南方医科大学学报

南方医科大学学报 ›› 2023, Vol. 43 ›› Issue (10): 1752-1760.doi: 10.12122/j.issn.1673-4254.2023.10.13

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铅暴露加重Aβ1-42处理的小胶质细胞活化和小胶质细胞中的铜离子蓄积

陈丽旋，黄定帮，郑 刚，孟晓静   

  1. 南方医科大学公共卫生学院职业卫生与职业医学系，广东 广州 510515；空军军医大学军事预防医学系，特殊作业环境危害评估与防治教育部重点实验室，陕西 西安 710032
  • 出版日期:2023-10-20 发布日期:2023-11-02

Lead exposure aggravates Aβ1-42-induced microglial activation and copper ion accumulation in microglial cells

CHEN Lixuan, HUANG Dingbang, ZHENG Gang, MENG Xiaojing   

  1. Department of Occupational Health and Occupational Medicine, School of Public Health, Southern Medical University, Guangzhou 510515, China; Department of Military Preventive Medicine, Air Force Military Medical University, Ministry of Education Key Laboratory of Hazard Assessment and Control in Special Operational Environment, Xi'an 710032, China
  • Online:2023-10-20 Published:2023-11-02

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摘要： 目的 探讨铜离子在铅（Pb）加重Aβ1-42处理的小胶质细胞活化及神经元损伤中的作用，以揭示铅加重阿尔兹海默症（AD）样变的调控机制。方法 将BV2细胞分为Control组（不予任何处理）、Aβ1-42组（5、10、15、20 μmol/L Aβ1-42处理12 h）、Pb组（5、10、15、20 μmol/L Pb处理12 h）和Aβ1-42+Pb组（10 μmol/L Aβ1-42+10 μmol/L Pb处理12 h）。CCK8检测细胞活力，相差显微镜观察细胞形态学改变，细胞免疫荧光检测iNOS释放及氧化应激情况，ELISA检测小胶质细胞的炎性因子释放情况，Western blot检测CTR1和ATP7A蛋白表达量，ICP-MS检测细胞铜含量。结果 15 μmol/L和20 μmol/L的 Aβ1-42处理BV2细胞12 h可降低其生长活力（P<0.05和P<0.01）；与对照组相比，10 μmol/L Aβ1-42处理BV2细胞12 h可引起iNOS、炎性因子TNF-α和IL-6释放增加（P<0.05）；10 μmol/L Aβ1-42联合15 μmol/L和20 μmol/L的铅处理BV2细胞可影响其生长活力（P<0.05）；与10 μmol/LAβ1-42单独处理组相比，10 μmol/L 铅与10 μmol/L Aβ1-42联合处理BV2细胞12h可使激活的小胶质细胞增多，具体表现为BV2细胞形态呈现胞体增大、突起增多且细长，iNOS、炎性因子TNF-α和IL-6、IL-1β释放增加（P<0.05），细胞ROS释放增多，细胞内铜离子蓄积（P<0.001），铜转运蛋白CTR1表达水平上升（P<0.05）；BV2细胞的激活明显降低了海马神经元HT22细胞生长活力（P<0.001）；与Aβ1-42+Pb组相比，在使用抗氧化剂NAC和铜螯合剂TM后，激活的小胶质细胞減少，且对神经元细胞的生长活力损伤有所减轻（P<0.05）。结论 铅暴露加重了Aβ1-42处理的小胶质细胞造成的神经元损伤，与铅加重Aβ1-42处理的小胶质细胞铜离子蓄积，氧化应激，炎性因子释放增加，引发小胶质细胞活化有关。

关键词: 铅；Aβ1-42；小胶质细胞；铜离子

Abstract: Objective To investigate the effect of lead (Pb) exposure on Aβ1-42-induced microglial activation and copper ion accumulation in microglial cells and explore the regulatory mechanism of Pb-induced aggravation of Alzheimer's disease (AD)-like pathology. Methods Cultured microglial BV2 cells were treated with different concentrations of Aβ1-42, lead acetate or their combination for 12 h, and the changes in cell viability and morphology were evaluated. Immunofluorescence assay was performed to detect iNOS and oxidative stress level in the treated cells, and the release of inflammatory factors was detected using ELISA. Western blotting and inductively coupled plasma-mass spectrometry (ICP-MS) were used to detect the expressions of CTR1 and ATP7A proteins and copper content in the cells. Results Treatment with 15 and 20 μmol/L Aβ1-42 for 12 h significantly lowered the viability of BV2 cells. Treatment with Aβ1- 42 at 10 μmol/L for 12 h obviously increased the release of iNOS, TNF-α and IL-6 in the cells (P<0.05), and its combination with 15 or 20 μmol/L lead acetate more strongly lowered BV2 cell viability (P<0.05). Compared with 10 μmol/L Aβ1- 42 treatment alone, 10 μmol/L Aβ1- 42 combined with 10 μmol/L lead acetate for 12 h caused more obvious microglial activation, as manifested by enlarged cell bodies, increased cell protrusions and elongation, enhanced release of iNOS, TNF-α, IL-6, IL-1β and ROS, and increased intracellular copper ion accumulation and expression of copper transporter CTR1 (P<0.05). Compared with the conditioned medium from activated BV2 cells, which caused obvious injuries in hippocampal neuron HT22 cells (P<0.001), the medium from BV2 cells treated with NAC and the copper ion chelating agent TM caused milder injuries in HT22 cells (P<0.05). Conclusion Lead exposure aggravates neuronal damage caused by Aβ1- 42-treated microglial cells by increasing copper ion accumulation, oxidative stress, and inflammatory factor release to trigger microglial activation.

Key words: lead exposure; Aβ1-42; microglia; copper ions