亚砷酸钠诱导SH-SY5Y细胞铁死亡的规律  

Ferroptosis patterns in SH-SY5Y cells induced by sodium arsenite

作　　者：夏双爽 田凤㛃 任学科 王慧[1] 李玲 穆箭兵 郑金平[1,3] XIA Shuang-shuang;TIAN Feng-jie;REN Xue-ke;WANG Hui;LI Ling;MU Jian-bing;ZHENG Jin-ping(Department of Toxicology,School of Public Heath,Shanxi Medical University,Taiyuan,Shanxi 030001,China;Laboratory of Malaria and Vector Research,National Institute of Allergy and Infectious Diseases,National Institute of Health,Rockville,MD 20852,USA;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]美国国立卫生研究院过敏与传染病研究所疟疾和媒介研究实验室,美国马里兰州罗克韦尔20852 [3]长治医学院公共卫生与预防医学系,山西长治046000

出　　处：《环境与职业医学》2020年第5期468-473,共6页Journal of Environmental and Occupational Medicine

基　　金：国家自然科学基金项目(30872137);山西省重点研发计划国际科技合作项目(201703D421021)。

摘　　要：[背景]砷可通过诱导神经元丢失导致神经毒性,但其是否可诱导神经细胞铁死亡尚不清楚。[目的]探讨亚砷酸钠(NaAsO_(2))诱导人神经母细胞瘤细胞SH-SY5Y铁死亡发生规律,为研究NaAsO_(2)神经毒性发生机制提供依据。[方法]选用SH-SY5Y细胞,将实验分为空白对照组(加入正常培养基)、阳性对照组(加入终浓度为10μmol·L^(-1)的铁死亡诱导剂Erastin)、NaAsO_(2)染毒组(NaAsO_(2)终浓度分别为20、40、80μmol·L^(-1))、抑制剂组[终浓度20μmol·L^(-1)凋亡抑制剂Z-VAD-FMK,10μmol·L^(-1)特异性铁死亡抑制剂Fer-1,100μmol·L^(-1)特异性铁死亡抑制剂去铁胺(DFO),20μmol·L^(-1)坏死性凋亡抑制剂Nec-1]、干预组(在阳性对照组及NaAsO_(2)各染毒组基础上分别加入4种抑制剂,剂量同抑制剂组),共25组,染毒24 h。实验重复3次。采用CCK-8法检测各组细胞存活率;试剂盒检测细胞内丙二醛(MDA)含量、超氧化物歧化酶(SOD)活性、谷胱甘肽(GSH)含量、谷胱甘肽过氧化物酶(GPXS)活性及Fe^(2+)含量;流式细胞仪检测脂质活性氧(ROS)水平。[结果]随NaAsO_(2)染毒剂量增加,SH-SY5Y细胞存活率呈下降趋势(b=-0.984,P<0.001);20、40、80μmol·L^(-1) NaAsO_(2)染毒组及Erastin组细胞存活率[(85.15±1.32)%、(72.63±2.67)%、(65.28±1.71)%、(74.34±2.07)%]均明显低于对照组存活率(P<0.01)。Z-VAD-FMK干预使20、40、80μmol·L^(-1) NaAsO_(2)染毒组及Erastin处理组细胞存活率上升至(88.30±1.92)%、(81.72±2.43)%、(77.72±1.05)%、(85.28±1.97)%(P<0.05),脂质ROS、MDA含量下降(P<0.05),SOD活性上升(P<0.05),但GSH含量、GPXS活性、Fe^(2+)含量均无明显变化(P>0.05)。Fer-1、DFO干预使40μmol·L^(-1)和80μmol·L^(-1) NaAsO_(2)染毒组、Erastin处理组细胞存活率分别上升至(86.33±2.31)%、(82.24±1.24)%、(88.76±2.87)%和(82.83±2.55)%、(79.66±0.67)%、(87.38±1.23)%(P<0.01),脂质ROS水平、MDA下降(P<0.05),SOD活性上升(P<0.05),同时GSH含量、GPXS活性上升(P[Background]Arsenic can cause neurotoxicity by inducing neuronal loss, but it is not clear whether it can induce ferroptosis in nerve cells.[Objective]This experiment is designed to study the ferroptosis patterns in SH-SY5Y human neuroblastoma cells, and provide insights to study the mechanism underlying neurotoxicity induced by sodium arsenite (NaAsO_(2)).[Methods]SH-SY5Y cells were divided into a blank control group (with normal medium), a positive control group (with a final concentration of 10 μmol·L^(-1) ferroptosis inducer Eratin), three NaAsO_(2) exposure groups (with a final concentration of 20, 40, 80 μmol·L^(-1) NaAsO_(2), respectively), four inhibitor groups[with a final concentration of 20 μmol·L^(-1) apoptosis inhibitor Z-VAD-FMK, 10 μmol·L^(-1) specific ferroptosis inhibitor Fer-1, 100 μmol·L^(-1) specific ferroptosis inhibitor deferoxamine (DFO), and 20 μmol·L^(-1) necrotic apoptosis inhibitor Nec-1, respectively], and sixteen intervention groups (four inhibitors were added to a group with the same positive control treatment and three groups with the same NaAsO_(2) exposure treatments respectively at the same dose of the inhibitor groups). A total of 25 groups were exposed following the designed protocol for 24 h. The experiment was repeated three times. CCK-8 method was used to detect cell survival rate of each group; corresponding kits were used to detect malondialdehyde (MDA) content, superoxide dismutase (SOD) activity, glutathione (GSH) content, glutathione peroxidase (GPXS) activity, and Fe^(2+) content; flow cytometry was used to detect lipid reactive oxygen species (ROS) level.[Results]The survival rate of SH-SY5Y cells showed a decreasing trend with the increasing NaAsO_(2) exposure concentration (b=-0.984, P < 0.001). The cell survival rates of the 20, 40, and 80 μmol·L^(-1) NaAsO_(2) exposure groups and the Erastin group were (85.15±1.32)%, (72.63±2.67)%, (65.28±1.71)%, and (74.34±2.07)%, respectively, and were lower than that of the control group (P < 0.01). The Z-VADF

关 键 词：亚砷酸钠 人神经母细胞瘤细胞 铁死亡 

分 类 号：R114[医药卫生—卫生毒理学]