肿瘤坏死因子-α诱导连接蛋白表达和分布在脓毒症大鼠心房颤动发生中的作用

Role of tumor necrosis factor-α in regulating the expression and distribution of connexin in the development of atrial fibrillation in rats with sepsis

摘要

目的 观察肿瘤坏死因子-α（tumor necrosis factor-α，TNF-α）对脓毒症大鼠连接蛋白43（conenxin 43，Cx43）和连接蛋白40（conenxin 40，Cx40）表达和分布调控作用，并探讨其与心房颤动（atrial fibrillation，AF）发生的关系。 方法 42只SD大鼠随机分为3组，分别为脓毒症组、给药组、假手术组，每组14只。通过盲肠结扎穿孔术制备脓毒症动物模型，给药组于术前6 h、术后24 h及术后48 h泵入TNF-α螯合剂重组人Ⅱ型肿瘤坏死因子受体-抗体融合蛋白。3组术后48 h均应用程序刺激诱发房颤，记录各组诱发情况。通过酶联免疫吸附试验（enzyme-linked immunosorbent assay，ELISA）检测血清中TNF-α浓度，应用蛋白免疫印迹法检测心房肌组织TNF-α、Cx43、Cx40的蛋白表达水平，应用激光共聚焦显微镜观察TNF-α、Cx43、Cx40的分布情况。 结果 假手术组、脓毒症组及给药组AF诱发率分别为7.14%、71.42%和21.40%，AF持续诱发时间分别为（79.21±4.10） s、（348.64±11.89） s和（294.50±37.28） s。通过ELISA检测假手术组、脓毒症组和给药组的血清中TNF-α的浓度分别为74.149 pg/mL、104.497 pg/mL和89.059 pg/mL。通过Western blot检测假手术组、脓毒症组和给药组的TNF-α蛋白表达水平分别为1.731±0.417、3.153±0.576和2.543±0.861，Cx40蛋白表达水平分别为1.207±0.230、0.970±0.170和1.010±0.164，Cx43蛋白表达水平分别为1.579±0.158、1.306±0.462和1.324±0.295。相对于假手术组，脓毒症组AF诱发率升高（F=5.394，P=0.003）且AF持续时间延长（F=335.577，P<0.001），血清TNF-α浓度升高（F=25.733，P<0.001），心房肌组织蛋白TNF-α表达增加（F=17.130，P<0.001），Cx40和Cx43表达降低（F=6.215，P=0.045；F=3.001，P=0.035），激光共聚焦显微镜观察心房肌Cx40及Cx43分布紊乱；相对于脓毒症组，给药组AF诱发率降低（χ²=1.292，P=0.028），且血清TNF-α浓度降低（F=18.192，P=0.004），心房肌组织蛋白TNF-α表达减少（F=28.078，P=0.017），心房肌组织Cx40和Cx43分布紊乱有所减轻。 结论 脓毒症大鼠过表达的TNF-α可以诱导心房肌Cx43及Cx40表达异常和分布重构，其在脓毒症诱导AF的发展中可能发挥重要作用。

Abstract

Objective To investigate the regulatory effect of tumor necrosis factor-α（TNF-α） on the expression and distribution of connexin 43 （Cx43） and connexin 40（Cx40） in rats with sepsis，as well as its association with the development of atrial fibrillation （AF）. Methods A total of 42 Sprague-Dawley rats were randomly divided into sepsis group，drug group，and sham-operation group，with 14 rats in each group. The method of cecal ligation and puncture was used to establish an animal model of sepsis，and the rats in the drug group were given the pumping of rhTNFR：FC at 6 hours before surgery and at 24 and 48 hours after surgery. Programmed electrical stimulation was imposed on all three groups at 48 hours after surgery. ELISA was used to measure the serum level of TNF-α，Western blotting was used to measure the protein expression levels of TNF-α，Cx43，and Cx40 in atrial muscle tissue，and a laser scanning confocal microscope was used to observe the distribution of TNF-α，Cx43，and Cx40. Results The induction rate of AF was 7.14% in the sham-operation group，71.42% in the sepsis group，and 21.40% in the drug group，with a duration of AF induction of 79.21±4.10 seconds，348.64±11.89 seconds，and 294.50±37.28 seconds，respectively. ELISA showed that the serum concentration of TNF-α was 74.149 pg/mL in the sham-operation group，104.497 pg/mL in the sepsis group，and 89.059 pg/mL in the drug group，and Western blot showed that in these three groups，the protein expression levels of TNF-α were 1.731±0.417，3.153±0.576，and 2.543±0.861，respectively，the protein expression levels of Cx40 were 1.207±0.230，0.970±0.170，and 1.010±0.164，respectively，and the protein expression levels of Cx43 were 1.579±0.158，1.306±0.462，and 1.324±0.295，respectively. Compared with the sham-operation group，the sepsis group had significant increases in the induction rate of AF（F=5.394，P=0.003），the duration of AF（F=335.577，P<0.001），the serum concentration of TNF-α（F=25.733，P<0.001），and the protein expression level of TNF-α in atrial muscle tissue（F=17.130，P<0.001） and significant reductions in the expression levels of Cx40 and Cx43（F=6.215 and 3.001，P=0.045 and 0.035），with disordered distribution of Cx40 and Cx43 in atrial muscle observed by the laser scanning confocal microscope. Compared with the sepsis group，the drug group had significant reductions in the induction rate of AF（χ²=1.292，P=0.028），the serum concentration of TNF-α（F=18.192，P=0.004），and the protein expression level of TNF-α in atrial muscle tissue（F=28.078，P=0.017），as well as alleviation of the disordered distribution of Cx40 and Cx43 in atrial muscle tissue. Conclusion Overexpression of TNF-α can induce the abnormal expression and distribution remodeling of Cx43 and Cx40 in atrial muscle of rats with sepsis，and it may play an important role in the development of AF induced by sepsis.

关键词

脓毒症 / 心房颤动 / 肿瘤坏死因子-α / 连接蛋白40 / 连接蛋白43

Key words

sepsis / atrial fibrillation / tumor necrosis factor-α / connexin 40 / connexin 43

基金资助

2022年度承德市科技计划资助项目(202204A074)

2021年河北省政府资助临床医学优秀人才培养资助项目