【作者】 杨萍；

【导师】 贾钰华；

【作者基本信息】 南方医科大学 ， 中西医结合临床， 2010， 博士

【摘要】 一、目的：心肌缺血后,尽早恢复血流是防治缺血性损伤的基本措施。近年来,采用溶栓疗法(thrombolytic therapy)、冠脉动脉成形术(coronary angioplasty)等多种新技术使缺血心肌重新恢复了血流,此乃再灌注。及早、完全和持续恢复血流灌注可以挽救缺血心肌,但与此同时,再灌注本身也可对心肌产生不利影响,即心肌缺血再灌注损伤(myocardial ischemia/reperfusion injury, MI/RI)。再灌注损伤可导致心脏功能障碍,影响患者的生活质量甚至预后。因此探索心肌缺血再灌注损伤的防治措施及其机制具有重要的现实意义。本课题组的前期蛋白质组学研究显示,Prohibitin(PHB)蛋白在缺血再灌注损伤组和正常组间存在差异表达,与国外研究结果不谋而合。但是,到目前为止,对于PHB蛋白功能的研究大多局限于肿瘤学方面,其在心肌细胞中的功能仍然知之甚少,为此,有必要探讨PHB蛋白在心肌细胞损伤中的作用及机制。丹参是具有多种心血管活性的中药,临床广泛应用于心血管疾病的防治。丹参酮ⅡA(TanshinoneⅡA)为丹参酮中含量最高的脂溶性活性成分。本课题以丹参酮ⅡA为研究对象,旨在了解丹参酮ⅡA对心肌缺血再灌注损伤的防治作用,并在细胞水平从抗氧化能力、拮抗钙超载和能量代谢方面探讨其对心肌细胞的保护作用,并了解其对PHB蛋白的调控作用。二、方法：动物水平：雄性Wistar大鼠随机分成5组,假手术组(SH组)、模型组(MI／R组)、丹参酮ⅡA高剂量组、丹参酮ⅡA中剂量组、丹参酮ⅡA低剂量组。SH组大鼠灌服生理盐水,手术时进行冠脉穿线但不结扎；MI／R组大鼠灌服生理盐水,手术时结扎冠状动脉左前降支；丹参酮ⅡA高、中、低剂量组大鼠分别灌服丹参酮ⅡA药液20mg/kg·d、10 mg/kg·d、5 mg/kg·d,2/日。采用经典的冠脉结扎再松开的办法制备动物模型。结扎冠状动脉左前降支15min,然后松开再灌注30min。连续观察记录Ⅱ导联心电图,并进行室性心律失常评分。通过多导生理记录仪同步记录左室收缩压(LVSP),左室舒张末压(LVEDP)、左心室上升最大速率(+dp/dtmax)、左心室下降最大速率(-dp/dtmax)、心率(HR),以评定心功能。采用用伊文思蓝+氯化三苯硝基四氮唑红双重染色及称重法测量心肌梗死范围。手术结束后处死动物取左心室,将标本分为两部分,一部分浸入10%福尔马林溶液,一部分冻存于-80℃备用。常规HE染色观察心肌组织形态学改变；采用TUNEL法检测心肌细胞凋亡；分别采用免疫组化法及免疫印迹法检测心肌PHB蛋白的表达。细胞水平：采用差数贴壁法体外分离培养新生乳鼠心肌细胞,以200μmol／L过氧化氢(H2O2)作用2h模拟心肌细胞氧化应激模型,分别以丹参酮ⅡA高剂量(1×10-4mol/L)、中剂量(5×10-5mol/L)、低剂量(1×10-5mol/L)在造模前干预24h。倒置相差显微镜观察心肌细胞形态学改变,采用MTT法检测细胞活力,流式细胞仪检测细胞凋亡率、细胞内游离钙浓度([Ca2+]i)及心肌细胞线粒体膜电位(MMP)的变化,检测心肌细胞膜Na+-K+-ATP酶、Ca2+-Mg2+-ATP酶活性,并检测心肌细胞总抗氧化能力(T-AOC)、LDH活性、MDA含量、SOD活力、GSH-PX活力、CAT活力。分别采用免疫组化法及免疫印迹法检测心肌PHB蛋白的表达。通过转染PHB siRNA后并检测上述各指标的变化,研究PHB蛋白功能。统计分析：实验数据采用SPSS13.0统计软件进行分析。计量资料数据以x±s表示。对数据进行正态性检验和方差齐性检验。若符合正态分布和方差齐性,则采用方差分析。若不符合正态分布和方差齐性,则采用秩和检验。方差齐性时两两比较采用LSD法,方差不齐时两两比较采用Tamhane’s T2法。多组计数资料的比较(各组AV评分)采用完全随机设计资料的Kruskal-Wallis检验方法。各实验组不同缺血及再灌注时间点血流动力学指标采用重复测量方差分析。检验显著性水准a=0.05。三、结果：(1)丹参酮ⅡA抗缺血再灌注损伤作用。①室性心律失常评分。SH组大鼠偶见室性早搏,未见其他心律失常；MI/R组均发生心律失常,大部分在冠脉再通1分钟左右开始出现,以频发室早、室速、室颤等为主,其中2只大鼠因室颤死亡；丹参酮ⅡA各剂量组均可见不同程度心律失常。心律失常评分由高到底依次为MI／R组、TanⅡA低剂量组、TanⅡA中剂量组、TanⅡA高剂量组、SH组。经统计学检验,各组间心律失常评分有显著差异(x2=29.118,P=0.000)。②心肌梗死面积测定。MI／R组大鼠出现明显的心肌缺血及梗死表现,心肌梗死面积最大；丹参酮ⅡA各剂量组梗死面积呈浓度依赖性减小。经统计学检验,各组间心肌梗死面积差异有统计学意义(F=95.049,P=0.000)。③血液动力学测定。缺血及再灌注损伤时各组间大鼠心率(HR)、左室收缩末压(LVSP)、左室收缩及舒张最大速率(±dp/dtmax)由高到底依次为SH组、丹参酮ⅡA高、中、低剂量组、MI／R组,而左室舒张末压(LVEDP)由低到高依次为SH组、丹参酮ⅡA高、中、低剂量组、MI／R组。经统计学分析,缺血及再灌注时各组间大鼠血流动力学参数HR、LVSP、LVEDSP、±dp/dtmax差异均有统计学意义(P均<0.05),以上结果表明缺血-再灌注损伤时大鼠整体心脏收缩及舒张功能均受损,丹参酮ⅡA可呈剂量依赖性改善大鼠收缩及舒张功能。不同缺血和再灌注时间点血流动力学检测结果表明,大鼠HR、LVSP、±dp/dtmax随着缺血及再灌注时间的延长而降低,而LVEDP则随着缺血及再灌注时间的延长而增加,尤以再灌注期变化更为显著,表明缺血-再灌注时间长短显著影响大鼠血流动力学改变,再灌注损伤较缺血更为严重。④组织学观察。SH组大鼠心肌细胞排列整齐,着色均匀,胞膜完整,无变性、坏死等改变；MI／R组大鼠心肌细胞排列紊乱,着色不均匀,部分区域心肌细胞浊肿；丹参酮ⅡA各剂量组心肌细胞变性与坏死改变程度较MI／R组呈剂量依赖性减轻。⑤细胞凋亡指数。MI／R组有大量凋亡细胞,凋亡细胞的细胞核内呈现较强的TUNEL阳性染色。丹参酮ⅡA呈剂量依赖性降低心肌细胞凋亡指数,各组间凋亡指数差异有统计学意义(x2=186.968,P=0.000)。(2)丹参酮ⅡA抗过氧化氢损伤的作用及机制。①筛选H2O2及丹参酮ⅡA浓度。不同浓度H2O2(50-800μmmol/L)作用于心肌细胞2h后,细胞活力呈浓度依赖性降低。以浓度为1×10-5mol/L、5×10-5mol/L、1×10-4mol/L丹参酮ⅡA预处理细胞24h后,细胞活力与正常对照组比较差异无统计学意义(P均>0.05),而丹参酮ⅡA浓度为5×10-4mol/L时,细胞活力较正常对照组显著降低(P=0.003)。②细胞形态学改变：倒置显微镜下见正常心肌细胞培养3天后呈梭形或菱形,细胞伸出伪足并交织成网,搏动同步化。200μmmol/L H2O2作用2h后,大量心肌细胞伪足缩短或消失,胞浆浓缩,核固缩,搏动减弱。丹参酮ⅡA预处理后心肌细胞形态得到明显改善。③对细胞凋亡相关指标的影响。模型组细胞凋亡率和细胞内钙离子浓度较正常对照组显著增高,线粒体膜电位显著降低(P均=0.000)。心肌细胞经不同浓度丹参酮ⅡA预先处理后,细胞凋亡率和细胞内钙离子浓度呈浓度依赖性降低,而线粒体膜电位则呈浓度依赖性升高,与模型组比较,差异有统计学意义(P均=0.000)。④对氧化平衡体系的影响。模型组心肌细胞总抗氧化能力(T-AOC)、SOD活性、GSH-PX活性、CAT活性显著降低,而LDH活性、MDA含量则显著增加；丹参酮ⅡA可呈剂量依赖性增加T-AOC、SOD活性、GSH-PX活性、CAT活性,降低LDH活性、MDA含量,各组间比较差异具有统计学意义(P均=0.000)。⑤心肌细胞经H202处理2h后,心肌细胞Na+-K+-ATP酶、Ca2+-Mg2+-ATP酶活性较正常对照组显著降低；心肌细胞经各浓度丹参酮ⅡA预先处理后,细胞Na+-K+-ATP酶、Ca2+-Mg2+-ATP酶活性呈浓度依赖性升高,各组间差异具有统计学意义(P均=0.000)。(3)PHB蛋白功能。①PHB siRNA转染效率。不同浓度PHB siRNA(5 nM-30 nM)转染效率呈浓度依赖性增高,差异具有统计学意义(F=295.323,P=0.000)。心肌细胞转染PHB siRNA后,PHB蛋白表达下降了74.11±3.23%。②PHB siRNA转染对心肌细胞功能的影响。PHB siRNA干扰PHB蛋白表达后,心肌细胞凋亡率、细胞内钙浓度显著增高,线粒体膜电位则显著降低(P均<0.05)；心肌细胞总抗氧化能力、SOD活性、GSH-PX活性、CAT活性显著降低,而LDH活性、MDA含量则显著增加(P均=0.000)；细胞Na+-K+-ATP酶、Ca2+-Mg2+-ATP酶活性较正常对照组显著降低(P均=0.000)。(4)缺血再灌注损伤、氧化应激及丹参酮ⅡA对PHB蛋白表达的影响。在动物水平,MI／R组PHB蛋白阳性表达较SH组显著增高(P=0.000)；与MI／R组比较,丹参酮ⅡA可呈剂量依赖性降低PHB蛋白表达,各组间差异有统计学意义(P=0.000)。在细胞水平,H202所致的氧化应激模型组PHB蛋白表达较正常对照组也显著增加(P均=0.000)；与模型组比较,丹参酮ⅡA可呈剂量依赖性降低PHB表达,各组间差异有统计学意义(P)均=0.000)。四、结论：(1)心肌缺血再灌注损伤容易诱发室性心律失常,可导致心肌组织结构的破坏,心功能的下降。丹参酮ⅡA对大鼠缺血再灌注损伤具有良好的保护作用,可以明显降低心律失常的严重程度,防止心肌组织结构的破坏、改善心功能、抑制细胞凋亡。(2)过氧化氢介导的心肌损伤可导致氧化平衡体系的破坏、能量代谢障碍、细胞内钙超载、细胞凋亡等病理改变。丹参酮ⅡA可以抑制过氧化氢诱导的心肌细胞损伤,这可能与其增强细胞抗氧化酶活力,降低脂质过氧化、促进细胞能量合成、抑制细胞内钙超载有关。(3)PHB蛋白具有显著的抗氧化作用,并可增加ATP酶活性、抑制细胞钙超载、抑制心肌细胞凋亡作用。(4)PHB蛋白在心肌缺血再灌注损伤及心肌细胞氧化损伤时呈高表达,其表达增加可能为心肌细胞内源性抗损伤作用的代偿性反应。丹参酮ⅡA可显著降低心肌缺血再灌注损伤时及氧化损伤时心肌细胞中PHB蛋白表达水平,其机制可能是通过抗氧化作用减轻心肌细胞的氧化应激损伤,从而减少心肌细胞代偿性的PHB蛋白表达。更多还原

【Abstract】 OBJECTIVE:It is generally acknowledged that restoring blood perfusion could be the basic measure in prevention of ischemic damage. Recently, thrombolytic therapy, coronary angioplasty and some other new techniques make ischemic tissues and organs restore blood perfusion. Although timely reperfusion of acute ischemic myocardium is essential for myocardial salvage, reperfusion may also result in a unique form of myocardial damage, which called (myocardial ischemia/reperfusion injury, MI/RI). MI/RI result in cardiac disfunction, which bad lead to life quality and prognosis in patients.For this reason, it is of great practical improtance to explore effective measure in prevention of MI/RI and the mechanisms.In our previous proteomics study, we found that Prohibitin protein was in hight expression in the MI/R heart compared with the control hearts, the result was consistent with foreign research. However, the study of PHB function was mostly focused on oncology, the specific cellular function of PHB in myocardial cells has not been clearly elucidated.Salvia miltiorrhiza Bunge has long been used for prevention and treatment of cardiovascular diseases in China. TanshinoneⅡA is one of the major lipid-soluble pharmacologic constituents of of Salvia miltiorrhiza Bunge.The goal of this study was to investigate the protective effect of tanshinoneⅡA in MI/RI. In order to study the machanism of tanshinoneⅡA, we focus our study on antioxidation, antagonizing calcium overload, promoting energy synthesis and the regulation of PHBMETHOD:Animal level:Male Wistar rats were randomly divided into 5 groups, they were Sham operated group (SH group), myocardial ischemia-reperfusion group (MI/R group), high-dose tanshinoneⅡA group, middle-dose tanshinoneⅡA group and low-dose tanshinoneⅡA group. Rats in both SH group and MI/R group were drenched with normal sodium, rats in different doses of tanshinoneⅡA group were drenched with different doses of tanshinoneⅡA physic liquor(20,10,5mg/kg`d), all of administration was performed twice a day. The animal model was conducted by ligating the left anterior descending coronary artery for 15 min and then loosening for 30min.Ⅱlead ECG was continously observed and recorded in order to conduct ventricular arrhythmia score. LVSP, LVEDP,+dp/dtmax,-dp/dtmax and HR was simultaneous recorded by multilead physiological instrument. The size of myocardial infarction was determined by double-staning of evans blue and TTC and weighing. After the operation, took out of the heart, washed it in normal sodium, separated the left ventricle and divided it into two parts. One part was dipped into 10% neutral formaldehyde for fixation, another part was stored in refrigerator with the temperature of -80℃.HE dyeing was used to observe the morphous changes of myocardium in each group. Myocardial cell apoptosis was determined by TUNEL method, and the expression of PHB was determined by immunohistochemical method and western blotting method. Cellular level:Primary cultured neonate rat myocardial cell was cultured in medium with 200μmol/L hydrogen peroxide to simulate oxidative stress.In different doses of TanshinoneⅡA group, the medium was supplemented with different concentrations of TanshinoneⅡA in advangce(1×10-4mol/L,5×10-5mol/L,1×10-5mol/L) for 24 h. The morphological changes of myocardial cell was observed by inverted phase contrast microscope. Cell viability was determined by MTT method.apoptosis rate was determined by Annexin V/PI bivariate dyeing, and it was detected by flow cytometric analysis,so as the level of [Ca2+]i and mitochondrial membrane potential.Besides, Na+-K+-ATPase and Ca2+-Mg2+-ATPase, T-AOC, LDH, MDA, SOD, GSH-PX, CAT were also evaluated in each group.The expression of PHB was determined by immunohistochemical method and western blotting method. siRNA PHB interference was transfected to myocardial cell in order to study the function of PHB.Statistical analysis:The data was analyzed by SPSS13.0. measurement data was presented as the means±SEM. Test of normality and homogeneity of variance was done in each group. If datas accorded with normality and homogeneity of variance, statistical analysis was made by one-way ANOVA followed by LSD test, otherwise statistical analysis was made by rank sum test, and multiple comparison was made by Tamhane’s T2.Multiple enumeration data was analyzed by Kruskal-Wallis. Hemodynamics parameters in different time points was analyzed by repetitive measurement and analysis of variance. Level of significanceα=0.05RESULT:(1)Effect of Tanshinone II A on cardiac function in myocardial ischemia/reperfusion injured rats.①Ventricular arrhythmia score. Rats in SH group appeared with premature ventricualr contraction occasionally. Howerer, all rats in MI/R group developed arrhythmia, mainly including premature ventricualr contraction, ventricular tachycardia and ventricular fibrillation,2 rats in the group die of ventricular fibrillation. In different doses of TanshinoneⅡA group, there were occurrence of arrhythmia at different degree. The ventricular arrhythmia score in each group in the sequence from high to low was MI/R group, low-dose of Tanshinone II A, middle-dose of TanshinoneⅡA, high-dose of Tanshinone II A, SH group. The difference of AV score in all the group was significant(x2=29.118, P=0.000).②The area of myocardial infarction. Rats in MI/R group suffered from serious myocardial ischemia and myocardial infarction, so the infarction size was the largest in all the group. TanshinoneⅡA presented decreasing infarction size in a dose dependent manner. The difference of infarction size in all the group was significant(F=95.049,P=0.000).③Hemodynamics parameters.HR, LVSP, and±dp/dtmax was significantly decreased in the sequence of SH group, TanⅡA high dose group, TanⅡA middle dose group, TanⅡA low dose group and MI/R group, while LVEDP was significantl increased in the same sequence. In different doses of TanshinoneⅡA groups, the hemodynamics parameters were improved in a dose dependent manner. The difference of hemodynamics parameters in all the group was significant(each P<0.05).HR, LVSP, and±dp/dtmax was significantly decreased with prolongation of time of ischemia and reperfusion, while LVEDP was significantl increased with prolongation of time of ischemia and reperfusion, the difference of hemodynamics parameters between each time spots was significant(each P<0.05).④Observation of histology.myocardium structure in SH group was characterized with well-arranged myocardial cells, well-distributed HE dyeing and integrated cell membrane. While in MI/R group, the myocardial cells were chaotic, HE dyeing was uneven,cells in some parts of the heart were cloudy swelling, the cardiac muscle fibers was unclear or disappeared.In different doses of TanshinoneⅡA groups, some myocardial cells developed degeneration and necrosis, but it was not severe, the improvement of structure was dose dependent.⑤Cell apoptosis index. There were a large number of apoptotic cells in MI/R group. The apoptotic cells characterized with srtong TUNEL positive staining. Cell apoptosis index in different TanshinoneⅡA groups was significantly increased dose dependently. The difference of cell apoptosis index in all the group was was significant(x2=186.968,P=0.000).(2) Antioxidation of TanshinoneⅡA and the mechanism.①The optimum concentration of H2O2 and TanshinoneⅡA. Myocardial cells were exposed to various concentrations of H2O2 (50-800μmol/L)for 2 h. Cell cytotoxicity was determined by MTT assay, the result was that H2O2 significantly inhibited the viability of both cell in a dose-dependent manner. When myocardial cells were exposed to TanshinoneⅡA at the concentration of 1×10-5mol/L,5×10-5mol/L,1×10-4mol/L for 24 h, there was no statistical significance in cell viability compared with normal control group(each P>0.05).But when the contration was 5×10-4mol/L, cell viability was significantly decreased(P=0.003).②orphological changes of cardiocytes. Normal cardiocyte were in fusiform or rhomubus shapes, with spurious leg interlaced like a net, cell beat simultaneously, cardiocytes treated with H2O2 for 2 h beat weakly, with spurious leg shortened or dispeared, endochylema condensed. The appearance of cardiocytes in TanshinoneⅡA groups had been improved.③Cell apoptosis related indexes.Apoptosis rate, intracellular concentration of calcium was significantly increased, and mitochondrial membrane potential levels was significantly down-regulated by treatment with H2O2 (200μmol/L) for 2h. Pretreatment with TanshinoneⅡA for 24 h provided significant protection against down-regulation of apoptosis rate, intracellular concentration of calcium caused by H2O2, while the mitochondrial membrane potential levels was significantly up-regulated.④The effect of TanshinoneⅡA on oxidation balance system. T-AOC,SOD activity, GSH-PXA activity, CAT activity was significantly decreased in model group, while LDH activity and MDA content was significanltly increased. T-AOC, SOD activity, GSH-PXA activity and CAT activity was significantly increased in TanshinoneⅡA groups in a dose dependent manner, and LDH activity and MDA content was significanltly decreased in the manner. The difference in all groups was significant(each P=0.000).⑤Na+-K+-ATPase, and Ca2+-Mg2+-ATPase was significantly decreased in model group, while pretreatment with TanshinoneⅡA for 24 h provided significant increasement of Na+-K+-ATPase, and Ca2+-Mg2+-ATPase in a dose dependent manner. The difference in all group was significant(each P=0.000).(3)The function of PHB.①siRNA transfection efficiency. The transfection efficiency of siRNA PHB was significantly increased depended on siRNA PHB concentration(F=295.323,P=0.000).The expression of prohibitin reduced by 74.11±3.23% in condition of siRNA PHB.Besides, apoptosis rate, [Ca2+]i, LDH activity and MDA content was significantly increased, and MMP levels(P<0.05),T-AOC, SOD activity, GSH-PXA activity, CAT activity, Na+-K+-ATPase and Ca2+-Mg2+-ATPasewas significantly decreased(each P=0.000).(4)Effect of MI/R, oxidative stress and tanshinoneⅡA on PHB protein expression in myocardial cells.At animal level, the expression of PHB was remarkably increased in MI/R group, and tanshinoneⅡA attenuated PHB expression in a dose dependent manner, the difference in all groups was significant(P=0.000).At cell level, the expression of prohibitin increased remarkably under oxidative stress conditions, the increase was attenuated by tanshinoneⅡA too, the difference in all groups was significant(P=0.000).CONCLUSION(1)Myocardial ischemia reperfusion injury could result in ventricular arrhythmia, damage on myocardial structure, cardiac dysfunction. TanshinoneⅡA show satisfactory protection against the above damage by MI/RI.(2) Hydrogen peroxide mediated myocardial injury through oxidative imbalance, energy metabolic disturbance, intracellular calcium overload, apoptosis and so on. TanshinoneⅡA protect myocardial cell against injury probably by strengthen the activity of antioxidant enzyme and energy synthesis, and inhibiting lipid peroxidation and intracellular calcium overload.(3)PHB showed multiple effect on myocardial cells, such as remarkable antioxidation, enhancing ATPase activity, and inhibition of intracellular calcium overload and apoptosis.(4) PHB protein was in high expression in condition of MI/RI and oxidative stress.TanshinoneⅡA decreased the expression of PHB probably through the inhibition of oxidative damage, so that compensatory increase by oxidative stress may be inhibited.更多还原