The study combined an in vivo rat model with sophisticated cellular assays to dissect the pathology. Male Sprague-Dawley rats received intraperitoneal injections of ferric ammonium citrate three times per week for four consecutive weeks, establishing systemic iron overload confirmed by Prussian blue staining of liver and femoral tissues and by elevated serum iron levels. High-resolution micro-computed tomography of the femora revealed devastating structural consequences: bone volume fraction was significantly decreased, trabecular number reduced, trabecular separation widened, and the structure model index elevated, indicating a transition from robust plate-like trabeculae to fragile rod-like struts. Cortical thickness also showed a declining trend. Serum biochemical analysis revealed significantly decreased levels of osteocalcin, alkaline phosphatase, and vitamin D, confirming that iron overload had fundamentally altered bone metabolic status.

Histological examination corroborated these quantitative findings. Hematoxylin and eosin staining showed sparse trabeculae with enlarged inter-trabecular spaces, while Masson’s trichrome staining revealed reduced mature bone matrix and loosely structured collagen networks, suggesting disrupted bone turnover balance and potentially enhanced resorption. Tartrate-resistant acid phosphatase staining demonstrated that both the number and average area of osteoclasts were increased in iron-overloaded rats, and immunofluorescence for MMP9, an osteoclast marker protein, was significantly elevated. Conversely, the osteogenic markers osteopontin and forkhead box protein O1 were markedly weakened in bone tissue, indicating that iron overload simultaneously suppressed bone formation and promoted bone resorption.

To identify the cell death mechanism underlying osteoblast depletion, the researchers examined key ferroptosis regulators in bone tissue. The pro-ferroptotic protein ACSL4 was significantly increased, while the anti-ferroptotic proteins GPX4 and SLC7A11 were significantly decreased, collectively indicating enhanced lipid peroxidation and impaired cellular defense against ferroptotic death. These molecular shifts provide a mechanistic explanation for the observed reductions in osteogenic markers and impaired bone formation. Simultaneously, the M1 macrophage marker CD86 was significantly elevated while the M2 marker arginase-1 was markedly decreased, and serum levels of the pro-inflammatory cytokine TNF-α were increased while the anti-inflammatory cytokine IL-10 was reduced, confirming that iron overload drives both local and systemic inflammatory responses.

In vitro experiments using MC3T3-E1 mouse embryonic osteoblast precursor cells confirmed the ferroptotic mechanism. Treatment with 300 micromolar ferric ammonium citrate for 48 hours reduced cell viability to approximately 70 percent of control levels and was selected as the standard iron overload condition. Flow cytometry revealed significant G0/G1 phase cell cycle arrest, which was effectively reversed by pretreatment with the ferroptosis inhibitor ferrostatin-1 or the iron chelator deferoxamine. Critically, neither the pan-caspase apoptosis inhibitor Z-VAD-FMK nor the autophagy inhibitor hydroxychloroquine provided statistically significant protection, confirming that ferroptosis, rather than apoptosis or autophagic death, is the dominant pathway of iron overload-induced osteoblast injury.

Biochemical assays painted a detailed portrait of the ferroptotic cascade. Iron-overloaded cells exhibited dramatically increased malondialdehyde content, a lipid peroxidation end product, alongside markedly depleted reduced glutathione and substantially elevated intracellular ferrous ion levels. Quantitative real-time PCR and Western blot analyses revealed significant downregulation of GPX4 and SLC7A11, the core antioxidant defense proteins, while the iron uptake receptor TFRC was significantly upregulated. Intracellular reactive oxygen species levels, measured by the DCFH-DA fluorescent probe, increased sharply under iron overload and were significantly attenuated by ferroptosis inhibition or iron chelation. Transmission electron microscopy revealed the ultrastructural hallmarks of ferroptotic mitochondria: significant atrophy, increased membrane density, and reduced cristae, contrasting sharply with the normal morphology observed in control cells.

Transcriptome sequencing identified a critical regulatory hub in this pathological cascade. Differential expression analysis revealed 873 downregulated and 724 upregulated genes following iron overload, with gene set enrichment analysis confirming significant activation of ferroptosis-related pathways. Among eight candidate genes validated by quantitative PCR, only Atp6v0d2, which encodes a V-ATPase subunit regulating lysosomal acidification and intracellular iron homeostasis, satisfied the stringent criteria of concordant expression trends and simultaneous reversal by both ferroptosis inhibition and iron chelation. Plasmid-mediated knockdown of Atp6v0d2 using short hairpin RNA constructs reduced iron overload-induced reactive oxygen species generation by 56 and 53 percent in the two knockdown lines, decreased malondialdehyde levels by 45 and 40 percent, and substantially restored alkaline phosphatase activity and mineralized nodule formation, demonstrating that Atp6v0d2 is a central mediator of iron overload-induced ferroptosis in preosteoblastic cells.

The second arm of the dual-pathway model involves macrophage-driven inflammatory disruption. RAW264.7 macrophages treated with iron overload showed dose-dependent increases in the CD86/CD206 ratio, indicating polarization toward the pro-inflammatory M1 phenotype. Transcriptional analysis revealed upregulation of M1 markers including CD86, inducible nitric oxide synthase, IL-6, and TNF-α, alongside downregulation of M2 markers. Conditioned medium from these iron-overloaded macrophages, termed inflammatory conditioned medium, exerted devastating effects on MC3T3-E1 cells: it induced S-phase cell cycle arrest, promoted apoptosis at rates exceeding 56 percent, and severely impaired osteogenic differentiation and mineralization. Notably, these effects were not reversed by ferroptosis inhibition or iron chelation, indicating that the mechanism is distinct from direct iron toxicity and operates through soluble inflammatory mediators.

Transcriptomic analysis of inflammatory conditioned medium-treated cells revealed dramatic enrichment of immune and inflammatory pathways, with the IL-17 signaling pathway showing the highest enrichment significance among all inflammation-related gene sets. A small-molecule IL-17 pathway antagonist, which disrupts the IL-17A/IL-17RA interaction by binding the central dimer interface pocket, was deployed to test causality. Pretreatment with 331 nanomolar antagonist for 6 hours significantly reversed the inhibitory effect of inflammatory conditioned medium on cell viability, reduced apoptosis rates from 66.91 to 32.85 percent, attenuated reactive oxygen species generation by 31.4 percent, and partially restored osteogenic differentiation and mineralization. On the osteoclast side, the antagonist reduced the average osteoclast area by approximately 59.8 percent and bone resorption area by 44.4 percent, confirming that IL-17 signaling drives both osteoblast suppression and osteoclast hyperactivation through the canonical NF-κB pathway, as evidenced by reduced IκBα expression alongside increased phosphorylated P65 and phosphorylated IκBα.

The implications of this dual-pathway model extend well beyond academic understanding. Current anti-resorptive agents, including bisphosphonates and RANKL monoclonal antibodies such as denosumab, constitute the mainstay of clinical osteoporosis management but carry risks of osteonecrosis of the jaw and atypical fractures with long-term use. The identification of Atp6v0d2 and IL-17 signaling as core mediators offers a dual-target strategy that may simultaneously attenuate ferroptotic osteoblast death and suppress excessive osteoclast activation. The researchers acknowledge that their conclusions remain at the level of functional validation, lacking complementary overexpression experiments, dynamic lysosomal pH monitoring, and ferritinophagy flux analysis. Moreover, all mechanistic experiments used the MC3T3-E1 preosteoblast cell line rather than primary osteoblasts, and the acute ferric ammonium citrate administration model differs from the chronic iron accumulation observed in clinical scenarios. Only male rats were included, leaving potential estrogen-iron interactions unexplored. Nevertheless, the convergence of ferroptosis biology and osteoimmunology in this study provides a compelling framework for understanding bone metabolic dysregulation under iron overload conditions and may ultimately inform combination therapeutic strategies for a patient population with few pharmacological options.

Subject of Research: Iron overload disrupts bone homeostasis via dual pathways: inducing ferroptosis of preosteoblastic cells and shaping a proinflammatory immune milieu

Article Title: Iron overload disrupts bone homeostasis via dual pathways: inducing ferroptosis of preosteoblastic cells and shaping a proinflammatory immune milieu

Article References: Pan, Y., Zhao, R., Zhang, C., Shi, F., Wu, H., & Xiao, D. (2026). Iron overload disrupts bone homeostasis via dual pathways: inducing ferroptosis of preosteoblastic cells and shaping a proinflammatory immune milieu. Frontiers in Pharmacology, 17, Article 1905654. https://doi.org/10.3389/fphar.2026.1905654

Image Credits: AI Generated

DOI: 10.3389/fphar.2026.1905654

Keywords: Iron, overload, disrupts, bone, homeostasis, dual, pathways, inducing, ferroptosis, preosteoblastic, cells, shaping