中介素抗大鼠缺血再灌注室性心律失常的作用及其机制探讨

Effects of intermedin on ventricular arrhythmia in rats during ischemia and reperfusion

目的观察中介素(IMD)对大鼠缺血再灌注室性心律失常、缝隙连接蛋白43(Cx43)蛋白表达和磷酸化状态的影响,探讨其可能的机制。方法清洁级健康成年雄性Sprague-Dawley(SD)大鼠40只,随机分为4组,每组10只:(1)假手术组,只在冠状动脉左前降支(LAD)下方穿线,不结扎;(2)缺血再灌注组,结扎LAD缺血30min再灌注120 min;(3)IMD组,于缺血前15 min经尾静脉注射IMD(20nmol/kg),结扎LAD缺血30min再灌注120min;(4)Chelerythrine Chloride(CHE)组,于缺血前15min经尾静脉注射IMD(20nmol/kg)和蛋白激酶C(PKC)抑制剂CHE(20nmol/kg),结扎LAD缺血30 min再灌注120 min。监测Ⅱ导联心电图变化,记录缺血再灌注全程的室性早搏(VPC)次数、室性心动过速(VT)总持续时间和心室颤动(VF)总持续时间,并进行室性心律失常评分。采用H-E染色在光学显微镜下观察心肌组织病理损伤情况。应用Western印迹法分析心室肌Cx43蛋白表达及其磷酸化水平。结果缺血再灌注组大鼠在缺血和再灌注期间,VPC次数显著多于假手术组(P<0.05),VT和VF总持续时间均显著长于假手术组(P值均<0.05),室性心律失常评分显著高于假手术组(P<0.05)。IMD组大鼠VF和VT总持续时间均显著短于缺血再灌注组(P值均<0.05),室性心律失常评分显著低于缺血再灌注组(P<0.05),VPC次数与缺血再灌注组的差异无统计学意义(P=0.067)。CHE组大鼠的VT和VF总持续时间均显著长于IMD组(P值均<0.05),室性心律失常评分显著高于IMD组(P<0.05),VPC次数与IMD组的差异无统计学意义(P=0.219)。CHE组室性心律失常各项指标与缺血再灌注组的差异均无统计学意义(P值均>0.05)。心肌H-E染色光学显微镜下可见,假手术组心肌细胞排列整齐,心肌纤维完整,染色均匀,无变性、坏死等改变;缺血再灌注组心肌细胞体积增大,细胞间质肿胀严重,心肌纤维不规则排列,出现大面积纤维断裂、坏死,可见收缩带形成,并有中性粒细胞浸润;IMD组和CHE组与缺血再灌注组相比,组织形态得到明显改善,细胞间质肿胀显著减轻,心肌纤维排列相对规则,有少量中性粒细胞浸润。Western印迹法结果显示,缺血再灌注组的兔抗磷酸化Cx43抗体(p-Cx43)蛋白相对表达量显著低于假手术组(P<0.05);IMD组的p-Cx43蛋白相对表达量显著高于缺血再灌注组(P<0.05);CHE组的pCx43蛋白相对表达量与缺血再灌注组的差异无统计学意义(P>0.05),但显著低于IMD组(P<0.05)。缺血再灌注组的小鼠抗去磷酸化Cx43抗体(Np-Cx43)蛋白相对表达量显著高于假手术组(P<0.05);IMD组的Np-Cx43蛋白相对表达量显著低于缺血再灌注组(P<0.05);CHE组的Np-Cx43蛋白相对表达量显著高于IMD组(P<0.05),与缺血再灌注组的差异无统计学意义(P>0.05)。结论 IMD可通过活化PKC信号通路,调节Cx43蛋白磷酸化状态,发挥抗缺血和再灌注室性心律失常的作用。

Objective To observe the effect of intermedin （IMD） on ventricular arrhythmia and the expression and phosphorylation of connexin 43（Cx43） in rats during ischemia and reperfusion, and then investigate the underlying mechanisms. Methods Forty clean healthy male adult Sprague-Dawley （SD） rats were randomly divided into 4 groups （n = 10）： （1）sham operation group, in which the animals were only subjected to threading under the left anterior descending coronary artery （LAD） without l igation; （2）ischemia and reperfusion group, in which the ischemia was induced by ligation of LAD for 30 minutes and reperfusion by relaxing the thread for 120 minutes; （3）IMD group, in which the animals were treated with 20 nmol/kg IMD through caudal vein prior to ischemia 15 minutes and then subjected to ischemia for 30 minutes and reperfusion for 120 minutes; （3）chelerythrine chloride （CHE, a protein kinase C inhibitor） group; in which the animals were treated with20 nmol/kg IMD and 20 nmol/kg CHE through caudal vein prior to ischemia 15 minutes and then subjected to ischemia for 30 minutes and reperfusion for 120 minutes. We monitored the Ⅱ leading electrocardiogram to record the frequency of ventricular premature contraction （VPC）, the duration of ventricular tachycardia （VT） and ventricular fibrillation （VF） during the whole process of ischemia and reperfusion. The ventricular arrhythmia score was determined. Hematoxylin- Eosin （H-E） staining was used to observe the myocardial injury under the light microscope. The expression and phosphorylation of Cx43 were evaluated by Western blot. Results During ischemia and reperfusion, the frequency of VPC and ventricular arrhythmia score in the ischemia and reperfusion group were significantly higher than those in the sham operation group （both P〈0. 05）, and the duration of VT and VF in the ischemia and reperfusion group were significantly longer than those in the sham operation group （both P〈0.05）. The duration of VT and VF in the IMD group were significantly shorter than those in the ischemia and reperfusion group （both P〈0.05）, and the ventricular arrhythmia score in the IMD group was significantly lower than that in the ischemia and reperfusion group （ P〈0.05）, but there was no significant difference in the frequency of VPC between the two groups （ P = 0. 067）. The duration of VT and VF in the CHE group were significantly longer than those in the IMD group （both P〈0.05）, and the ventricular arrhythmia score in the CHE group was significantly higher than that in the IMD group （ P〈0.05）, but there was no significant difference in the frequency of VPC between the two groups （P = 0. 219）. There was no significant difference in any indexes of ventricular arrhythmias between the CHE group and the ischemia and reperfusion group （all P〈0.05）. The H-E staining showed that myocardial cells in the sham operation group arranged well with intact cardiac muscle fibers, even dyeing but without any degeneration or necrosis; in the ischemia and reperfusion group, the myocardial cell enlarged with mesenchymal dropsy and cardiac muscle fibers arranged irregularly with large area of myocardial fiber fracture and necrosis, and the ischemic contraction band and neutrophil infiltration could be observed; compared with the ischemia and reperfusion group, histo!ogical changes in the IMD group and CHE group were significantly improved, the mesenchymal dropsy was deceased and cardiac muscle fibers arranged relatively regularly with a little of neutrophil infiltration. The relative expression of rabbit anti-phosphorylated Cx43 antibody （p-Cx43） significantly decreased in the ischemia and reperfusion group compared with the sham operation group （ P 〈 0. 05）. The relative expression level of p-Cx43 in the IMD group was significantly higher than that in the ischemia and reperfusion group （P〈0.05）. The relative expression of p-Cx43 in the CHE group was not significantly different from the ischemia and reperfusion group （P〉0. 05）, but significantly decreased when compared with the IMD group （P〈0. 05）. The relative expression of mouse anti-non-phosphorylated Cx43 antibody （Np-Cx43） in the ischemia and reperfusion group was significantly higher than that in the sham operation group （P〈0.05）. The relative expression of Np-Cx43 in the IMD group was significantly lower than that in the ischemia and reperfusion group （P〈0. 05）. The relative Np-Cx43 expression in the CHE group was significantly higher than that in the IMD group （P〈0.05）, but no significant difference was found between the CHE group and the ischemia and reperfusion group （ P 〉 0. 05）. Conclusion Intermedin antagonizes ventricular arrhythmias during myocardial ischemia and reperfusion via regulating the phosphorylation of Cx43 induced by activating PKC pathway.