【摘要】 目的研究硝酸镧〔La（NO₃）₃〕在引起人神经母细胞瘤（SK-N-SH）细胞毒性中氧化应激和自噬的作用机制。方法 La（NO₃）₃0.125,0.25,0.5,1.0,2.0和4.0 mmol·L^-1处理SK-N-SH细胞24 h,MTT法检测细胞存活率。La（NO₃）₃ 0.5,1.0和2.0 mmol·L^-1处理细胞24 h,流式细胞术检测细胞内活性氧（ROS）含量和线粒体膜电位,激光共聚焦显微镜检测细胞自噬体数量,Western印迹法检测微管相关蛋白1轻链3（LC3）-Ⅱ/Ⅰ比值,P62,Parkin和PTEN诱导假定激酶1（PINK1）蛋白表达水平。结果 MTT结果显示,与细胞对照组相比,La（NO₃）₃≤0.5 mmol·L^-1对细胞存活率无显著影响,La（NO₃）₃1.0,2.0和4.0 mmol·L^-1组细胞存活率均显著降低（P<0.05,P<0.01）。流式细胞术结果显示,与细胞对照组相比,La（NO₃）₃0.5 mmol·L^-1组细胞ROS含量无明显差异,La（NO₃）₃1.0和2.0 mmol·L^-1组细胞ROS含量均显著升高（P<0.05,P<0.01）,La（NO₃）₃0.5,1.0和2.0 mmol·L^-1组线粒体膜电位均显著降低（P<0.05,P<0.01）。激光共聚焦结果显示,与细胞对照组相比,La（NO₃）₃0.5,1.0和2.0 mmol·L^-1组细胞自噬体数量均显著增加（P<0.05,P<0.01）。Western印迹结果显示,与细胞对照组相比,La（NO₃）₃0.5,1.0和2.0 mmol·L^-1组LC3-Ⅱ/Ⅰ比值、Parkin和PINK1蛋白表达均显著升高（P<0.05）,P62蛋白表达均显著降低（P<0.05）。结论La（NO₃）₃可诱导SK-N-SH细胞发生氧化应激、线粒体功能障碍,同时可诱导SK-N-SH细胞产生细胞自噬和线粒体自噬。更多还原

【Abstract】 OBJECTIVE To explore the role of oxidative stress and autophogy in cytotoxicities of lanthanum nitrate[La（NO₃）₃]on human neuroblastoma（SK-N-SH） cells. METHODS SK-N-SH cells were treated with La（NO₃）₃ 0.125, 0.25, 0.5, 1.0, 2.0 and 4.0 mmol·L^-1 for 24 h. The cell viability was detected by MTT assay. The cells were treated with La（NO₃）₃ 0.5, 1.0 and 2.0 mmol·L^-1 for 24 h. The level of reactive oxygen species（ROS） and the mitochondrial membrane potential were measured by flow cytometry. The number of autophagosomes was determined under a laser scanning confocal microscope, and the protein expressions of micro-tubule associated protein 1 light chain 3（LC3）, P62, Parkin and PTEN-induced putative kinase 1（PINK1） were assessed by Western blotting. RESULTS Compared with the cell control group, La（NO₃）₃≤0.5 mmol·L^-1 did not affect cell viability, but the cell viability of La（NO₃）₃1.0, 2.0 and 4.0 mmol·L^-1 decreased significantly（P<0.05, P<0.01）. There was no significant difference in the level of ROS between cell control and La（NO₃）₃0.5 mmol·L^-1 group, but the level of ROS increased significantly in La（NO₃）₃1.0 and 2.0 mmol·L^-1 groups（P<0.05, P<0.01）. The levels of mitochondrial membrane potential of La（NO₃）₃0.5, 1.0 and 2.0 mmol·L^-1 groups were significantly reduced compared with the cell control group（P<0.05, P<0.01）. LSCM results showed that the number of autophagosomes of La（NO₃）₃0.5, 1.0 and 2.0 mmol·L^-1 groups increased significantly compared with the cell control group（P<0.05, P<0.01）. Western blotting results showed that in La（NO₃）₃0.5, 1.0 and2.0 mmol·L^-1 groups, the ratio of LC3-Ⅱ/Ⅰprotein was increased, protein expressions of Parkin and PINK1（P<0.05） were up-regulated, but the expression of P62（P<0.05） was down-regulated. CONCLUSION La（NO₃）₃ could induce oxidative stress, mitochondrial dysfunction, cell autophagy and mitochondrial autophagy in SK-N-SH cells.更多还原