Toll样受体4/核转录因子-κB信号通路在 猪心肺复苏后心肌功能障碍中的作用

Role of Toll like receptor 4/nuclear factor-κB signal pathway on the pathogenesis of acute myocardial dysfunction after cardiopulmonary resuscitation in pigs

目的：探讨Toll样受体4/核转录因子-κB（TLR4/NF-κB）信号通路在心搏骤停-心肺复苏（CA-CPR）猪心肌功能障碍中的作用。方法按照随机数字表法将26只猪分为假手术组（Sham，n＝6）、CA-CPR12h组（n＝10）和CA-CPR24h组（n＝10）。采用心内膜电刺激方法致颤，8min后给予CPR；Sham组仅进行麻醉、气管插管。观察各组动物复苏前后平均动脉压（MAP）、心排血量（CO）等血流动力学变化及心肌细胞形态学及超微结构改变；采用酶联免疫吸附试验（ELISA）检测血清肿瘤坏死因子-α（TNF-α）和白细胞介素-6（IL-6）含量；采用蛋白质免疫印迹试验（WesternBlot）检测心肌细胞TLR4/NF-κB蛋白表达；采用实时荧光定量反转录-聚合酶链反应（RT-PCR）检测心肌细胞TLR4/NF-κBmRNA表达。结果 CA-CPR组猪血流动力学紊乱，心肌细胞严重损伤。与Sham组比较，CA-CPR12h和24h组猪自主循环恢复（ROSC）0.5h血清TNF-α明显升高（pg/L：62.49±6.66、48.39±2.37比10.75±0.74，均P＜0.05），2h达高峰（pg/L：70.93±5.51、66.03±2.60比10.87±0.91，均P＜0.05）后逐渐下降；ROSC0.5h血清IL-6显著高于Sham组（pg/L：14.42±1.99、11.23±1.12比8.75±0.74，均P＜0.05），12h达高峰（pg/L：36.50±2.91、38.15±1.26比8.88±0.62，均P＜0.05）后逐渐下降。CA-CPR12h和24h组猪心肌细胞TLR4和NF-κB的蛋白表达较Sham组明显增加〔TLR4蛋白（灰度值）：0.11±0.03、0.24±0.05比0.05±0.02，NF-κB蛋白（灰度值）：0.27±0.04、0.24±0.03比0.09±0.02，均P＜0.05〕，TLR4mRNA表达分别为Sham组的（9.93±1.07）倍和（9.21±1.27）倍，NF-κBmRNA表达分别为Sham组的（4.44±0.96）倍和（6.09±0.81）倍（均P＜0.01）。结论 TLR4/NF-κB信号通路的激活可能是CA-CPR后心肌功能障碍的病理机制之一。

Objective To investigate the role of Toll like receptor 4/nuclear factor-κB （TLR4/NF-κB） signal pathway on myocardial dysfunction after cardiac arrest-cardiopulmonary resuscitation （CA-CPR） in animal model. Methods Twenty-six pigs were randomly divided into sham group （n = 6）, CA-CPR 12 hours group （n = 10） and CA-CPR 24 hours group （n = 10）. The model of CA-CPR was reproduced by endocardial electrical stimulation for 8 minutes followed by CPR, and the pigs in sham group were only given anesthesia and tracheal intubation. The changes in hemodynamics including mean arterial pressure （MAP） and cardiac output （CO）, as well as morphology and ultrastructure of myocardial cells were observed before and after CPR. The levels of serum tumor necrosis factor-α （TNF-α） and interleukin-6 （IL-6） were determined by enzyme linked immunosorbent assay （ELISA）, and protein and mRNA expressions of TLR4/NF-κB in the myocardium were determined by Western Blot and real-time fluorescence quantitative reverse transcription-polymerase chain reaction （RT-PCR）, respectively. Results Hemodynamic disturbance and myocardial serious injury were observed in CA-CPR groups. Compared with sham group, the levels of serum TNF-α were markedly increased 0.5 hour after return of spontaneous circulation （ROSC） in CA-CPR 12 hours and 24 hours groups （pg/L： 62.49±6.66, 48.39±2.37 vs. 10.75±0.74, both P 〈 0.05）, and peaked at 2 hours （pg/L： 70.93±5.51, 66.03±2.60 vs. 10.87±0.91, both P 〈 0.05） followed by a gradual decline. The levels of serum IL-6 at 0.5 hours after ROSC in CA-CPR 12 hours and 24 hours groups were markedly higher than those of sham group （pg/L： 14.42±1.99, 11.23±1.12 vs. 8.75±0.74, both P 〈 0.05）, and peaked at 12 hours （pg/L： 36.50±2.91, 38.15±1.26 vs. 8.88±0.62, both P 〈 0.05） followed by a gradual decline. The protein expressions of TLR4 and NF-κB in the myocardium were significantly increased in CA-CPR 12 hours and 24 hours groups as compared with sham group [TLR4 protein （gray value）： 0.11±0.03, 0.24±0.05 vs. 0.05±0.02; NF-κB protein （gray value）： 0.27±0.04, 0.24±0.03 vs. 0.09±0.02, all P 〈 0.05]. The mRNA levels of TLR4 in CA-CPR 12 hours and 24 hours groups were increased by approximately （9.93±1.07） folds and （9.21±1.27） folds of sham group respectively, and NF-κB mRNA expressions were increased by （4.44±0.96） folds and （6.09±0.81） folds of sham group respectively （all P 〈 0.01）. Conclusion Activation of TLR4/NF-κB signal pathway may be one of the main pathological mechanisms of post resuscitation myocardial injury in a porcine model of CA-CPR.