金属对SH-SY5Y细胞氧化应激及A蛋白沉积影响

朱敬丽; 吴越; 路赵硕; 王声远; 姚建; 王鑫鑫; 王德才

doi:10.11847/zgggws2017-33-11-13

摘要: 目的探讨铜、铁、锌、铝金属对人神经母细胞瘤细胞（SH-SY5Y）的氧化应激及β淀粉样蛋白（Aβ）沉积影响，为预防老年性痴呆提供依据。方法采用不同剂量的铜、铁、锌、铝分别作用于SH-SY5Y细胞，通过噻唑蓝（MTT）法检测细胞生长活性，黄嘌呤氧化酶法检测细胞内超氧化物歧化酶（SOD）活力，比色法测定谷胱甘肽过氧化物酶（GSH-Px）活力，硫代巴比妥酸法检测脂质过氧化产物丙二醛含量，2'，7'-二氯荧光黄双乙酸盐（DCFH-DA）探针法检测细胞内活性氧（ROS）水平，酶联免疫（ELISA）试剂盒法测定细胞内Aβ_1-42蛋白含量。结果铜、铁、锌、铝呈剂量依赖性地抑制细胞活性，50、200、400 μmol/L CuSO₄组SH-SY5Y细胞存活率分别为90.47%、74.81%、64.97%，1、2、4 mmol/L FeSO₄、AlCl₃组SH-SY5Y细胞存活率分别为93.08%、78.28%、56.10%和92.21%、85.30%、62.72%，50、100、200 μmol/L ZnSO₄组SH-SY5Y细胞存活率分别为91.76%、76.51%、61.27%；与对照组比较，各剂量金属组SH-SY5Y细胞存活率均明显下降，差异有统计学意义（P<0.05）；与对照组比较，各剂量铜、铁、锌、铝组SH-SY5Y细胞抗氧化酶SOD、GSH-Px活性明显下降，ROS水平和丙二醛、Aβ_1-42含量明显增加，差异均有统计学意义（P<0.05）。结论铜、铁、锌、铝可致神经细胞毒性作用和Aβ蛋白沉积，其机制可能与金属诱导细胞内ROS产生并导致氧化损伤有关。

Abstract: Objective To study the effects of copper (Cu),iron (Fe),zinc (Zn),and aluminum (Al) exposure on oxidative stress and beta amyloid (Aβ) protein deposition in human neuroblastoma cell line (SH-SY5Y) and to provide evidences for senile dementia prevention.Methods SH-SY5Y cells were treated with various doses of Cu,Fe,Zn,and Al,respectively.The viability of the cells was determined with 3-4,5-dimedthylthiazol-2,5-diphenyltetrazolium bromide (MTT) assay.The activity of superoxide dismutase (SOD) in cultured SH-SY5Y cells was detected with xanthine oxidase method;the activity of glutathione peroxidase (GSH-PX) was measured with colorimetry;the content of malondialdehyde (MDA) in culture supematant fluid was assessed with thiobituric acid reaction (TBA) method.Cellular reactive oxygen species (ROS) level was detected using fluorescent probe of 2',7'-dichlorofluorescein diacetate (DCFH-DA).The content of Aβ_1-42 was measured by enzyme-linked immunosorbent assay (ELISA).Results Cu,Fe,Zn,and Al could suppress the viability of SH-SY5Y cells in a concentration-dependent manner.When treated with copper sulfate (CuSO₄) at the dosages of 50,200,and 400 μmol/L,the survival rates of the SH-SY5Y cells were 90.47%,74.81%,and 64.97%;at the dosages of 1,2,and 4 mmol/L,the survival rates of the SH-SY5Y cells were 93.08%,78.28%,and 56.10% when exposed to ferrous sulfate (FeSO₄) and 2.21%,85.30%,and 62.72% when exposed to aluminum chloride (AlCl₃);the survival rates of the the SH-SY5Y cells were 91.76%,76.51%,and 61.27% when treated with zinc sulfate (ZnSO₄) at the dosages of 50,100,and 200 μmol/L,respectively.The survival rates of the SH-SY5Y cells exposed to various concentrations of the four metallic compounds were all significantly lower than that of the blank control group (P<0.05 for all).When treated with the four metallic compounds,the intracellular activity of SOD decreased but the intracellular ROS increased significantly;the activity of GSH-PX declined but the content of MDA and Aβ_1-42 increased significantly in culture supematant fluid compared to those of the control (all P<0.05).Conclusion Copper,iron,zinc,and aluminum compounds could induce damage in SH-SY5Y cells and result in deposition of Aβ protein by ROS generation and oxidative stress pathway.

金属对SH-SY5Y细胞氧化应激及A蛋白沉积影响

Impacts of metal elements on oxidative stress and A protein deposition in SH-SY5Y cells