铅诱导离体神经干细胞氧化应激和铜蓄积及MitoQ的保护作用

Lead-induced oxidative stress and copper overload in neural stem cells in vitro and the protective effects of MitoQ

目的研究铅离子(Pb^(2+))暴露对神经干细胞(NSC)氧化应激和诱导铜蓄积的作用,探讨MitoQ的抗氧化保护作用。方法C17.2神经干细胞用2.5、5、10、20μmol/L Pb(Ac)2分别暴露24 h和48 h,建立低剂量Pb^(2+)暴露体外模型,并设置对照组[0μmol/L Pb(Ac)2]。MTT法检测细胞活力;活性氧荧光探针(DCFH-DA)检测细胞活性氧自由基(ROS)含量;TBA法测定细胞丙二醛(MDA)含量;黄嘌呤氧化酶法测定细胞内超氧化物歧化酶1(SOD1)的活性;原子吸收分光光度法检测细胞铜吸收;Western blotting检测细胞内铜转运体CTR1、ATP7A和ATP7B蛋白水平。结果与对照组相比,2.5~20μmol/L Pb^(2+)暴露24 h或48 h后,对细胞活力产生显著的抑制作用,而细胞ROS和MDA水平、SOD1活性均显著高于对照组(P<0.01,P<0.05);不同浓度Pb^(2+)暴露后细胞内铜水平显著增高(P<0.05),CTR1蛋白水平增高(P<0.05),而ATP7A、ATP7B蛋白表达均显著降低(P<0.01,P<0.05)。使用抗氧化剂MitoQ处理细胞,可以逆转Pb^(2+)暴露所诱导的细胞的ROS、MDA和SOD1水平变化(P<0.01),且伴随细胞活力的显著回升。结论Pb^(2+)暴露可能通过诱导C17.2 NSC铜蓄积,促进氧化应激和细胞活力降低;抗氧化剂MitoQ可以抑制Pb^(2+)引起的氧化应激,起到显著的细胞保护作用。

Objective To study the effects of lead ion(Pb^(2+))exposure on oxidative stress and copper overload in neural stem cells(NSC),and to explore the antioxidative protection of MitoQ.Methods C17.2 neural stem cells were treated with 2.5,5,10,and 20μmol/L Pb(Ac)2 for 24 h and 48 h,respectively,to establish a low-dose Pb^(2+)exposure model in vitro,and the control group[0μmol/L Pb(Ac)2]was set.Cell viability was detected by MTT assay.The content of reactive oxygen species(ROS)was measured by DCFH-DA fluorescent probe,and the content of malondialdehyde(MDA)was determined by TBA method.The activity of superoxide dismutase 1(SOD1)was determined by xanthine oxidase method.Copper absorption was detected by atomic absorption spectrophotometry.The protein levels of CTR1,ATP7A and ATP7B were detected by Western blotting.Results Compared with the control group,2.5-20μmol/L Pb^(2+)exposure for 24 h or 48 h significantly inhibited cell viability,while the levels of ROS and MDA and the activity of SOD1 were significantly higher than those of the control group(P<0.01,P<0.05).After Pb^(2+)exposure,the intracellular copper level significantly increased(P<0.05),CTR1 protein level increased(P<0.05),while ATP7A and ATP7B protein level significantly decreased(P<0.01,P<0.05).Treatment with antioxidant MitoQ could reverse the changes of ROS,MDA and SOD1 induced by Pb^(2+)(P<0.01),with a significant increase in cell viability.Conclusion Pb^(2+)exposure may promote oxidative stress and decrease cell viability by inducing copper overload in C17.2 NSC.The antioxidant MitoQ can protect the cells from oxidative stress induced by Pb^(2+)exposure and promote the recovery of cell viability.