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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 observe the effects of Sijunzi Decoction on calcium ion regulation of polyamine signal pathway in small intestine epithelium of IEC-6 cells and to explore the mechanism of Sijunzi Decoction in promoting the repair of gastrointestinal mucosal injury. Methods: The normal control group, positive control group, Sijunzi Decoction low, medium and high dose groups (40,80,160 mg / L) were established in IEC-6 cell experiment. The model group (α-difluoride Methyl ornithine, and DFMO). DFMO was added into each treatment group with the test drug; the cell migration model was made by scratch method; the cell migration was observed by inverted phase contrast microscope; the intracellular polyamines (spermidine and spermine ). The mRNA and protein expression of TRPC1 were detected by RT-q PCR and Western Blot respectively. The protein expression of phospholipase C-γ1 (PLC-γ1) was detected by Western Blot and enzyme-linked immunosorbent assay The intracellular free calcium concentration ([Ca ~ (2 +)] cyt) was detected by flow cytometry. The calcium-free culture medium was prepared by substituting the calcium ion-free cell culture solution for the conventional Calcium cell culture solution. Results: Compared with the normal control group, Sijunzi decoction could promote cell migration, increase intracellular spermine and spermine content, increase TRPC1 mRNA and protein expression, increase PLC-γ1 protein expression and IP3 content, and increase [Ca (P <0.05, P <0.01). Compared with the model group, each dose of Sijunzi Decoction could reverse the inhibition of DFMO-induced cell migration, the decrease of polyamine content in cells, the expression of TRPC1 mRNA and protein (P <0.05, P <0.01) .Compared with normal control group (calcium-containing culture), calcium-free culture could induce cell migration (P <0.01). Compared with normal control group (no calcium culture), Sijunzi decoction could improve cell migration inhibition induced by calcium free culture (P <0.05, P <0.01), but could not restore cell migration normal level. Conclusion: Sijunzi Decoction promotes cell migration and its role in polyamine regulatory signaling pathway, of which regulation of Ca ~ (2+) is the key index.