双氢青蒿素诱导肿瘤细胞铁死亡及其机制研究  

Ferroptosis-induced effect and mechanism of dihydroartemisinin on tumor cells

作　　者：费伟东 叶轶青[1] 陈玥[1] 吴晓东[2] 宋倩倩[1] 姚瑶 郑彩虹[1] FEI Wei-dong;YE Yi-qing;CHEN Yue;WU Xiao-dong;SONG Qian-qian;YAO Yao;ZHENG Cai-hong(Department of Pharmacy,Women's Hospital,School of Medicine,Zhejiang University,Hangzhou 310006,China;Department of Gynecologic Oncology,Women’s Hospital,School of Medicine,Zhejiang University,Hangzhou 310006,China)

机构地区：[1]浙江大学医学院附属妇产科医院药剂科,浙江杭州310006 [2]浙江大学医学院附属妇产科医院妇科肿瘤,浙江杭州310006

出　　处：《中草药》2020年第13期3473-3481,共9页Chinese Traditional and Herbal Drugs

基　　金：国家自然科学基金资助项目(81873838);浙江省自然科学基金(LQ20H300002);浙江省自然科学基金(LY16H160025);浙江省医药卫生科技项目(2018KY429)。

摘　　要：目的阐明双氢青蒿素(DHA)诱导肿瘤细胞铁死亡的作用及其机制。方法利用3,3′,5,5′-四甲基联苯胺(TMB)检测DHA与FeSO4体外芬顿样(Fenton)反应生成氧自由基(·OH)的能力;MTT法检测DHA对人肝癌HepG2细胞的毒性(包括FeSO4与去铁胺预处理组)。MTT法考察谷胱甘肽(GSH)与铁死亡抑制剂(Fer-1)对DHA细胞毒性的影响;采用DCFH-DA染料考察DHA(包括FeSO4预处理组)诱导的细胞内活性氧的生成能力;采用C11-BODIPY581/591与DiO分别考察DHA(包括FeSO4预处理组)对细胞内脂质过氧化物生成能力以及细胞膜结构的影响;利用谷胱甘肽过氧化物酶4(GPX-4)试剂盒测定DHA(包括FeSO4预处理组)对HepG2细胞内GPX-4活性的影响。结果 Fe2+能够催化DHA发生芬顿样反应并生成·OH;DHA的半数抑制浓度(IC50)为(39.96±8.78)μmol/L,FeSO4与去铁胺分别能够增加或者降低DHA的细胞毒性;DHA处理后细胞内活性氧含量与脂质过氧化物含量升高,细胞形态变大,细胞膜呈散点状分布并呈现解离状态。FeSO4预处理组与DHA组相比较进一步增加细胞内活性氧含量与脂质过氧化物含量,并且细胞膜形态完全破坏。FeSO4能够增强DHA对GPX-4活性的抑制作用。结论 DHA通过芬顿样反应升高细胞内活性氧而最终诱导肿瘤细胞铁死亡。另外,外源性铁可以加速DHA发生芬顿样反应进而加速肿瘤细胞铁死亡的发生与发展。Objective To explore the effect and mechanism of dihydroartemisinin(DHA) in inducing ferroptosis of tumor cells. Methods 3,3′,5,5′-Tetramethylbenzidine was used to detect the oxygen free radicals(·OH) formed by DHA and FeSO4 in vitro. The cytotoxicity of DHA on HepG2 cells was detected by MTT method(including FeSO4 or deferoxamine pretreated groups). MTT assay was used to investigate the influence of glutathione(GSH) and inhibitor(Fer-1) on cytotoxicity of DHA;DCFH-DA dye was used to investigate intracellular reactive oxygen species induced by DHA(including FeSO4 pretreated groups). C11-BODIPY581/591 and DiO dye were used to examine the influence of DHA(including FeSO4 pretreated groups) on intracellular lipid peroxide formation and cell membrane structure;Glutathione peroxidase assay kit was used to explore the influence of DHA(including FeSO4 pretreated groups) on intracellular activity GPX-4 in HepG2 cells. Results Fenton-like reaction occurred between DHA and Fe2+, and ·OH was produced during the reaction. The half-inhibitory concentration(IC50) of DHA was(39.96 ± 8.78) μmol/L. FeSO4 and deferoxamine could increase or decrease the cytotoxicity of DHA, respectively. After treated with DHA, the intracellular content of reactive oxygen species and lipid peroxide was increased, the cell morphology became larger, and the cell membrane was broken. Compared with the DHA treated group, the FeSO4 pretreated group further increased the intracellular reactive oxygen species and lipid peroxide content, and the cell membrane morphology was completely destroyed. FeSO4 could also enhance the inhibitory effect of DHA on GPX-4 activity. Conclusion DHA increases intracellular reactive oxygen species through Fenton-like reaction and ultimately induces ferroptosis of tumor cells. In addition, exogenous iron can accelerate the Fenton-like reaction of DHA and accelerate the occurrence and development of ferroptosis of tumor cells.

关 键 词：双氢青蒿素 铁死亡 芬顿样反应 氧自由基 肿瘤细胞 外源性铁 谷胱甘肽过氧化物酶4 

分 类 号：R285.5[医药卫生—中药学]