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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 observe the effect of Sijunzi Decoction on the migration of IEC-6 cells and the potassium channel protein and membrane potential of polyamine signaling pathway in small intestine so as to explore the mechanism of Sijunzi Decoction in the repair of gastrointestinal mucosal injury. Methods: The IEC-6 cell migration model was established by scratch method. The effects of α-dioxymethylene (DFMO, polyamine synthesis inhibitor) or 4-aminopyridine (4-AP, potassium channel inhibitor) The effects of Sijunzi Decoction on the migration of IEC-6 cells were tested by flow cytometry. The effects of Sijunzi decoction on the expression of potassium channel protein kv1.1 mRNA and protein in IEC-6 cells were detected by fluorescence quantitative PCR and Western blot respectively. The effect of Sijunzi Decoction on membrane potential of IEC-6 cells was examined. RESULTS: Compared with the blank control group, Sijunzi Decoction (40,80,160 mg / L) could promote the migration of IEC-6 cells and increase the expression of kv1.1 mRNA and protein and the hyperpolarization of membrane potential in IEC-6 cells P <0.05 or P <0.01). Compared with the DFMO model group, Sijunzi Decoction (40,80,160 mg / L) could reverse the reduction of DFMO-induced cell migration, the decrease of kv1.1 mRNA and protein expression and the increase of membrane potential (P <0.05 or P <0.01). Compared with the 4-AP model group, Sijunzi Decoction (20,40,80 mg / L) could reverse the inhibition of cell migration induced by 4-AP, while kv1.1 mRNA And decreased protein expression (P <0.05 or P <0.01). Conclusion: Sijunzi Decoction can promote the migration of IEC-6 cells, and its mechanism is related to the effects of polyamine signaling pathway on the expression of potassium channel protein and cell membrane potential.