Ulinastatin preconditioning attenuates inflammatory reaction of hepatic ischemia reperfusion injury in rats via high mobility group box 1(HMGB1) inhibition

Abstract

Objective It has been found that ulinastatin (UTI) can attenuate hepatic injury in a rat model of ischemia reperfusion (IR), but the specific mechanism is unclear. This study aims to investigate possible pathomechanism of ulinastatin in reducing the inflammatory response after hepatic IR. Methods A male sprague-dawley(SD) rat model of hepatic ischemia reperfusion injury was used. The rats were randomly divided into 4 groups on average, which were 0.9% saline and IR group as control, ulinastatin preconditioning (UPC) group, UPC+rHMGB1 (recombinant HMGB1) group and UPC +anti-HMGB1 group. Serum aminotransferases, TNF-α, IL-1 and Myeloperoxidase (MPO) levels were measured. Histopathology examination and apoptotic cell detection and the different expression of HMGB1 protein were also assessed. Results Serum levels of aminotransferases, cytokines and hepatic MPO in UPC and UPC+anti-HMGB1 groups were significantly lower than those in control group (p<0.05). Decreased histologic damage and apoptosis were also seen in these two groups (p<0.05). Conclusions HMGB1 expressions in UPC and UPC+anti-HMGB1 groups were significantly lower than those in the two control groups (p<0.05), pretreatment with ulinastatin attenuated liver IR injury by reducing HMGB1 expression through its anti-inflammatory effects.

Keywords: High mobility group box 1 protein (HMGB1); Ischemia reperfusion; Preconditioning; Ulinastatin.

Figure 1

Serum aminotransferases, cytokines, and hepatic myeloperoxidase among different groups. Figure A showed serum ALT and AST levels significantly increased when 20 IU/ml of l.5ml/kg of ulinastatin pretreated ( UPC ) group or combined with 100μg of anti-HMGB1 (UPC+Anti-HMGB1 group) compared to those in control (p<0.05), but there was no significant difference between control group and UPC+rHMGB1 group (p>0.05). As showed in figure B and C, significantly increasing serum cytokines TNF-α and IL-1 levels were found in the control group and the UPC+rHMGB1 group compared with UPC group and UPC+Anti-HMGB1 group (p<0.05). At 120 min after reperfusion, rats in UPC and UPC plus Anti-HMGB1 had significantly reduced levels of myeloperoxidase (MDA) in liver homogenates compared to those in control as showed in figure D. Values are mean ± SD of 10 animals in each group. * P < 0.05 vs. Control.

Figure 2

Effect of ulinastatin preconditioning (UPC) on attenuating liver ischemia reperfusion injury. Liver tissue was processed with H&E staining for light microscopy. Photograph depicts typical pattern of focal necrosis (black arrows) after ischemic degeneration of hepatocytes around the central venous area. Areas of necrosis were significantly lower in UPC and Anti-HMGB1 treated groups than in control. Magnification: 200×. Scale bar = 200μm. Hepatocyte apoptosis was determined by immunohistochemical analysis by microscopy after TUNEL staining. Photograph depicts typical pattern of apoptotic cells (black arrows). Percentage of apoptotic cells was significantly lower in UPC and Anti-HMGB1 treated groups than in control group. Magnification: 200×. Scale bar = 200μm.

Figure 3

Immunohistological result of HMGB1 protein expression in groups. Hepatic HMGB1 positive cells were defined as stained with brown in cytoplasm (black arrows). It showed a decrease in HMGB1 expression in UPC and Anti-HMGB1 groups compared with control. Magnification: 400×. Scale bar = 100μm