Huang Xi, Feng Jinhua, Xu Qiaoyi, Tang Ri, Peng Yawen, Yang Wenyu, Yang Xinyi, Xing Shunpeng, Qian Guojun, Gao Yuan, Mei Shuya, He Zhengyu
Department of Critical Care Medicine, Ren Ji Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China; Key Laboratory of Anesthesiology (Shanghai Jiao Tong University), Ministry of Education, Shanghai, China.
Department of Critical Care Medicine, Ren Ji Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China.
J Adv Res. 2025 Jul 28. doi: 10.1016/j.jare.2025.07.043.
Mechanical ventilation (MV) is essential for treating respiratory failure but can paradoxically lead to pulmonary fibrosis. The mechanisms of MV-induced pulmonary fibrosis (MVPF) remain poorly understood. Ferritinophagy, a novel autophagic process, regulates ferroptosis and the release of ferritin-containing extracellular vesicles (EVs), both of which may contribute to MVPF.
This study aimed to investigate the mechanism of ferritinophagy, as well as how ferritin-containing EVs contribute to intercellular communication during MVPF progression.
A mouse MVPF model was established using high tidal volume ventilation. Lung tissues were analyzed via single-cell RNA sequencing (scRNA-seq). Mechanical stretch (MS) was applied to alveolar epithelial cells (AECs) in vitro. Fluorescence recovery after photobleaching (FRAP) analysis was used to capture ferritin phase separation in live cells. Ferritinophagy and ferroptosis were assessed via key molecular markers. Chloroquine and AAV-mediated knockdown of AGTR1 and NCOA4 were used to inhibit ferritinophagy. EVs were isolated by ultracentrifugation and evaluated by immunoblotting and uptake assays.
scRNA-seq revealed iron metabolism dysregulation and downregulation of ferroptosis-suppressor genes (Gpx4 and Fth) in AECs after MV. The ANG II/AGTR1 axis initiated ferritinophagy, leading to iron overload and subsequent ferroptosis. NCOA4-mediated ferritin phase separation under MS promoted ferritinophagy in AECs.Inhibition of ferritinophagy effectively reduced ferroptosis and alleviated MVPF. Moreover, ferritin-containing EVs released from injured AECs due to ferritinophagy can be assimilated by fibroblasts, resulting in fibroblast activation and extracellular matrix (ECM) accumulation through iron overload.
MV induces ANG II/AGTR1-mediated ferritinophagy and ferroptosis in AECs. NCOA4-driven ferritin phase separation promotes ferritinophagy under mechanical stress. Ferritin-containing EVs from damaged AECs activate fibroblasts, exacerbating MVPF. Our findings underscore the pivotal role of iron metabolism dysregulation in biomechanically induced programmed cell death and intercellular communication, and reveal potential therapeutic targets for the prevention and treatment of MVPF.
机械通气（MV）是治疗呼吸衰竭的关键手段，但反常的是，它可能导致肺纤维化。MV诱导的肺纤维化（MVPF）的机制仍不清楚。铁蛋白自噬是一种新的自噬过程，可调节铁死亡以及含铁蛋白的细胞外囊泡（EV）的释放，这两者都可能与MVPF有关。
本研究旨在探讨铁蛋白自噬的机制，以及含铁血蛋白的EV在MVPF进展过程中如何促进细胞间通讯。
采用高潮气量通气建立小鼠MVPF模型。通过单细胞RNA测序（scRNA-seq）分析肺组织。在体外对肺泡上皮细胞（AEC）施加机械拉伸（MS）。采用光漂白后荧光恢复（FRAP）分析来捕获活细胞中铁蛋白的相分离。通过关键分子标记评估铁蛋白自噬和铁死亡。使用氯喹以及AAV介导的AGTR1和NCOA4基因敲低来抑制铁蛋白自噬。通过超速离心分离EV，并通过免疫印迹和摄取试验进行评估。
scRNA-seq显示MV后AEC中铁代谢失调以及铁死亡抑制基因（Gpx4和Fth）下调。血管紧张素II/AGTR1轴启动铁蛋白自噬，导致铁过载和随后的铁死亡。MS下NCOA4介导的铁蛋白相分离促进了AEC中的铁蛋白自噬。抑制铁蛋白自噬可有效减少铁死亡并减轻MVPF。此外，由于铁蛋白自噬从受损AEC释放的含铁血蛋白的EV可被成纤维细胞摄取，导致成纤维细胞活化和细胞外基质（ECM）通过铁过载而积累。
MV诱导AEC中血管紧张素II/AGTR1介导的铁蛋白自噬和铁死亡。NCOA4驱动的铁蛋白相分离在机械应力下促进铁蛋白自噬。来自受损AEC的含铁血蛋白的EV激活成纤维细胞，加剧MVPF。我们的研究结果强调了铁代谢失调在生物力学诱导的程序性细胞死亡和细胞间通讯中的关键作用，并揭示了预防和治疗MVPF的潜在治疗靶点。
