Ursolic Acid Promotes Robust Tolerance To Cardiac Allografts In Mice

PartⅠThe Role of Ursolic Acid on NF-κB Nuclear Translocation and T Cell ActivationObjective:To investigate the influence of ursolic acid on NF-κB nuclear translocation and T cell activation trigged by CD3+CD28 mono-antibodies.Methods:T cell of B6 mice was isolated by FACS. For in vitro activation, T cells were stimulated by plate-bound anti-CD3 (2μg/ml) and soluble anti-CD28 (1μg/ml) mono-antibodies. For ursolic acid inhibition assay, T cells were pre-incubated with different concentrations of ursolic acid (5,10,25μM) for 2h before stimulating by CD3/CD28 mono-antibodies. NF-KBp65 activity was detected 24 hours later. The frequency of CD25 and CD69 on T cell surface was detected 48 hours later. IL-2 level was detected 48 hours later.Results:The purity of isolated T cell is above 95%(determined by FACS analysis of CD3+ cell surface expression). Ursolic acid dose-dependently inhibited TCR-triggered NF-κB nuclear translocation. The frequency of CD25+ T cell and CD69+ T cell was significantly down-regulated by 25μM urolic acid (p<0.05, VS mono-antibodies stimulation).IL-2 was down-regulated to 894.8±80.3 ng/ml,650.2±62.2 ng/ml and 428.1±39.9 ng/ml (p<0.01, VS mono-antibodies stimulation).Conclusion:Ursolic acid dose-dependently inhibited TCR-triggered NF-κB nuclear translocation and T cell activation.PartⅡUrsolic acid monotherapy prolongs cardiac Allografts survival in miceObjective:To investigate the effect of ursolic acid on cardiac allograft survival in mice and its detailed mechanisms.Methods:The murine abdominal cardiac transplant model from BALB/c (H-2d) to C57BL/6 (H-2b) was built. Ursolic acid was responded with 0.5% carboxymethyl cellulose, and was administered by gastric irrigation. Ursolic acid was given from 1 day before transplant. The recipients were left untreated or administered with 0.5% carboxymethyl cellulose or with different dosages of ursolic acid (10mg/(kg.day),20mg/(kg.day) and 20mg/(kg.day)). Graft beating was monitored by daily palpation The survival of cardiac grafts was observed. Histology was performed to estimate the severity of rejection. CD4+ T cell and CD8+ T cell infiltrated into cardiac grafts were detected by immunohistochemistry. Cytokine profile of lymphocytes was analyzed by real time PCR.Results:The median survival time (MST) of cardiac allografts in blank control and vehicle control was 7.4±0.3days and 7.8±0.5days (p>0.05, n=5). For ursolic acid treatment group (10mg/kg/day), the median survival time (MST) of cardiac allografts was 23.8±1.3 days, which was significantly longer than the vehicle control group (p<0.05). Furthermore, ursolic acid at dosage of 20mg/kg/day and 30mg/kg/day prolonged MST to 36.8±0.6days (p <0.05, VS vehicle control group) and 68.5±2.8 days (p<0.05, VS vehicle control group). The prolonged survival was associated with reduced infiltration of CD4 and CD8 lymphocytes in allografts. Furthermore, the intragraft transcriptional profiling in ursolic acid-treated group showed reduction IFN-y mRNA but unaltered IL-4 mRNA.Conclusion:Ursolic acid monotherapy significantly prolonged the survival of cardiac allograft in mice. This may related to its suppression of T cell infiltration and induction of Thl/Th2 bias. PartⅢUrsolic acid and DST Lead to Transplant Tolerance in Mice cardiac TransplantationObjective:To investigate treatments of mice with a brief course of ursolic acid plus donor specific transfusion (DST) on day 0 can induce transplant tolerance.Methods:A fully allogenic BALB/c (H-2d) to C57BL/6 (H-2b) mice cardiac transplant model was established. Cardiac recipients were left untreated or given either ursolic acid, DST, ursolic acid plus DST or control vehicle (0.5% carboxymethyl cellulose). Ursolic acid (20mg/kg/day) was administered from day-1 to day 14. For DST, 1×107 donor strain (BALB/c) splenocytes was administered intravenously just post transplantation. For secondary cardiac transplant, LTS (mice with long-term-surviving grafts) were selected as recipients, and C3H or BALB/c were selected as donors. The adoptive transfer protocols include (1) 1×107 B6 lymphocytes or 1×107 LTS lymphocytes into B6/RAG-/- recipients which had been transplanted 25 days ago with BALB/c cardiac allografts. (2) 1×107 B6 lymphocytes and 1×107 LTS lymphocytes into B6/RAG-/- recipients that had been transplanted 25 days ago with BALB/c cardiac allografts. (3) 1x10'Rej lymphocytes into B6/RAG-/- recipients that had been transplanted 25 days ago with BALB/c cardiac allografts. (4) 1×107 Rej lymphocytes into LTS. The survival of cardiac grafts were monitored. The allo-response of splenocytes were detected by INF-y ELISPOT. The frequency of CD4+CD25+Foxp3+Teg in recipients 7days post-transplant and LTS were also detected.Results:When DST given alone to B6 recipient immediately after transplantation with BALB/c hearts, these grafts were promptly rejected with a similar tempo to those transplanted into untreated controls (p>0.05,8±0.7 days VS 7.4±0.3 days). Ursolic acid monotherapy (20mg/kg/day, from day-1 to day 14) prolonged the survivial of allografts to 36.8±0.6 days (p<0.05 vs. control vehicle (7.8±05 days)). ursolic acid together with DST on day 0 led to 84.6% of grafts surviving for more than 150 days. LTS did not reject the second BALB/c heart for more than 100 days, they all rejected the C3H hearts within 14 days. IFN-γELISPOT also showed LTS splenocytes had attenuated response to BALB/c antigen in vitro, had the same response to C3H antigen as that in the control group. The frequency of CD4+CD25+Foxp3+Teg in ursolic acid and DST treated mice is 6.5% 7 days post-transplant(p>0.05, VS untreated control 5.1%). The frequency of CD4+CD25+Foxp3+Teg in LTS is 4.9%(p>0.05, VS untreated control 5.1%).Conclusion:Protocol-treated mice involving a brief course of ursolic acid can achieve donor specific transplantation tolerance. It related to the depletion of donor alloreactive T cells.