目的 运用网络药理学结合实验验证探讨白头翁汤治疗结直肠癌的作用机制。方法 在中药系统药理学分析平台(traditional Chinese medicine systems pharmacology database and analysis plat-form，TCMSP)数据库中寻找与白头翁、黄连、黄柏、秦皮相关的活性分子及靶点。在人类基因数据库(GeneCards)、靶点数据库(therapeutic target database，TTD)、孟德尔遗传数据库(online mende-lian inheritance in man，OMIM)搜索结直肠癌相关靶点。将白头翁汤有效成分的靶点与结直肠癌靶点取交集获得共同靶点，利用Cytoscape 3。7。2 软件构建"药物-成分-靶点-疾病"网络。采用蛋白质互作数据库(search tool for recurring instances of neighbouring genes，STRING)平台进行蛋白质相互作用(protein-protein interaction networks，PPI)网络的构建与分析。同时分别利用DAVID数据库(database for annotation，visualization and integrated discovery)和Bioconductor中的R包clus-terProfiler进行基因本体论(gene ontology，GO)富集和京都基因与基因组百科全书(Kyoto encyclo-pedia of genes and gnomes，KEGG)通路富集分析。采用噻唑蓝溴化四唑(methylthiazolyldiphenyl-tetrazolium bromide，MTT)实验检测白头翁汤处理过的人结肠癌细胞SW480 增殖情况，同时在细胞水平利用蛋白质印迹法(Western-blotting)和定量聚合酶链反应(quantitative polymerase chain re-action，q-PCR)对网络药理学预测出的核心靶点及通路进行初步验证。结果 从白头翁汤中筛选得到53 个活性成分，作用于结直肠癌的交集靶点 157 个。网络药理学分析显示白头翁汤中的槲皮素、β-谷甾醇等活性成分通过作用于关键靶点原癌基因JUN(Jun proto-oncogene，JUN)、蛋白激酶B1(protein kinase B1，Akt1)、丝裂原活化蛋白激酶 1(mitogen-activated protein kinase 1，MAPK1)等以及调控白介素17(interleukin 17，IL-17)信号通路、肿瘤坏死因子(tumor necrosis factor，TNF)信号通路、缺氧诱导因子-1(hypoxia-inducible factor 1，HIF-1)信号通路等信号通路治疗结直肠癌。细胞实验证实白头翁汤能够有效抑制SW480 细胞增殖、下调靶点蛋白p-Akt和p-p38 MAPK的表达、抑制IL-17、TNF-α、HIF-1α的mRNA水平。结论 白头翁汤能够通过调控AKT1、MAPK1等靶点以及调控IL-17、TNF、HIF-1 等信号通路，从而治疗结直肠癌。
Analysis of the mechanism of Baitouweng Decoction in the treatment of colorectal cancer based on network pharmacology combined with experimental verification
Objective To explore the mechanism of Baitouweng Decoction in the treatment of colorectal cancer,using network pharmacology combined with experimental verification.Methods Active molecules and targets related to Pulsatilla chinensis,Coptis chinensis,Phellodendron and Qinpi were searched in TCMSP database of Chinese medicine system pharmacological analysis platform.Colorectal cancer-related targets were searched in the Human Gene Database(GeneCards),Target Database(TTD)and Mendelian Genetic Database(OMIM).The targets of the effective components of Baitouweng Decoction and the targets of colorectal cancer were intersected to obtain a common target.Cytoscape 3.7.2 software was used to construct a drug-component-target-disease network.The STRING platform was used to construct and analyze the protein-protein interaction network(PPI).At the same time,the DAVID database and the R package clusterProfiler in Bioconductor were used for GO enrichment and KEGG signal pathway analysis.The MTT experiment was used to detect the proliferation of human colon cancer cells SW480 treated with Baitouweng Decoction,and the core targets and pathways predicted by network pharmacology were preliminarily verified by Western-blotting and q-PCR at the cell level.Results 53 active ingredients were screened from Baitouweng Decoction,which acted on 157 intersection targets for colorectal cancer.Network pharmacology analysis showed that the active ingredients such as quercetin and β-sitosterol in Baitouweng Decoction act on key targets,JUN,AKT1,MAPK1,etc.,and regulation of IL-17,TNF,HIF-1 and other signaling pathways to treat colorectal cancer.Cell experiments confirmed that Baitouweng Decoction can effectively inhibit the proliferation of SW480 cells,down-regulate the expression of target proteins p-Akt and p-p38 MAPK,and inhibit the mRNA levels of IL-17,TNF-α and HIF-1α.Conclusion Baitouweng Decoction can treat colorectal cancer by regulating AKT1,MAPK1 and other targets and regulating IL-17,TNF,HIF-1 and other signaling pathways.
network pharmacologyBaitouweng Decoctioncolorectal cancermechanism of actionexperimental verification
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