气-液界面染毒系统中不同暴露流量对汽油发动机尾气致肺细胞氧化应激相关蛋白表达的影响  

Effects of different exposure flow rates in air-liquid interface exposure systems on the expression of oxidative stress-related proteins in lung cells induced by gasoline engine exhaust

作　　者：李国梁 鱼涛[1] 张雪艳[1] 裴梦娟 晋瑜 李斌[1] 宾萍[1] LI Guo-liang;YU Tao;ZHANG Xue-yan;PEI Meng-juan;JIN Yu;LI Bin;BIN Ping(National Institute of Occupational Health and Poison Control,Chinese Center for Disease Control and Prevention,Beijing 100050,China)

机构地区：[1]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：《环境卫生学杂志》2024年第5期367-377,共11页JOURNAL OF ENVIRONMENTAL HYGIENE

基　　金：国家自然科学基金面上项目(82073596,81472955)。

摘　　要：目的采用气-液界面(air-liquid interface,ALI)培养和暴露系统,探索二轮摩托车来源的汽油发动机尾气(gasoline engine exhaust,GEE)对人肺细胞氧化应激相关蛋白表达的影响。方法实验分为空白对照组、洁净空气组、GEE组。除了空白对照组,其余各组采用气-液界面体外染毒技术,分别在暴露流量为10、15和25 mL/min,37℃水浴条件下,对生长在气-液界面插件多孔膜上的人支气管上皮细胞BEAS-2B和人肺癌A549细胞持续暴露60 min。利用Cell Counting Kit-8(CCK-8)法评价细胞相对存活率。采用2′,7′-二氯荧光黄双乙酸盐(2′,7′-dichloro-dihydro-fluoresceindiacetate,DCFH-DA)探针检测细胞内活性氧(reactive oxygen species,ROS)水平。Annexin V-FITC细胞凋亡检测试剂盒检测GEE对BEAS-2B和A549细胞凋亡及坏死率的影响。蛋白质免疫印迹法检测全细胞裂解物中核因子红细胞2相关因子2(nuclear factor erythroid-2-related factor 2,Nrf2)和血红素氧合酶1(heme oxygenase-1,HO-1)蛋白表达水平。结果在10、15和25 mL/min三种暴露流量下,随着暴露流量增大,GEE暴露组的2种细胞相对存活率逐渐降低、胞内ROS生成量和细胞凋亡率逐渐升高,且与洁净空气组相比,存在统计学上的显著性差异(P<0.05)。BEAS-2B和A549细胞相对存活率、胞内活性氧水平、早期凋亡率、晚期凋亡及坏死率和总凋亡率在GEE暴露和暴露流量上存在主效应,且GEE暴露与暴露流量存在交互效应(P<0.01)。在GEE暴露组,BEAS-2B细胞Nrf2和HO-1蛋白表达下降,A549细胞Nrf2和HO-1蛋白表达水平升高。BEAS-2B细胞Nrf2蛋白表达仅在暴露流量主效应上差异有统计学意义(P<0.01)。BEAS-2B细胞HO-1蛋白表达仅在GEE主效应上差异有统计学意义(P<0.01)。A549细胞Nrf2蛋白表达在GEE主效应、暴露流量主效应和交互效应上差异均有统计学意义(P<0.05)。A549细胞HO-1蛋白表达则在GEE主效应、暴露流量主效应和交互效应上差异均无统Objective To investigate the effect of gasoline engine exhaust(GEE)from two-wheel motorcycles on the expression of oxidative stress-related proteins in human lung cells using an air-liquid interface(ALI)culture and exposure system.Methods There were three groups in the experiment,including blank control group,clean air group,and GEE group.Except for the blank control group,the in vitro ALI exposure technology was used in the other groups.Human bronchial epithelial cells BEAS-2B and human lung cancer cells A549 cultured on porous membranes in the ALI were exposed to clean air or GEE at the exposure flow rate of 10,15,and 25 mL/min for 60 min in a 37℃water bath,respectively.The cell relative viability was evaluated using the Cell Counting Kit-8.The level of reactive oxygen species(ROS)was measured by a 2′,7′-dichloro-dihydro-fluoresceindiacetate probe.Annexin V-FITC apoptosis detection kit was used to determine the apoptosis and necrosis rates of BEAS-2B and A549 cells after GEE treatment.The expression levels of nuclear factor erythroid-2-related factor 2(Nrf2)and heme oxygenase1(HO-1)proteins in whole cell lysates were determined by Western blotting.Results Under the three flow rates of 10,15,and 25 mL/min,the cell relative viability of BEAS-2B and A549 cells in the GEE group decreased gradually,and the intracellular ROS generation and apoptosis rate increased gradually with the increasing flow rate;there were significant differences compared with the clean air group(P<0.05).There were main effects of GEE exposure and exposure flow on the cell relative viability,intracellular ROS generation,early apoptosis rate,late apoptosis and necrosis rate,and total apoptosis rate of BEAS-2B and A549 cells,and there was an interaction effect between GEE exposure and exposure flow(P<0.01).GEE exposure resulted in decreased expression of Nrf2 and HO-1 proteins in BEAS-2B cells and increased expression of Nrf2 and HO-1 proteins in A549 cells.The expression of Nrf2 protein in BEAS-2B cells was significantly different only i

关 键 词：汽油发动机尾气 气-液界面 肺细胞毒性 核因子红细胞2相关因子2(Nrf2) 血红素氧合酶1(HO-1) 

分 类 号：R114[医药卫生—卫生毒理学]