Isoflurane preconditioning at clinically relevant doses induce protective effects of heme oxygenase-1 on hepatic ischemia reperfusion in rats

Abstract

Background: Activation of heme oxygenase-1 (HO-1) has been proved to reduce damages to the liver in ischemia reperfusion injury. The objective of present study was to determine whether clinic relevant doses of isoflurane treatment could be sufficient to activate HO-1 inducing, which confers protective effect against hepatic ischemia-reperfusion injury.

Methods: The hepatic artery and portal vein to the left and the median liver lobes of forty male Sprague-Dawley rats were occluded for 60 minutes. Reperfusion was allowed for 4 hours before the animal subjects were sacrificed. Six groups (n = 12) were included in the study. A negative control group received sham operation and positive control group a standard ischemia-reperfusion regimen. The third group was pretreated with isoflurane prior to the ischemia-reperfusion. The fourth group received an HO-1 inhibitor zinc protoporphyrin (Znpp) prior to the isoflurane pretreatment and the ischemia-reperfusion. The fifth group received Znpp alone before ischemia-reperfusion procedure, and the sixth group was administrated with a HO-1 inducer hemin prior to IR. HO-1 in the liver was measured using an enzymatic activity assay, a Western blot analysis, as well as immunohistochemical method. Extent of liver damage was estimated by determination of the serum transaminases, liver lipid peroxidation and hepatic histology. Infiltration of the liver by neutrophils was measured using a myeloperoxidase activity assay. TNFα mRNA in the liver was measured using RT-PCR.

Results: Isoflurane pretreatment significantly attenuated the hepatic injuries and inflammatory responses caused by the ischemia reperfusion. Selectively inhibiting HO-1 with ZnPP completed blocked the protective effects of isoflurane. Inducing HO-1 with hemin alone produced protective effects similar in magnitude to that of isoflurane.

Conclusions: Clinic relevant doses of isoflurane attenuate ischemia reperfusion injury in rats by increasing the HO-1 expression and activity.

Figure 1

Effect of isoflurane preconditioning on liver heme oxygenase-1(HO-1) after ischemia reperfusion injury (IR) (n = 12 each group). (A) The HO-1 activity in the liver. Both isoflurane and hemin pretreatment augmented the HO-1 activity response, while administration of the HO-1 inhibitor zinc protoporphyrin (Znpp), prior to the isoflurane pretreatment or Znpp alone significantly decreased the HO-1 activity (p < 0.05, vs. IR). (B) Western blot analysis of HO-1in liver. Western blotting showed an increase in HO-1 protein expression in the groups of isoflurane and hemin (* P < 0.05 vs. IR). (C) Immunohistochemical detection of HO-1 in the liver. Hepatic HO-1 positive cells were defined as stained with brown in cytoplasm (black arrows). There was an increase in HO-1 expression in isoflurane and hemin pretreated groups compared in IR, it also showed a decrease in HO-1protein expression in both Znpp pretreated groups compared with isoflurane group (Magnification: 400×. Scale bar = 100 μm).

Figure 2

Effects of isoflurane preconditioning on hepatic histology after ischemia reperfusion (IR) procedure (n = 12 in each group). (A) Hepatic tissue histological changes were processed with H&E staining for light microscopy examination. Photograph depicted typical pattern of focal necrosis (black arrows) after ischemic degeneration of hepatocytes around the central venous area. More seriously necrosis was shown in both zinc protoporphyrin (Znpp) pretreated and IR groups compared in sham group. Magnification: 200 × Scale bar = 200 μm. (B) There was no obvious necrosis in sham group. Areas of necrosis were significantly lower in isoflurane and hemin pretreated groups than in IR group (* P < 0.05 vs. IR).