硫化氢缓释供体ADT后处理对大鼠在体心肌缺血再灌注损伤的保护—AMPK介导的自噬流在其中的作用

发布时间：2018-01-31 17:43

  本文关键词： 心肌缺血再灌注损伤 腺苷酸活化蛋白激酶 硫化氢 自噬流 活性氧　出处：《苏州大学》2015年硕士论文　论文类型：学位论文

【摘要】：目的观察硫化氢缓释供体ADT后处理是否对大鼠在体心肌缺血再灌注(I/R)损伤具有保护作用,探讨腺苷酸活化蛋白激酶(AMPK)介导的自噬流在其中的作用,研究硫化氢后处理心肌保护的机制。方法成年雄性SD大鼠202只,建立急性大鼠在体心肌I/R损伤模型。随机分为9组:假手术组(Sham组)、单纯缺血再灌注对照组(I/R+vehicle组)、缓释硫化氢供体ADT后处理组(I/R+ADT组)、单纯ADT对照组(Sham+ADT组)、AMPK抑制剂Compound c组(I/R+ADT+CC组)、Compound c溶剂二甲基亚砜对照组(I/R+ADT+DMSO组)、氯喹溶剂生理盐水对照组(I/R+ADT+NS组),自噬流抑制剂氯喹组(I/R+ADT+CQ),单纯氯喹对照组(I/R+CQ组)。除Sham和Sham+ADT组外,其余各组均缺血30 min,再灌注4 h。Sham,I/R+vehicle和I/R+CQ组于缺血末再灌注开始即刻腹腔注射ADT溶剂(10%DMSO+玉米油),其余各组均注射ADT(50 mg/kg);I/R+ADT+CC和I/R+ADT+DMSO组于再灌注开始即刻分别静脉给予Compound c(250μg/kg)和等体积DMSO;I/R+ADT+CQ和I/R+CQ组于手术开始前1 h腹腔注射氯喹(10 mg/kg),I/R+ADT+NS组注射等体积生理盐水。再灌注4 h末,提取心脏,采用氯化三苯基四氮唑染色法(TTC法)测定心肌梗死范围并通过苏木精-伊红染色法观察心肌细胞形态学变化;采用原位二氢乙啶(DHE)染色法检测活性氧(ROS)的生成。采用免疫印迹法(Western blot技术)检测p-AMPK/t-AMPK、p-S6/t-S6、Beclin-1、LAMP-2、LC3Ⅱ/Ⅰ及P62蛋白表达水平。结果与Sham组和Sham+ADT组比较,各组心肌梗死范围增大,心肌细胞损伤增加,ROS生成增多,Beclin-1、LC3Ⅱ/Ⅰ和P62蛋白表达上调,LAMP-2蛋白表达下调(P0.05)。与I/R+vehicle组比较,I/R+ADT组心肌梗死范围减小,心肌细胞损伤和ROS生成减少,p-AMPK/AMPK及LAMP-2蛋白表达上调,p-S6/S6、Beclin-1、LC3Ⅱ/Ⅰ及P62蛋白表达下调(P0.05)。与I/R+ADT组比较,I/R+ADT+CC组心肌梗死范围增加,细胞损伤和ROS生成增加,p-AMPK/AMPK及LAMP-2蛋白表达下调,p-S6/S6、Beclin-1、LC3Ⅱ/Ⅰ及P62蛋白表达上调(P0.05)。与I/R+ADT组比较,I/R+ADT+CQ组心肌梗死范围增加,LAMP-2蛋白表达下调,LC3Ⅱ/Ⅰ和P62蛋白表达上调(P0.05)。结论硫化氢缓释供体ADT对大鼠在体心肌I/R损伤具有保护作用,可能是通过激活AMPK,减少再灌注期间自噬体清除的破坏,保护了自噬流,进而抑制再灌注期间细胞内ROS的生成和细胞死亡。
[Abstract]:Objective to investigate the protective effect of hydrogen sulfide (H2S) sustained release donor (ADT) post treatment on myocardial ischemia reperfusion injury in rats. To investigate the role of adenylate activated protein kinase (AMPK) -mediated autophagy and to study the mechanism of myocardial protection after hydrogen sulfide treatment. Methods 202 adult male SD rats were enrolled in this study. Acute myocardial I / R injury model was established in rats. The rats were randomly divided into 9 groups: sham group (sham group) and I / R vehicle group (ischemia reperfusion control group). Sustained release hydrogen sulfide donor ADT post-treatment group (I / R ADT group, ADT control group, Sham ADT group). AMPK inhibitor Compound c group was treated with I / R ADT CC group and I / R ADT DMSO group was treated with dimethyl sulfoxide (DMSO). Chloroquine solvent saline control group (I / R ADT NS group), autophagy inhibitor chloroquine group (I / R ADT CQ). The control group of chloroquine was treated with I / R CQ group, except Sham and Sham ADT group, all of them were subjected to ischemia for 30 min and reperfusion for 4 h 路Sham. The I / R vehicle and I / R CQ groups were intraperitoneally injected with ADT solvent 10 corn oil immediately after ischemia and reperfusion. All the other groups were injected with ADT(50 mg / kg; The I / R ADT CC and I / R ADT DMSO groups were given Compound cor 250 渭 g / kg and iso-volume DMSO respectively at the beginning of reperfusion. In the I / R ADT CQ and I / R CQ groups, 10 mg / kg chloroquine was injected intraperitoneally 1 hour before the operation. I / R ADT NS group was injected with normal saline of the same volume. The heart was extracted at the end of 4 h after reperfusion. The size of myocardial infarction was measured by TTC method and the morphological changes of myocardial cells were observed by hematoxylin-eosin staining. The formation of reactive oxygen species (Ros) was detected by in situ dihydroethidime (DHEH) staining, and the p-AMPK / t-AMPK was detected by Western blotting (Western blot). The expression levels of p-S6 / t-S6 / Beclin-1 LAMP-2LC3 鈪，

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