Objective: To investigate the effect of manganese chloride (MnCl(2)) or 1-methyl-4-phenylpyridinium (MPP (+)) on oxidative stress and autophagy in human neuroblastomaSK-N-SH cells and the mechanism of the neurotoxicity of manganese. Methods: SK-N-SH cells were treated with MnCl(2) or MPP(+) at doses of 0.062 5, 0.125, 0.25, 0.5, 1.0, and 2.0 mmol/L for 24 hours, and MTT assay was used to measure cell viability. The cells weretreated with MnCl(2) or MPP(+) at doses of 0.125, 0.25, and 0.5 mmol/L for 24 hours, and flow cytometry was used to measure the content of reactive oxygen species (ROS) in cells, a laser scanning confocal microscope was used to observe autophagosome in cells, and Western blot was used to measure the expression of autophagy-related proteins P62 and LC3-II/LC3-I. Results: Compared with the control group, the 0.0625-2.0 mmol/L MnCl(2) and 0.125-2.0 mmol/L MPP (+) treatment groups had significant reductions in the viability of SK-N-SH cells, and the 0.25-2.0 mmol/L MnCl(2) treatment groups had significantly lower viability than the groups treated with the same doses of MPP(+) (all P<0.05) . Compared with the control group, the 0.125-0.25 mmol/L MnCl(2) and 0.125-0.5 mmol/L MPP(+) treatment groups had significant increases in the content of ROS, and the 0.25-0.5 mmol/L MPP(+) treatment groups had significantly higher content of ROS than the groups treated with the same doses of MnCl(2) (all P<0.05) . Compared with the control group, the 0.25-0.5 mmol/L MnCl(2) andMPP(+) treatment groups had significant increases in autophagy-related proteins LC3-II/LC3-I and significant reductions in P62 expression; the 0.125-0.5 mmol/L MPP(+) treatment groups had significantly higher LC3-II/LC3-I than the groups treated with the same doses of MnCl(2), and the 0.125 and 0.25 mmol/L MPP (+) treatment groups had significantly lower P62 expression than the groups treated with the same doses of MnCl(2) (all P<0.05) . Conclusion: Both MnCl(2) and MPP(+) can induce oxidative stress and autophagy in SK-N-SH cells, and MPP(+) has a significantly greater inductive effect on autophagy of SK-N-SH cells than MnCl(2).
目的： 研究锰和1-甲基-4-苯基吡啶（MPP(+)）引起人神经母细胞瘤（SK-N-SH）细胞氧化应激和自噬的异同，进一步探讨锰神经毒性机制。 方法： 以0.062 5、0.125、0.25、0.5、1.0和2.0 mmol/L MnCl(2)和MPP(+)分别处理SK-N-SH细胞24 h，采用3-（4，5-二甲基噻唑-2）-2，5-二苯基四氮唑溴盐（MTT）试验检测细胞存活率；0.125、0.25、0.5 mmol/L MnCl(2)和MPP(+)分别处理细胞24 h，采用流式细胞仪检测细胞内活性氧（ROS）含量，激光共聚焦显微镜观察细胞自噬体，蛋白免疫印迹（Western blotting）法检测自噬相关蛋白P62和LC3-Ⅱ/LC3-Ⅰ表达。 结果： 与对照组比较，0.062 5~2.0 mmol/L MnCl(2)和0.125~2.0 mmol/L MPP(+)处理组SK-N-SH细胞存活率均明显降低，且0.25~2.0 mmol/L MnCl(2)处理组SK-N-SH细胞存活率明显低于相同剂量MPP(+)处理组（均P<0.05）；与对照组比较，0.125~0.25 mmol/L MnCl(2)和0.125~0.5 mmol/L MPP(+)处理组SK-N-SH细胞内ROS含量均明显增加，0.25~0.5 mmol/L MPP(+)处理组细胞内ROS含量明显高于相同剂量MnCl(2)处理组（均P<0.05）；与对照组比较，0.25~0.5 mmol/L MnCl(2)和MPP(+)处理组细胞自噬相关蛋白LC3-Ⅱ/LC3-Ⅰ值均明显增加，P62表达均明显下降，且0.125~0.5 mmol/L MPP(+)处理组LC3-Ⅱ/LC3-Ⅰ值明显高于相同剂量MnCl(2)处理组，0.125和0.25 mmol/L MPP(+)处理组P62表达水平明显低于相同剂量MnCl(2)处理组（均P<0.05）。 结论： MnCl(2)和MPP(+)均可引起SK-N-SH细胞氧化应激，诱导自噬，且MPP(+)对SK-N-SH细胞自噬的诱导作用明显高于MnCl(2)。.
Keywords: Autophagy; MPP(+); Manganese; Oxidative stress; SK-N-SH cells.