汽油发动机尾气全组分气-液界面暴露致A549细胞的毒性效应  

Toxic effects of whole-component of gasoline engine exhaust on A549 cells by air-liquid interface exposure

作　　者：裴梦娟 晋瑜 宾萍[1] 周玉梅 李国梁 鱼涛[1] 张雪艳[1] 赵玮[1] 郑敏[1] 李斌[1] PEI Meng-juan;JIN Yu;BIN Ping;ZHOU Yu-mei;LI Guo-liang;YU Tao;ZHANG Xue-yan;ZHAO Wei;ZHENG Min;LI Bin(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：《毒理学杂志》2025年第1期11-17,26,共8页Journal of Toxicology

基　　金：国家自然科学基金(82073596,81472955)。

摘　　要：目的应用气-液界面(air-liquid interface,ALI)暴露系统评价汽油发动机尾气(gasoline engine exhaust,GEE)全组分暴露致A549细胞的毒性效应。方法以洁净空气作为对照组,以3个GEE浓度(V_(GEE)∶V_(洁净空气)=1∶10、1∶5和未稀释GEE)作为暴露组,分别设为低、中和高剂量组。利用ALI暴露系统,以10 ml/min作为暴露流量,在37℃水浴条件下,对A549细胞进行1 h的持续暴露。采用CCK-8(Cell Counting Kit-8)检测法、乳酸脱氢酶(lactate dehydrogenase,LDH)释放法、中性红摄入法(neutral red uptake,NRU)和噻唑蓝(MTT)法检测A549细胞的相对存活率。同时采用DCFH-DA探针检测细胞内活性氧(reactive oxygen species,ROS)生成量,Annexin V-FITC细胞凋亡检测试剂盒检测A549细胞凋亡及坏死率,酶联免疫吸附法(Enzyme Linked ImmunoSorbent Assay,ELISA)测定细胞培养上清液中白介素-1β(Interleukin-1β,IL-1β)、IL-6和IL-8水平。结果与洁净空气组相比,高剂量GEE组在CCK-8法、LDH释放法和MTT法检测中细胞存活率明显降低,差异有统计学意义(P<0.05);低、中和高剂量组中A549细胞胞内ROS生成量均明显升高,差异有统计学意义(F=19.799,P<0.05);中和高剂量组中细胞早期凋亡率明显升高,差异有统计学意义(F=7.072,P<0.05);低剂量组中A549细胞IL-1β和IL-6分泌水平明显升高,差异有统计学意义(P<0.05);未发现其余GEE暴露组的指标与洁净空气组比较的差异有统计学意义(P>0.05)。结论GEE的毒性效应主要表现为引起A549细胞的细胞膜损伤、线粒体损伤、氧化应激、细胞凋亡及促炎症效应,且随着染毒浓度的升高,GEE对A549细胞的毒性增强。Objective To evaluate the toxic effects of whole-component of gasoline engine exhaust(GEE)on A549 cells by an air-liquid interface(ALI)exposure system.Methods Clean air was used as the control,and three GEE concentrations(V_(GEE)∶V_(clean air)=1∶10,1∶5,and undiluted GEE)were used as the exposure,which were set as low-,middle-,and high-dose groups,respectively.The A549 cells were continuously exposed to clean air or GEE for 1 h at a flow rate of 10 ml/min in 37℃using the ALI exposure system.The relative survival rates of A549 cells were detected by the Cell Counting Kit-8(CCK-8)assay,lactate dehydrogenase(LDH)release assay,neutral red uptake(NRU)assay,and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT)assay.Meanwhile,production of intracellular reactive oxygen species(ROS),apoptosis and necrosis,and the levels of interleukin-1β(IL-1β),IL-6,and IL-8 in the cell culture supernatant were measured using a DCFH-DA probe,Annexin V-FITC apoptosis detection kit,and the enzyme-linked immunosorbent assay(ELISA),respectively.Results Compared with the clean air group,the cell survival rates detected by the CCK-8 assay,LDH release assay and MTT assay in the high-dose GEE group were significantly decreased,and the differences were statistically significant(P<0.05).The intracellular ROS production of A549 cells in the low-,middle-,and high-dose groups were statistically increased,and the differences were all statistically significant(F=19.799,P<0.05).The early apoptosis rates of A549 cells in the middle-and high-dose groups were significantly increased,and the differences were statistically significant(F=7.072,P<0.05).The secretion levels of IL-1βand IL-6 of A549 cells in the low-dose group were significantly increased,and the differences were statistically significant(P<0.05).No statistically significant differences were found between the remaining GEE exposure groups and the clean air group(P>0.05).Conclusion Toxic effects of whole-component gasoline engine exhaust on A549 cells could be manifested

关 键 词：汽油发动机尾气 气-液界面 A549细胞 细胞毒性 

分 类 号：R114[医药卫生—卫生毒理学] R99[医药卫生—公共卫生与预防医学]