Effects Of Sinomenine And Methotrexate On Joint Damage Of Collagen Induced Arthritis In Rats And Osteoclast Differentiation In Vitro

ObjectiveTo investigate the effects of Sinomenine(SIN) and Methotrexate(MTX) on arthritic inflammation and tissue invasion of collagen induced arthritis in rats. To investigate the effects of SIN and MTX on osteoclast differentiation from human peripheral blood monocyte in vitro, and to elucidate the potential mechanisms for this combination treatment in Rheumatoid Arthritis(RA).MethodsArthritis was induced by immunizing with type II collagen(C II). SD female rats were immunized on day1and14. Successfully modeling rats were randomized into model group, SIN group(120mg/kg/d, intragastric administration), MTX group(1mg/kg/w, intraperitoneal injection) and SIN+MTX group. Treatment was initiated on day21. The severity of arthritis was evaluated by arthritis index(AI). Histological analysis of the ankle joints was used to assess the pathological changes of the arthritis.Peripheral blood mononuclear cells (PBMC) were collected from healthy volunteers. Adherent PBMC were used as monocytes in the culture system. 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(1000,100,10μM) and MTX(10μM) respectively or combined. After15days’incubation, the cells were stained for tartrate-resistant acid phosphatase (TRAP) to confirm the formation of osteoclasts. For experiments of matrix metalloproteinase (MMP)-9, culture supernatants were collected on day15and the concentrations of these enzymes were measured using a MMP-9ELISA kit. Adherent PBMC were stimulated with RANKL (60ng/ml) and M-CSF (20ng/ml) in the presence or absence of different drugs. After24hours, Real-time PCR technique was used to determine the expressions of NFATc1(the key transcription factor in osteoclast differentiation) at transcription level.ResultsThe first clear onset of clinical signs of arthritis was demonstrated by ankle joint swelling, and the arthritis index reached the summit at day20approximately. Joint swelling in paws increased more in control group than in SIN group, MTX group or SIN+MTX group(P<0.05). In the medium and latter stage of treatment, the arthritis index of SIN+MTX group was lower than that of SIN group or MTX group. Histopathology of the ankle joints indicated that the combination treatment can inhibit the synovial inflammation and tissue damage effectively in rats with arthritis.After15days’culture of peripheral blood mononuclear cells in vitro, TRAP positive large nultinuclear cells could be found. Combination group(SIN1000μM+MTX10μM) markedly inhibited those changes (P<0.05). Compared with the control group, SIN group or MTX group, combination group had a significantly decreased level of MMP9(P<0.05). After stimulating for24hours, combination group(SIN1000μM+MTX10μM) significantly down-regulated the expression of NFATcl mRNA (P<0.05).ConclusionsThe combination of SIN and MTX suppressed the CIA models’joint inflammation obviously, and also delayed the progress of joint damage. These two drugs may have the cooperative effect. The combination of SIN and MTX suppressed OC differentiation in vitro, and this is possibly one of the mechanisms for the combination treatment inhibiting joint damage in RA.