Mechanism Of Inhibitory Effect Of Anemonin On Inflammatory Bone Loss Induced By Bacterial Endotoxin

Normal bone re-modelling depends on the balance of osteoclast-mediated bone resorption and osteoblast-mediated bone formation.Osteoclasts are the exclusive cells which are responsible for bone resorption.The increased number of osteoclasts and enhanced bone resorption lead to a variety of metabolic bone diseases,such as osteoporosis and Paget's.One promising strategy for treating osteoporosis is the inhibition of excessive bone resorbing activity-mediated by osteoclasts.It has been reported that anemonin(ANE)exhibits anti-tumor,enhancing immunity,anti-oxidation,antibacterial and anti-inflammation activities.However,the role of ANE in mediating osteoclastogenesis is unknown.This study aimed to investigate whether ANE could suppress RANKL-induced osteoclastogenesis and mitigate inflammatory bone loss in LPS-treated mice model.Firstly,RANKL-induced differentiation of BMMs into osteoclasts was used as in vitro model.The proliferative toxicity of ANE on BMMs was detected by CCK-8assay;the effect of ANE on osteoclast formation and bone resorption was detected by TRAP staining and bone resorption assay,respectively;and the effect of ANE on F-actin ring formation of osteoclasts was detected by immunofluorescence experiment.All data showed that ANE could inhibit the differentiation of osteoclasts induced by RANKL in a concentration-dependent manner.At the concentration of 10 ?M,ANE had no obvious cytotoxicity,and it could inhibit RANKL-induced the formation of F-actin ring and the bone resorption of osteoclasts.To determine the mechanism of ANE in regulating osteoclast formation,the expression levels of osteoclast-related genes affected by ANE were detected by RT-PCR,and the effects of ANE on master transcription factor NFATc1-related pathways including NF-?B,MAPKs and PLC?2/GSK-3? were detected by Western blotting.The results showed that ANE suppressed RANKL-stimulated osteoclast differentiation and bone resorption function by down-regulating the expression of osteoclast master transcriptor NFATc1,as well as its upstream transcriptor c-Fos,by decreasing NF-?B p65 subunit and ERK1/2 phosphorylation.Interestingly,ANE didnot change the phosphorylation and degradation of I?B-? and activation of JNK and p38 MAPKs.However,ANE inhibited the phosphorylation of MSK-1,the downstream target of ERK1/2 and p38 MAPK,which can phosphorylate NF-?B p65 subunit.These results implicated that ANE might suppress NF-?B activition via modulation of ERK1/2 mediated NF-?B phosphorylation.Moreover,ANE treatment did not affect phoshphorylation of GSK-3? and PLC?2.In addition,RT-PCR results showed that ANE could down-regulate the mRNA expression of osteoclast-specific genes including DC-STAMP,?v-Integrin and Atp6v0d2.ANE directly suppressed NFATc1 transcription by inhibition of Blimp-1 expression,and the subsequent enhancement of the expression of NFATc1 negative regulators,Bcl-6 and IRF-8.Finally,the effect of ANE on bone tissue of mice was studied using LPS-induced inflammatory bone loss in vivo model.Micro-CT showed that ANE treatment significantly improved trabecular bone parameters.HE and TRAP staining assay showed that ANE treatment can effectively alleviate LPS-induced the destruction of bone tissue structure and osteoclast activation in mice.In summary,these data indicated that ANE can attenuate RANKL-induced osteoclastogenesis and ameliorate LPS-induced inflammatory bone loss in mice through modulation of NFATc1 via ERK1/2-mediated NF-?B phosphorylation and Blimp1 signal pathway.ANE may provide new treatment options for osteoclast-related diseases.