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目的探讨交通相关PM_(2.5)能否改变iNOS基因DNA启动子区甲基化水平从而调控大鼠哮喘加重中NO的释放水平。方法选择45只雄性SD大鼠,用卵清蛋白致敏、激发建立大鼠哮喘模型,在激发阶段以气管滴注染毒不同剂量(1.5、6、24 mg/kg)PM_(2.5),构建PM_(2.5)刺激的哮喘加重模型。实验大鼠随机分为5组,分别为生理盐水对照组、哮喘组及PM_(2.5)低、中、高剂量刺激哮喘组。收集支气管肺泡灌洗液(BALF),计数细胞总数和嗜酸性粒细胞所占百分比;制作肺组织病理切片,以HE染色观察病理变化;以甲基化阳性对照标准曲线方法测定iNOS基因DNA启动子区甲基化水平,以qRTPCR测定iNOS mRNA水平,以硝酸还原酶法测定BALF中NO含量。结果哮喘组及PM_(2.5)低、中、高剂量刺激哮喘组大鼠的左肺脏器系数、BALF中嗜酸性粒细胞百分比、肺组织iNOS mRNA水平、BALF中NO含量均高于生理盐水对照组,肺组织iNOS基因DNA启动子区甲基化水平低于生理盐水对照组,差异均有统计学意义(P<0.05);PM_(2.5)低、中、高剂量刺激哮喘组大鼠的BALF中嗜酸性粒细胞百分比、NO含量均高于哮喘组,肺组织iNOS基因DNA启动子区甲基化水平和BALF中细胞总数低于哮喘组,差异均有统计学意义(P<0.05)。大鼠肺组织iNOS基因DNA启动子甲基化水平与iNOS mRNA水平呈负相关(r=-0.556,P=0.003),BALF中NO含量与iNOS mRNA水平呈正相关(r=0.446,P=0.025)。结论交通相关PM_(2.5)可降低iNOS基因DNA启动子区甲基化水平,后者可能参与调控大鼠哮喘加重中NO释放水平。
Objective To investigate whether traffic related PM_ (2.5) can alter the methylation level of iNOS gene DNA promoter to regulate the release of NO in rats with exacerbation of asthma. Methods Forty-five male Sprague-Dawley rats were selected and sensitized with ovalbumin to establish a rat model of asthma. PM-2.5 (1.5, 6, 24 mg / kg) was injected intratracheally PM_ (2.5) -stimulated asthma exacerbation model. The rats were randomly divided into 5 groups: normal saline control group, asthma group and PM 2.5 low, medium and high dose stimulation asthma group. The bronchoalveolar lavage fluid (BALF) was collected and the total number of cells and the percentage of eosinophils were counted. Pathological sections of lung tissue were made and pathological changes were observed by HE staining. The iNOS gene DNA promoter was determined by methylation-positive standard curve The level of iNOS mRNA was determined by qRTPCR. The content of NO in BALF was determined by nitrate reductase method. Results The left lung coefficient, the percentage of eosinophils in BALF, the level of iNOS mRNA in lung tissue and the content of NO in BALF in asthmatic rats and PM 2.5 low, medium and high doses were higher than those in saline control group , The DNA methylation level of iNOS gene promoter in lung tissue was lower than that in saline control group (P <0.05), PM_ (2.5) low, middle and high doses of BALF in asthmatic rats The percentage of eosinophils and the content of NO in asthma group were higher than those in asthma group. The DNA methylation level of iNOS gene in lung tissue and the total cell number in BALF were lower than those in asthma group (P <0.05). The promoter methylation level of iNOS gene was negatively correlated with iNOS mRNA level in lung tissue (r = -0.556, P = 0.003). There was a positive correlation between NO level and iNOS mRNA level in BALF (r = 0.446, . Conclusion Traffic-related PM 2.5 can reduce the methylation level of DNA promoter in iNOS gene, which may be involved in the regulation of NO release in asthmatic rats.