Rho-kinase和PARP在心肌缺血/再灌注损伤中对心肌细胞凋亡的影响及作用机制的研究
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摘要
研究背景
冠心病已成为我国城乡居民致死的主要原因之一,每年大约70万人死于冠心病,约占全部心血管病死亡的四分之一。再灌注疗法,即尽快恢复心肌缺血区的血供,已成为治疗急性缺血性心脏病的主要方法,然而,Braunwald和Kloner指出再灌注是一把“双刃剑”,因为再灌注本身不仅可能加速心肌损伤,并能引起新的心肌损伤,这也就是我们所说的心脏缺血再灌注(Myocardial ischemia-reperfusion,I/R)损伤。器官移植功能障碍、中风、心肌梗死、休克等都可以导致缺血再灌注损伤。再灌注过程伴随着心肌的生化、结构和功能的变化,决定着心肌细胞的存活和死亡。再灌注能使心脏收缩功能减弱,导致心律失常的发作。有实验证据表明,再灌注引起的心肌细胞不可逆性损伤可导致心肌细胞坏死和凋亡。在缺血再灌注治疗中,如何改善心功能、降低心律失常发生、延迟坏死发作和减少梗死面积,具有十分重要的临床意义。因此,缺血再灌注损伤的研究越来越受到学者们的重视。
近年来,Rho激酶(Rho-kinase,ROCK)和多ADP核糖聚合酶(poly(ADP-ribose) polymerase,PARP)在心脏缺血再灌注所致心肌细胞凋亡中的研究越来越多。
ROCK是一类分子量为160KD的丝-苏氨酸蛋白激酶家族,是小G蛋白Rho的下游效应物。Rho激酶由氨基末端激酶催化结构域、卷曲螺旋结构域、羧基末端PH结构域和半氨酸富集结构域组成。ROCK有两种亚型,即ROCKI和ROCKⅡ。ROCK可以介导下游一系列蛋白的磷酸化/脱磷酸化反应,目前已经发现数十个ROCK蛋白底物,如肌球蛋白轻链磷酸酶(MLCP)、肌球蛋白磷酸酶(MYPT-1)、LIM和PTEN等,其中最具特称意义的ROCK的下游底物是MLCP和MYPT-1。ROCK广泛参与多种生命活动,如调节细胞收缩、生长、分化、代谢、迁移、凋亡和基因表达。近期研究表明,ROCK与多种疾病如冠心病、动脉粥样硬化、高血压、肺动脉高压、心力衰竭、糖尿病、中风及恶性肿瘤等的发生发展密切相关。应用ROCK抑制剂Y-27632、法舒地尔和他汀类药物的临床研究都表明,ROCK是多种疾病的一个潜在治疗靶点。
多ADP核糖聚合酶是存在于真核细胞生物中细胞传导通路中的一个家族酶,现已证明其具有18个超家族,包括PARP-1、PARP-2、PARP-3等等,其中PARP-1的表达谱最广。多ADP核糖聚合酶不仅参与很多重要的细胞通路,还有助于维持染色体结构和基因组的稳定性,检测和修复损伤DNA。DNA修复,基因重组,氧化应激,与DNA结合药物以及蛋白与蛋白的相互作用都能引起多ADP核糖聚合酶活化。然而,多ADP核糖聚合酶过度激活导致NAD+耗竭,减慢糖酵解、电子转移和ATP形成,最终导致细胞功能障碍或者死亡。研究表明,多ADP核糖聚合酶在心脏缺血再灌注损伤、心肌炎、心力衰竭、休克及动脉粥样硬化等多种疾病中起重要作用。
我们的前期研究发现,ROCK和PARP在大鼠心脏缺血再灌注损伤中都发挥了重要作用,我们还对它们各自的作用机制做了进一步探讨,发现ROCK/JNK/AIF信号通路和PARP/JNK/AIF信号通路都是介导大鼠心脏I/R损伤的重要的通路,但是ROCK和PARP之间是否具有相互作用呢?它们之间的相互作用机制又是什么?这些问题构成了本研究的研究目的。
在本研究中,我们拟通过建立心肌细胞I/R模型和大鼠心脏I/R模型,从体外试验和体内试验二方面进行深入研究,旨在明确ROCK和PARP在心脏I/R损伤中的是否存在相互作用?如果二者之间存在相互作用,那么,具体作用如何?二者之间通过何种信号通路发挥作用?希望藉此研究,明确ROCK和PARP在心脏I/R损伤中的相互作用及其机制,从而为缺血性心脏病提供新的治疗途径。本研究共分三部分进行：
第一部分体外试验：ROCK和PARP在心肌细胞缺血再灌注损伤中的关系及其对心肌细胞凋亡的影响；
第二部分体外试验：ROCK和PARP在心肌细胞缺血再灌注损伤中的相互作用及其机制。
第三部分体内试验：ROCK和PARP在大鼠心脏缺血再灌注损伤中的相互作用及其机制。
第一部分体外试验：ROCK和PARP在心肌细胞缺血再灌注损伤中的关系及其对心肌细胞凋亡的影响
研究目的
本研究拟培养大鼠乳鼠原代心肌细胞,体外模拟I/R模型,应用Y27632和3-aminobenzamide(3-AB)分别抑制ROCK和PARP的活性,测定心肌细胞的凋亡,明确ROCK和PARP在心肌细胞缺血再灌注损伤中的相互作用。
研究方法
1.原代培养心肌细胞：wistar大鼠乳鼠(2日龄),取心脏,并把心脏剪成1mm3的碎块,胰蛋白酶和Ⅱ型胶原酶分次消化,收集细胞,差速贴壁去除成纤维细胞后将未贴壁的心肌细胞种板,37度孵箱中培养。
2.心肌细胞模拟I/R模型的建立和分组：以无糖缺血盐缓冲液置换培养液,置于37度密闭的孵箱中培养,再给95% N2和5% CO2的混合气体以2L/min的流速通气置换空气。缺氧、缺血孵化后更换新鲜培养基继续孵化造成心肌细胞模拟缺血/再灌注损伤模型。分为以下四组：对照组、I/R组+DMSO组、I/R+Y27632组和I/R+3-AB组。
3. ROCK和PARP表达的检测：通过Western检测p-MYPT-1和PARP的表达。MYPT-1是ROCK的特异性底物,MYPT-1的磷酸化水平可以表示ROCK的活性。
4.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)试剂盒检测心肌细胞的凋亡。
研究结果
1.ROCK和PARP均参与了I/R损伤,二者之间存在相互作用：在I/R模型中,p-MYPT-1(磷酸化的MYPT-1)和PARP表达明显增高；应用ROCK抑制剂Y27632后,p-MYPT-1和PARP表达均降低；
应用PARP抑制剂3-AB后,PARP表达降低,而p-MYPT-1表达无明显变化。证明ROCK和PARP均参与了I/R损伤,并且说明PARP是ROCK的下游分子。
2.抑制ROCK和PARP的活性均可减少心肌细胞凋亡率：
在对照组中,TUNEL染色阴性。
在I/R+DMSO组中,TUNEL染色阳性细胞占全部细胞的46.5±1.2%。
在I/R+ Y27632组中,TUNEL染色阳性细胞占全部细胞的16±0.5%,与I/R+DMSO组相比差异有显著性(P<0.05),说明在I/R大鼠中抑制ROCK活性能显著减少心肌细胞凋亡率。
在I/R+3-AB组中,TUNEL染色阳性细胞占全部细胞的20±0.3%,与I/R+DMSO组相比相比差异有显著性(P<0.05),说明在I/R中抑制PARP能显著减少心肌细胞凋亡率。
证明ROCK和PARP在缺血/再灌注损伤中发挥重要作用,抑制ROCK和PARP活性,均可显著降低缺血/再灌注损伤所致的心肌细胞凋亡。
结论
1.体外模拟心肌细胞I/R模型中,ROCK和PARP活性明显增加,心肌细胞凋亡率明显升高。
2.体外模拟心肌细胞I/R模型中,抑制ROCK和PARP活性均可减少心肌细胞的凋亡率。
3.体外模拟心肌细胞I/R模型中,ROCK和PARP之间具有相互作用,ROCK可能通过调节PARP活性而参与I/R损伤中细胞凋亡的发生。
第二部分体外试验：ROCK和PARP在心肌细胞缺血再灌注损伤中的相互作用及其机制
研究目的
利用乳鼠原代心肌细胞体外建立心肌细胞模拟I/R模型,应用抑制剂Y27632、、LY294002、Ac-DEVD-CHO、3-AB分别抑制ROCK、Akt、caspase-3和PARP活性,测定心肌细胞的凋亡,探讨ROCK和PARP在缺血再灌注中的作用及其作用机制。
研究方法
1.心肌细胞原代培养：wistar大鼠乳鼠(2日龄),取心脏,并把心脏剪成1mm3的碎块,胰酶和Ⅱ型胶原酶分次消化,收集细胞,差速贴壁去除成纤维细胞后将未贴壁的心肌细胞中板,37度孵箱中培养。
2.体外建立心肌细胞模拟I/R模型：以无糖缺血盐缓冲液置换培养液,置于37度密闭的孵箱中培养,再给95% N2和5% CO2的混合气体以2L/min的流速通气置换空气。缺氧、缺血孵化后更换新鲜培养基继续孵化造成心肌细胞模拟缺血/再灌注损伤模型。分为以下六组：对照组、I/R+DMSO组、I/R+Y27632组、I/R+LY294002组、I/R+Ac-DEVD-CHO组和I/R+3-AB组。
3. ROCK、Akt、caspase-3和PARP活性的检测：ROCK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。Western检测Akt、p-Akt、cleaved-Caspase-3和PARP的表达。
4.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)试剂盒检测各组心肌细胞的凋亡。
研究结果
1.与对照组相比,心肌细胞I/R损伤中p-MYPT-1、cleaved-Caspase-3、PARP表达增加,p-Akt水平降低。
证明心肌细胞I/R损伤可增加ROCK、Caspase-3、PARP活性,而降低Akt的磷酸化水平。
2. I/R+DMSO组心肌细胞凋亡率为60±1.68%；
I/R+Y27632组心肌细胞凋亡减少至24±0.52%,与I/R+DMSO组相比差异有显著性(P<0.05)；
I/R+Ac-DEVD-CHO组心肌细胞凋亡减少至32±1.24%,与I/R+DMSO组相比差异有显著性(P<0.05)；
I/R+3-AB组心肌细胞凋亡减少至36±0.65%,与I/R+DMSO组相比差异有显著性(P<0.05)。
证明在I/R中抑制ROCK、Caspase-3和PARP活性能显著减少心肌细胞凋亡。
3.在I/R+DMSO组p-MYPT-1表达增加,磷酸化Akt表达下降,应用ROCK抑制剂Y27632后磷酸化Akt的表达增高,而应用Akt抑制剂LY294002后,p-MYPT-1表达无明显变化。
证明磷酸化Akt的表达与ROCK有关,Akt是ROCK的下游分子。
4.在I/R+DMSO组p-MYPT-1和cleaved-Caspase-3表达增多,应用ROCK抑制剂Y27632,cleaved-Caspase-3表达减少,而应用Caspase-3抑制剂Ac-DEVD-CHO后,p-MYPT-1的表达无明显变化。
证明Caspase-3是ROCK的下游分子。
5.在I/R+DMSO组磷酸化Akt的表达降低,cleaved-Caspase-3表达增多,抑制Akt活性后cleaved-Caspase-3表达明显增多,而抑制Caspase-3后,磷酸化Akt的表达无明显变化。
证明Caspase-3是Akt的下游分子。
6.在I/R+DMSO组cleaved-Caspase-3和PARP表达增多,PARP的裂解片段(24KD)也明显增多。抑制Caspase-3后,PARP活性降低,PARP的裂解片段减少,而抑制PARP后,cleaved-Caspase-3的表达无明显变化。
证明PARP是Caspase-3的下游分子。
结论
1.心肌细胞模拟I/R模型中,ROCK.Caspase-3和PARP活性增加,而Akt的磷酸化水平降低。抑制ROCK.Caspase-3和PARP活性,能降低心肌细胞凋亡率。
2.ROCK/Akt/caspase-3/PARP信号通路是介导心肌细胞I/R损伤的一条重要的信号通路。
第三部分体内试验：ROCK和PARP在大鼠心脏缺血再灌注损伤中的相互作用及其机制
研究目的
利用wistar大鼠I/R模型,应用抑制剂fasudil.LY294002.Ac-DEVD-CHO.3-AB分别抑制ROCK.Akt.caspase-3和PARP活性,测定心肌梗死面积和心肌细胞的凋亡,探讨ROCK和PARP在缺血再灌注中的作用及其作用机制。进一步验证体外试验得出的结论。
研究方法
1.大鼠心脏I/R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型。分为以下六组：对照组、I/R+DMSO组、I/R+fasudil组、I/R+ LY294002组、I/R+Ac-DEVD-CHO组和I/R+3-AB组。每组各15只大鼠。
2. ROCK、Akt、caspase-3和PARP活性的检测：ROCK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。Western检测Akt、p-Akt、cleaved-Caspase-3和PARP的表达。
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.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。
研究结果
1.与对照组相比,心脏I/R损伤中p-MYPT-1、cleaved-Caspase-3、PARP表达增加,p-Akt水平降低。
说明心脏I/R损伤可增加ROCK、Caspase-3、PARP活性,而降低Akt的磷酸化水平。
2.抑制ROCK、Caspase-3和PARP后能减少大鼠心肌梗死面积：
I/R+DMSO组大鼠的心肌梗死面积占整个左心室面积的60.22±1%,
I/R+fasudil组大鼠的心肌梗死面积占整个左心室面积的30.5±1.2%,与I/R+DMSO组相比差异有显著性(P<0.05)；
I/R+Ac-DEVD-CHO组大鼠的心肌梗死面积占整个左心室面积的24.9±0.8%,与I/R+DMSO组相比差异有显著性(P<0.05)；
I/R+3-AB组大鼠的心肌梗死面积占整个左心室面积的29.1±0.5%,与I/R+DMSO组相比差异有显著性(P<0.05)。
证明在I/R中分别抑制ROCK、Caspase-3和PARP活性均能显著减少心肌梗死面积。
3.在I/R+DMSO组p-MYPT-1表达增加,磷酸化Akt表达下降,应用ROCK抑制剂fasudil后磷酸化Akt的表达增高,而应用Akt抑制剂LY294002后,p-MYPT-1表达无明显变化。
证明磷酸化Akt的表达与ROCK有关,Akt是ROCK的下游分子。
4.在I/R+DMSO组p-MYPT-1和cleaved-Caspase-3表达增多,应用ROCK抑制剂fasudil,cleaved-Caspase-3表达减少。而应用Caspase-3抑制剂Ac-DEVD-CHO后,p-MYPT-1的表达无明显变化。
证明Caspase-3是ROCK的下游分子。
5.在I/R+DMSO组磷酸化Akt的表达降低,cleaved-Caspase-3表达增多,抑制Akt活性后cleaved-Caspase-3表达明显增多,而抑制Caspase-3后,磷酸化Akt的表达无明显变化。
证明Caspase-3是Akt的下游分子。
6.在(?)[/R+DMSO组cleaved-Caspase-3和PARP表达增多,PARP的裂解片段(24KD)也明显增多。抑制Caspase-3后,PARP活性降低,PARP的裂解片段减少,而抑制PARP后,cleaved-Caspase-3的表达无明显变化。
证明PARP是Caspase-3的下游分子。
结论：体内试验得出了与体外试验相同的结论：
1.大鼠心脏I/R损伤模型中,ROCK、Caspase-3和PARP活性增加,而Akt的磷酸化水平降低。抑制ROCK、Caspase-3和PARP活性,能显著减少大鼠心肌梗死面积。
2. Rho-kinase/Akt/caspase-3/PARP信号通路是介导大鼠心肌细胞I/R损伤的一条重要的信号通路。
创新性及局限性
1.创新点
(1)提出并证实了ROCK和PARP在心脏缺血再灌注造成的心肌细胞凋亡中具有相互作用。
(2)提出并证实了ROCK/Akt/caspase-3/PARP信号通路是介导心肌细胞I/R损伤的一条重要的信号通路。
2.局限性
(1)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。
(2)由于时间及条件的限制,未能过表达Akt,以研究其在组织及细胞水平上的保护性效应。
Background
Coronary heart disease has been one major cause of disability and death in our country, approximately 700000 people die of coronary heart disease every year, accounting for about one quarter of all death caused by cardiovascular diseases. Reperfusion therapy, which can make the return of blood supply as soon as possible to the ischemic region of the myocardium, has been the chief treatment of acute myocardial ischemia. However, Braunwald and Kloner point out that reperfusion is a "double edged sword", because reperfusion itself can accelerate myocardial damage and can produce additional myocardial injury, which we called the Ischemia/reperfusion (I/R) injury. Ischemia/reperfusion (I/R) injury can be induced by organ transplant dysfunction, stroke, myocardial infarction, shock and so on. Reperfusion is followed by myocardial biochemical, structural, and functional changes and may determine myocardial cell survival and death. Reperfusion can lead to diminished cardiac contractile function and arrhythmia. There is experimental evidence that irreversible myocardial cell injury originated by reperfusion can result in cell necrosis and apoptosis. How to improve myocardial function, decrease the arrhythmogenesis, delay the episode of necrosis, and decrease the infarction size during ischemia/reperfusion injury is of great clinical significance. Therefore, increasingly importance has been attached to ischemia/reperfusion injury.
Recently, the research of Rho-kinase and poly (ADP-ribose) polymerase in myocardial cell apoptosis caused by ischemia/reperfusion injury has been more and more popular. Rho-kinases (ROCKs) are serine/threonine kinases with a molecular weight of 160 KD and are downstream effectors of the small GTPase Rho. ROCKs consist of an amino-terminal kinase domain, a mid-coiled-coil-forming lesion, the Pleckstrin homology (PH) motif and a carboxy-terminal cysteine-rich domain. There are two isoforms of ROCK:ROCK1 and ROCK2. ROCKs implicated in many kinds of vital movement, including the regulation of cellular contraction, growth, division, metabolism, migration apoptosis and gene expression. According to scientific survey, Rho-kinase is closely related to the development of a wide range of diseases such as coronary heart disease, atherosclerosis, hypertension, pulmonary hypertension, heart failure, diabetes, stroke, cancer and so on. Rho-kinase can mediate a series of Phosphorylation/phosphorylation reaction, dozens of substrates of ROCK have been identified, including:the myosin phosphatase-targeting subunit 1(MYPT1) and the myosin-binding subunit of myosin light chain phosphatase (MLCP), LIM, PTEN, and so on. The first characterized targets of ROCK are MLC and MYPT1. Evidence from clinical use of ROCK inhibitors, such asY-27632, fasudil and statins, supports the hypothesis that ROCK is a potential therapeutic target.
Poly (ADP-ribose) polymerases are resided in eukaryotes, and they are a family of cell signaling enzymes. There are 18 superfamily, including PARP-1、PARP-2、PARP-3, etc. PARP-1 is the most abundant subtype of poly (ADP-ribose) polymerases. Poly (ADP-ribose) polymerase (PARP) is involved in many important cellular pathways, and it can contribute to chromosomal structure and genomic stability, detects and repairs the damage of DNA. DNA repair, gene rearrangements, oxidative stress, DNA binding drugs and protein-protein interactions can cause the activation of Poly (ADP-ribose) polymerase. However, poly (ADP-ribose) polymerase overactivation can exhausts NAD+, steps down the rate of glycolysis, electron transport, and ATP formation, finally leads to functional impairment or death of cells. Clinical data indicates that, poly (ADP-ribose) polymerase plays an important role in many diseases such as myocardial ischemia/reperfusion injury, myocarditis, heart failure, shock, atherosclerosis and so on.
In our previous study, we found that both Rho-kinase and PARP play an important role in rat myocardial ischemia/reperfusion injury, and we further study the mechanism of them. We found that both ROCK/JNK/AIF pathway and PARP /JNK/AIF pathway are new pathways of cardiac myocyte injury. But whether there are interactions between Rho-kinase and PARP, what are the mechanisms of their interaction. In our study, we establish myocardial cell I/R model and the rat heart I/R model, investigate the interaction of Rho-kinase and PARP, identify the signaling pathway between them, so that we can provide a new therapeutic approach for ischemic heart disease.
This study includes there parts:
PartⅠIn vitro:The relationship and effect of Rho-kinase and PARP on cell apoptosis in the primary myocardial cell model of I/R
PartⅡIn vitro: The interaction and mechanism of Rho-kinase and PARP on the I/R model of primary cultured myocardial cell
PartⅢIn vivo:The interaction and mechanism of Rho-kinase and PARP on the rat I/R injury model
PartⅠIn vitro:The relationship and effect of Rho-kinase and PARP on cell apoptosis in the primary myocardial cell model of I/R
Objective
To investigate the interaction of Rho-kinase and PARP on I/R
Methods
1. Primary cultures of myocardial cells:obtain the heart of neonatal wistar rat, shred to 1mm3, and then digest with trypsin and collagenaseⅡ. After differential adhesion, put the myocardial cells in incubator.
2. Establish the hypoxia-reoxygenation injury model of myocardial cell, The following experimental groups were studied. (1) control group; (2) I/R+DMSOgroup; (3) I/R+Y27632group; (4) I/R+3-AB group.
3. Detect the activity of Rho-kinase and PARP: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 and PARP activity.
4. Evaluate the apoptotis of myocardial cells, the TUNEL technique was used.
Results
1. Rho-kinase and PARP both play an important role in I/R injury, there are interaction between them:Phospho-MYPT-1 and PARP increased during the hypoxia-reoxygenation injury model. Administration of inhibitors of Rho-kinase can depress the expression of Phospho-MYPT-1 and PARP. Administration of inhibitors of PARP can depress the expression of PARP, but do not effect the expression of Phospho-MYPT-1.This indicate that Rho-kinase and PARP both play an important role in I/R injury, and PARP is the downstream molecules of Rho-kinase.
2. The inhibition of Rho-kinase and PARP can both decrease the apoptosis of myocardial cells:The apoptosis ratio of myocardial cells was 46.5±1.2% in I/R +DMSO group. Administration of Y27632, the apoptosis ratio was 16±0.5%(P＜0.05 vs I/R+DMSO group); Administration of 3-AB, the apoptosis ratio was 20±0.3%(P <0.05 vs I/R+DMSO group), demonstrating that inhibition of Rho-kinase and PARP activity reduces myocardial cells apoptosis in rat heart I/R injury.
Conclusion
1. Rho-kinase and PARP activities increased in the hypoxia-reoxygenation injury model, they can aggravate the apoptosis of myocardial cells.
2. Rho-kinase and PARP inhibition in the hypoxia-reoxygenation injury can both reduce the apoptosis of myocardial cells.
3. There are interactions between Rho-kinase and PARP in the hypoxia-reoxygenation injury. By regulation the activity of PARP and Rho-kinase can reduce the apoptosis of myocardial cells.
Part II In vitro:The interaction and mechanism of Rho-kinase and PARP on the I/R model of primary cultured myocardial cells
Objective
To investigate the effect of Rho-kinase, Akt, caspase-3 and PARP in I/R
Methods
1. Primary cultures of myocardial cells from the neonatal wistar rat
2. Establish the hypoxia-reoxygenation injury model of myocardial cell. The following experimental groups were studied. (1) control group; (2) I/R+DMSO group; (3) I/R+Y27632 group; (4) I/R+LY294002 group; (5) I/R+Ac-DEVD-CHO group; (6) I/R+3-AB group.
3. Detect the activity of Rho-kinase, Akt, caspase-3 and PARP:Rho-kinase activity was assessed by examining phosphorylation of MYPT-1. Caspase-3 activity was assessed by examining the cleaved-Caspase-3. Western blot analysis was performed to evaluate Akt, p-Akt, cleaved-caspase-3 and PARP activity.
4. Evaluate the apoptosis of myocardial cells, the TUNEL technique was used.
Results
1. The expression of p-MYPT-1.. cleaved-Caspase-3 and PARP were increased in myocardial cell I/R model, but the expression of p-Akt was degraded. This shows that the activity of Rho-kinase、Caspase-3 and PARP increased in myocardial cell I/R model, phosphorylation of Akt was degraded.
2. The apoptosis ratio of myocardial cell is 60±1.68% I/R+DMSO group. The apoptosis ratio of myocardial cell is significantly reduced to 24±0.52% in I/R+ Y27632 group (P<0.05 vs I/R+DMSO group). The apoptosis ratio of myocardial cell is reduced to 32±1.24% in I/R+LY294002 group (P<0.05 vs I/R+DMSO group). The apoptosis ratio of myocardial cell is reduced to 36±0.65% in I/R+3-AB group (P <0.05 vs I/R+DMSO group). This suggests that inhibition of Rho-kinase, Caspase-3 and PARP activity reduces cell apoptosis.
3. The expression of p-MYPT-1 increased in I/R+DMSO group, but Phospho-Akt decreased in I/R+DMSO group. After administration of Y27632, the expression of Phospho-Akt was increased. After administration Akt inhibitor, the expression of p-MYPT-1 did not change significantly. This indicates Akt is the downstream molecules of Rho-kinase.
4. The expression of p-MYPT-1 and cleaved-Caspase-3 were increased in I/R +DMSO group. After administration of Rho-kinase inhibitor, the expression of cleaved-Caspase-3 was decreased. After administration of Caspase-3inhibitor, the expression of p-MYPT-1 did not change significantly. This indicates that Caspase-3 is the downstream molecules of Rho-kinase.
5. The expression of p-Akt was decreased in I/R+DMSO group, and the expression of cleaved-Caspase-3 were increased. After administration of Akt inhibitor, the expression of cleaved-Caspase-3 was increased. After administration of Caspase-3 inhibitor, the expression of p-Akt did not change significantly. This indicates that Caspase-3 is the downstream molecules of Akt.
6. The expression of PARP and cleaved-Caspase-3 were increased in I/R+ DMSO group, and the Cleavage fragments (24KD) of PARP was increased too. After administration of Caspase-3-inhibitor, the activity of PARP was decreased, the Cleavage fragments (24KD) of PARP were decreased. After administration of PARP inhibitor, the expression of cleaved-Caspase-3 did not change significantly. This indicates that PARP is the downstream molecules of Caspase-3.
Conclusion
1. In I/R model, the activity of Rho-kinase、Caspase-3 and PARP were increased, but phosphorylation of Akt was degraded. Inhibition the activity of Rho-kinase、Caspase-3 and PARP can reduce myocardial infarct size and the ratio of myocardial cell apoptosis.
2. Rho-kinase/Akt/caspase-3/PARP pathway may be a new pathway of heart I/R injury.
PartⅢIn vivo:The interaction and mechanism of Rho-kinase and PARP on the rat I/R injury model
Objective
To investigate the effect of Rho-kinase, Akt, caspase-3 and PARP in I/R
Methods
1. The rat model of I/R:The left anterior descending branch (LAD) of the left coronary artery was occluded and loosened to establish rat heart I/R model. The following experimental groups were studied:(1) control group (n=20) (2) I/R+DMSO group (n=20); (3) I/R+fasudil group (n=20); (4) I/R+LY294002 group (n=20); (5) I/R+Ac-DEVD-CHO group (n=20); (6) I/R+3-AB (n=20).
2. Detect the activity of Rho-kinase, Akt, caspase-3 and PARP:Rho-kinase activity was assessed by examining phosphorylation of MYPT-1. Caspase-3 activity was assessed by examining the cleaved-Caspase-3. Western blot analysis was performed to evaluate Akt, p-Akt and PARP 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. Evaluate the apoptosis of myocardial cells, the TUNEL technique was used.
Results
1. The expression of p-MYPT-1、cleaved-Caspase-3 and PARP were increased in the I/R model, but the expression of p-Akt was degraded. This shows that the activity of Rho-kinase、Caspase-3 and PARP increased in I/R model, phosphorylation of Akt was degraded.
2. Inhibition of Rho-kinase, Caspase-3 and PARP activity reduced myocardial infarct size. The infarct sizes of the heart were 60.22±1% in I/R+DMSO group, 30.5±1.2% in I/R+fasudil group,24.9±0.8% in I/R+LY294002 group and 29.1±0.5% in I/R+3-AB group(P<0.05 vs I/R+DMSO group). This indicates that inhibition of Rho-kinase, Caspase-3 and PARP can reduce myocardial infarct size.
3. The expression of p-MYPT-1 increased in I/R+DMSO group, but Phospho-Akt decreased in I/R+DMSO group. After administration of Y27632, the expression of Phospho-Akt was increased. After administration Akt inhibitor, the expression of p-MYPT-1 did not change significantly. This indicates Akt is the downstream molecules of Rho-kinase.
4. The expression of p-MYPT-1 and cleaved-Caspase-3 were increased in I/R +DMSO group. After administration of Rho-kinase inhibitor, the expression of cleaved-Caspase-3 was decreased. After administration of Caspase-3 inhibitor, the expression of p-MYPT-1 did not change significantly. This indicates that Caspase-3 is the downstream molecules of Rho-kinase.
5. The expression of p-Akt was decreased in I/R+DMSO group, and the expression of cleaved-Caspase-3 were increased. After administration of Akt inhibitor, the expression of cleaved-Caspase-3 was increased. After administration of Caspase-3 inhibitor, the expression of p-Akt did not change significantly. This indicates that Caspase-3 is the downstream molecules of Akt.
6. The expression of PARP and cleaved-Caspase-3 were increased in I/R+DMSO group, and the Cleavage fragments (24KD) of PARP was increased too. After administration of Caspase-3 inhibitor, the activity of PARP was decreased, the Cleavage fragments (24KD) of PARP were decreased. After administration of PARP inhibitor, the expression of cleaved-Caspase-3 did not change significantly. This indicates that PARP is the downstream molecules of Caspase-3.
Conclusion
1. In rat I/R model, the activity of Rho-kinase ^ Caspase-3 and PARP were increased, but phosphorylation of Akt was degraded. Inhibition the activity of Rho-kinase-. Caspase-3 and PARP can reduce myocardial infarct size.
2. Rho-kinase/Akt/caspase-3 /PARP pathway may be a new pathway of rat heart I/R injury.
冠心病已成为我国城乡居民致死的主要原因之一,每年大约70万人死于冠心病,约占全部心血管病死亡的四分之一。再灌注疗法,即尽快恢复心肌缺血区的血供,已成为治疗急性缺血性心脏病的主要方法,然而,Braunwald和Kloner指出再灌注是一把“双刃剑”,因为再灌注本身不仅可能加速心肌损伤,并能引起新的心肌损伤,这也就是我们所说的心脏缺血再灌注(Myocardial ischemia-reperfusion,I/R)损伤。器官移植功能障碍、中风、心肌梗死、休克等都可以导致缺血再灌注损伤。再灌注过程伴随着心肌的生化、结构和功能的变化,决定着心肌细胞的存活和死亡。再灌注能使心脏收缩功能减弱,导致心律失常的发作。有实验证据表明,再灌注引起的心肌细胞不可逆性损伤可导致心肌细胞坏死和凋亡。在缺血再灌注治疗中,如何改善心功能、降低心律失常发生、延迟坏死发作和减少梗死面积,具有十分重要的临床意义。因此,缺血再灌注损伤的研究越来越受到学者们的重视。
近年来,Rho激酶(Rho-kinase,ROCK)和多ADP核糖聚合酶(poly(ADP-ribose) polymerase,PARP)在心脏缺血再灌注所致心肌细胞凋亡中的研究越来越多。
ROCK是一类分子量为160KD的丝-苏氨酸蛋白激酶家族,是小G蛋白Rho的下游效应物。Rho激酶由氨基末端激酶催化结构域、卷曲螺旋结构域、羧基末端PH结构域和半氨酸富集结构域组成。ROCK有两种亚型,即ROCKI和ROCKⅡ。ROCK可以介导下游一系列蛋白的磷酸化/脱磷酸化反应,目前已经发现数十个ROCK蛋白底物,如肌球蛋白轻链磷酸酶(MLCP)、肌球蛋白磷酸酶(MYPT-1)、LIM和PTEN等,其中最具特称意义的ROCK的下游底物是MLCP和MYPT-1。ROCK广泛参与多种生命活动,如调节细胞收缩、生长、分化、代谢、迁移、凋亡和基因表达。近期研究表明,ROCK与多种疾病如冠心病、动脉粥样硬化、高血压、肺动脉高压、心力衰竭、糖尿病、中风及恶性肿瘤等的发生发展密切相关。应用ROCK抑制剂Y-27632、法舒地尔和他汀类药物的临床研究都表明,ROCK是多种疾病的一个潜在治疗靶点。
多ADP核糖聚合酶是存在于真核细胞生物中细胞传导通路中的一个家族酶,现已证明其具有18个超家族,包括PARP-1、PARP-2、PARP-3等等,其中PARP-1的表达谱最广。多ADP核糖聚合酶不仅参与很多重要的细胞通路,还有助于维持染色体结构和基因组的稳定性,检测和修复损伤DNA。DNA修复,基因重组,氧化应激,与DNA结合药物以及蛋白与蛋白的相互作用都能引起多ADP核糖聚合酶活化。然而,多ADP核糖聚合酶过度激活导致NAD+耗竭,减慢糖酵解、电子转移和ATP形成,最终导致细胞功能障碍或者死亡。研究表明,多ADP核糖聚合酶在心脏缺血再灌注损伤、心肌炎、心力衰竭、休克及动脉粥样硬化等多种疾病中起重要作用。
我们的前期研究发现,ROCK和PARP在大鼠心脏缺血再灌注损伤中都发挥了重要作用,我们还对它们各自的作用机制做了进一步探讨,发现ROCK/JNK/AIF信号通路和PARP/JNK/AIF信号通路都是介导大鼠心脏I/R损伤的重要的通路,但是ROCK和PARP之间是否具有相互作用呢?它们之间的相互作用机制又是什么?这些问题构成了本研究的研究目的。
在本研究中,我们拟通过建立心肌细胞I/R模型和大鼠心脏I/R模型,从体外试验和体内试验二方面进行深入研究,旨在明确ROCK和PARP在心脏I/R损伤中的是否存在相互作用?如果二者之间存在相互作用,那么,具体作用如何?二者之间通过何种信号通路发挥作用?希望藉此研究,明确ROCK和PARP在心脏I/R损伤中的相互作用及其机制,从而为缺血性心脏病提供新的治疗途径。本研究共分三部分进行：
第一部分体外试验：ROCK和PARP在心肌细胞缺血再灌注损伤中的关系及其对心肌细胞凋亡的影响；
第二部分体外试验：ROCK和PARP在心肌细胞缺血再灌注损伤中的相互作用及其机制。
第三部分体内试验：ROCK和PARP在大鼠心脏缺血再灌注损伤中的相互作用及其机制。
第一部分体外试验：ROCK和PARP在心肌细胞缺血再灌注损伤中的关系及其对心肌细胞凋亡的影响
研究目的
本研究拟培养大鼠乳鼠原代心肌细胞,体外模拟I/R模型,应用Y27632和3-aminobenzamide(3-AB)分别抑制ROCK和PARP的活性,测定心肌细胞的凋亡,明确ROCK和PARP在心肌细胞缺血再灌注损伤中的相互作用。
研究方法
1.原代培养心肌细胞：wistar大鼠乳鼠(2日龄),取心脏,并把心脏剪成1mm3的碎块,胰蛋白酶和Ⅱ型胶原酶分次消化,收集细胞,差速贴壁去除成纤维细胞后将未贴壁的心肌细胞种板,37度孵箱中培养。
2.心肌细胞模拟I/R模型的建立和分组：以无糖缺血盐缓冲液置换培养液,置于37度密闭的孵箱中培养,再给95% N2和5% CO2的混合气体以2L/min的流速通气置换空气。缺氧、缺血孵化后更换新鲜培养基继续孵化造成心肌细胞模拟缺血/再灌注损伤模型。分为以下四组：对照组、I/R组+DMSO组、I/R+Y27632组和I/R+3-AB组。
3. ROCK和PARP表达的检测：通过Western检测p-MYPT-1和PARP的表达。MYPT-1是ROCK的特异性底物,MYPT-1的磷酸化水平可以表示ROCK的活性。
4.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)试剂盒检测心肌细胞的凋亡。
研究结果
1.ROCK和PARP均参与了I/R损伤,二者之间存在相互作用：在I/R模型中,p-MYPT-1(磷酸化的MYPT-1)和PARP表达明显增高；应用ROCK抑制剂Y27632后,p-MYPT-1和PARP表达均降低；
应用PARP抑制剂3-AB后,PARP表达降低,而p-MYPT-1表达无明显变化。证明ROCK和PARP均参与了I/R损伤,并且说明PARP是ROCK的下游分子。
2.抑制ROCK和PARP的活性均可减少心肌细胞凋亡率：
在对照组中,TUNEL染色阴性。
在I/R+DMSO组中,TUNEL染色阳性细胞占全部细胞的46.5±1.2%。
在I/R+ Y27632组中,TUNEL染色阳性细胞占全部细胞的16±0.5%,与I/R+DMSO组相比差异有显著性(P<0.05),说明在I/R大鼠中抑制ROCK活性能显著减少心肌细胞凋亡率。
在I/R+3-AB组中,TUNEL染色阳性细胞占全部细胞的20±0.3%,与I/R+DMSO组相比相比差异有显著性(P<0.05),说明在I/R中抑制PARP能显著减少心肌细胞凋亡率。
证明ROCK和PARP在缺血/再灌注损伤中发挥重要作用,抑制ROCK和PARP活性,均可显著降低缺血/再灌注损伤所致的心肌细胞凋亡。
结论
1.体外模拟心肌细胞I/R模型中,ROCK和PARP活性明显增加,心肌细胞凋亡率明显升高。
2.体外模拟心肌细胞I/R模型中,抑制ROCK和PARP活性均可减少心肌细胞的凋亡率。
3.体外模拟心肌细胞I/R模型中,ROCK和PARP之间具有相互作用,ROCK可能通过调节PARP活性而参与I/R损伤中细胞凋亡的发生。
第二部分体外试验：ROCK和PARP在心肌细胞缺血再灌注损伤中的相互作用及其机制
研究目的
利用乳鼠原代心肌细胞体外建立心肌细胞模拟I/R模型,应用抑制剂Y27632、、LY294002、Ac-DEVD-CHO、3-AB分别抑制ROCK、Akt、caspase-3和PARP活性,测定心肌细胞的凋亡,探讨ROCK和PARP在缺血再灌注中的作用及其作用机制。
研究方法
1.心肌细胞原代培养：wistar大鼠乳鼠(2日龄),取心脏,并把心脏剪成1mm3的碎块,胰酶和Ⅱ型胶原酶分次消化,收集细胞,差速贴壁去除成纤维细胞后将未贴壁的心肌细胞中板,37度孵箱中培养。
2.体外建立心肌细胞模拟I/R模型：以无糖缺血盐缓冲液置换培养液,置于37度密闭的孵箱中培养,再给95% N2和5% CO2的混合气体以2L/min的流速通气置换空气。缺氧、缺血孵化后更换新鲜培养基继续孵化造成心肌细胞模拟缺血/再灌注损伤模型。分为以下六组：对照组、I/R+DMSO组、I/R+Y27632组、I/R+LY294002组、I/R+Ac-DEVD-CHO组和I/R+3-AB组。
3. ROCK、Akt、caspase-3和PARP活性的检测：ROCK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。Western检测Akt、p-Akt、cleaved-Caspase-3和PARP的表达。
4.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)试剂盒检测各组心肌细胞的凋亡。
研究结果
1.与对照组相比,心肌细胞I/R损伤中p-MYPT-1、cleaved-Caspase-3、PARP表达增加,p-Akt水平降低。
证明心肌细胞I/R损伤可增加ROCK、Caspase-3、PARP活性,而降低Akt的磷酸化水平。
2. I/R+DMSO组心肌细胞凋亡率为60±1.68%；
I/R+Y27632组心肌细胞凋亡减少至24±0.52%,与I/R+DMSO组相比差异有显著性(P<0.05)；
I/R+Ac-DEVD-CHO组心肌细胞凋亡减少至32±1.24%,与I/R+DMSO组相比差异有显著性(P<0.05)；
I/R+3-AB组心肌细胞凋亡减少至36±0.65%,与I/R+DMSO组相比差异有显著性(P<0.05)。
证明在I/R中抑制ROCK、Caspase-3和PARP活性能显著减少心肌细胞凋亡。
3.在I/R+DMSO组p-MYPT-1表达增加,磷酸化Akt表达下降,应用ROCK抑制剂Y27632后磷酸化Akt的表达增高,而应用Akt抑制剂LY294002后,p-MYPT-1表达无明显变化。
证明磷酸化Akt的表达与ROCK有关,Akt是ROCK的下游分子。
4.在I/R+DMSO组p-MYPT-1和cleaved-Caspase-3表达增多,应用ROCK抑制剂Y27632,cleaved-Caspase-3表达减少,而应用Caspase-3抑制剂Ac-DEVD-CHO后,p-MYPT-1的表达无明显变化。
证明Caspase-3是ROCK的下游分子。
5.在I/R+DMSO组磷酸化Akt的表达降低,cleaved-Caspase-3表达增多,抑制Akt活性后cleaved-Caspase-3表达明显增多,而抑制Caspase-3后,磷酸化Akt的表达无明显变化。
证明Caspase-3是Akt的下游分子。
6.在I/R+DMSO组cleaved-Caspase-3和PARP表达增多,PARP的裂解片段(24KD)也明显增多。抑制Caspase-3后,PARP活性降低,PARP的裂解片段减少,而抑制PARP后,cleaved-Caspase-3的表达无明显变化。
证明PARP是Caspase-3的下游分子。
结论
1.心肌细胞模拟I/R模型中,ROCK.Caspase-3和PARP活性增加,而Akt的磷酸化水平降低。抑制ROCK.Caspase-3和PARP活性,能降低心肌细胞凋亡率。
2.ROCK/Akt/caspase-3/PARP信号通路是介导心肌细胞I/R损伤的一条重要的信号通路。
第三部分体内试验：ROCK和PARP在大鼠心脏缺血再灌注损伤中的相互作用及其机制
研究目的
利用wistar大鼠I/R模型,应用抑制剂fasudil.LY294002.Ac-DEVD-CHO.3-AB分别抑制ROCK.Akt.caspase-3和PARP活性,测定心肌梗死面积和心肌细胞的凋亡,探讨ROCK和PARP在缺血再灌注中的作用及其作用机制。进一步验证体外试验得出的结论。
研究方法
1.大鼠心脏I/R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型。分为以下六组：对照组、I/R+DMSO组、I/R+fasudil组、I/R+ LY294002组、I/R+Ac-DEVD-CHO组和I/R+3-AB组。每组各15只大鼠。
2. ROCK、Akt、caspase-3和PARP活性的检测：ROCK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。Western检测Akt、p-Akt、cleaved-Caspase-3和PARP的表达。
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.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。
研究结果
1.与对照组相比,心脏I/R损伤中p-MYPT-1、cleaved-Caspase-3、PARP表达增加,p-Akt水平降低。
说明心脏I/R损伤可增加ROCK、Caspase-3、PARP活性,而降低Akt的磷酸化水平。
2.抑制ROCK、Caspase-3和PARP后能减少大鼠心肌梗死面积：
I/R+DMSO组大鼠的心肌梗死面积占整个左心室面积的60.22±1%,
I/R+fasudil组大鼠的心肌梗死面积占整个左心室面积的30.5±1.2%,与I/R+DMSO组相比差异有显著性(P<0.05)；
I/R+Ac-DEVD-CHO组大鼠的心肌梗死面积占整个左心室面积的24.9±0.8%,与I/R+DMSO组相比差异有显著性(P<0.05)；
I/R+3-AB组大鼠的心肌梗死面积占整个左心室面积的29.1±0.5%,与I/R+DMSO组相比差异有显著性(P<0.05)。
证明在I/R中分别抑制ROCK、Caspase-3和PARP活性均能显著减少心肌梗死面积。
3.在I/R+DMSO组p-MYPT-1表达增加,磷酸化Akt表达下降,应用ROCK抑制剂fasudil后磷酸化Akt的表达增高,而应用Akt抑制剂LY294002后,p-MYPT-1表达无明显变化。
证明磷酸化Akt的表达与ROCK有关,Akt是ROCK的下游分子。
4.在I/R+DMSO组p-MYPT-1和cleaved-Caspase-3表达增多,应用ROCK抑制剂fasudil,cleaved-Caspase-3表达减少。而应用Caspase-3抑制剂Ac-DEVD-CHO后,p-MYPT-1的表达无明显变化。
证明Caspase-3是ROCK的下游分子。
5.在I/R+DMSO组磷酸化Akt的表达降低,cleaved-Caspase-3表达增多,抑制Akt活性后cleaved-Caspase-3表达明显增多,而抑制Caspase-3后,磷酸化Akt的表达无明显变化。
证明Caspase-3是Akt的下游分子。
6.在(?)[/R+DMSO组cleaved-Caspase-3和PARP表达增多,PARP的裂解片段(24KD)也明显增多。抑制Caspase-3后,PARP活性降低,PARP的裂解片段减少,而抑制PARP后,cleaved-Caspase-3的表达无明显变化。
证明PARP是Caspase-3的下游分子。
结论：体内试验得出了与体外试验相同的结论：
1.大鼠心脏I/R损伤模型中,ROCK、Caspase-3和PARP活性增加,而Akt的磷酸化水平降低。抑制ROCK、Caspase-3和PARP活性,能显著减少大鼠心肌梗死面积。
2. Rho-kinase/Akt/caspase-3/PARP信号通路是介导大鼠心肌细胞I/R损伤的一条重要的信号通路。
创新性及局限性
1.创新点
(1)提出并证实了ROCK和PARP在心脏缺血再灌注造成的心肌细胞凋亡中具有相互作用。
(2)提出并证实了ROCK/Akt/caspase-3/PARP信号通路是介导心肌细胞I/R损伤的一条重要的信号通路。
2.局限性
(1)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。
(2)由于时间及条件的限制,未能过表达Akt,以研究其在组织及细胞水平上的保护性效应。
Background
Coronary heart disease has been one major cause of disability and death in our country, approximately 700000 people die of coronary heart disease every year, accounting for about one quarter of all death caused by cardiovascular diseases. Reperfusion therapy, which can make the return of blood supply as soon as possible to the ischemic region of the myocardium, has been the chief treatment of acute myocardial ischemia. However, Braunwald and Kloner point out that reperfusion is a "double edged sword", because reperfusion itself can accelerate myocardial damage and can produce additional myocardial injury, which we called the Ischemia/reperfusion (I/R) injury. Ischemia/reperfusion (I/R) injury can be induced by organ transplant dysfunction, stroke, myocardial infarction, shock and so on. Reperfusion is followed by myocardial biochemical, structural, and functional changes and may determine myocardial cell survival and death. Reperfusion can lead to diminished cardiac contractile function and arrhythmia. There is experimental evidence that irreversible myocardial cell injury originated by reperfusion can result in cell necrosis and apoptosis. How to improve myocardial function, decrease the arrhythmogenesis, delay the episode of necrosis, and decrease the infarction size during ischemia/reperfusion injury is of great clinical significance. Therefore, increasingly importance has been attached to ischemia/reperfusion injury.
Recently, the research of Rho-kinase and poly (ADP-ribose) polymerase in myocardial cell apoptosis caused by ischemia/reperfusion injury has been more and more popular. Rho-kinases (ROCKs) are serine/threonine kinases with a molecular weight of 160 KD and are downstream effectors of the small GTPase Rho. ROCKs consist of an amino-terminal kinase domain, a mid-coiled-coil-forming lesion, the Pleckstrin homology (PH) motif and a carboxy-terminal cysteine-rich domain. There are two isoforms of ROCK:ROCK1 and ROCK2. ROCKs implicated in many kinds of vital movement, including the regulation of cellular contraction, growth, division, metabolism, migration apoptosis and gene expression. According to scientific survey, Rho-kinase is closely related to the development of a wide range of diseases such as coronary heart disease, atherosclerosis, hypertension, pulmonary hypertension, heart failure, diabetes, stroke, cancer and so on. Rho-kinase can mediate a series of Phosphorylation/phosphorylation reaction, dozens of substrates of ROCK have been identified, including:the myosin phosphatase-targeting subunit 1(MYPT1) and the myosin-binding subunit of myosin light chain phosphatase (MLCP), LIM, PTEN, and so on. The first characterized targets of ROCK are MLC and MYPT1. Evidence from clinical use of ROCK inhibitors, such asY-27632, fasudil and statins, supports the hypothesis that ROCK is a potential therapeutic target.
Poly (ADP-ribose) polymerases are resided in eukaryotes, and they are a family of cell signaling enzymes. There are 18 superfamily, including PARP-1、PARP-2、PARP-3, etc. PARP-1 is the most abundant subtype of poly (ADP-ribose) polymerases. Poly (ADP-ribose) polymerase (PARP) is involved in many important cellular pathways, and it can contribute to chromosomal structure and genomic stability, detects and repairs the damage of DNA. DNA repair, gene rearrangements, oxidative stress, DNA binding drugs and protein-protein interactions can cause the activation of Poly (ADP-ribose) polymerase. However, poly (ADP-ribose) polymerase overactivation can exhausts NAD+, steps down the rate of glycolysis, electron transport, and ATP formation, finally leads to functional impairment or death of cells. Clinical data indicates that, poly (ADP-ribose) polymerase plays an important role in many diseases such as myocardial ischemia/reperfusion injury, myocarditis, heart failure, shock, atherosclerosis and so on.
In our previous study, we found that both Rho-kinase and PARP play an important role in rat myocardial ischemia/reperfusion injury, and we further study the mechanism of them. We found that both ROCK/JNK/AIF pathway and PARP /JNK/AIF pathway are new pathways of cardiac myocyte injury. But whether there are interactions between Rho-kinase and PARP, what are the mechanisms of their interaction. In our study, we establish myocardial cell I/R model and the rat heart I/R model, investigate the interaction of Rho-kinase and PARP, identify the signaling pathway between them, so that we can provide a new therapeutic approach for ischemic heart disease.
This study includes there parts:
PartⅠIn vitro:The relationship and effect of Rho-kinase and PARP on cell apoptosis in the primary myocardial cell model of I/R
PartⅡIn vitro: The interaction and mechanism of Rho-kinase and PARP on the I/R model of primary cultured myocardial cell
PartⅢIn vivo:The interaction and mechanism of Rho-kinase and PARP on the rat I/R injury model
PartⅠIn vitro:The relationship and effect of Rho-kinase and PARP on cell apoptosis in the primary myocardial cell model of I/R
Objective
To investigate the interaction of Rho-kinase and PARP on I/R
Methods
1. Primary cultures of myocardial cells:obtain the heart of neonatal wistar rat, shred to 1mm3, and then digest with trypsin and collagenaseⅡ. After differential adhesion, put the myocardial cells in incubator.
2. Establish the hypoxia-reoxygenation injury model of myocardial cell, The following experimental groups were studied. (1) control group; (2) I/R+DMSOgroup; (3) I/R+Y27632group; (4) I/R+3-AB group.
3. Detect the activity of Rho-kinase and PARP: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 and PARP activity.
4. Evaluate the apoptotis of myocardial cells, the TUNEL technique was used.
Results
1. Rho-kinase and PARP both play an important role in I/R injury, there are interaction between them:Phospho-MYPT-1 and PARP increased during the hypoxia-reoxygenation injury model. Administration of inhibitors of Rho-kinase can depress the expression of Phospho-MYPT-1 and PARP. Administration of inhibitors of PARP can depress the expression of PARP, but do not effect the expression of Phospho-MYPT-1.This indicate that Rho-kinase and PARP both play an important role in I/R injury, and PARP is the downstream molecules of Rho-kinase.
2. The inhibition of Rho-kinase and PARP can both decrease the apoptosis of myocardial cells:The apoptosis ratio of myocardial cells was 46.5±1.2% in I/R +DMSO group. Administration of Y27632, the apoptosis ratio was 16±0.5%(P＜0.05 vs I/R+DMSO group); Administration of 3-AB, the apoptosis ratio was 20±0.3%(P <0.05 vs I/R+DMSO group), demonstrating that inhibition of Rho-kinase and PARP activity reduces myocardial cells apoptosis in rat heart I/R injury.
Conclusion
1. Rho-kinase and PARP activities increased in the hypoxia-reoxygenation injury model, they can aggravate the apoptosis of myocardial cells.
2. Rho-kinase and PARP inhibition in the hypoxia-reoxygenation injury can both reduce the apoptosis of myocardial cells.
3. There are interactions between Rho-kinase and PARP in the hypoxia-reoxygenation injury. By regulation the activity of PARP and Rho-kinase can reduce the apoptosis of myocardial cells.
Part II In vitro:The interaction and mechanism of Rho-kinase and PARP on the I/R model of primary cultured myocardial cells
Objective
To investigate the effect of Rho-kinase, Akt, caspase-3 and PARP in I/R
Methods
1. Primary cultures of myocardial cells from the neonatal wistar rat
2. Establish the hypoxia-reoxygenation injury model of myocardial cell. The following experimental groups were studied. (1) control group; (2) I/R+DMSO group; (3) I/R+Y27632 group; (4) I/R+LY294002 group; (5) I/R+Ac-DEVD-CHO group; (6) I/R+3-AB group.
3. Detect the activity of Rho-kinase, Akt, caspase-3 and PARP:Rho-kinase activity was assessed by examining phosphorylation of MYPT-1. Caspase-3 activity was assessed by examining the cleaved-Caspase-3. Western blot analysis was performed to evaluate Akt, p-Akt, cleaved-caspase-3 and PARP activity.
4. Evaluate the apoptosis of myocardial cells, the TUNEL technique was used.
Results
1. The expression of p-MYPT-1.. cleaved-Caspase-3 and PARP were increased in myocardial cell I/R model, but the expression of p-Akt was degraded. This shows that the activity of Rho-kinase、Caspase-3 and PARP increased in myocardial cell I/R model, phosphorylation of Akt was degraded.
2. The apoptosis ratio of myocardial cell is 60±1.68% I/R+DMSO group. The apoptosis ratio of myocardial cell is significantly reduced to 24±0.52% in I/R+ Y27632 group (P<0.05 vs I/R+DMSO group). The apoptosis ratio of myocardial cell is reduced to 32±1.24% in I/R+LY294002 group (P<0.05 vs I/R+DMSO group). The apoptosis ratio of myocardial cell is reduced to 36±0.65% in I/R+3-AB group (P <0.05 vs I/R+DMSO group). This suggests that inhibition of Rho-kinase, Caspase-3 and PARP activity reduces cell apoptosis.
3. The expression of p-MYPT-1 increased in I/R+DMSO group, but Phospho-Akt decreased in I/R+DMSO group. After administration of Y27632, the expression of Phospho-Akt was increased. After administration Akt inhibitor, the expression of p-MYPT-1 did not change significantly. This indicates Akt is the downstream molecules of Rho-kinase.
4. The expression of p-MYPT-1 and cleaved-Caspase-3 were increased in I/R +DMSO group. After administration of Rho-kinase inhibitor, the expression of cleaved-Caspase-3 was decreased. After administration of Caspase-3inhibitor, the expression of p-MYPT-1 did not change significantly. This indicates that Caspase-3 is the downstream molecules of Rho-kinase.
5. The expression of p-Akt was decreased in I/R+DMSO group, and the expression of cleaved-Caspase-3 were increased. After administration of Akt inhibitor, the expression of cleaved-Caspase-3 was increased. After administration of Caspase-3 inhibitor, the expression of p-Akt did not change significantly. This indicates that Caspase-3 is the downstream molecules of Akt.
6. The expression of PARP and cleaved-Caspase-3 were increased in I/R+ DMSO group, and the Cleavage fragments (24KD) of PARP was increased too. After administration of Caspase-3-inhibitor, the activity of PARP was decreased, the Cleavage fragments (24KD) of PARP were decreased. After administration of PARP inhibitor, the expression of cleaved-Caspase-3 did not change significantly. This indicates that PARP is the downstream molecules of Caspase-3.
Conclusion
1. In I/R model, the activity of Rho-kinase、Caspase-3 and PARP were increased, but phosphorylation of Akt was degraded. Inhibition the activity of Rho-kinase、Caspase-3 and PARP can reduce myocardial infarct size and the ratio of myocardial cell apoptosis.
2. Rho-kinase/Akt/caspase-3/PARP pathway may be a new pathway of heart I/R injury.
PartⅢIn vivo:The interaction and mechanism of Rho-kinase and PARP on the rat I/R injury model
Objective
To investigate the effect of Rho-kinase, Akt, caspase-3 and PARP in I/R
Methods
1. The rat model of I/R:The left anterior descending branch (LAD) of the left coronary artery was occluded and loosened to establish rat heart I/R model. The following experimental groups were studied:(1) control group (n=20) (2) I/R+DMSO group (n=20); (3) I/R+fasudil group (n=20); (4) I/R+LY294002 group (n=20); (5) I/R+Ac-DEVD-CHO group (n=20); (6) I/R+3-AB (n=20).
2. Detect the activity of Rho-kinase, Akt, caspase-3 and PARP:Rho-kinase activity was assessed by examining phosphorylation of MYPT-1. Caspase-3 activity was assessed by examining the cleaved-Caspase-3. Western blot analysis was performed to evaluate Akt, p-Akt and PARP 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. Evaluate the apoptosis of myocardial cells, the TUNEL technique was used.
Results
1. The expression of p-MYPT-1、cleaved-Caspase-3 and PARP were increased in the I/R model, but the expression of p-Akt was degraded. This shows that the activity of Rho-kinase、Caspase-3 and PARP increased in I/R model, phosphorylation of Akt was degraded.
2. Inhibition of Rho-kinase, Caspase-3 and PARP activity reduced myocardial infarct size. The infarct sizes of the heart were 60.22±1% in I/R+DMSO group, 30.5±1.2% in I/R+fasudil group,24.9±0.8% in I/R+LY294002 group and 29.1±0.5% in I/R+3-AB group(P<0.05 vs I/R+DMSO group). This indicates that inhibition of Rho-kinase, Caspase-3 and PARP can reduce myocardial infarct size.
3. The expression of p-MYPT-1 increased in I/R+DMSO group, but Phospho-Akt decreased in I/R+DMSO group. After administration of Y27632, the expression of Phospho-Akt was increased. After administration Akt inhibitor, the expression of p-MYPT-1 did not change significantly. This indicates Akt is the downstream molecules of Rho-kinase.
4. The expression of p-MYPT-1 and cleaved-Caspase-3 were increased in I/R +DMSO group. After administration of Rho-kinase inhibitor, the expression of cleaved-Caspase-3 was decreased. After administration of Caspase-3 inhibitor, the expression of p-MYPT-1 did not change significantly. This indicates that Caspase-3 is the downstream molecules of Rho-kinase.
5. The expression of p-Akt was decreased in I/R+DMSO group, and the expression of cleaved-Caspase-3 were increased. After administration of Akt inhibitor, the expression of cleaved-Caspase-3 was increased. After administration of Caspase-3 inhibitor, the expression of p-Akt did not change significantly. This indicates that Caspase-3 is the downstream molecules of Akt.
6. The expression of PARP and cleaved-Caspase-3 were increased in I/R+DMSO group, and the Cleavage fragments (24KD) of PARP was increased too. After administration of Caspase-3 inhibitor, the activity of PARP was decreased, the Cleavage fragments (24KD) of PARP were decreased. After administration of PARP inhibitor, the expression of cleaved-Caspase-3 did not change significantly. This indicates that PARP is the downstream molecules of Caspase-3.
Conclusion
1. In rat I/R model, the activity of Rho-kinase ^ Caspase-3 and PARP were increased, but phosphorylation of Akt was degraded. Inhibition the activity of Rho-kinase-. Caspase-3 and PARP can reduce myocardial infarct size.
2. Rho-kinase/Akt/caspase-3 /PARP pathway may be a new pathway of rat heart I/R injury.
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