淫羊藿次苷Ⅱ促进大鼠骨髓间充质干细胞成骨性分化过程中iNOS活性和NO生成量的变化

The changes of iNOS and NO in the osteogenic differentiation process of rat bone marrow stromal cells promoted by icariside Ⅱ

研究淫羊藿次苷II(icariside II,ICS II)对大鼠体外培养骨髓间充质干细胞(rat bone marrow stromal cells,rBMSCs)成骨性分化过程中诱导性一氧化氮合酶(induced nitric oxide synthase,iNOS)表达及NO生成的影响。贴壁筛选法体外培养rBMSCs,待铺满80%皿底时,进行成骨性诱导培养,同时采用1×10-5 mol.L-1 ICS II进行药物干预,比较ICS II组、L-NAME组、ICS II+L-NAME组和不加药的对照组之间的iNOS的活性、NO生成量,对比各组之间的成骨性指标,包括碱性磷酸酶活性、碱性磷酸酶阳性克隆数(CFU-FALP)及钙化结节数量。提取总RNA,实时荧光定量RCR(real-time PCR)检测Osterix(OSX)、Runx-2及iNOS mRNA的表达情况;同时提取总蛋白,Western blotting法检测Ⅰ型胶原蛋白和iNOS的分泌量。ICS II可显著增强碱性磷酸酶(ALP)活性,增加钙化结节和CFU-FALP数量,与成骨性分化相关的因子OSX和Runx-2的基因表达量也显著升高,同时I型胶原的分泌量也明显增多,但这些效应均可被iNOS的特异性抑制剂L-NAME所抑制。ICS II可显著促进rBMSCs的成骨性分化,但采用L-NAME进行阻断后,随着iNOS和NO表达的降低,成骨性分化的指标随之降低,提示ICS II是通过提高iNOS活性,促进NO的生成来刺激rBMSCs的成骨性分化的。

This study is to investigate the effects on the expression of iNOS and production of NO in the osteogenic differentiation process of rat bone marrow stromal cells（rBMSCs） by icariside II.rBMSCs were cultured by adherence screening method.When the culture dishes were covered with 80% cells,the osteogenic induced cultures were adopted.Icariside II was supplemented into the culture at 1×10-5 mol-L-1.The activity of iNOS,content of NO and osteogenic differentiation markers including alkaline phosphatase（ALP） activity,CFU-FALP and mineralized bone nodules were compared among the icariside II-supplemented group,L-NMAE group,icariside II ＋ L-NAME group and the control.Total RNA was isolated and the gene expression of iNOS,Osterix and Runx-2 was investigated by real-time PCR.Total protein was also isolated and the secretion of iNOS and collagenⅠ was examined by Western blotting.Icariside II can significantly improved ALP activity,CFU-FALP amount and mineralized nodules.Besides,the mRNA level of factors related to the osteogenic differentiation includes Osterix and Runx-2 also enhanced.The secretion of collagen I also promoted significantly.But all of these effects can be inhibited by L-NAME which can specifically inhibit the activity of iNOS.Icariside II enhances the osteogenic differentiation of rBMSCs significantly,but if the activity of iNOS was blocked by L-NAME,the osteogenic differentiation markers decrease accompanied with iNOS and NO decrease,suggesting that icariside II stimulates the osteogenic differentiation via enhancing the activity of iNOS and promoting the generation of NO.