基于铁死亡机制的谷氨酸NMDA受体过度激活在肺纤维化发生发展中作用的再研究

Pulmonary fibrosis (PF) is an inexorably progressive lung disease with few available treatments. Alveolar epithelial cell injury is considered as one of the key points during the development of PF, but the underlying mechanism involved in this process remains to be elucidated. Based on the results from the applicant’s previous research project that the activation of NMDAR aggravated the PF, and according to the significant preliminary results that the BLM induced PF and the activation of NMDAR both resulted in the iron overload in lung epithelial cells, applicant for the first time put forward the hypothesis that the ferroptosis of the lung epithelial cell is involved in the excessive activation of NMDAR induced PF. This application will explore: (1) the role of ferroptosis and iron overload of alveolar type II epithelial cells in the NMDAR induced PF in specific NMDAR knockout mice; (2) the effect of NMDAR on the iron transportation, iron overload and ferroptosis in the lung epithelial cells, and find out the cell membrane mechanism of NMDAR activation mediated iron overload in vitro; (3) the effect of NMDAR activation on the mitochondrial dysfunction, and to reveal that the mitochondria is the key target of NMDAR activation induced ferroptosis in lung epithelial cells by using mitochondrial transplant, therefore to provide new thinking for revealing subcellular mechanism of the NMDAR activation induced alveolar epithelial cell injury, and also offer new preventive and therapeutic options for PF. The biological significance of ferroptosis in the development of PF is so far not been studied.

肺纤维化严重危害人们健康。肺上皮细胞损伤是肺纤维化发生的始动环节。肺上皮损伤的机制远未阐明。申请者在前一课题发现肺NMDA受体激活加重肺纤维化的基础上，结合前期关于博莱霉素肺纤维化和NMDA肺损伤均引起肺上皮细胞铁超载的初步发现，创新性提出从肺上皮细胞铁死亡角度对NMDA受体过度激活促肺纤维化发展的机制进行再研究。本申请设计：①在肺泡II型上皮细胞NMDA受体敲除小鼠研究NMDA受体在介导肺纤维化时II型细胞铁超载和铁死亡中的作用；②在细胞水平探讨NMDA受体对肺上皮细胞铁转运、铁超载及铁死亡的影响，查明介导细胞铁超载的相关膜转运环节；③研究NMDA受体激活对线粒体功能的影响，并采用线粒体功能剔除和移植揭示线粒体是NMDA受体激活引起肺上皮细胞铁死亡的关键靶标，为揭示NMDA受体激活损伤肺上皮细胞的亚细胞机制及肺纤维化防治的研究提供启示。铁死亡在肺纤维化发生中的作用迄今未见报道。

肺纤维化严重危害人们身体健康，是多种急性和慢性肺损伤的最后共同结局，但发病机制远未阐明。铁死亡（Ferroptosis）是新近发现一种铁依赖性的脂质过氧化驱动的非凋亡形式的调节性细胞死亡。铁死亡与肺纤维化发生的关系目前未见报道。申请者基于前期研究，从肺泡上皮细胞铁死亡的角度来探讨谷氨酸NMDA受体（NMDAR）激活促进肺纤维化发生的机制及意义，为临床肺纤维化防治的研究提供新的启示。为此，开展四方面的研究：.（1）在整体博来霉素（BLM）肺纤维化模型首次发现，模型小鼠肺组织存在多种跨膜铁转运相关蛋白的表达改变、肺组织铁沉积和肺泡Ⅱ型上皮细胞（ATII）铁死亡。铁死亡抑制剂去铁胺（DFO）可以有效减轻BLM诱导的小鼠肺纤维化程度。表明肺内铁代谢紊乱和ATII细胞铁死亡是BLM致肺纤维化发生发展的重要机制。.（2）在离体培养水平首次证实BLM可诱导肺泡上皮MLE-12细胞铁代谢紊乱、铁沉积、线粒体功能损伤及铁死亡，DFO可以减轻BLM诱导的小鼠肺上皮细胞相应变化，减轻BLM诱导的铁死亡。.（3）NMDAR激活可引起肺内铁代谢紊乱和ATII细胞铁死亡的发生，是促进肺纤维化发生发展的重要机制。NMDAR的激活可导致肺泡上皮细胞铁过载和铁死亡的发生，nNOS 是介导NMDAR激活所致肺泡上皮细胞铁代谢紊乱的重要信号分子。.（4）在离体水平证实，肺泡上皮线粒体铁过载和线粒体功能障碍是BLM致肺泡上皮损伤的重要机制。移植健康线粒体可挽救 BLM诱导的MLE-12细胞线粒体铁沉积、mtDNA损伤和细胞死亡。此外，PGE2-EP4/ FBXL5信号通路对IREB2/MFRN2/线粒体铁沉积轴的影响参与调控BLM致肺泡上皮损伤的过程，为揭示NMDA受体激活损伤肺上皮细胞的亚细胞机制及肺纤维化防治的研究提供启示。.上述研究首次表明，肺纤维化发生过程中存在肺内铁代谢紊乱和ATII细胞铁死亡。在BLM致肺纤维的过程中，通过NMDA受体激活介导肺泡上皮铁代谢紊乱、铁过载、线粒体损伤和铁死亡的发生，是导致ATII细胞铁死亡的重要机制；减轻肺泡上皮线粒体铁过载、线粒体功能障碍和铁死亡是肺纤维化防治的潜在靶点，是课题组既往有关NMDA受体激活是肺纤维化发生的重要机制的进一步深入，为将NMDA受体阻断剂应用于肺纤维化的防治提供了新的证据，同时本研究还展现了去铁胺抗肺纤维化的潜在应用前景。