【作者】 郭文超；

【导师】 牛凡；

【作者基本信息】 山西医科大学 ， 内科学， 2010， 硕士

【摘要】 研究背景：随着介入治疗的迅速发展,心肌缺血再灌注损伤成为人们研究的热点之一。目前,缺血再灌注损伤发生的机制尚未完全阐明,氧自由基生成、血管内皮损伤、钙超载和白细胞激活已较为公认,但并不能解释全部现象。最近的研究显示,心肌缺血再灌注损伤还与炎性因子产生、细胞凋亡、粘附分子表达及核转录因子调控等机制有关。有研究显示再灌注启动了一个典型的炎症反应,多形核中性粒细胞的浸润,已成为心肌缺血再灌注后的主要问题。Toll样受体4是新近发现的天然免疫中的细胞跨膜受体及病原模式识别受体,其除了参与病原微生物触发的炎症反应外,有研究提示TLR-4也参与了缺血组织损伤的炎症反应。在正常生理情况下,内皮细胞通过组成型一氧化氮合酶持续合成少量一氧化氮,对维持正常的血管内皮功能起重要作用。近年发现NO减少亦是PMN激活的一个重要因素,心肌缺血再灌注早期可导致内皮细胞功能障碍,内源性NO合成减少,抗PMN作用减弱,加重心肌缺血再灌注损伤。目的：培哚普利在动物实验有抗心肌缺血再灌注损伤作用,减轻心肌缺血再灌注损伤引起的心肌细胞结构的改变、心肌出血、心肌顿抑以及心律失常等,但其心肌保护作用的具体机制尚待进一步研究。本研究分两部分,第一部分观察培哚普利对缺血再灌注早期Toll样受体4蛋白的影响,探讨培哚普利对心肌缺血再灌注损伤保护作用与Toll样受体4蛋白介导的炎症反应的关系；第二部分腹腔注射NO非选择性阻断剂(L-NAME),观察培哚普利对心肌缺血再灌注损伤保护作用与NO的关系,初步探讨培哚普利对心肌缺血再灌注损伤的可能保护机制。第一部分：方法：30只Wistar大鼠随机分为3组：假手术组,缺血再灌注组及培哚普利组。培哚普利组灌服培哚普利4mg/kg,每日1次,共7天。假手术组和缺血再灌注组分别灌服相应体积的生理盐水。采用大鼠左冠状动脉前降支结扎法建立大鼠缺血再灌注模型,缺血30分钟,再灌注1小时。于实验结束时,取出心脏于4%多聚甲醛固定、石蜡包埋切片,HE染色观察心肌病理形态学变化,按TUNEL法检测凋亡细胞,以S-P免疫组化法检测TLR4蛋白表达。结果：①与缺血再灌注组相比,培哚普利组炎症损伤减轻；②培哚普利组心肌细胞凋亡指数明显低于缺血再灌注组但高于假手术组(P<0.05);③培哚普利组心肌组织Toll样受体4蛋白平均积分光密度值低于缺血再灌注组但高于假手术组(P<0.05)。第二部分：方法：40只Wistar大鼠随机分为4组：假手术组,缺血再灌注组,培哚普利组及联合用药组。假手术组和缺血再灌注组灌服和腹腔注射等量的生理盐水,假手术组只在左冠状动脉前降支下穿线,不结扎；培哚普利组灌服培哚普利4mg/kg,腹腔注射等量的生理盐水；联合用药组灌服培哚普利4mg/kg,腹腔注射L-NAME 15mg/kg;每日1次,共7天。结扎大鼠左冠状动脉前降支,建立大鼠缺血再灌注模型。测定血流动力学参数,左室收缩压(LVSP)、左室舒张末压(LVEDP)、左室内压最大上升速率(+dP／dTmax)及左室内压最大下降速率(-dP／dTmax)。光镜观察心肌形态学改变,TUNEL法检测细胞凋亡指数。结果：①缺血前四组动物LVSP、LVEDP、+dP/dTmax、-dP/dTmax,均无统计学差异(P>0.05)；假手术组在缺血30 min及再灌注30 min,60min时血流动力学平稳,无统计学差异(P>0.05)；缺血30 min后与缺血前相比三组动物LVSP、+dP/dTmax及-dP/dTmax均明显下降(P<0.05),但后三组间无统计学差异(P>0.05)；再灌注30 min,60min后缺血再灌注组及联合用药组LVSP+dP/dTmax及-dP／dTmax相对稳定,培哚普利组LVSP逐渐回升,与培哚普利组相比缺血再灌注组和联合用药组有统计学差异(P<0.05)。缺血30 min后三组动物LVEDP均明显上升,与缺血前相比有统计学差异(P<0.05),后三组间无统计学差异(P>0.05),再灌注30 min,60min后缺血再灌注组及联合用药组LVEDP无明显变化,培哚普利组略有下降与缺血再灌注组及联合用药组相比有统计学意义(P<0.05)(详见表2-2)。②假手术组散在心肌纤维肿胀、变性,间质轻度水肿。缺血再灌注组及联合用药组大鼠心肌纤维断裂坏死,大量炎性细胞浸润,而培哚普利组出血、坏死损伤轻微,有少量炎性细胞浸润。③与缺血再灌注组及联合用药组相比,培哚普利可减轻心肌细胞凋亡数(P<0.05),培哚普利组及联合用药组凋亡细胞数也有统计学差异(P<0.05),均明显高于假手术组(P<0.05)。结论：①缺血再灌注过程中存在细胞凋亡,细胞凋亡和Toll样受体4的表达是一个动态的过程。培哚普利可减少在体大鼠心肌缺血再灌注心肌细胞调亡指数,可能机制之一是通过减少Toll样受体4蛋白而减轻白细胞介素-6,TNF-α等表达,减轻炎症反应,保护缺血再灌注心肌。②培哚普利的心肌保护作用可部分被腹腔注射NO非选择性抑制剂L-NAME所抑制,提示培哚普利对心急缺血再灌注损伤的保护作用可能与NO有关。更多还原

【Abstract】 Background:Myocardial ischemia-reperfusion injury has been one of the study focus with the rapid development of percutaneous coronary intervention. At present, the mechanism of ischemia-reperfusion injury has not entirely beem clarified. That oxygen free radical generation, vascular endothelial injury, calcium overload and leukocyte activation may be the major mechanism, but it can’t not explain all the phenomena. Recent studies have shown that myocardial ischemia-reperfusion injury is related with the inflammatory factor production, apoptosis, adhesion molecule expression and regulation of nuclear factors and other mechanisms etc. Studies have shown that reperfusion initiated a typical inflammatory response, polymorpho-nuclear neutrophil infiltration,this became the major problem in myocardial ischemia-reperfusion.Toll-like receptor 4 is a newly discovered transmembrane receptor and pathogen pattern recognition receptors in the innate immune cells, addition to participating in the inflammatory response triggered by pathogenic micro-organisms, there are research suggests that TLR-4 may also be involved in the inflammatory response of the ischemic tissue. The endothelial cell continuously synthasize a small amount of nitric oxide by constitutive nitric oxide synthase under normal physiological circumstances. This plays an important role in the maintenance of normal vascular endothelial function. There are studies showed that the reduction of NO is an important factor in PMN activation in recent years. It can lead to endothelial cell dysfunction in myocardial ischemia reperfusion, then endogenous NO synthesis reduced, weakening the role of anti-PMN and increasing the myocardial ischemia-reperfusion injury.Objective:Perindopril has the effect of anti-myocardial ischemia-reperfusion injury in animal experiments, it can reduce myocardial ischemia-reperfusion injury caused by the changes in the structure of myocardial cells, myocardial hemorrhage, myocardial stunning, and arrhythmias, etc. The specific mechanism of myocardial protection remains to be further studied.This study can be divided into two parts:The first study was undertaken to study the effect of Perindopril on the expression of toll like receptor-4 in rats with early myocardial ischemia-reperfusion injury and to explor the possible mechanisms of Perindopril on inhibition of myocardial ischemia-reperfusion injury. The second part was to determine the relationship between Perindopril on myocardial ischemia-reperfusion injury and the NO protective effect on myocardial ischemia-reperfusion injury by intraperitoneal injection of non-selective NO inhibitor (L-NAME), to explore the early protection mechanism of perindopril on myocardial ischemia reperfusion injury.Part one: Methods:Thirty Wistar rats were randomly divided into 3 group:sham operation group, myocardial IR group and perindopril group. In the perindopril group,The rats were treated with perindopril 4mg/kg,once a day,for 7 days and equal quantity of normal saline were treated in IR group and sham-operated group. The left anterior descending artery in Wister rats was ligated to establish IR model. Wistar rats were subjected to 30 minutes of coronary ligation, followed by 1 hours of reperfusion.Morphology of myocardial tissue was observed with optics microscope, the numbers of apoptotic cardiomyocyte was determined by Tunnel staining and TLR4 expressions was detected by immunohistochemistry.Result:①Compared with IR group, Inflammatory injury in perindopril group was significantly lower than that in IR group;②Cardiomyocyte apoptosis index (AI) in perindopril group was significantly lower than that in IR group but higher than that in the sham-operated group(P< 0.05);③TLR-4 in perindopril group was significantly lower than that in the IR group but higher than that in the sham-operated group(P< 0.05).Part Two:Methods:40 Wistar rats were randomly divided into four groups:sham operation group, ischemic reperfusion group, perindopril group and combination group. Sham operation group and ischemic reperfusion group were drenched equal quantity of normal saline;perindopril group were drenched perindopril 4mg/kg, cobination group were drenched perindopril 4mg/kg, intraperitoneal injection of L-NAME 15mg/kg; once a day,7 days. The left anterior descending coronary artery in Wistar rats were ligated to establish ischemia-reperfusion model, the determination of hemodynamic parameters, left ventricular systolic pressure (LVSP), left ventricular end-diastolic pressure (LVEDP), left ventricular pressure maximum rate of rise and fall (+dP/dTmax and-dP/dTmax) were measured. Morphology of myocardial tissue was observed with optics microscope, the number of apoptotic cardiomyovyte were determined by Tunnel staining.Result:(1) LVSP、LVEDP、+dP/dTmax、-dP/dTmax were no statistically significant differences among the four groups of animals before the LAD in Wistar rats were ligated (P> 0.05); There were no statistically significant differences when the LAD in Wistar rats were ligated 30 min and that were reperfused 30 min,60min(P< 0.05); There were significantly decreased among the late three groups of animals when the LAD in Wistar rats were ligated 30 min among the late three groups of animals(P< 0.05) and there were no statistically significant differences among the late three groups of animals(P>0.05); There were no statistically significant differences in ischemic reperfusion group and and combination group when the LAD in Wistar rats were reperfused 30 min,60min(P>0.05); there were increased significantly in perindopril group when the LAD in Wistar rats were reperfused 30 min,60min(P< 0.05), ischemic reperfusion group and combination group were no statistically significant differences (P>0.05)。LVEDP in the late three groups were significantly increased when the LAD in Wistar rats were ligated 30 min and there were no statistically significant differences among the three groups of animals(P> 0.05); LVEDP were no significant change in ischemic reperfusion group and combination group when the LAD in Wistar rats were reperfused 30 min,60min(P> 0.05); LVEDP decreased slightly in the perindopril group,there was statistically significant in perindopril group with ischemia-reperfusion group and the combination group and combination group (P<0.05); (2) Myocardial fibers swelling degeneration and interstitial edema were little found in sham operation group. Myocardial fibers fracture, necrosis and lots of inflammatory cells were found in the ischemia-reperfusion group and combined group, but hemorrhage and necrosis were attenuated and a little inflammatory cells infiltrating were found in perindopril group. (3) Compared with ischemic reperfusion group and combined group, perindopril can significantly reduce myocardial cell apoptosis(P< 0.05), there were statistically significant difference in perindopril group and combination group (P< 0.05), there were statistically significant difference in sham operation group and ischemic reperfusion group, perindopril group, combination group (P< 0.05).Conclusion:(1) Apoptosis can be found in cardiocytes during ischemia-reperfusion. Myocardial apoptosis and the expression of Toll-like receptor 4 are dynamic changing in ischemia-reperfusion injury. Perindopril could reduce cardiomyocyte apoptosis following ischemia-reperfusion, The mechan-ism may be by inhibiting the expression of inflammatory response which was induced by Toll-like receptor 4.(2)Perindopril could reduce cardiomyocyte apoptosis following ischemia reperfusion, the myocardial protective effect of perindopril can partially be inhibited by L-NAME, a non-selective NO inhibitor.its relationship between NO and the myocardial protective effect were obvious,but the exat mechanism remains to be further studied.更多还原