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

  • Phytomedicine. 2026 Jul 25:157:158331. doi: 10.1016/j.phymed.2026.158331.
Shian Hu  1 ,  Junhong Li  1 ,  Yayun Zhang  2 ,  Jianwen Li  1 ,  Shiheng Wang  1 ,  Yan Mou  3 ,  Jian Liu  1 ,  Xuying Sun  1 ,  Peixuan Hu  4 ,  Honglei Kang  5 ,  Meipeng Zhu  6
Affiliations
  • 1. Department of Orthopedics, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China.
  • 2. Department of Trauma Surgery, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China.
  • 3. Department of Gastroenterology, Wuhan Red Cross Hospital, Wuhan 430015, China.
  • 4. Department of Orthopedics, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China. Electronic address: [email protected].
  • 5. Department of Orthopedics, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China. Electronic address: [email protected].
  • 6. Department of Pediatric Surgery, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China. Electronic address: [email protected].
PMID: 42229383 DOI: 10.1016/j.phymed.2026.158331
Abstract

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.

Keywords
Autophagy; Handelin; Lipocalin-2; NF-κB; Osteoclast; Osteoporosis.
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