Crebanine protects against ovariectomy-induced bone loss by targeting Sirt1 to interfere with NF-κB acetylation and ROS activity

Osteoporosis, the most prevalent skeletal disorder, is primarily driven by aberrantly increased osteoclast formation and/or activity. Targeting hyperactive osteoclasts remains the cornerstone of current therapeutic strategies. Crebanine (Cre), a natural isoquinoline-derived alkaloid with diverse pharmacological activities, has not yet been explored for osteoporosis treatment. This study aimed to evaluate the therapeutic potential of Cre against ovariectomy (OVX)-induced osteoporosis and elucidate its underlying mechanisms. Cre dose-dependently inhibited in vitro osteoclast differentiation, actin ring formation, and bone resorption by downregulating nuclear factor of activated T cells 1 (NFATc1) and key osteoclast-related genes. Simultaneously, Cre enhanced osteoblast differentiation and mineralization, upregulated osteoblast marker genes, and restored hydrogen peroxide-impaired alkaline phosphatase (ALP) activity impaired by hydrogen peroxide, indicating dual regulation of bone remodeling. Mechanistically, Cre activated sirtuin 1 (Sirt1), promoting p65 deacetylation, inactivated IκB kinase (IKK), and stabilized IκBα, thus inhibiting nuclear factor-kappaB (NF-κB) signaling. Additionally, Cre reduced reactive oxygen species (ROS) by upregulating antioxidant enzymes (heme oxygenase-1 (HO-1), catalase) and suppressing nicotinamide adenine dinucleotide phosphate (NADPH) oxidases (NOX1/4). Furthermore, Cre specifically bound to the predicted site of receptor activator of NF-κB (RANK), blocking RANK ligand (RANKL)-RANK interaction and disrupting downstream protein kinase B (Akt) and mitogen-activated protein kinase (MAPK) signaling pathways. In the OVX mouse model, Cre significantly attenuated bone loss and osteoclastogenesis. Crucially, Cre showed no toxicity in liver or kidney function tests. Collectively, these findings demonstrate that Cre exerts dual therapeutic effects, inhibiting osteoclastogenesis via Sirt1-mediated NF-κB/ROS suppression and promoting osteoblast activity, providing a promising therapeutic strategy for osteoporosis. 1. Crebanine inhibits osteoclast formation via Sirt1-mediated suppression of NF-κB and ROS pathways while enhancing osteoblast activity, balancing bone remodeling. 2. Activates Sirt1 to deacetylate NF-κB (p65) and stabilize IκBα, blocking osteoclastogenesis and oxidative stress. 3. Direct Sirt1/RANK binding confirmed by SPR and docking, disrupting RANKL signaling and downstream Akt/MAPK pathways.