Study Of Amentoflavone On RANKL Induced Osteoclastogenesis And Ti Particles Induced Mouse Calvarial Osteolysis

Objection: In the present study,we exploring the effect of amentoflavone on osteoclastogenesis,osteolysis and the possible signaling mechanism.Methods:1.0 The expression of osteoclast and inflammatory factor in periprosthetic membrane that patients with revision.The expression of osteoclast and inflammatory factor of synovitis in the joint capsule for the primary hip arthroplasty.The expression of osteoclast and inflammatory factor in periprosthetic membrane that patients with revision.2.1 The effect of amentoflavone on osteoclastogenesis and function of osteoclasts.CCK-8 assays were used to clarify the effect of used concenstrarions of amentoflavone on cell cytotoxicity.TRAP staining assay were used to clarify the effect of amentoflavone on osteoclast generation.F-actin stainng of osteoclasts were used to explore the effect of amentoflavone on osteoclast maturation.Bone absorption aassay were used to grope for the action of amentoflavone on bone destruction function of costeoclast.2.2 The effect of amentoflavone on Ti particles induced osteolysis.The Micro-CT scanning and histological analysis were performed to clarify the action of amentoflavone on mouse calvarial osteolysis form macroscopic and microcosmic in vivo.3.0 The mechanism of amentoflavone on RANKL induced osteoclastogenesis.Western-blot assay were used to validate the relationship between amentoflavone depressed osteoclastogenesis and signaling pathways of MAPK and NF-?B.Results:1.0 There are more inflammatory cytokines and osteoclasts in revision patients.The staining of human specimens showed that more inflammatory cytokines and osteoclasts in revision patients than those in primary joint replacement.The analysis found that the expression of key factors TRAF-6,TNF-alpha,staining positive osteoclasts are more.2.1 Amentoflavone suppressed osteoclastogenesis,F-actin ring formation,and bone absorption without cytotoxicity.CCK-8 assay results demonstrated that the concentrations of amentoflavone that were used in the present study without cytotoxicity.Tartrate-resistant acid phosphatase(TRAP)is characteristic to express in osteoclast.TRAP staining demonstrated that amentoflavone can inhibit the formation of osteoclast.F-actins were the typical cytoskeleton osteoclast and the symbol of mature osteoclasts.In the bone slice resorption study shown that amentoflavone can suppress the osteoclasts function of bone destruction and resorption.2.2 Amentoflavone can suppress the osteolysis that were induced by Ti particles.As the following amplified scanning and HE and TRAP staining shown that the mouse calvarial osteolysis induced by Ti particles presented reparation after treated with amentoflavone.The Micro-CT scanning were performed to clarify the action of amentoflavone on mouse calvarial osteolysis form macroscopic in vivo.The HE and TRAP staining shown that amentoflavone can suppress the osteoclastogenesis and osteolysis in vivo.3.0 NF-?B and MAPKs signaling pathways maybe the mechanism of amentoflavone on RANKL induced osteoclastogenesis.As results of western-blot shown that amentoflavone suppressed the relative proteins of NF-?B and MAPKs signaling pathways,and the key transcription factors of NFATc1 and c-fos.Conclutions:There are more osteoclasts in revision cases,verified that osteoclasts play an important role in aseptic osteolysis;The present study suggests that amentoflavone derived from plants could inhibit osteoclast-ogenesis and titanium wear debris-induced osteolysis via suppressing NF-?B and MAPKs signaling pathways.