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.

探究高迁移率族蛋白B1（high mobility group box 1 protein, HMGB1）与5-羟色胺受体7（5-hydroxytryptamine receptor 7, 5-HT7R）对小鼠抑郁样行为的调控作用及潜在机制。 将5-HT7R敲除（5-HT7R^−/−）小鼠与同窝野生型（WT）小鼠分为以下4组：WT/空白病毒（AAV-Scramble）、5-HT7R^−/−/AAV-Scramble、WT/HMGB1过表达病毒（AAV-HMGB1）和5-HT7R^−/−/AAV-HMGB1组，对AAV-Scramble组与AAV-HMGB1组小鼠分别进行空白病毒与AAV-HMGB1海马区注射。3周后通过抑郁样行为学评价小鼠的抑郁状态，利用免疫荧光染色检测小鼠海马区的神经元损伤，使用商业试剂盒测定小鼠海马区还原性谷胱甘肽与氧化型谷胱甘肽比值（GSH/GSSG）、Fe²⁺与丙二醛（MDA）的水平以及超氧化物歧化酶（SOD）的活性。此外，通过Western blot和ELISA检测小鼠海马区5-HT7R/环磷酸腺苷（cAMP）/蛋白激酶A（PKA）与核因子E2相关因子2（Nrf2）/胱氨酸-谷氨酸交换体（xCT）/谷胱甘肽过氧化物酶4（GPX4）信号通路水平，采用免疫荧光双染检测小鼠海马区M2型小胶质细胞铁死亡。最后，利用qRT-PCR检测小鼠海马区促炎因子〔白细胞介素（IL）-1β、肿瘤坏死因子-α（TNF-α）〕与抗炎因子〔IL-10、精氨酸酶-1（Arg-1）〕的mRNA水平。 与AAV-Scramble组相比，海马区HMGB1过表达诱导小鼠抑郁样行为与神经元损伤（P<0.01），而5-HT7R敲除减轻上述病理表型（P<0.01）。与AAV-Scramble组相比，HMGB1过表达降低小鼠海马区GSH/GSSG比值与SOD活性（均P<0.01），同时上调Fe²⁺与MDA水平（均P<0.01），而5-HT7R敲除改善上述铁死亡相关指标（P<0.01）。与AAV-Scramble组相比，HMGB1过表达上调小鼠海马区5-HT7R表达（P<0.01），同时下调cAMP/PKA和Nrf2/xCT/GPX4信号通路（P<0.01），而5-HT7R敲除改善上述信号通路蛋白的降低（P<0.01）。与AAV-Scramble组相比，HMGB1过表达上调小鼠海马区小胶质细胞上5-HT7R与铁蛋白重链（FTH）的表达（P<0.01），下调Nrf2的表达（P<0.01），同时诱导FTH与CD206的共定位（P<0.01）；而5-HT7R敲除逆转上述小胶质细胞上FTH与Nrf2的表达变化（P<0.01），同时降低FTH与CD206的共定位（P<0.01）。此外，HMGB1过表达诱导小鼠海马区小胶质细胞极化为M1型与M2型（P<0.01），上调促炎因子（IL-1β、TNF-α）与抗炎因子（IL-10、Arg-1）mRNA水平（P<0.01），而5-HT7R敲除减轻上述小胶质细胞M1型极化以及降低上述促炎因子的mRNA水平（P<0.01），同时促进小胶质细胞M2型极化以及上调上述抗炎因子的mRNA水平（P<0.01）。 HMGB1通过激活5-HT7R，下调cAMP/PKA/AKT/Nrf2/GPX4信号通路，进而介导M2型小胶质细胞铁死亡，最终促进小鼠抑郁样行为。

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.