低剂量铜暴露对HT22神经细胞氧化应激和凋亡的诱导作用

Induction of low-dose copper exposure on oxidative stress and apoptosis in HT22 neuronal cells

目的研究铜离子(Cu^(2+))暴露对海马神经元细胞氧化应激和凋亡的诱导作用,探讨Cu^(2+)对细胞抗氧化系统的影响。方法将小鼠海马神经元细胞系HT22分别用1.25、2.5、5、10、20μmol/L CuCl_(2)染毒24 h,建立低剂量Cu^(2+)暴露体外模型,并设置对照组(0μmol/L CuCl_(2))。CCK-8法测定细胞活力;Annexin V-FIFC/PI染色结合流式细胞术检测细胞凋亡;Rho-123染色结合流式细胞术检测线粒体膜电位;化学发光法检测三磷酸腺苷含量;DCFH-DA染色结合流式细胞术检测细胞内活性氧(ROS)含量;TBA法测定细胞丙二醛(MDA)含量;DTNB速率比色法、黄嘌呤氧化酶法和紫外分光光度法分别测定细胞谷胱甘肽(GSH)水平、超氧化物歧化酶(SOD)和过氧化氢酶(CAT)活性。结果与对照组相比,2.5~20μmol/L Cu^(2+)暴露可引起细胞活力显著抑制(P<0.05);细胞凋亡率随Cu^(2+)浓度增加而呈增高趋势,其中5~20μmol/L Cu^(2+)暴露后凋亡率显著增高(P<0.01);此外,5μmol/L及以上浓度Cu^(2+)刺激后细胞内ROS水平和MDA含量明显增多(P<0.05),GSH水平和SOD活性显著降低(P<0.05),而CAT活性只在20μmol/L Cu^(2+)暴露组有显著降低(P<0.05)。结论低剂量Cu^(2+)暴露引起HT22细胞活力下降,凋亡增多,细胞内氧化产物水平升高,且抗氧化能力降低,提示低剂量Cu^(2+)暴露可引起神经元损伤,而氧化还原水平紊乱可能是其主要机制。

Objective To study the induction of copper ion(Cu^(2+))exposure on oxidative stress and apoptosis in hippocampal neurons,and to investigate the effect of Cu^(2+)on the cellular antioxidant system.Methods HT22 mouse hippocampal neuronal cell line was treated with 1.25,2.5,5,10,and 20μmol/L CuCl_(2) for 24 h to establish a low-dose Cu^(2+)exposure model in vitro,and the control group(0μmol/L CuCl_(2))was set.Then,cell viability was determined by CCK-8,cell apoptosis by Annexin V-FIFC/PI staining combined with flow cytometry,mitochondrial membrane potential by Rho-123 staining and flow cytometry,ATP by chemiluminescence,intracellular reactive oxygen species(ROS)by DCFH-DA staining and flow cytometry,MDA by TBA,and glutathione(GSH)levels,superoxide dismutase(SOD)and catalase(CAT)activity by DTNB colorimetric method,xanthine oxidase and UV spectrophotometry,respectively.Results Compared with the control group,Cu^(2+)exposure of 2.5–20μmol/L caused significant inhibition of cell viability(P<0.05).The apoptotic rate increased with the increase of Cu^(2+)concentration,and it increased significantly after 5–20μmol/L Cu^(2+)exposure(P<0.05).In addition,we observed an significant increase of ROS and MDA,as well as an significant decrease of GSH levels and SOD activity in cells after Cu^(2+)exposure of 5μmol/L and above(P<0.05),whereas CAT activity was significantly reduced only in 20μmol/L Cu^(2+)exposure group(P<0.05).Conclusion Low-dose Cu^(2+)exposure results in decreased cell viability,increased apoptosis and intracellular oxidation product level,and decreased antioxidant capacity of HT22 cells.Therefore,this study indicates that low-dose Cu^(2+)exposure causes neuronal damage,and redox imbalance may be the main mechanism.