Mechanism Investigation In Regulatory Effects Of Catalpol On Osteoclastogenesis And Osteoclast-derived Bone Loss

Bone remodeling is a predominant physiological process to sustain the integrity of bone structure and function.The homeostasis of bone remodeling is delicately regulated by osteoclast-derived bone resorption and osteoblast-derived bone formation.Aberrant activation of osteoclastic bone resorption is responsible for many bone diseases,such as osteoporosis,rheumatoid arthritis,periprosthetic osteolysis,and periodontitis.Although many drugs targeting osteoclasts have been developed during the last decades,the adverse effects and high cost limit their clinical application.Therefore,further alternative therapeutic strategies are required for the treatment of osteoclast-related bone diseases.Catalpol is a bioactive iridoid extracted from a traditional herbal medicine Rehmannia glutinosa,which has been used to treat osteoporosis clinically in China for decades.Several studies have revealed that catalpol exhibits various pharmacological properties,including anti-inflammatory,antioxidant,antidiabetic,and antitumor effects.In recent studies,catalpol was show to inhibit LPS-induced inflammatory responses via NF-?B signaling pathway in BV2 microglia or alveolar macrophage,and to suppress AKT signaling pathway in HCT116 or T24 cancer cells.However,the effects of catalpol on osteoclastogenesis and pathological bone destruction have not yet beenfully evaluated.In this study,we assessed the effects of catalpol on RANKL-induced osteoclastogenesis and elucidate the underlying molecular mechanisms during RANKL/RANK signaling.In addition,we investigated the therapeutic potential of catalpol on ovariectomized(OVX)-or lipopolysaccharide(LPS)-induced bone loss in mouse models.To determine the effects of catalpol on osteoclastogenesis,BMMs and RAW264.7cells were induced to become osteoclasts by RANKL in the presence of different concentrations of catalpol.Our data showed that catalpol inhibited RANKL-induced osteoclast formation,especially at the early stage.We next investigated the effects of catalpol on bone-resorptive function of mature osteoclasts in vitro.The results showed that catalpol could inhibit F-actin formation and bone resorption.To elucidate the mechanisms underlying catalpol-mediated inhibition of osteoclast formation and function,we further investigated the main signaling pathways involved in RANKL/RANK signaling cascade.The investigation showed that catalpol upregulated PTEN activity by reducing its ubiquitination and degradation,subsequently suppressing RANKL-induced NF-?B and AKT signaling pathways,leading to an inhibition on NFATc1 induction.Based on our findings that catalpol inhibited osteoclastogenesis in vitro,LPS-and OVX-induced bone loss mouse models were established to evaluate whether catalpol has a protective effect on inflammation or estrogen deficiency-induced osteoporosis in vivo through its anti-osteoclastogenic effect.Micro-CT with 3-dimensional reconstruction images showed extensive bone destruction in the femurs of mice which underwent LPS injection or ovariectomy surgery.However,catalpol treatment protected mice from LPS-and OVX-induced bone loss.TRAP staining indicated that the administration of catalpol to LPS-and OVX-induced mice markedly reduced the number of osteoclasts in the areas of trabecular bone.Our findings suggested thatcatalpol prevents LPS-induced bone erosion and OVX-mediated bone loss by modulating osteoclast activity.In conclusion,for the first time,our study suggests that catalpol suppresses RANKL-induced osteoclastogenesis by suppression of NF-?B and AKT signaling pathways.These inhibitory effects of catalpol are suggested to be mediated by the inhibition of PTEN ubiquitination and degradation.Furthermore,catalpol protects against LPS-and OVX-induced bone loss in vivo through inhibiting osteoclast activity.These results suggest that catalpol might be developed as a promising candidate for treating osteoclast-related bone diseases.