PM_(2.5)对慢性阻塞性肺疾病小鼠肺组织CFTR表达及功能的影响

Effect of PM_(2.5)on the expression and function of cystic fibrosis transmembrane conductance regulator in lung tissue of chronic obstructive pulmonary disease mice

目的观察慢性阻塞性肺疾病急性加重(AECOPD)小鼠模型中肺功能、病理变化、炎症因子及相关炎性变化,探究气道上皮细胞中囊性纤维化跨膜传导调节体(CFTR)在该过程中的表达。方法本研究为动物实验研究。选取健康雄性C57BL/6小鼠24只,6~8周龄,SPF级,随机分为3组:空白对照组(n=8)、烟草烟雾暴露组(CS组,n=8)、烟草烟雾暴露+细颗粒物(PM_(2.5))干预组(CS+PM_(2.5)组,n=8)。采用烟草烟雾暴露90 d后PM_(2.5)干预3 d的方法建立AECOPD小鼠模型,建模结束后,使用BUXCO小动物有创肺功能仪检测小鼠肺功能;肺组织切片行HE染色评估肺组织病理变化;酶联免疫吸附试验法检测小鼠支气管肺泡灌洗液中炎症因子白细胞介素6(IL-6)、肿瘤坏死因子α、KC及黏蛋白MUC5AC、MUC5B水平,评估小鼠肺部炎症及黏液分泌情况;短路电流技术测定气道上皮组织CFTR功能;提取肺组织蛋白,蛋白质印迹法检测肺组织中CFTR蛋白水平变化。细胞实验:采用烟草烟雾提取物、PM_(2.5)共同处理16HBE细胞,收集细胞培养液并检测其中IL-6和IL-8水平;蛋白质印迹免疫荧光染色检测CFTR的表达;测定PM_(2.5)对细胞内Cl-浓度的影响。结果(1)PM_(2.5)促进烟草烟雾暴露导致的小鼠气道炎症、黏液高分泌、肺功能降低以及气道上皮细胞CFTR表达和功能下调(均P<0.05);(2)PM_(2.5)干预导致烟草烟雾提取物诱导的16HBE细胞中CFTR表达进一步降低,细胞内Cl-浓度进一步增加(均P<0.05)。结论PM_(2.5)诱导的AECOPD小鼠模型中气道上皮细胞CFTR表达及功能进一步下降。

Objective To study the lung functions,pathological changes,inflammatory factors,and related inflammatory changes in acute exacerbation of chronic obstructive pulmonary disease(AECOPD)mice model,and to study the expression of cystic fibrosis transmembrane conductance regulator(CFTR)in airway epithelial cells.Methods This was an animal experimental study.Twenty-four healthy C57B/6 male mice were randomly divided into 3 groups:control group(CTL)(8 mice),CS exposure group(8 mice),and PM_(2.5)and CS exposure group(8 mice).AECOPD mouse model was established by a 90-day CS exposure with 3-day PM_(2.5)challenge.Lung functions were assessed by using Buxco lung function measurement system.Lung tissue slices were subjected to H&E staining for pathological evaluation.The levels of inflammatory factors of interleukin-6(IL-6),tumor necrosis factor-α(TNF-α),keratinocyte chemoattractant(KC),Mucin-5ac(Muc5c),and Muc5b in bronchial alveolar lavage fluid(BLAF)were counted by ELISA,so as to assess the inflammatory and mucus secretion condition.Functions of CFTR in airway epithelial tissue were assessed by a short circuit current technology.Lung tissue proteins were extracted to detect the changes of CFTR protein levels in lung tissue by Western blotting.Cell experiment:16HBE cells were treated by both CS and PM_(2.5).The cell culture medium was collected to assay the IL-6 and IL-8 levels.The expression of CFTR was determined by Western blot immunofluorescence staining.The effects of PM_(2.5)on chloride ion concentration were investigated.Results(1)PM_(2.5)promoted lung inflammation,caused mucus hypersecretion,decreased lung function,and down-regulated expression and function of CFTR in airway epithelial cells induced by CS exposure in mice(all P<0.05).(2)PM_(2.5)decreased the expression of CFTR and increased the concentration of intracellular Cl-in 16HBE cells induced by CSE(all P<0.05).Conclusions The expression and function of CFTR in airway epithelial cells further decrease in the AECOPD mouse model induced by PM_(2.5).