【作者】 张楠；

【导师】 张建新；

【作者基本信息】 河北医科大学 ， 药理学， 2014， 硕士

【摘要】 心肌缺血损伤，从而继发的心律失常和收缩功能减退是导致临床上各种类型冠状动脉粥样硬化性心脏病患者血流动力学不稳定、循环衰竭、甚至猝死的重要原因。但心肌缺血后，其损伤和抗损伤的机制，至今尚未完全了解。硫化氢(hydrogen sulfide,H2S)—作为继一氧化氮(nitric oxide,NO)和一氧化碳(carbon monoxide,CO)后的第三种重要的气体化学递质和细胞内信号分子，在哺乳动物循环系统内，主要由胱硫醚-γ-裂解酶(cystathionine-γ-lyase，CSE)催化L-半胱氨酸代谢而产生。近年的研究已表明，内源性H2S能够通过抑制炎症反应、减轻钙超载、减少细胞凋亡、缩小心肌梗死面积，挽救存活心肌，恢复心肌功能，且能抑制血管平滑肌细胞增生，逆转血管重构，促进受损内皮细胞修复。然而，其对急性心肌缺血损伤后线粒体功能的影响尚未见报道。本研究通过建立离体大鼠急性心肌缺血损伤模型，观察大鼠在不同时间内源性H2S含量和CSE活性的变化，在此基础上给予H2S的供体—硫氢化钠（sodium hydrosulfide，NaHS），观察其对心肌缺血损伤后线粒体功能的影响。第一部分离体大鼠心脏缺血心肌内源性硫化氢/胱硫醚-γ-裂解酶体系的变化目的：观察离体大鼠心脏急性心肌缺血过程中不同时间内源性H2S含量和CSE活性的变化。方法：健康雄性SD大鼠(250-300g)共160只，随机分为6组：①、假手术组；②、缺血30min组；③、缺血1h组；④、缺血2h组；⑤、缺血3h组；⑥、缺血4h组。假手术组共80只(不同时间点各16只)，其余各组各16只。其中80只观察计算梗死体积，另80只测定其他指标。假手术组只穿线不结扎，缺血组结扎左冠状动脉前降支。分别于30min、1h、2h、3h、4h应用Powerlab/8s多导生理仪记录各组大鼠左室发展压(LVDP)、左室压力变化速率(±dp/dtmax)，测定冠脉流量(CF)；分别采用去蛋白法和亚甲基蓝法检测心肌组织中H2S含量和CSE的活性；以Evan′s蓝和TTC双染法对心肌组织切片染色，观察并计算心肌梗死体积。结果：1各组间血流动力学指标在灌流平衡期间无明显差异，与相应假手术组大鼠比较，缺血30min、1h、2h、3h、4h组大鼠LVDP、±dp/dtmax、CF明显降低（P<0.01）。2假手术组大鼠心肌组织中H2S含量和CSE活性在灌流平衡后30min～4h之间无明显变化。与相应假手术组大鼠比较，缺血30min时大鼠心肌组织中H2S含量和CSE活性无明显变化，但随着缺血时间的延长，缺血1h、2h、3h、4h大鼠心肌组织H2S含量和CSE活性均明显降低(P<0.05或P<0.01)。3与相应假手术组大鼠比较，缺血30min时大鼠心肌组织无明显梗死灶，缺血1h时可见明显梗死灶(P<0.01)，随着缺血时间的延长，缺血2h、3h、4h时梗死体积明显增大(P<0.01)。结论：心肌缺血2h后，随缺血时间延长，心肌组织中H2S含量和CSE活性明显降低，心肌组织梗死体积逐渐增大，提示H2S与CSE参与了离体大鼠急性心肌缺血损伤。第二部分硫化氢对离体大鼠心脏急性心肌缺血损伤后线粒体功能的影响目的：观察H2S对离体大鼠急性心肌缺血损伤后线粒体功能的影响。方法：健康成年雄性SD大鼠(250-300g)共80只，随机分为：①、假手术组；②、缺血模型组；③、缺血+NaHS低剂量组；④、缺血+NaHS中剂量组；⑤、缺血+NaHS高剂量组。每组16只，其中40只观察计算梗死体积，另40只测定其他指标。假手术组只穿线不结扎，缺血模型组结扎左冠状动脉前降支。NaHS低、中、高剂量组分别于心肌急性缺血2h时更换为5μmol/L，10μmol/L，20μmol/L的NaHS灌流液。应用Powerlab/8s多导生理仪记录各组大鼠左室发展压（LVDP）、左室压力变化速率（±dp/dtmax），测定冠脉流量(CF)。各组大鼠均于缺血4h时，检测心肌组织中H2S含量及CSE活性；观察给予NaHS后心肌梗死体积的变化；透射电镜观察心肌细胞超微结构变化。差速离心法提取心肌线粒体后，检测线粒体活力、膜肿胀度，以及线粒体总ATP酶、谷胱甘肽过氧化物酶(GSH-PX)、超氧化物岐化酶(SOD)和丙二醛(MDA)含量。结果：1与假手术组比较，模型组大鼠心功能指标LVDP、±dp/dtmax、CF明显降低（P<0.01）。与模型组比较，NaHS低、中、高剂量组大鼠心功能指标LVDP、±dp/dtmax、CF明显升高(P<0.05或P<0.01)。2与假手术组比较，模型组大鼠心肌组织中H2S含量和CSE活性明显降低（P<0.01）；与模型组比较，NaHS低、中、高剂量组大鼠心肌组织中H2S含量和CSE活性均明显升高（P<0.05或P<0.01）。3与假手术组比较，模型组大鼠心肌梗死明显（P<0.01）；与模型组比较，NaHS中、高剂量组大鼠心肌梗死体积明显减小（P<0.01）。4假手术组大鼠心肌细胞结构正常，心肌纤维排列整齐，嵴清晰，无水肿、空泡化；模型组大鼠心肌纤维排列紊乱，线粒体大量肿胀变形，嵴数量明显减少，断裂、溶解成空泡状，细胞核内染色体核边聚；与模型组相比，NaHS低、中、高剂量组大鼠心肌细胞肌丝、胞核、线粒体等的损伤程度明显减轻，且有剂量依赖关系。5与假手术组大鼠比较，模型组大鼠心肌线粒体膜肿胀，线粒体活力下降，心肌线粒体总ATP酶、GSH-PX、SOD活性明显降低(P<0.01)，MDA含量明显升高(P<0.01)；与模型组大鼠比较，缺血+NaHS低、中、高剂量组大鼠线粒体膜肿胀度减轻，活力有所恢复，心肌线粒体总ATP酶、GSH-PX、SOD活性升高，MDA含量降低(P<0.05或P<0.01)。结论：应用离体大鼠急性心肌缺血模型，观察了H2S供体NaHS对心肌缺血损伤及线粒体功能的影响，结果表明，NaHS可使H2S生成增加，CSE活性增强，明显缩小心肌梗死体积，升高线粒体活力，降低线粒体膜肿胀度，增强线粒体总ATP酶、GSH-PX、SOD活性，降低MDA含量，明显减轻心肌缺血组织损伤。更多还原

【Abstract】 Myocardial ischemia injury and the secondary arrhythmia, systolicfunction impairment are the reasons of various clinical types of coronaryatherosclerosis heart disease patients with hemodynamic instability,circulation failure, and even death. But after myocardial ischemia, themechanism of injury and resistance, is not yet fully understood. In recent years,as following the nitric oxide(NO) and carbon monoxide(CO) the third kind ofimportant chemical neurotransmitter and intracellular signalingmolecule-hydrogen sulfide (H2S),is mainly producted from l-cysteine andcystathionine-γ-lyase (CSE) within the mammalian circulatory system. It hasbeen shown that endogenous H2S can inhibit inflammation, reduce calciumoverload and cell apoptosis, protect mitochondria and other kinds of ways toreduce myocardial infarction area, save the survival myocardium, restorecardiac function, and can inhibit vascular smooth muscle cell proliferation,reverse vascular remodeling, promote restoration of the damaged endothelialcells. However, its relationship with the pathogenesis of cardiovasculardisease remains to be further studied. In the present experiment, we observedthe changes of H2S/CSE system in acute myocardial ischemia,and the effect ofH2S on myocardial ischemia injury and explored the possible mechanisms.Part1Change of endogenous hydrogen sulfide(H2S)/cystathionine-γ-lyase (CSE) in isolated ischemic heartsObjective: To observe the changes of H2S/CSE in isolated ischemichearts in rats.Methods:160male SD rats, weighing250-300g, were randomly dividedinto six groups.①sham group(n=80),②ischemia30min group(n=16),③ischemia1h group(n=16),④ischemia2h group(n=16),⑤ischemia3h group(n=16),⑥ischemia4h group(n=16). The rats in the sham group wereonly threaded without ligation, and the left anterior descending coronary arterywas ligated in rats of the ischemia group. The hemodynamic parameters, suchas the left ventricular developed pressure (LVDP),±dp/dtmaxand coronaryarterial flow(CF) were respectively recorded to evaluate the cardiac function.The content of H2S and the activity of CSE in cardiac tissue were respectivelydetermined. The infarct volumes of hearts were determined by dual stainingwith Evans-blue and TTC. Ischemic myocardial tissue area and volume wereobserved and calculated by image analysis system.Results:1There were no statistically differences in baseline cardiodynamic dataamong the experimental groups. Compared with those of the sham group,LVDP,±dp/dtmaxand CF were significantly decreased at30min、1h、2h、3h、4h after ischemia(P<0.01).2There were no statistically differences in the content of H2S and theactivity of CSE in cardiac tissue in the sham group from30min to4h afterstabilization. Compared with those of the sham group, there were nostatistically differences in the content of H2S and the activity of CSE incardiac tissue at30min after ischemia. However, during1h to4h afterischemia, the content of H2S and the activity of CSE in cardiac tissue weresignificantly decreased compared with those of the sham group(P<0.05orP<0.01).3Compared with those of the sham group, the infarct volumes weregreatly increased at1h to4h after ischemia (P<0.01).Conclusion: After ischemia for2h, the content of H2S and the activity ofCSE in cardiac tissue were significantly decreased with larger infarct volumes.It could be concluded that H2S and CSE were involved in myocardial ischemiainjury in isolated hearts in rats.Part2Effects of Hydrogen Sulfide on Myocardial Ischemia mitochondrialInjury in RatsObjective: To study the effect of H2S on myocardial ischemia and explore its possible mechanisms from the function of mitochondria in rats.Methods: Eighty male SD rats, weighing250-300g, were randomlydivided into five groups.①sham group(n=16),②model group(n=16),③lowdose group of NaHS(n=16),④middle dose group of NaHS(n=16),⑤high dosegroup of NaHS(n=16). The rats in the sham group were only threaded withoutligation, and the left anterior descending coronary artery was ligated in rats ofthe ischemia group. The normal perfusate was replaced with NaHS perfusate（5μmol/L，10μmol/L，20μmol/L）accordingly in low dose group of NaHS,middle dose group of NaHS and high dose group of NaHS at2h afterischemia. The left ventricular developed pressure (LVDP),±dp/dtmaxandcoronary arterial flow(CF) were respectively recorded to evaluate the cardiacfunction. The content of H2S and the activity of CSE in cardiac tissue wererespectively determined at4h after ischemia. The infarcted volume of thehearts in each group rats were observed and calculated by image analysissystem. The ultrastructural alterations of myocardium were observed byelectric microscope. The mitochondria were prepared by differentialcentrifugation. The swelling and activity of mitochondria were determined.The activities of ATPase， GSH-PXand SOD， and the contents ofmalondialdehyde (MDA) in myocardial mitochondria were respectivelymeasured.Results:1Compared with those of the sham group, LVDP,±dp/dtmax and CFwere significantly decreased in model group (P<0.01). Compared with thoseof the model group, LVDP,±dp/dtmaxand CF were significantly increased inNaHS low, middle and high dose groups (P<0.05or P<0.01).2Compared with those of the sham group, the content of H2S and theactivity of CSE in cardiac tissue were significantly decreased in model group(P<0.01).Compared with those of the model group, the content of H2S andthe activity of CSE in cardiac tissue were significantly increased in NaHS low,middle and high dose groups (P<0.05or P<0.01).3Compared with those of the sham group, the infarct volumes was significantly increased in model group(P<0.01). Compared with those of themodel group, the infarct volumes was significantly decreased in NaHS middleand high dose groups (P<0.01).4The ultrastructure of the myocardial cells exhibited regularmitochondria with uniform size, complete mitochondrial cristae, and intactnuclear membrane in the sham group,however,the myocardial cells werecharacterized by mitochondrial swelling, disappearance or deformation ofmitochondrial cristae, disruption of nuclear membrane, and nuclearcondensation in the model group. Compared with those of the model group,thepathological change in myofilaments, mitochondria and nucleus weresignificantly decreased in NaHS low, middle and high dose groups.5Compared with those of the sham group, the swelling of mitochondriawas markedly increased and the activity of mitochondria was significantlydecreased (P﹤0.01) and the activities of SOD, GSH-PXand ATPase weresignificantly decreased and the content of malondialdehyde (MDA) inmyocardial mitochondria was significantly increased(P﹤0.01) in model group.Compared with those of the model group, the activities of SOD, GSH-Px andATPase and the activity of myocardium mitochondria were significantlyenhanced(P <0.05or P <0.01),the mitochondria swelling was significantlyameliorated and the content of MDA was significantly decreased(P﹤0.01) inNaHS low, middle and high dose groups.Conclusion: It could be concluded that H2S has a beneficial ischemiatissue protection against the ischemia injury in rats by improving the functionof myocardial mitochondria, antagonizing oxidative stress in myocardialmitochondria and protecting mitochondrial structure and redox function,which may be one of the mechanisms of ameliorating ischemia injury.更多还原