促红细胞生成素在大鼠心肌缺血再灌注损伤中的保护性作用研究
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摘要
研究背景目的:
冠心病是导致人类死亡的主要原因,全球每年约有380万男性和340万女性死于冠心病。在急性心肌梗死后,采用溶栓治疗、经皮冠状动脉介入术(percutaneous coronary intervention,PCI)或冠状动脉旁路移植术(coronary arterybypass graft,CABG)早期成功恢复心肌再灌注是缩小梗死范围和改善临床转归的最有效方法。但在动物实验和临床观察中发现,恢复血液再灌注后,部分缺血动物或患者细胞功能代谢障碍及结构破坏反而加重,因此将这种血液再灌注使缺血性损伤进一步加重的现象称为缺血再灌注损伤(ischemia reperfusion injury,IRI),它反常地减少了心肌再灌注的有益作用。急性心肌梗死动物模型研究提示,致死性再灌注损伤最多可占心肌梗死最终体积的50%,因此,防治心肌缺血再灌注损伤可以提高急性心肌梗死再灌注疗法的临床疗效,有关方面的研究已成为热点,但迄今为止还没有可用于临床的药物。
心肌缺血再灌注损伤可以产生4种类型的心功能障碍:①心肌顿抑,是指心脏在冠脉血流已恢复正常或接近正常后继续存在机械性功能障碍,是一种可逆性损伤,通常在数天或数周后恢复;②无复流现象,是指梗死相关冠脉再通时微血管血流遇到阻抗,缺血区域不能得到有效的再灌注;③再灌注心律失常;④致死性再灌注损伤。有学者研究发现:心肌缺血再灌注不仅能导致细胞坏死,而且还能引起细胞凋亡,也就是说细胞凋亡参与了心肌缺血再灌注损伤过程。目前认为,心肌缺血再灌注损伤的发生可能与氧反常、钙反常、pH反常和炎症反应等因素有关,而细胞凋亡是心肌缺血再灌注损伤的重要病理生理机制。在心肌缺血再灌注损伤中,细胞凋亡对维持组织形态、机能稳定具有十分重要的意义;然而,细胞凋亡的异常增加会加重心肌的破坏。再灌注时,同时存在的细胞坏死和凋亡导致心肌细胞数量减少和心脏功能的下降,共同影响着心肌缺血再灌注损伤的结果。没有被吞噬作用消除的凋亡细胞,受到活性氧攻击后,发生坏死是有可能的。细胞坏死和凋亡作为心肌缺血再灌注损伤中细胞死亡的两种方式,并不是一成不变的,研究已证实,ATP耗竭或炎症反应均可促使凋亡向坏死演变。为了了解凋亡对心脏的影响,学者通过各种方法抑制凋亡来观察心脏结构和功能的变化,结果证明,抑制心肌细胞凋亡能够减少缺血再灌注后的心肌梗死体积几乎达到50%-70%,改善心功能。目前认为心肌缺血再灌注损伤中产生的氧反常、钙反常等生化改变以及由此导致的线粒体损伤,三者单独启动或联合作用是许多凋亡诱导因素的共同通道,三者互相联系,互为因果。这些因素并不能直接引起细胞凋亡,而是通过一定的信号传递方式激活生存或死亡相关基因,然后将信号传递到核内切酶,而执行死亡的功能。因此阻断心肌缺血再灌注损伤中心肌细胞凋亡的信号转导能够阻止凋亡程序的执行,减少心肌细胞凋亡,防治心肌缺血再灌注损伤,重新恢复心肌细胞的功能。
促红细胞生成素(erythropoietin,EPO)是一种能刺激骨髓造血的唾液糖蛋白类激素,主要由肾皮质和髓质交界处的球旁细胞合成。1985年,人们利用基因重组技术成功合成重组人红细胞生成素(recombinant human erythropoietin,rHuEPO),用于治疗各种类型的贫血。内源性EPO分子量为34KD,rHuEPO分子量为30.4KD,其理化性质和生物学活性与内源性EPO相同。静脉给予rHuEPO的半衰期为4-12小时,皮下注射rHuEPO的生物利用度为23-42%。EPO是通过与靶细胞膜表面的促红细胞生成素受体(erythropoietin receptor,EPOR)结合而发挥作用的,而EPO受体广泛表达于人体内的非造血组织,包括肝脏、大脑、子宫、血管内皮细胞、平滑肌细胞、心肌细胞等。
研究发现,EPO具有抗凋亡、抗氧化、促血管生成、抗炎和促干细胞迁移等作用,在细胞的缺氧复氧实验和心脏的离体灌注实验以及动物缺血前给药的实验中均能减轻细胞的缺氧复氧损伤或缺血再灌注损伤,具有心肌保护作用;其中关于EPO抗凋亡作用的研究较为广泛和深入:EPO与在心血管系统有广泛表达的EPOR结合,激活多条信号通道介导抗凋亡作用,其中又以对典型的抗凋亡信号通道--磷脂酰肌醇-3激酶(phosphatidyl inositol 3-kinase,PI3K)/AKt通道的研究较为多见,并且获得多数人认可。但是,既往EPO的心肌保护试验多为细胞的缺氧复氧实验或动物心脏离体灌注实验,在体实验EPO多在心肌缺血前给药,这可能与EPO需要充分的吸收和作用时间等因素有关,因为EPO的抗凋亡等作用需要一定的时间才能体现出来,这些研究结果显然不适用于急性心肌梗死的病人。因此,需要有实验来证明EPO缺血后给药是否也有心肌保护作用,关于这方面的研究比较罕见。
本研究中我们通过建立大鼠的在体心脏缺血再灌注模型来模拟急性心肌梗死再灌注治疗过程中心肌缺血再灌注损伤的病理生理变化,EPO于缺血后再灌注前静脉注射给药模拟临床干预,旨在观察其是否还具有抗凋亡等作用,保护心肌,并进一步探求其作用机制(主要是与PI3K/AKt信号转导通路的关系),为临床上有效控制心肌缺血再灌注损伤开辟新的思路。本课题分三个部分进行:第一部分观察EPO缺血后再灌注前静脉用药能否保护心肌,重点观察其对心肌显微结构、心肌损伤标志物和再灌注心律失常的影响;第二部分分别从基因水平和蛋白质水平观察EPO的抗凋亡作用,进一步探索其心肌保护作用机制;第三部分观察EPO的抗氧化作用。
研究方法:
第一部分:以左冠状动脉前降支(LAD)穿线结扎法制备心肌缺血模型,松开结扎线造成再灌注。45只SD大鼠随机分成5组:①假手术(SHAM)组(5只):仅开胸,穿线不结扎,开胸前30分钟给予0.5ml二甲基亚砜(dimethylsulfoxide,DMSO)、60分钟后0.5mlNS尾静脉注射,开胸穿线观察3.5小时后处死;②缺血再灌注(IR)组(10只):结扎30分钟,再灌注3小时后处死,开胸前30分钟给予0.5ml DMSO、再灌注前给予0.5ml NS尾静脉注射;③PI3K/Akt途径的高选择性阻断剂LY294002(LY)组(10只):方法同前,开胸前30分钟给予0.3mg/kgLY294002溶于0.5ml DMSO,再灌注前给予0.5mlNS尾静脉注射;④促红细胞生成素(EPO)组(10只):方法同前,开胸前30分钟给予0.5mlDMSO、再灌注前给予EPO1000U/kg溶于0.5ml NS尾静脉注射;⑤促红细胞生成素+LY294002(E=PO+LY)组(10只):方法同前,开胸前30分钟给予0.3mg/kgLY294002溶于0.5ml DMSO,再灌注前给予EPO 1000U/kg溶于0.5mlNS,尾静脉注射。标本收集:实验结束时处死动物,在心底部将心脏与大血管及组织离断,取出心脏,去除左右心房,按检测所需剪取受损心肌,同法切割成三部分:一部分用3%戊二醛固定,作为组织切片备用,以电镜观察心肌细胞超微结构;一部分置液氮冻存,作为检测mRNA用;一部分用精密电子天平称取记录质量,剪碎匀浆,置于-40℃冰箱保存,作为第三部分SOD、MDA检测用。术前断尾、灌注结束后断头各留取1m1血于-70℃保存,检验肌酸磷酸激酶同功酶MB(CK-MB)和肌钙蛋白I(cTnI)水平;观察心电图Ⅱ导联心律失常发生情况,根据Ravingerova T方法进行室性心律失常(ventricular arrhythmias,VA)评分。大鼠操作流程如下:
麻醉大鼠→采血,尾静脉注射DMSO或LY+DMSO溶液→30分钟后开胸,结扎LAD→结扎LAD30分钟后尾静脉注射NS或EPO+NS溶液→剪开结扎线,开始再灌注→再灌注3小时后断头采血,采集心肌标本。
第二部分:仍然采用第一部分的实验动物,但是,由于第一部分中的LY294002组大鼠的心肌缺血再灌注损伤明显加重,所以第二部分把该组删除,剩下SHAM、IR、EPO和EPO+LY四个组,共计35只大鼠。标本收集同第一部分。以末端脱氧核苷酸转移酶(TdT酶)介导的带荧光的脱氧三磷酸尿苷(dUTP)缺口末端标记法(TdT-mediated dUTP nick end labeling,TUNEL)检测凋亡细胞,计算细胞凋亡指数(apoptotic index,AI);以电镜观察凋亡小体及其变化;RT-PCR法对bcl-2、bax和caspase-3 mRNA进行定量分析,计算样品Ct值及相对拷贝数。
第三部分:实验动物及分组同第二部分,用第一部分制成的心肌匀浆,以硫代巴比妥酸显色法(TBAmethod)测定丙二醛(malondialdehyde,MDA)的含量;以黄嘌呤氧化酶法(xanthine oxidase method)测定超氧化物歧化酶(superoxidedismutase,SOD的含量。
结果:第一部分:
1、透射电镜检查结果显示:除SHAM组以外,其它各组心肌可见心梗溶解性坏死、再灌注肌溶解性坏死,心肌细胞呈弥漫性肿胀,不同程度的肌丝断裂、缺失,肌节挛缩,线粒体肿胀,肌浆网扩张等超微结构变化,但是EPO组较IR、LY和EPO+LY组损伤程度明显减轻,而LY组心肌破坏程度最重。
2、血清CK-MB和cTnI:与术前相比,术后各组血清CK-MB和cTnI水平均显著升高(P＜0.05),反映SHAM在开胸后也有肌肉组织损伤,其余各组心肌组织坏死,提示冠脉结扎成功;其中EPO组明显低于IR、LY和EPO+LY组(P＜0.001),各组水平又以SHAM组最低(P＜0.001),LY组最高(P＜0.001),差异有统计学意义。
3、再灌注心律失常:45只大鼠中32只发生心律失常,多出现在结扎LAD后5分钟左右和再灌注早期,包括室性早博、室性心动过速和心室纤颤等;结扎LAD后的心律失常为短暂性,无持续至再灌注阶段;SHAM组有3只出现＜5次的室性早搏,均出现在开胸1小时后(其它大鼠的再灌注期),故也作心律失常评分,以便对照。40只缺血再灌注大鼠中29只发生再灌注心律失常,其中,IR组9只、LY组10只,EPO组3只,EPO+LY组7只;LY组有2只死亡。心律失常评分EPO组明显低于IR、LY和EPO+LY组(P＜0.05),其中又以LY组最高(P＜0.05),差异有统计学意义。第二部分:
1、电镜观察心肌细胞凋亡小体:可见除了SHAM组外,其余各组均有不同程度的细胞心肌结构破坏。EPO组较IR组心肌细胞中凋亡小体减少,EPO+LY组较IR组心肌细胞中凋亡小体减少不明显。
2、心肌细胞凋亡指数:与SHAM组比较,EPO组心肌细胞凋亡指数升高(P＜0.001),但明显低于IR和EPO+LY组(P＜0.001),差异有统计学意义。3、bcl-2、bax和caspase-3的mRNA水平:EPO组的bcl-2 mRNA水平,明显高于SHAM、IR和EPO+LY组(P＜0.001);EPO组的caspase-3和bax水平较SHAM组增高(P＜0.001),但较IR和EPO+LY组明显降低(P＜0.001);EPO组的bcl-2/bax比值高于SHAM、IR和EPO+LY组(P＜0.001),差异有统计学意义。第三部分:
1、心肌组织SOD含量:SHAM组＞EPO组＞EPO+LY组＞IR组,差异有统计学意义(P＜0.001)。
2、心肌组织MDA含量:SHAM组＜EPO组＜EPO+LY组＜IR组,差异有统计学意义(P＜0.001)。
结论:
1、EPO缺血后再灌注前静脉用药能明显减轻大鼠缺血再灌注后的心肌细胞超微结构的破坏,显著地减少了心肌损伤标志物CK-MB和cTnI的漏出,减轻再灌注心律失常,从而表明EPO能抑制心肌细胞缺血再灌注损伤。
2、EPO缺血后再灌注前静脉用药能减少心肌细胞凋亡,但是这种作用可被P13K/Akt阻断剂所减弱,具体表现为:①降低促凋亡基因bax的表达,促进抑制凋亡基因bcl-2的表达,直接抗凋亡;②抑制caspase-3基因的表达,阻止凋亡程序的执行;③激活PI3K/Akt抗凋亡信号道路,减少心肌细胞凋亡。
3、心肌缺血再灌注能降低抗氧化酶SOD的含量,升高反映细胞受氧自由基攻击严重程度的MDA的含量;EPO缺血后再灌注前静脉用药能升高心肌组织SOD含量,降低MDA含量,从而显示其能减少氧自由基生成,减轻氧化应激。
4、PI3K/Akt信号通路参与介导促红细胞生成素对大鼠缺血再灌注损伤心肌的保护作用。
Background and objectives:
Coronary heart disease is the leading cause of death worldwide,and 3.8 million men and 3.4 million women die of the disease each year.After an acute myocardial infarction,early and successful myocardial reperfusion with the use of thrombolytic therapy or primary percutaneous coronary intervention(PCI) or coronary artery bypass graft(CABG) is the most effective strategy for reducing the size of a myocardial infarct and improving the clinical outcome.But according to the studies in animal and clinical observations,we can find that,in some animals or patients, the cell dysbolism and structural damage became more serious after ischemia-reperfusion.The phenomenon was called ischemia-reperfusion injury(IRI). The process of restoring blood flow to the ischemic myocardium also can induce injury.This phenomenon,termed myocardial ischemia-reperfusion injury,can paradoxically reduce the beneficial effects of myocardial reperfusion.Studies in animal models of acute myocardial infarction suggest mat lethal reperfusion injury accounts for up to 50%of the final size of a myocardial infarct.Therefore,to prevent or cure myocardial ischemia-reperfusion injury may improve the clinical outcome of acute myocardial infarction which was treated by reperfusion therapy.It has been a hot spot of medical science study,but,there is no drug for clinical use yet.
The injury to the heart during myocardial reperfusion causes four types of cardiac dysfunction.The first type is myocardial stunning,a term denoting the“mechanical dysfunction that persists after reperfusion despite the absence of irreversible damage and despite restoration of normal or near-normal coronary flow.”The myocardium usually recovers from this reversible form of injury after several days or weeks.The second type of cardiac dysfunction is no-reflow phenomenon, which was originally defined as the“inability to reperfuse a previously ischemic region.”It refers to the impedance of microvascular blood flow encountered during opening of the infarct-related coronary artery.The third type of cardiac dysfunction is reperfusion arrhythmias.The last type of cardiac dysfunction is lethal reperfusion injury.Not only cellular necrosis but also cardiomyocyte apoptosis were found in myocardium after ischemia-reperfusion in studies,and cardiomyocyte apoptosis contributed to myocardial ischemia-reperfusion injury.As we know,the mechanism of myocardial ischemia-reperfusion injury maybe relate to oxygen paradox、calcium paradox、pH paradox and inflammation,and the cardiomyocyte apoptosis is an important kind of pathomechanism.Cardiomyocyte apoptosis is very important for the body to maintain stableness of tissue structure and function during myocardial ischemia-reperfusion injury.But abnormally increase of apoptosis would severely destroy the myocardium.The decrease of myocardium cells and the cardiac function disorder could be induced by cellular necrosis and cardiomyocyte apoptosis during reperfusion,they contributed to the outcome of myocardial ischemia-reperfusion injury all togeter.The apoptotic cells which had not been phagocytosed could turn to necrosis after being attacked by reactive oxygen species(ROS).There are two types of cell death which are necrosis and apoptosis during myocardial ischemia-reperfusion,but they are not inflexible,it has been proved that apoptosis could turn to necrosis while ATP was run-down or there was an inflammation.To comprehend the effect of apoptosis to myocardial ischemiareperfusion injury,apoptosis was inhibited by all kinds of methods by researchers, and the change of the structure and function of the heart was observed,the results proved that inhibition of apoptosis could diminish the myocardial infarct size almost to 50%-70%.As we known,oxygen paradox、calcium paradox and secondary mitochodria damage can induce cell apoptosis,they work all together or by oneself, they are connected with each other,everyone is cause and effect of each other.But these factors can not induce cell apoptosis directly,they activate survival or death gene by signal transmission,then the signal transmit to the incision enzyme intranuclear to perform the function of death.To block the signal transduction of cardiomyocyte apoptosis induced by myocardial ischemia-reperfusion may inhibit cardiomyocyte apoptosis,prevent or cure myocardial ischemia-reperfusion injury, and renew the function of cardiomyocyte.
Erythropoietin(EPO) is a kind of sialoglycoprotein hormone,which can stimulate the marrow to produce red cell.EPO is chief made by the juxtaglomerular cells which located at the juncture between the cortex and medulla of kidney. Recombinant human Epo(rHuEpo) which had been manufactured to therapy all kinds of anemia had been synthetized artificially in 1985.Endogenous EPO has a molecular weight of 34 kDa,and the molecular weight of rHuEPO is 30.4 kDa.They have the same physico-chemical property and biologic activity.rHuEPO has a demiperiod of 4-12 hours while it was used by intravenous injection,the bioavailability of rHuEPO is 23%-42%while it was used by hypodermic injection.To produce a marked effect, the binding of EPO and EPO receptors on the membrane of target cells is necessary. EPO receptors have been discovered in numerous non-hematopoietic tissues including liver、cerebrum、womb、endothelial cell、smooth muscle cell、 myocardium,and so on.Studies had found that EPO could inhibit cell apoptosis, attenuate oxidative stress,exhibit angiogenic potential,attenuate inflammation, promote stem cell migration,and protect the myocardium from ischemia-reperfusion injury in anoxia/reoxygenation models,Langendorff-perfused rat models or when it was administrated before ischemia in rats.The mechanism how EPO inhibited cell apoptosis has been studied widely and deeply.The binding of EPO and EPO receptors which were widely expressed in cardiovascular system could activate some signal transduction pathways that mediated the inhibition of apoptosis.The phosphatidylinositol-3-kinase(PI3K)/Akt pathway was one of the pathways which had been studied widely,and it was well known that the pathway mediated the inhibition of apoptosis.The earlier studies on the cardioprotection of EPO usually used anoxia/reoxygenation models or Langendorff-perfused models,and EPO was administrated before ischemia in animal studies,it maybe relate to that it need some time for EPO to be absorbed and to produce a marked effect.Obviously,the results of these researchs can not be applied to the patients who suffer from acute myocardial infarction.Therefore,it is necessary to prove the cardioprotection of EPO when it is administrated between ischemia and reperfusion,similar study is rare.
In this study,we try to set up myocardial ischemia-reperfusion models in rats to simulate the pathophysiologic changes in patients which suffer from acute myocardial infarction and were treated with reperfusion therapy,and EPO was administrated by intravenous injection between ischemia and reperfusion,the whole procedure was similar to clinical treatment.The aim of this study is to demonstrate if EPO can inhibit apoptosis and protect myocardium from ischemia-reperfusion injury,discuss the mechanism involved,chiefly find out the relation between the protection and PI3K pathway,and find new evidence to protect myocardium from ischemiareperfusion injury.Our study includes three parts:The first part is to find out if EPO can protect myocardium from ischemia-reperfusion injury when it was administrated by intravenous injection between ischemia and reperfusion,especially to explore the effect of EPO on the change of cardiac ultrastructure、the marker of myocardial damage and the occurrence of arrhythmia after reperfusion.The second part is to demonstrate if EPO can inhibit apoptosis in both gene and protein,discuss the mechanism involved.The last part is to demonstrate if EPO could antioxidation.
Methods:
The first part of this study:The left anterior descending branch of coronary artery(LAD) of rats was ligated for 30 minutes and then loosed for 3 hours to establishischemia/reperfusion heart model.45 SD rats were randomly divided into 5 groups:The sham operation group(group SHAM),and 4 experimental groups:The ischemia/reperfusion group(group IR),the highly selected blocker of the PI3K/Akt pathway LY294002 group(group LY),the EPO group(group EPO) and the EPO plus LY294002 group(group EPO+LY).①SHAM group(5 rats):chest was opened,a suture was passed under the LAD,but the LAD was not ligated,0.5ml dimethyl sulfoxide(DMSO) was injected via vena caudalis before opening chest,0.5ml NS was injected an hour later,the rat was executed 3.5 h after opening chest;②IR group(10 rats):chest was opened,the LAD was ligated by a suture,0.5ml DMSO was injected via vena caudalis 30 min before opening chest,and 0.5ml NS was injected before reperfusion,the rat was executed after the LAD was ligated for 30 min with subsequent 3h reperfusion.③LY group(10 rats):the same surgical procedure like that of IR group,0.3mg/kg LY294002 which was resolved into 0.5 ml DMSO was injected 30 min before opening chest,and 0.5ml NS was injected before reperfusion.④EPO group(10 rats):the same surgical procedure like that of IR group,0.5ml DMSO was injected 30 min before opening chest,and 1000u/kg EPO which was resolved into 0.5 ml NS was injected before reperfusion.⑤EPO+LY group(10 rats):the same surgical procedure like that of IR group,0.3mg/kg LY294002 was injected 30 min before opening chest,and 1000u/kg EPO was injected before reperfusion.After the experiment had been done,we killed the rats by decapitation,mutilated the great vessels and tissues from the cardiac base,took out of the heart,removed the atriums,cut the heart to take the damaged cardiac muscles according to the need of detection,divided the cardiac muscles in three parts,the first part was fixed with 3%glutaral,the second part was kept in liquid nitrogen,the third part was kept in icebox at 40℃below zero.We observed the occurrence of arrhythmia in electrocardiogram of leadⅡ,and scored the ventricular arrhythmias (VA) according to the method of Ravingerova T.The changes of cardiac ultrastructure were examed by transmission electron microscope.The levels of serum CK-MB and cTnI were detected just before DMSO injection and after reperfusion.The procedure was just as below:
To anesthetize rat→to collect blood sample,DMSO or LY+DMSO was injected via vena caudalis→to open chest 30minutes later,and ligate LAD→NS or EPO+NS was injected via vena caudalis 30 minutes after ligating→reperfusion began with ligation released→to collect blood sample 3 hours after reperfusion and decapitation,to collect myocardium sample.
The second part of this study:The models and samples were the same as the first part,but in the first part,it had been proved that LY294002 could obviously aggravate the myocardial ischemia-reperfusion injury,so we deleted the LY group in second part.There were 35 SD rats which were divided into four groups in the second part: group SHAM,group IR,group EPO and group EPO+LY.Myocardial cells apoptosis were estimated by in situ nick end labeling(TUNEL) method,to calculate the apoptotic index(AI).The cardiac ultrastructure were examed by transmission electron microscope in order to observe the apoptotic bodies.bcl-2、box and caspase-3 mRNA transcription were detected by RT-PCR,to calculate the Ct values and relative copy numbers of samples.
The third part of this study:The models,the groups were the same as the second part,the damaged cardiac muscles were collected in the second part to detect the content of malondialdehyde(MDA) in myocardium tissues by thiobarbituric acid (TBA) method,and the content of superoxide dismutase(SOD) in myocardium tissues was detected by xanthine oxidase method.
Results:
The first part of this study:
1、The ultrastructural changes of ischemia-reperfused myocardium:The ultrastructural changes of ischemia-reperfused myocardium were observeded by transmission electron microscopy.Except the SHAM group,diffuse swelling of myocardial cells,fragmentation and depletion of myofilament,contraction of muscle rod,swelling of mitochondria,and expansion of sarcoplasmic reticulum were observed in IR、LY、EPO and EPO+LY groups,but these changes were significantly lightened in EPO group,and obviously aggravated in LY group.
2、The levels of serum CK-MB and cTnI:The levels after operation were increased in all groups significantly(P＜0.05) comparing to the preoperative levels.The results reflected the necrosis of myocardium,and the coronary artery was ligated successfully.The muscles were damaged during the operation in the SHAM group. The levels in EPO group were significantlylower than those in IR、LY and EPO+LY groups(P＜0.001);The levels in LY group were higher than those in other groups (P＜0.001),and the levels in SHAM group were lower than those in other groups (P＜0.001).
3、Effect to reperfusion arrhythmia:Arrhythmia was found in 40 rats,arrhythmia occurred mainly at about 5 min after the ligation of LAD and the beginning of reperfusion.The arrhythmia appeared in rats including ventricular premature beat、ventricular tachycardia(VT) and ventricular fibrillation(VF).The arrhythmia occurred after the ligation of LAD was transient,it did not last to the period of reperfusion.There were 3 rats appeared ventricular premature beat which less than 5 times,the arrhythmia occurred at an hour after the opening of chest while other rats were reperfused,so we also scored the VA in SHAM group in order to compare with other groups.There were 37 rats appeared reperfusion arrhythmia among 40 rats which had been reperfused,reperfusion arrhythmia were found in all the rats in IR、LY and EPO+LY group,and 7 rats in EPO group.There were 2 dead rats in LY group. The VA score in EPO group was more than that in SHAM group,but it was significantly less than those in IR、LY and EPO+LY groups(P＜0.05).The score in LY group was the highest(P＜0.05).
The second part of this study:
1、The apoptosis of myocytes:The apoptosis of myocytes was observeded by transmission electron microscopy.Disorganization of myocardium in IR、EPO and EPO+LY groups was observed.The apoptotic bodies in EPO group was more than that in SHAM group,but it was less than those in IR and EPO+LY groups.
2、The apoptotic index of myocardium cell:The AI in EPO group was significantly higher than that in SHAM group(P＜0.001),but it was markedly lower than those in IR and EPO+LY groups(P＜0.001).
3、The mRNA levels of bcl-2、bax and caspase-3:the mRNA level of bcl-2 in EPO group was significantlyhigher than those in SHAM、IR and EPO+LY groups (P＜0.001).The mRNA levels of box and caspase-3 in EPO group were higher than those in SHAM group(P＜0.001),but they were markedly lower than those in IR and EPO+LY groups(P＜0.001).The ratio of bcl-2/bax in EPO group was significantly higher than those in EPO、IR and EPO+LY groups(P＜0.001).
The third part of this study:
1、The content of SOD in myocardium tissues:the content of SOD in myocardium tissues in EPO group was less than that in SHAM group,but it was more than those in IR and EPO+LY groups,and the content in IR group was less than that in EPO+LY group,all differences between groups were significantly(P＜0.001).
2、The content of MDA in myocardium tissues:the content of MDA in myocardium tissues in EPO group was more than that in SHAM group,but it was less than those in IR and EPO+LY groups,and the content in IR group was more than that in EPO+LY group,all differences between groups were significantly(P＜0.001).
Conclusion:
1、When EPO was administrated by intravenous injection between ischemia and reperfusion,it could significantly lighten the destruction of cardiac ultrastructure, decrease the leakage of CK-MB and cTnI which are the markers of myocardial damage,and lighten reperfusion arrhythmia.The results indicated that EPO could attenuate myocardial ischemia reperfusion injury.
2、When EPO was administrated by intravenous injection between ischemia and reperfusion,it could inhibit the apoptosis of myocardium cells,but the effect could be attenuated by LY294002.In a word,EPO could:①down-regulate the mRNA expression of bax which can promote apoptosis,up-regulate the mRNA expression of bcl-2 which can inhibit apoptosis,and prevent apoptosis directly;②down-regulate the mRNA expression of caspase-3,prevent the execution of the apoptotic procedure;③activate the anti-apoptosis pathway of PI3K,inhibit the apoptosis of myocardium cells.
3、Myocardial ischemia reperfusion could decrease the content of SOD which is antioxidase,increase the content of MDA which reflect the severity of the cells attacked by oxyradical.When EPO was administrated by intravenous injection between ischemia and reperfusion,it could increase the content of SOD and decrease the content of MDA in myocardium tissues,decrease the production of oxyradical, and attenuate oxidative stress.
4、The cardioprotection by EPO required the PI3K pathway.
冠心病是导致人类死亡的主要原因,全球每年约有380万男性和340万女性死于冠心病。在急性心肌梗死后,采用溶栓治疗、经皮冠状动脉介入术(percutaneous coronary intervention,PCI)或冠状动脉旁路移植术(coronary arterybypass graft,CABG)早期成功恢复心肌再灌注是缩小梗死范围和改善临床转归的最有效方法。但在动物实验和临床观察中发现,恢复血液再灌注后,部分缺血动物或患者细胞功能代谢障碍及结构破坏反而加重,因此将这种血液再灌注使缺血性损伤进一步加重的现象称为缺血再灌注损伤(ischemia reperfusion injury,IRI),它反常地减少了心肌再灌注的有益作用。急性心肌梗死动物模型研究提示,致死性再灌注损伤最多可占心肌梗死最终体积的50%,因此,防治心肌缺血再灌注损伤可以提高急性心肌梗死再灌注疗法的临床疗效,有关方面的研究已成为热点,但迄今为止还没有可用于临床的药物。
心肌缺血再灌注损伤可以产生4种类型的心功能障碍:①心肌顿抑,是指心脏在冠脉血流已恢复正常或接近正常后继续存在机械性功能障碍,是一种可逆性损伤,通常在数天或数周后恢复;②无复流现象,是指梗死相关冠脉再通时微血管血流遇到阻抗,缺血区域不能得到有效的再灌注;③再灌注心律失常;④致死性再灌注损伤。有学者研究发现:心肌缺血再灌注不仅能导致细胞坏死,而且还能引起细胞凋亡,也就是说细胞凋亡参与了心肌缺血再灌注损伤过程。目前认为,心肌缺血再灌注损伤的发生可能与氧反常、钙反常、pH反常和炎症反应等因素有关,而细胞凋亡是心肌缺血再灌注损伤的重要病理生理机制。在心肌缺血再灌注损伤中,细胞凋亡对维持组织形态、机能稳定具有十分重要的意义;然而,细胞凋亡的异常增加会加重心肌的破坏。再灌注时,同时存在的细胞坏死和凋亡导致心肌细胞数量减少和心脏功能的下降,共同影响着心肌缺血再灌注损伤的结果。没有被吞噬作用消除的凋亡细胞,受到活性氧攻击后,发生坏死是有可能的。细胞坏死和凋亡作为心肌缺血再灌注损伤中细胞死亡的两种方式,并不是一成不变的,研究已证实,ATP耗竭或炎症反应均可促使凋亡向坏死演变。为了了解凋亡对心脏的影响,学者通过各种方法抑制凋亡来观察心脏结构和功能的变化,结果证明,抑制心肌细胞凋亡能够减少缺血再灌注后的心肌梗死体积几乎达到50%-70%,改善心功能。目前认为心肌缺血再灌注损伤中产生的氧反常、钙反常等生化改变以及由此导致的线粒体损伤,三者单独启动或联合作用是许多凋亡诱导因素的共同通道,三者互相联系,互为因果。这些因素并不能直接引起细胞凋亡,而是通过一定的信号传递方式激活生存或死亡相关基因,然后将信号传递到核内切酶,而执行死亡的功能。因此阻断心肌缺血再灌注损伤中心肌细胞凋亡的信号转导能够阻止凋亡程序的执行,减少心肌细胞凋亡,防治心肌缺血再灌注损伤,重新恢复心肌细胞的功能。
促红细胞生成素(erythropoietin,EPO)是一种能刺激骨髓造血的唾液糖蛋白类激素,主要由肾皮质和髓质交界处的球旁细胞合成。1985年,人们利用基因重组技术成功合成重组人红细胞生成素(recombinant human erythropoietin,rHuEPO),用于治疗各种类型的贫血。内源性EPO分子量为34KD,rHuEPO分子量为30.4KD,其理化性质和生物学活性与内源性EPO相同。静脉给予rHuEPO的半衰期为4-12小时,皮下注射rHuEPO的生物利用度为23-42%。EPO是通过与靶细胞膜表面的促红细胞生成素受体(erythropoietin receptor,EPOR)结合而发挥作用的,而EPO受体广泛表达于人体内的非造血组织,包括肝脏、大脑、子宫、血管内皮细胞、平滑肌细胞、心肌细胞等。
研究发现,EPO具有抗凋亡、抗氧化、促血管生成、抗炎和促干细胞迁移等作用,在细胞的缺氧复氧实验和心脏的离体灌注实验以及动物缺血前给药的实验中均能减轻细胞的缺氧复氧损伤或缺血再灌注损伤,具有心肌保护作用;其中关于EPO抗凋亡作用的研究较为广泛和深入:EPO与在心血管系统有广泛表达的EPOR结合,激活多条信号通道介导抗凋亡作用,其中又以对典型的抗凋亡信号通道--磷脂酰肌醇-3激酶(phosphatidyl inositol 3-kinase,PI3K)/AKt通道的研究较为多见,并且获得多数人认可。但是,既往EPO的心肌保护试验多为细胞的缺氧复氧实验或动物心脏离体灌注实验,在体实验EPO多在心肌缺血前给药,这可能与EPO需要充分的吸收和作用时间等因素有关,因为EPO的抗凋亡等作用需要一定的时间才能体现出来,这些研究结果显然不适用于急性心肌梗死的病人。因此,需要有实验来证明EPO缺血后给药是否也有心肌保护作用,关于这方面的研究比较罕见。
本研究中我们通过建立大鼠的在体心脏缺血再灌注模型来模拟急性心肌梗死再灌注治疗过程中心肌缺血再灌注损伤的病理生理变化,EPO于缺血后再灌注前静脉注射给药模拟临床干预,旨在观察其是否还具有抗凋亡等作用,保护心肌,并进一步探求其作用机制(主要是与PI3K/AKt信号转导通路的关系),为临床上有效控制心肌缺血再灌注损伤开辟新的思路。本课题分三个部分进行:第一部分观察EPO缺血后再灌注前静脉用药能否保护心肌,重点观察其对心肌显微结构、心肌损伤标志物和再灌注心律失常的影响;第二部分分别从基因水平和蛋白质水平观察EPO的抗凋亡作用,进一步探索其心肌保护作用机制;第三部分观察EPO的抗氧化作用。
研究方法:
第一部分:以左冠状动脉前降支(LAD)穿线结扎法制备心肌缺血模型,松开结扎线造成再灌注。45只SD大鼠随机分成5组:①假手术(SHAM)组(5只):仅开胸,穿线不结扎,开胸前30分钟给予0.5ml二甲基亚砜(dimethylsulfoxide,DMSO)、60分钟后0.5mlNS尾静脉注射,开胸穿线观察3.5小时后处死;②缺血再灌注(IR)组(10只):结扎30分钟,再灌注3小时后处死,开胸前30分钟给予0.5ml DMSO、再灌注前给予0.5ml NS尾静脉注射;③PI3K/Akt途径的高选择性阻断剂LY294002(LY)组(10只):方法同前,开胸前30分钟给予0.3mg/kgLY294002溶于0.5ml DMSO,再灌注前给予0.5mlNS尾静脉注射;④促红细胞生成素(EPO)组(10只):方法同前,开胸前30分钟给予0.5mlDMSO、再灌注前给予EPO1000U/kg溶于0.5ml NS尾静脉注射;⑤促红细胞生成素+LY294002(E=PO+LY)组(10只):方法同前,开胸前30分钟给予0.3mg/kgLY294002溶于0.5ml DMSO,再灌注前给予EPO 1000U/kg溶于0.5mlNS,尾静脉注射。标本收集:实验结束时处死动物,在心底部将心脏与大血管及组织离断,取出心脏,去除左右心房,按检测所需剪取受损心肌,同法切割成三部分:一部分用3%戊二醛固定,作为组织切片备用,以电镜观察心肌细胞超微结构;一部分置液氮冻存,作为检测mRNA用;一部分用精密电子天平称取记录质量,剪碎匀浆,置于-40℃冰箱保存,作为第三部分SOD、MDA检测用。术前断尾、灌注结束后断头各留取1m1血于-70℃保存,检验肌酸磷酸激酶同功酶MB(CK-MB)和肌钙蛋白I(cTnI)水平;观察心电图Ⅱ导联心律失常发生情况,根据Ravingerova T方法进行室性心律失常(ventricular arrhythmias,VA)评分。大鼠操作流程如下:
麻醉大鼠→采血,尾静脉注射DMSO或LY+DMSO溶液→30分钟后开胸,结扎LAD→结扎LAD30分钟后尾静脉注射NS或EPO+NS溶液→剪开结扎线,开始再灌注→再灌注3小时后断头采血,采集心肌标本。
第二部分:仍然采用第一部分的实验动物,但是,由于第一部分中的LY294002组大鼠的心肌缺血再灌注损伤明显加重,所以第二部分把该组删除,剩下SHAM、IR、EPO和EPO+LY四个组,共计35只大鼠。标本收集同第一部分。以末端脱氧核苷酸转移酶(TdT酶)介导的带荧光的脱氧三磷酸尿苷(dUTP)缺口末端标记法(TdT-mediated dUTP nick end labeling,TUNEL)检测凋亡细胞,计算细胞凋亡指数(apoptotic index,AI);以电镜观察凋亡小体及其变化;RT-PCR法对bcl-2、bax和caspase-3 mRNA进行定量分析,计算样品Ct值及相对拷贝数。
第三部分:实验动物及分组同第二部分,用第一部分制成的心肌匀浆,以硫代巴比妥酸显色法(TBAmethod)测定丙二醛(malondialdehyde,MDA)的含量;以黄嘌呤氧化酶法(xanthine oxidase method)测定超氧化物歧化酶(superoxidedismutase,SOD的含量。
结果:第一部分:
1、透射电镜检查结果显示:除SHAM组以外,其它各组心肌可见心梗溶解性坏死、再灌注肌溶解性坏死,心肌细胞呈弥漫性肿胀,不同程度的肌丝断裂、缺失,肌节挛缩,线粒体肿胀,肌浆网扩张等超微结构变化,但是EPO组较IR、LY和EPO+LY组损伤程度明显减轻,而LY组心肌破坏程度最重。
2、血清CK-MB和cTnI:与术前相比,术后各组血清CK-MB和cTnI水平均显著升高(P＜0.05),反映SHAM在开胸后也有肌肉组织损伤,其余各组心肌组织坏死,提示冠脉结扎成功;其中EPO组明显低于IR、LY和EPO+LY组(P＜0.001),各组水平又以SHAM组最低(P＜0.001),LY组最高(P＜0.001),差异有统计学意义。
3、再灌注心律失常:45只大鼠中32只发生心律失常,多出现在结扎LAD后5分钟左右和再灌注早期,包括室性早博、室性心动过速和心室纤颤等;结扎LAD后的心律失常为短暂性,无持续至再灌注阶段;SHAM组有3只出现＜5次的室性早搏,均出现在开胸1小时后(其它大鼠的再灌注期),故也作心律失常评分,以便对照。40只缺血再灌注大鼠中29只发生再灌注心律失常,其中,IR组9只、LY组10只,EPO组3只,EPO+LY组7只;LY组有2只死亡。心律失常评分EPO组明显低于IR、LY和EPO+LY组(P＜0.05),其中又以LY组最高(P＜0.05),差异有统计学意义。第二部分:
1、电镜观察心肌细胞凋亡小体:可见除了SHAM组外,其余各组均有不同程度的细胞心肌结构破坏。EPO组较IR组心肌细胞中凋亡小体减少,EPO+LY组较IR组心肌细胞中凋亡小体减少不明显。
2、心肌细胞凋亡指数:与SHAM组比较,EPO组心肌细胞凋亡指数升高(P＜0.001),但明显低于IR和EPO+LY组(P＜0.001),差异有统计学意义。3、bcl-2、bax和caspase-3的mRNA水平:EPO组的bcl-2 mRNA水平,明显高于SHAM、IR和EPO+LY组(P＜0.001);EPO组的caspase-3和bax水平较SHAM组增高(P＜0.001),但较IR和EPO+LY组明显降低(P＜0.001);EPO组的bcl-2/bax比值高于SHAM、IR和EPO+LY组(P＜0.001),差异有统计学意义。第三部分:
1、心肌组织SOD含量:SHAM组＞EPO组＞EPO+LY组＞IR组,差异有统计学意义(P＜0.001)。
2、心肌组织MDA含量:SHAM组＜EPO组＜EPO+LY组＜IR组,差异有统计学意义(P＜0.001)。
结论:
1、EPO缺血后再灌注前静脉用药能明显减轻大鼠缺血再灌注后的心肌细胞超微结构的破坏,显著地减少了心肌损伤标志物CK-MB和cTnI的漏出,减轻再灌注心律失常,从而表明EPO能抑制心肌细胞缺血再灌注损伤。
2、EPO缺血后再灌注前静脉用药能减少心肌细胞凋亡,但是这种作用可被P13K/Akt阻断剂所减弱,具体表现为:①降低促凋亡基因bax的表达,促进抑制凋亡基因bcl-2的表达,直接抗凋亡;②抑制caspase-3基因的表达,阻止凋亡程序的执行;③激活PI3K/Akt抗凋亡信号道路,减少心肌细胞凋亡。
3、心肌缺血再灌注能降低抗氧化酶SOD的含量,升高反映细胞受氧自由基攻击严重程度的MDA的含量;EPO缺血后再灌注前静脉用药能升高心肌组织SOD含量,降低MDA含量,从而显示其能减少氧自由基生成,减轻氧化应激。
4、PI3K/Akt信号通路参与介导促红细胞生成素对大鼠缺血再灌注损伤心肌的保护作用。
Background and objectives:
Coronary heart disease is the leading cause of death worldwide,and 3.8 million men and 3.4 million women die of the disease each year.After an acute myocardial infarction,early and successful myocardial reperfusion with the use of thrombolytic therapy or primary percutaneous coronary intervention(PCI) or coronary artery bypass graft(CABG) is the most effective strategy for reducing the size of a myocardial infarct and improving the clinical outcome.But according to the studies in animal and clinical observations,we can find that,in some animals or patients, the cell dysbolism and structural damage became more serious after ischemia-reperfusion.The phenomenon was called ischemia-reperfusion injury(IRI). The process of restoring blood flow to the ischemic myocardium also can induce injury.This phenomenon,termed myocardial ischemia-reperfusion injury,can paradoxically reduce the beneficial effects of myocardial reperfusion.Studies in animal models of acute myocardial infarction suggest mat lethal reperfusion injury accounts for up to 50%of the final size of a myocardial infarct.Therefore,to prevent or cure myocardial ischemia-reperfusion injury may improve the clinical outcome of acute myocardial infarction which was treated by reperfusion therapy.It has been a hot spot of medical science study,but,there is no drug for clinical use yet.
The injury to the heart during myocardial reperfusion causes four types of cardiac dysfunction.The first type is myocardial stunning,a term denoting the“mechanical dysfunction that persists after reperfusion despite the absence of irreversible damage and despite restoration of normal or near-normal coronary flow.”The myocardium usually recovers from this reversible form of injury after several days or weeks.The second type of cardiac dysfunction is no-reflow phenomenon, which was originally defined as the“inability to reperfuse a previously ischemic region.”It refers to the impedance of microvascular blood flow encountered during opening of the infarct-related coronary artery.The third type of cardiac dysfunction is reperfusion arrhythmias.The last type of cardiac dysfunction is lethal reperfusion injury.Not only cellular necrosis but also cardiomyocyte apoptosis were found in myocardium after ischemia-reperfusion in studies,and cardiomyocyte apoptosis contributed to myocardial ischemia-reperfusion injury.As we know,the mechanism of myocardial ischemia-reperfusion injury maybe relate to oxygen paradox、calcium paradox、pH paradox and inflammation,and the cardiomyocyte apoptosis is an important kind of pathomechanism.Cardiomyocyte apoptosis is very important for the body to maintain stableness of tissue structure and function during myocardial ischemia-reperfusion injury.But abnormally increase of apoptosis would severely destroy the myocardium.The decrease of myocardium cells and the cardiac function disorder could be induced by cellular necrosis and cardiomyocyte apoptosis during reperfusion,they contributed to the outcome of myocardial ischemia-reperfusion injury all togeter.The apoptotic cells which had not been phagocytosed could turn to necrosis after being attacked by reactive oxygen species(ROS).There are two types of cell death which are necrosis and apoptosis during myocardial ischemia-reperfusion,but they are not inflexible,it has been proved that apoptosis could turn to necrosis while ATP was run-down or there was an inflammation.To comprehend the effect of apoptosis to myocardial ischemiareperfusion injury,apoptosis was inhibited by all kinds of methods by researchers, and the change of the structure and function of the heart was observed,the results proved that inhibition of apoptosis could diminish the myocardial infarct size almost to 50%-70%.As we known,oxygen paradox、calcium paradox and secondary mitochodria damage can induce cell apoptosis,they work all together or by oneself, they are connected with each other,everyone is cause and effect of each other.But these factors can not induce cell apoptosis directly,they activate survival or death gene by signal transmission,then the signal transmit to the incision enzyme intranuclear to perform the function of death.To block the signal transduction of cardiomyocyte apoptosis induced by myocardial ischemia-reperfusion may inhibit cardiomyocyte apoptosis,prevent or cure myocardial ischemia-reperfusion injury, and renew the function of cardiomyocyte.
Erythropoietin(EPO) is a kind of sialoglycoprotein hormone,which can stimulate the marrow to produce red cell.EPO is chief made by the juxtaglomerular cells which located at the juncture between the cortex and medulla of kidney. Recombinant human Epo(rHuEpo) which had been manufactured to therapy all kinds of anemia had been synthetized artificially in 1985.Endogenous EPO has a molecular weight of 34 kDa,and the molecular weight of rHuEPO is 30.4 kDa.They have the same physico-chemical property and biologic activity.rHuEPO has a demiperiod of 4-12 hours while it was used by intravenous injection,the bioavailability of rHuEPO is 23%-42%while it was used by hypodermic injection.To produce a marked effect, the binding of EPO and EPO receptors on the membrane of target cells is necessary. EPO receptors have been discovered in numerous non-hematopoietic tissues including liver、cerebrum、womb、endothelial cell、smooth muscle cell、 myocardium,and so on.Studies had found that EPO could inhibit cell apoptosis, attenuate oxidative stress,exhibit angiogenic potential,attenuate inflammation, promote stem cell migration,and protect the myocardium from ischemia-reperfusion injury in anoxia/reoxygenation models,Langendorff-perfused rat models or when it was administrated before ischemia in rats.The mechanism how EPO inhibited cell apoptosis has been studied widely and deeply.The binding of EPO and EPO receptors which were widely expressed in cardiovascular system could activate some signal transduction pathways that mediated the inhibition of apoptosis.The phosphatidylinositol-3-kinase(PI3K)/Akt pathway was one of the pathways which had been studied widely,and it was well known that the pathway mediated the inhibition of apoptosis.The earlier studies on the cardioprotection of EPO usually used anoxia/reoxygenation models or Langendorff-perfused models,and EPO was administrated before ischemia in animal studies,it maybe relate to that it need some time for EPO to be absorbed and to produce a marked effect.Obviously,the results of these researchs can not be applied to the patients who suffer from acute myocardial infarction.Therefore,it is necessary to prove the cardioprotection of EPO when it is administrated between ischemia and reperfusion,similar study is rare.
In this study,we try to set up myocardial ischemia-reperfusion models in rats to simulate the pathophysiologic changes in patients which suffer from acute myocardial infarction and were treated with reperfusion therapy,and EPO was administrated by intravenous injection between ischemia and reperfusion,the whole procedure was similar to clinical treatment.The aim of this study is to demonstrate if EPO can inhibit apoptosis and protect myocardium from ischemia-reperfusion injury,discuss the mechanism involved,chiefly find out the relation between the protection and PI3K pathway,and find new evidence to protect myocardium from ischemiareperfusion injury.Our study includes three parts:The first part is to find out if EPO can protect myocardium from ischemia-reperfusion injury when it was administrated by intravenous injection between ischemia and reperfusion,especially to explore the effect of EPO on the change of cardiac ultrastructure、the marker of myocardial damage and the occurrence of arrhythmia after reperfusion.The second part is to demonstrate if EPO can inhibit apoptosis in both gene and protein,discuss the mechanism involved.The last part is to demonstrate if EPO could antioxidation.
Methods:
The first part of this study:The left anterior descending branch of coronary artery(LAD) of rats was ligated for 30 minutes and then loosed for 3 hours to establishischemia/reperfusion heart model.45 SD rats were randomly divided into 5 groups:The sham operation group(group SHAM),and 4 experimental groups:The ischemia/reperfusion group(group IR),the highly selected blocker of the PI3K/Akt pathway LY294002 group(group LY),the EPO group(group EPO) and the EPO plus LY294002 group(group EPO+LY).①SHAM group(5 rats):chest was opened,a suture was passed under the LAD,but the LAD was not ligated,0.5ml dimethyl sulfoxide(DMSO) was injected via vena caudalis before opening chest,0.5ml NS was injected an hour later,the rat was executed 3.5 h after opening chest;②IR group(10 rats):chest was opened,the LAD was ligated by a suture,0.5ml DMSO was injected via vena caudalis 30 min before opening chest,and 0.5ml NS was injected before reperfusion,the rat was executed after the LAD was ligated for 30 min with subsequent 3h reperfusion.③LY group(10 rats):the same surgical procedure like that of IR group,0.3mg/kg LY294002 which was resolved into 0.5 ml DMSO was injected 30 min before opening chest,and 0.5ml NS was injected before reperfusion.④EPO group(10 rats):the same surgical procedure like that of IR group,0.5ml DMSO was injected 30 min before opening chest,and 1000u/kg EPO which was resolved into 0.5 ml NS was injected before reperfusion.⑤EPO+LY group(10 rats):the same surgical procedure like that of IR group,0.3mg/kg LY294002 was injected 30 min before opening chest,and 1000u/kg EPO was injected before reperfusion.After the experiment had been done,we killed the rats by decapitation,mutilated the great vessels and tissues from the cardiac base,took out of the heart,removed the atriums,cut the heart to take the damaged cardiac muscles according to the need of detection,divided the cardiac muscles in three parts,the first part was fixed with 3%glutaral,the second part was kept in liquid nitrogen,the third part was kept in icebox at 40℃below zero.We observed the occurrence of arrhythmia in electrocardiogram of leadⅡ,and scored the ventricular arrhythmias (VA) according to the method of Ravingerova T.The changes of cardiac ultrastructure were examed by transmission electron microscope.The levels of serum CK-MB and cTnI were detected just before DMSO injection and after reperfusion.The procedure was just as below:
To anesthetize rat→to collect blood sample,DMSO or LY+DMSO was injected via vena caudalis→to open chest 30minutes later,and ligate LAD→NS or EPO+NS was injected via vena caudalis 30 minutes after ligating→reperfusion began with ligation released→to collect blood sample 3 hours after reperfusion and decapitation,to collect myocardium sample.
The second part of this study:The models and samples were the same as the first part,but in the first part,it had been proved that LY294002 could obviously aggravate the myocardial ischemia-reperfusion injury,so we deleted the LY group in second part.There were 35 SD rats which were divided into four groups in the second part: group SHAM,group IR,group EPO and group EPO+LY.Myocardial cells apoptosis were estimated by in situ nick end labeling(TUNEL) method,to calculate the apoptotic index(AI).The cardiac ultrastructure were examed by transmission electron microscope in order to observe the apoptotic bodies.bcl-2、box and caspase-3 mRNA transcription were detected by RT-PCR,to calculate the Ct values and relative copy numbers of samples.
The third part of this study:The models,the groups were the same as the second part,the damaged cardiac muscles were collected in the second part to detect the content of malondialdehyde(MDA) in myocardium tissues by thiobarbituric acid (TBA) method,and the content of superoxide dismutase(SOD) in myocardium tissues was detected by xanthine oxidase method.
Results:
The first part of this study:
1、The ultrastructural changes of ischemia-reperfused myocardium:The ultrastructural changes of ischemia-reperfused myocardium were observeded by transmission electron microscopy.Except the SHAM group,diffuse swelling of myocardial cells,fragmentation and depletion of myofilament,contraction of muscle rod,swelling of mitochondria,and expansion of sarcoplasmic reticulum were observed in IR、LY、EPO and EPO+LY groups,but these changes were significantly lightened in EPO group,and obviously aggravated in LY group.
2、The levels of serum CK-MB and cTnI:The levels after operation were increased in all groups significantly(P＜0.05) comparing to the preoperative levels.The results reflected the necrosis of myocardium,and the coronary artery was ligated successfully.The muscles were damaged during the operation in the SHAM group. The levels in EPO group were significantlylower than those in IR、LY and EPO+LY groups(P＜0.001);The levels in LY group were higher than those in other groups (P＜0.001),and the levels in SHAM group were lower than those in other groups (P＜0.001).
3、Effect to reperfusion arrhythmia:Arrhythmia was found in 40 rats,arrhythmia occurred mainly at about 5 min after the ligation of LAD and the beginning of reperfusion.The arrhythmia appeared in rats including ventricular premature beat、ventricular tachycardia(VT) and ventricular fibrillation(VF).The arrhythmia occurred after the ligation of LAD was transient,it did not last to the period of reperfusion.There were 3 rats appeared ventricular premature beat which less than 5 times,the arrhythmia occurred at an hour after the opening of chest while other rats were reperfused,so we also scored the VA in SHAM group in order to compare with other groups.There were 37 rats appeared reperfusion arrhythmia among 40 rats which had been reperfused,reperfusion arrhythmia were found in all the rats in IR、LY and EPO+LY group,and 7 rats in EPO group.There were 2 dead rats in LY group. The VA score in EPO group was more than that in SHAM group,but it was significantly less than those in IR、LY and EPO+LY groups(P＜0.05).The score in LY group was the highest(P＜0.05).
The second part of this study:
1、The apoptosis of myocytes:The apoptosis of myocytes was observeded by transmission electron microscopy.Disorganization of myocardium in IR、EPO and EPO+LY groups was observed.The apoptotic bodies in EPO group was more than that in SHAM group,but it was less than those in IR and EPO+LY groups.
2、The apoptotic index of myocardium cell:The AI in EPO group was significantly higher than that in SHAM group(P＜0.001),but it was markedly lower than those in IR and EPO+LY groups(P＜0.001).
3、The mRNA levels of bcl-2、bax and caspase-3:the mRNA level of bcl-2 in EPO group was significantlyhigher than those in SHAM、IR and EPO+LY groups (P＜0.001).The mRNA levels of box and caspase-3 in EPO group were higher than those in SHAM group(P＜0.001),but they were markedly lower than those in IR and EPO+LY groups(P＜0.001).The ratio of bcl-2/bax in EPO group was significantly higher than those in EPO、IR and EPO+LY groups(P＜0.001).
The third part of this study:
1、The content of SOD in myocardium tissues:the content of SOD in myocardium tissues in EPO group was less than that in SHAM group,but it was more than those in IR and EPO+LY groups,and the content in IR group was less than that in EPO+LY group,all differences between groups were significantly(P＜0.001).
2、The content of MDA in myocardium tissues:the content of MDA in myocardium tissues in EPO group was more than that in SHAM group,but it was less than those in IR and EPO+LY groups,and the content in IR group was more than that in EPO+LY group,all differences between groups were significantly(P＜0.001).
Conclusion:
1、When EPO was administrated by intravenous injection between ischemia and reperfusion,it could significantly lighten the destruction of cardiac ultrastructure, decrease the leakage of CK-MB and cTnI which are the markers of myocardial damage,and lighten reperfusion arrhythmia.The results indicated that EPO could attenuate myocardial ischemia reperfusion injury.
2、When EPO was administrated by intravenous injection between ischemia and reperfusion,it could inhibit the apoptosis of myocardium cells,but the effect could be attenuated by LY294002.In a word,EPO could:①down-regulate the mRNA expression of bax which can promote apoptosis,up-regulate the mRNA expression of bcl-2 which can inhibit apoptosis,and prevent apoptosis directly;②down-regulate the mRNA expression of caspase-3,prevent the execution of the apoptotic procedure;③activate the anti-apoptosis pathway of PI3K,inhibit the apoptosis of myocardium cells.
3、Myocardial ischemia reperfusion could decrease the content of SOD which is antioxidase,increase the content of MDA which reflect the severity of the cells attacked by oxyradical.When EPO was administrated by intravenous injection between ischemia and reperfusion,it could increase the content of SOD and decrease the content of MDA in myocardium tissues,decrease the production of oxyradical, and attenuate oxidative stress.
4、The cardioprotection by EPO required the PI3K pathway.
引文
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[1]Yellon DM,Hausenloy DJ.Myocardial reperfusion injury[J].N Engl J Med, 2007;357(11):1121-1135.
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