核因子κB通路参与缺氧诱导心肌细胞分泌肿瘤坏死因子α  

Nuclear factor kappaB signaling pathway regulates tumor necrosis factor alpha expression of cardiomyocytes induced by hypoxia

作　　者：邓玲艳[1] 余娴[1] 王丹[1] 厉娜[1] 高兴丽[1] 王敏[1] 廖玉华[1] 

机构地区：[1]等中对棼大誊协母医院心内科华中科技大学同济医学院心血管病研究所,武汉430022

出　　处：《临床心血管病杂志》2012年第4期298-301,共4页Journal of Clinical Cardiology

基　　金：国家“973”资助项目(No:2007CB512005)

摘　　要：目的:探讨缺氧刺激能否诱导体外培养心肌细胞分泌肿瘤坏死因子α(TNF-α),核因子κB(NF-κB)通路是否参与其中,并发挥调节作用。方法:1%O2物理缺氧刺激原代SD大鼠乳鼠心肌细胞,在不同时间点Real-time PCR检测TNF-αmRNA水平;ELISA检测培养上清中TNF-α蛋白水平;Western blot检测胞质NF-κB相关通路蛋白IKKα,IKKβ,IKKBα蛋白及磷酸化蛋白水平以及胞核NF-κB p65蛋白表达;电泳迁移率实验检测NF-κB DNA结合能力。缺氧同时给予NF-κB抑制剂四氢化吡咯二硫代氨基甲酸酯(PDTC)刺激心肌细胞,检测细胞核p65蛋白表达,TNF-αmRNA及蛋白水平。结果:缺氧诱导原代心肌细胞表达TNF-α,TNF-αmRNA水平从缺氧1h开始升高,12h及24h达峰值;其蛋白分泌从缺氧6h开始升高,12h和24h达峰值。缺氧激活NF-κB通路:缺氧5min胞质pIKKα/IKKβ表达开始上调,30min达峰值,持续6h;IKKBα表达缺氧5min开始降低,而pIKKBα缺氧30min开始增加;胞核p65蛋白水平缺氧5min上调,1h达峰值,持续6h;NF-κB DNA结合能力缺氧5min后开始增强。PDTC抑制NF-κB活性,有效抑制TNF-αmRNA及其蛋白表达。结论:缺氧通过激活NF-κB通路,诱导心肌细胞自分泌TNF-α,NF-κB在缺氧诱导心肌细胞自分泌TNF-α过程中起正向调节作用。Objective：To investigate whether hypoxia could induce secretion of TNF-α in primary neonatal rat cardiomyocytes,and the role of NF-κB signaling pathway in the process.Method：Primary neonatal rat cardiomyocytes were cultured in a hypoxic incubator（containing 1% O2） for various periods of time.TNF-α mRNA expression and protein secretion were detected by real-time PCR and enzyme-linked immunosorbent assay respectively.Protein levels of IKKα,IKKβ,IκBα,pIKKα/β,pIκBα in cytoplasmic extracts and NF-κB p65 in nuclear extracts were detected by western blot.The DNA-binding activity of NF-κB was measured by electrophoretic mobility shift assay.Pyrrolidine dithiocarbamate（PDTC）,the specific inhibitor of NF-κB,was added to the hypoxic cultured cardiomyocytes to block the effect of NF-κB.Expression of NF-κB p65 in nuclear extracts,and mRNA and protein levels of TNF-α were detected.Result：Hypoxia could induce TNF-α mRNA expression and protein secretion.TNF-α mRNA increased slightly as early as 1 h and peaked at 12 h and 24 h.TNF-α secretion became detectable at 6 h and peaked at 12 h and 24 h.pIKKα/β was detectable at 5 min,peaked at 30 min and lasted for 6 h.Significant elevation of pIKKBα was observed at 30 min,and degradation of IKKBα began at 5 min after hypoxia.Translocation of NF-κB p65 into cell nucleus was detectable as early as 5 min.Meanwhile,the DNA-binding activity of NF-κB was consistent with the variation of p65 in cell nucleus.Expression of p65 was inhibited effectively by PDTC after hypoxia,and PDTC also suppressed mRNA levels and protein secretion of TNF-α after 12-hour hypoxia.Conclusion：Hypoxia induces expression of TNF-α and activates the NF-κB pathway in primary cardiomyocytes.NF-κB participates in the upregulation of hypoxia induced TNF-α expression.

关 键 词：冠心病 核因子ΚB 肿瘤坏死因子Α 心肌细胞 缺氧 

分 类 号：R541.4[医药卫生—心血管疾病]