淫羊藿苷对MC3T3-E1细胞增殖分化的影响及机制研究

Effects of icariin on proliferation and differentiation of MC3T3-E1 cells and its mechanism

目的研究淫羊藿苷(ICA)对小鼠颅顶前成骨细胞(MC3T3-E1)增殖、分化的影响,以及骨形态发生蛋白2(BMP-2)/Smads信号通路在成骨细胞增殖、分化中的作用。方法用含不同浓度ICA(0、10、20、40 ng/ml)的完全培养液分别培养MC3T3-E1细胞48、72、96 h。HE染色法观察ICA对MC3T3-E1细胞形态的影响;分别用CCK-8及碱性磷酸酶(ALP)检测试剂盒检测ICA对MC3T3-E1细胞增殖活性和细胞ALP活性的影响;Western blot检测ICA对MC3T3-E1细胞BMP-2、骨形态发生蛋白2受体(BMPR-2)、Smad4和Smad1/5/8蛋白表达水平的影响。向细胞培养液中加入BMP抑制剂头蛋白(Noggin,100 ng/ml)阻断BMP-2/Smads信号通路后,再次检测上述各实验指标的变化。结果 ICA对MC3T3-E1细胞形态无明显影响;ICA(10、20 ng/ml)可增强MC3T3-E1细胞增殖活性( P 均<0.01)。ICA(10、20 ng/ml)可增强MC3T3-E1细胞ALP活性( P 均<0.01)。阻断BMP-2/Smads信号通路后,ICA促细胞增殖作用( P <0.01)及促分化作用( P <0.01)明显下降。ICA(10、20 ng/ml)可提高MC3T3-E1细胞BMP-2、BMPR-2、Smad4和Smad1/5/8蛋白表达水平( P 均<0.01)。当阻断BMP-2/Smads信号通路后,ICA促BMP-2、BMPR-2、Smad4和Smad1/5/8蛋白表达作用均受到抑制。结论 ICA对MC3T3-E1细胞形态无明显影响,ICA可能是通过上调MC3T3-E1前体成骨细胞BMP-2蛋白的表达,激活BMP-2/Smads信号通路,启动下游成骨相关基因的表达,从而促进细胞的增殖、分化。

Objective To evaluate the role of icariin (ICA) in the proliferation and differentiation of MC3T3-E1 cells and to explore the effects of the BMP-2/Smads signal pathway on the proliferation and differentiation of the cells. Methods Icariin with different concentrations (0,10,20 and 40 ng/ml) was used to modify the pre-osteoblastic MC3T3-E1 cells for 48,72 and 96 h,and the proliferation and morphology of the cells were evaluated by cell counting kit-8 (CCK-8) and microscopy,respectively.The levels of alkaline phosphatase(ALP) was evaluated by alkaline phosphatase assay kit.Bone morphogenic protein-2 (BMP-2),bone morphogenic protein receptor-2 (BMPR-2),Smad4,Smadl/5/8 proteins expression levels were obtained by Western blot.After the BMP-2/Smads signal pathway was blocked by the BMP antagonist (Noggin)(100 ng/ml),the changes of the above indexes were detected again. Results In this study,we found that different concentrations of icariin have no effects on the morphology of MC3T3-E1 cells.CCK-8 assay showed that cell viability in the group with 10 and 20 ng/ml icariin was higher than those in the control group (ICA,0 ng/ml ).ALP assay showed that ALP viability in the groups with 10 and 20 ng/ml icariin was higher than those in the control group.Western blot analysis showed that expression levels of BMP-2,BMPR-2,Smad4 and Smadl/5/8 proteins in the 10 and 20 ng/ml icariin groups were higher than those in the control group.Blockage of BMP-2/Smads signaling inhibited the effect of icariin on the proliferation and differentiation.Furthermore,after the BMP-2/Smads signaling was blocked,icariin promoted expression levels of BMP-2,BMPR-2,Smad4 and Smadl/5/8 proteins as significantly decreased. Conclusion These results showed that icariin had no effect on the morphology of MC3T3-E1 cells.ICA may up-regulate the expression of BMP-2 protein in MC3T3-E1 precursor osteoblasts,activate BMP-2/Smads signaling pathway,and initiate the expression of downstream osteogenic related genes,thereby promoting cell proliferation and differentiation.