Bilirubin Rinse: A Simple Protectant Against the Rat Liver Graft Injury Mimicking Heme Oxygenase-1 Preconditioning

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Abstract
Heme oxygenase (HO)–1 preconditioning through genetic or pharmacologic interventions was shown experimentally to improve posttransplant outcome of liver grafts. However, its clinical application requires careful consideration because of the complexity and economic costs of the procedures. This study aimed to examine if graft preconditioning with HO–1 could be substituted by a simple treatment with heme–degrading products such as bilirubin. Rats were pretreated with or without hemin, an HO–1 inducer for preconditioning. Their livers were harvested as grafts in University of Wisconsin (UW) solution for 16 hours at 4°C and followed by reperfusion ex vivo or by transplantation in vivo. The control grafts were also treated with a rinse buffer containing varied concentrations of unconjugated bilirubin with different time intervals. The HO–1–preconditioned grafts ex vivo exhibited a marked improvement of bile output and cell injury that was cancelled by blocking HO with zinc protoporphyrin–IX. The aggravation of the graft viability by the inhibitor was repressed by supplementation of bilirubin but not by that of carbon monoxide. Furthermore, a short–term rinse treatment with micromolar levels of bilirubin attenuated biliary dysfunction and cell injury of the grafts both ex vivo and in vivo even without HO–1 preconditioning. The protective effects of HO–1 preconditioning or bilirubin rinse appeared to involve its inhibitory effects on lipid peroxidation in hepatocytes. In conclusion, these results suggest that bilirubin rinse serves as a simple strategy to ameliorate hyperacute oxidative stress and hepatobiliary dysfunction of the transplanted grafts, mimicking effects of HO–1–mediated preconditioning.