基于转录组测序和网络药理学探究青蒿琥酯干预U87和U251脑胶质瘤细胞的药效差异机制  

Exploring the potential mechanism of artesunate in intervening with U87 cells and U251 cells with distinct therapeutic effects on the basis of transcriptome sequencing and network pharmacology

作　　者：李涛 毛霞[2] 张彦琼 林娜[2] SATO Takashi MIZUNO Koji OKUYAMA Katsuki 黄丰 LI Tao;MAO Xia;ZHANG Yan-qiong;LIN Na;SATO Takashi;MIZUNO Koji;OKUYAMA Katsuki;HUANG Feng(School of Chinese Materia Medica,Yunnan University of Chinese Medicine,Kunming 650500,China;Institute of Chinese Materia Medica,China Academy of Chinese Medical Sciences,Beijing 100700,China;Department of Biochemistry,Tokyo University of Pharmacy and Life Sciences,Tokyo 192-0392,Japan)

机构地区：[1]云南中医药大学中药学院,云南昆明650500 [2]中国中医科学院中药研究所,北京100700 [3]东京药科大学生物化学系,东京都八王子市192-0392

出　　处：《药学学报》2023年第6期1475-1483,共9页Acta Pharmaceutica Sinica

基　　金：国家自然科学基金资助项目(8210143224);中国中医科学院优秀青年科技人才(创新类)(ZZ15-YQ-029)。

摘　　要：青蒿琥酯具有干预脑胶质瘤的潜能,但其药理作用机制尚不明确。本研究首先基于U87和U251两种人源脑胶质瘤细胞,探究青蒿琥酯对细胞活性、增殖和凋亡的影响,发现青蒿琥酯对U87细胞活性和增殖能力的抑制作用强于对U251细胞;Hoechst和TUNEL细胞凋亡染色实验也发现,青蒿琥酯可以显著促进U87细胞的凋亡(P <0.05),而仅大剂量的青蒿琥酯可促进U251细胞的凋亡(P <0.01)。接着,利用青蒿琥酯处理前后的U87和U251细胞裂解液,通过转录组测序检测和差异数据分析,分别获得青蒿琥酯敏感性和非敏感性细胞株的差异基因集以及青蒿琥酯效应相关基因集,旨在挖掘青蒿琥酯对两类细胞敏感性不一的分子机制。通过构建上述各对比组差异基因互作网络和计算网络拓扑特征值,筛选药效相关的关键网络靶标;生物功能富集分析结果表明,上述关键网络靶标显著富集于多条与脑胶质瘤病理环节相关的信号通路,其中,与细胞凋亡相关的转录激活因子4 (ATF4)-DNA损伤诱导转录因子3 (DDIT3)-聚腺苷二磷酸核糖聚合酶1 (PARP1)信号轴的富集显著性最高。分子对接结果也表明,青蒿琥酯与ATF4和DDIT3均具有较好的结合能力。本研究初步揭示了青蒿琥酯干预U87脑胶质瘤细胞的潜能与其抑制肿瘤细胞内异常活化的ATF4-DDIT3-PARP1信号轴,进而诱导细胞凋亡有关;同时也表明了PARP1可能是U251细胞对青蒿琥酯产生耐药的重要靶标。相关结果为青蒿琥酯治疗脑胶质瘤,以及解决脑胶质瘤治疗药物耐药的现状提供了新的实验依据。Artesunate possesses the potential of intervening with glioma,however,its pharmacological mechanisms remain unclarified.Firstly,the effects of artesunate on cell activity,proliferation and apoptosis of U87 and U251 human glioma cells were explored.It was found that artesunate exerted stronger inhibitory effects on the activity and proliferation of U87 cells than U251 cells.It could significantly promote apoptosis in U87 cells(P<0.05),while only high dose of artesunate can promote that of U251 cells(P<0.01),detected by Hoechst and TUNEL cell apoptosis staining.Further,the differential expression gene sets between artesunate-sensitive and non-sensitive cell line,as well the therapeutic effects-related genes of artesunate were obtained through transcriptome sequencing and differential data analysis by using the lysates of U87 and U251 cells before and after artesunate treatment,aiming to explore the molecular mechanism of distinct artesunate sensitivity to two types of cells.Then,key putative targets that related to therapeutic effects were screened by constructing the interaction network of differential genes of three above comparison groups,and calculating their topological characteristics.Pathway enrichment analysis showed that those key putative targets were significantly enriched in several signaling pathways that were closely associated with the main pathological changes of glioma,among which apoptosis-related activating transcription factor 4(ATF4)-DNA damage induced transcript 3(DDIT3)-polyadenosine diphosphate ribose polymerase 1(PARP1)signaling axis was the most enriched in.Molecular docking indicated that artesunate had fine binding affinities with ATF4 and DDIT3.Above all,this study preliminarily revealed that ATF4-DDIT3-PARP1 signaling axis is the target pathway of artesunate intervening with U87 glioma cells,and PARP1 may be an important gene for U251 cells to develop resistance to artesunate.Our results not only provide fundamental experimental evidence for artesunate as a potential therapeutic drug i

关 键 词：青蒿琥酯 脑胶质瘤 转录组测序 网络药理学 药理机制 

分 类 号：R966[医药卫生—药理学]