Arsenic Trioxide Combining Leflunomide Activates Nrf2-ARE-HO-1 Signaling Pathway and Protects Heart Xenografts

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.