Combination Methotrexate And Cyclophosphamide Therapy According To Cell Cycle On Rats With Collagen Induced Arthritis

BackgroundRheumatoid Arthritis is a chronic inflammatory disease characterized by proliferative and invasive synovitis.RA is not necessarily a benign disease and many patients have a poor outcome as joint damage results in functional decline. Combination therapy is becoming increasingly popular in treatment of RA. However,the choice of combining DMARDs can not be expected effective in daily practice.To expore new combination therapy is very important.Based on successful combination therapy in leukocythemia,we first combined methotrexate with low dose cyclophosphamide to treat RA patients.MTX as prototype agent inRA is a cell cycle special stage agent,while CTX as a cell cycle nonspecial stage agent. During the last decade,the new combination therapy had been proved more effective and safetive through we carried on clinicl practice.ObjectiveFrom clinical practice to theory,to explore effects and possible mechanisms of combination therapy with methotrexate and low dose cyclophosphamide intermittently,by observing histopathology stained with hematoxylin and eosin, proinflammaton cytokines,and cyclin D1 expression as well as apoptosis of synoviocytes in collagen induced arthritis rats.It is hoped that the overall prognosis and outcome of RA would be favorably improved accordingly by exploring new combination therapy on RA. MethodsAfter established CIA on female Wistar rats successfully,they were randomly divided into CIA model group,Low-dose MTX treatment group(0.9mg/kg/w), High-dose MTX treatment group(2.7mg/kg/w),Low-dose CTX treatment group(24mg/kg/21d),and Low-dose MTX+Low-dose CTX treatment group (MTX 0.9mg/kg/w,CTX 24mg/kg/21d).All of the rats were sacrificed 24 weeks after the first immunization.Treatment effects were assessed clinically, radiologically,and histologically.Daily clinical evaluations of each paw welling were assessed by arthritis index.At the end of the experiment,the specimens of ankle joints were exposed,fixed,decalcified,wrapped and cut into slices.All slices were stained with hematoxylin and eosin to evaluate synovial inflammation and bone erosive changes.The serum TNF-αand IL-1 levels were test by ELISA. The rates of synoviocyte apoptosis were detected with terminal deoxynucleotidyl transferase-mediated nick end labeling(TUNEL)method.The rates of p53 mutations expression were examined by immunohistochemistry method.The Cyclin D1 mRNA levels were examined by situ hybridization method.Results1.CIA successfully established.CIA models were established in 83.3%of rats,presenting clinical,radiological, and histological characters of RA.A chronic inflammatory arthritis developed in 75 of 90 rats with typeⅡcollagen.Roentgenographys mirrored the progression from soft tissue swelling,articular bone erosion to bone ankylosis.Histopathologic sections of ankle joints showed marked a chronic proliferative synovitis which secondarily destroyed articular cartilage and bone.2.24.weeks after the first immunization,21weeks after the treatment,the improvement of arthritis index of combination treatment group was higher than other groups,which showed statistically significant difference versus CIA model group,Low-dose MTX group,Low-dose CTX group(P＜0.05).Combination treatment group versus High-dose MTX group,showed no statistically significant differences.3.Compared with the normal control group,HE staining showed that the layers of synoviocytes of the CIA group were increased to 6-10,and the arrangement of synoviocytes was disordered and heavy inflammatory of cell infiltration was found in the CIA group.In the single treatment group,Low-dose MTX group and Low-dose CTX group,the layers of synoviocytes were increased to 3-4, and medium amount of inflammatory of cell infiltration was found.In the combination treatment group and High-dose MTX group,the layers of synoviocytes were nearly normal,and light amount of inflammatory of cell infiltration was found.4.ELISA 6 weeks after the first immunization,rat serum TNF-αlevel was higher in CIA model group than that in normal control group(P＜0.05).Rat serum TNF-αlevel of 24 weeks after the first immunization,which was higher than that of 6 weeks in CIA model group(P＜0.05).YNF-αlevel of 24weeks,which was decreased in combination treatment group than single treatment groups,Low-dose MTX group and Low-dose CTX group,which showed statistically significant (P＜0.05).While combination treatment group versus High-dose MTX group showed no statistically significant differences.As was rat serum IL-1 level.5.Compared to CIA control group,synoviocyte apotosis in the treatment groups was increased markedly.The combination treatment group was higher than other groups,which showed statistically significant(P＜0.05).Intensity of p53 mutations positive expression of synoviocyte in CIA model group was significantlty higher than that in the normal group.In additon,the synoviocyte expression of p53 mutations in the treatment groups was decreased markedly, which did not return to normal level.The improvement of p53 mutations of the combination treatment group was higher than the single treatment groups, Low-dose MTX and CTX group,which showed statistically significant(P＜0.05).While the combination treatment group versus High-dose MTX group showed no statistically significant difference.6.It was found that the mRNA level of cyelin D1 in CIA synovial lining layer was significantly higher than that of normal control,which suggests that in situ proliferation of synoviocyte might contribute to the thickening of rheumatoid synovial lining layer.The synovial expression intensity of the mRNA level of cyclin D1 in the treatment groups was decreased markedly.The improvement of the mRNA level of cyclin D1 of the combination treatment group was higher than other groups,which showed statistically significant(P＜0.05).ConclusionsThis is the first research of effects of MTX combined with CTX intermitently according to cell cycle with CIA.Combination therapy was more effective than single-agent therapy.The results suggest combination cell cycle special stage agent MTX with cell cycle nonspecial stage agent CTX may be synergistic in the treatment of RA.MTX and CTX can inhibit proinflammation cytokines TNF-αand IL-1,inhibition synoviocyte differentiation and induce synoviocyte apotosis.Action of TNF-αpersists in acute and chronic inflammation stage of CIA model.Inhibition elevated TNF-αand IL-1 level,enhanced synovial apotosis and inhibition of the syn0vial express and action of cyclin D1 mRNA maybe important mechanisms of combination MTX and CTX treatment according to cell cycle in treating RA.