Study On The Role Of Streptolysin O Derived From Streptococcus Pyogenes On Osteoclasts Differentiation And Bone Resorption Function

Background:Bone is a dynamic organ undergoing continual remodeling to grow,heal damage,and regulate calcium and phosphate metabolism in the body.Bone infection is a serious complication in orthopedics field.Osteoclasts are the sole type of cells bearing a function of bone resorption.Overactivated osteoclasts during bone infections such as bacterial septic,osteomyelitis and implant infections could cause pathological bone destruction,consequently,result in bone non-union and delayed fracture healing.GAS is one of the most important bacterial strains which causing bone infection such as septic arthritis and osteomyelitis,as well as involved in the inflammatory destruction of joints and bones.GAS bone infections are likely to be a continuing and probably increasing problem,and understanding of the interaction of GAS with bone is central to the development of the novel therapeutic strategies for treating antibiotic-resistant and persistent infections.Streptolysin O(SLO)is a well characterized and considered as an important virulence factor being produced nearly by all clinical GAS isolates and overexpressed in invasive infections.However,the exact role of SLO on bone destruction induced by GAS largely remained unknown.Objectives:In this study,Raw264.7 cells as osteoclast precursors were used to establish osteoclast differentiation model in vitro.And then we investigate the effect of SLO on the proliferation,differentiation,fusion,bone resorption activity,mark genes of OCs and intracellular singnal pathway.This may provide cellular and molecular biology data for later targeted treatment of Streptococcus pyogenes-induced bone infections.Materials and Methods:Raw264.7 was used as osteoclast precursors.RANKL and M-CSF were chosed to induce osteoclast differentiation from Raw264.7 cells.Osteoclast differentiation was assessed by TRAP staining after stimulated by different dosages of SLO(0,0.25,0.5,1,2.5μg/ml).Osteoclast fusion was assessed by Actin Cytoskeleton and Focal Adhesion Staining,the cell size and number of nuclei was counted.Bone-resorbing activity was evaluated by osteo assay surface 96-well plates with different concentrations of SLO for 5 days.The expression of osteoclast-specific genes like TRAP,CTR,Integrinβ3,ATP6v0d2,DC-STAMP and MMP9 were evaluated by RT-PCR.The expression of IκBα,p-IκBα,p65,p-p65,Bax,Bcl-2,Caspase-3,c-fos and NFATc1 proteins were evaluated by Western blot.Flow cytometry was used to evaluate the cell apoptosis assay of osteoclast.ResultIn our study,we discovered that SLO decreased RANKL-stimulated osteoclast number,average number of nuclei and osteoclast resorption activity,downgraduated the expression of osteoclastogenesis-associated marker genes including TRAP,CTR,Integrinβ3,ATP6v0d2,DC-STAMP and MMP9,the protein expression of c-fos and NFATc1.It was demonstrated that SLO inhibited RANKL-induced NF-κB pathways by inhibiting phosphorylation of IκBα and p65.Meantime the apoptosis of osteoclast was increased throngh flow cytometry and Bax/Bcl-2/Caspase-3 pathway involved in apoptosis was actived at protein level.ConclusionThese findings provided evidence the SLO deviced from GAS inhibited osteoclast formation,fusion,function through suppressing NF-κB signaling pathway.Meantime SLO induced osteoclast apoptosis by Bax/Bcl-2/Caspase-3 pathway.