Division of Gastroenterology-Hepatology, Department of Internal Medicine, Maastricht University Medical Centre+, P. Debyelaan 25 6229 HX, Maastricht, the Netherlands.
School of Nutrition and Translational Research in Metabolism, Maastricht University, Maastricht, the Netherlands.
J Mol Med (Berl). 2021 Jun;99(6):805-815. doi: 10.1007/s00109-021-02045-7. Epub 2021 Feb 11.
Corticosteroids (CS), first-line therapeutics for Crohn's disease (CD) with moderate or severe disease activity, were found to restore intestinal permeability in CD patients, whereas the underlying molecular events are still largely unknown. This study aimed to investigate the effect and mechanisms of CS prednisolone on epithelial barrier using CD patient-derived intestinal organoids. 3D intestinal organoids were generated from colon biopsies of inactive CD patients. To mimic the inflammatory microenvironment, a mixture of cytokines containing TNF-α, IFN-γ, and IL-1β were added to the organoid culture with or without pre-incubation of prednisolone or mifepristone. Epithelial permeability of the organoids was assessed by FITC-D4 flux from the basal to luminal compartment using confocal microscopy. Expression of junctional components were analyzed by qRT-PCR, immunofluorescence staining, and western blot. Activity of signaling pathways were analyzed using western blot. Exposure of the cytokines significantly disrupted epithelial barrier of the intestinal organoids, which was partially restored by prednisolone. On the molecular level, the cytokine mixture resulted in a significant reduction in E-cadherin and ILDR-1, an increase in CLDN-2, MLCK, and STAT1 phosphorylation, whereas prednisolone ameliorated the abovementioned effects induced by the cytokine mixture. This study demonstrates that prednisolone confers a direct effect in tightening the epithelial barrier, identifies novel junctional targets regulated by prednisolone, and underscores intestinal barrier restoration as a potential mechanism that contributes to the clinical efficacy of prednisolone in CD patients. KEY MESSAGES: Prednisolone confers a direct preventive effect against cytokine-induced barrier dysfunction. Prednisolone regulates the expression of CLDN-2, E-cadherin, and ILDR-1. The effect of prednisolone is GR-, MLCK-, and STAT1-dependent.
皮质类固醇（CS）是治疗中重度活动期克罗恩病（CD）的一线药物，可恢复 CD 患者的肠道通透性，但其潜在的分子事件仍知之甚少。本研究旨在使用源自 CD 患者的肠类器官研究 CS 泼尼松龙对上皮屏障的影响和机制。从无活性 CD 患者的结肠活检中生成 3D 肠类器官。为了模拟炎症微环境，将包含 TNF-α、IFN-γ 和 IL-1β 的细胞因子混合物添加到类器官培养物中，同时或不预先孵育泼尼松龙或米非司酮。使用共聚焦显微镜通过 FITC-D4 从基底到腔室的通量评估类器官的上皮通透性。通过 qRT-PCR、免疫荧光染色和 Western blot 分析连接成分的表达。使用 Western blot 分析信号通路的活性。细胞因子的暴露显著破坏了肠类器官的上皮屏障，泼尼松龙部分恢复了这一屏障。在分子水平上，细胞因子混合物导致 E-钙粘蛋白和 ILDR-1 显著减少，CLDN-2、MLCK 和 STAT1 磷酸化增加，而泼尼松龙改善了细胞因子混合物引起的上述效应。本研究表明，泼尼松龙具有直接的紧致上皮屏障作用，确定了泼尼松龙调节的新的连接靶点，并强调了肠道屏障的恢复可能是泼尼松龙在 CD 患者中发挥临床疗效的潜在机制。
泼尼松龙对细胞因子诱导的屏障功能障碍具有直接的预防作用。
泼尼松龙调节 CLDN-2、E-钙粘蛋白和 ILDR-1 的表达。
泼尼松龙的作用依赖于 GR、MLCK 和 STAT1。
