Investigate The Effect Of Sinomenine Combined With Methotrexate In Treating Rheumatoid Arthritis And Explore Its Mechanism Of Action

ObjectiveTo observe the effects of sinomenine combined with methotrexate in treating rheumatoid arthritis and explorer its mechanism of action.Method.1in vivoThe collagen-induced arthritis (CIA) rat model was established, which were divided into five groups randomly as follow:model group (MODEL, n=8);medium dose sinomenine group (m-SIN, n=8,120mg/kg.d), intragastric administration of sinomenine; low dose sinomenine group (low, SIN,1-SIN, n=8,60mg/kg.d), intragastric administration of sinomenine; methotrexate group (MTX, n=8), lmg/kg.w, intraperitoneal injection;methotrexate combined with low dose sinomenine (1-SIN+MTX, n=8), sinomenine120mg/kg.d by gavage and intraperitoneal injection of methotrexate lmg/kg.w; methotrexate Combined with medium dose sinomenine (m-SIN+MTX, n=8), sinomenine120mg/kg.d by gavage and intraperitoneal injection of methotrexate lmg/kg.w;Another12rats as blank control group(CONTROL), gavage volume of saline.Once three days weight rats after second immunization, fixed two observers to grade arthritis index (AI) score; Blood routine detected leukocyte count、platelet count-, Hb and biochemical indexes of rats; serum was detected for alanine aminotransferase and alkaline phosphatase activity;Analysis OPG, RANKL, IL-17, IL-1a, IL-6, MMP-1, MMP-3, MMP-13secretion in rats’serum; HE staining rat’s ankle, observe inflammatory cell infiltration, proliferation of synovial cells, cartilage and bone destruction degree of erosionall of all the groups.2In vitroFLS were isolated from the synovium of rheumatoid arthritis patients;Different treatment groups’proliferation of RA synovial fibroblast were detected by MMT method;Curative effect of different treatment groups were evaluated by The Jin’s formula;Extracted total RNA from synovial fibroblast;Synovial fibroblasts’RNA reverse transcription by RT-RCR;REAL TIME-PCR detected the expression of OPG and RANKL gene of different treatment groups.Result1in vivo45successful model rats, according to the arthritis index (AI),were divided into MTX group, m-SIN group, m-SIN+MTX group,1-SIN group,1-SIN+MTX group,9rats each group.Rats’weight and Joint swelling of MTX group, m-SIN group, m-SIN+MTX group and1-SIN+MTX group were under good control (P<0.05). Model rats’WBC, PLT, Hb levels were higher than the control group (P<0.05), WBC and Hb level of MTX group and m-SIN group reduced(P<0.05); WBC, PLT, HB level of1-SIN group had no significantly difference with MODEL group; m-SIN+MTX group could significantly reduce WBC, PLT, Hb levels(P<0.05),1-SIN+MTX group’s WBC, PLT levels had decreased (P<0.05). All administered group’s AKP level significantly decreased(P<0.05),in which m-SIN+MTX group’s AKP level was significantly lower than other groups (P<0.05). MTX group, m-SIN+MTX group and1-sin+MTX group could significantly reduce serum IL-6levels(P<0.05), and the two-drug combination group’s IL-6levels were significantly lower than single drug group(P<0.05). m-SIN+MTX group’s IL-la level had significantly decreased(P <0.05).1-SIN, m-SIN as well as the MTX group had failed to reduce the level of IL-17; m-SIN+MTX group,1-SIN+MTX group’s IL-17level had decreased significantly (P<0.05).1-SIN group of rats MMP-1, MMP-3, MMP-13levels had no significantly difference with the model group, m-SIN group could only reduce rats’ MMP-3level (P<0.05), while MMP-1, MMP-3, MMP-13levels of m-SIN+MTX group,1-SIN+MTX group and MTX group were significantly lower than MODEL group(P<0.05).The level of RANKL in MTX group had significantly decreased (P<0.05), while the level of OPG and O/R value had no significantly difference with MODEL group (P>0.05); the level of RANKL and value of O/R of1-SIN group had no significantly difference compared with model group, either (P>0.05). The RANKL level of m-SINgroup、m-SIN+MTX group and1-SIN+MTX group had significantly decreased, while the level of OPG and O/R value had significantly increased (P<0.05).The level of RANKL、OPG and O/R value of m-SIN+MTX group had significantly difference compared with other administration groups.HE staining shows:1-SIN+MTX group, MTX group could control ankle’s inflammation, inhibite the formation of pannus, but failed to effectively protect the cartilage and subchondral bone;1-SIN group could not effectively inhibite the inflammation of osteoclastogenesis; m-SIN group could control inflammation and prevent cartilage from erosion in certain degree and inhibit osteoclastogenesis at the same time; m-SIN+MTX group could not only control inflammation, but also significantly reduce the formation of osteoclast,in order to protect the bone.2in vitroUse type II collagenase to extract rheumatoid arthritis (RA) patients’synovial fibroblasts(FLS).3-4generation of FLS cell showed hastypical morphology, which proliferated fast, were used in the experiment.MTT shows that sinlmtx4group (5mg/mlsin+0.001mg/mlmtx), sin2mtx2group (2mg/mlsin+0.1mg/mlmtx), sin3mtx4group (lmg/mlsin+0.001mg/mlmtx) could significantly inhibit the proliferation of synovial cells compared with the control group (P<0.05).The Jin’s formula was to evaluate combined groups’synergy. The Q’value of sin2mtx2group (2mg/mlsin+0.1mg/mlmtx) was6.42, significantly higher than the other groups, indicated that the medium dose of SIN combined with low dose MTX could play a significant synergistic effect.RANKL, OPG expression of FLS cells on12h and48h had no significant difference. Sin2mtx2(2mg/mlsin+0.01mg/mlmtx) could significantly reduce the expression of RANKL and up-regulate OPG expression.Conclusion:1in vivo experiments showed that, compared with other treatment groups, SIN combined MTX could cure RA better; while compared with1-SIN+MTX group, m-SIN+MTX group could significantly reduce joint swelling of CIA rats,decrease RA disease activity by reducing white blood cell、platelet count and alkaline phosphatase activity; exerted anti-inflammatory effects by decreasing IL-1a, IL-6, IL-17level; directly and indirectly decrease the level of MMP-1、MMP-3、MMP-13, protected cartilage damage; inhibited osteoclastogenesis by down-regulating RANKL expression, up-regulating OPG expression and OPG/RANKL ratio, inhibited bone destruction. As two drugs combined could increase ALT level, the dosage of two drugs should be adjusted at any time in order to prevent the occurrence of adverse reactions2in vitro experiment showed that, medium dose SIN combined with low dose MTX had synergistic effects, which could significantly inhibit proliferation of RA synovial cells, down-regulate RANKL expression and up-regulate OPG expression, block osteoclastogenesis, inhibit bone destruction.