氢气抗氧化应激对哮喘气道黏膜固有免疫的影响及机制研究

Few methods were approached to the treatment of severe asthma and the effectiveness is not satisfactory. To explore more effective and safe treatments is an urgent clinical need to resolve this difficult problem. The literatures have reported that the innate immune deficiency of the airway epithelium occurred in severe asthma patients, which is related to the imbalance of oxidation and antioxidation caused by oxidative stress. High concentration hydrogen gas has been proved to be a new method of antioxidative stress treatment. The preliminary results of our group showed that the inhalation of hydrogen gas had significant antiasthmatic effect in OVA-sensitized asthmatic mice model safely; however the specific mechanism is unknown. There is no literature report about the effect of inhalation of high concentration hydrogen gas on the innate immunity of the airway mucosa in asthma and on the severe asthma patients. This study will investigate the effect of hydrogen gas inhalation on the airway mucosal innate immunity of asthmatic animal model in vivo, and of hydrogen gas on human bronchial epithelial cells line (16HBEs) sensitized by house dust mite antigen and primary bronchial epithelial cells (PBECs) from severe asthma patients in vitro. The innate immune responses to hydrogen gas will be detected from three aspects (including physical epithelial barrier, chemical immune responses, the antioxidative defense function) by using a variety of molecular biology methods through the novel hydrogen gas-containing cell culture system. The results will be helpful to clarify and reveal the related molecular mechanism of antioxidant intervention of hydrogen gas on the protection of airway mucosal innate immunity and to provide a necessary theoretical foundation for inhalation therapy to antioxidative stress to achieve clinical translation from bench to bedside in the future.

重度哮喘临床治疗手段有限且效果不理想，探讨更多有效安全的治疗措施成为临床上的迫切需要。重度哮喘患者气道黏膜存在固有免疫功能缺陷，且与氧化应激引起的氧化抗氧化失衡有关。高浓度氢气摄入成为抗氧化应激治疗的一种新方法。本课题前期研究结果显示吸入高浓度氢气对哮喘小鼠模型具有安全显著的抗哮喘作用，而具体机制不明。吸入高浓度氢气对哮喘气道黏膜免疫功能的影响以及氢气对哮喘患者作用如何尚未见报道。本课题拟选择哮喘动物模型、尘螨抗原致敏人气道上皮细胞株、哮喘患者原代气道上皮细胞为研究对象，应用细胞培养氢气共孵育系统，采用跨膜电阻抗、ELISA、流式细胞术、荧光免疫标记等多种分子生物学方法从气道黏膜物理屏障、化学免疫应答、抗氧化防御等三方面探讨氢气对气道黏膜固有免疫的影响，通过在体和体外实验揭示和阐明氢气抗氧化应激对哮喘气道黏膜固有免疫功能影响的分子机制，为吸入性抗氧化应激治疗实现转化、走向临床奠定理论基础。

1.氢分子通过抑制IL-33/ILC2轴减轻哮喘。目的：本研究旨在探讨分子氢气分子在哮喘发病机制中的作用。方法：建立OVA致敏哮喘小鼠模型，用氢气吸入干预哮喘小鼠。采用分子生物学方法检测E-cadherin、ZO-1、caspase3和9、NF-kB、IL-33和ST2的表达、IL-33启动子活性、ILC2细胞群、差异表达的miRNA。结果：OVA可显著提高哮喘小鼠血清和BALF中IL-33及其他alarmin和II型细胞因子的水平，H2可显著抑制哮喘小鼠血清和BALF中IL-33和其他alarmin及II型细胞因子的水平。OVA上调NF-κB（p65）和ST2的表达，H2抑制其表达。在OVA诱导的哮喘小鼠中，ILC2的数量显著增加，H2抑制了这种增加。哮喘小鼠气道组织中E-cadherin和ZO-1水平显著低于对照组，H2治疗后其水平恢复。H2可减轻HDM诱导的16HBE细胞凋亡、IL-33和ST2的上调及IL-33启动子活性的升高。在HDM和HDM+H2处理的16HBE细胞中发现了一组差异表达的miRNA。结论：H2能有效抑制变应原诱导的哮喘，可作为治疗哮喘和其他Ⅱ型炎症的方法。.2. 慢阻肺大鼠肺泡巨噬细胞来源外泌体对气道上皮细胞增殖能力的影响及机制研究.目的：为探讨慢阻肺大鼠动物模型肺泡巨噬细胞来源外泌体对气道上皮细胞(16HBE)的影响及其分子机制。方法：该研究通过烟熏和脂多糖诱导建立慢阻肺大鼠动物模型，提取肺泡巨噬细胞与16HBE共培养。采用分子生物学实验检测16HBE的增殖和迁移能力、黏蛋白和促炎介质表达、肺泡巨噬细胞外泌体差异表达的miRNA，在16HBE中转染该miRNA及靶基因的siRNA后对16HBE功能的影响。结果：慢阻肺大鼠肺泡巨噬细胞增强了气道上皮细胞（16HBE）的增殖和迁移能力，促进16HBE黏蛋白和炎症介质TNF-α、IL-6的表达。miR-380在肺泡巨噬细胞外泌体中明显上调。miR-380明显增强16HBE的增殖和和迁移能力，促进16HBE黏蛋白的表达，抑制CFTR表达。结论：该研究表明慢阻肺大鼠肺泡巨噬细胞可增强气道上皮细胞（16HBE）的增殖和迁移能力，促进其黏蛋白及促炎介质的表达；16HBE的增殖和和迁移能力的增强可能与慢阻肺肺泡巨噬细胞外泌体向16HBE递送miR-380，负调控CFTR基因抑制CFTR表达有关。