淫羊藿苷通过雌激素受体和p38MAPK信号诱导MC3T3-E1Subclone14细胞的分化

摘要

目的 观察淫羊藿苷(ICA)对MC3T3-E1Subclone14前体成骨细胞株活力、分化的影响,以及雌激素受体(ER)信号、p38MAPK信号在分化过程中的作用.方法 WST-8方法检测MC3T3-E1Subclone14细胞活力；pNPP法检测细胞碱性磷酸酶活性(ALP)；ELISA检测Ⅰ型胶原(Col Ⅰ)和骨钙素(BGP)；Western印迹法检测p38MAPK的蛋白磷酸化；并分别用ICI182780阻断ER受体或SB203580阻断p38 MAPK信号后检测ICA对细胞ALP、Col Ⅰ、BGP的影响；Western印迹法检测ICI182780阻断ER受体信号后,ICA对p38MAPK蛋白磷酸化的影响.结果 ICA(10-7、10-6、10-5 mol/L)对细胞活力与对照组比较,在统计学上无显著差异(P＞0.05)；ICA可以浓度依赖性的提高细胞的ALP、ColⅠ和BGP和矿化结节数量(P＜0.01,P＜0.05)；ICI182780阻断ER受体信号后,10-5 mol/L浓度的ICA促细胞分化的特性明显下降(P＜0.01)；ICA可以浓度组依赖性的提高细胞p38MAPK的蛋白磷酸的水平(P＜0.01);SB203580阻断p38MAPK信号后,10-5mol/L浓度的ICA促分化的特性下降(P＜0.01)；ICI182780阻断ER受体信号后,10-5 mol/L浓度ICA促p38MAPK磷酸化明显减弱(P＜0.01).结论 ICA可以促进MC3T3-E1 Subclone14细胞分化,ER受体信号、p38MAPK信号在促分化过程中起着重要作用,ER受体信号通路在p38MAPK信号通路的上游.%Objective To observe the effects of Icariin on the proliferation and differentiation of MC3T3-ElSubclonel4, a pre-osteo-blasts cell line, and observe the role of estrogen receptor (ER) and p38MAPK signaling pathways during the differentiation. Methods WST-8 method was used to observe cell viability, pNPP method was used to observe alkaline phosphatase (ALP) activity, ELISA kit was used to observe type I collagen and osteocalcin activity and Western-blot was used to observe p38MAPK protein phosphorylation after treatment with different concentration of Icariin. After ER signaling pathways was blocked by ICI182780 or p38MAPK signaling pathways was blocked by SB2O358O, ALP, type I collagen and osteocalcin of MC3T3-ElSubclonel4 cell were observed after treatment with Icariin. Westem-blot was used to observe p38MAPK protein phosphorylation after ER signaling pathways was blocked by ICI182780 after treatment with Icariin. Results Cell viability of Icariin groups had no difference with control group after 48 and 72 h (P>0.05). ALP, type I collagen , osteocalcin and mineralized nodus of Icariin groups were higher than those of control group ( P < 0. 01, P < 0.05 ). ALP, type I collagen and osteocalcin of experimental group (Icariin + 1CI182780) were less than those of Icariin group (P <0.01). Icariin increased the protein relative expression of phospho-p38MAPK in a concentration-dependent manner (P <0.05, P <0.01). ALP, type I collagen and osteocalcin of experimental group (Icariin + SB203580) were less than those of Icariin group (f<0.01, P<0.05). p38MAPK protein phosphorylation of Icariin-induced was decreased significantly after ER signaling pathways was blocked by IC1182780. Conclusions Icariin can increase differentiation of MC3T3-E1 Subclonel4 cells via the ER and p38MAPK signaling. ER signaling may be upstream of p38MAPK signaling pathways in the differentiation process.