| Objective To explore the synergistic effect of total flavonoids of epimedium in combination with bone nutrients on the treatment of osteoporosis and to discuss the potential mechanism of combination therapy. Methods A postmenopausal osteoporosis rat model was established by removing the ovaries. Seven rats in each group were set up in the sham-operated group (Sham), model group (OVX), estradiol valerate group (EV), total flavonoid extract of epimedium extract group (T), and total flavonoid extract of epimedium extract with bone nutrients group (TG). After 8 weeks of the treatment, serum bone metabolism markers and inflammatory factors were measured using enzyme-linked immunosorbent assay (ELISA). Micro-CT and biomechanical analysis were used to determine bone density and microstructure. Hematoxylin-eosin staining (H&E) and tartrate resistant acid phosphatase staining (TRAP) were used to determine bone pathological damage and osteoclastic bone resorption. Immunohistochemistry and real-time fluorescence quantitative polymerase chain reaction (qRT-PCR) were used to determine gene transcriptions and expressions of TLR4, PI3K, and AKT in the bone. Results Compared with the OVX group, the EV, T, and TG groups showed an increased number of bone trabeculae, neat arrangement and more complete structure, significantly improved serum bone metabolic indexes, serum inflammatory factors, bone trabecular parameters, bone biomechanical parameters and the number of osteoclasts (P<0.05), and significantly higher positive regions and mRNA expressions of PI3K and AKT (P<0.05). In addition, the expressions of TLR4 and mRNA were significantly lower (P<0.05). Compared to those in the T group, the pharmacodynamic effects were more significant in the TG group (P<0.05), with significantly lower TLR4 mRNA expression (P<0.05) and significantly higher PI3K mRNA expression (P<0.05). Conclusion Total flavonoids of epimedium combined with bone nutrients synergistically exerts pharmacological effects on the TLR4/PI3K/AKT signaling pathway to alleviate the symptoms of osteoporosis in OVX rats. |