刘艳菊1,2,,,
郭青云1,
杨峥1,
钟震宇1,
李俊芳1,
单云芳1,
孟玉萍1,
王欣欣2
1. 北京麋鹿生态实验中心, 北京 100076;
2. 北京市科学技术研究院分析测试研究所(北京市理化分析测试中心), 北京 100089
作者简介: 程志斌(1983-),男,硕士,副研究员,研究方向为环境毒理和动物生态学,E-mail:czb@milupark.org.cn.
通讯作者: 刘艳菊,liuyanju@hotmail.com ;
基金项目: 国家自然科学基金资助项目（41475133）中图分类号: X171.5
Subchronic Exposure to Diesel Engine Exhaust Induced Pulmonary Inflammation Response, Oxidative Stress and Cell Apoptosis in Mice
Cheng Zhibin1,Liu Yanju1,2,,,
Guo Qingyun1,
Yang Zheng1,
Zhong Zhenyu1,
Li Junfang1,
Shan Yunfang1,
Meng Yuping1,
Wang Xinxin2
1. Beijing Milu Ecological Research Center, Beijing 100076, China;
2. Institute of Analysis and Testing, Beijing Academy of Science and Technology(Beijing Center for Physical and Chemical Analysis), Beijing 100089, China
Corresponding author: Liu Yanju,liuyanju@hotmail.com ;
CLC number: X171.5
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摘要:柴油车尾气暴露对动物呼吸系统和其他器官造成损害，其长期或短期暴露的健康风险受到关注，然而柴油车尾气长期暴露引起的生物毒性机制仍不清楚。本研究通过自制暴露箱模拟柴油车尾气环境，研究了每日不同暴露时间条件下（0.5、1和2 h），长期暴露（95 d）对雄性小鼠肺组织炎症反应、氧化损伤和细胞凋亡的作用，探讨长期暴露于柴油车尾气对成年雄性小鼠肺组织损伤的生物毒性及机制，评估柴油车尾气的毒性效应。结果表明：（1）亚慢性暴露箱内柴油车尾气PM2.5中，有机碳（OC）浓度最高，占所测PM2.5总化学物质浓度的51.95%；其次是元素碳（EC），占45.78%；再次是阴离子和阳离子，分别占1.29%和0.95%；暴露箱内NO2浓度为3.705 mg·m-3，CO浓度为104.087 mg·m-3，均超过《环境空气质量标准》（GB3095—2012）的标准。（2）组织病理观察结果表明，3个柴油暴露组肺充血均比对照组严重，大量淋巴细胞和中性粒细胞浸润，肺泡间质增生明显，损伤程度随时间呈梯度加重。（3）TUNEL染色实验结果显示，1 h柴油暴露组的细胞凋亡率显著高于对照组和0.5 h柴油暴露组。（4）与对照组相比，1 h柴油暴露组和2 h柴油暴露组的乳酸脱氢酶（LDH）均显著升高，说明柴油车尾气亚慢性暴露引起小鼠肺部炎症损伤；1 h柴油暴露组和2 h柴油暴露组的总抗氧化能力（T-AOC）活性、2 h柴油暴露组的谷胱甘肽过氧化物酶（GSH-Px）活性均显著下降，说明柴油车尾气亚慢性暴露可引起小鼠肺组织氧化应激。（5）与对照组相比，3个柴油暴露组的Bax和Bcl-2的表达水平及Bcl-2/Bax指数虽然无显著性差异，但柴油暴露组Bax和Bcl-2表达及Bcl-2/Bax指数均异常。这说明，柴油车尾气亚慢性暴露会导致小鼠肺组织病理损伤，且损伤程度随着暴露时间的增加而增加；导致细胞凋亡率升高，且存在时间效应。进一步探讨机制发现，柴油车尾气亚慢性暴露会导致小鼠肺组织LDH活性显著异常，T-AOC、GSH-Px的活性显著降低，造成细胞凋亡，引起凋亡蛋白Bax和Bcl-2表达水平出现异常；化学成分分析表明，这可能与柴油车尾气中高浓度NO2、CO和多环芳烃（PAHs）等有毒气体及其细颗粒物中高浓度的OC、EC等污染物的作用有关。
关键词: 柴油车尾气/
小鼠/
亚慢性暴露/
炎症反应/
氧化损伤/
细胞凋亡
Abstract:Exposure to diesel engine exhaust (DEE) causes damage to the animal respiratory system and other organs, and the health risks of long-term or short-term exposure are of concern. However, the biotoxicology mechanism caused by long-term exposure to DEPs remains unclear. This study aims to explore the biotoxicity and mechanism of long-term exposure to DEPs on lung tissue in adult male mice and to assess the toxic effects of diesel vehicle exhaust. We exposed adult male mice to DEE via homemade chambers over a period of 95 d and assessed the effects of lung inflammation, oxidative damage and cell death (apoptosis). We created three exposure groups (DEEG), with mice in each group being exposed for 0.5, 1 and 2 h respectively each day, and one blank control group (BCG). The results show that: (1) Among the chemical species of PM2.5 analyzed in the subchronic exposure chamber, the organic carbon (OC) concentration is the highest, accounting for 51.95% of the total mass of PM2.5, followed by elemental carbon (EC), accounting for 45.78%, and then anion and cation, accounting for 1.29% and 0.95%, respectively. Concentrations of NO2 and CO are 3.705 mg·m-3 and 104.087 mg·m-3 respectively, both exceeding the Ambient Air Quality Standards (GB3095—2012) in China. (2) Histopathological observation showed that the pulmonary congestion was more severe in the three DEEGs than that of the BCG, including a large number of lymphocytes and neutrophils infiltrated, and obvious alveolar interstitial hyperplasia, with the degree of damage increased gradually with time. (3) TUNEL staining showed that the apoptotic index (AI) of the 1 h DEEG was significantly higher than that of the BCG and 0.5 h DEEG. (4) Compared with the BCG, lactate dehydrogenase (LDH) levels in the 1 h DEEG and 2 h DEEG significantly increased, indicating that exposure to diesel exhaust had induced mouse lung inflammation damage. In addition, total antioxidant capacity (T-AOC) activity in 1 h DEEG and 2 h DEEG and glutathione peroxidase (GSH-Px) activity in 2 h DEEG significantly declined, indicating that exposure to diesel exhaust had induced mouse lung oxidative stress. (5) Though there were no significant difference between DEEGs and BCG, the expression levels of Bax and Bcl-2 as well as the Bcl-2/Bax ratios were distinct in the three DEEGs. We concluded that subchronic exposure to diesel engine exhaust does lead to pathological injury of lung tissue in mice, and the degree of injury increases with exposure time, which leads to higher rates of apoptosis. Further investigation identified several mechanisms of pathology, including significantly elevated LDH activity in mouse lung tissue, significantly decreased activity of T-AOC, GSH-Px, resulting in cell apoptosis, and abnormal expression of apoptotic protein Bax and Bcl-2. Chemical composition analysis shows that this may be related to the high concentration of toxic gases such as NO2, CO, polycyclic aromatic hydrocarbons (PAHs) and high concentration of OC, EC and other pollutants in fine diesel exhaust particles.
Key words:diesel engine exhaust/
mice/
subchronic exposure/
inflammatory factor/
oxidative stress/
apoptosis.
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