Study Of Bone Destruction Inhibition Effect And Mechanisms In Rheumatoid Arthritis Treated With Sinomenine And Methotrexate

ObjectiveTo investigate the effects and Mechanisms of Bone Destruction Inhibition on Rheumatoid Arthritis Treated with Sinomenine and MethotrexateMethodsPart oneThe collagen-induced arthritis (CIA) rat model was established, which were divided into five groups randomly as follow, normal group, model group (NS, i g), SIN group (120mg/kg.d, intragastric administration), MTX group (lmg/kg.w, intraperitoneal injection) and SIN+MTX group (SIN,120mg/kg.d, plus MTX, lmg/kg.w). Treatment was initiated on day21.Arthritis were evaluated by arthritis index(AI) and the expressions of OPG, RANKL, IL-17, IL-1α, IL-6, MMP-1, MMP-3and MMP-13in serum were detected by ELISA. The degree of pathogenicity was indicated by H&E dyeing in joint. Immunohistochemistry of RANKL and OPN was determined on synovium in different groups.Part twoFLS were isolated from the synovium of rheumatoid arthritis patients and cultured in vitro. FLS were incubated with different concentrations of SIN(1,0.1,0.01,0.001mg/ml) and MTX(1,0.1,0.01,0.001mg/ml) respectively or combined. MTT was applied to detect the growth of FLS. The flow cytometry was applied to detect the apoptosis of FLS. The expression of FLS RANKL mRNA and OPG mRNA was observed by semi-quantified RT-PCR.Part threePeripheral blood mononuclear cells (PBMC) were collected from healthy volunteers. Cell proliferation was detected through MTT assay when interfered with different concentration of SIN(1000,100,10uM) and MTX(10uM) single or combination use. Cells were incubated in a-MEM with M-CSF (20ng/ml) and RANKL (60ng/ml) to induce the osteoclastogenesis. The osteoclastogenesis was interfered with different concentrations of SIN(1000,100,10uM) and MTX(10uM) respectively or combined. MMP-9concentration in cell supernate was determined through ELISA assay in different treatment group.After incubation for15days the cells were stained for tartrate-resistant acid phosphatase (TRAP) to confirm the formation of osteoclasts. After24hours,Real-time PCR technique was used to determine the expressions of NFATclResultsPart oneThe swelling of joint occurred on day21obviously, and then lightened gradually, and the swelling became obvious again on day35. It was found that SIN and MTX can suppress the secondary paw swelling and pain response significantly. Compared with model group. the index of arthritis in CIA rats down-regulated on day46(P<0.05).Compared with model group,in MTX group and SIN+MTX group the level of IL-1α was significantly lower(P<0.05). In SIN+MTX group the level of IL-la and IL-17was lower than that in MTX group (P<0.05).It was found that the expression levels of MMP-3and MMP-13in SIN+MTX group were significantly lower than model group (P<0.05); In SIN+MTX group, the level of MMP-3was lower than that in MTX group (P<0.05).Compared with model group, OPG concentration and OPG/RANKL ratio in SIN group, MTX group or SIN+MTX group were higher, and in MTX group the RANKL concentration was obviously down-regulated(P<0.05);compared with MTX group, OPG/RANKL ratio in SIN+MTX group was significantly higher(P<0.05).Ankle joints harvested after experiment were stained with H&E. Inflammation factor infiltration was obvious in model group, which had permeated into the cavity of the joints. Moreover, rats treated with SIN showed bone erosion and pannus. The combination treatment can inhibit the synovial inflammation and tissue damage effectively in rats with arthritis.OPN and RANKL protein expression on FLS were significantely increased in model group than that in control group. However, Compared with model group,the protein expression was noteablely lower in MTX+SIN group(P<0.001); compared with MTX group, OPN and RANKL protein expression in SIN+MTX group were significantly lower(P<0.05).Part twoThe combination of SIN and MTX (SIN0.1mg/ml+MTX0.1mg/ml) presented significant inhibitory action on FLS proliferation compared with controlled groups and other groups (P<0.05).The apoptosis ratio of FLS incubated with MTX0.1mg/ml plus SIN0.1mg/ml was markedly increased compared with controlled groups (P<0.05).The expression of RANKL mRNA was reduced while the expression of OPG mRNA was enhanced in FLS when incubated with MTX0.1mg/ml plus SIN0.1mg/ml (P<0.05).Part threeMTT result indicated that cell proliferation was not affected either single or combined treatment after48h in different group (P>0.05)ELISA result suggested that when MTX combined with0.1M SIN MMP-9secreted from osteoclasts was reduced. However, there was no difference in other lower concentration group, which was confirmed through TRAP dyeing results. Mature OC number in0.1M SIN and MTX group was obviously less than that in control group and MTX group on16d (P<0.05)RT-PCR result indicated that NFATcl expression in OC was decreased treated with0.1M SIN and MTX after24h.ConclusionPart oneThe joint swelling were controlled in CIA rats model treated with SIN and MTX. The expression level of OPG concentration and OPG/RANKL ratio was significantly enhanced, and RANKL, IL-17, MMP-3and MMP-13in CIA rat model serum could be decreased in treatment with combination of SIN and MTX. SIN and MTX also inhibited the formation of pannus and cell infiltration, and down-regulated the expression of RANKL and OPN in FLS, which indicated that SIN and MTX could decrease cartilage and bone destruction jointly in RA.Part twoSIN and MTX have a synergistic effect in inhibiting FLS, which can be the mechanism of treating RA bone damage.Part threeOC differentiation was suppressed by SIN and MTX through reducing NFATcl expression, MMP-9secretion. Thus, bone destruction was inhibited.