Cucumber seed polypeptides regulate RANKL-induced osteoclastogenesis through OPG/RANKL/RANK and NF-κB

Abstract

Postmenopausal osteoporosis (PMOP) is a common disease that endangers the health of elderly women. Cucumber seeds have shown excellent therapeutic effects on PMOP, but the mechanism of cucumber seed peptide (CSP) remains unclear. The expression levels of NF-κB and osteoclast-related genes were detected by RT-qPCR. The levels of apoptosis-related proteins were detected by Western blotting. Nuclear translocation of NF-κB p65 and osteoclast formation were detected by immunofluorescence and tartrate-resistant acid phosphatase (TRAP) staining, respectively. ELISA was used to detect the expression levels of OPG, M-CSF, and RANKL. Hematoxylin–eosin (H&E) and TRAP staining were used to observe the effects of CSP on bone formation. In RAW264.7 cells, CSP (0.4 mg/L, 4 mg/L, and 40 mg/L) effectively inhibited the expression of osteoclast-related genes (Cathepsin-K, MT1-MMP, MMP-9, and TRAP). TRAP-positive multinucleated giant cells gradually decreased. Furthermore, NF-κB pathway activation downstream of RANK was inhibited. In bone marrow stromal cells (BMSCs), the expression levels of M-CSF and RANKL gradually decreased, and OPG gradually increased with increasing CSP concentrations. Treatment of RAW264.7 cells with pyrrolidine dithiocarbamate (PDTC, an inhibitor of NF-κB) prevented the formation of osteoclasts. Treatment with different concentrations of CSP effectively decreased the levels of RANKL and M-CSF in rat serum and increased the expression of OPG in the oophorectomy (OVX) rat model. Furthermore, different concentrations of CSP could ameliorate the loss of bone structure and inhibit the formation of osteoclasts in rats. CSP inhibits osteoclastogenesis by regulating the OPG/RANKL/RANK pathway and inhibiting the NF-kB pathway.