Induction of heme oxygenase-1 protects mouse liver from apoptotic ischemia/reperfusion injury

Abstract

Ischemia/reperfusion (I/R) injury is the main cause of primary graft dysfunction of liver allografts. Cobalt-protoporphyrin (CoPP)-dependent induction of heme oxygenase (HO)-1 has been shown to protect the liver from I/R injury. This study analyzes the apoptotic mechanisms of HO-1-mediated cytoprotection in mouse liver exposed to I/R injury. HO-1 induction was achieved by the administration of CoPP (1.5 mg/kg body weight i.p.). Mice were studied in in vivo model of hepatic segmental (70 %) ischemia for 60 min and reperfusion injury. Mice were randomly allocated to four main experimental groups (n = 10 each): (1) A control group undergoing sham operation. (2) Similar to group 1 but with the administration of CoPP 72 h before the operation. (3) Mice undergoing in vivo hepatic I/R. (4) Similar to group 3 but with the administration of CoPP 72 h before ischemia induction. When compared with the I/R mice group, in the I/R+CoPP mice group, the increased hepatic expression of HO-1 was associated with a significant reduction in liver enzyme levels, fewer apoptotic hepatocytes cells were identified by morphological criteria and by immunohistochemistry for caspase-3, there was a decreased mean number of proliferating cells (positively stained for Ki67), and a reduced hepatic expression of: C/EBP homologous protein (an index of endoplasmic reticulum stress), the NF-κB's regulated genes (CIAP2, MCP-1 and IL-6), and increased hepatic expression of IκBa (the inhibitory protein of NF-κB). HO-1 over-expression plays a pivotal role in reducing the hepatic apoptotic IR injury. HO-1 may serve as a potential target for therapeutic intervention in hepatic I/R injury during liver transplantation.

Fig. 1

A statistically significant difference was noted in mean serum ALT/AST/LDH levels following the induction of I/R, compared with sham operated mice. In CoPP pretreated mice following the induction of I/R, the increase in serum liver enzymes level was significantly less compared with non-treated mice

Fig. 2

Pre-treatment with CoPP significantly increased hepatic HO-1 expression (p = 0.032) (a). ER stress pathway was up-regulated following I/R as demonstrated by the significant increase in the hepatic expression of the CHOP protein (p <0.05). Pre-treatment with CoPP decreased CHOP protein expression p <0.05 (b)

Fig. 3

a IkB (inhibitory proteins of NF-κB) hepatic expression was significantly lower following I/R (p = 0.05) compared with sham operated mice. b Liver pretreated with CoPP had significantly higher hepatic expression of IkB (p = 0.01)

Fig. 4

NF-κB pathway was stimulated by the induction of hepatic ischemia, as is demonstrated by the increase in the hepatic expression of the three targeted NF-κB’s genes: CIAP2, MCP-1 and IL-6. CoPP pre-treatment significantly decreased a CIAP2 (p = 0.00001), b MCP-1 (p = 0.0001), and c IL-6 hepatic (p = 0.0005) expression

Fig. 5

Histologic examination of sections from liver tissue stained with hematoxylin-eosin revealed damage in the I/R untreated livers manifested by many acidophilic bodies and groups of necrotic hepatocytes (black line delineating area of coagulative necrosis) (a). Following the pre-administration of CoPP only occasional apoptotic bodies were found (arrows) (b)

Fig. 6

Sections from I/R livers were highly positively stained for the activated form of caspase-3. Scattered positive nuclei and cytoplasmic staining were noted in hepatocytes as well as sinusoidal cells (a). In the I/R pretreated-CoPP group, only rare cells stained for caspase-3 activity (b). I/R livers showed a significant increase in the mean number of apoptotic cells (positively stained for caspase-3) (p = 0.013) (c). Sections from I/R livers were highly positively stained for Ki67 as a proliferation index (d). In livers pre treated with CoPP, fewer hepatocytes stained for Ki67 (e). Although I/R livers showed an increase in the mean number of proliferating cells (positively stained for Ki67) compared with livers pre treated with CoPP (p = NS)