摘要
目的采用网络药理学和分子对接技术预测莪术-三棱(Curcumae Rhizoma-Sparganii Rhizoma,CR-SR)药对调控血管生成抗肺癌作用机制,并通过斑马鱼模型进行初步验证。方法采用网络药理学方法筛选CR和SR的活性成分及其靶点,分别与从OMIM database和GeneCards数据库筛选得到的肺癌和抗血管生成靶点取交集,取交集靶点构建药对-肺癌和药对-抗血管生成的蛋白质-蛋白质相互作用(Proteinprotein interaction,PPI)网络,筛选药对-肺癌和药对-抗血管生成的共有靶点和核心靶点。通过Metascape数据库对药对-肺癌和药对-抗血管生成的核心靶点进行基因本体(Gene ontology,GO)功能、京都基因与基因组的百科全书(Kyoto encyclopedia of genes and genomes,KEGG)通路富集分析。采用分子对接技术评价核心靶点与活性成分的结合能力,分析结合作用力及参与的氨基酸残基。体外采用CCK-8法探讨CR-SR药对、CR和SR对人脐静脉内皮(HUVECs)细胞活力的影响。将斑马鱼胚胎随机分为空白对照组、不同浓度药对及单味药组、阳性药对照组,72 h后统计各组斑马鱼节间血管数,采用qRT-PCR技术检测血管生成相关基因VEGFA、VEGFR2、VEGFR3、EGFR等mRNA表达水平。结果网络药理学分析筛选获得药对-肺癌共同靶点106个,药对-抗血管生成共同靶点130个,其中有85个完全相同;药对-肺癌的GO功能富集分析得到1648个条目,KEGG通路富集分析得到186条信号通路;药对-抗血管生成的GO功能富集分析得到1844个条目,KEGG通路富集分析得到188条信号通路。分子对接结果显示靶点与成分的亲和作用较好,之间的作用力主要包括氢键作用和疏水作用。体外细胞实验表明两药配伍作为药对使用可增强对HUVECs细胞活力的抑制作用。斑马鱼实验的结果表明,药对和单味药的毒性顺序为CR>CR-SR>SR,抗血管生成活性顺序为CR-SR>SR>CR;qRT-PCR结果显示,药对及单味药能显著降低血管生成相关基因VEGFA、VEGFR2、EGFR、MMP9等的表达水平。结论药对及单味药抗肺癌和抗血管生成作用于多个相同靶点调节多条相同信号通路,且配伍使用能够增强抗血管生成作用,药对可能通过调控VEGFA、VEGFR2、EGFR等血管生成相关基因以发挥抗血管生成作用从而治疗肺癌。
Objective To predict the mechanism of herbal couple Curcumae Rhizoma-Sparganii Rhizoma(CR-SR)in modulation of angiogenesis against lung cancer based on network pharmacology and molecular docking technology,and validate by zebrafish model.Methods The active ingredients and potential targets for anti-lung cancer and antiangiogenesis of CR and SR were screened by network pharmacology.The targets were intersected with those screened from the OMIM database and GeneCards database for lung cancer and antiangiogenesis.Herbal couple-lung cancer and herbal couple-antiangiogenesis of protein-protein interaction(PPI)network was constructed by taking intersecting targets to screen the common and core targets of the herbal couple in lung cancer and anti-angiogenesis.Herbal couple-lung cancer and herbal couple-antiangiogenesis of Gene ontology(GO)function and Kyoto encyclopedia of genes and genomes(KEGG)pathway enrichment analyses were performed by Metascape database.The binding ability and the amino acid residues involved of core targets to major components were evaluated by molecular docking technique.In vitro,CCK-8 method was applied to investigate the effects of herbal couple and single drugs on the cell viability of human umbilical vein endothelial cells(HUVECs).Zebrafish embryos were randomly divided into blank control group,different concentration of drug pairs and single drug group,and positive drug control group,and the number of intersegmental vessels of zebrafish in each group was counted after 72 hour.The mRNA expression levels of angiogenesis-related genes,VEGFA,VEGFR2,VEGFR3,EGFR,etc.,were detected by qRT-PCR.Results 106 herbal couple-lung cancer common targets and 130 herbal couple-antiangiogenesis common targets were screened by network pharmacology.Meanwhile,85 of targets were identical.GO function enrichment analyses of herbal couple-lung cancer resulted in 1648 GO analysis entries,KEGG pathway enrichment analyses resulted in 186 signaling pathways.GO function enrichment analyses herbal couple-antiangiogenesis resulted in 1844 GO analysis entries,KEGG pathway enrichment analyses resulted in 188 signaling pathways.The molecular docking results showed a better affinity between the target and the components,and the forces between them mainly included hydrogen bonding and hydrophobic interactions.In vitro cellular experiments demonstrated that the two drugs were used as a drug pair to enhance the inhibitory effect on the cell viability of HUVECs.The zebrafish experiments indicated that the toxicity order of herbal couple and single drugs was CR>CR-SR>SR.The results of transgenic zebrafish vascular fluorescence model confirmed that CR-SR and single drugs had anti-angiogenic activity,with the anti-angiogenic activity order of herbal couple and single drugs was CR-SR>SR>CR.The results of qRT-PCR showed that CR-SR drug pairs and single drugs significantly reduced the expression levels of angiogenesis-related genes VEGFA,VEGFR2,EGFR,MMP9,etc.,and had anti angiogenic effects.Conclusion CR-SR and single drugs had anti-lung cancer effects on multiple identical targets and regulated multiple identical signaling pathways,and their combination had a synergistic effect.The treatment of lung cancer may be through the regulation of angiogenesis-related target VEGFA,VEGFR2,EGFR,etc.,in order to play an anti-angiogenic effect.
作者
黎明霞
刘伟锐
孙梦豫
刘薇
李仙仙
王秀环
折改梅
LI Mingxia;LIU Weirui;SUN Mengyu;LIU Wei;LI Xianxian;WANG Xiuhuan;SHE Gaimei(School of Chinese Materia Medica,Beijing University of Chinese Medicine,Beijing 100029,China;Haikou Pharmaceutical Factory Co.,Hainan 570311,China;Beijing Huilongguan hospital,Beijing 100096,China)
出处
《世界科学技术-中医药现代化》
北大核心
2025年第5期1485-1500,共16页
Modernization of Traditional Chinese Medicine and Materia Medica-World Science and Technology
关键词
莪术-三棱
网络药理学
分子对接
斑马鱼
血管生成
肺癌
Curcumae Rhizoma-Sparganii Rhizoma
Network pharmacology
Molecular docking
Zebrafish
Angiogenesis
Lung cancer
