四君子汤含药血清对IEC-6细胞增殖多胺/HuR信号通路的影响

Effects of Sijunzi decoction-containing serum on polyamine/HuR signaling pathway during IEC-6 cell proliferation

目的研究四君子汤(Sijunzi decoction-containing serum,)对小肠上皮细胞(IEC-6)增殖多胺/HuR信号通路生长相关基因蛋白表达及细胞周期的影响,探讨其对肠黏膜损伤修复的机制。方法SD大鼠制备四君子汤含药血清(Sijunzi decoction-containing serum,SJZD),免疫荧光法及免疫印迹法分析HuR蛋白在胞浆及胞核表达情况,RT-PCR法检测激活转录因子2(activating transcription factor-2,ATF-2),JunD,周期蛋白依赖性激酶4(cyclin dependent kinase 4,CDK4)mRNA表达,免疫印迹法检测HuR、ATF-2、JunD及CDK4蛋白表达,流式细胞仪分析细胞周期分布。结果与空白组比较,SZJD组ATF-2及JunD mRNA及蛋白表达降低,CDK4 mRNA及蛋白表达升高,G_(0)/G_(1)期细胞占比减少,S期细胞占比增加;α-二氟甲基鸟氨酸(DFMO)组细胞核HuR蛋白水平减少,细胞浆HuR蛋白水平增加,ATF-2及JunD mRNA及蛋白表达上调,Cdk4 mRNA及蛋白表达下调,G_(0)/G_(1)期细胞占比增加,S期细胞占比减少;SJZD组可拮抗DFMO的上述作用。结论SJZD作用于肠上皮细胞多胺/HuR信号通路,影响生长相关基因蛋白ATF-2、JunD及CDK4表达,可能是其促进肠黏膜损伤修复的机制之一。

Aim To investigate the effect of Sijunzi Decoction on mRNA and protein expression related to growth and cell cycle in polyamine/HuR signaling pathway during small intestinal epithelial cell(IEC-6)proliferation,and to explore its mechanism on intestinal mucosal injury repair.Methods Sijunzi Decoction-containing serum(SJZD)was prepared from SD rats,the expression of HuR protein in cytoplasm and nucleus was analyzed by immunofluorescence and Western blot,the mRNA level of activating transcription factor-2(ATF-2),JunD and cyclin dependent kinase 4(CDK4)were determined by real-time fluorescent quantitative PCR(RT-PCR),Western blot was used to detect protein level of HuR,ATF-2,JunD and CDK4,and flow cytometry was applied to analyse cell cycle distribution.Results Compared with the control group,the mRNA and protein expression of ATF-2 and JunD decreased,while the expression of Cdk4 mRNA and protein increased in SZJD group,and the proportion of G 0/G 1 phase cells decreased,while the proportion of S phase cells increased in SZJD group.Compared with the control group,α-difluorommethylornithine(DFMO)reduced the level of HuR protein in nucleus,enhanced the level of HuR protein in cytoplasm,up-regulated the mRNA and protein expressions of ATF-2 and JunD,down-regulated the expression of Cdk4 mRNA and protein,ascended the proportion of G_(0)/G_(1) phase cells,and descended the proportion of S phase cells.SJZD could antagonize the role of DFMO as described above.Conclusions Sijunzi Decoction acts on the polyamines/HuR signaling pathway in intestinal epithelial cells and regulates the expression of growth-related genes and proteins,such as ATF-2,JunD and CDK4,which may be one of the mechanisms of Sijunzi Decoction in promoting the repair of intestinal mucosal injury.