基于网络药理学和体外实验研究苔黑酚葡萄糖苷治疗骨质疏松的分子机制  

Mechanism of Orcinol Glucoside in Treatment of Osteoporosis:An Exploration Based on Network Pharmacology and in Vitro Validation

作　　者：刘霞[1,2] 黄明春[2] 张小琼[1,2] 李杰 张梅[1] LIU Xia;HUANG Ming-chun;ZHANG Xiao-qiong;LI Jie;ZHANG Mei(State Key Laboratory of Southwestern Chinese Medicine Resources,School of Pharmacy,Chengdu University of Traditional Chinese Medicine(TCM),Chengdu 611137,China;Chongqing Hospital of TCM,Chongqing 400021,China)

机构地区：[1]成都中医药大学药学院,省部共建西南特色中药资源国家重点实验室,成都611137 [2]重庆市中医院,重庆400021

出　　处：《中国实验方剂学杂志》2022年第1期197-203,共7页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：国家自然科学基金项目(81774202);成都中医药大学2020年度“杏林学者”医院专项(YYZX2020059);2019年度“杏林学者”学科人才科研提升计划项目(YYZX2019053)。

摘　　要：目的:基于网络药理学预测苔黑酚葡萄糖苷(OG)治疗骨质疏松的作用靶点及相关信号通路,应用分子对接和体外实验验证,从而揭示其潜在作用机制。方法:通过Similarity ensemble approach(SEA),SwissTargetPrediction数据库获取OG药物靶点;通过疾病相关的基因与突变位点数据库(DisGeNET),GeneCards数据库获取疾病靶点,交叉分析筛选出OG与骨质疏松共同作用靶点。利用STRING构建蛋白质-蛋白质相互作用(PPI)网络,随后利用Cytoscape 3.7.2软件中的CytoNCA插件进行拓扑分析筛选出核心靶点。其次,通过g:Profiler基因注释网站对所得共同靶点进行基因本体(GO)与京都基因与基因组百科全书(KEGG)富集分析。最后,AutoDock Vina软件用来进行分子对接以及体外细胞实验验证OG的抗骨质疏松作用。结果:OG与骨质疏松的相关靶点73个,其中关键靶点14个。GO和KEGG功能富集分析结果表明,OG治疗骨质疏松涉及259个细胞生物学过程,主要包括有机氮化合物的代谢、细胞群增殖、蛋白质代谢过程、应激反应的调节、化学反应等。其作用机制可能与糖基化终末产物/糖基化终末产物受体(AGE/RAGE),白细胞介素-17(IL-17),磷脂酰肌醇3-激酶/蛋白激酶B(PI3K/Akt)等信号通路相关。分子对接提示OG与关键靶点细胞周期蛋白D;(CCND1或Cyclin D;),周期蛋白依赖性激酶4(CDK4)的结合能最低且相近。流式细胞仪检测结果显示,与正常组比较,OG组G0/G1期细胞比例下降(P<0.01),S期细胞比例明显增加(P<0.01);蛋白免疫印迹法(Western blot)结果显示,与正常组比较,OG能上调Cyclin D_(1),CDK4蛋白的表达(P<0.05,P<0.01)。结论:OG治疗骨质疏松具有多靶点-多通路的特点,作用机制可能通过上调细胞周期关键蛋白Cyclin D_(1),CDK4的表达改变细胞周期,从而促进细胞增殖。Objective: To predict the therapeutic targets and related signaling pathways of orcinol glucoside(OG)in the treatment of osteoporosis by network pharmacology,and further clarify its mechanisms based on molecular docking and in vitro cell model. Method:The pharmacological targets of OG were obtained from Similarity ensemble approach(SEA)and SwissTargetPrediction,and the targets related to osteoporosis from DisGeNET and GeneCards. The cross-analysis was conducted to screen the common targets between OG and osteoporosis. STRING was used to construct the protein-protein interaction(PPI)network,followed by topology analysis using CytoNCA plug-in of Cytoscape 3.7.2 to screen out the core targets. The obtained common targets were subjected to gene ontology(GO)and Kyoto encyclopedia of genes and genomes(KEGG)analysis by g:Profiler. AutoDock Vina was utilized for molecular docking,and the in vitro cell experiments were then carried out for verifying the mechanism of OG in treating osteoporosis. Result:A total of 73 targets related to OG and osteoporosis were harvested,among which 14 were proved to be key targets by topological analysis.GO and KEGG functional enrichment analysis yielded 259 cell biological processes, mainly involving organonitrogen compound metabolic process, cell population proliferation, protein metabolic process,regulation of response to stress,and response to chemicals. Its mechanism of action might be related to advanced glycation end-product(AGE)-AGE receptor(RAGE)signaling pathway,interleukin-17(IL-17)signaling pathway, and phosphatidylinositol 3-kinase(PI3 K)/protein kinase B(Akt) signaling pathway. Molecular docking indicated that the binding energies of OG to Cyclin D;(CCND1)and cyclin-dependent kinase 4(CDK4)were the lowest and similar. The results of flow cytometry showed that compared with the normal group,OG group exhibited decreased proportion of cells in G0/G1 phase(P<0.01)and decreased proportion of cells in S phase(P<0.01). As demonstrated by Western blot,compared with the normal group,OG

关 键 词：苔黑酚葡萄糖苷 骨质疏松 网络药理学 分子对接 体外验证 

分 类 号：R285[医药卫生—中药学] R289[医药卫生—中医学]