摘要
基于网络药理学、分子对接和体外实验探讨熟地黄治疗骨关节炎的作用机制。研究首先采用核因子κB受体活化因子配体(receptor activator of nucleus factor-κB ligand,RANKL)及巨噬细胞集落刺激因子(macrophage colony-stimulating factor,M-CSF)诱导RAW264.7细胞构建破骨细胞模型评价熟地黄对破骨细胞模型抗酒石酸酸性磷酸酶(TRAP)活性的影响,对熟地黄治疗骨关节炎的药效进行验证。之后通过TCMSP数据库及文献检索,获得熟地黄活性成分,通过SwissTargetPrediction数据库获得活性成分潜在作用靶点,通过OMIM、TTD、GeneCards及DisGeNET数据库收集骨关节炎疾病靶点。利用Venny平台获得熟地黄与骨关节炎交集靶点,使用Cytoscape 3.8.2构建交集靶点蛋白互作网络,基于拓扑算法获得关键靶标。使用DAVID数据库对交集靶点进行GO(Gene Ontology)功能分析和KEGG(Kyoto Encyclopedia of Genes and Genomes)通路富集分析。RT-qPCR法检测熟地黄对破骨细胞中关键靶点基因表达的影响。最后通过分子对接对成分与关键靶点之间的活性进行验证。结果显示,熟地黄可以有效抑制破骨细胞中的TRAP活性,从而抑制破骨细胞的骨吸收,对骨关节炎起治疗作用。网络药理学结果表明,熟地黄共有14个活性成分,作用靶点与骨关节炎交集靶点126个。网络药理学富集结果筛选到生物过程432条,信号通路139条。靶点网络拓扑分析依据degree得到关键靶点原癌基因酪氨酸蛋白激酶(proto-oncogene tyrosine-protein kinase Src,SRC)、信号转导和转录活化因子3(signal transducer and activator of transcription 3,STAT3)、转录因子p65(transcription factor p65,RELA)等。SRC在破骨细胞中呈现高表达,能够加速骨关节炎的发生。因此选择SRC进行后续实验验证。结果显示,熟地黄能够降低破骨细胞中SRC的基因表达水平。分子对接结果显示毛蕊花糖苷、异类叶升麻苷、棉子糖与SRC有较好的结合活性,可能为熟地黄治疗骨关节炎的关键成分。综上,熟地黄可以通过作用于SRC,抑制破骨细胞的骨吸收,平衡关节软骨及软骨下骨的骨代谢,从而对骨关节炎起治疗作用。此外,熟地黄可能通过STAT3、RELA等靶点,通过PI3K-AKT信号通路、IL-17信号通路等相关通路,对骨关节炎发挥整体疗效。
The mechanism of Rehmanniae Radix Praeparata against osteoarthritis was investigated based on network pharmacology,molecular docking,and in vitro experiments in the present study.Osteoclast models were established via receptor activator of nuclear factor-κB ligand(RANKL)and macrophage colony-stimulating factor(M-CSF)inducing RAW264.7 cells.Further,the influence of Rehmanniae Radix Praeparata on the activity of tartrate-resistant acid phosphatase(TRAP)was evaluated and the efficacy of Rehmanniae Radix Praeparata in the treatment of osteoarthritis was verified.The active components of Rehmanniae Radix Praeparata were obtained from Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP)and literature,and the potential targets of the components were collected from SwissTargetPrediction.Osteoarthritis disease targets were searched in Online Mendelian Inheritance in Man(OMIM),Therapeutic Target Database(TTD),GeneCards,and DisGeNET.The intersection targets of Rehmanniae Radix Praeparata and osteoarthritis were obtained by Venny platform.The protein-protein interaction(PPI)network was constructed by Cytoscape 3.8.2,and key targets were obtained based on topology algorithm.The Database for Annotation,Visualization and Integrated Discovery(DAVID)was used to perform Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment analysis.Finally,the mRNA expression of the key targets was determined by RT-qPCR and the binding activity between the components and key targets was validated by molecular docking.The results showed that Rehmanniae Radix Prae-parata inhibited the TRAP activity,thus inhibiting bone resorption by osteoclasts and treating osteoarthritis.By network pharmacology,14 active components of Rehmanniae Radix Praeparata and 126 intersection targets were obtained.The network pharmacology enrichment results revealed 432 biological processes and 139 signaling pathways.Key targets such as proto-oncogene tyrosine-protein kinase Src(SRC),signal transducer and activator of transcription 3(STAT3)and transcription factor p65(RELA)were obtained according to the degree in topological analysis.SRC was highly expressed in osteoclasts,which accelerated the development of osteoarthritis.Therefore,SRC was selected for subsequent verification,and Rehmanniae Radix Praeparata decreased the gene expression level of SRC.The molecular docking showed that acteoside,isoacteoside,raffinose had good bonding activity with SRC,suggesting that they might be the critical components in treating osteoarthritis.In conclusion,Rehmanniae Radix Praeparata can inhibit bone resorption by osteoclasts and balance the metabolism of articular cartilage and subchondral bone via acting on SRC,thus playing a therapeutic role in osteoarthritis.In addition,Rehmanniae Radix Praeparata may exert overall efficacy on osteoarthritis through other targets such as STAT3 and RELA,and other related pathways such as PI3 K-AKT and IL-17 signaling pathways.
作者
凤雯晴
刘凯洋
张嘉宁
李勇枝
刘军莲
卢建秋
张燕玲
FENG Wen-qing;LIU Kai-yang;ZHANG Jia-ning;LI Yong-zhi;LIU Jun-lian;LU Jian-qiu;ZHANG Yan-ling(State Administration of Traditional Chinese Medicine,Engineering Research Center of Traditional Chinese Medicine-Information,School of Chinese Materia Medica,Beijing University of Chinese Medicine,Beijing 102488,China;China Astronaut Research and Training Center,Beijing 100094,China)
出处
《中国中药杂志》
CAS
CSCD
北大核心
2022年第19期5336-5343,共8页
China Journal of Chinese Materia Medica
基金
航天医学实验项目(HYZHXM05002)。
关键词
骨关节炎
熟地黄
网络药理学
分子对接
osteoarthritis
Rehmanniae Radix Praeparata
network pharmacology
molecular docking
作者简介
凤雯晴,硕士研究生,E-mail:essie1020@163.com;通信作者:卢建秋,研究员,E-mail:lujq@vip.sina.com;通信作者:张燕玲,研究员,E-mail:zhangyanling@bucm.edu.cn。
