Nuclear factor-κB decoy oligodeoxynucleotides attenuates ischemia/reperfusion injury in rat liver graft

AIM: To evaluate the protective effect of NF-kappa B decoy oligodeoxynuleotides (ODNs) on ischemia/reperfusion (I/R) injury in rat liver graft. METHODS: Orthotopic syngeneic rat liver transplantation was performed with 3 h of cold preservation of liver graft in University of Wisconsin solution containing phosphorothioated double-stranded NF-kappa B decoy ODNs or scrambled ODNs. NF-kappa B decoy ODNs or scrambled ODNs were injected intravenously into donor and recipient rats 6 h and 1 h before operation, respectively. Recipients were sacrificed 0h to 16 h after liver graft reperfusion. NF-kappa B activity in the liver graft was analyzed by electrophoretic mobility shift assay (EMSA). Hepatic mRNA expression of TNF-alpha, IFN-gamma and intercellular adhesion molecule-1(ICAM-1) were determined by semiquantitative RT-PCR. Serum levels of TNF-alpha and IFN-gamma were measured by enzyme-linked immunosorbent assays (ELISA). Serum level of alanine transaminase (ALT) was measured using a diagnostic kit. Liver graft myeloperoxidase (MPO) content was assessed. RESULTS: NF-kappa B activation in liver graft was induced in a time -dependent fashion, and NF-kappa B remained activated for 16 h after graft reperfusion. NF-kappa B activation in liver graft was significant at 2 to 8 h and slightly decreased at 16 h after graft reperfusion. Administration of NF-kappa B decoy ODNs significantly suppressed NF-kappa B activation as well as mRNA expression of TNF-alpha, IFN-gamma and ICAM-1 in the liver graft. The hepatic NF-kappa B DNA binding activity [presented as integral optical density (IOD) value] in the NF-kappa B decoy ODNs treatment group rat was significantly lower than that of the I/R group rat (2.16 +/- 0.78 vs 36.78 +/- 6.35 and 3.06 +/- 0.84 vs 47.62 +/- 8.71 for IOD value after 4 h and 8 h of reperfusion, respectively, P < 0.001). The hepatic mRNA expression level of TNF-alpha, IFN-gamma and ICAM-1 [presented as percent of beta-actin mRNA(%)] in the NF-kappa B decoy ODNs treatment group rat was significantly lower than that of the I/R group rat (8.31 +/- 3.48 vs 46.37 +/- 10.65 and 7.46 +/- 3.72 vs 74.82 +/- 12.25 for hepatic TNF-alpha mRNA, 5.58 +/- 2.16 vs 50.46 +/- 9.35 and 6.47 +/- 2.53 vs 69.72 +/- 13.41 for hepatic IFN-gamma mRNA, 6.79 +/- 2.83 vs 46.23 +/- 8.74 and 5.28 +/- 2.46 vs 67.44 +/- 10.12 for hepatic ICAM-1 mRNA expression after 4 hours and 8 h of reperfusion, respectively, P < 0.001). Administration of NF-kappa B decoy ODNs almost completely abolished the increase of serum level of TNF-alpha and IFN-gamma induced by hepatic ischemia/reperfusion, the serum level (pg/ml) of TNF-alpha and IFN-gamma in the NF-kappa B decoy ODNs treatment group rat was significantly lower than that of the I/R group rat (42.7 +/- 13.6 vs 176.7 +/- 15.8 and 48.4 +/- 15.1 vs 216.8 +/- 17.6 for TNF-alpha level, 31.5 +/- 12.1 vs 102.1 +/- 14.5 and 40.2 +/- 13. 5 vs 118.6 +/- 16.7 for IFN-gamma level after 4 hours and 8 h of reperfusion, respectively, P < 0.001). Liver graft neutrophil recruitment indicated by MPO content and hepatocellular injury indicated by serum ALT level were significantly reduced by NF-kappa B decoy ODNs, the hepatic MPO content (A655) and serum ALT level (IU/L) in the NF-kappa B decoy ODNs treatment group rat was significantly lower than that of the I/R group rat (0.17 +/- 0.07 vs 1.12 +/- 0.25 and 0.46 +/- 0.17 vs 1.46 +/- 0.32 for hepatic MPO content, 71.7 +/- 33.2 vs 286.1 +/- 49.6 and 84.3 +/- 39.7 vs 467.8 +/- 62.3 for ALT level after 4 hours and 8 h of reperfusion, respectively, P < 0.001). CONCLUSION: The data suggest that NF-kappa B decoy ODNs protects against I/R injury in liver graft by suppressing NF-kappa B activation and subsequent expression of proinflammatory mediators. (C) 2005 The WJG Press and Elsevier Inc. All rights reserved.