Hydromorphone ameliorates endotoxin-induced acute lung injury by suppressing ferroptosis via Nrf2/HO-1 signaling pathway

Background Endotoxin-induced acute lung injury (ALI) features uncontrolled pulmonary inflammation with high mortality. Ferroptosis, an iron-dependent immunogenic cell death, contributes to ALI pathogenesis. While the Nrf2/HO-1 pathway mitigates ALI by regulating oxidative stress, hydromorphone (HM)-a clinical opioid analgesic-alleviates ALI through incompletely defined mechanisms. This study investigated HM's protective effects in lipopolysaccharide (LPS)-induced ALI models. Methods LPS was administered to mice and MH-S alveolar macrophages with/without HM. Lung pathology was evaluated via H&E and Masson staining. Pro-inflammatory cytokines were measured by ELISA. Ferroptosis markers were assessed using Western blot, PCR, immunohistochemistry, and immunofluorescence. Apoptosis and mitochondrial membrane potential (JC-1 probe) were analyzed by flow cytometry. Mitochondrial ultrastructure was examined via transmission electron microscopy. Nrf2 knockout mice and siRNA-transfected cells determined pathway involvement. Results HM significantly attenuated LPS-induced ALI. The LPS + HM group exhibited reduced pulmonary edema, inflammatory cell infiltration, and fibrosis vs LPS group, along with decreased pro-inflammatory cytokines. HM suppressed oxidative stress and ferroptosis, evidenced by elevated GSH, SOD, and GPX4 levels/expression, but reduced GSSG, MDA, Fe²⁺, LPO, PTGS2, and 4-HNE in LPS + HM group versus LPS controls. HM treatment reversed mitochondrial membrane potential collapse and ameliorated mitochondrial damage. Crucially, HM's protection depended on Nrf2/HO-1 activation, as LPS + HM treatment significantly upregulated Nrf2 and HO-1. Experiments also confirmed that HM's anti-ferroptotic and lung-protective effects require intact Nrf2/HO-1 signaling. Conclusion HM protects against endotoxin-induced ALI by inhibiting ferroptosis via activating the Nrf2/HO-1 pathway.