The Mechanism Of Scutellarein In The Inflammatory Osteolysis Disorders

Background and aims: Osteoporosis,arthritis,Peget’s disease,bone tumor,periprosthetic joint infection and periprosthetic loosening have a common characteristic of osteolysis,which are characterized by enhanced osteoclastic bone resorptive function.There are many kinds of cells that can promote the bone formation,but osteoclast is the unique cells that can do bone resorption.At present,there are several kinds of drugs used to prevent and treat osteolysis induced by inflammation.However,based on the effect of side effects and its curative effect,it is very urgent to develop drugs that can inhibit osteolysis induced by inflammation and have less side effects and better curative effect.Chinese herbal monomer is a natural active product,among which most of the flavonoids have anti-inflammatory effects,and have the advantages of small side effects,appropriate price and so on,which has become the treatment of anti-inflammatory bone loss drugs.Scutellarein(Scu)is an active component of Erigeron breviscapus.It belongs to flavonoids and has anti-inflammatory and anti-tumor biological effects.In this study,we found that Scu could inhibit osteoclast formation and bone resorption in vitro and inhibit LPS-induced inflammatory bone loss in vivo.Experimental methods:(1)In vitro experiment: 1)Bone-marrow macrophages(BMMs)were obtained from femurs and tibias of 6-week-old C57/BL6 mice,to detect the effect of scutellarein(Scu)on BMMs proliferation stimulated by M-CSF.Selecting the concentration of Scu has non-toxic on BMMs proliferation to detect scu on osteoclastic differentiation by RANKL-induced.TRAcP-positive multinucleated cells with more than 3 nuclei were calculated as osteoclast cells after TRAcP staining.The effects of osteoclastic number and morphology was observed.2)To investigate the effects of Scu on osteoclastic bone resorptive function,hydroxyapatite coated was used to detect the pit area.To further explore the mechanism of bone resorption,we designed the F-actin ring staining assay.3)During the differentiation of BMMs into osteoclasts,different concentrations of Scu were treated,and real-time PCR was used to detect the effects of Scu on the expression of osteoclast-specific genes.4)To explore the molecular mechanism of the effect of Scu on osteoclastic formation and bone resorptive function,we used Western Blot to detect the signal pathway of osteoclastic formation and differentiation and bone resorptive function.The proteins of two classical signal pathway,MAPK and NF-κB pathway,were detected to determine on which signaling pathway of Scu was acting.(2)In vivo experiments: 24 eight-week-old male C57/BL6 mice were randomly divided into four equal groups: Sham group,LPS group,LPS with Scu at low dose(2.5 mg/kg body weight)and high dose(5 mg/kg body weight).After 7 days of treatment,the calvaria of mice were analysed by microCT and histomorphology analysis.Result:(1)In vitro experiment: 1)Scu could inhibit RANKL-induced osteoclastic differentiation and formation.2)Scu could inhibited boneresorptive funtion of osteoclast.3)Scu could suppress the expression of osteoclast specific genes by RANKL-induced.4)Scu could attenuate NF-κB pathway to affect osteoclastogenesis.(2)In vivo experiment: Scu could reduce the bone loss of osteolysis inudced by LPS.Conclusion: Together our data demonstrated that scutellarein inhibits RANKL-induced osteoclastogenesis by suppressing the NF-κB,c-Fos and NFATc1 signaling pathway and that it is a promising agent for the treatment of osteolytic diseases.