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

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。

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

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.