【作者】 韩辉；

【导师】 赵文增；

【作者基本信息】 郑州大学 ， 外科学， 2007， 硕士

【摘要】 背景良好的血液循环是组织细胞获得充足的氧和营养物质供应并排出代谢产物的基本保证，各种原因造成的组织血液灌流量减少可使细胞发生缺血性损伤，尽早恢复组织的血液灌流是减轻缺血性损伤的根本措施。近年来，溶栓疗法、动脉搭桥术、心脏外科体外循环、断肢再植和器官移植等方法的建立和推广应用，使许多组织、器官缺血后重新得到血液灌注。再灌注具有两重性。多数情况下，缺血后再灌注使组织、器官功能得到恢复，损伤的结构得到修复，患者病情好转、康复。但是有时缺血后再灌注不仅不能使组织、器官功能恢复，反而加重组织、器官的功能障碍和结构损伤，即缺血再灌注损伤(ischemia reperfusion injury，IRI)，表现为心肌坏死、心律失常、心肌顿抑以及包括“无复流现象”的微血管内皮损伤。再灌注损伤的发生机制复杂，包括氧自由基大量产生、中性粒细胞与内皮细胞相互作用、细胞凋亡、胞内钙超载和三磷酸腺苷(adenosine triphosphate，ATP)产生不足等。随着研究深入，人们逐渐发现炎症反应在缺血再灌注损伤中也扮演着重要的角色。心肌缺血预适应(ischemic preconditioning，IPC)指反复几次短暂的心肌缺血再灌注，可对抗随后的较长时间持续缺血所造成的心肌损伤。其保护效应主要包括缩小IRI后心肌梗死范围，减少室性心律失常发生和促进心功能恢复。药理性预适应(pharmacological preconditioning，PPC)是应用药物模拟或激发机体内源性物质而诱导心肌保护，目前已成为一种研究趋势，将有利于开发抗心肌缺血药物和进一步探讨心肌预适应的机制。曲美他嗪(trimetazidine，TMZ)是一种治疗缺血性心脏病、具有抗氧化效应的代谢类药物，已广泛应用于临床，许多实验和临床研究已经证实，它能通过增强葡萄糖氧化代谢、减轻细胞内酸中毒和高钙血症、限制炎症反应以及减少氧自由基生成等抗缺血再灌注损伤，保护心肌。左旋精氨酸(L-Arginine，L-Arg)是一氧化氮(nitrogen monoxidum，NO)的前体物质，NO是具有广泛生物学效应的自由基，它在心血管系统能维持血管平滑肌的紧张度、抑制血小板聚集、黏附和调节血管平滑肌细胞增生，从而减轻IRI。目前，关于TMZ和L-Arg抗缺血再灌注损伤的报道较多，但有关TMZ对再灌注损伤中炎症反应作用的报道国内尚未见到，而且TMZ与L-Arg抗心肌再灌注损伤的机制不同，联合应用是否对心肌保护有协同作用目前尚无定论。目的本实验拟探讨曲美他嗪和左旋精氨酸联合应用抗大鼠心肌缺血再灌注损伤的作用及其机制。方法雄性健康成年SD大鼠40只，体重180-220克。采用随机区组设计将动物随机分为4组：对照组(A组)、曲美他嗪组(B组)、左旋精氨酸组(C组)和联合用药组(D组)，每组各10例，各组均采用冠脉结扎法制作IRI模型，缺血30min，再灌注90min，记录动物心电图。根据L-Arg和TMZ作用机制的不同，B组于实验前6天，每日给予生理盐水稀释的TMZ10mg／kg灌胃，C组于缺血前30min腹腔注射5％L-Arg300mg／kg，D组联合应用上述两种药物。分别于缺血前、再灌注30min、60min、90min后，取大鼠静脉血2ml，测血浆肿瘤坏死因子(tumor necrosis factor，TNF-α)的含量，实验结束后处死大鼠，取出心脏，制成10％心肌组织生理盐水匀浆，检测超氧化物歧化酶(superoxide dismutase，SOD)、丙二醛(malonaldehyde，MDA)、ATP。观察比较各组心律失常发生情况以及心肌组织内血管细胞黏附分子1(vascular cell adhesion molecule，VCAM-1)表达现象。对上述数据进行统计分析，组间比较采用方差分析，组内比较采用t检验。P＜0.05为差异具有显著性，P＜0.01为差异具有非常显著性。结果1再灌注30min、60min、90min，各组血浆TNF-α水平较缺血前均升高(P＜0.05)；在同一时点，药物干预各组与对照组比较，TNF-α水平均降低(P＜0.05或P＜0.01)，而且D组与B组、C组相比较降低显著(P＜0.05)。2药物干预组与对照组比较，可显著降低心肌组织内MDA的上升幅度，提高SOD活性和ATP含量，降低心律失常的发生和持续时间，减少VCAM-1的表达(P＜0.05或P＜0.01)，并且D组与B组、C组比较有显著差异性(P＜0.05)。结论1炎症反应参与大鼠心肌缺血再灌注损伤。2曲美他嗪和左旋精氨酸可通过限制再灌注损伤中炎症反应、增加心肌能量供应等保护大鼠缺血再灌注损伤心肌。3两药联合应用优于两药单用。更多还原

【Abstract】 Background:Several investigations indicated that reperfusion of ischemia myocardium induced ischemia reperfusion injury (IRI). This injury includes myocardial necrosis, arrhythmia, myocardial stunning and microvascular endothelial dysfunction including the no-reflow phenomenon. Myocardial reperfusion injury is a multifarious process including high lever of oxygen free radicals, neutrophil-endothelium interactions, apoptosis, intracellular calcium overload and low lever of ATP. As the development of investigations, people found gradually that inflammation also played a very important role in IRI.Preconditioning the heart with a brief period of ischemia followed by reperfusion renders it very resistant to injury from a subsequent prolonged ischemia. The protection by preconditioning mainly lies in limiting infarct size, reducing the incidence of ventricular arrhythmia and improving post ischemic cardiac function. Pharmacological preconditioning protection against heart injury is trigged by medicines which can mimic beneficial effects of cardiac ischemic preconditioning. That would be helpful to probe the mechanism of IPC and develop novel access to treatment of the cardiovascular diseases.Trimetazidine (TMZ), an antioxidant agent and metabolism medicine, has been extensively used in coronary artery disease and other cardiovascular disease, several experimental and clinical studies have demonstrated that it also exerts a cytoprotective effect, limiting IRI, through several mechanisms of action: potentiation of oxidative glucose metabolism, reduction of the degree of intracellular acidosis and hypercalcaemia, and attenuation of the inflammatory response and OFR production. L-Arginine (L-Arg) is a precursor of Nitric oxide (NO), NO is a free radical with extensive biological effect. It can maintain the tonicity of vascular smooth muscle, suppress platelet aggregation and adhesion, and regulate proliferation of vascular smooth muscle cell in cardio-vascular system, thus limit IRI. At present, the lots of reports about their limitation to IRI have emerged, but the effect of TMZ on inflammatory was blank in our country. Furthermore, the mechanism of TMZ on limiting IRI is different with L-Arg. It has not defined whether the combination of them would do more profit to myocardial preservation.Objective:This experiment plans to investigate the protective effects and mechanism of TMZ plus L-Arg on myocardial ischemia reperfusion injury in rat.Methods:Experiments were performed on adult male SD rats weighing 180-220g. They were randomly divided into four groups: the control group (A), the TMZ treated group (B), the L-Arg treated group (C) and the TMZ plus L-Arg group(D), 10 in each group. Myocardial ischemia-reperfusion injury models were made by ligating the coronary artery for 30min followed by reperfusion for 90 min. The electrocardiogram (ECG) was registered. Before establishment of ischemia reperfusion model, rats in group B were fed with 10mg/kg TMZ for six days. L-Arg (300mg/kg) was administered into peritoneal 30min before ischemia in C group, and both were applied in D group.Venous blood was collected before ischemia and at 30min、60min、90min after reperfusion, and then, tumor necrosis factor (TNF-α) was detected. After experiment, rats were executed and the hearts were taken out. Heart 10% tissue homogenate was made and then superoxide dismutase (SOD), malondialdehyde (MDA), ATP, was detected. The occurrence of arrhythmia and the generation of myocardial vascular cell adhesion molecule (VCAM-1) were compared among groups.Data was presented with statistical analysis, and analysis of Variances was used to determine changed between groups, Student’s test was used to determine changed within group. Results were significant when P<0.05 and even significant when P<0.01. Results:1 The level of TNF-αin blood plasma 60min after reperfusion was higher than before ischemia in group A (P<0.05). Compared with the group A, the treated groups could lower the level of TNF-α(P<0.05 or P<0.01).And the difference was significant when the group D contrasted with the group B and the group C (P<0.05).2 The treated groups could significant lower the rising tendency of MDA; depress the occurrence and duration of arrhythmia; enhance the activity of SOD and the level of ATP; reduce the express of VCAM-1 (P<0.05 or P<0.01). And the difference was significant when the group D contrasted with the group B and the group C (P<0.05).Conclusions:1 Inflammation is one of the reasons causing myocardial ischemia reperfusion injury.2 Trimetazidine、L-arginine has notable protective effect on myocardial ischemia reperfusion injury through limiting the inflammation and enhancing ATP.3 Trimetazidine plus L-arginine is better than using the 2 medicines separately.更多还原