| 摘要: |
| 目的 观察黄芪多糖对大鼠小肠隐窝上皮IEC-6细胞迁移过程中多胺信号通路Ca2+调节指标的影响，并探讨黄芪促进胃肠黏膜损伤修复的作用机制。方法 采用划痕法建造细胞迁移模型并观察黄芪多糖在无钙培养时对细胞迁移的影响；采用qPCR法检测经典瞬时受体电位通道1（TRPC1）、基质交感分子（STIM）1、STIM2 mRNA表达；采用免疫荧光法检测STIM1蛋白的分布及表达；采用蛋白质印迹法检测TRPC1、STIM1和STIM2蛋白表达；采用免疫沉淀法检测STIM1/TRPC1、STIM1/STIM2蛋白复合体的表达。结果 无钙培养（去除胞外Ca2+内流来源）减弱了黄芪多糖促进细胞迁移的作用（P<0.01）。黄芪多糖能提高TRPC1 mRNA和蛋白的表达（P<0.05或P<0.01）、逆转二氟甲基鸟氨酸（DFMO）所致的TRPC1 mRNA和蛋白表达降低（P<0.01）；黄芪多糖能促进STIM1向胞膜移位并提高其表达水平，改善DFMO所致的STIM1向胞膜移位延迟及表达抑制；黄芪多糖能提高STIM1 mRNA和蛋白表达、逆转DFMO对STIM1 mRNA和蛋白表达的抑制（P<0.05或P<0.01）；黄芪多糖能降低STIM2 mRNA和蛋白表达，逆转DFMO对STIM2 mRNA和蛋白表达的提高（P<0.05或P<0.01）。黄芪多糖能提高STIM1/TRPC1的表达水平，逆转DFMO对STIM1/TRPC1表达的抑制，还可通过提高复合体中STIM1表达水平、降低STIM2表达水平调节STIM1/STIM2表达，并能拮抗DFMO对STIM1/STIM2表达的影响。结论 黄芪多糖促进IEC-6细胞迁移的作用与其影响Ca2+调节蛋白及蛋白复合体表达有关。 |
| 关键词: 黄芪多糖 肠上皮细胞 细胞迁移 多胺 Ca2+调节 |
| DOI：10.16781/j.CN31-2187/R.20210760 |
| 投稿时间：2021-03-08修订日期：2022-12-04 |
| 基金项目:国家自然科学基金（81673940），广州市科技计划项目（201607010335），广州中医药大学一流学科重点项目（［2020］62号）. |
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| Astragalus polysaccharides promoting migration of rat intestinal epithelial cells via Ca2+ regulatory proteins and their complexes |
| ZENG Dan,WANG Guan-yu,LI Zhi-jin,WU Ting-ting,HU Ling,LI Ru-liu* |
(Pi-wei Institute, Science and Technology Innovation Center, Guangzhou University of Chinese Medicine, Guangzhou 510405, Guangdong, China
*Corresponding author) |
| Abstract: |
| Objective To observe the effect of Astragalus polysaccharides on the Ca2+ regulatory indexes of polyamine signaling pathway during the migration of rat intestinal epithelial cell line IEC-6 cells, so as to explore the mechanism by which Astragalus membranaceus promoting the repair of gastrointestinal mucosal injury. Methods The cell migration model was established by scratch method and the effect of Astragalus polysaccharide on cell migration was observed in calcium-free culture. The expression of transient receptor potential channel 1 (TRPC1), stromal interaction molecule (STIM)1 and STIM2 mRNA was determined by quantitative polymerase chain reaction. The distribution and expression of STIM1 protein were detected by immunofluorescence. The expression of TRPC1, STIM1 and STIM2 was detected by Western blotting. The expression of STIM1/TRPC1 and STIM1/STIM2 complexes was detected by immunoprecipitation. Results The promoting effect of Astragalus polysaccharides on cell migration was attenuated in calcium-free culture (removing the source of extracellular Ca2+ influx) (P<0.01). Astragalus polysaccharides had promoting effects on TRPC1 mRNA and protein expression (P<0.05 or P<0.01), and had reversal effects on TRPC1 mRNA and protein expression in difluoromethylornithine (DFMO) treated cells (P<0.01). Astragalus polysaccharide promoted the translocation of STIM1 to the plasma membrane and increased STIM1 protein expression, and reversed the translocation of STIM1 to the plasma membrane and the inhibition effect which was found in DFMO-treated cells. Astragalus polysaccharides increased the expression of STIM1 mRNA and protein and reversed the inhibition effects on STIM1 mRNA and protein expression induced by DFMO (P<0.05 or P<0.01). In addition, Astragalus polysaccharides reduced the expression of STIM2 mRNA and protein and reversed the increased expression of STIM2 mRNA and protein induced by DFMO (P<0.05 or P<0.01). Astragalus polysaccharides increased the expression of STIM1/TRPC1 complex and reversed the inhibition effect of DFMO on STIM1/TRPC1 complex expression, modulated the expression of STIM1/STIM2 by increasing STIM1 expression and reducing STIM2 expression, and antagonized the effect of DFMO on STIM1/STIM2 expression. Conclusion The role of Astragalus polysaccharides in promoting the migration of IEC-6 cells is related to its effect on the expression of Ca2+ regulatory protein and protein complex. |
| Key words: Astragalus polysaccharides intestinal epithelial cells cell migration polyamine Ca2+ regulation |
