七氟醚预处理对大鼠离体心脏缺血再灌注损伤时自噬的影响及PI3K/Akt信号通路在其中的作用

摘要

目的 评价七氟醚预处理对大鼠离体心脏缺血再灌注损伤时自噬的影响及磷脂酰肌醇3-激酶/丝氨酸苏氨酸蛋白激酶(PI3K/Akt)信号通路在其中的作用.方法 健康成年雄性Wistar大鼠,6～8周龄,体重250 ～ 280 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84个,采用随机数字表法,将其分为7组(n=12):空白对照组(NC组)K-H液持续灌注180min;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120 min;七氟醚预处理组(S+I/R组)K-H液灌注10 min,2.5％七氟醚预充饱和的K-H液灌注15 min,K-H液洗脱5min,全心缺血30min,再灌注120 min;空白对照+渥曼青霉素组(NC+W组)开胸前30 min大鼠腹腔注射渥曼青霉素(PI3K抑制剂)15 μg/kg,其余处理同NC组;缺血再灌注+渥曼青霉素组(I/R+W组)开胸前30 min大鼠腹腔注射渥曼青霉素15 μg/kg,其余处理同I/R组;七氟醚预处理+渥曼青霉素组(S+ I/R+W组)开胸前30 min大鼠腹腔注射渥曼青霉素15 μg/kg,其余处理同S+I/R组;溶媒组(S+ I/R+D组)开胸前30 min大鼠腹腔注射DMSO 1.5 ml/kg,其余处理同S+ I/R组.于平衡末及再灌注末记录HR、左心室内压力上升/下降最大速率(±dp/dtmax)、左心室发展压(LVDP)、左心室舒张末期压(LVEDP).于再灌注末收集冠脉流出液检测乳酸脱氢酶(LDH)活性,取心肌,计算心肌梗死体积百分比;采用Westernblot法检测自噬标记物微管相关蛋白1轻链3-Ⅱ(LC3-Ⅱ)以及Akt、磷酸化Akt (p-Akt)、雷帕霉素靶蛋白(mTOR)、磷酸化mTOR(p-mTOR)蛋白表达水平.结果 与NC组比较,NC+W组血流动力学指标差异无统计学意义(P＞0.05),其余各组HR、±dp/dtmax、LVDP降低,LVEDP、心肌梗死体积、冠脉流出液LDH活性升高,心肌LC3-Ⅱ表达上调,p-Akt及p-mTOR表达下调(P＜0.05).与I/R组比较,S+I/R组、S+ I/R+D组HR、±dp/dtmax、LVDP增加,LVEDP、心肌梗死体积、冠脉流出液LDH活性降低,心肌LC3-Ⅱ表达下调,p-Akt及p-mTOR表达上调(P＜0.05),S+ I/R+W组各项指标差异无统计学意义(P＞0.05).与S+I/R组比较,S+ I/R+W组HR、±dp/dtmax、LVDP降低,LVEDP、心肌梗死体积、冠脉流出液LDH活性升高,心肌LC3-Ⅱ表达上调,p-Akt及p-mTOR表达下调(P＜0.05),S+I/R+D组各项指标差异无统计学意义(P＞0.05).各组总Akt及mTOR表达水平比较差异无统计学意义(P＞0.05).结论 七氟醚预处理可通过激活PI3K/Akt信号通路增强下游mTOR活性来降低缺血再灌注期间心肌细胞的自噬水平,从而发挥心肌保护作用,减轻大鼠离体心脏缺血再灌注损伤.%Objective To evaluate the effect of sevoflurane preconditioning on autophagy during ischemiareperfusion (I/R) injury to isolated rat hearts and the role of PI3K/Akt signaling pathway.Methods Healthy adult male Wistar rats,aged 6-8 weeks,weighing 250-280 g,were anesthetized.Their hearts were excised and perfused in a Langendorff apparatus.Eighty-four isolated rat hearts with I/R injury were randomly divided into 7 groups (n =12 each):normal control group (NC group),I/R group,sevoflurane preconditioning group (S + I/R group),normal control plus wortmannin group (NC + W group),I/R plus wortmannin group (I/R + W group),sevoflurane preconditioning plus wortmannin group (S + I/R + W group),and solvent group (S + I/R + D group).In NC group,the hearts were continuously perfused with K-H solution for 180 min.In I/R group,the hearts were perfused with K-H solution for 30 min,and then subjected to ischemia for 30 min followed by 120 min of reperfusion.In S+ I/R group,the hearts were perfused with K-H solution for 10 min,and then with K-H solution saturated with 2.5％ sevoflurane for 15 min,followed by 5 min washout with K-H solution before ischemia.In NC + W group,wortmannin (PI3K inhibitor) 15 μg/kg was injected intraperitoneally at 30 min before chest opening,and the other procedures were similar to those previously described in group NC.In I/R + W group,wortmarnin 15 μg/kg was injected intraperitoneally at 30 min before chest opening,and the other procedures were similar to those previously described in group I/R.In S + I/R + W group,wortmannin 15 μg/kg was injected intraperitoneally at 30 min before chest opening,and the other procedures were similar to those previously described in S + I/R group.In S + I/R + D group,dimethyl sulfoxide 1.5 ml/kg was injected intraperitoneally at 30 min before chest opening,and the other procedures were similar to those previously described in group S + I/R.The HR,± dp/dtmax,left ventricular developed pressure (LVDP) and left ventricular end-diastolic pressure (LVEDP) were recorded at the end of equilibration and reperfusion.At the end of reperfusion,coronary effluent was collected to detect lactate dehydrogenase (LDH) activity,and myocardial specimens were obtained to calculate the percentage of myocardial infract size.The expression of autophagy marker LC3-Ⅱ and Akt,phosphor-Akt (p-Akt),mammalian target of rapamycin (mTOR),and phosphor-mTOR (p-mTOR) was determined by Western blot.Results Compared with NC group,no significant change was found in the parameters of hemodynamics in NC + W group,and HR,± dp/dtmax and LVDP were significantly decreased,LVEDP,myocardial infract size,and LDH activity were increased,LC3-Ⅱ expression was up-regulated,and the expression of p-Akt and p-mTOR was down-regulated in the other groups.Compared with group I/R,HR,± dp/dtmax,and LVDP were significantly increased,LVEDP,myocardial infract size,and LDH activity were decreased,LC3-Ⅱ expression was downregulated,and the expression of p-Akt and p-mTOR was up-regulated in S + I/R and S + I/R + D groups,and no significant change was found in each parameter in S+ I/R+ W group.Compared with S + I/R group,HR,± dp/ dtmax and LVDP were significantly decreased,LVEDP,myocardial infract size and LDH activity were increased,LC3-Ⅱ expression was up-regulated,and the expression of p-Akt and p-mTOR was down-regulated in S + I/R + W group,and no significant change was found in each parameter in S + I/R + D group.Conclusion Sevoflurane preconditioning can decrease autophagy of myocardial cells during I/R through activating PI3K/Akt signaling pathway and enhancing mTOR activity in the downstream,thus protecting isolated rat hearts against I/R injury.