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

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

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

摘要

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。

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.