【作者】 王高频；

【导师】 孙英贤；

【作者基本信息】 中国医科大学 ， 内科学， 2007， 博士

【摘要】 前言随着急诊溶栓、经皮冠状动脉腔内成形术、冠脉搭桥术等再灌注治疗的发展，心肌缺血再灌注（Ischemia／Reperfusion，I／R）损伤已成为阻碍缺血心肌从再灌注治疗中获得最佳疗效的主要难题。许多基础和临床研究表明，I／R损伤的机制包括触发物质—中介物质—效应物质三个环节，目前多数研究是针对触发物质和效应物质，对中介物质即细胞内信号通路研究较少。探讨细胞内信号通路与I／R损伤之间的关系，有助于从分子水平研究I／R损伤的机制，从而为治疗I／R损伤提供新的作用靶点。心肌I／R损伤由复杂的细胞信号网络调节，涉及多条细胞内信号通路，并在多层次和诸多环节存在交互作用。近年来研究显示，Janus激酶信号转导子与转录激活子（Janus kinase signal transducers and activators of transcription，JAK-STAT）通路与心肌I／R损伤有关，但这方面的研究尚少。目前存在的问题是：①心肌缺血再灌注时，STAT家族的哪些成员被激活，激活的时程如何?不同的家族成员对心脏起保护还是有害作用?②JAK-STAT通路激活通过什么机制对心脏发挥作用还不清楚；③药物预处理的心脏保护作用是否与该通路有关?这些问题还有待进一步研究。肾素血管紧张素系统（RAS）在心肌I／R损伤中起重要作用。大量的动物实验表明，血管紧张素Ⅱ（AngⅡ）在心肌I／R时大量释放，并通过G蛋白偶联的AT1受体引起心肌再灌注损伤，AT1受体拮抗剂具有抗再灌注损伤的心脏保护作用，但也有相反的结果，尤其对心肌细胞凋亡的影响还存在争议。JAK-STAT通路主要经由G蛋白偶联受体和细胞因子受体被激活，在心肌I／R时，AT1受体拮抗剂的心脏保护作用是否与JAK-STAT通路有关还不十分清楚。凋亡是I／R时心肌细胞死亡的主要机制之一。有证据表明，RAS与JAK-STAT通路均与IR损伤中发生的心肌细胞凋亡有着紧密的联系。凋亡的产生是从细胞外信号—细胞内信号通路—凋亡相关基因和酶的复杂过程。Bcl-2、Bax和caspases-3是控制凋亡发生的相关基因和酶。RAS—JAK-STAT通路—Bcl-2、Bax、caspase-3在三个不同的层面上对细胞凋亡产生影响，三者在心肌I／R时发生怎样的变化?之间是否存在联系?并通过怎样的方式起作用还不清楚。本研究采用Langendorff离体心肌缺血再灌注模型，探讨：①JAK-STAT通路的主要成员SIAT1和STAT3在心肌I／R损伤过程中是否被激活，及激活的时程变化规律；②JAK-STAT通路在心肌I／R中被激活的功能作用，及AT1受体拮抗剂伊贝沙坦抗心肌I／R损伤的作用是否与JAK-STAT通路有关；③心肌I／R时凋亡相关基因和酶的变化，及与其上游RAS和JAK-SIAT通路的关系。为临床防治心肌FR损伤提供实验基础和理论依据。方法1、JAK-SIAT通路在大鼠离体心肌I／R中激活时程的研究采用Langendorff离体心肌缺血再灌注模型，42只雄性Wistar大鼠分为7组，每组6只：正常对照组、缺血30min组（R0）、缺血再灌注5min组（R5）、缺血再灌注15min组（R15）、缺血再灌注30min组（R30）、缺血再灌注60min组（R60）和缺血再灌注120min组（R120）。正常对照组用改良Krebs-Henseleit（KH）液持续灌流180min；缺血及再灌注组用改良的KH液灌流平衡30min后，全心停灌30min，分别再灌注0min、5min、15min、30min、60min、120min。动态监测左室发展压（LVDP）、左室压力最大上升速率(dp／dt_max)；GCA亮绿染色观察心肌坏死程度；免疫组化法定位p-STAT1和p-STAT3蛋白表达及核易位；免疫印迹法检测p-STAT1，p-STAT3和t-STAT1，t-STAT3的蛋白表达。2、伊贝沙坦通过JAK-STAT通路对大鼠心肌I／R损伤的保护作用采用Langendorff离体心肌缺血再灌注模型，65只雄性Wistar大鼠分为5组，每组13只：正常对照组、缺血再灌注组、AG490组、伊贝沙坦预处理组、伊贝沙坦后处理组。动态监测LVDP和dp／dtmax变化；再灌注120min TTC染色计算心肌梗死面积；再灌注30min免疫印迹法检测p-STAT1，p-STAT3和t-STAT1，t-STAT3的蛋白表达。3、伊贝沙坦对大鼠心肌I／R损伤细胞凋亡及相关基因表达的影响采用Langendorff离体心肌缺血再灌注模型，25只雄性Wistar大鼠分为5组，每组5只：正常对照组、缺血再灌注组、AG490组、伊贝沙坦预处理组、伊贝沙坦后处理组。TUNNEL法检测心肌细胞凋亡指数（AI），RT-PCR法检测Bcl-2、Bax的mRNA表达，免疫印迹法检测Bcl-2、Bax和Caspase-3蛋白表达。结果1、JAK-STAT通路在大鼠离体心肌I／R中激活时程的研究①心功能监测结果显示：KH液灌流30min后，心功能指标LVDP和dp／dt_max，随着再灌注时间的延长呈进行性下降，再灌注120min降至最低(p＜0.01=。②GCA亮绿染色显示：坏死心肌纤维随再灌注时间延长逐渐增多，再灌注120min最明显。③免疫组化定位p-S7ATs核易位显示：正常对照组仅在胞浆内呈现少量p-STAT1和p-SIAT3棕色淡染颗粒；与对照阻比，胞浆内p-STAT1棕色颗粒在缺血30min无明显增多，再灌注5min开始增多，并向胞核内积聚、浓染，发生核易位，再灌注30min最明显；胞浆内p-STAT3棕色颗粒在缺血30min即明显增多，并向胞核内易位，再灌注期未见进一步增多。④免疫印迹法半定量p-STATs和t-STAT3蛋白表达显示：正常对照组p-STAT1和p-STAT3蛋白少量表达；与正常对照组比，p-STAT1蛋白表达在缺血30min未见明显增加，随再灌注时间延长表达逐渐增多，再灌注30min时达高峰，而p-STAT3蛋白表达在缺血30min即明显增加，随再灌注时间延长未见表达进一步增加；t-STAT1和t-STAT3蛋白表达在缺血和再灌注期间均无变化。⑤相关分析显示：p-STAT1与p-STAT3比值与LVDP和dp／dt_max均呈显著负相关（r=—0.894，—0.886，p＜0.001）。2、伊贝沙坦通过JAK-STAT通路对大鼠心肌I／R损伤的保护作用①与正常对照组比，I／R组心肌梗死面积明显增加（p＜0.01），心功能指标LVDP和dp／dtmax均下降（p＜0.01），p-STAT1和p-STAT3蛋白表达均增多（p＜0.01），以p-STAT1蛋白表达升高更为明显，约为正常对照组4倍，p-STAT1和p-STAT3比值上调，而t-STAT1和t-STAT3蛋白表达无变化。②与I／R组比，应用AG490、伊贝沙坦预处理及后处理使心肌梗死面积均缩小（p＜0.01），LVDP和dp／dt_max)均明显升高（p＜0.01），p-STAT1和p-SIAT3的蛋白表达明显减少（p＜0.01和p＜0.05），以p-STAT1表达降低更为明显，p-STAT1与p-SIAT3比值下调。③AG490组较伊贝沙坦预处理和后处理组降低p-STAT1蛋白表达的作用更为明显（p＜0.05），三种处理方式对t-STAT1和t-STAT3蛋白表达无影响。，3、伊贝沙坦对大鼠离体心肌I／R损伤凋亡及相关基因表达的影响①与正常对照组比，I／R组心肌细胞凋亡指数显著增加（p＜0.01），Bcl-2的mRNA及蛋白表达无明显变化，Bax的mRNA及蛋白表达均增加（P＜0.01），Caspase-3活性蛋白表达也增加了近4倍（p＜0.01）。②与I／R组比，应用AG490、伊贝沙坦预处理和后处理使心肌细胞凋亡指数下降（p＜0.01），Bcl-2的mRNA及蛋白表达均升高（p＜0.05），Bax的mRNA及蛋白表达均显著下降（p＜0.01），Caspase-3活性蛋白表达明显降低（p＜0.01）。结论（1）心肌缺血再灌注可激活JAK-STAT通路。（2）阻断JAK-STAT通路减轻心肌I／R损伤，提示该通路促进心肌I／R损伤。（3）伊贝沙坦具有抗心肌I／R损伤作用，其机制与阻断JAK-STAT通路，下调促凋亡基因Bax，上调抑制凋亡基因Bcl-2，降低Caspase-3活性有关。更多还原

【Abstract】 The Role of JAK-STAT Signal Pathway in Ischemia/ Reperfusion Injury and Mechanism of Cardioprotection by IrbesartanIntroductionWith the development of emergency thrombolysis, percutaneous tranluminal coronary angioplasty（PTCA） and coronary artery bypass graft（CABG）, myocardial Ischemia/Reperfusion（I/R） injury has been becoming the major problem which hinders ischemic myocardium acquiring the best therapeutic effect by reperfusion therapy. Many basic and clinical studies showed that the mechanism of ischemia/reperfusion injury included three links of triggering substance, medial substance and effector, most of the studies aimed directly at trigging substance and effector, however there are not satisfactory therapeutic measure. This results, in part, from our limited tmderstanding of medial substance, which was cellular signal pathway. It is conduced to invest the pathogenesy of I/R injury at the level of molecular level to explore the relationship between cellular signal transduction pathway and I/R injury, then supplies new effective target for the treatment of I/R injury.The influence of I/R injury on the heart is mediated by a complex cellular signal network consisting of multiple cellular signal transduction pathway, and existes interaction in multistrata and polynucleation. Recent studies demonstrated that Janus kinase signal transducers and activators of transcdption（JAK-STAT） pathway related to I/R injury, howerver there were little study on it. The problem at present are as following: First, which members of STAT family are activated when I/R occur, and how is the time course of activating? The effect of different family members on heart is protective or harmful? Second, it was not clear that how the JAK-STAT signal pathway exerted its effect on heart. Third, If cardioprotection of the medicine preconditioning is involved in this signal pathway? All these questions are still to be further studied.Renin angiotensin systerm（RAS） plays an important role in myocardial I/R injury. Lots of animal experiments demonstrated that angiotoninⅡreleased largely during I/R, and caused I/R injury by AT1 receptor coupling with G protein. AT1 receptor antagonist had the role of cardioprotection angist I/R injury, but also existed adverse results. Especially, it was dispute about its effect on apoptosis. JAK-STAT pathway is activates mainly by G protein couple receper and cytokine receper. It was not very clear that if role of AT1 receptor antagonist in protection against I/R injury is related to the JAK-STAT pathway.Apoptosis has become increasingly recognized as one of the mechanisms of cell death during myocardial I/R injury. It had shown that RAS and the JAK-STAT pathway were involed tightly in myocardial apoptosis during I/R injury. Apoptosis is a complex process from ecto-signal to cyto- sigal pathway, then to apoptotic related gene and enzyme. Bcl-2, Bax and caspases-3 are apoptotic related gene and enzyme controlling apoptosis. RAS, the JAK-STAT pathway and Bcl-2, Bax, caspase-3 affect on apoptosis in three different decks respectively. It is not clear how change they occur during I/R, what relationship are among them, and what manner they play a part in.The study applied Langendorff isolated myocardial ischemia/reperfusion model, to explore the following questions. First, if the main number STAT1 and STAT3 of the JAK-STAT signal pathway is activated during I/R injury and the changing regularity of time course? Second, the role of the JAK-STAT pathway in the I/R injury; and if cardiprotection by AT1 receper antagonist. Final, the changes of apoptotic related gene and enzyme, and the relationship with RAS and the JAK-STAT pathway during I/R injury. These would supply experimental and theory basement for clinical therapy of I/R injuryMethods1. The study of the activating time course of the JAK-STAT signal pathway during myocardial I/R injury of rats The Langendorff isolated myocardial ischemia/reperfusion model was applied. 42 male Wistar rats were divided randomly into 7 groups, and 6 ones for every group. The groups were control group, ischemia30min group（R0）, ischemia/reperfusion5min group, （R5） ischemia/reperfusionl 5min group（R15）, ischemia/reperfusion30min group（R30）, ischemia/reperfusion60min group（R60）, and ischemia/reperfusion120min group（R120）. In control group, isolated rats hearts were perfuse with modified Krebs-Henseleit（KH） buffer for 180 min, in ischemia/reperfusion group, hearts were perfusion with KH buffer for 30min, followed by 30minutes of globle ischemia and 0minute, 5minutes, 15minutes, 30minutes, 60minutes and 120 minutes of reperfusion respectively. The left ventricular developed pressure（LVDP） and Left ventficular first derivative of developed pressure(dp/dt_max) were monitored dynamicly; The extent of myocardial necrosis was observed by GCA bright green staining; the phosphorylated STAT1 and STAT3 proteins were located by immunohistochemical method; and the total as well as the phosphorylated STAT1 and STAT3 protein expression were detected by immunoblotting.2. The protective role of Irbesartan against myocardial I/R injury of rats through the JAK-STAT signal pathwayThe Langendorff isolated heart perfusing model was applied. 65 male Wistar rats were randomly divided into 5 groups, and 13 ones for each group. The groups were control group, ischemia/reperfusion group, AG490 group, Irbesartan preconditioning group and Irbesartan postcinditioning group. The change of LVDP and dp/dt_max were monitored dynamicly; The myocardial infarct size was measured by TTC staining after 120 min of reperfusion; The total and phosphorylated STAT1 and STAT3 protein expression were detected by immunoblotting after 30min of reperfusion.3. The effect of Irbesartan on apoptosis and the related gene expression during myocardial I/Rinjury of ratsThe Langendorff isolated heart perfusing model was applied. 25 male Wistar rats were randomly divided into 5 groups, and 5 ones for each group. The groups were control group, ischemia/reperfusion group, AG490 group, Irbesartan preconditioning group and Irbesartan postcinditioning group respectively. The myocardial apoptotic index was detected by TUNNEL method; The mRNA expression of Bcl-2 and Bax was detected by RT-PCR method; The protein expression of Bcl-2, Bax and Caspase-3 was detected by immunoblotting method.Results1. The study of the activating time course of the JAK-STAT signal pathway during myocardial I/R injury of rats①With the prolongation of reperfusing time, The values of LVDP and dp/dt_max decreased gradually after KH buffer perfusion for 30min, and decreased to the lowest level after 120 min of reperfusion（p＜0.01）.②extent of myocardial necrosis stained by GCA bright green increased gradually with the prolongation of reperfusing time, and the most obvious result appeared after reperfusion for 120 min.③The immunohistochemical localization demonstrated that in control group, protein of p-STAT1 and p-STAT3 appeared the light brown grana in the cytoplasm. Compared with control group, brown grana of p-STAT1 in the cytoplasm did not increase after 30min of ischemia, and it began to rise and translocated to nucleus after 5min of reperfusion, brown grana of p-STAT1 reached peak afer 30min of reperfusion. Howerver, brown grana of p-STAT3 in the cytoplasm increased remarkablely and translocated to nucleus after 30min of ischemia, and did not increased again during reperfusion.④The Westeron-blot demonstrated that the protein of p-STAT1 and p-STAT3 expressed slightly in control group. Compared with control group, the protein expression of p-STAT1 didn’t increase obviously after 30min of ischemia, and it began to increase after 5min of reperfusion, then reached the peak after 30 min of reperfusion. While the protein expression of p-STAT3 increased significantly after 30min of ischemia, and it did not incrase again during reperfusion. The t-STAT1 and t-STAT3 had no change in the period of ischemia and reperfusion.⑤The proportionality of p-STAT1 and p-STAT3 was correlate negtively with LVDP and dp/dt_max（r=—0.894,—0.886, p＜0.001）.2. The protective role of Irbesartan against myocardial I/R injury of rats through the JAK-STAT signal pathway①Compared with control group, myocardial infarct size was increased obviously in I/R group（p＜0.01）, the values of LVDP and dp/dtmax were all decreased（p＜0.01）, and the protein expression of p-STAT1 and p-STAT3 were all increased（p＜0.01）, especially, the protein expression of p-STAT1 approached 4 times of that in control group, and the proportionality of p-STAT1/p-STAT3 upregulated. The total STAT1 and STAT3 had no change.②Compared with I/R group, AG490, Irbesartan preconditioning and postconditioning reduced myocardial infarct size （p＜0.01）, and the values for both LVDP and dp/dt_max were elevated markedly（p＜0.01）, At the same time, all the three could block the increasing of protein expression of p-STAT1 and p-STAT3（p＜0.05 or p＜0.01）, and the proportionality of the two down-regulated; The three had no effect on the protein expression of total STAT1 and STAT3.3. The effect of Irbesartan on apoptosis and the related gene expression during myocardial I/Rinjury of rats①Compared with control group, the myocardial apoptotic index increased significantly in I/R group （p＜0.01）; The mRNA and protein expression of Bax were all increased （p＜0.01）, while the mRNA and protein expression of Bcl-2 had no change. the proportionality of Bax and Bcl-2 up-regulated; The protein expression of active Caspase-3 also increased about 4 times （p＜0.01）.②Compared with I/R group, AG490, Irbesartan preconditioning and postconditioning caused a marked lowering of myocardial apoptotic index（p＜0.01）; The mRNA and protein expression of Bax all decreased（p＜0.01）, While the mRNA and protein expression of Bcl-2 all increased（p＜0.05）. The proportionality of Bax and Bcl-2 down-regulated; the protein expression of active Caspase-3 decreased, too（p＜0.01）. Conclusion（1） JAK-STAT signal pathway was activated during myocardial I/R.（2） The blockage of JAK-STAT signal pathway lightened myocardial I/R injury, which suggested that the activation of this pathway can promot myocardial I/R injury.（3） The role of cardioprotection was taken on by Irbesartan during myocardial I/R. Its mechanism was related to the blockage of JAK-STAT signal pathway, down-regulating of the expression of Bax, up-regulatating of the expression Bcl-2, and the activity decrease of Caspase-3.更多还原