Carbon monoxide-releasing molecule-2 (CORM-2) attenuates acute hepatic ischemia reperfusion injury in rats

Abstract

Background: Hepatic ischemia-reperfusion injury (I/Ri) is a serious complication occurring during liver surgery that may lead to liver failure. Hepatic I/Ri induces formation of reactive oxygen species, hepatocyte apoptosis, and release of pro-inflammatory cytokines, which together causes liver damage and organ dysfunction. A potential strategy to alleviate hepatic I/Ri is to exploit the potent anti-inflammatory and cytoprotective effects of carbon monoxide (CO) by application of so-called CO-releasing molecules (CORMs). Here, we assessed whether CO released from CORM-2 protects against hepatic I/Ri in a rat model.

Methods: Forty male Wistar rats were randomly assigned into four groups (n = 10). Sham group underwent a sham operation and received saline. I/R group underwent hepatic I/R procedure by partial clamping of portal structures to the left and median lobes with a microvascular clip for 60 minutes, yielding approximately 70% hepatic ischemia and subsequently received saline. CORM-2 group underwent the same procedure and received 8 mg/kg of CORM-2 at time of reperfusion. iCORM-2 group underwent the same procedure and received iCORM-2 (8 mg/kg), which does not release CO. Therapeutic effects of CORM-2 on hepatic I/Ri was assessed by measuring serum damage markers AST and ALT, liver histology score, TUNEL-scoring of apoptotic cells, NFkB-activity in nuclear liver extracts, serum levels of pro-inflammatory cytokines TNF-alpha and IL-6, and hepatic neutrophil infiltration.

Results: A single systemic infusion with CORM-2 protected the liver from I/Ri as evidenced by a reduction in serum AST/ALT levels and an improved liver histology score. Treatment with CORM-2 also up-regulated expression of the anti-apoptotic protein Bcl-2, down-regulated caspase-3 activation, and significantly reduced the levels of apoptosis after I/Ri. Furthermore, treatment with CORM-2 significantly inhibited the activity of the pro-inflammatory transcription factor NF-kappaB as measured in nuclear extracts of liver homogenates. Moreover, CORM-2 treatment resulted in reduced serum levels of pro-inflammatory cytokines TNF-alpha and IL-6 and down-regulation of the adhesion molecule ICAM-1 in the endothelial cells of liver. In line with these findings, CORM-2 treatment reduced the accumulation of neutrophils in the liver upon I/Ri. Similar treatment with an inactive variant of CORM-2 (iCORM-2) did not have any beneficial effect on the extent of liver I/Ri.

Conclusions: CORM-2 treatment at the time of reperfusion had several distinct beneficial effects on severity of hepatic I/Ri that may be of therapeutic value for the prevention of tissue damage as a result of I/Ri during hepatic surgery.

Figure 1

CORM-2 attenuates hepatic ischemia reperfusion injury (I/Ri). Serum levels of A ALT and B AST of rats subjected to 60 min. of ischemia followed by 6 h of reperfusion. C Liver histology damage scoring of sham-operated rats, I/Ri rats, CORM-2 treated rats and iCORM-2 treated rats. *P < 0.05 vs. I/R group.

Figure 2

CORM-2 treatment inhibits apoptosis in hepatic I/Ri. Hepatocyte apoptosis was visualised by TUNEL staining in biopsies of A Sham-operated rats, B I/Ri-treated rats, C CORM-2 treated rats, D iCORM-2 treated rats. E. Graph of the apoptotic index in the various treatment groups. *P < 0.05 vs. I/R group.

Figure 3

CORM-2 modulates pro- and anti-apoptotic proteins and decreases neutrophil infiltration. A Western blot analysis of the expression of ICAM-1, caspase-3, Bcl-2 and HO-1 in sham-operated, I/Ri operated, CORM-2 treated and iCORM-2 treated rats. B The relative expression levels of ICAM-1 and caspase-3 or C Bcl-2 and HO-1 were analysed with densitometry analysis in relation to β-actin. D Serum levels of TNF-α and E IL-6 of rats subjected to 60 min. of ischemia followed by 6 h of reperfusion. F Liver tissue MPO activity for each group was determined. *P < 0.05 vs. I/R group.

Figure 4

CORM-2 blocks pro-inflammatory NF-κB signalling in vivo. NF-κB activation was determined using A ELISA-based TransAM NF-κB p65 kit and B visualised using western blot followed by C densitometry analysis.*P < 0.05 vs. I/R group.