【作者】 施珊珊；

【导师】 赵正言； 舒强；

【作者基本信息】 浙江大学 ， 儿科学， 2009， 博士

【摘要】 研究背景:缺血和再灌注可严重破坏心肌细胞超微结构,阻碍心肌组织能量代谢,导致心肌细胞凋亡和坏死,降低缺血再灌注损伤后心肌收缩和舒张功能。预处理（缺血或药物）是目前研究最为广泛,用于减轻心肌缺血再灌注损伤的主要手段。本研究通过建立大鼠离体心脏Langendorff模型,以重组人促红细胞生成素预灌注作为处理手段,观察rhEPO预处理对心肌缺血再灌注损伤的保护作用;同时,通过测定磷酸化PKCε、p38MAPK和ERK1/2的表达,探讨rhEPO心肌保护的可能分子机制。第一部分促红细胞生成素预处理对大鼠缺血再灌注损伤后心功能的保护作用目的:研究rhEPO预处理对心肌缺血再灌注损伤后心肌收缩和舒张功能以及细胞活性的影响。方法:本实验采用全心缺血30分钟,再灌注120分钟模拟心肌缺血再灌注模型（I/R模型）。分别测定I/R组、rhEPO（10IU/ml）+I/R组、chelerythrine+rhEPO+I/R组、SB203580+rhEPO+I/R组、PD98059+rhEPO+I/R组、glibenclamide+rhEPO+I/R组和L-NAME+rhEPO+I/R组（共7组）缺血前、再灌注30分钟、再灌注60分钟及再灌注120分钟时的LVDP、RPP、+dP/dt_max、-dP/dt_max和CF以评测心肌收缩和舒张功能;应用MTT比色法测定各组心肌缺血再灌注损伤后心肌细胞活度。结果:1.RhEPO预处理大鼠心脏再灌注30分钟的LVDP、RPP和+dP/dt_max,60、120分钟的LVDP、RPP、+dP/dt_max和CF值以及OD550均较I/R组显著升高。2.Chelerythrine和rhEPO联合预处理后,大鼠心脏再灌注30分钟的LVDP、RPP和-dP/dt_max,60、120分钟时的LVDP、RPP、±dP/dt_max以及心肌细胞活性均较rhEPO处理组显著下降。SB203580或PD98059与rhEPO联合预处理后,再灌注30分钟的LVDP、RPP和±dP/dt_max,60、120分钟的LVDP、RPP、±dP/dt_max和CF以及OD550均较rhEPO处理组显著下降。3.Glibenclamide和rhEPO联合应用后,大鼠心脏再灌注30分钟的LVDP、RPP和±dP/dt_max,60、120分钟的LVDP、RPP、±dP/dt_max和CF以及OD550均较rhEPO处理组显著下降。4.L-NAME和rhEPO联合应用后,大鼠心肌其再灌注30分钟、60分钟及120分钟的LVDP、RPP、±dP/dt_max、CF以及OD550各项指标均较rhEPO处理组显著下降。结论:RhEPO预处理能显著提高大鼠心肌缺血再灌注损伤的左心收缩和舒张功能,增加冠脉流量并提高心肌细胞存活率,其机制与PKCε、p38MAPK或ERK1/2的磷酸化激活有关,抑制PKC、p38MAPK或ERK1/2途径活化均可完全阻断rhEPO上述心肌保护作用。RhEPO预处理的心肌保护作用还与K_ATP通道的活化和开放有关,抑制K_ATP通道的开放可完全阻断rhEPO的心肌保护作用。此外,NO的合成和释放也是介导rhEPO预处理心肌保护的必要因素,阻断NOS、抑制NO的合成和释放可完全阻断rhEPO的心肌保护作用。第二部分促红细胞生成素预处理对大鼠缺血再灌注损伤的急性心肌保护的机制研究目的:探讨rhEPO预处理心肌保护的相关分子机制。方法:采用Western-blot技术分别测定对照组、rhEPO（10IU/ml）预处理、Chelerythrine+rhEPO预处理、SB203580+rhEPO预处理、PD98059+rhEPO预处理、Glibenclamidee+rhEPO预处理和L-NAME+rhEPO预处理（共7组）后心肌组织磷酸化PKCε、p38MAPK和ERK1/2的表达。结果:1.RhEPO（10IU/ml）预灌注10分钟后,心肌细胞颗粒成份（主要为线粒体、细胞核和胞膜成份）内PKCε含量较对照组显著升高,而细胞浆内PKCε含量却显著减少。同时,磷酸化p38MAPK和磷酸化ERK1/2也均较对照组显著升高。2.PKC抑制剂chelerythrine可完全阻断PKCε活化和转位,心肌细胞颗粒成份内PKcε含量较rhEPO处理组显著减少并回落至基础水平。而p38MAPK抑制剂SB203580和ERK1/2抑制剂PD98059对PKCε的活化和转位无显著影响。P38MAPK抑制剂SB203580可完全抑制磷酸化p38MAPK表达;chelerythrine可部分抑制磷酸化p38MAPK表达;而PD98059对p38MAPK磷酸化表达无显著影响。ERK1/2抑制剂PD98059可完全抑制磷酸化ERK1/2的表达;chelerythrine可部分抑制磷酸化ERK1/2的表达;而SB203580对ERK1/2的磷酸化表达无显著影响。3.广谱K_ATP通道抑制剂glibenclamide与rhEPO联合灌注对PKCε的活化和转位无显著影响,却能部分阻断磷酸化p38MAPK和ERK1/2的表达。4.广谱一氧化氮合酶抑制剂L-NAME与rhEPO联合灌注均能部分减少PKCε、p38MAPK和ERK1/2的磷酸化激活。结论:RhEPO（10U/ml）预灌注可快速磷酸化激活PKCε,并促使其由细胞浆向细胞颗粒成份内转移,并快速磷酸化激活p38MAPK和ERK1/2。PKCs是p38MAPK和ERK1/2的上游激酶,阻断PKCε可部分减少磷酸化p38MAPK和ERK1/2的表达。抑制K_ATP通道的活化和开放并不影响PKCε的活化和转位,却能部分阻断p38MAPK和ERK1/2的磷酸化表达。可见,PKCε可能是K_ATP通道的上游激酶;而p38MAPK和ERK1/2是K_ATP通道的下游激酶,p38MAPK和ERK1/2的磷酸化活化存在K_ATP通道依赖和非依赖途径。NOS合酶抑制剂可部分抑制PKCε的活化和转位,同时也能部分减少磷酸化p38MAPK和ERK1/2的表达。可见,PKCε、p38MAPK和ERK1/2的激活可能存在NO依赖和非依赖途径。更多还原

【Abstract】 Background:Myocardial ischemia reperfusion injury significantly depressed myocardial contractile and diastolic function for myocardiocyte necrosis and apoptosis,which were resulted from breakdown of ultrastructural organization and disorder of energy metabolism.Ischemia preconditioning or pharmacological preconditioning was used extensively in order to palliating the myocardial injury suffering from ischemia and reperfusion injury in both clinical and experimental settings.The purpose of this study was to determined the cardioprotective effect of rhEPO preconditioning on the isolated rat heart,and to identify the possible molecular mechanisms underlying the cardioprotection of rhEPO preconditioning by detecting the phosphorylated PKCε、p38MAPK and ERK1/2. PartⅠThe Cardioproteetive Effect of Human Recombinant Erythropoietin on the Cardiac Function of Isolated Rat Heart Suffering from Ischemia Reperfusion InjuryObjective:To investigate the effect of rhEPO preconditioning on the post-ischemic contractile and diastolic function and the cell viability after myocardial ischemia reperfusion injury.Methods:Hearts from normoxic rat（n=8/group） were isolated and perfused in the Langendorff mode.The hearts were perfused with rhEPO（10.0IU/ml） for 15 minutes prior to 30 minutes ischemia and 120 minutes reperfusion.The experimental protocol used was shown in Fig.1 PartⅠ.For mechanism studies,the hearts were pretreated with PKC inhibitor（chelerythrine）,p38MAPK inhibitor（SB203580）,ERK1/2 inhibitor （PD98059）,potassium channel blockers（glibenclamide） or nitric oxide synthase inhibitors（L-NAME） for 15 minutes alone followed by 15 minutes in combination with rhEPO prior to ischemia..The left ventricular contractile and diastolic function(LVDP、RPP、+dP/dt_max、-dP/dt_max) and the coronary flow（CF） were recorded under steady state conditions prior to global no-flow ischemia and at 30、60、120min of reperfusion,and the cell viability was detected by MTT assay.Results:1.RhEPO significantly increased the LVDP、RPP、+dP/dt_max、-dP/dt_max but no effect on the CF at 30 minutes of reperfusion,while the LVDP、RPP、+dP/dt_max、-dP/dt_max and CF were all increased significantly at 60 and 120 minutes of reperfusion compared with I/R group.The cell viability was also increased significantly compared with I/R group.2.Inhibiting the activation and traslocation of PKCεwith chelerythrine abolished the cardioprotective effects of rhEPO.The LVDP、RPP、-dP/dt_max at 30 minutes of reperfusion,the LVDP、RPP、+dP/dt_max at 60 and 120 minutes of reperfusion,and the cell viability were all decreased significantly compare with rhEPO group.At the same time,in group pretreated with SB203580 or PD98059 following by rhEPO,the LVDP、RPP、±dP/dt_max at 30 minutes,the LVDP、RPP、±dP/dt_max、CF at 60 and 120 minutes, and the cell viability were also decreased significantly compared with rhEPO group.3.The LVDP、RPP、±dP/dt_max at 30 minutes of reperfusion,the LVDP、RPP、±dP/dt_max、CF at 60 and 120 minutes of reperfusion,and the cell viability were all decreased in group pretreated with K_ATP channel inhibitor glibenclamide following by rhEPO perfusion compared with rhEPO group.4.The inhibitor of NOS（L-NAME） can also abolish the cardioprotection of rhEPO preconditioning.The LVDP、RPP、+dP/dt_max、-dP/dt_max and CF at 30、60、120 minutes of reperfusion and the cell viability were all decreased in group pretreated with L-NAME following with rhEPO perfusion compared with rhEPO group.Conclusions:Our study demonstrated that rhEPO preconditioning increased the left ventricular post-ischemia contractile and diastolic function and coronary flow,as well as the cell viability after I/R injury.The mechanisms underlying the cardioprotection possibly associated with the phosphorylation of PKCε、p38MAPK and ERK1/2、for inhibiting the activity of PKCε、p38MAPK or ERK1/2 could abolish the cardioprotective effect of rhEPO absolutely.Besides,the opening of K_ATP channel and the synthesizing of NO might also involve in the cardioprotection of rhEPO preconditioning,for inhibiting the K_ATP channel or the NOS could abolish the cardioprotective effects of rhEPO absolutely as well.PartⅡThe Possible Molecular Mechanisms Underlying the Cardioprotection of Human Recombinant Erythropoietin on the Isolated Rat Heart Suffering from Ischemia Reperfusion InjuryObjective:To identify the possible molecular mechanisms underlying the cardioprotection of rhEPO preconditioning by detecting the phosphorylated PKCε、p38MAPK and ERK1/2.Methods:Hearts from rats were isolated and aerobically perfused with K-H buffer for 30 minutes,then perfused with erythropoietin（10IU/ml） for 10 minutes.To investigating the molecular mechanism of cardioprotection of rhEPO,the hearts were pretreated with PKCεinhibitor（chelerythrine）,p38MAPK inhibitor（SB203580）,ERK1/2 inhibitor （PD98059）,potassium channel blockers（glibenclamide） or nitric oxide synthase inhibitor（L-NAME） for 10 minutes alone followed by 10 minutes in combination with rhEPO.Then the free wall of left ventricle was excised and immediately stored in liquid nitrogen for western blot analysis.Frozen myocardial tissue samples were powdered in a precooled stainless steel mortar and pestle.The powdered tissue was then processed to obtain cell lysates or fractionated to obtain cytosolic and particulate fractions.Equal amounts of protein was analyzed by SDS-PAGE and Western blotting by using either isoform-specific antibodies for phospho-PKCεdetection both in cytosolic and particulate fraction or specific antibodies against phosphorylated and nonphsophorylated of p38 MAPK and ERK1/2 in cell lysates.The blots were developed by ECL.Results:1.The analysis of cytosolic and particulate fractions revealed that in the rhEPO preconditioning（10IU/ml） hearts,PKCεwas activated and translocated from cytosolic fraction to particulate fraction after perfused with rhEPO for 10 minutes.And the expression of phosphorylation of p38MAPK and ERK1/2 in total cell lysate was increased significantly compared with control group as well.2.Administration of chelerythrine before rhEPO inhibited the activation and translocation of PKCε,and PKCεlocated in the particular fraction was decreased significantly compared with rhEPO group.While the SB203580 or PD98059 had no effect on the activation and translocation of PKCε.SB203580 pretreating before rhEPO abolished the phosphorylation and activation of p38MAPK.Pretreatment with chelerythrine before rhEPO could partially inhibit the phosphorylation and activation of p38MAPK.While PD98059 had no effect on the phosphorylation and activation of p38MAPK.PD98059 inhibited the phosphorylation and activation of ERK1/2,and chelerythrine could also partially inhibit the phosphorylation and activation of ERK1/2. While SB203580 had no effect on the phosphorylation and activation of ERK1/2.3.Pretreatment with glibenclamide followed by rhEPO resulted in partially inhibiting the phosphorylation and activation of p38MAPK and ERK1/2,but had no effect on the activation and translocation of PKCε. 4.Pretreament with L-NAME following by rhEPO partially inhibited the phosphorylation and activation of p38MAPK and ERK1/2,and partially inhibited the activation and translocation of PKCεas well.Conclusion:The activation and translocation of PKCεwas induced soon after pretreated with rhEPO（10IU/ml）.And treatment with rhEPO also phosphorylated p38MAPK and ERK1/2 rapidly.PKCεwas the upstream kinase both of p38MAPK and ERK1/2,and blocking the activation of PKCεpartially inhibited the phosphorylation of p38MAPK and ERK1/2.PKCεwas the upstream kinase of K_ATP channel,and inhibiting the activation and opening of K_ATP channel had no effect on the activation and translocation of PKCε.In the contrary,p38MAPK and ERK1/2 were the downstream kinases of K_ATP channel,and blocking the opening of K_ATP channel partially inhibited the phosphorylation of p38MAPK and ERK1/2.So we concluded that the activation and phosphorylation of p38MAPK and ERK1/2 were K_ATP channel-dependent and K_ATP channel-independent.Inhibition the production of NO partially abolished the activation and phosphorylation of PKCε,and partially inhibited the activation and phosphorylation of p38MAPK and ERK1/2.The activation of PKCε、p38MAPK and ERK1/2 were all NO-dependent and NO-independent.更多还原