李文燕 郭亚楠 郑昕蕊 张红霞 于丽 江洪

[摘要] 目的 探讨孕期PM2.5暴露影响子代大脑发育中HMGB1/NF-κB p65通路的作用。 方法 40只昆明小鼠孕鼠随机分5组，每组8只。气管滴注法向对照组注30 μL PBS，PM2.5低、中、高剂量组各注0.25920、1.56695、3.45600 μg/μL PM2.5混悬液30 μL，孕期每3天注1次，共7次。空白组无处理。ELISA法检测子代大脑皮层肿瘤坏死因子-α（TNF-α）、γ-干扰素（IFN-γ）、高迁移率族蛋白1（HMGB1）、核因子κB p65（NF-κB p65）浓度；Western blot、RT-PCR法检测HMGB1、NF-κB p65蛋白及mRNA变化。 结果 ELISA示与对照组相比，高、中剂量组IFN-γ、NF-κB p65升高而HMGB1下降（P < 0.05），高剂量组TNF-α升高（P < 0.05）。Western blot示高、中剂量组与对照组相比，HMGB1下降且NF-κB p65上升（P < 0.05）。PCR示HMGB1 mRNA无变化，高剂量组NF-κB p65 mRNA高于对照组（P < 0.05）。 结论 孕期PM2.5暴露使子代鼠大脑皮层产生炎症，HMGB1/NF-κB通路可能起重要作用。

[关键词] 大脑皮层；PM2.5；γ-干扰素；肿瘤坏死因子-α；高迁移率族蛋白1；核因子κB p65；子代鼠

[中图分类号] R715.3 [文献标识码] A [文章编号] 1673-7210（2018）04（a）-0022-04

[Abstract] Objective To investigate the mechanism of HMGB1/NF-κB signal pathway affecting cerebral development of offspring after exposure to PM2.5 during pregnancy. Methods Fourty pregnant Kunming mice were randomly divided into 5 groups， 8 rats in each group. PM2.5 low-dose group， medium-dose group and high-dose group were instilled 30 μL PM2.5 suspension of 0.25920， 1.56695， 3.45600 μg/μL respectively by improved technique for rapid intratracheal instillation. The control group was instilled PBS of the same volume. The blank group received no treatments. Mice were instilled tracheally once every 3 days for 7 times during pregnancy. ELISA was used to measure the levels of TNF-α， IFN-γ， HMGB1 and NF-κB p65 in the cerebral cortex of offspring mice. Western blot and RT-PCR were used to detect the protein and mRNA expression of HMGB1 and NF-κB p65. Results ELISA showed that compared with the control group， the levels of IFN-γ， NF-κB p65 and HMGB1 in the high and middle dose groups were significantly lower （P < 0.05）； high-dose group TNF-α increased （P < 0.05）. Western blot showed that HMGB1 decreased and NF-κB p65 increased in the high and middle dose groups compared with the control group （P < 0.05）. PCR showed HMGB1 mRNA unchanged； high-dose group NF-κB p65 mRNA was higher than the control group （P < 0.05）. Conclusion PM2.5 exposure during pregnancy may cause inflammation in the cerebral cortex of offspring mice， and HMGB1/NF-κB pathway may play an important role.

[Key words] Cerebral cortex； PM2.5； IFN-γ； TNF-α； HMGB1； NF-κB p65； Offspring mice

大氣细颗粒物（AD≤2.5 μm，PM2.5）成分复杂，可吸附细菌、病毒、重金属等。PM2.5通过呼吸进入机体经血液循环及胎盘影响孕妇和新生儿[1]。炎性反应是孕期PM2.5暴露影响子代认知的首要机制[2]。本课题组前期发现孕期PM2.5暴露使子代鼠心、脾、肝等脏器结构紊乱，出现炎症等病理改变[3]。目前关于PM2.5对子代脑发育影响机制的研究尚少。本研究通过检测孕期PM2.5暴露后子代鼠大脑皮层TNF-α、IFN-γ、HMGB1、NF-κB p65变化，评估孕期空气污染对胎儿神经系统的毒性作用。

1 材料与方法

1.1 实验动物与模型制备

雌性昆明小鼠40只，6～8周龄，体重（34±2）g，购自青岛动物实验中心，生产许可证号：SCXK（鲁）20140001。按动物伦理学标准处理动物。室温25℃，自由饮水、进食。雌雄比2∶1合笼，次日查有阴栓者记为孕第1天。孕鼠随机分为空白组、对照组及PM2.5低、中、高剂量组，每组8只。空白组无处理；对照组注30 μL PBS；PM2.5低、中、高剂量组各注30 μL浓度为0.25920、1.56695、3.45600 μg/μL的PM2.5混悬液，采用气管滴注法孕期每3天（即孕第1、4、7、10、13、16、19天）滴注，共7次。

1.2 PM2.5的采集和處理