摘要
目的:从网络药理学、分子对接及细胞验证实验探讨黑沙蒿有效物质群(HSH)治疗类风湿关节炎(RA)作用机制。方法:利用Pubchem(化合物数据库)、BATMAN-TCM(中药分子机制生物信息学分析工具)、DisGeNet(疾病基因关联网络数据库)、GeneCards(基因卡片数据库)、NCBI(美国国家生物技术信息中心)、OMIM(在线人类孟德尔遗传数据库)、PharmGKB(药理基因组知识库)、GEO(基因表达综合数据库)等数据库及R语言获得HSH核心成分和成分靶点、疾病靶点及HSH治疗RA的潜在靶点。建立蛋白-蛋白相互作用(PPI)网络,进行基因本体(GO)和京都基因和基因组百科全书(KEGG)富集分析,选取核心成分和核心靶点进行分子对接。采用细胞计数试剂盒-8(CCK-8)和CFSE标记法检测HSH的无毒浓度和作用浓度。采用流式细胞技术、流式液相多重蛋白定量技术(CBA)、逆转录PCR(RT-PCR)等实验技术检测白细胞介素(IL)-17/T细胞受体信号通路相关细胞、关键性炎症因子及蛋白的变化。结果:对羟基肉桂酸、邻羟基肉桂酸、o-hydroxycapillene等3个核心成分与RA的关联度最高,其成分靶点有90个;经疾病相关靶点及RA差异表达基因交集获得875个RA疾病靶点。将RA疾病靶点与成分靶点相交,得到36个RA的潜在靶点;经PPI构建获取5个核心靶点,核心靶点与核心成分之间具有一定的结合活性;GO和KEGG富集分析显示IL-17/T细胞受体信号通路与HSH抗RA密切相关,HSH参与炎症反应及细胞因子产生和调节等多种生物过程,进而调控机体炎症和免疫反应发挥抗RA的作用。HSH的作用浓度为16μg/mL,HSH能下调辅助性T细胞1型(Th1)水平,上调调节性T细胞(Treg)水平,降低Th17/Treg的比值,也能显著降低IL-21、IL-6、IL-2、γ干扰素(IFN-γ)、转化生长因子β1(TGF-β1)水平。RT-PCR实验结果显示,HSH显著下调维甲酸相关孤儿受体γt(RORγt)mRNA的表达,而叉头翼状螺旋转录因子(Foxp3)mRNA的表达升高无显著性差异。结论:HSH对RA具有良好的治疗作用,其作用机制可能与降低Th1,升高Treg,调节Th17/Treg的平衡和降低IL-21、IL-6、IL-2、IFN-γ、TGF-β1及RORγt表达水平有关。
Objective:To explore the mechanism of the effective substance groups from Artemisia ordosica Krasch in the treatment of rheumatoid arthritis(RA)by network pharmacology,molecular docking and cell validation experiments.Methods:The databases such as Pubchem,BATMAN-TCM,DisGeNet,GeneCards NCBI,OMIM,PharmGKB,GEO,and R language were used to obtain HSH core components and component targets,disease targets,and potential targets for HSH treatment of RA.The protein-protein interaction(PPI)network was established and the gene ontology(GO)and Kyoto encyclopedia of genes and genomes(KEGG)enrichment analysis were conducted to select core components and core targets for molecular docking.The CCK-8 and CFSE labeling method were used to detect the non-toxic concentration and effective concentration of HSH.The experimental techniques such as flow cytometry,CBA,and RT-PCR were used to detect changes in cells,key inflammatory factors,and proteins related to the interleukin(IL)-17/T cell receptor signaling pathway.Results:The three core components,namely hydroxycinnamic acid,o-hydroxycinnamic acid,and o-hydroxycapillene,had the highest correlation with RA,with 90 target points.Totally 875 RA disease targets were obtained by intersecting disease-related targets and differentiable expressed genes in RA.Intersection of RA disease targets with component targets resulted in 36 potential targets for RA.A total of 5 core targets were obtained through PPI construction,and there was a certain binding activity between the core targets and the core components.GO and KEGG enrichment analysis results showed that the IL-17/T cell receptor signaling pathway was closely related to the anti-RA effect of HSH,which also participated in regulating various biological processes such as inflammatory response,cytokine production,and regulation,thereby regulating the body's inflammation and immune response to exert anti-RA effects.The effective concentration of HSH was 16μg/mL.HSH could down-regulate Th1 levels,up-regulate Treg levels,reduced Th17/Treg ratios,and significantly reduced the levels of IL-21,IL-6,IL-2,interferon-γ(IFN-γ),and transforming growth factor-β1(TGF-β1).The RT-PCR experiment results showed that HSH significantly down-regulated the expression of RORγt mRNA,while there was no significant difference in the expression of Foxp3 mRNA.Conclusions:HSH has good therapeutic effect on RA,and its mechanism may be related to reducing Th1,increasing Treg,regulating Th17/Treg balance,and reducing the expression levels of IL-21,IL-6,IL-2,IFN-γ,TGF-β1,and RORγt.
作者
呼格吉乐
王青虎
张小峰
康秀荣
高明霞
Hugejile;WANG Qinghu;ZHANG Xiaofeng;KANG Xiurong;GAO Mingxia(College of Traditional Mongolian Medicine,Inner Mongolia University for Nationalities,Tongliao Inner Mongolia 028000,China;Tongliao Vocational College,Tongliao Inner Mongolia 028000,China)
出处
《中医药导报》
2025年第8期31-40,52,共11页
Guiding Journal of Traditional Chinese Medicine and Pharmacy
基金
内蒙古自治区自然科学基金项目(2023MS08027)
内蒙古自治区教育厅一流学科科研专项项目子课题(YLXKZX-NMD-002)。
关键词
类风湿关节炎
黑沙蒿
有效物质群
网络药理学
分子对接
细胞验证实验
rheumatoid arthritis
Artemisia ordosica Krasch
effective substance groups
network pharmacology
molecular docking
cell validation experiment
