Cajaninstilbene Acid targets TNF-α-mediated mitochondrial translocation to restore osteogenic-osteoclastic coupling equilibrium and ameliorate estrogen-deficient osteoporosis

CSA ameliorates estrogen-deficient osteoporosis without hepatotoxicity or nephrotoxicity. • CSA bidirectionally inhibits osteoclastogenesis and promotes osteogenesis. • CSA directly targets TNF-α to restore bone coupling balance. • CSA upregulates MIRO1 and promotes mitochondrial transfer from osteoblasts to osteoclasts. Background Oestrogen-deficient osteoporosis features an imbalanced bone formation–resorption coupling with excessive osteoclast activation and impaired osteoblast function. Compounds that bidirectionally regulate bone metabolism remain rare among natural small molecules. Cajaninstilbene acid (CSA) from Cajanus cajan leaves exerts regulatory effects on bone metabolism and anti-inflammatory activity, but its mechanism in restoring bone homeostasis via intercellular mitochondrial transfer remains unclear. Purpose To investigate the therapeutic effect of CSA on oestrogen-deficient osteoporosis and reveal its molecular mechanism by targeting TNF-α-mediated mitochondrial transfer to rebalance osteogenesis and osteoclastogenesis. Materials Ovariectomised (OVX) mice were used as the estrogen-deficient osteoporosis model and treated with Cajaninstilbene Acid (CSA) for 6 weeks. Bone microstructure and metabolism were evaluated by micro-computed tomography (micro-CT), histological staining and immunohistochemistry (IHC). Primary bone marrow macrophages (BMMs) and bone marrow mesenchymal stem cells (BMSCs) were cultured for osteoclastic and osteogenic differentiation. Differentiation was assessed by tartrate-resistant acid phosphatase (TRAcP), alkaline phosphatase (ALP) and alizarin red staining (ARS). Key targets were screened by transcriptomic analysis and validated by molecular docking and surface plasmon resonance (SPR). Mitochondrial transfer and activity were observed by co-culture, fluorescence labelling and JC-1 staining. Protein and gene expression were determined by Western blot (WB) and quantitative real-time polymerase chain reaction (qPCR). Results CSA significantly increased bone mineral density and improved trabecular microarchitecture in OVX mice without obvious hepatorenal toxicity. In vitro, CSA dose-dependently suppressed RANKL-induced osteoclastogenesis by downregulating NFATc1 and CTSK, while enhancing osteoblast differentiation by upregulating RUNX2, OCN and OPG. Mechanistically, CSA directly bound and inhibited TNF-α, upregulated the mitochondrial transporter MIRO1, and restored mitochondrial transfer from osteoblasts to osteoclasts, reversing the disrupted bone coupling balance. Conclusion CSA bidirectionally inhibits bone resorption and promotes bone formation by blocking TNF-α signalling and restoring MIRO1-mediated mitochondrial transfer, effectively ameliorating oestrogen-deficient osteoporosis. This study provides pharmacological support and novel mechanistic insights for CSA as a promising natural candidate for osteoporosis treatment. Download: Download high-res image (189KB) Download: Download full-size image