s2、s3组心肌线粒体细胞色素c释放减少，胞浆细胞色素c的量明显降低（P〈0．05或0．01）。结论七氟烷预处理能够通过抑制心肌线粒体细胞色素c释放到胞浆，降低心肌细胞凋亡率，减轻心肌I／RI。

Objective To explore the effect of sevoflurane preconditioning on cardiocyte apoptosis and cytochrome C release from mitochondria during ischemia/reperfusion in isolated rat hearts. Methods Forty male Sprague-Dawley rats weighing 250 g-300 g were randomly divided into 4 groups （n=10 for each group）： control group （C）; 3 different concentrations of sevoflurane preconditioning groups of 0.75% （S 1 ）, 1.5% （ S2 ）, 3.0% （ S3 ） sevoflurane respectively. All of the isolated rat hearts were retrogradely perfused via aorta with Krebs-Henseleit （K-H） solution on Langendorff apparatus. The isolated hearts were subjected to ischemia for 30 min followed by 60 min reperfusion. Hearts in the SI ,S2,S3 groups were preconditioned by perfusing with K-H solution which containing 0.75%, 1.5%, 3.0% sevoflurane respectively for 10 min and then followed by 5 rain K-H solution washout before ischemia. The preconditioning procedure was repeated twice. Hemodynamics of the hearts were recorded after equilibration （baseline values）, immediately before ischemia, 30 and 60 min after reperfusion respectively. Apoptotic myocardial cells were detected by terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling （TUNEL） and the level of cytochrome C expression in myocardial cytosol and mitochondria was measured by Western Blot at the end of reperfusion. Results At the end of 30 and 60 min reperfusion, left ventricular end-diastolic pressure （LVEDP） was significantly lower and left ventricular developed pressure （LVDP） was significantly higher in S1, S2, S3 groups than those in C group （P〈0.05）. Apoptotic index decreased significantly in S1,S2, S3 groups compared with C group at the end of reperfusion （P〈0.05, P〈0.01） and the apoptotic index of C,S1,S2,S3 groups was （33.10±2.57）%, （28.03±3.11）%, （25.20±3.04）%, （24.06±2.58）% respectively. Cytochrome C level increased significantly in mitochondria but decreased significantly in cytosol in S1,S2, S3 groups as compared with C group （P〈0.05, P〈0.01 ）. Conclusions Sevoflurane preconditioning decreased cardioeyte apoptosis, protected the heart against ischemia/reperfusion injury by attenuatiing the release of cvtochrome C from mitochondria to cvtosol.