目的 探讨姜黄素对骨髓间充质干细胞(bone marrow-derived mesenchymal stem cells,BMSCs)成骨分化的潜在调控机制.方法 通过网络药理学方法获取姜黄素调控成骨分化的靶点基因,构建蛋白质作用网络(protein-protein interactions,PPI),并进行基因本体(Gene Ontology,GO)、京都基因与基因组百科全书(Kyoto Encyclopedia of Genes and Genomes,KEGG)通路富集分析及分子对接处理.采用CCK-8法及细胞克隆筛选姜黄素药物浓度,进一步采用 ALP染色及活性测定、AR染色及定量分析检测姜黄素对成骨分化的宏观调控作用.采用RT-qPCR法及Western blotting法检测Wnt/β-catenin信号通路相关mRNA及蛋白的微观表达差异.结果 筛选出姜黄素调控成骨分化的靶点基因 92 个,主要涉及细胞内部对化学刺激、含氧化合物的反应等生物过程.其中,有 3 个靶点基因作用于Wnt/β-catenin信号通路,且均可以与姜黄素良好对接.此外,低浓度姜黄素(5、10 μmol)对大鼠骨髓间充质干细胞(rat bone marrow-derived mesenchymal stem cells,rBMSCs)增殖无明显毒性且可增强碱性磷酸酶活性、增加钙盐沉积量(P<0.05),上调成骨相关基因及蛋白的表达(P<0.05),从而促进rBMSCs成骨分化.而高浓度姜黄素(15 μmol)对rBMSCs成骨分化过程有潜在的抑制作用.结论 姜黄素可以通过调控Wnt/β-catenin信号通路影响成骨相关基因的表达,进而调控BMSCs成骨分化过程.
Mechanism of curcumin regulating bone formation via the Wnt/β-catenin signaling pathway
Objective To explore the potential regulatory mechanism of curcumin on osteogenic differentiation of bone marrow-derived mesenchymal stem cells(BMSCs).Methods The target genes of curcumin regulating osteogenic dif-ferentiation were obtained through network pharmacology,and a protein-protein interaction network(PPI)was con-structed.Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment analysis and mo-lecular docking were performed.CCK-8 and cell cloning were used to screen the concentration of curcumin.ALP stai-ning and activity assay,AR staining and quantitative analysis were used to evaluate the macro-regulatory effect of curcu-min on osteogenic differentiation.RT-qPCR and Western blotting were used to detect the micro-expression differences of mRNA and protein related to the Wnt/β-catenin gignaling pathway.Results A total of 92 target genes for curcumin regulation of osteogenic differentiation were screened,mainly involving biological processes such as the intracellular response to chemical stimuli and the response to oxidative compounds.Three target genes acted on the Wnt/β-catenin signaling pathway and could be effectively docked with curcumin.In addition,low concentrations of curcumin(5,10 μmol)showed no obvious toxicity to rat bone marrow-derived mesenchymal stem cells(rBMSCs),enhanced alkaline phosphatase activity,increased calcium deposition(P<0.05),up-regulated the expression of osteogenic-related genes and proteins(P<0.05),and promoted osteogenic differentiation of rBMSCs.However,high concentration of cur-cumin(15 μmol)had a potential inhibitory effect on the osteogenic differentiation of rBMSCs.Conclusion Curcumin can regulate the expression of osteogenic-related genes by modulating the Wnt/β-catenin signaling pathway,thereby regulating the osteogenic differentiation of BMSCs.
万方数据
维普
