Dihydrobaicalein suppresses osteoclast differentiation and mitochondrial transfer to prevent postmenopausal osteoporosis.

Abstract

Background: Postmenopausal osteoporosis (PMOP) is a prevalent metabolic bone disorder driven primarily by excessive osteoclast (OC) activation and enhanced bone resorption. The current therapeutic agents for PMOP remain limited by adverse effects associated with long-term use, underscoring the need for novel pharmacological alternatives with favorable safety profiles. Dihydrobaicalein (DIH), a polyhydroxylated flavanone compound, has demonstrated promising anti-inflammatory, antioxidant, and immunomodulatory activities,yet its therapeutic potential and mechanisms of action in PMOP treatment remain largely unexplored.

Purpose: This study aimed to evaluate the therapeutic efficacy of DIH against PMOP and elucidate the underlying mechanisms from multiple perspectives, including multitarget signaling pathway regulation, modulation of energy metabolism, and inhibition of tunneling nanotube (TNT)-mediated intercellular mitochondrial transfer.

Study design: An integrative approach combining in vivo pharmacological evaluation in an ovariectomized (OVX) mouse model with comprehensive in vitro mechanistic studies using receptor activator of nuclear factor kappa-B ligand (RANKL)-induced osteoclastogenesis was employed.

Methods: For in vivo experiments, OVX mice were randomized into sham, OVX vehicle, estradiol (E₂) positive control, and low- and high-dose DIH (5 and 10 mg/kg) groups, and were treated by intragastric administration for 8 weeks. Bone microarchitecture, histomorphology, mechanical properties, bone metabolic markers, and systemic safety were evaluated. For in vitro experiments, bone marrow-derived macrophages were stimulated with RANKL to induce osteoclast differentiation. The effects of DIH on osteoclast differentiation, bone-resorptive function, and osteogenic differentiation were assessed using cytochemical staining, functional assays, RT-qPCR, Western blotting, and RNA sequencing. Direct molecular targets of DIH were identified through surface plasmon resonance, cellular thermal shift assay, biolayer interferometry, and molecular docking/dynamics simulations. Osteoclast energy metabolism and mitochondrial function were evaluated using a Seahorse extracellular flux analyzer, metabolite quantification, and mitochondrial membrane potential detection. Finally, a coculture system of Mito-DsRed-labeled BMSCs and Mito-EGFP-labeled RAW264.7 cells, together with a Transwell model, was established to investigate the effect of DIH on TNT-mediated intercellular mitochondrial transfer and apoptosis-related signaling.

Results: In vivo, DIH dose-dependently improved bone microarchitecture, restored mechanical properties, and normalized bone metabolic markers in OVX mice, with high-dose DIH (10 mg/kg) achieving efficacy comparable to that of E₂ and showing no evident systemic toxicity. Histological and Western blot analyses confirmed reduced osteoclast numbers and downregulated expression of osteoclast differentiation markers in bone tissue. In vitro, DIH dose-dependently suppressed RANKL-induced osteoclast formation-mainly during the early and middle stages-as well as F-actin ring assembly and bone resorption, while exerting no significant effect on osteogenic differentiation. Mechanistically, DIH directly bound to RANK, RANKL, p38, and p65, competitively disrupted the RANK-RANKL interaction, and concurrently suppressed downstream MAPK, canonical/noncanonical NF-κB, and PI3K/AKT signaling. Energy metabolism analyses revealed that DIH attenuated the RANKL-induced increase in oxidative phosphorylation and glycolysis, reduced mitochondrial membrane potential, and modulated the SIRT1/AMPK/SIRT3 axis. Coculture experiments further demonstrated bidirectional mitochondrial transfer between BMSCs and RAW264.7 cells through TNTs, which was dose-dependently blocked by DIH, thereby abrogating the antiapoptotic protection, upregulating pro-apoptotic proteins, and inducing progressive mitochondrial ultrastructural damage in osteoclast precursors.

Conclusion: DIH exerts multi-target, multi-mechanism bone-protective effects by directly targeting RANK, RANKL, p38, and p65 to suppress osteoclast differentiation, disrupting osteoclast metabolic reprogramming, and blocking TNT-mediated intercellular mitochondrial transfer. Through these coordinated actions, DIH significantly ameliorates bone loss in OVX mice with a favorable safety profile, highlighting its potential as a promising therapeutic candidate for postmenopausal osteoporosis.