四君子汤含药血清对IEC-6细胞增殖及RhoA、p21、Cdk2表达的影响

Effects of Sijunzi Decoction-medicated serum on IEC-6 cell proliferation and RhoA, p21 and Cdk2 expressions

目的 探究四君子汤含药血清对IEC-6细胞增殖及Rho GTP酶(RhoA)、p21、细胞周期蛋白依赖性激酶2(Cdk2)表达的影响。方法 分别制备大鼠空白血清及四君子汤含药血清,MTT法检测IEC-6细胞增殖,RT-qPCR法检测p21、Cdk2 mRNA表达,Western blot法检测RhoA、p21、Cdk2及磷酸化Cdk2(p-Cdk2)蛋白表达。结果 与对照组比较,10%、20%四君子汤含药血清可促进给药24 h后细胞增殖(P<0.01),提高RhoA蛋白表达,Cdk2 mRNA和蛋白表达(P<0.05,P<0.01);各剂量四君子汤含药血清可促进给药48、72 h后细胞增殖(P<0.01),降低p21 mRNA和蛋白表达(P<0.01),升高p-Cdk2蛋白表达(P<0.05,P<0.01)。与DFMO组比较,各剂量四君子汤含药血清可促进给药48 h后细胞增殖(P<0.01),提高RhoA及p-Cdk2蛋白表达(P<0.01);10%、20%四君子汤含药血清可促进给药72 h后细胞增殖(P<0.01),降低p21 mRNA和蛋白表达(P<0.05,P<0.01),提高Cdk2 mRNA和蛋白表达(P<0.05,P<0.01)。结论 四君子汤含药血清可促进正常小肠上皮细胞增殖,其机制与影响多胺调控RhoA、p21、Cdk2表达有关。

AIM To investigate the effects of Sijunzi Decoction-medicated serum on IEC-6 cell proliferation and expressions of Rho GTPases(RhoA),p21 and cyclin-dependent kinase 2(Cdk2).METHODS The SD rats serum with or without Sijunzi Decoction were both subjected to the detection of IEC-6 cell proliferation by MTT assay;the test of mRNA expressions of p21 and Cdk2 by RT-qPCR;the determination of protein expressions of RhoA,p21,Cdk2 and phospho-Cdk2(p-Cdk2)by Western blot.RESULTS Compared with the control group,the groups given 10%and 20%Sijunzi Decoction-medicated serum shared accelerated cell proliferation 24 h after administration(P<0.01),increased RhoA protein expression,and mRNA and protein expressions of Cdk2(P<0.05,P<0.01);all the groups given Sijunzi Decoction-medicated serum displayed promoted cell proliferation 48 h and 72 h after administration(P<0.01),decreased mRNA and protein expressions of p21(P<0.01)and improved p-Cdk2 protein expression(P<0.05,P<0.01).Compared with DFMO group,Sijunzi Decoction-medicated serum at all doses brought forth promoted cell proliferation 48 h after administration(P<0.01)and increased protein expressions of RhoA and p-Cdk2(P<0.01),which at 10%and 20%concentrations increased cell proliferation 72 h after administration(P<0.01),inhibited p21 mRNA and protein expressions and enhanced mRNA and protein expressions of Cdk2(P<0.05,P<0.01).CONCLUSION Sijunzi Decoction-medicated serum promote the proliferation of normal small intestinal epithelial cells,which may be related to the impact of its polyamines on the regulation of RhoA,p21 and Cdk2 expressions.