基于miR-584调控内质网应激途径安胃汤干预胃黏膜肠上皮化生作用机制研究

Exploring the Mechanism of An-wei Decoction Intervention on Gastric Intestinal Epithelial Metaplasia Through miR-584 Regulation of Endoplasmic Reticulum Stress Pathway

目的探讨安胃汤基于miR-584调控内质网应激途径干预胃黏膜肠上皮化生细胞的作用机制。方法运用鹅去氧胆酸诱导人胃黏膜GES-1细胞系构建胃黏膜肠上皮化生细胞模型;实验动物分为正常组、模型组、阴性对照组、安胃汤组,在此研究基础上设置miR 584 inhibitor组、miR 584 inhibitor NC组、miR 584 mimics组、miR 584 mimics NC组,分别进行瞬时转染后给予安胃汤含药血清干预。采用激光扫描共聚焦显微镜观察ER-Tracker Red变化,蛋白免疫印迹法检测内质网应激相关蛋白Caspase 12、GRP78、ATF6、PERK、CHOP表达情况和肠上皮化生相关蛋白CDX2、MUC2、KLF4、SOX2的表达。结果安胃汤作用于胃黏膜肠上皮化生细胞,下调CDX2、MUC2、KLF4蛋白表达(P<0.01),上调SOX2表达(P<0.01),显著增强内质网相对荧光强度(P<0.01),显著上调肠上皮化生细胞中内质网应激相关蛋白Caspase 12、GRP78、ATF6、PERK、CHOP的表达(P<0.01);转染后给药的胃黏膜肠上皮化生细胞,miR-584 inhibitor组CDX2、MUC2、KLF4蛋白表达显著下调(P<0.01),SOX2表达显著上调(P<0.01),miR-584 inhibitor组内质网相对荧光强度显著上调(P<0.05),内质网应激相关蛋白表达显著上调(P<0.01)。结论减低肠上皮化生细胞miR-584表达并给予安胃汤干预后可促进内质网应激的发生,安胃汤可通过miRNA-584调控激活内质网应激途径改善胃黏膜肠上皮化生。

Objective Explore the mechanism of Anwei Decoction's intervention on gastric mucosal intestinal metaplasia cells based on miR-584 regulation of the endoplasmic reticulum stress pathway.Methods A human gastric mucosal cell line GES-1 was induced by deoxycholic acid of goose to construct a model of gastric mucosal intestinal metaplasia cells.The cells were initially divided into four groups:GES-1 group,Model group,Negative control group,and An-wei Decoction group.Additional groups were established,including miR 584 inhibitor group,miR 584 inhibitor NC group,miR 584 mimics group,and miR 584 mimics NC group.Each group underwent transient transfection and was treated with Anwei Decoction-containing serum.Changes in ER-Tracker Red were observed using a laser scanning confocal microscope.Western blot was used to detect the expression levels of endoplasmic reticulum stress-related proteins,including Caspase 12,GRP78,ATF6,PERK,and CHOP,as well as gastric intestinal metaplasia-related proteins,including CDX2,MUC2,KLF4,and SOX2.Results Anwei decoction acts on gastric intestinal metaplasia cells,downregulating the expression of CDX2,MUC2,KLF4 protein(P<0.01)and upregulating the expression of SOX2(P<0.01).It significantly enhances the relative fluorescence intensity of the ER(P<0.01)and upregulates the expression of endoplasmic reticulum stress-related protein Caspase 12,GRP78,ATF6,PERK,CHOP(P<0.01)in gastric intestinal metaplasia cells.In transfected and treated A cells,the protein expression of CDX2,MUC2,and KLF4 are significantly downregulated(P<0.01),while the expression of SOX2 is significantly upregulated(P<0.01)in the miR-584 inhibitor group.The relative fluorescence intensity of the ER is significantly increased(P<0.05),and the expression of B-related proteins is significantly upregulated(P<0.01)in the miR-584 inhibitor group.Conclusion Reducing the expression of miR-584 in gastric intestinal metaplasia cells and intervening with Anwei decoction can promote the occurrence of ER stress.Anwei decoction can improve gastric intestinal metaplasia by regulating the activation of the ER stress pathway through miRNA-584.