Handelin inhibits osteoclastogenesis and bone loss by targeting lipocalin-2 and restoring autophagy to suppress NF-κB signaling.

BACKGROUND

Excessive osteoclastogenesis is a pivotal pathological process in osteoporosis. Identifying compounds that can effectively inhibit osteoclastogenesis without toxicity is of great therapeutic interest. Handelin, a guaianolide dimer from Chrysanthemum indicum and other Chrysanthemum spp., possesses known anti-inflammatory and antioxidant properties, yet its role in osteoclastogenesis remains unclear. This study aims to investigate the role and mechanism of Handelin in osteoclastogenesis.

METHODS

The effects of Handelin on osteoclast differentiation and function were assessed using Cell Counting Kit-8 (CCK-8), tartrate-resistant acid phosphatase (TRAP) and F-actin ring staining, bone pit assays, real-time quantitative PCR (RT-qPCR), and western blot in vitro. An ovariectomized (OVX) mouse model was employed for in vivo validation, evaluated by Micro-CT, histological staining, and ELISA assays. RNA sequencing (RNA-seq), Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Set Enrichment Analysis (GSEA) analysis, molecular docking, surface plasmon resonance (SPR), cellular thermal shift assay (CETSA), co-immunoprecipitation, immunofluorescence, and autophagy flux assays were employed for mechanistic investigation. Rescue experiments were conducted using recombinant lipocalin-2 (rLCN2) protein and sequestosome-1 (p62) overexpression.

RESULTS

Handelin potently inhibited RANKL-induced osteoclast differentiation and bone resorption in vitro without cytotoxicity and alleviated OVX-induced bone loss in vivo. RNA-seq revealed Handelin downregulated LCN2 and activated autophagy while inhibiting the NF-κB pathway. Handelin directly bounded LCN2, reduced its expression at both mRNA and protein levels in cells and tissues, and restored autophagic flux, weakening the interaction between p62-TRAF6 to block NF-κB signaling. The anti-osteoclastogenic effects of Handelin were partially reversed by rLCN2 supplementation or p62 overexpression. Notably, Handelin did not impair osteogenic differentiation.

CONCLUSION

This study identifies Handelin as a novel inhibitor of osteoclastogenesis that targets the LCN2-autophagy pathway and suppresses NF-κB signaling, highlighting its potential as a therapeutic agent for osteoporosis and related bone diseases.