DPEP1 mediates regulation of mitochondrial quality control via FOXO1/ALDH1L2 axis to attenuate ferroptosis in pulmonary endothelial cells to alleviate sepsis-associated acute lung injury

Sepsis is a leading cause of acute lung injury worldwide; however, the contribution of ferroptosis, an iron-dependent form of regulated cell death, to sepsis-associated acute lung injury (SALI) remains poorly understood. In this study, we established in vitro and in vivo models of SALI using lipopolysaccharide (LPS) to explore the underlying cellular and molecular mechanisms. Human pulmonary microvascular endothelial cells or mice were treated according to experimental groupings with the mitochondrial division inhibitor 1 (Mdivi-1), the autophagy inhibitor 3-methyladenine (3-MA), the specific forkhead box O1 (FOXO1) inhibitor AS1842856 (AS18), phosphoinositide 3-kinase/protein kinase B (PI3K/AKT) signaling pathway activator Insulin-like Growth Factor I (IGF-1) or dipeptidase 1 (DPEP1) short hairpin RNA. Ferroptosis, mitochondrial quality control (MQC), and inflammatory responses were evaluated using hematoxylin and eosin staining, immunofluorescence, lung wet/dry weight ratio, enzyme-linked immunosorbent assay, western blotting, reverse transcription quantitative polymerase chain reaction, chromatin immunoprecipitation quantitative polymerase chain reaction, and RNA sequencing. DPEP1 knockdown significantly attenuated LPS-induced inflammation, oxidative stress, and ferroptosis in pulmonary endothelial cells. In LPS-treated human pulmonary microvascular endothelial cells, DPEP1 knockdown preserved MQC, as demonstrated by a shift from mitochondrial fission to fusion, reduced mitophagy, and improved fatty acid β-oxidation. Mechanistically, RNA sequencing analysis revealed that DPEP1 knockdown inhibited the phosphoinositide 3-kinase/protein kinase B signaling pathway, decreasing FOXO1 phosphorylation and promoting its nuclear translocation. This led to upregulation of aldehyde dehydrogenase 1 family member L2 transcription, improvement of fatty acid β-oxidation, stabilization of mitochondrial quality, and reduction of ferroptosis. Inhibition of FOXO1 with AS18 reversed these protective effects. Furthermore, combining DPEP1 knockdown with Mdivi-1 or 3-MA synergistically suppressed ferroptosis and oxidative stress. Overall, the results of this study demonstrate that DPEP1 modulates MQC likely through the FOXO1/ALDH1L2 axis to counteract ferroptosis. Targeting DPEP1 via knockdown offers a promising therapeutic approach for SALI, and when combined with Mdivi-1 and 3-MA, this strategy produces a synergistic protective effect against ferroptosis.