铅暴露诱导铜蓄积对小鼠神经元氧化应激的促进作用  

Contribution of copper overload to lead-induced oxidative stress in neurons of mice

作　　者：王德胜 郑刚 王涛 赵再华 陈景元 Wang Desheng;Zheng Gang;Wang Tao;Zhao Zaihua;Chen Jingyuan(Medical School of Chinese PLA,Bejing 100853;Chinese PLA General Hospital,Beijing 100853;School of Military Preventive Medicine and the Ministry of Education Key Lab of Hazard Assessment and Control in Special Operational Environment,Air Force Medical University,Xian 710032,China)

机构地区：[1]中国人民解放军医学院,北京100853 [2]中国人民解放军总医院,北京100853 [3]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,西安710032

出　　处：《神经解剖学杂志》2022年第4期367-373,共7页Chinese Journal of Neuroanatomy

基　　金：国家自然科学基金(81920108030,81773378)。

摘　　要：目的:探讨铅(Pb)暴露诱导小鼠神经元铜(Cu)蓄积,促进细胞氧化应激损伤的机制。方法:C57BL/6小鼠饮用300 ppm醋酸铅水溶液2个月建立铅暴露动物模型;小鼠海马神经元细胞系HT22细胞暴露于0~100μmol/L醋酸铅24 h,采用噻唑蓝(MTT)法筛选适宜的铅作用剂量,建立铅暴露细胞模型。石墨炉原子吸收分光光度法(AAS)检测铅暴露后小鼠脑组织和HT22细胞铜水平变化;Western Blot法检测铅暴露对小鼠脑组织和HT22细胞中铜转运蛋白1(CTR1)、铜转运ATP酶α肽(ATP7A)、铜转运ATP酶β肽(ATP7B)表达水平的影响;2′,7′-二氯荧光素二乙酸酯(DCFH-DA)荧光探针法、硫代巴比妥酸比色法(TBA)分别测定铅暴露后HT22细胞内活性氧(ROS)和丙二醛(MDA)活性变化情况,并观察特异性铜螯合剂四硫代钼酸铵(TTM)的保护作用。结果:与对照组相比,铅暴露组小鼠血铜水平降低,而皮质和海马组织铜水平均显著升高(P<0.05);以5、10μmol/L铅暴露24 h后,HT22细胞的铜吸收呈剂量-依赖性增高(P<0.05)。铅暴露后小鼠脑组织和HT22细胞中CTR1蛋白水平显著增高,而ATP7A和ATP7B蛋白水平显著降低(P<0.05)。铅暴露引起HT22细胞ROS、MDA水平较对照组呈剂量-依赖性升高(P<0.05);而TTM的处理逆转了铅暴露引起的氧化应激(P<0.05)。结论:铅暴露可能通过影响神经元铜转运蛋白水平导致细胞铜蓄积,进而诱导细胞氧化应激损伤。Objective: To investigate the mechanisms by which lead(Pb) exposure induces copper accumulation, and prompts oxidative stress in neurons of mice. Methods: C57 BL/6 mice were fed 300 ppm lead acetate solution for 2 months to establish the animal models of lead exposure. HT22 cells, a mice hippocampal neuronal cell line, were exposed to 0~100 μmol/L lead acetate for 24 h to establish the cell model, and the proper lead doses were chosen based on the results from the methyl thiazolyl tetrazolium(MTT) assay. The lead and copper levels in the brain tissues of mice and HT22 cells were determined by atomic absorption spectrophotometry(AAS). Western Blot was employed to detect the changes of copper transporter 1(CTR1), copper transporting ATPase alpha polypeptide(ATP7 A) and copper transporting ATPase beta polypeptide(ATP7 B) in brain tissues of mice and HT22 cells followed by lead exposure. The changes of intracellular reactive oxygen species(ROS) and malondialdehyde(MDA) levels following the lead-exposed HT22 cells were measured by the fluorescence probe assay, 2′,7′-Dichlorofluorescin diacetate(DCFH-DA), and thiobarbituric acid colorimetry(TBA), respectively. The protective effects of ammonium tetrametholybdate(TTM), a specific copper chelating agent, was also determined. Results: Despite of the dropped blood copper levels in the lead-treated mice, the copper levels in both the brain tissues and HT22 cells following lead exposure significantly increased when compared to their controls(P<0.05). When exposed to 5 and 10 μmol/L lead for 24 h, the copper levels of HT22 cells increased in a dose-dependent manner(P<0.05). Western Blot showed that the lead exposure resulted in significantly up-regulated CTR1 protein levels, but decreased ATP7 A and ATP7 B protein levels in both the mice brain and HT22 cells, as compared with their controls(P<0.05). Lead treatment induced a dose-dependent elevation of ROS and MDA levels in HT22 cells, while TTM reversed the lead-induced increase of both levels of ROS and MDA(P<0.05). Co

关 键 词：铅 铜蓄积 铜转运蛋白 氧化应激 过氧化损伤 小鼠 

分 类 号：X503.1[环境科学与工程—环境工程]