四君子汤多糖对IEC-6细胞迁移、钾通道蛋白及膜电位的影响

Effect of Sijunzi Decoction Polysaccharide on IEC-6 Cell Migration,Potassium Channel and Membrane Potential

目的:观察四君子汤多糖对小肠上皮IEC-6细胞迁移及多胺调控信号通路钾通道蛋白和膜电位的影响,探讨四君子汤修复胃肠黏膜损伤的作用机制。方法:划痕法建立IEC-6细胞迁移模型,观察正常培养及α-二氧甲基乌氨酸(DFMO,多胺合成抑制剂)或4-氨基吡啶(4-AP,钾通道抑制剂)负荷下四君子汤多糖对IEC-6细胞迁移的影响;分别以荧光定量PCR法和Western blot法检测四君子汤多糖对IEC-6细胞钾通道蛋白kv1.1 mRNA和蛋白表达的影响;流式细胞仪检测四君子汤多糖对IEC-6细胞膜电位的影响。结果:与空白对照组比较,四君子汤多糖(40、80、160 mg/L)可促进IEC-6细胞迁移,提高IEC-6细胞kv1.1 mRNA和蛋白表达水平及细胞膜电位超极化水平(P<0.05或P<0.01);与DFMO模型组比较,四君子汤多糖(40、80、160 mg/L)可逆转DFMO所致细胞迁移数减少、kv1.1 mRNA和蛋白表达的降低以及细胞膜电位去极化(P<0.05或P<0.01);与4-AP模型组比较,四君子汤多糖(20、40、80 mg/L)可逆转4-AP所致细胞迁移的抑制、kv1.1 mRNA和蛋白表达的降低(P<0.05或P<0.01)。结论:四君子汤多糖可促进IEC-6细胞迁移,作用机制与影响多胺调控信号通路钾通道蛋白表达和细胞膜电位有关。

Objective： To investigate the effect of Sijunzi decoction polysaccharide（ SJZDP） on intestinal epithelial cells（ IEC-6）cell migration and polyamine signaling pathway potassium channel during intestinal epithelial cell migration,and to explore the mechanism of SJZDP on promoting gastrointestinal mucosal restitution after wounding. Methods： Cell migration model was established by scratch damage,and then the effect of SJZDP normal cultured or with difluoromethylornithine（ DFMO） and 4-aminopyridine（ 4-AP） on IEC-6 cell migration was observed and calculated on this wounding model. The effect of SJZDP on expression of IEC-6 cell kv1. 1 mRNA and protein levels were detected by RT-q PCR and Western blot analysis,respectively. The Effects of SJZDP on IEC-6 cell membrane potential were detected by flow cytometry. Results： The results showed that treatment with SJZDP（ 40,80,160 mg / L） caused a promotion of IEC-6 cell migration,and increased of expression of in IEC-6 cell kv1. 1 mRNA and protein significantly（ P〈0.05 or P〈0.01） compared with normal control group. In addition,SJZDP（ 40,80,160 mg / L） increased cell membrane potential which resulted in cell membrane hyperpolarization compared with normal control group（ P〈0.05 or P〈0.01）. SJZDP（ 40,80,160 mg / L） reversed the inhibition of cell migration was reduced,kv1. 1 mRNA,kv1. 1 protein expression,and cell membrane potential were decreased by polyamines synthesis inhibitor DFMO compared with DFMO model group（ P〈0.05 or P〈0.01）. SJZDP（ 20,40,80 mg / L） reversed the inhibition of cell migration,kv1. 1 protein and mRNA levels expression were decreased by potassium channel inhibitor 4-AP compared with 4-AP model group（ P〈0.05 or P〈0.01）. Conclusion： These results indicate that the effect of SJZDP on promoting IEC-6 cell migration may be related to its influence on polyamine signaling pathway potassium channel and cell membrane potential.