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中国精品科技期刊2020

穿心莲内酯对人神经胶质瘤U251细胞的抑制作用及机制研究

陈伟鸿 吴琪琪 黄桔 龙文清 蒋艳平 周越菡

陈伟鸿,吴琪琪,黄桔,等. 穿心莲内酯对人神经胶质瘤U251细胞的抑制作用及机制研究[J]. 食品工业科技,2022,43(23):360−370. doi:  10.13386/j.issn1002-0306.2022020202
引用本文: 陈伟鸿,吴琪琪,黄桔,等. 穿心莲内酯对人神经胶质瘤U251细胞的抑制作用及机制研究[J]. 食品工业科技,2022,43(23):360−370. doi:  10.13386/j.issn1002-0306.2022020202
CHEN Weihong, WU Qiqi, HUANG Ju, et al. Anti-glioma Effect and Mechanism of Andrographolide on Human U251 Glioma Cells[J]. Science and Technology of Food Industry, 2022, 43(23): 360−370. (in Chinese with English abstract). doi:  10.13386/j.issn1002-0306.2022020202
Citation: CHEN Weihong, WU Qiqi, HUANG Ju, et al. Anti-glioma Effect and Mechanism of Andrographolide on Human U251 Glioma Cells[J]. Science and Technology of Food Industry, 2022, 43(23): 360−370. (in Chinese with English abstract). doi:  10.13386/j.issn1002-0306.2022020202

穿心莲内酯对人神经胶质瘤U251细胞的抑制作用及机制研究

doi: 10.13386/j.issn1002-0306.2022020202
基金项目: 国家自然科学基金项目(81660031、81360090);桂林医学院国家级大学生创新创业训练计划项目(202010601013)。
详细信息
    作者简介:

    陈伟鸿(1996−),男,硕士研究生,研究方向:肿瘤药理学,E-mail:honggg111@163.com

    通讯作者:

    周越菡(1985−),女,博士,教授,研究方向:肿瘤药理学,E-mail:yuehanzhou2012@163.com

  • 中图分类号: R285.5

Anti-glioma Effect and Mechanism of Andrographolide on Human U251 Glioma Cells

  • 摘要: 目的:应用网络药理学、生物信息学方法,结合体外实验,研究穿心莲内酯(Andrographolide,AND)对人神经胶质瘤U251细胞的抑制作用及机制。方法:通过网络药理学手段,根据AND的结构预测其神经胶质瘤相关靶点。应用生物信息学方法,筛选出枢纽基因进行基因本体(Gene Ontology,GO)分子功能及基因组百科全书(Kyoto Encyclopedia of Genes and Genomes,KEGG)富集分析以确定与其相关的信号通路,再进行分子对接,研究AND与上述靶点的结合活性,最后进行基因富集分析(Gene Set Enrichment Analysis,GSEA),通过神经胶质瘤样本和正常样本的比较,分析枢纽基因的表达差异并获得其Kaplan-Meier生存曲线。在上述基础上,在人源神经胶质瘤细胞U251观察AND对其生长的影响及形态变化,并检测AND在蛋白质水平对PI3K/AKT通路的影响。结果:通过网络药理学手段获得AND的40个神经胶质瘤相关靶点,并应用生物信息学方法筛选出10个枢纽基因,AND与其有较强的结合活性。GO、KEGG富集分析结果显示,上述枢纽基因参与神经元凋亡过程的调控、活性氧代谢等生物过程,且与PI3K/AKT等信号通路有关。GSEA富集分析提示PI3K/AKT-mTOR等信号通路在神经胶质瘤干细胞中显著上调。枢纽基因中HSP90AA1、HSP90AB1、RHOA、CDK2、MET在神经胶质瘤样本与正常样本间存在显著差异,Kaplan-Meier生存曲线显示低表达的枢纽基因BCL2L1、CDK2、SOD2、MET有较高的生存率。体外研究发现,AND时间、浓度依赖性地抑制U251细胞的生长。在AND的作用下,U251细胞出现变圆、皱缩,并显著降低PI3K/AKT通路相关蛋白p-PI3K、p-AKT、p-PI3K/PI3K、p-AKT/AKT蛋白的表达。结论:AND可抑制人神经胶质瘤U251细胞增殖,其作用机制与PI3K/AKT信号通路相关。
  • 图  1  AND的2D(左图)、3D(右图)结构

    Figure  1.  The 2D (left) and 3D (right) structures of AND

    图  2  神经胶质瘤相关基因与AND作用靶点基因交集与交集基因名

    Figure  2.  The intersection of glioma-associated genes with AND target genes and the name of the intersection genes

    注:红色代表AND的作用靶点,蓝色代表神经胶质瘤相关基因。

    图  3  PPI网络构建及核心靶点的筛选过程

    Figure  3.  PPI network construction and core target screening process

    图  4  枢纽基因GO、KEGG功能富集分析结果

    Figure  4.  Results of GO and KEGG functional enrichment analysis of hub genes

    图  5  AND与SOD2(左)、CDK2(右)分子对接结果示意图

    Figure  5.  Schematic diagram of molecules docking results of AND with SOD2 (left) and CDK2 (right)

    图  6  GSEA功能富集分析结果(上调和下调前三位)

    Figure  6.  GSEA functional enrichment analysis results (top 3 in up-regulation and down-regulation)

    图  7  枢纽基因在正常组织与神经胶质瘤组织中的表达量

    Figure  7.  Expression levels of hub genes in normal and glioma tissues

    注:与正常组相比,**表示极显著P<0.01,***表示高度显著P<0.001。

    图  8  枢纽基因在样本中的Kaplan-Meier生存曲线

    Figure  8.  Kaplan-Meier survival curves of hub genes

    注:红、蓝线表示高低风险组的生存率;红色、蓝色虚线代表95%置信区间。

    图  9  不同浓度的AND对U251细胞活力的影响

    Figure  9.  Effects of different concentrations of AND on U251 cell viability

    注:与control组相比,*表示差异显著P<0.05,**表示差异极显著P<0.01,***表示差异高度显著P<0.001。

    图  10  6.25 μmol/L AND作用于U251细胞24、48、72 h后的形态(100×)

    Figure  10.  Morphology of U251 cells after the administration of 6.25 μmol/L AND for 24, 48 and 72 h (100×)

    图  11  12.5 μmol/L的AND作用48 h对U251细胞PI3K/AKT信号通路的影响

    Figure  11.  Effects of 12.5 μmol/L AND for 48 h on PI3K/AKT signaling pathway in U251 cells

    图  12  AND作用于U251细胞后PI3K/AKT信号通路Western blot结果灰度值分析

    Figure  12.  Grayscale analysis of PI3K/AKT signaling pathway Western blot results after the action of AND on U251 cells

    注:与对照组相比,*表示差异显著P<0.05,**表示差异极显著P<0.01,***表示差异高度显著P<0.001。

    表  1  数据库信息

    Table  1.   Database information

    数据库名称数据库地址
    PubChemhttps://pubchem.ncbi.nlm.nih.gov/
    PharmMapperhttp://www.lilab-ecust.cn/PharmMapper/
    Uniprothttps://www.uniprot.org/
    GeneCardshttps://www.GeneCards.org/
    STRINGhttps://string-db.org/
    DAVIDhttps://david.ncifcrf.gov/
    GEOhttps://www.ncbi.nlm.nih.gov/geo/
    TCGAhttps://portal.gdc.cancer.gov/
    GEPIAhttp://gepia.cancer-pku.cn/index.htmL
    下载: 导出CSV

    表  2  枢纽基因的蛋白信息与分子对接最低结合能

    Table  2.   Protein information of hub genes and the lowest binding energy for molecular docking

    基因名称PDB数据库ID蛋白质名称结合能(kcal·mol−1
    HSP90AA16TN5Heat shock protein HSP 90-alpha−8.0
    MAPK146SP9Mitogen-activated protein kinase 14−8.4
    BCL2L17JGWBcl-2-like protein 1−9.2
    IGF1R1TGRInsulin-like growth factor IA−7.1
    RHOA2CE2GTPASE HRAS−6.8
    HSP90AB16N8YHeat shock protein HSP 90-beta−8.5
    PARP15LX6Poly [ADP-ribose] polymerase 10−9.5
    CDK26Q4GCyclin-dependent kinase 2−8.3
    MET5D8VHigh-potential iron-sulfur protein−10.1
    SOD21PL4Superoxide dismutase [Mn], mitochondrial−8.3
    下载: 导出CSV
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  • 收稿日期:  2022-02-23
  • 网络出版日期:  2022-10-21
  • 刊出日期:  2022-11-23

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