Division of General Surgery, Peking University First Hospital, Peking University, Beijing 100034, China.
Mediators Inflamm. 2019 Jul 3;2019:5796491. doi: 10.1155/2019/5796491. eCollection 2019.
Escherichia coli Nissle 1917 (EcN), a kind of probiotic, has been reported to have a protective effect on the intestinal barrier function and can ameliorate certain gastrointestinal disorders. In this study, the potential protective effect of EcN on the intestinal barrier function in a septic mouse model induced by cecal ligation and puncture (CLP) operation was investigated. FITC-Dextran 4,000 Da (FD-4) flux and the expression levels of tight junction (TJ) proteins were measured to evaluate the protective effect of EcN on the intestinal barrier function. Then, Caco-2 monolayers were utilized to further investigate the protective effect of the EcN supernatant (EcN) on the barrier dysfunction induced by TNF- and IFN- in vitro; the plasma level of both the cytokines increased significantly during sepsis. Transepithelial electrical resistance (TEER) and FD-4 transmembrane flux were measured, and the localization of ZO-1 and Occludin was investigated by immunofluorescence. The expression of MLCK and the phosphorylation of MLC were detected by western blot. The activation of NF-B was explored by immunofluorescence, and CHIP assays were performed to investigate the conjunction of NF-B with the promoter of MLCK. The results indicated that EcN protected the intestinal barrier function in sepsis by ameliorating the altered expression and localization of TJ proteins and inhibiting the NF-B-mediated activation of the MLCK-P-MLC signaling pathway which might be one of the mechanisms underlying the effect of EcN.
大肠杆菌 Nissle 1917（EcN）是一种益生菌，据报道对肠道屏障功能具有保护作用，并能改善某些胃肠道疾病。在本研究中，我们研究了 EcN 对盲肠结扎穿刺（CLP）手术诱导的脓毒症小鼠模型肠道屏障功能的潜在保护作用。通过测量 FITC-葡聚糖 4000（FD-4）通量和紧密连接（TJ）蛋白的表达水平，评估 EcN 对肠道屏障功能的保护作用。然后，我们利用 Caco-2 单层细胞进一步研究 EcN 上清液（EcN）对 TNF-α和 IFN-γ体外诱导的屏障功能障碍的保护作用；脓毒症期间这两种细胞因子的血浆水平显著升高。通过测量跨上皮电阻（TEER）和 FD-4 跨膜通量，并通过免疫荧光法研究 ZO-1 和 Occludin 的定位，评估了 EcN 对屏障功能障碍的保护作用。通过 Western blot 检测 MLCK 的表达和 MLC 的磷酸化。通过免疫荧光法探索 NF-B 的激活，并通过 CHIP 测定法研究 NF-B 与 MLCK 启动子的结合。结果表明，EcN 通过改善 TJ 蛋白的改变表达和定位，抑制 NF-B 介导的 MLCK-P-MLC 信号通路的激活，从而保护脓毒症中的肠道屏障功能，这可能是 EcN 作用的机制之一。
