Fabp5 Is the Key Regulator Mediating γ‐ CEHC Differentiation in Osteoblasts and Osteoclasts

Osteoporosis is closely linked to oxidative stress and inflammation, positioning the vitamin E metabolite γ-CEHC, known for its robust antioxidant and anti-inflammatory properties, as a promising therapeutic agent. However, its molecular targets have remained largely unknown. In this study, we characterized the protein targets of γ-CEHC and clarified its role in regulating bone metabolism using an ovariectomized (OVX) mouse model and in vitro assays. Bone morphological analysis and histomorphometry demonstrated that γ-CEHC improves osteoporosis in OVX mice by inhibiting osteoclast differentiation and enhancing osteoblast differentiation. To identify the underlying mechanisms, we employed isothermal thermal proteome profiling (TPP) to map γ-CEHC-interacting proteins, followed by Gene Ontology (GO) and KEGG enrichment analyses. Our findings identified fatty acid-binding protein 5 (Fabp5) as a core target. The direct and specific binding between γ-CEHC and Fabp5 was confirmed through cellular thermal shift assays (CETSA), molecular docking-suggesting hydrogen bonding with Thr63-and Surface Plasmon Resonance (SPR) which showed a strong binding affinity (Kd = 5.24 μM). Furthermore, γ-CEHC was found to suppress LPS-induced M1 macrophage activation and promote M2 polarization, thereby reducing reactive oxygen species (ROS) levels and restoring bone remodeling homeostasis. This study is the first to systematically elucidate the molecular mechanisms of γ-CEHC in bone metabolism, revealing that it acts as a highly selective ligand for Fabp5. These findings provide a novel mechanistic basis for using γ-CEHC and targeting Fabp5 in the treatment of osteoporosis.