The Study Of Mechanisms Of Protective Effect Of Rg1 Against Arthritis By Inhibiting Osteoclast Differentiation And Maturation In CIA Mice

Objective: Although it is well established that Rg1 protects tissue structure and functions by inhibiting local inflammatory reaction, the mechanism is poorly understood. The current research explored the therapeutic mechanism of Rg1 on osteoarthritis by in vitro studies of inhibitory effect of Rg1 on RANKL-induced osteoclast differentiation of RAW264.7 cells and in vivo studies of therapeutic efficacy of Rg1 on osteoarthritis in a collagen-induced arthritis(CIA) mice model.Methods: In vitro experiments: mouse bone marrow-derived RAW264.7 cells were cultivated in medium containing RANKL to induce osteoclast differentiation. Effects of different concentrations of Rg1 on osteoclast differentiation were studied. Tartrate-resistant acid phosphatase(TRAP) activity and bone resorption area were measured after osteoclast formation. Expression levels of several genes related to osteoclast function such as TRAP, CTSK, CTR and MMP-9 were quantified using real-time RT-PCR. Furthermore, expression levels of essential transcription factors for osteoclast development including c-Fos, c-Jun and NFATc1, and phosphorylation levels of NF –κB and MAPK signaling pathways were examined by Western blot. In vivo experiments: The severity of arthritis in CIA mice receiving inTRAPeritoneal injections of Rg1 or placebo was scored. Articular cartilages were further subjected to HE staining, Alcian blue staining and TRAP staining to evaluate the effects of Rg1 on cartilage destruction and osteoclast differentiation and maturation in CIA mice.Results: In vitro experiments: Rg1 dose-dependently inhibited TRAP activity in RANKL-induced osteoclasts, the number of osteoclasts and osteoclast resorption area. Rg1 also significantly suppressed RANKL-induced gene expression including TRAP,CTSK,MMP9,CTR, phosphorylation levels of NF-κB p65 and MAPK(ERK, JNK, p38), as well as expression of critical transcription factors c-Fos, c-Jun and NFATc1 in RANK signaling pathway. In vivo experiments: Rg1 dramatically decreased arthritis scores in CIA mice and effectively controlled symptoms of inflammatory arthritis. Pathologic analysis demonstrated that Rg1 significantly attenuated pathological changes in CIA mice. Pronounced reduction in synovial hyperblastosis, angiogenesis and inflammatory cell invasion were observed in CIA mice after Rg1 therapy. Alcian blue staining illustrated that mice treated with Rg1 had significantly lighter destruction in articular cartilage. TRAP immunohistochemistry staining demonstrated a significant reduction of TRAP activity in articular cartilage in proximal interphalangeal joints and ankle joints in Rg1-treated mice.Conclusion: The present study revealed that Rg1 reduced inflammatory destruction of osteoarthritis by inhibiting differentialtion and maturation of osteoclasts in CIA mice.