吸入炭黑气溶胶对小鼠肺组织氧化和抗氧化系统的影响  

Effects of inhalation of carbon black aerosols on oxidation and antioxidant system in lung tissue of mice

作　　者：孟涛 宋佳阳 杨墨[3] 尉杰忠[1,2] 段化伟[3] MENG Tao;SONG Jia-yang;YANG Mo;YU Jie-zhong;DUAN Hua-wei(School of medicine,Shanxi Datong University,Datong 037009,China;First Hospital of Shanxi Datong University,Datong 037006,China;National Institute of Occupational Health and Poison Control,Chinese Center for Disease Control and Prevention,Beijing 100050,China)

机构地区：[1]山西大同大学医学院,山西大同037009 [2]山西大同大学附属第一医院,山西大同037006 [3]中国疾病预防控制中心职业卫生与中毒控制所,北京100050

出　　处：《中国药理学与毒理学杂志》2020年第5期368-375,共8页Chinese Journal of Pharmacology and Toxicology

基　　金：国家自然科学基金(81502845);国家自然科学基金(81573124);国家重点研发计划(2017YFC0211604);山西省应用基础研究项目(201801D221372);山西省高等学校科技创新项目(2019L0761);大同市科技局重点研发计划(2019105);山西大同大学博士科研启动经费项目(2018-B-15);山西大同大学附属第一医院横向合作项目(HX-201927)。

摘　　要：目的探讨反复吸入炭黑气溶胶对小鼠肺组织氧化应激水平和抗氧化因子核转录因子红系2相关因子2(Nrf2)及其调控的抗氧化酶水平的影响,分析持续暴露后肺组织氧化应激和氧化防御的关系。方法C57BL/6小鼠置于动式全身吸入柜中,每天6 h吸入炭黑气溶胶15.06和29.61 mg·m^-3连续14 d。末次染毒后,光镜和电镜下观察肺组织病理结构的变化;ELISA法测定肺组织超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)和过氧化氢酶(CAT)活性及丙二醛(MDA)水平;化学荧光法检测肺组织单细胞内活性氧(ROS)水平;荧光定量PCR和Western印迹法检测肺组织Nrf2及抗氧化酶亚铁血红素加氧酶1(HO-1)、NAD(P)H醌氧化还原酶1(NQO-1)和谷氨酰半胱氨酸连接酶催化亚基(GCLC)mRNA和蛋白表达水平。结果炭黑气溶胶15.06和29.61 mg·m^-3组小鼠肺组织细胞出现炭黑颗粒沉积、毛细血管充血、炎细胞浸润、肺泡和支气管上皮损伤及肺间隔增宽等改变,且肺损伤评分分别为正常对照组的2.96和4.02倍(P<0.01)。与正常对照组相比,炭黑气溶胶15.06和29.61 mg·m^-3组肺组织SOD,GSH-Px和CAT活性明显下降,细胞内ROS和肺组织MDA水平明显升高(P<0.05);肺组织抗氧化酶HO-1,NQO-1和GCLC mRNA表达显著上调(P<0.05),Nrf2 mRNA表达无显著差异;Nrf2蛋白总体水平未见显著变化,但胞质Nrf2蛋白水平显著下调而胞核Nrf2蛋白水平显著上调(P<0.05);HO-1,NQO-1和GCLC蛋白水平均显著上调(P<0.05)。相关分析显示,HO-1,NQO-1和GCLC mRNA水平与细胞内ROS水平呈正相关,相关系数分别为0.785,0.699和0.720(P<0.01)。结论持续吸入炭黑气溶胶可造成小鼠肺组织细胞内炭黑颗粒的沉积,诱导肺组织的氧化应激,并促使Nrf2从胞质转位到胞核,上调抗氧化酶HO-1,NQO-1和GCLC的表达。OBJECTIVE To investigate the effects of repeated inhalation of carbon black(CB)aero⁃sols on the levels of oxidative stress and the levels of antioxidant factor nuclear factor erythroid 2-related factor 2(Nrf2)and its regulation of antioxidant enzymes,and to analyze the relationship between oxida⁃tive stress and oxidative defense in lung tissue of mice after continuous exposure.METHODS C57BL/6 mice inhaled CB aerosols 15.06 and 29.61 mg·m^-3 for 6 h per day for a continuous exposure of 14 d in a whole-body inhalation device.The pathological changes in lung tissue in mice were observed under the light microscope and electron microscope after the last exposure.ELISA was used to determine the levels of oxidative stress indicators including the activity of superoxide dismutase(SOD),glutathione peroxi⁃dase(GSH-Px)and catalase(CAT)as well as the levels of malondialdehyde(MDA)in lung tissue.Chemical fluorescence assay was performed to measure the intracellular levels of reactive oxygen species(ROS).In addition,the mRNA and protein levels of Nrf2 and antioxidant enzymes including heme oxygen⁃ase-1(HO-1),NAD(P)H quinone oxidoreductase-1(NQO-1)and glutamate-cysteine ligase catalytic subunit(GCLC)in lung tissue were determined by fluorescent quantitative PCR and Western blotting.RESULTS After inhalation of CB aerosols 15.06 and 29.61 mg·m^-3 for 14 d,the black particles were found in the lung tissue and cells of mice,and various pathological changes including capillary conges⁃tion,inflammatory cell exudation,alveolar epithelium and bronchial epithelium cell injury,and lung septal widening,were also observed.The pathological scores for lung injury of mice in CB aerosols 15.06 and 29.61 mg·m^-3 groups were 2.96 and 4.02 times of those in normal control group(P<0.01).Compared with normal control group,the activities of SOD,GSH-Px and CAT in lung tissue of mice in CB aero⁃sols 15.06 and 29.61 mg·m^-3 groups significantly decreased,while the intracellular ROS and MDA levels in lung tissues significantly increased(

关 键 词：炭黑 气溶胶 吸入染毒 NF-E2相关因子 抗氧化通路 氧化应激 呼吸系统 

分 类 号：R827.12[医药卫生—临床医学]