荜茇酰胺诱导胶质瘤细胞铁死亡的机制  

Mechanism of piperlongumine-induced ferroptosis in glioma cells

作　　者：仇建婷 史记[3] 郭堂军 朴浩哲 QIU Jianting;SHI Ji;GUO Tangjun;PIAO Haozhe(Liaoning University of Traditional Chinese Medicine,Liaoning Shenyang 110847,China;The People's Hospital of Liaoning Province,Liaoning Shenyang 110013,China;Dalian Medical University,Liaoning Dalian 116044,China)

机构地区：[1]辽宁中医药大学,辽宁沈阳110847 [2]辽宁省人民医院,辽宁沈阳110013 [3]大连医科大学,辽宁大连116044

出　　处：《现代肿瘤医学》2024年第22期4262-4272,共11页Journal of Modern Oncology

基　　金：辽宁省沈阳市科技计划项目(编号:20-205-4-003)。

摘　　要：目的:通过体外实验探索荜茇酰胺(piperlongumine,PL)对胶质母细胞瘤(glioblastoma,GBM)的增殖抑制作用及GBM细胞死亡的主要方式、分子机制。方法:用CCK-8试剂盒检测PL对胶质瘤LN229和A172细胞活力的影响;平板克隆实验检测PL对胶质瘤细胞增殖的抑制作用;电子显微镜观察线粒体结构变化;免疫荧光Ki67染色实验检测细胞增殖能力;免疫荧光实验检测4-HNE水平;用相应化验试剂盒检测GSH、MDA水平;流式细胞术检测细胞内ROS水平;CCK-8实验检测各种细胞死亡抑制剂对PL抑制胶质瘤细胞增殖的回复效果;流式细胞术凋亡试剂盒检测Fer-1的回复效果;生信分析寻找靶基因及分子对接评价;WB检测铁死亡相关蛋白;RT-qPCR检测mRNA水平;质粒转染过表达EZH2基因;CHIP实验及qPCR检测H3K27me3蛋白与Keap1 DNA之间的相互作用。结果:PL能抑制胶质瘤细胞LN229和A172的细胞活力;抑制胶质瘤细胞增殖;超微结构显示细胞内线粒体变小,双层膜密度增高;细胞内GSH水平下降,ROS、MDA、4-HNE水平增高;铁死亡抑制剂Fer-1及Lip-1能够回复PL对胶质瘤细胞增殖的抑制作用;荜茇酰胺小分子配体与靶蛋白大分子EZH2结合稳定;铁死亡相关蛋白PTGS2水平增高,Nrf2、xCT、GPX4水平降低。PL靶蛋白EZH2降低,其下游蛋白H3K27me3降低,Nrf2的上游蛋白Keap1增加;EZH2和Nrf2的mRNA水平无明显变化,而Keap1的mRNA水平增加;过表达EZH2后,H3K27me3、Keap1和Nrf2水平呈现相应的变化;CHIP实验表明H3K27me3蛋白与Keap1 DNA存在相互作用。结论:荜茇酰胺通过一个新的EZH2/H3K27me3/Keap1/Nrf2通路诱导胶质瘤细胞铁死亡,有望为GBM的治疗提供一个有潜力的药物。Objective:To explore the proliferation inhibition effect of piperlongumine(PL)on glioblastoma(GBM)in vitro experiments and the main ways,molecular mechanisms of GBM cells death.Methods:Use the CCK-8 assay kit to detect the effect of PL on the cell viability of glioma LN229 and A172.Conduct a colony formation experiment to detect the inhibitory effect of PL on glioma cell proliferation.Observe mitochondrial structural changes through electron microscopy.Immunofluorescence Ki67 staining experiment was used to detect cell proliferation ability.Immunofluorescence experiment was used to detect 4-HNE levels.Detect GSH and MDA levels using corresponding assay kits.Flow cytometry was used to detect intracellular ROS levels.CCK-8 assay was used to detect the recovery effect of various cell death inhibitors on PL-inhibited glioma cell proliferation.Flow cytometry apoptosis assay kit was used to detect the recovery effect of Fer-1.Bioinformatics analysis was used to find target genes and molecular docking evaluation.WB was used to detect iron death-related proteins.RT-qPCR was used to detect mRNA levels.The overexpressing EZH2 gene was transfected.CHIP experiment and qPCR was used to detect the interaction between H3K27me3 protein and Keap1 DNA.Results:PL can inhibit the cell viability of glioma cells LN229 and A172 and inhibit the proliferation of glioma cells.Ultrastructure showed that mitochondria in cells shrunk,and the double membrane density increased.Intracellular GSH levels decreased,while ROS,MDA,and 4-HNE levels increased.Ferroptosis inhibitors Fer-1 and Lip-1 can restore the inhibitory effect of PL on the proliferation of glioma cells.The small molecule ligand of PL is stable in binding with target protein macromolecule EZH2.Ferroptosis-related protein PTGS2 levels increased,Nrf2,xCT,GPX4 levels decreased.PL target protein EZH2 decreased,and its downstream protein H3K27me3 decreased,Nrf2 upstream protein Keap1 increased.EZH2 and Nrf2 mRNA levels showed no significant changes,while Keap1 mRNA levels increase.After

关 键 词：荜茇酰胺 胶质母细胞瘤 铁死亡 EZH2 

分 类 号：R730.264[医药卫生—肿瘤]