【作者】 刘丹；

【导师】 张吉翔；

【作者基本信息】 南昌大学 ， 外科学， 2009， 博士

【摘要】 上世纪中叶人们发现,某些情况下组织器官缺血后恢复血流,不但不能减轻损伤反而导致损伤进一步加重和器官功能障碍。这种缺血组织在恢复血液灌注后,超微结构、代谢、功能以及电生理方面发生进一步损伤的现象被称为缺血/再灌注损伤(ischemia/reperfusion injury, IRI)。近年来,临床医生发现在休克治疗、心肺脑复苏、心绞痛冠脉解痉、心脑血管栓塞再通、体外循环心内直视手术、经皮腔内冠状动脉成形术、脏器和肢体移植、外科烧伤植皮等情况下均有IRI发生,是造成手术后病人病情恶化的一个重要原因。如何减少再灌注所造成的损伤已成为当前临床相关学科,特别是心胸外科的研究重点。基因治疗通过将基因导入机体并发挥作用,可以赋予心肌抗急性心肌损伤的能力,并且没有手术带给病人的二次打击,必将为防治心肌急性损伤带来新的曙光。原癌基因是一类编码关键性调控蛋白的正常细胞基因,在细胞的生存、生长、发育、分化过程中具有重要的生理功能,当原癌基因被激活有可能引起细胞生长过度活跃和/或细胞死亡受抑制。凋亡是急性损伤过程中心肌细胞丢失造成心肌受损的重要原因。原癌基因的运用使受损的心肌组织得到有效的修复并达到逆转心脏衰竭的目的。已有文献报道,心肌的缺血性损伤,通过原癌基因src, ras, vav酪氨酸激酶所介导的途径而得以修复,从而阻止了心力衰竭的发生。原癌基因丝/苏氨酸激酶Pim-3在结构上与Pim-1、Pim-2同属Pim激酶家族。该家族各成员在激酶结构域上高度同源,其生物学功能也存在显著的重叠。与其它丝/苏氨酸蛋白激酶如PI3K-Akt、mTOR等一样,Pim激酶家族能使在细胞生长通路中起关键作用的蛋白质分子内的丝/苏氨酸残基磷酸化,并通过不同于PI3K-Akt-mTOR的另一条通路,促进细胞的生存和繁殖。已证实多种生长因子、激素和细胞生长通路如JAK/STAT、PI3K、Akt等可在不同水平介导Pim的表达。因此,Pim激酶在细胞生长的调控中起重要作用,并对细胞凋亡有明显的抑制作用。有实验证明,Pim-3基因在脑组织缺血损伤周围出现早期高水平的表达。在组织缺血早期,许多基因的表达被显著抑制,但Pim-3基因表达则被早期诱导,提示它可能对细胞的生存和功能恢复有重要意义。受此启发,本课题拟探讨在心肌IRI时Pim-3基因是否具有抗凋亡和对心肌细胞生存具有保护作用。本工作的总体研究思路为:首先,采用原代培养的乳鼠心肌细胞,建立缺氧/复氧(anoxia/ reoxygenation, A/R)损伤及缺氧预适应(anoxia preconditioning, APC)保护模型,在用多种经典、公认的检测方法确认已造成急性心肌损伤及保护作用的基础上,采用分子生物学检测方法,研究Pim-3 mRNA以及蛋白表达的变化规律。接着,构建Pim-3表达质粒,采用基因转染技术处理心肌细胞。在确认Pim-3在细胞内高表达后,采用多种经典、公认的检测方法研究Pim-3在急性A/R损伤模型与APC保护模型的作用,阐明Pim-3基因表达在急性心肌细胞损伤时的意义。然后,在明确Pim-3能对抗心肌A/R损伤的基础上,选定心肌细胞中一条重要的信号转导通路MAPKs通路,MAPKs包括3种主要的亚家族: ERK1/2, JNK和p38 MAPK,研究究竟是哪一个或哪几个MAPKs亚家族介导了心肌细胞急性损伤时Pim-3的表达。最后,探讨Pim-3对抗心肌急性A/R损伤的机制,从细胞凋亡的线粒体信号途径入手,阐明Pim-3对抗心肌急性损伤,发挥保护作用的机制。第一部分:Pim-3在心肌细胞急性A/R损伤时的表达及意义目的建立大鼠乳鼠原代培养心肌细胞急性A/R损伤及APC保护模型,运用多种经典、公认的生理、生化等检测指标,在确认模型已成功的基础上,采用RT-PCR、Western Blotting等分子生物学检测方法来探讨Pim-3的表达及变化规律;构建pcDNA3.1/Pim-3基因表达质粒,将其转染入心肌细胞,探讨原癌基因Pim-3对心肌细胞急性缺氧/复氧(A/R)损伤的保护作用。方法实验共分5组,每组重复6次。①Control组;②A/R组;③APC+A/R组;④空载质粒pcDNA3.1+A/R组;⑤pcDNA3.1/Pim-3+A/R组。实验结束后测定Pim-3 mRNA及蛋白表达水平(RT-PCR、Western-blotting法)的改变,同时检测培养液中乳酸脱氢酶(LDH)活性、四唑盐(MTT)比色试验测定细胞存活率、TUNEL法检测细胞凋亡。结果:1.心肌细胞Pim-3 mRNA的表达A/R组Pim-3 mRNA表达(0.37±0.052)较Control(对照)组(0.05±0.008)明显升高(p<0.01),APC+A/R组(0.75±0.094)则进一步升高(p<0.01),pcDNA3.1+A/R组Pim-3基因mRNA表达与A/R组相似,无显著性差异(p>0.05),pcDNA3.1/Pim-3+A/R组Pim-3 mRNA表达(0.82±0.11)与A/R组相比则进一步升高(p<0.01)。2.心肌细胞Pim-3蛋白的表达A/R组Pim-3蛋白表达(1.07±0.24)较Control(对照)组(0.3±0.05)明显升高(p<0.01),APC+A/R组(2.31±0.37)则进一步升高(p<0.01),pcDNA3.1+A/R组Pim-3蛋白表达与A/R组相似(p>0.05),pcDNA3.1/Pim-3+A/R组Pim-3表达(2.57±0.41)则进一步升高(p<0.01)。3. Pim-3基因对心肌细胞A/R损伤后细胞存活率、LDH的影响A/R组的细胞存活率为36.7±6.7%,LDH值为27.1±5.0,与Control(对照)组比较细胞存活率明显降低,LDH升高(p<0.01); APC+A/R组细胞存活率为80.3±7.8%,LDH值为8.4±1.2,pcDNA3.1/Pim-3+A/R组与APC+A/R组的细胞存活率和LDH值相似,无显著性差异,但是上述两组与A/R组相比细胞存活率分别升高、LDH均降低(p<0.01)。提示心肌细胞转染Pim-3之后能明显对抗A/R损伤所致的细胞存活率下降和LDH值升高。4. Pim-3基因对心肌细胞A/R损伤后凋亡的影响A/R组的心肌细胞有30.4±4.5%出现凋亡,Control组出现的的凋亡细胞很少(p<0.01);APC+A/R组、pcDNA3.1/Pim-3+A/R组凋亡的心肌细胞明显减少,凋亡指数分别是14.7±2.7%和17.0±3.2%,与A/R组比较差异性显著(p<0.01)。提示心肌细胞转染Pim-3之后能明显减少A/R损伤所致的细胞凋亡。讨论与结论心肌细胞凋亡是A/R损伤的一个重要特征,APC是目前已知的最为强大的对抗A/R损伤的方法,研究证实,APC对抗A/R损伤的机制主要是上调一些内源性保护蛋白,这些蛋白能够抑制心肌细胞的凋亡,从而发挥心肌保护作用。原癌基因丝/苏氨酸激酶Pim-3在结构上与Pim-1、Pim-2同属Pim激酶家族。大量的实验研究已证实,Pim激酶的上调与细胞凋亡的抑制存在明显的相关性。Pim-3基因在脑组织缺血损伤周围出现早期高水平的表达,在组织缺血早期,许多基因的表达被显著抑制的情况下,Pim-3基因表达却被诱导,表达上调,提示可能对细胞的生存和功能恢复有重要意义。因此,本研究采用原代培养新生大鼠的心肌细胞,建立A/R损伤、APC保护模型,结果显示缺氧预适应能明显增加Pim-3的表达,构建pcDNA3.1/ Pim-3基因表达质粒,转染入心肌细胞,结果显示外源性的Pim-3基因的导入能对抗A/R所致的急性损伤,发挥类似于缺氧预适应的保护作用。第二部分:p38 MAPK通路介导Pim-3在心肌细胞急性损伤时的表达目的建立大鼠乳鼠原代培养心肌细胞急性A/R损伤及APC保护模型,用多种经典、公认的生理、生化及超微结构改变等检测指标,在确认已产生抗急性A/R损伤的保护作用基础上,分别给予特异性阻断剂U0126、SP600125及SB203850阻断MAPKs通路中的ERK1/2、JNK、p38 MAPK三个亚家族。采用Western Blotting等分子生物学检测方法,检测MAPKs通路中ERK1/2、JNK、p38 MAPK三个亚家族磷酸化蛋白表达水平及Pim-3蛋白的表达水平,研究MAPKs通路在介导原癌基因Pim-3对抗心肌急性损伤中的作用。方法实验共分6组,每组重复6次。①Control组;②A/R组;③APC+A/R组;④U0126+APC+A/R组;⑤SP600125+APC+A/R组;⑥SB203850+APC+A/R组。在缺氧预处理前分别用终浓度为10μM SB203850、U0126、SP600125与细胞孵育30 min,然后再行APC及A/R处理。实验结束后测定MAPKs通路中ERK1/2、JNK、p38 MAPK磷酸化蛋白表达水平及Pim-3蛋白的表达水平,同时检测培养液中乳酸脱氢酶(LDH)活性、四唑盐(MTT)比色试验测定细胞存活率、TUNEL法检测细胞凋亡。结果1. MAPKs通路对Pim-3表达的影响使用特异性的磷酸化MAPKs抗体分别检测phospho-ERK1/2, phospho-p38 MAPK和phospho-JNK的蛋白表达。与Control组相比,A/R组的phospho-ERK1/2, phospho-p38 MAPK蛋白水平分别升高了2.3和3.2倍(p<0.01),并且,APC组进一步升高了2.6和3.2倍(p<0.01)。phospho-JNK在A/R组比Control组升高了1.4倍(p<0.05),但是,APC并没有使phospho-JNK表达进一步升高。当分别给予这三个通路的阻断剂,由APC或A/R所诱导的磷酸化水平的升高全部被取消,证明U0126、SP600125和SB203580均能特异性地阻断ERK1/2、JNK和p38 MAPK的活化。而APC所诱导的Pim-3表达的升高只在p38 MAPK通路被阻断后明显下调(p<0.01),证明Pim-3的表达与p38 MAPK的活化有关,而与ERK1/2、JNK无关。因此以下结果主要研究p38 MAPK通路介导Pim-3表达对心肌的保护作用。2. SB203850对心肌细胞A/R损伤后细胞存活率、LDH的影响A/R组与Control组比较细胞存活率明显降低,LDH升高(p<0.01); APC+A/R组与A/R组相比细胞存活率分别升高、LDH均降低(p<0.01)。而给予p38 MAPK通路特异性的阻断剂之后,APC的保护作用被取消3. SB203850对心肌细胞A/R损伤后凋亡的影响A/R组的心肌细胞有32.4±4.5%出现凋亡,Control组出现的的凋亡细胞很少(p<0.01),APC+A/R组的凋亡指数是13.8±2.6%,与A/R组比较差异性显著(p<0.01)。而给予p38 MAPK通路特异性的阻断剂之后,凋亡指数为26.7±4.1%,与APC+A/R组相比差异性显著(p<0.01)。讨论与结论在前期实验证实缺氧预适应能明显增加Pim-3的表达,从而对抗心肌细胞的A/R损伤之后,在这一部分我们进一步探讨了缺氧预适应中Pim-3表达上调所涉及的心肌细胞信号传导通路。多项研究证明, MAPKs是缺血、缺氧、牵张、激素、生长因子和细胞因子等多种细胞外刺激诱导基因表达、细胞增殖等核反应的共同通路或汇聚点,多种细胞外刺激(包括缺血、应激、激素、细胞因子、生长因子等),无论是通过G蛋白或是酪氨酸蛋白激酶的活化,都可激活Raf→MAPK激酶(MAPK kinase)→MAPK的磷酸化连锁反应,经过MAPK的核转位,引起转录因子的磷酸化,调节原癌基因和应激蛋白基因的表达,促进有关蛋白质合成增加,完成对细胞外刺激的反应。MAPKs包括3种主要的亚家族: ERK1/2, JNK和p38 MAPK。引起ERK1/2激活的主要是生长因子等有丝分裂刺激,导致细胞增殖、分化等核反应;JNK and p38 MAPK则在缺血缺氧、应激、紫外线、细胞因子等刺激下被激活,从而影响不同病理条件下细胞的生物学行为和组织器官的功能。但是这三条通路在细胞中的确切作用还不是很清楚。在本实验中,分别给予这三条通路的特异性阻断剂阻断由APC所诱导的活化,结果发现阻断ERK1/2和JNK的活化对Pim-3的表达没有明显的影响,而阻断了p38 MAPK的活化,Pim-3的表达则明显下降,提示由APC所诱导的Pim-3的表达升高与p38 MAPK通路有关。第三部分:细胞凋亡的线粒体信号通路在Pim-3抗心肌急性损伤中的作用目的建立大鼠乳鼠原代培养心肌细胞急性A/R损伤模型,用多种经典、公认的生理、生化等检测指标,确认已产生急性A/R损伤的基础上,将已构建好的pcDNA3.1/Pim-3表达质粒导入心肌细胞中,探讨原癌基因Pim-3对抗心肌急性损伤的作用是否与线粒体信号转导途径介导的细胞色素C(Cyt C)的释放有关。方法实验共分4组,每组重复6次。①Control组;②A/R组;③pcDNA3.1+A/R组;④pcDNA3.1/Pim-3+A/R组。将已构建好的pcDNA3.1和pcDNA3.1/Pim-3质粒导入心肌细胞中,实验结束后,检测培养液中乳酸脱氢酶(LDH)活性,四唑盐(MTT)比色试验测定细胞存活率,TUNEL法检测细胞凋亡,分光光度法检测心肌线粒体渗透性转换,蛋白印迹检测Pim-3蛋白表达水平,线粒体、胞浆Cyt C动态变化和caspase-3活性。结果1.心肌细胞Pim-3蛋白的表达A/R组Pim-3蛋白表达(1.31±0.22)较Control组(0.25±0.04)升高,pcDNA3.1+A/R组蛋白表达与A/R组无显著性差异(p>0.05) ,pcDNA3.1/Pim-3+A/R组Pim-3表达(2.89±0.48)则进一步升高(p<0.01)。2. Pim-3基因转染对心肌细胞A/R损伤后细胞存活率、LDH的影响A/R组与Control组比较细胞存活率明显降低,LDH升高(p<0.01),pcDNA3.1+A/R组细胞存活率、LDH值分别为40.1±6.7%和30.2±5.3,与A/R组相比,无显著性差异,pcDNA3.1/Pim-3+A/R组与A/R组相比则细胞存活率升高、LDH降低,差异有显著性(p<0.01)。3. Pim-3基因转染对心肌细胞A/R损伤后凋亡的影响A/R组的心肌细胞有31.2±5.2%出现凋亡,Control组出现的凋亡细胞很少(p<0.01);pcDNA3.1/Pim-3+A/R组凋亡的心肌细胞明显减少,凋亡指数是18.7±3.2% ,与A/R组比较差异性显著(p<0.01)。4. Pim-3基因转染对心肌细胞A/R损伤后线粒体渗透性转换的影响在A/R组,mPTP开放明显,ODA540下降绝对值(ΔODA540)与Control组比较明显增加,差异有统计学意义(p<0.01);pcDNA3.1+A/R组ΔODA540与A/R组比较无显著性差异(p>0.05);pcDNA3.1/Pim-3+A/R组mPTP开放较A/R组明显被抑制,ΔODA540与A/R组相比明显减少(p<0.01)(见Fig.13)。5. Pim-3基因转染对心肌细胞A/R损伤后Cyt C从线粒体向胞浆释放的影响在Control组Cyt C主要存在于线粒体,胞浆中含量很少,而在A/R组,Cyt C则由线粒体大量释放至胞浆中,pcDNA3.1/Pim-3+A/R组则明显抑制Cyt C由线粒体向胞浆的释放,与A/R组比较差异性显著(p<0.01)。6. Pim-3基因转染对心肌细胞A/R损伤后caspase-3水平的影响在A/R组caspase-3(1.47±0.16)较Control组(0.52±0.09)明显升高(p<0.01),pcDNA3.1+A/R组caspase-3表达(1.65±0.17)与A/R组比较无显著性差异(p>0.05),pcDNA3.1/Pim-3+A/R组caspase-3水平则明显下降(p<0.01)。讨论与结论本研究在证实Pim-3基因转染入心肌细胞后,逆转了A/R所致的心肌急性损伤的基础上,对Pim-3基因产生心肌保护作用的机制进行探讨。凋亡是心肌细胞A/R损伤过程中心肌细胞丢失造成心肌受损的重要原因,Pim-3作为原癌基因具有对抗凋亡的能力,我们推测Pim-3抗急性心肌损伤是通过抑制凋亡而起作用的。凋亡发生的分子生物学机制是相当复杂的,近年来,线粒体信号途径介导的细胞凋亡机制研究倍受关注,认为凋亡过程中线粒体可能起着中心调控作用,通过使mPTP过度开放,造成线粒体肿胀,外膜破裂,释放Cyt C至胞浆中导致凋亡发生。本研究结果表明,心肌细胞在遭受A/R损伤时,线粒体渗透性转换增大,Cyt C由线粒体向胞浆大量释放,而Pim-3转染组则明显抑制了线粒体的肿胀,从而抑制了Cyt C由线粒体向胞浆的释放,而且改善了心肌细胞的各项指标,起到了心肌保护作用。这些数据证明, mPTP的开放,细胞色素C从线粒体释放至胞质是引发凋亡的关键步骤。当凋亡刺激因子引起细胞色素C从线粒体膜间隙释放到胞浆后,启动caspase级联反应,整个过程为一正反馈,活化的caspases能对其底物进行特异的切割,DNA片段化,细胞发生凋亡。在caspase网络中caspase-3是细胞凋亡的主要执行者,被称为细胞凋亡的“终结者”。本实验发现A/R损伤伴有caspase-3的激活,而Pim-3转染组则抑制了caspase-3的激活,证明Pim-3的抗凋亡作用是通过线粒体途径抑制caspase网络而产生的。更多还原

【Abstract】 It was reported in the middle of the last century that under certain circumstances , reperfusion of blood to ischemia tissues and organs not only fail to alleviate the damage but also led to further aggravate the injury and dysfunction. The phenomenon that the ultrastructure, metabolism, functions and electrophysiological of ischemia organizations got further injury after reperfusion was named ischemia / reperfusion injury. Recently, clinicians have found that IRI occured in shock therapy, cardiopulmonary-cerebral resuscitation, coronary spasm angina pectoris, cardiovascular and cerebrovascular embolization recanalization, off-pump open heart surgery, percutaneous transluminal coronary angioplasty, organ and limb transplants, skin burns and other surgical cases, and it could be one of the important reasons why post-operative patients got condition deteriorated. How to protecte organs from IRI has now become the focus of clinical research, in particular to cardiothoracic Surgery. Through transfection certain gene into body, gene therapy can confer cadiocyte the ability to resist acute myocardial injury, meanwhile avoid the second-hit of surgery. Gene therapy could be a new and hopeful way to prevent and treat the acute myocardial injury.Proto-oncogene is a kind of normal cell gene that encodes a key regulatory protein. When proto-oncogene is activated, it will not only promote cell proliferation but also inhibit cell apoptosis. Apoptosis is an important contributory factor in the loss and damage of cardiomyocytes when the myocardium is subjected to acute anoxia or ischemic injury. The fundamental cellular mechanism behind apoptosis can best be described as a balance between anti-apoptotic and pro-apoptotic activities. Activation of the endogenous proto-oncogene or introduction of the exogenous one may fundamentally repair myocardial injury, through stimulation of anti-apoptotic factors. Some studies have shown that myocardial ischemic injury can be repaired by the proto-oncogene src, ras, vav-mediated pathway, futher to prevent the occurrence of heart failure. Therefore, proto-oncogene plays an important role in the treatment of myocardial damage, which inspires people to put the role of the proto-oncogene into the treatment of acute myocardial injury.A proto-oncogene Pim kinase belongs to a serine/threoine protein kinase family consisting of Pim-1, -2 and -3. The three members are well conserved in vertebrates and show high degrees of sequence and structural similarities. There is also overlap and redundancy in their biological functions. Such other serine/threoine protein kinases as PI3K-Akt or mTOR, Pim kinases can phosphorylate serine/threonine residue that regulate apoptosis and play important roles in the regulation of cell growth and surviva by other pathway differented from PI3K-Akt-mTOR. The expression of Pim kinase can be mediated by many growth factors, hormone and cell growth pathway such as JAK/STAT, PI3K and Akt, so it has been implicated in stimulating cell growth, inhibiting cell apoptosis and promoting cell cycle progressioN. Pim-3 was found to be overexpressed in ischemic brain tissue, which indicated that Pim-3 may play an important role in cell survival and functional recovery. Given that Pim-3 could prevent apoptosis and promote cell survival, we assumed that Pim-3 might play a protective effect on the myocardium subjected to I/R injury. Based on the above hypothesis, we design the experiment as follows. Firstly, the A/R and anoxia preconditioning (APC) models of primary cultured neonatal rat myocytes were established, AND we examined the expressions of mRNA and protein of Pim-3. WE then cloned Pim-3 expression vector and transfected it into rat cardiomyocytes and examined Pim-3 expression in rat cardiomyocytes, especially in the cells subjected to A/R injury in order to explore the protective role of Pim-3 in acute cardiomyocyte injury.Secondly, we studied the role of three major MAPK pathways, p38 MAPK, JNK, and ERK1/2, in order to evaluate the molecular mechanism underlying Pim-3 overexpression. Thirdly, we also investigate the protective effect of proto-oncogene Pim-3 on cardiomyocytes aganist anoxia–reoxygenation(A/R) injury and the relation of this process with THE release of cytochrome c mediated by mitochondrial signal transduction pathways. PartⅠThe Expression and significance of Pim-3 in cardiomyocytes subjected to acute damage.Objective:We established the A/R and anoxia preconditioning (APC) models of primary cultured neonatal rat myocytes and detected expression of Pim-3 by RT-PCR and Western blotting, and then we cloned Pim-3 expression vector and transfected it into rat cardiomyocytes in order to study the protective effect of proto-oncogene Pim-3 on cardiomyocytes aganist anoxia–reoxygenation(A/R) injury.Methods:The primarily neonatal rat ventricular cardiomyocytes were randomly divided into 5 groups: (1) control group; (2) A/R group; (3) APC+A/R group; (4) pcDNA3.1+A/R group; (5) pcDNA3.1/Pim-3+A/R group. Expression of Pim-3 was detected by RT-PCR and Western blotting. LDH and CK-MB activity, MTT and TUNEL were detected after treatment.Results:1. The expression of Pim-3 mRNAThe expression level of Pim-3 mRNA showed a significant increase in A/R group compared with that of the control group (p<0.01). The expression level of Pim-3 mRNA was further increased in APC+A/R group, compared with that of A/R group (p<0.01). In pcDNA3.1/Pim-3+A/R group, the expression level of Pim-3 mRNA was remarkably elevated compared to that of A/R group(p<0.01). However, there was little change of the expression level in pcDNA3.1+A/R group(p>0.05).2. The expression of Pim-3 proteinThe expression level of Pim-3 protein showed a significant increase in A/R group compared with that of the control group (p<0.01). The expression level of Pim-3 protein was further increased in APC+A/R group, compared with that of A/R group (p<0.01). In pcDNA3.1/Pim-3+A/R group, the expression level of Pim-3 protein was remarkably elevated compared to that of A/R group(p<0.01). However, there was little change of the expression levels in pcDNA3.1+A/R group(p>0.05).3. Protective Effects of Pim-3 against A/R on Cell Viability and LDH ActivityThe viabilities of cardiomyocytes in the control and A/R groups were 92.4±8.8 % and 36.7±6.1%, respectively. Consistent with viability, LDH activity levels in the cultured medium were 4.0±0.7 for the control cells and 27.1±5.0 for the A/R-treated cells with a significant difference between them (p<0.01). In both groups of APC+A/R and pcDNA3.1/Pim-3+A/R, the levels of LDH activity were noticeably lower than those of A/R group; while the viability of ventricular myocytes was significantly increased compared with those of A/R group. The transfection of pcDNA3.1 into cardiomyocytes, however, showed little protective effect on the cells. This suggests that APC could induce cardioprotection and transfection of Pim-3 gene could mimic APC induced cardioprotection.4. Protective Effects of Pim-3 against A/R on Cardiomyocyte ApoptosisVery few TUNEL-positive cells were detected among cardiomyocytes from the control group. Numerous cardiomyocytes from A/R group presented as positive for TUNEL(p<0.01). In contrast, the number of TUNEL-positive cells was significantly reduced among cardiomyocytes of APC+A/R group; meanwhile, pcDNA3.1/Pim-3+A/R group showed an effect similar to that of APC+A/R group. There were 30.4±4.5% of cells that presented apoptosis in A/R group, whereas only 14.7±2.1% and 17.0±3.2% cardiomyocytes underwent apoptosis in APC+A/R and pcDNA3.1/Pim-3+A/R groups, respectively.Discussion and Conclusion:Cardiomyocyte apoptosis is the character for A/R injury. It is well known that APC is the most powerful means against A/R injury. APC has been investigated for many years; yet its physiological mechanisms of action are still not completely understood. There are growing evidences indicating that the protection is dependent on de novo protein synthesis. These proteins can inhibit apoptosis of myocardial cells, which play a protective role. Pim family is composed of at least three members: Pim-1, Pim-2, and Pim-3. Anti-apoptotic effects of Pim-1 and Pim-2 have been demonstrated earlier in several independent experimental systems. Because both Pim-1 and Pim-2 can induce anti-apoptotic effects, as a member of the Pim family, Pim-3 has been intriguing to investigators who tried to investigate if Pim-3 is involved in cell cycle regulation or anti-apoptosis through phosphorylating some substrates that regulate apoptosis. Pim-3 was found to be overexpressed in ischemic brain tissue, which indicated that Pim-3 may play an important role in cell survival and functional recovery. In this study, we confirmed that APC induced an over-expression of Pim-3 in parallel with the protection of cardiomyocytes against A/R injury. Furthermore, we demonstrated that transfection of Pim-3 into cardiomyocytes was able to mimic myocardial protection similar to that of APC. PartⅡThe Expression of Pim-3 in Acute Myocardial Injury Mediated by p38 MAPK Signal PathwayObjective: we established the A/R and anoxia preconditioning (APC) models of primary cultured neonatal rat myocytes and treated cardiomyocytes with U0126, SP600125 and SB203580 separately at 30 minutes prior to APC to inhibit the activities of ERK1/2, JNK and p38 MAPK, respectively. The aim is to investigate the role of mitogen-activated protein kinases (MAPKs) pathways and the molecular mechanism by which the proto-oncogene Pim-3 would protect cardiomyocyte against anoxia/reoxygenation(A/R) injury.Methods:The primarily neonatal rat ventricular cardiomyocytes were randomly divided into 6 groups: control group; A/R group; APC+A/R group; SB203850+APC+A/R group; U0126+APC+A/R group; SP600125+APC+A/R group. The cells were pre-incubated with U0126(ERK1/2 inhibitor), SP600125(SAPK/JNK inhibitor), or SB203580(p38 MAPK) at 10μmol/L concentration for 30 min before the APC. The activities of p38 MAPK, JNK and ERK1/2 were detected by Western blotting. The viability of cardiomyocytes was assayed by MTT and the apoptosis of cardiomyocyte was performed by TUNEL.Results:1. The Effects of MAPKs Signal Pathway on the Expression of Pim-3Alterations in the activity of MAPKs in cardiomyocytes were detected by Western blotting using phospho-specific antibodies against ERK1/2, p38 MAPK, and JNK respectively. At the same time, levels of total ERK1/2, p38 MAPK, and JNK proteins were also examined in parallel with anti-ERK1/2 antibody, anti-p38 MAPK antibody, and anti-JNK antibody. As seen in Figure 4, the cardiomyocytes showed 2.3- and 3.2-fold increases in ERK1/2 and p38 MAPK activities as compared to those of the control group (p<0.01) at 3 h after A/R. Meanwhile, in APC+A/R group, ERK1/2 and p38 MAPK activities showed further increases up to 6.7- and 10.1-fold compared to those of the control group (p<0.01). At the same time, cardiomyocytes of A/R group, compared to the control group, showed an approximately 1.4-fold increase in JNK activity (p<0.05). However, APC did not induce any further increase in JNK activity as compared with A/R group. U0126, SB203580, and SP600125, given at 30 min before APC, abolished the increased expression of ERK1/2, p38 MAPK, and JNK proteins induced by APC or A/R, respectively. Subsequently, we detected the expression level of Pim-3 protein after treating cardiomyotes with U0126, SB203580, and SP600125 respectively. The results showed that the expression level of Pim-3 protein significantly decreased when the p38 MAPK signal pathway was inhibited.2. The Effects of SB203850 on Cell Viability and LDH ActivityThe viability of cardiomyocytes in the A/R group was remarkably decreases compared to the control group, while LDH in the A/R group was significant increases compared to the control group(p<0.01). Once administrated with p38 MAPK inhibitor SB203850, the protective effects of APC was abolished.3. The Effects of SB203850 on Cardiomyocyte ApoptosisVery few TUNEL-positive cells were detected among cardiomyocytes from the control group. Numerous cardiomyocytes from A/R group presented as positive for TUNEL(p<0.01). In contrast, the number of TUNEL-positive cells was significantly reduced among cardiomyocytes of APC+A/R group. There were 32.4±4.5% of cells that presented apoptosis in A/R group, whereas only 13.8±2.6% cardiomyocytes underwent apoptosis in APC+A/R group(p<0.01). Once administrated with p38 MAPK inhibitor SB203850, 26.7±4.1% cardiomyocytes underwent apoptosis (p<0.01).Discussion and Conclusion:We have confirmed that the expression of Pim-3 could be significantly increased induced by APC and then play a protective role in cardiomyocytes against A/R injury in the part 1 of the research project. In this part, we studied the cellular signal pathway involved in the expression of Pim-3 induced by APC. In cardiomyocytes, the intracellular signaling mechanisms that mediate anoxic preconditioning require the presence of one or more members of MAPK cascades. These members are involved in the activation of genes that play important roles in the regulation of cellular responses occurring during cell proliferation and stress. A variety of extracellular stimuli (ischemia, stress, hormones, cytokines, growth factors, etc.), whether it is through the G protein or tyrosine kinase activation, can activate the Raf→MAPK kinase (MAPK kinase)→MAPK phosphorylation chain reaction. The nuclear translocation of MAPK can cause phosphorylation of transcription factors to regulate the expression of proto-oncogenes, stress protein gene and then promote protein synthesis induced by the extracellular stimuli. The principal MAPKs investigated in cardiac myocytes are ERK1/2, JNK and p38 MAPK. ERK1/2 is potently activated by hypertrophic stimuli, whereas JNK and p38 MAPK are activated by cellular stresses such as oxidative stress. However, there is a cross talk between JNK and p38 MAPK that results in the activation of cellular stresses when they are activated by hypertrophic stimuli and ERK1/2. The contribution of each pathway to the overall cardiac myocyte response is currently not entirely clear. In the present study, we treated cardiomyocytes with U0126, SP600125 and SB203580 separately at 30 minutes prior to APC to inhibit the activities of ERK1/2, JNK and p38 MAPK, respectively. We found that the inhibition of p38 MAPKs resulted in the abolishment of Pim-3 up-regulation in cardiomyocytes, which is induced by APC; while inhibition of either ERK1/2 or JNK did not show any effect on the Pim-3 expression. These findings indicate that over-expression of Pim-3 gene induced by APC may be regulated through the p38 MAPK signaling pathway. Part III The Role of Mitochondrial Apoptosis Signaling pathway in Pim-3 Protecting Against Acute Myocardial InjuryObjective: we established the A/R and anoxia preconditioning (APC) models of primary cultured neonatal rat myocytes and cloned Pim-3 expression vector and transfected it into rat cardiomyocytes in order to investigate the protective effect of proto-oncogene Pim-3 on cardiomyocytes aganist anoxia–reoxygenation(A/R) injury and the relation of this process with release of cytochrome c mediated by mitochondrial signal transduction pathways.Methods:The primarily neonatal rat ventricular cardiomyocytes were randomly divided into 4 groups: control group; A/R group; pcDNA3.1+A/R group; pcDNA3.1/Pim-3+A/R group. LDH activity, MTT and TUNEL were detected after treatment. Mitochondrial swelling was assayed by spectrophotometer. The expressions of Pim-3 and caspase-3 and the release of cytochrome c from mitochondria to cytoplasm were detected by Western blotting.Results:1. The expression of Pim-3 proteinThe expression level of Pim-3 protein showed a significant increase in A/R group compared with that of the control group (p<0.01). The expression level of Pim-3 protein was further increased in pcDNA3.1/Pim-3+A/R group, compared with that of A/R group (p<0.01).2. The Effects of Pim-3 on Cell Viability and LDH ActivityThe viability of cardiomyocytes in the A/R group was remarkably decreased and LDH activity was remarkably increased compared with those of control group (p<0.01). In the group of pcDNA3.1/Pim-3+A/R, the levels of LDH activity were noticeably lower than those of A/R group; while the viability of ventricular myocytes was significantly increased compared with those of A/R group (p<0.01). The transfection of pcDNA3.1 into cardiomyocytes, however, showed little protective effect on the cells.3. The Effects of Pim-3 on Cardiomyocyte ApoptosisThere were about 31.2±5.2% cardiomyocytes from A/R group presented as positive for TUNEL. Very few TUNEL-positive cells were detected among cardiomyocytes from the control group(p<0.01). In contrast, the number of TUNEL-positive cells was significantly reduced among cardiomyocytes of pcDNA3.1/Pim-3+A/R group, only 18.7±3.2% cardiomyocytes underwent apoptosis pcDNA3.1/Pim-3+A/R groups (p<0.01).4. The Effects of Pim-3 on Mitochondrial SwellingA/R induced damage to the inner mitochondrial membrane can be assessed by the classic swelling techniques, which monitor the net influx of the osmotic support associated with a non-specific increase in membrane permeability. It was shown that A/R induced mitochondrial swelling as revealed by the large decrease in absorbance of the mitochondrial suspension at 520 nm. However, Pim-3 inhibited the swelling process (P < 0.01).5. The Effects of Pim-3 on Cytochrome c Release from mitochondria to cytoplasmThe overall amount of cytochrome c in whole-cell extracts of cardiomyocytes remained at the same level. In control group, there was a low level of cytochrome c expression observed in the cytoplasmic fraction. However, the cardiomyocytes treated with A/R showed a higher expression level of cytochrome c in the cytosolic fraction when cells were undergoing apoptosis, which was concurrent with a lower level of cytochrome c in the mitochondrial fraction. In pcDNA3.1/Pim-3+A/R group, majority of cytochrome c was found to be retained in the mitochondrial fraction, indicating the low level of mitochondrial apoptosis in these cells(p<0.01).6. The Effects of Pim-3 on the activity of caspase-3The expression level of caspase-3 protein showed a significant increase in A/R group compared with that of the control group (p<0.01). However there was little change of the expression levels between pcDNA3.1+A/R and A/R group. In contrast, the expression levels of caspase-3 in pcDNA3.1/Pim-3+A/R group dramatically declined as compared to that of A/R group (P<0.01).Discussion and Conclusion:In this experiment, we demonstrated that transfection of Pim-3 into cardiomyocytes could effectively protect cardiomyocytes against A/R injury, and further investigated the mechanism of Pim-3 protective effects. Apoptosis is an important contributory factor in the loss and damage of cardiomyocytes when the myocardium is subjected to acute anoxia or ischemic injury. The fundamental cellular mechanism behind apoptosis can best be described as a balance between anti-apoptotic and pro-apoptotic activities. As a member of the Pim family, Pim-3 has been intriguing to investigators who tried to investigate if Pim-3 may fundamentally repair myocardium damage, through its anti-apoptotic effects. The mechanism of apoptosis is very complex. Recently, more investigations showed that Mitochondria should play a critical role in apoptosis in response to many stimuli. Mitochondria release cytochrome c into the cytosol, in response to an excessive open of mPTP. Therefore, cytochrome c release from the intermembrane space is supposed to be the determining factors in the final step to apoptosis. Our data indicated that the cardiomyocytes treated with A/R showed a higher expression level of cytochrome c in the cytosolic fraction when cells were undergoing apoptosis, which was concurrent with a lower level of cytochrome c in the mitochondrial fraction. However, transfection of cardiomyocytes with exogenous Pim-3 significantly lessened the release of cytochrome c from the mitochondria into the cytosol, which confirms that Pim-3 indeed has a cardio-protective effect by anti-apoptosis. The release of cytochrome c acts as an essential cofactor in the activation of the dormant killer proteases. Cytochrome c is released following the increase of mitochondrial membrane permeabilization. Cytochrome c can activate caspase cascade, the whole process is a positive feedback. The activation of caspases can carry out their function, mainly specific substrate cutting, DNA fragmentation, and cell apoptosis. Caspase-3 is the main executors of apoptosis and is known as the "terminator of apoptosis”. Our results showed that caspase-3 was activated when cardiomyocytes subjected to A/R injury, while Pim-3 could inhibit the activity of caspase-3. In conclusion, the mechanism of Pim-3 anti-apoptosis is involved in mitochondrial apoptosis signaling pathway.更多还原