Supplementary file 1_Astragaloside IV modulates oxidative stress and osteoimmune–Wnt signaling in ovariectomized rats: an integrated study of RNA sequencing, molecular docking, and experimental validation.docx

BackgroundPostmenopausal 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.
MethodsFemale 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).
ResultsOVX 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.
ConclusionAS-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.