Background: Compelling evidence has implicated osteoblast ferroptosis as a critical contributor to the pathogenesis of glucocorticoid-induced osteoporosis (GIOP). However, the underlying regulatory mechanisms remain poorly understood. Methods: In the glucocorticoids (GCs)-induced GIOP rat model, both osteoblast dysfunction and ferroptosis markers were assessed. Ferroptosis was inhibited with deferoxamine (DFO). Transcriptomic profiling was performed to analyze the correlation between mitophagy and SIRT3 levels. In MC3T3-E1 cells exposed to GCs, we examined key mitophagy markers PINK1 and PARKIN, mitochondrial function, and SIRT3 expression. Treatments included DFO, mitophagy inhibitors (Mdivi-1), and SIRT3 agonists (Nicotinamide riboside chloride). Results: In the GIOP rat model, significant osteoblast dysfunction and elevated ferroptosis markers were observed. Although DFO treatment inhibited ferroptosis, it failed to restore osteogenesis, suggesting the involvement of additional regulatory mechanisms in osteogenic function regulation. Transcriptomic profiling highlighted a robust correlation between mitophagy and SIRT3 levels in GIOP. In GC-exposed MC3T3-E1 cells, key mitophagy markers PINK1 and PARKIN were upregulated, mitochondrial function was impaired, and SIRT3 expression was significantly reduced. Notably, while DFO treatment did not restore mitochondrial homeostasis, the application of Mdivi-1 (mitophagy inhibitor) and Nicotinamide riboside chloride (SIRT3 agonists) effectively alleviated ferroptosis and restored mitochondrial function. Conclusions: SIRT3 regulates ferroptosis by inhibiting excessive mitophagy in osteoblasts, providing a novel mechanistic pathway for mitigating GIOP. These findings suggest that SIRT3 represents a critical regulator of mitophagy-dependent ferroptosis and a potential therapeutic target for GIOP.

Treatment of steroid-induced osteonecrosis of the femoral head (SIONFH) is challenging. Due to the limited understanding of its molecular mechanisms, investigating the potential mechanisms of ferroptosis will shed light on SIONFH and provide directions for treating this disease. The GSE123568 dataset was utilized to apply various bioinformatics methodologies to identify ferroptosis-related hub genes (FRHGs). Subsequently, the importance of these genes and the reliability of the results were confirmed using protein data-independent acquisition (DIA) and cell experiments. Finally, we assessed the correlation between FRHG expression and immune cell infiltration. Thirty-one hub genes were identified and validated by constructing a protein-protein interaction network and subsequent screening using experimentally determined interactions. These 31 hub genes were enriched in immunity, the AMPK signaling pathway, and the Toll-like receptor signaling pathway. Next, we identified a diagnostic marker comprising two ferroptosis-related genes, NCF2 and SLC2A1. The differential expression of these two genes in healthy and necrotic regions was confirmed by protein DIA analysis. Cell experiments verified the link between FRHGs and ferroptosis and preliminarily explored the potential mechanism of the antioxidant vitexin in promoting osteogenic differentiation in cells. The diagnostic efficiency of these two markers was confirmed by receiver operating characteristic curve (ROC) curves, yielding an area under the curve of 1.0. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) indicated enrichment of FRHGs in the superoxide anion and HIF-1 signaling pathways. A significant correlation was observed between FRHGs and various immune cell populations. NCF2 and SLC2A1 are promising ferroptosis-related diagnostic biomarkers of SIONFH. Concurrently, we embarked on a preliminary investigation to elucidate the potential mechanism underlying the promotion of osteogenic differentiation by the antioxidant vitexin. Moreover, these biomarkers are associated with distinct immune cell populations.

Immune cells exert influence on skeletal homeostasis, and re-establishing metabolic equilibrium is central to the management of steroid-induced osteonecrosis of the femoral head (SIONFH). However, the osteoimmune mechanisms underpinning this pathology remain largely under-researched. This study aimed to delineate these mechanisms, thereby informing early therapeutic strategies to restore bone homeostasis. We identified the key immune cell subsets associated with SIONFH, and investigated their upstream regulatory factors and downstream effector molecules by integrating multiomics analyses (samples derived from human peripheral blood, femoral head tissues, and rat peripheral blood), mining of the GSE123568 dataset, and performing in vivo and in vitro experiments. Clinical transcriptomics revealed enrichment of immune-related pathways, with ssGSEA consistently highlighting activated CD8⁺ T cells and Th1 cells. Correlation analyses demonstrated a stronger association between T cells and osteoblasts than osteoclasts (r = 0.8728 vs r = -0.065). Staphylococcal enterotoxin C2 (sec2) activated T cells both in vitro and in vivo, thereby augmenting osteogenic markers, enhancing bone marrow stromal cell (BMSC) differentiation, and elevating T cell-derived interferon gamma (IFNγ). Mechanistically, IFNγ facilitated signal transducer and activator of transcription 1 (STAT1)-runt-related transcription factor 2 (Runx2) dissociation and Runx2 nuclear translocation, thereby potentiating osteogenesis. Multiomics and functional studies identified neuropilin 1 (NRP1) as a critical IFNγ/T cell-associated protein, downregulated by staphylococcal enterotoxin C2 (sec2). Pharmacological activation of NRP1 with certepetide validated its role in modulating IFNγ secretion and downstream signalling. Glucocorticoids suppress IFNγ production in T cells and weaken NRP1/IFNγ-driven osteogenic differentiation. Ultimately, sec2 reshaped antigen-presenting cell-mediated regulation of the T cell-NRP1/IFNγ axis, promoting bone formation and mitigating SIONFH. Glucocorticoids suppress T cell-derived IFNγ via NRP1 upregulation, thereby impairing Runx2 nuclear translocation and BMSC osteogenesis. Sec2 counteracts this cascade through antigen-presenting cell modulation, restoring the NRP1/IFNγ axis to preserve femoral head integrity.

Background: Glucocorticoid-induced oxidative stress and impaired osteogenic differentiation are key pathological features of steroid-induced osteonecrosis of the femoral head (SIONFH); however, the molecular mechanisms linking oxidative stress to impaired osteogenesis remain poorly understood. Methods: We integrated multi-omics sequencing data from clinical samples and mouse models to identify key genes associated with OS and osteogenic differentiation in SIONFH. Functional enrichment and interaction network analyses were performed to explore the underlying mechanisms. In vitro and in vivo experiments were further conducted to evaluate the effects of recombinant SUZ12 protein on osteogenic differentiation and SIONFH. Results: Suppressor of zeste 12 protein (SUZ12) emerged as a potential hub gene linking oxidative stress to impaired osteogenesis. Functional enrichment and interaction network analyses suggested that SUZ12 may contribute to SIONFH pathogenesis through its involvement in polycomb repressive complex 2 (PRC2). Mechanistically, oxidative stress downregulated SUZ12 expression. In vitro and in vivo experiments demonstrated that treatment with recombinant SUZ12 protein impaired osteogenic differentiation and exacerbated SIONFH. Conclusions: Collectively, our findings identify SUZ12 as a candidate mediator linking oxidative stress to impaired osteogenesis in SIONFH and reveal a previously unrecognized mechanism whereby oxidative stress promotes SIONFH progression by downregulating SUZ12 and subsequently suppressing osteogenic differentiation. These findings advance our understanding of oxidative stress-mediated osteogenic impairment in SIONFH and highlight SUZ12 as a potential therapeutic target.

Background: Osteonecrosis of the femoral head (ONFH) is a challenging global health issue with an unclear pathogenesis, complicating the development of effective treatment strategies. Bone marrow edema (BME) is a critical imaging indicator of ONFH progression, yet its underlying mechanisms remain poorly understood. Methods: Bioinformatics was employed to identify gene characteristic of BME in ONFH patients. Expression of PTGS2 was validated in these patients through Western blot and ELISA assays. Clinical relevance was assessed by analyzing the correlation between PTGS2 expression levels and pain severity as well as the timing of total hip replacement surgery. In addition to GSE74089, we externally validated PTGS2 in an independent GEO cohort (GSE123568, human serum; GPL15207) using single‐gene receiver operating characteristic (ROC) analysis. Results: Six hundred and eighty‐eight overlapping targets were identified for ONFH and BME, with 15 key targets shared with XLGBC, including PTGS2, IGFBP3, MCL1, TNF, F7, PLA2G4A, PRKCA, MMP1, and PTGER3. GO and KEGG enrichment analyses indicated that XLGBC exerts its effects mainly through pathways related to inflammation, pain, angiogenesis, and bone metabolism, notably involving VEGF signaling, arachidonic acid metabolism, and MAPK pathways. Molecular docking revealed strong binding between XLGBC compounds and the target genes. ELISA results indicated that higher PTGS2 levels correlated with increased pain severity in ONFH patients, and Western blot analysis showed significantly elevated PTGS2 in ONFH patients compared to controls, with levels decreasing after XLGBC treatment. Patients with higher PTGS2 expression showed shorter times to hip replacement surgery, suggesting faster disease progression. In the external cohort (GSE123568), PTGS2 showed good diagnostic discrimination for ONFH versus controls (AUC = 0.86), supporting the robustness of our bioinformatics findings. Conclusions: PTGS2 is an important gene in ONFH with BME, influencing pain and disease progression. Monitoring PTGS2 expression may help to assess symptom severity and inform surgical timing in ONFH patients.