Rho-kinase在心肌缺血/再灌注损伤和缺血预适应中对细胞凋亡的影响及其调控机制
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摘要
研究背景
心肌长时间缺血可以导致组织损伤以及细胞死亡,冠状动脉再通是缓解缺血损伤的关键,但较长时间缺血后的再灌注却可导致更为严重的损伤,即心脏缺血／再灌注损伤(Ischemia and reperfusion Injury, I/R), I/R的概念于1960年被Jennings等第一次提出。I／R是冠脉再通术(溶栓、PTCA或搭桥术)后的重要的并发症。如何做到既保证尽早恢复缺血组织的血流灌注,又减轻甚至消除再灌注损伤的发生便成为缺血性心脏病防治中的重要课题。有效防治再灌注损伤的重要前提是阐明其发病机制。心肌在遭受一次或多次反复的短暂缺血再灌注后,会表达出一种对随后而来的一次长时间的严重缺血损伤的抵抗能力的提高,这种现象称为缺血预适应(ischemic preconditioning, IPC), IPC于1986年首次被Murry等发现。经过预适应的心肌能够缩小心肌梗死的面积,改善心肌收缩力,保护冠状动脉内皮和心肌细胞的超微结构,保护微循环,降低再灌注导致的心律失常的发生率,更快地使心肌从再灌注诱导的心肌顿抑中恢复,是一种内源性自我保护方式。
Rho-kinase是一类丝-苏氨酸蛋白家族,可作为小G蛋白RhoA的直接下游效应物,参与多种细胞生物功能,如：平滑肌收缩、肌动蛋白细胞骨架组装、细胞黏附、移动、基因表达等。Rho-kinase以两种同源性极高的异构体形式存在：ROCK-1(或ROKβ、p160ROCK)和ROCK-2(或ROKα)。ROCK-1位于18号染色体,含有1354个氨基酸,ROCK-2位于12号染色体,含有1388个氨基酸。二者有65%的同源性,而在激酶结构域则有92%的同源性。Y-27632和fasudil是ROCK的选择性抑制剂,作用于其ATP依赖激酶区,可同时抑制ROCK-1和ROCK-2。Rho-kinase可以介导下游一系列磷酸化／脱磷酸化反应,已有20多种下游蛋白被发现。如：MLC、MYPT-1 ERM、PTEN、IRS1 NHE1、LIM激酶等。最具特征意义的Rho-kinase底物是MLC (myosin light chain)和MYPT-1 (myosin binding subunit of MLC phosphorylation, MLC磷酸酶的肌球蛋白结合亚基)。Rho-kinase可以通过作用于MLC或MYPT1来增加胞浆内MLC的磷酸化,促进肌动蛋白微丝骨架的聚合。
最近研究发现,Rho-kinase参与了变异性心绞痛、心衰、心肌缺血再灌注损伤等心血管疾病。初步研究表明Rho-kinase在心脏I／R损伤中活性增加,而在IPC中活性可能降低,但是其中发生机制却远未清楚。例如Rho-kinase调控哪些细胞因子,通过哪些信号途径发挥作用,以及在IPC中Rho-kinase活性是否会降低,Rho-kinase活性降低的机制如何,这些问题构成了本研究课题的内容。在本研究中,我们拟建立大鼠I／R和IPC模型,明确Rho-kinase活性在IPC和I／R中是否不同,Rho-kinase对心肌梗死面积、心肌细胞凋亡的影响以及对细胞凋亡有关信号通路的调节；明确IPC中ERK-MAPK是否能通过下调Rho-kinase活性起到抗心肌细胞凋亡作用。通过以上研究,进一步明确Rho-kinase在心脏I/R和IPC中所起的作用及其机制。这对于阐明I／R和IPC的发病机制,为心肌保护提供新的治疗途径具有重要的意义。
本研究共分三部分进行：
第一部分Rho-kinase在心脏缺血再灌注损伤和心肌缺血预适应中的表达及其对细胞凋亡的影响；
第二部分心脏缺血再灌注损伤中Rho-kinase对AIF的影响及调节机制；
第三部分心肌缺血预适应中ERK对Rho-kinase的调节。
第一部分Rho-kinase在心脏缺血再灌注损伤和心肌缺血预适应中的表达及其对细胞凋亡的影响研究目的
本部分研究拟建立大鼠I／R和IPC模型,应用药物fasudil抑制Rho-kinase活性,测定心肌梗死面积、心肌细胞的凋亡,明确Rho-kinase在心脏I／R和IPC中的作用。研究方法
1.大鼠心脏I／R和IPC模型的建立和分组：采用开胸结扎和松开冠状动脉的方法建立大鼠心脏I／R和IPC型。分为以下四组：对照组(18只)、I／R组(18只)、I/R+fasudil组(18只)和IPC组(18只)
2. Rho-kinase活性的检测：Rho-kinase的活性通过Western检测其特异性底物MYPT-1的磷酸化水平来表示。
3.心肌梗死面积检测：采用伊文思蓝染色鉴别非缺血区与缺血危险区(AAR),硝基四氮唑蓝(NBT)染色鉴别梗死心肌与非梗死心肌。以称重法计算心肌缺血危险区(伊文思蓝未染区),梗死区(NBT未染区)的重量,心肌梗死范围的大小以梗死区重量占缺血危险区重量的百分比表示。
公式为：心肌梗死范围=NBT未染区／伊文思蓝未染区×100%
4.大鼠心肌细胞凋亡的检测：采用末端脱氧核苷酸转移酶介导的末端标记技术(TUNEL)试剂盒检测各组大鼠心肌细胞的凋亡。按照试剂盒说明书操作。标记前用DNA酶处理切片做阳性对照,用标记液代替TdT酶反应液做阴性对照。每张切片随机选取10个视野(×400倍),计数凋亡细胞个数和所有细胞个数,以凋亡细胞个数／所有细胞个数反映各组心肌细胞凋亡的情况。
5. Caspase-3活性的检测：Caspase-3的活性通过Western检测其裂解产物cleaved-Caspase-3的表达水平来表示。研究结果
1.p-MYPT-1(磷酸化的MYPT-1)在I／R中大量表达,而在IPC中活性降低。在I／R中应用Rho-kinase抑制剂fasudil后,p-MYPT-1表达降低：对照组中只有微弱的p-MYPT-1表达。说明Rho-kinase在I／R中活性增加,而IPC和fasudil均抑制了Rho-kinase的活性。
2. Rho-kinase活性的抑制可减少大鼠心肌梗死面积：I／R组大鼠的心肌梗死面积占危险区心肌面积的60.3±4.08%, I/R+fasudil组大鼠的,心肌梗死面积／AAR减少至38.62±2.66%,说明在I／R大鼠中抑制Rho-kinase活性后能显著减少心肌梗死面积。在IPC中大鼠的心肌梗死面积降至29.16±1.08%,说明IPC可能通过降低Rho-kinase活性而明显缩小心肌梗死面积。
3. Rho-kinase活性的抑制可减少大鼠心肌细胞凋亡率：在对照组大鼠心肌中无TUNEL染色阳性细胞。在I／R组大鼠心肌中,TUNEL染色阳性细胞占全部细胞的33.87±1.57%,说明I／R可引起心肌细胞凋亡增加。在I/R+fasudil组大鼠心肌中,TUNEL染色阳性细胞占全部细胞的17.05±4.22%,说明在I／R大鼠中抑制Rho-kinase活性能减少心肌细胞凋亡率。在IPC中,细胞凋亡率为17.29±0.84%,说明IPC可能通过降低Rho-kinase活性而明显降低心肌细胞凋亡率。
4. Rho-kinase活性的抑制降低Caspase-3活性：在I／R中cleaved-Caspase-3大量表达,在IPC和I/R+fasudil组中其表达分别减少了约65.3%和58.5%,说明IPC和fasudil在抑制Rho-kinase活性同时抑制了Caspase-3的活性。
结论
1.在大鼠心肌I/R损伤中Rho-kinase活性明显增加,加重了心脏的损伤、增加了心肌细胞凋亡。
2.在大鼠心肌I/R损伤中抑制Rho-kinase活性后能减少心肌梗死面积和心肌细胞的凋亡率,减轻心肌细胞的损伤程度。
3.IPC中Rho-kinase活性降低,心肌梗死面积和心肌细胞凋亡减轻,具有心脏保护作用。
第二部分心脏缺血再灌注损伤中Rho-kinase对AIF的影响及调节机制
研究目的
探讨在大鼠心脏I／R中Rho-kinase对AIF的影响及调节机制。(Rho-kinase/JNK/AIF信号通路在心脏I／R损伤中的作用)研究方法
1.大鼠心脏I／R损伤模型的建立,分为以下5组：对照组(18只)I/R+NS组(18只)、I/R+fasudil组(18只)、I/R+SP600125(JNK抑制剂)组(18只)和I/R+DMSO组(18只)。
2.心肌梗死面积检测,计算梗死心肌面积占危险区(AAR)心肌面积百分比。
3.TUNEL法检测细胞凋亡。
4.采用Western Blot检测各组大鼠心肌中Rho-kinase、JNK、AIF的活性变化。Rho-kinase活性通过检测MYPT-1的磷酸化水平来表示,通过检测JNK的磷酸化蛋白的水平来反映JNK的活性,通过分别检测AIF的线粒体和细胞核中的蛋白水平来反映AIF的活性。研究结果
1.心脏I／R损伤中Rho-kinase、JNK、AIF活性增加：与对照组相比,I/R+NS组中p-MYPT-1和p-JNK蛋白表达明显增加。I/R+NS组中AIF在细胞核中的表达增多,在线粒体中的表达减少,AIF从线粒体到细胞核的转位增加。说明心脏I／R损伤可增加Rho-kinase、JNK、AIF活性。
2.抑制Rho-kinase或JNK后减少大鼠心肌梗死面积：I/R+NS组和I/R+DMSO组大鼠的心肌梗死面积分别占整个左心室面积的59.89±3.83%和61.64±3.3%,两者相比无显著性差异。I/R+fasudil组大鼠的心肌梗死面积／AAR减少至38.62±2.66%,说明在I／R中抑制Rho-kinase活性能显著减少心肌梗死面积。I/R+SP600125组大鼠的心肌梗死面积／AAR减少至41.1±2.57%,说明在I／R中抑制JNK活性能显著减少心肌梗死面积。
3.抑制Rho-kinase或JNK后减少大鼠心肌细胞凋亡：I/R+NS组和I/R+DMSO组大鼠的心肌细胞凋亡分别为32.78±5.01%和34.45±3.73%,两者相比无显著性差异。I/R+fasudil组大鼠的心肌细胞凋亡减少至17.05±4.22%,说明在I／R中抑制Rho-kinase活性能显著减少心肌细胞凋亡。I/R+SP600125组大鼠的心肌细胞凋亡减少至16.25±3.29%,说明在I／R中抑制JNK活性能显著减少心肌细胞凋亡。
4.抑制Rho-kinase活性可降低JNK活性：在I／R组大鼠心肌中磷酸化JNK表达增多,应用fasudil后磷酸化JNK的表达下降,说明磷酸化JNK的表达与Rho-kinase有关,JNK是Rho-kinase的下游分子。
5.在I/R+DMSO组p-MYPT-1表达明显增加,应用JNK抑制剂SP600125后,p-MYPT-1表达无明显变化。说明p-MYPT-1的表达与JNK活性无关,进一步证实JNK是Rho-kinase的下游分子。
6.抑制Rho-kinase活性可减少AIF从线粒体到心肌细胞核的转位：在I／R组大鼠心肌细胞核中AIF的表达增多,抑制Rho-kinase活性后其表达减少；另一方面,在I／R组大鼠心肌细胞线粒体中AIF的表达减少,而抑制Rho-kinase活性后其表达增多。说明抑制Rho-kinase活性可减少AIF从线粒体向细胞核的转移,证实AIF是Rho-kinase的下游分子。
7.抑制JNK活性可减少AIF的从线粒体到心肌细胞核的转位：在I／R组大鼠心肌细胞核中AIF的表达增多,抑制JNK活性后其表达减少；另一方面,在I／R组大鼠心肌细胞线粒体中AIF的表达减少,而抑制JNK活性后其表达增多。说明抑制JNK可以减少AIF从线粒体向细胞核的转移,证实AIF是JNK的下游分子。
结果证实,在大鼠I／R损伤模型中,Rho-kinase调节AIF从线粒体向细胞核的转移,在这个过程中,JNK起了重要的作用。Rho-kinase通过JNK来发挥调节AIF的作用。Rho-kinase/JNK/AIF信号通路是介导大鼠心脏I/R损伤的一条重要的通路。
结论
1.I／R损伤模型中,Rho-kinase调节AIF从线粒体向细胞核的转移,在这个过程中,JNK起了重要的作用。Rho-kinase通过JNK来发挥调节AIF的作用。
2. Rho-kinase/JNK/AIF信号通路是介导心脏I／R损伤的一条重要的通路。
第三部分心肌缺血预适应中ERK对Rho-kinase的调节
研究目的
探讨在心肌缺血预适应中ERK是否可通过下调Rho-kinase活性起到心肌保护作用。
研究方法
1.大鼠心脏I/R和IPC模型的建立,分为以下6组：I／R组(18只)IPC组(18只)、IPC+PD98059 (ERK抑制剂)组(18只)、IPC+fasudil组(18只)、IPC+PD98059+fasudil组(18只)和I/R+DMSO组(18只)。
2.心肌梗死面积检测,计算梗塞心肌面积占危险区(AAR)心肌面积百分比。
3. TUNEL检测细胞凋亡。
4. Western Blot分别检测ERK. RhoA. Rho-kinase、ROCK1、ROCK2、Csapase-3活性变化。通过检测ERK1/2磷酸化蛋白的表达水平来检测其活性。RhoA被激活后转位至细胞膜,我们通过分别检测细胞膜和细胞浆的表达来反映RhoA活性。
研究结果
1.IPC降低心肌细胞凋亡、Caspase-3活性和心肌梗死面积：I／R组大鼠心肌细胞凋亡率为33.87±1.57%,IPC组大鼠的心肌细胞凋亡率降低至17.29±0.84%。与I／R组相比,IPC组Capsase-3 cleavage蛋白表达大约减少65%,Caspase-3活性明显降低。I／R组大鼠心肌梗死面积为60.53±4.08%,而在IPC组心肌梗死面积降至29.16±1.08%。IPC+DMSO组大鼠的心肌细胞凋亡率和心肌梗死面积分别为30.06±0.79%和18.46±0.92%,与IPC组相比二者无明显差异。结果表明IPC可明显降低大鼠心脏缺血再灌注损伤造成的心肌细胞凋亡和心肌梗死面积
2.IPC对ERK1/2的作用：免疫组化检测到IPC组磷酸化的ERK1/2表达明显增加；Western Blot同样检测到IPC组ERK1/2的磷酸化大致增加了1.8倍。说明IPC可明显增加的ERK1/2的活性。
3.IPC对RhoA、Rho-kinase、ROCK1、ROCK2的作用：RhoA蛋白的活性通过RhoA的膜转位来表示,I／R组与IPC组表达无明显差异。ROCK1蛋白表达在I／R组明显增加而在IPC组降低,ROCK1 mRNA表达水平在I／R组明显增加,而在IPC组降低。ROCK2表达在I／R组和IPC组均增加。Rho-kinase活性通过检测其特异性底物MYPT-1的磷酸化水平来表示,与I／R组相比,IPC组MYPT-1的磷酸化减低了49%。结果说明心肌IPC可明显降低大鼠I／R损伤造成的Rho-kinase活性和ROCK1表达增加,而对RhoA活性和ROCK2表达则无明显作用。
4.IPC中抑制ERK1/2后增加心肌细胞凋亡、Caspase-3活性和心肌梗死面积：IPC+PD98059组大鼠心肌细胞凋亡率为29.83±0.7%,与IPC组相比明显增加；IPC+PD98059组的Capsase-3 cleavage蛋白表达大概增加2.1倍,Caspase-3活性明显增加(P<0.05 vs IPC组);IPC+PD98059组大鼠心肌梗死面积为44.88±0.7%,与IPC组相比明显增加。结果表明IPC中应用PD98059抑制.ERK1/2活性可明显增加I／R损伤造成的心肌细胞凋亡和心肌梗死面积。
5.心肌IPC中ERK1/2抑制Rho-kinase活性：在IPC中,ERK1/2活性增加而Rho-kinase活性则明显降低。在IPC+PD98059组,MYPT-1的磷酸化水平和ROCK1蛋白表达水平均明显增加(P<0.05 vs IPC组),ROCK1 mRNA表达水平也明显增加。结果表明IPC中应用PD98059抑制ERK1/2可明显增加Rho-kinase活性和ROCK1表达。但是,IPC+fasudil组的ERK1/2磷酸化水平与IPC组相比,二者无明显差异；在IPC+PD98059组,ERK1/2磷酸化水平明显降低；而在IPC+PD98059+fasudil组ERK1/2磷酸化水平与IPC+PD98059组相比,二者无明显差异。结果说明应用Rho-kinase抑制剂后并不能改变ERK1/2的活性水平。
6.心肌IPC中抑制Rho-kinase可对抗由ERK1/2抑制所致的心肌细胞凋亡增加：与IPC+PD98059组相比,IPC+PD98059+fasudil组心肌细胞凋亡减少了22%而Capsase-3活性水平降低了30%。心肌梗死面积减少了12%左右。我们的结果表明IPC中,抑制Rho-kinase活性可部分减少由ERK1/2活性抑制所致的心肌细胞凋亡,进一步说明IPC中,ERK1/2信号可以下调Rho-kinase活性,起到保护心肌的作用。
结论
1.心肌缺血预适应中,激活的ERK信号可以下调Rho-kinase,从而减少心肌梗死面积和心肌细胞的凋亡率,减轻心肌细胞的损伤程度,具有心脏保护作用。
2.‘在此过程中RhoA并未参与,ERK主要通过下调ROCK1蛋白水平和基因水平来发挥作用。
创新性及局限性
1.创新点
(1)提出并证实了Rho-kinase可通过Rho-kinase/JNK/AIF途径参与心肌缺血再灌注造成的心肌细胞凋亡。
(2)提出并证实了心肌缺血预适应中ERK-MAPK信号通路可通过下调Rho-kinase活性降低细胞凋亡,并发现在此过程中RhoA并未参与,ERK主要通过下调ROCK1蛋白水平和基因水平来发挥作用。
2.局限性
(1)限于时间,本研究未能在细胞水平上研究细胞信号通路,这是本研究目前正在进行的内容。
(2)由于时间及条件的限制,未能应用基因敲除和RNA干扰等技术在组织及细胞水平上研究。
Background
With the improvement of people's standard of living, cardiovascular disease has now become a great threat to human life and health. The main cause of death of cardiovascular diseases is coronary artery disease. The best treatment of coronary heart disease is the resumption of blood perfusion of myocardium as soon as possible. However, both animal experiments and clinical studies have found that the damage of myocardial cell function and structural became worse with the restoration of blood supply. We call the pathophysiological state ischemia-reperfusion injury (I/R). Reperfusion involved in the clinical practice such as reperfusion myocardial infarction after coronary artery bypass grafting, heart transplants, heart resuscitation of cardiac arrest and cardiopulmonary bypass heart surgery. Ischemic preconditioning (IPC) has been exploited as a powerful endogenous form of cardioprotection. IPC was first discovered by Murry and associates, who demonstrated that a brief period of repetitive cardiac I/R exerts a protective effect against subsequent lethal periods of ischemia. IPC was found to similarly reduce cytosolic and mitochondrial Ca2+ overloading, to augment postischemic functional recovery, and to decrease infarct size. And IPC is known to protect cardiomyocyte apoptosis during reperfusion.
The ubiquitously expressed Rho-kinase, a serinethreonine kinase, has been identified as one of the effectors of the small GTP-binding protein Rho. Rho-kinase plays crucial roles in various cellular functions, and mediates cellular events such as changes in cell morphology, cell motility, focal adhesions, and cytokinesis. The Rho-kinase family contains two members:ROCK1 (also called ROKβor p160ROCK) and ROCK2 (also known as ROKa), which share 65% overall identity and 92% identity in the kinase domain. Two relatively selective Rho-kinase inhibitors, Y27632 and fasudil, bind to the kinase domain and inhibit ROCK1 and ROCK2 with similar potency. Rho-kinase phosphorylates a variety of protein substrates at serine or threonine residues. More than 20 Rho-kinase substrates have been identified. The first characterized targets of ROCK are myosin light chain (MLC) and the myosin binding subunit of MLC phosphatase (MYPT1). Rho-kinase can increase MLC phosphorylation through direct effect on MLC or indirectly by inactivating MLC phosphatase. The increased MLC phosphorylation results in stimulation of actomyosin contractility.
Accumulating evidences have demonstrated that Rho-kinase plays an important role in many major cardiovascular diseases such as hypertension, heart failure, myocardial infarction and atherosclerosis. Recent animal studies suggest that inhibition of Rho-kinase protects the heart against I/R injury. Previous studies have also demonstrated IPC caused a substantial decrease of Rho-kinase activation during sustained ischemia and reduced infarct size. However, little is known about the mechanism of Rho-kinase increased in I/R and reversed in IPC. Therefore, the aim of this study was to elucidate the mechanism of decreased Rho-kinase activity in IPC. We established IPC and I/R model in vivo and vitro. The activation of Rho-kinase, cardiomyocyte apoptosis, area of myocardial infarction and related signaling pathway were detected.
This study includes three parts:
PartⅠThe effect of Rho-kinase on I/R and IPC
PartⅡThe effect of Rho-kinase on AIF signaling pathway in I/R
PartⅢThe effect of ERK1/2 on Rho-kinase in IPC
Part I The effect of Rho-kinase on cell apoptosis in I/R and IPC
Objective
To investigate the effect of Rho-kinase on I/R and IPC
Methods
1. Animal preparation:The left anterior descending branch (LAD) of the left coronary artery was occluded and loosened to establish rat heart IPC and I/R model. The following experimental groups were studied. (1) control group (n=18); (2) I/R group (n=18); (3) I/R+fasudil(inhibitor of Rho-kinase) group (n=18); (4) IPC group (n=18).
2. Rho-kinase activity was assessed by examining phosphorylation of MYPT-1, a well established Rho-kinase specific substrate. Western blot analysis was performed to evaluate Rho-kinase activity.
3. Determination of myocardial infarct size:Evans blue and Nitro blue tetrazolium was used to evaluate the infracted and noninfarcted areas. Infarct size was expressed as the percentage of the area at risk(AAR).
4. To evaluate apoptotic activity, the TUNEL technique was used. TUNEL-positive cells were determined by randomly counting 10 fields of the section and were expressed as a percentage of normal nuclei.
5. Activation of Caspase-3 is a hallmark of apoptotic cell death and Caspase-3 cleavage is indicative of its activation. Caspase-3 cleavage was determined by Western blot analysis.
Results
1. Phospho-MYPT-1 increased during the I/R protocol. This increase in MYPT-1 phosphorylation was reversed in IPC protocol and I/R+fasudil protocol, demonstraying that Rho-kinase activity decreased in IPC.
2. Rho-kinase inhibition decreased the infarct size of the heart. The infarct size of the heart was 59.89±3.89% in I/R group. Administration of fasudil, the infarct size was 38.62±2.66%, demonstraying that inhibition of Rho-kinase activity reduces myocardial infarct size in rat heart I/R injury. In IPC, the myocardial infarct size was 29.16±1.08% (P<0.05 vs I/R group). These data suggest that myocardial infarct size was attenuated in IPC.
3. Inhibition of Rho-kinase reduced cardiomyocyte apoptosis. No TUNEL positive cells were found in the control group. The number of TUNEL positive cells were significantly increased in I/R group (32.78±5.1%). After fasudil was used, the TUNEL positive cells reduced to 17.05±4.2% (P<0.05 vs I/R group). The TUNEL positive cells were significantly reduced to 17.29±0.84% in IPC group (P<0.05 vs I/R group).
Conclusion
1. Rho-kinase activity increased in I/R. The overexpression of Rho-kinase in rat heart I/R protocol can aggravate the heart damage.
2. Rho-kinase inhibition in I/R has the cardiacprotective effect via reduced the apoptosis of cardiomyocytes and infarct size.
3. Rho-kinase activity increased in I/R and reversed in IPC. IPC has the cardiacprotective effect via reduced the apoptosis of cardiomyocytes and infarct size.
PartⅡThe effect of Rho-kinase on AIF signaling pathway in I/R
Objective
To investigate the effect of Rho-kinase on AIF signaling pathway in I/R
Methods
1. Animal preparation:The left anterior descending branch (LAD) of the left coronary artery was occluded and loosened to establish rat heart IPC and I/R model. The following experimental groups were studied. (1) control group (n=18); (2) I/R+NS group (n=18); (3) I/R+fasudil (inhibitor of Rho-kinase) group (n=18); (4) IPC+SP600125 (inhibitor of JNK) group (n=18); (5) I/R+DMSO group (n=18).
2. Determination of myocardial infarct size:Evans blue and Nitro blue tetrazolium was used to evaluate the infracted and noninfarcted areas. Infarct size was expressed as the percentage of the area at risk(AAR).
3. To evaluate apoptotic activity, the TUNEL technique was used. TUNEL-positive cells were determined by randomly counting 10 fields of the section and were expressed as a percentage of normal nuclei.
4. Western blot analysis was performed to evaluate Rho-kinase, JNK, and AIF activity.
Results
1. Rho kinase, JNK and AIF were activated in the rat I/R model. Western blot analysis was performed to evaluate whether Rho-kinase, JNK and AIF can be activated in the myocardial I/R rat model. Rho-kinase activity was assessed by examining phosphorylation of MYPT-1, a well established Rho-kinase specific substrate. JNK activity was assessed by examining phosphorylation of JNK. Both phospho-MYPT1 and phospho-JNK increased in I/R. AIF activity was assessed by nuclear translocation of AIF. The activation of AIF increased in nuclear fractions while it decreased in mitochondria fractions. The results indicate that AIF translocated to the nucleus in the I/R rat model.
2. Inhibition of Rho-kinase or JNK activity reduced myocardial infarct size. Administration of fasudil or SP600125 caused significant reduction of AAR and infarct size. The infarct sizes of the heart were 38.62±2.66% in I/R+fasudil group and 41.1±2.57% in I/R+SP600125 group respectively. These data suggest that inhibition of Rho-kinase or JNK activity reduces myocardial infarct size in rat heart I/R injury.
3. Inhibition of Rho-kinase or JNK activity reduced cell apoptosis of the heart. The number of TUNEL positive cells was significantly increased in I/R+NS group (32.78±5.1%). The TUNEL positive cells were significantly reduced to 17.05±4.2% in I/R+fasudil group. The number of TUNEL positive cells was significantly increased in I/R+DMSO group (34.45±3.73%). The TUNEL positive cells were significantly reduced to 16.25±3.29% in I/R+SP600125 group. These data suggest that inhibition of Rho-kinase or JNK activity activity reduces cell apoptosis.
4. Inhibition of Rho-kinase activity reduced the activation of JNK. Phospho-JNK increased after reperfusion in I/R group. The activation of phospho-JNK was significantly attenuated in I/R+fasudil group.
5. Inhibition of Rho-kinase or JNK activity reduces the AIF translocation: AIF increased after reperfusion in nuclear fractions. The activation of AIF was significantly attenuated by inhibition of Rho-kinase or JNK activity. On the contrary, AIF decreased in mitochondria fractions after reperfusion. Inhibition of Rho-kinase or JNK activity could increase the activation of AIF in mitochondria fractions. These data suggest that inhibition of Rho-kinase or JNK activity was able to reduce the mitochondrial-nuclear translocation of AIF.
Conclusion
1. JNK may be downstream of Rho-kinase activation and JNK may be required for Rho-kinase mediates AIF translocation in a rat model of myocardial ischemia and reperfusion.
2. Rho-kinase/JNK/AIF pathway may be a new pathway of cardiac myocyte injury.
PartⅢThe effect of ERK1/2 on Rho-kinase in IPC
Objective
To investigate the mechanism of decreased Rho-kinase activity in IPC
Methods
1. Animal preparation:(1) I/R group (n=18); (2) PC group (n=18); (3) IPC+PD98059 group (n=18); (4) IPC+fasudil (n=18); (5) IPC+PD98059+fasudil group (n=18); (6) IPC+DMSO (n=18).
2. Determination of myocardial infarct size:Evans blue and Nitro blue tetrazolium was used to evaluate the infracted and noninfarcted areas. Infarct size was expressed as the percentage of the area at risk(AAR).
3. To evaluate apoptotic activity, the TUNEL technique was used. TUNEL-positive cells were determined by randomly counting 10 fields of the section and were expressed as a percentage of normal nuclei.
4. Western blot analysis was performed to evaluate ERK, Rho-kinase, Caspase-3 activity and ROCK1, ROCK2 expression.
5. Immunohistochemistry Staining was performed to evaluate phospho-ERK1/2.
6. Real-time quantitative reverse transcription polymerase chain reaction (RT-PCR) was performed to evaluate ROCK1 mRNA.
Results
1. Effect of IPC on cardiomyocyte apoptosis, Caspase-3 cleavage and infarct size. TUNEL positive cells were expressed as a percentage of normal nuclei. The number of TUNEL positive cells was 33.87±1.57% in I/R group. The TUNEL positive cells were significantly reduced to 17.29±0.84% in IPC group. Activation of Caspase-3 is a hallmark of apoptotic cell death and Caspase-3 cleavage is indicative of its activation. IPC resulted in a 65% reduction in the amount of Capsase-3 cleavage (P<0.05 vs I/R group). Capsase-3 activity attenuated in IPC group. The infarct size of the heart was 60.53±4.08% in I/R group. In IPC, the myocardial infarct size was 29.16±1.08%. These values showed no significant differences between IPC and IPC+DMSO groups.
2. Effect of IPC on activity of ERK1/2. Phospho-ERK1/2 expression increased clearly in the heart in IPC. IPC resulted in an immediate increase in ERK1/2 phosphorylation. The increase in the phosphorylation of ERK1/2 was about 1.8-fold (P<0.05 vs I/R group).
3. Effect of IPC on activity of RhoA, ROCK1, ROCK2 and Rho-kinase. RhoA protein was determined by immunoblotting of cytosolic and membrane fractions. In all groups, RhoA protein was detected in both cytosolic and membrane fractions. An enhanced RhoA translocation to the membrane was detected in the I/R rat model. But no difference was observed between the I/R and IPC groups. ROCK1 and ROCK2 were also determined by western blot analysis. ROCK1 increased in the I/R group and attenuated in IPC group. ROCK2 increased in the I/R group, but there were no significant differences between the I/R and IPC groups. RT-PCR was also used to evaluate ROCK1 activity. A significant increase in mRNA expression of ROCK1 was detected in the I/R group, and the expression was decreased by IPC. Rho-kinase activity was assessed by examining phosphorylation of MYPT-1, a well established Rho-kinase specific substrate. IPC resulted in a 49% reduction in MYPT-1 phosphorylation and a 54% reduction in ROCK1 (P<0.05 vs I/R group).
4. Inhibition of ERK1/2 increased cardiomyocyte apoptosis, Caspase-3 activity and infarct size during IPC. The TUNEL positive cells were significantly increased with administration of PD98059 (29.83±0.7%) in IPC+PD98059 group (P<0.05 vs IPC group). The increase in the amount of cleaved Caspase-3 in IPC+PD98059 group was about 2.1-fold (P<0.05 vs IPC group). The infarct size of the heart was 44.88±0.7% in IPC+PD98059 group (P<0.05 vs IPC group).
5. ERK1/2 opposed Rho-kinase during IPC. ERK1/2 was strongly activated and Rho-kinase activity decreased in IPC. Inhibition of ERK1/2 in IPC could lead to upregulation of MYPT-1 phosphorylation and the amount of ROCK1. Inhibition of ERK1/2 in IPC also could lead to upregulation of ROCK1 mRNA. In the IPC+fasudil group, phospho-ERK1/2 expression was similar to the IPC group. In the IPC+PD98059 group, phospho-ERK1/2 expression significantly decreased. And phospho-ERK1/2 expression was not recovered in the IPC+PD98059+fasudil group.
6. Inhibition of Rho-kinase rescued the effect of inhibition of ERK1/2 on apoptosis. Fasudil treatment reversed cell apoptosis caused by treatment with PD98059 in IPC. The number of TUNEL positive cells was 23.3±0.67% in IPC+PD98059+fasudil group. The amount of cleaved Caspase-3 was also significantly reduced in IPC+PD98059+fasudil group. Fasudil resulted in a 22% reduction in cardiomyocyte apoptosis and a 30% reduction in cleaved Caspase-3 in IPC+PD98059+fasudil group (p<0.05 vs IPC+PD98059 group). Additionally, the infarct size of the heart was 39.44±0.92% in IPC+PD98059+fasudil group. The combination of PD98059 and fasudil also resulted in a 12% reduction in myocardial infarct size (p<0.05 vs IPC+PD98059 group).
Conclusion
1. ERK-MAPK signaling is required in IPC to oppose the Rho-kinase signaling on cardiomyocyte apoptosis.
2. Overpression of RhoA in I/R might not be changed by preconditioning.
3. ROCK1 may be the major Rho-kinase which is opposed by ERK-MAPK signaling in IPC.
心肌长时间缺血可以导致组织损伤以及细胞死亡,冠状动脉再通是缓解缺血损伤的关键,但较长时间缺血后的再灌注却可导致更为严重的损伤,即心脏缺血／再灌注损伤(Ischemia and reperfusion Injury, I/R), I/R的概念于1960年被Jennings等第一次提出。I／R是冠脉再通术(溶栓、PTCA或搭桥术)后的重要的并发症。如何做到既保证尽早恢复缺血组织的血流灌注,又减轻甚至消除再灌注损伤的发生便成为缺血性心脏病防治中的重要课题。有效防治再灌注损伤的重要前提是阐明其发病机制。心肌在遭受一次或多次反复的短暂缺血再灌注后,会表达出一种对随后而来的一次长时间的严重缺血损伤的抵抗能力的提高,这种现象称为缺血预适应(ischemic preconditioning, IPC), IPC于1986年首次被Murry等发现。经过预适应的心肌能够缩小心肌梗死的面积,改善心肌收缩力,保护冠状动脉内皮和心肌细胞的超微结构,保护微循环,降低再灌注导致的心律失常的发生率,更快地使心肌从再灌注诱导的心肌顿抑中恢复,是一种内源性自我保护方式。
Rho-kinase是一类丝-苏氨酸蛋白家族,可作为小G蛋白RhoA的直接下游效应物,参与多种细胞生物功能,如：平滑肌收缩、肌动蛋白细胞骨架组装、细胞黏附、移动、基因表达等。Rho-kinase以两种同源性极高的异构体形式存在：ROCK-1(或ROKβ、p160ROCK)和ROCK-2(或ROKα)。ROCK-1位于18号染色体,含有1354个氨基酸,ROCK-2位于12号染色体,含有1388个氨基酸。二者有65%的同源性,而在激酶结构域则有92%的同源性。Y-27632和fasudil是ROCK的选择性抑制剂,作用于其ATP依赖激酶区,可同时抑制ROCK-1和ROCK-2。Rho-kinase可以介导下游一系列磷酸化／脱磷酸化反应,已有20多种下游蛋白被发现。如：MLC、MYPT-1 ERM、PTEN、IRS1 NHE1、LIM激酶等。最具特征意义的Rho-kinase底物是MLC (myosin light chain)和MYPT-1 (myosin binding subunit of MLC phosphorylation, MLC磷酸酶的肌球蛋白结合亚基)。Rho-kinase可以通过作用于MLC或MYPT1来增加胞浆内MLC的磷酸化,促进肌动蛋白微丝骨架的聚合。
最近研究发现,Rho-kinase参与了变异性心绞痛、心衰、心肌缺血再灌注损伤等心血管疾病。初步研究表明Rho-kinase在心脏I／R损伤中活性增加,而在IPC中活性可能降低,但是其中发生机制却远未清楚。例如Rho-kinase调控哪些细胞因子,通过哪些信号途径发挥作用,以及在IPC中Rho-kinase活性是否会降低,Rho-kinase活性降低的机制如何,这些问题构成了本研究课题的内容。在本研究中,我们拟建立大鼠I／R和IPC模型,明确Rho-kinase活性在IPC和I／R中是否不同,Rho-kinase对心肌梗死面积、心肌细胞凋亡的影响以及对细胞凋亡有关信号通路的调节；明确IPC中ERK-MAPK是否能通过下调Rho-kinase活性起到抗心肌细胞凋亡作用。通过以上研究,进一步明确Rho-kinase在心脏I/R和IPC中所起的作用及其机制。这对于阐明I／R和IPC的发病机制,为心肌保护提供新的治疗途径具有重要的意义。
本研究共分三部分进行：
第一部分Rho-kinase在心脏缺血再灌注损伤和心肌缺血预适应中的表达及其对细胞凋亡的影响；
第二部分心脏缺血再灌注损伤中Rho-kinase对AIF的影响及调节机制；
第三部分心肌缺血预适应中ERK对Rho-kinase的调节。
第一部分Rho-kinase在心脏缺血再灌注损伤和心肌缺血预适应中的表达及其对细胞凋亡的影响研究目的
本部分研究拟建立大鼠I／R和IPC模型,应用药物fasudil抑制Rho-kinase活性,测定心肌梗死面积、心肌细胞的凋亡,明确Rho-kinase在心脏I／R和IPC中的作用。研究方法
1.大鼠心脏I／R和IPC模型的建立和分组：采用开胸结扎和松开冠状动脉的方法建立大鼠心脏I／R和IPC型。分为以下四组：对照组(18只)、I／R组(18只)、I/R+fasudil组(18只)和IPC组(18只)
2. Rho-kinase活性的检测：Rho-kinase的活性通过Western检测其特异性底物MYPT-1的磷酸化水平来表示。
3.心肌梗死面积检测：采用伊文思蓝染色鉴别非缺血区与缺血危险区(AAR),硝基四氮唑蓝(NBT)染色鉴别梗死心肌与非梗死心肌。以称重法计算心肌缺血危险区(伊文思蓝未染区),梗死区(NBT未染区)的重量,心肌梗死范围的大小以梗死区重量占缺血危险区重量的百分比表示。
公式为：心肌梗死范围=NBT未染区／伊文思蓝未染区×100%
4.大鼠心肌细胞凋亡的检测：采用末端脱氧核苷酸转移酶介导的末端标记技术(TUNEL)试剂盒检测各组大鼠心肌细胞的凋亡。按照试剂盒说明书操作。标记前用DNA酶处理切片做阳性对照,用标记液代替TdT酶反应液做阴性对照。每张切片随机选取10个视野(×400倍),计数凋亡细胞个数和所有细胞个数,以凋亡细胞个数／所有细胞个数反映各组心肌细胞凋亡的情况。
5. Caspase-3活性的检测：Caspase-3的活性通过Western检测其裂解产物cleaved-Caspase-3的表达水平来表示。研究结果
1.p-MYPT-1(磷酸化的MYPT-1)在I／R中大量表达,而在IPC中活性降低。在I／R中应用Rho-kinase抑制剂fasudil后,p-MYPT-1表达降低：对照组中只有微弱的p-MYPT-1表达。说明Rho-kinase在I／R中活性增加,而IPC和fasudil均抑制了Rho-kinase的活性。
2. Rho-kinase活性的抑制可减少大鼠心肌梗死面积：I／R组大鼠的心肌梗死面积占危险区心肌面积的60.3±4.08%, I/R+fasudil组大鼠的,心肌梗死面积／AAR减少至38.62±2.66%,说明在I／R大鼠中抑制Rho-kinase活性后能显著减少心肌梗死面积。在IPC中大鼠的心肌梗死面积降至29.16±1.08%,说明IPC可能通过降低Rho-kinase活性而明显缩小心肌梗死面积。
3. Rho-kinase活性的抑制可减少大鼠心肌细胞凋亡率：在对照组大鼠心肌中无TUNEL染色阳性细胞。在I／R组大鼠心肌中,TUNEL染色阳性细胞占全部细胞的33.87±1.57%,说明I／R可引起心肌细胞凋亡增加。在I/R+fasudil组大鼠心肌中,TUNEL染色阳性细胞占全部细胞的17.05±4.22%,说明在I／R大鼠中抑制Rho-kinase活性能减少心肌细胞凋亡率。在IPC中,细胞凋亡率为17.29±0.84%,说明IPC可能通过降低Rho-kinase活性而明显降低心肌细胞凋亡率。
4. Rho-kinase活性的抑制降低Caspase-3活性：在I／R中cleaved-Caspase-3大量表达,在IPC和I/R+fasudil组中其表达分别减少了约65.3%和58.5%,说明IPC和fasudil在抑制Rho-kinase活性同时抑制了Caspase-3的活性。
结论
1.在大鼠心肌I/R损伤中Rho-kinase活性明显增加,加重了心脏的损伤、增加了心肌细胞凋亡。
2.在大鼠心肌I/R损伤中抑制Rho-kinase活性后能减少心肌梗死面积和心肌细胞的凋亡率,减轻心肌细胞的损伤程度。
3.IPC中Rho-kinase活性降低,心肌梗死面积和心肌细胞凋亡减轻,具有心脏保护作用。
第二部分心脏缺血再灌注损伤中Rho-kinase对AIF的影响及调节机制
研究目的
探讨在大鼠心脏I／R中Rho-kinase对AIF的影响及调节机制。(Rho-kinase/JNK/AIF信号通路在心脏I／R损伤中的作用)研究方法
1.大鼠心脏I／R损伤模型的建立,分为以下5组：对照组(18只)I/R+NS组(18只)、I/R+fasudil组(18只)、I/R+SP600125(JNK抑制剂)组(18只)和I/R+DMSO组(18只)。
2.心肌梗死面积检测,计算梗死心肌面积占危险区(AAR)心肌面积百分比。
3.TUNEL法检测细胞凋亡。
4.采用Western Blot检测各组大鼠心肌中Rho-kinase、JNK、AIF的活性变化。Rho-kinase活性通过检测MYPT-1的磷酸化水平来表示,通过检测JNK的磷酸化蛋白的水平来反映JNK的活性,通过分别检测AIF的线粒体和细胞核中的蛋白水平来反映AIF的活性。研究结果
1.心脏I／R损伤中Rho-kinase、JNK、AIF活性增加：与对照组相比,I/R+NS组中p-MYPT-1和p-JNK蛋白表达明显增加。I/R+NS组中AIF在细胞核中的表达增多,在线粒体中的表达减少,AIF从线粒体到细胞核的转位增加。说明心脏I／R损伤可增加Rho-kinase、JNK、AIF活性。
2.抑制Rho-kinase或JNK后减少大鼠心肌梗死面积：I/R+NS组和I/R+DMSO组大鼠的心肌梗死面积分别占整个左心室面积的59.89±3.83%和61.64±3.3%,两者相比无显著性差异。I/R+fasudil组大鼠的心肌梗死面积／AAR减少至38.62±2.66%,说明在I／R中抑制Rho-kinase活性能显著减少心肌梗死面积。I/R+SP600125组大鼠的心肌梗死面积／AAR减少至41.1±2.57%,说明在I／R中抑制JNK活性能显著减少心肌梗死面积。
3.抑制Rho-kinase或JNK后减少大鼠心肌细胞凋亡：I/R+NS组和I/R+DMSO组大鼠的心肌细胞凋亡分别为32.78±5.01%和34.45±3.73%,两者相比无显著性差异。I/R+fasudil组大鼠的心肌细胞凋亡减少至17.05±4.22%,说明在I／R中抑制Rho-kinase活性能显著减少心肌细胞凋亡。I/R+SP600125组大鼠的心肌细胞凋亡减少至16.25±3.29%,说明在I／R中抑制JNK活性能显著减少心肌细胞凋亡。
4.抑制Rho-kinase活性可降低JNK活性：在I／R组大鼠心肌中磷酸化JNK表达增多,应用fasudil后磷酸化JNK的表达下降,说明磷酸化JNK的表达与Rho-kinase有关,JNK是Rho-kinase的下游分子。
5.在I/R+DMSO组p-MYPT-1表达明显增加,应用JNK抑制剂SP600125后,p-MYPT-1表达无明显变化。说明p-MYPT-1的表达与JNK活性无关,进一步证实JNK是Rho-kinase的下游分子。
6.抑制Rho-kinase活性可减少AIF从线粒体到心肌细胞核的转位：在I／R组大鼠心肌细胞核中AIF的表达增多,抑制Rho-kinase活性后其表达减少；另一方面,在I／R组大鼠心肌细胞线粒体中AIF的表达减少,而抑制Rho-kinase活性后其表达增多。说明抑制Rho-kinase活性可减少AIF从线粒体向细胞核的转移,证实AIF是Rho-kinase的下游分子。
7.抑制JNK活性可减少AIF的从线粒体到心肌细胞核的转位：在I／R组大鼠心肌细胞核中AIF的表达增多,抑制JNK活性后其表达减少；另一方面,在I／R组大鼠心肌细胞线粒体中AIF的表达减少,而抑制JNK活性后其表达增多。说明抑制JNK可以减少AIF从线粒体向细胞核的转移,证实AIF是JNK的下游分子。
结果证实,在大鼠I／R损伤模型中,Rho-kinase调节AIF从线粒体向细胞核的转移,在这个过程中,JNK起了重要的作用。Rho-kinase通过JNK来发挥调节AIF的作用。Rho-kinase/JNK/AIF信号通路是介导大鼠心脏I/R损伤的一条重要的通路。
结论
1.I／R损伤模型中,Rho-kinase调节AIF从线粒体向细胞核的转移,在这个过程中,JNK起了重要的作用。Rho-kinase通过JNK来发挥调节AIF的作用。
2. Rho-kinase/JNK/AIF信号通路是介导心脏I／R损伤的一条重要的通路。
第三部分心肌缺血预适应中ERK对Rho-kinase的调节
研究目的
探讨在心肌缺血预适应中ERK是否可通过下调Rho-kinase活性起到心肌保护作用。
研究方法
1.大鼠心脏I/R和IPC模型的建立,分为以下6组：I／R组(18只)IPC组(18只)、IPC+PD98059 (ERK抑制剂)组(18只)、IPC+fasudil组(18只)、IPC+PD98059+fasudil组(18只)和I/R+DMSO组(18只)。
2.心肌梗死面积检测,计算梗塞心肌面积占危险区(AAR)心肌面积百分比。
3. TUNEL检测细胞凋亡。
4. Western Blot分别检测ERK. RhoA. Rho-kinase、ROCK1、ROCK2、Csapase-3活性变化。通过检测ERK1/2磷酸化蛋白的表达水平来检测其活性。RhoA被激活后转位至细胞膜,我们通过分别检测细胞膜和细胞浆的表达来反映RhoA活性。
研究结果
1.IPC降低心肌细胞凋亡、Caspase-3活性和心肌梗死面积：I／R组大鼠心肌细胞凋亡率为33.87±1.57%,IPC组大鼠的心肌细胞凋亡率降低至17.29±0.84%。与I／R组相比,IPC组Capsase-3 cleavage蛋白表达大约减少65%,Caspase-3活性明显降低。I／R组大鼠心肌梗死面积为60.53±4.08%,而在IPC组心肌梗死面积降至29.16±1.08%。IPC+DMSO组大鼠的心肌细胞凋亡率和心肌梗死面积分别为30.06±0.79%和18.46±0.92%,与IPC组相比二者无明显差异。结果表明IPC可明显降低大鼠心脏缺血再灌注损伤造成的心肌细胞凋亡和心肌梗死面积
2.IPC对ERK1/2的作用：免疫组化检测到IPC组磷酸化的ERK1/2表达明显增加；Western Blot同样检测到IPC组ERK1/2的磷酸化大致增加了1.8倍。说明IPC可明显增加的ERK1/2的活性。
3.IPC对RhoA、Rho-kinase、ROCK1、ROCK2的作用：RhoA蛋白的活性通过RhoA的膜转位来表示,I／R组与IPC组表达无明显差异。ROCK1蛋白表达在I／R组明显增加而在IPC组降低,ROCK1 mRNA表达水平在I／R组明显增加,而在IPC组降低。ROCK2表达在I／R组和IPC组均增加。Rho-kinase活性通过检测其特异性底物MYPT-1的磷酸化水平来表示,与I／R组相比,IPC组MYPT-1的磷酸化减低了49%。结果说明心肌IPC可明显降低大鼠I／R损伤造成的Rho-kinase活性和ROCK1表达增加,而对RhoA活性和ROCK2表达则无明显作用。
4.IPC中抑制ERK1/2后增加心肌细胞凋亡、Caspase-3活性和心肌梗死面积：IPC+PD98059组大鼠心肌细胞凋亡率为29.83±0.7%,与IPC组相比明显增加；IPC+PD98059组的Capsase-3 cleavage蛋白表达大概增加2.1倍,Caspase-3活性明显增加(P<0.05 vs IPC组);IPC+PD98059组大鼠心肌梗死面积为44.88±0.7%,与IPC组相比明显增加。结果表明IPC中应用PD98059抑制.ERK1/2活性可明显增加I／R损伤造成的心肌细胞凋亡和心肌梗死面积。
5.心肌IPC中ERK1/2抑制Rho-kinase活性：在IPC中,ERK1/2活性增加而Rho-kinase活性则明显降低。在IPC+PD98059组,MYPT-1的磷酸化水平和ROCK1蛋白表达水平均明显增加(P<0.05 vs IPC组),ROCK1 mRNA表达水平也明显增加。结果表明IPC中应用PD98059抑制ERK1/2可明显增加Rho-kinase活性和ROCK1表达。但是,IPC+fasudil组的ERK1/2磷酸化水平与IPC组相比,二者无明显差异；在IPC+PD98059组,ERK1/2磷酸化水平明显降低；而在IPC+PD98059+fasudil组ERK1/2磷酸化水平与IPC+PD98059组相比,二者无明显差异。结果说明应用Rho-kinase抑制剂后并不能改变ERK1/2的活性水平。
6.心肌IPC中抑制Rho-kinase可对抗由ERK1/2抑制所致的心肌细胞凋亡增加：与IPC+PD98059组相比,IPC+PD98059+fasudil组心肌细胞凋亡减少了22%而Capsase-3活性水平降低了30%。心肌梗死面积减少了12%左右。我们的结果表明IPC中,抑制Rho-kinase活性可部分减少由ERK1/2活性抑制所致的心肌细胞凋亡,进一步说明IPC中,ERK1/2信号可以下调Rho-kinase活性,起到保护心肌的作用。
结论
1.心肌缺血预适应中,激活的ERK信号可以下调Rho-kinase,从而减少心肌梗死面积和心肌细胞的凋亡率,减轻心肌细胞的损伤程度,具有心脏保护作用。
2.‘在此过程中RhoA并未参与,ERK主要通过下调ROCK1蛋白水平和基因水平来发挥作用。
创新性及局限性
1.创新点
(1)提出并证实了Rho-kinase可通过Rho-kinase/JNK/AIF途径参与心肌缺血再灌注造成的心肌细胞凋亡。
(2)提出并证实了心肌缺血预适应中ERK-MAPK信号通路可通过下调Rho-kinase活性降低细胞凋亡,并发现在此过程中RhoA并未参与,ERK主要通过下调ROCK1蛋白水平和基因水平来发挥作用。
2.局限性
(1)限于时间,本研究未能在细胞水平上研究细胞信号通路,这是本研究目前正在进行的内容。
(2)由于时间及条件的限制,未能应用基因敲除和RNA干扰等技术在组织及细胞水平上研究。
Background
With the improvement of people's standard of living, cardiovascular disease has now become a great threat to human life and health. The main cause of death of cardiovascular diseases is coronary artery disease. The best treatment of coronary heart disease is the resumption of blood perfusion of myocardium as soon as possible. However, both animal experiments and clinical studies have found that the damage of myocardial cell function and structural became worse with the restoration of blood supply. We call the pathophysiological state ischemia-reperfusion injury (I/R). Reperfusion involved in the clinical practice such as reperfusion myocardial infarction after coronary artery bypass grafting, heart transplants, heart resuscitation of cardiac arrest and cardiopulmonary bypass heart surgery. Ischemic preconditioning (IPC) has been exploited as a powerful endogenous form of cardioprotection. IPC was first discovered by Murry and associates, who demonstrated that a brief period of repetitive cardiac I/R exerts a protective effect against subsequent lethal periods of ischemia. IPC was found to similarly reduce cytosolic and mitochondrial Ca2+ overloading, to augment postischemic functional recovery, and to decrease infarct size. And IPC is known to protect cardiomyocyte apoptosis during reperfusion.
The ubiquitously expressed Rho-kinase, a serinethreonine kinase, has been identified as one of the effectors of the small GTP-binding protein Rho. Rho-kinase plays crucial roles in various cellular functions, and mediates cellular events such as changes in cell morphology, cell motility, focal adhesions, and cytokinesis. The Rho-kinase family contains two members:ROCK1 (also called ROKβor p160ROCK) and ROCK2 (also known as ROKa), which share 65% overall identity and 92% identity in the kinase domain. Two relatively selective Rho-kinase inhibitors, Y27632 and fasudil, bind to the kinase domain and inhibit ROCK1 and ROCK2 with similar potency. Rho-kinase phosphorylates a variety of protein substrates at serine or threonine residues. More than 20 Rho-kinase substrates have been identified. The first characterized targets of ROCK are myosin light chain (MLC) and the myosin binding subunit of MLC phosphatase (MYPT1). Rho-kinase can increase MLC phosphorylation through direct effect on MLC or indirectly by inactivating MLC phosphatase. The increased MLC phosphorylation results in stimulation of actomyosin contractility.
Accumulating evidences have demonstrated that Rho-kinase plays an important role in many major cardiovascular diseases such as hypertension, heart failure, myocardial infarction and atherosclerosis. Recent animal studies suggest that inhibition of Rho-kinase protects the heart against I/R injury. Previous studies have also demonstrated IPC caused a substantial decrease of Rho-kinase activation during sustained ischemia and reduced infarct size. However, little is known about the mechanism of Rho-kinase increased in I/R and reversed in IPC. Therefore, the aim of this study was to elucidate the mechanism of decreased Rho-kinase activity in IPC. We established IPC and I/R model in vivo and vitro. The activation of Rho-kinase, cardiomyocyte apoptosis, area of myocardial infarction and related signaling pathway were detected.
This study includes three parts:
PartⅠThe effect of Rho-kinase on I/R and IPC
PartⅡThe effect of Rho-kinase on AIF signaling pathway in I/R
PartⅢThe effect of ERK1/2 on Rho-kinase in IPC
Part I The effect of Rho-kinase on cell apoptosis in I/R and IPC
Objective
To investigate the effect of Rho-kinase on I/R and IPC
Methods
1. Animal preparation:The left anterior descending branch (LAD) of the left coronary artery was occluded and loosened to establish rat heart IPC and I/R model. The following experimental groups were studied. (1) control group (n=18); (2) I/R group (n=18); (3) I/R+fasudil(inhibitor of Rho-kinase) group (n=18); (4) IPC group (n=18).
2. Rho-kinase activity was assessed by examining phosphorylation of MYPT-1, a well established Rho-kinase specific substrate. Western blot analysis was performed to evaluate Rho-kinase activity.
3. Determination of myocardial infarct size:Evans blue and Nitro blue tetrazolium was used to evaluate the infracted and noninfarcted areas. Infarct size was expressed as the percentage of the area at risk(AAR).
4. To evaluate apoptotic activity, the TUNEL technique was used. TUNEL-positive cells were determined by randomly counting 10 fields of the section and were expressed as a percentage of normal nuclei.
5. Activation of Caspase-3 is a hallmark of apoptotic cell death and Caspase-3 cleavage is indicative of its activation. Caspase-3 cleavage was determined by Western blot analysis.
Results
1. Phospho-MYPT-1 increased during the I/R protocol. This increase in MYPT-1 phosphorylation was reversed in IPC protocol and I/R+fasudil protocol, demonstraying that Rho-kinase activity decreased in IPC.
2. Rho-kinase inhibition decreased the infarct size of the heart. The infarct size of the heart was 59.89±3.89% in I/R group. Administration of fasudil, the infarct size was 38.62±2.66%, demonstraying that inhibition of Rho-kinase activity reduces myocardial infarct size in rat heart I/R injury. In IPC, the myocardial infarct size was 29.16±1.08% (P<0.05 vs I/R group). These data suggest that myocardial infarct size was attenuated in IPC.
3. Inhibition of Rho-kinase reduced cardiomyocyte apoptosis. No TUNEL positive cells were found in the control group. The number of TUNEL positive cells were significantly increased in I/R group (32.78±5.1%). After fasudil was used, the TUNEL positive cells reduced to 17.05±4.2% (P<0.05 vs I/R group). The TUNEL positive cells were significantly reduced to 17.29±0.84% in IPC group (P<0.05 vs I/R group).
Conclusion
1. Rho-kinase activity increased in I/R. The overexpression of Rho-kinase in rat heart I/R protocol can aggravate the heart damage.
2. Rho-kinase inhibition in I/R has the cardiacprotective effect via reduced the apoptosis of cardiomyocytes and infarct size.
3. Rho-kinase activity increased in I/R and reversed in IPC. IPC has the cardiacprotective effect via reduced the apoptosis of cardiomyocytes and infarct size.
PartⅡThe effect of Rho-kinase on AIF signaling pathway in I/R
Objective
To investigate the effect of Rho-kinase on AIF signaling pathway in I/R
Methods
1. Animal preparation:The left anterior descending branch (LAD) of the left coronary artery was occluded and loosened to establish rat heart IPC and I/R model. The following experimental groups were studied. (1) control group (n=18); (2) I/R+NS group (n=18); (3) I/R+fasudil (inhibitor of Rho-kinase) group (n=18); (4) IPC+SP600125 (inhibitor of JNK) group (n=18); (5) I/R+DMSO group (n=18).
2. Determination of myocardial infarct size:Evans blue and Nitro blue tetrazolium was used to evaluate the infracted and noninfarcted areas. Infarct size was expressed as the percentage of the area at risk(AAR).
3. To evaluate apoptotic activity, the TUNEL technique was used. TUNEL-positive cells were determined by randomly counting 10 fields of the section and were expressed as a percentage of normal nuclei.
4. Western blot analysis was performed to evaluate Rho-kinase, JNK, and AIF activity.
Results
1. Rho kinase, JNK and AIF were activated in the rat I/R model. Western blot analysis was performed to evaluate whether Rho-kinase, JNK and AIF can be activated in the myocardial I/R rat model. Rho-kinase activity was assessed by examining phosphorylation of MYPT-1, a well established Rho-kinase specific substrate. JNK activity was assessed by examining phosphorylation of JNK. Both phospho-MYPT1 and phospho-JNK increased in I/R. AIF activity was assessed by nuclear translocation of AIF. The activation of AIF increased in nuclear fractions while it decreased in mitochondria fractions. The results indicate that AIF translocated to the nucleus in the I/R rat model.
2. Inhibition of Rho-kinase or JNK activity reduced myocardial infarct size. Administration of fasudil or SP600125 caused significant reduction of AAR and infarct size. The infarct sizes of the heart were 38.62±2.66% in I/R+fasudil group and 41.1±2.57% in I/R+SP600125 group respectively. These data suggest that inhibition of Rho-kinase or JNK activity reduces myocardial infarct size in rat heart I/R injury.
3. Inhibition of Rho-kinase or JNK activity reduced cell apoptosis of the heart. The number of TUNEL positive cells was significantly increased in I/R+NS group (32.78±5.1%). The TUNEL positive cells were significantly reduced to 17.05±4.2% in I/R+fasudil group. The number of TUNEL positive cells was significantly increased in I/R+DMSO group (34.45±3.73%). The TUNEL positive cells were significantly reduced to 16.25±3.29% in I/R+SP600125 group. These data suggest that inhibition of Rho-kinase or JNK activity activity reduces cell apoptosis.
4. Inhibition of Rho-kinase activity reduced the activation of JNK. Phospho-JNK increased after reperfusion in I/R group. The activation of phospho-JNK was significantly attenuated in I/R+fasudil group.
5. Inhibition of Rho-kinase or JNK activity reduces the AIF translocation: AIF increased after reperfusion in nuclear fractions. The activation of AIF was significantly attenuated by inhibition of Rho-kinase or JNK activity. On the contrary, AIF decreased in mitochondria fractions after reperfusion. Inhibition of Rho-kinase or JNK activity could increase the activation of AIF in mitochondria fractions. These data suggest that inhibition of Rho-kinase or JNK activity was able to reduce the mitochondrial-nuclear translocation of AIF.
Conclusion
1. JNK may be downstream of Rho-kinase activation and JNK may be required for Rho-kinase mediates AIF translocation in a rat model of myocardial ischemia and reperfusion.
2. Rho-kinase/JNK/AIF pathway may be a new pathway of cardiac myocyte injury.
PartⅢThe effect of ERK1/2 on Rho-kinase in IPC
Objective
To investigate the mechanism of decreased Rho-kinase activity in IPC
Methods
1. Animal preparation:(1) I/R group (n=18); (2) PC group (n=18); (3) IPC+PD98059 group (n=18); (4) IPC+fasudil (n=18); (5) IPC+PD98059+fasudil group (n=18); (6) IPC+DMSO (n=18).
2. Determination of myocardial infarct size:Evans blue and Nitro blue tetrazolium was used to evaluate the infracted and noninfarcted areas. Infarct size was expressed as the percentage of the area at risk(AAR).
3. To evaluate apoptotic activity, the TUNEL technique was used. TUNEL-positive cells were determined by randomly counting 10 fields of the section and were expressed as a percentage of normal nuclei.
4. Western blot analysis was performed to evaluate ERK, Rho-kinase, Caspase-3 activity and ROCK1, ROCK2 expression.
5. Immunohistochemistry Staining was performed to evaluate phospho-ERK1/2.
6. Real-time quantitative reverse transcription polymerase chain reaction (RT-PCR) was performed to evaluate ROCK1 mRNA.
Results
1. Effect of IPC on cardiomyocyte apoptosis, Caspase-3 cleavage and infarct size. TUNEL positive cells were expressed as a percentage of normal nuclei. The number of TUNEL positive cells was 33.87±1.57% in I/R group. The TUNEL positive cells were significantly reduced to 17.29±0.84% in IPC group. Activation of Caspase-3 is a hallmark of apoptotic cell death and Caspase-3 cleavage is indicative of its activation. IPC resulted in a 65% reduction in the amount of Capsase-3 cleavage (P<0.05 vs I/R group). Capsase-3 activity attenuated in IPC group. The infarct size of the heart was 60.53±4.08% in I/R group. In IPC, the myocardial infarct size was 29.16±1.08%. These values showed no significant differences between IPC and IPC+DMSO groups.
2. Effect of IPC on activity of ERK1/2. Phospho-ERK1/2 expression increased clearly in the heart in IPC. IPC resulted in an immediate increase in ERK1/2 phosphorylation. The increase in the phosphorylation of ERK1/2 was about 1.8-fold (P<0.05 vs I/R group).
3. Effect of IPC on activity of RhoA, ROCK1, ROCK2 and Rho-kinase. RhoA protein was determined by immunoblotting of cytosolic and membrane fractions. In all groups, RhoA protein was detected in both cytosolic and membrane fractions. An enhanced RhoA translocation to the membrane was detected in the I/R rat model. But no difference was observed between the I/R and IPC groups. ROCK1 and ROCK2 were also determined by western blot analysis. ROCK1 increased in the I/R group and attenuated in IPC group. ROCK2 increased in the I/R group, but there were no significant differences between the I/R and IPC groups. RT-PCR was also used to evaluate ROCK1 activity. A significant increase in mRNA expression of ROCK1 was detected in the I/R group, and the expression was decreased by IPC. Rho-kinase activity was assessed by examining phosphorylation of MYPT-1, a well established Rho-kinase specific substrate. IPC resulted in a 49% reduction in MYPT-1 phosphorylation and a 54% reduction in ROCK1 (P<0.05 vs I/R group).
4. Inhibition of ERK1/2 increased cardiomyocyte apoptosis, Caspase-3 activity and infarct size during IPC. The TUNEL positive cells were significantly increased with administration of PD98059 (29.83±0.7%) in IPC+PD98059 group (P<0.05 vs IPC group). The increase in the amount of cleaved Caspase-3 in IPC+PD98059 group was about 2.1-fold (P<0.05 vs IPC group). The infarct size of the heart was 44.88±0.7% in IPC+PD98059 group (P<0.05 vs IPC group).
5. ERK1/2 opposed Rho-kinase during IPC. ERK1/2 was strongly activated and Rho-kinase activity decreased in IPC. Inhibition of ERK1/2 in IPC could lead to upregulation of MYPT-1 phosphorylation and the amount of ROCK1. Inhibition of ERK1/2 in IPC also could lead to upregulation of ROCK1 mRNA. In the IPC+fasudil group, phospho-ERK1/2 expression was similar to the IPC group. In the IPC+PD98059 group, phospho-ERK1/2 expression significantly decreased. And phospho-ERK1/2 expression was not recovered in the IPC+PD98059+fasudil group.
6. Inhibition of Rho-kinase rescued the effect of inhibition of ERK1/2 on apoptosis. Fasudil treatment reversed cell apoptosis caused by treatment with PD98059 in IPC. The number of TUNEL positive cells was 23.3±0.67% in IPC+PD98059+fasudil group. The amount of cleaved Caspase-3 was also significantly reduced in IPC+PD98059+fasudil group. Fasudil resulted in a 22% reduction in cardiomyocyte apoptosis and a 30% reduction in cleaved Caspase-3 in IPC+PD98059+fasudil group (p<0.05 vs IPC+PD98059 group). Additionally, the infarct size of the heart was 39.44±0.92% in IPC+PD98059+fasudil group. The combination of PD98059 and fasudil also resulted in a 12% reduction in myocardial infarct size (p<0.05 vs IPC+PD98059 group).
Conclusion
1. ERK-MAPK signaling is required in IPC to oppose the Rho-kinase signaling on cardiomyocyte apoptosis.
2. Overpression of RhoA in I/R might not be changed by preconditioning.
3. ROCK1 may be the major Rho-kinase which is opposed by ERK-MAPK signaling in IPC.
引文
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11. Ji ES, Yue H, Wu YM, He RR. (2004) Effects of phytoestrogen genistein on myocardial ischemia/reperfusion injury and apoptosis in rabbits. Acta Pharmacol Sin.25:306-312.
12. Mohanty I, Arya DS, Gupta SK. (2006) Effect of Curcuma longa and Ocimum sanctum on myocardial apoptosis in experimentally induced myocardial ischemic-reperfusion injury. BMC Complement Altern Med.6:3.
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15. Tosello-Trampont AC, Nakada-Tsukui K, Ravichandran KS. (2003) Engulfment of apoptotic cells is negatively regulated by Rho-mediated signaling. JBiol Chem.278:49911-49919.
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20. Piazzolla D, Meissl K, Kucerova L, Rubiolo C, Baccarini M. (2005) Raf-1 sets the threshold of Fas sensitivity by modulating Rok-alpha signaling. J Cell Biol.171:1013-1022.
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26. Sugden PH, Clerk A. (2006) Oxidative stress and growth-regulating intracellular signaling pathways in cardiac myocytes. Antioxid Redox Signal. 8: 2111-2124.
27. Bueno OF, Molkentin JD. (2002) Involvement of extracellular signal-regulated kinases 1/2 in cardiac hypertrophy and cell death. Circ Res.91: 776-781.
28. Lips DJ, et al. (2004) MEK1-ERK2 signaling pathway protects myocardium from ischemic injury in vivo. Circulation.109:1938-1941.
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