Abstract
BACKGROUND: Postmenopausal osteoporosis (PMOP) is characterized by high-turnover bone loss and oxidative stress. Astragaloside IV (AS-IV) exhibits antioxidant and immunomodulatory activities, yet its skeletal effects and mechanistic basis in ovariectomized (OVX) models remain incompletely defined. METHODS: Female Sprague-Dawley rats underwent bilateral ovariectomy. Six weeks after surgery, OVX rats were randomized to model, positive control, or AS-IV treatment (20/40/80 mg/kg, gavage, 8 weeks), with sham-operated controls. Serum bone-turnover markers (CTX-I, OCN, PTH) and oxidative stress markers (SOD, MDA) were measured by ELISA. Femora were assessed by micro-CT (BMD, BV/TV, Tb.N, Tb.Sp) and H&E staining. RNA sequencing was performed followed by GO/KEGG enrichment and WGCNA to identify hub genes, and molecular docking was used to evaluate AS-IV-target interactions. Multiplex immunofluorescence in the femoral trabecular bone quantified Lep, Ptgs2, Gfap, Igfbp2, Wnt1, β-catenin, and NF-κB p65 region (mean fluorescence intensity, normalized to Sham). RESULTS: OVX rats developed a clear high-turnover phenotype with trabecular rarefaction, reflected by higher CTX-I and MDA and lower SOD. AS-IV produced a dose-related improvement in these readouts-CTX-I fell, oxidative stress was eased (SOD increased and MDA decreased), and OCN and PTH moved back toward control levels. Histological analysis showed AS-IV treatment partially mitigated the trabecular deterioration observed in OVX rats. However, micro-CT analysis showed no statistically significant differences in trabecular bone structure between AS-IV-treated groups and OVX controls (p > 0.05). RNA-seq identified osteoimmune and Wnt-centered programs and nominated hub genes including Lep, Ptgs2, Gfap, Igfbp2, Il22ra2, Wnt10a, and Wnt1. In femoral trabecular bone, multiplex immunofluorescence revealed increased NF-κB p65 and decreased Wnt1 and Gfap signals in OVX rats compared to Sham. High-dose AS-IV significantly reduced NF-κB p65 and partly restored Wnt1 and Gfap. However, Ptgs2 showed no significant differences across groups, and Igfbp2 and β-catenin were not significantly rescued within this sampling window. Molecular docking suggested favorable binding between AS-IV and selected targets, supporting its multitarget profile. CONCLUSION: AS-IV modulates oxidative stress and osteoimmune-Wnt signaling in OVX rats, with directional improvements in bone turnover markers but without significant micro-CT structural changes. This work provides a foundation for hypothesis-driven investigation of AS-IV in PMOP.