The Effect And Mechanism Of Decoy Receptor 3 On Osteoclastogenesis And Fibroblast-like Synovial Cells In Rheumatoid Arthritis

Objectives:To explore the effect and mechanism of decoy receptor 3(Dc R3)on osteoclastogenesis and fibroblast-like synovial cells(FLS)in patients with rheumatoid arthritis(RA).Methods:Part one: the effect and mechanism of Dc R3 on osteoclast differentiation1.To explore the effect of Dc R3 on osteoclast differentiation,we used different concentrations of Dc R3 to induce RAW 264.7 cell lines to differentiate into osteoclasts,then the numbers of osteoclasts were calculated after the identification of using tartrate resistant acid phosphatase(TRAP)staining.2.In order to verify the impact of Dc R3 on osteoclast differentiation induced by RANKL/M-CSF,RAW 264.7 cells were treated with RANKL/M-CSF alone or with 10μg/ml Dc R3 to induce osteoclast differentiation,then the numbers of osteoclasts were calculated after the identification of using TRAP staining.3.Through Western Blot to detect the molecules involved in Dc R3 signaling.Part two: the effect of Dc R3 on fibroblast-like synovial cells1.Using q RT-PCR and ELISA to detect the relative expression and release of Dc R3 in FLS.2.After treated with different concentrations of Dc R3,the relative expression of IL-6,IL-8 and MMP-3 in RA-FLS and OA-FLS were detected through q RT-PCR;and the release of TNF-α in RA-FLS and OA-FLS were detected using ELISA.Results:Part one: the effect and mechanism of Dc R3 on osteoclast differentiation1.DcR3 induced osteoclast differentiation: Dc R3 induced RAW 264.7 cell lines to differentiate to osteoclast in a dose-dependent manner and increased the numbers of TRAP positive osteoclasts.2.DcR3 enhanced M-CSF-induced osteoclast differentiation: As for RANKL induced osteoclast formation,Dc R3 could not significantly increase the numbers of TRAP positive osteoclasts.As for M-CSF,Dc R3 could increase the numbers of TRAP positive osteoclasts.3.ERK is involved in Dc R3 signaling: Dc R3 could enhance osteoclast differentiate by increasing the phosphorylation of ERK.Meanwhile,it could also prompt M-CSF induced osteoclast differentiation by increasing the phosphorylation of ERK.Part two: the effect of Dc R3 on fibroblast-like synovial cells1.Compared with OA-FLS,RA-FLS had higher expression of Dc R3 m RNA and elevated levels of Dc R3 protein concentrations in the cell supernatant.2.DcR3 could increase the relative expression of IL-6 and IL-8 m RNA both in RA-FLS and OA-FLS,and it could induce FLS cell to release TNF-α.Conclusion:DcR3 could independently enhance osteoclast differentiation by increasing the phosphorylation of ERK,it also could enhance the effect of M-CSF induced osteoclast differentiation.In addition,RA-FLS had higher expression and elevated levels of Dc R3,and Dc R3 could induce FLS to express IL-6 and IL-8 and release TNF-α.Collectively,Dc R3 could enhance osteoclast differentiation and induce FLS to express and release inflammatory cytokines,thus involving in RA.