黑骨藤靶向TNF-α抗类风湿关节炎的药效物质基础分析  

Effective Components of Periploca forrestii Against Rheumatoid Arthritis by Targeting TNF-α

作　　者：曹陶涛 杨畅[1] 孙佳[1] 王永林[1] 刘春花[3] 李勇军[3] 何彬[3] 刘亭[1] CAO Tao-tao;YANG Chang;SUN Jia;WANG Yong-lin;LIU Chun-hua;LI Yong-jun;HE Bin;LIU Ting(Provincial Key Laboratory of Pharmaceutics in Guizhou Province,State Key Laboratory of Functions and Applications of Medicinal Plants,Guizhou Medical University,Guiyang 550004,China;School of Pharmaceutical Sciences,Guizhou Medical University,Guiyang 550004,China;Engineering Research Center for Development and Application of Ethnic Medicine and Traditional Chinese Medicine,Ministry of Education,Guizhou Medical University,Guiyang 550004,China)

机构地区：[1]贵州医科大学贵州省药物制剂重点实验室,省部共建药用植物功效与利用国家重点实验室,贵阳550004 [2]贵州医科大学药学院,贵阳550004 [3]贵州医科大学民族药与中药开发应用教育部工程研究中心,贵阳550004

出　　处：《中国实验方剂学杂志》2022年第3期187-195,共9页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：国家自然科学基金项目(82060703,U1812403-05);贵州省普通高等学校科技拔尖人才项目(黔教合KY字[2021]033);贵州省优秀青年科技人才项目(黔科合平台人才[2021]5619号)。

摘　　要：目的:应用网络药理学和实验验证,分析黑骨藤靶向肿瘤坏死因子-α(TNF-α)治疗类风湿关节炎的药效物质基础。方法:课题组前期研究发现黑骨藤醇提组分(CDLF,CQAF)具有显著抗类风湿关节炎(RA)活性,并鉴定出10个具有抗RA的活性单体。应用酶联免疫吸附测定法(ELISA),以白细胞介素-6(IL-6),一氧化氮(NO),白细胞介素-1β(IL-1β)和前列腺素E(2 PGE2)为指标比较活性单体,CDLF和CQAF组分抗RA活性;网络药理学分析黑骨藤抗RA可能的分子机制;TNF-α分子对接、表面等离子分子互作(SPR)技术和TNF-α诱导L929损伤模型验证黑骨藤化学单体靶向TNF-α能力。结果:ELISA发现,CDLF和CQAF组分抗RA活性明显高于鉴定出的10个活性单体;网络药理学分析,发现黑骨藤抗RA的核心靶点为信号传导及转录因子3(STAT3),肿瘤坏死因子(TNF)和IL-6,基因本体(GO)富集有胶原分解代谢、炎症反应,核转录因子(NF)-κB转录因子活性的正调控,B细胞增殖的正调控等,基于京都基因与基因组百科全书(EKGG)富集有TNF信号通路,磷脂酰肌醇3-激酶(PI3K)/蛋白激酶B(Akt)信号通路,NF-κB信号通路,Toll样受体信号通路,丝裂原活化蛋白激酶(MAPK)信号通路等;TNF-α分子对接,SPR技术和TNF-α诱导L929损伤模型实验发现,CDLF和CQAF组分具有较好的结合并拮抗TNF-α的活性,但从两组分中分离鉴定的活性成分,并没有表现出与CDLF和CQAF组分一致的抗TNF-α活性。结论:黑骨藤的CDLF和CQAF组分可能通过靶向TNF-α治疗RA,实验发现已分离的化学成分与TNF-α结合活性低于CDLF和CQAF组分,同时课题组在黑骨藤药材中分离到最低质量分数为0.25 ng·g^(-1)的化学成分,说明与TNF-α结合而产生抗RA活性成分可能为痕量成分。Objective:To analyze the effective components of Periploca forrestii against rheumatoid arthritis(RA)by targeting tumor necrosis factor(TNF)-αbased on network pharmacology and experimental verification.Method:The preliminary research of the research group found that the alcohol extracts of P.forrestii(CDLF and CQAF)had significant anti-RA activities,and 10 monomers with such activities were identified.The anti-RA activities of active monomers,CDLF,and CQAF were compared by the enzyme-linked immunosorbent assay(ELISA)with interleukin(IL)-6,nitric oxide(NO),IL-1β,and prostaglandin E(2 PGE2)as indicators.Network pharmacology was employed to analyze the possible molecular mechanism of P.forrestii against RA.The targeting ability of P.forrestii chemical monomers to TNF-αwas verified by TNF-αmolecular docking,surface plasmon resonance(SPR),and TNF-α-induced L929 injury model.Result:ELISA showed that the anti-RA activities of CDLF and CQAF were significantly stronger than those of identified 10 active monomers.Network pharmacology analysis showed that the core targets of P.forrestii against RA were signal transducer and activator of transcription protein 3(STAT3),TNF,and IL-6.Gene Ontology(GO)analysis revealed collagen catabolism,inflammatory response,positive regulation of nuclear factor kappa-B(NF-κB)transcription factor activity,and positive regulation of B cell proliferation.Kyoto Encyclopedia of Genes and Genomes(EKGG)pathway enrichment analysis demonstrated TNF signaling pathway,phosphoinositide 3-kinase(PI3K)/protein kinase B(Akt)signaling pathway,NF-κB signaling pathway,Toll-like receptor signaling pathway,mitogen-activated protein kinase(MAPK)signaling pathway,etc.Verification experiments by TNF-αmolecular docking,SPR,and TNF-α-induced L929 injury model found that CDLF and CQAF had good binding activities and could manifestly antagonize TNF-α.However,the active components separated and identified from CDLF and CQAF did not show the same anti-TNF-αactivity.Conclusion:The CDLF and CQAF of P.forrestii m

关 键 词：类风湿关节炎 黑骨藤 网络药理学 肿瘤坏死因子(TNF)-α 分子对接 

分 类 号：R285[医药卫生—中药学] R289[医药卫生—中医学]