醋酸铅暴露致小胶质细胞系BV-2细胞铁死亡的机制研究  

Mechanisms of ferroptosis in microglial cell line BV-2 cells after lead acetate exposure

作　　者：赵宇威 王伟轩 师凡 付治嘉 吴桐 张艳淑 ZHAO Yuwei;WANG Weixuan;SHI Fan;FU Zhijia;WU Tong;ZHANG Yanshu(School of Public Heath,North China University of Science and Technology,Tangshan,Hebei 063210,China;School of Medical Sciences,North China University of Science and Technology,Tangshan,Hebei 063210,China;Laboratory Animal Center,North China University of Science and Technology,Tangshan,Hebei 063210,China)

机构地区：[1]华北理工大学公共卫生学院,河北唐山063210 [2]华北理工大学基础医学院,河北唐山063210 [3]华北理工大学实验动物中心,河北唐山063210

出　　处：《环境与职业医学》2022年第8期895-901,共7页Journal of Environmental and Occupational Medicine

基　　金：河北省自然基金(H2020209250);河北省教育厅课题(ZD2020113,QN2021121);国家自然基金(82073598);河北省研究生创新资助项目(CXZZBS2022111)。

摘　　要：[背景]铅暴露可诱导小胶质细胞死亡,但是机制尚不清楚。铁死亡是新发现的一种细胞死亡方式,在铅暴露导致的小胶质细胞死亡中的作用还未见报道。[目的]探讨铁死亡在铅暴露致小胶质细胞死亡中的作用,以期为铅的神经毒性机制研究提供理论依据。[方法]小胶质细胞系BV-2细胞与0、10、20、40μmol·L^(-1)醋酸铅共同培养24 h,40μmol·L^(-1)醋酸铅组中加入铁螯合剂(DFO),即为40+DFO组;用倒置显微镜观察铅暴露后BV-2细胞形态的变化;组织铁试剂盒、谷胱甘肽试剂盒分别检测细胞内铁、谷胱甘肽(GSH);流式细胞术检测脂质活性氧(ROS)荧光强度。Western Blotting、qPCR检测谷胱甘肽过氧化物酶4(GPX4)、溶质载体家族7成员11(SLC7A11)、转铁蛋白受体1(TFR-1)、二价金属转运蛋白1(DMT1)、膜铁转运蛋白1(FPN1)蛋白及mRNA的表达。[结果]与对照组比较,随着铅染毒剂量的增加,BV-2细胞数量减少,且形态呈大而圆的阿米巴状;10、20、40μmol·L^(-1)醋酸铅组BV-2细胞内铁的水平分别为(1.08±0.04)、(1.29±0.03)、(1.72±0.10)mg·g^(–1)(以蛋白计,后同),均高于对照组(P<0.05),且40+DFO组细胞内铁水平为(1.34±0.10)mg·g^(–1),低于40μmol·L^(-1)醋酸铅组的(1.72±0.03)mg·g^(–1)(P<0.05);与对照组比较,10、20、40μmol·L^(-1)醋酸铅组的BV-2细胞中TFR-1、DMT1蛋白和mRNA表达均增加,差异有统计学意义(P<0.05),且40μmol·L^(-1)醋酸铅组尤为显著;FPN1蛋白表达无明显变化,但10、20、40μmol·L^(–1)醋酸铅组BV-2细胞中FPN1 mRNA的表达均明显下降(P<0.05)。与对照组比较,三个铅染毒组BV-2细胞内GSH水平均下降,脂质ROS水平升高;相比于40μmol·L^(−1)醋酸铅组,40+DFO组中GSH升高了12.30%,脂质ROS含量下降了13.00%(P<0.05)。10、20、40μmol·L^(−1)醋酸铅组中GPX4蛋白表达分别降低为对照组的50.00%、35.00%、17.00%,同时GPX4 mRNA表达也下降;20、40μmol·L^(−1)醋酸铅组中SLC7 A11蛋白[Background]Lead exposure induces microglial cell death,of which the mechanism is unclear.Ferroptosis is a new death form and its role in microglia death has not been reported.[Objective]To investigate the role of ferroptosis in microglia following lead exposure in order to provide a theoretical basis for the mechanism of lead neurotoxicity.[Methods]Microglial cell line BV-2 cells were co-cultured with 0,10,20 and 40μmol·L^(−1) lead acetate for 24 h.The 40μmol·L^(−1) lead acetate group with iron chelator(DFO)was named the 40+DFO group.Changes in BV-2 cell morphology after lead exposure were observed under an inverted microscope;tissue iron kit and glutathione kit were used to detect intracellular iron and glutathione(GSH)respectively;flow cytometry was applied to detect lipid reactive oxygen species(lipid ROS)immunofluorescence intensity.Western blotting and qPCR were adopted to detect the expressions of glutathione peroxidase 4(GPX4),solute carrier family 7 member 11(SLC7A11),transferrin receptor 1(TFR-1),divalent metal transporter 1(DMT1),ferroportin 1(FPN1)protein and mRNA.[Results]Compared with the control group,the number of BV-2 cells decreased with increasing doses of lead and the cells showed a large,round amoeboid shape.The intracellular levels of iron of BV-2 cells were(1.08±0.04),(1.29±0.03),and(1.72±0.10)mg·g^(–1)(calculated by protein,thereafter)in the 10,20,and 40μmol·L^(−1) lead acetate groups,respectively,significantly higher than that in the control group(P<0.05),and the intracellular level of iron in the 40+DFO group,(1.34±0.10)mg·g^(–1),was lower than that in the 40μmol·L^(−1) lead acetate group,(1.72±0.03)mg·g^(–1)(P<0.05).Compared with the control group,the TFR-1 and DMT1 protein and mRNA expressions were increased in BV-2 cells in the 10,20,40μmol·L^(−1) lead acetate groups(P<0.05),especially in the 40μmol·L^(−1) lead acetate group;the FPN1 protein expression did not change significantly,but the FPN1 mRNA expressions in BV-2 cells in the 10,20,40μmol·L

关 键 词：铅 BV-2细胞 铁死亡 氧化损伤 铁转运 

分 类 号：R114[医药卫生—卫生毒理学]