Mechanistic Role of the IL-1β/c-Fos/NFATc1 Signaling Axis in Echinococcal Infection-Elicited Osteoclastogenesis and Pathological Osteolysis: A Prospective Controlled Trial

Mechanistic Role of the IL-1β/c-Fos/NFATc1 Signaling Axis in Echinococcal Infection-Elicited Osteoclastogenesis and Pathological Osteolysis: A Prospective Controlled Trial | 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 Mechanistic Role of the IL-1β/c-Fos/NFATc1 Signaling Axis in Echinococcal Infection-Elicited Osteoclastogenesis and Pathological Osteolysis: A Prospective Controlled Trial Yelinaer Ayiheng, Wuluhan Mahan, Xie Zengru This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7059251/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 26 Nov, 2025 Read the published version in Journal of Orthopaedic Surgery and Research → Version 1 posted 8 You are reading this latest preprint version Abstract Background Effective therapies for devastating osteolysis in osseous echinococcosis remain elusive, necessitating mechanistic exploration. To elucidate the molecular mechanism by which echinococcal infection promotes osteoclast differentiation and activation via the IL-1β/c-Fos/NFATc1 signaling axis in the pathological osteolysis of osseous echinococcosis. Methods Retrospective RT‒qPCR analysis was used to quantify the mRNA expression of osteoclastogenesis-associated inflammatory factors (TNF-α, IL-1β, IL-6, and IL-8) in bone cyst tissues from 21 osseous echinococcosis patients versus histologically normal bone adjacent to tumors in 21 matched bone tumor controls. Murine RAW 264.7 monocytes/macrophages were divided into the following groups: (i) Untreated control; (ii) Osteoclast induction (100 ng/mL RANKL + 25 ng/mL M-CSF); (iii-v) induction + hydatid antigen B (25/50/75 ng/mL); and (vi) induction + antigen B (75 ng/mL) + an IL-1β antagonist (canakinumab, 10 ng/mL). TRAP staining revealed osteoclasts (≥3 nuclei), with the percentage of positive cells calculated across ≥5 random fields. ELISA was used to measure cytokine levels in the supernatants; Western blotting was used to quantify c-Fos, NFATc1, cathepsin K, and MMP9 expression. The results Compared with control tissues, bone cyst tissues presented elevated TNF-α, IL-1β, IL-6, and IL-8 mRNA levels (P < 0.05). Antigen B (25–75 ng/mL) dose-dependently increased the number of TRAP⁺ cells and the levels of inflammatory cytokines compared with those in the induction group (P < 0.05). c-Fos, NFATc1, Cathepsin K, and MMP9 were upregulated in the induction and antigen B (75 ng/mL) groups compared with the control group (P < 0.05), with further elevation in the antigen B (75 ng/mL) group compared with the induction group (P < 0.05). Compared with antigen B (75 ng/mL), canakinumab reversed these protein increases (P < 0.05). Conclusion Echinococcal infection promotes pathological osteoclastogenesis and osteolysis through IL-1β/c-Fos/NFATc1 signaling activation. Echinococcus granulosus osseous echinococcosis pathological osteolysis osteoclast IL-1β c-Fos/NFATc1 signaling axis Figures Figure 1 Figure 2 Figure 3 Introduction Cystic echinococcosis (CE) is a zoonotic infection caused by the larval stage of the Echinococcus granulosus sensu lato species complex, which includes E. granulosus sensu stricto, E. felidis, E. equinus, E. ortleppi, and E. canadensis . With the exception of Antarctica, CE occurs globally and is highly endemic in the Mediterranean Basin, Middle East, Central Asia, China, the Russian Federation, and regions of northern and eastern Africa [1]. CE lesions most frequently develop in the liver (70%) and lungs (20%), although virtually any anatomical site may be affected (spleen, kidneys, heart, central nervous system, bones, etc.). Osseous echinococcosis accounts for an estimated 0.5–4% of all CE cases [2]. This skeletal manifestation typically involves a single bone, with the spine (40–50%) representing the most common site, followed by the pelvis (20–25%), long bones, including the femur (15%) and tibia (10%), and, less frequently, the skull, sternum, scapulae, and phalanges. Osseous CE causes severe disability due to its destructive growth pattern, which mimics locally aggressive bone lesions [3]. In China, CEs are distributed across more than ten provinces in the western, central, eastern, and northeastern regions, with the highest endemicity in Xinjiang, Inner Mongolia, Qinghai, and Gansu [4]. Osseous hydatid cysts predominantly develop in vascular spongy bone and the metaphyseal regions of long bones. Within the trabecular bone matrix, multiple CE cysts form interconnected tunnels resembling a "rat-bite" or "moth-eaten" radiographic pattern, frequently leading to pathological fractures. Histopathologically, these lesions exhibit cancer-like invasive growth characteristics without neo-osteogenesis. The cysts lack a fibrous capsule (laminated ectocyst) and demonstrate exogenous expansion; rupture results in leakage of cyst fluid, causing osteolytic destruction. Progressive cyst enlargement directly activates osteoclasts, ultimately leading to the disruption of cortical bone or articular cartilage, pathological fractures or dislocations, and secondary cyst formation in surrounding soft tissues. This is often accompanied by neurological compromise, joint infections, and fistula formation, resulting in high rates of functional impairment [5]. Currently, no satisfactory pharmacotherapy exists for osseous echinococcosis. Surgical intervention remains the primary therapeutic approach and is typically combined with systemic albendazole therapy [2]. The rigidity of bone tissue, compounded by extensive destruction of osseous and/or periarticular soft tissues from cyst dissemination, renders surgery technically challenging. Intraoperative containment to prevent parasitic dissemination into surrounding structures is critical. However, owing to the infiltrative nature of the infection, complete parasite eradication cannot be guaranteed surgically, resulting in recurrence rates as high as 48% [6]. Adjuvant radiation therapy significantly improves patient outcomes and substantially reduces the risk of recurrence [7]. Novel therapeutic approaches remain imperative for this disease. Proinflammatory cytokines—including TNF-α, IL-1β, IL-6, and IL-8—activate osteoclast precursor differentiation into osteoclasts (OCs) [8]. In this study, we conducted a retrospective analysis of osteoclastogenic inflammatory factor expression in surgically obtained bone cyst tissues from 21 osseous echinococcosis patients treated in our orthopedic department and compared them with control samples of histologically normal bone tissue adjacent to tumors from 21 matched bone tumor patients (archived in our pathology department). Furthermore, murine monocyte/macrophage RAW 264.7 cells were stimulated in vitro with hydatid antigen B protein, with subsequent intervention using the IL-1β antagonist canakinumab. This experimental approach aims to elucidate the molecular mechanisms by which the IL-1β/c-Fos/NFATc1 signaling axis mediates osteoclast differentiation and activation during pathological osteolysis in hydatid infection. 1 Experimental Materials and Methods 1.1 Experimental material The murine monocyte/macrophage RAW 264.7 cell line was procured from Procell Life Science & Technology Co., Ltd. (Wuhan, China). RNA extraction was performed via the RNAprep Pure Tissue Total RNA Kit (Cat# DP431; TIANGEN Biotech, Beijing). For complementary DNA synthesis, FastKing gDNA Dispelling RT SuperMix (Cat# KR116; TIANGEN) was used. Quantitative PCR analysis was performed with FastFire qPCR PreMix (SYBR Green) (Cat# FP207; TIANGEN). The hydatid antigen B protein (Cat# YDYM007; Yuduo Bio, Shanghai) and the IL-1β antagonist canakinumab (Cat# Ab170512; Aladdin Biochemical Technology, Shanghai) were commercially sourced. The primary antibodies used included the following: anti-c-Fos monoclonal antibody (Cat# MA5-15055), anti-NFATc1 monoclonal antibody (Cat# MA5-32686), anti-Cathepsin K polyclonal antibody (Cat# PA5-14270), anti-MMP9 polyclonal antibody (Cat# PA5-13199), and anti-GAPDH monoclonal antibody (Cat# 39-8600). For secondary detection, HRP-conjugated goat anti-rabbit IgG (Cat# 31460) was used, and all the antibodies were obtained from Thermo Fisher Scientific (Waltham, MA, USA). 1.2 methodologies (1) Clinical patient samples A retrospective analysis was conducted on 21 histopathologically confirmed bone cyst tissue samples obtained via surgical biopsy from osseous echinococcosis patients treated in our orthopedic department between January 2015 and December 2024, with 21 matched control samples of histologically normal bone tissue adjacent to tumors sourced from bone tumor patients in our pathology department. Differential diagnosis distinguishing bone tumors from osseous echinococcosis was established through comprehensive assessment of detailed patient histories, clinical symptomatology, multimodal imaging [X-ray, computed tomography (CT), and magnetic resonance imaging (MRI)], and serological immunogold analysis measuring serum echinococcal antibody (Ab) levels, with patient clinical characteristics detailed in Table 1. Table 1. Clinical information for patients with bone hydatid disease Information Sex(n) 21 male 12 female 9 Age (years, mean ± standard deviation) (range) 40.7±10.1（28-53） Infected bone site Clavicle 2 Femur 2 Ilium 8 Rib 肋骨 3 Sacrum 2 Vertebra 4 Disease Duration, years (Mean ± SD) 3.5±1.7 (2) RT‒qPCR analysis Bone tissue samples were homogenized in liquid nitrogen via a cryogenic tissue homogenizer with the addition of TRIzol reagent at a ratio of 1 mL per mg of tissue. Total RNA was extracted via the RNAprep Pure Tissue Total RNA Kit (TIANGEN), followed by genomic DNA removal and cDNA synthesis via FastKing gDNA Dispelling RT SuperMix (TIANGEN). Quantitative reverse transcription PCR (RT‒qPCR) amplification was performed with FastFire qPCR PreMix (SYBR Green; TIANGEN) under the following primer conditions (5’→3’ orientation): TNF-α forward-GACGCCACATCCCCTGACA, reverse-CGAGGAGGCGCTCCCCAAGA; IL-1β forward-GTTCTTTGAAGCTGATGGCC, reverse-GTTGTTGTGGCCATGGACAA; IL-6 forward-CCAGGAGAAGATTCCAAAGA, reverse-CCTGAGAAAGGAGACATGTA; and IL-8 forward-GTTTTTGAAGAGGGCTGAGA, reverse-GGGTTGCCAGATGCAATAC. (3) Cell culture The murine monocyte/macrophage RAW 264.7 cell line was maintained in complete DMEM supplemented with 10% fetal bovine serum under standard culture conditions (37°C, 5% CO₂, humidified atmosphere). (4) Cellular experimental groupings and treatments For experimental interventions, the cells were divided into six groups: (i) control group: basal culture without treatment; (ii) osteoclastogenesis induction group: continuous 9-day stimulation with 100 ng/ml RANKL and 25 ng/ml macrophage colony-stimulating factor (M-CSF) to drive osteoclast differentiation [9]; (iii) induction + hydatid antigen B (25 ng/ml) group: cotreatment with RANKL/M-CSF plus 25 ng/ml hydatid antigen B protein for 9 days; (iv) induction + hydatid antigen B (50 ng/ml) group: cotreatment with RANKL/M-CSF plus 50 ng/ml antigen B; (v) induction + hydatid antigen B (75 ng/ml) group: cotreatment with RANKL/M-CSF plus 75 ng/ml antigen B; and (vi) induction + hydatid antigen B (75 ng/ml) + IL-1β antagonist group: cotreatment with RANKL/M-CSF, 75 ng/ml antigen B, and 10 ng/ml canakinumab (anti-IL-1β monoclonal antibody) for 9 days [10]. All induction protocols aimed to promote osteoclast differentiation. (5) TRAP staining TRAP activity in adherent cell cultures was assessed via an acid phosphatase assay kit. Cells exhibiting TRAP-positive staining with three or more nuclei were identified as osteoclasts. The percentage of TRAP-positive cells was quantified by counting stained cells in a minimum of five randomly selected microscopic fields via light microscopy and was calculated as follows: (number of TRAP-positive cells ÷ total cells per field) × 100%. (6) ELISA The concentrations of TNF-α, IL-1β, IL-6, and IL-8 in the cell culture supernatants were quantified via species-specific anti-mouse ELISA kits according to the manufacturer's protocols. (7) Western blot Total protein was extracted by adding 1 ml of RIPA lysis buffer supplemented with protease inhibitors to 6-well plate cultures, followed by conventional SDS‒PAGE. Proteins were transferred to PVDF membranes via semidry electrophoretic transfer, blocked with 5% skim milk, and incubated with primary antibody working solution at 4°C overnight. After the addition of the HRP-conjugated secondary antibody working solution for 1 h at room temperature, the target bands were visualized via an enhanced chemiluminescence (ECL) substrate. The antibody dilutions used were as follows: c-Fos (1:1500), NFATc1 (1:1500), Cathepsin K (1:1500), MMP-9 (1:1500), and GAPDH (1:1500) and an HRP-IgG secondary antibody (1:5000). (8) Analytics Statistical analysis was performed with GraphPad Prism version 8. Continuous data are presented as the means ± standard deviations (means ± SDss). Student's t test was used to compare differences between two groups; one-way analysis of variance (ANOVA) with Tukey's post hoc test was used for multiple group comparisons. Statistical significance was set at P < 0.05. 2 Results 2.1 Osteoclastogenesis-associated inflammatory factors are highly expressed in bone cyst tissue As shown in Table 2, the relative mRNA expression levels of the osteoclastogenesis-associated inflammatory factors TNF-α, IL-1β, IL-6, and IL-8 were greater in the bone cyst tissue than in the adjacent normal bone tissue (P < 0.05). Table 2. Statistical results of the determination of the expression of osteoclast differentiation-associated inflammatory factors in paracancerous bone tissues and bone cyst tissues are presented in Table (mean±SD). Group TNF-α mRNA Relative Expression Level IL-1β mRNA Relative Expression Level Adjacent Normal Bone Tissue （ n=21 ） 1.00±0.11 1.00±0.13 Bone Cyst Tissue （ n=21 ） 37.52±1.11 28.44±0.76 t 150.00 163.10 P <0.01 <0.01 Table 2. Continued: Expression of Osteoclast Differentiation-Associated Inflammatory Factors in Adjacent Normal Bone Tissue and Bone Cyst Tissue (Mean ± SD) Group IL-6 mRNA Relative Expression Level IL-8 mRNA Relative Expression Level Adjacent Normal Bone Tissue （ n=21 ） 1.00±0.12 1.00±0.16 Bone Cyst Tissue （ n=21 ） 17.36±0.82 5.56±0.45 t 90.46 43.75 P <0.01 <0.01 2.2 EgAgB enhances osteoclast differentiation in RAW 264.7 cells. As shown in Figure 1 and Table 3, TRAP staining revealed significant increases in the percentage of TRAP-positive cells in the induction + EgAgB group: Concentrations 1, 2, and 3 versus the induction group (P < 0.05); in the induction + EgAgB group: Concentrations 2 and 3 versus Concentration 1 (P < 0.05); and in the induction + EgAgB group: Concentration 3 versus Concentration 2 (P < 0.05). Table 3. Statistical results of the TRAP staining assay for the differentiation of RAW 264.7 cells into osteoclasts (mean±SD, n=6) Group Percentage of TRAP-Positive Cells(%) Induction Group 22.0±1.4 Induction+EgAgB: Concentration 1 41.4±1.2 a Induction+EgAgB: Concentration 2 57.8±1.8 ab Induction+EgAgB: Concentration 3 83.2±2.2 abc F 1405.00 P <0.01 Notes: ⁰Significantly different vs. induction group, P < 0.05; ᵇSignificantly different vs. Induction + EgAgB: Concentration 1, P < 0.05; ᶜSignificantly different vs. Induction + EgAgB: Concentration 2, P < 0.05. 2.3 EgAgB Stimulation Upregulates Secretion of Osteoclastogenesis-Associated Inflammatory Factors in RAW 264.7 Cells As shown in Table 4, significantly elevated levels of TNF-α, IL-1β, IL-6, and IL-8 were observed in the Induction Group, Induction + EgAgB: Concentrations 1, 2, and 3 compared with those in the Control Group (P < 0.05); furthermore, all three antigen concentration groups presented higher levels of these inflammatory factors than did the Induction Group alone (P < 0.05), with Concentrations 2 and 3 showing increased levels versus Concentration 1 (P < 0.05) and Concentration 3 demonstrating further elevation versus Concentration 2 (P < 0.05). Table 4. Statistical results for the measurement of the levels of osteoclast differentiation-related inflammatory factors in the supernatants of RAW 264.7 cell cultures (mean±SD, n=6) Group TNF-α （ ng/ml ） IL-1β （ ng/ml ） Control Group 16.47±0.52 21.22±0.43 Induction Group 123.64±1.11 a 56.53±0.71 a Induction+EgAgB: Concentration 1 144.35±1.34 ab 88.45±0.84 ab Induction+EgAgB: Concentration 2 158.36±1.41 abc 95.76±0.85 abc Induction+EgAgB: Concentration 3 174.48±1.55 abcd 102.34±0.91 abcd F 15319.00 11637.00 P <0.01 <0.01 Notes: ⁰Significantly different vs. control group, P < 0.05; ᵃSignificantly different from the induction group, P < 0.05; ᵇSignificantly different vs. Induction + EgAgB: Concentration 1, P < 0.05; ᶜSignificantly different vs. Induction + EgAgB: Concentration 2, P < 0.05. Table 4. Continued: Levels of Osteoclast Differentiation-Associated Inflammatory Factors in Culture Supernatants of RAW 264.7 Cells (Mean ± SD; n= 6) Group IL-6 （ ng/ml ） IL-8 （ ng/ml ） Control Group 31.33±0.37 25.82±0.32 Induction Group 58.53±0.42 a 61.23±0.24 a Induction+EgAgB: Concentration 1 63.52±0.53 ab 66.37±0.28 ab Induction+EgAgB: Concentration 2 67.44±0.56 abc 70.12±0.30 abc Induction+EgAgB: Concentration 3 72.35±0.61 abcd 73.46±0.35 abcd F 6065.00 24819.00 P <0.01 <0.01 Notes: ᵃSignificantly different from the control group, P < 0.05; ᵇSignificantly different from the induction group, P < 0.05; ᶜSignificantly different vs. Induction + EgAgB: Concentration 1, P < 0.05; ᵈSignificantly different vs. Induction + EgAgB: Concentration 2, P < 0.05. 2.4 EgAgB Stimulation Upregulates the c-Fos/NFATc1 Signaling Axis in RAW 264.7 Cells As demonstrated in Fig. 2 and Table 5, significant upregulation of (1) the key osteoclastogenic transcription factor axis c-Fos/NFATc1 and (2) the signature protease markers cathepsin K and MMP-9—was observed in both the induction group and the induction + EgAgB:Concentration 3 group compared with the control group (P < 0.05). Furthermore, the Induction + EgAgB:Concentration 3 group presented elevated expression of these markers compared with the Induction Group alone (P < 0.05). Table 5. Expression Analysis of the Key Osteoclastogenic Transcription Factor Axis c-Fos/NFATc1 in RAW 264.7 Cells (Mean±SD, n=6) Group c-Fos Relative Expression NFATc1 Relative Expression 1 Control Group 1.00±0.12 1.00±0.10 2 Induction Group 1.72±0.14 1.64±0.12 3 Induction+EgAgB: Concentration 3 2.23±0.15 2.58±0.16 F 121.70 227.40 P <0.01 <0.01 Notes: ᵃSignificantly different from the control group, P < 0.05; ᵇSignificantly different from the induction group, P < 0.05. Table 5. Continued: Expression Analysis of the Key Osteoclastogenic Transcription Factor Axis c-Fos/NFATc1 in RAW 264.7 Cells (Mean±SD, n=6) Group Cathepsin K Relative Expression MMP9 Relative Expression 1 Control Group 1.00±0.11 1.00±0.10 2 Induction Group 2.12±0.14 1.52±0.13 3 Induction+EgAgB: Concentration 3 2.87±0.16 2.34±0.14 F 278.20 176.70 P <0.01 <0.01 Notes: ᵃSignificantly different from the control group, P < 0.05 ᵇSignificantly different from the induction group, P < 0.05 2.5 IL-1β Antagonism Downregulates c-Fos/NFATc1/Cathepsin K/MMP-9 Expression in EgAgB-Stimulated RAW 264.7 Cells As shown in Fig. 3 and Table 6, significant upregulation of (1) the key osteoclastogenic transcription factor axis c-Fos/NFATc1 and (2) the signature proteases cathepsin K and MMP-9 was observed in the Induction+EgAgB:Concentration 3 group compared with the Induction group (P<0.05). Conversely, the induction+EgAgB:Concentration 3+IL-1β antagonist group presented marked downregulation of these markers compared with the induction+EgAgB:Concentration 3 group (P<0.05). Table 6. Statistical analysis of c-Fos/NFATc1/Cathepsin K/MMP-9 expression levels in RAW 264.7 cells (means±SDs, n=6) Group c-Fos Relative Expression NFATc1 Relative Expression 1 Control Group 1.00±0.12 1.00±0.11 2 Induction Group 1.75±0.14 a 1.66±0.12 a 3 Induction+EgAgB: Concentration 3 2.36±0.13 ab 2.41±0.14 ab 4 Induction+EgAgB:Concentration 3 +IL-1β Antagonist 1.78±0.11 ac 1.65±0.13 ac F 118.40 126.60 P <0.01 <0.01 Notes: ᵃSignificantly different from the control group, P < 0.05; ᵇSignificantly different from the induction group, P < 0.05; ᶜSignificantly different from Induction + EgAgB: Concentration 3, P < 0.05. Table 6. Continued: Statistical analysis of c-Fos/NFATc1/Cathepsin K/MMP-9 expression levels in RAW 264.7 cells (means±SDs, n=6) Group Cathepsin K Relative Expression MMP9 Relative Expression 1 Control Group 1.00±0.10 1.00±0.12 2 Induction Group 2.10±0.12 a 1.55±0.14 a 3 Induction+EgAgB: Concentration 3 2.45±0.14 ab 2.67±0.21 ab 4 Induction+EgAgB:Concentration 3 +IL-1β Antagonist 2.08±0.11 ac 1.57±0.13 ac F 168.90 123.80 P <0.01 <0.01 Notes: ᵃSignificantly different from the control group, P < 0.05; ᵇSignificantly different from the induction group, P < 0.05; ᶜSignificantly different from Induction + EgAgB: Concentration 3, P < 0.05. 3 Discussion Increased osteoclast (OC) differentiation and activity are key events driving bone loss and joint destruction. OCs are multinucleated cells with three or more nuclei that are typically located on trabecular bone surfaces. Their cytoplasm contains high concentrations of vesicles and vacuoles filled with lysosomal acid phosphatase [stained by tartrate-resistant acid phosphatase (TRAP)] and cathepsin K [ 11 ]. Activated OCs are characterized by the formation of a specialized cell membrane called the ruffled border, which opposes the bone surface and facilitates the secretion of acids and lysosomal enzymes for bone resorption [ 12 ]. Concurrently, these cells exhibit elevated expression of matrix metalloproteinases (MMPs) 8 and 9 [ 13 ]. Receptor activator of nuclear factor-kappa B ligand (RANKL), a member of the tumor necrosis factor (TNF) superfamily, binds to its receptor RANK. In conjunction with macrophage colony-stimulating factor (M-CSF) stimulation, the RANKL–RANK interaction induces terminal differentiation, fusion, and activation of osteoclasts (OCs) [ 9 ]. However, RANK lacks intrinsic kinase activity to mediate downstream signaling; consequently, RANKL binding recruits various molecules, including TNF receptor-associated factors (TRAFs) and kinases, such as transforming growth factor-β-activated kinase 1 (TAK1). This recruitment subsequently activates the transcription factors NF-κB, c-Fos, and nuclear factor of activated T cells cytoplasmic 1 (NFATc1), all of which are essential for OC differentiation [ 14 ]. Proinflammatory cytokines, including TNF-α, IL-1β, IL-6, and IL-8, can activate the differentiation of osteoclast precursors into osteoclasts (OCs) [ 8 ]. Like, but independent of RANKL, TNF-α recruits TNF receptor-associated factors (TRAFs), subsequently activating the transcription factors NF-κB, c-Fos, and nuclear factor of activated T cells cytoplasmic 1 (NFATc1) to induce OC differentiation [ 14 ]. Inhibition of G protein-coupled receptor 120 (GPR120) suppresses TNF-α-activated OC differentiation [ 15 ]. IL-6 and TNF-α synergistically activate osteocyte-mediated OC differentiation and activation, mediating the pathological progression of osteoimmunology through complex mechanisms in rheumatoid arthritis and postmenopausal osteoporosis [ 16 ]. The IL-6/RANKL axis is crucial for the osteocyte-mediated activation of OC differentiation [ 17 ]. Elevated levels of IL-8 promote breast cancer bone metastasis, and inhibition of IL-8 suppresses this pathological process [ 18 ]. IL-1β is another critical proinflammatory cytokine that plays a pivotal role in pathological bone erosion. It enhances NF-κB-mediated osteoclast (OC) differentiation through mechanisms independent of both RANKL and TRAF6. IL-1β activates OC differentiation and bone resorption via a PKCθ/NF-κB/IL-1β-dependent positive feedback loop [ 19 ]. Treatment with IL-1β upregulates the expression of inducible nitric oxide synthase (iNOS), insulin-like growth factor 2 (IGF-2), and the chemokines CX3CL1 and CXCL7 in mouse bone marrow stem cells, thereby promoting the differentiation of nonosteoclastic cells into OCs during bone erosion in rheumatoid arthritis [ 20 ]. The NLRP3 inflammasome accelerates osteolysis and bone remodeling in osteoporosis by upregulating IL-1β, whereas blockade of the NLRP3/IL-1β signaling axis attenuates this pathological process [ 21 ]. After entering bone tissue, Echinococcus granulosus cysts (CEs) fail to elicit a typical demarcating reactive fibrous capsule from the host. Instead, CEs invade adjacent bone regions in a manner analogous to that of bone tumors [ 2 ]. On computed tomography (CT) scans, CE-induced skeletal destruction exhibits a "moth-eaten" pattern, with lesional areas demonstrating a "rat-bite" morphological appearance [ 22 ]. CE enhances osteolytic bone erosion by activating osteoclasts (OCs) through Nrf2-mediated suppression, which elevates oxidative stress and inflammation at bone resorption surfaces [ 23 ]. The tissues surrounding CE-induced bone erosions show increased inflammation, dysregulated osteoimmune responses, and abnormally elevated OC activity [ 24 ]. Following CE infection, patients test positive for antibodies against E. granulosus cyst fluid (EgCF), E. granulosus protoscolex (EgP), and semipurified hydatid cyst fluid native antigen B (EgB). In the present study, analysis of the mRNA expression levels of osteoclast differentiation-associated inflammatory factors in CE bone cyst tissues revealed elevated relative expression levels of TNF-α, IL-1β, IL-6, and IL-8 compared with those in adjacent nonlesioned bone tissue. Stimulation with hydatid antigen B protein upregulated the secretion of osteoclastogenic inflammatory factors (TNF-α, IL-1β, IL-6, and IL-8) in RAW 264.7 cells and enhanced their differentiation into osteoclasts, concurrently increasing the intracellular expression of c-Fos, NFATc1, cathepsin K, and MMP-9. Treatment with an IL-1β antagonist suppressed hydatid antigen B-induced expression of c-Fos, NFATc1, cathepsin K, and MMP-9 in RAW 264.7 cells. These findings suggest that IL-1β antagonism may inhibit pathological osteolytic destruction in skeletal echinococcosis. Overall, while this study has not delineated whether TNF-α or IL-1β plays a predominant role—or whether they act synergistically—in pathological osteolytic destruction during CE infection, our findings establish that Echinococcus granulosus infection promotes osteoclast differentiation and activation in osseous echinococcosis through the IL-1β/c-Fos/NFATc1 signaling axis. Declarations Ethics approval and consent to participate The study was performed in line with the principles of the Declaration of Helsinki. Approval was granted by the Ethics Committee of The 1nd Affiliated Hospital of Xinjiang Medical University before the study began. The written informed consent has been obtained from the participants involved. Consent for publication Not applicable. Availability of data and materials The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. Competing interests The authors declare no competing interests. Funding This study was supported by the Funding District Project of the National Natural Science Foundation of China (No:82260409) and Xinjiang Uygur Autonomous Region Natural Science Foundation Key Projects (No:2021D01D19). Authors' contributions YLNE AYH, WLH MH: Study design, data collection, formal analysis, writing – original draft. XZR: Funding acquisition, Writing – review & editing. All authors read and approved the final manuscript. Acknowledgements Not Applicable. References Lundström-Stadelmann B, Rostami A, Frey CF, et al. Human alveolar echinococcosis-global, regional, and national annual incidence and prevalence rates. Clin Microbiol Infect. 2025;31(7):1139–45. 10.1016/j.cmi.2025.01.034 . Monge-Maillo B, Lopez-Velez R. Cystic echinococcosis of the bone. Curr Opin Infect Dis. 2023;36(5):341–7. 10.1097/QCO.0000000000000951 . Cattaneo L, Manciulli T, Cretu CM, et al. Cystic Echinococcosis of the Bone: A European Multicenter Study. Am J Trop Med Hyg. 2019;100(3):617–21. 10.4269/ajtmh.18-0758 . Ma C, Huang JY, Luo XF, Xie ZR. Advances in the study of the diagnosis and treatment of osseous echinococcosis.2021;16(5):608–13. 10.13350/j.cjpb.210522 Chang YS, Yang LH, He X et al. 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The effect of IL-1β inhibitor canakinumab (Ilaris®) on IL-6 production in human skeletal muscle cells. PLoS ONE. 2025;20(3):e0316110. 10.1371/journal.pone.0316110 . Fu Yonghua,Wang Yuqian,Li Ji,et al. Mechanism of necroptosis of osteocytes in up-regulating biglycan to promote the activation of osteoclasts in postmenopausal osteoporosis. Chin J Osteoporos,2025;31(1):8–14. 10.3969/j.issn.1006-7108.2025.01.002 Søe K, Delaisse JM, Borggaard XG. Osteoclast formation at the bone marrow/bone surface interface: Importance of structural elements, matrix, and intercellular communication. Semin Cell Dev Biol. 2021;112:8–15. 10.1016/j.semcdb.2020.05.016 . Ye Q, Xu H, Liu S, et al. Apoptotic extracellular vesicles alleviate Pg-LPS induced inflammatory responses of macrophages via AMPK/SIRT1/NF-κB pathway and inhibit osteoclast formation. J Periodontol. 2022;93(11):1738–51. 10.1002/JPER.21-0657 . Yao Z, Getting SJ, Locke IC. Regulation of TNF-Induced Osteoclast Differentiation. Cells. 2021;11(1):132. 10.3390/cells11010132 . Ma J, Kitaura H, Ogawa S, et al. Docosahexaenoic acid inhibits TNF-α-induced osteoclast formation and orthodontic tooth movement through GPR120. Front Immunol. 2023;13:929690. 10.3389/fimmu.2022.929690 . Wang T, He C. TNF-α and IL-6: The Link between Immune and Bone System. Curr Drug Targets. 2020;21(3):213–27. 10.2174/1389450120666190821161259 . Kim HJ, Kim HJ, Choi Y, et al. Zoledronate Enhances Osteocyte-Mediated Osteoclast Differentiation by IL-6/RANKL Axis. Int J Mol Sci. 2021;20(6):1467. 10.3390/ijms20061467 . Wei C, Shi M, Wang Z, et al. Epiberberine inhibits bone metastatic breast cancer-induced osteolysis. J Ethnopharmacol. 2024;327:118039. 10.1016/j.jep.2024.118039 . Wang Q, Lei Z, Wang Z, et al. PKCθ Regulates Pituitary Adenoma Bone Invasion by Activating Osteoclast in NF-κB/IL-1β-Dependent Manner. Cancers (Basel). 2023;15(5):1624. 10.3390/cancers15051624 . Otsuka Y, Kondo T, Aoki H, et al. IL-1β promotes osteoclastogenesis by increasing the expression of IGF2 and chemokines in non-osteoclastic cells. J Pharmacol Sci. 2023;151(1):1–8. 10.1016/j.jphs.2022.10.007 . Chen W, Tang P, Fan S, et al. A Novel Inhibitor INF 39 Promotes Osteogenesis via Blocking the NLRP3/IL-1 β Axis. Biomed Res Int. 2022;2022:7250578. 10.1155/2022/7250578 . Barth TFE, Casulli A. Morphological Characteristics of Alveolar and Cystic Echinococcosis Lesions in Human Liver and Bone. Pathogens. 2021;10(10):1326. 10.3390/pathogens10101326 . Huang Y, Huang Y, Xiao J, et al. Mechanisms of Nrf2 suppression and Camkk1 upregulation in Echinococcus granulosus-induced bone loss. Int J Biol Macromol. 2025;288:138521. 10.1016/j.ijbiomac.2024.138521 . Sun H, Wang S, Tan W, et al. Echinococcus granulosus promotes bone resorption by increasing osteoclasts differentiation. Acta Trop. 2023;248:107027. 10.1016/j.actatropica.2023.107027 . Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 26 Nov, 2025 Read the published version in Journal of Orthopaedic Surgery and Research → Version 1 posted Editorial decision: Revision requested 28 Jul, 2025 Reviews received at journal 26 Jul, 2025 Reviewers agreed at journal 26 Jul, 2025 Reviewers agreed at journal 21 Jul, 2025 Reviewers invited by journal 17 Jul, 2025 Editor assigned by journal 08 Jul, 2025 Submission checks completed at journal 08 Jul, 2025 First submitted to journal 06 Jul, 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. 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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-7059251","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":488605253,"identity":"b19f40b9-1c4b-4371-9961-06098e2a5232","order_by":0,"name":"Yelinaer Ayiheng","email":"","orcid":"","institution":"The First Affiliated Hospital of Xinjiang Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yelinaer","middleName":"","lastName":"Ayiheng","suffix":""},{"id":488605254,"identity":"bd2f6631-4467-4b34-931d-27b1e83e52a7","order_by":1,"name":"Wuluhan Mahan","email":"","orcid":"","institution":"The First Affiliated Hospital of Xinjiang Medical University","correspondingAuthor":false,"prefix":"","firstName":"Wuluhan","middleName":"","lastName":"Mahan","suffix":""},{"id":488605255,"identity":"e3f160ed-384d-411d-9a34-b820a0c6c707","order_by":2,"name":"Xie Zengru","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAtklEQVRIiWNgGAWjYDACZijNz8x88AFpWiTb2ZINSLPN4DyPmQBxKo8zH/7wcce9xM2HGcwYGGpsoglqkWxmSzCceaY4cdthhrQHDMfSchsIaeFn5jFI5m1LAGk5bsDYcJiwFjZm/g+H/wK1bG5mbJMgSgvQFkag4oTEDczMbMRpAfrFmLG3LcF4xmE2ZoMEYvxicP7w4w8/2xJk+/vPf3zwocaGsBZUkECa8lEwCkbBKBgFuAAA0L07VtFhBFQAAAAASUVORK5CYII=","orcid":"","institution":"The First Affiliated Hospital of Xinjiang Medical University","correspondingAuthor":true,"prefix":"","firstName":"Xie","middleName":"","lastName":"Zengru","suffix":""}],"badges":[],"createdAt":"2025-07-06 17:23:18","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7059251/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7059251/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s13018-025-06505-5","type":"published","date":"2025-11-26T15:57:01+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":87379216,"identity":"ab3bdb5e-672a-4769-a13b-834e9f70caa4","added_by":"auto","created_at":"2025-07-23 08:25:05","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":55872,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTRAP staining revealed significant osteoclast differentiation in RAW 264.7 cells, with dose-dependent increases in TRAP⁺ multinucleated cells following antigen B stimulation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNotes: a. Induction group b. Induction + EgAgB: Concentration 1 c. Induction + EgAgB: Concentration 2 d. Induction + EgAgB: Concentration 3\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7059251/v1/b9063e717fc1cb5a40e30799.jpeg"},{"id":87379212,"identity":"87b9b007-86aa-4a42-9604-b8cd628b8fb7","added_by":"auto","created_at":"2025-07-23 08:25:05","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":144719,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExpression Analysis of the Key Osteoclastogenic Transcription Factor Axis c-Fos/NFATc1 in RAW 264.7 Cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNotes: a. Control Group b. Induction Group c. Induction + EgAgB: Concentration 3\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7059251/v1/24448ff8f419f950518d392b.png"},{"id":87379209,"identity":"9d652e0f-3bcf-4a02-a65e-7fb90ee3b42b","added_by":"auto","created_at":"2025-07-23 08:25:05","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":40437,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExpression levels of c-Fos/NFATc1/Cathepsin K/MMP-9 in RAW 264.7 cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNotes:\u003c/p\u003e\n\u003cp\u003e1. Control Group\u003c/p\u003e\n\u003cp\u003e2. Induction Group\u003c/p\u003e\n\u003cp\u003e3. Induction + EgAgB: Concentration 3\u003c/p\u003e\n\u003cp\u003e4. Induction + EgAgB: Concentration 3 + IL-1β antagonist\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7059251/v1/ca77e1b12b1bb3508517bdff.jpeg"},{"id":97178332,"identity":"5eb0da70-25fd-441a-8e24-200fdae443e8","added_by":"auto","created_at":"2025-12-01 16:08:04","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2402878,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7059251/v1/e90d3c4c-dd16-47b0-bccb-24af0e502642.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Mechanistic Role of the IL-1β/c-Fos/NFATc1 Signaling Axis in Echinococcal Infection-Elicited Osteoclastogenesis and Pathological Osteolysis: A Prospective Controlled Trial","fulltext":[{"header":"Introduction","content":"\u003cp\u003eCystic echinococcosis (CE) is a zoonotic infection caused by the larval stage of the Echinococcus granulosus sensu lato species complex, which includes E. granulosus sensu stricto, E. felidis, E. equinus, E. ortleppi, and \u003cem\u003eE. canadensis\u003c/em\u003e. With the exception of Antarctica, CE occurs globally and is highly endemic in the Mediterranean Basin, Middle East, Central Asia, China, the Russian Federation, and regions of northern and eastern Africa [1]. CE lesions most frequently develop in the liver (70%) and lungs (20%), although virtually any anatomical site may be affected (spleen, kidneys, heart, central nervous system, bones, etc.). Osseous echinococcosis accounts for an estimated 0.5–4% of all CE cases [2]. This skeletal manifestation typically involves a single bone, with the spine (40–50%) representing the most common site, followed by the pelvis (20–25%), long bones, including the femur (15%) and tibia (10%), and, less frequently, the skull, sternum, scapulae, and phalanges. Osseous CE causes severe disability due to its destructive growth pattern, which mimics locally aggressive bone lesions [3]. In China, CEs are distributed across more than ten provinces in the western, central, eastern, and northeastern regions, with the highest endemicity in Xinjiang, Inner Mongolia, Qinghai, and Gansu [4].\u003c/p\u003e\n\u003cp\u003eOsseous hydatid cysts predominantly develop in vascular spongy bone and the metaphyseal regions of long bones. Within the trabecular bone matrix, multiple CE cysts form interconnected tunnels resembling a \"rat-bite\" or \"moth-eaten\" radiographic pattern, frequently leading to pathological fractures. Histopathologically, these lesions exhibit cancer-like invasive growth characteristics without neo-osteogenesis. The cysts lack a fibrous capsule (laminated ectocyst) and demonstrate exogenous expansion; rupture results in leakage of cyst fluid, causing osteolytic destruction. Progressive cyst enlargement directly activates osteoclasts, ultimately leading to the disruption of cortical bone or articular cartilage, pathological fractures or dislocations, and secondary cyst formation in surrounding soft tissues. This is often accompanied by neurological compromise, joint infections, and fistula formation, resulting in high rates of functional impairment [5].\u003c/p\u003e\n\u003cp\u003eCurrently, no satisfactory pharmacotherapy exists for osseous echinococcosis. Surgical intervention remains the primary therapeutic approach and is typically combined with systemic albendazole therapy [2]. The rigidity of bone tissue, compounded by extensive destruction of osseous and/or periarticular soft tissues from cyst dissemination, renders surgery technically challenging. Intraoperative containment to prevent parasitic dissemination into surrounding structures is critical. However, owing to the infiltrative nature of the infection, complete parasite eradication cannot be guaranteed surgically, resulting in recurrence rates as high as 48% [6]. Adjuvant radiation therapy significantly improves patient outcomes and substantially reduces the risk of recurrence [7]. Novel therapeutic approaches remain imperative for this disease.\u003c/p\u003e\n\u003cp\u003eProinflammatory cytokines—including TNF-α, IL-1β, IL-6, and IL-8—activate osteoclast precursor differentiation into osteoclasts (OCs) [8]. In this study, we conducted a retrospective analysis of osteoclastogenic inflammatory factor expression in surgically obtained bone cyst tissues from 21 osseous echinococcosis patients treated in our orthopedic department and compared them with control samples of histologically normal bone tissue adjacent to tumors from 21 matched bone tumor patients (archived in our pathology department). Furthermore, murine monocyte/macrophage RAW 264.7 cells were stimulated in vitro with hydatid antigen B protein, with subsequent intervention using the IL-1β antagonist canakinumab. This experimental approach aims to elucidate the molecular mechanisms by which the IL-1β/c-Fos/NFATc1 signaling axis mediates osteoclast differentiation and activation during pathological osteolysis in hydatid infection.\u003c/p\u003e"},{"header":"1 Experimental Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003e1.1 Experimental material\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe murine monocyte/macrophage RAW 264.7 cell line was procured from Procell Life Science \u0026amp; Technology Co., Ltd. (Wuhan, China). RNA extraction was performed via the RNAprep Pure Tissue Total RNA Kit (Cat# DP431; TIANGEN Biotech, Beijing). For complementary DNA synthesis, FastKing gDNA Dispelling RT SuperMix (Cat# KR116; TIANGEN) was used. Quantitative PCR analysis was performed with FastFire qPCR PreMix (SYBR Green) (Cat# FP207; TIANGEN). The hydatid antigen B protein (Cat# YDYM007; Yuduo Bio, Shanghai) and the IL-1\u0026beta; antagonist canakinumab (Cat# Ab170512; Aladdin Biochemical Technology, Shanghai) were commercially sourced. The primary antibodies used included the following: anti-c-Fos monoclonal antibody (Cat# MA5-15055), anti-NFATc1 monoclonal antibody (Cat# MA5-32686), anti-Cathepsin K polyclonal antibody (Cat# PA5-14270), anti-MMP9 polyclonal antibody (Cat# PA5-13199), and anti-GAPDH monoclonal antibody (Cat# 39-8600). For secondary detection, HRP-conjugated goat anti-rabbit IgG (Cat# 31460) was used, and all the antibodies were obtained from Thermo Fisher Scientific (Waltham, MA, USA).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e1.2 methodologies\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(1) Clinical patient samples\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA retrospective analysis was conducted on 21 histopathologically confirmed bone cyst tissue samples obtained via surgical biopsy from osseous echinococcosis patients treated in our orthopedic department between January 2015 and December 2024, with 21 matched control samples of histologically normal bone tissue adjacent to tumors sourced from bone tumor patients in our pathology department. Differential diagnosis distinguishing bone tumors from osseous echinococcosis was established through comprehensive assessment of detailed patient histories, clinical symptomatology, multimodal imaging [X-ray, computed tomography (CT), and magnetic resonance imaging (MRI)], and serological immunogold analysis measuring serum echinococcal antibody (Ab) levels, with patient clinical characteristics detailed in Table 1.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 1. Clinical information for patients with bone hydatid disease\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 199px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 151px;\"\u003e\n \u003cp\u003eInformation\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 199px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSex(n)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 151px;\"\u003e\n \u003cp\u003e21\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 199px;\"\u003e\n \u003cp\u003e\u003cstrong\u003emale\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 151px;\"\u003e\n \u003cp\u003e12\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 199px;\"\u003e\n \u003cp\u003e\u003cstrong\u003efemale\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 151px;\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 199px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eAge (years, mean \u0026plusmn; standard deviation)\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(range)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 151px;\"\u003e\n \u003cp\u003e40.7\u0026plusmn;10.1（28-53）\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 199px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eInfected bone site\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 151px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 199px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eClavicle\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 151px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 199px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eFemur\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 151px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 199px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eIlium\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 151px;\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 199px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eRib\u003c/strong\u003e\u003cstrong\u003e肋骨\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 151px;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 199px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSacrum\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 151px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 199px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eVertebra\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 151px;\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 199px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eDisease Duration, years (Mean \u0026plusmn; SD)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 151px;\"\u003e\n \u003cp\u003e3.5\u0026plusmn;1.7\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003e(2) RT‒qPCR analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBone tissue samples were homogenized in liquid nitrogen via a cryogenic tissue homogenizer with the addition of TRIzol reagent at a ratio of 1 mL per mg of tissue. Total RNA was extracted via the RNAprep Pure Tissue Total RNA Kit (TIANGEN), followed by genomic DNA removal and cDNA synthesis via FastKing gDNA Dispelling RT SuperMix (TIANGEN). Quantitative reverse transcription PCR (RT‒qPCR) amplification was performed with FastFire qPCR PreMix (SYBR Green; TIANGEN) under the following primer conditions (5\u0026rsquo;\u0026rarr;3\u0026rsquo; orientation): TNF-\u0026alpha; forward-GACGCCACATCCCCTGACA, reverse-CGAGGAGGCGCTCCCCAAGA; IL-1\u0026beta; forward-GTTCTTTGAAGCTGATGGCC, reverse-GTTGTTGTGGCCATGGACAA; IL-6 forward-CCAGGAGAAGATTCCAAAGA, reverse-CCTGAGAAAGGAGACATGTA; and IL-8 forward-GTTTTTGAAGAGGGCTGAGA, reverse-GGGTTGCCAGATGCAATAC.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(3) Cell culture\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe murine monocyte/macrophage RAW 264.7 cell line was maintained in complete DMEM supplemented with 10% fetal bovine serum under standard culture conditions (37\u0026deg;C, 5% CO₂, humidified atmosphere).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(4) Cellular experimental groupings and treatments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor experimental interventions, the cells were divided into six groups: (i) control group: basal culture without treatment; (ii) osteoclastogenesis induction group: continuous 9-day stimulation with 100 ng/ml RANKL and 25 ng/ml macrophage colony-stimulating factor (M-CSF) to drive osteoclast differentiation [9]; (iii) induction + hydatid antigen B (25 ng/ml) group: cotreatment with RANKL/M-CSF plus 25 ng/ml hydatid antigen B protein for 9 days; (iv) induction + hydatid antigen B (50 ng/ml) group: cotreatment with RANKL/M-CSF plus 50 ng/ml antigen B; (v) induction + hydatid antigen B (75 ng/ml) group: cotreatment with RANKL/M-CSF plus 75 ng/ml antigen B; and (vi) induction + hydatid antigen B (75 ng/ml) + IL-1\u0026beta; antagonist group: cotreatment with RANKL/M-CSF, 75 ng/ml antigen B, and 10 ng/ml canakinumab (anti-IL-1\u0026beta; monoclonal antibody) for 9 days [10]. All induction protocols aimed to promote osteoclast differentiation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(5) TRAP staining\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTRAP activity in adherent cell cultures was assessed via an acid phosphatase assay kit. Cells exhibiting TRAP-positive staining with three or more nuclei were identified as osteoclasts. The percentage of TRAP-positive cells was quantified by counting stained cells in a minimum of five randomly selected microscopic fields via light microscopy and was calculated as follows: (number of TRAP-positive cells \u0026divide; total cells per field) \u0026times; 100%.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(6) ELISA\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe concentrations of TNF-\u0026alpha;, IL-1\u0026beta;, IL-6, and IL-8 in the cell culture supernatants were quantified via species-specific anti-mouse ELISA kits according to the manufacturer\u0026apos;s protocols.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(7) Western blot\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTotal protein was extracted by adding 1 ml of RIPA lysis buffer supplemented with protease inhibitors to 6-well plate cultures, followed by conventional SDS‒PAGE. Proteins were transferred to PVDF membranes via semidry electrophoretic transfer, blocked with 5% skim milk, and incubated with primary antibody working solution at 4\u0026deg;C overnight. After the addition of the HRP-conjugated secondary antibody working solution for 1 h at room temperature, the target bands were visualized via an enhanced chemiluminescence (ECL) substrate. The antibody dilutions used were as follows: c-Fos (1:1500), NFATc1 (1:1500), Cathepsin K (1:1500), MMP-9 (1:1500), and GAPDH (1:1500) and an HRP-IgG secondary antibody (1:5000).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(8) Analytics\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStatistical analysis was performed with GraphPad Prism version 8. Continuous data are presented as the means \u0026plusmn; standard deviations (means \u0026plusmn; SDss). Student\u0026apos;s t test was used to compare differences between two groups; one-way analysis of variance (ANOVA) with Tukey\u0026apos;s post hoc test was used for multiple group comparisons. Statistical significance was set at P \u0026lt; 0.05.\u003c/p\u003e"},{"header":"2 Results","content":"\u003cp\u003e\u003cstrong\u003e2.1 Osteoclastogenesis-associated inflammatory factors are highly expressed in bone cyst tissue\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAs shown in Table 2, the relative mRNA expression levels of the osteoclastogenesis-associated inflammatory factors TNF-\u0026alpha;, IL-1\u0026beta;, IL-6, and IL-8 were greater in the bone cyst tissue than in the adjacent normal bone tissue (P \u0026lt; 0.05).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2. Statistical results of the determination of the expression of osteoclast differentiation-associated inflammatory factors in paracancerous bone tissues and bone cyst tissues are presented in Table (mean\u0026plusmn;SD).\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 123px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eGroup\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 114px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTNF-\u0026alpha; mRNA\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eRelative Expression Level\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 134px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eIL-1\u0026beta; mRNA\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eRelative Expression Level\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 123px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eAdjacent Normal Bone Tissue\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e（\u003c/strong\u003e\u003cstrong\u003en=21\u003c/strong\u003e\u003cstrong\u003e）\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 114px;\"\u003e\n \u003cp\u003e1.00\u0026plusmn;0.11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 134px;\"\u003e\n \u003cp\u003e1.00\u0026plusmn;0.13\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 123px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eBone Cyst Tissue\u003c/strong\u003e\u003cstrong\u003e（\u003c/strong\u003e\u003cstrong\u003en=21\u003c/strong\u003e\u003cstrong\u003e）\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 114px;\"\u003e\n \u003cp\u003e37.52\u0026plusmn;1.11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 134px;\"\u003e\n \u003cp\u003e28.44\u0026plusmn;0.76\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 123px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003et\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 114px;\"\u003e\n \u003cp\u003e150.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 134px;\"\u003e\n \u003cp\u003e163.10\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 123px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eP\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 114px;\"\u003e\n \u003cp\u003e\u0026lt;0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 134px;\"\u003e\n \u003cp\u003e\u0026lt;0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2. Continued: Expression of Osteoclast Differentiation-Associated Inflammatory Factors in Adjacent Normal Bone Tissue and Bone Cyst Tissue (Mean \u0026plusmn; SD)\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 123px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eGroup\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 114px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eIL-6 mRNA\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eRelative Expression Level\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eIL-8 mRNA\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eRelative Expression Level\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 123px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eAdjacent Normal Bone Tissue\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e（\u003c/strong\u003e\u003cstrong\u003en=21\u003c/strong\u003e\u003cstrong\u003e）\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 114px;\"\u003e\n \u003cp\u003e1.00\u0026plusmn;0.12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e1.00\u0026plusmn;0.16\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 123px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eBone Cyst Tissue\u003c/strong\u003e\u003cstrong\u003e（\u003c/strong\u003e\u003cstrong\u003en=21\u003c/strong\u003e\u003cstrong\u003e）\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 114px;\"\u003e\n \u003cp\u003e17.36\u0026plusmn;0.82\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e5.56\u0026plusmn;0.45\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 123px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003et\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 114px;\"\u003e\n \u003cp\u003e90.46\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e43.75\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 123px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eP\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 114px;\"\u003e\n \u003cp\u003e\u0026lt;0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e\u0026lt;0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003e2.2 EgAgB enhances osteoclast differentiation in RAW 264.7 cells.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAs shown in Figure 1 and Table 3, TRAP staining revealed significant increases in the percentage of TRAP-positive cells in the induction + EgAgB group: Concentrations 1, 2, and 3 versus the induction group (P \u0026lt; 0.05); in the induction + EgAgB group: Concentrations 2 and 3 versus Concentration 1 (P \u0026lt; 0.05); and in the induction + EgAgB group: Concentration 3 versus Concentration 2 (P \u0026lt; 0.05).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 3. Statistical results of the TRAP staining assay for the differentiation of RAW 264.7 cells into osteoclasts (mean\u0026plusmn;SD, n=6)\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 159px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eGroup\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 127px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePercentage of TRAP-Positive Cells(%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 159px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eInduction Group\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 127px;\"\u003e\n \u003cp\u003e22.0\u0026plusmn;1.4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 159px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eInduction+EgAgB: Concentration 1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 127px;\"\u003e\n \u003cp\u003e41.4\u0026plusmn;1.2\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 159px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eInduction+EgAgB: Concentration 2\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 127px;\"\u003e\n \u003cp\u003e57.8\u0026plusmn;1.8\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 159px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eInduction+EgAgB: Concentration 3\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 127px;\"\u003e\n \u003cp\u003e83.2\u0026plusmn;2.2\u003csup\u003eabc\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 159px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eF\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 127px;\"\u003e\n \u003cp\u003e1405.00\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 159px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eP\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 127px;\"\u003e\n \u003cp\u003e\u0026lt;0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eNotes:\u003c/p\u003e\n\u003cp\u003e⁰Significantly different vs. induction group, P \u0026lt; 0.05;\u003c/p\u003e\n\u003cp\u003eᵇSignificantly different vs. Induction + EgAgB: Concentration 1, P \u0026lt; 0.05;\u003c/p\u003e\n\u003cp\u003eᶜSignificantly different vs. Induction + EgAgB: Concentration 2, P \u0026lt; 0.05.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.3 EgAgB Stimulation Upregulates Secretion of Osteoclastogenesis-Associated Inflammatory Factors in RAW 264.7 Cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAs shown in Table 4, significantly elevated levels of TNF-\u0026alpha;, IL-1\u0026beta;, IL-6, and IL-8 were observed in the Induction Group, Induction + EgAgB: Concentrations 1, 2, and 3 compared with those in the Control Group (P \u0026lt; 0.05); furthermore, all three antigen concentration groups presented higher levels of these inflammatory factors than did the Induction Group alone (P \u0026lt; 0.05), with Concentrations 2 and 3 showing increased levels versus Concentration 1 (P \u0026lt; 0.05) and Concentration 3 demonstrating further elevation versus Concentration 2 (P \u0026lt; 0.05).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 4. Statistical results for the measurement of the levels of osteoclast differentiation-related inflammatory factors in the supernatants of RAW 264.7 cell cultures (mean\u0026plusmn;SD, n=6)\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 137px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eGroup\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 124px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTNF-\u0026alpha;\u003c/strong\u003e\u003cstrong\u003e（\u003c/strong\u003e\u003cstrong\u003eng/ml\u003c/strong\u003e\u003cstrong\u003e）\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 146px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eIL-1\u0026beta;\u003c/strong\u003e\u003cstrong\u003e（\u003c/strong\u003e\u003cstrong\u003eng/ml\u003c/strong\u003e\u003cstrong\u003e）\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 137px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eControl Group\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 124px;\"\u003e\n \u003cp\u003e16.47\u0026plusmn;0.52\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 146px;\"\u003e\n \u003cp\u003e21.22\u0026plusmn;0.43\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 137px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eInduction Group\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 124px;\"\u003e\n \u003cp\u003e123.64\u0026plusmn;1.11\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 146px;\"\u003e\n \u003cp\u003e56.53\u0026plusmn;0.71\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 137px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eInduction+EgAgB: Concentration 1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 124px;\"\u003e\n \u003cp\u003e144.35\u0026plusmn;1.34\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 146px;\"\u003e\n \u003cp\u003e88.45\u0026plusmn;0.84\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 137px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eInduction+EgAgB: Concentration 2\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 124px;\"\u003e\n \u003cp\u003e158.36\u0026plusmn;1.41\u003csup\u003eabc\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 146px;\"\u003e\n \u003cp\u003e95.76\u0026plusmn;0.85\u003csup\u003eabc\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 137px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eInduction+EgAgB: Concentration 3\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 124px;\"\u003e\n \u003cp\u003e174.48\u0026plusmn;1.55\u003csup\u003eabcd\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 146px;\"\u003e\n \u003cp\u003e102.34\u0026plusmn;0.91\u003csup\u003eabcd\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 137px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eF\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 124px;\"\u003e\n \u003cp\u003e15319.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 146px;\"\u003e\n \u003cp\u003e11637.00\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 137px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eP\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 124px;\"\u003e\n \u003cp\u003e\u0026lt;0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 146px;\"\u003e\n \u003cp\u003e\u0026lt;0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eNotes:\u003c/p\u003e\n\u003cp\u003e⁰Significantly different vs. control group, P \u0026lt; 0.05;\u003c/p\u003e\n\u003cp\u003eᵃSignificantly different from the induction group, P \u0026lt; 0.05;\u003c/p\u003e\n\u003cp\u003eᵇSignificantly different vs. Induction + EgAgB: Concentration 1, P \u0026lt; 0.05;\u003c/p\u003e\n\u003cp\u003eᶜSignificantly different vs. Induction + EgAgB: Concentration 2, P \u0026lt; 0.05.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 4. Continued: Levels of Osteoclast Differentiation-Associated Inflammatory Factors in Culture Supernatants of RAW 264.7 Cells (Mean \u0026plusmn; SD; n= 6)\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 161px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eGroup\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 124px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eIL-6\u003c/strong\u003e\u003cstrong\u003e（\u003c/strong\u003e\u003cstrong\u003eng/ml\u003c/strong\u003e\u003cstrong\u003e）\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 122px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eIL-8\u003c/strong\u003e\u003cstrong\u003e（\u003c/strong\u003e\u003cstrong\u003eng/ml\u003c/strong\u003e\u003cstrong\u003e）\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 161px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eControl Group\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 124px;\"\u003e\n \u003cp\u003e31.33\u0026plusmn;0.37\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 122px;\"\u003e\n \u003cp\u003e25.82\u0026plusmn;0.32\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 161px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eInduction Group\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 124px;\"\u003e\n \u003cp\u003e58.53\u0026plusmn;0.42\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 122px;\"\u003e\n \u003cp\u003e61.23\u0026plusmn;0.24\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 161px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eInduction+EgAgB: Concentration 1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 124px;\"\u003e\n \u003cp\u003e63.52\u0026plusmn;0.53\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 122px;\"\u003e\n \u003cp\u003e66.37\u0026plusmn;0.28\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 161px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eInduction+EgAgB: Concentration 2\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 124px;\"\u003e\n \u003cp\u003e67.44\u0026plusmn;0.56\u003csup\u003eabc\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 122px;\"\u003e\n \u003cp\u003e70.12\u0026plusmn;0.30\u003csup\u003eabc\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 161px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eInduction+EgAgB: Concentration 3\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 124px;\"\u003e\n \u003cp\u003e72.35\u0026plusmn;0.61\u003csup\u003eabcd\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 122px;\"\u003e\n \u003cp\u003e73.46\u0026plusmn;0.35\u003csup\u003eabcd\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 161px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eF\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 124px;\"\u003e\n \u003cp\u003e6065.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 122px;\"\u003e\n \u003cp\u003e24819.00\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 161px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eP\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 124px;\"\u003e\n \u003cp\u003e\u0026lt;0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 122px;\"\u003e\n \u003cp\u003e\u0026lt;0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eNotes:\u003c/p\u003e\n\u003cp\u003eᵃSignificantly different from the control group, P \u0026lt; 0.05;\u003c/p\u003e\n\u003cp\u003eᵇSignificantly different from the induction group, P \u0026lt; 0.05;\u003c/p\u003e\n\u003cp\u003eᶜSignificantly different vs. Induction + EgAgB: Concentration 1, P \u0026lt; 0.05;\u003c/p\u003e\n\u003cp\u003eᵈSignificantly different vs. Induction + EgAgB: Concentration 2, P \u0026lt; 0.05.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.4 EgAgB Stimulation Upregulates the c-Fos/NFATc1 Signaling Axis in RAW 264.7 Cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAs demonstrated in Fig. 2 and Table 5, significant upregulation of (1) the key osteoclastogenic transcription factor axis c-Fos/NFATc1 and (2) the signature protease markers cathepsin K and MMP-9\u0026mdash;was observed in both the induction group and the induction + EgAgB:Concentration 3 group compared with the control group (P \u0026lt; 0.05). Furthermore, the Induction + EgAgB:Concentration 3 group presented elevated expression of these markers compared with the Induction Group alone (P \u0026lt; 0.05).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 5. Expression Analysis of the Key Osteoclastogenic Transcription Factor Axis c-Fos/NFATc1 in RAW 264.7 Cells (Mean\u0026plusmn;SD, n=6)\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 161px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eGroup\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 124px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ec-Fos Relative Expression\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 122px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eNFATc1 Relative Expression\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 161px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e1 Control Group\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 124px;\"\u003e\n \u003cp\u003e1.00\u0026plusmn;0.12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 122px;\"\u003e\n \u003cp\u003e1.00\u0026plusmn;0.10\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 161px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e2 Induction Group\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 124px;\"\u003e\n \u003cp\u003e1.72\u0026plusmn;0.14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 122px;\"\u003e\n \u003cp\u003e1.64\u0026plusmn;0.12\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 161px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e3 Induction+EgAgB: Concentration 3\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 124px;\"\u003e\n \u003cp\u003e2.23\u0026plusmn;0.15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 122px;\"\u003e\n \u003cp\u003e2.58\u0026plusmn;0.16\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 161px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eF\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 124px;\"\u003e\n \u003cp\u003e121.70\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 122px;\"\u003e\n \u003cp\u003e227.40\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 161px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eP\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 124px;\"\u003e\n \u003cp\u003e\u0026lt;0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 122px;\"\u003e\n \u003cp\u003e\u0026lt;0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eNotes:\u003c/p\u003e\n\u003cp\u003eᵃSignificantly different from the control group, P \u0026lt; 0.05;\u003c/p\u003e\n\u003cp\u003eᵇSignificantly different from the induction group, P \u0026lt; 0.05.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 5. Continued: Expression Analysis of the Key Osteoclastogenic Transcription Factor Axis c-Fos/NFATc1 in RAW 264.7 Cells (Mean\u0026plusmn;SD, n=6)\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eGroup\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 143px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCathepsin K\u003c/strong\u003e\u003cstrong\u003eRelative Expression\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 140px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMMP9 Relative Expression\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e1 Control Group\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 143px;\"\u003e\n \u003cp\u003e1.00\u0026plusmn;0.11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 140px;\"\u003e\n \u003cp\u003e1.00\u0026plusmn;0.10\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e2 Induction Group\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 143px;\"\u003e\n \u003cp\u003e2.12\u0026plusmn;0.14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 140px;\"\u003e\n \u003cp\u003e1.52\u0026plusmn;0.13\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e3 Induction+EgAgB: Concentration 3\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 143px;\"\u003e\n \u003cp\u003e2.87\u0026plusmn;0.16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 140px;\"\u003e\n \u003cp\u003e2.34\u0026plusmn;0.14\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eF\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 143px;\"\u003e\n \u003cp\u003e278.20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 140px;\"\u003e\n \u003cp\u003e176.70\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eP\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 143px;\"\u003e\n \u003cp\u003e\u0026lt;0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 140px;\"\u003e\n \u003cp\u003e\u0026lt;0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eNotes:\u003c/p\u003e\n\u003cp\u003eᵃSignificantly different from the control group, P \u0026lt; 0.05\u003c/p\u003e\n\u003cp\u003eᵇSignificantly different from the induction group, P \u0026lt; 0.05\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.5 IL-1\u0026beta; Antagonism Downregulates c-Fos/NFATc1/Cathepsin K/MMP-9 Expression in EgAgB-Stimulated RAW 264.7 Cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAs shown in Fig. 3 and Table 6, significant upregulation of (1) the key osteoclastogenic transcription factor axis c-Fos/NFATc1 and (2) the signature proteases cathepsin K and MMP-9 was observed in the Induction+EgAgB:Concentration 3 group compared with the Induction group (P\u0026lt;0.05). Conversely, the induction+EgAgB:Concentration 3+IL-1\u0026beta; antagonist group presented marked downregulation of these markers compared with the induction+EgAgB:Concentration 3 group (P\u0026lt;0.05).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 6. Statistical analysis of c-Fos/NFATc1/Cathepsin K/MMP-9 expression levels in RAW 264.7 cells (means\u0026plusmn;SDs, n=6)\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 173px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eGroup\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ec-Fos Relative Expression\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 122px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eNFATc1 Relative Expression\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 173px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e1 Control Group\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003e1.00\u0026plusmn;0.12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 122px;\"\u003e\n \u003cp\u003e1.00\u0026plusmn;0.11\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 173px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e2 Induction Group\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003e1.75\u0026plusmn;0.14\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 122px;\"\u003e\n \u003cp\u003e1.66\u0026plusmn;0.12\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 173px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e3 Induction+EgAgB: Concentration 3\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003e2.36\u0026plusmn;0.13\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 122px;\"\u003e\n \u003cp\u003e2.41\u0026plusmn;0.14\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 173px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e4 Induction+EgAgB:Concentration 3 +IL-1\u0026beta; Antagonist\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003e1.78\u0026plusmn;0.11\u003csup\u003eac\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 122px;\"\u003e\n \u003cp\u003e1.65\u0026plusmn;0.13\u003csup\u003eac\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 173px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eF\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003e118.40\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 122px;\"\u003e\n \u003cp\u003e126.60\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 173px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eP\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003e\u0026lt;0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 122px;\"\u003e\n \u003cp\u003e\u0026lt;0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eNotes:\u003c/p\u003e\n\u003cp\u003eᵃSignificantly different from the control group, P \u0026lt; 0.05;\u003c/p\u003e\n\u003cp\u003eᵇSignificantly different from the induction group, P \u0026lt; 0.05;\u003c/p\u003e\n\u003cp\u003eᶜSignificantly different from Induction + EgAgB: Concentration 3, P \u0026lt; 0.05.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 6. Continued: Statistical analysis of c-Fos/NFATc1/Cathepsin K/MMP-9 expression levels in RAW 264.7 cells (means\u0026plusmn;SDs, n=6)\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 173px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eGroup\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 134px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCathepsin K Relative Expression\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 111px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMMP9 Relative Expression\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 173px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e1 Control Group\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 134px;\"\u003e\n \u003cp\u003e1.00\u0026plusmn;0.10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 111px;\"\u003e\n \u003cp\u003e1.00\u0026plusmn;0.12\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 173px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e2 Induction Group\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 134px;\"\u003e\n \u003cp\u003e2.10\u0026plusmn;0.12\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 111px;\"\u003e\n \u003cp\u003e1.55\u0026plusmn;0.14\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 173px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e3 Induction+EgAgB: Concentration 3\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 134px;\"\u003e\n \u003cp\u003e2.45\u0026plusmn;0.14\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 111px;\"\u003e\n \u003cp\u003e2.67\u0026plusmn;0.21\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 173px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e4 Induction+EgAgB:Concentration 3 +IL-1\u0026beta; Antagonist\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 134px;\"\u003e\n \u003cp\u003e2.08\u0026plusmn;0.11\u003csup\u003eac\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 111px;\"\u003e\n \u003cp\u003e1.57\u0026plusmn;0.13\u003csup\u003eac\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 173px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eF\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 134px;\"\u003e\n \u003cp\u003e168.90\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 111px;\"\u003e\n \u003cp\u003e123.80\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 173px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eP\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 134px;\"\u003e\n \u003cp\u003e\u0026lt;0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 111px;\"\u003e\n \u003cp\u003e\u0026lt;0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eNotes:\u003c/p\u003e\n\u003cp\u003eᵃSignificantly different from the control group, P \u0026lt; 0.05;\u003c/p\u003e\n\u003cp\u003eᵇSignificantly different from the induction group, P \u0026lt; 0.05;\u003c/p\u003e\n\u003cp\u003eᶜSignificantly different from Induction + EgAgB: Concentration 3, P \u0026lt; 0.05.\u003c/p\u003e"},{"header":"3 Discussion","content":"\u003cp\u003eIncreased osteoclast (OC) differentiation and activity are key events driving bone loss and joint destruction. OCs are multinucleated cells with three or more nuclei that are typically located on trabecular bone surfaces. Their cytoplasm contains high concentrations of vesicles and vacuoles filled with lysosomal acid phosphatase [stained by tartrate-resistant acid phosphatase (TRAP)] and cathepsin K [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Activated OCs are characterized by the formation of a specialized cell membrane called the ruffled border, which opposes the bone surface and facilitates the secretion of acids and lysosomal enzymes for bone resorption [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Concurrently, these cells exhibit elevated expression of matrix metalloproteinases (MMPs) 8 and 9 [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eReceptor activator of nuclear factor-kappa B ligand (RANKL), a member of the tumor necrosis factor (TNF) superfamily, binds to its receptor RANK. In conjunction with macrophage colony-stimulating factor (M-CSF) stimulation, the RANKL\u0026ndash;RANK interaction induces terminal differentiation, fusion, and activation of osteoclasts (OCs) [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. However, RANK lacks intrinsic kinase activity to mediate downstream signaling; consequently, RANKL binding recruits various molecules, including TNF receptor-associated factors (TRAFs) and kinases, such as transforming growth factor-β-activated kinase 1 (TAK1). This recruitment subsequently activates the transcription factors NF-κB, c-Fos, and nuclear factor of activated T cells cytoplasmic 1 (NFATc1), all of which are essential for OC differentiation [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eProinflammatory cytokines, including TNF-α, IL-1β, IL-6, and IL-8, can activate the differentiation of osteoclast precursors into osteoclasts (OCs) [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Like, but independent of RANKL, TNF-α recruits TNF receptor-associated factors (TRAFs), subsequently activating the transcription factors NF-κB, c-Fos, and nuclear factor of activated T cells cytoplasmic 1 (NFATc1) to induce OC differentiation [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Inhibition of G protein-coupled receptor 120 (GPR120) suppresses TNF-α-activated OC differentiation [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. IL-6 and TNF-α synergistically activate osteocyte-mediated OC differentiation and activation, mediating the pathological progression of osteoimmunology through complex mechanisms in rheumatoid arthritis and postmenopausal osteoporosis [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. The IL-6/RANKL axis is crucial for the osteocyte-mediated activation of OC differentiation [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Elevated levels of IL-8 promote breast cancer bone metastasis, and inhibition of IL-8 suppresses this pathological process [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIL-1β is another critical proinflammatory cytokine that plays a pivotal role in pathological bone erosion. It enhances NF-κB-mediated osteoclast (OC) differentiation through mechanisms independent of both RANKL and TRAF6. IL-1β activates OC differentiation and bone resorption via a PKCθ/NF-κB/IL-1β-dependent positive feedback loop [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Treatment with IL-1β upregulates the expression of inducible nitric oxide synthase (iNOS), insulin-like growth factor 2 (IGF-2), and the chemokines CX3CL1 and CXCL7 in mouse bone marrow stem cells, thereby promoting the differentiation of nonosteoclastic cells into OCs during bone erosion in rheumatoid arthritis [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. The NLRP3 inflammasome accelerates osteolysis and bone remodeling in osteoporosis by upregulating IL-1β, whereas blockade of the NLRP3/IL-1β signaling axis attenuates this pathological process [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eAfter entering bone tissue, Echinococcus granulosus cysts (CEs) fail to elicit a typical demarcating reactive fibrous capsule from the host. Instead, CEs invade adjacent bone regions in a manner analogous to that of bone tumors [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. On computed tomography (CT) scans, CE-induced skeletal destruction exhibits a \"moth-eaten\" pattern, with lesional areas demonstrating a \"rat-bite\" morphological appearance [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. CE enhances osteolytic bone erosion by activating osteoclasts (OCs) through Nrf2-mediated suppression, which elevates oxidative stress and inflammation at bone resorption surfaces [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. The tissues surrounding CE-induced bone erosions show increased inflammation, dysregulated osteoimmune responses, and abnormally elevated OC activity [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Following CE infection, patients test positive for antibodies against E. granulosus cyst fluid (EgCF), E. granulosus protoscolex (EgP), and semipurified hydatid cyst fluid native antigen B (EgB). In the present study, analysis of the mRNA expression levels of osteoclast differentiation-associated inflammatory factors in CE bone cyst tissues revealed elevated relative expression levels of TNF-α, IL-1β, IL-6, and IL-8 compared with those in adjacent nonlesioned bone tissue. Stimulation with hydatid antigen B protein upregulated the secretion of osteoclastogenic inflammatory factors (TNF-α, IL-1β, IL-6, and IL-8) in RAW 264.7 cells and enhanced their differentiation into osteoclasts, concurrently increasing the intracellular expression of c-Fos, NFATc1, cathepsin K, and MMP-9. Treatment with an IL-1β antagonist suppressed hydatid antigen B-induced expression of c-Fos, NFATc1, cathepsin K, and MMP-9 in RAW 264.7 cells. These findings suggest that IL-1β antagonism may inhibit pathological osteolytic destruction in skeletal echinococcosis.\u003c/p\u003e\u003cp\u003eOverall, while this study has not delineated whether TNF-α or IL-1β plays a predominant role\u0026mdash;or whether they act synergistically\u0026mdash;in pathological osteolytic destruction during CE infection, our findings establish that Echinococcus granulosus infection promotes osteoclast differentiation and activation in osseous echinococcosis through the IL-1β/c-Fos/NFATc1 signaling axis.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study was performed in line with the principles of the Declaration of\u003c/p\u003e\n\u003cp\u003eHelsinki. Approval was granted by the Ethics Committee of The 1nd Affiliated\u003c/p\u003e\n\u003cp\u003eHospital of Xinjiang Medical University before the study began. The written\u003c/p\u003e\n\u003cp\u003einformed consent has been obtained from the participants involved.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analyzed during the current study are available from\u003c/p\u003e\n\u003cp\u003ethe corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by the Funding District Project of the National Natural Science Foundation of China (No:82260409) and Xinjiang Uygur Autonomous Region Natural Science Foundation Key Projects (No:2021D01D19).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors' contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eYLNE AYH, WLH MH: Study design, data collection, formal analysis, writing\u0026nbsp;–\u0026nbsp;original draft. XZR: Funding acquisition, Writing – review \u0026amp; editing. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot Applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eLundstr\u0026ouml;m-Stadelmann B, Rostami A, Frey CF, et al. Human alveolar echinococcosis-global, regional, and national annual incidence and prevalence rates. Clin Microbiol Infect. 2025;31(7):1139\u0026ndash;45. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.cmi.2025.01.034\u003c/span\u003e\u003cspan address=\"10.1016/j.cmi.2025.01.034\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMonge-Maillo B, Lopez-Velez R. Cystic echinococcosis of the bone. 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Acta Trop. 2023;248:107027. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.actatropica.2023.107027\u003c/span\u003e\u003cspan address=\"10.1016/j.actatropica.2023.107027\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\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":"Echinococcus granulosus, osseous echinococcosis, pathological osteolysis, osteoclast, IL-1β, c-Fos/NFATc1 signaling axis","lastPublishedDoi":"10.21203/rs.3.rs-7059251/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7059251/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground \u003c/strong\u003eEffective therapies for devastating osteolysis in osseous echinococcosis remain elusive, necessitating mechanistic exploration. To elucidate the molecular mechanism by which echinococcal infection promotes osteoclast differentiation and activation via the IL-1β/c-Fos/NFATc1 signaling axis in the pathological osteolysis of osseous echinococcosis.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods\u003c/strong\u003e Retrospective RT‒qPCR analysis was used to quantify the mRNA expression of osteoclastogenesis-associated inflammatory factors (TNF-α, IL-1β, IL-6, and IL-8) in bone cyst tissues from 21 osseous echinococcosis patients versus histologically normal bone adjacent to tumors in 21 matched bone tumor controls. Murine RAW 264.7 monocytes/macrophages were divided into the following groups: (i) Untreated control; (ii) Osteoclast induction (100 ng/mL RANKL + 25 ng/mL M-CSF); (iii-v) induction + hydatid antigen B (25/50/75 ng/mL); and (vi) induction + antigen B (75 ng/mL) + an IL-1β antagonist (canakinumab, 10 ng/mL). TRAP staining revealed osteoclasts (≥3 nuclei), with the percentage of positive cells calculated across ≥5 random fields. ELISA was used to measure cytokine levels in the supernatants; Western blotting was used to quantify c-Fos, NFATc1, cathepsin K, and MMP9 expression. The results\u003cstrong\u003e \u003c/strong\u003eCompared with control tissues, bone cyst tissues presented elevated TNF-α, IL-1β, IL-6, and IL-8 mRNA levels (P \u0026lt; 0.05). Antigen B (25–75 ng/mL) dose-dependently increased the number of TRAP⁺ cells and the levels of inflammatory cytokines compared with those in the induction group (P \u0026lt; 0.05). c-Fos, NFATc1, Cathepsin K, and MMP9 were upregulated in the induction and antigen B (75 ng/mL) groups compared with the control group (P \u0026lt; 0.05), with further elevation in the antigen B (75 ng/mL) group compared with the induction group (P \u0026lt; 0.05). Compared with antigen B (75 ng/mL), canakinumab reversed these protein increases (P \u0026lt; 0.05).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion\u003c/strong\u003e \u003cem\u003eEchinococcal infection promotes pathological osteoclastogenesis and osteolysis through IL-1β/c-Fos/NFATc1 signaling activation.\u003c/em\u003e\u003c/p\u003e","manuscriptTitle":"Mechanistic Role of the IL-1β/c-Fos/NFATc1 Signaling Axis in Echinococcal Infection-Elicited Osteoclastogenesis and Pathological Osteolysis: A Prospective Controlled Trial","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-23 08:25:00","doi":"10.21203/rs.3.rs-7059251/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-07-28T16:38:42+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-07-26T14:44:48+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"68272908253857372857118420578792828580","date":"2025-07-26T12:29:01+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"95259216936054314528404358307870048929","date":"2025-07-21T08:17:29+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-07-17T10:44:25+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-07-09T03:43:17+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-07-08T07:07:42+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Orthopaedic Surgery and Research","date":"2025-07-06T17:20:03+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":"282f0f7d-3584-4ffa-9da4-decb847da704","owner":[],"postedDate":"July 23rd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-12-01T16:00:47+00:00","versionOfRecord":{"articleIdentity":"rs-7059251","link":"https://doi.org/10.1186/s13018-025-06505-5","journal":{"identity":"journal-of-orthopaedic-surgery-and-research","isVorOnly":false,"title":"Journal of Orthopaedic Surgery and Research"},"publishedOn":"2025-11-26 15:57:01","publishedOnDateReadable":"November 26th, 2025"},"versionCreatedAt":"2025-07-23 08:25:00","video":"","vorDoi":"10.1186/s13018-025-06505-5","vorDoiUrl":"https://doi.org/10.1186/s13018-025-06505-5","workflowStages":[]},"version":"v1","identity":"rs-7059251","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7059251","identity":"rs-7059251","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}