Extracellular vesicles from acute pancreatitis promote acute lung injury by driving ferroptosis via targeting SOD2

Abstract

Acute pancreatitis-associated acute lung injury (AP-ALI) is a major cause of mortality in AP, yet its precise mechanisms remain unclear, limiting targeted therapies. Extracellular vesicles (EVs) are emerging as key mediators of intercellular communication in inflammatory diseases. This study investigated the role of circulating EVs and their cargo in AP-ALI, focusing on the novel mechanism of ferroptosis in lung epithelial cells. A rat model of AP-ALI was established via retrograde bile duct infusion of sodium taurocholate. EVs were isolated from the plasma of control and AP-ALI rats. The role of EVs was assessed by administering them to AP-ALI rats, with or without the exosome inhibitor GW4869. Ferroptosis was evaluated using transmission electron microscopy, biochemical assays (GSH, MDA, Fe²⁺), and Western blotting for GPX4 and COX-2, C11-BODIPY staining for lipid peroxidation, and inhibitor-based cell death specificity assays. miRNA sequencing of plasma EVs identified differentially expressed miRNAs. The function of miR-200c-3p was validated in vitro in rat lung epithelial L2 cells using mimics and inhibitors, and in vivo using agomirs and antagomirs. Target prediction and luciferase reporter assays identified SOD2 as a direct target of miR-200c-3p. Plasma-derived EVs from AP-ALI rats exacerbated lung injury, inflammation, and pancreatic damage when administered to recipient AP-ALI rats, effects attenuated by GW4869. These EVs induced characteristic features of ferroptosis in lung tissue, including shrunken mitochondria, decreased GSH, increased MDA and Fe²⁺, downregulated GPX4, and upregulated COX-2, which were rescued by the ferroptosis inhibitor Fer-1. miRNA profiling revealed miR-200c-3p was significantly enriched in AP-ALI EVs. In vitro, AP-ALI EVs or miR-200c-3p overexpression promoted ferroptosis in L2 cells, while miR-200c-3p inhibition had the opposite effect. Mechanistically, miR-200c-3p directly targeted and suppressed SOD2. In vivo, miR-200c-3p agomir worsened AP-ALI and ferroptosis, whereas its antagomir alleviated injury and ferroptosis markers. Our findings demonstrate that circulating EVs from AP-ALI rats carry miR-200c-3p, which is delivered to lung epithelial cells, where it targets and inhibits SOD2, thereby promoting ferroptosis and exacerbating lung injury. This EV-miR-200c-3p-SOD2-ferroptosis axis unveils a novel mechanism in AP-ALI pathogenesis and suggests potential therapeutic targets.