赵宇威, 王伟轩, 师凡, 付治嘉, 吴桐, 张艳淑. 醋酸铅暴露致小胶质细胞系BV-2细胞铁死亡的机制研究[J]. 环境与职业医学, 2022, 39(8): 895-901. DOI: 10.11836/JEOM21531
引用本文: 赵宇威, 王伟轩, 师凡, 付治嘉, 吴桐, 张艳淑. 醋酸铅暴露致小胶质细胞系BV-2细胞铁死亡的机制研究[J]. 环境与职业医学, 2022, 39(8): 895-901. DOI: 10.11836/JEOM21531
ZHAO Yuwei, WANG Weixuan, SHI Fan, FU Zhijia, WU Tong, ZHANG Yanshu. Mechanisms of ferroptosis in microglial cell line BV-2 cells after lead acetate exposure[J]. Journal of Environmental and Occupational Medicine, 2022, 39(8): 895-901. DOI: 10.11836/JEOM21531
Citation: ZHAO Yuwei, WANG Weixuan, SHI Fan, FU Zhijia, WU Tong, ZHANG Yanshu. Mechanisms of ferroptosis in microglial cell line BV-2 cells after lead acetate exposure[J]. Journal of Environmental and Occupational Medicine, 2022, 39(8): 895-901. DOI: 10.11836/JEOM21531

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

  • 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、40 μmol·L−1醋酸铅共同培养24 h,40 μmol·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、40 μmol·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40 μmol·L−1醋酸铅组的(1.72±0.03) mg·g−1P<0.05);与对照组比较,10、20、40 μmol·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40 μmol·L−1醋酸铅组尤为显著;FPN1蛋白表达无明显变化,但10、20、40 μmol·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40 μmol·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、40 μmol·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、40 μmol·L−1醋酸铅组中SLC7 A11蛋白和mRNA表达均低于对照组,其中,40 μmol·L−1醋酸铅组下降最为明显(P<0.05)。

    结论

    铅暴露诱导BV-2细胞发生铁死亡,铁转运失衡及氧化损伤可能参与了铅诱导BV-2细胞铁死亡的发生发展。

     

    Abstract: 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 40 μmol·L−1 lead acetate for 24 h. The 40 μmol·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 40 μmol·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 40 μmol·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, 40 μmol·L−1 lead acetate groups (P<0.05), especially in the 40 μmol·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, 40 μmol·L−1 lead acetate groups were significantly decreased (P<0.05). Compared with the control group, the intracellular GSH level decreased and the lipid ROS content increased in all three lead acetate groups; compared with the 40 μmol·L−1 lead acetate group, the GSH level increased by 12.30% and the lipid ROS content decreased by 13.00% in the 40+DFO group (P<0.05). The expressions of GPX4 protein were reduced to 50.00%, 35.00%, and 17.00% of that of the control group in the 10, 20, and 40 μmol·L−1 lead acetate groups respectively, while the expressions of GPX4 mRNA were also significantly reduced; the expressions of SLC7A11 protein and mRNA in the 20 and 40 μmol·L−1 lead acetate groups were lower than that in the control group, with the most significant decrease in the 40 μmol·L−1 lead acetate group (P<0.05).

    Conclusion

    Lead exposure could induce ferroptosis in BV-2 cells, in which iron transport imbalance and oxidative damage might be involved.

     

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