心肌顿抑中的氧化应激作用及氨基胍干预的效果

Role of Oxidative Stress in the Pathogenesis of Myocardial Stunning in Dogs and Effects of Aminoguanidine

背景氧化应激的超氧阴离子(O-.2)可与细胞一氧化氮合酶(NOS)释出的一氧化氮(NO)结合,生成过氧亚硝酸阴离子(ONOO-)。ONOO-是氧化应激各种产物[如O2.-、H2O2、羟自由基(.OH)等]之一,统称为反应性氧族(ROS)。ROS在心肌顿抑中的作用研究不多,氨基胍作为NOS抑制剂对ROS的作用也不清楚。目的探讨ONOO-在心肌顿抑发生中的作用和机制以及氨基胍的干预作用。方法24条雄性杂种犬,随机分为4组:1)短顿抑组[左前降支冠状动脉(LAD)阻断15min/再灌注120min];2)长顿抑组(LAD阻断60min/再灌注120min);3)氨基胍组[LAD阻断60min/再灌注120min加一氧化氮合酶抑制剂氨基胍(100mg/kg)干预];4)假手术组。在不同观察时间点测定超声心功能和冠状静脉窦血浆NO浓度。实验完毕后心肌标本行电镜检查,并行硝基酪氨酸免疫组化检查以证实是否有ONOO-生成。结果1)LAD结扎后缺血心肌节段收缩期增厚百分率和左室射血分数显著下降,缺血心肌节段表现为矛盾运动;再灌注开始后心肌节段收缩功能和左室射血分数呈进行性改善,短顿抑组和氨基胍组心功能的恢复快于长顿抑组。2)短顿抑组和长顿抑组再灌注期血浆NO浓度明显升高,氨基胍组再灌注期血浆NO浓度无显著升高。3)短顿抑组顿抑心肌硝基酪氨酸免疫组化染色见阳性染色的心肌细胞灶;长顿抑组见较大、较多的强阳性染色心肌细胞灶,主要是胞浆尤其横纹处染色较深;氨基胍组顿抑心肌偶见心肌细胞弱阳性染色。4)透射电镜观察发现,短顿抑组心肌细胞偶见线粒体轻度脱颗粒;长顿抑组心肌细胞部分肌丝断裂,收缩带溶解,线粒体肿胀、脱颗粒,胞质水肿;氨基胍组心肌超微结构保存良好。结论1)顿抑心肌生成NO增多伴ONOO-形成;2)ONOO-主要攻击的蛋白质对象是肌丝上的蛋白质;3)氨基胍抑制顿抑心肌过多的NO生成,显著减少ONOO-形成,并对顿抑心肌的超微结构和功能有明显保护作用。

Back ground Superoxide anion produced by oxidative stress combined with nitric oxide（NO）released by cellular NO synthase（NOS）generates peroxynitrite（ONOO^-）which was one of reactive oxygen species.Objective To study the role of peroxynitrite in the pathogenesis of myocardial stunning（MS）and the effects of aminoguanidine（AMD）.Methods Twenty-four dogs were randomly divided into four groups：（1）Short-time myocardical stunning（MS）group：the animals underwent 15 min occlusion of left anterior descending coronary artery（LAD）,followed by 120 min of reperfusion.（2）Long-time MS group：60 min of LAD occlusion,followed by 120 min of reperfusion.（3）AMD group：Dogs were subjected to the same ischemia/reperfusion management as Long-MS group,but AMD was administrated（total 100 mg/kg）.（4）Sham-operation group.Left ventricular function was evaluated by echocardiography and blood samples were taken from coronary venous sinus for the examination of NO at different time points.The stunned myocardium was examined immunohistochemically and electronic microscope.Results（1）Systolic thickening of stunned myocardium region and left ventricular ejection fraction were markedly declined during LAD occlusion in the 3 experimental groups,but progressively improved with the time of reperfusion.The improvement was faster in short-MS and AMD groups than in long-MS group.（2）Plasma NO concentrations in coronary venous sinus were greatly increased during reperfusion in short-and long-MS group,while no significant elevation was found in AMD group.（3）A marked increase in the immunoreactivity to nitrotyrosine,a specific ＂footprint＂ of peroxynitrite,was shown in the stunned myocardium both in short-and long-MS group,with larger and stronger positive foci in long-MS group.The positive staining was located in myocardial cytoplasm,especially at myofibrils and contractile bands.Treatment with AMD significantly reduced the nitrotyrosine staining in the stunned myocardium.（4）No ultrastructural changes of myocardial cells were noted in stunned myocardium in short-MS group except a slight loss of mitochondrial granules.On the contrary,myocyte edema,myofilament fractures,granule loss and swelling of mitochondrias were significant in long-MS group.AMD significantly ameliorated the ultrastructural abnormalities.Conclusion Myocardial stunning was associated peroxynitrite with production which mainly deteriorate the contractile proteins of myofibrils.AMD significantly inhibit the peroxynitrite formation leading to attenuation of the ultrastructural and functional injuries of stunned myocardium.