【作者】 赵改霞；

【导师】 高海青；

【作者基本信息】 山东大学 ， 老年医学， 2010， 博士

【摘要】 研究背景早期再灌注治疗对于缺血心肌的存活至关重要。然而,再灌注治疗被Braunwald和Kloner称为“双刃剑”,因为再灌注本身可以导致加速的心肌损伤和缺血单独导致的心肌损伤之外的新生的心肌损伤。这就导致了一系列再灌注相关的病理过程,统称为再灌注损伤(reperfusion injury)。再灌注损伤在各种各样的再灌注治疗中都可以观察到,包括经皮冠状动脉介入治疗(percutaneous coronary intervention, PCI)、溶栓治疗和冠状动脉旁路移植手术。再灌注损伤的不同临床表现包括心肌坏死、心肌凋亡、再灌注性心律失常(reperfusion arrhythmias)、心肌顿抑和内皮细胞功能障碍、微血管功能障碍包括无复流现象。然而,再灌注性心律失常尤其是再灌注导致的室性心动过速简称室速(ventricular tachycardia, VT)和心室纤颤简称室颤(ventricular fibrillation,VF)仍然是再灌注后猝死的最主要原因。因此,必须尽最大努力减少再灌注性心律失常引起的猝死事件。再灌注性心律失常的分子机制包括心肌传导恢复的异质性和非完全再灌注的抵抗期、折返机制、异常激动和由Ca2+超载、自由基引起的损伤。再灌注性心律失常到目前为止仍然没有得到令人满意的治疗。葡萄子原花青素(grape seed proanthocyanidin extracts, GSPE)是从葡萄籽中获得的一种天然的具有生物活性的多酚类黄酮复合物,具有显著的自由基清除能力。GSPE起药理作用的主要成分是低聚体原花青素。众所周知,自由基和氧化应激在一系列心血管疾病的病理生理过程中有着举足轻重的作用,包括心衰、心脏瓣膜病、心肌病、心肌肥大、冠状动脉粥样硬化性心脏病。由于GSPE出色的抗氧化能力,GSPE表现出一定的心血管保护作用,包括抗动脉粥样硬化、降低血压、调节血脂等。国内外研究发现,GSPE还具有保护心肌缺血再灌注损伤的作用。实验和临床研究发现,缺血再灌注损伤主要与氧自由基损伤及钙超载相关。GSPE作为一种强大的抗氧化剂,具有出色的自由基清除能力,对于心肌缺血再灌注损伤有着一定的保护作用,它可以促进再灌注后心功能的恢复,减少心肌梗死面积,抑制心肌细胞的凋亡等。在研究GSPE保护缺血再灌注损伤的实验中,人们发现,GSPE具有抗再灌注性心律失常的作用。研究表明,在离体大鼠心脏缺血再灌注模型中,GSPE可以降低再灌注引起的VT、VF的发生。然而,此保护作用的分子机制目前还不十分清楚。心脏组织的比较蛋白质组学研究能够提供心肌对于各种心脏性疾病所作反应的特定早期分子机制,因此,比较蛋白质组学研究对于心血管疾病的特效、有效的诊断和治疗可能有着重要的启示。随着蛋白质组学技术的发展,一种新的比较蛋白质组学技术-同位素标记相对和绝对定量技术(isobaric tags for relative and absolute quantification, iTRAQ)被开发出来。iTRAQ技术是一种新的、功能强大的可以同时对四种样品进行绝对和相对定量研究的方法。iTRAQ试剂是可以与氨基酸N端及赖氨酸侧链反应的胺标记同重元素。在质谱图中,任何一种iTRAQ试剂标记的不同样本中的同一肽段表现为相同的质荷比。而在串联质谱中,报道离子表现为不同质荷比(114-117)的峰,因此,根据波峰的高度及面积,就可以得到蛋白质的定量信息。虽然荧光差异凝胶电泳(difference gel electrophoresis, DIGE)和同位素亲和标记(isotope-coded affinity tags, ICAT)技术已经广泛应用于蛋白质研究,但是iTRAQ作为一种新的蛋白质绝对和相对定量技术,具有很好的精确性和重复性,弥补了DIGE及ICAT的不足,迅速被广大学者接受。GSPE抗再灌注性室性心律失常作用的分子机制可能涉及到减少自由基损伤、减轻钙超载等多个不同的分子机制,目前仍不十分清楚,还需要进一步的研究。目前已知的资料中,有关GSPE对大鼠在体缺血再灌注性心律失常的作用并没有文献报道。不同种属决定了他们对于缺血、再灌注和组织损伤程度耐受度的不同,考虑到这一方面,相比犬和人类,由于缺乏功能性冠状动脉侧支,大鼠心脏对于缺血再灌注损伤更为敏感。因此,本实验构建大鼠在体心脏缺血再灌注模型,观察GSPE抗大鼠在体心肌再灌注性室性心律失常的作用。此模型模拟临床上心肌梗死或者冠状动脉内血栓形成的环境,对于再灌注性室性心律失常较为敏感,且具有较高的可复制性。蛋白质组学技术日趋完善与成熟,因而,借助先进的分子生物学手段,研究GSPE对缺血再灌注心律失常的保护作用,深入探讨GSPE在细胞水平的生物学作用机制,寻找其作用靶蛋白和靶位相关蛋白,进一步明确临床对缺血再灌注性心律失常病理生理学机制的认识,寻找药物治疗的新靶位,开辟新的心血管疾病治疗途径具有重要的理论和实际意义。研究目的(1)本实验通过建立大鼠在体心肌缺血再灌注模型,观察GSPE对大鼠在体心肌缺血再灌注损伤引起的再灌注性室性心律失常的保护作用。(2)应用比较蛋白质组学方法-iTRAQ,研究缺血心肌的差异蛋白质的表达,阐明GSPE抗再灌注性心律失常的分子机制,并希翼找到此保护作用的关键蛋白质,发现新的有抗再灌注性心律失常作用的药物靶点。研究方法1、建立大鼠在体心脏缺血再灌注模型雄性wistar大鼠66只,体重为180g-200g,随机分到3组：假手术组(sham)22只,缺血再灌注组(IR)22只,GSPE治疗组(GSPE)22只。GSPE组用GSPE溶液灌胃(200mg／kg·d)共4周,Sham组和IR组用相同体积的生理盐水灌胃共4周。IR组和GSPE组行冠状动脉左前降支(left anterior descending coronary artery, LAD)结扎术形成心肌缺血,结扎30min后松开活结恢复血流,形成再灌注模型,再灌流120min。Sham组只穿线不结扎。应用BL410生物信号采集系统记录整个实验期间ECG,采用再灌注期间的心律失常数据进行数据分析。实验结束后,立即处死大鼠,迅速取出心脏。切取心尖部分电镜观察心肌超微结构改变。IR组和GSPE组各取6只大鼠心脏,用NBT染色行心肌梗死面积测定。2、比较蛋白质组学-iTRAQ分析再灌注结束后,立即取出心脏,取心尖部分放入液氮罐冷冻保存以进行后期的蛋白质组学实验。将心肌组织进行差速离心,以富集蛋白,然后用trypsin进行蛋白消化得到肽段。用iTRAQ试剂标记消化后的肽段,114标记假手术组,116标记GSPE组,118标记IR组。将标记的肽段混合后运用强阳离子交换柱(strong cation exchange, SCX)和C18柱进行分级分离,最后用MALDI-TOF/TOF和microQ-TOF进行质谱鉴定。研究结果1、再灌注性VT、VF发生率本实验证实,在大鼠在体心肌缺血再灌注模型中,GSPE能够显著地降低再灌注引起的VT、VF的发生率。Sham组无心律失常发生。再灌注导致的VF从IR组的64%降至GSPE组的17%(P＜0.05)。再灌注导致的VT发生率从IR组的91%降至GSPE组的33%(P＜0.01)。2、透射电镜观察心肌微结构透射电镜观察到GSPE具有明显的心肌保护作用,同GSPE组的心肌组织相比,IR组的心肌组织表现出更多的受损特征。Sham组的标本表现出了正常心肌细胞的特征。IR组的标本观察到明显的细胞核分布的不正常,心肌纤维的断裂和缺失也可以观察到,很多排列紊乱的线粒体代替了心肌纤维,还可以观察到线粒体空泡化。而在GSPE组,可以观察到明显的改善,心肌细胞受损的表现减少,只有少部分的线粒体和细胞核表现不正常,心肌细胞得到了保护。3、心肌梗死面积测定实验结果发现,GSPE能够显著减少缺血再灌注心肌梗死面积。心肌梗死面积从IR组的26.7%±2.3%下降至GSPE组的13.3%±3.3%(P＜0.01)4、差异蛋白的鉴定将心肌组织进行差速离心富集蛋白,然后用trypsin进行蛋白消化得到肽段。用iTRAQ试剂标记消化后的肽段,114标记假手术组, 116标记GSPE组,118标记IR组。将标记的肽段混合后运用强阳离子交换柱和C18柱进行分级分离,最后用MALDI-TOF/TOF和microQ-TOF进行质谱鉴定。最终发现91个差异蛋白点,其中两个或两个以上肽段进行定量鉴定的蛋白有67个,采用单个肽段进行定量鉴定的蛋白有24个。分别用DATA ANALYSIS 4.0和WARP-LC软件分析质谱数据,MASCOT搜索质谱匹配蛋白标本差异表达蛋白点,在鉴定出的91个蛋白中,以sham组为对照,43种蛋白质的表达在IR组下降,而在GSPE组中则被显著上调；以sham组为对照,21种蛋白质的表达在IR组上升,而在GSPE组则被明显下调；余下的27种蛋白在GSPE组与IR组的变化不明显。这91个差异蛋白的功能涉及到离子转运、脂肪酸的氧化、钙离子结合、细胞粘附、凋亡的调节和信号转导等,这预示着这些过程可能参与了GSPE抗再灌注损伤。结论1、本实验构建大鼠在体心脏缺血再灌注损伤模型,首次观察到GSPE具有抗大鼠在体心脏再灌注性室性心律失常VT、VF的保护作用。此模型模拟临床上心肌梗死或者冠状动脉内血栓形成的环境,提示GSPE有可能成为有效的抗再灌注性心律失常药物。2、比较蛋白质组学-iTRAQ,具有较好的精确性、重复性和定量效果,能够对多组样本同时进行比较分析,可以用于研究GSPE抗再灌注性心律失常作用的分子机制,找到此保护作用的关键蛋白,发现新的有抗再灌注性心律失常作用的药物靶点。3、应用iTRAQ,鉴定出91个差异蛋白,其功能涉及到离子转运、脂肪酸的氧化、钙离子结合、细胞粘附、凋亡的调节和信号转导等,这预示着这些过程可能参与了GSPE抗再灌注损伤。4、在这些差异蛋白中,以sham组为对照,Na+/k+-ATPaseα1亚基和HSP60的表达在IR组明显下降,在GSPE组则显著地提高,这2个蛋白可能是GSPE抗再灌注性心律失常作用的关键蛋白。Na+/K+-ATPaseα1亚基的增加可能导致Na+/K+-ATP酶活性的升高,继而引起心肌细胞内Ca2+超载的减少；提高再灌注后心肌细胞的HSP60的表达,可能会抑制再灌注后心肌细胞的凋亡。GSPE可能通过这2个途径最终减少了再灌注性心律失常的发生。更多还原

【Abstract】 Research backgroundEarly reperfusion is crucial for the survival of ischemic myocardium. However, reperfusion has been referred by Braunwald and Kloner as the "double edged sword" because reperfusion itself may lead to accelerated and additional myocardial injury beyond that generated by ischemia alone. This results in a spectrum of reperfusion-associated pathologies, collectively called "reperfusion injury". Reperfusion injury has been observed in each of reperfusion therapies including percutaneous coronary intervention (PCI), thrombolysis and coronary bypass grafting. The different clinical manifestations of this injury include myocardial necrosis, myocardial apoptosis, reperfusion arrhythmias, myocardial stunning and endothelial-and microvascular dysfunction including the no-reflow phenomenon. However, VT and VF induced by reperfusion remain the most important causes of sudden death following spontaneous restoration of antegrade flow. Therefore, every effort must be made to minimize sudden cardiac death caused by reperfusion arrhythmias. The mechanisms of reperfusion arrhythmias may include heterogeneous recovery of conduction and a refractory period of incomplete reperfusion, reentry, abnormal automaticities, and activities triggered by Ca2+ overload and free radicals. However, the details of the mechanisms remain unclear and reperfusion arrhythmia has not received satisfactory treatment.GSPE is a natural, standardized, water-ethanol extract from red grape seeds which has great free radical scavenging ability. The main pharmacological effective components of GSPE are oligomeric proanthocyanidins. Free radicals and oxidative stress play a crucial role in the pathophysiology of abroad spectrum of cardiovascular diseases including congestive heart failure, valvular heart disease, cardiomyopathy, hypertrophy, atherosclerosis and ischemic heart disease. A series of studies were conducted using GSPE to demonstrate its cardioprotective ability in animals and humans.Proteomics is the large scale study of proteins, usually by biochemical methods. Many studies of cardiovascular disease and cancer have used proteomics techniques. Nonetheless, proteomics has already revolutionized the discovery process for antimicrobial drugs by accelerating target identification and evaluation, assay development and the follow-up support for medicinal chemistry programs. It is important to note that proteomics techniques could discover not only novel biomarkers, but also proteins that may actively participate in cardiovascular process. Proteomic studies of heart tissues can provide new insights into the specific early molecular mechanisms’that underlie the responses to IR injury and therefore may have vital implications for the specificity and efficacy of diagnosis and treatment. Our previous work had shown GSPE reduced reperfusion-induced arrhythmias in rabbits. In isolated rat hearts GSPE had been proved to reduce the incidence of reperfusion-induced VF and VT. However the mechanism of this protection against reperfusion-induced VT, VF by GSPE remained unclear. With the development of proteomic techniques a quantitative proteomics- isobaric tags for relative and absolute quantification (iTRAQ) was introduced. iTRAQ employs primary amine reactive isobaric tags to derivatize peptides at the N-termini and lysine side-chains. Therefore, theoretically, iTRAQ labels most of the peptide fragments in a digested mixture. Although the peptides labeled with any of the iTRAQ reagents are indistinguishable in single MS analysis, tag fragmentation during MS/MS analysis produces reporter ions (m/z=114,115,116, and 117), which provide quantitative information upon integration of the peak areas.An important starting point for drug discovery is to supply a validated therapeutic target, and the proteomic study of GSPE using iTRAQ on reperfusion arrhythmias may yet provide insights into the mechanism of reperfusion-induced ventricular arrhythmias, and provide new starting points for antiarrhythmia drugs.Aims1. We illustrated the ischemia/reperfusion modal in rats in vivo to observe whether GSPE could reduce the accidents of reperfusion-induced VT, VF.2. We applied iTRAQ combined with mass spectrometry to identify alterations in the expression of proteins in myocardial tissue in order to elucidate the potential molecular mechanism of the protective effects of GSPE on reperfusion-induced VT, VF and find potential new drug targets for reperfusion arrhythmias.Materials and methods1. IR Protocol in Rats in vivo Sixty six rats were randomly assigned to three groups:Sham group (n=22), IR group (n=22) and GSPE group (n=22). Twenty two Rats in GSPE group were fed with GSPE (200 mg/kg·d for 4 weeks, intragastricly) while the other 44 rats were given 0.9% NaCl intragastricly only for 4 weeks. Rats (280-340g) were anaesthetized with 20% Urethane(0.5ml/100g, i.p.). Body temperature was routinely monitored via a rectal thermometer using a heated operating platform and appropriate heating lamps. Subdermal electrodes were placed to allow the determination of a leadⅡelectrocardiogram (ECG) which was recorded continuously throughout the experiment by the BL410 multichannel acquisition system (Chengdu Taimeng, China) for physiological signals. The incidence of reperfusion-induced VF and VT during reperfusion was analyzed. A 4-0 polypropylene suture through a 3-mm long polyethylene tube (PE-10) was then placed around the left anterior descending coronary artery (LAD), proximal to its main branching point. Then the LAD was ligated with the 4-0 suture. The coronary artery was occluded by tightening of the occluder. After 30min of ischemia, in the IR and GSPE groups, the suture around the LAD was removed and the occurrence of reperfusion was assessed by the observation of blood flow in the epicardial coronary arteries through a surgical microscope. The ischemic myocardium was reperfused for 120min. Sham group were made following an identical procedure but without the actual tying of the polypropylene suture. At the end of each experiment, the heart was excised. Ultrathin sections cut from the ischemic area were stained with uranylacetate and lead citrate and were examined with an H-800 electron microscope (Hitachi, Japan). With NBT stain, infarct sizes of IR group and GSPE group were assessed.2. iTRAQ ProteomicsThe proteins-enriched fractions were digested by tripsin. The resulting complex peptide mixtures were labeled with iTRAQ reagents (114 for the peptides of Sham group,116 for the peptides of GSPE group, and 118 for the peptides of IR group respectively), and then separated by SCX and C18 fractionation. And 6 SCX fractions were analyzed by MALDI-TOF/TOF and microQ-TOF.Results1. Reduction of the Incidence of Reperfusion-induced VT, VF by GSPE Our results demonstrated that GSPE can significantly reduce the incidence of reperfusion-induced VT, VF in rats in vivo. There was no arrhythmia happened in Sham group. The incidence of reperfusion-induced VF was reduced from 64% in rats without GSPE-treated (IR group) to 17% in rats treated with GSPE (GSPE group) (P< 0.05). The incidence of reperfusion-induced VT was reduced from 91% in rats without GSPE-treated to 33% in rats with GSPE-treated (P<0.01).2. Results of Transmission Electron Microscopic ObservationSpecimens from sham group showed normal features:the cardiac cells were rich in mitochondria arranged between myofibrils in rows with myofibrils arranged evenly and normal intercalated disks. The sarcomere was of the same length. Nuclear membrane was integrated and chromatin was normally distributed. In IR group, the structure abnormalities of nuclear were apparently observed. Disarrangement, breakage and local absence of myofibrils were observed. Numerous disarranged mitochondria substituted for myofibrils. Mitochondria vacuolization and crista loss were observed. In GSPE group there were more apparent differences. The protective effect was acquired in that there were only moderate mitochondrial and nucleolus abnormalities in cardiac myocytes.3. Infarct size Infarct size was noticeably reduced in the GSPE-fed group [13.3%±3.3%) as compared to the IR group (26.7%±2.3%) (P<0.01).4. Identification of ProteinsWe identified 91 differential proteins among which the expression of 53 proteins was significantly changed. The expression of 21 proteins were increased in IR group when compared to sham group, while these were significantly down-regulated in GSPE group and the expression of 43 proteins were decreased in IR group when compared to sham group while these were significantly up-regulated in GSPE group. The function of all 91 proteins consists of ion transport, fatty acid oxygenation, calcium binding, cellular adhesion, regulation of apoptosis and signal transport which indicated that these functions maybe involved in the protective effects of GSPE on ischemia/reperfusion injury.Conclusions1. Our results demonstrated for the first time that GSPE can significantly reduce the incidence of reperfusion-induced VT, VF in rats in vivo. Since ischemia/reperfusion models of rat in vivo mimic clinical myocardial infarction or intracoronary thrombolysis or angioplasty situations, it is expected that GSPE may become effective drugs against reperfusion-induced arrhythmias.2. Comparative proteome-iTRAQ is more accurate and reproducible which can be used to elucidate the potential molecular mechanism of the protective effects of GSPE on reperfusion-induced VT, VF and find potential new drug targets for reperfusion arrhythmias.3. Using we identified 91 differential proteins. The function of all 91 proteins consists of ion transport, fatty acid oxygenation, calcium binding, cellular adhesion, regulation of apoptosis and signal transport which indicated that these functions maybe involved in the protective effects of GSPE on ischemia/reperfusion injury.4. We found that Na+/K+ ATPaseα1 subunit and HSP60 were decreased in IR group while it was significantly increased in GSPE group compared to sham group. These two proteins may be the key proteins involved in the protective effects of GSPE on reperfusion-induced arrhythmias. This change in subunit distribution may lead to the increase of Na+/K+-ATPase activity in GSPE treated rats which would ultimately protect hearts from reperfusion arrhythmia injury. The protection of increased expression of HSP60 in GSPE group maybe involved in against apoptotic cell death which in turn resulted in the reduction of the accidents of reperfusion-induced VT, VF in rats.更多还原