BACKGROUND Intestinal epithelial barrier dysfunction, which involves myosin light chain kinase (MLCK) activation, contributes to the occurrence and progression of inflammation in inflammatory bowel disease (IBD). Wogonoside helps maintain intestinal homeostasis in mice with dextran sulfate sodium (DSS)-induced colitis, but it is unclear whether it modulates intestinal barrier function. PURPOSE Here, we demonstrate that wogonoside protects against intestinal barrier dysfunction in colitis via the MLCK/pMLC2 pathway both in vivo and in vitro. METHODS Caco-2 cell monolayers treated with the proinflammatory cytokine TNF-α showed barrier dysfunction and were assessed in the absence and presence of wogonoside for various physiological, morphological, and biochemical parameters. Colitis was induced by 3% DSS in mice, which were used as an animal model to explore the pharmacodynamics of wogonoside. We detected MLCK/pMLC2 pathway proteins via western blot analysis, assessed the cytokines IL-13 and IFN-γ via ELISA, tested bacterial translocation via fluorescence in situ hybridization (FISH) and a proper sampling of secondary lymphoid organs for bacterial culture. In addition, the docking affinity of wogonoside and MLCK was observed with DS2.5 software. RESULTS Wogonoside alleviated the disruption of transepithelial electrical resistance (TER) in TNF-α exposured Caco-2 cell; FITC-dextran hyperpermeability; loss of the tight junction (TJ) proteins occludin, ZO-1 and claudin-1 in Caco-2 cell monolayers; and bacterial translocation in colitic mice. Moreover, wogonoside reduced the levels of the proinflammatory cytokines IL-13 and IFN-γ to maintain intestinal immune homeostasis. Transmission electron microscopy (TEM) confirmed that wogonoside ameliorated the destruction of intestinal epithelial TJs. Wogonoside not only inhibited the cytoskeletal F-actin rearrangement induced by TNF-α, stabilized the cytoskeletal structure, suppressed MLCK protein expression, and reduced MLC2 phosphorylation. In addition, the results of molecular docking analysis showed that wogonoside had a high affinity for MLCK and formed hydrogen bonds with the amino acid residue LYS261 and π bonds with LYS229. CONCLUSION Collectively, our study indicates that wogonoside alleviates colitis by protecting against intestinal barrier dysfunction, and the potential mechanism may involve regulation of TJs via the MLCK/pMLC2 signaling pathway. Meanwhile, our study also explains the success of S. baicalensis in the treatment of ulcerative colitis (UC).

中文翻译：

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Wogonoside通过改善MLCK / pMLC2途径的肠上皮屏障功能来缓解结肠炎。

背景技术涉及肌球蛋白轻链激酶（MLCK）活化的肠上皮屏障功能障碍促成炎性肠病（IBD）中炎症的发生和发展。Wogonoside有助于维持硫酸右旋糖酐钠（DSS）引起的结肠炎的小鼠肠道稳态，但尚不清楚它是否能调节肠道屏障功能。目的在这里，我们证明了在体内和体外，wogonoside通过MLCK / pMLC2途径可以预防结肠炎的肠屏障功能障碍。方法用促炎性细胞因子TNF-α处理的Caco-2细胞单层显示屏障功能障碍，并在不存在和存在伍格诺甙的情况下评估其各种生理学，形态学和生化指标。3％DSS诱发小鼠结肠炎，它们被用作动物模型，以探索牛磺酸皂苷的药效学。我们通过蛋白质印迹分析检测了MLCK / pMLC2途径蛋白，通过ELISA评估了细胞因子IL-13和IFN-γ，通过荧光原位杂交（FISH）测试了细菌易位，并对细菌培养的次级淋巴器官进行了适当采样。另外，用DS2.5软件观察到了窝草皂甙和MLCK的对接亲和力。结果Wogonoside减轻了TNF-α暴露的Caco-2细胞的跨上皮电阻（TER）的破坏。FITC-葡聚糖高通透性；Caco-2细胞单层中紧密连接（TJ）蛋白occludin，ZO-1和claudin-1的丢失；和细菌在结肠小鼠中的易位。此外，牛磺酸皂甙降低了促炎细胞因子IL-13和IFN-γ的水平，以维持肠道免疫稳态。透射电子显微镜（TEM）证实，牛磺酸皂甙改善了肠上皮TJ的破坏。Wogonoside不仅抑制TNF-α诱导的细胞骨架F-肌动蛋白重排，稳定细胞骨架结构，抑制MLCK蛋白表达，并减少MLC2磷酸化。另外，分子对接分析的结果表明，牛磺酸皂苷对MLCK具有高亲和力，并且与氨基酸残基LYS261形成氢键，与LYS229形成π键。结论总体而言，我们的研究表明，牛磺酸皂甙可通过预防肠道屏障功能障碍来缓解结肠炎，潜在的机制可能涉及通过MLCK / pMLC2信号通路调节TJ。同时，我们的研究还解释了黄ical对溃疡性结肠炎（UC）的成功治疗。