淫羊藿苷通过PI3K/AKT-eNOS信号途径促进大鼠骨髓基质细胞的成骨性分化

ICA promotes osteogenic differentiation of rat bone marrow stromal cells via PI3K/AKT-eNOS pathway

目的研究淫羊藿苷(Icariin,ICA)促进体外培养大鼠骨髓基质细胞(rat bone marrow stromal cells,rBMSCs)成骨性分化的信号传导机制。方法贴壁分离法培养rBMSCs,传至第2代后用流式法鉴定细胞纯度并用于机制研究。细胞铺满皿底后分为ICA组(1×10-5mol.L-1)、LY294002组(5×10-5 mol.L-1)、ICA+LY294002组和空白对照组。不同处理24 h后分别用Real-time PCR法检测ALP、eNOS和iNOS的基因表达水平,用Western blot法检测p-AKT、eNOS和iNOS蛋白表达量,同时测定TNOS、iNOS活性和NO生成量。结果基因表达和蛋白质检测结果均显示,10-5mol.L-1ICA明显提高rBMSCs的ALP表达水平(P<0.01),PI3K的特异性阻断剂LY294002可抑制ICA对ALP的提高作用,亦明显降低p-AKT的表达水平。ICA可提高rBMSCs的eNOS和iNOS表达水平,但LY294002只能抑制eNOS的增加,对iNOS无影响。TNOS和NO与eNOS和ALP的变化趋势一致,iNOS活性不受LY294002的影响。结论 ICA通过PI3K/AKT-eNOS途径促进rBMSCs的成骨性分化。

Aim To investigate the pathway mecha- nism of icariin （ICA） promoting osteogenic differentia- tion of rat bone marrow stromal cells （rBMSCs）. Methods rBMSCs were isolated and subcultured in second generation that was identified by flow cytome- try. The subcultured rBMSCs were randomly divided into four groups： ICA group （1 ×10-5mol·L-1）, LY294002 group （5 ×10-5mol·L-1）, ICA ＋ LY294002 group and control group. The mRNA ex- pression levels were detected by quantitative real-time RT-PCR after 24 hours. The protein expression levels of p-AKT, eNOS and iNOS were detected by Western blot. The activities of TNOS and iNOS, the NO contents were measured by commercial regents. Results The mRNA expression level of ALP in ICA group was significantly improved （ P 〈 0. 01 ） , while LY294002 strongly inhibited the increase of ALP expression level, and decreased the p-AKT protein expression level. ICA increased the expression levels of both eNOS and iNOS. LY294002 inhibited the increase of eNOS, but had no obvious effect on iNOS. The change tendency of TNOS and NO content was similar to eNOS and ALP. Conclu- sion ICA promotes the osteogenic differentiation of rBMSCs through PI3K/AKT-eNOS pathway.