Abstract
BACKGROUND: Increasing evidence suggests that ferroptosis plays a pivotal role in Staphylococcus aureus (S. aureus)-induced osteomyelitis. However, the regulatory mechanisms underlying ferroptosis-related genes (FRGs) in osteomyelitis remain poorly understood. This study aimed to identify key FRGs and elucidate their regulatory roles in osteomyelitis. METHODS: Key FRGs were identified using least absolute shrinkage and selection operator (LASSO) logistic regression and support vector machine-recursive feature elimination (SVM-RFE) algorithms based on transcriptomic data from the Gene Expression Omnibus (GEO) database. Bone marrow mesenchymal stromal cells (BMSCs) were isolated, characterized, and infected with S. aureus protein A (SpA) to construct an in vitro model. Cell viability, osteogenic differentiation, inflammation, and gene expression were assessed using Cell Counting Kit-8 (CCK-8), Alizarin Red S staining, enzyme-linked immunosorbent assay (ELISA), and quantitative reverse transcription polymerase chain reaction (qRT-PCR). In vivo, a mouse model of S. aureus-induced osteomyelitis was established, and the role of KLF2 was examined by micro-computed tomography (micro-CT), ELISA, histological staining, immunohistochemistry, immunofluorescence, and qRT-PCR. RESULTS: A total of 683 differentially expressed FRGs were identified. Ten candidate biomarkers were screened using LASSO and SVM-RFE, of which TXN, KLF2, HSPA8, CCT3, and AKR1C3 were consistently validated across training and validation datasets. These genes were associated with immune regulation, protein synthesis, and multiple ribosome- and metabolism-related pathways. In vitro, SpA treatment increased inflammation response, reduced BMSC proliferation and osteogenic differentiation, upregulated HSPA8, TXN, and CCT3, and downregulated KLF2 and its putative downstream target GPX4. In vivo, KLF2 overexpression alleviated S. aureus-induced bone loss, inflammation, and ferroptosis, while promoting angiogenesis and osteogenesis, in part through modulation of GPX4. CONCLUSION: This study highlights KLF2 as a potential protective factor in S. aureus-induced osteomyelitis, possibly by regulating GPX4 and ferroptosis.