强骨康疏方通过RANKL/RANK/OPG信号通路调控破骨细胞分化而抑制类风湿关节炎骨破坏  

Qianggu-Kangshu formula inhibits rheumatoid arthritis bone destruc⁃tion by regulating osteoclast differentiation via RANKL/RANK/OPG sig⁃naling pathway

作　　者：江露 张宗星 李玮怡 刘道忠 包卓玛 聂青云 袁林 JIANG Lu;ZHANG Zongxing;LI Weiyi;LIU Daozhong;BAO Zhuoma;NIE Qingyun;YUAN Lin(Hubei Provincial Key Laboratory of Occurrence and Intervention of Rheumatic Diseases,Hubei Minzu University,Enshi 445000,China;Health Science Center,Hubei Minzu University,Enshi 445000,China;Hubei Provincial Clinical Medical Research Center for Nephropathy,Enshi 445000,China;Traditional Chinese Medicine Hospital of Shizhu,Chonqing 409199,China)

机构地区：[1]湖北民族大学风湿性疾病发生与干预湖北省重点实验室,湖北恩施445000 [2]湖北民族大学医学部,湖北恩施445000 [3]湖北省肾脏病临床医学研究中心,湖北恩施445000 [4]石柱土家族自治县中医院,重庆409199

出　　处：《中国病理生理杂志》2025年第1期123-135,共13页Chinese Journal of Pathophysiology

基　　金：国家自然科学基金资助项目(No.82460827,No.81860757);湖北民族大学研究生教育创新项目(No.MYK2023067)。

摘　　要：目的:基于网络药理学及分子对接技术探讨强骨康疏方(Qianggu-Kangshu formula,QGKSF)治疗类风湿关节炎(rheumatoid arthritis,RA)骨破坏的作用机制并利用细胞实验进行验证。方法:通过TCMSP数据库及文献检索获取QGKSF的主要活性成分及潜在靶点;使用OMIM数据库和GeneCards数据库检索与RA骨破坏相关的靶点,利用Venny 2.1.0筛选QGKSF抗RA骨破坏的靶点和靶点数量;通过STRING数据库构建潜在作用靶点的蛋白质-蛋白质相互作用网络,并利用Cytoscape软件进行拓扑分析筛选核心靶点;通过Metascape系统将筛选的QGKSF抗RA骨破坏的靶点进行进行京都基因与基因组百科全书(Kyoto Encyclopedia of Genes and Genomes,KEGG)和基因本体(Gene Ontology,GO)富集分析;使用AutoDock和PyMOL软件对药物的核心成分与核心蛋白进行分子对接验证。体外培养小鼠RAW264.7巨噬细胞,使用CCK-8法检测细胞活力;抗酒石酸酸性磷酸酶(tartrateresistant acid phosphatase,TRAP)染色观察各组TRAP阳性多核细胞的数量;测定TRAP酶活性以反映细胞酶活性;鬼笔环肽染色检测F-肌动蛋白环形成情况;Western blot检测活化T细胞核因子1(nuclear factor of activated T cells 1,NFATc1)、TRAP、组织蛋白酶K(cathepsin K,CTSK)、c-Fos、基质金属蛋白酶9(matrix metalloproteinase 9,MMP9)、骨保护素(osteoprotegerin,OPG)、核因子κB受体活化因子(receptor activator of nuclear factorκB,RANK)、RANK配体(RANK ligand,RANKL)和磷酸化蛋白激酶B(phosphorylated protein kinase B,p-AKT)蛋白水平。结果:从QGKSF中共筛选出136种有效活性成分及126个有效成分靶点;QGKSF抗RA骨破坏的靶点有207个,其中175个核心靶点。GO富集得到199条通路,筛选出与破骨细胞相关的通路共20条,主要有磷脂酰肌醇3-激酶/AKT信号通路和破骨细胞分化等。TRAP染色、酶活性测定及鬼笔环肽染色结果显示,与model组相比,QGKSF和氨甲蝶呤(methotrexate,MTX)组的阳性细胞形成减少,酶活AIM:To investigate the mechanism of Qianggu-Kangshu formula(QGKSF)in the treatment of rheumatoid arthritis(RA)bone destruction based on network pharmacology,molecular docking,and cell experiments.METHODS:The main active ingredients and potential targets of QGKSF were obtained through TCMSP database and literature search.OMIM database and GeneCards database were used to search the targets related to RA bone destruction,and Venny 2.1.0 was employed to screen the intersected targets of QGKSF and RA bone destruction.The protein-protein interaction network of potential intersected targets was constructed by STRING database,and topological analysis was carried out by Cytoscape software to screen core targets.The screened targets of QGKSF related to RA bone destruction were evaluated through the Metascape system.Kyoto Encyclopedia of Genes and Genomes(KEGG)and Gene Ontology were applied to complete enrichment analysis.AutoDock and PyMOL software was used to carry out molecular docking between the core components and the core proteins.Mouse RAW264.7 macrophages were cultured in vitro,and the cell viability was detected by CCK-8 assay.The number of tartrate-resistant acid phosphatase(TRAP)positive multinucleated cells in each group was calculated by TRAP staining.The enzyme activity of the cells was evaluated by determination of TRAP activity.F-actin ring formation was detected by phalloidin staining.Western blot analysis was conducted to detect the protein levels of nuclear factor of activated T cells 1(NFATc1),TRAP,cathepsin K(CTSK),c-Fos,matrix metalloproteinase 9(MMP9),osteoprotegerin(OPG),receptor activator of nuclear factorκB(RANK),RANK ligand(RANKL)and phosphorylated protein kinase B(p-AKT).RESULTS:A total of 136 active ingredients and 126 targets were selected correlated with QGKSF,whil 207 intersected targets of QGKSF and RA bone destruction were screened,175 of which were core targets.There were 199 pathways obtained by GO enrichment,and 20 pathways related to osteoclasts were screened out,including phosphatidy

关 键 词：强骨康疏方 类风湿关节炎 破骨细胞 RANKL/RANK/OPG信号通路 

分 类 号：R285.5[医药卫生—中药学] R593.22[医药卫生—中医学] R363