肿瘤坏死因子α抑制剂通过激活Notch1信号通路减轻创伤小鼠心肌再灌注损伤  

Tumor necrosis factor-α inhibitor protects against myocardial ischemia/reperfusion injury via Notch1 mediated inhibition of oxidative/nitrative stress in traumatic mice

作　　者：汪雄[1] 白婧[3] 薛强[3] 宋晓峰[2] 邱琛茗 李秀川[1] 裴海峰[1] 

机构地区：[1]成都军区总医院心内科,610083 [2]成都军区总医院医疗保健科,610083 [3]第四军医大学西京医院心内科

出　　处：《中华心血管病杂志》2016年第2期156-160,共5页Chinese Journal of Cardiology

基　　金：基金项目：国家自然科学基金（81500208）;全军医学科技青年培育项目（14QNP050）;四川省科技支持计划（2015JY0277）

摘　　要：目的 研究肿瘤坏死因子α（TNF-α）抑制剂对创伤后心肌缺血再灌注（ML/R）损伤的影响,探讨跨膜蛋白受体Notch1在其中的作用.方法 健康雄性c57小鼠,10～ 12周龄.建立创伤合并MI/R小鼠模型后分为5组,即溶剂对照组、依那西普组、乱码RNA对照组、Notch1 siRNA组和依那西普＋ Notch1 siRNA组,每组8只小鼠.采用Noble-Collip鼓建立创伤小鼠模型.创伤5d后,通过心肌点注射特异性siRNA（20 μg）建立Notch1降低小鼠模型.创伤7d后,建立MI/R小鼠模型,即麻醉小鼠后经左胸前第4、5肋间暴露心脏,结扎冠状动脉左前降支,缺血30 min后,解开结扎线实现再灌注.需要依那西普干预的小鼠均是在再灌注前10 min腹腔注射依那西普8 mg/kg.ELISA法检测血浆中TNF-α和肌钙蛋白I（cTnI）含量以及心肌组织中硝基酪氨酸水平.再灌注24 h后,利用小动物超声系统测定小鼠左心室射血分数（LVEF）.伊文思蓝和2,3,5-氯化三苯基四氮唑（TTC）双染缺血再灌注心肌组织,计算梗死心肌面积比值.采用caspase-3试剂盒测定心肌caspase-3活性.蛋白印迹法测定心肌TNF-α蛋白和Notch1胞内片段（Notch1 ICD）含量.化学发光法测定心肌超氧阴离子含量.结果 （1）依那西普组和溶剂对照组小鼠cTnI、LVEF、心肌梗死面积和心肌caspase-3活性的检测结果：依那西普组小鼠cTnI的含量明显低于溶剂对照组（P＜0.01）,LVEF明显高于溶剂对照组（P＜0.01）,心肌梗死面积明显低于溶剂对照组（P＜0.01）.依那西普组小鼠心肌组织中caspase-3活性明显低于溶剂对照组（P＜0.01）.（2）依那西普组和溶剂对照组小鼠心肌中TNF-α蛋白和Notch1 ICD含量及血浆TNF-α浓度的检测结果：依那西普组小鼠心肌中TNF-α蛋白含量明显低于溶剂对照组（P＜0.01）,且血浆中TNF-α浓度亦明显低于溶剂对照组（P＜0.01）.而依那西普组小鼠心肌中Notch1 ICD含量则明显高于溶剂对照组（P＜0.Objective To test the effects of TNF-α inhibitor Etanercept on myocardial ischemia/ reperfusion （MI/R） injury in posttraumatic mice,and explore related mechanisms.Methods Traumatic mouse model was established with Noble-Collip drum.Five days after trauma,Notch1 was knocked down by intramyocardial injection of Notch1 small interfering RNAs （siRNA） or scrambled siRNA （20 μg）.Seven days after trauma,mice were subjected to MI/R （30 minutes ischemia followed by reperfusion）.Sham operation was similarly performed without coronary artery ligation.Ten minutes before reperfusion,mice received Etanercept （8 mg/kg,i.p.）.ELISA was used to detect plasma levels of TNF-α and troponin I （cTnI） and myocardial nitrotyrosine content.Twenty-four hours after reperfusion,left ventricular ejection fraction （LVEF） was measured by echocardiography.Infarct size was determined by Evans blue/2,3,5-triphenyl tetrazolium chloride （TTC） double staining.Cardiac caspase-3 activity was detected using a caspase-3 kit.Myocardial TNF-α and Notchl intracellular domains （Notch1 ICD） expressions were determined by Western blot.Chemiluminescence was used to assess myocardial superoxide anion content.Results （1） Compared to vehicle group,Etanercept treatment significantly reduced cTnl content,infarct size and caspase-3 activity （all P 〈0.01）,while obviously increased LVEF （P 〈0.01）.（2） Etanercept treatment also significantly reduced plasma and myocardial TNF-α contents （P 〈 0.01）,whereas markedly increased myocardial Notch1 ICD content （P 〈0.05）.（3） Compared to scrambled siRNA group,Notch1 deficiency significantly increased cTnI content,infarct size and caspase-3 activity （P 〈 0.05）,whereas obviously reduced LVEF （P 〈 0.05）.（4） Etanercept significantly reduced myocardial superoxide anion and nitrotyrosine content （P 〈 0.01）,which was reversed by downregulation of Notch1 （P 〈 0.05）.Conclusions TNF-α inhibitor Etanercept can alleviate MI/R injury after

关 键 词：心肌再灌注损伤 肿瘤坏死因子Α 受体 NOTCHL 

分 类 号：R542.2[医药卫生—心血管疾病]