溶酶体在锰致SK-N-SH细胞毒性中的作用

The role of lysosomes in manganese-induced toxicity in SK-N-SH cells

目的探讨溶酶体在锰致人神经母细胞瘤(SK-N-SH)细胞毒性中的作用。方法以浓度分别为0、0.0625、0.125、0.25、0.5、1.0、2.0和4.0mmol/LMnCl2处理SK-N-SH细胞24h,通过3-(4,5-二甲基噻唑-2)-2,5-二苯基四氮唑溴盐(MTT)法检测细胞存活率。以0、0.125、0.25、0.5和1.0mmol/LMnCl2处理细胞24h,激光共聚焦显微镜观察红色荧光探针标记的溶酶体,蛋白免疫印迹(Westernblot)法检测溶酶体膜相关蛋白1(LAMP1)、组织蛋白酶D(CTSD)表达水平,CTSD活力荧光测定试剂盒检测CTSD活力。结果与对照组比较,0.5~4.0mmol/LMnCl2处理组SK-N-SH细胞存活率明显降低,0.5~1.0mmol/LMnCl2处理组SK-N-SH细胞相对荧光强度明显增加,差异均有统计学意义(P<0.01);与对照组比较,0.125~0.5mmol/LMnCl2处理组SK-N-SH细胞LAMP1蛋白表达水平明显增加,0.125、0.25mmol/LMnCl2处理组SK-N-SH细胞成熟型CTSD(m-CTSD)蛋白表达水平明显增加,1.0mmol/LMnCl2处理组m-CTSD蛋白明显下降,差异均有统计学意义(P<0.01);与对照组比较,各MnCl2处理组CTSD活力差异均无统计学意义(P>0.05)。结论MnCl2可引起SK-N-SH细胞毒性,溶酶体在锰低剂量时发挥正常功能,但在锰高剂量时受损。溶酶体作为自噬下游发挥降解功能的细胞器,可能参与了锰的神经毒性机制。

Objective To investigate the role of lysosomes in manganese-induced toxicity in human neuroblastoma SK-N-SH cells. Methods SK-N-SH cells were treated with MnCl2 at doses of 0.062 5, 0.125, 0.25, 0.5, 1.0, 2.0 and 4.0 mmol/L for 24 h, and the cell viability was detected by MTT assay. Cells were treated with MnCl2 at doses of 0.125, 0.25, 0.5 and 1.0mmol/L for 24 h, and lysosomes labeled with lysotracker red were observed by laser confocal microscopy, the expression levels of LAMP1 and CTSD were detected by western blot, and CTSD activity was detected by Cathepsin D Activity Fluorometric Assay Kit. Results Compared with the control group, the survival rates of SK-N-SH cells were decreased significantly in the 0.5-4.0 mmol/L MnCl2 treatment groups (P<0.01), the relative fluorescence intensities of 0.5 and 1.0 mmol/L MnCl2 treatment groups were increased (P<0.01). Compared with the control group, the 0.125-0.5 mmol/L MnCl2 treatment groups had significant increase in the the expression of LAMP1 (P<0.01). Compared with the control group, the expression of m-CTSD was significantly increased at the does of 0.125-0.25 mmol/L MnCl2, while it was decreased at the does of 1.0 mmol/L (P<0.01). Otherwise, it wasn’t observed significant difference of the activity of CTSD between different MnCl2 treatment groups. Conclusion MnCl2 could cause cytotoxicity in SK-N-SH cells. Lysosomes may play a normal function at low doses of manganese, but they may be damaged at high doses of manganese. As an organelle that can degradate substrates in autophagy, lysosomes participate in the neurotoxic mechanism of manganese.