Wang X, Bai J, Xue Q, Song X F, Qiu C M, Li X C, Pei H F
Department of Cardiology, Chengdu Military General Hospital, Chengdu 610083, China.
Zhonghua Xin Xue Guan Bing Za Zhi. 2016 Feb;44(2):156-60. doi: 10.3760/cma.j.issn.0253-3758.2016.02.014.
To test the effects of TNF-α inhibitor Etanercept on myocardial ischemia/reperfusion (MI/R) injury in posttraumatic mice, and explore related mechanisms.
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 Notch1 intracellular domains (Notch1 ICD) expressions were determined by Western blot. Chemiluminescence was used to assess myocardial superoxide anion content.
(1) Compared to vehicle group, Etanercept treatment significantly reduced cTnI 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).
TNF-α inhibitor Etanercept can alleviate MI/R injury after trauma by reducing myocardial oxidative/nitrative stress via activating Notch1 signaling pathway.
检测肿瘤坏死因子-α（TNF-α）抑制剂依那西普对创伤后小鼠心肌缺血/再灌注（MI/R）损伤的影响，并探讨相关机制。
采用Noble-Collip鼓建立创伤小鼠模型。创伤后5天，通过心肌内注射Notch1小干扰RNA（siRNA）或乱序siRNA（20μg）敲低Notch1。创伤后7天，对小鼠进行MI/R（缺血30分钟后再灌注）。同样进行假手术，不结扎冠状动脉。再灌注前10分钟，小鼠腹腔注射依那西普（8mg/kg）。采用酶联免疫吸附测定（ELISA）检测血浆TNF-α、肌钙蛋白I（cTnI）水平及心肌硝基酪氨酸含量。再灌注24小时后，通过超声心动图测量左心室射血分数（LVEF）。采用伊文思蓝/2,3,5-氯化三苯基四氮唑（TTC）双重染色法测定梗死面积。使用caspase-3试剂盒检测心肌caspase-3活性。采用蛋白质免疫印迹法检测心肌TNF-α和Notch1细胞内结构域（Notch1 ICD）表达。采用化学发光法评估心肌超氧阴离子含量。
（1）与溶剂组相比，依那西普治疗显著降低了cTnI含量、梗死面积和caspase-3活性（均P<0.01），同时明显提高了LVEF（P<0.01）。（2）依那西普治疗还显著降低了血浆和心肌TNF-α含量（P<0.01），而显著增加了心肌Notch1 ICD含量（P<0.05）。（3）与乱序siRNA组相比，Notch1缺陷显著增加了cTnI含量、梗死面积和caspase-3活性（P<0.05），而明显降低了LVEF（P<0.05）。（4）依那西普显著降低了心肌超氧阴离子和硝基酪氨酸含量（P<0.01），Notch1下调可逆转这一作用（P<0.05）。
TNF-α抑制剂依那西普可通过激活Notch1信号通路减轻心肌氧化/硝化应激，从而减轻创伤后MI/R损伤。
