Objective:To investigate the molecular mechanisms underlying the effects of arsenic trioxide(As2O3) in combination with leflunomide on the hamster-to-rat heart xenotransplant.Methods:Transplantation of LVG hamster hearts to Lewis rats was performed by anastomosis of vessels in the neck using end-to-end anastomosis with a non-suture cuff technique.Four groups of recipient rats (n=6 in each) were treated with normal saline (control),As2O3[5 mg/(kg·day) intraperitoneally],leflunomide[5 mg/(kg·d) orally],or leflunomide[5 mg/(kg·d)+As2O3[5 mg/(kg·d)]in combination.Donor hearts and/or rat spleens were harvested and analyzed 4 days after transplantation.Quantitative reverse-transcription polymerase chain reaction and Western blot analysis were performed to detect the expression of the nuclear factor erythroid-derived factor 2-related factor (Nrf2) and its target gene heme oxygenase-1 (HO-1),Treg cell marker fork-head Box P3(FOXP3),apoptosis-associated proteins Bcl-2,Bax,and cleaved caspase-3.Immunohistochemical staining was used to detect the levels of inflammatory natural killer cell and macrophage infiltration,intercellular cell adhesion molecule-1 (ICAM-1) and complement C3.Results:Expression of Nrf2-ARE-HO-1 signaling pathway was upregulated in heart xenografts in rats treated with As2O3 plus leflunomide compared with control rats or rats treated with either drug alone (P＜0.01),and this was accompanied by an increased Treg cells in the recipient rat spleen (P＜0.01).In contrast,the expressions of Bax,cleaved caspase-3,ICAM-1,and complement C3,and infiltration of inflammatory cells in the xenografts were inhibited by As2O3 plus leflunomide treatment (P＜0.01).Conclusion:Combination treatment with As2O3 and leflunomide protected hamster heart-xenografts in recipient rats.