Iron overload-induced ferroptosis of osteoblasts as a potential therapeutic target for osteoporosis

Iron overload-induced ferroptosis of osteoblasts as a potential therapeutic target for osteoporosis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Iron overload-induced ferroptosis of osteoblasts as a potential therapeutic target for osteoporosis Shuangqing Li, Shan Wan, Yanting He, Baochen Chong, Zhili Xia, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6072367/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Osteoporosis is a systemic skeletal disorder marked by reduced bone mass, compromised bone microarchitecture, heightened fragility, and an increased risk of fractures. Fractures resulting from osteoporosis are a leading cause of mortality and disability among the elderly. Ferroptosis is an emerging form of programmed cell death that occurs due to unregulated iron-dependent lipid peroxidation. Our study reveals that high-iron exposure triggers ferroptosis in osteoblasts through the FTH1/FTL pathway, as demonstrated by both in vivo and in vitro experiments. Ferroptotic osteoblasts initiate a co-stimulatory pathway that fosters osteoclast differentiation, culminating in an osteoporotic phenotype in mice. We propose that the high-iron intervention in mice could be utilized as a novel model for replicating clinical osteoporosis, and that inhibiting ferroptosis in osteoblasts may represent a promising therapeutic strategy for the treatment of osteoporosis. Overall, our findings offer fresh perspectives on the pathogenesis of OP and identify potential new targets for the clinical management of this condition. Health sciences/Diseases/Endocrine system and metabolic diseases Biological sciences/Chemical biology/Biophysical chemistry Osteoporotic fracture ferroptosis osteoblast proteomics phosphoproteomics Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Osteoporosis (OP), a systemic bone disease, is defined by diminished bone density, microarchitectural degradation, augmented brittleness, and an elevated propensity for fractures(1–3). With the intensifying aging population in China, the incidence of osteoporosis has surged markedly. National epidemiological studies reveal that 32% of individuals aged over 65 in China suffer from osteoporosis, affecting approximately 90 million people at present(4). Osteoporotic fractures, or fragility fractures are grave outcomes of osteoporosis, inflicting substantial harm and serving as major contributors to morbidity and mortality among the elderly, despite the paucity of efficacious pharmaceuticals and interventions. Within a year post-hip fracture, 20% of patients succumb to assorted complications, around half become incapacitated, and their quality of life deteriorates considerably(5). The healthcare costs associated with osteoporosis and fractures exact a considerable financial toll on families and society alike(6). Hence, elucidating the attributes of skeletal senescence and unveiling its underlying molecular mechanisms, identifying novel targets for modulating bone metabolism, and devising innovative therapeutics grounded in these discoveries are imperative endeavors aimed at forestalling disability in the geriatric demographic and enhancing their quality of life. Iron holds a pivotal position in living organisms, and its metabolic equilibrium is intricately linked to a myriad of diseases(7–14). The burgeoning advancements in omics technologies have recently furnished novel vistas and methodologies for investigating iron metabolism. Proteomics, for instance, delves into the expression, modification, and functional aspects of proteins, thereby unveiling the metamorphoses in iron metabolism-associated proteins across diverse physiological and pathological milieus. Investigations have unearthed that both iron surfeit and insufficiency engender intricate proteomic shifts, underscoring the paramountcy of proteomics in the realm of iron metabolism research. Metabolomics zeroes in on the holistic fluctuations in bodily metabolites to pinpoint metabolic aberrations precipitated by iron excess or dearth. Research divulges that iron surplus, or deficit is inextricably bound to alterations in plasma metabolic profiles, notably influencing the metabolism of branched-chain amino acids, heme catabolism derivatives, fatty acids, and cholesterol. Iron homeostasis exerts a profound impact on the epigenetic embellishments of proteins, encompassing DNA methylation, histone modifications, and miRNA. Disruptions in iron homeostasis may catalyze anomalous epigenetic modifications of proteins, thereby instigating related maladies(7–9). Ferroptosis, a nascent form of cellular demise(15, 16), intertwines with the pathophysiological trajectories of sundry ailments(17–20). The deployment of omics techniques to scrutinize ferroptosis furnishes a vital instrument for comprehending the nexus between iron metabolism and cell death(21). In summation, the utilization of proteomics, metabolomics, and post-translational modification omics has enriched our comprehension of iron metabolism and proffers novel insights and strategies for diagnosing and managing disorders affiliated with iron homeostasis imbalances. In this study, we collected femoral neck bone tissues from patients with different bone mineral density (BMD) levels and observed that as BMD declined from normal to osteopenia and then to osteoporosis, there was a progressive accumulation of iron within the bone tissue. This raises the question of whether this accumulated iron might influence bone metabolism by modulating the phosphorylation of proteins integral to this process. To explore this further, we subjected mice to a high-iron intervention and observed that the elevated iron levels impaired bone microarchitecture, suppressed osteoblast differentiation, enhanced osteoclast differentiation, markedly increased the iron content within the bone tissue, and induced ferroptosis in osteoblasts. Moreover, when we cultured primary bone marrow cells from mice under high-iron conditions, we witnessed a suppression of osteogenic differentiation, a promotion of osteoclastic differentiation, and the occurrence of ferroptosis in the primary osteoblasts. Proteomics and phosphoproteomics analyses uncovered a significant role of FTH1/FTL in mediating ferroptosis in osteoblasts and facilitating osteoclast differentiation, possibly through a co-stimulatory pathway in response to high-iron exposure. Drawing on these discoveries, we suggest that iron overload-induced ferroptosis in osteoblasts may represent a potential therapeutic target for the treatment of osteoporosis. Material and Methods Collection and Grouping Methods of Clinical Samples The collection and use of clinical samples were approved by the Biomedical Ethics Committee of West China Hospital of Sichuan University (Ethics Approval No: 2023 − 1307). For short, we recruited patients diagnosed with unilateral femoral neck fracture who required hip replacement surgery. Each patient underwent a BMD examination using a dual-energy X-ray absorptiometry (DXA, GE Healthcare, Lunar iDXA, ME + 212243) 1–3 days before the hip replacement surgery. Based on the T-scores of the lumbar spine (L1-L4) as measured by DXA, the samples were divided into three groups: normal bone density (T-score ≥ -1.0), osteopenia (-1.0 > T-score > -2.5), and osteoporosis (T-score ≤ -2.5). Exclusion criteria ruled out individuals with tumor, hip dysplasia, infection, necrosis of the femoral head, osteomalacia, renal insufficiency, history of hip surgery, coagulation disorders, history of anti-osteoporosis treatment, and diseases or medications affecting bone metabolism, such as parathyroid, thyroid, adrenal disease, diabetes, as well as treatment with steroid or bisphosphonate. Following informed consent, the study comprised femoral neck bone tissues from 13 participants with normal BMD, 12 with osteopenia, and 15 with osteoporosis. The mean T-scores for the lumbar spines (L1-L4) across the three groups were − 0.07 ± 0.77, -1.10 ± 1.34, and − 2.97 ± 0.91, respectively (Table 1 ). Table 1 Demographics and characteristics of the clinical samples Characteristics Normal N = 13 Osteopenia N = 12 Osteoporosis N = 15 P value Age, years, mean ± SD 47.4 ± 11.2 64.6 ± 13.7 74.1 ± 12.0 < 0.001 Male, N (%) 7 (53.8%) 7 (58.3%) 0 (0%) 0.002 Female, N (%) 6 (46.2%) 5 (41.7%) 15 (100%) 0.002 BMI, kg/m 2 , mean ± SD 24.4 ± 3.1 24.9 ± 2.8 22.3 ± 3.0 0.063 BMD T-score, mean ± SD Lumbar spine -0.07 ± 0.77 -1.10 ± 1.34 -2.97 ± 0.91 < 0.001 Total hip -0.07 ± 0.79 -1.41 ± 0.65 -2.30 ± 0.98 < 0.001 Femoral neck 0.08 ± 1.08 -1.07 ± 1.31 -2.28 ± 1.51 < 0.001 Detection of Iron Content Accurately weigh a predetermined quantity of bone tissue and deposit it into a microwave digestion vessel. Introduce 2mL of concentrated nitric acid into the vessel, then position both the digestion vessel and its corresponding support liner within the microwave digestion apparatus. Initiate the microwave-assisted digestion process at 120°C for a duration of 60min. Upon completion of digestion, carefully decant the resultant solution into a beaker, subject it to evaporation to remove excess acid, followed by a reflux procedure lasting 60min. Subsequently, dilute the solution to achieve a final volume of 25mL using ultrapure water, and quantify the iron concentration via inductively coupled plasma mass spectrometry (ICP-MS). For osteoblast samples exposed to high iron intervention, isolate 300µg of protein, which should be diluted to a consistent volume utilizing RIPA lysis buffer, and subsequently concentrated until complete dryness is attained. Thereafter, incorporate 2mL of nitric acid into the dried sample and proceed with the identical sequential steps as those employed for the bone tissue analysis. Animal Experiments All animal experiments were conducted following the guidelines set forth by the Institutional Animal Care and Use Committee of West China Hospital, Sichuan University (Approval No. 20240311021), adhering strictly to the principles outlined in the Guide for the Care and Use of Laboratory Animals. Seven-week-old female C57BL/6 mice were purchased from GemPharmatech Co., Ltd. (Nanjing, China) and maintained in a standard light-dark cycle and provided with free access to water and the corresponding diet. After one week of adaptive feeding, the mice were randomized into two distinct dietary cohorts: a standard diet (SD) group and a rich-iron diet (RFeD) group, comprising 20 mice per cohort. The SD group received a diet supplemented with 37ppm iron, whereas the RFeD group was administered a diet enriched with 350ppm iron. At the conclusion of one and two months of dietary intervention, ten mice from each group were euthanized for the purposed of collecting biological specimens. Throughout the experimental phase, the body weight and caloric consumption of the mice were recorded biweekly. Micro-computed tomography (µCT) analysis Post-intervention with either RFeD or SD, the right femur of the mice was harvested. Careful excision of soft tissues including muscles and fasciae was performed, followed by thorough rinsing of the femurs with chilled phosphate-buffered saline (PBS). Subsequently, the bones were preserved via overnight fixation in paraformaldehyde. High-resolution µCT scans were executed employing a vivaCT80 scanner (Scanco Medical, Switzerland), facilitated by the Analytical and Testing Center at Sichuan University. Scan settings comprised an energy/intensity of 55kV and 145µA, resulting in 8W power output, with a voxel resolution of 10µm. The scanned area encompassed the metaphysis and diaphysis of the right femur. Regions of interest (ROIs) were delineated to include 100 layers of trabecular bone situated directly beneath the growth plate, along with 100 layers of cortical bone derived from the diaphysis. Three-dimensional reconstructions and analyses were carried out utilizing Scanco’s proprietary analytical software suite, applying lower and upper threshold values of 170 and 1000, respectively. Parameters assessed for trabecular bone encompassed bone volume fraction (BV/TV), trabecular number (Tb.N), trabecular thickness (Tb.Th), trabecular separation (Tb.Sp), connectivity density (Conn.D), and structural model index (SMI). For cortical bone, evaluated metrics consisted of cortical BV/TV, cortical thickness (Ct.Th), and the ratio of bone surface area to bone volume (BS/BV). Histopathological Analysis Following the intervention with either RFeD or SD, the left tibia of the mice was collected. Meticulous removal of soft tissues, such as muscles and fasciae, was undertaken, followed by a thorough rinse of the tibias with chilled PBS. Subsequently, the bones underwent preservation through overnight fixation in paraformaldehyde. On the following day, the fixed bone tissue was washed under running water for 10 minutes, then immersed in EDTA decalcification solution for 21 days, with the solution being replaced every 3 days. Post-decalcification, the bone tissue was embedded in paraffin and sliced into sections measuring 5µm in thickness. These sections were stained with hematoxylin and eosin (HE) and tartrate-resistant acid phosphatase (TRAP, Wako TRAP/ALP staining kit, Wako Pure Chemical Industries, Ltd., Japan, Catalog No. 294-67001) to examine the impact of RFeD intervention on osteoblast and osteoclast differentiation. Immunohistochemical staining was conducted to assess the expression of ferritin using an anti-ferritin antibody (Abcam, British, Catalog No. ab183781). Additionally, the TUNEL assay kit (Promega, Wisconsin, United States, Catalog No. G3250) was utilized to investigate the apoptosis of bone cells. Serum Ferritin Detection Serum ferritin concentrations in mice were quantified utilizing an enzyme-linked immunosorbent assay (ELISA) kit provided by Kenuodi Biotechnology Co., Ltd. (Quanzhou, China; Catalog No. SU-B21204), adhering meticulously to the manufacturer’s protocols. The resultant ferritin levels were derived from the established standard curve. Lipid Peroxidation-MDA Detection To assess lipid peroxidation in plasma, bone, and osteoblasts of the mice, we employed the Lipid Peroxidation Assay Kit (Beyotime, S0131, Shanghai, China). Following the acquisition of bone tissue and osteoblast protein, the protein concentration was measured via the BCA method. Plasma samples were utilized directly for malondialdehyde (MDA) quantification. The protocol for MDA detection was rigorously adhered to the guidelines provided by the manufacturer. Culture and Induction of Mouse Primary Bone Marrow Cells The culture and induction of mouse primary bone marrow cells were carried out as described in previous studies(22–26). Briefly, the isolated bone marrow cells were resuspended in aMEM medium containing 10% fetal bovine serum (FBS, Gibco). These cells were cultured in an incubator maintained at 37°C with 5% CO 2 . On the following day, the non-adherent cells were gently collected, centrifuged, and subsequently resuspended in fresh aMEM medium containing 10% FBS and 30ng/mL of recombinant mouse macrophage colony-stimulating factor (M-CSF, R&D System, Minnesota, United States, Catalog No. 416-ML-010). After an additional 24-hour incubation period, the adherent cells obtained were identified as bone marrow-derived macrophages (BMDMs). These BMDMs could be subjected to further osteoclast differentiation induction, with or without the inclusion of high iron (at a concentration of 5µM). Meanwhile, the adherent cells remaining in the initial culture dish were designated as bone marrow mesenchymal stem cells (BMSCs, P0). Upon subsequent passaging and purification processes (approximately reaching P3), these BMSCs became suitable for osteoblast differentiation induction, again with or without the addition of high iron (at a concentration of 20µM). TRAP, ALP Staining and Relative Quantification of ALP Activity First, digested the adherent BMDMs, centrifuged the resulting suspension, and then resuspended the cells in aMEM medium supplemented with 10% FBS and 30ng/mL of recombinant mouse M-CSF. Next, seeded the cells into 48-well plates and 6-well plates at cell densities of 30 000 cells per well and 200 000 cells per well, respectively. On the following day, initiated osteoclast differentiation by treating the cells with 30ng/mL of mouse M-CSF and 50ng/mL of recombinant mouse receptor activator for nuclear factor κB ligand (RANKL, R&D system, Catalog No. 462-TEC-010), companied with or without high iron (5µM). Replaced the culture medium every two days and observed the development of fused multinucleated osteoclasts under a microscope. Seven days post-induction, we performed TRAP staining on the osteoclasts residing in the 48-well plates using the TRAP staining kit (Sigma-Aldrich, Missouri, United States, 387A-1KT). Captured digital images of the stained osteoclasts. Utilized ImageJ software (developed by the National Institutes of Health) to quantify the area occupied by multinucleated (> 3 nuclei) TRAP-positive osteoclasts. Subsequently, we employed Prism software for statistical analysis and graphical representation of the data. Meanwhile, the osteoclasts cultivated in the 6-well plates can be lysed to extract proteins for assessing the impact of high iron exposure on the expression of NFATc1, a pivotal protein involved in osteoclast differentiation. Analogously, once BMSCs reach passage three (P3), they should be digested, centrifuged, and then resuspended in aMEM medium enriched with 10% FBS. Plated the cells at densities of 50 000 cells per well in 24-well plates and 200 000 cells per well in 6-well plates. Commence osteoblast differentiation by exposing the cells to 50µg/mL ascorbic acid and 10mM β-glycerophosphate, with or without high iron supplementation (20µM). As previously mentioned, refreshed the culture medium every two days throughout the experiment. Upon completion of seven days of induction, we conducted alkaline phosphatase (ALP) staining on the osteoblasts within the 24-well plates using the ALP staining kit (Sigma-Aldrich, 86R-1KT). Record visual documentation of the stained osteoblasts. Measure the relative ALP activity using appropriate methods. Finally, harvest the osteoblasts from the 6-well plates for protein extraction to evaluate the influence of high iron administration on the expression of RUNX2, which is crucial for osteoblast differentiation. TUNEL Assay, C11-BODIPY and FerroOrange Staining The apoptosis of osteoblasts in high-iron-treated mice was assessed using a One-Step TUNEL Apoptosis Detection Kit (Beyotime, C1086) strictly according to the manufacturer’s instructions. Briefly, primary mouse BMSCs were subjected to osteogenic induction and high-iron intervention, then fixed with 4% paraformaldehyde for 30min. After washing with PBS, the cells were permeabilized with PBS containing 0.3% Triton X-100 at room temperature for 5min. Subsequently, 50µL TUNEL working solution was added, and the samples were incubated at 37°C in the dark for 60min. After washing with PBS, the cells were stained with Hoechst 33342 (Thermo Scientific, Massachusetts, United States, 62249) solution at room temperature for 10min. Lipid peroxidation was evaluated using a C11-BODIPY probe (Beyotime, S0043). Following osteogenic induction and high-iron treatment, the osteoblasts were incubated with C11-BODIPY working solution (2µmol/L) at 37°C in the dark for 15min. After washing with PBS, the cells were stained with Hoechst 33342 solution at room temperature for 10min. Intracellular Fe²⁺ levels were detected using a FerroOrange probe (Dojindo, Tokyo, Japan, F374). After osteogenic induction and high-iron treatment, the osteoblasts were washed with PBS and incubated with FerroOrange working solution (1µmol/L) at 37°C in the dark for 30min, followed by staining with Hoechst 33342 solution at room temperature for 10min. Images of the above stained cells were acquired and analyzed using a high-content imaging system (Opera Phenix Plus, PerkinElmer). Western Blot Mature osteoblasts (OBs) and osteoclasts (OCs) of the mice were lysed using RIPA lysis buffer containing a mixture of protease and phosphatase inhibitors (Bimake, Shanghai, China). After lysis, the protein concentration was determined using the Pierce™ BCA method (Thermo Scientific), and all samples were adjusted to the same protein concentration with 4× SDS. Equal amounts of protein samples were loaded onto 10% SDS-PAGE precast gels (Shanghai EpiZyme Biotechnology, Shanghai, China) and subsequently transferred to PVDF membranes (Millipore, Massachusetts, United States). The PVDF membranes were blocked in PBS/Tween containing 5% nonfat milk at room temperature for 1h, followed by incubation with primary antibodies (RUNX2 (1:1000, CST, Massachusetts, United States, 12556s), NFATc1 (1:500, Santa Cruz Biotechnology, California, United States, sc-7294), GPX4 (1:2000, Proteintech, Illinois, United States, 67763-1-Ig), and GAPDH (1:5000, Proteintech, 60004-1-Ig)) overnight at 4°C. The membranes were then incubated with HRP-conjugated goat anti-mouse secondary antibodies (1:5000, Proteintech, SA00001-1, SA00001-2) at room temperature for 1h. Finally, signals were detected using HRP substrate peroxide solution (Millipore) and HRP substrate luminol solution (Millipore), and the signal bands were visualized using an ultra-high sensitivity chemiluminescence imaging system (Minichemi). For protein extraction from mouse bone tissue, an appropriate amount of bone tissue should first be quickly placed in liquid nitrogen for grinding. After grinding, the powder was transferred into an EP tube containing an appropriate amount of RIPA lysis buffer. Subsequent steps were performed as described previously. In addition, the full and uncropped western blots were uploaded as “Supplemental Material”. LC-MS/MS Analyses The detailed steps of protein extraction from mice primary OBs and OCs were described in the western blot section. 100µg of protein from each sample was then reduced using tris (2-carboxyethyl) phosphine (TCEP), alkylated with iodoacetamide (IAA), precipitated with CH 3 OH, H 2 O and CHCl 3 (CH 3 OH: H 2 O: CHCl 3 : protein = 4:3:1:1), and digested with trypsin before tandem mass tag (TMT)-10 (Catalog No. 90110, Thermo Fisher Scientific, Waltham, MA) labeling consistent with the previous studies(21, 24, 27, 28). After desalting, the samples underwent phosphoprotein fractionation. The fractionated samples were enriched for phosphopeptides using PureCube Fe-NTA Agarose Beads (Catalog No. 31403-Fe, Cube Biotech, Monheim, Germany) according to the manufacturer’s instructions, and the enriched phosphopeptides were desalted with C18 ZipTips and analyzed by an Nano EASY-nLC 1200 liquid chromatography system LC instrument coupled with a Q Exactive HF-X high-resolution mass spectrometer (Serial No. SN06464L, Thermo Fisher Scientific). For protein quantification, 50µg of peptides from the remaining sample after phosphopeptides enrichment was used for HPLC fractionation and LC-MS/MS analysis using a Nano EASY-nLC 1200 liquid chromatography system LC instrument coupled with a Q Exactive Plus high-resolution mass spectrometer (Serial No. SN03572L, Thermo Fisher Scientific). Due to the large number of samples, to ensure instrument stability and comparability between samples during the analysis, a quality control (QC) sample was injected every three runs. The correlation between QC samples was monitored to assess instrument status and data reliability. The mass spectrometry raw data was processed using the MaxQuant (version 1.6) search engine. Bioinformatics Analysis The principal component analysis (PCA) of proteome and phosphoproteome was performed on protein abundance. Differentially expressed proteins (DEPs) were analyzed using R package, limma and edgeR, with a fold change greater than 1.2 and a statistically significant adjusted P-value less than 0.05. Gene ontology (GO) and kyoto encyclopedia of genes and genomes (KEGG) analysis were conducted to identify the biological functions of the DEPs and the pathways in which they are involved. Statistical Analysis Statistical analysis and plotting were performed using GraphPad Prism 10.1.1 software. All quantitative data were presented as mean ± standard error (Mean ± SEM). Correlation analysis was conducted using either Pearson or Spearman correlation analysis. Comparisons between two groups were made using a two-sided t-test . One-way ANOVA was used for multiple comparisons, and Tukey’s multiple comparison test was performed when significant differences were detected. A p -value ˂ 0.05 was considered statistically significant. Results Significant negative correlation between iron content and bone mineral density in clinical bone tissue. Literature reviews indicate that plasma ferritin levels in osteoporotic patients are markedly elevated compared to healthy individuals(29, 30). To elucidate the alterations in iron content within bone tissue during the progression of bone loss-from normal BMD to low bone mass and eventually to osteoporosis-we assessed the iron content in femoral neck bone tissue obtained from clinical specimens. Our findings demonstrated that reductions in BMD were associated with iron accumulation in bone tissue (Fig. 1a). Notably, there existed a significant negative correlation between the iron content in bone tissue and BMD (Fig. 1b). The impact of accumulated iron in bone tissue on bone health remains unclear, as does the mechanism by which iron regulates bone metabolism. To address these gaps, we employed high-iron interventions in mice and their primary bone marrow cells to delve deeper into these inquiries. RFeD induced osteoporosis in vivo . To examine the effects of iron accumulation in bone tissue on bone phenotype and metabolism, we administered SD and RFeD to mice, respectively (Fig. 2a). Following one-month and two-month treatments, ten mice per group were euthanized. µCT scans of the right femurs revealed that one month of RFeD intervention had already compromised the trabecular bone microarchitecture in mice. Prolonging the RFeD regimen to two months maintained this deterioration. Notably, there was a reduction in trabecular BV/TV, Tb.N, Tb.Th, and Conn.D, alongside an elevation in Tb.Sp and SMI (Fig. 2b-c). Furthermore, while one month of RFeD intervention did not disrupt the cortical bone microarchitecture in the mid-diaphysis of the femur, extending the dietary intervention to two months led to RFeD-induced impairment of the cortical bone microarchitecture. This impairment was evidenced by a decline in cortical BV/TV and Ct.Th, accompanied by an increase in BS/BV (Supplementary Fig. 1). To elucidate the pathological mechanisms behind the severe bone microarchitecture deterioration observed in mice following RFeD intervention, we conducted TRAP and HE staining on the left tibia of mice to assess the effects of RFeD intervention on osteoclast and osteoblast differentiation. Our findings demonstrated that RFeD intervention significantly augmented the TRAP-positive area within the bone tissue (Fig. 2d), thereby indicating an increase in the surface area occupied by osteoclasts (Oc.S/BS, Fig. 2e). In contrast, HE staining disclosed a substantial reduction in the number of osteoblasts present on the surface of the trabecular bone (Ob.N/BS) in mice subjected to RFeD intervention (Fig. 2f-g). Moreover, we investigated the expression of NFATc1, a critical regulator of osteoclastic differentiation, and RUNX2, a pivotal transcription factor involved in osteogenic differentiation, in mouse bone tissue post-corresponding dietary intervention. Our analyses revealed a dramatic upregulation of NFATc1 expression in mouse bone tissue (Fig. 2h-i), whereas the expression of RUNX2 exhibited a marked decrease following RFeD intervention (Fig. 2j-k). Collectively, these results underscored that RFeD intervention facilitated osteoclastic differentiation while concurrently inhibiting osteogenic differentiation, ultimately leading to osteoporosis in vivo . RFeD induced osteoblast ferroptosis in vivo . Subsequently, we assessed the serum ferritin levels and discovered a notable elevation in the serum ferritin content of mice subjected to RFeD intervention (Fig. 3a), which corresponded with the immunohistochemical staining of ferritin in bone tissue (Fig. 3b-c). Upon measuring the iron content in bone tissue, we observed a significant augmentation in the iron content of bone tissue in mice following RFeD intervention (Fig. 3d-e), and there was a pronounced negative correlation between bone tissue iron content and BMD (Fig. 3f-g). The TUNEL assay of bone tissue revealed apoptosis of osteoblasts (Fig. 3h-i), and the expression of the ferroptosis inhibitor-glutathione peroxidase 4 (GPX4) diminished considerably post-RFeD intervention (Fig. 3j-k). Furthermore, the content of the lipid oxidation marker-MDA in both plasma and bone tissue were significantly elevated (Fig. 3l-m). These findings collectively imply that RFeD intervention induces osteoblast ferroptosis in vivo . Iron overload impeded osteogenic differentiation while concurrently promoting osteoclastic differentiation in vitro . To explore the impact of high iron supplementation on osteogenic/osteoclast differentiation, we cultured mouse primary bone marrow cells and treated them with or without high iron (5/20µM (NH₄)₂Fe(SO₄)₂, Fig. 4a). Following a seven-day induction period, we initially assessed the iron concentration within the cells. Notably, the iron level in primary osteoblasts markedly escalated upon exposure to high iron conditions (20µM (NH₄)₂Fe(SO₄)₂, Fig. 4b). Subsequently, we elucidated the influence of high iron administration on osteogenic and osteoclastic differentiation through ALP and TRAP staining, alongside examining the protein expressions of RUNX2 and NFATc1. Our findings indicated that treating with 20µM (NH₄)₂Fe(SO₄)₂ substantially hindered the differentiation of mouse primary BMSCs into osteoblasts (Fig. 4c-d). Conversely, administering 5µM (NH₄)₂Fe(SO₄)₂ significantly enhanced the differentiation of mouse primary bone marrow cells into osteoclasts (Fig. 4e-f). The protein expression patterns of RUNX2 (Fig. 4g-h) and NFATc1 (Fig. 4i-j) mirrored the outcomes of the histopathological staining analyses. Iron overload induced osteoblast ferroptosis in vitro . Ferroptosis typically manifests as a range of morphological and biochemical alterations, including iron accumulation, mitochondrial contraction, and lipid peroxidation(20). To confirm the occurrence of ferroptosis, TUNEL assay, C11-BODIPY staining and FerroOrange staining were conducted on mice primary osteoblasts subjected to high iron treatment. The TUNEL assay verified a substantial increase in cell death among primary osteoblasts post-high iron exposure (Fig. 5a-b). Intracellular lipid peroxides were assayed using the C11-BODIPY fluorescent probe, revealing pronounced lipid peroxidation in primary osteoblasts following high iron treatment (Fig. 5a, c). A similar pattern was noted in the generation of MDA, the terminal product of lipid peroxidation (Fig. 5e). FerroOrange, a Fe 2+ -specific probe, exhibited a marked rise in fluorescence intensity under high iron conditions (Fig. 5a, d). Lastly, the protein expression level of GPX4 was notably reduced in mice primary osteoblasts exposed to high iron (Fig. 5f-g). These findings collectively demonstrate that high iron intervention induces ferroptosis in osteoblasts in vitro . Iron overload triggered ferroptosis in osteoblasts and activated osteoclast differentiation. To elucidate the underlying mechanisms behind the suppression of osteogenic differentiation and stimulation of osteoclast differentiation in primary bone marrow cells after high-iron intervention, we undertook proteomic and phosphoproteomic analyses on the primary cells (Fig. 6a-c). Our findings demonstrated a pronounced activation of the “ferroptosis” signaling pathway within osteoblasts following high-iron intervention (Fig. 6d-f), whereas “osteoclast differentiation” was distinctly augmented in osteoclasts (Fig. 6g-i). Subsequent phosphoproteomic investigation disclosed a significant amplification of the “actin cytoskeleton organization” signaling pathway in both osteoblasts and osteoclasts post-intervention (Fig. 6j-l), which was essential for the migration of osteoblasts and osteoclasts to sites of active bone metabolism, enabling them to perform their osteogenic and osteoclastic functions. Extensive analysis revealed a substantial upregulation of FTH1/FTL protein expression within the ferroptosis signaling pathway of osteoblasts (Fig. 6m). Ferroptotic osteoblasts subsequently initiated the co-stimulatory pathway, thereby promoting osteoclast differentiation, culminating in an osteoporotic phenotype in mice (Fig. 6n). Collectively, our findings suggest that high-iron intervention results in iron accumulation within the bone tissue of mice. The accumulated iron induces ferroptosis in osteoblasts via signaling pathways such as FTH1/FTL. Ferroptotic osteoblasts activate the co-stimulatory pathway, which further promotes osteoclast differentiation, ultimately leading to the development of osteoporosis. Specifically, iron overload disrupts the equilibrium between bone formation and resorption, resulting in heightened osteoclast activity and bone loss. Discussion and Conclusion In this article, our literature review revealed that clinical osteoporotic patients exhibit significantly elevated serum ferritin levels compared to healthy individuals(29, 30), alongside a notable increase in bone iron content. A striking inverse relationship exists between bone tissue iron content and BMD. Subsequent studies using RFeD intervention on mice demonstrated pronounced impairment of bone microarchitecture, a substantial reduction in osteoblast numbers, and a marked increase in osteoclast surface area. Further investigation indicated that post-RFeD intervention, both serum ferritin levels and bone iron content rose significantly. Consistently, the iron concentration in mouse bone tissue exhibited a negative correlation with BMD following RFeD intervention. Additionally, RFeD intervention led to significant apoptosis of bone cells, with a notable downregulation of the ferroptosis inhibitor-GPX4 in bone tissue. Concurrently, the level of the lipid oxidation end product-MDA in plasma and bone tissue escalated dramatically. In addition to the evident destruction of bone microarchitecture, ferroptosis of osteoblasts represents a crucial discovery within this research. Ferroptosis denotes a novel form of programmed cell death triggered by unchecked iron-dependent lipid peroxidation(15, 16, 31, 32). Distinct from other cell death modalities, ferroptosis exhibits distinctive biological hallmarks such as iron accumulation, augmented lipid peroxidation products, and diminished GPX4 expression(33–36). Extensive research indicates that ferroptosis is intricately linked to a myriad of disorders, encompassing metabolic diseases, neurodegenerative ailments, ischemia-reperfusion injuries, cardiomyopathies, and the immunotherapeutic responses against cancer, among others(33, 34, 37–39). Consequently, targeting ferroptosis could emerge as a potent therapeutic avenue for managing associated diseases(18). Preserving osteoblast vitality is widely regarded as an efficacious approach to sustaining skeletal well-being(40–42). Previous investigations predominantly attributed caspase-dependent apoptosis and necrosis as the predominant modes of cell demise. For instance, Emerton et al . documented that estrogen insufficiency amplifies osteocyte apoptosis, thereby diminishing bone volume(43). Nonetheless, the present study unveils that excessive lipid peroxidation potentially constitutes the principal inducer of osteoblast damage under high iron conditions, intimating a close association between ferroptosis and its underlying molecular mechanisms. Next, we subjected mice primary bone marrow cells to high-iron intervention and found that this intervention inhibited osteogenic differentiation while promoting osteoclastic differentiation. Our subsequent investigations revealed that high-iron exposure resulted in a marked elevation in mortality, lipid oxidation, and intracellular Fe 2+ levels among primary osteoblasts in mice. Simultaneously, there was a pronounced reduction in GPX4 expression within osteoblasts. These findings suggest that high-iron intervention triggers a surge in free, labile iron within cells, which catalyzes the Fenton reaction. Consequently, accumulated fatty acids interact with reactive oxygen species (ROS) to generate lipid peroxides and lipid peroxyl radicals(44, 45). An abundance of lipid peroxides subsequently oxidizes cellular membranes and organelles, liberating additional ROS from compromised mitochondria. This process establishes a detrimental feedback loop of ferroptosis, culminating in the demise of a greater number of osteoblasts. Iron plays a crucial role in maintaining normal physiological functions across organisms, and dysregulation of iron metabolism is implicated in the pathogenesis of numerous diseases(7, 14). In recent years, advancements in omics technologies have furnished novel insights and methodologies for investigating iron homeostasis. Particularly, the implementation of proteomics and phosphoproteomics techniques has substantially enhanced our comprehension of iron homeostasis and associated metabolic disorders. Ultimately, we conducted proteomic and phosphoproteomic analyses on mice primary bone marrow cells following high-iron treatment. We identified that the “ferroptosis” signaling pathway was prominently enriched in osteoblasts, whereas “osteoclast differentiation” was distinctly activated in osteoclasts. Through comprehensive examination, we observed a substantial upregulation of FTH1/FTL protein expression within the osteoblast ferroptosis signaling cascade. This implies that excessive iron might facilitate ferroptosis in osteoblasts via signaling routes such as FTH1/FTL(46). Ferroptotic osteoblasts subsequently engage the co-stimulatory pathway, thereby promoting osteoclast differentiation and contributing to the onset and progression of osteoporosis(19). Hence, it stands to reason that disrupting the ferroptosis vicious cycle in osteoblasts could represent a promising therapeutic strategy for managing clinical osteoporosis. Finally, despite these findings, our study does have certain limitations. First, obtaining clinical bone tissue samples presents challenges. The high inorganic content of bone tissue complicates protein extraction. Additionally, research focusing on the proteomics and post-translational modifications of human bone remains limited. However, analyzing human bone tissue would provide valuable validation of our conclusions. Consequently, we plan to extend our omics studies to include clinical bone tissue in future work. Furthermore, although we established that high-iron intervention triggers significant ferroptosis in osteoblasts, we did not delve into the downstream pathways associated with ferroptosis. We intend to pursue a deeper investigation into these pathways. Lastly, our current observations were confined to the effects of high-iron intervention on mice and their primary bone marrow cells, both in vivo and in vitro . For our upcoming research, we aim to administer a chelating agent to high-iron-intervened mice and their primary cells. This approach will allow us to assess whether blocking the ferroptosis cycle can partially or fully reverse the bone metabolic phenotype in both mice and primary cells. In summary, through comprehensive in vivo and in vitro experimentation, we demonstrated that high-iron exposure triggers ferroptosis in mice osteoblasts, thereby hindering osteogenic differentiation and augmenting osteoclastic activity. This cascade of events compromises bone microarchitecture and culminates in the development of an osteoporotic phenotype in mice. Building upon these findings, we posit that RFeD intervention in mice offers a promising model for recapitulating osteoporosis in clinical scenarios, with ferroptosis in osteoblasts emerging as a plausible therapeutic avenue for managing osteoporosis. Future endeavors should concentrate on evaluating the efficacy of iron-chelating therapies in iron-overloaded mice and their primary cells. Subsequent investigations should emphasize the potential clinical applications of this therapeutic strategy. Declarations Acknowledgments The authors would like to thank Dr. Li Chen from Analytical & Testing Center of Sichuan University for her help with micro-CT scanning. Competing Interests The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Availability of Data The mass spectrometry proteomics and phosphoproteomics data have been deposited to the ProteomeXchange Consortium (https://proteomecentral.proteomexchange.org) via the iProX partner repository(47, 48) with the dataset identifier PXD060747. All data underlying this article will be shared on reasonable request to the corresponding authors. Funding This work was supported by grants from the Sichuan Science and Technology Program to JWX (2023YFS0233), the Science and Technology Department of Sichuan Province (2024NSFSC1623) and the Postdoctor Research Fund of West China Hospital, Sichuan University (2024HXBH129). References Guha I, Nadeem SA, Zhang X, DiCamillo PA, Levy SM, Wang G, et al. Deep learning-based harmonization of trabecular bone microstructures between high- and low-resolution CT imaging. Med Phys. 2024. Xu J, Cai X, Miao Z, Yan Y, Chen D, Yang ZX, et al. Proteome-wide profiling reveals dysregulated molecular features and accelerated aging in osteoporosis: A 9.8-year prospective study. Aging Cell. 2024;23(2):e14035. Ensrud KE, Crandall CJ. Osteoporosis. Ann Intern Med. 2017;167(3):Itc17-itc32. Wang L, Yu W, Yin X, Cui L, Tang S, Jiang N, et al. Prevalence of Osteoporosis and Fracture in China: The China Osteoporosis Prevalence Study. JAMA Netw Open. 2021;4(8):e2121106. Wang O, Hu Y, Gong S, Xue Q, Deng Z, Wang L, et al. A survey of outcomes and management of patients post fragility fractures in China. Osteoporosis international : a journal established as result of cooperation between the European Foundation for Osteoporosis and the National Osteoporosis Foundation of the USA. 2015;26(11):2631–2640. Briggs AM, Cross MJ, Hoy DG, Sànchez-Riera L, Blyth FM, Woolf AD, et al. Musculoskeletal Health Conditions Represent a Global Threat to Healthy Aging: A Report for the 2015 World Health Organization World Report on Ageing and Health. Gerontologist. 2016;56 Suppl 2:S243-255. Li GF, Pan YZ, Sirois P, Li K, Xu YJ. Iron homeostasis in osteoporosis and its clinical implications. Osteoporosis international : a journal established as result of cooperation between the European Foundation for Osteoporosis and the National Osteoporosis Foundation of the USA. 2012;23(10):2403–2408. Aschemeyer S, Qiao B, Stefanova D, Valore EV, Sek AC, Ruwe TA, et al. Structure-function analysis of ferroportin defines the binding site and an alternative mechanism of action of hepcidin. Blood. 2018;131(8):899–910. Yuan Y, Fang Y, Zhu L, Gu Y, Li L, Qian J, et al. Deterioration of hematopoietic autophagy is linked to osteoporosis. Aging Cell. 2020;19(5):e13114. Ledesma-Colunga MG, Weidner H, Vujic Spasic M, Hofbauer LC, Baschant U, Rauner M. Shaping the bone through iron and iron-related proteins. Semin Hematol. 2021;58(3):188–200. Zhang H, Yang F, Cao Z, Xu Y, Wang M. The influence of iron on bone metabolism disorders. Osteoporosis international : a journal established as result of cooperation between the European Foundation for Osteoporosis and the National Osteoporosis Foundation of the USA. 2024;35(2):243–253. Wang F, Lv H, Zhao B, Zhou L, Wang S, Luo J, et al. Iron and leukemia: new insights for future treatments. J Exp Clin Cancer Res. 2019;38(1):406. Jiang X, Stockwell BR, Conrad M. Ferroptosis: mechanisms, biology and role in disease. Nat Rev Mol Cell Biol. 2021;22(4):266–282. Muckenthaler MU, Rivella S, Hentze MW, Galy B. A Red Carpet for Iron Metabolism. Cell. 2017;168(3):344–361. Yang WS, Stockwell BR. Synthetic lethal screening identifies compounds activating iron-dependent, nonapoptotic cell death in oncogenic-RAS-harboring cancer cells. Chem Biol. 2008;15(3):234–245. Dixon SJ, Lemberg KM, Lamprecht MR, Skouta R, Zaitsev EM, Gleason CE, et al. Ferroptosis: an iron-dependent form of nonapoptotic cell death. Cell. 2012;149(5):1060–1072. Jiang Z, Wang H, Qi G, Jiang C, Chen K, Yan Z. Iron overload-induced ferroptosis of osteoblasts inhibits osteogenesis and promotes osteoporosis: An in vitro and in vivo study. IUBMB Life. 2022;74(11):1052–1069. Yang Y, Lin Y, Wang M, Yuan K, Wang Q, Mu P, et al. Targeting ferroptosis suppresses osteocyte glucolipotoxicity and alleviates diabetic osteoporosis. Bone Res. 2022;10(1):26. Jiang Z, Qi G, He X, Yu Y, Cao Y, Zhang C, et al. Ferroptosis in Osteocytes as a Target for Protection Against Postmenopausal Osteoporosis. Adv Sci (Weinh). 2024;11(12):e2307388. Wu X, Fang X, Lu F, Chen Q, Liu J, Zheng L. An update on the role of ferroptosis in the pathogenesis of osteoporosis. EFORT Open Rev. 2024;9(8):712–722. Wang A, Zhang H, Li G, Chen B, Li J, Zhang T, et al. Deciphering core proteins of osteoporosis with iron accumulation by proteomics in human bone. Front Endocrinol (Lausanne). 2022;13:961903. Zhang J, Zheng L, Wang Z, Pei H, Hu W, Nie J, et al. Lowering iron level protects against bone loss in focally irradiated and contralateral femurs through distinct mechanisms. Bone. 2019;120:50–60. Hu Y, Zhang Y, Ni CY, Chen CY, Rao SS, Yin H, et al. Human umbilical cord mesenchymal stromal cells-derived extracellular vesicles exert potent bone protective effects by CLEC11A-mediated regulation of bone metabolism. Theranostics. 2020;10(5):2293–2308. Xia W, Xie J, Cai Z, Liu X, Wen J, Cui ZK, et al. Damaged brain accelerates bone healing by releasing small extracellular vesicles that target osteoprogenitors. Nat Commun. 2021;12(1):6043. Lin L, Guo Z, He E, Long X, Wang D, Zhang Y, et al. SIRT2 regulates extracellular vesicle-mediated liver-bone communication. Nat Metab. 2023;5(5):821–841. Zhang W, Zhou X, Hou W, Chen E, Ye C, Chen M, et al. Reversing the imbalance in bone homeostasis via sustained release of SIRT-1 agonist to promote bone healing under osteoporotic condition. Bioact Mater. 2023;19:429–443. Zhang R, Hu M, Liu Y, Li W, Xu Z, He S, et al. Integrative Omics Uncovers Low Tumorous Magnesium Content as A Driver Factor of Colorectal Cancer. Genomics Proteomics Bioinformatics. 2024;22(4). Wang X, Luo Y, He S, Lu Y, Gong Y, Gao L, et al. Age-, sex- and proximal-distal-resolved multi-omics identifies regulators of intestinal aging in non-human primates. Nat Aging. 2024;4(3):414–433. Fung EB, Harmatz PR, Milet M, Coates TD, Thompson AA, Ranalli M, et al. Fracture prevalence and relationship to endocrinopathy in iron overloaded patients with sickle cell disease and thalassemia. Bone. 2008;43(1):162–168. Valenti L, Varenna M, Fracanzani AL, Rossi V, Fargion S, Sinigaglia L. Association between iron overload and osteoporosis in patients with hereditary hemochromatosis. Osteoporosis international : a journal established as result of cooperation between the European Foundation for Osteoporosis and the National Osteoporosis Foundation of the USA. 2009;20(4):549–555. Hu Y, Wang Y, Liu S, Wang H. The Potential Roles of Ferroptosis in Pathophysiology and Treatment of Musculoskeletal Diseases-Opportunities, Challenges, and Perspectives. J Clin Med. 2023;12(6). Yao X, Sun K, Yu S, Luo J, Guo J, Lin J, et al. Chondrocyte ferroptosis contribute to the progression of osteoarthritis. J Orthop Translat. 2021;27:33–43. Stockwell BR, Friedmann Angeli JP, Bayir H, Bush AI, Conrad M, Dixon SJ, et al. Ferroptosis: A Regulated Cell Death Nexus Linking Metabolism, Redox Biology, and Disease. Cell. 2017;171(2):273–285. Tang D, Chen X, Kang R, Kroemer G. Ferroptosis: molecular mechanisms and health implications. Cell Res. 2021;31(2):107–125. Hadian K, Stockwell BR. SnapShot: Ferroptosis. Cell. 2020;181(5):1188–1188.e1181. Lee H, Zandkarimi F, Zhang Y, Meena JK, Kim J, Zhuang L, et al. Energy-stress-mediated AMPK activation inhibits ferroptosis. Nat Cell Biol. 2020;22(2):225–234. Fang X, Wang H, Han D, Xie E, Yang X, Wei J, et al. Ferroptosis as a target for protection against cardiomyopathy. Proc Natl Acad Sci U S A. 2019;116(7):2672–2680. Liang C, Zhang X, Yang M, Dong X. Recent Progress in Ferroptosis Inducers for Cancer Therapy. Adv Mater. 2019;31(51):e1904197. Maher P, Currais A, Schubert D. Using the Oxytosis/Ferroptosis Pathway to Understand and Treat Age-Associated Neurodegenerative Diseases. Cell Chem Biol. 2020;27(12):1456–1471. Han Y, You X, Xing W, Zhang Z, Zou W. Paracrine and endocrine actions of bone-the functions of secretory proteins from osteoblasts, osteocytes, and osteoclasts. Bone Res. 2018;6:16. Maurel DB, Matsumoto T, Vallejo JA, Johnson ML, Dallas SL, Kitase Y, et al. Characterization of a novel murine Sost ER(T2) Cre model targeting osteocytes. Bone Res. 2019;7:6. Li MCM, Chow SKH, Wong RMY, Qin L, Cheung WH. The role of osteocytes-specific molecular mechanism in regulation of mechanotransduction - A systematic review. J Orthop Translat. 2021;29:1–9. Emerton KB, Hu B, Woo AA, Sinofsky A, Hernandez C, Majeska RJ, et al. Osteocyte apoptosis and control of bone resorption following ovariectomy in mice. Bone. 2010;46(3):577–583. D'Autréaux B, Toledano MB. ROS as signalling molecules: mechanisms that generate specificity in ROS homeostasis. Nat Rev Mol Cell Biol. 2007;8(10):813–824. Holmström KM, Finkel T. Cellular mechanisms and physiological consequences of redox-dependent signalling. Nat Rev Mol Cell Biol. 2014;15(6):411–421. Liu J, Ren Z, Yang L, Zhu L, Li Y, Bie C, et al. The NSUN5-FTH1/FTL pathway mediates ferroptosis in bone marrow-derived mesenchymal stem cells. Cell Death Discov. 2022;8(1):99. Ma J, Chen T, Wu S, Yang C, Bai M, Shu K, et al. iProX: an integrated proteome resource. Nucleic Acids Res. 2019;47(D1):D1211-d1217. Chen T, Ma J, Liu Y, Chen Z, Xiao N, Lu Y, et al. iProX in 2021: connecting proteomics data sharing with big data. Nucleic Acids Res. 2022;50(D1):D1522-d1527. Additional Declarations There is no duality of interest Supplementary Files SupplementalMaterial.docx Original western blots SupplementalFigure1.jpg Supplemental Figure 1 Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6072367","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":420169923,"identity":"1be1b29d-6edd-4e7f-b015-72a7f066dd56","order_by":0,"name":"Shuangqing Li","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAvklEQVRIiWNgGAWjYBACPgYGNgYGngQ5NvbmA8RpYYNqMebjOZZAihaGhMR5EjkKRGrhP7ztMY9MWnobQw4Dw4+KbURokUgrN5zBk5PbxnD2AGPPmdvEaOExk/jAU5HbxtiXwMzYRowW/jNmEgk8FelszDwGRGphyAHZkpPAxka0Fom0MskZPGmGbTxsCQeJ8gs/MMSkeXuS5eXnPz744EcFEVqAwICBsQfCOkCUerAWhh/Eqh0Fo2AUjIIRCQA2OjIcIqKm+wAAAABJRU5ErkJggg==","orcid":"","institution":"West China Hospital，Sichuan University","correspondingAuthor":true,"prefix":"","firstName":"Shuangqing","middleName":"","lastName":"Li","suffix":""},{"id":420169924,"identity":"09d0c0df-0ad7-468b-875d-7b31184623a5","order_by":1,"name":"Shan Wan","email":"","orcid":"","institution":"West China Hospital, Sichuan University","correspondingAuthor":false,"prefix":"","firstName":"Shan","middleName":"","lastName":"Wan","suffix":""},{"id":420169925,"identity":"abc66346-f257-40ea-b4f5-db58623eb7c1","order_by":2,"name":"Yanting He","email":"","orcid":"","institution":"West China Hospital, Sichuan University","correspondingAuthor":false,"prefix":"","firstName":"Yanting","middleName":"","lastName":"He","suffix":""},{"id":420169926,"identity":"43a14c4d-deb6-4f8f-b34d-177cb361a699","order_by":3,"name":"Baochen Chong","email":"","orcid":"","institution":"West China Hospital, Sichuan University","correspondingAuthor":false,"prefix":"","firstName":"Baochen","middleName":"","lastName":"Chong","suffix":""},{"id":420169927,"identity":"0ad633e6-f90c-4b92-b9e8-66530d5e51b7","order_by":4,"name":"Zhili Xia","email":"","orcid":"","institution":"Lanzhou University","correspondingAuthor":false,"prefix":"","firstName":"Zhili","middleName":"","lastName":"Xia","suffix":""},{"id":420169928,"identity":"75351dde-0bee-4efa-a941-dd136b40e3a8","order_by":5,"name":"Yijie Qiu","email":"","orcid":"","institution":"West China Hospital, Sichuan University","correspondingAuthor":false,"prefix":"","firstName":"Yijie","middleName":"","lastName":"Qiu","suffix":""},{"id":420169929,"identity":"f1064a09-d77c-426b-bd49-a20f08e83608","order_by":6,"name":"Rou Zhang","email":"","orcid":"","institution":"West China Hospital, Sichuan University","correspondingAuthor":false,"prefix":"","firstName":"Rou","middleName":"","lastName":"Zhang","suffix":""},{"id":420169930,"identity":"989d85d1-7e5a-4c2f-8c85-de8705d3565e","order_by":7,"name":"Meng Hu","email":"","orcid":"","institution":"West China Hospital, Sichuan University","correspondingAuthor":false,"prefix":"","firstName":"Meng","middleName":"","lastName":"Hu","suffix":""},{"id":420169931,"identity":"e3a34aaa-e7fd-4442-b9ef-e5e9edd4c5e6","order_by":8,"name":"Zhenmei An","email":"","orcid":"","institution":"West China Hospital, Sichuan University","correspondingAuthor":false,"prefix":"","firstName":"Zhenmei","middleName":"","lastName":"An","suffix":""},{"id":420169932,"identity":"99eb68e8-86d1-4ac1-9781-6e6ba56fff4f","order_by":9,"name":"Jinwei Xie","email":"","orcid":"","institution":"West China Hospital, Sichuan University","correspondingAuthor":false,"prefix":"","firstName":"Jinwei","middleName":"","lastName":"Xie","suffix":""}],"badges":[],"createdAt":"2025-02-20 13:32:28","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6072367/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6072367/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":80942135,"identity":"f51551c9-76f6-4381-9764-71a6d93086b7","added_by":"auto","created_at":"2025-04-19 11:27:29","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":88032,"visible":true,"origin":"","legend":"\u003cp\u003eLegend not included with this version\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6072367/v1/94ae6dc69d5d2a95970ee6d7.jpg"},{"id":80942137,"identity":"effae22f-7bfd-40a8-b98f-b37b9b86b1c2","added_by":"auto","created_at":"2025-04-19 11:27:29","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":735356,"visible":true,"origin":"","legend":"\u003cp\u003eLegend not included with this version\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6072367/v1/0c510b07b172ee2761f492f8.jpg"},{"id":80942140,"identity":"a44f9ace-04ff-49bc-86af-e71b2460fd88","added_by":"auto","created_at":"2025-04-19 11:27:29","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":440548,"visible":true,"origin":"","legend":"\u003cp\u003eLegend not included with this version\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6072367/v1/5726b3df767e4630885e5610.jpg"},{"id":80942152,"identity":"e5c3abb7-8940-48fd-9342-5a0158e6e470","added_by":"auto","created_at":"2025-04-19 11:27:30","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":378208,"visible":true,"origin":"","legend":"\u003cp\u003eLegend not included with this version\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6072367/v1/a0a4201202809fee0abe52ce.jpg"},{"id":80942143,"identity":"17530412-4d12-42d7-ae9d-29ed08c918f1","added_by":"auto","created_at":"2025-04-19 11:27:29","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":518010,"visible":true,"origin":"","legend":"\u003cp\u003eLegend not included with this version\u003c/p\u003e","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6072367/v1/9e12c718d36f6a46f9dfaedb.jpg"},{"id":80942148,"identity":"8965a8ea-000f-4397-b1ae-dc08aee1d4fd","added_by":"auto","created_at":"2025-04-19 11:27:30","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1534192,"visible":true,"origin":"","legend":"\u003cp\u003eLegend not included with this version\u003c/p\u003e","description":"","filename":"Figure6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6072367/v1/95db6a9efe26e57ac4531eb0.jpg"},{"id":86171580,"identity":"1583357d-e8e2-4a37-bb08-fa70f2663124","added_by":"auto","created_at":"2025-07-07 14:34:46","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4612224,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6072367/v1/6e69e226-61cf-492a-9ebf-360a1088b6af.pdf"},{"id":80942486,"identity":"61c124d2-f1a1-40b1-a0a6-c41a6e8cdbde","added_by":"auto","created_at":"2025-04-19 11:35:29","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":4267709,"visible":true,"origin":"","legend":"Original western blots","description":"","filename":"SupplementalMaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-6072367/v1/6843e724eabb4f1a457f3a95.docx"},{"id":80942763,"identity":"df6c882d-30b9-4898-bebb-f9c9bca30aa9","added_by":"auto","created_at":"2025-04-19 11:43:29","extension":"jpg","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":184987,"visible":true,"origin":"","legend":"Supplemental Figure 1","description":"","filename":"SupplementalFigure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6072367/v1/b335084fa2c1c82b326b445b.jpg"}],"financialInterests":"There is no duality of interest","formattedTitle":"Iron overload-induced ferroptosis of osteoblasts as a potential therapeutic target for osteoporosis","fulltext":[{"header":"Introduction","content":"\u003cp\u003eOsteoporosis (OP), a systemic bone disease, is defined by diminished bone density, microarchitectural degradation, augmented brittleness, and an elevated propensity for fractures(1\u0026ndash;3). With the intensifying aging population in China, the incidence of osteoporosis has surged markedly. National epidemiological studies reveal that 32% of individuals aged over 65 in China suffer from osteoporosis, affecting approximately 90\u0026nbsp;million people at present(4). Osteoporotic fractures, or fragility fractures are grave outcomes of osteoporosis, inflicting substantial harm and serving as major contributors to morbidity and mortality among the elderly, despite the paucity of efficacious pharmaceuticals and interventions. Within a year post-hip fracture, 20% of patients succumb to assorted complications, around half become incapacitated, and their quality of life deteriorates considerably(5). The healthcare costs associated with osteoporosis and fractures exact a considerable financial toll on families and society alike(6). Hence, elucidating the attributes of skeletal senescence and unveiling its underlying molecular mechanisms, identifying novel targets for modulating bone metabolism, and devising innovative therapeutics grounded in these discoveries are imperative endeavors aimed at forestalling disability in the geriatric demographic and enhancing their quality of life.\u003c/p\u003e \u003cp\u003eIron holds a pivotal position in living organisms, and its metabolic equilibrium is intricately linked to a myriad of diseases(7\u0026ndash;14). The burgeoning advancements in omics technologies have recently furnished novel vistas and methodologies for investigating iron metabolism. Proteomics, for instance, delves into the expression, modification, and functional aspects of proteins, thereby unveiling the metamorphoses in iron metabolism-associated proteins across diverse physiological and pathological milieus. Investigations have unearthed that both iron surfeit and insufficiency engender intricate proteomic shifts, underscoring the paramountcy of proteomics in the realm of iron metabolism research. Metabolomics zeroes in on the holistic fluctuations in bodily metabolites to pinpoint metabolic aberrations precipitated by iron excess or dearth. Research divulges that iron surplus, or deficit is inextricably bound to alterations in plasma metabolic profiles, notably influencing the metabolism of branched-chain amino acids, heme catabolism derivatives, fatty acids, and cholesterol. Iron homeostasis exerts a profound impact on the epigenetic embellishments of proteins, encompassing DNA methylation, histone modifications, and miRNA. Disruptions in iron homeostasis may catalyze anomalous epigenetic modifications of proteins, thereby instigating related maladies(7\u0026ndash;9). Ferroptosis, a nascent form of cellular demise(15, 16), intertwines with the pathophysiological trajectories of sundry ailments(17\u0026ndash;20). The deployment of omics techniques to scrutinize ferroptosis furnishes a vital instrument for comprehending the nexus between iron metabolism and cell death(21). In summation, the utilization of proteomics, metabolomics, and post-translational modification omics has enriched our comprehension of iron metabolism and proffers novel insights and strategies for diagnosing and managing disorders affiliated with iron homeostasis imbalances.\u003c/p\u003e \u003cp\u003eIn this study, we collected femoral neck bone tissues from patients with different bone mineral density (BMD) levels and observed that as BMD declined from normal to osteopenia and then to osteoporosis, there was a progressive accumulation of iron within the bone tissue. This raises the question of whether this accumulated iron might influence bone metabolism by modulating the phosphorylation of proteins integral to this process. To explore this further, we subjected mice to a high-iron intervention and observed that the elevated iron levels impaired bone microarchitecture, suppressed osteoblast differentiation, enhanced osteoclast differentiation, markedly increased the iron content within the bone tissue, and induced ferroptosis in osteoblasts. Moreover, when we cultured primary bone marrow cells from mice under high-iron conditions, we witnessed a suppression of osteogenic differentiation, a promotion of osteoclastic differentiation, and the occurrence of ferroptosis in the primary osteoblasts. Proteomics and phosphoproteomics analyses uncovered a significant role of FTH1/FTL in mediating ferroptosis in osteoblasts and facilitating osteoclast differentiation, possibly through a co-stimulatory pathway in response to high-iron exposure. Drawing on these discoveries, we suggest that iron overload-induced ferroptosis in osteoblasts may represent a potential therapeutic target for the treatment of osteoporosis.\u003c/p\u003e"},{"header":"Material and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eCollection and Grouping Methods of Clinical Samples\u003c/h2\u003e \u003cp\u003e The collection and use of clinical samples were approved by the Biomedical Ethics Committee of West China Hospital of Sichuan University (Ethics Approval No: 2023\u0026thinsp;\u0026minus;\u0026thinsp;1307). For short, we recruited patients diagnosed with unilateral femoral neck fracture who required hip replacement surgery. Each patient underwent a BMD examination using a dual-energy X-ray absorptiometry (DXA, GE Healthcare, Lunar iDXA, ME\u0026thinsp;+\u0026thinsp;212243) 1\u0026ndash;3 days before the hip replacement surgery. Based on the T-scores of the lumbar spine (L1-L4) as measured by DXA, the samples were divided into three groups: normal bone density (T-score \u0026ge; -1.0), osteopenia (-1.0\u0026thinsp;\u0026gt;\u0026thinsp;T-score \u0026gt; -2.5), and osteoporosis (T-score \u0026le; -2.5). Exclusion criteria ruled out individuals with tumor, hip dysplasia, infection, necrosis of the femoral head, osteomalacia, renal insufficiency, history of hip surgery, coagulation disorders, history of anti-osteoporosis treatment, and diseases or medications affecting bone metabolism, such as parathyroid, thyroid, adrenal disease, diabetes, as well as treatment with steroid or bisphosphonate. Following informed consent, the study comprised femoral neck bone tissues from 13 participants with normal BMD, 12 with osteopenia, and 15 with osteoporosis. The mean T-scores for the lumbar spines (L1-L4) across the three groups were \u0026minus;\u0026thinsp;0.07\u0026thinsp;\u0026plusmn;\u0026thinsp;0.77, -1.10\u0026thinsp;\u0026plusmn;\u0026thinsp;1.34, and \u0026minus;\u0026thinsp;2.97\u0026thinsp;\u0026plusmn;\u0026thinsp;0.91, respectively (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003e\u003cb\u003eDemographics and characteristics of the clinical samples\u003c/b\u003e\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCharacteristics\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNormal\u003c/p\u003e \u003cp\u003eN\u0026thinsp;=\u0026thinsp;13\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eOsteopenia\u003c/p\u003e \u003cp\u003eN\u0026thinsp;=\u0026thinsp;12\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eOsteoporosis\u003c/p\u003e \u003cp\u003eN\u0026thinsp;=\u0026thinsp;15\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eP\u003c/em\u003e value\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAge, years, mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e47.4\u0026thinsp;\u0026plusmn;\u0026thinsp;11.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e64.6\u0026thinsp;\u0026plusmn;\u0026thinsp;13.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e74.1\u0026thinsp;\u0026plusmn;\u0026thinsp;12.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMale, N (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7 (53.8%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7 (58.3%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.002\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFemale, N (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6 (46.2%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5 (41.7%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e15 (100%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.002\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBMI, kg/m\u003csup\u003e2\u003c/sup\u003e, mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e24.4\u0026thinsp;\u0026plusmn;\u0026thinsp;3.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e24.9\u0026thinsp;\u0026plusmn;\u0026thinsp;2.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e22.3\u0026thinsp;\u0026plusmn;\u0026thinsp;3.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.063\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBMD T-score, mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"4\" nameend=\"c5\" namest=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLumbar spine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-0.07\u0026thinsp;\u0026plusmn;\u0026thinsp;0.77\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-1.10\u0026thinsp;\u0026plusmn;\u0026thinsp;1.34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-2.97\u0026thinsp;\u0026plusmn;\u0026thinsp;0.91\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTotal hip\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-0.07\u0026thinsp;\u0026plusmn;\u0026thinsp;0.79\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-1.41\u0026thinsp;\u0026plusmn;\u0026thinsp;0.65\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-2.30\u0026thinsp;\u0026plusmn;\u0026thinsp;0.98\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFemoral neck\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.08\u0026thinsp;\u0026plusmn;\u0026thinsp;1.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-1.07\u0026thinsp;\u0026plusmn;\u0026thinsp;1.31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-2.28\u0026thinsp;\u0026plusmn;\u0026thinsp;1.51\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eDetection of Iron Content\u003c/h3\u003e\n\u003cp\u003eAccurately weigh a predetermined quantity of bone tissue and deposit it into a microwave digestion vessel. Introduce 2mL of concentrated nitric acid into the vessel, then position both the digestion vessel and its corresponding support liner within the microwave digestion apparatus. Initiate the microwave-assisted digestion process at 120\u0026deg;C for a duration of 60min. Upon completion of digestion, carefully decant the resultant solution into a beaker, subject it to evaporation to remove excess acid, followed by a reflux procedure lasting 60min. Subsequently, dilute the solution to achieve a final volume of 25mL using ultrapure water, and quantify the iron concentration via inductively coupled plasma mass spectrometry (ICP-MS).\u003c/p\u003e \u003cp\u003eFor osteoblast samples exposed to high iron intervention, isolate 300\u0026micro;g of protein, which should be diluted to a consistent volume utilizing RIPA lysis buffer, and subsequently concentrated until complete dryness is attained. Thereafter, incorporate 2mL of nitric acid into the dried sample and proceed with the identical sequential steps as those employed for the bone tissue analysis.\u003c/p\u003e\n\u003ch3\u003eAnimal Experiments\u003c/h3\u003e\n\u003cp\u003e All animal experiments were conducted following the guidelines set forth by the Institutional Animal Care and Use Committee of West China Hospital, Sichuan University (Approval No. 20240311021), adhering strictly to the principles outlined in the Guide for the Care and Use of Laboratory Animals. Seven-week-old female C57BL/6 mice were purchased from GemPharmatech Co., Ltd. (Nanjing, China) and maintained in a standard light-dark cycle and provided with free access to water and the corresponding diet.\u003c/p\u003e \u003cp\u003eAfter one week of adaptive feeding, the mice were randomized into two distinct dietary cohorts: a standard diet (SD) group and a rich-iron diet (RFeD) group, comprising 20 mice per cohort. The SD group received a diet supplemented with 37ppm iron, whereas the RFeD group was administered a diet enriched with 350ppm iron. At the conclusion of one and two months of dietary intervention, ten mice from each group were euthanized for the purposed of collecting biological specimens. Throughout the experimental phase, the body weight and caloric consumption of the mice were recorded biweekly.\u003c/p\u003e\n\u003ch3\u003eMicro-computed tomography (µCT) analysis\u003c/h3\u003e\n\u003cp\u003ePost-intervention with either RFeD or SD, the right femur of the mice was harvested. Careful excision of soft tissues including muscles and fasciae was performed, followed by thorough rinsing of the femurs with chilled phosphate-buffered saline (PBS). Subsequently, the bones were preserved \u003cem\u003evia\u003c/em\u003e overnight fixation in paraformaldehyde. High-resolution \u0026micro;CT scans were executed employing a vivaCT80 scanner (Scanco Medical, Switzerland), facilitated by the Analytical and Testing Center at Sichuan University. Scan settings comprised an energy/intensity of 55kV and 145\u0026micro;A, resulting in 8W power output, with a voxel resolution of 10\u0026micro;m. The scanned area encompassed the metaphysis and diaphysis of the right femur. Regions of interest (ROIs) were delineated to include 100 layers of trabecular bone situated directly beneath the growth plate, along with 100 layers of cortical bone derived from the diaphysis. Three-dimensional reconstructions and analyses were carried out utilizing Scanco\u0026rsquo;s proprietary analytical software suite, applying lower and upper threshold values of 170 and 1000, respectively. Parameters assessed for trabecular bone encompassed bone volume fraction (BV/TV), trabecular number (Tb.N), trabecular thickness (Tb.Th), trabecular separation (Tb.Sp), connectivity density (Conn.D), and structural model index (SMI). For cortical bone, evaluated metrics consisted of cortical BV/TV, cortical thickness (Ct.Th), and the ratio of bone surface area to bone volume (BS/BV).\u003c/p\u003e\n\u003ch3\u003eHistopathological Analysis\u003c/h3\u003e\n\u003cp\u003eFollowing the intervention with either RFeD or SD, the left tibia of the mice was collected. Meticulous removal of soft tissues, such as muscles and fasciae, was undertaken, followed by a thorough rinse of the tibias with chilled PBS. Subsequently, the bones underwent preservation through overnight fixation in paraformaldehyde. On the following day, the fixed bone tissue was washed under running water for 10 minutes, then immersed in EDTA decalcification solution for 21 days, with the solution being replaced every 3 days. Post-decalcification, the bone tissue was embedded in paraffin and sliced into sections measuring 5\u0026micro;m in thickness. These sections were stained with hematoxylin and eosin (HE) and tartrate-resistant acid phosphatase (TRAP, Wako TRAP/ALP staining kit, Wako Pure Chemical Industries, Ltd., Japan, Catalog No. 294-67001) to examine the impact of RFeD intervention on osteoblast and osteoclast differentiation. Immunohistochemical staining was conducted to assess the expression of ferritin using an anti-ferritin antibody (Abcam, British, Catalog No. ab183781). Additionally, the TUNEL assay kit (Promega, Wisconsin, United States, Catalog No. G3250) was utilized to investigate the apoptosis of bone cells.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eSerum Ferritin Detection\u003c/h2\u003e \u003cp\u003eSerum ferritin concentrations in mice were quantified utilizing an enzyme-linked immunosorbent assay (ELISA) kit provided by Kenuodi Biotechnology Co., Ltd. (Quanzhou, China; Catalog No. SU-B21204), adhering meticulously to the manufacturer\u0026rsquo;s protocols. The resultant ferritin levels were derived from the established standard curve.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eLipid Peroxidation-MDA Detection\u003c/h3\u003e\n\u003cp\u003eTo assess lipid peroxidation in plasma, bone, and osteoblasts of the mice, we employed the Lipid Peroxidation Assay Kit (Beyotime, S0131, Shanghai, China). Following the acquisition of bone tissue and osteoblast protein, the protein concentration was measured \u003cem\u003evia\u003c/em\u003e the BCA method. Plasma samples were utilized directly for malondialdehyde (MDA) quantification. The protocol for MDA detection was rigorously adhered to the guidelines provided by the manufacturer.\u003c/p\u003e\n\u003ch3\u003eCulture and Induction of Mouse Primary Bone Marrow Cells\u003c/h3\u003e\n\u003cp\u003eThe culture and induction of mouse primary bone marrow cells were carried out as described in previous studies(22\u0026ndash;26). Briefly, the isolated bone marrow cells were resuspended in aMEM medium containing 10% fetal bovine serum (FBS, Gibco). These cells were cultured in an incubator maintained at 37\u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e. On the following day, the non-adherent cells were gently collected, centrifuged, and subsequently resuspended in fresh aMEM medium containing 10% FBS and 30ng/mL of recombinant mouse macrophage colony-stimulating factor (M-CSF, R\u0026amp;D System, Minnesota, United States, Catalog No. 416-ML-010). After an additional 24-hour incubation period, the adherent cells obtained were identified as bone marrow-derived macrophages (BMDMs). These BMDMs could be subjected to further osteoclast differentiation induction, with or without the inclusion of high iron (at a concentration of 5\u0026micro;M). Meanwhile, the adherent cells remaining in the initial culture dish were designated as bone marrow mesenchymal stem cells (BMSCs, P0). Upon subsequent passaging and purification processes (approximately reaching P3), these BMSCs became suitable for osteoblast differentiation induction, again with or without the addition of high iron (at a concentration of 20\u0026micro;M).\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eTRAP, ALP Staining and Relative Quantification of ALP Activity\u003c/h2\u003e \u003cp\u003eFirst, digested the adherent BMDMs, centrifuged the resulting suspension, and then resuspended the cells in aMEM medium supplemented with 10% FBS and 30ng/mL of recombinant mouse M-CSF. Next, seeded the cells into 48-well plates and 6-well plates at cell densities of 30 000 cells per well and 200 000 cells per well, respectively. On the following day, initiated osteoclast differentiation by treating the cells with 30ng/mL of mouse M-CSF and 50ng/mL of recombinant mouse receptor activator for nuclear factor κB ligand (RANKL, R\u0026amp;D system, Catalog No. 462-TEC-010), companied with or without high iron (5\u0026micro;M). Replaced the culture medium every two days and observed the development of fused multinucleated osteoclasts under a microscope.\u003c/p\u003e \u003cp\u003eSeven days post-induction, we performed TRAP staining on the osteoclasts residing in the 48-well plates using the TRAP staining kit (Sigma-Aldrich, Missouri, United States, 387A-1KT). Captured digital images of the stained osteoclasts. Utilized ImageJ software (developed by the National Institutes of Health) to quantify the area occupied by multinucleated (\u0026gt;\u0026thinsp;3 nuclei) TRAP-positive osteoclasts. Subsequently, we employed Prism software for statistical analysis and graphical representation of the data. Meanwhile, the osteoclasts cultivated in the 6-well plates can be lysed to extract proteins for assessing the impact of high iron exposure on the expression of NFATc1, a pivotal protein involved in osteoclast differentiation.\u003c/p\u003e \u003cp\u003eAnalogously, once BMSCs reach passage three (P3), they should be digested, centrifuged, and then resuspended in aMEM medium enriched with 10% FBS. Plated the cells at densities of 50 000 cells per well in 24-well plates and 200 000 cells per well in 6-well plates. Commence osteoblast differentiation by exposing the cells to 50\u0026micro;g/mL ascorbic acid and 10mM β-glycerophosphate, with or without high iron supplementation (20\u0026micro;M). As previously mentioned, refreshed the culture medium every two days throughout the experiment.\u003c/p\u003e \u003cp\u003eUpon completion of seven days of induction, we conducted alkaline phosphatase (ALP) staining on the osteoblasts within the 24-well plates using the ALP staining kit (Sigma-Aldrich, 86R-1KT). Record visual documentation of the stained osteoblasts. Measure the relative ALP activity using appropriate methods. Finally, harvest the osteoblasts from the 6-well plates for protein extraction to evaluate the influence of high iron administration on the expression of RUNX2, which is crucial for osteoblast differentiation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eTUNEL Assay, C11-BODIPY and FerroOrange Staining\u003c/h2\u003e \u003cp\u003eThe apoptosis of osteoblasts in high-iron-treated mice was assessed using a One-Step TUNEL Apoptosis Detection Kit (Beyotime, C1086) strictly according to the manufacturer\u0026rsquo;s instructions. Briefly, primary mouse BMSCs were subjected to osteogenic induction and high-iron intervention, then fixed with 4% paraformaldehyde for 30min. After washing with PBS, the cells were permeabilized with PBS containing 0.3% Triton X-100 at room temperature for 5min. Subsequently, 50\u0026micro;L TUNEL working solution was added, and the samples were incubated at 37\u0026deg;C in the dark for 60min. After washing with PBS, the cells were stained with Hoechst 33342 (Thermo Scientific, Massachusetts, United States, 62249) solution at room temperature for 10min.\u003c/p\u003e \u003cp\u003eLipid peroxidation was evaluated using a C11-BODIPY probe (Beyotime, S0043). Following osteogenic induction and high-iron treatment, the osteoblasts were incubated with C11-BODIPY working solution (2\u0026micro;mol/L) at 37\u0026deg;C in the dark for 15min. After washing with PBS, the cells were stained with Hoechst 33342 solution at room temperature for 10min. Intracellular Fe\u0026sup2;⁺ levels were detected using a FerroOrange probe (Dojindo, Tokyo, Japan, F374). After osteogenic induction and high-iron treatment, the osteoblasts were washed with PBS and incubated with FerroOrange working solution (1\u0026micro;mol/L) at 37\u0026deg;C in the dark for 30min, followed by staining with Hoechst 33342 solution at room temperature for 10min. Images of the above stained cells were acquired and analyzed using a high-content imaging system (Opera Phenix Plus, PerkinElmer).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eWestern Blot\u003c/h2\u003e \u003cp\u003eMature osteoblasts (OBs) and osteoclasts (OCs) of the mice were lysed using RIPA lysis buffer containing a mixture of protease and phosphatase inhibitors (Bimake, Shanghai, China). After lysis, the protein concentration was determined using the Pierce\u0026trade; BCA method (Thermo Scientific), and all samples were adjusted to the same protein concentration with 4\u0026times; SDS. Equal amounts of protein samples were loaded onto 10% SDS-PAGE precast gels (Shanghai EpiZyme Biotechnology, Shanghai, China) and subsequently transferred to PVDF membranes (Millipore, Massachusetts, United States). The PVDF membranes were blocked in PBS/Tween containing 5% nonfat milk at room temperature for 1h, followed by incubation with primary antibodies (RUNX2 (1:1000, CST, Massachusetts, United States, 12556s), NFATc1 (1:500, Santa Cruz Biotechnology, California, United States, sc-7294), GPX4 (1:2000, Proteintech, Illinois, United States, 67763-1-Ig), and GAPDH (1:5000, Proteintech, 60004-1-Ig)) overnight at 4\u0026deg;C. The membranes were then incubated with HRP-conjugated goat anti-mouse secondary antibodies (1:5000, Proteintech, SA00001-1, SA00001-2) at room temperature for 1h. Finally, signals were detected using HRP substrate peroxide solution (Millipore) and HRP substrate luminol solution (Millipore), and the signal bands were visualized using an ultra-high sensitivity chemiluminescence imaging system (Minichemi). For protein extraction from mouse bone tissue, an appropriate amount of bone tissue should first be quickly placed in liquid nitrogen for grinding. After grinding, the powder was transferred into an EP tube containing an appropriate amount of RIPA lysis buffer. Subsequent steps were performed as described previously. In addition, the full and uncropped western blots were uploaded as \u0026ldquo;Supplemental Material\u0026rdquo;.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eLC-MS/MS Analyses\u003c/h2\u003e \u003cp\u003eThe detailed steps of protein extraction from mice primary OBs and OCs were described in the western blot section. 100\u0026micro;g of protein from each sample was then reduced using tris (2-carboxyethyl) phosphine (TCEP), alkylated with iodoacetamide (IAA), precipitated with CH\u003csub\u003e3\u003c/sub\u003eOH, H\u003csub\u003e2\u003c/sub\u003eO and CHCl\u003csub\u003e3\u003c/sub\u003e (CH\u003csub\u003e3\u003c/sub\u003eOH: H\u003csub\u003e2\u003c/sub\u003eO: CHCl\u003csub\u003e3\u003c/sub\u003e: protein\u0026thinsp;=\u0026thinsp;4:3:1:1), and digested with trypsin before tandem mass tag (TMT)-10 (Catalog No. 90110, Thermo Fisher Scientific, Waltham, MA) labeling consistent with the previous studies(21, 24, 27, 28). After desalting, the samples underwent phosphoprotein fractionation. The fractionated samples were enriched for phosphopeptides using PureCube Fe-NTA Agarose Beads (Catalog No. 31403-Fe, Cube Biotech, Monheim, Germany) according to the manufacturer\u0026rsquo;s instructions, and the enriched phosphopeptides were desalted with C18 ZipTips and analyzed by an Nano EASY-nLC 1200 liquid chromatography system LC instrument coupled with a Q Exactive HF-X high-resolution mass spectrometer (Serial No. SN06464L, Thermo Fisher Scientific). For protein quantification, 50\u0026micro;g of peptides from the remaining sample after phosphopeptides enrichment was used for HPLC fractionation and LC-MS/MS analysis using a Nano EASY-nLC 1200 liquid chromatography system LC instrument coupled with a Q Exactive Plus high-resolution mass spectrometer (Serial No. SN03572L, Thermo Fisher Scientific). Due to the large number of samples, to ensure instrument stability and comparability between samples during the analysis, a quality control (QC) sample was injected every three runs. The correlation between QC samples was monitored to assess instrument status and data reliability. The mass spectrometry raw data was processed using the MaxQuant (version 1.6) search engine.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eBioinformatics Analysis\u003c/h2\u003e \u003cp\u003eThe principal component analysis (PCA) of proteome and phosphoproteome was performed on protein abundance. Differentially expressed proteins (DEPs) were analyzed using R package, limma and edgeR, with a fold change greater than 1.2 and a statistically significant adjusted P-value less than 0.05. Gene ontology (GO) and kyoto encyclopedia of genes and genomes (KEGG) analysis were conducted to identify the biological functions of the DEPs and the pathways in which they are involved.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eStatistical analysis and plotting were performed using GraphPad Prism 10.1.1 software. All quantitative data were presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error (Mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM). Correlation analysis was conducted using either Pearson or Spearman correlation analysis. Comparisons between two groups were made using a two-sided \u003cem\u003et-test\u003c/em\u003e. One-way ANOVA was used for multiple comparisons, and Tukey\u0026rsquo;s multiple comparison test was performed when significant differences were detected. A \u003cem\u003ep\u003c/em\u003e-value ˂ 0.05 was considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eSignificant negative correlation between iron content and bone mineral density in clinical bone tissue.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eLiterature reviews indicate that plasma ferritin levels in osteoporotic patients are markedly elevated compared to healthy individuals(29, 30). To elucidate the alterations in iron content within bone tissue during the progression of bone loss-from normal BMD to low bone mass and eventually to osteoporosis-we assessed the iron content in femoral neck bone tissue obtained from clinical specimens. Our findings demonstrated that reductions in BMD were associated with iron accumulation in bone tissue (Fig.\u0026nbsp;1a). Notably, there existed a significant negative correlation between the iron content in bone tissue and BMD (Fig.\u0026nbsp;1b). The impact of accumulated iron in bone tissue on bone health remains unclear, as does the mechanism by which iron regulates bone metabolism. To address these gaps, we employed high-iron interventions in mice and their primary bone marrow cells to delve deeper into these inquiries.\u003c/p\u003e \u003cp\u003e \u003cb\u003eRFeD induced osteoporosis\u003c/b\u003e \u003cb\u003ein vivo\u003c/b\u003e.\u003c/p\u003e \u003cp\u003eTo examine the effects of iron accumulation in bone tissue on bone phenotype and metabolism, we administered SD and RFeD to mice, respectively (Fig.\u0026nbsp;2a). Following one-month and two-month treatments, ten mice per group were euthanized. \u0026micro;CT scans of the right femurs revealed that one month of RFeD intervention had already compromised the trabecular bone microarchitecture in mice. Prolonging the RFeD regimen to two months maintained this deterioration. Notably, there was a reduction in trabecular BV/TV, Tb.N, Tb.Th, and Conn.D, alongside an elevation in Tb.Sp and SMI (Fig.\u0026nbsp;2b-c). Furthermore, while one month of RFeD intervention did not disrupt the cortical bone microarchitecture in the mid-diaphysis of the femur, extending the dietary intervention to two months led to RFeD-induced impairment of the cortical bone microarchitecture. This impairment was evidenced by a decline in cortical BV/TV and Ct.Th, accompanied by an increase in BS/BV (Supplementary Fig.\u0026nbsp;1).\u003c/p\u003e \u003cp\u003eTo elucidate the pathological mechanisms behind the severe bone microarchitecture deterioration observed in mice following RFeD intervention, we conducted TRAP and HE staining on the left tibia of mice to assess the effects of RFeD intervention on osteoclast and osteoblast differentiation. Our findings demonstrated that RFeD intervention significantly augmented the TRAP-positive area within the bone tissue (Fig.\u0026nbsp;2d), thereby indicating an increase in the surface area occupied by osteoclasts (Oc.S/BS, Fig.\u0026nbsp;2e). In contrast, HE staining disclosed a substantial reduction in the number of osteoblasts present on the surface of the trabecular bone (Ob.N/BS) in mice subjected to RFeD intervention (Fig.\u0026nbsp;2f-g). Moreover, we investigated the expression of NFATc1, a critical regulator of osteoclastic differentiation, and RUNX2, a pivotal transcription factor involved in osteogenic differentiation, in mouse bone tissue post-corresponding dietary intervention. Our analyses revealed a dramatic upregulation of NFATc1 expression in mouse bone tissue (Fig.\u0026nbsp;2h-i), whereas the expression of RUNX2 exhibited a marked decrease following RFeD intervention (Fig.\u0026nbsp;2j-k). Collectively, these results underscored that RFeD intervention facilitated osteoclastic differentiation while concurrently inhibiting osteogenic differentiation, ultimately leading to osteoporosis \u003cem\u003ein vivo\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003eRFeD induced osteoblast ferroptosis\u003c/b\u003e \u003cb\u003ein vivo\u003c/b\u003e.\u003c/p\u003e \u003cp\u003eSubsequently, we assessed the serum ferritin levels and discovered a notable elevation in the serum ferritin content of mice subjected to RFeD intervention (Fig.\u0026nbsp;3a), which corresponded with the immunohistochemical staining of ferritin in bone tissue (Fig.\u0026nbsp;3b-c). Upon measuring the iron content in bone tissue, we observed a significant augmentation in the iron content of bone tissue in mice following RFeD intervention (Fig.\u0026nbsp;3d-e), and there was a pronounced negative correlation between bone tissue iron content and BMD (Fig.\u0026nbsp;3f-g). The TUNEL assay of bone tissue revealed apoptosis of osteoblasts (Fig.\u0026nbsp;3h-i), and the expression of the ferroptosis inhibitor-glutathione peroxidase 4 (GPX4) diminished considerably post-RFeD intervention (Fig.\u0026nbsp;3j-k). Furthermore, the content of the lipid oxidation marker-MDA in both plasma and bone tissue were significantly elevated (Fig.\u0026nbsp;3l-m). These findings collectively imply that RFeD intervention induces osteoblast ferroptosis \u003cem\u003ein vivo\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003eIron overload impeded osteogenic differentiation while concurrently promoting osteoclastic differentiation\u003c/b\u003e \u003cb\u003ein vitro\u003c/b\u003e.\u003c/p\u003e \u003cp\u003eTo explore the impact of high iron supplementation on osteogenic/osteoclast differentiation, we cultured mouse primary bone marrow cells and treated them with or without high iron (5/20\u0026micro;M (NH₄)₂Fe(SO₄)₂, Fig.\u0026nbsp;4a). Following a seven-day induction period, we initially assessed the iron concentration within the cells. Notably, the iron level in primary osteoblasts markedly escalated upon exposure to high iron conditions (20\u0026micro;M (NH₄)₂Fe(SO₄)₂, Fig.\u0026nbsp;4b). Subsequently, we elucidated the influence of high iron administration on osteogenic and osteoclastic differentiation through ALP and TRAP staining, alongside examining the protein expressions of RUNX2 and NFATc1. Our findings indicated that treating with 20\u0026micro;M (NH₄)₂Fe(SO₄)₂ substantially hindered the differentiation of mouse primary BMSCs into osteoblasts (Fig.\u0026nbsp;4c-d). Conversely, administering 5\u0026micro;M (NH₄)₂Fe(SO₄)₂ significantly enhanced the differentiation of mouse primary bone marrow cells into osteoclasts (Fig.\u0026nbsp;4e-f). The protein expression patterns of RUNX2 (Fig.\u0026nbsp;4g-h) and NFATc1 (Fig.\u0026nbsp;4i-j) mirrored the outcomes of the histopathological staining analyses.\u003c/p\u003e \u003cp\u003e \u003cb\u003eIron overload induced osteoblast ferroptosis\u003c/b\u003e \u003cb\u003ein vitro\u003c/b\u003e.\u003c/p\u003e \u003cp\u003eFerroptosis typically manifests as a range of morphological and biochemical alterations, including iron accumulation, mitochondrial contraction, and lipid peroxidation(20). To confirm the occurrence of ferroptosis, TUNEL assay, C11-BODIPY staining and FerroOrange staining were conducted on mice primary osteoblasts subjected to high iron treatment. The TUNEL assay verified a substantial increase in cell death among primary osteoblasts post-high iron exposure (Fig.\u0026nbsp;5a-b). Intracellular lipid peroxides were assayed using the C11-BODIPY fluorescent probe, revealing pronounced lipid peroxidation in primary osteoblasts following high iron treatment (Fig.\u0026nbsp;5a, c). A similar pattern was noted in the generation of MDA, the terminal product of lipid peroxidation (Fig.\u0026nbsp;5e). FerroOrange, a Fe\u003csup\u003e2+\u003c/sup\u003e-specific probe, exhibited a marked rise in fluorescence intensity under high iron conditions (Fig.\u0026nbsp;5a, d). Lastly, the protein expression level of GPX4 was notably reduced in mice primary osteoblasts exposed to high iron (Fig.\u0026nbsp;5f-g). These findings collectively demonstrate that high iron intervention induces ferroptosis in osteoblasts \u003cem\u003ein vitro\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003eIron overload triggered ferroptosis in osteoblasts and activated osteoclast differentiation.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo elucidate the underlying mechanisms behind the suppression of osteogenic differentiation and stimulation of osteoclast differentiation in primary bone marrow cells after high-iron intervention, we undertook proteomic and phosphoproteomic analyses on the primary cells (Fig.\u0026nbsp;6a-c). Our findings demonstrated a pronounced activation of the \u0026ldquo;ferroptosis\u0026rdquo; signaling pathway within osteoblasts following high-iron intervention (Fig.\u0026nbsp;6d-f), whereas \u0026ldquo;osteoclast differentiation\u0026rdquo; was distinctly augmented in osteoclasts (Fig.\u0026nbsp;6g-i). Subsequent phosphoproteomic investigation disclosed a significant amplification of the \u0026ldquo;actin cytoskeleton organization\u0026rdquo; signaling pathway in both osteoblasts and osteoclasts post-intervention (Fig.\u0026nbsp;6j-l), which was essential for the migration of osteoblasts and osteoclasts to sites of active bone metabolism, enabling them to perform their osteogenic and osteoclastic functions. Extensive analysis revealed a substantial upregulation of FTH1/FTL protein expression within the ferroptosis signaling pathway of osteoblasts (Fig.\u0026nbsp;6m). Ferroptotic osteoblasts subsequently initiated the co-stimulatory pathway, thereby promoting osteoclast differentiation, culminating in an osteoporotic phenotype in mice (Fig.\u0026nbsp;6n). Collectively, our findings suggest that high-iron intervention results in iron accumulation within the bone tissue of mice. The accumulated iron induces ferroptosis in osteoblasts \u003cem\u003evia\u003c/em\u003e signaling pathways such as FTH1/FTL. Ferroptotic osteoblasts activate the co-stimulatory pathway, which further promotes osteoclast differentiation, ultimately leading to the development of osteoporosis. Specifically, iron overload disrupts the equilibrium between bone formation and resorption, resulting in heightened osteoclast activity and bone loss.\u003c/p\u003e "},{"header":"Discussion and Conclusion","content":"\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003cp\u003eIn this article, our literature review revealed that clinical osteoporotic patients exhibit significantly elevated serum ferritin levels compared to healthy individuals(29, 30), alongside a notable increase in bone iron content. A striking inverse relationship exists between bone tissue iron content and BMD. Subsequent studies using RFeD intervention on mice demonstrated pronounced impairment of bone microarchitecture, a substantial reduction in osteoblast numbers, and a marked increase in osteoclast surface area. Further investigation indicated that post-RFeD intervention, both serum ferritin levels and bone iron content rose significantly. Consistently, the iron concentration in mouse bone tissue exhibited a negative correlation with BMD following RFeD intervention. Additionally, RFeD intervention led to significant apoptosis of bone cells, with a notable downregulation of the ferroptosis inhibitor-GPX4 in bone tissue. Concurrently, the level of the lipid oxidation end product-MDA in plasma and bone tissue escalated dramatically.\u003c/p\u003e \u003cp\u003eIn addition to the evident destruction of bone microarchitecture, ferroptosis of osteoblasts represents a crucial discovery within this research. Ferroptosis denotes a novel form of programmed cell death triggered by unchecked iron-dependent lipid peroxidation(15, 16, 31, 32). Distinct from other cell death modalities, ferroptosis exhibits distinctive biological hallmarks such as iron accumulation, augmented lipid peroxidation products, and diminished GPX4 expression(33\u0026ndash;36). Extensive research indicates that ferroptosis is intricately linked to a myriad of disorders, encompassing metabolic diseases, neurodegenerative ailments, ischemia-reperfusion injuries, cardiomyopathies, and the immunotherapeutic responses against cancer, among others(33, 34, 37\u0026ndash;39). Consequently, targeting ferroptosis could emerge as a potent therapeutic avenue for managing associated diseases(18). Preserving osteoblast vitality is widely regarded as an efficacious approach to sustaining skeletal well-being(40\u0026ndash;42). Previous investigations predominantly attributed caspase-dependent apoptosis and necrosis as the predominant modes of cell demise. For instance, Emerton \u003cem\u003eet al\u003c/em\u003e. documented that estrogen insufficiency amplifies osteocyte apoptosis, thereby diminishing bone volume(43). Nonetheless, the present study unveils that excessive lipid peroxidation potentially constitutes the principal inducer of osteoblast damage under high iron conditions, intimating a close association between ferroptosis and its underlying molecular mechanisms.\u003c/p\u003e \u003cp\u003eNext, we subjected mice primary bone marrow cells to high-iron intervention and found that this intervention inhibited osteogenic differentiation while promoting osteoclastic differentiation. Our subsequent investigations revealed that high-iron exposure resulted in a marked elevation in mortality, lipid oxidation, and intracellular Fe\u003csup\u003e2+\u003c/sup\u003e levels among primary osteoblasts in mice. Simultaneously, there was a pronounced reduction in GPX4 expression within osteoblasts. These findings suggest that high-iron intervention triggers a surge in free, labile iron within cells, which catalyzes the Fenton reaction. Consequently, accumulated fatty acids interact with reactive oxygen species (ROS) to generate lipid peroxides and lipid peroxyl radicals(44, 45). An abundance of lipid peroxides subsequently oxidizes cellular membranes and organelles, liberating additional ROS from compromised mitochondria. This process establishes a detrimental feedback loop of ferroptosis, culminating in the demise of a greater number of osteoblasts.\u003c/p\u003e \u003cp\u003eIron plays a crucial role in maintaining normal physiological functions across organisms, and dysregulation of iron metabolism is implicated in the pathogenesis of numerous diseases(7, 14). In recent years, advancements in omics technologies have furnished novel insights and methodologies for investigating iron homeostasis. Particularly, the implementation of proteomics and phosphoproteomics techniques has substantially enhanced our comprehension of iron homeostasis and associated metabolic disorders. Ultimately, we conducted proteomic and phosphoproteomic analyses on mice primary bone marrow cells following high-iron treatment. We identified that the \u0026ldquo;ferroptosis\u0026rdquo; signaling pathway was prominently enriched in osteoblasts, whereas \u0026ldquo;osteoclast differentiation\u0026rdquo; was distinctly activated in osteoclasts. Through comprehensive examination, we observed a substantial upregulation of FTH1/FTL protein expression within the osteoblast ferroptosis signaling cascade. This implies that excessive iron might facilitate ferroptosis in osteoblasts \u003cem\u003evia\u003c/em\u003e signaling routes such as FTH1/FTL(46). Ferroptotic osteoblasts subsequently engage the co-stimulatory pathway, thereby promoting osteoclast differentiation and contributing to the onset and progression of osteoporosis(19). Hence, it stands to reason that disrupting the ferroptosis \u003cem\u003evicious cycle\u003c/em\u003e in osteoblasts could represent a promising therapeutic strategy for managing clinical osteoporosis.\u003c/p\u003e \u003cp\u003eFinally, despite these findings, our study does have certain limitations. First, obtaining clinical bone tissue samples presents challenges. The high inorganic content of bone tissue complicates protein extraction. Additionally, research focusing on the proteomics and post-translational modifications of human bone remains limited. However, analyzing human bone tissue would provide valuable validation of our conclusions. Consequently, we plan to extend our omics studies to include clinical bone tissue in future work. Furthermore, although we established that high-iron intervention triggers significant ferroptosis in osteoblasts, we did not delve into the downstream pathways associated with ferroptosis. We intend to pursue a deeper investigation into these pathways. Lastly, our current observations were confined to the effects of high-iron intervention on mice and their primary bone marrow cells, both \u003cem\u003ein vivo\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e. For our upcoming research, we aim to administer a chelating agent to high-iron-intervened mice and their primary cells. This approach will allow us to assess whether blocking the ferroptosis cycle can partially or fully reverse the bone metabolic phenotype in both mice and primary cells.\u003c/p\u003e \u003cp\u003eIn summary, through comprehensive \u003cem\u003ein vivo\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e experimentation, we demonstrated that high-iron exposure triggers ferroptosis in mice osteoblasts, thereby hindering osteogenic differentiation and augmenting osteoclastic activity. This cascade of events compromises bone microarchitecture and culminates in the development of an osteoporotic phenotype in mice. Building upon these findings, we posit that RFeD intervention in mice offers a promising model for recapitulating osteoporosis in clinical scenarios, with ferroptosis in osteoblasts emerging as a plausible therapeutic avenue for managing osteoporosis. Future endeavors should concentrate on evaluating the efficacy of iron-chelating therapies in iron-overloaded mice and their primary cells. Subsequent investigations should emphasize the potential clinical applications of this therapeutic strategy.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe\u0026nbsp;authors\u0026nbsp;would\u0026nbsp;like\u0026nbsp;to\u0026nbsp;thank\u0026nbsp;Dr.\u0026nbsp;Li\u0026nbsp;Chen\u0026nbsp;from\u0026nbsp;Analytical\u0026nbsp;\u0026amp;\u0026nbsp;Testing\u0026nbsp;Center of Sichuan\u0026nbsp;University\u0026nbsp;for\u0026nbsp;her\u0026nbsp;help\u0026nbsp;with\u0026nbsp;micro-CT\u0026nbsp;scanning.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of Data\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe mass spectrometry proteomics and phosphoproteomics data have been deposited to the ProteomeXchange Consortium (https://proteomecentral.proteomexchange.org) \u003cem\u003evia\u003c/em\u003e the iProX partner repository(47, 48) with the dataset identifier PXD060747. All data underlying this article will be shared on reasonable request to the corresponding authors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by grants from the Sichuan Science and Technology Program to JWX (2023YFS0233), the Science and Technology Department of Sichuan Province (2024NSFSC1623) and the Postdoctor Research Fund of West China Hospital, Sichuan University (2024HXBH129).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eGuha I, Nadeem SA, Zhang X, DiCamillo PA, Levy SM, Wang G, \u003cem\u003eet al.\u003c/em\u003e Deep learning-based harmonization of trabecular bone microstructures between high- and low-resolution CT imaging. Med Phys. 2024.\u003c/li\u003e\n\u003cli\u003eXu J, Cai X, Miao Z, Yan Y, Chen D, Yang ZX, \u003cem\u003eet al.\u003c/em\u003e Proteome-wide profiling reveals dysregulated molecular features and accelerated aging in osteoporosis: A 9.8-year prospective study. Aging Cell. 2024;23(2):e14035.\u003c/li\u003e\n\u003cli\u003eEnsrud KE, Crandall CJ. Osteoporosis. Ann Intern Med. 2017;167(3):Itc17-itc32.\u003c/li\u003e\n\u003cli\u003eWang L, Yu W, Yin X, Cui L, Tang S, Jiang N, \u003cem\u003eet al.\u003c/em\u003e Prevalence of Osteoporosis and Fracture in China: The China Osteoporosis Prevalence Study. JAMA Netw Open. 2021;4(8):e2121106.\u003c/li\u003e\n\u003cli\u003eWang O, Hu Y, Gong S, Xue Q, Deng Z, Wang L, \u003cem\u003eet al.\u003c/em\u003e A survey of outcomes and management of patients post fragility fractures in China. Osteoporosis international : a journal established as result of cooperation between the European Foundation for Osteoporosis and the National Osteoporosis Foundation of the USA. 2015;26(11):2631\u0026ndash;2640.\u003c/li\u003e\n\u003cli\u003eBriggs AM, Cross MJ, Hoy DG, S\u0026agrave;nchez-Riera L, Blyth FM, Woolf AD, \u003cem\u003eet al.\u003c/em\u003e Musculoskeletal Health Conditions Represent a Global Threat to Healthy Aging: A Report for the 2015 World Health Organization World Report on Ageing and Health. Gerontologist. 2016;56 Suppl 2:S243-255.\u003c/li\u003e\n\u003cli\u003eLi GF, Pan YZ, Sirois P, Li K, Xu YJ. Iron homeostasis in osteoporosis and its clinical implications. Osteoporosis international : a journal established as result of cooperation between the European Foundation for Osteoporosis and the National Osteoporosis Foundation of the USA. 2012;23(10):2403\u0026ndash;2408.\u003c/li\u003e\n\u003cli\u003eAschemeyer S, Qiao B, Stefanova D, Valore EV, Sek AC, Ruwe TA, \u003cem\u003eet al.\u003c/em\u003e Structure-function analysis of ferroportin defines the binding site and an alternative mechanism of action of hepcidin. Blood. 2018;131(8):899\u0026ndash;910.\u003c/li\u003e\n\u003cli\u003eYuan Y, Fang Y, Zhu L, Gu Y, Li L, Qian J, \u003cem\u003eet al.\u003c/em\u003e Deterioration of hematopoietic autophagy is linked to osteoporosis. Aging Cell. 2020;19(5):e13114.\u003c/li\u003e\n\u003cli\u003eLedesma-Colunga MG, Weidner H, Vujic Spasic M, Hofbauer LC, Baschant U, Rauner M. Shaping the bone through iron and iron-related proteins. Semin Hematol. 2021;58(3):188\u0026ndash;200.\u003c/li\u003e\n\u003cli\u003eZhang H, Yang F, Cao Z, Xu Y, Wang M. The influence of iron on bone metabolism disorders. Osteoporosis international : a journal established as result of cooperation between the European Foundation for Osteoporosis and the National Osteoporosis Foundation of the USA. 2024;35(2):243\u0026ndash;253.\u003c/li\u003e\n\u003cli\u003eWang F, Lv H, Zhao B, Zhou L, Wang S, Luo J, \u003cem\u003eet al.\u003c/em\u003e Iron and leukemia: new insights for future treatments. J Exp Clin Cancer Res. 2019;38(1):406.\u003c/li\u003e\n\u003cli\u003eJiang X, Stockwell BR, Conrad M. Ferroptosis: mechanisms, biology and role in disease. Nat Rev Mol Cell Biol. 2021;22(4):266\u0026ndash;282.\u003c/li\u003e\n\u003cli\u003eMuckenthaler MU, Rivella S, Hentze MW, Galy B. A Red Carpet for Iron Metabolism. Cell. 2017;168(3):344\u0026ndash;361.\u003c/li\u003e\n\u003cli\u003eYang WS, Stockwell BR. Synthetic lethal screening identifies compounds activating iron-dependent, nonapoptotic cell death in oncogenic-RAS-harboring cancer cells. Chem Biol. 2008;15(3):234\u0026ndash;245.\u003c/li\u003e\n\u003cli\u003eDixon SJ, Lemberg KM, Lamprecht MR, Skouta R, Zaitsev EM, Gleason CE, \u003cem\u003eet al.\u003c/em\u003e Ferroptosis: an iron-dependent form of nonapoptotic cell death. Cell. 2012;149(5):1060\u0026ndash;1072.\u003c/li\u003e\n\u003cli\u003eJiang Z, Wang H, Qi G, Jiang C, Chen K, Yan Z. Iron overload-induced ferroptosis of osteoblasts inhibits osteogenesis and promotes osteoporosis: An in vitro and in vivo study. IUBMB Life. 2022;74(11):1052\u0026ndash;1069.\u003c/li\u003e\n\u003cli\u003eYang Y, Lin Y, Wang M, Yuan K, Wang Q, Mu P, \u003cem\u003eet al.\u003c/em\u003e Targeting ferroptosis suppresses osteocyte glucolipotoxicity and alleviates diabetic osteoporosis. Bone Res. 2022;10(1):26.\u003c/li\u003e\n\u003cli\u003eJiang Z, Qi G, He X, Yu Y, Cao Y, Zhang C, \u003cem\u003eet al.\u003c/em\u003e Ferroptosis in Osteocytes as a Target for Protection Against Postmenopausal Osteoporosis. Adv Sci (Weinh). 2024;11(12):e2307388.\u003c/li\u003e\n\u003cli\u003eWu X, Fang X, Lu F, Chen Q, Liu J, Zheng L. An update on the role of ferroptosis in the pathogenesis of osteoporosis. EFORT Open Rev. 2024;9(8):712\u0026ndash;722.\u003c/li\u003e\n\u003cli\u003eWang A, Zhang H, Li G, Chen B, Li J, Zhang T, \u003cem\u003eet al.\u003c/em\u003e Deciphering core proteins of osteoporosis with iron accumulation by proteomics in human bone. Front Endocrinol (Lausanne). 2022;13:961903.\u003c/li\u003e\n\u003cli\u003eZhang J, Zheng L, Wang Z, Pei H, Hu W, Nie J, \u003cem\u003eet al.\u003c/em\u003e Lowering iron level protects against bone loss in focally irradiated and contralateral femurs through distinct mechanisms. Bone. 2019;120:50\u0026ndash;60.\u003c/li\u003e\n\u003cli\u003eHu Y, Zhang Y, Ni CY, Chen CY, Rao SS, Yin H, \u003cem\u003eet al.\u003c/em\u003e Human umbilical cord mesenchymal stromal cells-derived extracellular vesicles exert potent bone protective effects by CLEC11A-mediated regulation of bone metabolism. Theranostics. 2020;10(5):2293\u0026ndash;2308.\u003c/li\u003e\n\u003cli\u003eXia W, Xie J, Cai Z, Liu X, Wen J, Cui ZK, \u003cem\u003eet al.\u003c/em\u003e Damaged brain accelerates bone healing by releasing small extracellular vesicles that target osteoprogenitors. Nat Commun. 2021;12(1):6043.\u003c/li\u003e\n\u003cli\u003eLin L, Guo Z, He E, Long X, Wang D, Zhang Y, \u003cem\u003eet al.\u003c/em\u003e SIRT2 regulates extracellular vesicle-mediated liver-bone communication. Nat Metab. 2023;5(5):821\u0026ndash;841.\u003c/li\u003e\n\u003cli\u003eZhang W, Zhou X, Hou W, Chen E, Ye C, Chen M, \u003cem\u003eet al.\u003c/em\u003e Reversing the imbalance in bone homeostasis via sustained release of SIRT-1 agonist to promote bone healing under osteoporotic condition. Bioact Mater. 2023;19:429\u0026ndash;443.\u003c/li\u003e\n\u003cli\u003eZhang R, Hu M, Liu Y, Li W, Xu Z, He S, \u003cem\u003eet al.\u003c/em\u003e Integrative Omics Uncovers Low Tumorous Magnesium Content as A Driver Factor of Colorectal Cancer. Genomics Proteomics Bioinformatics. 2024;22(4).\u003c/li\u003e\n\u003cli\u003eWang X, Luo Y, He S, Lu Y, Gong Y, Gao L, \u003cem\u003eet al.\u003c/em\u003e Age-, sex- and proximal-distal-resolved multi-omics identifies regulators of intestinal aging in non-human primates. Nat Aging. 2024;4(3):414\u0026ndash;433.\u003c/li\u003e\n\u003cli\u003eFung EB, Harmatz PR, Milet M, Coates TD, Thompson AA, Ranalli M, \u003cem\u003eet al.\u003c/em\u003e Fracture prevalence and relationship to endocrinopathy in iron overloaded patients with sickle cell disease and thalassemia. Bone. 2008;43(1):162\u0026ndash;168.\u003c/li\u003e\n\u003cli\u003eValenti L, Varenna M, Fracanzani AL, Rossi V, Fargion S, Sinigaglia L. Association between iron overload and osteoporosis in patients with hereditary hemochromatosis. Osteoporosis international : a journal established as result of cooperation between the European Foundation for Osteoporosis and the National Osteoporosis Foundation of the USA. 2009;20(4):549\u0026ndash;555.\u003c/li\u003e\n\u003cli\u003eHu Y, Wang Y, Liu S, Wang H. The Potential Roles of Ferroptosis in Pathophysiology and Treatment of Musculoskeletal Diseases-Opportunities, Challenges, and Perspectives. J Clin Med. 2023;12(6).\u003c/li\u003e\n\u003cli\u003eYao X, Sun K, Yu S, Luo J, Guo J, Lin J, \u003cem\u003eet al.\u003c/em\u003e Chondrocyte ferroptosis contribute to the progression of osteoarthritis. J Orthop Translat. 2021;27:33\u0026ndash;43.\u003c/li\u003e\n\u003cli\u003eStockwell BR, Friedmann Angeli JP, Bayir H, Bush AI, Conrad M, Dixon SJ, \u003cem\u003eet al.\u003c/em\u003e Ferroptosis: A Regulated Cell Death Nexus Linking Metabolism, Redox Biology, and Disease. Cell. 2017;171(2):273\u0026ndash;285.\u003c/li\u003e\n\u003cli\u003eTang D, Chen X, Kang R, Kroemer G. Ferroptosis: molecular mechanisms and health implications. Cell Res. 2021;31(2):107\u0026ndash;125.\u003c/li\u003e\n\u003cli\u003eHadian K, Stockwell BR. SnapShot: Ferroptosis. Cell. 2020;181(5):1188\u0026ndash;1188.e1181.\u003c/li\u003e\n\u003cli\u003eLee H, Zandkarimi F, Zhang Y, Meena JK, Kim J, Zhuang L, \u003cem\u003eet al.\u003c/em\u003e Energy-stress-mediated AMPK activation inhibits ferroptosis. Nat Cell Biol. 2020;22(2):225\u0026ndash;234.\u003c/li\u003e\n\u003cli\u003eFang X, Wang H, Han D, Xie E, Yang X, Wei J, \u003cem\u003eet al.\u003c/em\u003e Ferroptosis as a target for protection against cardiomyopathy. Proc Natl Acad Sci U S A. 2019;116(7):2672\u0026ndash;2680.\u003c/li\u003e\n\u003cli\u003eLiang C, Zhang X, Yang M, Dong X. Recent Progress in Ferroptosis Inducers for Cancer Therapy. Adv Mater. 2019;31(51):e1904197.\u003c/li\u003e\n\u003cli\u003eMaher P, Currais A, Schubert D. Using the Oxytosis/Ferroptosis Pathway to Understand and Treat Age-Associated Neurodegenerative Diseases. Cell Chem Biol. 2020;27(12):1456\u0026ndash;1471.\u003c/li\u003e\n\u003cli\u003eHan Y, You X, Xing W, Zhang Z, Zou W. Paracrine and endocrine actions of bone-the functions of secretory proteins from osteoblasts, osteocytes, and osteoclasts. Bone Res. 2018;6:16.\u003c/li\u003e\n\u003cli\u003eMaurel DB, Matsumoto T, Vallejo JA, Johnson ML, Dallas SL, Kitase Y, \u003cem\u003eet al.\u003c/em\u003e Characterization of a novel murine Sost ER(T2) Cre model targeting osteocytes. Bone Res. 2019;7:6.\u003c/li\u003e\n\u003cli\u003eLi MCM, Chow SKH, Wong RMY, Qin L, Cheung WH. The role of osteocytes-specific molecular mechanism in regulation of mechanotransduction - A systematic review. J Orthop Translat. 2021;29:1\u0026ndash;9.\u003c/li\u003e\n\u003cli\u003eEmerton KB, Hu B, Woo AA, Sinofsky A, Hernandez C, Majeska RJ, \u003cem\u003eet al.\u003c/em\u003e Osteocyte apoptosis and control of bone resorption following ovariectomy in mice. Bone. 2010;46(3):577\u0026ndash;583.\u003c/li\u003e\n\u003cli\u003eD'Autr\u0026eacute;aux B, Toledano MB. ROS as signalling molecules: mechanisms that generate specificity in ROS homeostasis. Nat Rev Mol Cell Biol. 2007;8(10):813\u0026ndash;824.\u003c/li\u003e\n\u003cli\u003eHolmstr\u0026ouml;m KM, Finkel T. Cellular mechanisms and physiological consequences of redox-dependent signalling. Nat Rev Mol Cell Biol. 2014;15(6):411\u0026ndash;421.\u003c/li\u003e\n\u003cli\u003eLiu J, Ren Z, Yang L, Zhu L, Li Y, Bie C, \u003cem\u003eet al.\u003c/em\u003e The NSUN5-FTH1/FTL pathway mediates ferroptosis in bone marrow-derived mesenchymal stem cells. Cell Death Discov. 2022;8(1):99.\u003c/li\u003e\n\u003cli\u003eMa J, Chen T, Wu S, Yang C, Bai M, Shu K, \u003cem\u003eet al.\u003c/em\u003e iProX: an integrated proteome resource. Nucleic Acids Res. 2019;47(D1):D1211-d1217.\u003c/li\u003e\n\u003cli\u003eChen T, Ma J, Liu Y, Chen Z, Xiao N, Lu Y, \u003cem\u003eet al.\u003c/em\u003e iProX in 2021: connecting proteomics data sharing with big data. Nucleic Acids Res. 2022;50(D1):D1522-d1527.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Osteoporotic fracture, ferroptosis, osteoblast, proteomics, phosphoproteomics","lastPublishedDoi":"10.21203/rs.3.rs-6072367/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6072367/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eOsteoporosis is a systemic skeletal disorder marked by reduced bone mass, compromised bone microarchitecture, heightened fragility, and an increased risk of fractures. Fractures resulting from osteoporosis are a leading cause of mortality and disability among the elderly. Ferroptosis is an emerging form of programmed cell death that occurs due to unregulated iron-dependent lipid peroxidation. Our study reveals that high-iron exposure triggers ferroptosis in osteoblasts through the FTH1/FTL pathway, as demonstrated by both \u003cem\u003ein vivo\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e experiments. Ferroptotic osteoblasts initiate a co-stimulatory pathway that fosters osteoclast differentiation, culminating in an osteoporotic phenotype in mice. We propose that the high-iron intervention in mice could be utilized as a novel model for replicating clinical osteoporosis, and that inhibiting ferroptosis in osteoblasts may represent a promising therapeutic strategy for the treatment of osteoporosis. Overall, our findings offer fresh perspectives on the pathogenesis of OP and identify potential new targets for the clinical management of this condition.\u003c/p\u003e","manuscriptTitle":"Iron overload-induced ferroptosis of osteoblasts as a potential therapeutic target for osteoporosis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-19 11:27:24","doi":"10.21203/rs.3.rs-6072367/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"c7d413c2-1775-4411-b736-182416637a0b","owner":[],"postedDate":"April 19th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":44789598,"name":"Health sciences/Diseases/Endocrine system and metabolic diseases"},{"id":44789599,"name":"Biological sciences/Chemical biology/Biophysical chemistry"}],"tags":[],"updatedAt":"2025-07-07T14:26:38+00:00","versionOfRecord":[],"versionCreatedAt":"2025-04-19 11:27:24","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6072367","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6072367","identity":"rs-6072367","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}