【作者】 赵胜；

【导师】 冯国清； 翁世艾；

【作者基本信息】 郑州大学 ， 药理学， 2004， 硕士

【摘要】 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缺血再灌注（ischemia／reperfusion，I／R）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索心肌预适应的机制。反应性氧族（reactive oxygen species，ROS）是I／R损伤的重要因素之一，Ca²⁺-ATP酶和Na⁺-K⁺-ATP酶受ROS攻击而活性下降，从而使Ca²⁺外流减少，内流增加；肌浆膜上Ca²⁺-ATP酶受抑而阻滞肌浆网自心肌细胞胞浆中摄钙，终使胞浆内钙超载发生。内源性抗氧化酶能阻滞或延缓对蛋白质、碳氢化合物、脂质及DNA等亚细胞物质的氧化作用。内源性一氧化氮（NO）在心肌预适应延迟保护中有十分重要作用，它既是延迟保护的触发者，也是执行者。作为触发者的NO可能由内皮型NO合成酶（eNOS）合成，而作为效应者的NO则可能由iNOS合成。近年来中药减轻心肌I／R损伤的研究取得较大进展，其中地奥心血康是一种有效的抗心肌缺血药物，已广泛应用于临床心肌缺血性疾病的治疗。巴戟天提取物可提高脑、肝组织的抗氧化酶水平，但在心肌保护方面尚未见报导。本研究以巴戟天水提物预适应大鼠为模型，地奥心血康作为阳性对照药物，来观察巴戟天对大鼠心肌缺血再灌注损伤保护作用。 研究方法 巴戟天水提物配成含生药0.45g／ml、0.15g／ml、0.05g／ml三种浓度药液。地奥心血康胶囊，取其内容物以0.5％羧甲基纤维素钠配制成7mg／ml混悬溶液。96只Wistar大鼠，雌雄各半，随机分为假手术（sham operation，Sham）组、心肌缺血／再灌注（myocardial ischemia／reperfusion，I／R）模型组，阳性药物对郑州大学2（）04硕士学位论文巴戟天对大鼠心肌缺血再灌注损伤的防护作用照即地奥心血康（DK）组及巴戟天（RMO）4.5雏g、1，5留kg、0.5留kg三个剂量组，共6组，每组16只。DK组及RMO三个剂量组分别灌胃给予0.7叭g地奥心血康混悬液和三种相应浓度的巴戟天药液，l次/天，共10天，Sham组及FR组给予同容积去离子水灌胃10天。末次给药24h后，各组动物均接受开胸、sham组动物心脏只挂线不结扎，结扎余组动物左冠状动脉前降支（LAD）造成缺血30min和松扎再灌注90min处理。记录所有动物心电图、测定血流动力学指标;各组均有一半动物推注伊文思蓝，取出心脏，心脏标本沿左室长轴切成1.omm厚的薄片，经TTC染色、10%甲醛固定，用解剖刀剔取不同颜色组织，分别称重，测定缺血和梗死范围;另一半动物则取出心脏，制成10%心肌组织生理盐水匀浆，测定soD、e灯、osH一Px、MDA、No、Nos、Na十一K几灯Pase及C扩几ATpase等生化指标。 结果 1血流动力学参数LAD结扎前各血流动力学指标各组间差异无显著性口今0.05)。与sham组相比血组LAD结扎后各观测时间LVSP和士dp/dt max的数值均降低尤以再灌注期显著护<0.01)，与呱组相比DK与R为10 4.5叭g组多数观测时间数值明显升高（P<0.05，p<0，01）。再灌注期LVEDP: DK和RMo4.5叭g组较血组明显降低（P<0.05）。以上指标DK与RMO 4.sg/kg组间无显著差异（羚0.05）O 2心律失常评分值除RMO 0.5岁kg组，余各手术组在缺血期及再灌注期心律失常评分值均低于眼组，其中再灌注期DK及RMo4.5留吃组差异显著（275士1.00，2.75士l.06vs3.sl士098，P<0.01）。 3心肌缺血和心肌梗死范围心肌缺血范围分别为呱组（33.30士0.98）%，DK组（32.85士0.91）%，RMO4.sg/kg组（34.01士一43）o/o，l.sg/kg组（32.97士1.48）%和0.5妙g组（33.54士0.93）%，经统计学检验差异无显著性（P>o.05），梗死范围DK[（1 1.30士1.18）%l和RMO4.sg/吨[（12.44士0.91）%1及1.sg/kg组〔（19.72士1.64）%l均明显低于呱组[（ 21.42士1.28）%，p<0.01，P<O.05〕，且RMO在05一4.sg/kg范围内心肌梗死范围与剂量呈负相关性（r=一0.811）。 4 SOD、C灯、 GSH一Px及MDA的变化1/R组心肌SOD，CAI几GSH-Px活力显著低于Sham组，MDA含量则显著高于sham组（P<0.01）。而DK，RMO郑州大学2004硕十学位论文巴戟天对大鼠心肌缺血再灌注损伤的防护作用4.5叭g及1 .5叭g组SOD，CAT， GSH一Px活力显著高于呱组，MDA显著低于FR组（P<0.01）。RMO对CAT、GSH一Px及MDA的影响在0.5一4.5叭g范围内有剂量依赖性（二0.889，0.855，一0.944）。 5 NO、iNOS及T一NOS的变化 NO:与sharn组相比眼组心肌NO水平显著降低（P<0.01）;而与I瓜组相比，DK及RM。三个剂量组均升高，以DK，RMO 4.5叭g和1 .5叭g组差异有显著性（尸切.01）。 汹05:与Sh印旧组比较，呱组心肌iNOS活力无显著差异（外0.05）;与血组相比RMO 4.sg/kg、l.sg/kg组iNOS活力显著升高护<0.01)。 T一05:与sham组相比呱组心肌T一05活力显著升高护<0.01);与皿组比较DK组明显降低（P<0.01），RMo 4.5叭g组明显升高（P<0.05），RMO 1.5叭g与0.59瓜g组差异无显著性（外0.05）。 6 Na+一K+一ATpase及c扩九ATPase的变化与更多还原

【Abstract】 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 postischemic 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. There are two main hypotheses, namely oxidative stress and Ca2+-overload, which have been proposed to explain the pathogenesis of ischemia-reperfusion injury. Oxidative stress is usually associated with increased formation of reactive oxygen species(ROS), which may result in a depression in the sarcolemmal Ca2+-pump ATPase and Na+-K+-ATPase activities; these changes lead to decrease Ca2+-efflux and to increase Ca2+-influx respectively. ROS can also depress the sarcoplasmic reticulum Ca2+-pump ATPase and thus inhibit Ca2+ sequestration from the cytoplasm in cardiomyocytes. The depression in Ca 2+-regulatory mechanism by ROS ultimately results in intracellar Ca2+ overload. All endogenous antioxidants may act in concert to inhibit or delay the oxidative damage to subcellular proteins, carbohydrates, lipids and DNA. Endogenous nitric oxide(NO) has been reported to play an important role in late preconditioning, that is to say, it serves both as a trigger and a mediator. Two different NOS isforms are sequentially involved in the pathophysiologic cascade of late preconditioning, with eNOSgenerating the NO which acts as a trigger, and iNOS then generating the NO which acts as a mediator which protects against ischemia. Recently, there has been great development in Chinese medicine to protect myocardium against ischemia reperfusion injury. Di-ao-xin-xue-kang (DK), an effective Chineses medicine has been being used in clinical treatment of myocardial ischemic disease. The extract of Radix moridae officinalls (RMO) has been reported to increase antioxidants in brain and liver but there’s had no report in heart. We preconditioned rat hearts with aqueous extract of RMO and with DK as positive control drug to investigate the effect of RMO on myocardial ischemia reperfusion injury.Methods: Three different concentrations of aqueous extract of RMO: 0.45g/ml, 0.15g/ml and 0.05g/ml were prepared and DK Capsule inclusion was mixed with 0.5% sodium carboxymethyl cellulose into suspension with the concentration of 7mg/ml. 96 Wistar rats (half in male and female) were randomly divided into 6 groups, namely sham operation (Sham) group, myocardial ischemia/reperfusion (I/R) model group, positive control drug: DK group, and three different doses of RMO: 4.5g/kg, 1.5g/kg, 0.5g/kg groups. The rats in DK and three different doses of RMO groups were infused intragastricly with the DK suspension 0.7g/(kg-d) and the three concentration of RMO 4.5g/ (kg-d), 1.5g/ (kg-d), 0.5g/ (kg-d) accordingly for 10 days, while the same volume of deionized water infused intragastricly in Sham and I/R model groups. After 24 hours of the last intragastric infusion, all rats were subjected to openning chest then the left anterior desending coronary arteries (LAD) were ligated for 30min and undamped for 90min except the rats with only thread-drawing in Sham group. Hemodynamics, ischemic zone size and infarct size (IZS and IS) of myocardium and scores of ventricular arrhythmia were measured. One half rats of every group were injected Evans blue then the hearts were taken out and the atria and right ventricle were removed. After refrigeration, the frozen heart was sliced into 5-6 sections, and the slices were incubated in 1% triphenyltetrazolium chloride (TTC). The slices were immersed in 10% formalin overnight. The infarcted myocardium was dissected from the IZS under the illumination of a dissecting microscope. IS, IZS and left ventricle were weighed. Heart更多还原