山莨菪碱对心搏骤停复苏猪心室肌缝隙连接蛋白43表达的影响

Effect of anisodamine on myocardial connexin 43 expression in pig after resuscitation from cardiac arrest

目的：探讨山莨菪碱对心搏骤停猪复苏后心室肌缝隙连接蛋白43（Cx43）表达的影响。方法选择健康家猪进行实验，按随机数字表法分为假手术组、肾上腺素复苏组（对照组）与山莨菪碱复苏组（实验组，复苏时加用山莨菪碱0.4 mg/kg），每组5只。电刺激诱导心室纤颤（室颤）并维持8 min后开始心肺复苏（CPR），复苏成功后24 h留取左心室样本。经免疫荧光法观察心室肌Cx43的表达与分布，反转录-聚合酶链反应（RT-PCR）检测Cx43 mRNA表达，蛋白质免疫印迹试验（Western Blot）检测Cx43、磷酸化Cx43（p-Cx43）蛋白表达。结果假手术组心室肌Cx43阳性表达分布均匀，多位于心肌细胞的端-端连接部位，少数位于细胞的侧-侧连接部位；对照组Cx43阳性表达较假手术组显著减少，信号强度明显下降（4.35±2.10比10.02±3.66， P＜0.01）；实验组Cx43阳性表达在端-端连接与侧-侧连接分布较规则，信号强度明显高于对照组（7.91±2.54比4.35±2.10，P＜0.05），但明显低于假手术组（7.91±2.54比10.02±3.66，P＜0.05）。对照组和实验组Cx43的mRNA与蛋白表达均较假手术组显著降低〔Cx43 mRNA（A值）：0.32±0.05、0.32±0.03比0.48±0.07；Cx43蛋白（A值）：0.43±0.03、0.50±0.07比0.65±0.04，均P＜0.01〕，实验组与对照组比较差异均无统计学意义（均P＞0.05）。对照组p-Cx43蛋白表达明显低于假手术组（A值：0.22±0.03比0.37±0.06，P＜0.01）；实验组p-Cx43蛋白表达高于对照组（A值：0.29±0.07比0.22±0.03，P＜0.01），而与假手术组比较差异无统计学意义（P＞0.05）。假手术组、对照组、实验组p-Cx43/Cx43比值差异无统计学意义（0.57±0.09、0.51±0.05、0.58±0.06，均P＞0.05）。结论山莨菪碱可以纠正心搏骤停猪复苏后心室肌Cx43表达的异常，维护正常的心脏传导功能。

ObjectiveTo investigate the effect of anisodamine on the expression of connexin 43 （Cx43） in swine ventricular myocardium after resuscitation from cardiac arrest.Methods The experiment was conducted on healthy pigs, and they were randomly divided into three groups, namely sham group, epinephrine group （control group） and anisodamine group （experimental group, animals were resuscitated combined with injection of 0.4 mg/kg of anisodamine）, with 5 pigs in each group. Model of ventricular fibrillation was reproduced by alternating current challenge, and cardiopulmonary resuscitation （CPR） was performed 8 minutes after cardiac arrest. Left ventricular myocardium was harvested at 24 hours after restoration of spontaneous circulation. The expression and distribution of Cx43 were observed by immunofluorescence, Cx43 mRNA expression was assessed with reverse transcription-polymerase chain reaction （RT-PCR）, and the protein expressions of Cx43 and phosphorylation of Cx43 （p-Cx43） were analyzed by Western Blot.Results The positive expression of Cx43 in ventricular muscle was distributed uniformly, mostly at the end-to-end linkage of myocardial cells, with a few side-to-side linkage in sham group. The positive expression of Cx43 in control group was significantly weaker than that in the sham group, and the signal intensity was significantly declined （4.35± 2.10 vs. 10.02± 3.66,P〈 0.01）. The positive expression of Cx43 at the end-to-end linkage and side-to-side linkage was irregular in experimental group, and the signal intensity was obviously higher than that in the control group （7.91±2.54 vs. 4.35± 2.10,P〈 0.05）, but it was significantly weaker than that in the sham group （7.91±2.54 vs. 10.02±3.66,P〈 0.05）. For control group and experimental group, the Cx43 mRNA and protein expressions were significantly lower than those of the sham group [Cx43 mRNA （A value）： 0.32±0.05, 0.32±0.03 vs. 0.48±0.07; Cx43 protein （A value）： 0.43±0.03, 0.50±0.07 vs. 0.65±0.04, allP〈 0.01], and there were no significant differences between experimental group and control group （allP〉 0.05）. The p-Cx43 protein expression of control group was significantly lower than that of the sham group （A value： 0.22±0.03 vs. 0.37±0.06,P〈 0.01）, and it was significantly higher in the experiment group than that in the control group （A value： 0.29±0.07 vs. 0.22±0.03, P〈 0.01）, but there was no significant difference with the sham group （P〉 0.05）. No significant difference in p-Cx43/Cx43 was found among sham, control, and experiment groups （0.57±0.09, 0.51±0.05, 0.58±0.06, all P〉 0.05）.ConclusionAnisodamine can improve the abnormal expression of Cx43 in ventricular muscle of pigs with cardiac arrest, which may be related to the protection effect of anisodamine on cardiac conduction.