淫羊藿次苷Ⅱ通过激活雌激素信号通路促进骨髓间充质干细胞的成骨性分化

Promotion of the osteogenic differentiation of rat bone marrow stromal cells via estrogen signal pathway by icariside Ⅱ

目的研究淫羊藿次苷Ⅱ(icarisideⅡ,ICSⅡ)对大鼠体外培养骨髓间充质干细胞(rat bone marrow stromal cells,rBMSCs)成骨性分化过程中雌激素信号通路的影响。方法贴壁筛选法体外培养rBMSCs,采用1×10-5 mol.L-1的ICSⅡ进行药物干预,比较ICSⅡ组、ICI组(ICI 182.78)、ICSⅡ+ICI组、Estrogen组、Esreogen+ICI组和不加药的对照组之间雌激素通路相关因子(包括ERα、PR和PS-2)的表达量,同时对比各组之间的成骨性指标,包括碱性磷酸酶活性、骨钙素和Ⅰ型胶原分泌量及钙盐沉积量。提取TotalRNA,Real Time RT-PCR检测Runx-2、Osterix(OSX)、ERα、PR、及PS-2 mRNA的表达情况,同时提取总蛋白,Westernblot法检测Ⅰ型胶原蛋白、ERα、PR和PS-2的分泌量。结果 ICSⅡ可明显增强碱性磷酸酶(ALP)活性,促进骨钙素、Ⅰ型胶原蛋白的分泌和钙盐沉积,与成骨性分化相关的因子OSX和Runx-2的基因表达量也升高,但这些效应均可被雌激素通路的特异性阻断剂ICI 182.780所抑制。结论 ICSⅡ可促进rBMSCs的成骨性分化,但采用ICI 182.780阻断雌激素信号通路后,成骨性分化的指标随之降低,提示ICSⅡ是通过激活雌激素信号通路来促进rBMSCs成骨性分化的。

Aim To investigate the effects on the estrogen signal pathway in the osteogenic differentiation process of rat bone marrow stromal cells（rBMSCs） by icariside Ⅱ.Methods rBMSCs were cultured by adherence screening method,icariside Ⅱ was supplemented into the culture at 10-5 mol·L-1.The expression of estrogen signal pathway＇s factors（ERα,PR,PS-2） and osteogenic differentiation markers including alkaline phosphatase（ALP） activity,osteocalcin and calcium deposition were compared among the icariside Ⅱ-supplemented group,ICI group,icariside Ⅱ＋ICI group,estrogen group,estrogen＋ICI group and the control.Total RNA was isolated and the gene expression of ERα,PR,PS-2,Osterix and Runx-2 were investigated by Real Time RT-PCR.Total protein was also isolated and the secretion of ERα,PR,PS-2 and collagenⅠ were examined by Western blot.Results Icariside Ⅱ significantly improved ALP activity,calcium deposition and the secretion of collagen Ⅰ and osteocalcin.Besides,the mRNA level of factors related to the osteogenic differentiation including Osterix and Runx-2 also enhanced.But all of these effects were inhibited by ICI 182.780 which could specifically inhibit the estrogen signal pathway.Conclusions Icariside Ⅱ enhances the osteogenic differentiation of rBMSCs significantly,but the osteogenic differentiation markers decrease accompanied with the estrogen signal pathway inhibited by ICI 182.780,suggesting that icariside Ⅱ stimulates the osteogenic differentiation via activating the estrogen signal pathway.