The Role Of Celastrol On Osteoclastogenesis And Bone Erosion In Collagen-Induced Arthritis

Objectives:To determine the effects of Celastrol on osteoclastogenesis in vitro and on bone erosion in vivo in collagen-induced arthritis (CIA).Methods:1. In vitro osteoclastogenesis assays were performed using RAW264.7as osteoclast precursors. Osteoclast formation was evaluated by counting positive cells for tartrate-resistant acid phosphatase (TRAP) staining. Quantitative gene expression analysis for osteoclastic markers was performed by Real time quantitative PCR. Expression of NFATcl, c-Jun and c-Fos, which are known to be essential for osteoclastogenesis,and phosphorylation of NF-κB p65、 MAPK signals in RAW264.7cells by Celastrol were examined. Activity of bone resorption was tested by pit formation assay in the presence or absence of Celastrol..2.Prepare CIA mice by immunizing DBA1/J mice using Type II Collagen and complete Freund’s adjuvant. Mice received daily intraperitoneal (IP) injections (0.2m L) of either celastrol diluted solutions or carrier only as a control,over the course of14days. Arthritis score is recorded.After that,their blood is collected,the serums are separated and the TRAP in the serums are detected using enzyme-linked immunosorbent assay.At the same time their joints are collected to be observed with a Micro-CT and then made into sections to be inspected for pathology purpose. Quantitative gene expression analysis for osteoclastic markers was performed by Real time quantitative PCR.Results:1.Celastrol inhibited the differentiation of Raw264.7to osteoclast in a dose-dependent manner. The osteoclast formation was completely blocked with0.3μM of Celastrol. Celastrol reduced the RANKL-induced expression of NFATcl, c-Jun and c-Fos, in RAW264.7, also decreased the RANKL-induced expression of phosphorylation of NF-κB p65and MAPK resulting in inhibited osteoclast formation. Celastrol inhibited the bone-resorbing activity of mature osteoclasts measured by Osteologic disks.2. Administration of Celastrol completely blocked CIA development and delayed its progression. Histology of joints from mice treated with PBS showed severe infiltration of inflammatory cells and bone erosion, whereas treatment with Celastrol suppressed these changes.Furthermore, treatment with Celastrol at the onset of arthritis significantly inhibited the progress of joint inflammation. Administration of Celastrol also suppressed the expression level of NFATc1, c-Jun and c-Fos in the joint tissues and reduced the levels of TRAP in the serums.Conclusion:1. Celastrol directly inhibits the differentiation and bone resportion of Raw264.7to osteoclast.2. Celastrol reduced the RANKL-induced expression of NFATcl, c-Jun and c-Fos, in RAW264.7, also decreased the RANKL-induced expression of phosphorylation of NF-κB p65and MAPK(ERK、JNK、p38).3. Celastrol can suppress the inflammation and bone resportion of CIA mice via down-regulate the expression of NFATc1, c-Jun and c-Fos in inhibited osteoclast differentiation and funciton.