基于网络药理学及体外实验探究野蚕豆根抗氧化活性及其作用机制

Antioxidant activity and mechanism of Centranthera grandiflora Benth roots based on network pharmacology and in vitro experiments

利用网络药理学和体外实验探讨野蚕豆根抗氧化活性物质基础及作用机制。首先,采用DPPH法、ABTS法、清除羟自由基法对野蚕豆根进行体外抗氧化实验验证;通过TCMSP数据库和文献搜索筛选出野蚕豆根中的活性物质,同时收集其作用靶点;借助OMIM、GeneCards数据库获得氧化应激相关疾病靶点;将野蚕豆根成分靶点与氧化应激靶点取交集,通过STRING数据库构建蛋白互作网络(protein-protein interaction networks,PPI);利用R语言对交集靶点进行基因本体(GO,Gene Ontology)富集分析和京都基因与基因组百科全书(Kyoto Encyclopedia of Genes and Genomes,KEGG)富集分析;借助AutoDock 1.5.7软件对核心成分及关键靶点基因进行分子对接,通过Pymol进行可视化操作;利用蛋白质免疫印迹(Western blot)检测进行机制验证。结果显示,野蚕豆根提取物对DPPH自由基、ABTS自由基和羟基自由基有一定的清除能力,其半数抑制浓度(IC_(50))分别为0.166、0.054和0.180 mg/mL;从野蚕豆根中共筛选出22个活性成分,86个成分靶点以及84个交集靶点,其中包括STAT3、HSP90AA1、HDAC1等23个核心靶点;GO富集分析共得到了3386个条目,主要包括碳水化合物结合、核受体活性等分子功能。KEGG富集分析共得到了211个条目,主要包括PI3K-Akt信号通路、化学致癌-受体激活等通路;分子对接结果显示,野蚕豆根中活性成分与核心靶点间结合效应较强,结合能均小于-1.2 kcal/mol;Western blot结果表明,野蚕豆根提取物能明显上调p-PI3K蛋白及p-Akt蛋白的表达。本研究初步表明野蚕豆根提取物可通过激活PI3K-Akt信号通路从而发挥抗氧化作用,为后期野蚕豆根抗氧化活性的研究与应用提供理论依据及参考。

To explore the antioxidant activity and mechanism of Centranthera grandiflora Benth roots based on network pharmacology and in vitro experiments.Antioxidant activity were evaluated by DPPH radical scavenging assay,ABTS radical scavenging assay,and hydroxyl radical scavenging method.The TCMSP database,and literature mining were used to collect active ingredients of C.grandiflora roots,target genes related to oxidative stress were collected through the OMIM and GeneCards database,and protein interaction networks(PPI)were constructed through the STRING database.GO and KEGG enrichment analyses were performed on the potential targets using the R language.Molecular docking of key active ingredients with potential targets using AutoDock 1.5.7 software.The results showed that C.grandiflora roots extract had a certain ability on DPPH,ABTS,and hydroxyl radicals scavenging,with IC_(50) values of 0.166,0.054 and 0.180 mg/mL,respectively.Twenty-two active ingredients of C.grandiflora roots,86 target genes,and 84 intersection targets were collected,including STAT3,HSP90AA1,HDAC1,etc.Go function enrichment analysis found 3386 items,including molecular functions such as carbohydrate binding and nuclear receptor activity.KEGG pathway enrichment analysis found 211 items(P<0.05),involving PI3K/AKT,and other signaling pathways related to C.grandiflora roots.The molecular docking results showed that the predicted key component,showed good binding to the core targets,binding energies<-1.2 kcal/mol.The Western blot results showed that the extract of C.grandiflora roots significantly upregulated the expression of p-PI3K and p-Akt proteins.The above results suggest that the extract of C.grandiflora roots can exert antioxidant activity by activating the PI3K-Akt signaling pathway.This study provides a reference for the study on the clinical use and application of C.grandiflora roots.