基于网络药理学研究瑶药黑老虎三萜类成分对类风湿关节炎的作用机制及试验验证  

Mechanism of action and experimental verification of triterpenoids from the Yao ethnomedicine Kadsura coccinea on the rheumatoid arthritis based on network pharmacology

作　　者：李会影 刘实琪 李东慧 张烁 姚雨轩 苏维[1] 罗洁 王炜[1] 杨玉佩[1] LI Huiying;LIU Shiqi;LI Donghui;ZHANG Shuo;YAO Yuxuan;SU Wei;LUO Jie;WANG Wei;YANG Yupei(TCM and Ethnomedicine Innovation&Development International Laboratory,School of Pharmacy,Hunan University of Chinese Medicine,Changsha,Hunan 410208,China)

机构地区：[1]湖南中医药大学中医药民族医药国际联合实验室,湖南长沙410208

出　　处：《湖南中医药大学学报》2024年第10期1834-1844,共11页Journal of Hunan University of Chinese Medicine

基　　金：国家自然科学基金项目(82174078);湖南省自然科学基金青年项目(2024JJ6344,2024JJ6347);2023年度湖南省大学生创新创业训练计划一般项目(S202310541070);2023年度大学本科生科研创新基金项目(2023BKS105)。

摘　　要：目的基于网络药理学和实验研究探讨瑶药黑老虎Kadsura coccinea(Lem.)A.C.Smith治疗类风湿关节炎(rheumatoid arthritis,RA)的作用机制。方法采用硅胶、C18反相柱色谱及半制备高效液相分离技术对黑老虎的有效成分进行分离纯化;采用波谱技术对分离得到的化合物进行结构鉴定;通过Swiss Target Prediction数据库筛选黑老虎成分靶点,在GeneCards数据库和MalaCards数据库挖掘RA相关靶点,进而筛选黑老虎作用于RA的靶点;制作蛋白质-蛋白质相互作用(protein-protein interaction,PPI)网络取其交集,并通过GO分析和KEGG富集分析;采用MTT试剂盒检测化合物对肿瘤坏死因子(tumor necrosis factor,TNF)-α和白细胞介素(interleukin,IL)-6的释放情况,以及蛋白免疫印迹实验对预测结果进行验证。结果从黑老虎中提取分离出6个三萜类化合物,分别鉴定为heilaohuacid A(1),heilaohuacid G(2),seco-coccinic acid F(3),seco-coccinic acid G(4),schisandronic acid(5)和schisanlactone B(6)。MTT实验结果是化合物2,3,4,6能抑制RA-FLS细胞增殖,其IC50值为7.52~8.85μmol。6个化合物在浓度为10μmol和20μmol时均能抑制炎症因子TNF-α和IL-6的释放(P<0.05,P<0.01)。网络药理学预测得到109个共同作用靶标,通过与691个RA疾病相关靶标进行交集映射,获得其干预RA靶标38个,主要为丝氨酸/苏氨酸蛋白激酶、TNF、丝裂原活化蛋白激酶1、前列腺素内过氧化物合酶2、丝裂原活化蛋白激酶14等。GO分析和KEGG富集分析得关键通路为白细胞介素(interleukin,IL)-17信号通路和TNF信号通路等。化合物3在浓度为20μmol能上调核因子κB抑制因子α(recombinant inhibitory subunit of NF kappa B-α,IκB-α)的表达水平(P<0.05)。结论本研究从网络药理学角度预测瑶药黑老虎三萜成分治疗RA的作用机制,初步证实其可上调IκB-α蛋白的表达,为瑶药黑老虎治疗RA提供了科学依据。Objective To explore the mechanism of action of the Yao ethnomedicine Kadsura coccinea(Lem.)A.C.Smith in treating rheumatoid arthritis(RA)based on network pharmacology and experimental research.Methods The active compounds of Kadsura coccinea were separated and purified by silica gel,C18 reversed-phase column chromatography,and semi-preparative high-performance liquid chromatography.The structures of the compounds obtained from the separation were identified using spectroscopic techniques.The targets of Kadsura coccinea components were screened through the Swiss Target Prediction database,and RA-related targets were excavated from the GeneCards and MalaCards databases to further identify the targets of Kadsura coccinea acting on RA.A protein-protein interaction(PPI)network was constructed to find the intersections,followed by GO analysis and KEGG enrichment analysis.The MTT(thiazolyl blue)assay kit was used to detect the release of tumor necrosis factor(TNF)-α and interleukin(IL)-6 by the compounds,and the prediction results were verified through Western blot.Results Six triterpenoids were extracted and separated from Kadsura coccine and identified as heilaohuacid A(1),heilaohuacid G(2),seco-coccinic acid F(3),seco-coccinic acid G(4),schisandronic acid(5),and schisanlactone B(6).The MTT assay results demonstrated that compounds 2,3,4,and 6 could inhibit the proliferation of RA-FLS cells,with IC50 values of 7.52~8.85μmol.Six compounds suppressed the release of inflammatory cytokines TNF-α and IL-6 at concentrations of 10 and 20μmol(P<0.05,P<0.01).A total of 109 co-acting targets were predicted by network pharmacology,which were mapped against 691 RA-related targets to obtain 38 intervention targets for RA.These targets primarily included serine/threonine protein kinases,TNF,mitogen-activated protein kinase 1(MAPK1),prostaglandin-endoperoxide synthase 2(COX-2),and mitogen-activated protein kinase 14(MAPK14).The key pathways identified through GO analysis and KEGG enrichment analysis were interleukin(IL)-17 sign

关 键 词：瑶药 黑老虎 三萜 类风湿关节炎 网络药理学 

分 类 号：R285.5[医药卫生—中药学]