NLRP3炎症小体介导Th17/Treg失衡在哮喘小鼠气道炎症中的作用

Role of NOD-like receptor family pyrin domain containing 3 inflammasome in airway inflammation in asthmatic mice by mediating T helper 17/regulatory T cell imbalance

摘要

目的 探讨NLR家族的Pyrin域蛋白3（NLR family，pyrin domain containing protein 3，NLRP3）炎症小体介导Th17/Treg失衡在哮喘小鼠气道炎症中的作用及其机制。 方法 将32只BALB/c雌性小鼠随机分为正常对照组（NS组）、哮喘模型组（AS组）、MCC950干预组（MC组）及地塞米松组（Dex组）。AS组予卵清白蛋白（OVA）致敏和激发。MC组和Dex组分别予以MCC 950 和地塞米松干预。末次激发24 h后麻醉、放血，制备支气管肺泡灌洗液（bronchoalveolar lavage fluid，BALF）细胞计数和ELISA检测IL-17A、IL-1β、IL-10、IL-18、IL-33、IL-35浓度；流式细胞技术检测小鼠外周血、脾、肺组织CD4细胞IL-17（CD4⁺IL-17⁺） 和CD4⁺CD25⁺CD127^low占CD4⁺细胞比例分别反映辅助性T细胞17（Th17）和调节性T淋巴细胞（Treg）水平；免疫组织化学（immunohistochemistry，IHC）观察肺组织NLRP3、Caspase-1蛋白水平。 结果 与NS组比较，AS组BALF细胞计数、IL-17A、IL-1β、IL-18及IL-33浓度均升高（P<0.01），而IL-10、IL-35浓度降低（P<0.05），外周血、肺及脾组织Th17比例升高（P<0.01），而Treg比例降低（P<0.01），肺组织NLRP3、Caspase-1蛋白水平升高（P<0.01）。与AS组比较，MC组及Dex组BALF细胞计数、IL-17A、IL-1β、IL-18及IL-33浓度均降低（P<0.01），IL-10、IL-35浓度升高（P<0.05）；外周血、肺及脾组织Th17比例降低（P<0.01），Treg比例升高（P<0.01），肺组织NLRP3、Caspase-1蛋白水平降低（P<0.01）。 结论 NLRP3炎症小体可介导Th17/Treg失衡，参与哮喘气道炎症的发生，MCC950可调节Th17/Treg平衡，减轻哮喘气道炎症。

Abstract

Objective To investigate the role and mechanism of action of NOD-like receptor family pyrin domain containing 3 （NLRP3） inflammasome in airway inflammation in asthmatic mice by mediating T helper 17（Th17）/regulatory T（Treg） cell imbalance. Methods A total of 32 female BALB/c mice were randomly divided into normal control group（NS group），asthma model group（AS group），MCC950 intervention group（MC group），and dexamethasone group（Dex group）. The mice in the AS group were given ovalbumin for sensitization and challenge，and those in the MC group and the Dex group were given MCC950 and dexamethasone，respectively. At 24 hours after the last challenge，the mice were anesthetized and exsanguinated，and bronchoalveolar lavage fluid（BALF） was prepared for cell counting； ELISA was used to measure the concentrations of interleukin-1A（IL-17A），interleukin-1β（IL-1β），interleukin-10（IL-10），interleukin-18（IL-18），interleukin-33（IL-33），and interleukin-35 （IL-35）； flow cytometry was used to measure the proportion of CD4⁺IL-17⁺ cells and CD4⁺CD25⁺CD127^low cells among CD4⁺ cells in peripheral blood，spleen，and lung tissue，which reflected the level of Th17 and Treg cells，respectively；immunohistochemistry was used to observe the protein levels of NLRP3 and Caspase-1 in lung tissue. Results Compared with the NS group，the AS group had significant increases in BALF cell count and the concentrations of IL-17A，IL-1β，IL-18，and IL-33 （P<0.01），significant reductions in the concentrations of IL-10 and IL-35 （P<0.05），a significant increase in the proportion of Th17 cells and a significant reduction in the proportion of Treg cells in peripheral blood，lung，and spleen tissue （P<0.01），and significant increases in the protein levels of NLRP3 and caspase-1 in lung tissue（P<0.01）. Compared with the AS group，the MC group and Dex group had significant reductions in BALF cell count and the concentrations of IL-17A，IL-1β，IL-18，and IL-33（P<0.01），significant increases in the concentrations of IL-10 and IL-35（P<0.05），a significant reduction in the proportion of Th17 cells and a significant increase in the proportion of Treg cells in peripheral blood，lung，and spleen tissue（P<0.01），and significant reductions in the protein levels of NLRP3 and caspase-1 in lung tissue（P<0.01）. Conclusion NLRP3 inflammasome may participate in the onset of airway inflammation in asthma by mediating Th17/Treg imbalance，while MCC950 can regulate Th17/Treg balance and alleviate airway inflammation in asthma.

关键词

支气管哮喘 / NLR家族的Pyrin域蛋白3 / Th17/Treg / 气道慢性炎症

Key words

bronchial asthma / NOD-like receptor family pyrin domain containing 3 / T helper 17/regulatory T cells / chronic airway inflammation

基金资助

遵义市科技计划资助项目(遵市科合HZ（2021）24号)