Mechanisms of tumor necrosis factor-alpha-induced leaks in intestine epithelial barrier
Mechanisms of tumor necrosis factor-alpha-induced leaks in intestine epithelial barrier
复制标题
肿瘤坏死因子-α诱导肠上皮屏障渗漏的机制
DOI：
10.1016/j.cyto.2012.04.008
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发表时间：
2012-08-01
期刊：
影响因子：
3.8
通讯作者：
Yin, Fei
中科院分区：
文献类型：
--
作者：
He, Fang;Peng, Jing;Yin, Fei
目的：探讨肿瘤坏死因子- α (tnf - α)诱导人肠上皮细胞紧密连接（IT）和通透性改变的信号机制。方法：为了证实tnf - α通过破坏紧密连接诱导上皮屏障高通透性，将Caco-2细胞暴露于tnf - α中，监测上皮通透性（通过TER法）、f -肌动蛋白动力学（通过罗丹明-phalloidin染色）和紧密连接蛋白表达（通过western blot）的变化。此外，为了确保NF-kappa B参与调控机制，我们用DNMu-I kappa B α或对照质粒转染Caco-2细胞，重复上述实验，并通过荧光素酶报告基因法检测tnf - α对NF-kappa B的激活作用。最后，我们采用显性阴性质粒和敲低方法来研究NF-kappa B/肌球蛋白轻链激酶(MLCK)/肌球蛋白轻链磷酸化（pMLC）途径在tnf -a介导的损伤中的潜在重要性。结果：tnf - α可引起nf - κ B活化、f -肌动蛋白重排、紧密连接破坏和屏障功能障碍。这些作用通过抑制NF-kappa B而得到缓解。tnf - α诱导的MLCK转录和MLC磷酸化的增加晚于NF-kappa B的激活，这可以通过灭活和删除NF-kappa B来抑制。结论：tnf - α通过破坏Tjs诱导肠上皮细胞高通透性，部分通过MLCK上调，其中NF-kappa B是MLCK的上游正向调节因子。(C) 2012 Elsevier Ltd.版权所有。
Purpose: The aim of this study was to investigate the signaling mechanisms surrounding changes in tight junction (IT and the permeability of human intestinal epithelial cell induced by tumor necrosis factor-alpha (TNF-alpha).Methods: To confirm that TNF-alpha induces epithelial barrier hyperpermeability by disrupting tight junction, Caco-2 cells were exposed to TNF-alpha, and changes in epithelial permeability (via TER assay), F-actin dynamics (via Rhodamine-phalloidin staining) and tight junction protein expression (via western blot) were monitored. Moreover, to ensure that NF-kappa B participated in the regulatory mechanisms, Caco-2 cells were transfected with DNMu-I kappa B alpha or control plasmids, the above experiments were repeated and the activation effect of TNF-alpha on NF-kappa B was detected by luciferase reporter assays. Lastly, we took dominant negative plasmid and knockdown approaches to investigate the potential importance of the NF-kappa B/myosin light chain kinase (MLCK)/myosin light chain phosphorylation (pMLC) pathways in TNF-a-mediated damage.Result: TNF-alpha could cause NF-kappa B activation, F-actin rearrangement, tight junction disruption and barrier dysfunction. These effects were alleviated by inhibiting NF-kappa B. TNF-alpha induced increase of MLCK transcription and MLC phosphorylation act later than NF-kappa B activation, which could be suppressed both by inactivating and deleting NF-kappa B.Conclusions: TNF-alpha induces intestinal epithelial cell hyperpermeability by disrupting Tjs, in part through MLCK upregulation, in which NF-kappa B is the positive upstream regulator for MLCK. (C) 2012 Elsevier Ltd. All rights reserved.