臭氧氧化应激对小鼠急性过敏性气道炎症所致气道高反应性和黏液分泌的影响

Effects of ozone oxidative stress on the airway hyperresponsiveness and mucus production in mice with acute allergic airway inflammation

目的 探讨臭氧对过敏性支气管哮喘（简称哮喘）小鼠气道高反应性（AHR）、气道炎症和黏液分泌等病理生理特征的影响.方法 将28只小鼠按随机数字表法随机分为健康对照组、臭氧对照组、哮喘组和臭氧组,每组7只.哮喘组和臭氧组采用卵清白蛋白（OVA）致敏激发建模,健康对照组采用生理盐水;臭氧对照组和臭氧组在对应时间点连续暴露于2.0 ppm臭氧3h,对照组暴露于过滤空气3h.暴露后,检测各组小鼠肺功能、BALF炎症细胞计数、上清液炎症因子及肺组织病理,RT-PCR检测肺组织炎症因子及黏液基因表达,比色法检测肺组织氧化代谢物水平等.多组间比较采用单因素方差分析,两两比较采用LSD检验.结果 臭氧组小鼠的LogPC100Penh（与AHR呈反比）低于哮喘组（分别为0.22±0.09和0.50±0.19,t＝-3.06,P=0.006）.与哮喘组比较,臭氧组BALF中性粒细胞数量[分别为（0.80±0.21）× 10^3/L和（0.15 ±0.06）×10^3/L,=3.63,P=0.019]、低分子量透明质酸[分别为（111±17） μg/L和（35±18） μg/L,t =5.12,P =0.000]、肿瘤坏死因子-α（TNF-α）[分别为（155±30） μg/L和（86±19） μg/L,t=2.15,P=0.044]、IL-13[分别为（65±11） μg/L和（33±20） μg/L,t=2.95,P=0.008]均较高.臭氧组血管周围炎症评分（2.79±0.10）高于哮喘组（1.92±0.23,t=3.91,P=0.000）.与哮喘组比较,臭氧组小鼠肺组织TNF-α mRNA（分别为7.0±1.5和3.57±1.20,t=2.65,P=0.014）、CXCL-1 mRNA（分别为7.0±1.1和2.5±1.0,t=4.12,P=0.000）表达上调,臭氧组IL-17 mRNA的表达（28.8±2.0）明显高于臭氧对照组（4.5±2.0,t=12.4,P=0.000）及哮喘组（16.4±1.7,t =6.33,P=0.000）.臭氧组PAS染色阳性细胞的百分比以及单位面积的气道上皮表面潴留黏液的容积均高于哮喘组[分别为（76±9）％和（56±14）％,t=8.14,P＝0.000;（721±87） nl/mm^2和（272±185） nl/mm^2,=5.78,P=0.000].臭氧组肺组织MUC5ac mRNA表达显著高于哮喘组（分别为15.4±2.6和7.0±1.9,=4.37,P=0.002）及臭氧对照组（0.60±0.18,t =8.27,P=0.000）.臭氧组小鼠气道上皮细胞密度低于哮喘组（分别为82±22和116±15,t=-10.1,P=0.000）而BALF白蛋白水平则高于该组[（45±6）g/L和（33±4）g/L,t=3.89,P=0.001].结论 臭氧吸入加重过敏性哮喘小鼠的AHR、气道及肺部炎症、气道上皮损伤以及黏液分泌.

Objective To explore the impact of ozone on the airway hyperresponsinveness （AHR）,airway inflammation and mucus production in an allergic asthma mouse model.Methods Twenty-eight female BALB/c mice were randomly divided into 4 equal groups：healthy control,ozone control,asthma model,and ozone intervention.For asthma model establishing,the mice were sensitized and challenged with ovalbumin,while the controls received saline.For ozone exposure,the mice were exposed to 2.0 ppm ozone for 3 hrs,while the control treatment group exposed to filtered air for 3 hrs.Some measurements were performed 24 hrs after the exposure,including AHR,pulmonary inflammation,mucus secretion,epithelial barrier function,and the level of oxidant stress.Results Compared with the asthma model group,mice in the ozone intervention group exhibited lower LogPC100Penh （0.22 ±0.09 vs 0.50 ±0.19,t =3.06,P =0.006）,higher bronchoalveolar lavage （BAL） neutrophil numbers [（0.80 ± 0.21） × 10^3/L vs （0.15 ± 0.06） × 10^3/L,t =3.63,P =0.019] and BAL concentration of lower molecular weight hyaluronan （LMW-HA） [（111 ±17） μg/Lvs （35 ±18） μg/L,t=5.12 P=0.000],TNF-α[（155 ±30） μg/Lvs （86±19） μg/L,t=2.15,P=0.044] and IL-13[（65±11） μg/Lvs （33 ±20） μg/L,t=2.95,P=0.008].Mice in the ozone intervention group showed higher lung pathological inflammation score （2.80 ± 0.10 vs 1.92 ±0.23,t =3.91,P =0.000） and upregulated expressions of TNF-α mRNA （7.0 ± 1.5 vs 3.57±1.20,t=2.65,P=0.014）,CXCL-1 mRNA （7.0±1.1 vs2.5±1.0,t=4.12,P=0.000） and IL-17 mRNA （28.8 ±5.2 vs 16.4 ±4.4,t =6.33,P =0.000）.Ozone exposure on the asthmatic mice also caused higher percentage of PAS positive-staining epithelial cells [（76.2 ± 8.7） % vs （55.8 ± 14.4） %,t =8.14,P =0.000] and higher epithelial surface mucus volume [（721 ± 584） nl/mm^2 vs （272 ± 185） nl/mm^2,t =5.78,P =0.000] as well as the MUC5ac mRNA expression （15.4 ±4.6 vs 7.0 ± 1.9,t =4.37,P =0.000）.Besides,ozone exposure in the asthma model decreased epithelial cell density （82 ± 22 vs 116 ± 15,t =-10.1,P =0.000）,while increased the BAL concentration of albumin [（45 ±6） g/L vs （33 ± 4） g/L,t =3.89,P =0.001].Conclusions Ozone exaggerates AHR and pulmonary inflammation,and causes damages in epithelial cells and promotes the production of epithelial mucus.