Clonorchis sinensis Crude Antigen Suppresses Osteoclast Differentiation via Modulation of the NF-κB and MAPK Signaling Pathway.

BACKGROUND: Clonorchis sinensis crude antigen (CsCA) is recognized for its immunomodulatory capacity in host-pathogen interactions and immune-related pathologies, its direct impact on bone remodeling cells remains underexplored. Although prior studies demonstrate CsCA-mediated suppression of inflammatory bone formation in ankylosing spondylitis, the specific regulatory effects on osteoclastogenesis and associated molecular mechanisms remain elusive. This study aimed to investigate the impact of CsCA on osteoclast differentiation and its potential molecular mechanisms. METHODS: The cytotoxicity of CsCA on mouse bone marrow-derived macrophages (BMMs) was evaluated using the CCK-8 assay. At the established non-cytotoxic dose, the effects of CsCA on osteoclast morphology and activity were observed via TRAcP staining and F-actin ring formation assay. Expression of osteoclast differentiation-related genes and proteins was analyzed by combined RT-qPCR and Western Blot. RNA-seq was performed to identify CsCA-regulated signaling pathways during osteoclast differentiation, followed by validation of key pathway components through Western Blot and immunofluorescence. RESULTS: CsCA exhibited no significant cytotoxicity on BMMs at concentrations ≤ 40 μg/mL. TRAcP staining revealed a significant reduction in osteoclast numbers in CsCA-treated groups. F-actin ring formation assays demonstrated abnormal cytoskeletal structures. RT-PCR results showed significantly downregulated expression of osteoclast differentiation-related genes, including NFATc1, c-Fos, Acp5, CTSK and MMP-9. Western Blot confirmed reduced protein expression levels of NFATc1 and c-Fos. RNA-seq analysis combined with experimental validation by Western Blot and immunofluorescence confirmed that CsCA primarily inhibits osteoclast differentiation through the NF-κB and MAPK signaling pathways. CONCLUSIONS: CsCA inhibits the formation and function of osteoclasts by suppressing the expression of key genes involved in osteoclast differentiation through the inhibition of the NF-κB and MAPK signaling pathways. This study elucidates the mechanism by which CsCA regulates osteoclasts and suggests its translational potential in preventing and treating hyper-resorptive bone diseases such as osteoporosis and bone metastasis.