线粒体氧化应激在染铅小胶质细胞炎性活化中的作用研究

Effect of mitochondrial oxidative stress on lead-exposed inflammatory activation of microglia

目的:探讨线粒体氧化应激在铅(Pb)导致的小胶质细胞炎症反应中的作用及机制。方法:将BV2小胶质细胞分为4组,分别用0μmol/L(对照组)、1μmol/L、5μmol/L、10μmol/L乙酸铅处理BV2细胞,实时荧光定量PCR(RT-qPCR)检测小胶质细胞白细胞介素(IL)-6、IL-8、肿瘤坏死因子(TNF)-α mRNA的表达水平,免疫荧光观察小胶质细胞激活状态以及线粒体活性氧(mtROS)表达,JC-1染色检测线粒体膜电位;将C57BL/6小鼠分为对照组、Pb染毒组(Pb组)(100 ppm)、MitoTEMPO处理组(MitoTEMPO组)(5 mg/kg)、MitoTEMPO联合Pb处理组(MitoTEMPO+Pb组),酶联免疫吸附实验(ELISA)法测定小鼠血清及大脑皮层炎症因子IL-1β和TNF-α水平,蛋白质免疫印迹(western blotting)法测定小鼠海马NLRP3、Caspase-1、IL-1β蛋白水平。结果:Pb可使BV2小胶质细胞激活,与对照组相比,Pb组的IL-6、IL-8和TNF-α mRNA水平出现剂量依赖性升高,mtROS增高,线粒体膜电位下降;MitoTEMPO可抑制Pb导致的小鼠海马区小胶质细胞活化,血清及大脑皮层炎症因子IL-1β和TNF-α水平下降,海马NLRP3、Caspase-1、IL-1β蛋白水平增高(均P<0.05)。结论:Pb导致线粒体氧化应激,可使小胶质细胞激活,其机制可能与NLRP3炎症小体活化有关。

Objective:To investigate the role and mechanism of mitochondrial oxidative stress in lead(Pb)-in-duced microglial inflammation.Methods:BV2 microglia cells were divided into 4 groups and treated with 0μmol/L(control group),1μmol/L,5μmol/L and 10μmol/L lead acetate,respectively.The mRNA expression lev-els of interleukin(IL)-6,IL-8and tumor necrosis factor(TNF-α)in microglia were detected by reverse transcrip-tion-quantitative PCR(RT-qPCR).The activation state of microglia and the expression of mitochondrial reactive oxygen species(mtROS)were observed by immunofluorescence,and the mitochondrial membrane potential was detected by JC-1.C57BL/6 mice were divided into control group,Pb exposure group(Pb group)(100 ppm),Mito-TEMPO treatment group(MitoTEMPO group)(5 mg/kg),MitoTEMPO combined with Pb treatment group(Mito-TEMPO+Pb group),and the levels of serum and cerebral cortex inflammatory factors IL-1βand TNF-αwere de-termined by enzyme-linked immunosorbent assay(ELISA).The levels of NLRP3,Caspase-1 and IL-1βin the hippocampus of mice were determined by western blotting.Results:Pb could activate BV2 microglia cells.Com-pared with the control group,the levels of IL-6,IL-8and TNF-αmRNA in the Pb group were increased in a dose-dependent manner,mtROS was increased,and mitochondrial membrane potential was decreased.MitoTEMPO could inhibit the activation of microglia in hippocampus of mice,decrease the levels of IL-1βand TNF-αin se-rum and cerebral cortex,and increase the levels of NLRP3,Caspase-1 and IL-1βproteins in hippocampus(all P<0.05).Conclusion:Pb induces mitochondrial oxidative stress,which can activate microglia.The mechanism may be related to NLRP3 inflammasome activation.