氯化锰致人骨髓神经母细胞瘤细胞株线粒体损伤及对多巴胺分泌和<i>PARK2</i>表达的影响

范希敏; 罗英; 许洁; 吴芹; 刘杰; 范奇元

doi:10.13213/j.cnki.jeom.2017.17123

氯化锰致人骨髓神经母细胞瘤细胞株线粒体损伤及对多巴胺分泌和PARK2表达的影响

Effects of manganese chloride on mitochondrial damage, dopamine secretion, and expression of PARK2 in human bone marrow neuroblastoma cells

摘要

摘要:
目的研究氯化锰对人骨髓神经母细胞瘤细胞株（SH-SY5Y）线粒体损伤、氧化应激、多巴胺分泌及PARK2表达的影响。

方法 0、100、300、500μmol/L浓度氯化锰染毒SH-SY5Y细胞24 h后，用MTT法测细胞抑制率（反映线粒体损伤情况），石墨炉原子吸收光谱法测定细胞内锰浓度，高度水溶性四唑盐（WST-1）法测定细胞内超氧化物歧化酶（SOD）活性，硫代巴比妥酸法测定细胞内丙二醛（MDA）含量，反相高效液相色谱-荧光法测定细胞内多巴胺（DA）含量，实时荧光定量-PCR检测PARK2 mRNA表达，蛋白免疫印迹法检测Parkin蛋白表达。

结果与对照组比较，MnCl₂浓度为300、500 μmol/L时，细胞抑制率（线粒体损伤）增高（P<0.01）。与对照组比较，染锰组细胞内锰浓度升高（P<0.05或P<0.01）。与对照组比较，MnCl₂浓度为300、500 μmol/L时，SOD活性和DA含量降低（P<0.01），MDA含量升高（P<0.01）；细胞PARK2 mRNA表达和Parkin蛋白表达降低（P<0.01）。相关性分析显示，PARK2 mRNA表达与细胞抑制率（线粒体损伤）、细胞内锰浓度及MDA含量呈负相关，r值分别为-0.872、-0.880、-0.862（均P<0.01）；PARK2 mRNA表达与SOD活性、DA含量以及Parkin蛋白表达呈正相关，r值分别为0.879、0.859、0.809（均P<0.01）。

结论氯化锰暴露可引起SH-SY5Y细胞的线粒体损伤、氧化应激、DA分泌减少和PARK2表达下降

Abstract:
Objective To study the effects of manganese chloride (MnCl₂) on mitochondrial damage, oxidative stress, secretion of dopamine, and expression of PARK2 in human bone marrow neuroblastoma cells (SH-SY5Y).

Methods SH-SY5Y cells were exposed to MnCl₂ (0, 100, 300, and 500 μmol/L) for 24 h. MTT assay was used to measure in hibition rate of the cells (mitochondrial damage), graphite furnace atomic absorption spectrometry for intracellular manganese concentration, water soluble tetrazolium salt (WST-1) assay for superoxide dismutase (SOD) activity, thiobarbituric acid assay for malondialdehyde (MDA) level, reverse phase high performance liquid chromatography-fluorometry for dopamine (DA) level, RT-PCR for the expression of PARK2 mRNA, and Western blot for the expression of Parkin protein, respectively.

Results The cell inhibition rates (mitochondrial damage) of the 300 and 500 μmol/L MnCl₂ groups were increased compared with the control group (P < 0.01). The intracellular manganese concentration of the SH-SY5Y cells was increased in the MnCl₂ groups compared with the control group (P < 0.05 or P < 0.01). Compared with the control group, decreasing SOD activities and DA levels and increasing MDA levels were observed in the 300 and 500 μmol/L MnCl₂ groups (P < 0.01). At the same time, the expressions of PARK2 mRNA and Parkin protein were also decreased (P < 0.01). The results of correlation analysis revealed that the expression of PARK2 mRNA was inversely correlated with cell inhibition rate (mitochondrial damage) (r=-0.872), in tracellular manganese concentration (r=-0.880), and MDA level (r=-0.862) (all Ps < 0.01), respectively; the expression of PARK2 mRNA was positively correlated with SOD activity (r=0.879), DA level (r=0.859), and the expression of Parkin protein (r=0.809) (all Ps < 0.01), respectively.

Conclusion MnCl₂ exposure could induce mitochondrial damage, oxidative stress, and decreased DA secretion and expression of PARK2 in SH-SY5Y cells.

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