慢性哮喘小鼠气道重塑情况观察及发生机制探讨

Observation of airway remodeling in mice with chronic asthma and its mechanism

目的 观察慢性哮喘小鼠气道重塑情况,并探讨其可能发生机制。方法 取小鼠16只,并随机分为哮喘组和对照组(各8只)。哮喘组制备慢性哮喘模型,于第0和7天腹腔注射致敏液,第2~12周用激发液雾化激发,每2周2次,连续2 d,每次30 min;对照组采用PBS替代致敏液和激化液;比较两组小鼠肺支气管厚度变化及支气管平滑肌厚度、基底膜下胶原沉积厚度和肺组织miR-3162-3p、β-catenin蛋白、LC3B-Ⅱ/Ⅰ蛋白相对表达量。取对数生长期哮喘小鼠气道平滑肌细胞,分成两组,对照组加入PBS,Mimic组转染微小RNA 3162-3p模拟物(miR-3162-3p mimic),比较两组LC3B-Ⅱ/Ⅰ蛋白相对表达量。结果 对照组马松三色染色结果显示肺泡间隔匀称、支气管结构清晰正常;哮喘组肺组织结构不均匀,偶见支气管上皮细胞疏散,支气管平滑肌肌层肥厚,上皮下纤维化明显增多,基底膜增厚,支气管周围蓝色胶原沉积加重。与对照组比较,哮喘组支气管平滑肌厚度、基底膜下胶原沉积厚度增加(P均<0.05)。与对照组比较,哮喘组肺组织miR-3162-3p、LC3B-Ⅱ/Ⅰ蛋白表达升高,β-catenin蛋白表达降低(P均<0.05)。与对照组比较,Mimic组气道平滑肌细胞LC3B-Ⅱ/Ⅰ蛋白表达增加(P<0.05)。结论 慢性哮喘小鼠肺支气管平滑肌显著增厚,基底膜下的胶原纤维明显沉积,其发生机制可能是miR-3162-3p通过下调β-catenin蛋白表达促进气道平滑肌细胞自噬水平上调。

Objective To observe the airway remodeling in mice with chronic asthma and to explore the possi-ble mechanism.Methods Sixteen mice were randomly divided into the asthma group and control group,with eight ineach group. The chronic asthma model was prepared in the asthma group:mice were injected intraperitoneally with sensiti-zation liquid on day 0 and 7,and then were challenged with atomized challenge liquid from week 2 to 12(twice every 2weeks for 2 consecutive days,30 minutes each time). In the control group,PBS was used instead of sensitization and chal-lenge liquid. The changes of pulmonary bronchial thickness,thickness of bronchial smooth muscle,thickness of collagendeposition under basement membrane,and the relative expression levels of miR-3162-3p,β-catenin protein and LC3B-Ⅱ/Ⅰproteins were compared. The airway smooth muscle cells of asthmatic mice in the logarithmic growth phase were dividedinto two groups. PBS was added to the control group,and miR-3162-3p mimic was transfected into the Mimic group,andthen the relative expression levels of LC3B-Ⅱ/Ⅰprotein in the two groups were compared.Results Masson trichromestaining results in the control group showed that the alveolar septa were evenly distributed and the bronchial architecturewas clear and normal. The lung tissue structure was uneven,bronchial epithelial cells were occasionally evacuated,thebronchial smooth muscle layer was hypertrophic,the subepithelial fibrosis significantly increased,the basement membranewas thickened,and the deposition of blue collagen around the bronchus was aggravated in the asthma group. Comparedwith the control group,the thickness of bronchial smooth muscle and the thickness of collagen deposition under base-ment membrane in the asthma group significantly increased(all P<0. 05). Compared with the control group,the expres-sion levels of miR-3162-3p and LC3B-Ⅱ/I proteins significantly increased whereas the β-catenin protein expression inlung tissue of asthmatic mice decreased. The expression of LC3B-Ⅱ/I protein in the asthma group was significantly higherthan that of the control group(P<0. 05).Conclusion The bronchial smooth muscle is obviously thickened and the colla-gens under basement membrane are significantly deposited. The occurrence mechanism may be that miR-3162-3p canpromote the up-regulation of autophagy level of airway smooth muscle cells by down-regulating the β-catenin proteinexpression.