肠三叶因子介导PI3K/Akt信号通路保护胃黏膜上皮细胞紧密连接

Protective effects of intestinal trefoil factor on gastric mucosal epithelial tight junction via activating PI3K/Akt signaling pathway

目的:探讨肠三叶因子对胃黏膜上皮细胞紧密连接的保护作用,并研究PI3K/Akt信号通路在其中的作用机制。方法:体外培养GES-1细胞,分别设正常对照组,LPS组,ITF组,LPS+ITF组,LPS+ITF+LY294002组,ITF+LY294002组。正常对照组:正常培养;LPS组:加入浓度为10mg/L的LPS;ITF组:加入浓度为100μg/L的ITF;LPS+ITF组:加入浓度为10mg/L的LPS,同时加入100μg/L的ITF;LPS+ITF+LY294002组:加入浓度为10mg/L的LPS、100μg/L的ITF,同时加入PI3K/Akt信号通路的抑制剂LY294002(15μM);ITF+LY294002组:加入100μg/L的ITF,同时加入15μM的LY294002。培养48h,采用Western blot检测ITF对PI3K/Akt信号通路的作用,采用免疫荧光和Western blot检测细胞紧密连接蛋白Occludin和ZO-1的变化情况。结果:Western blot检测结果说明,与对照组相比,ITF提高了pAkt蛋白的表达水平,而LY294002抑制了ITF激活的pAkt蛋白的表达,说明ITF可以通过激活PI3K/Akt信号通路来调控GES-1细胞的生理活动。免疫荧光和Western blot结果显示LPS导致GES-1细胞的紧密连接遭到破坏,降低紧密连接蛋白Occludin和ZO-1的表达水平,而ITF可以通过激活PI3K/Akt信号通路来保护GES-1细胞的紧密连接的完整性。结论:ITF保护胃黏膜上皮细胞紧密连接的完整性,提高紧密连接蛋白的表达水平,其发挥作用的主要分子机制是通过激活PI3K/Akt信号通路来实现的。

Objective：To explore the protective effects of intestinal trefoil factor （ITF） on gastric mucosal epithelial tight junction and to clarify the role of PI3K/Akt signaling. Method：Experiment groups were divided into 6 groups ,Control group; LPS group; ITF group; LPS＋ ITF group; LPS＋ ITF＋ LY294002 group; ITF＋ LY294002 group. Then the treated cells were cultured for 48 h and used in the following research. The expression of PI3K/ Akt signaling in GES-1 cells was analyzed Using western blot. Immunofluorescence staining and western blot were used to detect the expression of tight junction in GES-1 cells. Result：Western blot demonstrated that ITF could induce the increased expression of pAkt, but LY294002 inhibited the expression of pAkt. It indicated that ITF regu- lates GES-1 cells via activating PI3KA/Akt signaling. Immunofluorescence and western blot demonstrated that LPS led gastric mucosal epithelial tight junction damage and inhibited Occludin and ZO-1 expression. However, ITF could protect gastric mucosal epithelial tight junction integrity from LPS damage via activating PI3K/Akt signaling. Conclusion：ITF protects epithelial tight junction integrity,and its main molecular mechanisms may be accomplished by the activation of PI3K/Akt signaling.