Cannabinoid-2 receptor agonist HU-308 protects against hepatic ischemia/reperfusion injury by attenuating oxidative stress, inflammatory response, and apoptosis

Abstract

In this study, we have investigated the role of the cannabinoid CB(2) (CB(2)) receptor in an in vivo mouse model of hepatic ischemia/reperfusion (I/R) injury. In addition, we have assessed the role of the CB(2) receptor in TNF-alpha-induced ICAM-1 and VCAM-1 expression in human liver sinusoidal endothelial cells (HLSECs) and in the adhesion of human neutrophils to HLSECs in vitro. The potent CB(2) receptor agonist HU-308, given prior to the induction of I/R, significantly attenuated the extent of liver damage (measured by serum alanine aminotransferase and lactate dehydrogenase) and decreased serum and tissue TNF-alpha, MIP-1alpha, and MIP-2 levels, tissue lipid peroxidation, neutrophil infiltration, DNA fragmentation, and caspase 3 activity. The protective effect of HU-308 against liver damage was also preserved when given right after the ischemic episode. HU-308 also attenuated the TNF-alpha-induced ICAM-1 and VCAM-1 expression in HLSECs, which expressed CB(2) receptors, and the adhesion of human neutrophils to HLSECs in vitro. These findings suggest that selective CB(2) receptor agonists may represent a novel, protective strategy against I/R injury by attenuating oxidative stress, inflammatory response, and apoptosis.

Fig. 1

CB₂ agonist limits liver I/R-induced damage, lipid peroxidation, and neutrophil infiltration. (A) Serum transaminase ALT and (B) LDH levels; (C) liver MDA level and (D) MPO activity in sham or in mice exposed to 60/120 min I/R, pretreated with vehicle, HU-308 (10 mg/kg), AM251 (1 mg/kg) + HU-308, or AM630 (1 mg/kg) + HU-308 (n=7–12/each group; *, P<0.05, vs. I/R; ^#, P<0.05, versus I/R+HU-308).

Fig. 2

CB₂ receptor agonist decreases proinflammatory markers in serum and liver. (A and B) TNF-α, MIP-2, and MIP-1α and levels in serum and liver tissue, measured by ELISA in sham or in mice exposed to 60/120 min I/R, pretreated with vehicle, HU-308 (10 mg/kg), or AM630 (1 mg/kg) + HU-308 (n=6–12/each group; *, P<0.05, vs. I/R; ^#, P<0.05, vs. I/R+HU-308).

Fig. 3

CB₂ receptor agonist decreases I/R-induced apoptosis. (A) Caspase 3 activity and (B) DNA fragmentation in sham or in mice exposed to 60/120 min I/R, pretreated with vehicle, HU-308 (10 mg/kg), or AM630 (1 mg/kg) + HU-308 (n=6–8/each group; *, P<0.05, vs. I/R; ^#, P<0.05, vs. I/R+HU-308).

Fig. 4

CB₂ receptor agonist decreases histological damage and neutrophil infiltration 24 h following ischemia. Representative liver sections of sham mice, of mice exposed to 1 h/24 h I/R with vehicle, or HU-308 pretreatment. (A) H&E staining. (B) MPO staining (brown) contrastained with nuclear fast red. A similar histological profile was seen in three to four livers/group. The original scale bars indicate 50 μm.

Fig. 5

CB₂ receptor agonist decreases TNF-α-induced overexpression of ICAM-1 and VCAM-1 in HLSECs. (A) CB₂ receptor expression in HLSECs (RT-PCR). (B and C) ICAM-1 expression; (D and E) VCAM-1 expression (*, P<0.05, vs. TNF-α; ^#, P<0.05, vs. TNF-α+HU-308; n=9).

Fig. 6

CB₂ antagonist attenuates TNF-α-induced neutrophil adhesion to HLSECs. Representative images and quantification of human neutrophil adhesion to human liver endothelial cells. (*, P<0.05, vs. TNF-α; ^#, P<0.05, vs. TNF-α + HU-308; n=4).