Asperosaponin ? Protects Collagen-induced Arthritis From Bone Destruction By Inhibiting Osteoclastogenesis

Background and objectives:Rheumatoid arthritis(RA)is a common autoimmune disease.In the early stage,in the course of this disease,inflammatory cell infiltration first appears around the joints and then develops into bone erosion.Osteoclasts play an vital role in the pathological process of RA joint destruction.Therefore,drugs targeting osteoclasts to protect bone erosion maight become a feasible treatment strategy for RA.Asperosaponin ?(AVI),belongs to triterpenoid saponins,is the main active component of Dipsacus asper.Studies have shown that AVI can promote the proliferation and differentiation of osteoblasts,but its effect on osteoclasts has not been reported,and whether it can relieve related bone destruction diseases by inhibiting osteoclasts is still unknown.In this study,RANKL-induced osteoclasts culture system in vitro and animal model of collagen-induced arthritis in vivo were established respectively to investigate the effects of AVI on osteoclasts and its potential mechanism.Methods:1.In vivo,collagen-induced arthritis mouse model was established,and methotrexate was used as a positive control drug to explore the effect of AVI on joint swelling in mice through the foot swelling score;Micro-CT and HE staining were used to detect the effect of AVI on bone destruction in arthritis mice.The effects of AVI on serum TNF-a and IL-1? were detected by ELISA.TRAP staining,q-PCR and western-blot were used to investigate the effect of AVI on osteoclastogenesis and related genes(TRAP,MMP9,CtsK and ?3-Integrin)and proteins(p-FAK,p-Src,MMP9 and ?3-Integrin)in arthritis mice.2.In vitro,the model of RANKL induced primary bone marrow mononuclear cells differentiation into osteoclasts was established.The effect of AVI on proliferation toxicity of BMMs was detected by CCK8 assay.The effects of AVI on osteoclast formation and bone resorption were investigated by TRAP staining and bone resorption lacuna assay.The effect of AVI on RANKL induced osteoclast actin ring formation was detected by immunofluorescence assay.q-PCR and western-blot were used to detect the effect of A? on the expression of RANKL induced osteoclast formation related genes(TRAP,MMP9,CtsK and ?3-Integrin)and proteins(p-FAK,p-Src,MMP9 and ?3-Integrin).3.In terms of molecular mechanism,the effects of AVI on NF-?B(I?B-? and p65),MAPKs(p38,JNK,ERK 1/2),Akt,c-Fos and NFATcl related to osteoclast formation were detected by qRT-PCR,western-blot and immunofluorescence.Results:1.In vivo,A? can significantly alleviate the degree of joint swelling and bone erosion in arthritis mice,reduce the levels of TNF-a and IL-1?in serum,inhibit the formation of osteoclasts as well as down-regulation the expression levels of genes(TRAP,MMP9,CtsK and ?3-Integrin)and proteins(p-FAK,p-Src,MMP9 and?3-Integrin)which involved in osteoblast formation in arthritic mouse tissues.2.In vitro,A? can significantly inhibit RANKL induced osteoclast formation,bone resorption and actin ring formation,and down-regulate genes expression levels(TRAP,MMP9,CtsK and p3-Integrin)and protein expression levels(p-FAK,p-Src,MMP9 and ?3-Integrin)which involved in osteoclast formation.3.In terms of molecular mechanism,AVI can significantly inhibit the expression of c-Fos and NFATcl,inhibit the nuclear translocation of p65 in NF-?B but without affecting the ubiquitination degradation of I?B-?,inhibit the phosphorylation of Akt and the phosphorylation of JNK and p38 in the MAPK pathway without affecting the phosphorylation level of ERK.Conclusions:AVI may inhibit RANKL induced osteoclast formation and bone resorption activity in vitro and alleviate the bone destruction in collagen-induced arthritis mice in vivo by inhibiting Akt,nuclear translocation of p65 in NF-kB and phosphorylation of JNK and p38 in MAPK signal pathway,and then affect the activation of downstream c-Fos and NFATcl.