BENEFICIAL EFFECT OF CARBON MONOXIDE-INHALATION AGAINST ISCHEMIA/REPERFUSION INJURY AFTER RAT LIVER TRANSPLANTATION: POSSIBLE MECHANISM OF iNOS/NO PATHWAY BLOCKADE

Abstract

P84 Background: Heme oxygenase (HO)-1 is a pleiotropic molecule that has been shown to provide potent cytoprotection in numerous models of cellular stress. Carbon monoxide (CO), product of heme degradation by HO-1, has been shown to induce similar cytoprotection as HO-1 against ischemia/reperfusion (I/R) injury. On the contrary, roles of inducible nitric oxide synthase (iNOS), another stress inducible gene product, during I/R injury remain controversial. In this study, we examined whether CO would protect the liver graft against cold I/R injury associating with transplantation. Particular attention was paid to the influence of CO treatment on hepatic iNOS/NO pathway. Methods: Orthotopic syngeneic LEW rat liver transplantation (OLT) was performed with 18 hrs preservation in cold UW solution. Recipients were exposed to air or two different doses of CO (20 or 100 ppm) for 1 hr before and 24 hrs after OLT. Recipients were sacrificed 3-48 hrs for serum AST and NO levels, histopathology, and hepatic iNOS protein expression. Efficacy of CO was further analyzed in vitro primary rat hepatocyte culture system after stimulation with cytokine mixture (CM) (TNF-α 500U/ml, IL-1 200U/ml, IF-γ 100U/ml). iNOS protein, NO production and hepatocyte viability after CM stimulation were analyzed. Results: After OLT, CO-inhalation at 100 ppm effectively improved hepatic I/R injury. Serum AST levels and tissue necrosis were significantly reduced with 100 ppm CO, but not with 20 ppm, compared to air-treated controls. Hepatic iNOS protein expression was significantly decreased in CO-treated group as compared with air-treated controls at 3 and 48 hrs, associating with lower serum NO levels (Table, mean ± SE, n=3-6 animals per group, *P < 0.05). When rat hepatocytes were cultured in the presence of CO 100 ppm, CM-induced iNOS protein expression was markedly inhibited with significantly reduced NO levels at 24 hrs (20.3 ± 0.6 vs. 2.8 ± 1.8 μM in CO-group, p < 0.05). Hepatocyte viability also was increased with CO treatment at 24 hrs after CM stimulation (56.9 ± 0.02 vs. 89.9 ± 0.03 % in CO-group, p < 0.02). Conclusion: The results demonstrate that exogenous CO treatment efficiently ameliorates hepatic I/R injury. The possible mechanism, by which CO protects the liver against cold I/R, may include the down-regulation of iNOS/NO pathway. Thus, CO-inhalation treatment would be a novel therapeutic strategy to combat hepatic cold I/R injury.Figure