Celastrol Inhibit Osteoclast Differentiation By Regulating The Expression Of Selectin-P

Part 1 The expression of SELP in osteoclast differentiation.Objective:To study the expression of selectin-P during the osteoclast differentiationMethods:The osteoclast precursors RAW264.7 cells were stimulated with RANKL, and osteoclast formation was evaluated by tartrate-resistant acid phosphatase (TRAP) staining. The expression of SELP during the osteoclast differentiation was evaluated by RT-PCR and western blot method. Collagen-induced arthritis (CIA) mice were developed, and the RT-PCR was used to examine the expression of SELP in the primary cells of bone marrow during the pathogenic progress of CIA mice.Results:Previous microarray results showed that the expression of genes SOX5, SELP, Fcgr3a, Clr were significantly increased in osteoclasts. Moreover, RT-PCR confirmed that SELP showed the highest levels among the above genes..Furthermore, RT-PCR and western blot results showed that the SELP expression was increased at 5th day and 3rd day during the differentiation of oesteoclast precursors to osteoclast. SELP expression in the primary bone marrow cells from CIA mice in the 35th day and 45th day after the first induction was higher than that in the 11th day and 28th day.Conclusion:Selectin-P plays an important role in the progress of osteoclast differentiation.Part 2 The effect of SELP on osteoclast differentiationObjective:To study the effect of SELP on osteoclast differentiation.Methods:The RAW264.7 cells were induced to osteoclast by stimulating with RANKL afterdown-expression of SELP through lentiviral transfection, and then the expression of osteoclast surface landmark genes were tested by RT-PCRthe osteoclast formation was evaluated through TRAP staining for counting multinucleated cells.Results:The number and size of osteoclast was less in SELP down-expressed group than those in normal group. Moreover, the expression of osteoclast surface landmark genes was decreased in SELP down-expressed group compared to normal group.Conclusion:Reducing the expression of SELP in the process of osteoclast differentiation may be an important way to inhibit the bone erosion in RA.Part 3 The effect of Cel on the expression of SELP in the process of osteoclast differentiationObjective:To study the effect of Cel on the expression of SELP in the process of osteoclast differentiation.Methods:CCK8 method was employed to test the safe concentration of cel for the activity of RAW264.7 cells. The effect of Cel on osteoclast differentiation was evaluated by TRAP straining, and the expression of SELP was examined by RT-PCR in RANKL-induced RAW264.7 cells. In CIA mice, daily intraperitoneally injection of Cel (beginning on day 28 after first arthritis induction) at 2 mg/kg/day for 14 days, and mice were monitored daily in a blinded manner for activity, weight and signs of arthritis. RT-PCR was used to evaluate the effect of Cel on SELP expression in primary bone marrow from both Cel treated group and CIA group mice.Results:Cel (0.3 μM) significantly suppressed osteoclast differentiation and SELP expression in RANKL-cultured RAW264.7 cells. In CIA mice, Cel significantly reduced joint inflammation and SELP expression in bone marrow in Cel treated mice compared with CIA mice.Conclusion:Cel could reduce the mRNA expression of SELP, which may be a new important mechanism to inhibit osteoclast differentiation in RA.