基于网络药理学探讨瘀血痹片的抗炎作用机制

Anti-inflammatory Mechanism of Yuxuebi(瘀血痹)Tablet Based on Network Pharmacology

目的:以网络药理学探讨瘀血痹片的抗炎作用机制,并进行实验验证。方法:经由Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP)数据库、SwissTargetPrediction数据库、Uniprot数据库确定瘀血痹片11味中药的活性成分及作用靶点;于GeneCards数据库获取炎症相关靶点;将瘀血痹片作用靶点及炎症靶点在Venny 2.1中得到的交集靶点,通过STRING 11.0数据库建立蛋白质相互作用(PPI)网络模型,并于DAVID数据库对靶点富集分析;采用Cytoscape 3.7.2构建“瘀血痹片-潜在活性成分-靶点”网络图及“炎症靶点-通路”网络图。通过角叉菜胶致炎小鼠模型证明瘀血痹片抗炎作用,通过酶联免疫吸附(ELISA)法检测血清肿瘤坏死因子-α(TNF-α)、白介素-1β(IL-1β)、IL-6的含量,蛋白印迹(Western Blot)法检测ERK1/2、JNK、p38以及NF-κB p65蛋白的表达,进一步验证其作用机制。结果:通过网络药理学分析,瘀血痹片抗炎的核心成分包括槲皮素、β-谷甾醇、豆甾醇、山柰酚、木犀草素等,关键靶点包括PTGS2、RELA、TNF、MAPK1、MAPK14等,可能通过RNA聚合酶Ⅱ启动子对转录的正调控、炎症反应、一氧化氮生物合成过程的正调控、细胞对脂多糖的应答、脂多糖介导的信号通路等;T细胞受体信号通路、NOD样受体信号通路、类风湿关节炎、Toll样受体信号通路、TNF信号通路等发挥抗炎作用。在动物实验方面,与正常对照组相比,模型对照组小鼠足肿胀度显著增加,血清TNF-α、IL-1β及IL-6含量显著升高,小鼠足爪组织ERK1/2、JNK、p38及NF-κB p65蛋白表达显著上调(P<0.05或P<0.01);与模型对照组比较,瘀血痹片1.5 g/kg组小鼠在致炎后第6 h足肿胀度显著降低,血清炎症因子IL-1β及IL-6含量显著降低,小鼠足爪组织ERK1/2、JNK、p38及NF-κB p65蛋白表达显著下调(P<0.05或P<0.01)。结论:瘀血痹片可以降低角叉菜胶致炎小鼠模型足肿胀度及血清炎症因子水平,可能通过下调足爪组织ERK1/2、JNK、p38以及NF-κB p65蛋白表达,发挥抗炎作用。

Objective:To explore the anti-inflammatory mechanism of Yuxuebi(瘀血痹)tablet based on network pharmacology and to conduct experimental verification.Methods:The active ingredients and targets of 11 Chinese medicines in Yuxuebi tablet were searched from the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP),SwissTargetPrediction,and Uniprot,and the inflammation-related targets were obtained from GeneCards.Then,we employed Venny 2.1 to obtain the common targets shared by Yuxuebi tablet and inflammation,and established the protein-protein interaction(PPI)network via STRING 11.0.The Database for Annotation,Visualization and Integrated Discovery(DAVID)was used for the enrichment of targets,and Cytoscape 3.7.2 was used to construct the Yuxuebi tablet-active ingredient-target network and the target-pathway network.We established the carrageenan-induced paw swelling model to study the anti-inflammatory effect of Yuxuebi tablet,employed ELISA to determine the serum levels of TNF-α,IL-1β,and IL-6,and carried out Western blotting to detect the expression of ERK1/2,JNK,p38,and NF-κB p65.Results:The active ingredients of Yuxuebi tablet in the treatment of inflammation included quercetin,beta-sitosterol,stigmasterol,kaempferol,and luteolin,and the core targets included PTGS2,RELA,TNF,MAPK1,and MAPK14.Yuxuebi tablet may exert anti-inflammatory effect through the T cell receptor signaling pathway,NOD-like receptor signaling pathway,rheumatoid arthritis,Toll-like receptor signaling pathway,and TNF signaling pathway.Compared with those in the normal group,the mice in the model group had increased paw swelling degree,elevated serum levels of TNF-α,IL-1β,and IL-6,and up-regulated expression of JNK,ERK1/2,p38,and NF-κB p65(P<0.05 or P<0.01).Compared with the model group,1.50 g/kg Yuxuebi tablets ameliorated the paw swelling of mice 6 h after induction of inflammation,lowered the levels of IL-1βand IL-6 in serum,and down-regulated the expression of ERK1/2,JNK,p38,and NF-κB p65(P<0.05 or P<0.01).Conclusion:Yuxuebi tablet can alleviate the carrageenan-induced paw swelling,lower the serum levels of inflammatory cytokines,and down-regulate the expression of ERK1/2,JNK,p38,and NF-κB p65 in the paw tissue,thus exerting the anti-inflammatory effect.