药物后处理对离体大鼠心肌缺血再灌注损伤影响的对比研究及其对线粒体ALDH2作用的相关研究

发布时间：2018-09-16 20:55

【摘要】：背景:急性心肌梗死(Acute myocardial infarction)是指冠状动脉在急性的缺血和缺氧的情况下所引起的心肌细胞的坏死。临床上患者多有不能缓解的胸骨后疼痛,口服硝酸酯类药物以及休息后不能或者完全缓解,通常会出现心电图的动态改变,血清心肌酶以及心脏损伤标志物的升高,通常会并发一些恶性心律失常比如:室性心动过速、心室颤动,甚至心源性休克、心脏破裂等,猝死率50%。急性心肌梗死严重危害了人类的生命健康,目前成为全世界最常见的疾病。及早的恢复冠脉血流,是目前治疗的关键,但是在再灌注时会出现一系列的损伤作用。预处理虽可以显著减轻再灌注损伤,但是缺血的无法预测在临床上得不到很好的实施,从而限制了预处理的应用。2005年,Staat等[1]首次将缺血后处理应用于临床,后处理组与对照组相比减少了36%的心肌梗死的面积。本实验室已经证实缺血后处理可有效减少缺血再灌注性心律失常、改善心脏的泵功能、减少心肌梗死的面积[2]。同时,机械后处理的方式可以在再灌注损伤起到保护作用,但是同时可能会存在斑块的脱落、机械性血管壁的损伤以及栓塞等风险。药物后处理(Pharmacology postconditioning)在临床上可实施性强。目前药物后处理药物有多种,其中常见的有硝酸甘油、地尔硫?、腺苷、尼可地尔等。这四种药物分别通过不同的机制对心肌缺血再灌注引起的损伤起到了相应的保护作用,但既往对于这四种药物之间的治疗效果的差异研究甚少,本文旨在研究这四种药物后处理给药的方式在离体大鼠心肌缺血再灌注损伤的治疗效果的差异,以及探讨线粒体ALDH2是否参与其作用机制的过程,为临床上单独以及联合用药提供一定的理论基础。目的:1.观察硝酸甘油、地尔硫?、腺苷以及尼可地尔四种药物后处理对Wistar离体大鼠心肌缺血/再灌注后左心室心功能(LVDP、±dp/dtmax)、RA、心肌梗死面积的影响,并进行药物之间的对比。2.研究四种药物后处理对离体大鼠心肌缺血/再灌注过程中心室肌线粒体ALDH2表达的影响,探讨ALDH2是否参与药物作用机制的过程。方法:1.药物后处理对离体大鼠心肌缺血/再灌注损伤影响的对比的研究:将硝酸甘油(10-8mol/L)、地尔硫?(5μmo/L)、尼可地尔(200μmo/L)以及腺苷(100μmo/L),加入到Krebs-Henseleit(K-H)模拟液中,充分震荡搅匀后备用。将66只品种为Wistar大鼠(雄性)随机分为六组:正常组(N组)、缺血再灌注组(I-R组)、硝酸甘油+缺血再灌注组、地尔硫?+缺血再灌注组、尼可地尔+缺血再灌注组、腺苷+缺血再灌注组(各组n=11只)。应用Langendorff离体心脏灌流实验系统以及BL-420S生物机能实验系统。正常组(N组)持续灌流正常液150min;I-R组正常液稳定灌流30min后,结扎前降支30min,继以正常液再灌注90min;药物后处理组分别在再灌注即刻给予硝酸甘油、地尔硫?、尼可地尔)以及腺苷再灌注15min,继以正常液体灌流75min。用3-0号医用缝线的3/8弯针在左心耳根部下2mm处穿过心肌表层,在肺动脉圆锥旁出针[3]勾绕冠状动脉前降支,进针深度0.5-1mm,宽度为2-3mm[4]。待心脏稳定灌流30 min后,实验组套一球囊并结扎前降支与球囊,打一活结,球囊内加压至6KPa。此时会看到左室心脏表面开始失去原有的红润光泽而显灰白,左心室收缩活动减弱以至左心室内压力减半,通过这些现象初步判断结扎位置准确[5],心肌梗死模型建立。结扎30min后迅速松开球囊,打开结扎线,再灌注1.5h。最后将药物后处理组与单纯再灌注组大鼠的前降支重新结扎,2ml浓度1%Evans-blue从主动脉的根部逆行灌注心脏,用PBS液体冲洗后迅速置于1%TTC溶液中再染色,区分正常区(深蓝色)、缺血未梗死区(红色)及梗死区域(灰白色)。TTC染色后的心脏组织不能用于后续的分子生物学研究[6],组织标本分开留取。记录并分析各组左心室功能:左心室发展压(LVDP)、左室内压力最大上升/下降速率(±dp/dtmax)、再灌注心律失常(RA)、心肌梗死面积等参数。2.药物后处理对离体大鼠心肌缺血再灌注损伤心室肌线粒体ALDH2表达的影响:动物实验分组同第一部分,本部分采用第一部分离体灌流结束后留取的左心室心肌组织。其中Real time RT-PCR所用的组织在液氮中保存,Western blot方法所用的组织在-80℃冰箱中保存。实时荧光定量逆转录-聚合酶链反应(Realtime RT-PCR)检测线粒体ALDH2基因水平的表达。Western Blot检测线粒体ALDH2蛋白表达。结果:1.1I-R组再灌注30 min及45 min时LVDP均低于各药物后处理组(均P0.05)。在药物后处理组,再灌注30 min地尔硫?组LVDP[(92.68±5.09)mm Hg比(84.26±3.02)mm Hg比(83.35±2.88)mm Hg]、尼可地尔组[(88.95±1.75)mm Hg比(84.26±3.02)mm Hg比(83.35±2.88)mm Hg]与再灌注45 min地尔硫?组LVDP[(90.39±4.29)mm Hg比(82.09±4.24)mm Hg比(80.98±3.89)mm Hg]、尼可地尔组[(86.13±2.38)mm Hg比(82.09±4.24)mm Hg比(80.98±3.89)mm Hg]均高于硝酸甘油组与腺苷组,且地尔硫?组高于尼可地尔组,差异均有统计学意义(均P0.05),但硝酸甘油组与腺苷组比较,差异无统计学意义(P0.05)。1.2 I-R组再灌注30 min及45 min的±dp/dtmax均低于各药物后处理组(均P0.05)。地尔硫?组、尼可地尔组再灌注30 min及45 min的±dp/dtmax均高于硝酸甘油组、腺苷组,且地尔硫?组高于尼可地尔组,差异均有统计学意义(均P0.05)。但硝酸甘油组与腺苷组比较,差异无统计学意义(P0.05)。2.RA评分比较:I-R组评分[5(3,6),57.36]均明显高于各个药物后处理组:尼可地尔组[1(1,3),22.05],地尔硫?组[3(1,4),34.77],硝酸甘油组[4(1,4),45.41],腺苷组[2(1,3),23.14],差异均有统计学意义(均P0.05)。尼可地尔组RA评分最低,但尼可地尔组与腺苷组差异无统计学意义(P=0.771)。3.心肌梗死面积比较:硝酸甘油组(27.04±2.45)%、地尔硫?组(17.01±1.13)%、腺苷组(47.97±1.22)%以及尼可地尔组(34.95±1.25)%均小于I-R组(55.51±1.43)%,差异均有统计学意义(均P0.01)。各药物后处理组组间比较差异均有统计学意义(均P0.01),且地尔硫?组梗死面积最小。4.基因与蛋白表达的结果:在药物硝酸甘油、尼可地尔后处理组线粒体ALDH2m RNA表达分别为0.0657±0.0050、0.0587±0.0004,线粒体ALDH2 m RNA表达量分别为N组的0.7463±0.0726、0.8339±0.0808倍,与I-R组相比差异有统计学意义(P0.05,P0.05)。硝酸甘油与尼可地尔后处理线粒体ALDH2 m RNA表达差异无统计学意义(P0.05)。N组线粒体ALDH2蛋白处于高表达状态,I-R组蛋白表达降低。其中,腺苷、地尔硫?蛋白处于低表达状态,硝酸甘油以及尼可地尔蛋白较I-R组相比表达增加(P0.05)。结论:1.在心肌缺血再灌注时,硝酸甘油、地尔硫?、腺苷、尼可地尔四种药物均可改善缺血再灌注后的心脏泵功能、减少再灌注心律失常评分、缩小心肌梗死面积,从而减少心肌缺血再灌注损伤,对心肌起到保护作用。地尔硫?改善心脏泵功能能力更强;在后处理RA方面,尼可地尔以及腺苷效果更好;在减少心肌梗死面积方面,地尔硫?更优。本实验为临床单独以及联合用药方面提供一定的实验理论基础。2.腺苷、地尔硫?后处理不能通过上调线粒体ALDH2相关机制对缺血再灌注损伤的心肌起到保护作用。硝酸甘油、尼可地尔药物后处理可能通过上调线粒体ALDH2相关机制对缺血再灌注损伤的心肌起到保护作用。ALDH2可能是硝酸甘油以及尼可地尔发挥缺血再灌注损伤保护作用的机制之一。
[Abstract]:BACKGROUND: Acute myocardial infarction (AMI) refers to the necrosis of myocardial cells caused by acute ischemia and hypoxia of the coronary artery. Acute myocardial infarction (AMI) seriously endangers human life and health and is now the most common disease in the world. Pulse blood flow is the key to current treatment, but a series of injuries occur during reperfusion. Pretreatment can significantly reduce reperfusion injury, but the unpredictability of ischemia can not be well implemented in clinical practice, thus limiting the application of preconditioning. In 2005, Staat et al. [1] first applied ischemic postconditioning to clinical, posterior. Compared with the control group, the area of myocardial infarction was reduced by 36%. Our laboratory has proved that ischemic postconditioning can effectively reduce ischemia-reperfusion arrhythmia, improve cardiac pump function, and reduce the area of myocardial infarction [2]. Pharmacological postconditioning is clinically practicable. There are a variety of post-treatment drugs, including nitroglycerin, diltiazem, adenosine, nicorandil, etc. These four drugs are used to treat myocardium through different mechanisms. Ischemia-reperfusion injury plays a corresponding protective role, but there is little research on the difference of therapeutic effect between these four drugs. This paper aims to study the difference of therapeutic effect of these four drugs on myocardial ischemia-reperfusion injury in isolated rats by post-treatment, and to explore whether mitochondrial ALDH2 participates in it. Objective: 1. To observe the effects of nitroglycerin, diltiazem, adenosine and nicorandil on left ventricular function (LVDP, +dp/dtmax), RA and myocardial infarction area after myocardial ischemia/reperfusion in Wistar rats. To study the effects of four kinds of drug postconditioning on the expression of ALDH2 in ventricular myocardial mitochondria during myocardial ischemia/reperfusion in isolated rats, and to explore whether ALDH2 participates in the mechanism of drug action. Methods: 1. The comparative study of the effects of drug postconditioning on myocardial ischemia/reperfusion injury in isolated rats: Nitroglycerin (10-8 mol/L) Sixty-six Wistar rats (male) were randomly divided into six groups: normal group (N group), ischemia-reperfusion group (I-R group), nitroglycerin + ischemia-reperfusion group, diltiazem + ischemia-reperfusion group, diltiazem + ischemia-reperfusion group. Nicorandil + ischemia-reperfusion group, adenosine + ischemia-reperfusion group (n = 11 rats in each group). Langendorff isolated cardiac perfusion system and BL-420S biological function experimental system were used. Normal group (N group) was perfused for 150 minutes; normal fluid in I-R group was perfused steadily for 30 minutes, the anterior descending branch was ligated for 30 minutes, followed by normal fluid reperfusion for 90 minutes. In the treatment group, nitroglycerin, diltiazem and nicorandil were given immediately after reperfusion for 15 minutes and adenosine for 75 minutes. The needle was inserted into the left atrial appendage 2 mm below the root of the left atrial appendage with a 3/8 curved needle of medical suture No. 3-0. 2-3 mm [4]. After 30 minutes of stable cardiac perfusion, the experimental group set up a balloon and ligated the anterior descending branch and balloon, made a knot, and the balloon was pressurized to 6KPa. At this time, the left ventricular surface began to lose its original red luster and grey, left ventricular systolic activity weakened so that the pressure in the left ventricle halved. Through these phenomena, the ligation was preliminarily judged. Myocardial infarction model was established. After 30 minutes of ligation, the balloon was loosened, the ligation line was opened, and the anterior descending branch was ligated again in the drug treatment group and the reperfusion group for 1.5 hours. The TTC stained cardiac tissue could not be used for follow-up molecular biology studies [6], and the tissue samples were taken separately. Left ventricular function: left ventricular development pressure (LVDP), maximum increase/decrease rate of left ventricular pressure (+dp/dtmax), were recorded and analyzed. Reperfusion arrhythmias (RA), myocardial infarction area and other parameters. 2. Effects of drug postconditioning on the expression of ALDH2 in isolated rat ventricular myocardium after myocardial ischemia-reperfusion injury: Animal experimental group was the same as the first part, this part used the left ventricular myocardial tissue after the first part of perfusion in vitro. Tissues were preserved in liquid nitrogen, and the tissues used in Western blot were stored in a refrigerator at - 80 C. The expression of mitochondrial ALDH2 gene was detected by real-time fluorescence quantitative reverse transcription-polymerase chain reaction (Realtime RT-PCR). The expression of mitochondrial ALDH2 protein was detected by Western Blot. Results: LVDP in 1.1I-R group was lower than that after reperfusion for 30 min and 45 min. The treatment group (all P 0.05). In the drug treatment group, LVDP [(92.68 [(92.68 [(5.09) mm Hg ratio (84.26 [(92.68 [(5.09) mm Hg ratio (84.26 [(92.68 [(3.02) mm Hg ratio (84.26 [(3.02) mm Hg ratio (83.35 [2.88) mm Hg ratio (83.35 [2.88) mm Hg ratio (83.35 [2.88) mm Hg ratio (83.35 [(83.29) mm Hg ratio] Hg ratio (83.39 [2.88) mm Hg ratio (83.35 [2.88) mm Hg ratio (83.35 [2.mm Hg ratio (80.98) The ratio of nicorandil group [(86.13+2.38)mm Hg to (82.09+4.24)mm Hg to (80.98+3.89)mm Hg] was higher than that of nitroglycerin group and adenosine group, and that of diltiazem group was higher than that of nicorandil group (all P 0.05), but there was no significant difference between nitroglycerin group and adenosine group (P 0.05). The (+) DP / dtmax at 30 min and 45 min of reperfusion in nicorandil group were higher than those in nitroglycerin group and adenosine group, and the (+) DP / dtmax in nicorandil group was higher than that in nicorandil group (all P 0.05). However, there was no significant difference between nitroglycerin group and adenosine group. The scores of I-R group [5 (3,6), 57.36] were significantly higher than those of each drug treatment group: nicorandil group [1 (1,3), 22.05], diltiazem group [3 (1,4), 34.77], nitroglycerin group [4 (1,4), 45.41], adenosine group [2 (1,3), 23.14], the difference was statistically significant (all P 0.05). There was no significant difference in myocardial infarction size between glycoside group and I-R group (P = 0.771). 3. Comparison of myocardial infarction size between nitroglycerin group (27.04 (+ 2.45)), diltiazem group (17.01 (+ 1.13)), adenosine group (47.97 (+ 1.22)) and nicorandil group (34.95 (+ 1.25)) was significantly smaller than that of I-R group (55.51 (+ 1.43)). There were significant differences among the post-treatment groups (all P 0.01). The expression of ALDH2 m RNA in mitochondria was 0.0657 (+ 0.0050) and 0.0587 (+ 0.0004) respectively in nitroglycerin and nicorandil treatment groups, and the expression of ALDH2 m RNA in mitochondria was 0.7463 (+ 0.0726) and 0.8339 (+ 0.08088) times that in N group, respectively. There was no significant difference in the expression of mitochondrial ALDH2 m RNA between nitroglycerin and nicorandil post-treatment (P 0.05). The expression of mitochondrial ALDH2 protein was high in N group, but decreased in I-R group. Conclusion: 1. Nitroglycerin, diltiazem, adenosine and nicorandil can improve cardiac pump function, reduce reperfusion arrhythmia score and myocardial infarction area during myocardial ischemia and reperfusion, thereby reducing myocardial ischemia and reperfusion injury and protecting myocardium. Ertiazem? Is more effective in improving cardiac pump function; nicorandil and adenosine are more effective in treating RA; diltiazem? Is better in reducing myocardial infarction area. This study provides a theoretical basis for clinical use of either alone or in combination. 2. Adenosine, diltiazem? Post-processing can not up-regulate mitochondrial ALDH2-related. Nitroglycerin and nicorandil postconditioning may protect the myocardium from ischemia-reperfusion injury by up-regulating the related mechanisms of mitochondrial ALDH2. ALDH2 may be one of the mechanisms of nitroglycerin and nicorandil in protecting the myocardium from ischemia-reperfusion injury.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R542.22

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