黄芪多糖对IEC-6细胞迁移多胺信号通路相关指标的影响

Effects of polysaccharides from Astragalus membranaceuson polyamines mediated signaling pathway during intestinal epithelial cell(IEC-6) migration

目的:观察黄芪多糖对大鼠小肠上皮细胞(IEC-6)迁移多胺信号通路的影响,探讨益气健脾中药黄芪促进胃肠黏膜损伤修复的作用机制。方法:在划痕损伤致细胞迁移模型上观察黄芪多糖对细胞迁移的影响;流式细胞仪检测细胞膜电位(Em)和细胞内游离钙离子水平([Ca2+]cyt);Western blot法检测钾通道蛋白Kv1.1和RhoGTPaes(Cdc42、Rac1、Rho A)蛋白表达。结果:黄芪多糖(20、40、80mg/L,下同)有以下作用:(1)促进细胞迁移(P<0.01),逆转α-二氟甲基鸟氨酸(DFMO)所致的细胞迁移抑制(P<0.01);(2)提高细胞迁移过程Kv1.1蛋白表达、逆转DFMO所致的Kv1.1蛋白表达抑制(P<0.05,P<0.01);(3)促进Em超极化、逆转DFMO所致的Em去极化(P<0.05,P<0.01);(4)提高细胞迁移过程[Ca2+]cyt、逆转DFMO所致的[Ca2+]cyt降低(P<0.01);(5)提高细胞迁移过程Cdc42、Rac1、Rho A蛋白表达、逆转DFMO的抑制作用(P<0.05,P<0.01)。结论:黄芪多糖促进IEC-6细胞迁移与影响多胺信号通路相关指标有关。

Objective: To investigate the effects of polysaccharides from Astragalus membranaceus(APS) on polyamines mediated signaling pathway during rats' intestinal epithelial cell(IEC-6) migration, and to explore the mechanism of APS on promoting gastrointestinal mucosal repair after injury. Methods: Cell migration was observed based on the cell migration model of IEC-6 cells induced by a single-edge razor blade. The cell membrane potential(Em) and the cytosolic free Ca2+ concentration([Ca2+]cyt) were determined by flow cytometry. The protein expressions of Kv1.1 and Rho-GTPases(Rho A, Cdc42, Rac1) were assessed by Western Blot. Results:(1)Compared with the control group, administrations of 20, 40 and 80mg/L dosage of APS caused an acceleration in cell migration(P<0.01), and reversed inhibition of cell migration induced by polyamines inhibitor DFMO(P<0.01).(2)Treatment with APS caused an elevation on the protein expression of Kv1.1, and the inhibitory expression of Kv1.1 protein induced by DFMO was reversed(P<0.05, P<0.01).(3)Treatment with APS could promote hyperpolarization of Em and reverse the Em depolarization induced by DFMO(P<0.05, P<0.01).(4)Treatment with APS could promote [Ca2+]cyt, and reverse the decrease of [Ca2+]cyt induced by DFMO(P<0.01).(5)APS could increase the protein expressions of Cdc42, Rac1 and Rho A, and reverse the inhibitory effect of DFMO(P<0.05, P<0.01). Conclusion: APS promoting intestinal epithelial cell migration is related to its role of regulating the related targets in polyamine mediated signaling pathway.