Cyclophosphamide And Methotrexate In Combination Inhibit RANKL Expression In Fibroblast-like Synoviocytes From Patients With Rheumatoid Arthritis Via Suppression Of The JAK2/STAT3 And P38MAPK Signaling Pathway

Background:Rheumatoid arthritis(RA)is a chronic autoimmune disease characterized by persistent polyarticular synovitis,synovial hyperplasia and pannus formation,eventually resulting in the irreversible destruction of cartilage and bone as well as disability in the absence of adequate treatment.Fibroblast-like synoviocytes(FLS)are the most common cell type at the pannus-cartilage junction and contribute to joint destruction through their production of cytokines,chemokines,and matrix degrading molecules and by migrating and invading joint cartilage.FLS,which are stimulated by interleukin(IL)-6,TNF-?,and IL-17,can produce receptor activator of nuclear factor-?B ligand(RANKL)in the inflamed joints of patients with RA.RANKL exerts its functions by binding to its unique receptor RANK,regulating the process of osteoclastogenesis and further inducing inflammatory bone loss in RA.The early diagnosis of RA and prompt initiation of disease-modifying anti-rheumatic drugs(DMARDs)therapy are key factors to prevent or minimize damage.Conventional combination therapy in RA has a long-term radiographic benefit compared with monotherapy.Methotrexate(MTX)is a classic DMARD.Although conventional combination therapy is efficacious for most patients with RA,many still do not respond to the current therapies.Therefore,there is a need for novel combination regimens that better target the cellular processes involved in RA pathogenesis.Preliminary studies have suggested that the cell cycle-nonspecific drug cyclophosphamide(CTX)in combination with the cell cycle-specific drug MTX has a synergistic effect in a rat model of RA.FLS have important roles in the synovial pathological process as a major source of RANKL production in patients with RA.Therefore,decreasing or blocking the expression of RANKL on FLS may be crucial for the regulation of osteoclast differentiation and the prevention of bone erosion in RA.In the present study,we investigated the effect of4-hydroperoxy cyclophosphamide(4-H-CTX),an active metabolite of CTX,in combination with MTX on the expression of RANKL in an inflammatory context using human RA-FLS stimulated with IL-6/soluble IL-6 receptor(sIL-6R),and the possible mechanism of the associated signal transduction pathway.Objective:In this study,the effects of CTX combined with MTX on RANKL expression in FLS from patients with RA and the mechanisms underlying these effects were examined.FLS were stimulated by the IL-6/sIL-6R complex.RANKL,STAT3,and p38MAPK expression levels were assessed by quantitative reverse transcription-polymerase chain reaction(qRT-PCR),western blotting,and enzyme-linked immunosorbent assays in FLS treated with CTX and/or MTX.Immunofluorescent staining was performed to identify the effects of CTX and/or MTX on RANKL expression.Methods:Synovial tissues were obtained from patients with active RA(five women and one man)who were undergoing knee replacement surgery.FLS were incubated in the established cell culture matrix.Cells from passages four to six were used in the following experiments.Part one:Effects of CTX and/or MTX on the expression of RANKL in RA-FLS induced by IL-6/sIL-6R complex.(1)Identification of FLS from patients with RA.RA-FLS were identified and characterized by flow cytometry and microscopy.(2)Expression of RANKL mRNA in RA-FLS stimulated with IL-6/sIL-6R at various concentrations and incubation times.To determine the appropriate concentration of IL-6/sIL-6R and the suitable incubation time,RA-FLS(1×10~5/ml)were cultured in six-well plates and treated with 0,10,25,50,100,and 200 ng/ml IL-6/sIL-6R for 1 h,and RA-FLS were stimulated with 100 ng/ml IL-6/sIL-6R for various incubation times(0,6,24,48,72,and 96 h).Cells were harvested and total RNA was extracted from the cells using the E.Z.N.A.Total RNA Kit.(3)The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide(MTT)assay was used to determine the cell viability of RA-FLS under the proper concentration of 4-H-CTX and/or MTX.(4)Effects of CTX and/or MTX on the expression of RANKL in RA-FLS induced by IL-6/sIL-6R complex.FLS cells were divided into five groups:the control group,IL-6/sIL-6R group,CTX group,MTX group,CTX combined with MTX group.Western blotting,qRT-PCR,and immunofluorescent staining was performed to identify the inhibitory effects of CTX and/or MTX on RANKL.Part two:To determine whether the JAK2/STAT3 or p38MAPK signaling pathway plays an important role in the IL-6/s IL-6R-induced upregulation of RANKL expression in RA-FLS,and analyze the effects of CTX and/or MTX on the activation of STAT3 and p38MAPK in FLS stimulated by IL-6/sIL-6R.(1)IL-6/sIL-6R-induced RANKL upregulation in RA-FLS require the activation of STAT3 and p38 signaling by phosphorylation.FLS cells were divided into four groups:the control group,IL-6/sIL-6R group,the JAK2/STAT3 inhibitor AG490 group,the p38MAPK inhibitor SB203580group.Western blotting was performed to identify the inhibitory effects of AG490 or SB203580 on RANKL expression and signaling protein phosphorylation.(2)CTX and/or MTX can inhibit the expression of RANKL through suppressing the phosphorylation of STAT3 and p38MAPK in FLS induced by IL-6/sIL-6R.FLS cells were divided into five groups:the control group,IL-6/sIL-6R group,CTX group,MTX group,CTX combined with MTX group.The expression of RANKL and the phosphorylation of STAT3 and p38was determined by cell-based enzyme-linked immunosorbent assay(ELISA).Results:(1)The culture system established in our laboratory was reliable.A majority of FLS at passages one and two in cultures under light microscopy appeared polygonal or fusiform,with prominent nucleoli and frequent mitotic figures.While FLS at passages five in cultures was spindle and well distributed.Flow cytometry revealed that,after in vitro culture,RA-derived FLS at passage four essentially expressed cadherin-11.But CD14 or HLA class II expression was not detected in FLS at passage four,suggesting that macrophages were eliminated from the culture system of RA-FLS.(2)The treatment duration and concentration of IL-6/sIL-6R were optimized to induce RANKL expression in RA-FLS.The results showed that stimulation with 100ng/ml IL-6/sIL-6R significantly increased RANKL mRNA levels and the expression of RANKL mRNA was stable and high at 72 and 96 h.The results of MTT showed that treatment with 4-H-CTX(1?g/ml),MTX(100 nM),and 4-H-CTX(1?g/ml)combined with MTX(100 nM)did not significantly suppress cell viability during the incubation time of 72 h.Therefore,the treatment duration and concentration of drugs above were used in subsequent analyses.(3)RA-FLS stimulation with IL-6/sIL-6R at 100 ng/ml led to a prominent increase in RANKL mRNA.Additionally,the expression of RANKL mRNA was stable and high at 72and 96 h.As compared with the control group,the expression of RANKL protein and mRNA increased in FLS induced with IL-6/sIL-6R.An immunofluorescence assay showed that the number of RANKL-positive cells increased after treatment with IL-6/s IL-6R compared to the number observed in controls.(4)In a qRT-PCR analysis of RANKL expression,after pretreatment of cultured FLS with IL-6/sIL-6R,incubation with CTX and/or MTX led to a significant reduction in RANKL at the mRNA(P<0.01 vs.IL-6/sIL-6R-treated FLS,and P<0.05 vs.CTX combined with MTX).In contrast,CTX and MTX significantly increased the levels of OPG mRNA(P<0.05 vs.IL-6/sIL-6R-treated FLS,and P<0.05 vs.CTX combined with MTX).The inhibitory effect of CTX combined with MTX on RANKL expression was more prominent than that of CTX alone or MTX alone.OPG mRNA expression was increased by CTX combined MTX when compared to treatment with MTX alone.A two-way classification ANOVA for 2×2 factorial design was used to determine the interaction effects of CTX and MTX on RANKL expression in RA-FLS.The results showed that CTX and MTX had an synergistic inhibitory effect on the RANKL mRNA expression(F=33.932,P<0.001).In a western blot analysis of RANKL expression,CTX and MTX treatment inhibited RANKL expression in FLS stimulated with IL-6/sIL-6R(P<0.01 vs.IL-6/sIL-6R-treated FLS,and P<0.05 vs.CTX combined with MTX).In contrast,OPG expression increased following CTX and MTX treatment(P<0.05 vs.IL-6/sIL-6R-treated FLS).The inhibitory effect of CTX combined with MTX on RANKL protein expression was more prominent than that of CTX alone or MTX alone.OPG protein expression was increased by CTX combined with MTX when compared to IL-6/sIL-6R-treated FLS.CTX and MTX had an synergistic inhibitory effect on the RANKL protein expression in RA-FLS(F=16.265,P<0.001).An immunofluorescence assay showed that the number of RANKL-positive cells decreased after treatment with CTX and/or MTX compared to the number observed in IL-6/sIL-6R group.The number of RANKL-positive cells in CTX combined with MTX group was the least.(5)Treatment of FLS with IL-6/sIL-6R(100 ng/ml for both)for 1 h increased the expression of p-STAT3,p-p38MAPK,and RANKL.In FLS treated with the JAK2/STAT3inhibitor AG490,the expression levels of p-STAT3 and RANKL were significantly lower than those of IL-6/sIL-6R-treated FLS.In FLS treated with the p38MAPK inhibitor SB203580,the expression levels of p-p38MAPK and RANKL were significantly lower than those of IL-6/s IL-6R-treated FLS.Cell-based ELISA showed that the expression of p-STAT3,p-p38 and RANKL increased in FLS induced with IL-6/sIL-6R as compared to the blank control FLS(P<0.01or P<0.05).CTX and/or MTX both led to a significant reduction in the phosphorylation of STAT3 and p38MAPK and the expression of RANKL(P<0.01 or P<0.05).p-STAT3 and p-p38MAPK expression tended to be lower in FLS treated with the CTX-MTX combination than in FLS treated with CTX or MTX alone,although this difference was not significant(P>0.05).The inhibitory effect of the CTX-MTX combination on RANKL expression was more prominent than that of CTX or MTX alone(P<0.05).A two-way classification ANOVA for 2×2 factorial design was used to determine the interaction effects of CTX and MTX on RANKL,STAT3,and p38 expression in RA-FLS.The results showed that CTX and MTX had an synergistic inhibitory effect on the RANKL and p-STAT3(F=57.871,P<0.001;F=16.477,P=0.001).Conclusions:(1)RA-FLS incubated with IL-6/sIL-6R complex significantly upregulates the expression of RANKL.(2)CTX and/or MTX can markedly suppress the expression of RANKL in IL-6/sIL-6R-induced FLS.(3)IL-6/sIL-6R-induced RANKL upregulation in FLS requires the activation of the STAT3 and p38 signaling pathways by phosphorylation.(4)CTX and MTX can inhibit the expression of RANKL in FLS stimulated with IL-6/sIL-6R via the JAK2/STAT3 and p38MAPK signaling pathways.(5)CTX and MTX had an synergistic inhibitory effect on the RANKL and p-STAT3expression in RA-FLS.