Inhibition of ferroptosis by serine protease inhibitor attenuates acute respiratory distress syndrome

Background: Acute respiratory distress syndrome (ARDS), characterized by high mortality, involves multiple molecular programs, notably ferroptosis—a form of immunogenic cell death driven by iron overload and lipid peroxidation. Ulinastatin (UTI), a serine protease inhibitor, shows clinical efficacy in ARDS, but its underlying mechanism remains unclear. We aim to identify potential molecular targets in this process to promote clinical translation in ARDS treatment. Methods: We performed RNA sequencing (RNA-seq) on lung tissues from LPS-induced ARDS mice revealed significant enrichment of ferroptosis-related pathways in UTI-treated ARDS mice, prompting the hypothesis that UTI mitigates ARDS by suppressing ferroptosis. Using LPS-induced murine ARDS, we assessed UTI's therapeutic effects via histopathology, qRT-PCR, RNA sequencing, and molecular assays. Ferroptosis biomarkers (iron, MDA, GSH), key proteins (GPX4, KEAP1, NRF2), and inflammatory cytokines were evaluated. In vitro, HUVEC and MLE-12 were used to investigate the molecular mechanisms by which UTI's functions via ferroptosis. Molecular docking explored UTI-KEAP1/NRF2 interactions. Results: UTI significantly attenuated lung injury, reduced inflammatory cytokines (IL-1β, IL-6, TNF-α), and restored hepatic/renal function in LPS-challenged mice. Transcriptomics revealed ferroptosis as a top enriched pathway suppressed by UTI. Mechanistically, in both HUVEC and MLE-12 cells, UTI attenuated LPS-induced increases in labile iron, MDA, and lipid ROS levels. Additionally, UTI suppressed KEAP1 expression while activating NRF2, an effect comparable to that of ferroptosis inhibitors. Consequently, GPX4 expression was upregulated, suggesting a potential anti-ferroptotic mechanism. Conclusion: Inhibition of ferroptosis is a novel mechanism underpinning UTI's lung-protective effect against ARDS. UTI potentially regulates the KEAP1-NRF2 interaction through direct binding to KEAP1, offering a new molecular-level explanation for its mechanism of action.