Anti-glioma Effect and Mechanism of Andrographolide on Human U251 Glioma Cells
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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。
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关键词:
- 穿心莲内酯 /
- U251 /
- PI3K/AKT信号通路 /
- 网络药理学 /
- 生物信息学
Abstract: Objective: To investigate the anti-glioma effect and mechanism of AND on human glioma U251 cells via network pharmacology, bioinformatics and in vitro experiments. Methods: Network pharmacology was used to predict glioma-related targets based on the structure of AND. The hub genes were selected by bioinformatics, analyzed by gene ontology (GO) molecular function and kyoto encyclopedia of genes and genomes enrichment analysis (KEGG) to determine their related signaling pathways. Molecular docking was conducted to study the binding activity of AND with the above targets. Finally, gene set enrichment analysis (GSEA) was performed, analyzing the expression differences of hub genes and the Kaplan-Meier survival curve by comparing glioma samples with normal samples. On the above basis, the effects of AND on the growth and morphological changes of human glioma U251 cells were observed, the effects of AND on the PI3K/AKT pathway were detected. Results: A total of 40 glioma related targets of AND were identified by network pharmacology, and 10 hub genes were screened out by bioinformatics method. AND had high binding activity to hub genes. GO and KEGG enrichment analysis results showed that the hub genes were related to biological processes such as the regulation of neuronal apoptosis, reactive oxygen metabolism, and were related to PI3K/AKT signaling pathways. GSEA enrichment analysis suggested that PI3K/AKT-mTOR and other signaling pathways were significantly up-regulated in glioma cells. Among the hub genes, HSP90AA1, HSP90AB1, RHOA, CDK2 and MET showed significant differences between glioma samples and normal samples. Kaplan-Meier survival curve showed that low expression of genes like BCL2L1, CDK2, SOD2 and MET had higher survival rates. In the in vitro studies, AND inhibited the growth of U251 cells in both time-dependent and dose-dependent manners. After the administration of AND, U251 cells became round and shriveled, which significantly decreased the expression of p-PI3K, p-AKT, p-PI3K/PI3K, p-AKT/AKT to the PI3K/AKT pathway. Conclusion: AND can inhibit the proliferation of human glioma U251 cells, which mechanism is related to the PI3K/AKT signaling pathway.-
Key words:
- andrographolide /
- U251 /
- PI3K/AKT signaling pathway /
- network pharmacology /
- bioinformatics
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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 PubChem https://pubchem.ncbi.nlm.nih.gov/ PharmMapper http://www.lilab-ecust.cn/PharmMapper/ Uniprot https://www.uniprot.org/ GeneCards https://www.GeneCards.org/ STRING https://string-db.org/ DAVID https://david.ncifcrf.gov/ GEO https://www.ncbi.nlm.nih.gov/geo/ TCGA https://portal.gdc.cancer.gov/ GEPIA http://gepia.cancer-pku.cn/index.htmL 
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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） HSP90AA1 6TN5 Heat shock protein HSP 90-alpha −8.0 MAPK14 6SP9 Mitogen-activated protein kinase 14 −8.4 BCL2L1 7JGW Bcl-2-like protein 1 −9.2 IGF1R 1TGR Insulin-like growth factor IA −7.1 RHOA 2CE2 GTPASE HRAS −6.8 HSP90AB1 6N8Y Heat shock protein HSP 90-beta −8.5 PARP1 5LX6 Poly [ADP-ribose] polymerase 10 −9.5 CDK2 6Q4G Cyclin-dependent kinase 2 −8.3 MET 5D8V High-potential iron-sulfur protein −10.1 SOD2 1PL4 Superoxide dismutase [Mn], mitochondrial −8.3
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