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藜芦胺对人胶质母细胞瘤细胞U251增殖的影响及机制                                                            Δ



          曹梓珍 ,张 琳,傅若秋,赵祎博,陈 翔,陈剑鸿(陆军特色医学中心药剂科,重庆 400042)
                                                       #
                *
          中图分类号  R965      文献标志码  A      文章编号  1001-0408(2023)22-2734-06
          DOI  10.6039/j.issn.1001-0408.2023.22.08

          摘   要  目的  探究藜芦胺(VTM)对人胶质母细胞瘤(GBM)细胞U251增殖的影响及其潜在机制。方法  借助网络药理学方法,
          筛选VTM干预GBM的铁死亡相关交集靶点,并进行基因本体、京都基因和基因组百科全书富集分析。以U251细胞为对象,采用
          CCK-8法、细胞克隆形成实验、细胞形态变化观察、2′,7′-二氯荧光素二乙酸酯(DCFH-DA)荧光探针法、FerroOrange荧光探针法、
          Western blot实验对VTM抑制U251细胞增殖的作用及可能机制进行验证。结果  共筛选出VTM干预GBM的铁死亡相关交集靶
          点462个,富集于氧化应激、细胞凋亡等生物学过程,胞质囊泡、线粒体膜等细胞成分,影响二价铁离子(Fe )结合、DNA转录过程
                                                                                            2+
          等分子功能,以及铁死亡、磷脂酰肌醇3激酶/蛋白激酶B信号通路等。40、60、80、100、120、140 μmol/L的VTM均可显著降低细胞
          存活率(P<0.01);40、80、120 μmol/L的VTM可使细胞萎缩、细胞核破碎,可显著抑制其克隆形成,显著提高其活性氧(ROS)、Fe                          2+
          水平,并可不同程度地上调核转录因子红系2相关因子2(Nrf2)、血红素加氧酶1(HO-1)蛋白的表达,下调谷胱甘肽过氧化物酶4
         (GPX4)蛋白的表达(P<0.05或P<0.01)。结论  VTM可抑制U251细胞的增殖,促进细胞内ROS和Fe 的蓄积,从而诱导铁死亡,
                                                                                         2+
          上述作用可能与调控Nrf2/HO-1/GPX4信号通路有关。
          关键词  藜芦胺;胶质母细胞瘤;铁死亡;核转录因子红系2相关因子2/血红素加氧酶1/谷胱甘肽过氧化物酶4信号通路


          Effects and mechanism of veratramine on the proliferation of human glioblastoma U251 cells
          CAO Zizhen,ZHANG Lin,FU Ruoqiu,ZHAO Yibo,CHEN Xiang,CHEN Jianhong(Dept.  of  Pharmacy, Army
          Medical Center of PLA, Chongqing 400042, China)

          ABSTRACT    OBJECTIVE To explore the effects and potential mechanism of veratramine (VTM) on the proliferation of human
          glioblastoma  U251  cells.  METHODS  The  network  pharmacology  methods  were  adopted  to  screen  the  targets  of  ferroptosis  related
          to  the  effects  of  VTM  on  glioblastoma,  and  to  conduct  gene  ontology  and  Kyoto  Encyclopedia  of  Genes  and  Genosomes
          enrichment  analysis.  Using  U251  cells  as  the  object,  CCK-8  assay,  the  observation  of  cell  morphological  changes,  DCFH-DA
          fluorescence  probe  method,  FerroOrange  fluorescence  probe  method  and  Western  blot  assay  were  used  to  validate  the  inhibitory
          effects  of  VTM  on  U251  cell  proliferation  and  its  possible  mechanism.  RESULTS  Totally  462  targets  of  ferroptosis  related  to  the
          effects  of  VTM  on  glioblastoma  were  screened  out;  they  mainly  enriched  in  biological  processes  such  as  oxidative  stress  and
          apoptosis,  and  cellular  components  such  as  cytoplasmic  vesicles  and  mitochondrial  membranes;  they  affected  molecular  functions
          such as iron ion (Fe ) binding and DNA transcription processes, as well as iron death and phosphoinositide 3-kinase/protein kinase
                         2+
          B  signaling  pathways.  VTM  with  40,  60,  80,  100,  120  and  140  μmol/L   could  significantly  reduce  the  cell  survival  rate (P<
          0.01);  VTM  with  40,  80  and  120  μmol/L  could  cause  cell  atrophy  and  nuclear  fragmentation,  significantly  inhibit  the  clone
          formation,  increase  the  levels  of  intracellular  reactive  oxygen  species (ROS)  and  Fe   levels,  increase  the  expressions  of  nuclear
                                                                           2+
          factor-erythroid  2-related  factor  2 (Nrf2)  and  heme  oxygenase  1 (HO-1)  protein  to  different  extents,  while  down-regulate  the
          expression of glutathione peroxidase 4 (GPX4) protein (P<0.05 or P<0.01). CONCLUSIONS VTM can inhibit the proliferation
          of  U251  cells,  and  promote  the  accumulation  of  intracellular  ROS  and  Fe ,  thus  inducing  ferroptosis;  its  mechanism  might  be
                                                                    2+
          related to the regulation of the Nrf2/HO-1/GPX4 signaling pathway.
          KEYWORDS     veratramine; glioblastoma; ferroptosis; Nrf2/HO-1/GPX4 signaling pathway


              胶质母细胞瘤(glioblastoma,GBM)又称恶性胶质                  中枢神经系统肿瘤的33%,具有恶性程度高、致死率高、
          瘤,是中枢神经系统常见的原发性恶性肿瘤,约占所有                            复发率高的特点       [1―2] 。目前,GBM 的临床治疗以手术治
                                                              疗联合放疗及替莫唑胺化疗的传统疗法为主,但该疗法
                                                                                                     [2]
              Δ 基金项目 重 庆 市 研 究 生 教 育“ 课 程 思 政 ”示 范 项 目(No.    不良反应大,患者易出现化疗耐药且预后较差 ,因此迫
          YKCSZ23057);重庆市卫生计生委中医药科技项目(No.ZY201801004)         切需要寻找高效、低毒的辅助药物或替代疗法。
             *第一作者 药师,硕士研究生。研究方向:药理学。E-mail:                      近年来,天然产物在肿瘤研究领域扮演了重要角
          1129457183@qq.com
                                                              色。藜芦胺(veratramine,VTM)为甾体生物碱类成分,
              # 通信作者 主任药师,教授,博士生导师。研究方向:抗感染与化
          疗药物的分子机制、天然植物药作用机制。电话:023-68746956。E-               是百合科植物藜芦的有效成分之一。体外研究表明,
          mail:chenjh-110@263.net                             VTM可通过诱导肿瘤细胞自噬、凋亡来抑制其增殖                     [3―4] 。


          · 2734 ·    China Pharmacy  2023 Vol. 34  No. 22                            中国药房  2023年第34卷第22期
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