雷帕霉素减弱七氟烷预处理对心肌缺血／再灌损伤的保护作用

Rapamycin decreases the protective effective of sevoflurane preconditioning on myocardial ischemia/reperfusion injury

目的观察自噬增强剂雷帕霉素（rapamycin，RAPA）对七氟烷预处理离体大鼠心脏缺血／再灌注损伤（ischemia/reperfusion injury，I／RI）保护作用的影响，探讨自噬在七氟烷预处理对心肌I／RI保护中的作用。方法36只健康成年雄性Wistar大鼠，体重250g～280g，采用随机数字表法分为3组（每组12只）：缺血，再灌注（ischemia/reperfusion，I／R）组，七氟烷预处理组（S＋UR组），七氟烷预处理＋RAPA组（S＋I/R＋RAPA组）。采用Langendorff灌注装置制备离体心脏I／R模型，缺血30min、再灌注120min，记录平衡末及再灌注末的左室舒张末期压（left ventricular diastolic pressure，LVEDP）、左室发展压（left ventricular developed pressure，LVDP）、左心室内压最大上升/下降速率（the maximum rate of increase or decrease of left ventricular pressure，＋dp／dtmax）、心率（heartrate，HR）.再灌注120min后，收集冠状动脉流出液测乳酸脱氢酶（lactate dehydrogenase，LDH）活性；取心肌，计算心肌梗死面积百分比。Western blot半定量检测自噬标记物微管相关蛋白1轻链3（microtubule-associated protein1light3，LC3）、RAPA靶蛋白（target of rapamycin，mTOR）及磷酸化mTOR（phosphorylated mTOR，p-mTOR）蛋白表达量。结果再灌注末，与I／R组比较，S＋I／R组心功能明显改善（P〈0．05），心肌梗死面积[（47±6）％、（29±5）％]、冠状动脉流出液LDH活性[（29±5）U／L、（19±4）U／L]均降低，心肌LC3-Ⅱ表达下调，p-mTOR表达上调（P〈0．05）；S＋I／R＋RAPA组各指标与I／R组比较，差异无统计学意义。与S＋FR组比较，S＋FR＋RAPA组心功能各项指标呈损害眭变化（P〈0．05）、梗死面积增大[（29±5）％、（46±3）％]、冠状动脉流出液LDH活性[（19±4）U／L、（27±5）U／LI均增加（P〈0．05），LC3-Ⅱ表达水平增加，p-mTOR表达降低。结论RAPA能够减弱七氟烷预处理对I／RI产生的保护作用，可能与其降低p-mTOR表达，减弱对自噬的抑制作用有关。

Objective To study the effects of rapamycin （RAPA） on sevoflurane preconditioning during myocardial ischemia/ reperfusion（I/R） in rats. Methods Thirty-six healthy adult male Wistar rats, 250 g-280 g, were randomly divided into three groups （n=12）： group I/R, group sevoflurane preconditioning（group S＋I/R）, group sevoflurane preconditioning plus RAPA（group S＋I/R＋RAPA）. The hearts were excised and perfused in a langendorff apparatus. The left ventricular diastolic pressure （LVEDP）, left ventricular developed pressure （LVDP）, the maximum rate of increase or decrease of left ventricular pressure （＋dp/dtmax）, heart rate （HR） were recorded at 30 min of equilibrium and 120 min of reperfusion respectively. Myocardial infarct size and lactate dehydrogenase（LDH） levels were examined by 2,3,5-triphenyltetrazolium chloride （Trc） staining and enzyme standard method at 120 min of reperfusion. Expressions of microtubule-associated protein 1 light 3 （LC3）, target of rapamycin （mTOR）, phosphorylated mTOR （p-mTOR） were determined by Western blotting. Results After reperfusion, compared with that in group I/R, the cardiac function in group S＋I/R was improved （P〈0.05）, the myocardial infarct size [（47±6）%, （29±5）%], LDH levels [（29±5） U/L, （19±4） U/L] and the expressions of LC3-Ⅱ were decreased（P〈0.05 ）, while the expression of p-roTOR increased in group S＋I/R（P〈0.05 ）. Compared with that in group S＋I/R, the cardiac function in group S＋I/R＋RAPA was worse（P〈0.05 ）, the myocardial infarct size[ （29±5）%, （46±3）% ], LDH levels[ （19±4） U/L, （27±5） U/L] and the expressions of LC3-Ⅱ were increased（P〈0.05 ）, while the expression of p-roTOR decreased （P〈0.05） in group S＋I/R＋RAPA （P〈0.05）. Conclusions Rapamycin could decrease the protective effective of sevoflurane preconditioning and may be related to p-mTOR and autophagy.