摘要
目的通过网络药理学预测、动物实验验证探讨四妙散干预类风湿关节炎(RA)的潜在核心靶点及其作用机制。方法从中药系统药理学数据库与分析平台(TCMSP)获得四妙散的活性成分及其对应的靶点,在通用蛋白质资源(UniProt)数据库进行比对;从人类基因数据库(GeneCards)、在线人类孟德尔遗传数据库(OMIM)、治疗靶点数据库(TTD)、DrugBank和疾病基因网络数据库(DisGeNet)筛选RA相关靶点;在蛋白质相互作用检索数据库(STRING)中将四妙散和RA的共同靶点构建蛋白质相互作用(PPI)网络,并在注释、可视化和综合发现数据库(DAVID)通过基因本体论(GO)功能注释及京都基因与基因组百科全书(KEGG)信号通路富集分析进行基因注释及功能富集分析;构建中药活性成分-疾病靶点-信号通路网络图,预测四妙散治疗RA的作用机制;将核心靶点芳香烃受体(AHR)与四妙散中的核心活性成分进行分子对接,预测靶向AHR的中药成分。在动物实验中,将30只雌性SPF级C57/BL小鼠分为正常组、模型组、甲氨蝶呤组(1.52 mg/kg,3 d灌胃1次)及四妙散组(12.48 g/kg,每天灌胃1次),干预30 d。测量小鼠踝关节直径,并进行关节炎指数评分。HE染色法观察小鼠踝关节组织炎性反应,免疫组织化学法(IHC)检测小鼠踝关节细胞色素P4501A1(CYP1A1)、核因子κB的p65亚基(p65)及磷酸化p65(p-p65)蛋白表达,多重荧光免疫组织化学法(mIHC)检测小鼠踝关节组织叉头框蛋白P3(FOXP3)、白细胞介素-17A(IL-17A)的蛋白表达。结果从TCMSP数据库筛选出四妙散的41个活性成分,得到228个靶点;从GeneCards、OMIM、TTD、DrugBank和DisGeNet数据库得到1207个RA疾病靶点;将药物靶点与疾病靶点汇总,得到94个共同靶点;通过GO功能注释及KEGG信号通路富集分析,得到612条GO富集条目、143条KEGG信号通路;将AHR配体结合结构域与Degree值排名前10位的药物活性成分(槲皮素、豆甾醇、汉黄芩素、β-谷甾醇、山柰酚、黄芩素等)分别进行分子对接,发现豆甾醇、β-谷甾醇、四氢小檗碱及异延胡索单酚碱与AHR蛋白的结合较稳定。动物实验结果表明,与模型组比较,四妙散组和甲氨蝶呤组小鼠关节肿胀程度和关节炎指数评分降低(P<0.01);IHC结果表明,相较于模型组小鼠,四妙散组和甲氨蝶呤组小鼠踝关节CYP1A1蛋白表达增加,p65、p-p65蛋白表达降低(P<0.05,P<0.01);mIHC结果表明,相较于模型组小鼠,四妙散组和甲氨蝶呤组小鼠踝关节IL-17A蛋白表达降低、FOXP3蛋白表达增加(P<0.05,P<0.01)。结论四妙散对RA模型小鼠的关节炎症具有较好的干预作用,AHR是其干预的核心靶点之一,四妙散可能通过激活AHR,减少p65的磷酸化从而抑制局部炎症过度应答。同时,四妙散可抑制IL-17A蛋白的表达,并促进FOXP3蛋白的表达,提示其对T细胞免疫平衡具有调节作用,而AHR可能是其调控辅助性T细胞17/调节性T细胞平衡的关键靶点。
Objective To investigate the potential core target and its mechanism of Simiao San(SMS)in the treatment of rheumatoid arthritis(RA)using network pharmacology and animal experiments.Methods Active components and corresponding SMS targets were obtained from the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP)and cross-referenced with the Universal Protein(UniProt)database.RA-related targets were screened from The Human Gene Database(GeneCards),Online Mendelian Inheritance in Man(OMIM),Therapeutic Target Database(TTD),DrugBank,and Disease Gene Network(DisGeNet).Protein-protein interaction(PPI)networks were constructed for shared targets between SMS and RA using Search Tool for the Retrieval of Interacting Genes/Proteins(STRING),followed by Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment analyses via The Database for Annotation,Visualization and Integrated Discovery(DAVID).A“herb active component-disease target-signaling pathway”network was established to predict the mechanism of SMS in RA treatment.Molecular docking was performed between aryl hydrocarbon receptor(AHR)and the core active components of SMS to identify AHR-targeting constituents.For animal experiments,30 female SPF-grade C57/BL mice were randomly divided into normal,model,methotrexate(1.52 mg/kg,every 3 days),and SMS(12.48 g/kg,daily)groups with a 30-day intervention.Ankle diameter and arthritis index scores were measured.HE staining was used to assess joint inflammation,whereas immunohistochemistry(IHC)was used to measure cytochrome P4501A1(CYP1A1),nuclear factor kappa B subunit p65(p65),and phosphorylated p65(p-p65)protein expression levels.Multiplex immunofluorescence(mIHC)was used to evaluate forkhead box protein P3(FOXP3)and interleukin-17A(IL-17A)protein expression.Results Forty-one active components and 228 targets of SMS were identified from TCMSP,whereas 1,207 RA-related targets were extracted from GeneCards,OMIM,TTD,DrugBank,and DisGeNet.Ninety-four overlapping targets were analyzed,yielding 612 GO terms and 143 KEGG pathways.Molecular docking of the ligand-binding domain of AHR with the top 10 Degree values of compounds of SMS(quercetin,stigmasterol,wogonin,beta-sitosterol,kaempferol,baicalein,et al.)revealed that stigmasterol,beta-sitosterol,(S)-canadine,and isocorypalmine was able to bind to AHR stably.In vivo,compared to the model group,the mice of the SMS and methotrexate groups joint swelling and arthritis index scores reduced(P<0.01).IHC indicated elevated CYP1A1 protein and decreased p65 and p-p65 protein levels in the SMS and methotrexate groups(P<0.05,P<0.01).mIHC demonstrated reduced IL-17A and increased FOXP3 protein expression in the SMS and methotrexate groups(P<0.05,P<0.01).Conclusion SMS alleviates joint inflammation in RA mice,potentially by targeting AHR,one of the core targets.SMS may suppress excessive inflammatory responses by activating AHR and inhibiting p65 phosphorylation.Additionally,SMS modulates the helper T cells 17/regulatory T cells balance by downregulating IL-17A and upregulating FOXP3.These results suggest that AHR is a key mediator in T-cell immune regulation.
作者
孙钰禾
蒋海旭
许杰
张红林
赵子涵
陆清怡
SUN Yuhe;JIANG Haixu;XU Jie;ZHANG Honglin;ZHAO Zihan;LU Qingyi(School of Life Sciences,Beijing University of Chinese Medicine,Beijing 100029,China;School of Chinese Materia Medica,Beijing University of Chinese Medicine,Beijing 102488,China)
出处
《北京中医药大学学报》
北大核心
2025年第8期1067-1080,共14页
Journal of Beijing University of Traditional Chinese Medicine
基金
国家自然科学基金项目(No.82205068)
北京中医药大学“揭榜挂帅”项目(No.2023-JYB-JBQN-019)。
关键词
四妙散
类风湿关节炎
网络药理学
芳香烃受体
小鼠
Simiao San
rheumatoid arthritis
network pharmacology
aryl hydrocarbon receptor
mice
