Fer-1和DFO对Erastin诱导人神经母细胞瘤细胞损伤的保护作用  

Protective effects of Fer-1 and DFO on Erastin-induced injury in human neuroblastoma cells

作　　者：郭璨璨 李朝菲 夏双爽 任学科 吕懿[1] 郑金平[1,2] GUO Can-can;LI Zhao-fei;XIA Shuang-shuang;REN Xue-ke;LV Yi;ZHENG Jin-ping(Department of Toxicology,School of Public Heath,Shanxi Medical University,Taiyuan Shanxi 030001,China;Department of Public Health and Preventive Medicine,Changzhi Medical College,Changzhi Shanxi 046000,China)

机构地区：[1]山西医科大学公共卫生学院卫生毒理学教研室,山西太原030001 [2]长治医学院公共卫生与预防医学系,山西长治046000

出　　处：《毒理学杂志》2023年第1期1-5,11,共6页Journal of Toxicology

基　　金：国家自然科学基金(30872137);山西省重点研发计划(国际合作,201703D421021);山西省“1331工程”提质增效项目(202155221)。

摘　　要：目的 研究Erastin对人神经母细胞瘤细胞(SH-SY5Y)的损伤作用及Ferrostatin-1(Fer-1)和Deferoxamine(DFO)对细胞损伤的保护效果,为Fer-1和DFO应用于神经退行性疾病提供依据。方法 将SH-SY5Y细胞分为溶剂对照组(DMSO)、Erastin组(10μmol/L)、Fer-1组(10μmol/L)、DFO组(100μmol/L)、Erastin+Fer-1组(10μmol/L Erastin+10μmol/L Fer-1)和Erastin+DFO组(10μmol/L Erastin+100μmol/L DFO),染毒24 h。CCK-8法检测细胞活力;显微镜观察细胞形态结构;透射电子显微镜观察细胞线粒体结构变化;流式细胞仪检测细胞脂质ROS水平;试剂盒检测细胞丙二醛(MDA)含量、超氧化物歧化酶(SOD)活力、谷胱甘肽(GSH)含量、谷胱甘肽过氧化物酶(GSH-Px)活力、细胞铁含量;Western blot法检测谷胱甘肽过氧化物酶4(GPX4)、人胱氨酸/谷氨酸转运蛋白(SLC7a11)和突触素(SYN)蛋白表达。结果 Erastin暴露可导致细胞存活率降低至73.19%±3.51%,明显低于对照组100%±0.00%(P<0.01);细胞形态不规则,突触减少,SYN蛋白表达明显降低(P<0.01);细胞线粒体明显皱缩,膜增厚,线粒体嵴减少。与对照组相比,Erastin暴露组细胞GSH含量、GSH-Px的活力、SOD的活力明显降低(P<0.01),脂质ROS含量、MDA含量、细胞内铁离子水平明显升高(P<0.01),GPX4、SLC7a11蛋白表达水平明显降低(P<0.01)。与Erastin暴露组相比,Erastin+Fer-1组、Erastin+DFO组细胞的存活率明显提高(P<0.01),不规则形态和突触缺失的细胞数目减少,SYN蛋白表达水平明显增加(P<0.01);异常形态线粒体数量减少,线粒体形态结构趋于正常,脂质活性氧(ROS)、MDA和细胞铁离子水平明显下降(P<0.01),GSH含量、GSH-Px和SOD活力明显提升(P<0.01),GPX4、SLC7a11蛋白表达水平明显增加(P<0.01)。结论 Erastin可以诱导SH-SY5Y细胞损伤及铁死亡;Fer-1和DFO通过抑制细胞铁死亡而起到细胞保护作用。Objective To investigate the damage effect of Erastin on human neuroblastoma cells(SH-SY5Y) and the protective effect of Ferrostatin-1(Fer-1) and Deferoxamine(DFO) on cell damage, and provide evidence for the application of Fer-1 and DFO in the prevention and treatment of neurodegenerative diseases. Methods SH-SY5Y cells were divided into solvent control group(DMSO), Erastin group(10 μmol/L), Fer-1 group(10 μmol/L), DFO group(100 μmol/L), Erastin+Fer-1 group(10 μmol/L Erastin+10 μmol/L Fer-1) and Erastin+DFO group(10 μmol/L Erastin+100 μmol/L DFO). All groups were treated for 24 h. CCK-8 method was used to detect cell viability;cell morphology was observed with a microscope;changes in cellular mitochondrial structure were observed with transmission electron microscope;flow cytometry was used to detect cell lipid ROS level;kits were used to detect cell malondialdehyde(MDA) content, superoxide dismutase(SOD) activity, glutathione(GSH) content, glutathione peroxidase(GSH-PX) activity, iron content. Western blot was used to detect proteins( SYN, SLC7a11, GPX4). Results SH-SY5Y cells were treated with 10 μmol/L Erastin for 24 h, and the cell viability was 73.19%±3.51%, which was significantly lower than the control group(P<0.01);decreased cell number, irregular morphology, reduced synapses, and reduced SYN protein expression electron microscopy showed that erastin could make cell mitochondria smaller, mitochondrial membrane density thicken, and mitochondrial cristae reduce;compared with the control group, the content of GSH, and the activities of GSH-Px and SOD reduced significantly(P<0.01), and the content of MDA, lipid ROS, and intracellular iron levels increased significantly in the erastin-exposed group(P<0.01), SLC7a11, GPX4 reduced significantly(P<0.01). Compared with the erastin-exposed group, the cell viabilities in the Erastin+Fer-1 group and Erastin+DFO group increased significantly(P<0.01). The results of cells morphology and mitochondrial morphology tended to be normal;The levels of lipid ROS, MDA

关 键 词：Erastin 人神经母细胞瘤细胞 铁死亡 突触 保护作用 

分 类 号：R99[医药卫生—毒理学] R114[医药卫生—药学]