| Objective: Through the effect of UBC13 on collagen-induced arthritis mice,the role of UBC13 on Treg cells and Th17 cells were investigated,which laid a foundation for the development of new RA drugs.Methods: 1.Establishment of a rheumatoid joint mouse model: Forty male DBA1 mice,6 weeks old,were randomly divided into UBC13 low dose group,UBC13 medium dose group,UBC13 high dose group,model group and blank group.The model group and each UBC13 treated groups mice were injected subcutaneously with collagen at the base of the tail on the 0 day,then the mice were boosted on the 21 st day to establish CIA model.After the 21 st day,all UBC13 dose group were injected subcutaneously with UBC13 recombinant protein every day,and the model group was injected with equal volume normal saline.After 56 days of secondary immunization,the mice were dissected?2.HE staining was used to observe joint damage,cartilage loss and inflammatory cell infiltration in mice.The therapeutic effect of UBC13 was evaluated by pathological section of knee joint.3.The number of Treg cells and Th17 cells in the spleen was determined by flow cytometry,evaluating the effect of UBC13 on the balance of Treg cells/Th17 cells.4.Western-Blot was used to detect the expression of I?B? and p-TAK1 in mouse thymus to evaluate activation of IKB and TAK1 by UBC13 protein.5.The m RNA levels of Foxp3,RORc,IL-17 and IL-23 in mouse spleen were detected by qRT-PCR.The effects of UBC13 protein on the expression of transcription factors and cytokines in Treg cells and Th17 cells were analyzed.Results: 1.Blank group mice have normal activities and no depilation;after the second immunization,the joint swelling in the model group is gradually obvious,unable to move with depilation;The hair color luster and joint swelling of the mice in each dose of UBC13 group were significantly reduced compared with the model group.Compared with the model group,the arthritis index of the UBC13 high-dose group was lighter in 0~30 d,while no significant difference of the arthritis index in the UBC13 low-dose group and the middle-dose group(P>0.05).2.The results of HE staining showed that the articular cartilage cavity of the normal group had intact structure,no lymphocyte infiltration and cartilage loss.The model group had severe hyperplasia of synovial tissue,lymphocyte infiltration,cartilage severe deletion;Pathological features of joint injury in UBC13 treatment groups were alleviated,compared with the model group.3.The T cell flow cytometry in the mouse spleen showed that the number of CD4+CD25+Foxp3+ cells in the spleen of the model group decreased(p<0.05)while the number of CD4+IL-17+ cells increased(p<0.05)compared with the blank group.CD4+CD25+Foxp3+ cells increased(p<0.05)and the number of CD4+IL-17+ cells decreased(p<0.05)in UBC13 dose groups compared with the model group.4.Western-Blot results of mice thymus tissue showed that p-TAK1 and I?B increased in the model group compared with the blank group(p>0.05).Compared with the model group,the expression of p-TAK1 and I?B? were increased in the UBC13 treatment groups,in a dose-dependent manner(p <0.05).5.The results of qRT-PCR in mice spleen showed that compared with the blank group,the expression levels of transcription factors and inflammatory factors in Th17 cells were increased(p<0.05).The expression levels of ROR?t,IL-17 and IL-23 decreased(p<0.05)in each UBC13 dose groups.Conclusion: 1.UBC13 treatment can inhibit arthritis in CIA mice and reduce joint pathological changes.2.UBC13 promotes the activation of TAK1 and IKK in CIA mice in a dose-dependent manner.3.UBC13 treatment affects the balance of peripheral Treg cells/Th17 cells in CIA mice by inhibiting the expression of Th17 cell transcription factors and cytokines,among UBC13 treatment groups the high dose group show the best consequent. |
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