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

AIM: To evaluate the protective effect of NF-kappaB 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-kappaB decoy ODNs or scrambled ODNs. NF-kappaB 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-kappaB 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-kappaB activation in liver graft was induced in a time-dependent manner, and NF-kappaB remained activated for 16 h after graft reperfusion. NF-kappaB activation in liver graft was significant at 2 to 8 h and slightly decreased at 16 h after graft reperfusion. Administration of NF-kappaB decoy ODNs significantly suppressed NF-kappaB activation as well as mRNA expression of TNF-alpha, IFN-gamma and ICAM-1 in the liver graft. The hepatic NF-kappaB DNA binding activity [presented as integral optical density (IOD) value] in the NF-kappaB 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-alpha, IFN-gamma and ICAM-1 [presented as percent of beta-actin mRNA (%)] in the NF-kappaB 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 and 8 h of reperfusion, respectively, P<0.001). Administration of NF-kappaB 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-kappaB 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 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-kappaB decoy ODNs, the hepatic MPO content (A655) and serum ALT level (IU/L) in the NF-kappaB 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-kappaB decoy ODNs protects against I/R injury in liver graft by suppressing NF-kappaB activation and subsequent expression of proinflammatory mediators.

[Effects of escharectomy during burn shock stage on the mRNA expression of IFN-gamma and IL-4 in spleen T lymphocytes in rats after thermal injury].

OBJECTIVE: To determine the serum level and mRNA expression of type-1/type-2 cytokines of T lymphocytes in spleens of rats after thermal injury and to investigate the effects of escharectomy during burn shock stage on IFN-gamma and IL-4. METHODS: One hundred and sixty male Wistar rats were randomized into four groups. In group A, animals were not subjected to escharectomy. In groups B, C and D, escharectomy and skin allograft were performed at 8, 24, 96 hours postburn (PB) respectively. At 4, 12, 24, 48, 96, 120 and 168 hours PB, animals were killed and blood and spleens samples were harvested. ELISA was applied to determine the concentration of IFN-gamma and IL-4 in serum. The expression pattern of IFN-gamma and IL-4 were observed at mRNA level in T lymphocytes isolated from spleen by RT-PCR. RESULTS: The serum level of IFN-gamma and IL-4 rose rapidly and significantly after scald injury, expression of IFN-gamma and IL-4 mRNA in rats' T lymphocyte were also up-regulated spontaneously. The serum level of IFN-gamma and its mRNA expression began to rise within 4 hours PB, peaking at 24 hours PB. Whereas IL-4 and its mRNA expression showed a persistent elevation. Thereby leading to a dominant tendency of Th2 cytokine response on 7 d PB. In group A all above parameters revealed most obvious changes compared with controls, then ranked in group D, B and C. CONCLUSION: Escharectomy during burn shock stage is helpful to decrease the harmful over expression of Th2-type lymphocyte after severe thermal injury.

[Effects of the escharectomy during burn shock stage on expression of glucose translator-4 mRNA in skeletal muscle and adipose tissue].

OBJECTIVE: To investigate the effect of the escharectomy during burn shock stage on expression of glucose translator-4 (GLUT4) mRNA in skeletal muscle and adipose tissue. METHODS: 30% TBSA scalded rats were employed. Escharectomy were conducted at 8 h, 24 h, 168 h after burns respectively. Insulin, glucagon, cortisol and glucose levels in serum were analyzed. RT-PCR were employed to analyze GLUT4 mRNA expression in skeletal muscle and adipose tissue. RESULTS: Glucagon, cortisol and glucose levels in serum were declined in groups which escharectomy were conducted during burn shock stage. GLUT4 mRNA expression in both skeletal muscle and adipose tissue were downregulated after burns and escharectomy conducted during burn shock stage made it restored to near normal. CONCLUSION: GLUT4 mRNA expression will declined after major burns in skeletal muscle and adipose tissue. Escharectomy during shock stage could make it upregulated, which will be helpful to improve glucose metabolism and hypermetabolism after major burns.

[Escharectomy during burn shock on Th1/Th2 polarization of helper T lymphocytes in rats after thermal injury].

OBJECTIVE: To determine the type-1/type-2 cytokine response of helper T lymphocytes after thermal injury, and to investigate the effects of escharectomy during burn shock stage on the polarization of Th cells. METHODS: One hundred and sixty male Wistar rats were randomized into four groups. In group A, animals were subjected to a 30 percent full-thickness thermal injury without escharectomy. In groups B, C and D, escharectomy and skin allograft were done at 8, 24, and 96 hours postburn, respectively. At 4, 12, 24, 48, 96, 120 and 168 hours postburn, animals were killed and bloods samples as well as spleens were harvested. Enzyme linked immunoadsorbent assay (ELISA) was applied to determine interferon-gamma(IFN-gamma) and interleukin-4(IL-4) levels in blood and spleen tissues. RESULTS: Levels of IFN-gamma and IL-4 rapidly and significantly were increased after scald injury. IFN-gamma levels began to rise within 4 hours postburn, peaking at 24 hours later. IL-4 showed a persistent elevation up to 168 hours postburn, thereby leading to a dominant tendency of Th2 cytokine response on postburn day 7. In group A, all above parameters revealed most obvious changes compared with controls, then ranked in group D, B and C. CONCLUSION: Escharectomy during burn shock stage is helpful to prevent the shift to Th2 cell response after severe thermal injury.