INTRODUCTION. We hypothesize that ischemia/reperfusion (I/R)-induced nuclear factor-kappa B (NF-kB) activation enhances iNOS expression, resulting in subsequent damage to the microcirculation in reperfused muscle due to excessive NO production from iNOS. This study investigated the effects of pyrrolidine dithiocarbamate (PDTC), a selective NF-kB inhibitor, on microcirculation and iNOS expression in skeletal muscle following I/R.

METHODS. Rats weighing 90-110 g were received PDTC (150mg/kg) or phosphate-buffered saline (PBS, 0.2ml) 15 min prior to reperfusion. The left cremaster muscle of each animal underwent 5 h ischemia and 1.5 h reperfusion. During reperfusion, overall muscle blood flow was measured with laser Doppler flowmetry at 10 min intervals and the expression of iNOS mRNA and protein and NF-kB (p65) protein was determined by using real-time PCR technique and Western blot analysis.

RESULTS. At 10 min of reperfusion, the mean blood flow was only 47±5.6% (mean±SE) of baseline in controls and 79±13% in the PDTC-treated group. There was a significant difference between the two groups (p<0.05). Blood flow gradually increased to close to the baseline level (97±8%) at 50 min of reperfusion and remained at that level throughout the experiment, with a maximum of 102±7.5% at 90 min of reperfusion. In contrast, blood flow in the PDTC-treated group rapidly increased to 98±13% of baseline at 20 min of reperfusion and remained at the level of over baseline throughout the experiment. The highest level of the blood flow was 137±9% of baseline at 80 min of reperfusion. When compared to controls, the blood flow was significantly greater in the PDTC-treated group at 10-30 min and 70-90 min. Expression of iNOS mRNA and protein in controls had 34-fold and 13-fold increase from contralateral normal muscle expression, respectively, but only 14-fold and 1.2-fold increase in the PDTC group, respectively. There was significant difference in both mRNA and protein expression between the two groups. Expression of p65 protein in controls was 22-26% of normal in the cytoplasm and 114-138% in the nucleus. These values were 52-71% and 96-115% in the PDTC group, respectively.

DISCUSSION. Our data have shown that inhibition of NF-kB by PDTC improves microcirculation and reduces upregulated iNOS expression in reperfused skeletal muscle. The results support our hypothesis and indicate that NF-kB activation plays an important role in exaggerate I/R injury. Thus, inhibition of NF-kB activation may be a therapeutic intervention for reperfusion injury.