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

[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 m RNA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、500μmol/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、500μmol/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 m RNA表达和Parkin蛋白表达降低(P<0.01)。相关性分析显示,PARK2 m RNA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 m RNA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。

[ Objective ] To study the effects of manganese chloride （MnC12） 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 MnC12 （0, 100, 300, and 500 μmol/L） for 24h. MTT assay was used to measure inhibition 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 MnCI2 groups were increased compared with the control group （P 〈 0.01）. The intracellular manganese concentration of the SH-SY5Y cells was increased in the MnC12 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 Ixmol/L MnCI2 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）, intracellular 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.