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首页> 外文期刊>Cellular Physiology and Biochemistry >Potential Regulatory Effects of Corticotropin-Releasing Factor on Tight Junction-Related Intestinal Epithelial Permeability are Partially Mediated by CK8 Upregulation
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Potential Regulatory Effects of Corticotropin-Releasing Factor on Tight Junction-Related Intestinal Epithelial Permeability are Partially Mediated by CK8 Upregulation

机译：促肾上腺皮质激素释放因子对紧密连接相关的肠上皮通透性的潜在调节作用部分由CK8上调介导

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>Background/Aims: Intestinal permeability and stress have been implicated in the pathophysiology of irritable bowel syndrome (IBS). Cytokeratin 8 (CK8), for the first time, has been shown to mediate corticotropin-releasing factor (CRF)-induced changes in intestinal permeability in animal models of IBS. In this study, we investigated the regulatory effects of CRF on the permeability of human intestinal epithelial cells through the CK8-mediated tight junction. Methods: The expression levels of corticotropin-releasing factor receptor 1 (CRFR1) and corticotropin-releasing factor receptor 2 (CRFR2) on the HT29 cell surface were determined by immunofluorescence, RT-PCR, and Western blotting. After treatment with 100 nM CRF for 72 h, the translocation of FITC-labelled dextran was measured in a transwell chamber; the structural changes of tight junctions were observed under transmission electron microscopy; the expression levels of CK8, F-actin and tight junction proteins ZO-1, claudin-1, and occludin were detected by immunoblotting and immunofluorescence. The activity of RhoA was detected by immunoprecipitation. Furthermore, the effects of CRF on intestinal epithelial permeability were examined in CK8-silenced HT29 cells, which were constructed by shRNA interference. Results: CRF treatment increased FITC-labelled dextran permeability, caused the opening of tight junctions, induced increased fluorescence intensity of CK8 and decreased the intensities of ZO-1, claudin-1, and occludin, together with structural disruption. The expression levels of F-actin, occludin, claudin-1, and ZO-1 were downregulated. RhoA activity peaked at 30 min after CRF treatment. CRF-induced increased permeability, and downregulation of claudin-1 and occludin were not blocked by CK8 silencing. Nevertheless, CK8 silencing blocked the effects of CRF regarding the decrease in the expression levels of F-action and ZO-1 and increase in RhoA activity. Conclusion: CRF may increase intestinal epithelial permeability by upregulating CK8 expression, activating the RhoA signalling pathway, promoting intestinal epithelial actin remodelling, and decreasing the expression of the tight junction protein ZO-1. Other CK8-independent pathways may be involved in the downregulation of claudin-1 and occludin, which might also contribute to increased intestinal epithelial permeability.
机译：> 背景/目的： 肠通透性和压力与肠易激综合征（IBS）的病理生理有关。在IBS动物模型中，首次显示了细胞角蛋白8（CK8）介导促肾上腺皮质激素释放因子（CRF）引起的肠道通透性变化。在这项研究中，我们调查了CRF通过CK8介导的紧密连接对人肠上皮细胞通透性的调节作用。 方法： 通过免疫荧光，RT法测定HT29细胞表面促肾上腺皮质激素释放因子受体1（CRFR1）和促肾上腺皮质激素释放因子受体2（CRFR2）的表达水平。 -PCR和蛋白质印迹。用100 nM CRF处理72小时后，在transwell室中测量FITC标记的葡聚糖的易位;在透射电镜下观察到紧密连接的结构变化。通过免疫印迹和免疫荧光检测CK8，F-肌动蛋白和紧密连接蛋白ZO-1，claudin-1和occludin的表达水平。通过免疫沉淀检测RhoA的活性。此外，在shRNA干扰下构建的CK8沉默的HT29细胞中，研究了CRF对肠上皮通透性的影响。 结果： CRF处理可增加FITC标记的葡聚糖通透性，引起紧密连接的打开，诱导CK8的荧光强度增加，并降低ZO-1和claudin-1的强度。 ，和occludin，以及结构破坏。 F-肌动蛋白，闭合蛋白，claudin-1和ZO-1的表达水平下调。 CRF治疗后30分钟，RhoA活性达到峰值。 CRF诱导的通透性增加，以及CK8沉默并未阻止claudin-1和occludin的下调。然而，CK8沉默阻止了CRF对F-作用和ZO-1表达水平降低以及RhoA活性增加的影响。 结论： CRF可能通过上调CK8表达，激活RhoA信号传导途径，促进肠道上皮肌动蛋白重塑和降低紧密连接蛋白ZO-的表达来增加肠道上皮通透性。 1。其他不依赖CK8的途径可能与claudin-1和occludin的下调有关，这也可能导致肠道上皮通透性增加。

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