雷帕霉素对哮喘缓解期小鼠模型气道炎症的作用

摘要

目的 探讨雷帕霉素对哮喘缓解期小鼠气道炎症的作用与机制.方法 将50只Balb/c小鼠随机分为5组:正常对照组、哮喘缓解组、哮喘模型组、地塞米松组和雷帕霉素组,每组10只.采用卵蛋白致敏与激发制备哮喘模型,休息3周后,采用卵蛋白再次激发1周,在小鼠休息期间治疗组采用雷帕霉素或地塞米松干预.采用Buxco无创肺功能仪检测气道高反应性;Luminex测定肺泡灌洗液IL-4、IL-5、IL-13、IL-17及INF-γ水平;肺组织HE染色评价观察小鼠肺组织气道炎症;Western blot检测肺组织p-S6、S6、AKT、p-AKT(S473)蛋白含量;流式分析测定肺泡灌洗液CD4+CD25+Foxp3+Treg细胞百分率.结果 与正常组及哮喘缓解组比较:哮喘模型组气道反应性升高(P＜0.01),灌洗液IL-4、IL-5、IL-13及IL-17水平增加(均P＜0.01),肺组织气道炎症评分增高(P＜0.01),肺组织mTORC1信号通路下游蛋白p-S6蛋白增加(P＜0.01),灌洗液Treg细胞比例上升(P＜0.01);与模型组比较:雷帕霉素在乙酰甲胆碱浓度为6.25mg/ml时,可降低Penh值(P＜0.01),同时降低肺泡灌洗液IL-4水平(P＜0.01),并抑制肺组织炎症细胞浸润(P＜0.01),而肺组织mTORC1信号通路下游蛋白p-S6蛋白下降(P＜0.01),但雷帕霉素可降低肺泡灌洗液Treg细胞比例(P＜0.01).结论 在哮喘缓解期应用雷帕霉素,其可通过mTORC1信号通路显著抑制哮喘急性发作时气道炎症;在哮喘缓解期应用雷帕霉素,对控制哮喘再次发作具有积极作用.%Objective To investigate the effects of rapamycin on airway inflammation in mice with asthma during remission.Methods Fifty Balb/c mice were randomly divided into 5 groups:normal control,asthma,asthma in remission,dexamethasone and rapamycin groups with 10 mice in each group.The asthma model was induced by ovalbumin (OVA) sensitization and challenge;after 3-week interval the mice were challenged by OVA again.Dexamethasone,rapamycin or saline was given to 3 groups,respectively during 3-week interval.Airway reactivity was measured by Buxco's non-invasive system.Cytokines IL-4,IL-5,IL-13,IL-17 and INF-γ in bronchoalveolar lavage fluid (BALF) were assessed by Luminex method.Lung tissues were examined for inflammatory cell infiltration.The expression of p-S6,S6,AKT and p-AKT (S473) in lung tissue was examined by Western blot.CD4+CD25+Foxp3+ Treg cells in BALF was assessed by flow cytometry.Results Compared with the normal and remission groups,OVA re-expose significantly increased airway hyperresponsiveness(P＜0.01),elevated IL-4,IL-5,IL-13,IL-17 levels (P＜0.01),and reduced INF-γ in the BALF (P＜0.01);it also markedly increased inflammatory cells in lung tissue,increased Ttreg cells in BALF and decreased expression of p-S6(P＜0.01).Rapamycin significantly reduced airway hyperresponsiveness to aerosolized methacholine at 6.25mg/ml(P＜0.01),inhibited IL-4 level in BALF,and markedly decreased inflammatory infiltration in lung tissues(P＜0.01).Notably,rapamycin significantly inhibited the expression of p-S6;and also Treg cells in BALF were significantly reduced after rapamycin treatment.Conclusion Antiasthmatic effects of rapamycin during remission time are at least partially through mTORC1 signaling pathway,and rapamycin may be used for asthma patients in remission to control asthma attack.