中性粒细胞中HMGB1依赖性自噬对1,3-β-葡聚糖所致肺部炎症的免疫调控机制

The rapid development of modern industrial and agricultural production brings related occupational workers the sever exposure to the complex organic dust contaminated environment. The high-dose fugues contamination induced hypersensitivity pneumonitis draws an increasingly wide attention. The fundamental pathological characters are the inflammatory cells accumulation in lung parenchyma and the formation of granuloma. However, the mechanism of the immunological modulation during the pathology process is not still clearly demonstrated. Our previous research demonstrated the critical role of neutrophils in regulating the process of hypersensitivity pneumonitis. Autophagy contributes to maintain the dynamic balance in intercellular environment and is becoming the research hotspot. The transformation of neutrophil autophagy could directly affect its immune regulatory function. High mobility group box-1 protein (HMGB1) could induce autophagy and play a regulatory role in multiple diseases. However, the actual role of HMGB1 signal pathway in the development of hypersensitivity pneumonitis is not clear. The detailed mechanism of HMGB1-dependent autophagy in regulating neutrophils function need further investigation. Our project was aimed at illustrating the immune molecular mechanisms of the HMGB1 signal pathway and HMGB1-dependent autophagy of neutrophils in hypersensitivity pneumonitis at both cellular and molecular levels by using conditional knockout mice and neutralizing antibody. The major constructive composition of fugues 1-3-β-glucan will be used to setup mice pulmonary inflammation model. It is believed that if we demonstrated the molecular regulatory mechanisms of HMGB1-dependent autophagy of neutrophils in hypersensitivity pneumonitis successfully, the innovative immune molecular targets will be provided to contribute to the treatment and prognosis of occupational hypersensitivity pneumonitis.

现代工农业生产的飞速发展使相关职业劳动者长期暴露于复杂的有机粉尘污染环境。高浓度真菌污染所引起的过敏性肺炎等免疫性肺疾病受到越来越广泛的关注。其基本病理特征是肺间质炎性细胞浸润和肉芽肿。但目前其发病机制尚不清楚。前期研究表明中性粒细胞在过敏性肺炎中发挥重要的调控作用。近年来，自噬因可通过维持细胞内环境动态平衡调控细胞功能而成为研究热点。中性粒细胞自噬水平的变化直接影响其免疫调控功能的发挥。HMGB1可诱导细胞自噬并调控多种疾病。但HMGB1在过敏性肺炎中的作用尚不清楚，HMGB1依赖性自噬调控中性粒细胞功能的具体机制尚未明确。本实验拟以真菌细胞壁主要成分1,3-β-葡聚糖所致的小鼠肺部炎症为模型，利用条件性基因敲除及消除抗体等手段，从细胞、分子水平探讨HMGB1信号通路及中性粒细胞HMGB1依赖性自噬调控过敏性肺炎的免疫学分子机制。旨在为职业性过敏性肺炎的治疗及预后提供新的免疫分子靶标。

在职业环境中吸入被真菌等污染的有机粉尘而引起以弥漫性肺部炎症及肺间质肉芽肿为主要改变的一系列职业性肺部疾病，称为过敏性肺炎。过敏性肺炎的反复发生可发展为不可逆的肺组织纤维化。职业过程中接触有机粉尘不可避免，因此，早期诊断治疗对预防肺纤维化的发生极为重要。探究过敏性肺炎的发病机制，寻找有效的治疗靶点，对预防和控制过敏性肺炎具有十分重要的意义。. 本研究利用1,3-β-葡聚糖建立小鼠肺部炎症模型，结合小鼠原代中性粒细胞，小鼠上皮细胞系A549为研究对象，通过基因沉默、特异性抑制剂等方式干扰HMGB1的表达水平，探讨HMGB1依赖性自噬对过敏性肺炎的调控机制。结果表明，HMGB1依赖性自噬可抑制1,3-β-葡聚糖诱导的小鼠肺部炎症。沉默HMGB1对小鼠肺泡灌洗液中性粒细胞的数量影响最为显著。因此，结合动物体内实验和小鼠原代中性粒细胞体外培养，发现HMGB1依赖性自噬通过调控1,3-β-葡聚糖诱导的中性粒细胞的趋化、脱颗粒、吞噬、MPO的表达及NETs的分泌水平等多方面实现其对中性粒细胞功能的影响，从而抑制炎症反应。另一方面，本研究从肺组织最主要的效应细胞上皮细胞入手，结合小鼠体内研究和上皮细胞系体外培养实验，发现HMGB1可通过调控Beclin1泛素化水平，影响Beclin1和BCL2结合，进而增强1,3-β-葡聚糖诱导的肺上皮细胞自噬小体的成核阶段，从而促进肺上皮细胞自噬反应，发挥其对1,3-β-葡聚糖所致的肺部炎症的抑制作用。. 综合动物、细胞实验结果，本研究证实HMGB1参与抑制1,3-β-葡聚糖诱导的肺部炎症。HMGB1可通过调控中性粒细胞及上皮细胞自噬发挥其抑制炎症的作用。胞浆中的HMGB1通过影响Beclin1泛素化水平，调节Beclin1和BCL2结合，促进自噬小体的形成，促进受损细胞自噬。为明晰过敏性肺炎发病机制提供可靠的理论和实验依据。

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