A new cannabinoid CB2 receptor agonist HU-910 attenuates oxidative stress, inflammation and cell death associated with hepatic ischaemia/reperfusion injury

Abstract

Background and purpose: Cannabinoid CB(2) receptor activation has been reported to attenuate myocardial, cerebral and hepatic ischaemia-reperfusion (I/R) injury.

Experimental approach: We have investigated the effects of a novel CB(2) receptor agonist ((1S,4R)-2-(2,6-dimethoxy-4-(2-methyloctan-2-yl)phenyl)-7,7-dimethylbicyclo[2.2.1]hept-2-en-1-yl)methanol (HU-910) on liver injury induced by 1 h of ischaemia followed by 2, 6 or 24 h of reperfusion, using a well-established mouse model of segmental hepatic I/R.

Key results: Displacement of [(3) H]CP55940 by HU-910 from specific binding sites in CHO cell membranes transfected with human CB(2) or CB(1) receptors (hCB(1/2) ) yielded K(i) values of 6 nM and 1.4 µM respectively. HU-910 inhibited forskolin-stimulated cyclic AMP production by hCB(2) CHO cells (EC(50) = 162 nM) and yielded EC(50) of 26.4 nM in [(35) S]GTPγS binding assays using hCB(2) expressing CHO membranes. HU-910 given before ischaemia significantly attenuated levels of I/R-induced hepatic pro-inflammatory chemokines (CCL3 and CXCL2), TNF-α, inter-cellular adhesion molecule-1, neutrophil infiltration, oxidative stress and cell death. Some of the beneficial effect of HU-910 also persisted when given at the beginning of the reperfusion or 1 h after the ischaemic episode. Furthermore, HU-910 attenuated the bacterial endotoxin-triggered TNF-α production in isolated Kupffer cells and expression of adhesion molecules in primary human liver sinusoidal endothelial cells stimulated with TNF-α. Pretreatment with a CB(2) receptor antagonist attenuated the protective effects of HU-910, while pretreatment with a CB(1) antagonist tended to enhance them.

Conclusion and implications: HU-910 is a potent CB(2) receptor agonist which may exert protective effects in various diseases associated with inflammation and tissue injury.

Linked articles: This article is part of a themed section on Cannabinoids in Biology and Medicine. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2012.165.issue-8. To view Part I of Cannabinoids in Biology and Medicine visit http://dx.doi.org/10.1111/bph.2011.163.issue-7.

Figure 1

Chemical structure and representative graphs of binding assays of HU-910. (A) Chemical structure of HU-910. (B) Competition binding assays were performed with either CHO-CB₁ or CHO-CB₂ cellular membranes by displacement of 2.5 nM [³H]CP 55,940. (C) Representative graph of inhibition of forskolin (fsk)-stimulated cAMP accumulation in CHO 3HA hCB₂ cells by HU910. Data was normalized to the maximum response achieved by HU-308 in parallel stimulations. (D) GTPγS binding at CB₂. HU910 mediated GTPγS binding at CHO-CB₂ membranes. Data is shown as [³⁵S]GTPγS binding normalized to maximal HU-308 binding under the same experimental conditions. Values in graphs are means ± SEM, n= 4–5.

Figure 14

HU-910 attenuates the TNFα-induced adhesion molecules expression in human liver sinusoidal endothelial cells (HLSEC). Treatment of HLSEC cells with 50 ng·mL⁻¹ TNF-α for 6 h, markedly enhances the expression of adhesion molecules such as ICAM-1 and VCAM-1. HU-910 concentration-dependently (1 nM-10 µM) attenuates this enhanced expression of adhesion molecules ICAM-1 (A) and VCAM-1 (B). The CB₂ receptor antagonist SR144528 (SR2; 1 µM) attenuates this effect of HU-910 on adhesion molecule expression. Results are mean ± SEM, n= 5–6/group. *P < 0.05 vehicle (Veh) versus TNF-α (50 ng·mL⁻¹); #P < 0.05 TNF-α versus TNF-α+ HU-910 ± SR2; $P < 0.05 TNF-α+ HU-910 3 µM versus TNF-α+ HU-910 ± SR2.

Figure 2

HU-910 attenuates hepatic ischaemia/reperfusion (I/R) injury. (A,B) Serum transaminase alanine amino-transferase (ALT) (A) and aspartate amino-transferase (AST) (B) levels in sham operated mice treated with vehicle (Veh) or HU-910 (n = 4–5) or in mice exposed to 1 h of hepatic ischaemia followed by 2, 6 and 24 h of reperfusion with vehicle or HU-910 (3 mg·kg⁻¹) given before ischaemia (n= 6–10). Results are mean ± SEM. *P < 0.05 vehicle versus I/R 2, 6 and 24 h; #P < 0.05 I/R 2, 6 and 24 h versus corresponding I/R + HU-910.

Figure 3

HU-910 dose-dependently attenuates hepatic ischaemia/reperfusion (I/R) injury: role of CB_1/2 receptors. (A,B) Serum transaminase alanine amino-transferase (ALT) (A) and aspartate amino-transferase (AST) (B) levels in sham operated mice treated with vehicle (Veh) or SR144528 (SR2; 3 mg·kg⁻¹) or SR141716A (SR1; 3 and 10 mg·kg⁻¹) (n= 4–5) or in mice exposed to 1 h of hepatic ischaemia followed by 6 h of reperfusion treated with vehicle or HU-910 (0.3, 1, 3 or 10 mg·kg⁻¹) or SR144528 (3 mg·kg⁻¹) or SR141716A (3 and 10 mg·kg⁻¹) or their combinations (n= 4–8) given before ischaemia. Results are mean ± SEM. *P < 0.05 vehicle versus I/R 6 h; #P < 0.05 I/R 6 h versus I/R 6 h + SR1/HU-910/HU-910 + SR2; $P < 0.05 I/R 6 h + HU-910 (3 or 10 mg·kg⁻¹) versus corresponding I/R 6 h + SR2 + HU-910 or I/R 6 h + SR1 (10 mg·kg⁻¹) versus corresponding I/R 6 h + SR1 + HU-910 treatment.

Figure 4

HU-910 treatment at reperfusion or 1 h later attenuates hepatic ischaemia/reperfusion (I/R) injury. Serum transaminase alanine amino-transferase (ALT) and aspartate amino-transferase (AST) levels in mice exposed to 1 h of hepatic ischaemia followed by 6 h of reperfusion treated with vehicle or HU-910 (3 or 10 mg·kg⁻¹) right after the ischaemia (A) or 1 h after reperfusion (B). Results are mean ± SEM, n= 7–8. #P < 0.05 versus corresponding mice exposed to I/R for 6 h.

Figure 5

HU-910 attenuates histological damage at 24 h following ischaemia. Haematoxylin and eosin staining of representative liver sections of sham mice treated with vehicle (Sham), and mice exposed to 1 h of ischaemia followed by 24 h of reperfusion treated with vehicle [ischaemia/reperfusion (I/R) 24 h] or HU-910 3 mg·kg⁻¹ (I/R + HU-910) or combined HU-910 3 mg·kg⁻¹ and SR144528 (SR2; 3 mg·kg⁻¹) (I/R + SR2 + HU-910) given before ischaemia. Note the marked attenuation of the coagulation necrosis and inflammatory cell infiltration characteristic of I/R 24 h by HU-910, which was mostly prevented by pretreatment with SR144528. At this dose SR144528 alone had no effect on I/R-induced histopathological injury; likewise neither HU-910 nor SR144528 had effects on normal liver histology in sham controls (data not shown). Upper row of images depicts 200 × magnification, while the lower one 400 × magnification. A similar histological profile was seen in 3–5 livers per treatment group.

Figure 6

HU-910 attenuates the ischaemia/reperfusion (I/R)-induced increased neutrophil infiltration. (A) Myeloperoxidase (MPO) staining (brown) of representative liver sections of sham mice treated with vehicle (Sham), and mice exposed to 1 h of ischaemia followed by 24 h of reperfusion treated with vehicle (I/R) or HU-910 3 mg·kg⁻¹ (I/R + HU-910) or combined HU-910 3 mg·kg⁻¹ and SR144528 (SR2; 3 mg·kg⁻¹; I/R + SR2 + HU-910) given before ischaemia. Slides were counterstained by nuclear fast red. Note the marked attenuation of the MPO positive neutrophil infiltration (brown staining) characteristic of I/R 24 h by HU-910, which was largely attenuated by SR144528 pretreatment. At this dose SR144528 alone had no effect on I/R-induced increased MPO positive cell infiltration; neither HU-910 nor SR144528 had effects on normal livers in which the MPO positive infiltrating immune cells were practically absent (data not shown). Images are 400 × magnification. A similar histological profile was seen in three to five livers per treatment group. (B) Quantification of MPO staining in the liver sections. MPO positive cells were counted at 400 × magnification and expressed as MPO positive cells/field. Results are mean ± SEM, n= 4. *P < 0.05 vehicle in sham versus I/R 24 h; #P < 0.05 I/R 24 h versus I/R + HU910 ± SR2; $P < 0.05 I/R + HU910 versus I/R + HU910 + SR2.

Figure 7

HU-910 attenuates the ischaemia/reperfusion (I/R)-induced acute pro-inflammatory chemokine response in the liver. Real-time PCR shows significant increase of hepatic pro-inflammatory chemokine CCL3 (A), CXCL2 (B), CCL2 (C) mRNA levels at 2 h of reperfusion (I/R 2 h), and a gradual decrease by 6 and 24 h (I/R 6 h and I/R 24 h). Treatment with HU-910 at 3 mg·kg⁻¹ before ischaemia significantly attenuates the I/R-induced increased levels of these pro-inflammatory markers. This effect could be largely prevented by pretreatment with SR144528 (SR2; 3 mg·kg⁻¹). Results are mean ± SEM, n= 7–8. *P < 0.05 vehicle versus I/R 2, 6 or 24 h; #P < 0.05 I/R 2, 6 or 24 h versus corresponding I/R + HU-910 ± SR2 mice; $P < 0.05 I/R + HU-910 versus corresponding I/R + HU-910 + SR2.

Figure 8

HU-910 attenuates the ischemia/reperfusion (I/R)-induced acute pro-inflammatory cytokine and adhesion molecule response in the liver. Real-time PCR shows significant increase of pro-inflammatory cytokine TNF-α (A), and adhesion molecule ICAM-1 (B) mRNA level at 2 h of reperfusion (I/R 2 h), and a gradual decrease by 6 h and 24 h (I/R 6 h and I/R 24 h). Treatment with HU-910 at 3 mg·kg⁻¹ before ischaemia significantly attenuates the I/R-induced increased levels of these pro-inflammatory markers. This effect could be largely prevented by pretreatment with SR144528 (SR2; 3 mg·kg⁻¹) Results are mean ± SEM, n= 6–8/groups. *P < 0.05 vehicle versus I/R 2, 6 or 24 h; #P < 0.05 I/R 2, 6 or 24 h versus corresponding I/R + HU-910 ± SR2 mice; $P < 0.05 I/R + HU-910 versus corresponding I/R + HU-910 + SR2.

Figure 9

HU-910 attenuates the ischaemia/reperfusion (I/R)-induced increased oxidative stress and apoptotic cell death. (A) HNE adducts (a marker for lipid peroxidation/oxidative stress); (B) Oxidative modification of proteins measured by carbonyl adducts; (C,D) hepatic caspase 3/7 activity and DNA fragmentation (markers of apoptosis). All these markers show time-dependent increases following I/R injury peaking at 24 h of reperfusion. 3 mg·kg⁻¹ HU-910 treatment before ischaemia significantly attenuates these increases, which were mostly prevented by pretreatment with SR144528 (SR2; 3 mg·kg⁻¹). Results are mean ± SEM, n = 4–10/groups. *P < 0.05 vehicle versus I/R 2, 6 or 24 h; #P < 0.05 I/R 2, 6 or 24 h versus corresponding I/R + HU-910 ± SR2 mice; $P < 0.05 I/R + HU-910 versus corresponding I/R + HU-910 + SR2.

Figure 10

HU-910 treatment (administered after ischaemia) attenuates histological damage at 24 h following ischaemia. (A) Haematoxylin and eosin staining of representative liver sections of mice exposed to 1 h of ischaemia followed by 24 h of reperfusion treated right after the induction of the ischaemia with vehicle [ischaemia/reperfusion (I/R) 24 h] or HU-910 at 3 mg·kg⁻¹ (I/R + HU-910). Images are at 400 × magnification. A similar histological profile was seen in three to five livers/group. (B) Myeloperoxidase (MPO) staining (brown) of representative liver sections of mice exposed to 1 h of ischaemia followed by 24 h of reperfusion treated immediately after the induction of the ischaemia with vehicle (I/R 24 h) or HU-910 at 3 mg·kg⁻¹ (I/R + HU-910). Images are at 400 × magnification. A similar histological profile was seen in three to five livers per treatment group.

Figure 11

HU-910 treatment (administered after ischaemia) attenuates oxidative stress and cell death at 24 h following ischaemia. Ischaemia/reperfusion (I/R) induced significant increases at 24 h of reperfusion in hepatic oxidative stress markers [protein carbonyl (A) and HNE (B)] and apoptosis [caspase 3/7 activity (C) and DNA fragmentation (D)], which were attenuated by HU-910 3 mg·kg⁻¹ given right after the induction of ischaemia. Results are mean ± SEM, n= 5–6. *P < 0.05 vehicle versus I/R 24 h; #P < 0.05 I/R 24 h versus corresponding I/R + HU-910.

Figure 12

HU-910 (administered after ischaemia) attenuates the ischaemia/reperfusion (I/R)-induced pro-inflammatory response in the liver at 6 h following ischaemia. Real-time PCR shows significant increase of mRNA for chemokines CCL3 (A), CXCL2 (B), CCL2 (C), adhesion molecule ICAM-1 (D), and pro-inflammatory cytokine TNF-α (E) at 6 h of reperfusion (I/R 6 h). Treatment with HU-910 3 mg·kg⁻¹ given immediately after the induction of the ischaemia significantly attenuates the I/R-induced increased levels of these inflammatory markers. Results are mean ± SEM, n= 6–8. *P < 0.05 vehicle versus I/R 6 h; #P < 0.05 I/R 6 h versus corresponding I/R + HU-910.

Figure 13

HU-910 attenuates lipopolysaccharide (LPS)-induced TNF-α secretion in Kupffer cells. HU-910 (10 nM-10 µM) attenuates LPS-induced TNF-α secretion of Kupffer cells in a concentration-dependent manner. The CB₂ receptor antagonist SR144528 (SR2; 1 µM) attenuates the effects of 3 µM of HU-910. At higher concentrations SR144528 alone also shows some inhibitory effect on LPS-induced TNF-α secretion. Results are mean ± SEM, n= 6. *P < 0.05 vehicle (Veh) versus LPS (100 ng·mL⁻¹); #P < 0.05 LPS versus LPS + HU-910 ± SR2; $P < 0.05 LPS + HU-910 3 µM versus LPS + HU-910 ± SR2.