AQP5调控免疫源性及神经源性CGRP比例失调引发肺纤维化的机制研究

Pulmonary fibrosis is the typical pathological manifestation of interstitial lung disease (ILD). The accelerated apoptosis of alveolar epithelial cells (AEC) could induce irreversible pulmonary fibrosis. Our previous study has found that the high frequency mutation of aquaporin 5 (AQP5) which could downregulate the expression of AQP5 so as to cause the excessive apoptosis of AEC in ILD. We further found that AQP5 could regulate the expression of immunogenic and neurogenic calcitonin gene-related peptide (CGRP). However, the specific mechanism is still unknown. So we hypothesized that it is a key role of abnormal expression of AQP5 gene in regulating the maladjustment of the immune source and nerve source CGRP leading to pulmonary fibrosis. A series of studies will be carried out based on this hypothesis through the following three aspects: 1) To investigate the dose effect relationship between AQP5 and CGRP in patients with ILD. 2) To verify the relationship between AQP5 and CGRP and the effect of abnormal expression of AQP5 on cell cycle in alveolar epithelial cells, using lentivirus mediated siRNA transfected the human alveolar epithelial cells HPAEpiC to silence AQP5 gene expression; 3) To verify whether AQP5 could regulate the process of apoptosis on alveolar epithelial cells leading to pulmonary fibrosis through the ras-related C3 botulinum toxin substrate 1 (RAC1)-CGRP pathway, the model of CGRP gene knock-out rats will be used to promote the development of related research. As it is a key role of abnormal expression of AQP5 gene in regulating the maladjustment of the immune source and nerve source CGRP, which caused the excessive apoptosis of AEC leading to pulmonary fibrosis, this study would be a new exploration for the triggering mechanism with ILD.

间质性肺疾病（ILD）典型病理表现为肺纤维化。肺泡上皮细胞（AEC）加速凋亡可诱发不可逆的肺纤维化。我们前期发现ILD中水通道蛋白5（AQP5）高频突变、进而引发AQP5表达下调导致AEC过度凋亡，深入研究发现AQP5可调节免疫源性和神经源性降钙素基因相关肽（CGRP）表达，但具体机制未明。我们推测“AQP5调控免疫源性及神经源性CGRP比例失调引发肺纤维化”，为验证假说将通过1）检测患者AQP5与CGRP表达的量效关系；2）利用慢病毒介导的siRNA转染人肺泡上皮细胞株HPAEpiC沉默AQP5基因，验证AQP5与CGRP的表达关系及对AEC细胞周期的影响；3）利用CGRP敲除鼠探讨AQP5是否通过Ras相关的C3肉毒素底物1（RAC1）-CGRP通路调控AEC凋亡致肺纤维化。本研究将从AQP5调控免疫源性和神经源性CGRP比例失调促进AEC凋亡致肺纤维化的新视点探索ILD的触发机制。

间质性肺疾病（interstitial lung disease，ILD）是一大组以间质纤维化为共同结局的弥漫性致死性肺疾病，大多为罕见病，进行性纤维化表型患者肺功能持续恶化，中位生存期仅3年，由于发病机制不清，除肺移植外无法根治。现有研究集中在ILD的早期启动过程，认为2型免疫反应诱导肺泡上皮细胞（AEC）凋亡及不规则修复是其关键病理机制，但未能解释细胞凋亡的触发机制及纤维化持续有效进行的原因，此特有的瓶颈吸引了呼吸免疫学专家的广泛关注，探索该领域已成为当前的热点与难点。.我们研究初步发现ILD中M2型巨噬细胞、Th2主导的2型免疫反应激活导致AEC低表达水通道蛋白5（AQP5）而凋亡。接着以降钙素基因相关肽（CGRP）KO模型鼠重现肺纤维化病理表型为突破口，发现CGRP缺失可诱导巨噬细胞极化及Th1/Th2失衡、淋巴细胞亚群紊乱，伴AQP5表达下调，证实CGRP是肺泡上皮细胞的保护因子，但各环节的相互作用不清。接着利用博来霉素及CGRP敲除鼠模型证实低表达的CGRP通过TGF-β/P-Smad1/Smad4/AQP5通路激活2型免疫反应导致肺组织病态修复形成纤维化。深入研究发现肺纤维化进程中出现T淋巴细胞的参与，辅助性T细胞介导自身抗体的产生，其可能通过IL-4启动B细胞的克隆扩增和分化，益母草碱通过抑制RAG2负性调节肺纤维化肺组织中T细胞活性，可能对肺纤维化具有治疗效果。最后，我们在探索CGRP基因与表达方面发现，β-CGRP基因多态性与涎腺腺样囊性癌的遗传易感性有关，血清CGRP和β-CGRP可作为涎腺样囊性癌的新标志物。.因此，本项目从自身免疫紊乱导致肺纤维化的新角度，探索AEC凋亡及组织持续无效修复的根源，开展开拓性实验发掘关键蛋白分子CGRP及AQP5，阐明CGRP-AQP5调控AEC程序性凋亡引发肺纤维化的机制，这不仅对揭示全新的ILD发病理论具有重要科学意义，还引领ILD免疫分子机制探索的前沿，并提出CGRP及AQP5这二者可能可作为早期干预肺纤维化的全新诊疗靶标，因此是ILD早期精准治疗的新靶点。