ROS介导Nrf2/HO-1通路在纳米MnO2致BV2小胶质细胞氧化损伤中的作用
The role of ROS-mediated Nrf2/HO-1 pathway in oxidative damage of BV2 microglial cells induced by Nano MnO2
覃镡 1孙兴昌 2梁高峰 2常旭红 1朱慧科 1张佳豪 1张丹 1冯三畏 2孙应彪1
作者信息
- 1. 兰州大学公共卫生学院卫生毒理学系,甘肃兰州 730000
- 2. 兰州石化总医院职业病研究所,甘肃兰州 730060
- 折叠
摘要
目的 探讨ROS介导的Nrf2/HO-1通路在纳米(Nano)MnO2诱导的BV2小胶质细胞氧化损伤中的作用.方法 采用2.5、5.0和10.0 μg/ml Nano MnO2和活性氧(ROS)清除剂N-乙酰半胱氨酸(NAC)单独或联合处理BV2细胞12 h,分别建立细胞毒性和干预模型.采用DCFH-DA荧光探针检测细胞ROS水平,生化试剂盒检测细胞SOD、GSH-Px、MDA含量及·OH清除能力,Western blot及细胞免疫荧光技术检测细胞Nrf2/HO-1通路相关蛋白表达水平.结果 Nano MnO2可致BV2细胞活力降低,细胞膜损伤程度增加,并呈剂量依赖性(F细胞活力=126.648、FLDH=17.394,P＜0.05).与对照组相比,Nano MnO2组BV2细胞中ROS水平升高(F=16.584,P＜0.05),Nano MnO2高剂量组细胞SOD和GSH-Px活力明显降低(FSOD=17.394、FGSH-Px=16.584,P＜0.05);而Nano MnO2中和高剂量组细胞·OH清除能力降低,MDA含量明显增高(F.OH清除能力=5.352、FMDA=5.478,P＜0.05).Nano MnO2中和高剂量组BV2细胞中Nrf2蛋白磷酸化水平明显升高的同时也出现明显的Nrf2核易位现象,且HO-1蛋白表达水平亦明显升高(Fp-Nrf2=8.608、FHO-1=7.769,P＜0.05).采用ROS清除剂NAC处理BV2细胞后,Nano MnO2激活的BV2细胞Nrf2/HO-1通路被抑制(Fp-Nrf2=11.05、FHO-1=10.19,P＜0.05).结论 Nano MnO2可诱导BV2小胶质细胞内ROS过量生成而致氧化应激,进而通过活化Nrf2/HO-1通路而发挥抗氧化损伤作用,但其对细胞毒性主要以氧化损伤为主.
Abstract
Objective To investigate the mechanism of ROS-mediated Nrf2/HO-1 pathway in manganese dioxide nanoparticles(Nano MnO2)-induced oxidative damage in BV2 microglial cells.Methods BV2 cells were treated with 2.5,5.0,and 10.0 μg/ml Nano MnO2 and reactive oxygen species(ROS)scavenger N-acetylcysteine(NAC)alone or in combination for 12 h to establish the cytotoxicity and intervention models,respectively.The intracellular ROS level was detected by DCFH-DA fluorescent probe.Biochemical kits were used to measure the cellular SOD,GSH-Px and MDA contents,as well as the·OH scavenging ability.The cellular Nrf2/HO-1 pathway related protein levels were detected by Western blot and immunofluorescence assay.Results Nano MnO2 caused a dose-dependent decrease in BV2 microglial cells viability and an increase in cell membrane damage(Fcell viabi1ity=126.648,FLDH=17.394,P＜0.05).Compared with the control group,the ROS level was increased in BV2 microglial cells in the Nano MnO2 group(FROS=16.584,P＜0.05),and the SOD and GSH-Px activity was significantly decreased in the Nano MnO2 high-dose group(FSOD=17.394,FGSH-Px=16.584,P＜0.05),while the cellular·OH scavenging ability was decreased and the MDA content was significantly increased in the middle and high dose groups of Nano MnO2(F.OH scavenging ability=5.352,FMDA=5.478,P＜0.05).In the middle and high dose Nano MnO2 groups,the phosphorylation level of Nrf2 protein significantly increased.Additionally,the nuclear translocation phenomenon of Nrf2 appeared,and the expression level of HO-1 protein significantly increased in BV2 microglial cells(FNrf2=8.608,FHO-1=-7.769,P＜0.05).The Nrf2/HO-1 pathway activated by Nano MnO2 was inhibited after treatment of BV2 microglial cells with the ROS scavenger NAC(FNrf2=11.05,FHO-1=10.19,P＜0.05).Conclusion Nano MnO2 could increase ROS production to induce oxidative stress,which further activated Nrf2/HO-1 pathway to exert the effect of antioxidant damage.However,the toxicity of Nano MnO2 is mainly oxidative damage in BV2 microglial cells.
关键词
纳米MnO2/BV2小胶质细胞/ROS/Nrf2/HO-1通路/氧化损伤Key words
Nano manganese dioxide/BV2 microglial cells/Reactive oxygen species/Nrf2/HO-1 pathway/Oxidative damage引用本文复制引用
基金项目
甘肃省科技计划重点研发计划(21YF5FA113)
出版年
2024
NETL
NSTL
万方数据