Division of Trauma, Surgical Critical Care, and Burns, Department of Surgery, University of California San Diego Health Sciences, San Diego, California, United States of America.
PLoS One. 2013 Jul 1;8(7):e69042. doi: 10.1371/journal.pone.0069042. Print 2013.
Intestinal barrier failure may lead to systemic inflammation and distant organ injury in patients following severe injury. Enteric glia cells (EGCs) have been shown to play an important role in maintaining gut barrier integrity through secretion of S-Nitrosoglutathione (GSNO). We have recently shown than Vagal Nerve Stimulation (VNS) increases EGC activation, which was associated with improved gut barrier integrity. Thus, we sought to further study the mechanism by which EGCs prevent intestinal barrier breakdown utilizing an in vitro model. We postulated that EGCs, through the secretion of GSNO, would improve intestinal barrier function through improved expression and localization of intestinal tight junction proteins.
Epithelial cells were co-cultured with EGCs or incubated with GSNO and exposed to Cytomix (TNF-α, INF-γ, IL-1β) for 24 hours. Barrier function was assessed by permeability to 4kDa FITC-Dextran. Changes in tight junction proteins ZO-1, occludin, and phospho-MLC (P-MLC) were assessed by immunohistochemistry and immunoblot.
Co-culture of Cytomix-stimulated epithelial monolayers with EGCs prevented increases in permeability and improved expression and localization of occludin, ZO-1, and P-MLC. Further, treatment of epithelial monolayers with GSNO also prevented Cytomix-induced increases in permeability and exhibited a similar improvement in expression and localization of occludin, ZO-1, and P-MLC.
CONCLUSIONS & INFERENCES: The addition of EGCs, or their secreted mediator GSNO, prevents epithelial barrier failure after injury and improved expression of tight junction proteins. Thus, therapies that increase EGC activation, such as VNS, may be a novel strategy to limit barrier failure in patients following severe injury.
肠屏障功能衰竭可导致严重创伤患者全身炎症和远隔器官损伤。肠神经胶质细胞（EGC）通过分泌 S-亚硝基谷胱甘肽（GSNO），在维持肠道屏障完整性方面发挥重要作用。我们最近发现，迷走神经刺激（VNS）增加 EGC 激活，与改善肠道屏障完整性有关。因此，我们试图利用体外模型进一步研究 EGC 防止肠道屏障破坏的机制。我们推测，EGC 通过分泌 GSNO，通过改善肠道紧密连接蛋白的表达和定位，改善肠道屏障功能。
上皮细胞与 EGC 共培养或用 GSNO 孵育，然后用细胞因子混合物（TNF-α、IFN-γ、IL-1β）孵育 24 小时。通过 4kDa FITC-右旋糖酐的通透性评估屏障功能。通过免疫组化和免疫印迹评估紧密连接蛋白 ZO-1、occludin 和磷酸化肌球蛋白轻链（P-MLC）的变化。
共培养细胞因子刺激的上皮细胞单层与 EGC 可防止通透性增加，并改善 occludin、ZO-1 和 P-MLC 的表达和定位。此外，上皮细胞单层用 GSNO 处理也可防止细胞因子混合物诱导的通透性增加，并表现出 occludin、ZO-1 和 P-MLC 表达和定位的类似改善。
添加 EGC 或其分泌的介质 GSNO 可防止损伤后上皮屏障功能衰竭，并改善紧密连接蛋白的表达。因此，增加 EGC 激活的治疗方法，如 VNS，可能是限制严重创伤后患者屏障功能衰竭的一种新策略。
