基于网络药理学和细胞实验探讨杜仲+续断药对治疗骨质疏松症的作用机制

Mechanism of Eucommiae ulmoides and Radix Dipsaci in prevention and treatment of osteoporosis:An analysis based on network pharmacology and cell experiment

目的探讨杜仲+续断药对治疗骨质疏松症(osteoporosis,OP)的作用机制。方法从TCMSP、BATMAN-TCM平台检索及查阅文献获得杜仲+续断的活性成分。于GeneCards、OMIM查询OP相关基因。Cytoscape3.7.2软件构建化合物-靶点网络,STRING在线网站构建蛋白相互作用网络,在DAVID进行GO和KEGG富集分析。成骨细胞增殖、碱性磷酸酶表达和矿化结节形成实验检测杜仲+续断协同增效作用。Western blot法检测杜仲+续断对BMP2和Wnt信号通路相关蛋白表达的影响。结果获得活性成分杜仲44个,续断17个,杜仲+续断与OP交集靶点80个。GO富集分析得到52个生物过程、8个细胞组成、16个分子功能。KEGG通路富集分析得出6条主要信号通路。杜仲、续断显著促进成骨细胞增殖、碱性磷酸酶表达和矿化结节形成(P<0.01),杜仲主要调节Wnt信号通路(P<0.05或P<0.01),续断激活BMP2信号通路更显著(P<0.01),杜仲+续断作用显著提高(P<0.01)。结论杜仲、续断物质基础不同,但治疗OP作用的主要靶点和通路相同,杜仲+续断可通过BMP2和Wnt信号通路调节成骨细胞功能,两药合用治疗OP具有协同增效作用。

Aim To investigate the effect and mechanism of Eucommiae ulmoides and Radix Dipsaci in the treatment of osteoporosis.Methods The active ingredients of Eucommiae ulmoides and Radix Dipsaci were obtained by TCMSP,BATMAN-TCM platform and the literature.Osteoporosis related genes were collected by GeneCards and OMIM.The compound target network was constructed with Cytoscape 3.7.2 software.The protein interaction network was constructed with STRING online website.DAVID was used to perform gene ontology(GO)enrichment analysis and kyoto encyclopedia of genes and genomes(KEGG)pathway enrichment analysis.The synergistic effect of Eucommiae ulmoides and Radix Dipsaci was tested by osteoblast proliferation,alkaline phosphatase expression and mineralized nodule formation experiments.The protein expressions of BMP2 and Wnt signaling pathway were investigated by Western blotting.Results A total of44 active components of Eucommiae ulmoides and 17active components of Radix Dipsaci were screened,and80 targets were intersected by osteoporosis.GO analysis showed that the action mechanism of Eucommiae ulmoides and Radix Dipsaci in the treatment of osteoporosis was involved with 52 items of biological process,8 items of cell composition and 16 items of molecular function.The KEGG enrichment pathway was dominated by 6 major signaling pathways.Compared with the control group,the osteoblast proliferation,alkaline phosphatase expression and mineralized nodule formation significantly increased in Eucommiae ulmoides or Radix Dipsaci group(P<0.01).Western blotting showed that Eucommiae ulmoides mainly regulated the expression levels of Wnt signaling pathways(P<0.05or P<0.01),and Radix Dipsaci up-regulated the expression levels of BMP2 signaling pathways(P<0.01).Meanwhile,Eucommiae ulmoides and Radix Dipsaci significantly enhanced these effects,respectively(P<0.01).Conclusions Although Eucommiae ulmoides and Radix Dipsaci have different active components,their main targets and pathways are the same in the treatment of osteoporosis.Eucommiae ulmoides and Radix Dipsaci can regulate the function of osteoblasts through BMP2 and Wnt signaling pathways,and the combination of the two drugs in the treatment of osteoporosis has synergistic effect.