Raimondi Francesco, Santoro Pasquale, Barone Maria Vittoria, Pappacoda Serena, Barretta Maria Luisa, Nanayakkara Merlin, Apicella Carmela, Capasso Letizia, Paludetto Roberto
Division of Neonatology, Department of Pediatrics, "Federico II" University, Naples, Italy.
Am J Physiol Gastrointest Liver Physiol. 2008 Apr;294(4):G906-13. doi: 10.1152/ajpgi.00043.2007. Epub 2008 Jan 31.
Intestinal and systemic illnesses have been linked to increased gut permeability. Bile acids, whose luminal profile can be altered in human disease, modulate intestinal paracellular permeability. We investigated the mechanism by which selected bile acids increase gut permeability using a validated in vitro model. Human intestinal Caco-2 cells were grown in monolayers and challenged with a panel of bile acids. Transepithelial electrical resistance and luminal-to-basolateral fluxes of 10-kDa Cascade blue-conjugated dextran were used to monitor paracellular permeability. Immunoprecipitation and immunoblot analyses were employed to investigate the intracellular pathway. Redistribution of tight junction proteins was studied by confocal laser microscopy. Micromolar concentrations of cholic acid, deoxycholic acid (DCA), and chenodeoxycholic acid (CDCA) but not ursodeoxycholic acid decreased transepithelial electrical resistance and increased dextran flux in a reversible fashion. Coincubation of 50 muM CDCA or DCA with EGF, anti-EGF monoclonal antibody, or specific src inhibitor 4-Amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP-2) abolished the effect. A concentration of 50 muM of either CDCA or DCA also induced EGF receptor phosphorylation, occludin dephosphorylation, and occludin redistribution at the tight junction level in the same time frame and in a reversible fashion. We conclude that selected bile acids modulate intestinal permeability via EGF receptor autophosphorylation, occludin dephosphorylation, and rearrangement at the tight junction level. The effect is mediated by the src family kinases and is abolished by EGF treatment. These data also support the role of bile acids in the genesis of necrotizing enterocolitis and the protective effect of EGF treatment.
肠道疾病和全身性疾病与肠道通透性增加有关。胆汁酸在人类疾病中其肠腔内分布会发生改变，可调节肠道细胞旁通透性。我们使用经过验证的体外模型研究了特定胆汁酸增加肠道通透性的机制。人肠道Caco-2细胞长成单层，并接受一组胆汁酸的刺激。采用跨上皮电阻和10 kDa 级联蓝偶联葡聚糖的肠腔到基底外侧通量来监测细胞旁通透性。运用免疫沉淀和免疫印迹分析来研究细胞内途径。通过共聚焦激光显微镜研究紧密连接蛋白的重新分布。微摩尔浓度的胆酸、脱氧胆酸（DCA）和鹅脱氧胆酸（CDCA），但熊去氧胆酸不会以可逆方式降低跨上皮电阻并增加葡聚糖通量。50 μM 的 CDCA 或 DCA 与表皮生长因子（EGF）、抗 EGF 单克隆抗体或特异性 src 抑制剂4-氨基-5-（4-氯苯基）-7-（叔丁基）吡唑并[3,4-d]嘧啶（PP-2）共同孵育可消除该效应。50 μM 的 CDCA 或 DCA 浓度在同一时间范围内且以可逆方式还诱导了 EGF 受体磷酸化、闭合蛋白去磷酸化以及紧密连接水平上的闭合蛋白重新分布。我们得出结论，特定胆汁酸通过 EGF 受体自磷酸化、闭合蛋白去磷酸化以及紧密连接水平上的重排来调节肠道通透性。该效应由 src 家族激酶介导，并且 EGF 处理可消除该效应。这些数据也支持了胆汁酸在坏死性小肠结肠炎发生过程中的作用以及 EGF 治疗的保护作用。
