核因子B激活在大鼠冠状动脉微栓塞后的心肌组织中的作用及机制

Time course of myocardial NF-KB activation post coronary microembolization

目的探讨核因子-κB（NF-κB）激活在冠状动脉微栓塞（CME）后心肌组织中的作用及机制。方法清洁级雄性SD大鼠88只，其中64只经左心室内注射自体微血栓、同时短暂夹闭主动脉建立大鼠CME模型后，随机分为未治疗组及二硫代氨基甲酸吡咯烷（PDTC）干预组，分别于术后1、3、7、14d处死；余24只为假手术组。电泳迁移率改变分析（EMSA）检测不同时间点心肌组织NF-κBDNA结合活性，Westernblot测定肿瘤坏死因子仅（TNFα）、白细胞介素-6（IL-6）及细胞间黏附分子-1（ICAM-1）的表达水平，实时聚合酶链式反应检测TNFα、IL-6及ICA-1mRNA表达含量。结果NF-κBDNA结合活性在CME后1d增高，3d时达到高峰；7d时明显降低，14d时与假手术组无显著性差异。CME组在不同观察时间点心肌TNFα、IL-6、ICAM-1蛋白及基因的表达较假手术组均明显增强（P〈0．05）。NF-κBDNA结合活性与TNFα、IL-6、ICAM-13种细胞因子的mRNA表达水平均呈显著正相关（分别是r=0．72，P〈0．05；r=0．94，P〈0．01；r=0．62，P〈0．05）。NF-κB特异性抑制剂PDTC干预显著抑制CME后心肌中TNFα、IL-6及ICAM-1蛋白及mRNA的表达（P均〈0．05），同时也改善心功能。结论CME后NF-κB激活促使炎性因子TNFα、IL-6、ICAM-1在基因及蛋白水平明显上调，是诱导CME心肌炎症反应、心功能减低的重要早期事件。

Objective To investigate the time course of myocardial NF-κB activation and association with cardiac function and other pro-inflammation cytokines following coronary microembolizatiou （CME）. Methods CME was induced by homologous microthrombotic particle suspension injection into left ventricle with simultaneous short-term ascending aorta clamping. The CME rats were randomized to untreated group and pyrrolidine dithiocarbamate （PDTC, a specific NF-κB inhibitor） treated group （n = 32 respectively）. The rats were sacrificed on day 1, 3, 7 and 14 post-operationally （n = 8 each）. Twenty-four rats were sham-operated and served as controls. NF-κB DNA-binding activity was evaluated by electrophoretic mobility shift assay （ EMSA）, protein expressions of TNFα, IL-6 and ICAM-1 were analyzed by Western blotting, the dynamic aherations of TNFα, IL-6 and ICAM-1 mRNA were quantitatively assessed by Real-time PCR post hemodynamic measurements. Results NF-κB DNA-binding activity in CME group was significantly increased than that of sham group on day 1, peaked at day 3 and was similar as that in sham rats on day 14. The protein and mRNA expressions of TNFα, IL-6 and ICAM-1 were significantly increased in CME group at various time points compared those in sham rats. NF-κB DNA-binding activity positively correlated with mRNA expressions of TNFα, IL-6, ICAM-1, respectively （r = 0.72, P 〈 0.05 ; r = 0. 94, P 〈0. 01 ; r =0. 62, P 〈 0. 05 ）. PDTC significantly suppressed protein and mRNA expressions of TNFα, IL-6 and ICAM-1 （ P 〈 0. 05 ） and improved left ventricular function. Conclusion NF-κB activation post CME could upregulate the gene transcriptions of TNFα, IL-6, ICAM-1 and enhance inflammatory responses and aggravate left ventricular dysfunction.