Anti-osteoporosis effect of Semen Cuscutae in ovariectomized mice through inhibition of bone resorption by osteoclasts

Semen Cuscutae, called Tu-si-zi in Chinese, is a kind of dried mature seed in the Convolvulaceae family. It mainly distributes in China, Korea, Pakistan, Vietnam, India and Thailand. It is used as a kidney-tonifying drug for treatment of aging related diseases such as osteoporosis in traditional Chinese medicine. However, the exact mechanisms on bone resorption are poorly studied.The aim of this study was to investigate the potential effect of Semen Cuscutae on ovariectomy (OVX)-induced osteoporosis in mice and clarify the exact mechanisms by which Semen Cuscutae exert the anti-osteoporosis effect.Qualitative and quantitative analyses of Semen Cuscutae were performed by UPLC-Q-TOF-MS and HPLC-MS/MS, respectively. Changes in bone mineral density (BMD) induced by OVX in mice were measured by dual-energy X-ray absorptiometry and micro-computed tomography (μCT). Tartrate-resistant acid phosphatase (TRAP) staining as well as hematoxylin and eosin (HE) staining were used to observe bone microarchitectural changes. ELISA kits were used to assess the therapeutic effects of Semen Cuscutae on the serum levels of osteoprotegerin (OPG), tartrate-resistant acid phosphatase 5b (TRACP-5b), and receptor activator of nuclear factor-κB (RANKL). The effect of Semen Cuscutae on primary cell viability was assessed using CCK-8 and anti-tartrate phosphatase assays. TRAP staining and actin ring staining were used to observe the effect of Semen Cuscutae on osteoclast differentiation. Western blotting was used to measure the effects of Semen Cuscutae on expressions of NFATC1, c-Src kinase, and c-fos.Results from UPLC-Q-TOF-MS showed that the main components of Semen Cuscutae were flavonoid compounds that included quercitrin, quercetin, hyperoside, caffeic acid, rutin, chlorogenic acid, luteolin, apigenin, kaempferol, isoquercetin, cryptochlorogenic acid, isorhamnetin-3-O-glucoside, and astragalin. After the Semen Cuscutae extract was orally administered to OVX mice, bone density increased (P < 0.01) and bone microstructure was significantly improved (P < 0.01 or 0.05). Additionally, Semen Cuscutae exhibited a significant descending effect in the levels of serum TRACP-5b and RANKL, while there was a significant increase in OPG in the Semen Cuscutae group compared with the OVX group, especially at high doses. Moreover, we found that increasing of c-fos, c-Src kinase, and NFATC1 protein expressions were reversed by Semen Cuscutae in vitro and in vivo.Our results showed that Semen Cuscutae exhibited anti-osteoporosis effects through the c-fos/c-Src kinase/NFATC1 signaling pathway.