本文關(guān)鍵詞： 心肌缺血再灌注損傷 腺苷酸活化蛋白激酶 硫化氫 自噬流 活性氧　出處：《蘇州大學(xué)》2015年碩士論文　論文類型：學(xué)位論文

【摘要】：目的觀察硫化氫緩釋供體ADT后處理是否對大鼠在體心肌缺血再灌注(I/R)損傷具有保護(hù)作用,探討腺苷酸活化蛋白激酶(AMPK)介導(dǎo)的自噬流在其中的作用,研究硫化氫后處理心肌保護(hù)的機(jī)制。方法成年雄性SD大鼠202只,建立急性大鼠在體心肌I/R損傷模型。隨機(jī)分為9組:假手術(shù)組(Sham組)、單純?nèi)毖俟嘧φ战M(I/R+vehicle組)、緩釋硫化氫供體ADT后處理組(I/R+ADT組)、單純ADT對照組(Sham+ADT組)、AMPK抑制劑Compound c組(I/R+ADT+CC組)、Compound c溶劑二甲基亞砜對照組(I/R+ADT+DMSO組)、氯喹溶劑生理鹽水對照組(I/R+ADT+NS組),自噬流抑制劑氯喹組(I/R+ADT+CQ),單純氯喹對照組(I/R+CQ組)。除Sham和Sham+ADT組外,其余各組均缺血30 min,再灌注4 h。Sham,I/R+vehicle和I/R+CQ組于缺血末再灌注開始即刻腹腔注射ADT溶劑(10%DMSO+玉米油),其余各組均注射ADT(50 mg/kg);I/R+ADT+CC和I/R+ADT+DMSO組于再灌注開始即刻分別靜脈給予Compound c(250μg/kg)和等體積DMSO;I/R+ADT+CQ和I/R+CQ組于手術(shù)開始前1 h腹腔注射氯喹(10 mg/kg),I/R+ADT+NS組注射等體積生理鹽水。再灌注4 h末,提取心臟,采用氯化三苯基四氮唑染色法(TTC法)測定心肌梗死范圍并通過蘇木精-伊紅染色法觀察心肌細(xì)胞形態(tài)學(xué)變化;采用原位二氫乙啶(DHE)染色法檢測活性氧(ROS)的生成。采用免疫印跡法(Western blot技術(shù))檢測p-AMPK/t-AMPK、p-S6/t-S6、Beclin-1、LAMP-2、LC3Ⅱ/Ⅰ及P62蛋白表達(dá)水平。結(jié)果與Sham組和Sham+ADT組比較,各組心肌梗死范圍增大,心肌細(xì)胞損傷增加,ROS生成增多,Beclin-1、LC3Ⅱ/Ⅰ和P62蛋白表達(dá)上調(diào),LAMP-2蛋白表達(dá)下調(diào)(P0.05)。與I/R+vehicle組比較,I/R+ADT組心肌梗死范圍減小,心肌細(xì)胞損傷和ROS生成減少,p-AMPK/AMPK及LAMP-2蛋白表達(dá)上調(diào),p-S6/S6、Beclin-1、LC3Ⅱ/Ⅰ及P62蛋白表達(dá)下調(diào)(P0.05)。與I/R+ADT組比較,I/R+ADT+CC組心肌梗死范圍增加,細(xì)胞損傷和ROS生成增加,p-AMPK/AMPK及LAMP-2蛋白表達(dá)下調(diào),p-S6/S6、Beclin-1、LC3Ⅱ/Ⅰ及P62蛋白表達(dá)上調(diào)(P0.05)。與I/R+ADT組比較,I/R+ADT+CQ組心肌梗死范圍增加,LAMP-2蛋白表達(dá)下調(diào),LC3Ⅱ/Ⅰ和P62蛋白表達(dá)上調(diào)(P0.05)。結(jié)論硫化氫緩釋供體ADT對大鼠在體心肌I/R損傷具有保護(hù)作用,可能是通過激活A(yù)MPK,減少再灌注期間自噬體清除的破壞,保護(hù)了自噬流,進(jìn)而抑制再灌注期間細(xì)胞內(nèi)ROS的生成和細(xì)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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