芳香烃受体在雏鸡肺呼吸起始期吸入柴油机尾气致心肺毒性中的作用机制

Roles of aryl hydrocarbon receptors in diesel exhaust inhalation-induced cardiopulmonary toxicities during initiation of pulmonary respiration in chicken

[背景]由于研究模型的限制,肺呼吸起始期柴油机尾气的健康效应及机制研究较少,亟待阐明相关效应及机制,以期在生命早期采取保护性措施。[目的]应用团队首创的鸡胚气室吸入染毒模型,评价柴油机尾气在雏鸡肺呼吸起始期吸入暴露所引起的健康效应,并以特异性芳香烃受体抑制剂为工具,探讨芳香烃受体信号通路在其中所发挥的作用。[方法]受精鸡蛋按蛋重随机分为5组,每组15只鸡蛋:对照组、空气对照组、芳香烃受体抑制剂(PDM2)组、柴油机尾气组、柴油机尾气+芳香烃受体抑制剂(PDM2)组,鸡胚正常孵化至17 d后(ED17)以气室注射的方法应用芳香烃受体抑制剂,在18~19 d(ED18-19)行气室内柴油机尾气吸入染毒,空气对照组18~19 d(ED18-19)以气室吸入法吸入洁净的空气,对照组则无任何处理,继续孵化至出壳,26只雏鸡在孵化24 h内进行心电测量后处死,以组织病理学方法检测心肺毒性,Western blotting检测芳香烃受体通路信号分子细胞色素P450家族1A1(CYP1A1)及纤维化相关通路信号分子磷酸化SMAD同源物2(pSMAD2)表达水平的改变。其余29只雏鸡继续饲养至两周龄后进行同样处理。[结果]肺呼吸起始期气室吸入柴油机尾气染毒后,在孵化24 h内的雏鸡观测到右心室壁的增厚(220.3%,与对照组相比,下同)及心率的升高(17.4%),虽然肺组织尚未出现明显的纤维化改变,但CYP1A1蛋白表达水平升高81.3%及SMAD2的蛋白磷酸化水平升高71.6%。芳香烃受体抑制剂PDM2预处理有效地令上述效应幅度降低。饲养至两周龄后,肺呼吸起始期柴油机尾气吸入染毒仍令雏鸡右心室壁增厚(339.3%)、心率升高(18.9%),同时Masson染色结果提示显著的肺组织纤维化。芳香烃受体抑制剂PDM2预处理同样有效地令上述效应幅度降低。两周龄雏鸡肺组织中,肺呼吸起始期柴油机尾气暴露对CYP1A1表达差异无统计学意义,但SMAD2磷酸化水平出现下调。与此同时,芳香烃受体抑制剂PDM2处理也能降低两周龄雏鸡肺组织SMAD2磷酸化水平。[结论]在鸡胚气室吸入模型中,肺呼吸起始期柴油机尾气吸入暴露可导致持久的心肺损伤,其机制可能与芳香烃受体信号通路对pSMAD2的调控作用有关。

[Background]Due to the limited availability of established research models,very few studies addressed the health effects and underlying mechanisms following exposure to diesel exhaust during the initiation of pulmonary respiration.It is highly demanded to elucidate such health effects and underlying mechanisms,so as to exert protective measures during the early stages of life.[Objective]To evaluate the health effects of diesel exhaust very-early-in-life inhalation in hatchling chicken with a novel chicken embryo air cell inhalation exposure model,and to explore the potential roles of aryl hydrocarbon receptor signaling pathways in the observed effects with a specific aryl hydrocarbon receptor inhibitor.[Methods]Fertilized chicken eggs were assigned into five groups randomly(15 eggs per group):control group,air control group,aryl hydrocarbon receptor inhibitor(PDM2)group,diesel exhaust group,and diesel exhaust+aryl hydrocarbon receptor inhibitor(PDM2)group.Fertilized eggs were incubated with standard procedure.At embryonic day 17(ED17),aryl hydrocarbon receptor inhibitor was administered to the corresponding animals.During embryonic day 18-19(ED18-19),chicken embryos were exposed to diesel exhaust via air cell inhalation,then placed back to incubator until hatch.The air control group received clean air infusion during ED18-19,while the control group did not receive any treatment.Within 24 h post-hatch,26 hatchling chickens were anesthetized with sodium pentobarbital,subjected to electrocardiography,and sacrificed to harvest tissue samples of heart and lung.Cardiopulmonary toxicities were evaluated by histopathology,and potential changes in the protein expression levels of aryl hydrocarbon receptor pathway molecule cytochrome P450,family 1,subfamily A,polypeptide 1(CYP1A1)and fibrosis-related pathway molecule phosphorylated SMAD family member 2(pSMAD2)were assessed by Western blotting.The remaining 29 hatchling chickens were reared until two weeks old,and then subjected to identical treatments.[Results]The inhalation exposure to diesel exhaust at initiation of pulmonary respiration resulted in thickened right ventricular wall(by 220.3%relative to the control group,same hereafter)and elevated heart rate(17.4%)in one-day-old hatchling chickens.Although no remarkable fibrotic lesions were observed at this point,the expression levels of CYP1A1 and phosphorylation levels of SMAD2 in the lung tissues significantly increased(by 81.3%and 71.6%,respectively).Such changes were effectively abolished by the aryl hydrocarbon receptor inhibitor PDM2 pretreatment.In the two-week-old animals,the thickened right ventricular wall(by 339.3%)and elevated heart rate(by 18.9%)persisted,and significant fibrotic lesions were observed in the lung tissue samples under Masson staining.Again,the aryl hydrocarbon receptor inhibitor PDM2 pretreatment effectively abolished such changes.In addition,no statistically significant changes in CYP1A1 expression levels were observed in the two-week-old chicken lung samples,and a remarkable down-regulation of SMAD2 phosphorylation was observed.The aryl hydrocarbon receptor inhibitor PDM2 pretreatment independently decreased the phosphorylation levels of SMAD2 in the two-week-old chicken lung samples.[Conclusion]Inhalation exposure to diesel exhaust at initiation of pulmonary respiration could result in persistent cardiopulmonary injury in hatchling chickens,and the underlying mechanism might be associated with the regulation of pSMAD2 by the aryl hydrocarbon receptor signaling pathway.