目的 探究青蒿琥酯干预胶质母细胞瘤（glioblastoma，GBM）的药效及其潜在分子机制。方法 采用Cell Titer-Blue检测青蒿琥酯对U87和U251 2种人源胶质母细胞瘤细胞活性和增殖的影响；流式细胞术检测青蒿琥酯对2种细胞周期的调节作用；采用qRT-PCR和Western blotting检测青蒿琥酯对2种细胞自噬水平的影响。采用DNA甲基化联合网络药理学从表观遗传学层面研究青蒿琥酯干预GBM的靶标基因，并建立蛋白质-蛋白质相互作用网络，通过计算网络中各节点的网络拓扑特征值，筛选出青蒿琥酯干预GBM的核心靶点并进行生物功能富集分析；采用AutoDock v4.2.6和AutoDock Vina v1.2.0软件开展分子对接虚拟计算，考察青蒿琥酯与其候选关键靶点的结合能力，并采用Western blotting验证青蒿琥酯对2种脑胶质瘤细胞中关键靶点表达的影响。结果 青蒿琥酯可显著抑制U87细胞的活性和增殖（P＜0.05、0.01、0.001），而对U251细胞的药效不显著；高剂量青蒿琥酯可影响2种细胞周期分布；高剂量青蒿琥酯可显著提高U87细胞中3种自噬标志分子[自噬关键分子酵母Atg6同系物（ATG6 autophagy related 6 homolog，Beclin1）、泛素结合蛋白（ubiquitin-binding protein，p62）、微管相关蛋白1轻链3（microtubule-associated protein 1 light chain 3，LC3）]的mRNA表达水平（P＜0.001），而仅高剂量青蒿琥酯升高U251细胞中p62的mRNA表达水平（P＜0.05）；低、中、高剂量青蒿琥酯可显著提高U87细胞中Beclin1、p62、LC3B蛋白表达水平（P＜0.05、0.01），仅中、高剂量青蒿琥酯可以影响U251细胞中Beclin1、p62、LC3B蛋白表达。利用给药前后的U87与U251细胞进行DNA甲基化测序与网络药理学整合分析，在获得青蒿琥酯敏感型和耐药型脑胶质瘤细胞的差异基因集，以及青蒿琥酯干预2种细胞效应基因集的基础上，发现其干预2类脑胶质瘤细胞的关键网络靶标共同参与GBM恶性进展密切相关的血小板衍生生长因子受体α（platelet derived growth factor receptor α，PDGFRA）-大鼠肉瘤基因（rat sarcoma viral oncogene homolog，RAS）-B-Raf原癌基因（B-Raf proto-oncogene，BRAF）-丝裂原活化蛋白激酶激酶（mitogen-activated protein kinase kinase，MEK）-细胞外信号调节激酶（extracellular signal regulated kinase，ERK）通路，且青蒿琥酯与其直接靶标（PDGFRA、BRAF）具有较强的亲和力。Western blotting实验证实青蒿琥酯可以抑制U87和U251细胞中关键靶点PDGFRA和BRAF蛋白的表达（P＜0.05、0.01、0.001）。结论 青蒿琥酯可能通过靶向调控PDGFRA-RAS-BRAF-MEK-ERK信号通路，从而抑制GBM细胞的生长和扩散，为青蒿琥酯治疗GBM提供了体外实验依据，也为解决临床抗肿瘤药物的耐药问题提供了新思路和新方法。

Objective To explore the efficacy and mechanism of artesunate in intervening glioblastoma. Methods Cell Titer-Blue was used to detect the effect of artesunate on viability and proliferation of U87 and U251 human glioblastoma cells; Flow cytometry was used to detect the regulation effect of artesunate on two cell cycles; qRT-PCR and Western blotting were used to detect the effect of artesunate on autophagy level of two kinds of cells. Further, target genes of artesunate intervention in glioblastoma were studied from the epigenetic level by using the analysis strategy of DNA methylation combined with network pharmacology, and a protein-protein interaction network was established. By calculating the network topological eigenvalues of each node in network, core targets of artesunate intervention in glioblastoma were screened out and biological function enrichment analysis was carried out. Finally, AutoDock v4.2.6 and AutoDock Vina v1.2.0 software were used to carry out molecular docking virtual calculation, to investigate the binding ability of artesunate and its candidate key targets, and verify the influence of artesunate on the expressions of key targets in two types of glioma cells by Western blotting. Results Artesunate significantly inhibited the activity and proliferation of U87 cells (P < 0.05, 0.01, 0.001), but had no significant effect on U251 cells. High-dose artesunate could affect the distribution of two cell cycles. High-dose artesunate significantly increased the mRNA expression levels of three autophagy markers [ATG6 autophagy related 6 homolog (Beclin1), ubiquitin-binding protein (p62), microtubule-associated protein 1 light chain 3 (LC3)] in U87 cells (P < 0.001), while only high-dose artesunate increased the mRNA expression level of p62 in U251 cells (P < 0.05). Low-, medium- and high-dose of artesunate could significantly improve the expression levels of Beclin1, p62 and LC3B proteins in U87 cells (P < 0.05, 0.01), only medium- and high-dose of artesunate could affect Beclin1, p62 and LC3B protein expressions in U251 cells. Furthermore, DNA methylation sequencing and network pharmacologic integration analysis were performed on U87 and U251 cells before and after drug administration, on the basis of obtaining the differential gene sets of artesunate-sensitive and drug-resistant glioma cells, and effector gene sets of two types of cells after artesunate intervention, it was found that key network targets of two types of glioma cells were involved in platelet derived growth factor receptor α (PDGFRA)-rat sarcoma viral oncogene homolog (RAS)-B-Raf proto-oncogene (BRAF)-mitogen-activated protein kinase kinase (MEK)-extracellular signal regulated kinase (ERK) pathway, which were closely related to the malignant progression of glioblastoma, and artesunate had strong affinity with its direct targets (PDGFRA and BRAF). Western blotting analysis confirmed that artesunate could inhibit the expressions of PDGFRA and BRAF proteins in U87 and U251 cells (P < 0.05, 0.01, 0.001). Conclusion Artesunate may exert its medicinal effect on inhibiting the growth and proliferation of glioblastoma cells by targeting PDGFRA-RAS-BRAF-MEK-ERK signaling pathway, which provides an in vitro experimental basis for artesunate in treatment of glioblastoma, and also provides new ideas and methods for solving the drug resistance problem of clinical anti-tumor drugs.

R285.5

国家自然科学基金青年项目（8210143224）；中国中医科学院优秀青年科技人才（创新类）（ZZ15-YQ-029）