Calycosin, the main extract from the traditional Chinese medicine (TCM) Astragalus membranaceus, has demonstrated anti-osteoporotic properties in ovariectomized (OVX) mice. However, the specific pathways through which it prevents osteoporosis remain unexplored. This study aimed to investigate the pathways by which calycosin promotes autophagy in bone marrow mesenchymal stem cells (BMSCs) and alleviates ovariectomy-induced osteoporosis. Mice were divided into three groups: sham, OVX, and OVX + calycosin. Following a 12-week intervention period, assessments included analysis of bone microstructure, serum concentrations of LC3II and ALP, and evaluation of Trap expression in femoral tissue. Immunohistochemical staining was used to assess the expression levels of PI3K, Runx2, and Beclin-1 in bone tissue. Additionally, levels of Runx2, ALP, p-PI3K, PI3K, mTOR, p-mTOR, Beclin-1, and ULK1 were analyzed. Osteogenic differentiation of BMSCs was evaluated using ALP and Alizarin red staining. OVX significantly impaired BMSCs osteogenic differentiation, resulting in bone loss. In contrast, calycosin increased bone mass, promoted osteogenesis, and reduced cancellous bone loss. Parameters, such as BMD, BV/TV, Tb.N, and Tb.Th, were significantly higher in the OVX + calycosin group compared to the OVX group. Additionally, Tb.Sp was notably reduced in the OVX + calycosin group. Calycosin also upregulated levels of Runx2, ALP, p-PI3K, p-mTOR, ULK1, and Beclin-1. In cellular studies, calycosin promoted BMSCs osteogenesis under OVX conditions
however, this effect was inhibited by LY294002. Calycosin effectively combats bone loss and improves bone structure. Its mechanism likely involves the promotion of autophagy in osteoblasts, thereby stimulating BMSC osteogenic differentiation. This effect may be mediated through the PI3K/Akt/mTOR pathway. These findings suggest that calycosin has the potential to serve as an alternative therapy for treating osteoporosis.