核因子-κB抑制剂熊果酸延长小鼠心脏移植物存活时间

Ursolic Acid Monotherapy Prolongs Cardiac Allograft Survival in Mice

目的:研究熊果酸对同种异体小鼠心脏移植排斥反应的作用。方法:体外实验:CD3、CD28单抗刺激B6小鼠T细胞,实验组加入不同浓度的熊果酸。24h后检测P65核转位程度;48h后检测T细胞表面CD25、CD69表达情况。体内实验:熊果酸用0.5%羧甲基纤维素钠混悬。行BALB/c到B6的小鼠心脏移植。实验组使用10mg/(kg·d)、30mg/(kg·d)的熊果酸灌胃。对照组使用相同剂量的0.5%羧甲基纤维素钠灌胃。观察术后7d时移植心CD4+、CD8+T细胞浸润情况,IFN-γ、IL-4mRNA水平和移植心存活时间。结果:熊果酸剂量依赖性抑制P65核转位。25μmol/L的熊果酸可将T细胞表面的CD25下调92.4%,CD69下调89.3%。10mg/(kg·d)熊果酸可将移植心平均存活时间(MST)延长至(23.8±1.3)d[P<0.05,与对照组(7.4±0.3)d相比]。而30mg/(kg·d)的熊果酸可将MST延长至(68.5±2.8)d。熊果酸组移植心内CD4+和CD8+T细胞浸润数量显著减少(P<0.05,与对照组比较),且IFN-γmRNA水平显著下调(P<0.05,与对照组比较),而IL-4mRNA水平无明显变化。结论:熊果酸可抑制T细胞NF-κB活化。体内应用熊果酸能显著延缓同种异体移植心排斥时间,这与熊果酸抑制T细胞活化,下调Th1反应相关。

Objective： To investigate the effect of ursolic acid on cardiac allograft survival in mice. Methods： B6mice T cells were stimulated by anti-CD3 and anti-CD28 monoclonal antibody with or without ursolic acid. NF-κB P65 activity was detected 24 hours later. The frequency of CD25 and CD69 on T cell surface was also detected 48 hours later. The murine cardiac transplant model from BALB/c （H-2d） to C57BL/6 （H-2b） was built. The recipients were administered with or without different dosages of ursolic acid. The survival of cardiac grafts was observed. Histology and immunohistochemistry were performed to estimate the severity of rejection. Cytokine profile of lymphocytes was analyzed by real time PCR. Results： Ursolic acid dose-dependently inhibited TCR-triggered NF-κB nuclear translocation. The frequency of CD25 and CD69 was significantly downregulated by 25 μmol/L urolic acid （P0.05,vs vehicle control）. For ursolic acid treatment group （10 mg/／[kg／5d／]）, the median survival time （MST） of cardiac allografts was 23.8±1.3 days, which was significantly longer than the vehicle control group （P0.05, MST=7.4±0.5 d）. Furthermore, ursolic acid at dosage of 30 mg/（kg／5d） prolonged MST to 68.5±2.8 days. 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-γ mRNA but unaltered IL-4 mRNA. Conclusion： Ursolic acid inhibites NF-κB activation of T cell. Ursolic acid monotherapy could significantly prolong the survival of cardiac allograft in mice. This may relate to its suppression of T cell activation and induction of Th1/Th2 bias.