右美托咪定通过PI3K/Akt通路减弱大鼠离体心脏缺血/再灌注损伤中LC3表达

Dexmedetomidine decreases the expression of LC3 in isolated rat heart during ischemia-reperfusion injury through the PI3K/Akt pathway

目的观察右美托咪定(dexmedetomidine,Dex)对大鼠离体心脏缺血/再灌注损伤中微管相关蛋白1轻链3(microtubule-associated protein 1 light 3,LC3)及磷脂酰肌醇3-激酶/蛋白激酶B(phosphatidy-linositol 3-kinase/protein kinase B,PI3K/Akt)信号通路的影响,探讨Dex对心肌的保护作用。方法健康成年雄性SD大鼠48只,8~10周龄,体重250~300 g,采用Langendorff灌注装置制备离体心脏缺血/再灌注模型。取模型制备成功的心脏48个,采用随机数字表法将其分为4组(n=12):空白对照组(NC组),K-H液持续灌注120 min;缺血/再灌注组(I/R组),K-H液灌注30 min,全心缺血30 min,再灌注60 min;Dex预处理组(Dex+I/R组),K-H液灌注10 min后再用含有25μg/L Dex的K-H液灌注15 min,K-H液洗脱5 min,全心缺血30 min,再灌注60 min;Dex预处理+渥曼青霉素组(Dex+I/R+W组),开胸前30 min大鼠腹腔注射渥曼青霉素15μg/kg,其余处理同Dex+I/R组。于平衡末(K-H液灌注30 min时)及再灌注末记录心率、左心室内压力上升/下降速率最大值(the maximum rate of increase or decrease of left ventricular pressure,±dp/dtmax)、左心室发展压(left ventricular development pressure,LVDP)、左心室舒张末期压(left ventricular diastolic pressure,LVEDP)。于再灌注末收集冠状动脉流出液积,酶标仪法检测乳酸脱氢酶(lactate dehydrogenase,LDH)活性;取心肌组织,2,3,5-氯化三苯基四氮唑(2,3,5-triphenyltetrazoliumchlofide,TTC)染色法检测心肌梗死面积,计算心肌梗死面积百分比;采用Western blot法检测自噬标记物LC3以及Akt、磷酸化蛋白激酶B(phosphorylated Akt,p-Akt)、哺乳动物雷帕霉素靶蛋白(mammalian target of rapamycin,mTOR)、磷酸化哺乳动物雷帕霉素靶蛋白(phosphorylated mTOR,p-mTOR)表达量。结果与NC组比较,其余各组HR、±dp/dtmax、LVDP降低,LVEDP、心肌梗死面积、冠状动脉流出液LDH活性升高,心肌LC3-Ⅱ表达上调,p-Akt及p-mTOR表达下调(P均<0.05)。与I/R组比较,Dex+I/R组心率、±dp/dtmax、LVDP增加,LVEDP、心肌梗死面积、冠状动脉流出液LDH活性降低,心肌LC3-Ⅱ表达下调,p-Akt及p-mTOR表达上调(P均<0.05)。与Dex+I/R组比较,Dex+I/R+W组心率、±dp/dtmax、LVDP降低,LVEDP、心肌梗死面积、冠状动脉流出液LDH活性升高,心肌LC3-Ⅱ表达上调,p-Akt及p-mTOR表达下调(P均<0.05)。结论Dex预处理对离体大鼠心脏缺血/再灌注损伤具有保护作用,且该作用可能与PI3K/Akt信号通路激活,引起下游mTOR活性增强从而降低缺血/再灌注期间心肌细胞的自噬水平有关。

Objective To observe the effects of dexmedetomidine(Dex)on microtubule-associated protein 1 light 3(LC3)and phosphatidy-linositol 3-kinase/protein kinase B(PI3K/Akt)signal pathway in isolated rat heart during ischemia-reperfusion injury,and to explore the protective effects of Dex on myocardium.Methods A total of 48 healthy adult male SD rats(8−10 weeks old and weighing 250−300 g)were used to establish an isolated heart model of ischemia-reperfusion with Langendorff perfusion device.After successful modeling,a 48 rat hearts were divided into 4 groups(n=12),according to the random number table method:a blank control group(group NC,which was continuously perfused with K-H solution over 120 min),an ischemia-reperfusion group(group I/R,which was perfused with K-H solution over 30 min followed by global ischemia for 30 min,and reperfusion over 60 min),a Dex preconditioning group(group Dex+I/R,which was perfused with K-H solution over 10 min followed by infusion of K-H solution containing 25μg/L Dex for 15 min,elution of K-H solution for 5 min,ischemia for 30 min,and reperfusion for 60 min),a Dex preconditioning+Warman group(group Dex+I/R+W,which was intraperitoneally injected with 15μg/kg of Warman 30 min before thoracotomy,along with the similar treatments with group Dex+I/R).Their heart rate(HR),left ventricular diastolic pressure(LVEDP),left ventricular developed pressure(LVDP),the maximum rate of increase or decrease of left ventricular pressure(±dp/dtmax)were recorded at the end of equilibrium(after infusion over 30 min)and reperfusion.The coronary perfusion fluid was collected at the end of reperfusion and the level of lactate dehydrogenase(LDH)was examined by a microplate reader.The myocardiac tissues were taken and myocardial infarct size was measured by 2,3,5-triphenyltetrazolium chloride(TTC)staining,and the percentage of myocardial infarct size was calculated.The expression of autophagosomal marker LC3,Akt,phosphorylated Akt(p-Akt),mammalian target of rapamycin(mTOR),and phosphorylated mTOR(p-mTOR)was determined by Western blot.Results Compared with group NC,remarkable decreases in HR,±dp/dtmax,and LVDP and the levels of p-Akt and p-mTOR as well as increases in LVEDP,myocardial infarction size,and the activity of LDH in coronary perfusion fluid,and the level of myocardial LC3 were found in other groups(P<0.05).Compared with group I/R,remarkable increases in HR,±dp/dtmax,and LVDP and the levels of p-Akt and p-mTOR as well as decreases in LVEDP,myocardial infarction size,and the activity of LDH in coronary perfusion fluid,and the level of myocardial LC3 were found in group Dex+I/R(P<0.05).Compared with group Dex+I/R,remarkable decreases in HR,±dp/dtmax and LVDP and the levels of p-Akt and p-mTOR as well as increases in LVEDP,myocardial infarction size,and the activity of LDH in coronary perfusion fluid,and the level of myocardial LC3 were found in group Dex+I/R+W(P<0.05).Conclusions Dex preconditioning can protect ischemia-reperfusion injury in isolated rat heart,which may be related to decreased autophagy in cardiomyocytes during ischemia reperfusion period through activating PI3K/Akt signal pathway and enhancing downstream mTOR.