基于血清药物化学、网络药理学及细胞实验探究壮骨健膝方治疗膝骨关节炎的药效物质  

Study on the Pharmacodynamic Substances of Zhuanggu Jianxi Recipe in the Treatment of Knee Osteoarthritis Based on Serum Pharmacochemistry,Network Pharmacology and Cell Experiment

作　　者：肖艳 林震 郭洁梅 陈鹏[1] 张英杰[1] 张鹏[1] 苏友新[1] XIAO Yan;LIN Zhen;GUO Jiemei;CHEN Peng;ZHANG Yingjie;ZHANG Peng;SU Youxin(Fujian University of Traditional Chinese Medicine,Fuzhou 350122 Fujian,China;Fujian Health College,Fuzhou 350101 Fujian,China)

机构地区：[1]福建中医药大学,福建福州350122 [2]福建卫生职业技术学院,福建福州350101

出　　处：《中药新药与临床药理》2023年第3期357-366,共10页Traditional Chinese Drug Research and Clinical Pharmacology

基　　金：国家自然科学基金项目(81774347,82104681);福建省自然科学基金项目(2019J01091)。

摘　　要：目的探究并验证壮骨健膝方(骨碎补、杜仲、秦艽、独活等组成)治疗膝骨关节炎(knee osteoarthritis,KOA)的药效物质。方法基于壮骨健膝方药物成分自建数据库,采用超高效液相串联三重四级杆质谱技术(ultra performance liquid chromatography tandem mass spectrometry,UPLC-MS/MS)定性定量检测壮骨健膝方入血成分;通过TCMSP、SwissTargetPrediction、SEA和Batman-TCM数据库筛选入血成分靶点,以“knee osteoarthritis”为关键词在GeneCards、OMIM、Drugbank数据库搜索膝骨关节炎相关靶点,将二者取交集后,通过Cytoscape软件构建入血成分-膝骨关节炎作用靶点网络,STRING平台构建蛋白互作网络,寻找核心成分与靶点;通过DAVID数据库对靶点进行基因本体(GO)功能富集和京都基因与基因组百科全书(KEGG)通路富集分析;通过细胞实验,以核心成分在血清中的浓度干预炎症滑膜巨噬细胞、退变软骨细胞模型,ELISA法检测细胞上清中膝骨关节炎核心靶点指标的含量。结果在壮骨健膝方含药血清中定性定量检测出6种原型入血成分,各成分及浓度分别为龙胆苦苷(1002.02±51.60)ng·mL^(-1),佛手柑内酯(448.50±13.35)ng·mL^(-1),阿魏酸(225.60±9.61)ng·mL^(-1),獐牙菜苦苷(68.49±4.62)ng·mL^(-1),柚皮苷(2.34±0.09)ng·mL^(-1),柚皮素(1.69±0.07)ng·mL^(-1);6种入血成分靶点406个,膝骨关节炎疾病靶点2492个,交集靶点155个,度值排前3位的核心成分分别是阿魏酸、柚皮素、龙胆苦苷,核心靶点包括肿瘤坏死因子(tumor necrosis factor,TNF)、白细胞介素1β(interleukin-1 beta,IL-1β)、基质金属蛋白酶(matrix metalloproteinases,MMPs)、丝裂原活化蛋白激酶3(mitogen-activated protein kinase 3,MAPK3)等;功能富集分析表明,壮骨健膝方通过多途径发挥其在膝骨关节炎中的药理作用,主要涉及炎症反应、凋亡过程、脂多糖的反应等;细胞实验结果显示,与模型组比较,阿魏酸组、柚皮素组、龙胆苦苷组�Objective To explore and verify the pharmacodynamic substances of Zhuanggu Jianxi Recipe(ZJR,composed of Drynariae Rhizoma,Eucommia Cortex,Radix Gentiana Macrophyllae,Radix Angelicae Pubescentis,etc.)in treating knee osteoarthritis(KOA).Methods Based on the self-built database of drug components of ZJR,serum components of ZJR were qualitatively and quantitatively analyzed by ultra-performance liquid chromatography tandem mass spectrometry(UPLC-MS/MS). The targets of the components absorbed in serum were retrieved from TCMSP,SwissTargetPrediction,SEA and Batman-TCM database. Key search term of knee osteoarthritis was used to obtain targets related to KOA from the databases of GeneCards,OMIM and Drugbank. After taking the intersection,we constructed absorbed components-disease target network using Cytoscape and the protein-protein interaction(PPI)network of the common targets using SRTING to find out the core components and targets. Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment analysis were carried out by DAVID database. In cell experiments, synovial inflammatory macrophages and degenerative chondrocytes were intervened respectively by the core components screened out before, based on the concentration of each component in the serum,and ELISA was used to detect the level of KOA-related core targets.Results Six main components absorbed in ZJR-containing serum were qualitatively and quantitatively screened out. The components were gentiopicroside,bergamot lactone, ferulic acid, swertiamarin, naringin and naringenin, with the concentrations of(1 002.02 ±51.60)ng·mL^(-1),(448.50±13.35)ng·mL^(-1),(225.60±9.61)ng·mL^(-1),(68.49±4.62)ng·mL^(-1),(2.34±0.09)ng·mL^(-1),(1.69±0.07) ng·mL^(-1),respectively. There were 406 targets for the 6 absorbed components in serum,2 492 targets related to KOA, and 155 intersection targets. Top 3 core components in degree value were ferulic acid,naringenin, gentiopicroside. Core targets included tumor necrosis factor(TNF), interleukin-1 beta(IL-1�

关 键 词：壮骨健膝方 膝骨关节炎 血清药物化学 网络药理学 药效物质 

分 类 号：R285.5[医药卫生—中药学]