Combination Methotrexate、leflunomideand And Cyclophosphamide Therapy According To Cell Cycle On Rats With Collagen Induced Arthritis Effecting On Synovial CyclinD1Expression

[Background] Rheumatoid arthritis (RA) is a chronic erosive polyarthritis as the main manifestation of systemic autoimmune diseases. The combination therapy of disease modifying anti-rheumatic drugs (DMARDs) is still the mainstream of RA therpy. By more than10years of clinical and basic research, combination cycle specific drug methotrexate (MTX) and cycle non-specific drugs cyclophosphamide (CTX) according to cell cycle has confirmed a significant effect, and significantly reduced side effects. However, the pathogenesis of RA has not yet been fully elucidated, The RA synovial excessive proliferation newly discovered related to abnormal expression of cell cycle genes. Cell cycle proteins in the research of RA pathogenesis are new field. CyclinDl has be the most studied, cyclinDl isn’t expressed or expresses lower in most normal tissue, amplification gene rearrangements and mutations, resulting in increased expression product,often occur in tumor tissue. Whether the RA synovial excessive proliferation is relevant to cyclinD1mRNA high expression or not? Whether combination DMARDs therapy has synergies in downregulation the expression of cyclin Dl mRNA? The subject carried out the experimental study of animal models of RA, to explore synergies of the combination mechanism from cyclinDl angle.[Objective]To research the synergy and the mechanism of action of combination Methotrexate (MTX)、Leflunomide (LEF) and Cyclophosphamide (CTX) therapy according to Cell Cycle,from the perspective of the cyclin,on Rats with Collagen Induced Arthritis (CIA).[Methods]Establish collagen type Ⅱ-induced female SD rats CIA model. Randomly divided into normal control group, the CIA model control group, the MTX group (0.9mg.kg-1.w-1), the LEF therpy group (1.2mg.kg-1.d-1) and CTX therpy group (24mg. kg-1.3w-1), MTX United CTX therpy group (MTX:0.9mg.kg-1.w-1+the CTX:24mg.kg-1.3w-1), the LEF United CTX therpy group (LEF:1.2mg. kg-1.d-1+CTX:24mg.kg-1.3w-1), MTX United LEF therpy group (MTX:0.9mg.kg-1.w-1+the LEF:1.2mg.kg-1.d-1).Conduct experiments in vivo9weeks, administration starts after the primary immunization three weeks.Continuou to rate the degree of arthritis, measuring the right ankle joint diameter to calculate the arthritis index in the therpy process. All animals were sacrificed after6weeks of therpy, then fixed, decalcified, embedded, made of slices, then to observe the histopathological changes of the synovium of joints which were stained with HE in each group, and to score. Further detect the expression levels of the synovial cyclin D1mRNA through in situ hybridization. We represent the results by gray value,comparing gray value of groups.[Results]1. CIA rat models reached to a success rate of92.3%(72/78).Symptoms, radiological and pathological assessment confirmed that the model successfully for chronic arthritis.2. Arthritis index (AI):AI compared with model group of the therpy groups improved significantly (P<0.05); The degree of AI improvement:the MTX combined with CTX group>the LEF combined with CTX group> the LEF combined with MTX group> the MTX group> the LEF group> the CTXgroup;AI improvement was better in combination group than in monotherapy group (P<0.05); In combination group, AI improvement was superior in MTX combined with CTX group and in the LEF combined with CTX group than in the MTX combined LEF group (P<0.05);3. X-ray radiography:X-ray changes of each therpy group was lighter than the model group, the combination groups X-ray efficacy were better than the monotherapy group, There was no significant difference between the combination therapy groups, the monotherapy groups were also.4. HE joint pathological changes:synovial levels of the CIA model rats increased up to6-8layers, disorganized, and inflammatory cell infiltration, cartilage, bone tissue is severely eroded.It was in turn reduce on the improvement in the degree of synovitis, cartilage and bone erosion in the MTX combined with LEF group,the MTX group, the LEF group, the CTX group,no significant differences among the groups;Synovitis, cartilage and bone erosion degree of improvement was significantly in the MTX combined CTX group and in the LEF combined CTX group than in the MTX combined LEF grcu、the MTX group、the LEF group and the CTX group (P<0.05).5. Synovial cell cycle protein1) The results of synovial expression of cyclin D1mRNA in situ hybridization (molecular level):The synovial cyclin D1mRNA positive staining intensity of the CIA model rats were significantly higher than that of the normal control group (P<0.01).2) Compared with the CIA group,the synovial expression of cyclin D1mRNA in the therpy groups were downregulated after9weeks therpy:the MTX combined with CTX group>the LEF combined with CTX group> the LEF combined with MTX group> the MTX group> the LEF group> the CTX group. Compared with MTX combined with LEF group, the single drug group, MTX combined with CTX group, the LEF combined with CTX group was significantly (P<0.05). There was not significantly difference between MTX combined with LEF group, monotherapy groups.3) There are interactions in the MTX with the CTX and in the LEF with the CTX by analysis of variance of factorial design.The average gray value of MTX combined with CTX group, the LEF combined with CTX group were up to the highest, inhibit the expression of synovial cyclinDlmRNA mostly,prompting that there were synergistic effects in the combination MTX and CTX therapy; the combination LEF and CTX therapy.[Conclusion]1. In the CIA rat model:synovial expression of cyclin DlmRNA upregulation, resulting in synovial abnormal proliferation.2. The combined therapy groups are better than the monotherapy groups in improving the symptoms of arthritis.3.The MTX combined CTX group, the LEF combined with CTX group was better than the LEF combined with MTX group、the MTX group、the LEF group、the CTX group in improving the synovial pathological changes.4. There are interactions in the MTX with the CTX and in the LEF with the CTX by analysis of variance of factorial design, prompting that there were synergistic effects in the combination MTX and CTX therapy; the combination LEF and CTX therapy.