The Role Of NR4A1 In CSA Participated In Transplantation Immune Tolerance Resistance

Part ?:The role of CsA in transplantation immune tolerance resistanceObjective:To established the mice abdominal heterotopic heart transplantation model and explored the role of CsA in transplantation immune tolerance resistance.Methods:1:Establish Balb/c ? C57BL/6 mice cardiac allograft acute rejection model.The treatment group received CsA(10 mg/kg.qd)subcutaneous injection and the allografts survival were analyzed;2:The pathological changes of cardiac grafts were observed at 7 days after transplantation;3:The proportion of T-cell subpopulations and activation induced cell death in spleen were analyzed by flow cytometry;4:PCR and Western blot were performed to detect NR4A1 expression of grafts.Results:1:CsA protected the allografts and inhibited Thl cells proportion but also inhibited Treg subsets and T cell activation induced cell death.And CsA leaded to renal injury in mice;2:Local NR4A1 expression significantly elevated in cardiac grafts after transplantation.CsA reduced the protein expression of NR4A1.Conclusion:CsA had different effects in transplantation immune tolerance induced.Its inhibition of Treg and AICD resisted to induction of immune tolerance.CsA also leaded to renal injury in mice.Those negative effects may be associated with NR4A1 protein decrease.Part ?:Activation of NR4A1 antagonized immune tolerance resistance by cyclosporin AObjective:To explored the effect of NR4A1 on immune tolerance resistance by cyclosporin A.Methods:1:CD4 T lymphocytes separated by magnetic bead were treated with CsA and Csn-B.T cells differentiation and activation induced cell death were analyzed by flow cytometry;2:Mouse renal interstitial fibroblasts were cultured in vitro.To obverse the influence of Csn-B on the fibroblast phenotype transformation induced by CsA.3:Establishing the model of heart or skin transplantation in mice.Mice received CsA(10 mg/kg.qd)alone or combined with Csn-B(13 mg/kg.qod)and the allografts survival were analyzed.The proportion of T-cell subpopulations and activation induced cell death in spleens were analyzed by flow cytometry.4:Establishing the model of renal fibrosis in mice by CsA subcutaneous injection.Csn-B treatment group was received intraperitoneal injection of 13 mg/kg · qod.To observe the effect of Csn-B on renal damage and interstitial fibrosis induced by CsA.PCR was used to detect the expression of TGF-beta signaling pathway target gene.Results:1:Csn-B enhanced inhibition of Thl cells by CsA and restrained reducing of Treg subsets and activation induced cell death by CsA;2:Csn-B combination with cyclosporin A obviously prolonged the skin and heart graft survival and weakened T cell-mediated inflammation;3:Csn-B inhibited mouse renal interstitial fibroblasts phenotype transformation induced by CsA;4:Csn-B alleviated kidney damage and renal interstitial fibrosis induced by CsA.Conclusion:Activation of NR4A1 antagonized immune tolerance resistance by cyclosporin A and combination treatment obviously prolonged graft survival.Part ?:CsA regulated NR4A1 expression and NR4A1 regulated TGF-beta signaling pathways target genes expressionObjective:To explore the molecular mechanism of NR4A1 regulated by CsA and TGF-beta signaling pathways target genes regulated by NR4A1.Methods:1:Primary T cells and EL4 cells were detected NR4A1 expression changed by CsA;2:Immune co-precipitation was used to confirm NR4A1 and VHL protein interactions predicted by bioinformatics analysis;3:The expression of VHL was knockdown by small interference RNA to observe its role in the NR4A1 regulated by CsA;4:ELISA was used to detect TGF-beta expression level in mice kidney fibroblasts treated with CsA and Csn-B;5:the expression of TGF-beta signal pathway target genes in mice kidney interstitial fibroblasts treated with CsA and Csn-B was detected by PCR and Western blot.Results:1:CsA promote the degradation of NR4A1;2:CsA regulated expression of NR4A1 through VHL which interacted with NR4A1 protein;3:CsA promote TGF-beta expression in fibroblasts while Csn-B did not;4:Csn-B inhibit the expression of TGF-beta signal pathway target genes raised by CsA.Conclusion:CsA promote the degradation of NR4A1 by VHL.Activation of NR4A1 inhibited activation of TGF-beta signal pathway target genes by CsA.