四君子汤多糖对小肠上皮细胞迁移多胺信号通路钙离子调控的影响

Effects of Sijunzi Decoction polysaccharide on polyamine mediated calcium signaling pathway during intestinal epithelial cell migration

目的:观察四君子汤多糖对小肠上皮IEC-6细胞迁移多胺信号通路钙离子调控的影响,探讨四君子汤促进胃肠黏膜损伤修复的作用机制。方法:在IEC-6细胞实验中,设正常对照组,阳性对照组,四君子汤多糖低、中、高剂量组(40、80、160mg/L);负荷实验则设模型组(α-二氟甲基鸟氨酸,DFMO),各用药组在加受试药同时加入DFMO;划痕法制造细胞迁移模型;相差倒置显微镜观察细胞迁移情况;HPLC法测定细胞内多胺(精脒和精胺)含量;RT-q PCR和Western Blot法分别检测瞬时受体电位通道1(TRPC1)m RNA和蛋白表达;Western Blot法检测磷脂酶C-γ1(PLC-γ1)蛋白表达;酶联免疫法检测三磷酸肌醇(IP3)含量;流式细胞仪检测细胞内游离钙离子浓度([Ca^(2+)]cyt);无钙培养条件是以不含钙离子的细胞培养液代替常规的含钙细胞培养液。结果:与正常对照组比较,四君子汤多糖各剂量组可促进细胞迁移、增加细胞内精脒和精胺含量、提高TRPC1 m RNA及蛋白表达、提高PLC-γ1蛋白表达和IP3含量、增加[Ca^(2+)]cyt(P<0.05,P<0.01);与模型组比较,四君子汤多糖各剂量组可逆转DFMO所致的细胞迁移抑制、细胞多胺含量降低、TRPC1 m RNA和蛋白表达降低、PLC-γ1蛋白表达和IP3含量降低、[Ca^(2+)]cyt降低(P<0.05,P<0.01);与正常对照组(含钙培养)比较,无钙培养可致细胞迁移抑制(P<0.01);与正常对照组(无钙培养)比较,各剂量四君子汤多糖可改善无钙培养所致的细胞迁移抑制(P<0.05,P<0.01),但不能使细胞迁移恢复正常水平。结论:四君子汤多糖促进细胞迁移与其作用于多胺调控信号通路有关,其中对Ca^(2+)调控是其关键指标。

Objective： To investigate the effects of Sijunzi Decoction polysaccharide（SJZDP） on polyamine mediated calcium signaling pathway during intestinal epithelial cells（IEC-6） migration, and to explore the mechanism of SJZDP on promoting gastrointestinal mucosal restitution after wounding. Methods： IEC-6 cells were divided into normal control group, spermidine positive group, SJZDP low, middle, high dose groups（40, 80, 160mg/L）, and α-difluoromethylornithine（DFMO） model group. DFMO model group was added in DFMO, other groups were treated with positive drugs or SJZDP as well as DFMO. Cell migration model was established by scratch damage; cell migration was observed by phase contrast microscope, polyamines contents including spermidine（SPD） and spermine（SPM） were determined by HPLC; the expression of transient receptor potential canonical 1（TRPC1） m RNA and protein levels were detected by RT-q PCR and Western Blot respectively; the protein expression level of phospholipase C-γ1（PLC-γ1） was also detected by Western Blot; the content of inositol triphosphate（IP3） was detected by enzyme-linked immunosorbent assay（ELISA）; cytosolic free Ca^（2＋） concentration（[Ca^（2＋）]cyt） was detected by flow cytometry; under no calcium culture condition, normal cell culture medium with calcium was replaced with Ca^（2＋）-free medium. Results： SJZDP（40, 80, 160mg/L, the same below） could promote cell migration, increase cell SPD and SPM contents, improve TRPC1 m RNA and protein expression levels, enhance the PLC-γ1 protein level, and IP3 content, and cause an elevation of [Ca^（2＋）]cyt compared with normal control group（P〈0.05, P〈0.01）; compared with DFMO model group, SJZDP could reverse the inhibition of cell migration, cell polyamines contents, TRPC1 m RNA and protein expression level, PLC-γ1 protein expression level, IP3 content, and [Ca^（2＋）]cyt induced by DFMO（P〈0.05, P〈0.01）; compared with normal Ca^（2＋） medium control group, cell migration was inhibited by culturing with Ca^（2＋）-free medium（P〈0.01）, treatment with SJZDP improved the cell migration inhibited by culturing with Ca^（2＋）-free medium compared with Ca^（2＋）-free medium control group（P〈0.05, P〈0.01）, but could not return to the normal level. Conclusion： SJZDP accelerating cell migration was related to its effect on polyamines dependent signaling pathway, and calcium signaling pathway was the key indicator.