肺纤维化发生的新机制研究- - - - NMDA受体的作用

Pulmonary fibrosis is a serious health hazard common complication of the respiratory system, but the mechanism is far from being elucidated. Previous studies in our laboratory have suggested that a large number of the glutamate released during the bleomycin induced pulmonary fibrosis in the lungs and the activation of glutamate NMDA receptors in vitro could promote lung fibroblast proliferation and collagen synthesis. Based on these results, this project for the first time proposes the intrapulmonary excessive release of glutamate and NMDA receptor activation may be an important factor in the promotion of pulmonary fibrosis. Therefore, the project plans to examine the effect of NMDA receptor inhibitor, memantine, on the mice pulmonary fibrosis induced by bleomycin in vivo, a variety of techniques including constructing bone marrow GFP chimera mice， will be used to investigate the cell origin of fibroblasts in pulmonary fibrosis promoted by NMDA receptors; variety of research strategies, such as transcriptional regulation by the transcription factor activity microarray, EMSA, reporter gene, ChIP, siRNA, etc. will be used to explore the transcriptional regulatory mechanisms and intracellular signaling pathways of the NMDA receptor promoted lung epithelial cell EMT and the expression of the key regulatory gene to promote EMT, Snail1; to reveal the role of NMDA receptor activation in the occurrence of pulmonary fibrosis and the underlined cellular and molecular mechanisms; to confirm the activation of the NMDA receptor is a new important factor in promoting pulmonary fibrosis, provide a new target for the prevention and treatment of pulmonary fibrosis.

肺纤维化是严重危害健康的呼吸系统常见并发症，但发生机制远未阐明。申请者基于博莱霉素肺纤维化的发生时肺内有谷氨酸的大量释放及激活谷氨酸的NMDA受体在体外可促进肺成纤维细胞增殖和胶原合成的前期结果，首次提出肺内谷氨酸的过量释放及NMDA受体的激活可能是促进肺纤维化发生的重要因素。为此，本申请计划在整体水平观察NMDA受体阻断剂美金刚胺对博莱霉素肺纤维化程度的影响，并采用骨髓GFP嵌合小鼠等多种技术探讨NMDA受体促肺内成纤维细胞增生的细胞来源；还利用转录因子活性谱芯片、EMSA、报告基因、ChIP、siRNA等多种转录调控研究策略在体外探讨NMDA受体促肺上皮细胞EMT发生及促EMT关键调控基因Snail1表达的转录调控机制和胞内信号途径，揭示NMDA受体激活在肺纤维化发生中的作用及细胞分子机制，确认NMDA受体的激活是促进肺纤维化发生的新的重要因素，为肺纤维化防治的研究提供新的靶点。

肺纤维化是严重危害健康的呼吸系统常见并发症，但发生机制远未阐明，至今仍无特效的治疗方法。申请者基于自己有关博莱霉素肺纤维化的发生时肺内有谷氨酸的大量释放及激活谷氨酸的NMDA受体在体外可促进肺成纤维细胞增殖和胶原合成的前期结果，首次提出肺内谷氨酸的过量释放及NMDA受体的激活可能是促进肺纤维化发生的重要因素。为此，开展三个方面的研究：.1.本项目在整体水平证实，在博来霉素建模同时预防性给予NMDA受体阻断剂美金刚胺可减轻早期肺内炎症反应和后期肺纤维化程度；在建模14天时给予美金刚胺治疗性干预也可有效减轻后期小鼠博莱霉素肺纤维化程度，但其保护作用略低于预防性用药，表明NMDA受体激活参与博来霉素所致早期肺内炎症反应和后期肺纤维化的发生发展过程。.2.本项目在细胞水平证实，肺成纤维细胞和上皮细胞均有NMDA受体表达；NMDA受体激活可促进成纤维细胞增殖、胶原表达和向肌成纤维细胞转变，内源性谷氨酸通过NMDA受体参与介导TGFβ促成纤维细胞增殖；NMDA受体激活可促进肺泡上皮细胞EMT的发生；阻断NMDA受体可降低肺纤维化模型小鼠肺组织和骨髓细胞中fibrocyte含量，减少肺内成纤维细胞的来源。.3.本项目在转录调控水平证实， NMDA受体激活可与低剂量TGF-β1协同促进在EMT发生中发挥重要作用的Snail1基因的表达；通过成功构建不同长度重组报告基因质粒，分别证明Snail1基因启动子对TGF-β1应答区域和对NMDA应答区域分别位于不同区域。进一步采用EMSA技术证明，-869～-815bp可能是对NMDA刺激应答的关键启动子区域结合之一。.上述研究表明，NMDA受体激活在促进肺纤维化发生发展中的起有重要作用。NMDA受体激活促进肺纤维化发生发展的细胞机制与促进纤维细胞的活化和肺上皮细胞的EMT发生，促进肺内趋化因子的表达和肺内fibrocyte的募集有关。NMDA受体激活促进肺上皮细胞EMT的发生有赖于ERK介导促进Snail基因的表达增强。因此，NMDA受体的激活是促进肺纤维化发生的新的重要因素，为肺纤维化防治的研究提供新的靶点