RESUMO
Background: Shen-sui-tong-zhi formula (SSTZF) has been used to treat osteoporosis for decades and shows excellent clinical efficacy. This article aims to explore the optimal anti-osteoporotic ingredient and its precise mechanisms in mice models. Methods: In this study, we first screened the optimal anti-osteoporosis fraction of SSTZF extract in vivo, and then further explored the mechanism of its effects both in vivo and in vitro. Ten-week-old female C57BL/6J mice were administrated with each fraction of SSTZF. At 10 weeks after ovariectomy (OVX), femurs were collected for tissue analyses, including histology, micro-CT, biomechanical tests, and immunohistochemistry for ALP, FABP4, and ß-catenin. Additionally, we also evaluated the mRNA expression level of ALP and FABP4 and the protein expression level of ß-catenin after being treated with SSTZF extract in C3H10T1/2 cells. Moreover, we investigated the anti-osteoporosis effect of SSTZF extract on mice with ß-catenin conditional knockout in growth plate chondrocytes (ß-catenin Gli1ER mice) through µCT, histology, and immunohistochemistry analyzes. Results: At 10 weeks after treatment, osteoporosis-like phenotype were significantly ameliorated in SSTZF n-butanol extract (SSTZF-NB) group mice, as indicated by increased trabecular bone area and ALP content, and decreased lipid droplet area and FABP4 content. No such improvements were observed after being treated with other extracts, demonstrating that SSTZF-NB is the optimal anti-osteoporosis fraction. Additionally, the elevated ß-catenin was revealed in both OVX mice and C3H10T1/2 cells with SSTZF-NB administered. Furthermore, a significant osteoporosis-like phenotype was observed in ß-catenin Gli1ER mice as expected. However, SSTZF-NB failed to rescue the deterioration in ß-catenin Gli1ER mice, no significant re-upregulated ALP and downregulated FABP4 were observed after being treated with SSTZF-NB, demonstrating that SSTZF-NB prevents bone loss mainly via ß-catenin signaling. Conclusion: SSTZF-NB enhances osteogenesis mainly via activation of ß-catenin signaling in growth plate chondrocytes. SSTZF-NB is the optimal anti-osteoporosis fraction of SSTZF and it can be considered a salutary alternative therapeutic option for osteoporosis.
RESUMO
BACKGROUND: Bushenhuoxue formula (BSHXF) has shown excellent clinical effects on the treatment of osteoporosis in China. The aim of this study is to determine the anti-osteoporosis effects and precise molecular mechanisms of BSHXF on mouse models. METHODS: Ten-week-old female C57BL/6 J mice were subjected to ovariectomy and provided a daily treatment of BSHXF. At 8 weeks post-surgery, the femurs were harvested for tissue analyses including µCT, histology, qRT-PCR and immunohistochemical (IHC) staining of ß-catenin, ALP and FABP4. To investigate the role of ß-catenin in the anti-osteoporosis effects of BSHXF, relative experiments mentioned above were performed in ß-catenin conditional knockout mice. RESULTS: Ovariectomized (OVX) mice presented severe bone loss and excessive fat accumulation in the chondro-osseous junction underneath the growth plate, with decreased expression of ALP and increased expression of FABP4. BSHXF significantly recovered the OVX-induced abnormal osteogenesis and adipogenesis with the activation of ß-catenin in growth plate chondrocytes. Further, we generated growth plate chondrocyte-specific ß-catenin knockout (ß-cateninGli1ER) mice that exhibited bone loss and fat accumulation in the chondro-osseous junction, similar to the OVX mice. However, BSHXF failed to rescue the osteoporosis-like phenotype in ß-cateninGli1ER mice, indicating the anti-osteoporosis effects of BSHXF act mainly through ß-catenin signaling. No significant restoration of ALP and FABP4 was observed in ß-cateninGli1ER mice after the treatment of BSHXF. CONCLUSIONS: BSHXF attenuates osteoporosis by promoting osteogenic differentiation of growth plate chondrocytes mainly in ß-catenin-dependent manner. BSHXF is considered as a new candidate for the treatment of osteoporosis.
