Iguratimod Alleviates Ovariectomy-Induced Bone Loss Though Inhibition Of Osteoclastogenesis And Adipogenesis

Objective1.To observe the variation of bone trabecula and other tissue in ovariectomized mice after orally taking iguratimod.2.To investigate the effect of iguratimod on osteoclastogenesis and adipogenesis in vitro.Methods:1.C57/BL6 mice were bilateral ovariectomized or sham operated.Then mice were treated with iguraitmod(30 mg/kg/day)or placebo orally.6 weeks later,mice femurs were isolated and subjected to ?-CT analysis.Slices of the femur were performed H&E staining and TRAP staining.Serum levels of CTX-I were detected using ELISA.2.Bone marrow mononuclear cells(BMMCs)were isolated and cultured with RANKL and M-CSF to form osteoclasts.TRAP staining and a Corning Osteo Assay Surface were used to detect the effects of iguratimod on osteoclasts formation and function.The protein levels of PPAR-y,c-Fos,NFATcl and osteoclasts specific proteins were detected by Western blotting.Rosiglitazone was used to validate the effect of iguratimod on PPAR-?/c-Fos signaling.3.Bone marrow mesenchyma stem cells(MSCs)were obtained from Sprague-Dawley rats and cultured with adipogenic differentiation medium.Oil O staining was used to detect the effect of iguratimod on adipogenesis.The mRNA levels of adiponectin and adipsin were detected using qRT-PCR.Effect of iguratimod on PPAR-? activation was tested using qRT-PCR and immunoblotting.Results:1.Iguratimod prevented ovariectomy-induced trabecular bone loss and osteoclasts activation.The number of adipocytes was also suppressed by treatment of iguratimod.In addition,iguratimod also decreased serum levels of CTX-I.2.TRAP staining and bone pits formation showed that iguratimod significantly suppressed osteoclasts formation and function.Iguratimod also suppressed the formation of actin rings and the expression of osteoclasts marker proteins including c-Fos,NFATc1,cathepsin K,MMP-9 and TRAP.Furthermore,immunoblotting indicated that the activation of PPAR-?induced by RANKL was suppressed by iguratimod,which resulted in the downregulation of c-Fos and NFATcl.Treatment of rosiglitazone partly reversed the inhibitory effect of iguratimod on osteoclastogenesis.3.Oil red O staining showed that iguratimod suppressed adipogenesis.Iguratimod also decreased the upregulation of PPAR-y induced by adipogenic differentiation medium and subsequently suppressed the expression of adiponectin and adipsin.Conclusions:1.Iguratimod alleviated ovariectomy-induced trabecular bone loss.2.Iguratimod suppressed osteoclasts formation and function partly though the inhibition of PPAR-?/c-Fos signaling and the expression of downstream NFATcl and osteoclasts specific proteins.3.Iguratimod suppressed adipogenesis though the inhibition of PPAR-y expression.