钙调节蛋白在七氟烷后处理心脏保护中的机制探讨

Mechanism of calcium regulatory proteins in cardioprotection after sevoflurane postconditioning

目的探讨钙调节蛋白在七氟烷后处理(SevoPoC)心脏保护中的机制。方法选取30只SD大鼠建立离体心脏模型,血流动力学稳定30 min后,缺血30 min复灌60 min建立缺血再灌注损伤模型,完全随机分入以下3组,每组10只:①时间对照组(TC组):将离体心脏在恒流下灌注充分氧合的KH液120 min;②缺血再灌注组(I/RI组):将离体心脏在恒流下灌注充分氧合的KH液30 min后,停止灌注30 min,随后恢复灌注60 min;③七氟烷后处理组(SevoPoC组):将离体心脏在恒流下灌注充分氧合的KH液30 min后,停止灌注30 min,在复灌即刻使用含3%的七氟烷持续灌注10 min,然后使用氧合的KH液灌注50 min。各实验组均持续监测左心室舒张末期压力(LVEDP)、心率、左心室发展压(LVDP)、最大LVDP上升速率(+dp/dt)及最大LVDP下降速率。检测心肌梗死面积和冠状动脉流出液中心肌肌钙蛋白I(cTnl)水平。分别用蛋白印迹法和荧光实时定量聚合酶链反应(RT-PCR)方法检测L型钙通道(LTCCs)、兰尼碱受体2(RyR2)以及钠钙交换体1(NCXI)的蛋白和基因转录水平。结果 SevoPoC能明显加快LVEDP、RPP(心率与LVDP的乘积)和+dp/dt向基线水平恢复,SevoPoC组LVEDP水平低于,RPP及+dp/dt高于I/RI组(均P<0.05)。同时,SevoPoC可以降低心肌缺血再灌注损伤后cTnI的释放,SevoPoC组cTnI水平低于I/RI组[(0.060±0.003)mg/L比(0.110±0.004)mg/L](P<0.01)。NCX1相对表达水平I/RI组高于TC组[(0.59±0.09)比(0.35±0.05)],SevoPoC组(0.32±0.06)低于I/RI组(均P<0.01)。LTCCs和RyR2的表达各组间差异无统计学意义(P>0.05)。结论 SevoPoC可以提供心肌保护作用,表现在心功能恢复的增强、冠状动脉流出液cTnI释放以及心肌梗死面积的减少。其作用可能与其对NCXl的抑制有关。

Objective To discuss the mechanism of calcium regulatory proteins in cardiac protection after sevoflurane postconditioning （SevoPoC）. Methods Isolated hearts were separated from 30 SD rats to make ischemia-reperfusion injury（I/RI） models and they were randomly divided into 3 groups（ n = 10） ： （1）Time control （TC） group had continuous perfusion of oxygenated KH solution for 120 rain; （2）I/RI group had continuous perfusion of oxygenated KH solution for 30 min, stopped 30 min, and then reperfused for 60 min; （3）SevoPoC group had continuous perfusion of oxygenated KH solution for 30 min, stopped 30 min, and then had 3% sevoflurane perfusion for 10 rain and KH solution perfusion for 50 min. Left ventricular end-diastolic pressure（ LVEDP）, heart rate（HR）, left ventricular developed pressure （LVDP） , the maximum LVDP increase rate （ ＋ dp/dt） and the maximum LVDP decrease rate were observed. Myocardial infarct size was measured. Cardiac troponin （cTnI） in coronary outflow fluid was tested. Expressions of L-type Ca2 ＋ channels （ LTCCs）, ryanodine receptor 2 （ RyR2 ） and Na＋ -Ca2＋ exchanger isoform 1 （NCX1） were determined. Results LVEDP in SevoPoC group was significantly lower, RPP（ HR x LVDP） and ＋ dp/dt were significantly higher than those in I/RI group（P 〈0. 05 ）. Level of cTnI in SevoPoC group was significantly lower than that in I/RI group[ （0. 060 ±0. 003）mg/L vs （0. 110 ±0. 004） mg/L] （ P 〈 0. 01 ）. NCX1 expression level in I/RI group was significantly higher than that in TC group [ （0. 59 ± 0. 09） vs （0. 35 ±0. 05） ] ; NCX1 expression level in SevoPoC group（0. 32 ±0. 06） was significantly lower than that in I/RI group（ P 〈 0. 01 ）. Expressions of LTCCs and RyR2 showed no significant differences among groups （P 〉 0. 05 ）. Conclusion SevoPoC can promote cardiac function recovery, increase cTnI release in coronary outflow fluid and reduce infarction size ; the cardioprotection effect may be associated with NCX1 deactivation.