Background. Diazoxide, a selective mitochondria ATP-sensitive potassium (KATP) channel opener, has been reported to preserve the microvascular integrity of ischemia-reperfusion (I/R)-injured tissues. Our study aimed to investigate the effect of diazoxide on I/R-injured cremaster muscles, and to further elucidate the underlying mechanism.

Methods. Male Sprague Dawley rats were randomized (n=8 per group) into four groups: sham-operated control group, I/R group (4 hours of pudic epigastic artery ischemia followed by 2 hours of reperfusion), diazoxide + I/R group, and chelerythrine (PKC inhibitor) + diazoxide + I/R group. Intravital microscopy was used to observe leukocyte/endothelial cell interactions and quantify functional capillaries in rat cremaster muscle flaps. Biochemical examinations were implemented at the end of reperfusion periods. Protein and mRNA expressions of TNF-a, ICAM-1, PECAM-1, E-selectin, L-selectin, and P-selectin were determined by Western blotting, immunohistochemical analysis, and RTPCR.

Results. Microscopically, we observed that I/R markedly increased the number of rolling, adhering, and migrating leukocytes (Figure 1). Biochemically, we found that I/R significantly increased TNF-a and L-selectin expressions. However, we did not see any significant increases in ICAM-1, PECAM-1, E-selectin, and P-selectin expressions in this group. In the diazoxide + I/R group, we found that diazoxide indeed reduced the number of I/R-induced leukocyte rolling, adhesion, and migration (Figure 1). Further more, we observed that diazoxide downregulated I/R-induced L-selectin expression (Figure 2) but had no effect on TNF-a expression. Finally, in the chelerythrine + diazoxide + I/R group, we observed that diazoxide¢s protective effects were antagonized by the addition of chelerythrine.

Conclusion. The ability of diazoxide to ameliorate microcirculatory disturbances appears to be related to its ability to downregulate L-selectin expression. Our results indicated that diazoxide¢s protective mechanism against I/R-induced injury operates through a PKC-dependent pathway.