摘要
目的:采用网络药理学、分子对接及细胞实验探讨至真方抗结直肠癌(colorectal cancer,CRC)的作用机制。方法:利用TCMSP数据库检索至真方的活性成分和作用靶点,GeneCards数据库检索CRC靶点,并获取两者的交集靶点。通过STRING数据库构建蛋白质-蛋白质相互作用(protein-protein interaction,PPI)网络,根据拓扑分析筛选核心靶点。采用Cytoscape 3.7.2软件构建“中药-活性成分-靶点-疾病”网络,根据拓扑分析筛选关键成分。借助R软件进行京都基因与基因组百科全书(Kyoto Encyclopedia of Genes and Genomes,KEGG)富集分析。采用AutoDock软件对关键成分和核心靶点进行分子对接验证。将SW620细胞分为对照组(CTR)、至真方低剂量组(ZZF-L,65 mg·L^(-1))及至真方高剂量组(ZZF-H,440 mg·L^(-1)),干预48 h。采用RT-qPCR法检测细胞蛋白激酶B(protein kinase B,AKT1)、肿瘤蛋白p53(tumor protein 53,TP53)基因表达水平;Western blot法检测细胞磷脂酰肌醇3-激酶(phosphatidylinositol-3-kinase,PI3K)、p-PI3K、AKT、p-AKT、p53蛋白表达水平。结果:至真方活性成分41个,作用靶点225个。CRC靶点10317个,其与至真方的交集靶点193个。核心靶点包括AKT1、TP53、IL-6等,关键成分包括槲皮素、木犀草素等。KEGG通路主要包括PI3K/AKT信号通路、p53信号通路等。关键成分(槲皮素、木犀草素)与核心靶点(AKT、TP53)的结合能均小于-5.0 kJ·mol^(-1)。与CTR组比较,ZZF-H组细胞AKT1 mRNA、TP53 mRNA水平升高(P<0.05)。与CTR组比较,ZZF-H组细胞p-PI3K、p-AKT蛋白表达水平降低,AKT、p53蛋白表达水平升高(P<0.05)。结论:至真方可能通过槲皮素、木犀草素调控PI3K/AKT、p53信号通路,作用于AKT、p53等靶点,从而发挥抗CRC的作用。
Objective:To investigate the mechanism of action of Zhizhen Formula against colorectal cancer(CRC)by network pharmacology,molecular docking and cell experiments.Methods:The active ingredients and targets of Zhizhen Formula were retrieved by TCMSP database,and CRC targets were retrieved by GeneCards database,and the intersection targets of the two were obtained.A protein-protein interaction(PPI)network was constructed through the STRING database,and the core targets were screened according to topological analysis.Cytoscape 3.7.2 software was used to construct a"traditional Chinese medicine-active ingredient-target-disease"network,and key components were screened according to topological analysis.Enrichment analysis of the Kyoto Encyclopedia of Genes and Genomes(KEGG)was performed using R software.AutoDock software was used to verify the molecular docking of key components and core targets.SW620 cells were divided into control group(CTR)and Zhizhen Formula low-dose group(ZZF-L,65 mg·L^(-1))and Zhizhen Formula high-dose group(ZZF-H,440 mg·L^(-1)),and the intervention was 48 h.RT-qPCR was used to detect the expression levels of protein kinase B(AKT1)and tumor protein p53(TP53).Western blot was used to detect the expression levels of phosphatidylinositol-3-kinase(PI3K),p-PI3K,AKT,p-AKT and p53 proteins.Results:There were 41 active ingredients and 225 targets of Zhizhen Formula.There were 10,317 CRC targets,and 193 intersecting targets with Zhizhen Formula.The core targets include AKT1,TP53,IL-6,etc.,and the key components include quercetin,luteolin,etc.The KEGG pathway mainly includes PI3K/AKT signaling pathway,p53 signaling pathway,etc.The binding energy of the key components(quercetin and luteolin)and the core targets(AKT,TP53)was less than-5.0 kJ·mol^(-1).Compared with the CTR group,the levels of AKT1 mRNA and TP53 mRNA in the ZZF-H group were increased(P<0.05).Compared with the CTR group,the expression levels of p-PI3K and p-AKT proteins in the ZZF-H group decreased,and the expression levels of AKT and p53 proteins increased(P<0.05).Conclusion:Zhizhen Formula may regulate PI3K/AKT and p53 signaling pathways through quercetin and luteolin,and act on AKT,p53 and other targets,thereby exerting anti-CRC effects.
作者
韩惠杰
李永静
赵永波
刘辉
王松坡
HAN Huijie;LI Yongjing;ZHAO Yongbo;LIU Hui;WANG Songpo(Department of Traditional Chinese Medicine,The First People′s Hospital of Shanghai,Shanghai China 200080;Department of Traditional Chinese Medicine,Shuguang Hospital Affiliated to Shanghai University of Traditional Chinese Medicine,Shanghai China 201203;Department of Neurology,The First People′s Hospital of Shanghai,Shanghai China 200080)
出处
《中医学报》
2025年第9期1968-1976,共9页
Acta Chinese Medicine
基金
第五批全国中医临床优秀人才研修项目{国中医药人教函〔2022〕1号}
上海市进一步加快中医药事业发展三年行动计划项目(张氏内科流派脾胃病传承创新团队)(2021LPTD-010)
上海申康医院发展中心临床三年行动计划资助项目(SHDC2020CR4049)。
