淫羊藿黄酮类主要成分促进骨髓间充质干细胞向成骨细胞增殖分化作用及机制的影响  

Promotive Effect of Epimedii Folium Flavonoids on Proliferation and Differentiation of BMSCs into Osteoblasts

作　　者：许静[1] 张晶晶[2] 郭非非 李欣[1] 张毅[2] 杨洪军[2] 吴宏伟[2] 张琼[1] 

机构地区：[1]山西医科大学药学院,太原030001 [2]中国中医科学院中药研究所,北京100700

出　　处：《中国实验方剂学杂志》2017年第14期113-120,共8页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：国家科技支撑计划项目(2011BAI07B08)

摘　　要：目的:探讨淫羊藿黄酮类主要成分朝藿定A,朝藿定B,朝藿定C,淫羊藿苷,淫羊藿次苷对骨髓间充质干细胞(BMSCs)向成骨细胞分化的促进作用,并对其作用机制进行预测。方法:采用全骨髓细胞贴壁法分离纯化SD大鼠BMSCs做原代培养。实验分为朝藿定A+成骨诱导培养基(1×10^(-8)mol·L^(-1))组,朝藿定B+成骨诱导培养基(1×10^(-8)mol·L^(-1))组,朝藿定C+成骨诱导培养基(1×10^(-8)mol·L^(-1))组,淫羊藿苷+成骨诱导培养基(1×10^(-8)mol·L^(-1))组,淫羊藿次苷组+成骨诱导培养基组及成骨诱导培养基组(空白组)6组。加入药物进行干预14 d后,采用定性与定量相结合的方法,通过茜素红染色、细胞骨架染色、碱性磷酸酶定量分析,对淫羊藿5种黄酮类主要成分促进骨髓间充质干细胞向成骨细胞分化作用进行药效学评价;在此基础上,基于BATMAN-TCM网络药理学研究平台,对相关靶点和通路进行预测分析,探讨其潜在作用机制。结果:BMSCs在作用浓度为1×10^(-8)mol·L^(-1)的药物浓度作用下诱导14 d,通过细胞骨架染色,各给药组均可明显观察到多角形成骨细胞增多;通过茜素红染色,各给药组可明显观察到大量的矿化结节的形成;通过碱性磷酸酶含量测定,与空白组比较,各给药组碱性磷酸酶含量升高,其中朝藿定C,淫羊藿苷组有显著性差异(P<0.05),淫羊藿次苷组具有极显著性差异(P<0.01)。同时网络药理学分析表明,5种黄酮类成分在促进增殖和分化方面共得到33个潜在作用靶点,并富集分析得到8条信号通路,其中activation of MAPK activity,estrogen signaling pathway已有实验报道。结论:淫羊藿黄酮类主要成分朝藿定A,朝藿定B,朝藿定C,淫羊藿苷,淫羊藿次苷均具有促进BMSCs向成骨方向分化的作用,其中朝藿定C,淫羊藿苷,淫羊藿次苷的作用效果显著,网络药理学分析结果从整体上阐释了多成分的作用机制,为进一步实验验证提供参考。Objective： To investigate the effects of main Epimedii Folium flavonoids （epimedin A, epimedin B, epimedin C, icariin and icarisid I ） on promoting BMSCs proliferation and differentiation into osteoblasts and to predict the mechanism of them. Method： BMSCs in SD rats were isolated and purified by whole bone marrow adherence method for primary culture. The experiment was divided into 6 groups： epimedin A ＋osteogenic medium （1 × 10^-8 mol·L^-1） , epimedin B ＋ osteogenic medium （1 × 10^-8 mol·L^-1） , epimedin C ＋ osteogenic medium （1 × 10^-8 mol·L^-1）, icariin ＋ osteogenic medium （1 × 10^-8 mol·L^-1）, icariside group ＋ osteogenic medium （blank group）. After drug intervention for 14 days, qualitative and quantitative methods were combined for pharmacodynamic evaluation, of the above 5 kinds of Epimedii Folium flavonoids by alizarin red staining, cytoskeleton staining and alkaline phosphatase assay. Meanwhile, the potential targets and pathways were predicted and analyzed based on BATMAN-TCM network pharmacology research platform to investigate the action mechanisms. Result： BMSCs were treated with 1 × 10^-8 mol·L^-1 drugs for 14 d, and polygonal osteoblasts were increased in various groups by cytoskeleton staining; in alizarin red staining, a large number of mineralized nodules were formed in various treatment groups; in Alkaline phosphatase assay, the content of alkaline phosphatase was increased in various treatment groups as compared with the blank group, particularly, the groups of epimedin C （P〈0.05）, icariin （P 〈0.05）, and icarisid I （P 〈0.01） showed significant changes. As for network pharmacology analysis, 33 potential targets and 8 pathways about proliferation and differentiation were obtained. Among them, activation of MAPK activity and estrogen signaling pathway had been verified by the relative experimental reports. Conclusion： All of the above 5 kinds of Epimedii Folium flavonoids could promote BMSCs proliferation and differentiation

关 键 词：淫羊藿 淫羊藿黄酮 骨髓间充质干细胞 成骨细胞 分子机制 网络药理学 

分 类 号：R285.5[医药卫生—中药学]