Sarsasapogenin Inhibits Osteoclastogenesis And Prevents LPS-Induced Osteolysis

Background: Bone homeostasis is a kind of dynamic balance in which the body maintains bone mass within the normal range through various adjustments.It is dually regulated by osteoclasts(Osteoclasts OCs)and osteoblasts.Osteoclasts play an important role.The overactivity of osteoclasts during bone remodeling is closely related to many osteolytic diseases such as osteoporosis,cancer,prosthesis loosening after joint replacement,and bone destruction related to osteoarthritis.Osteoclasts are huge fusion cells with many nuclei,and they exert bone resorption activity.Therefore they are of great significance in bone loss.The increasing number of osteoclasts and the enhancement of bone resorption have significant impacts on osteoclast-related osteolytic diseases.Objective: The current treatments for osteolytic diseases have limitations,and various drugs for the treatment of osteolytic diseases also have more or less negatives.Many scholars have conducted research on natural compounds to treat osteoporosis.Sarsasapogenin is a sapogenin present in the Chinese medical herb Anemarrhena asphodeloides Bunge.Sarsasapogenin has obvious anti-tumor and anti-inflammatory effects.It has been found to inhibit NF-κB and MAPK signal transduction.However,no studies have found its specific role in the field of osteoclasts and bone resorption.Therefore,we guessed whether sarsasapogenin also has an anti-osteoclast effect.This study was to explore whether sarsasapogenin can inhibit osteoclasts and whether it has a preventive effect on osteolysis,and study its pathways and mechanisms in osteoclasts.Methods:The inhibitory effects of sarsasapogenin on osteoclasts and the bone loss in the body were explained by these three aspects: cytology,molecular and zoology.In vitro experiments: The whole bone of 6-8 week old C57/BL6 mice was used to get BMMs.The MTT kit was used to determine the proliferation of BMMs after the processing of sarsasapogenin.When the safe concentration was determined,TRAP staining was used to explore the effect of sarsasapogenin on cell differentiation.F-actin staining,which further explained the sarsasapogenin influence on osteoclast differentiation.The resorption area on the bone slice was used to calculate the degree of bone resorption,with the purpose of detecting the effect of sarsasapogenin on the resorption function of osteoclasts.RT-q PCR confirmed the further inhibitory effect of sarsasapogenin on osteoclast-specific gene expression.Western Blot was used to study protein changes in related signal transduction.In vivo experiment: We constructed an LPS-induced osteolysis model.Twenty-four 8-week-old healthy male wild-type C57/BL6 mice were randomly and equally distributed.The mice are divided into sham,LPS,Low concentration or high concentration sarsasapogenin group.Samples were collected,then they were analyzed for bone mass analysis(micro-CT).H&E staining,TRAP staining and OCs count on bone surface were used for histomorphological analysis.By studying the inhibition of sarsasapogenin on osteoclasts and the rescue of bone resorption in the body,we can study whether sarsasapogenin can be used as a new,safe and reliable clinical drug to treat osteolytic diseases such as osteoporosis.Results: Osteoclast formation was inhibited by sarsasapogenin,and the inhibition was dose-dependent.At the same time,the inhibition occured at an early stage.Sarsasapogenin inhibited F-actin ring and bone resorption in vitro.Sarsasapogenin also inhibited the related gene expression of osteoclasts in vitro.At the same time,sarsasapogenin attenuated the RANKL-induced NF-κB and JNK/MAPK pathways,thereby inhibiting the expression of NFATc1,a transcription factor that is particularly critical for osteoclasts.In the in vivo data,Quantitative analysis showed that as the concentration of sarsasapogenin increased,BV/TV increased,while the porosity decreased significantly.Other analysis further showed that under the intervention of sarsasapogenin,TRAPpositive cells number of the bone surface was also reduced.Conclusion: Our results were consistent: sarsasapogenin treatment can inhibit osteoclast-related activities.It can alleviate bone loss in an LPS-mediated osteolysis model.Sarsasapogenin can inhibit the differentiation and resorption of osteoclasts through NF-κB and JNK/MAPK signaling pathways,thereby significantly prevent bone loss in vivo.Therefore,we inferred that sarsasapogenin can be used as a new therapeutic agent to treat osteolytic diseases.