三妙丸治疗类风湿关节炎主要活性成分及作用机制的探讨  

Main active components and mechanism of Sanmiao Pills in treating rheumatoid arthritis

作　　者：夏仕林 未怡 江小慢 许静 李雅 祝宇龙 王言 邓然[2] 吴虹 XIA Shi-lin;WEI Yi;JIANG Xiao-man;XU Jing;LI Ya;ZHU Yu-long;WANG Yan;DENG Ran;WU Hong(School of Pharmacy,Anhui University of Chinese Medicine,Hefei 230012,China;School of Integrated Chinese and Western Medicine,Anhui University of Chinese Medicine,Hefei 230012,China;Anhui Provincial Key Laboratory of Traditional Chinese Medicine Formula,Hefei 230012,China)

机构地区：[1]安徽中医药大学药学院,安徽合肥230012 [2]安徽中医药大学中西医结合学院,安徽合肥230012 [3]安徽省中药复方重点实验室,安徽合肥230012

出　　处：《中国中药杂志》2024年第19期5204-5217,共14页China Journal of Chinese Materia Medica

基　　金：国家自然科学基金面上项目(82374117,81874360,81473400);安徽省高等学校科学研究项目重大项目(2022AH050453);安徽省自然科学基金青年科学基金项目(2308085QH300);安徽省高等学校科学研究项目(2022AH050520)。

摘　　要：利用UPLC-Q-TOF-MS技术分析三妙丸化学成分,通过网络药理学和动物实验研究三妙丸治疗类风湿性关节炎(RA)的作用机制。利用UPLC-Q-TOF-MS技术检测三妙丸所含化学成分,结合DisGeNET、SwissTargetPrediction等数据库筛选类风湿关节炎与三妙丸化学成分的交集靶点,构建蛋白-蛋白相互作用(protein-protein interaction,PPI)网络。将关键靶点导入MATESCAPE平台进行基因本体论(Gene Ontology,GO)分析和京都基因与基因组百科全书(Kyoto Encyclopedia of Genes and Genomes,KEGG)通路注释,结合成分、靶点和通路,利用Cytoscape 3.8软件构建“成分-靶点-通路”网络,预测三妙丸治疗类风湿性关节炎的主要活性成分、关键靶点及通路。建立HPLC-DAD法同时测定三妙丸中小檗碱、苍术内酯-Ⅰ、苍术内酯-Ⅱ、人参皂苷Ro、三七皂苷R_(1)的含量。建立大鼠胶原诱导性关节炎(collagen-induced arthritis,CIA)模型,进行行为学实验,HE检测SMP对大鼠滑膜病理形态的影响,Western blot检测磷脂酰肌醇3-激酶(phosphatidylinositol 3-kinase,PI3K)/蛋白激酶B(protein kinase B,Akt)通路的变化。结果鉴定出三妙丸化学成分共126个。化学成分与疾病共有交集靶点332个,根据PPI网络结果筛选排名前十的为核心靶点,其中包括AKT1、肿瘤坏死因子(TNF)等,KEGG富集结果显示主要涉及PI3K/AKT样受体通路等20条典型通路。5种成分在各自范围内线性关系良好,RSD≤2%。动物实验研究结果显示,三妙丸有效改善CIA大鼠滑膜异常增生,降低滑膜PI3K和p-AKT蛋白表达水平。综上所述,三妙丸可能通过调控PI3K/AKT通路,缓解RA病情。UPLC-Q-TOF-MS was employed to analyze the chemical components of Sanmiao Pills(SMP)and network pharmacology and animal experiments were employed to investigate the mechanism of SMP in treating rheumatoid arthritis(RA).DisGeNET and SwissTargetPrediction were used to identify the overlapping targets between RA and SMP.Subsequently,a protein-protein interaction(PPI)network was built based on the key targets.The identified targets were further subjected to Gene Ontology(GO)analysis and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway annotation in MATESCAPE.Cytoscape 3.8 was used to build the"component-target-pathway"network,on the basis of which the main active components,key targets,and pathways involved in the treatment of RA with SMP were predicted.Additionally,an HPLC-DAD method was established to simultaneously determine the content of berberine,atractylolideⅠ,atractylolideⅡ,ginsenoside Ro,and notoginsenoside R_(1) in SMP.Furthermore,a rat model of collagen-induced arthritis(CIA)was established and the behavior test was carried out.Hematoxylin-eosin(HE)staining was conducted to reveal the pathological changes in the synovial tissue.Western blot was employed to determine the expression levels of proteins in the phosphatidylinositol 3-kinase(PI3K)/protein kinase B(AKT)pathway.A total of 126 chemical components were identified in SMP,and 332 targets were shared by SMP and RA.The PPI network predicted the key targets including AKT1 and TNF,which were mainly involved in 20 pathways including the PI3K/AKT pathway.Five components showed good linear relationships within the test concentration ranges,with RSD≤2%.The animal experiments revealed that SMP ameliorated synovial hyperplasia and down-regulated the expression levels of PI3K and p-AKT in CIA rats.The findings suggest that SMP may alleviate RA by modulating the PI3K/AKT pathway.

关 键 词：UPLC-Q-TOF-MS 三妙丸 网络药理学 类风湿关节炎 PI3K/AKT 

分 类 号：R285.5[医药卫生—中药学]