ZM-306416 prevents ovariectomy-induced bone loss by promoting osteoblastogenesis and inhibiting osteoclastogenesis

ZM-306416 prevents ovariectomy-induced bone loss by promoting osteoblastogenesis and inhibiting osteoclastogenesis | 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 Research Article ZM-306416 prevents ovariectomy-induced bone loss by promoting osteoblastogenesis and inhibiting osteoclastogenesis Yicheng Li, Shuo Shi, Dejian Yang, Lianhui Zhao, Tianyu Chen, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7828854/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 31 Jan, 2026 Read the published version in Journal of Orthopaedic Surgery and Research → Version 1 posted 8 You are reading this latest preprint version Abstract Background Osteoporosis affects millions of people worldwide, and current medications such as bisphosphonates and denosumab are not effective enough to reverse bone loss. Moreover, these treatments have drawbacks, including jaw osteonecrosis and skin eczema. Hence, there is an urgent need for new drugs to treat osteoporosis. Methods Drug library screening was performed via alkaline phosphatase (ALP) staining in osteoblasts to identify potential candidates for osteoporosis treatment. qPCR, Western blotting, ALP staining, alizarin red staining, and tartrate-resistant acid phosphatase (TRAP) staining were conducted to assess the impact of ZM-306416 (ZM) on osteoblast and osteoclast differentiation in vitro. Additionally, RNA sequencing and pathway analysis were carried out to explore the underlying molecular mechanisms involved. Micro-CT scanning and immunostaining were used to determine bone phenotypes in vivo. Results Drug library screening revealed that ZM enhances ALP activity in osteoblasts, indicating its potential as a pro-osteogenic agent. ZM exerts dual effects by promoting osteoblast differentiation through the Wnt/β-catenin signaling pathway and simultaneously inhibiting osteoclast differentiation through the NF-κB and MAPK signaling pathways. In an OVX mouse model, ZM effectively prevents bone loss by stimulating osteoblast formation and inhibiting osteoclast development. Conclusions Our study revealed that ZM has a dual anti-osteoporosis effect by promoting osteoblastogenesis and inhibiting osteoclastogenesis, which is mediated by activation of the Wnt/β-catenin signaling pathway and suppression of the NF-κB/MAPK cascades. These findings suggest that ZM could be a promising therapeutic agent for alleviating osteoporosis. ZM-306416 drug library screening osteoporosis osteoblast osteoclast Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1 Introduction Adult bone undergoes continuous remodeling to maintain bone homeostasis and strength, which involves osteoblast-mediated bone formation and osteoclast-mediated bone resorption ( 1 – 3 ). Osteoblasts, the chief bone-making cells, arise from bone marrow mesenchymal stromal cells and secrete specific extracellular proteins (e.g., osteocalcin, alkaline phosphatase, and type I collagen) to facilitate mineralization of the extracellular matrix ( 4 ). In contrast, osteoclasts, specialized multinucleated cells derived from monocytes-macrophages under stimulation by macrophage colony-stimulating factor (M-CSF) and receptor activator of NF-κB ligand (RANKL), absorb old bone ( 5 ). With advancing age and estrogen deficiency, an imbalance in bone remodeling occurs, leading to increased bone resorption and insufficient bone formation, culminating in osteoporosis—a metabolic bone disease characterized by reduced bone mass and impaired microarchitecture, resulting in increased fracture risk ( 6 ). The prevalence of osteoporosis has been steadily increasing due to the aging population, with approximately 200 million individuals affected worldwide, thus presenting a significant healthcare challenge ( 7 , 8 ). Common treatments for osteoporosis include bisphosphonates, RANKL antibodies, monoclonal sclerostin antibodies, and parathyroid hormone receptor agonists ( 9 ). Parathyroid hormone receptor agonists increase osteoblast activity and inhibit osteoclast recruitment, whereas other treatments target osteoclast resorption. Nonetheless, these treatments have limitations and side effects ( 10 – 15 ). For example, long-term bisphosphonate use has been associated with the development of atypical femur fractures and osteonecrosis of the jaw ( 15 ). RANKL antibodies also have side effects, such as skin eczema, flatulence, cellulite inflammation, and osteonecrosis of the jaw ( 16 ). Consequently, there is an urgent need for novel therapeutic interventions for osteoporosis. High-throughput screening of bioactive compound libraries has proven effective for identifying bone metabolism regulators. A study screened 512 small molecules and identified higenamine, which promotes osteogenesis via the IQGAP1/SMAD4 signaling pathway and prevents bone loss in murine models of osteoporosis. This work directly validates library screening as a robust tool for discovering bone-anabolic agents ( 17 ). Another study developed an aptamer-based competitive screening assay to evaluate 96 compounds (including FDA-approved drugs) and identified 6 sclerostin inhibitors that enhance osteoblastic activity. This work further confirms the reliability of library-based screening for targeting key bone regulatory molecules ( 18 ). In this study, we performed ALP staining screening on a bioactive compound library (L1022P, APExBIO) using osteoblasts. Our analysis revealed that ZM-306416 (referred to as ZM), an EGFR inhibitor, is a potential enhancer of osteoblast differentiation. Therefore, our objective was to investigate the effects of ZM on both osteoblast and osteoclast differentiation and explore its therapeutic potential for osteoporosis. 2 Materials and methods 2.1 Mice and ovariectomy murine model C57BL/6 mice, aged 10 to 12 weeks, were obtained from the Laboratory Animal Research Centre of Southern Medical University and housed at a temperature of 20–22°C and a humidity of 30–70%, with four or five mice per cage. All animal care protocols and experiments were reviewed and approved by the Southern Medical University Laboratory Animal Ethics Committee, and this study was compliant with all relevant ethical regulations regarding animal research. The mice were divided into three groups: the OVX group (n = 6), the OVX + ZM group (0.5 mg/kg, n = 6), and the sham group (n = 6). For anaesthesia during bilateral OVX, tribromoethanol anaesthetic (1.25% tribromoethanol solution) was used. It was prepared by weighing 2.5 g of tribromoethanol powder, dissolving it in 5 mL of tert-amyl alcohol, and diluting to 200 mL with deionized water. The solution was magnetically stirred until homogeneous, protected from light with tin foil, and stored at 4°C. The injection dosage was 0.3 ml/20 g body weight via intraperitoneal injection, ensuring adequate anaesthesia while minimizing toxicity. The OVX group and the OVX + ZM group underwent bilateral OVX after being anaesthetized with this tribromoethanol anaesthetic, whereas the sham group only had fat tissue near the ovary removed. The ZM group was injected with 0.5 mg/kg ZM peritoneally, whereas the Sham group and the OVX group were injected with corn oil twice a week for 8 weeks. In the 9th week, all the mice were euthanized by cervical dislocation (a humane and rapid method that minimally interferes with tissue analysis, consistent with ethical animal research guidelines), and the femurs were isolated after excess tissue was removed for histological and morphological evaluation, micro-CT, and microscopic examination. 2.2 Primary osteoblast isolation and culture Primary osteoblasts were harvested from the calvaria of 3-day-old C57BL/6 mice as described previously ( 19 ). Specifically, the excess soft tissue was removed, after which the calvaria was cut into small bone fragments via sterile scissors. The bone fragments were fully digested with 0.25% trypsin for 10 minutes and 0.2% type II collagenase for 90 minutes. Primary osteoblasts were then collected by gently pipetting the digested fragments. The cells were then suspended in complete α-MEM (containing 10% fetal bovine serum) and incubated at 37°C in a 5% CO2 incubator. 2.3 BMM isolation and culture BMMs were generated from the bone marrow of 6-week-old C57BL/6 male mice as described previously ( 20 ). The bone marrow cells were then obtained by flushing the bones with a 1 mL syringe. The cells were subsequently suspended in complete α-MEM supplemented with 10% fetal bovine serum and 30 ng/mL M-CSF. The cells were cultured at 37°C in a 5% CO2 incubator. After three days, the adherent cells were identified as bone marrow-derived macrophages. 2.4 CCK-8 assay The cells were seeded into 96-well plates with a liquid volume of 200 µL per well and counted via a cell counting plate, resulting in approximately 8,000 cells per well. Four replicate wells were set up. The 96-well plates were then incubated overnight at 37°C in a 5% CO2 incubator to allow the cells to adhere. Subsequently, 20 µL of CCK-8 solution was added to each well. A blank control group was also established, where the same volumes of cell culture medium and CCK-8 solution were added, but no cells were present. The 96-well plates were further incubated, and the absorbance was measured at 450 nm after 0.5, 1, 2, and 4 hours via a microplate reader (BIO-TEK, Synergy HTX). On the basis of pre-experimental data, the 2-hour time point was selected for final analysis, as it yielded absorbance values within the linear range (0.8–1.2 OD) for all groups, ensuring accurate quantification of viable cells without signal saturation or underdetection. The OD values for each group at this time point were recorded to calculate cell viability. 2.5 ALP staining and alizarin red staining Osteoblasts were cultured in osteogenic induction medium (OIM; 50 µg/mL ascorbic acid and 5 mM sodium β-glycerophosphate) for approximately 7 days, and the OIM was changed every 2 days to maintain stable concentrations of osteogenic factors and minimize the accumulation of metabolic byproducts. The cells were subsequently fixed with 4% paraformaldehyde for 20 minutes. A BCIP/NBT staining working solution was prepared following the provided instructions. ALP staining was performed at room temperature to stain the cells for 10–30 minutes. Finally, the 6-well plate was scanned via an EPSON Perfection V800 scanner (EPSON, Tokyo, Japan), and the resulting images were stored for subsequent analysis. After a 21-day induction period with the osteogenic induction solution, the cells were fixed with 4% paraformaldehyde for 20 minutes and incubated with a 1% alizarin red staining solution, which was prepared according to the instructions. The cells were stained with alizarin red solution for approximately 5–10 minutes at 37°C. The 6-well plate was then scanned via a scanner, and the resulting images were stored for analysis. 2.6 TRAP staining Bone marrow-derived macrophages were induced with complete α-MEM (containing 10% FBS) supplemented with 30 ng/mL M-CSF and 100 ng/mL RANKL. After 7 days of induction, multinucleated osteoclasts were observed under a microscope. To perform TRAP staining, the staining solution was prepared following the provided instructions. The cells were then incubated with TRAP staining solution at 37°C for 30–60 minutes in the dark. The cells were subsequently washed 1–2 times with deionized water. Finally, the cells were observed, and pictures were taken under a microscope. Osteoclasts were identified as TRAP-positive cells with more than 3 nuclei. The ability to form osteoclasts was determined by analysing the number of TRAP-positive cells in each group. 2.7 Western blotting (WB) The cells were seeded in 6-well plates at a density of 4 × 10⁵ cells per well and cultured overnight. After treatment, the cells were lysed on ice for 10 minutes in RIPA lysis buffer containing protease inhibitors to extract total protein. The total protein was separated by SDS‒PAGE and transferred to polyvinylidene fluoride (PVDF) membranes. Following blocking with skim milk for 1 hour, the membranes were incubated overnight at 4°C with the following primary antibodies: anti-OSX (1:1000, BOSTER, China); anti-RUNX2, anti-CTSK, and anti-NFATC1 (1:200, Santa Cruz, USA); anti-Tubulin (1:5000, Affinity, China); anti-ERK, anti-p-ERK, anti-P38, anti-P-P38, anti-P65, anti-P-P65, anti-JNK, and anti-P-JNK (1:1000, Cell Signaling, USA); anti-β-actin (1:20000, Proteintech, China); and anti-β-catenin (1:1000, Abmart, China). The membranes were subsequently washed three times with TBST (5 minutes each) and incubated with specific secondary antibodies: goat anti-mouse IgG H&L (HRP) pAb (1:20000, PTM Bio, China) and goat anti-rabbit IgG H&L (HRP) pAb (1:10000, PTM Bio, China) for 1 hour at room temperature. After three additional washes with TBST, the membranes were incubated with chemiluminescent substrate (Abkine, Cat. No. BMP3010) and visualized via the GeneSys capture system. 2.8 Real-time quantitative PCR (qPCR) Total RNA was isolated from the cells via TRIzol Reagent (Cat. No. 9109; Takara Biotechnology) following the manufacturer's protocol. The reverse transcription reaction system (Cat. RR036A, Takara Biotechnology) was then prepared according to the manufacturer's instructions. The system was subsequently placed into the reverse transcription PCR instrument to generate complementary DNA (cDNA). The obtained cDNA was used for qPCR with ChamQ SYBR qPCR Master Mix (Cat. No. Q311-02, Vazyme Biotech) in a LightCycler96 instrument (Roche Molecular Biochemicals). The relative quantification of gene expression was performed via the comparative threshold method. Changes in mRNA expression levels were calculated after normalization of the values to those of the GAPDH housekeeping gene. The primers used are listed in Table S1 . 2.9 Immunohistochemistry The femurs were separated from the mice and fixed in 4% paraformaldehyde for 24 hours. After decalcification in 10% EDTA (pH 8.0) for 21 days, the decalcified tissues were embedded in paraffin and prepared as 4 µm sagittal-oriented sections for histological analysis. The paraffin sections were then incubated at 65°C for 2 hours. After the paraffin sections were dewaxed and hydrated, antigen retrieval was performed. Next, the paraffin sections were incubated overnight at 4°C with a primary antibody against osteocalcin (OCN), followed by incubation with secondary antibodies for 1 hour at room temperature. Finally, diaminobenzidine (DAB) was used to visualize the positive cells, and the samples were observed and photographed via an Olympus BX51 microscope. Immunohistochemical staining was evaluated by the number of positive cells per bone perimeter (B.Pm). 2.10 Micro-CT analysis Micro-CT analysis was conducted using a CT40 scanner (Scanco Medical AG, Bassersorf, Switzerland) to examine the femoral structure. The scanning conditions were set to 55 kV, 145 µA, and a voxel size of 10 µm (optimal for trabecular bone resolution in murine femurs). Image segmentation was performed using a threshold range of 220–1000 HU, which effectively distinguishes mineralized bone tissue from soft tissue and marrow in mouse femurs. For the analysis of bone trabeculae, 200 section planes were scanned from the distal femoral growth plate towards the proximal end. The analysis of cortical bone involved scanning from the middle of the femur towards the proximal end. The parameters analysed included the trabecular bone density (Tb. BMD, mg HA/ccm), bone trabecular volume fraction (BV/TV, %), bone trabecular number (Tb. N, mm⁻¹), bone trabecular thickness (Tb. Th, mm), bone trabecular separation/porosity (Tb. Sp, mm), cortical bone density (Ct. BMD, mg HA/ccm), and cortical bone thickness (Ct. Th, mm). 2.11 ELISA Blood was collected from the mice via eyeball extraction and left at room temperature for 2 hours. After centrifugation at 1000 × g for 20 minutes, the supernatant was collected for serum preparation. The serum was stored at -80°C. The serum sample and the CTX-1 ELISA kit were subsequently brought to room temperature for equilibration. Next, the procedures to measure the levels of CTX-1 in the serum of each group were carried out according to the instructions provided with the ELISA kits. 2.12 Hematoxylin and eosin (HE) staining The paraffin-embedded sections were stained with HE. The sections were observed and photographed using an Olympus BX51 microscope. Subsequently, histological analysis was conducted via ImageJ software. 2.13 RNA sequencing and pathway analysis Primary osteoblasts were treated with or without 10 µM ZM in osteogenic induction medium for 6 days, and the cells were divided into a control group and an intervention group. Concomitantly, bone marrow-derived macrophages were treated with or without 10 µM ZM in complete α-MEM (containing 10% FBS) supplemented with 30 ng/mL M-CSF and 100 ng/mL RANKL for 2 days, and the cells were also divided into a control group and an intervention group. Total RNA was extracted via TRIzol Reagent (Cat. No. 9109; Takara Biotechnology) following the manufacturer’s instructions, and three samples per group were used. The RNA concentration and purity were quantified via a UV spectrophotometer (NanoDropOne, Thermo Fisher Scientific, USA). After RNA extraction, the raw sequencing reads were filtered via Fastp to remove low-quality and adapter-containing reads. Clean reads were aligned to the mouse reference genome (mm10) with HISAT2. Gene read counts were generated by FeatureCounts. Differentially expressed genes (DEGs) were identified via DESeq2, with the criteria of |log₂-fold change (FC)| >1 and adjusted P value < 0.05. The Kyoto Encyclopedia of Genes and Genomes (KEGG; http://www.genome.ad.jp/kegg ) was employed for pathway analysis via enriched R packages. The terms or pathways were mapped and visualized by selecting the top 20 according to the number of genes for which they were enriched. The original data were uploaded to the GEO database ( https://www.ncbi.nlm.nih.gov/ , GSE307058, GSE307904). 2.14 Key Reagents ZM-306416 (ZM) was purchased from APExBIO Technology LLC (Houston, USA; Catalogue No. A8684; CAS No. 690206-97-4). The compound has a purity ≥ 95% (verified by HPLC, as confirmed by the batch-specific certificate of analysis). ZM was dissolved in DMSO to prepare a 10 mM stock solution (solubility ≥ 33.4 mg/mL in DMSO, insoluble in water) and stored at -20°C according to the manufacturer’s recommendations. ICG-001 was purchased from Selleck Chemicals (Catalogue No. S2662). It was dissolved in DMSO to prepare a 10 mM stock solution and stored at -20°C. For in vitro experiments, a working concentration of 10 µM was used, as determined on the basis of references indicating that this concentration effectively inhibits Wnt/β-catenin signaling ( 21 ). 2.15 Drug Library Screening Primary osteoblasts were isolated from the calvaria of 3-day-old C57BL/6 mice and seeded in 24-well plates at a density of 5 × 10⁴ cells per well. Following 24 hours of adherence in α-MEM supplemented with 10% fetal bovine serum (FBS), the cells were treated with compounds from the DiscoveryProbe™ Bioactive Compound Library Plus (Cat. No. L1022P, APExBIO, USA). The compounds were diluted in osteogenic induction medium (OIM) to a final concentration of 10 µM. Each compound was administered in triplicate (n = 3). The cells were cultured in OIM for 7 days, and the medium was changed every 48 hours. Alkaline phosphatase (ALP) staining was performed via BCIP/NBT kits according to the manufacturer’s instructions. The staining intensity was semiquantified via ImageJ software, and the compounds were evaluated relative to 0.1% dimethyl sulfoxide (DMSO) vehicle controls. 2.16 Statistical analysis The experiments were conducted a minimum of three times, and the results are presented as the mean ± SEM. Data analysis was performed via GraphPad Prism 10.1.2. Student's t- test was used to compare two groups, whereas one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison test was used to compare differences between three or more groups. A p value of less than 0.05 was considered statistically significant for all the experiments. 3 Results 3.1 ZM promotes osteoblast differentiation In a drug screening assay using a bioactive compound library (L1022P, APExBIO), our results demonstrated that ZM significantly enhanced alkaline phosphatase (ALP) activity (Fig. 1 A). To verify the role of ZM in osteoblast differentiation, primary mouse osteoblasts were treated with varying concentrations of ZM, and the differentiation phenotypes were analysed. Initially, a CCK8 assay was conducted to detect the effect of ZM on osteoblast proliferation. The findings indicated that concentrations ranging from 1.25 to 10 µM had no significant effect on osteoblast proliferation (Fig. 1 B). A concentration of 10 µM was chosen for subsequent cell experiments. Following 7 days of osteogenic induction, the expression of the osteoblast differentiation markers Runx2 , Alpl , and Bglap was assessed. We found that ZM markedly upregulated these osteogenic differentiation marker genes (Fig. 1 C). Consistent with the mRNA findings, the protein levels of OSX and RUNX2 were also significantly upregulated (Fig. 1 D). On the 7th day of osteogenic induction, ALP staining was subsequently performed, followed by alizarin red staining on the 14th day. The results demonstrated that ZM increased alkaline phosphatase activity and calcium deposition in primary osteoblasts (Fig. 1 E and 1 F). Overall, these findings highlight that ZM effectively promotes osteoblast differentiation in vitro without affecting cell growth. 3.2 ZM inhibits osteoclast differentiation Considering the coupling role of osteoblasts and osteoclasts in bone remodeling and the development of osteoporosis, we aimed to examine whether ZM could inhibit osteoclast differentiation. We initially assessed the effect of ZM on BMM proliferation and found no significant effect (Fig. 2 A). Following a 3-day period of M-CSF and RANKL stimulation, we evaluated the mRNA and protein levels of osteoclast differentiation markers. Notably, ZM inhibited both the mRNA and protein levels of these markers in a concentration-dependent manner (Fig. 2 B and 2 C). M-CSF and RANKL induced the formation of numerous TRAP-positive multinucleated osteoclasts from BMMs (Fig. 2 D). However, ZM at a concentration of 10 µM inhibited M-CSF and RANKL-induced osteoclastogenesis almost completely (Fig. 2 D). Collectively, these findings demonstrate the in vitro inhibitory effect of ZM on osteoclast differentiation. 3.3 ZM prevents OVX-induced bone loss in vivo To investigate the potential of ZM in restoring bone loss in vivo, we conducted a study to determine whether it could prevent the established bone loss caused by OVX in mice. We observed that ovariectomy-induced estrogen deprivation resulted in a decrease in the size and weight of the mouse uterus (Fig. 3 B). Since estrogen deficiency causes an imbalance in bone formation and bone resorption in postmenopausal women, leading to bone loss and osteoporosis, and our results indicate that ZM stimulates osteogenesis and inhibits osteoclastogenesis, we hypothesized that ZM treatment could prevent estrogen deficiency-induced bone loss. For this purpose, OVX mice were intraperitoneally injected with ZM (Fig. 3 A). Micro-CT analysis of the distal femoral metaphysis and diaphysis revealed that OVX mice presented noticeable loss of trabecular and cortical bone (Fig. 3 C, 3 D). We found that bone mineral density (BMD), bone volume per tissue volume (BV/TV), the trabecular number (Tb.N), the trabecular thickness (Tb.Th), and the trabecular bone area were significantly reduced in mice 8 weeks after OVX (Figs. 3 C, 3 D, and 4 A). Conversely, trabecular spacing (Tb.Sp) and the number of TRAP-positive osteoclasts were significantly increased, indicating severe bone loss in the femur as a result of OVX (Figs. 3 C and 4 B). The serum levels of C-terminal telopeptide of collagen type I (CTX-1), a marker for bone resorption, were significantly greater in OVX mice than in control mice (Fig. 3 E). These results indicated severe bone loss in the female mice as a result of OVX. To assess the therapeutic potential of ZM, we administered either vehicle or ZM (0.5 mg/kg) intraperitoneally to OVX mice twice a week for 8 weeks. We subsequently conducted micro-CT analysis and histological staining of femurs from these mice. Compared with vehicle-treated OVX mice, OVX mice treated with ZM presented substantial bone restoration and significantly greater BMD, BV/TV, Tb.N, Tb.Th and trabecular bone area (Figs. 3 C, 3 D, and 4 A). Histomorphometric analysis further revealed that the number of osteoblasts (N.Ocn/B. Pm) was increased in ZM-treated OVX mice, whereas the osteoclast surface was significantly smaller and the number of osteoclasts (N.Oc/B. Pm) was lower than that in vehicle-treated OVX mice (Fig. 4 B). Taken together, our results suggest that ZM treatment is a potential therapeutic approach for postmenopausal bone loss. 3.4 ZM regulates osteoblast and osteoclast differentiation through the Wnt/β-catenin signaling pathway and NF-κB/MAPK signaling, respectively. To elucidate the molecular mechanisms by which ZM modulates osteoblast and osteoclast differentiation, we performed transcriptomic and biochemical analyses. KEGG pathway enrichment analysis of differentially expressed genes (DEGs) in osteoblasts revealed significant enrichment of the Wnt signaling pathway, indicating that this pathway plays a critical role in ZM-mediated regulation of osteoblastogenesis (Fig. 5 A). Numerous studies have confirmed that canonical activation of the Wnt/β-catenin pathway is essential for osteoblastogenesis and bone formation ( 22 ). To test the hypothesis that ZM promotes osteogenesis by activating the Wnt/β-catenin pathway, we examined the protein levels of β-catenin (a key mediator of canonical Wnt signaling) and RUNX2 (a downstream transcription factor indicative of osteogenic commitment) in osteoblasts treated with ZM ( 23 , 24 ). Western blot analysis revealed significant upregulation of both β-catenin and RUNX2 in ZM-treated osteoblasts compared with OIM-treated controls (Fig. 5 B). To further validate the involvement of the Wnt/β-catenin signaling pathway, we co-treated primary osteoblasts with ZM and ICG-001, a specific inhibitor of the canonical Wnt/β-catenin pathway, in OIM, ensuring that the experiments were conducted under conditions that mimic the osteogenic microenvironment ( 25 , 26 ). As expected, ICG-001 significantly attenuated the ZM-induced upregulation of β-catenin and RUNX2 (Fig. 5 B), confirming that the osteogenic effect of ZM is mediated through canonical Wnt/β-catenin activation. This effect was further validated by ALP staining, where ZM-treated osteoblasts presented stronger ALP activity (darker staining) than OIM-treated controls did, whereas co-treatment with ICG-001 diminished this enhancement (lighter staining), confirming that the osteogenic effect of ZM relies on canonical Wnt/β-catenin activation (Fig. 5 C). For osteoclasts, we performed parallel KEGG pathway analysis to identify signaling pathways involved in ZM-mediated regulation. This analysis revealed significant enrichment in the "osteoclast differentiation" pathway (Fig. 5 D), a pathway well- established to be tightly regulated by the NF-κB and MAPK signaling cascades. These cascades are recognized as core mediators of osteoclastogenesis downstream of RANKL-RANK activation ( 27 , 28 ), which aligns with our focus on deciphering how ZM inhibits RANKL-induced osteoclast differentiation and prompting us to investigate their involvement. Thus, we examined the phosphorylation levels of NF-κB (p65) and MAPK family members (p38, JNK, and ERK), which are crucial and fundamental downstream of RANKL–RANK signaling ( 28 ). As expected, co-stimulation with M-CSF and RANKL triggered rapid phosphorylation of NF-κB (p65) and MAPKs (Fig. 5 E). Notably, treatment of BMMs with ZM significantly suppressed M-CSF and RANKL-induced phosphorylation of p65, p38, ERK, and JNK (Fig. 5 E), indicating that ZM targets these signaling cascades to exert its inhibitory effect on osteoclast differentiation. Taken together, these findings demonstrate that ZM promotes osteoblast differentiation by activating the canonical Wnt/β-catenin pathway and inhibits osteoclastogenesis by suppressing NF-κB/MAPK signaling, supporting our conclusion that ZM regulates bone remodeling via these two distinct pathways. 4 Discussion In this study, we performed high-throughput drug library screening and identified ZM as a potential candidate that promotes osteoblast differentiation. Notably, ZM has dual effects: it not only enhances osteoblast differentiation but also inhibits osteoclast differentiation. Consistent with these in vitro findings, ZM was further shown to prevent OVX-induced bone loss in vivo, highlighting its potential to regulate bone remodeling bidirectionally. This identification of ZM relied on high-throughput drug library screening, which laid the foundation for our subsequent mechanistic studies. High-throughput drug library screening is a valuable approach in drug discovery and development. It offers several advantages, including rapid screening of a large number of compounds, which significantly reduces the time compared with traditional methods. This enables researchers to rapidly identify potential drug candidates, explore new targets, and discover drugs for previously unexplored therapeutic areas. However, high-throughput screening requires substantial manpower and material resources. Despite these limitations, it remains a valuable tool in the early stages of drug discovery when used in conjunction with other approaches. After confirming the initial activity of ZM by screening, we further explored its molecular mechanisms through in vitro experiments. Through in vitro experiments, we demonstrated that ZM activates the Wnt/β-catenin signaling pathway, as evidenced by KEGG enrichment of Wnt signaling-related genes in ZM-treated osteoblasts, alongside upregulation of β-catenin (a central mediator of canonical Wnt signaling) and RUNX2 (a master transcription factor driving osteogenic commitment). Furthermore, the use of ICG-001, a specific inhibitor of the Wnt/β-catenin signaling pathway, inhibited osteoblast differentiation, confirming that activation of the Wnt/β-catenin signaling pathway is indispensable for regulating osteoblast differentiation. Functional validation via ALP staining further confirmed that ZM enhances osteogenic commitment through this cascade. Moreover, ZM inhibits osteoclast differentiation by targeting nuclear factor κB (NF-κB) and mitogen-activated protein kinase (MAPK) signaling. KEGG analysis revealed significant enrichment in the “osteoclast differentiation” pathway in ZM-treated osteoclast precursors, a pathway tightly regulated by the NF-κB and MAPK cascades downstream of RANKL-RANK activation. We confirmed that ZM suppresses the RANKL-induced phosphorylation of p65 (NF-κB) and MAPKs (p38, JNK, and ERK), which aligns with their known role as core mediators of osteoclastogenesis ( 29 , 30 ). To contextualize the dual effects of ZM, we compared it with those of clinically approved dual-acting anti-osteoporosis agents. For example, romosozumab, a sclerostin antibody, promotes bone formation via Wnt pathway activation and inhibits resorption through downstream effects. In contrast, ZM modulates both the Wnt/β-catenin pathway (to increase osteogenesis) and the NF-κB/MAPK pathway (to suppress osteoclastogenesis) directly, suggesting a distinct mechanistic profile that may confer unique therapeutic advantages. This distinct mechanism of action of ZM addresses a longstanding gap in current osteoporosis treatment, as most existing drugs lack dual effects on bone formation and resorption. For several years, the medical community has been conducting research on therapies that aim to increase bone mass by stimulating new bone formation while also inhibiting bone resorption. However, most drugs currently used to treat osteoporosis focus only on inhibiting bone resorption and lack the dual effects of stimulating bone formation ( 6 , 9 , 31 ). Our findings present a potential promising candidate for the development of anti-osteoporosis drugs. The results of our study suggest that ZM may be an effective therapeutic agent for osteoporosis by simultaneously inhibiting bone resorption and promoting bone formation. However, this study has limitations that should be acknowledged. First, we did not assess the long-term toxicity of ZM, which is critical for evaluating its translational potential. Second, we only tested ZM in an OVX mouse model and did not evaluate its efficacy in aged mouse models or models of glucocorticoid-induced osteoporosis, which are scenarios that are highly relevant to clinical osteoporosis presentations. 5 Conclusion Our study revealed that ZM has a dual anti-osteoporosis effect by promoting osteoblastogenesis and inhibiting osteoclastogenesis via the activation of Wnt/β-catenin signaling and suppression of NF-κB/MAPK cascades. These findings suggest that ZM could be a promising therapeutic agent for alleviating osteoporosis. Declarations 6 Data availability statement The raw RNA-seq data were deposited in the NCBI Gene Expression Omnibus (GEO) database under accession numbers GSE307058 and GSE307904. Further inquiries regarding the data can be directed to the corresponding author. 7 Ethics statement All animal care protocols and experiments were reviewed and approved by the Southern Medical University Laboratory Animal Ethics Committee (Approval No. LAEC-21-123), and this study was compliant with all relevant ethical regulations regarding animal research. 8 Author contributions YL: Data curation, Formal analysis, Investigation, Writing – original draft. SS: Data curation, Formal analysis, Resources, Writing – original draft. DY: Formal analysis, Writing – review and editing. LZ: Investigation, Writing – review and editing. TC: Funding acquisition, Writing – review and editing. KX: Software, Methodology, Writing – original draft. WL: Data curation, Writing – original draft. JC: Funding acquisition, Project administration, Resources, Writing –resources, writing, review and editing. JC: Conceptualization, Funding acquisition, Project administration, Writing – review and editing. 9 Funding This work was supported by the general project of the Anhui Province Outstanding Young Talents Support Program for Universities (Grant No. gxyq2022009); the Anhui Institute of Translational Medicine (Grant No. 2022zhyx-C90); the Medical Scientific Research Foundation of Guangdong Province, China (Grant No. B2025168); and the Longhua District Medical and Health Institution 2025 District-Level Scientific Research Project (Grant No. 2025036). 10 Acknowledgements We especially appreciate the support of Wenquan Liang and Tianyu Chen and, of course, all the participants. 11 Conflicts of interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as potential conflicts of interest. 12 Consent for publication Not applicable. References Bolamperti S, Villa I, Rubinacci A. Bone remodeling: an operational process ensuring survival and bone mechanical competence. Bone research. 2022;10(1):48. Salhotra A, Shah HN, Levi B, Longaker MT. Mechanisms of bone development and repair. Nature reviews Molecular cell biology. 2020;21(11):696-711. Zaidi M. Skeletal remodeling in health and disease. Nature Medicine. 2007;13(7):791-801. Long F. Building strong bones: molecular regulation of the osteoblast lineage. Nature Reviews Molecular Cell Biology. 2012;13(1):27-38. Henriksen K, Bollerslev J, Everts V, Karsdal MA. Osteoclast Activity and Subtypes as a Function of Physiology and Pathology—Implications for Future Treatments of Osteoporosis. Endocrine Reviews. 2011;32(1):31-63. Ensrud KE, Crandall CJ. Osteoporosis. Annals of internal medicine. 2024;177(1):Itc1-itc16. Kanis JA, Cooper C, Rizzoli R, Reginster JY. European guidance for the diagnosis and management of osteoporosis in postmenopausal women. Osteoporosis international : a journal established as result of cooperation between the European Foundation for Osteoporosis and the National Osteoporosis Foundation of the USA. 2019;30(1):3-44. Reginster J-Y, Burlet N. Osteoporosis: A still increasing prevalence. Bone. 2006;38(2, Supplement 1):4-9. Compston JE, McClung MR, Leslie WD. Osteoporosis. Lancet (London, England). 2019;393(10169):364-76. Curry SJ, Krist AH, Owens DK, Barry MJ, Caughey AB, Davidson KW, et al. Screening for Osteoporosis to Prevent Fractures US Preventive Services Task Force Recommendation Statement. JAMA-JOURNAL OF THE AMERICAN MEDICAL ASSOCIATION. 2018;319(24):2521-31. Khosla S, Hofbauer LC. Osteoporosis treatment: recent developments and ongoing challenges. The lancet Diabetes & endocrinology. 2017;5(11):898-907. Miller PD, Hattersley G, Riis BJ, Williams GC, Lau E, Russo LA, et al. Effect of Abaloparatide vs Placebo on New Vertebral Fractures in Postmenopausal Women With Osteoporosis A Randomized Clinical Trial. JAMA-JOURNAL OF THE AMERICAN MEDICAL ASSOCIATION. 2016;316(7):722-33. Peugh J, Khalil A, Chan MR, Hansen KE. Teriparatide Treatment for Hypercalcemia Associated With Adynamic Bone Disease. JBMR plus. 2019;3(7):e10176-e. Nicholson WK, Silverstein M, Wong JB, Chelmow D, Coker TR, Davis EM, et al. Screening for Osteoporosis to Prevent Fractures US Preventive Services Task Force Recommendation Statement. JAMA-JOURNAL OF THE AMERICAN MEDICAL ASSOCIATION. 2025;333(6):498-508. Wang M, Wu Y-F, Girgis CM. Bisphosphonate Drug Holidays: Evidence From Clinical Trials and Real-World Studies. JBMR plus. 2022;6(6):e10629-e. Zhang N, Zhang ZK, Yu Y, Zhuo Z, Zhang G, Zhang BT. Pros and Cons of Denosumab Treatment for Osteoporosis and Implication for RANKL Aptamer Therapy. Frontiers in cell and developmental biology. 2020;8:325. Dong H, Liu R, Zou K, Jin Z, Kang J, Zhang Y, et al. Higenamine Promotes Osteogenesis Via IQGAP1/SMAD4 Signaling Pathway and Prevents Age- and Estrogen-Dependent Bone Loss in Mice. Journal of Bone and Mineral Research. 2023;38(5):775-91. Lee CC, Hung CM, Chen CH, Hsu YC, Huang YP, Huang TB, et al. Novel Aptamer-Based Small-Molecule Drug Screening Assay to Identify Potential Sclerostin Inhibitors against Osteoporosis. International journal of molecular sciences. 2021;22(15). Liu Z, Liang W, Kang D, Chen Q, Ouyang Z, Yan H, et al. Increased Osteoblastic Cxcl9 Contributes to the Uncoupled Bone Formation and Resorption in Postmenopausal Osteoporosis. Clinical interventions in aging. 2020;15:1201-12. Liang W, Chen Q, Cheng S, Wei R, Li Y, Yao C, et al. Skin chronological aging drives age-related bone loss via secretion of cystatin-A. Nature Aging. 2022;2(10):906-22. Ping Z, Hu X, Wang L, Shi J, Tao Y, Wu X, et al. Melatonin attenuates titanium particle-induced osteolysis via activation of Wnt/β-catenin signaling pathway. Acta Biomaterialia. 2017;51:513-25. Hu L, Chen W, Qian A, Li YP. Wnt/β-catenin signaling components and mechanisms in bone formation, homeostasis, and disease. Bone research. 2024;12(1):39. Clevers H, Nusse R. Wnt/β-Catenin Signaling and Disease. Cell. 2012;149(6):1192-205. Zhu S, Chen W, Masson A, Li YP. Cell signaling and transcriptional regulation of osteoblast lineage commitment, differentiation, bone formation, and homeostasis. Cell discovery. 2024;10(1):71. McMillan M, Kahn M. Investigating Wnt signaling: a chemogenomic safari. Drug discovery today. 2005;10(21):1467-74. Yang MH, Basappa B, Deveshegowda SN, Ravish A, Mohan A, Nagaraja O, et al. A novel drug prejudice scaffold-imidazopyridine-conjugate can promote cell death in a colorectal cancer model by binding to β-catenin and suppressing the Wnt signaling pathway. Journal of advanced research. 2025;72:615-32. Arnst J, Jing Z, Cohen C, Ha SW, Viggeswarapu M, Beck GR, Jr. Bioactive silica nanoparticles target autophagy, NF-κB, and MAPK pathways to inhibit osteoclastogenesis. Biomaterials. 2023;301:122238. Park JH, Lee NK, Lee SY. Current Understanding of RANK Signaling in Osteoclast Differentiation and Maturation. Molecules and cells. 2017;40(10):706-13. Cheng HM, Xing M, Zhou YP, Zhang W, Liu Z, Li L, et al. HSP90β promotes osteoclastogenesis by dual-activation of cholesterol synthesis and NF-κB signaling. Cell death and differentiation. 2023;30(3):673-86. Lee DK, Jin X, Choi PR, Cui Y, Che X, Lee S, et al. Phospholipase C β4 promotes RANKL-dependent osteoclastogenesis by interacting with MKK3 and p38 MAPK. Experimental & molecular medicine. 2025;57(2):323-34. Roux C, Briot K. Addressing the crisis in the treatment of osteoporosis. Nature Reviews Rheumatology. 2018;14(2):67-8. Additional Declarations No competing interests reported. Supplementary Files TableS1.NucleotidesequencesofprimersusedforqPCR.docx UncroppedWesternBlots.zip Cite Share Download PDF Status: Published Journal Publication published 31 Jan, 2026 Read the published version in Journal of Orthopaedic Surgery and Research → Version 1 posted Editorial decision: Revision requested 14 Dec, 2025 Reviews received at journal 09 Dec, 2025 Reviewers agreed at journal 08 Dec, 2025 Reviewers agreed at journal 19 Nov, 2025 Reviewers invited by journal 13 Nov, 2025 Editor assigned by journal 23 Oct, 2025 Submission checks completed at journal 22 Oct, 2025 First submitted to journal 17 Oct, 2025 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-7828854","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":549349401,"identity":"c1214d91-291f-447d-9a6b-422fa31c7e3d","order_by":0,"name":"Yicheng Li","email":"","orcid":"","institution":"Department of Rehabilitation Medicine, Shenzhen Longhua District Central Hospital","correspondingAuthor":false,"prefix":"","firstName":"Yicheng","middleName":"","lastName":"Li","suffix":""},{"id":549349402,"identity":"4c5dbffe-35e7-499f-82bb-5a121db93293","order_by":1,"name":"Shuo Shi","email":"","orcid":"","institution":"School of Basic Medical Sciences, Anhui Medical University","correspondingAuthor":false,"prefix":"","firstName":"Shuo","middleName":"","lastName":"Shi","suffix":""},{"id":549349403,"identity":"beb67ac1-c4d9-4da4-b788-eee99733c929","order_by":2,"name":"Dejian Yang","email":"","orcid":"","institution":"Department of Anatomy, School of Basic Medical Sciences, Southern Medical University","correspondingAuthor":false,"prefix":"","firstName":"Dejian","middleName":"","lastName":"Yang","suffix":""},{"id":549349404,"identity":"d3f247c6-f10b-4051-a52a-e2486a908c0e","order_by":3,"name":"Lianhui Zhao","email":"","orcid":"","institution":"School of Basic Medical Sciences, Anhui Medical University","correspondingAuthor":false,"prefix":"","firstName":"Lianhui","middleName":"","lastName":"Zhao","suffix":""},{"id":549349405,"identity":"c00426ac-b0ba-4c58-9115-9e0e1b3122de","order_by":4,"name":"Tianyu Chen","email":"","orcid":"","institution":"Department of Orthopedics, The Third Affiliated Hospital of Southern Medical University","correspondingAuthor":false,"prefix":"","firstName":"Tianyu","middleName":"","lastName":"Chen","suffix":""},{"id":549349406,"identity":"862b9ece-86f0-4846-aab0-d9141930ffe2","order_by":5,"name":"Kangshuai Xu","email":"","orcid":"","institution":"Department of Trauma Orthopedics, Jinhua Municipal Central Hospital","correspondingAuthor":false,"prefix":"","firstName":"Kangshuai","middleName":"","lastName":"Xu","suffix":""},{"id":549349407,"identity":"ff3e26ee-a3b1-43b7-b8a4-e685dd787820","order_by":6,"name":"Wenquan Liang","email":"","orcid":"","institution":"Department of Anatomy, School of Basic Medical Sciences, Southern Medical University","correspondingAuthor":false,"prefix":"","firstName":"Wenquan","middleName":"","lastName":"Liang","suffix":""},{"id":549349408,"identity":"d933b5f0-6751-421e-be9f-939a89db731a","order_by":7,"name":"Junqi Chen","email":"","orcid":"","institution":"Department of Rehabilitation Medicine, The Third Affiliated Hospital of Southern Medical University","correspondingAuthor":false,"prefix":"","firstName":"Junqi","middleName":"","lastName":"Chen","suffix":""},{"id":549349409,"identity":"0be6c49b-883b-4a3e-872e-eee314d2e403","order_by":8,"name":"Jun Chang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABDUlEQVRIiWNgGAWjYDACCRDBA8TsjQ0HPlQcAAseeECUFp7DBx/OOHMAzD6QQFALmJGWbMzbBtHCgE8L/+zmZw+/yBzOY2DIMZOcOe+OnL3Y4YdAW+zkdBtwWHLnmLmxDM/hYgaGM2YSH7c9M+aRTjMAakk2NjuAXYuBRIKZtATP4cQGxh6gLdsOJ/ZIJ4C0HEjchlNL+jeIFmYeM2neOSAt6R8IaAF64QNICxsb0PsNIC05+G2RuJFTJs3Ak57YwMMMDORjh415bucUHEgwwO0X/hnp2yR/9lgnNsg/BEZlzWE59tnpmz98qLCTw6UFBJh5exgY7FEVGOBWDgKMP37gVzAKRsEoGAUjHAAADcBk9yg3XyYAAAAASUVORK5CYII=","orcid":"","institution":"School of Basic Medical Sciences, Anhui Medical University","correspondingAuthor":true,"prefix":"","firstName":"Jun","middleName":"","lastName":"Chang","suffix":""}],"badges":[],"createdAt":"2025-10-10 16:08:22","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7828854/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7828854/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s13018-025-06654-7","type":"published","date":"2026-01-31T15:58:29+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":96709565,"identity":"b2f4da92-aaeb-4714-b83f-60bc5b730cea","added_by":"auto","created_at":"2025-11-25 10:09:16","extension":"tif","order_by":0,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":11510716,"visible":true,"origin":"","legend":"","description":"","filename":"Figure1.tif","url":"https://assets-eu.researchsquare.com/files/rs-7828854/v1/2caa4925e56a9fd65e0a9c18.tif"},{"id":96651376,"identity":"a4ee6fef-46f5-4daa-a0b1-da90e2da7cf3","added_by":"auto","created_at":"2025-11-24 16:14:27","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":84699,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.docx","url":"https://assets-eu.researchsquare.com/files/rs-7828854/v1/6d01a7edcba071c97ed584e6.docx"},{"id":96651377,"identity":"fc05eea4-ddc2-4ef4-af38-19b6eac28764","added_by":"auto","created_at":"2025-11-24 16:14:27","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":17662,"visible":true,"origin":"","legend":"","description":"","filename":"TableS1.NucleotidesequencesofprimersusedforqPCR.docx","url":"https://assets-eu.researchsquare.com/files/rs-7828854/v1/66b0cd311540eab392559a57.docx"},{"id":96651391,"identity":"b213f43d-706d-4ca0-90c0-fd647d5c4fb7","added_by":"auto","created_at":"2025-11-24 16:14:28","extension":"tif","order_by":3,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":5938492,"visible":true,"origin":"","legend":"","description":"","filename":"Figure2.tif","url":"https://assets-eu.researchsquare.com/files/rs-7828854/v1/c6c0db9cf7e90b17061249dd.tif"},{"id":96651394,"identity":"b6926b3c-3749-49b3-b6d3-c02d1675b4c7","added_by":"auto","created_at":"2025-11-24 16:14:28","extension":"tif","order_by":4,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":9062564,"visible":true,"origin":"","legend":"","description":"","filename":"Figure3.tif","url":"https://assets-eu.researchsquare.com/files/rs-7828854/v1/5649fdeed84c5e090f7f07d5.tif"},{"id":96651388,"identity":"e3a7083c-2a9e-47b8-b1eb-33acb2349114","added_by":"auto","created_at":"2025-11-24 16:14:28","extension":"tif","order_by":5,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":12269500,"visible":true,"origin":"","legend":"","description":"","filename":"Figure4.tif","url":"https://assets-eu.researchsquare.com/files/rs-7828854/v1/dbc76ebf5df538789d1626f4.tif"},{"id":96651385,"identity":"b918341c-7167-48a2-8ea3-78dd415f8004","added_by":"auto","created_at":"2025-11-24 16:14:28","extension":"tif","order_by":6,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":10383904,"visible":true,"origin":"","legend":"","description":"","filename":"Figure5.tif","url":"https://assets-eu.researchsquare.com/files/rs-7828854/v1/2bb018d6aaba239dd5f2e21a.tif"},{"id":96651387,"identity":"2e5dcac9-ebd6-4655-b4fb-fd50920cd020","added_by":"auto","created_at":"2025-11-24 16:14:28","extension":"json","order_by":7,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":10297,"visible":true,"origin":"","legend":"","description":"","filename":"c79393fdc3a844a7b8664793ff053260.json","url":"https://assets-eu.researchsquare.com/files/rs-7828854/v1/151a29f534c357b191186ddc.json"},{"id":96651398,"identity":"4c67ac41-0605-432a-ad9d-d7686a06addf","added_by":"auto","created_at":"2025-11-24 16:14:28","extension":"zip","order_by":8,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":25490301,"visible":true,"origin":"","legend":"","description":"","filename":"UncroppedWesternBlots.zip","url":"https://assets-eu.researchsquare.com/files/rs-7828854/v1/0a9c60287e4ec4ec0e2fe6bf.zip"},{"id":96651392,"identity":"e37da9ac-61b5-4c6a-a43e-f0e7035ef6f7","added_by":"auto","created_at":"2025-11-24 16:14:28","extension":"xml","order_by":9,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":100903,"visible":true,"origin":"","legend":"","description":"","filename":"c79393fdc3a844a7b8664793ff0532601enriched.xml","url":"https://assets-eu.researchsquare.com/files/rs-7828854/v1/bd18c98856c516bd79744c2d.xml"},{"id":96710663,"identity":"e9562d0a-a599-4009-bb00-2813c341ef67","added_by":"auto","created_at":"2025-11-25 10:11:03","extension":"tif","order_by":10,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":11510716,"visible":true,"origin":"","legend":"","description":"","filename":"Figure1.tif","url":"https://assets-eu.researchsquare.com/files/rs-7828854/v1/44a05b16c907036aad307517.tif"},{"id":96651390,"identity":"d7bfe854-3009-415a-b434-4d0e0c7b53e1","added_by":"auto","created_at":"2025-11-24 16:14:28","extension":"tif","order_by":11,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":5938492,"visible":true,"origin":"","legend":"","description":"","filename":"Figure2.tif","url":"https://assets-eu.researchsquare.com/files/rs-7828854/v1/337ae4f25702e3650d447892.tif"},{"id":96651396,"identity":"d3ae4aa9-ea43-4e61-b8f5-6428f4ec7ff7","added_by":"auto","created_at":"2025-11-24 16:14:28","extension":"tif","order_by":12,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":9062564,"visible":true,"origin":"","legend":"","description":"","filename":"Figure3.tif","url":"https://assets-eu.researchsquare.com/files/rs-7828854/v1/00718feb3958de7cdbfbcaa2.tif"},{"id":96709353,"identity":"798b8662-ca34-4c57-a2fb-8465c17c1af9","added_by":"auto","created_at":"2025-11-25 10:08:48","extension":"tif","order_by":13,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":12269500,"visible":true,"origin":"","legend":"","description":"","filename":"Figure4.tif","url":"https://assets-eu.researchsquare.com/files/rs-7828854/v1/5f7ec8381ec775980f4a3a93.tif"},{"id":96651401,"identity":"e5b56602-501d-4a8e-acde-4b7d8611b4ad","added_by":"auto","created_at":"2025-11-24 16:14:28","extension":"tif","order_by":14,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":10383904,"visible":true,"origin":"","legend":"","description":"","filename":"Figure5.tif","url":"https://assets-eu.researchsquare.com/files/rs-7828854/v1/6d13fdded3219e1272b82378.tif"},{"id":96651402,"identity":"eeda2cd1-343a-4e13-8d29-80df31d4d7d7","added_by":"auto","created_at":"2025-11-24 16:14:28","extension":"png","order_by":15,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":2744157,"visible":true,"origin":"","legend":"","description":"","filename":"OnlineFigure1.png","url":"https://assets-eu.researchsquare.com/files/rs-7828854/v1/fc958dfb44b41c35a1b67b10.png"},{"id":96709680,"identity":"02acda99-a920-4064-810a-0e328eccb0ee","added_by":"auto","created_at":"2025-11-25 10:09:31","extension":"png","order_by":16,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":1677564,"visible":true,"origin":"","legend":"","description":"","filename":"OnlineFigure2.png","url":"https://assets-eu.researchsquare.com/files/rs-7828854/v1/4af1d1bd0192733e563a4e88.png"},{"id":96710664,"identity":"8e54c06e-0cb7-4b3d-b9f8-34ec9fe618ee","added_by":"auto","created_at":"2025-11-25 10:11:03","extension":"png","order_by":17,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":1091123,"visible":true,"origin":"","legend":"","description":"","filename":"OnlineFigure3.png","url":"https://assets-eu.researchsquare.com/files/rs-7828854/v1/9d426d5b6437e94168dd892e.png"},{"id":96651389,"identity":"7c3a369d-27f9-40eb-b1d4-c1f0800a7ad8","added_by":"auto","created_at":"2025-11-24 16:14:28","extension":"png","order_by":18,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":4261921,"visible":true,"origin":"","legend":"","description":"","filename":"OnlineFigure4.png","url":"https://assets-eu.researchsquare.com/files/rs-7828854/v1/a2fe67a55f76d8ad1f02a025.png"},{"id":96651399,"identity":"da844b8a-584e-4e2b-a7bd-4f5c649a8ca7","added_by":"auto","created_at":"2025-11-24 16:14:28","extension":"png","order_by":19,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":1035774,"visible":true,"origin":"","legend":"","description":"","filename":"OnlineFigure5.png","url":"https://assets-eu.researchsquare.com/files/rs-7828854/v1/65f1a9f2e363f7307184a911.png"},{"id":96709360,"identity":"e6582801-4e73-4e49-b428-3516e652fc3f","added_by":"auto","created_at":"2025-11-25 10:08:48","extension":"xml","order_by":20,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":98564,"visible":true,"origin":"","legend":"","description":"","filename":"c79393fdc3a844a7b8664793ff0532601structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7828854/v1/a2d691287715e93ee6610992.xml"},{"id":96651404,"identity":"16ee8ce1-842b-435d-9260-43f4c1078cba","added_by":"auto","created_at":"2025-11-24 16:14:29","extension":"html","order_by":21,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":109034,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7828854/v1/1bdc849cca4aa0ca4d5e7bd4.html"},{"id":96710528,"identity":"614af14d-365a-432a-905c-e03cc305253f","added_by":"auto","created_at":"2025-11-25 10:10:50","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":4730652,"visible":true,"origin":"","legend":"\u003cp\u003eZM promotes osteoblast differentiation. (A) ALP staining of candidate drugs from the plate 1022P-17. The ZM and control groups are labelled in red. (B) CCK8 assay of ZM on primary mouse osteoblasts. (C) Primary mouse osteoblasts were cultured with ZM, and the mRNA levels of osteoblast differentiation markers were detected. (D) Primary mouse osteoblasts were cultured with ZM, and the protein levels of osteoblast differentiation markers were detected. (E) Primary mouse osteoblasts were cultured with ZM, and ALP staining was performed. (F) Primary mouse osteoblasts were cultured with ZM, and alizarin red staining was performed. NS, not significant; **, p\u0026lt;0.01; ***, p\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-7828854/v1/4a6e602f0a4e46d2baf658e7.png"},{"id":96651384,"identity":"63b8c539-f415-414b-9f2e-b6c4b49305b5","added_by":"auto","created_at":"2025-11-24 16:14:28","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2740994,"visible":true,"origin":"","legend":"\u003cp\u003eZM inhibits osteoclast differentiation. (A) CCK8 assay of ZM on BMMs. (B) BMMs were cultured with ZM, and the mRNA levels of osteoclast differentiation markers were detected. (C) BMMs were cultured with ZM, and the protein levels of osteoclast differentiation markers were detected. (D) BMMs were cultured with ZM, and TRAP staining was performed. NS, not significant; ***, p\u0026lt;0.001; ****, p\u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-7828854/v1/14cc8a865a0b48661f6a0118.png"},{"id":96651380,"identity":"16d0956b-1bfd-4fb8-881f-a107555b952f","added_by":"auto","created_at":"2025-11-24 16:14:28","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2004956,"visible":true,"origin":"","legend":"\u003cp\u003eZM prevents OVX-induced bone loss in vivo. (A) Schematic representation of the experimental design involving the intraperitoneal injection of ZM into OVX mice. (B) Representative image and weights of uteri from sham and OVX mice. (C) Representative micro-CT images and histomorphometric analysis of the metaphysis of femurs from sham and OVX mice. (D) Representative micro-CT images and histomorphometric analysis of the diaphysis of femurs from sham and OVX mice. (E) Serum levels of CTX-1 in sham and OVX mice. NS, not significant; *, p\u0026lt;0.05; **, p\u0026lt;0.01; ***, p\u0026lt;0.001; ****, p\u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-7828854/v1/226554e6c591122933bacddd.png"},{"id":96651382,"identity":"6a2de292-5dc9-43b5-a9e2-021d8a624643","added_by":"auto","created_at":"2025-11-24 16:14:28","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":6555243,"visible":true,"origin":"","legend":"\u003cp\u003eZM treatment promoted osteoblastogenesis and inhibited osteoclastogenesis in OVX mice. (A) HE staining and quantitative analysis of the metaphysis of femurs from sham and OVX mice. (B) Ocn and TRAP immunostaining and quantitative analysis of the metaphysis of femurs from sham and OVX mice. *, p\u0026lt;0.05; **, p\u0026lt;0.01.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-7828854/v1/ff35420eac62edf836fc29b2.png"},{"id":96651381,"identity":"f0697d50-7258-4489-8788-d815bb8fe351","added_by":"auto","created_at":"2025-11-24 16:14:28","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1786617,"visible":true,"origin":"","legend":"\u003cp\u003eZM regulates osteoblast and osteoclast differentiation through the Wnt/β-catenin signaling pathway and NF-κB/MAPK signaling, respectively. (A) KEGG pathway enrichment analysis of differentially expressed genes in osteoblasts (B) Primary mouse osteoblasts were cultured with OIM, ZM and ICG-001, and the protein levels of osteoblast differentiation markers were detected. (C) Primary mouse osteoblasts were cultured with OIM, ZM and ICG-001, and ALP staining was performed. (D) KEGG pathway enrichment analysis of differentially expressed genes in osteoclasts. (E) Phosphorylation levels of NF-κB (p65) and MAPK (p38, JNK, ERK) proteins in BMMs treated with M-CSF/RANKL ± ZM. NS, not significant; *, p\u0026lt;0.05; **, p\u0026lt;0.01; ***, p\u0026lt;0.001; ****, p\u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-7828854/v1/d1921fb497366df933f57666.png"},{"id":101691100,"identity":"a4e9c8b6-2668-41be-a0ed-1d0724f26848","added_by":"auto","created_at":"2026-02-02 16:12:13","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":17318260,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7828854/v1/e0192706-79ab-4f7f-a772-1306690d6060.pdf"},{"id":96709449,"identity":"89047566-bd5e-494e-896b-e61e192ba5d6","added_by":"auto","created_at":"2025-11-25 10:09:02","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":17662,"visible":true,"origin":"","legend":"","description":"","filename":"TableS1.NucleotidesequencesofprimersusedforqPCR.docx","url":"https://assets-eu.researchsquare.com/files/rs-7828854/v1/bec9fba3a494d979a340074c.docx"},{"id":96651405,"identity":"4b6283b6-51ff-4c55-84d3-ac6cf49fe4f5","added_by":"auto","created_at":"2025-11-24 16:14:29","extension":"zip","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":25490301,"visible":true,"origin":"","legend":"","description":"","filename":"UncroppedWesternBlots.zip","url":"https://assets-eu.researchsquare.com/files/rs-7828854/v1/b42ef758ede873dc2dc8ec0f.zip"}],"financialInterests":"No competing interests reported.","formattedTitle":"ZM-306416 prevents ovariectomy-induced bone loss by promoting osteoblastogenesis and inhibiting osteoclastogenesis","fulltext":[{"header":"1 Introduction","content":"\u003cp\u003eAdult bone undergoes continuous remodeling to maintain bone homeostasis and strength, which involves osteoblast-mediated bone formation and osteoclast-mediated bone resorption (\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). Osteoblasts, the chief bone-making cells, arise from bone marrow mesenchymal stromal cells and secrete specific extracellular proteins (e.g., osteocalcin, alkaline phosphatase, and type I collagen) to facilitate mineralization of the extracellular matrix (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). In contrast, osteoclasts, specialized multinucleated cells derived from monocytes-macrophages under stimulation by macrophage colony-stimulating factor (M-CSF) and receptor activator of NF-κB ligand (RANKL), absorb old bone (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eWith advancing age and estrogen deficiency, an imbalance in bone remodeling occurs, leading to increased bone resorption and insufficient bone formation, culminating in osteoporosis\u0026mdash;a metabolic bone disease characterized by reduced bone mass and impaired microarchitecture, resulting in increased fracture risk (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). The prevalence of osteoporosis has been steadily increasing due to the aging population, with approximately 200\u0026nbsp;million individuals affected worldwide, thus presenting a significant healthcare challenge (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eCommon treatments for osteoporosis include bisphosphonates, RANKL antibodies, monoclonal sclerostin antibodies, and parathyroid hormone receptor agonists (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). Parathyroid hormone receptor agonists increase osteoblast activity and inhibit osteoclast recruitment, whereas other treatments target osteoclast resorption. Nonetheless, these treatments have limitations and side effects (\u003cspan additionalcitationids=\"CR11 CR12 CR13 CR14\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). For example, long-term bisphosphonate use has been associated with the development of atypical femur fractures and osteonecrosis of the jaw (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). RANKL antibodies also have side effects, such as skin eczema, flatulence, cellulite inflammation, and osteonecrosis of the jaw (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). Consequently, there is an urgent need for novel therapeutic interventions for osteoporosis. High-throughput screening of bioactive compound libraries has proven effective for identifying bone metabolism regulators. A study screened 512 small molecules and identified higenamine, which promotes osteogenesis via the IQGAP1/SMAD4 signaling pathway and prevents bone loss in murine models of osteoporosis. This work directly validates library screening as a robust tool for discovering bone-anabolic agents (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). Another study developed an aptamer-based competitive screening assay to evaluate 96 compounds (including FDA-approved drugs) and identified 6 sclerostin inhibitors that enhance osteoblastic activity. This work further confirms the reliability of library-based screening for targeting key bone regulatory molecules (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIn this study, we performed ALP staining screening on a bioactive compound library (L1022P, APExBIO) using osteoblasts. Our analysis revealed that ZM-306416 (referred to as ZM), an EGFR inhibitor, is a potential enhancer of osteoblast differentiation. Therefore, our objective was to investigate the effects of ZM on both osteoblast and osteoclast differentiation and explore its therapeutic potential for osteoporosis.\u003c/p\u003e"},{"header":"2 Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1 Mice and ovariectomy murine model\u003c/h2\u003e\u003cp\u003eC57BL/6 mice, aged 10 to 12 weeks, were obtained from the Laboratory Animal Research Centre of Southern Medical University and housed at a temperature of 20\u0026ndash;22\u0026deg;C and a humidity of 30\u0026ndash;70%, with four or five mice per cage. All animal care protocols and experiments were reviewed and approved by the Southern Medical University Laboratory Animal Ethics Committee, and this study was compliant with all relevant ethical regulations regarding animal research. The mice were divided into three groups: the OVX group (n\u0026thinsp;=\u0026thinsp;6), the OVX\u0026thinsp;+\u0026thinsp;ZM group (0.5 mg/kg, n\u0026thinsp;=\u0026thinsp;6), and the sham group (n\u0026thinsp;=\u0026thinsp;6). For anaesthesia during bilateral OVX, tribromoethanol anaesthetic (1.25% tribromoethanol solution) was used. It was prepared by weighing 2.5 g of tribromoethanol powder, dissolving it in 5 mL of tert-amyl alcohol, and diluting to 200 mL with deionized water. The solution was magnetically stirred until homogeneous, protected from light with tin foil, and stored at 4\u0026deg;C. The injection dosage was 0.3 ml/20 g body weight via intraperitoneal injection, ensuring adequate anaesthesia while minimizing toxicity. The OVX group and the OVX\u0026thinsp;+\u0026thinsp;ZM group underwent bilateral OVX after being anaesthetized with this tribromoethanol anaesthetic, whereas the sham group only had fat tissue near the ovary removed. The ZM group was injected with 0.5 mg/kg ZM peritoneally, whereas the Sham group and the OVX group were injected with corn oil twice a week for 8 weeks. In the 9th week, all the mice were euthanized by cervical dislocation (a humane and rapid method that minimally interferes with tissue analysis, consistent with ethical animal research guidelines), and the femurs were isolated after excess tissue was removed for histological and morphological evaluation, micro-CT, and microscopic examination.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2 Primary osteoblast isolation and culture\u003c/h2\u003e\u003cp\u003ePrimary osteoblasts were harvested from the calvaria of 3-day-old C57BL/6 mice as described previously (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e). Specifically, the excess soft tissue was removed, after which the calvaria was cut into small bone fragments via sterile scissors. The bone fragments were fully digested with 0.25% trypsin for 10 minutes and 0.2% type II collagenase for 90 minutes. Primary osteoblasts were then collected by gently pipetting the digested fragments. The cells were then suspended in complete α-MEM (containing 10% fetal bovine serum) and incubated at 37\u0026deg;C in a 5% CO2 incubator.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3 BMM isolation and culture\u003c/h2\u003e\u003cp\u003eBMMs were generated from the bone marrow of 6-week-old C57BL/6 male mice as described previously (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). The bone marrow cells were then obtained by flushing the bones with a 1 mL syringe. The cells were subsequently suspended in complete α-MEM supplemented with 10% fetal bovine serum and 30 ng/mL M-CSF. The cells were cultured at 37\u0026deg;C in a 5% CO2 incubator. After three days, the adherent cells were identified as bone marrow-derived macrophages.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4 CCK-8 assay\u003c/h2\u003e\u003cp\u003eThe cells were seeded into 96-well plates with a liquid volume of 200 \u0026micro;L per well and counted via a cell counting plate, resulting in approximately 8,000 cells per well. Four replicate wells were set up. The 96-well plates were then incubated overnight at 37\u0026deg;C in a 5% CO2 incubator to allow the cells to adhere. Subsequently, 20 \u0026micro;L of CCK-8 solution was added to each well. A blank control group was also established, where the same volumes of cell culture medium and CCK-8 solution were added, but no cells were present. The 96-well plates were further incubated, and the absorbance was measured at 450 nm after 0.5, 1, 2, and 4 hours via a microplate reader (BIO-TEK, Synergy HTX). On the basis of pre-experimental data, the 2-hour time point was selected for final analysis, as it yielded absorbance values within the linear range (0.8\u0026ndash;1.2 OD) for all groups, ensuring accurate quantification of viable cells without signal saturation or underdetection. The OD values for each group at this time point were recorded to calculate cell viability.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.5 ALP staining and alizarin red staining\u003c/h2\u003e\u003cp\u003eOsteoblasts were cultured in osteogenic induction medium (OIM; 50 \u0026micro;g/mL ascorbic acid and 5 mM sodium β-glycerophosphate) for approximately 7 days, and the OIM was changed every 2 days to maintain stable concentrations of osteogenic factors and minimize the accumulation of metabolic byproducts. The cells were subsequently fixed with 4% paraformaldehyde for 20 minutes. A BCIP/NBT staining working solution was prepared following the provided instructions. ALP staining was performed at room temperature to stain the cells for 10\u0026ndash;30 minutes. Finally, the 6-well plate was scanned via an EPSON Perfection V800 scanner (EPSON, Tokyo, Japan), and the resulting images were stored for subsequent analysis. After a 21-day induction period with the osteogenic induction solution, the cells were fixed with 4% paraformaldehyde for 20 minutes and incubated with a 1% alizarin red staining solution, which was prepared according to the instructions. The cells were stained with alizarin red solution for approximately 5\u0026ndash;10 minutes at 37\u0026deg;C. The 6-well plate was then scanned via a scanner, and the resulting images were stored for analysis.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e2.6 TRAP staining\u003c/h2\u003e\u003cp\u003eBone marrow-derived macrophages were induced with complete α-MEM (containing 10% FBS) supplemented with 30 ng/mL M-CSF and 100 ng/mL RANKL. After 7 days of induction, multinucleated osteoclasts were observed under a microscope. To perform TRAP staining, the staining solution was prepared following the provided instructions. The cells were then incubated with TRAP staining solution at 37\u0026deg;C for 30\u0026ndash;60 minutes in the dark. The cells were subsequently washed 1\u0026ndash;2 times with deionized water. Finally, the cells were observed, and pictures were taken under a microscope. Osteoclasts were identified as TRAP-positive cells with more than 3 nuclei. The ability to form osteoclasts was determined by analysing the number of TRAP-positive cells in each group.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e2.7 Western blotting (WB)\u003c/h2\u003e\u003cp\u003eThe cells were seeded in 6-well plates at a density of 4 \u0026times; 10⁵ cells per well and cultured overnight. After treatment, the cells were lysed on ice for 10 minutes in RIPA lysis buffer containing protease inhibitors to extract total protein. The total protein was separated by SDS‒PAGE and transferred to polyvinylidene fluoride (PVDF) membranes. Following blocking with skim milk for 1 hour, the membranes were incubated overnight at 4\u0026deg;C with the following primary antibodies: anti-OSX (1:1000, BOSTER, China); anti-RUNX2, anti-CTSK, and anti-NFATC1 (1:200, Santa Cruz, USA); anti-Tubulin (1:5000, Affinity, China); anti-ERK, anti-p-ERK, anti-P38, anti-P-P38, anti-P65, anti-P-P65, anti-JNK, and anti-P-JNK (1:1000, Cell Signaling, USA); anti-β-actin (1:20000, Proteintech, China); and anti-β-catenin (1:1000, Abmart, China). The membranes were subsequently washed three times with TBST (5 minutes each) and incubated with specific secondary antibodies: goat anti-mouse IgG H\u0026amp;L (HRP) pAb (1:20000, PTM Bio, China) and goat anti-rabbit IgG H\u0026amp;L (HRP) pAb (1:10000, PTM Bio, China) for 1 hour at room temperature. After three additional washes with TBST, the membranes were incubated with chemiluminescent substrate (Abkine, Cat. No. BMP3010) and visualized via the GeneSys capture system.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e2.8 Real-time quantitative PCR (qPCR)\u003c/h2\u003e\u003cp\u003eTotal RNA was isolated from the cells via TRIzol Reagent (Cat. No. 9109; Takara Biotechnology) following the manufacturer's protocol. The reverse transcription reaction system (Cat. RR036A, Takara Biotechnology) was then prepared according to the manufacturer's instructions. The system was subsequently placed into the reverse transcription PCR instrument to generate complementary DNA (cDNA). The obtained cDNA was used for qPCR with ChamQ SYBR qPCR Master Mix (Cat. No. Q311-02, Vazyme Biotech) in a LightCycler96 instrument (Roche Molecular Biochemicals). The relative quantification of gene expression was performed via the comparative threshold method. Changes in mRNA expression levels were calculated after normalization of the values to those of the GAPDH housekeeping gene. The primers used are listed in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e2.9 Immunohistochemistry\u003c/h2\u003e\u003cp\u003eThe femurs were separated from the mice and fixed in 4% paraformaldehyde for 24 hours. After decalcification in 10% EDTA (pH 8.0) for 21 days, the decalcified tissues were embedded in paraffin and prepared as 4 \u0026micro;m sagittal-oriented sections for histological analysis. The paraffin sections were then incubated at 65\u0026deg;C for 2 hours. After the paraffin sections were dewaxed and hydrated, antigen retrieval was performed. Next, the paraffin sections were incubated overnight at 4\u0026deg;C with a primary antibody against osteocalcin (OCN), followed by incubation with secondary antibodies for 1 hour at room temperature. Finally, diaminobenzidine (DAB) was used to visualize the positive cells, and the samples were observed and photographed via an Olympus BX51 microscope. Immunohistochemical staining was evaluated by the number of positive cells per bone perimeter (B.Pm).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e2.10 Micro-CT analysis\u003c/h2\u003e\u003cp\u003eMicro-CT analysis was conducted using a CT40 scanner (Scanco Medical AG, Bassersorf, Switzerland) to examine the femoral structure. The scanning conditions were set to 55 kV, 145 \u0026micro;A, and a voxel size of 10 \u0026micro;m (optimal for trabecular bone resolution in murine femurs). Image segmentation was performed using a threshold range of 220\u0026ndash;1000 HU, which effectively distinguishes mineralized bone tissue from soft tissue and marrow in mouse femurs. For the analysis of bone trabeculae, 200 section planes were scanned from the distal femoral growth plate towards the proximal end. The analysis of cortical bone involved scanning from the middle of the femur towards the proximal end. The parameters analysed included the trabecular bone density (Tb. BMD, mg HA/ccm), bone trabecular volume fraction (BV/TV, %), bone trabecular number (Tb. N, mm⁻\u0026sup1;), bone trabecular thickness (Tb. Th, mm), bone trabecular separation/porosity (Tb. Sp, mm), cortical bone density (Ct. BMD, mg HA/ccm), and cortical bone thickness (Ct. Th, mm).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003e2.11 ELISA\u003c/h2\u003e\u003cp\u003eBlood was collected from the mice via eyeball extraction and left at room temperature for 2 hours. After centrifugation at 1000 \u0026times; g for 20 minutes, the supernatant was collected for serum preparation. The serum was stored at -80\u0026deg;C. The serum sample and the CTX-1 ELISA kit were subsequently brought to room temperature for equilibration. Next, the procedures to measure the levels of CTX-1 in the serum of each group were carried out according to the instructions provided with the ELISA kits.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003e2.12 Hematoxylin and eosin (HE) staining\u003c/h2\u003e\u003cp\u003eThe paraffin-embedded sections were stained with HE. The sections were observed and photographed using an Olympus BX51 microscope. Subsequently, histological analysis was conducted via ImageJ software.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003e2.13 RNA sequencing and pathway analysis\u003c/h2\u003e\u003cp\u003ePrimary osteoblasts were treated with or without 10 \u0026micro;M ZM in osteogenic induction medium for 6 days, and the cells were divided into a control group and an intervention group. Concomitantly, bone marrow-derived macrophages were treated with or without 10 \u0026micro;M ZM in complete α-MEM (containing 10% FBS) supplemented with 30 ng/mL M-CSF and 100 ng/mL RANKL for 2 days, and the cells were also divided into a control group and an intervention group. Total RNA was extracted via TRIzol Reagent (Cat. No. 9109; Takara Biotechnology) following the manufacturer\u0026rsquo;s instructions, and three samples per group were used. The RNA concentration and purity were quantified via a UV spectrophotometer (NanoDropOne, Thermo Fisher Scientific, USA). After RNA extraction, the raw sequencing reads were filtered via Fastp to remove low-quality and adapter-containing reads. Clean reads were aligned to the mouse reference genome (mm10) with HISAT2. Gene read counts were generated by FeatureCounts. Differentially expressed genes (DEGs) were identified via DESeq2, with the criteria of |log₂-fold change (FC)| \u0026gt;1 and adjusted P value\u0026thinsp;\u0026lt;\u0026thinsp;0.05. The Kyoto Encyclopedia of Genes and Genomes (KEGG; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.genome.ad.jp/kegg\u003c/span\u003e\u003cspan address=\"http://www.genome.ad.jp/kegg\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) was employed for pathway analysis via enriched R packages. The terms or pathways were mapped and visualized by selecting the top 20 according to the number of genes for which they were enriched. The original data were uploaded to the GEO database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/\u003c/span\u003e\u003cspan address=\"https://www.ncbi.nlm.nih.gov/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e, GSE307058, GSE307904).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003e2.14 Key Reagents\u003c/h2\u003e\u003cp\u003eZM-306416 (ZM) was purchased from APExBIO Technology LLC (Houston, USA; Catalogue No. A8684; CAS No. 690206-97-4). The compound has a purity\u0026thinsp;\u0026ge;\u0026thinsp;95% (verified by HPLC, as confirmed by the batch-specific certificate of analysis). ZM was dissolved in DMSO to prepare a 10 mM stock solution (solubility\u0026thinsp;\u0026ge;\u0026thinsp;33.4 mg/mL in DMSO, insoluble in water) and stored at -20\u0026deg;C according to the manufacturer\u0026rsquo;s recommendations. ICG-001 was purchased from Selleck Chemicals (Catalogue No. S2662). It was dissolved in DMSO to prepare a 10 mM stock solution and stored at -20\u0026deg;C. For in vitro experiments, a working concentration of 10 \u0026micro;M was used, as determined on the basis of references indicating that this concentration effectively inhibits Wnt/β-catenin signaling (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003e2.15 Drug Library Screening\u003c/h2\u003e\u003cp\u003ePrimary osteoblasts were isolated from the calvaria of 3-day-old C57BL/6 mice and seeded in 24-well plates at a density of 5 \u0026times; 10⁴ cells per well. Following 24 hours of adherence in α-MEM supplemented with 10% fetal bovine serum (FBS), the cells were treated with compounds from the DiscoveryProbe\u0026trade; Bioactive Compound Library Plus (Cat. No. L1022P, APExBIO, USA). The compounds were diluted in osteogenic induction medium (OIM) to a final concentration of 10 \u0026micro;M. Each compound was administered in triplicate (n\u0026thinsp;=\u0026thinsp;3). The cells were cultured in OIM for 7 days, and the medium was changed every 48 hours. Alkaline phosphatase (ALP) staining was performed via BCIP/NBT kits according to the manufacturer\u0026rsquo;s instructions. The staining intensity was semiquantified via ImageJ software, and the compounds were evaluated relative to 0.1% dimethyl sulfoxide (DMSO) vehicle controls.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003e2.16 Statistical analysis\u003c/h2\u003e\u003cp\u003eThe experiments were conducted a minimum of three times, and the results are presented as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM. Data analysis was performed via GraphPad Prism 10.1.2. Student's t- test was used to compare two groups, whereas one-way analysis of variance (ANOVA) followed by Tukey\u0026rsquo;s multiple comparison test was used to compare differences between three or more groups. A p value of less than 0.05 was considered statistically significant for all the experiments.\u003c/p\u003e\u003c/div\u003e"},{"header":"3 Results","content":"\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003e3.1 ZM promotes osteoblast differentiation\u003c/h2\u003e\u003cp\u003eIn a drug screening assay using a bioactive compound library (L1022P, APExBIO), our results demonstrated that ZM significantly enhanced alkaline phosphatase (ALP) activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). To verify the role of ZM in osteoblast differentiation, primary mouse osteoblasts were treated with varying concentrations of ZM, and the differentiation phenotypes were analysed. Initially, a CCK8 assay was conducted to detect the effect of ZM on osteoblast proliferation. The findings indicated that concentrations ranging from 1.25 to 10 \u0026micro;M had no significant effect on osteoblast proliferation (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). A concentration of 10 \u0026micro;M was chosen for subsequent cell experiments. Following 7 days of osteogenic induction, the expression of the osteoblast differentiation markers \u003cem\u003eRunx2\u003c/em\u003e, \u003cem\u003eAlpl\u003c/em\u003e, and \u003cem\u003eBglap\u003c/em\u003e was assessed. We found that ZM markedly upregulated these osteogenic differentiation marker genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). Consistent with the mRNA findings, the protein levels of OSX and RUNX2 were also significantly upregulated (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). On the 7th day of osteogenic induction, ALP staining was subsequently performed, followed by alizarin red staining on the 14th day. The results demonstrated that ZM increased alkaline phosphatase activity and calcium deposition in primary osteoblasts (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF). Overall, these findings highlight that ZM effectively promotes osteoblast differentiation in vitro without affecting cell growth.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\u003ch2\u003e3.2 ZM inhibits osteoclast differentiation\u003c/h2\u003e\u003cp\u003eConsidering the coupling role of osteoblasts and osteoclasts in bone remodeling and the development of osteoporosis, we aimed to examine whether ZM could inhibit osteoclast differentiation. We initially assessed the effect of ZM on BMM proliferation and found no significant effect (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Following a 3-day period of M-CSF and RANKL stimulation, we evaluated the mRNA and protein levels of osteoclast differentiation markers. Notably, ZM inhibited both the mRNA and protein levels of these markers in a concentration-dependent manner (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). M-CSF and RANKL induced the formation of numerous TRAP-positive multinucleated osteoclasts from BMMs (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). However, ZM at a concentration of 10 \u0026micro;M inhibited M-CSF and RANKL-induced osteoclastogenesis almost completely (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). Collectively, these findings demonstrate the in vitro inhibitory effect of ZM on osteoclast differentiation.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\u003ch2\u003e3.3 ZM prevents OVX-induced bone loss in vivo\u003c/h2\u003e\u003cp\u003eTo investigate the potential of ZM in restoring bone loss in vivo, we conducted a study to determine whether it could prevent the established bone loss caused by OVX in mice. We observed that ovariectomy-induced estrogen deprivation resulted in a decrease in the size and weight of the mouse uterus (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Since estrogen deficiency causes an imbalance in bone formation and bone resorption in postmenopausal women, leading to bone loss and osteoporosis, and our results indicate that ZM stimulates osteogenesis and inhibits osteoclastogenesis, we hypothesized that ZM treatment could prevent estrogen deficiency-induced bone loss. For this purpose, OVX mice were intraperitoneally injected with ZM (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Micro-CT analysis of the distal femoral metaphysis and diaphysis revealed that OVX mice presented noticeable loss of trabecular and cortical bone (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). We found that bone mineral density (BMD), bone volume per tissue volume (BV/TV), the trabecular number (Tb.N), the trabecular thickness (Tb.Th), and the trabecular bone area were significantly reduced in mice 8 weeks after OVX (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD, and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Conversely, trabecular spacing (Tb.Sp) and the number of TRAP-positive osteoclasts were significantly increased, indicating severe bone loss in the femur as a result of OVX (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). The serum levels of C-terminal telopeptide of collagen type I (CTX-1), a marker for bone resorption, were significantly greater in OVX mice than in control mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). These results indicated severe bone loss in the female mice as a result of OVX. To assess the therapeutic potential of ZM, we administered either vehicle or ZM (0.5 mg/kg) intraperitoneally to OVX mice twice a week for 8 weeks. We subsequently conducted micro-CT analysis and histological staining of femurs from these mice. Compared with vehicle-treated OVX mice, OVX mice treated with ZM presented substantial bone restoration and significantly greater BMD, BV/TV, Tb.N, Tb.Th and trabecular bone area (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD, and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Histomorphometric analysis further revealed that the number of osteoblasts (N.Ocn/B. Pm) was increased in ZM-treated OVX mice, whereas the osteoclast surface was significantly smaller and the number of osteoclasts (N.Oc/B. Pm) was lower than that in vehicle-treated OVX mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Taken together, our results suggest that ZM treatment is a potential therapeutic approach for postmenopausal bone loss.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec23\" class=\"Section2\"\u003e\u003ch2\u003e3.4 ZM regulates osteoblast and osteoclast differentiation through the Wnt/β-catenin signaling pathway and NF-κB/MAPK signaling, respectively.\u003c/h2\u003e\u003cp\u003eTo elucidate the molecular mechanisms by which ZM modulates osteoblast and osteoclast differentiation, we performed transcriptomic and biochemical analyses. KEGG pathway enrichment analysis of differentially expressed genes (DEGs) in osteoblasts revealed significant enrichment of the Wnt signaling pathway, indicating that this pathway plays a critical role in ZM-mediated regulation of osteoblastogenesis (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Numerous studies have confirmed that canonical activation of the Wnt/β-catenin pathway is essential for osteoblastogenesis and bone formation (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e). To test the hypothesis that ZM promotes osteogenesis by activating the Wnt/β-catenin pathway, we examined the protein levels of β-catenin (a key mediator of canonical Wnt signaling) and RUNX2 (a downstream transcription factor indicative of osteogenic commitment) in osteoblasts treated with ZM (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e). Western blot analysis revealed significant upregulation of both β-catenin and RUNX2 in ZM-treated osteoblasts compared with OIM-treated controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). To further validate the involvement of the Wnt/β-catenin signaling pathway, we co-treated primary osteoblasts with ZM and ICG-001, a specific inhibitor of the canonical Wnt/β-catenin pathway, in OIM, ensuring that the experiments were conducted under conditions that mimic the osteogenic microenvironment (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e). As expected, ICG-001 significantly attenuated the ZM-induced upregulation of β-catenin and RUNX2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB), confirming that the osteogenic effect of ZM is mediated through canonical Wnt/β-catenin activation. This effect was further validated by ALP staining, where ZM-treated osteoblasts presented stronger ALP activity (darker staining) than OIM-treated controls did, whereas co-treatment with ICG-001 diminished this enhancement (lighter staining), confirming that the osteogenic effect of ZM relies on canonical Wnt/β-catenin activation (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eFor osteoclasts, we performed parallel KEGG pathway analysis to identify signaling pathways involved in ZM-mediated regulation. This analysis revealed significant enrichment in the \"osteoclast differentiation\" pathway (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD), a pathway well- established to be tightly regulated by the NF-κB and MAPK signaling cascades. These cascades are recognized as core mediators of osteoclastogenesis downstream of RANKL-RANK activation (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e), which aligns with our focus on deciphering how ZM inhibits RANKL-induced osteoclast differentiation and prompting us to investigate their involvement. Thus, we examined the phosphorylation levels of NF-κB (p65) and MAPK family members (p38, JNK, and ERK), which are crucial and fundamental downstream of RANKL\u0026ndash;RANK signaling (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e). As expected, co-stimulation with M-CSF and RANKL triggered rapid phosphorylation of NF-κB (p65) and MAPKs (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE). Notably, treatment of BMMs with ZM significantly suppressed M-CSF and RANKL-induced phosphorylation of p65, p38, ERK, and JNK (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE), indicating that ZM targets these signaling cascades to exert its inhibitory effect on osteoclast differentiation.\u003c/p\u003e\u003cp\u003eTaken together, these findings demonstrate that ZM promotes osteoblast differentiation by activating the canonical Wnt/β-catenin pathway and inhibits osteoclastogenesis by suppressing NF-κB/MAPK signaling, supporting our conclusion that ZM regulates bone remodeling via these two distinct pathways.\u003c/p\u003e\u003c/div\u003e"},{"header":"4 Discussion","content":"\u003cp\u003eIn this study, we performed high-throughput drug library screening and identified ZM as a potential candidate that promotes osteoblast differentiation. Notably, ZM has dual effects: it not only enhances osteoblast differentiation but also inhibits osteoclast differentiation. Consistent with these in vitro findings, ZM was further shown to prevent OVX-induced bone loss in vivo, highlighting its potential to regulate bone remodeling bidirectionally. This identification of ZM relied on high-throughput drug library screening, which laid the foundation for our subsequent mechanistic studies.\u003c/p\u003e\u003cp\u003eHigh-throughput drug library screening is a valuable approach in drug discovery and development. It offers several advantages, including rapid screening of a large number of compounds, which significantly reduces the time compared with traditional methods. This enables researchers to rapidly identify potential drug candidates, explore new targets, and discover drugs for previously unexplored therapeutic areas. However, high-throughput screening requires substantial manpower and material resources. Despite these limitations, it remains a valuable tool in the early stages of drug discovery when used in conjunction with other approaches. After confirming the initial activity of ZM by screening, we further explored its molecular mechanisms through in vitro experiments.\u003c/p\u003e\u003cp\u003eThrough in vitro experiments, we demonstrated that ZM activates the Wnt/β-catenin signaling pathway, as evidenced by KEGG enrichment of Wnt signaling-related genes in ZM-treated osteoblasts, alongside upregulation of β-catenin (a central mediator of canonical Wnt signaling) and RUNX2 (a master transcription factor driving osteogenic commitment). Furthermore, the use of ICG-001, a specific inhibitor of the Wnt/β-catenin signaling pathway, inhibited osteoblast differentiation, confirming that activation of the Wnt/β-catenin signaling pathway is indispensable for regulating osteoblast differentiation. Functional validation via ALP staining further confirmed that ZM enhances osteogenic commitment through this cascade. Moreover, ZM inhibits osteoclast differentiation by targeting nuclear factor κB (NF-κB) and mitogen-activated protein kinase (MAPK) signaling. KEGG analysis revealed significant enrichment in the \u0026ldquo;osteoclast differentiation\u0026rdquo; pathway in ZM-treated osteoclast precursors, a pathway tightly regulated by the NF-κB and MAPK cascades downstream of RANKL-RANK activation. We confirmed that ZM suppresses the RANKL-induced phosphorylation of p65 (NF-κB) and MAPKs (p38, JNK, and ERK), which aligns with their known role as core mediators of osteoclastogenesis (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eTo contextualize the dual effects of ZM, we compared it with those of clinically approved dual-acting anti-osteoporosis agents. For example, romosozumab, a sclerostin antibody, promotes bone formation via Wnt pathway activation and inhibits resorption through downstream effects. In contrast, ZM modulates both the Wnt/β-catenin pathway (to increase osteogenesis) and the NF-κB/MAPK pathway (to suppress osteoclastogenesis) directly, suggesting a distinct mechanistic profile that may confer unique therapeutic advantages. This distinct mechanism of action of ZM addresses a longstanding gap in current osteoporosis treatment, as most existing drugs lack dual effects on bone formation and resorption.\u003c/p\u003e\u003cp\u003eFor several years, the medical community has been conducting research on therapies that aim to increase bone mass by stimulating new bone formation while also inhibiting bone resorption. However, most drugs currently used to treat osteoporosis focus only on inhibiting bone resorption and lack the dual effects of stimulating bone formation (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e). Our findings present a potential promising candidate for the development of anti-osteoporosis drugs. The results of our study suggest that ZM may be an effective therapeutic agent for osteoporosis by simultaneously inhibiting bone resorption and promoting bone formation.\u003c/p\u003e\u003cp\u003eHowever, this study has limitations that should be acknowledged. First, we did not assess the long-term toxicity of ZM, which is critical for evaluating its translational potential. Second, we only tested ZM in an OVX mouse model and did not evaluate its efficacy in aged mouse models or models of glucocorticoid-induced osteoporosis, which are scenarios that are highly relevant to clinical osteoporosis presentations.\u003c/p\u003e"},{"header":"5 Conclusion","content":"\u003cp\u003eOur study revealed that ZM has a dual anti-osteoporosis effect by promoting osteoblastogenesis and inhibiting osteoclastogenesis via the activation of Wnt/β-catenin signaling and suppression of NF-κB/MAPK cascades. These findings suggest that ZM could be a promising therapeutic agent for alleviating osteoporosis.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003e6\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eData availability statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe raw RNA-seq data were deposited in the NCBI Gene Expression Omnibus (GEO) database under accession numbers GSE307058 and GSE307904. Further inquiries regarding the data can be directed to the corresponding author.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e7\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eEthics statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll animal care protocols and experiments were reviewed and approved by the Southern Medical University Laboratory Animal Ethics Committee (Approval No. LAEC-21-123), and this study was compliant with all relevant ethical regulations regarding animal research.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e8\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eYL: Data curation, Formal analysis, Investigation, Writing \u0026ndash; original draft. SS: Data curation, Formal analysis, Resources, Writing \u0026ndash; original draft. DY: Formal analysis, Writing \u0026ndash; review and editing. LZ: Investigation, Writing \u0026ndash; review and editing. TC: Funding acquisition, Writing \u0026ndash; review and editing. KX: Software, Methodology, Writing \u0026ndash; original draft. WL: Data curation, Writing \u0026ndash; original draft. JC: Funding acquisition, Project administration, Resources, Writing \u0026ndash;resources, writing, review and editing. JC: Conceptualization, Funding acquisition, Project administration, Writing \u0026ndash; review and editing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e9\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the general project of the Anhui Province Outstanding Young Talents Support Program for Universities (Grant No. gxyq2022009); the Anhui Institute of Translational Medicine (Grant No. 2022zhyx-C90); the Medical Scientific Research Foundation of Guangdong Province, China (Grant No. B2025168); and the Longhua District Medical and Health Institution 2025 District-Level Scientific Research Project (Grant No. 2025036).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e10\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe especially appreciate the support of Wenquan Liang and Tianyu Chen and, of course, all the participants.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e11\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;Conflicts of interest\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 potential conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e12\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;Consent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBolamperti S, Villa I, Rubinacci A. Bone remodeling: an operational process ensuring survival and bone mechanical competence. Bone research. 2022;10(1):48.\u003c/li\u003e\n\u003cli\u003eSalhotra A, Shah HN, Levi B, Longaker MT. Mechanisms of bone development and repair. Nature reviews Molecular cell biology. 2020;21(11):696-711.\u003c/li\u003e\n\u003cli\u003eZaidi M. Skeletal remodeling in health and disease. Nature Medicine. 2007;13(7):791-801.\u003c/li\u003e\n\u003cli\u003eLong F. Building strong bones: molecular regulation of the osteoblast lineage. Nature Reviews Molecular Cell Biology. 2012;13(1):27-38.\u003c/li\u003e\n\u003cli\u003eHenriksen K, Bollerslev J, Everts V, Karsdal MA. Osteoclast Activity and Subtypes as a Function of Physiology and Pathology\u0026mdash;Implications for Future Treatments of Osteoporosis. Endocrine Reviews. 2011;32(1):31-63.\u003c/li\u003e\n\u003cli\u003eEnsrud KE, Crandall CJ. Osteoporosis. Annals of internal medicine. 2024;177(1):Itc1-itc16.\u003c/li\u003e\n\u003cli\u003eKanis JA, Cooper C, Rizzoli R, Reginster JY. European guidance for the diagnosis and management of osteoporosis in postmenopausal women. Osteoporosis international : a journal established as result of cooperation between the European Foundation for Osteoporosis and the National Osteoporosis Foundation of the USA. 2019;30(1):3-44.\u003c/li\u003e\n\u003cli\u003eReginster J-Y, Burlet N. Osteoporosis: A still increasing prevalence. Bone. 2006;38(2, Supplement 1):4-9.\u003c/li\u003e\n\u003cli\u003eCompston JE, McClung MR, Leslie WD. Osteoporosis. Lancet (London, England). 2019;393(10169):364-76.\u003c/li\u003e\n\u003cli\u003eCurry SJ, Krist AH, Owens DK, Barry MJ, Caughey AB, Davidson KW, et al. Screening for Osteoporosis to Prevent Fractures US Preventive Services Task Force Recommendation Statement. JAMA-JOURNAL OF THE AMERICAN MEDICAL ASSOCIATION. 2018;319(24):2521-31.\u003c/li\u003e\n\u003cli\u003eKhosla S, Hofbauer LC. Osteoporosis treatment: recent developments and ongoing challenges. The lancet Diabetes \u0026amp; endocrinology. 2017;5(11):898-907.\u003c/li\u003e\n\u003cli\u003eMiller PD, Hattersley G, Riis BJ, Williams GC, Lau E, Russo LA, et al. Effect of Abaloparatide vs Placebo on New Vertebral Fractures in Postmenopausal Women With Osteoporosis A Randomized Clinical Trial. JAMA-JOURNAL OF THE AMERICAN MEDICAL ASSOCIATION. 2016;316(7):722-33.\u003c/li\u003e\n\u003cli\u003ePeugh J, Khalil A, Chan MR, Hansen KE. Teriparatide Treatment for Hypercalcemia Associated With Adynamic Bone Disease. JBMR plus. 2019;3(7):e10176-e.\u003c/li\u003e\n\u003cli\u003eNicholson WK, Silverstein M, Wong JB, Chelmow D, Coker TR, Davis EM, et al. Screening for Osteoporosis to Prevent Fractures US Preventive Services Task Force Recommendation Statement. JAMA-JOURNAL OF THE AMERICAN MEDICAL ASSOCIATION. 2025;333(6):498-508.\u003c/li\u003e\n\u003cli\u003eWang M, Wu Y-F, Girgis CM. Bisphosphonate Drug Holidays: Evidence From Clinical Trials and Real-World Studies. JBMR plus. 2022;6(6):e10629-e.\u003c/li\u003e\n\u003cli\u003eZhang N, Zhang ZK, Yu Y, Zhuo Z, Zhang G, Zhang BT. Pros and Cons of Denosumab Treatment for Osteoporosis and Implication for RANKL Aptamer Therapy. Frontiers in cell and developmental biology. 2020;8:325.\u003c/li\u003e\n\u003cli\u003eDong H, Liu R, Zou K, Jin Z, Kang J, Zhang Y, et al. Higenamine Promotes Osteogenesis Via IQGAP1/SMAD4 Signaling Pathway and Prevents Age- and Estrogen-Dependent Bone Loss in Mice. Journal of Bone and Mineral Research. 2023;38(5):775-91.\u003c/li\u003e\n\u003cli\u003eLee CC, Hung CM, Chen CH, Hsu YC, Huang YP, Huang TB, et al. Novel Aptamer-Based Small-Molecule Drug Screening Assay to Identify Potential Sclerostin Inhibitors against Osteoporosis. International journal of molecular sciences. 2021;22(15).\u003c/li\u003e\n\u003cli\u003eLiu Z, Liang W, Kang D, Chen Q, Ouyang Z, Yan H, et al. Increased Osteoblastic Cxcl9 Contributes to the Uncoupled Bone Formation and Resorption in Postmenopausal Osteoporosis. Clinical interventions in aging. 2020;15:1201-12.\u003c/li\u003e\n\u003cli\u003eLiang W, Chen Q, Cheng S, Wei R, Li Y, Yao C, et al. Skin chronological aging drives age-related bone loss via secretion of cystatin-A. Nature Aging. 2022;2(10):906-22.\u003c/li\u003e\n\u003cli\u003ePing Z, Hu X, Wang L, Shi J, Tao Y, Wu X, et al. Melatonin attenuates titanium particle-induced osteolysis via activation of Wnt/\u0026beta;-catenin signaling pathway. Acta Biomaterialia. 2017;51:513-25.\u003c/li\u003e\n\u003cli\u003eHu L, Chen W, Qian A, Li YP. Wnt/\u0026beta;-catenin signaling components and mechanisms in bone formation, homeostasis, and disease. Bone research. 2024;12(1):39.\u003c/li\u003e\n\u003cli\u003eClevers H, Nusse R. Wnt/\u0026beta;-Catenin Signaling and Disease. Cell. 2012;149(6):1192-205.\u003c/li\u003e\n\u003cli\u003eZhu S, Chen W, Masson A, Li YP. Cell signaling and transcriptional regulation of osteoblast lineage commitment, differentiation, bone formation, and homeostasis. Cell discovery. 2024;10(1):71.\u003c/li\u003e\n\u003cli\u003eMcMillan M, Kahn M. Investigating Wnt signaling: a chemogenomic safari. Drug discovery today. 2005;10(21):1467-74.\u003c/li\u003e\n\u003cli\u003eYang MH, Basappa B, Deveshegowda SN, Ravish A, Mohan A, Nagaraja O, et al. A novel drug prejudice scaffold-imidazopyridine-conjugate can promote cell death in a colorectal cancer model by binding to \u0026beta;-catenin and suppressing the Wnt signaling pathway. Journal of advanced research. 2025;72:615-32.\u003c/li\u003e\n\u003cli\u003eArnst J, Jing Z, Cohen C, Ha SW, Viggeswarapu M, Beck GR, Jr. Bioactive silica nanoparticles target autophagy, NF-\u0026kappa;B, and MAPK pathways to inhibit osteoclastogenesis. Biomaterials. 2023;301:122238.\u003c/li\u003e\n\u003cli\u003ePark JH, Lee NK, Lee SY. Current Understanding of RANK Signaling in Osteoclast Differentiation and Maturation. Molecules and cells. 2017;40(10):706-13.\u003c/li\u003e\n\u003cli\u003eCheng HM, Xing M, Zhou YP, Zhang W, Liu Z, Li L, et al. HSP90\u0026beta; promotes osteoclastogenesis by dual-activation of cholesterol synthesis and NF-\u0026kappa;B signaling. Cell death and differentiation. 2023;30(3):673-86.\u003c/li\u003e\n\u003cli\u003eLee DK, Jin X, Choi PR, Cui Y, Che X, Lee S, et al. Phospholipase C \u0026beta;4 promotes RANKL-dependent osteoclastogenesis by interacting with MKK3 and p38 MAPK. Experimental \u0026amp; molecular medicine. 2025;57(2):323-34.\u003c/li\u003e\n\u003cli\u003eRoux C, Briot K. Addressing the crisis in the treatment of osteoporosis. Nature Reviews Rheumatology. 2018;14(2):67-8.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"journal-of-orthopaedic-surgery-and-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"josr","sideBox":"Learn more about [Journal of Orthopaedic Surgery and Research](http://josr-online.biomedcentral.com)","snPcode":"13018","submissionUrl":"https://submission.nature.com/new-submission/13018/3","title":"Journal of Orthopaedic Surgery and Research","twitterHandle":"@MSKmedBMC","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"ZM-306416, drug library screening, osteoporosis, osteoblast, osteoclast","lastPublishedDoi":"10.21203/rs.3.rs-7828854/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7828854/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e\u003cp\u003eOsteoporosis affects millions of people worldwide, and current medications such as bisphosphonates and denosumab are not effective enough to reverse bone loss. Moreover, these treatments have drawbacks, including jaw osteonecrosis and skin eczema. Hence, there is an urgent need for new drugs to treat osteoporosis.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e\u003cp\u003eDrug library screening was performed via alkaline phosphatase (ALP) staining in osteoblasts to identify potential candidates for osteoporosis treatment. qPCR, Western blotting, ALP staining, alizarin red staining, and tartrate-resistant acid phosphatase (TRAP) staining were conducted to assess the impact of ZM-306416 (ZM) on osteoblast and osteoclast differentiation in vitro. Additionally, RNA sequencing and pathway analysis were carried out to explore the underlying molecular mechanisms involved. Micro-CT scanning and immunostaining were used to determine bone phenotypes in vivo.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eDrug library screening revealed that ZM enhances ALP activity in osteoblasts, indicating its potential as a pro-osteogenic agent. ZM exerts dual effects by promoting osteoblast differentiation through the Wnt/β-catenin signaling pathway and simultaneously inhibiting osteoclast differentiation through the NF-κB and MAPK signaling pathways. In an OVX mouse model, ZM effectively prevents bone loss by stimulating osteoblast formation and inhibiting osteoclast development.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e\u003cp\u003eOur study revealed that ZM has a dual anti-osteoporosis effect by promoting osteoblastogenesis and inhibiting osteoclastogenesis, which is mediated by activation of the Wnt/β-catenin signaling pathway and suppression of the NF-κB/MAPK cascades. These findings suggest that ZM could be a promising therapeutic agent for alleviating osteoporosis.\u003c/p\u003e","manuscriptTitle":"ZM-306416 prevents ovariectomy-induced bone loss by promoting osteoblastogenesis and inhibiting osteoclastogenesis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-24 16:14:12","doi":"10.21203/rs.3.rs-7828854/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-12-14T11:49:26+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-09T23:09:15+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"249622642872768478064608145522056605765","date":"2025-12-08T15:51:02+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"270351752830927710485152810451838817100","date":"2025-11-20T02:51:50+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-11-13T09:49:12+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-10-23T10:20:22+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-10-22T12:09:46+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Orthopaedic Surgery and Research","date":"2025-10-17T11:40:18+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-orthopaedic-surgery-and-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"josr","sideBox":"Learn more about [Journal of Orthopaedic Surgery and Research](http://josr-online.biomedcentral.com)","snPcode":"13018","submissionUrl":"https://submission.nature.com/new-submission/13018/3","title":"Journal of Orthopaedic Surgery and Research","twitterHandle":"@MSKmedBMC","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"d7331530-7806-438c-b4d6-cc56e803817d","owner":[],"postedDate":"November 24th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-02-02T16:09:08+00:00","versionOfRecord":{"articleIdentity":"rs-7828854","link":"https://doi.org/10.1186/s13018-025-06654-7","journal":{"identity":"journal-of-orthopaedic-surgery-and-research","isVorOnly":false,"title":"Journal of Orthopaedic Surgery and Research"},"publishedOn":"2026-01-31 15:58:29","publishedOnDateReadable":"January 31st, 2026"},"versionCreatedAt":"2025-11-24 16:14:12","video":"","vorDoi":"10.1186/s13018-025-06654-7","vorDoiUrl":"https://doi.org/10.1186/s13018-025-06654-7","workflowStages":[]},"version":"v1","identity":"rs-7828854","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7828854","identity":"rs-7828854","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}