Diazoxide attenuates ischemia/reperfusion injury via upregulation of heme oxygenase-1 after liver transplantation in rats

Abstract

Aim: To evaluate the effects of diazoxide on ischemia/reperfusion (I/R)-injured hepatocytes and further elucidate its underlying mechanisms.

Methods: Male Sprague-Dawley rats were randomized (8 for donor and recipient per group) into five groups: I/R group (4 h of liver cold ischemia followed by 6 h of reperfusion); heme oxygenase-1 (HO-1) small interfering RNA (siRNA) group (injection of siRNA via donor portal vein 48 h prior to harvest); diazoxide (DZ) group (injection of DZ via donor portal vein 10 min prior to harvest); HO-1 siRNA + DZ group; and siRNA control group. Blood and liver samples were collected at 6 h after reperfusion. The mRNA expressions and protein levels of HO-1 were determined by reverse transcription polymerase chain reaction and Western blotting, and tissue morphology was examined by light and transmission electron microscopy. Serum transaminases level and cytokines concentration were also measured.

Results: We observed that a significant reduction of HO-1 mRNA and protein levels in HO-1 siRNA and HO-1 siRNA + DZ group when compared with I/R group, while the increases were prominent in the DZ group. Light and transmission electron microscopy indicated severe disruption of tissue with lobular distortion and mitochondrial cristae damage in the HO-1 siRNA and HO-1 siRNA + DZ groups compared with DZ group. Serum alanine aminotransferase, aspartate transaminase, tumor necrosis factor-α and interleukin-6 levels increased in the HO-1 siRNA and HO-1 siRNA + DZ groups, and decreased in the DZ group.

Conclusion: The protective effect of DZ may be induced by upregulation of HO-1. By inhibiting expression of HO-1, this protection pretreated with DZ was abolished.

Keywords: Diazoxide; Heme oxygenase-1; Ischemia/reperfusion injury; Liver transplantation; Rat.

Figure 1

Experimental protocol. DZ: Diazoxide; siRNA: Small interfering RNA.

Figure 2

Serum alanine aminotransferase and aspartate transaminase levels after reperfusion. ^aP < 0.05 vs ischemia/reperfusion (I/R) group, ^bP < 0.01 vs diazoxide (DZ) group. ALT: Alanine aminotransferase; AST: Aspartate transaminase.

Figure 3

Liver heme oxygenase-1 mRNA and protein levels at 6 h after transplantation. ^aP < 0.05 vs ischemia/reperfusion (I/R) group, ^bP < 0.01 vs diazoxide (DZ) group. A: Liver heme oxygenase-1 (HO-1) mRNA expression; B: Liver HO-1 protein levels.

Figure 4

Liver histology and ultrastructural changes of hepatocytes. A: Ischemia/reperfusion (I/R) treated livers showed vacuolization, nuclear fragmentation, sinusoidal congestion, and hepatocyte necrosis. However, histological tissue damage was aggravated in the small interfering RNA (siRNA) and siRNA + diazoxide (DZ) groups. In contrast, DZ treatment relieved liver damage (HE, × 400); B: The mitochondria and the smooth endoplasmic reticulum were dilated and even destroyed, and the mitochondrial cristae were disorganized in I/R treated hepatocytes.

Figure 5

Serum cytokine levels (interleuki-6/ tumor necrosis factor-α) were measured. ^aP < 0.05 vs ischemia/reperfusion (I/R) group, ^bP < 0.01 vs diazoxide (DZ) group. IL-6: Interleukin-6; TNF-α: Tumor necrosis factor-α.