Wogonoside alleviates colitis by improving intestinal epithelial barrier function via the MLCK/pMLC2 pathway
Wogonoside alleviates colitis by improving intestinal epithelial barrier function via the MLCK/pMLC2 pathway
复制标题
汉黄芩苷通过 MLCK/pMLC2 途径改善肠上皮屏障功能，从而缓解结肠炎
DOI：
10.1016/j.phymed.2020.153179
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发表时间：
2020-03-01
期刊：
影响因子：
7.9
通讯作者：
Luo Xia
中科院分区：
文献类型：
--
作者：
Huang Shaowei;Fu Yajun;Luo Xia
背景资料：肠上皮屏障功能障碍涉及肌球蛋白轻链激酶（MLCK）激活，有助于炎症性肠病（IBD）中炎症的发生和进展。汉黄芩苷有助于维持葡聚糖硫酸钠（DSS）诱导的结肠炎小鼠的肠道稳态，但尚不清楚它是否调节肠屏障功能。目的：在这里，我们证明汉黄芩苷通过MLCK/pMLC 2途径在体内和体外保护结肠炎中的肠屏障功能障碍。用促炎细胞因子TNF-α处理的Caco-2细胞单层显示屏障功能障碍，并在不存在和存在汉黄芩苷的情况下评估各种生理学，形态学，和生化参数。以3%DSS诱导小鼠结肠炎为动物模型，探讨汉黄芩苷的药效学。我们通过蛋白质印迹分析检测MLCK/pMLC 2通路蛋白，通过ELISA评估细胞因子IL-13和IFN-γ，通过荧光原位杂交（FISH）检测细菌易位，并适当取样次级淋巴器官进行细菌培养。结果：汉黄芩苷可减轻TNF-α对Caco-2细胞跨上皮电阻（TER）的破坏，减轻Caco-2细胞单层内FITC-葡聚糖通透性的增加，减轻紧密连接蛋白occludin、ZO-1和claudin-1的丢失，减轻结肠炎小鼠肠道细菌移位。此外，汉黄芩苷降低促炎细胞因子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，汉黄芩苷改善肠上皮TJ的破坏。汉黄芩苷不仅抑制TNF-α诱导的细胞骨架F-actin重排，稳定细胞骨架结构，抑制MLCK蛋白表达，并减少MLC 2磷酸化。分子对接分析结果表明，汉黄芩苷与MLCK具有较高的亲和力，并与氨基酸残基LYS 261形成氢键，与LYS 229形成π键。结论：汉黄芩苷抗结肠炎的机制可能是通过MLCK/pMLC 2信号通路调节TJ。同时，我们的研究也解释了S.黄芩治疗溃疡性结肠炎（UC）。
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-alpha 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-gamma 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-alpha 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-gamma 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-alpha, 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 pi 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).