Abstract
Acute lung injury (ALI) is a common and severe complication of sepsis with a high mortality rate. Ferroptosis, an iron-dependent form of cell death, contributes to lung injury. Homeobox A5 (HOXA5) is involved in the regulation of septic acute kidney damage; however, its function on ferroptosis in septic ALI remains unclear. An in vitro model of septic lung injury was established in the pulmonary epithelial cell line (MLE-12) via lipopolysaccharide (LPS) stimulation. Cell viability, ferrous iron (Fe(2+)) level, and cellular lipid reactive oxygen species (ROS) were determined with Cell Counting Kit-8 assay, iron assay kit, and BODIPY™ 665/676 molecular probe, respectively. HOXA5, ferroptosis suppressor protein 1 (FSP1), sirtuin 5 (SIRT5), and glutathione peroxidase 4 (GPX4) expressions were measured using western blotting and Real-Time Quantitative Polymerase Chain Reaction (RT-qPCR. Chromatin immunoprecipitation and luciferase reporter assays were performed to validate HOXA5 binding to the FSP1/GPX4 promoter, and regulation of SIRT5 on HOXA5 desuccinylation was confirmed through co-immunoprecipitation. LPS stimulation induced ferroptosis (reduced cell viability, elevated Fe(2+) and lipid ROS levels, and decreased GPX4 levels) and downregulated FSP1 and HOXA5 protein levels. HOXA5 overexpression neutralized LPS-induced ferroptosis. Moreover, LPS exposure inhibited HOXA5 binding to the FSP1 promoter, which was counteracted via HOXA5 overexpression. Furthermore, SIRT5 overexpression suppressed LPS-induced ferroptosis. In LPS-challenged MLE-12 cells, SIRT5-mediated HOXA5 desuccinylation was reduced. HOXA5 depletion neutralized the suppressive role of SIRT5 overexpression in LPS-induced ferroptosis. SIRT5-mediated HOXA5 desuccinylation inhibited LPS-induced ferroptosis by upregulating FSP1, which may offer a prospective therapeutic strategy for septic lung injury.