【作者】 田雪莹；

【导师】 于公元；

【作者基本信息】 天津医科大学 ， 生物化学与分子生物学， 2008， 硕士

【摘要】 目的:观察姜黄素对离体大鼠心脏血流动力学指标、乳酸脱氢酶（LDH）活性、心肌梗死面积、嘌呤核苷酸分解代谢产物的释放量、嘌呤核苷磷酸化酶（PNPase）活性以及心肌肌浆网Ca²⁺-ATP酶活性、Ca²⁺摄取和Ca²⁺释放的影响。探讨姜黄素对大鼠心肌缺血再灌注损伤的保护作用。方法:将16只Wistar大鼠随机分成2组,建立Langendorff离体心脏灌注模型。对照组用Krebs-Henseleit（K-H）液灌注,心脏保持持续工作状态20 min,缺血30 min,再灌注30 min;姜黄素组用加入姜黄素（0.2 mmol/L）的K-H液灌注,心脏保持持续工作状态20 min,缺血30 min,再灌注30 min。于缺血前检测血流动力学指标（心率、收缩压、冠状动脉流量、心输出量）。再灌注结束时,运用高效液相色谱技术检测主动脉与冠状动脉流出液中嘌呤核苷酸代谢产物（尿酸、次黄嘌呤、黄嘌呤、次黄苷、腺苷）的释放量和PNPase活性。采用紫外分光光度法测定心肌释放的LDH活性。用TTC染色法测定心肌梗死面积。用差速离心法提取心肌肌浆网,Folin-酚法测定蛋白质浓度。借助分光光度计观察心肌肌浆网Ca²⁺-ATP酶(Ca²⁺泵)摄取Ca²⁺和兰诺定受体（RyR）/Ca²⁺释放通道释放Ca²⁺。应用酶偶联分析法测定心肌肌浆网Ca²⁺-ATP酶的活性。结果:1姜黄素对大鼠心脏血流动力学指标、心肌释放的LDH活性和心肌梗死面积的影响对照组与姜黄素组的心率、收缩压和心输出量无差异（P＞0.05）,姜黄素组的冠状动脉流量高于对照组（P＜0.05）。姜黄素组心肌释放的LDH活性显著低于对照组（P＜0.01）。与对照组相比,姜黄素组心肌梗死面积缩小（P＜0.05）。2姜黄素对缺血再灌注损伤大鼠心肌嘌呤核苷酸代谢产物（尿酸、次黄嘌呤、黄嘌呤、次黄苷、腺苷）的释放量和PNPase活性的影响对照组与姜黄素组尿酸和黄嘌呤的释放量无差异（P＞0.05）。姜黄素组次黄嘌呤的释放量和PNPase活性低于对照组（P＜0.05）。姜黄素组次黄苷和腺苷的释放量高于对照组（P＜0.05）。3姜黄素对缺血再灌注损伤大鼠心肌肌浆网Ca²⁺摄取、Ca²⁺释放和Ca²⁺-ATP酶活性的影响姜黄素组心肌肌浆网Ca²⁺最大摄取量、Ca²⁺最大释放量和Ca²⁺-ATP酶活性均高于对照组（P＜0.05）。结论:姜黄素能够改善离体大鼠心脏的血流动力学指标,增加冠状动脉流量;缩小心肌梗死面积;减少缺血再灌注损伤心肌细胞LDH的释放,对缺血再灌注损伤心肌具有保护作用。姜黄素能够降低缺血再灌注心肌嘌呤碱基的释放量,减少高能磷酸键的消耗,改善缺血再灌注损伤心肌的能量代谢,从而对缺血再灌注损伤心肌起保护作用。姜黄素能够改善缺血再灌注损伤心肌肌浆网摄取与释放Ca²⁺的功能,提示姜黄素能够促进缺血后心肌细胞收缩和舒张功能的恢复,通过减轻心肌细胞Ca²⁺超载,起到保护缺血再灌注损伤心肌的作用。本研究结果证实了姜黄素对缺血再灌注损伤心肌具有保护作用。为进一步研究姜黄素保护缺血再灌注损伤心肌的机制提供新的线索。更多还原

【Abstract】 ObjectiveThe effects of curcumin on the hemodynamic parameters,lactate dehydrogenase （LDH）activity,myocardial infarct size,the release of purine nucleotide catabolites, purine nucleoside phosphorylase（PNPase）activity and the sarcoplasmic reticulum （SR）Ca²⁺-ATPase activity,Ca²⁺uptake,Ca²⁺release of isolated rat hearts were observed.To explore the protective effects of curcumin on myocardial ischemia reperfusion injury in rats.MethodsSixteen Wistar rats were randomly divided into tow groups.Isolated rat hearts were mounted on a Langendorff perfusion apparatus.Control group,isolated rat hearts were perfused with Krebs-Henseleit（K-H）perfusion buffer and maintained for 20 min of heart working,ischemia for 30 min and reperfused for 30 min. Curcumin group,isolated rat hearts were perfused with K-H buffer containing curcumin（0.2 mmol/L）and maintained for 20 min of heart working,ischemia for 30 min and reperfused for 30 min.Before ischemia the hemodynamic parameter（heart rate,systolic pressure,coronary artery flow,cardiac output）were observed.After the reperfusion,the purine nucleotide catabolites（uric acid,hypoxanthine,xanthine, inosine,adenosine）and PNPase activity of arteriae aorta and coronary artery effluent were assayed by HPLC.The LDH activity was determined by ultraviolet spectrophotometry.The myocardial infarct size was determined by the TTC staining method.The myocardial SR was extracted with differential centrifugation.The protein concentration was determined by Folin-Phenol method.The Ca²⁺uptake of myocardial SR Ca²⁺-ATPase(Ca²⁺pump)and the Ca²⁺release of Ryanodine receptor （RyR）/ Ca²⁺release channel were observed by spectrophotometry.The myocardial SR Ca²⁺-ATPase activity was determined by enzyme coupling assay.Results1 Effects of curcumin on the hemodynamics parameters,LDH activity and infarct size of rat heartsThe heart rate,systolic pressure,and cardiac output in curcumin group and control group had no difference（P＞0.05）.The coronary artery flow in curcumin group was higher than that in control group（P＜0.05）.The LDH activity in curcumin group was lower than that in control group（P＜0.01）.The myocardial infarct size in curcumin group was less than that in control group（P＜0.05）.2 Effects of curcumin on the release of purine nucleotide catabolites and PNPase activity of myocardial ischemia reperfusion injury in ratsThe release of uric acid and xanthine in curcumin group and control group had no difference（P＞0.05）.The release of hypoxanthine and PNPase activity in curcumin group were lower than those in control group（P＜0.05）.While the release of inosine and adenosine in curcumin group were higher than those in control group （P＜0.05）.3 Effects of curcurnin on myocardial SR Ca²⁺uptake,Ca²⁺release and Ca²⁺-ATPase activityThe maxium of Ca²⁺uptake and Ca²⁺release and the Ca²⁺-ATPase activity of myocardial SR in curcumin group were higher than those in control group（P＜0.05）.ConclusionsCurcumin can improve the hemodynamic parameters,increase the coronary artery flow and reduce the myocardial infarct size and the LDH release of myocardial cells.Consequently,curcumin can protect ischemia-reperfused myocardium.Curcumin can improve the energy metabolism of ischemia-reperfused myocardium by reducing the release of purine bases and the consumption of high-energy phosphate bond.Therefore,curcumin can protect ischemia-reperfused myocardium.The function of myocardial SR Ca²⁺uptake and Ca²⁺release was improved, indicating that curcumin can promote the recovery of contractile and diastolic function and reduce the cytosolic Ca²⁺overload.Consequently,curcumin can protect ischemia-reperfused myocardium.The results of this study indicate that curcumin can protect ischemia-reperfused myocardium.The findings might provide new clue for further researches on the protective mechanisms of curcumin on myocardial ischemia reperfusion injury.更多还原