【作者】 张国明；

【导师】 李天德； 王禹；

【作者基本信息】 中国人民解放军军医进修学院 ， 内科学， 2010， 博士

【摘要】 第一部分机械后适应中磷酸化p38／JNK变化及其对心肌再灌注损伤的影响目的探讨大鼠心肌缺血后适应中磷酸化p38/JNK MAPK的变化,并进一步分析其与心肌再灌注损伤的关系。方法70只雄性大鼠随机分为假手术组、缺血再灌注组(R／I)、机械后适应组(Post)、JNK抑制剂SP600125组(SP)、p38抑制剂SB203580组(SB)、p38／JNK激活剂Anisomycin+后适应组(Ani+Post)和p38／JNK激活剂Anisomycin (Ani)组七组。建立急性心肌梗死再灌注模型,根据分组在缺血结束前5min经颈静脉持续注射相应药物,缺血结束时注射完毕。在不同时间点处死大鼠分别测定心肌凋亡、坏死、梗死面积和凋亡途径分子的表达。结果SP和SB组较R／I组血流动力学指标显著改善,细胞凋亡和坏死减轻,心肌梗死面积减少,同时伴随凋亡信号分子降低(P<0.05)；SP和SB组同Post组相比上述指标均无明显差异。SP组P-JNK显著低于R／I组(P<0.05),同Post组相似(P>0.05)；SB组P-p38显著低于R／I组(P<0.05),同Post组相似(P>0.05)。Ani+Post组部分抵消了Post组的心肌保护效应,Post组各指标均显著优于Ani+Post组(P<0.05)；Ani组各项指标均与R／I组相似(P>0.05)。结论机械后适应可以抑制p38/JNK MAPK在心肌再灌注损伤中的磷酸化,并通过P-p38和P-JNK的减少抑制了细胞死亡受体途径和线粒体途径凋亡的发生。第二部分机械后适应改良方案-渐进延长再灌注模式减轻心肌缺血再灌注损伤及其机制的研究目的探讨后适应不同模式对大鼠急性心肌缺血再灌注损伤的影响并进一步研究其机制。方法60只雄性大鼠随机分为假手术组、缺血再灌注组(R／I)、后适应渐进缩短再灌注组(GDR)、后适应相等再灌注组(ER)和后适应渐进延长再灌注组(GIR)五组。建立急性心肌梗死再灌注和缺血后适应模型,根据分组给予不同后适应处理,在不同时间点处死大鼠分别测定心肌凋亡、坏死、梗死面积和凋亡途径分子的表达。结果三种后适应处理组较R／I组均可显著改善血流动力学指标、减轻细胞凋亡和坏死、限制心肌梗死面积；同时伴随P-p38,P-JNK等致凋亡分子表达显著减少(P<0.05)。上述变化GIR组最为明显,其次是ER组,GDR组最不显著。GIR组同ER组相比,除心肌梗死面积减轻未达显著性差异外(P>0.05),上述指标均明显优于ER组(P<0.05)。以上指标GIR组均显著优于GDR组(P<0.05)。结论后适应渐进延长再灌注模式能发挥更大程度的心肌保护作用,这可能与其更大程度激活ERK、抑制p38／JNK活化、抑制细胞死亡受体和线粒体凋亡途径相关。第三部分乳酸和低剂量依达拉奉联合药物后适应减轻心肌缺血再灌注损伤及其机制的研究目的探讨乳酸和低剂量氧自由基清除剂依达拉奉能否模拟后适应发挥有效的心肌保护作用并进一步研究其机制。方法108只雄性大鼠随机分为假手术组、缺血再灌注组(R／I)、后适应组(Post)、乳酸组(Lac)、低剂量依达拉奉组(Eda)、乳酸+低剂量依达拉奉组(Lac+Eda)六组。建立急性心肌梗死再灌注模型,在再灌注即刻使用微量注射器根据不同组别,在梗死边缘注射不同药物或生理盐水。测定再灌注10min后右心房血浆pH值,在不同时间点处死大鼠分别测定心肌凋亡、坏死、梗死面积和凋亡途径分子的表达。结果Lac和Lac+Eda组右心房血浆pH值较Post组相似(P>0.05)；Lac+Eda组再灌注1h和6h后线粒体吸光度较Post组相似(P>0.05),而Lac组在再灌注6h后期线粒体吸光度显著低于Post组(P<0.05)。Eda和Lac+Eda组MDA和SOD含量均与Post组相似(P>0.05)。Lac+Eda组较R／I组可显著改善血流动力学,减轻细胞凋亡和坏死,限制心肌梗死面积,同时伴随各凋亡途径分子表达显著减少(P<0.05)；且同Post组相比上述指标均无明显差异(P>0.05)。Lac和Eda组在上述指标方面较R／I组部分改善,但同Post组相比仍有明显差异(P<0.05)。结论局部注射乳酸能较好模拟后适应带来的酸中毒延长,局部注射低剂量依达拉奉能较好模拟后适应开始时合成的适量氧自由基；乳酸和低剂量依达拉奉联合使用可较好模拟后适应上游触发因子,有效减轻再灌注损伤。更多还原

【Abstract】 Part One:The Change of Phospho-p38/JNK and Its Influence on Myocardial Reperfusion Injury in Mechanical PostconditioningObjective:Study the change of Phospho-p38/JNK MAPK and its influence on myocardial reperfusion injury in mechanical postconditioning. Methods:70 rats were randomly divided into 7 groups:sham, reperfusion/injury(R/I), postconditioning (Post), SP600125 (SP), SB203580 (SB), Anisomycin+Post (Ani+Post) and Anisomycin (Ani). Results:Compared with R/I group, the level of serum marker, apoptotic index, infarct size, the expression of molecules leading to apoptosis in SP and SB group were all significantly lower (P< 0.05). The expression of P-JNK in SP group was lower than R/I group (P<0.05) and similar with Post group (P>0.05). The expression of P-p38 in SB group was lower than R/I group (P<0.05) and similar with Post group (P>0.05). On the other hand Ani+Post group lost cardioprotection partly, all variables in Ani group were similar with those in R/I group (P>0.05). Conclusion:Postconditioning could inhibit phosphorylation of p38/JNK MAPK, through which it attenuates cardiomyocyte apoptosis by both death receptor and mitochondria pathway.Part Two:Improved Algorithm-Gradual Increased Reperfusion in Postconditioning Plays Better Role in CardioprotectionObjective:Study cardioprotection of different postconditioning algorithm, and investigate the mechanism in the process. Methods:60 rats were randomly divided into 5 groups:sham, reperfusion/injury(R/I), postconditioning (Post), gradual decreased reperfusion (GDR), equal reperfusion(ER), and gradual increased reperfusion (GIR). Results:Compared with R/I group, the level of homodynamic variables, serum marker of myocardium, apoptotic index, infarct size, the expression molecules leading to apoptosis in GDR, ER and GIR group were lower significantly (P<0.05). The difference of GIR was the most, and then was ER and the last was GDR group. Compared with ER group, except for infarct size was similar (P>0.05), variables in GDR group were all much better (P <0.05). Conclusion:Gradual increased reperfusion algorithm of postconditioning could relieve reperfusion injury more significantly, this is probably because it could activate ERK and inhibit p38/JNK MAPK more seriously.Part Three:Pharmacological Postconditioning with Lactic Acid and Low Dose of Edaravone could Attenuate Myocardial Reperfusion InjuryObjective:The aim of this study was to test the hypothesis that pharmacological postconditioning with lactic acid and low dose of edaravone could mimic the upper trigger of mechanical postconditioning and relieve reperfusion injury. Methods:108 rats were randomly divided into 6 groups:sham, reperfusion/injury(R/I), postconditioning (Post), lactic acid (Lac), low dose of edaravone (Eda), and lactic acid+low dose of edaravone edaravone (Lac+Eda).Results:Compared with Post group, pH of blood from right atrium in Lac and Lac+Eda group were similar (P>0.05); and the level of MDA and SOD in Eda and Lac+Eda group were also similar (P>0.05). The level of homodynamic variables, serum marker of myocardium, apoptotic index, infarct size, the expression molecules leading to apoptosis in Lac+Eda group were all similar with Post group(P>0.05). Lac and Eda group could mimic the cardioprotection partly.Conclusion:Lactic acid injected in ischemic myocardium at the onset of reperfusion could mimic the prolonged local acidosis in postconditioning, and pharmacological postconditioning with lactic acid and low dose of edaravone could mimic the upper trigger of mechanical postconditioning and attenuate myocardial reperfusion injury.更多还原