Study On Inhibitary Effect And Mechanism Of The Inflammatory-osteoporosis Caused By Osteoclasts In Flavonoid Okanin

The interior of the animal skeleton is made up of a bone matrix.The maintenance of the bone matrix requires dynamic balance of bone formation by osteoblasts and bone resorption by osteoclasts.When the balance is disturbed,it can lead to a range of skeletal diseases.Over-activation of osteoblasts leads to osteosclerosis and bone spurs,while over-activation of osteoclasts leads to reduced bone density and increased brittleness of bones,such as rheumatoid arthritis and osteoporosis.Okanin is reported to be a flavonoid extracted in Kunlun Snowdrop with a wide range of pharmacological activities and biological activities.Studies have shown that Okanin has antioxidant,anti-hypertensive and anti-neuritis activities.However,the role of Okanin in the differentiation of osteoclasts and its bone resorption function remains unclear.In this study,we investigated the effects of Okanin on osteoclast differentiation,formation and osteoclast-mediated bone resorption function,and further clarified the underlying mechanism of okanin inhibiting osteoclastogenesis and investigated the potential therapeutic effects of okanin on osteoporosis using inflammatory bone loss mice model.Mature TRAP positive multinucleated osteoclasts were obtained by isolating and culturing mouse bone marrow derived mononuclear cells(BMMs)using culture medium containg M-CSF and RANKL.The cells were stimulated with different concentrations of Okanin to investigate the effects of Okanin on osteoclast differentiation,maturation and bone resorption.TRAP staining showed that Okanin significantly inhibited the formation of osteoclasts induced by RANKL;F-actin ring staining and bone resorption assays showed that Okanin affected the bone resorption function of osteoclasts by inhibiting the formation of F-actin rings in osteoclasts.Real-time PCR and Western Blotting were used to examine the effects of Okanin on the expression of genes related to osteoclast differentiation and maturation and the activation of key protein pathways.The results showed that in RANKL-stimulated BMMs,Okanin inhibited the induction of the key osteoclastogenic transcription factors c-Fos and NFATc1 and their downstream target genes including TRAP,ATP6v0d2 and MMP-9.Western Blotting further showed that Okanin treatment inhibited the phosphorylation of the NF-κB p65 subunit through the inhibition of IKKβ phosphorylation without altering the degradation of IκB-α and IKKαphosphorylation,implicated that okanin might directly suppressed NF-κB p65 phosphorylation through inhibtion of IKKβ activation.However,Okanin did not influence RANKL-induced phosphorylation of MAPKs(p38,JNK and ERK1/2).Furthermore,the results showed that Okanin inhibited Blimp1 expression and enhanced the expression of NFATc1 negative regulators such as Bcl-6,Maf-B and IRF-8,suggesting that Okanin may inhibit the transcriptional activity of NFATc1 through Blimp-1 signaling.To further investigate the potential therapeutic effects of okanin on osteoporosis in vivo,LPS-induced inflammatory bone loss mice model was established.As results showed,TRAP staining and HE staining of bone sections indicated that okanin treatment inhibited osteoclasts over-activation and attenuated bone destruction in LPS-treated mice.Micro-CT scanning further showed that okanin improved bone loss by LPS in mice.Taken together,in in vitro experiments,the results demonstrated that Okanin inhibits RANKL-induced expression of c-Fos and NFATc1 through RANKL-induced NF-κB early activation by inhibition of IKKβ-mediated NF-κB p65 phosphorylation,thereby suppressing the expression of the downstream genes of c-Fos and NFATc1 such as TRAP,ATP6v0d2 and MMP-9,hindering the formation of the F-actin ring and inhibiting the maturation and bone resorption of osteoclasts.Meanwhile,Okanin also suppressed the expression of Blimp1 and enhanced the expression of NFATc1 negative regulators such as Bcl-6,Maf-B and IRF-8,thereby suppressing the transcription activity of NFATc1.In in vivo experiments,Okanin inhibited LPS-induced hyperactivation of osteoclasts in an in vivo mouse model and ameliorated LPS-induced bone loss.Therefore,these results suggest that Okanin can be considered as a potential therapeutic agent against bone loss disorders mediated by osteoclast over-activation.