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基于UPLC-Orbitrap-MS^(2)、网络药理学及细胞实验验证探究芍药甘草汤治疗类风湿关节炎的作用机制研究

Mechanisms of Shaoyao Gancao Decoction in treatment of rheumatoid arthritis based on UPLC-Orbitrap-MS^(2),network pharmacology,and cellular experiment verification
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摘要 芍药甘草汤是临床治疗关节疾病如类风湿关节炎(rheumatoid arthritis,RA)的经典方剂,而其作用机制尚不清晰。该文拟采用化学、网络药理学分析并结合细胞实验探究其治疗RA的作用机制。采用UPLC-Orbitrap-MS2联用技术对芍药甘草汤(Shaoyao Gancao Decoction,SGD)及大鼠口服SGD后的含药血清进行定性分析,以鉴定SGD的化学成分及入血成分;采用SwissTargetPrediction、PharmMapper、GeneCards和DrugBank数据库获取该方入血成分治疗RA的潜在作用靶点,STRING数据平台构建靶点蛋白-蛋白相互作用(protein-protein interaction,PPI)网络,Metascape数据库进行GO功能富集分析和KEGG通路富集分析,利用分子对接技术及脂多糖(lipopolysaccharide,LPS)诱导的RAW264.7细胞进行体外验证。结果提示SGD中共指认到95个化合物,其中15个为原型吸收成分,包括7个黄酮类、5个萜类化合物、2个酚类化合物及1个其他类化合物;网络药理学分析表明甘草异黄烷酮、芹糖甘草苷、5-羟基阿魏酸、芍药内酯苷、常春藤皂苷元、芍药苷等为SGD治疗RA药效成分,SRC、MAPK、EGFR、HSP90AA1、STAT3等为SGD治疗RA的核心靶点,对NF-κB、PI3K-Akt、MAPK信号通路的调节作用为SGD抗RA的关键机制。分子对接结果显示6个核心成分和SRC、MAPK、NF-κB关键靶点具有较高亲和力。体外细胞实验显示,SGD可下调LPS诱导RAW264.7细胞炎症因子白细胞介素-1β(IL-1β)、环氧合酶-2(COX-2)、肿瘤坏死因子-α(TNF-α)的表达,Western blot分析结果显示,SGD可以显著降低NF-κB p65和p38 MAPK蛋白磷酸化水平,该研究为SGD治疗RA的活性成分及作用机制研究提供科学依据。 Shaoyao Gancao Decoction(SGD)is a classic formula used in the clinical treatment of joint diseases,such as rheumatoid arthritis(RA),though its mechanism of action remains unclear.This study aimed to explore the mechanism of SGD in treating RA through chemical and network pharmacology analyses,combined with cellular experiments.UPLC-Orbitrap-MS~2 was used to qualitatively analyze SGD and drug-containing serum of rats after oral administration of SGD,thereby identifying the chemical composition and plasma components of SGD.Potential targets for the plasma components in treating RA were identified using the SwissTargetPrediction,PharmMapper,GeneCards,and DrugBank databases,and a protein-protein interaction(PPI)network was constructed using the STRING data platform.GO functional enrichment and KEGG pathway enrichment analyses were conducted using the Metascape database.Molecular docking and lipopolysaccharide(LPS)-induced RAW264.7 cell experiments were utilized for in vitro validation.The results identified 95 compounds in SGD,including 15 prototypical absorbed components,i.e.,7 flavonoids,5 terpenoids,2 phenolic compounds,and 1 other compound.Network pharmacology analysis revealed that licoisoflavanone,liquiritin apioside,5-hydroxyferulic acid,albiflorin,hederagenin,and paeoniflorin were the pharmacodynamic components of SGD for treating RA.The core targets of SGD for RA treatment were identified as SRC,MAPK,EGFR,HSP90AA1,and STAT3,with regulation of the NF-κB,PI3K-Akt,and MAPK signaling pathways identified as key mechanisms for anti-RA effects of SGD.Molecular docking results showed that the six core components exhibited high affinity with the key targets SRC,MAPK,and NF-κB.In vitro cellular experiments demonstrated that SGD down-regulated the expression of inflammatory factors,including interleukin-1β(IL-1β),cyclooxygenase-2(COX-2),and tumor necrosis factor-α(TNF-α),in LPS-induced RAW264.7 cells.Western blot analysis revealed that SGD significantly reduced the phosphorylation levels of NF-κB p65 and p38 MAPK proteins.This study provides a scientific basis for further research into the active components and mechanisms of action of SGD in treating RA.
作者 竹蒙 郭辉 代丽萍 王智民 ZHU Meng;CUO Hui;DAI Li-ping;WANG Zi-min(Henan Collaborative Innovation Center for Research and Development on the Whole Industry Chain of Yu-Yao,Henan University of Chinese Medicine,Zhengzhou 450046,China;Henan University of Chinese Medicine,Zhengzhou 450046,China;National Engineering Laboratory for Quality Control Technology of Chinese Herbal Medicines,Institute of Chinese Materia Medica,China Academy of Chinese Medical Sciences,Beijing 100700,China)
出处 《中国中药杂志》 CAS CSCD 北大核心 2024年第22期6149-6164,共16页 China Journal of Chinese Materia Medica
基金 河南省科技研发计划联合基金(优势学科培育类)项目(222301420082) 2023年度河南省高校科技创新团队(23IRTSTHN028)。
关键词 芍药甘草汤 类风湿关节炎 UPLC-Oribtrap-MS^(2) 入血成分 网络药理学 Shaoyao Gancao Decoction rheumatoid arthritis UPLC-Oribtrap-MS^(2) plasma components network pharmacology
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