Zonula Occludens-1 alterations and enhanced intestinal permeability in methotrexate-treated rats
Zonula Occludens-1 alterations and enhanced intestinal permeability in methotrexate-treated rats
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DOI：
10.1007/s00280-010-1253-9
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发表时间：
2010-11-01
影响因子：
3
通讯作者：
Horie, Toshiharu
中科院分区：
文献类型：
--
作者：
Hamada, Kazuma;Shitara, Yoshihisa;Horie, Toshiharu
氨甲喋呤（MTX）介导的肠屏障功能破坏的分子机制尚未完全确定。上皮屏障功能在很大程度上由位于侧膜最顶端部分的多蛋白复合物决定，其被称为紧密连接（TJ）。在本研究中，我们检测了闭合小带-1（ZO-1）的改变，ZO-1是一种支架蛋白，在TJ的形成中起关键作用，以确定上皮屏障功能障碍的另一种分子机制。雄性Wistar大鼠口服MTX（15 mg kg（-1）），每日一次，持续3-5天。肠粘膜通透性测定采用体外外翻肠囊技术。通过髓过氧化物酶活性和活性氧的产生来评估粘液炎症。通过RT-PCR、Western blotting、免疫沉淀和免疫组织化学方法检测ZO-1表达、酪氨酸磷酸化和定位的改变。屏障功能研究显示，MTX处理4天后，大鼠肠道通透性增加，表现为异硫氰酸荧光素-葡聚糖通量增加。此外，粘膜炎症与肠通透性增强有关。ZO-1表达的定量分析表明，MTX治疗的大鼠中没有显着差异，而ZO-1的酪氨酸去磷酸化。此外，我们还检测到ZO-1免疫染色沿着肠绒毛顶膜的明显减少，这些结果表明，在MTX治疗的大鼠中，ZO-1的改变可能有助于TJ屏障的紊乱，从而导致肠通透性增加。
The molecular mechanisms that underlie the methotrexate (MTX)-mediated disruption of intestinal barrier function have not been fully characterized. Epithelial barrier function is determined in large part by a multiprotein complex located at the most apical part of the lateral membrane, which is referred to as a tight junction (TJ). In the present study, we examined the alteration of zonula occludens-1 (ZO-1), which is a scaffolding protein that plays a pivotal role in the formation of TJs, to identify an additional molecular mechanism for epithelial barrier dysfunction.Male Wistar rats were administered MTX (15 mg kg(-1)) orally once daily for 3-5 days. Intestinal mucosal permeability was determined using the in vitro everted intestinal sac technique. Mucosal inflammation was assessed by myeloperoxidase activity and production of reactive oxygen species. Altered expression, tyrosine phosphorylation, and localization of ZO-1 were evaluated by RT-PCR, Western blotting, immunoprecipitation, and immunohistochemistry.A barrier function study revealed increased intestinal permeability in rats treated with MTX for 4 days, as indicated by enhanced fluorescein isothiocyanate-dextran flux. In addition, mucosal inflammation was linked to enhanced intestinal permeability. Quantitative analysis of ZO-1 expression showed the absence of significant differences in MTX-treated rats, whereas tyrosine dephosphorylation of ZO-1 was observed. Moreover, we also detected an obvious reduction of ZO-1 immunostaining along the apical membrane of intestinal villi.These results indicate that, in MTX-treated rats, ZO-1 alterations may contribute to disturbance of the TJ barrier, which leads to enhanced intestinal permeability.