| Background:RA is a chronic inflammatory disease which primarily affects the joints and surrounding tissues. Joint pain and deformed in patients with rheumatoid arthritis had greatly effects on their society, family and spiritual life, wich also caused huge economic loss and social burden. The therapies of RA were combining two or more DMARDs in the worldwide and the typical combination was MTX with LEF. Confirmed by more than 10years’ clinical practice and basic research, cycle combined with MTX and CTX was better than that of MTX and LEF, and the side effects was significantly reduced[1-2]In recent years, a new kind of subgroup of T cell was discovered,which was named Th17. It could stimulate the release of inflammatory cytokines, such as IL-6,TNF-α,IL-1βthrough the secretion of IL-17. It could increase the systemic inflammatory response in RA and promoted the joint damage. The JAK-STAT3 pathway was involved in RA disease progression by adjusting the Th17 cells’ differentiation, proliferation and the secretion of cytokines. Animal experiments had confirmed that the cycle combined with MTX and CTX could restrain the differentiation of Th17 cells in mice. But it was indefinite whether the suppress of spleen Na?ve T cells’ differentiation to Th17 cell of CIA mice. Further explore will observe the possible mechanisms and target of the inhibition of the inflammatory response in RA.Objective:Clarify whether the therapeutic alliance of MTX and CTX synergistically suppress Th17cell’s differentiation of CIA through JAK-STAT3 passway.Methods:The study was an animal experiment, Includes two parte: in vivo and in vitro.In vivo experiment: 56 male DBA1 mice were selected and divided into two groups randomly: 10 cases in normal control group and 46 cases in CIA group. Then establish the CIA model, which was divided into 4 groups randomly include the CIA group, MTX group(1.5mg·kg-1·3d-1), CTX group(30mg·kg-1·10d-1) and MTX+CTX group(combination group, MTX: 1.5mg·kg-1·3d-1, CTX: 30mg·kg-1·10d-1). Three weeks after Strengthen immune, they were treated with medicine. During the treatment process, the left ankle swelling in mice and AI was monitored continuously. All of them were sacrificed at 11 weeks. Their knees and left ankle were sectioned and stained for HE to observed the pathological changes. Naive T cells was separated from spleen cells and cultured in 37℃5% CO2 incubator with IL-6, TGF-β1, IL-β1, IL-23 for 96 hours. The proportion of Th17 cells after training was detected by FCM, the expression of STAT3 level was detected by RT-PCR after the differentiation of Th17 cells.In vitro experiment: 20 CIA model was set up by male DBA1 mice which were executed in 7thweek. The Naive T cells were separated from the spleen lymphocytes and divided into 10 groups: blank group, Anti-CD3CD28 activation group, Th17 cell differentiation group, CTX group, the low concentration of MTX group(MTXL group), the middle concentration of MTX group(MTXM group), the high concentration of MTX group(MTXH group), MTXL+CTX group, MTXM+CTX group, which were cultured in 37 ℃5% CO2 incubator for 96 hours. The proportion of Th17 cells after training was detected by FCM, the expression of STAT3 level was detected by RT-PCR after the differentiation of Th17 cells.Results:1.In vivo studies(1) before treating, the joint swelling degree and AI in treatment groups compared with the CIA group, there was no statistically significant difference; after 4 weeks treatment,compared with the CIA group, the joint swelling degree and AI in treatment groups improved obviously, difference was statistically significant, there was no statistically significant difference between the treatment groups; after 11 weeks treatment, compared with the CIA group and CTX group, the joint swelling degree and AI in joint group was improved significantly, the difference was statistically significant; compared with the MTX group, the joint group improved significantly, but there was no statistically significant difference;(2) After 11 weeks treatment, the Th17 cells percentage in treatment groups significantly lower than the CIA group, the difference was statistically significant, the Th17 percentage in joint group was obviously lower than single drug groups, but there was no statistically significant difference;(3) After 11 weeks treatment, the expression of STAT3 m RNA in Th17 cells in treatment groups were lower than the CIA group, difference was statistically significant;the expression of STAT3 m RNA in Th17 cells in joint group was lower than the single drug groups, but there was no statistically significant difference.2. In vitro studies(1) After 96h’s culture, the percentage of Th17 cell in Th17 differentiation group, was highter than the the blank control group, the difference was statistically significant; the middle MTX group, high MTX group, low joint group compared with the blank control group, the difference was statistically significant; Low joint group compared with the single drug groups, Th17 cells percentage decrease after training, but there was no statistically significant difference;(2) After 96h’s culture, the expression of STAT3 m RNA in Th17 cells in Th17 differentiation group, was highter than the the blank control group, the difference was statistically significant; compared with the blank control group, the expression of STAT3 m RNA in Th17 cells in the middle MTX group, high MTX group, low joint group were lower, and the difference was statistically significant; Low joint group compared with the single drug groups, Th17 cells percentage decrease after training, but there was no statistically significant difference;Conclusions:Cycle Combined with Methotrexate and Cyclophosphamide may affect the differentiation of Th17 of CIA by JAK- STAT3 pathway. |
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