Mechanism of Compound Ziyin Granules in improving postmenopausal osteoporosis by modulating Wnt/β-catenin pathway.

This study investigates the therapeutic effect and underlying mechanism of Compound Ziyin Granules (CZYG) on postmenopausal osteoporosis (PMOP) induced by bilateral ovariectomy in rats. Six-month-old female SD rats were randomly divided into sham-operated (sham) group, ovariectomy (OVX) model group, high-, medium-, and low-dose CZYG groups, and alendronate sodium (AS) group. After 30 days of model establishment, treatment was administered by gavage once daily for 8 weeks, followed by sample collection. Enzyme-linked immunosorbent assay (ELISA) was used to measure serum levels of calcium ions, alkaline phosphatase (AKP), estrogen (E₂), osteoprotegerin (OPG), osteocalcin (BGP), tartrate-resistant acid phosphatase (TRAP), and type Ⅰ procollagen N-terminal propeptide (PINP). Hematoxylin-eosin (HE) staining was used to observe the histopathological changes in the femurs of rats, while micro-computed tomography (micro-CT) was used to analyze the microstructure of the distal femur. Western blot analysis was performed to measure the expression levels of bone metabolism-related proteins, including wingless-type MMTV integration site family member 2 (Wnt2), β-catenin, low-density lipoprotein receptor-related protein 5 (LRP5), glycogen synthase kinase-3β (GSK-3β). The mRNA expression levels of Wnt2, β-catenin, LRP5, GSK-3β, p-GSK-3β were determined by quantitative real-time PCR (qRT-PCR). Thirty days after bilateral ovariectomy, compared to the sham group, the OVX group showed significant increases in body weight and significant decreases in uterine coefficient. After 8 weeks of treatment, compared to the OVX group, CZYG (medium and high doses) and AS reduced body weight, with high-dose CZYG and AS significantly increasing the uterine coefficient. Serum levels of AKP and TRAP were significantly elevated, while levels of calcium, E₂, BGP, and OPG were significantly decreased in the OVX group. Compared to the OVX group, CZYG and AS significantly reduced serum levels of AKP and TRAP, while high-dose CZYG and AS notably increased the levels of E₂, BGP, OPG, and PINP. Micro-CT and HE staining results indicated that CZYG (medium and high doses) and AS significantly increased bone tissue volume, trabecular number, bone mineral density, and improved the microstructure of the femur. Compared to the OVX group, high-dose CZYG and AS significantly upregulated the protein and mRNA expression levels of Wnt2, β-catenin, and LRP5, and downregulated the phosphorylation level of p-GSK-3β. These results suggest that CZYG can improve PMOP by promoting estrogen secretion, improving bone metabolism indicators, increasing trabecular number and bone mineral density. Its mechanism may be related to the regulation of the Wnt/β-catenin signaling pathway.