Study On The Improvement Of Bone Destrcution Treated By Fengshi Qutong Capsule In Rheumatoid Arthritis

ObjectiveUsing collagen-induced arthritis（CIA）rat model and receptor activator of nuclear factorκB ligand（RANKL）in vitro induced osteoclast（OC）culture system,to observe the intervention effect of FSQTC on RA bone destruction and explore its influence on OC differentiation and activation function,as well as its regulatory effect on c-fos/NFATc1,the signal pathway of osteoclast differentiation induced by RANKL.Method（1）In vivo:establish the CIA rat model,FSQTC intervention was given different doses(0.25,0.5,1 g·kg^-1·d^-1)and methotrexate（MTX）was used as the positive control drug.To evaluate the therapeutic effect of FSQTC on arthritis in CIA rats by observing the clinical symptoms of arthritis and collecting the clinical scores and incidence.Through X-ray and micro-CT imaging changes and bone metrology statistics,the damage of FSQTC on inflammatory joint bone in CIA rats was evaluated.The number and fusion index of OC in inflammatory joints were observed by TRAP staining.The protein expression levels of OC-activated markers CTSK,MMP-9,β3-integrin and differentiation regulators RANKL and OPG in inflammatory joints were detected by Western Blot.（2）In vitro:mouse mononuclear macrophages leukemia cells（RAW264.7）were induced to form osteoclasts by RANKL,and the effect of FSQTC on RAW264.7 cell activity was detected by MTT method.TRAP staining was used to observe the formation of TRAP-positive polykaryocytes and OC fusion index.OC activation and skeletal changes were observed with actin ring staining.Western Blot,RT-pcr and immunofluorescence assay were used to detect the influence of FSQTC on the expression levels of OC activation markers（TRAP,CTSK,MMP-9 andβ3-integrin）and activation regulatory signaling molecules c-Fos,NFATc1 protein and gene.Results（1）FSQTC can obviously improve the symptoms of inflamed joint redness and deformity in CIA rats,and reduce the clinical score and morbidity.It also improved the degree of inflammatory joint imaging lesions and abnormal bone metrology indicators.FSQTC can reduce the degree of OC fusion in inflammatory joints,as well as the protein levels of OC activation markers CTSK,MMP-9,andβ3-integrin.In addition,FSQTC can reduce RANKL content,increase OPG expression level and reduce RANKL/OPG ratio.（2）FSQTC significantly inhibited the differentiation and activation of OC induced by RANKL and the formation of actin rings.It also decreased the protein and gene expression of OC activation markers TRAP,CTSK,MMP-9,andβ3-integrin,as well as the nuclear translocation and gene expression levels of c-fos and NFATc1.ConclusionIn this study,through CIA rat model and in vitro RANKL-induced OC culture system,the improvement effect of FSQTC on CIA bone destruction was proved.The mechanism of action can be summarized as down-regulation of RANKL level,elevation of OPG level,inhibition of c-Fos and NFATc1nucleation,inhibition of osteoclast formation,and reduction of bone destruction.This study revealed the molecular mechanism of FSQTC inhibiting osteoarthritis destruction,which provided a theoretical basis for the development and utilization of FSQTC as an anti-RA bone destruction drug.