维生素D调控Th17细胞自噬对激素抵抗型哮喘小鼠气道炎症的作用及机制研究

Steroid therapy is an important treatment for asthmatic patients in clinical practice. However, the patients with steroid-resistant (SR) asthma often respond poorly to the steroid treatment even with high dosages, making it a great concern in the asthma therapies. Neutrophil-dominated airway inflammation plays a key role in steroid-resistant asthma. IL-17A is one of the pro-inflammatory cytokines produced by Th17 cells, and it plays an important role in neutrophilic inflammation of airway in steroid-resistant asthmatics. Vitamin D has a suppressive effect on inflammation and it improves the sensitivity of steroid-resistant asthma to corticosteroid by inhibiting Th17-mediated autoimmunity and reducing IL-17A production. We found previously that vitamin D was able to alter the expression of autophagy-related proteins in the lung tissue in OVA induced asthmatic mice, we therefore speculate that it may inhibit the inflammatory response of steroid resistant asthma by regulating autophagy in Th17 cells. For this hypothesis, we plan to study the effects of vitamin D at different concentrations on airway inflammation and autophagy in lung tissues in steroid resistant asthmatic mice induced by exposing to ozone. We will also study autophagy-related protein expression in the Th17 cells isolated from animal spleen to further understand the impact of vitamin D on autophagy in asthma in vitro, and explore the molecular mechanism of vitamin D on autophagy by silencing autophagy-related proteins with shRNA before screening the possible signaling pathways. This study may reveal the role of vitamin D in steroid-resistant asthma and its mechanism, which may provide a novel target for the therapy of steroid resistance in asthma.

激素抵抗（SR）是哮喘治疗的难点，发病机制不明。氧化应激活化IL-17A致中性粒细胞性气道炎症在SR型哮喘中起重要作用。IL-17A由Th17细胞产生，是Th17细胞关键的促炎细胞因子，提示Th17细胞在SR型哮喘发病机制中起重要的作用。维生素D（VitD）可改善SR型哮喘对激素敏感性，且可调节Th17细胞反应，抑制IL-17A水平。我们前期发现VitD可导致哮喘小鼠肺组织中自噬相关蛋白表达变化，故推测其可能通过影响Th17细胞的自噬来抑制SR型哮喘的炎症反应。为此，本课题拟在前期建立成熟的臭氧诱导的SR型哮喘小鼠模型基础上，研究不同摄入浓度VitD对SR型哮喘小鼠气道炎症及自噬的影响，并利用体外分离Th17细胞，分别给予不同shRNA沉默相关自噬蛋白，探求VitD调控自噬的分子机制并尝试筛选信号通路，旨在揭示VitD在SR型哮喘中的作用及调控机制，为治疗SR型哮喘提供新思路。

氧化应激活化IL-17A致中性粒细胞性气道炎症在临床中主要表现非T2型哮喘，导致激素抵抗（SR），是哮喘治疗的难点，维生素D（VitD）可改善SR型哮喘对激素敏感性，且可调节Th17细胞反应，抑制IL-17A水平。本课题组首先将哮喘患者根据嗜酸粒细胞计数及FeNO水平分为T2型及非T2型，检测患者血清25(OH)D3水平、炎性细胞因子及肺功能。根据血清中VitD中位数分为VitD缺乏组（Lo）及充足组（Hi），结果发现在两种内型中，与VitD充足组相比，VitD缺乏组哮喘患者的第一秒用力呼气量占预测值（FEV1%pred）百分比均降低，与T2组相比，非T2组VitD缺乏患者的FEV1%pred下降更明显，但无统计学差异。在非T2型哮喘患者VitD缺乏状态下血清中的IL-6和IL-17A显著升高，IL-10显著下降，T2组VitD缺乏状态下血清中的IL-5和IL-6显著升高，提示VitD对T2和非T2型哮喘的影响和机制不同。同时，在OVA及OVA+臭氧（O3）两种哮喘模型中，与对照组相比，粘液高分泌、炎症、高反应性和气道重塑均存在，而OVA+O3组表现为严重的中性粒细胞浸润及Th17相关细胞因子（IL-6、IL-17A、IL-21）明显升高。同时为了进一步验证VitD在不同内型哮喘中的作用，BALB/c小鼠出生后分别饲喂VitD缺乏（LVD）、VitD充足（NVD）或VitD补充饮食（HVD），用OVA致敏/激发以建立“T2型”哮喘，并用OVA+O3致敏或激发以诱发“非T2型”哮喘。在小鼠模型中，与NVD组和HVD组相比，单独OVA或OVA+ O3共同暴露在LVD组诱导更高的相应的T2和非T2的气道炎症和较高的RL。然而，与“T2型”哮喘模型相比，VitD缺乏可显著增加 “非T2型”小鼠模型的中性粒细胞炎症和更高RL。与补充VitD的OVA介导组相比，VitD缺乏OVA介导组肺组织中包括LC3、Beclin-1、ATG5和NFκB p65的表达增加。BALF中LC3 mRNA的表达与炎性细胞数量和细胞因子有关。在体外，1,25(OH)2D3还以时间和剂量依赖性的方式调节AMs中LC3、Beclin-1、ATG5和NF-κB p65 mRNA的表达。缺乏VitD可加重过敏性气道炎症且和自噬蛋白表达相关，需要进一步探索机制。