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

AIM： To evaluate the protective effect of NF-kB decoy oligodeoxynucleotides （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-kB decoy ODNs or scrambled ODNs. NF-kB decoy ODNs or scrambled ODNs were injected intravenously into donor and recipient rats 6 and 1 h before operation, respectively. Recipients were killed 0 to 16 h after liver graft reperfusion. NF-kB activity in the liver graft was analyzed by electrophoretic mobility shift assay （EMSA）. Hepatic mRNA expression of TNF-α, IFN-γ and intercellular adhesion molecule-1 （ICAM-1） were determined by semiquantitative RT-PCR. Serum levels of TNF-α and IFN-γ 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-kB activation in liver graft was induced in a time-dependent manner, and NF-kB remained activated for 16 h after graft reperfusion. NF-kB activation in liver graft was significant at 2 to 8 h and slightly decreased at 16 h after graft reperfusion. Administration of NF-kB decoy ODNs significantly suppressed NF-kB activation as well as mRNA expression of TNF-α, IFN-γ, and ICAM-1 in the liver graft. The hepatic NF-kB DNA binding activity [presented as integral optical density （IOD） value] in the NF-kB 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 and 8 h of reperfusion, respectively, P〈0.001）. The hepatic mRNA expression level of TNF-α, IFN-γ and ICAM-1 rpresented as percent of β-actin mRNA （%）] in the NF-kB 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-α mRNA, 5.58±2.16 vs 50.46±9.35 and 6.47±2.53 vs 69.72±13.41 for hepatic IFN-γ 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 and 8 h of reperfusion, respectively, P〈0.001）. Administration of NF-kB decoy ODNs almost completely abolished the increase of serum level of TNF-α and IFN-γ induced by hepatic ischemia/reperfusion, the serum level （pg/mL） of TNF-α and in the NF-kB 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-α level, 31.5±12.1 vs 102.1±14.5 and 40.2±13.5 vs 118.6±16.7 for IFN-γ level after 4 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-kB decoy ODNs, the hepatic MPO content （A655） and serum ALT level （IU/L） in the NF-kB 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 and 8 h of reperfusion, respectively, P〈0.001）. CONCLUSION： The data suggest that NF-kB decoy ODNs protects against I/R injury in liver graft by suppressing NF-kB activation and subsequent expression of proinflammatory mediators.