Fracture-Induced Immunological Cascades Trigger Rapid Systemic bone lo

Introduction

The rapid increase in the risk of refracture shortly after an initial fracture, known as imminent fracture risk, is a critical concern.^1–5 Our study and previous early studies identified systemic bone loss after fracture as a significant contributor to this increased risk,^6,7 which may perpetuate a vicious cycle of fracture-systemic bone loss-refracture. Research has indicated that the extent of systemic bone loss correlates with age,⁷ sex,⁸ the severity of the initial fracture,⁶ and the skeletal location of bone loss.⁹ However, the specific mechanisms underlying bone loss after fracture remain unclear.

Inflammation plays an important role in bone loss, including postmenopausal osteoporosis^10–12 and osteoarthritis-associated bone loss.^13–15 Coincidentally, fracture healing can be divided into four phases, with the initial inflammatory phase being a crucial stage.¹⁶ Previous researchers have reported that excessive inflammatory response is detrimental to bone.^17–19 Additionally, clinical trials have demonstrated that targeting inflammation, including the use of IL-6 inhibitors or IL-6 receptor antibodies such as tocilizumab (TCZ), holds therapeutic potential for conditions like osteoarthritis and polymyalgia rheumatica (PMR).^20–23 Now, the rapid advancement in novel drug delivery materials offers innovative strategies for the targeted delivery of IL-6 therapeutics, holding substantial promise for applications in the treatment of associated diseases.^24–26

Furthermore, as a major molecule that regulates various systems in the body, such as heart health,^27,28 obesity,²⁹ cancer,³⁰ autoimmune disease³¹ and diabetes,³² the inflammatory cytokine IL-6, which is released most extensively and at the highest levels during the inflammatory phase, has been associated with various types of osteoporosis.^33–39 Whether the sharp increase in IL-6 during the early inflammatory phase of fracture healing, as reported in previous studies and our studies,^6,40–42 contributes to rapid systemic bone loss during fracture healing remains unknown. Additionally, the specific bone cell type targeted by IL-6 to regulate bone loss remains unclear. However, prior studies have explored the role of IL-6 in osteoclast and osteoblast.^34,43–47 Research has reported both the enhancing^48,49 and inhibitory effects^50,51 of IL-6 on osteoblast, but consensus remains elusive.^52,53 For osteoclast, further investigations suggest that IL-6 may not directly increase osteoclast activity^54,55 but instead requires osteoblast to indirectly promote RANKL, thereby increasing osteoclastic ability.^56,57 Combined with our previous findings that bone loss after fracture has no significant effect on osteoblast and considering the increase in osteoclast activity after fracture,⁶ IL-6 may not directly participate in osteoclastic and osteoblastic functions.

Osteocyte, which constitute more than 90–95% of all bone cells and can communicate rapidly,^58,59 play a crucial role in bone metabolism.^60–62 They influence various types of bone loss and participate in the crosstalk of other organs with bone,⁶³ which highlights their systemic importance.^48,64 The osteocyte-osteoclast axis is well established to enhance osteoclastic activity mediated by RANKL-induced bone loss.⁶⁵ We further investigated whether IL-6 can indirectly influence osteoclastic activity through osteocyte, leading to systemic bone loss after fracture.

STAT3 phosphorylation occurs downstream of IL-6 and has recently been discovered to closely regulate various pathways of bone metabolism, which leads to bone loss. Therefore, elucidating the role of the IL-6/ STAT3 phosphorylation pathway in regulating osteocyte would be highly beneficial for uncovering the mechanisms of bone loss after fracture.^66–68 In this study, we aimed to define the role of IL-6 in post-fracture bone loss and further delineate the mechanistic pathways through which IL-6 drives osteocyte-mediated osteoclastic bone resorption.

Materials and Methods

Experimental Design

A total of 102 C57BL/6J male mice, aged 12 weeks, were employed in this research. Male mice were chosen to eliminate the influence of estrogen on bone metabolism, in accordance with previous studies.^6,69,70 These mice were maintained under specific pathogen-free (SPF) conditions with a stable 12-hour light-dark cycle and had unrestricted access to both food and water. After a one-week adaptation phase, 57 mice were randomly distributed into three experimental groups, each comprising 19 individuals (The sample size in this study was determined based on the consistency of intergroup results observed in preliminary experiments and ethical considerations to minimize animal use, in accordance with previous studies).^71,72

The groups received distinct treatments: femoral fracture group, femoral fracture + MR16-1 group, and control group. To ensure effective blockade of the IL-6 receptor, we selected MR16-1 dosing (0.5 mg per animal, three times per week) as reported in previous literature.⁷³

Additionally, a separate animal experiment was conducted using another 45 mice. The mice were further divided into three groups: femoral fracture group, femoral fracture + STAT3 phosphorylation inhibitor (Stattic) group, and control group. Given that previous studies employed Stattic at doses of 10 mg/kg (three times per week)⁷⁴ and 20 mg/kg (daily).^75,76 We selected an intermediate dose of 15 mg/kg, administered three times per week, to effectively inhibit STAT3 phosphorylation while minimizing potential toxicity in mice.

Micro-computed tomography (µCT) imaging was conducted on the L5 vertebral body and the contralateral femur at post-fracture 4, 14, and 28 days to evaluate bone microstructure. Additionally, tartrate-resistant acid phosphatase (TRAP) staining was performed on the L5 vertebral body at the same time points to determine osteoclast activity. Sequential fluorescence labeling of the L5 vertebral body was used to investigate bone formation activity. To assess the number of P-STAT3-positive osteocyte and the expression level of RANKL, immunohistochemical analysis was performed. Blood samples were obtained at days 4, 14, and 28 post-fracture to examine systemic inflammatory responses (Figure 1).

Figure 1 Animal treatment protocol.The initial red arrow indicates that fracture surgery was performed on day 0, followed immediately by the first MR16-1 or Stattic injection. The second red arrow represents the administration of Sequential fluorescence labeling, including calcein green and alizarin red, for dynamic bone formation assessment. Black arrows denote time points for sample collection and analyses, including micro-CT, TRAP staining, inflammatory cytokine quantification, immunohistochemistry, and immunofluorescence. These evaluations were conducted at days 4, 14, and 28 post-fracture. Abbreviations: TRAP, tartrate-resistant acid phosphatase.

All experimental procedures followed ethical guidelines and were approved by the Experimental Animal Ethics Committee of Shandong Provincial Hospital Affiliated to Shandong First Medical University (Approval No. 2022–811).

Label-Free Proteomics

Label-free protein sequencing was performed using four vertebrae derived from two fractured mice and two non-fractured mice, respectively. The specific experimental procedure and data analysis are as follows:

Total Protein Extraction

Frozen tissue samples were placed on ice, and an appropriate volume of lysis buffer (8 M urea, 1% SDS) containing protease inhibitors was added to prevent protein degradation. The mixture was then sonicated for 2 minutes at low temperature, followed by lysis for 30 minutes. Subsequently, the samples were centrifuged at 12,000g at 4°C for 30 minutes. The protein concentration in the supernatant was determined using the Bicinchoninic Acid (BCA) assay following the manufacturer’s protocol (Pierce, Thermo, USA).

Protein Reduction, Alkylation, and Digestion

A total of 100 µg of protein was mixed with Triethylammonium Bicarbonate (TEAB) buffer to a final concentration of 100 mM. Tris(2-carboxyethyl)phosphine (TCEP) was then added to a final concentration of 10 mM, followed by incubation at 37°C for 60 minutes. Iodoacetamide (IAM) was subsequently introduced to a final concentration of 40 mM and incubated for 40 minutes at room temperature in the dark. To precipitate proteins, pre-chilled acetone (sample: acetone, v/v = 1:6) was added, and the mixture was incubated at −20 °C for 4 hours. After centrifugation at 10,000g for 20 minutes, the precipitate was collected and resuspended in 100 µL of 100 mM TEAB. Finally, trypsin digestion was performed overnight at 37 °C using a trypsin-to-protein mass ratio of 1:50.

Peptide Desalting and Quantification

After vacuum drying, peptides were resuspended in 0.1% trifluoroacetic acid (TFA), desalted using Oasis HLB cartridges, and vacuum dried again. Peptide concentrations were measured using a peptide quantification kit (Thermo, Cat. 23275). Prior to mass spectrometry analysis, loading buffer was added to each sample to achieve a final concentration of 0.25 µg/µL.

LC-MS/MS Analysis

Trypsin-digested peptides were analyzed using an EASY-nLC 1200 system (Thermo, USA) coupled to a Q Exactive HF-X quadrupole Orbitrap mass spectrometer (Thermo, USA) at Majorbio Bio-Pharm Technology Co. Ltd. (Shanghai, China). Chromatographic separation was performed on a C18-reversed phase column (75µm*25 cm, Thermo, USA), equilibrated with solvent A (2% acetonitrile with 0.1% formic acid) and solvent B (80% acetonitrile with 0.1% formic acid). Peptides were eluted using a gradient program: 5%-23% B (0–56 min), 23%-29% B (56–62 min), 29%-38% B (62–63 min), 38%-48% B (63–63.5 min), 48%-100% B (63.5–64 min), and 100% B (64–90 min). Separation was achieved at a flow rate of 300 nL/min.

The Q Exactive HF-X operated in data-dependent acquisition (DDA) mode, alternating between full-scan MS and MS/MS. Full MS scans (m/z 300–1500) were acquired in the Orbitrap at a resolution of 60,000. The automatic gain control (AGC) target was set to 3e6 with a maximum injection time of 20 ms. The top 20 most intense precursor ions were fragmented via higher-energy collision dissociation (HCD). MS/MS spectra were acquired at a resolution of 15,000 (m/z 100), with an AGC target of 1e5, a maximum fill time of 50 ms, and a dynamic exclusion duration of 18 seconds.

Protein Identification

MS/MS spectra were analyzed using Proteome Discoverer 2.4 software. Protein identification was based on the highest-scoring peptide mass match in the database. The search parameters included trypsin digestion with up to two missed cleavages, carbamidomethylation of cysteine residues as a fixed modification, and methionine oxidation and N-terminal acetylation as variable modifications. The false discovery rate (FDR) for peptide identification was set to ≤0.01. A minimum of one unique peptide was required for protein identification.

Statistical Analyses

Proteomic data analysis was performed using the Majorbio Cloud platform (https://cloud.majorbio.com). Differentially expressed proteins (DEPs) between groups were identified using the R package t-test,with a significance threshold set at P < 0.05 and fold change (FC) >1.2 or <0.83. Functional annotation of identified proteins was conducted using Gene Ontology (GO) (http://geneontology.org/) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis (http://www.genome.jp/kegg/). Enrichment analysis of DEPs for GO and KEGG pathways was also performed. Protein-protein interaction (PPI) networks were analyzed using the STRING v11.5 database.

Femoral Fracture

The femoral fracture model in mice was established based on published articles and our previous studies.^6,77 Briefly, mice were anesthetized, and the tibial plateau and femoral condyle were exposed under sterile conditions. A mid-diaphyseal femoral fracture was created using a surgical blade. The fracture site was continuously irrigated with cold saline, and the surrounding muscle was protected from damage. Intramedullary fixation was performed using a 25-gauge syringe needle. Finally, the muscles and skin were sutured with 5/0 silk. All femoral fractures were successfully created, with uncomplicated postoperative recovery observed in all cases. No signs of infection or complications were detected.

Micro Computed Tomography (μCT) Analysis

Micro computed tomography (μ CT) imaging was performed using an Inveon Multi-Modality system (viva CT 40, Scanco Medical, Switzerland) to evaluate the microstructural characteristics of the L5 vertebral body and contralateral femur. The scans were acquired with an effective voxel size of 10.5 μm, operating at 98 μA current, 100 keV voltage, and an exposure time of 1500 ms per step over 360 rotational increments.

For trabecular bone analysis in the distal femoral metaphysis, measurements were initiated from the metaphyseal growth plate and extended distally over 100 slices. In the L5 vertebral body, the analysis covered the region between the cranial and caudal growth plates. Key trabecular bone parameters, including bone volume fraction (BV/TV), trabecular thickness (Tb.Th), trabecular number (Tb.N), and trabecular separation (Tb.Sp), were automatically quantified.

Cortical bone assessment was conducted at the femoral mid-diaphysis. The region of interest encompassed 120 slices (equivalent to 720 µm) centered at the bone’s longitudinal midpoint. Parameters such as cortical bone area (Ct.Ar), total cross-sectional area (Tt.Ar), medullary area (Ma.Ar), cortical thickness (Cort.Th), and the cortical area-to-total cross-sectional area ratio (Ct.Ar/Tt.Ar) were measured.

Enzyme-Linked Immunosorbent Assay (ELISA)

The levels of IL-6, TNF-α and IL-1β serum were measured using commercially available immunoassay kits, following the manufacturer’s protocol (IL-6, KYY-0163M1, China; TNF-α, KYY-0132M1, China; IL-1β, KYY-0040M1, China).

TRAP Staining of L5 Vertebral Body

The L5 vertebral body was initially preserved in 4% paraformaldehyde for 24 hours. It was then subjected to decalcification in 10% EDTA at room temperature for 30 days. After completing the decalcification process, the samples were embedded longitudinally in paraffin. Thin sections, each measuring 5 µm in thickness, were prepared and stained using the TRAP staining system (Sigma-Aldrich, St. Louis, MO, USA) following the manufacturer’s guidelines.

The quantification of osteoclast was conducted using Image-Pro Plus 6.0 software (Media Cybernetics, Rockville, MD, USA), which measured both the number of osteoclast per bone surface and the osteoclast surface area relative to the bone surface.

Immunohistochemical Staining and Immunofluorescence Labelling

The vertebrae and fractured femurs were initially fixed in 4% paraformaldehyde overnight, followed by decalcification in 10% EDTA and subsequent paraffin embedding using standard procedures. Bone sections, each 5 µm thick, were prepared for immunohistochemical and immunofluorescence analysis. Immunohistochemistry was performed using antibodies targeting RANKL (Bs-0747R; BIOSS; 1:200) and P- STAT3 (GB150001; Servicebio; 1:200), while immunofluorescence staining utilized antibodies against OCN (GB11233; Servicebio; 1:200), IL-6 (GB11117; Servicebio; 1:300), and CTSK (GB111276; Servicebio; 1:500), all following the respective manufacturer’s protocols.

After staining, the sections were examined under a Nikon E800 microscope following diaminobenzidine (DAB) substrate treatment. The proportion of osteocyte positively expressing P-STAT3 within the vertebral cancellous bone (500 µm from the growth plate, covering an area of approximately 4 mm²) was quantified. And the optical density (OD) value of RANKL expression within the same region were measured. Negative control was included for osteocyte density assessment. Quantitative analysis was performed using ImageJ software (version 1.52, NIH, USA). For fluorescently stained tissue sections, the mean fluorescence intensity of Cathepsin K (CTSK) and osteocalcin (OCN) was quantified for each selected non-consecutive section.

Sequential Fluorescence Labelling

To evaluate new bone formation and remodeling, sequential fluorescence labeling was performed. Calcein green (20 mg/kg, Sigma-Aldrich) and Alizarin red (30 mg/kg, Sigma-Aldrich) were administered via tail vein injection 14 days and 7 days before euthanasia, respectively. The L5 vertebral bodies from each group were then fixed in 10% formalin for two weeks, followed by dehydration in a graded ethanol series under vacuum for 48 hours. Subsequently, the specimens were embedded in methyl methacrylate resin.

Undecalcified sections were processed by grinding and polishing to a final thickness of 40–60 µm to obtain longitudinal views of the vertebral body (HistoCore AUTOCUT, Leica Biosystems, Wetzlar, Germany). The mineral apposition rate (MAR) was determined using BioQuant software (BIOQUANT Image Analysis Corporation, Nashville, TN, USA) by measuring the average distance between the two fluorescent labels and dividing it by the time interval between injections. Additionally, the bone formation rate per bone surface (BFR/BS) and the mineralizing surface relative to the bone surface (MS/BS) were calculated.

Cell Cultures

The murine osteocytic MLO-Y4 cell line (University of Missouri, Kansas City, KS) was used with approval from the Institutional Research Ethics Committee of Shandong Provincial Hospital Affiliated to Shandong First Medical University. No contamination or phenotypic alterations were observed.

The MC3T3-E1 osteoblastic cell line was obtained from Procell Life Science & Technology Co., Ltd. (Wuhan, China) and cultured in α-MEM (Gibco, USA) supplemented with 10% FBS and 1% penicillin-streptomycin at 37°C in a humidified atmosphere containing 5% CO₂. The culture medium was refreshed every two days.

Isolation of Bone Marrow Cells

Twelve-week-old C57BL/6 mice were euthanized and immersed in 75% ethanol for 10 minutes. Femurs and tibias were aseptically isolated under sterile conditions and transferred to PBS. Bone marrow was flushed with α-MEM (Gibco, USA) until the bones turned white. The cell suspension was gently mixed, filtered through a 100 µm strainer, and centrifuged at 1200 rpm for 5 minutes. After discarding the supernatant, red blood cell lysis buffer (Solarbio, China) was added, and the sample was incubated on ice for 10 minutes. Cells were incubated at 37°C with 5% CO₂ overnight. Non-adherent cells were collected, resuspended in α-MEM containing 30 ng/mL M-CSF (BioLegend, USA) and 10% FBS, and incubated for 48 hours. Adherent cells were identified as bone marrow cells (BMMs).

Cell Transfection

MLO-Y4 cells were cultured on rat tail type I collagen-coated plates in α-MEM supplemented with 2.5% FBS, 2.5% calf serum, and 1% penicillin/streptomycin. And cells were seeded in 6-well plates and transfected with control siRNA or STAT3-specific siRNA (siSTAT3; RiboBio, China) using Lipofectamine 2000 transfection reagent (KYY-R Transfection Reagent, China) according to the manufacturer’s instructions.^78,79 After 48 hours, cells were treated with recombinant IL-6 for an additional 24 hours to assess downstream effects.

Indirect Coculture System

Osteocyte was seeded on collagen-coated glass plates in α-MEM with 10% FBS and 1% penicillin-streptomycin and incubated for 24 hours. To generate conditioned medium, MLO-Y4 cells were treated with 0, 10, or 50 ng/mL recombinant mouse IL-6 for 24 hours. In parallel, 5 μM Stattic (a small-molecule inhibitor that effectively blocks the activation, transcription, and DNA-binding functions of STAT3) was added to the 50 ng/mL IL-6group and incubated for an additional 6 hours. The dose of Stattic (5 μM) was selected according to published studies.⁸⁰ The control group did not receive Stattic.

Conditioned medium was collected and centrifuged, then mixed 1:1 with fresh BMM medium (containing 10% FBS and 1% penicillin/streptomycin). To induce osteoclast formation, mouse BMMs (8000 cells/well) were seeded in 96-well plates and cultured for 2 days in medium with 30 ng/mL M-CSF and 20 ng/mL Recombinant Mouse RANKL. The medium was then replaced with conditioned medium supplemented with 30 ng/mL M-CSF and 30 ng/mL RANKL (R&D Systems, Minneapolis, MN, USA) for indirect coculture. Medium were refreshed every two days, and TRAP staining was performed on day 7.

Then the conditioned medium (CM) from osteocyte was mixed 1:1 with osteogenic induction medium (Gibco, USA) and applied to MC3T3-E1 cells in an indirect coculture system to assess whether IL-6-treated osteocyte CM influences osteoblast activity.

TRAP Staining in Vitro

Following 30-min immobilization in 4% paraformaldehyde solution, cellular specimens underwent dual PBS rinses. Subsequently, samples were incubated with tartrate-resistant acid phosphatase (TRAP) staining solution (Sigma-Aldrich, St. Louis, MO, USA) under light-protected conditions for 50 min, followed by aqueous washing. Hematoxylin counterstaining was performed for 5 min, with subsequent 10 min bluing treatment using municipal water. After natural drying, cellular morphology was documented through an inverted microscope (Olympus IX53, Tokyo, Japan). In our analyses, mature osteoclasts were identified as TRAP-positive multinucleated cells (≥3 nuclei) directly contact with the bone surface.^44,65 Pre-osteoclast, including TRAP-positive mononuclear cells, were excluded from the quantification. Quantitative analysis of osteoclast surface area was conducted employing ImageJ software (version 1.53t, NIH, USA).

Western Blot Analysis

Protein lysates from primary osteocyte, osteoclast and osteoblast were analyzed by Western blotting. Cells were washed with cold PBS and lysed in RIPA buffer (Shanghai Epizyme Biomedical, China) containing phosphatase and protease inhibitors. Protein concentrations were measured using a BCA assay kit (Beyotime).

Equal amounts of protein were separated by SDS-PAGE, transferred to PVDF membranes (Millipore, USA), and blocked with 5% milk for 1.5 hours. Membranes were incubated overnight at 4°C with primary antibodies against p-STAT3 (CST, 9145T; 1:1000), RANKL (Proteintech, 23408-1-AP; 1:1000), β-actin (Proteintech, 66009–1; 1:20,000), GAPDH (Proteintech, 10494-1-AP,1:5000), NFATC1 (Proteintech, 66963–1; 1:5000), STAT3 (Abcam, ab68153; 1:1000), ALP (Proteintech,18507–1;1:1000), RUNX2 (Proteintech,82636–2;1:2000), and OCN (Wuhan, China; A6205; 1:1000) and CTSK (Wuhan, A1782; 1:300), followed by HRP-conjugated secondary antibodies (Boster, BA1056; 1:5000). Protein bands were visualized using enhanced chemiluminescence (ECL) and quantified with ImageJ software (NIH, Bethesda, MD), with target protein levels normalized to β-actin or GAPDH.

Cellular Immunofluorescence Staining

MLO-Y4 cells (10,000 cells per well) were seeded in 24-well plates, fixed using 4% paraformaldehyde (PFA) and 0.5% Triton X-100 to enhance membrane permeability at room temperature, and then rinsed with PBS. The samples were blocked with 5% bovine serum albumin (BSA) at room temperature, followed by overnight incubation with primary antibodies against p-STAT3 (Cell Signaling Technology, 9145 T; 1:200) and RANKL (Proteintech, 66610–1; 1:400). After the primary antibody incubation, the cells were exposed to secondary fluorescent antibodies in the dark for 1 hour. Following three PBS washes and a 10-minute incubation with DAPI, images were captured using a confocal microscope (Leica, Germany).

Statistical Analysis

Data are presented as mean ± standard deviation (SD), with all individual data points displayed in bar graphs. Statistical analyses were performed using GraphPad Prism 8.0 (GraphPad Software, San Diego, CA, USA). One-way analysis of variance (ANOVA) followed by Tukey’s post hoc test or unpaired Student’s t-test were used for comparisons among groups. A p-value < 0.05 was considered statistically significant. “***” indicates p < 0.001 for pathway enrichment significance.

Results

IL-6 Is Regionally and Systemically Upregulated After Femur Fracture and Can Be Neutralized by MR16-1

The label-free proteomics-based KEGG enrichment analysis of the vertebrae from fracture and control group revealed that the different proteins were enriched primarily in inflammatory pathways. Additionally, IL-6 was identified as a key differentially expressed protein (Figure 2A and B).

Figure 2 IL-6 levels rapidly increase both locally and systemically after femur fracture, and the use of MR16-1 blocks this increase. (A) KEGG enrichment analysis. Differentially expressed proteins were defined by t-test (p < 0.05, fold change >1.2 or <0.83); “***” indicates p < 0.001 for pathway enrichment significance. (n = 2 mice/group). (B) Protein interaction analysis. (C) Representative images showing the localization of IL-6 fluorescence at the fracture sites in three groups: saline-injected mice, MR16-1-injected mice, and control mice. The scale bar represents 250µm. (D) Serum IL-6 levels measured at 4 days, 2 weeks, and 4 weeks after fracture in mice treated with saline, MR16-1, and control (n = 5 mice/group). (E) Serum TNF-α levels measured at 4 days, 2 weeks, and 4 weeks after fracture in mice treated with saline, MR16-1, and control (n = 5 mice/group). (F) Serum IL-1β levels measured at 4 days, 2 weeks, and 4 weeks after fracture in mice treated with saline, MR16-1, and control (n = 5 mice/group). Error bars indicate standard deviations (SDs). p-values were calculated using one-way ANOVA followed by Tukey’s post hoc test.

Immunofluorescence revealed increased IL-6 expression at the femoral fracture site on 4 days after injury, which was significantly reduced in the fracture + MR16-1 group compared to control (Figure 2C).

Furthermore, serum IL-6 levels were significantly elevated in fracture group at 4 days, 2 weeks, and 4 weeks post-injury. Although no difference was observed at day 4, the fracture + MR16-1 group showed a marked reduction in IL-6 levels at 2 and 4 weeks compared to the fracture group (Figure 2D). Serum TNF-α and IL-1β levels were markedly elevated at 4 days, 2 weeks, and 4 weeks after fracture; however, MR16-1 treatment did not significantly alter these cytokines levels compared to the untreated group (Figure 2E and F).

Systemic Blockade of IL-6 by MR16-1 Reverses Bone Loss After Fracture

Representative 3D images of L5 trabecular bone microarchitecture were obtained at 4 days, 2 weeks, and 4 weeks post-injury for three groups: control, fracture + saline group and fracture + MR16-1 group (Figure 3A–I). Quantitative micro-CT analysis demonstrated that femur fracture led to a significant decrease in trabecular bone volume within the L5 vertebral body at all examined time points (4 days, 2 weeks, and 4 weeks). Nevertheless, administration of MR16-1 markedly enhanced bone structural parameters. At 4 days after femur fracture, the BV/TV of lumbar cancellous bone was significantly lower in fracture + saline group than control. Treatment with MR16-1 significantly increased BV/TV compared to fracture + saline group and showed elevated levels compared to control. Other parameters, including Tb.Th, Tb.N, and Tb.Sp, showed no significant differences among the three groups. (Figure 3J–M). At 2 weeks post-femur fracture, BV/TV and Tb.Th were significantly reduced compared to control. MR16-1 treatment restored these parameters to levels comparable with the saline group. (Figure 3N–Q). Four weeks after femur fracture, BV/TV and Tb.Th decreased significantly, while Tb.Sp increased compared to control. MR16-1 treatment reversed these changes, with no significant differences from control. (Figure 3R–U).

Figure 3 Rapid cancellous bone loss in the L5 vertebra occurs after femur fracture, and the use of MR16-1 alleviates bone loss.(A–I) Representative images of the 3D trabecular structure in the L5 vertebral body at 4 days, 2 weeks, and 4 weeks post-fracture in mice treated with saline, MR16-1, or the control group. (J–U) Quantitative analysis of trabecular microarchitecture changes in the L5 vertebral body at 4 days, 2 weeks, and 4 weeks post-fracture in mice treated with saline, MR16-1, or control (n = 5 per group). Error bars indicate standard deviations (SDs). p-values were calculated using one-way ANOVA followed by Tukey’s post hoc test. Abbreviations: BV/TV, bone volume/tissue volume; Tb.Th, trabecular thickness; Tb.N, trabecular number; Tb.Sp, trabecular separation.

For the contralateral femur diaphysis (Figure 4A–I), BV/TV at 4 days post-fracture was significantly higher in the fracture + MR16-1 group than in the fracture + saline group. (Figure 4J–M). By 2 weeks after fracture, both BV/TV and Tb.N showed a significant decline, Conversely, Tb.Sp was significantly elevated. However, BV/TV in the fracture + MR16-1 group remained significantly higher than that in the fracture + saline group and showed no significant difference from the control group. (Figure 4N–Q). At 4 weeks post-fracture, only BV/TV was significantly lower in fracture + saline group compared to control. MR16-1 treatment significantly increased BV/TV, with no other significant differences observed. (Figure 4R–U).

Figure 4 Fracture leads to bone loss in the contralateral distal femur metaphasis, and the use of MR16-1 can prevent bone loss.(A–I) Representative images showing the three-dimensional trabecular structure of the contralateral distal femoral metaphysis at 4 days, 2 weeks, and 4 weeks post-fracture in mice treated with saline, MR16-1, or the control group. (J–U) Quantitative analysis of changes in the trabecular microarchitecture of the distal femoral metaphysis at 4 days, 2 weeks, and 4 weeks after fracture in saline treated, MR16-1 treated, and control mice (n = 5 mice/group). Error bars represent standard deviations (SDs). p-values were obtained using one-way ANOVA followed by Tukey’s post hoc test. Abbreviations: BV/TV, bone volume/tissue volume; Tb.Th, trabecular thickness; Tb.N, trabecular number; Tb.Sp, trabecular separation.

Cortical bone assessments of the contralateral femur at 4 days, 2 weeks, and 4 weeks post-fracture showed no significant differences among groups (Figure 5A–I). These parameters included cortical thickness (Cort.Th), total tissue area (Tt.Ar), cortical bone area (Ct.Ar), the ratio of cortical area to total area (Ct.Ar/Tr.Ar), and medullary area (Ma.Ar) (Figure 5J–X).

Figure 5 The cortical bone of the contralateral femoral midshaft is unaffected after femur fracture, and the use of MR16-1 does not result in further changes. (A–I) Representative cross-sectional images of the contralateral femoral midshaft at 4 days, 2 weeks, and 4 weeks post-fracture in mice treated with saline, MR16-1, or the control group. (J–X) Quantitative µCT measurements of cortical thickness (Cort.Th), total cross-sectional area (Tt.Ar), cortical area (Ct.Ar), cortical area/total cross-sectional area (Ct.Ar/Tt.Ar), and medullary area (Ma.Ar) at 4 days, 2 weeks, and 4 weeks post-fracture in mice treated with saline, MR16-1, or control (n = 5 per group). Error bars represent standard deviations (SDs). p-values were calculated using one-way ANOVA followed by Tukey’s post hoc test. Abbreviations: Ct.Ar, cortical area, Tt.Ar, total cross-sectional area, Ma.Ar, medullary area, Cort.Th, cortical thickness, and Ct.Ar/Tt.Ar, cortical area/total cross-sectional area.

Osteoclast Activity Increases Rapidly and Dramatically After Fracture and Can Be Decreased by MR16-1

TRAP-stained images of the L5 vertebral body are shown for control, saline treated fracture, and MR16-1 treated fracture groups at 4 days (Figure 6A–C), 2 weeks (Figure 6D–F), and 4 weeks post-fracture (Figure 6G–I). At day 4 post-fracture, the saline treated fracture group showed significantly increased osteoclast numbers and surface resorption in L5 trabecular bone compared to control. These increases were markedly reduced in the fracture + MR16-1 group. (Figure 6J and K) Two and four weeks after fracture, osteoclast numbers and surface resorption per bone surface showed no significant differences among the three groups (Figure 6L and O).

Figure 6 Osteoclast activity increases promptly after fracture, and the use of MR16-1 can counteract this effect. (A–I) Representative TRAP staining images of trabecular bone within the L5 vertebral body from control mice and fractured mice treated with saline or MR16-1 at (A–C) 4 days, (D–F) 2 weeks, and (G–I) 4 weeks post-fracture. Scale bar = 250μm. (J and K) Mice with femur fractures exhibited a significantly higher number of osteoclast and resorbed surfaces compared to control mice at 4 days post-fracture, and these effects were reversed by MR16-1 treatment (n = 5 mice/group). (L–O) No significant differences were found at 2 and 4 weeks after fracture (n = 5 mice/group). Error bars represent standard deviations (SDs). p-values were calculated using one-way ANOVA followed by Tukey’s post hoc test.

Representative CTSK stained images of L5 trabecular bone at 4 days post-fracture were shown for MR16-1 or saline treated group. (Figure 7A) Four days after fracture, CTSK expression, a marker of osteoclast activity, was significantly upregulated in fractured mice, but was effectively suppressed by MR16-1 treatment (Figure 7B).

Figure 7 Osteoblast activity does not significantly change after fracture, whereas the increase in osteoclast activity can be reversed by MR16-1 or Stattic treatment.(A) CTSK staining in control, fracture, and MR16-1 treated mice. (B) Quantification of CTSK fluorescence intensity (n = 4 mice/group). (C) OCN staining in control, fracture, and MR16-1 treated mice. (D) Quantification of OCN fluorescence intensity (n = 4 mice/group). (E) CTSK staining in control, fracture, and Stattic treated mice. (F) Quantification of CTSK fluorescence intensity (n = 4 mice/group). (G) OCN staining in control, fracture, and Stattic treated mice. (H) Quantification of OCN fluorescence intensity (n = 4 mice/group). Nuclei were counterstained with DAPI (blue), and CTSK and OCN positive signals are shown in red. Scale bars: 250 µm. Error bars represent standard deviations (SDs). p-values were calculated using one-way ANOVA followed by Tukey’s post hoc test. (I) Sequential fluorescence labeling using calcein and alizarin red in the trabecular bone of the L5 vertebral body in fractured mice, fractured mice treated with MR16-1, and control mice. Scale bar = 50 μm. (J-L) Dynamic histomorphometric analysis of trabecular bone formation following fracture. (n = 4 mice/group). Error bars represent standard deviations (SDs). p-values were determined using one-way ANOVA followed by Tukey’s post hoc test. Abbreviations: BFR/BS, bone formation rate per bone surface; MAR, mineral apposition rate; MS/BS, mineralizing surface per bone surface.

Osteoblast Activity Remained Stable Following Fracture and Was Not Modulated by MR16-1 or Stattic

At 4 days post-fracture, osteocalcin expression in the L5 trabecular bone was evaluated by immunofluorescence in the control group, the fracture + saline group, and the fracture + MR16-1 group. No significant differences in osteocalcin expression were observed, and MR16-1 did not alter its levels. (Figure 7C and D).

To further evaluate the effects of STAT3 phosphorylation inhibition on osteoclast and osteoblast activity, immunofluorescence staining for CTSK and OCN was performed in L5 vertebrae at 4 days post-fracture. The results showed that CTSK expression was markedly elevated in the fracture + saline group, while Stattic treatment significantly reduced its levels (Figure 7E and F). However, OCN expression remained consistent across all groups. (Figure 7G and H).

Additionally, vertebrae images labeled sequentially with calcein and alizarin red were captured 4 days post-fracture in mice treated with MR16-1 or saline. (Figure 7I). The mineral apposition rate (MAR), mineralizing surface per bone surface (MS/BS), and bone formation rate per bone surface (BFR/BS), which reflect osteoblast activity, showed no significant differences among the three groups (Figure 7J–L).

P-STAT3+ Osteocyte and RANKL Expression are Dramatically Increased After Fracture and Can Be Rescued by MR16-1

Immunohistochemical images of P-STAT3 and RANKL staining in the L5 vertebral body at 4 days post femur fracture are shown for control, fracture + saline, and fracture + MR16-1 group. The percentage of P-STAT3⁺ osteocyte markedly increased after fracture, while MR16-1 treatment significantly reduced it in the lumbar vertebrae (Figure 8A and B). Similarly, RANKL expression was significantly reduced in the fracture + MR16-1 group relative to the fracture + saline group. (Figure 8C and D).

Figure 8 P-STAT3+ positive osteocyte and RANKL expression also increase after fracture, and these changes are attenuated by the use of MR16-1.(A) Representative image of osteocyte positively stained for P-STAT3 protein. Scale bar = 50 µm. (B) The percentage of P-STAT3+ osteocyte in control mice, fractured mice and fracture with MR16-1 treatment mice (n = 4 mice/group). (C) Immunohistochemical staining of RANKL in the cancellous bone of the L5 vertebra. Scale bar = 200 µm. (D) Quantification of RANKL levels of (C). (n = 4 mice/group) Error bars represent standard deviations (SDs). p-values were determined using one-way ANOVA followed by Tukey’s post hoc test.

IL-6 Stimulate Osteocyte to Secrete RANKL and Indirectly Regulates Osteocyte-Mediated Osteoclast Formation

To study the dynamic crosstalk between osteocyte and osteoclast under IL-6 stimulation, an indirect co-culture system was established. (Figure 9A). A gradual increase in RANKL expression was observed in osteocyte with increasing IL-6 concentrations (Figure 9B and C). Western blot results further revealed that osteoclast marker proteins, including CTSK and NFATC1, were significantly upregulated in the co-culture system (Figure 9D–F).

Figure 9 IL-6 can stimulate osteocyte to secrete RANKL and increase osteoclast formation in an indirect coculture system of osteocyte and osteoclast. (A) Schematic of the protocol for the indirect co-culture of osteocyte and osteoclast to investigate the role of IL-6. (B) Western blot analysis of RANKL expression in MLO-Y4 cells following IL-6 activation. (C) Quantification of RANKL protein levels from (B) (n = 3). (D) Western blot analysis of osteoclast-specific markers, including NFATC1 and CTSK, following indirect co-culture. (E and F) Quantification of NFATC1 and CTSK protein levels from (D) (n = 3). (G) Representative confocal microscopy images showing co-staining of p-STAT3 (red), RANKL (green), and DAPI (blue) in MLO-Y4 cells. Scale bars = 25 µm. (H-I) Quantitative analysis of p-STAT3 and RANKL fluorescence intensity in MLO-Y4 cells stimulated with 0, 10, or 50 ng/mL IL-6. Both p-STAT3 and RANKL levels increased in a dose-dependent manner following IL-6 treatment, with statistically significant differences observed between all groups. (n = 3). (G) TRAP staining to assess osteoclast differentiation. Scale bars = 200 μm. (K) Osteoclast area was quantified following treatment with conditioned media derived from MLO-Y4 cells stimulated with increasing concentrations (0, 10, or 50 ng/mL) of recombinant mouse IL-6, corresponding to CM1, CM2, and CM3, respectively (n = 3). Error bars represent±SDs. All data are representative of at least three independent experiments. Statistical analysis was performed using one-way ANOVA followed by Tukey’s post hoc test.

In vitro, immunofluorescence co-staining revealed that both P- STAT3 and RANKL expression in osteocyte were increased after IL-6 stimulation (Figure 9G–I). TRAP staining revealed that when osteoclast was further treated with culture medium from IL-6-treated osteocyte, the activity of osteoclast was distinctly increased with increasing concentrations of IL-6 (Figure 9J and K).

IL-6 Regulates RANKL Production by Osteocyte and Modulates Osteocyte-Mediated Osteoclast Activity Through STAT3 Phosphorylation

To investigate the role of STAT3 activation in osteocyte-mediated osteoclast formation, Stattic was used to inhibit STAT3 phosphorylation in the co-culture system (Figure 10A). IL-6 induces P-STAT3 expression in osteocyte, and this expression is reduced upon treatment with Stattic, as shown by Western blotting. Likewise, STAT3 phosphorylation inhibitor diminished the capacity of IL-6 to stimulate osteocyte to secrete RANKL (Figure 10B–D). In line with this, the expression of nuclear factor of activated T cell 1 (NFATC1) and CTSK, both markers of osteoclast activity, was elevated by IL-6 stimulation but was reversed following Stattic treatment (Figure 10E–G). Co-immunofluorescence analysis demonstrated that IL-6 upregulated P- STAT3 and RANKL expression in osteocyte, while Stattic treatment effectively suppressed both proteins (Figure 10H–J). TRAP staining further demonstrated that Stattic suppressed IL-6-mediated osteocyte-driven osteoclast activity (Figure 10K and L).

Figure 10 IL-6 stimulates osteocyte to release RANKL, which indirectly increases osteoclast activity and relies on the phosphorylation of STAT3.(A) Experimental setup for the indirect coculture of osteocyte and osteoclast to investigate the role of STAT3. (B) Western blot analysis showing the expression of RANKL and phosphorylated STAT3 (P-STAT3) in MLO-Y4 cells following IL-6 stimulation and Stattic treatment. (C and D) Quantification of P-STAT3 and RANKL protein levels from (B). (n = 3). (E) Western blot analysis of osteoclastic marker genes, NFATC1 and CTSK, after indirect coculture with Stattic. (F and G) Quantification of NFATC1 and CTSK protein levels from (E). (n = 3). (H) Representative confocal microscopy images displaying co-staining of P-STAT3 (red), RANKL (green), and DAPI (blue). Scale bars = 25 µm. (I and J) Quantitative fluorescence analysis of p-STAT3 and RANKL expression in MLO-Y4 cells treated with IL-6 (50 ng/mL) in the presence or absence of Stattic (5 μM). IL-6 stimulation markedly increased p-STAT3 and RANKL levels, whereas co-treatment with Stattic significantly reduced both signals (n = 3). (K) TRAP staining to assess osteoclast differentiation. Scale bars = 200 μm. (K) Quantification of osteoclast area from (I). (n = 3). CM1 and CM3 were conditioned media collected from MLO-Y4 cells treated with 0 and 50 ng/mL recombinant mouse IL-6, respectively. CM4 was derived from MLO-Y4 cells treated with 50 ng/mL recombinant mouse IL-6 in combination with 5 µM Stattic. Error bars represent ± SDs. Data are representative of at least three independent experiments and were analyzed using one-way ANOVA followed by Tukey’s post hoc test.

IL-6/STAT3 Signaling Regulates Osteocyte-Mediated Osteoclastogenesis without Impairing Osteoblast Activity

Osteocyte were transfected with specific siSTAT3 to silence the STAT3 gene. Western blot results showed that STAT3 expression was significantly reduced in the siSTAT3 treated group compared to the control group (p < 0.05), indicating successful establishment of the osteocyte transfection model (Figure 11A and B). Additionally, an IL-6 stimulation experiment was performed. Quantitative analysis by Western blot revealed that, under stimulation with 50 ng/mL IL-6, the expression of phosphorylated STAT3 was markedly decreased in STAT3 silenced osteocyte compared to siSTAT3 transfected cells without IL6 treatment (Figure 11C and D). Similarly, STAT3 knockdown weakened the ability of IL-6 to promote RANKL secretion by osteocyte (Figure 11E). Furthermore, Western blot analysis revealed comparable levels of ALP, RUNX2, and OCN in MC3T3-E1 cells treated with conditioned media, with no significant differences among groups (Figure 11F–I). To further validate these findings, MC3T3-E1 cells were treated with conditioned media from IL-6 + Stattic intervention group. ALP, RUNX2, and OCN expression remained comparable across all groups, and densitometric analysis confirmed no statistically significant differences, suggesting that IL-6/STAT3 activation in osteocyte selectively promotes osteoclastogenesis without affecting osteoblast activity (Figure 11J–M).

Figure 11 Osteocyte STAT3 knock-down attenuates IL-6-induced RANKL expression, whereas conditioned media from IL-6-activated osteocyte do not alter osteoblast activity. (A) Western blot confirming efficient STAT3 knockdown in MLO-Y4 cells. (B) Quantification of STAT3 protein levels (n = 3). Followed by an unpaired Student’s t-test. (C) Western blot analysis of p-STAT3 and RANKL in osteocyte treated with IL-6 (50 ng/mL), with or without STAT3 siRNA. (D and E) Quantification of p-STAT3 and RANKL signals (n = 3). (F) Western blot analysis of ALP, RUNX2, and OCN in MC3T3-E1 cells cultured with osteocyte-conditioned media (CM1, CM2, or CM3). CM1, CM2, and CM3 were derived from MLO-Y4 cells exposed to 0, 10, or 50 ng/mL IL-6, respectively. (G–I) Quantification of ALP, RUNX2, and OCN expression in CM1-CM3 groups. (n = 3). (J) Western blot analysis of ALP, RUNX2, and OCN in MC3T3-E1 cells treated with CM1, CM3, or CM4. CM4 represents conditioned medium from osteocyte stimulated with 50 ng/mL recombinant mouse IL-6 and 5 µM Stattic. (K–M) Corresponding densitometric analyses for ALP, RUNX2 and OCN in the CM1, CM3 and CM4 groups (n = 3). Error bars indicate ± SD. Data are representative of at least three independent experiments and were analyzed using one-way ANOVA followed by Tukey’s post hoc test.

Increased Osteoclast Activity After Fracture Can Also Be Reversed by Stattic

TRAP staining was performed on L5 vertebral trabecular bone sections from control group, fracture mice, and fracture + Stattic group, at 4 days (Supplementary Figure 1A–C), 2 weeks (Supplementary Figure 1D–F) and 4 weeks post-fracture (Supplementary Figure 1G–I). Similarly, at 4 days, we observed a significantly lower number of osteoclast and resorbed surfaces in femur fracture model mice treated with Stattic compared to those injected with saline (Supplementary Figure 1J and K). At the later time points, no notable differences were found in osteoclast count or surface resorption between the three groups (Supplementary Figure 1L–O).

Stattic Reverses Systemic Bone Loss After Fracture

Representative images of the three-dimensional trabecular bone microstructure in the L5 vertebral body were obtained at 4 days, 2 weeks, and 4 weeks under control conditions, post-femur fracture, and femur fracture with Stattic treatment, as shown in (Supplementary Figure 2A–I). Quantitative µCT analysis showed that femur fracture reduced trabecular bone volume in the L5 vertebral body at all time points (4 days, 2 weeks, and 4 weeks). Stattic treatment, however, significantly improved these bone parameters. At 4 days post-fracture, BV/TV in the L5 vertebral cancellous bone was lower in the fracture group than in control (Supplementary Figure 2J–M). Two weeks post-fracture, BV/TV and Tb.N decreased, while Tb.Sp increased markedly in the fracture group compared to control. Stattic treatment substantially improved BV/TV (Supplementary Figure 2N–Q). Four weeks after fracture, both BV/TV and Tb. Th were significantly decreased in the fracture group, but Stattic treatment alleviated this reduction, with values considerably higher than those in the fracture group (Supplementary Figure 2R–U).

Analysis of the contralateral femur diaphysis (Supplementary Figure 3A–I) showed no significant differences among the groups at 4 days post-fracture (Supplementary Figure 3J–M). However, at 2 weeks after fracture, the BV/TV, Tb. Th, and Tb. N were significantly reduced, and Tb. Sp was significantly elevated in the fracture group compared to control. Stattic treatment markedly improved BV/TV and Tb. Th (Supplementary Figure 3N–Q). At 4 weeks post-fracture, there was a dramatically decrease in Tb. N and an increase in Tb. Sp (Supplementary Figure 3R–U), but Stattic treatment mitigated these changes.

For the contralateral femur cortical shell, no significant differences were observed in cortical bone parameters, including Cort.th, Tt.Ar, Ct.Ar, Ct.Ar/Tt.Ar, and Ma. Ar, among the groups at 4 days, 2 weeks, and 4 weeks post-fracture (Supplementary Figure 4A–I, Supplementary Figure 4J–X).

Discussion

This study aimed to elucidate the immunological mechanism underlying systemic bone loss following fracture. We demonstrated that the inflammatory cytokine IL-6 plays a critical role in mediating interactions between the immune system and the skeletal system by influencing osteocyte to indirectly regulate osteoclast activity, thereby promoting bone resorption and contributing to systemic bone loss. Specifically, IL-6 is rapidly and abundantly released following fracture as a component of the early immune response. Acting on osteocyte, IL-6 promotes STAT3 phosphorylation and induces RANKL expression, thereby amplifying osteoclast mediated bone resorption and contributing to both local and systemic bone loss. These findings highlight the role of IL-6 dependent signaling in connecting immune activation with skeletal deterioration (Figure 12).

Figure 12 Summary diagram of IL-6–mediated STAT3 signaling in osteocytes promoting bone resorption following fracture. (A) Local fracture leads to the release of large amounts of IL-6 from various cells, including fibroblasts, macrophages, neutrophils, T lymphocytes, and endothelial cells. IL-6 binds to its receptor (IL-6R) on osteocyte, activating the STAT3 signaling pathway; (B) Activation of the STAT3 signaling pathway promotes RANKL expression, which in turn stimulates osteoclastogenesis, thereby contributing to systemic bone loss.

Our findings were further validated both in vitro and in vivo through pharmacological inhibition of IL-6 signaling using MR16-1 and suppression of STAT3 phosphorylation, both of which effectively reduced osteoclast activity and bone resorption. These findings deepen our understanding of the immune mechanisms driving post-fracture bone loss and highlight a potential immunotherapeutic approach to prevent rapid systemic bone deterioration.

IL-6 is considered a common and broadly impactful inflammatory cytokine, standing out as a distinctive and prominent “star” molecule among numerous inflammatory factors. It plays vital physiological and pathological roles across nearly all major organ systems, including the nervous, skeletal, respiratory, circulatory, and digestive systems^{29,35,81–86.}In the skeletal system, previous studies using various inflammation associated disease models have indirectly demonstrated the harmful effects of IL-6 on bone.^37,46,51,87 Research has also revealed that IL-6 can modulate bone related cells, such as osteoblast, osteoclast, and osteocyte, to closely regulate bone metabolism.^15,44,88,89 However, the importance of IL-6 in bone loss after fracture remains unclear.

This study demonstrated that IL-6 is rapidly and robustly elevated both locally and systemically following femur fracture. Meanwhile, serum levels of TNF-α and IL-1β were also markedly elevated. Similarly, the results align with clinical observations, where a significant increase in serum IL-6, TNF-⍺ and IL-1β levels has been reported after fracture.^90–92

When treated with an IL-6 receptor neutralizing antibody, serum IL-6 levels were reduced at 2 and 4 weeks. However, IL-6 levels remained unchanged at 4 days. This phenomenon may be driven by two primary causes. Firstly, because anti-IL-6 receptor antibodies inhibit IL-6R-mediated clearance of IL-6 from circulation, their administration does not result in an immediate decrease in serum IL-6 levels.⁹³ Secondly, repeated administration may lead to drug accumulation when dosing frequency exceeds the rate of metabolism or excretion, potentially enhancing drug effect.⁹⁴

In line with prior studies on the role of IL-6 in postmenopausal osteoporosis^33–36 and osteoarthritis related bone loss,^15,95 our results show that the use of MR16-1 could restore bone loss in the vertebral cancellous body after fracture. Interestingly, unlike IL-6, the levels of TNF-α and IL-1β did not decrease following MR16-1 treatment. The results indicate that TNF-α and IL-1β are not primary drivers of the observed phenotype, thereby underscoring the specific role of IL-6 in post-fracture bone loss. These findings suggest that, unlike other bone loss models involving multiple cytokines such as IL-6, IL-1β and TNF-α,^96–99 the acute and robust IL-6 response following fracture may have distinct effects on systemic bone loss.^100,101

Furthermore, our study showed that femur fracture induced rapid vertebral bone loss within 4 days, as evidenced by reduced BV/TV, which was restored by MR16-1 treatment. Two weeks after femur fracture, both BV/TV and Tb.Th were reduced.

IL-6 blockade effectively prevented bone loss at this stage. At 4 weeks post-fracture, BV/TV and Tb.Th were further reduced, while Tb.Sp increased. MR16-1 treatment restored Tb.Th and reduced Tb.Sp. The results clearly demonstrate that bone loss progressively worsens over the 4 weeks period following fracture. This finding stands in contrast to previous studies that reported no significant bone loss in young mice at 4 weeks post-fracture.⁷ In distal femoral cancellous bone, BV/TV and Tb.Th decreased while Tb.Sp increased at 2 weeks post-fracture. MR16-1 treatment reversed these changes. At 4 weeks, BV/TV was the only parameter that remained decreased, and the treatment effectively alleviated this reduction. Bone loss was observed in both the distal femur and vertebra, and MR16-1 treatment reduced bone loss in both regions. The specific microstructural parameters affected by bone loss and improved by treatment varied, likely due to the distinct anatomical characteristics of vertebral cancellous bone and distal femoral trabecular bone. This finding is consistent with previous research, which suggested that femur fracture can lead to compensatory bone mass increases in the contralateral femur, whereas such effects were not observed in the vertebral body.⁶

Furthermore, our findings and existing literature on post-fracture bone loss consistently indicate that bone loss predominantly occurs in trabecular bone,⁷³ which partially contrasts with some clinical studies reporting cortical bone loss following fractures.¹⁰² Previous studies have indicated that the trans-signaling pathway predominantly affects trabecular bone,⁸⁷ leading us to hypothesize that IL-6 may exert its effects through this pathway. However, our experiments primarily focused on investigating bone loss by employing an IL-6 receptor blocker, which inhibits both classical and trans-signaling pathways simultaneously.^73,103 While we have partially clarified the role of IL-6 in post-fracture bone loss, further investigation into the specific signaling pathways mediating this effect would be highly valuable. In addition, the discrepancy that bone loss predominantly occurs in trabecular bone may also attributed to the factors such as differences in bone turnover rates between mice and humans, and variations in anatomical and methodological approaches and so on.^104,105

Many studies have investigated the effects of IL-6 on bone metabolism, revealing its influence on osteoblast, osteocyte, and osteoclast.^34,43–47 Our research further revealed that the cause of bone loss after fracture may be related to the activation of osteoclast activity at 4 days after fracture, which is consistent with the findings of previous studies.⁶ At this time, immunohistochemistry showed that fracture increased the number of P-STAT3⁺ osteocyte and upregulated RANKL expression, which is similar to the clinical observations that elevated serum RANKL levels were reported following fracture.^106,107 These effects were reduced after blocking IL-6. Additionally, the use of MR16-1 reduced bone resorption to efficiently restore bone loss. However, osteoclast activation was rapid but transient, with no significant changes in bone resorption observed at 2 and 4 weeks post-fracture.

Osteocyte act as a command center to regulate both bone formation and resorption, with numerous studies highlighting their upstream regulatory role and pivotal function as the primary source of RANKL, which mediates osteoclastic activity most effectively.^58,59 Similarly, our findings indicate that IL-6 stimulation leads to increased RANKL production by osteocyte. When the medium from osteocyte continues to interact with osteoclast, osteoclast activity was increased in a gradual manner, which indicates that IL-6 can establish a connection between osteocyte and osteoclast, ultimately leading to bone loss. We also found that, in vitro, osteogenic markers such as Runx2, ALP, and OCN were not significantly affected by IL-6 or the STAT3 phosphorylation inhibitor in the co-culture system. Consistently, immunofluorescence staining and dynamic histomorphometry revealed no significant changes in OCN expression or osteogenic parameters (MAR, MS/BS) after MR16-1 or Stattic treatment. These findings suggest that osteoblast are not directly involved in IL-6 mediated rapid bone loss after fracture, and IL-6 may indirectly promote osteoclast activity via osteocyte, consistent with previous studies showing that IL-6 acts through osteocyte rather than directly targeting osteoclast or osteoblast.^64,65,108

In previous studies, IL-6 is widely accepted and confirmed to indirectly increase the expression of osteoclastic activity by acting on osteoblast.^65,109 Furthermore, IL-6 is believed to exert its effects by activating STAT3 phosphorylation through the CRP300 receptor on the osteoblast surface, thereby increasing RANKL expression.^56,57 The IL-6/STAT3 pathway is a key regulator of diverse physiological and pathological functions in multiple organs,^110–113 with p-STAT3 activation exerting notable influence on bone metabolism.^{38,68,114–116} To determine whether STAT3 phosphorylation is a critical step in IL-6-induced bone loss, we treated osteocyte with a STAT3 phosphorylation inhibitor to block phosphorylation. Our research revealed that IL-6 exerts its osteoclastic effects through osteocyte, specifically by increasing STAT3 phosphorylation and further increasing the expression of RANKL. When Stattic was used, the promotion of osteocyte RANKL expression by IL-6 and the activation of osteoclast in cocultures were significantly weakened. These findings further underscore the critical role of osteocyte in systemic bone loss after fracture. Additionally, intriguing findings from in vitro experiments indicate that IL-6 can increase RANKL expression without the addition of soluble antibodies, contradicting previous research suggesting that increased osteoclast expression requires soluble antibodies.⁶⁴

In vitro, osteocyte was treated with Stattic, a selective STAT3 inhibitor. Western blot analysis demonstrated that this pharmacological blockade markedly suppressed the expression of osteoclast markers CTSK and NFATC1, and significantly inhibited osteoclast formation, as evidenced by TRAP staining. To further validate these findings, STAT3 was specifically knocked down in osteocyte. Western blot analysis confirmed efficient knockdown, and subsequent IL-6 stimulation resulted in a decrease in STAT3 phosphorylation. Consistently, RANKL expression was also reduced following STAT3 silencing under IL-6 stimulation.

After blocking, all the increases in bone resorption caused by IL-6 were completely attenuated. Therefore, STAT3 is considered to play a critical role in IL-6-induced bone loss. To further support our hypothesis and validate the role of STAT3, in vivo experiments were performed, which revealed that STAT3 phosphorylation blockade prevented IL-6-induced systemic bone loss after fracture. STAT3 phosphorylation inhibition also blocked IL-6-induced bone resorption. Similar to the outcome using MR16-1, bone formation markers exhibited minimal changes. Furthermore, reduced numbers of STAT3-positive and RANKL-positive osteocyte in the vertebral cancellous bone suggest that STAT3 is a downstream molecule of IL-6-induced bone loss, which is consistent with previous research indicating that STAT3 deficiency or loss can lead to consistent recovery of bone mass.^{38,68,114–116}

However, given the broad inhibitory effects of Stattic on STAT3 signaling across various cell types,^34,117 which may result in unanticipated target-off effects, we intend to employ gene knockout mouse models in future experiments to delineate the mechanism with greater specificity.

Collectively, these in vivo and in vitro results highlight the crucial role of the IL-6/STAT3 phosphorylation pathway in the bone-regulatory function of osteocyte.

The harmful effects of systemic bone loss after fracture are significant. Currently, few drugs are available to treat fracture induced bone loss which was completely different from osteoporotic fractures, and the effectiveness of using anti-osteoporotic drugs for bone loss after fracture still needs further investigation.¹¹⁸ Interestingly, we found that blocking IL-6 can inhibit the phosphorylation of STAT3. More importantly, this blocking can reduce osteoclast activity by inhibiting the release of RANKL from osteocyte, thereby alleviating bone loss. Furthermore, treatment with Stattic resulted in reduced bone loss. These findings imply that targeting IL-6 or STAT3 inhibition may serve as a promising strategy to prevent bone loss and lower the risk of subsequent fractures. However, previous literature has reported conflicting results regarding the impact of IL-6 blockade on fracture healing. While some studies suggest that IL-6 inhibition promotes healing,⁴² others indicate it may impair the process.¹¹⁹ These discrepancies may be attributed to differences in the timing of IL-6 blockade, the fracture models used, and the specific time points assessed^42,119 Therefore, further investigation into the effects of IL-6 blockade on local fracture healing holds significant clinical relevance and research value.

Our study possesses several strengths. First, considering the significantly increased risk of recurrent fractures and the pronounced bone loss following an initial fracture, we dynamically analyzed the characteristics of bone loss at multiple time points. More importantly, we are among the first to elucidate the immunological mechanisms driving systemic bone loss post-fracture. We emphasize the critical immunoregulatory role of the inflammatory cytokine IL-6 in initiating rapid systemic bone loss. Additionally, our study highlights the pivotal function of osteocyte in post-fracture bone remodeling, clarifying how IL-6 influences osteocyte to modulate osteoclast activity. This finding advances the understanding of osteocyte-mediated immune regulation in bone metabolism. Furthermore, we established that the IL-6/STAT3 signaling axis is a key mediator of systemic bone loss. And we demonstrated that targeting this pathway with inhibitors effectively mitigates osteoclast activation and bone resorption. These insights provide potential immunotherapeutic strategies for preventing post-fracture bone loss and reducing the likelihood of recurrent fractures.

This study also has several limitations. First, while our hypotheses were examined in a murine model, species-specific differences between mice and humans may affect the translatability of the findings. Future studies should incorporate larger animal models (eg, minipigs or dogs) and human fracture samples to validate IL-6 pathway activation in clinical settings. Second, while we observed a rapid and robust increase in IL-6 following fracture, the upstream regulators of IL-6 in bone loss remain to be elucidated. Third, Stattic is a broad STAT3 inhibitor that may affect various STAT3-expressing cell types in vivo. As our current design does not fully control for this, future studies using STAT3 knockout mice are warranted to provide more precise mechanistic insights. Finally, we selected the 4-week time point for experimentation. This choice was based on the near completion of fracture healing during this period,^120,121 indicating its adequacy for mechanistic investigation. Nevertheless, extending the observation period to assess delayed effects remains valuable, and future studies will address this.

Conclusion

IL-6 drives rapid systemic bone loss after fracture by promoting STAT3 phosphorylation in osteocyte and upregulating RANKL, thereby enhancing osteoclast activity and bone resorption. These findings suggest that the IL-6/STAT3/RANKL axis may serve as a critical link between immune signaling and skeletal homeostasis, as well as a promising therapeutic target for preventing inflammation induced bone loss and fracture recurrence. Future studies should focus on translating these insights into clinical strategies for improving bone healing after fracture.

Data Sharing Statement

Data supporting the findings of this study can be obtained from the corresponding authors upon reasonable request.

Ethics Approval

All animal experiments were conducted in compliance with the ethical guidelines established by the Ethics Committee for the Care and Use of Laboratory Animals at Shandong Provincial Hospital Affiliated to Shandong First Medical University. The procedures were conducted in strict accordance with the internationally recognized Five Freedoms for the welfare of laboratory animals: freedom from hunger and thirst; freedom from discomfort; freedom from pain, injury, and disease; freedom from fear and distress; and freedom to express normal behavior.

Acknowledgments

The authors express their gratitude to AJE for their assistance in language editing of an earlier draft of this manuscript.

Author Contributions

All authors made a significant contribution to the work reported, whether that is in the conception, study design, execution, acquisition of data, analysis and interpretation, or in all these areas; took part in drafting, revising or critically reviewing the article; gave final approval of the version to be published; have agreed on the journal to which the article has been submitted; and agree to be accountable for all aspects of the work.

Funding

This study was funded by the National Natural Science Foundation of China (Project No. 82202701) and the Natural Science Foundation of Shandong Province (Grant No. ZR2022QH184).

Disclosure

The authors declare that they have no competing interests in this work.

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