黄芪多糖调控多胺介导的Ca^(2+)-RhoA通路促进小肠上皮细胞迁移研究

Promotion of Astragalus membranaceus polysaccharides on intestinal epithelial cell migration via polyamine mediated Ca^(2+)-Rho A signaling pathway

目的考察黄芪多糖对小肠上皮细胞(IEC-6)迁移及迁移过程细胞内多胺水平、细胞质游离钙离子([Ca2+]cyto)的量、细胞骨架蛋白Rho A表达的影响,探讨黄芪修复胃肠黏膜损伤的疗效机制。方法黄芪药材经水提醇沉、Sevage法去蛋白后得到黄芪粗多糖,运用DEAE纤维素柱分离得到黄芪多糖I、II、III、IV,黄芪多糖I以Sephadex LH-20凝胶柱色谱得到黄芪多糖V。Tips划痕法建立IEC-6细胞损伤迁移模型,柱前衍生高效液相色谱法测定多胺的量,流式细胞仪检测[Ca2+]cyto水平,Western blotting法检测Rho A蛋白表达。考察黄芪多糖对正常IEC-6细胞[未加α-二氟甲基鸟氨酸(DFMO)]和多胺耗竭IEC-6细胞(加入DFMO)迁移、多胺水平、[Ca2+]cyto的量及Rho A蛋白表达的影响。结果黄芪粗多糖、黄芪多糖I、黄芪多糖V(50、100、200 mg/L)均能促进IEC-6细胞迁移(P<0.01),并且可逆转多胺合成抑制剂DFMO所致的细胞迁移抑制效果(P<0.01);在正常(未加DFMO)或多胺耗竭情况(加DFMO),黄芪多糖V均可提高迁移过程细胞内多胺的量(P<0.01);黄芪多糖V可提高细胞迁移过程[Ca2+]cyto水平(P<0.01),并且可逆转DFMO所致的[Ca2+]cyto水平下降(P<0.01);黄芪多糖V(100、200 mg/L)可明显提高Rho A蛋白表达,同时可逆转DFMO所导致的Rho A蛋白表达抑制作用。结论黄芪多糖可加速胃肠黏膜损伤早期修复过程,其机制可能与增加黏膜上皮细胞多胺的量,提升[Ca2+]cyto,从而提高Rho A蛋白表达,进而促进胃肠黏膜上皮细胞迁移有关。

Objective To study the effect of Astragalus membranaceus polysaccharides （AMP） on migration,intracellular polyamines content,cytosolic free Ca2＋ concentration （[Ca2＋]cyto）,and RhoA protein expression of intestinal epithelial （IEC-6） cells,so as to explore the repairing mechanism of Astragalus membranaceus （AM） on gastrointestinal injury.Methods AM was extracted by water and precipitated by ethanol,AM crude polysaccharides were obtained after removing protein.AMP I,II,III,and IV were obtained by DEAE cellulose column chromatography.AMP V was further obtained by Sephadex LH-20 gel column chromatography from AMP I.Cell migration model was established by Tips scratch method;High performance liquid chromatography was used to determine the polyamines content;Flow cytometry was used to detect the[Ca2＋]cyto;Western blotting analysis was used to detect RhoA protein expression.The improving effect of AMP on migration,[Ca2＋]cyto,and RhoA protein expression of normal and polyamine-depleted IEC-6 cells was observed.Results AM crude polysaccharides,AMP I,and AMP V （100 mg/L or 200 mg/L） promoted cell migration and reversed the inhibition of cell migration induced by DFMO （P〈0.01）;AMP V increased the intracellular polyamines content in normal and polyamine-deficient IEC-6 cells;AMP V also enhanced[Ca2＋]cyto in the process of IEC-6 cell migration and reversed the reduction of[Ca2＋]cyto induced by DFMO （P〈0.01）;Further study suggested that AMP V increased RhoA protein expression in normal and polyamine-deficient IEC-6 cells （P〈0.01）.Conclusion These results indicate that the repairing effect of AM on the gastrointestinal mucosa damage may be related to its role of increasing polyamines content,then improving[Ca2＋]cyto and RhoA protein expression and thereby promoting cell migration.