Cell line generation and maintenance

The cDNA clones for human CB₁ and CB₂ receptors tagged with three haemagglutinin (HA) sequences were obtained from the Missouri S&T cDNA Resource Center (http://www.cdna.org) in cloning vector pcDNA3.1 + (pcDNA 3 × HA hCB_1/2). The vector containing the human CB₂ receptor was transfected directly into CHO-KI cells obtained from ATCC. The HA-tagged human CB₁ receptor sequence was subcloned into the pef4-V5-HisA vector with Kpn1 (Roche) and Pme1 (New England Biolabs) restriction enzymes and subsequently transfected into CHO-K1 cells using Lipofectamine 2000 according to the manufacturer’s instructions (Invitrogen). Cells were clonally isolated by limited dilution and screened by immunocytochemistry for expression of the HA tag. Clones expressing the HA tag were also screened by reverse transcription PCR to confirm expression of human CB₁ and human CB₂ receptor mRNA transcripts.

Cells were maintained in Dulbecco’s modified Eagle medium: nutrient mixture F-12 (DMEM/F12) media supplemented with 10% fetal bovine serum (FBS), 100 units·mL⁻¹penicillin and 100 µg·mL⁻¹ streptomycin and 2 mM L-glutamine. Transfected cell lines were maintained with additional 250 µg·mL⁻¹ zeocin for CHO-CB₁ transfected cells and 500 µg·mL⁻¹ G-418 for CHO-CB₂ transfected cells (all reagents obtained from Invitrogen).

Membrane preparation

Cells were grown to 90–100% confluence and harvested in ice-cold phosphate buffered saline with 5 mM EDTA. Cells were centrifuged at 200×g for 10 min and frozen at −80°C until required. Cell pellets were thawed with cold 0.32 M sucrose and homogenized with a glass homogenizer. The homogenate was centrifuged at 1000×g for 10 min at 4°C and the supernatant centrifuged in a Sorvall ultracentrifuge for 30 min at 100 000×g. The pellet was then washed in ice-cold Tris wash buffer and re- centrifuged twice more. The final pellet was resuspended in 50 mM Tris pH 7.5, 0.5 mM EDTA. Protein concentration was determined using the Dc protein assay kit (Bio-Rad, Hercules, CA, USA).

Competition binding assay

The K_d of CP 55,940 in the isolated CB₁ and CB₂ receptor expressing membranes was previously determined to be 2.3 nM and 1.5 nM respectively. Competition binding assays at 2.5 nM [³H]-CP 55,940 (PerkinElmer) were carried out to determine the K_i values for tested compounds. Membranes (5–10 µg) were incubated with radioligand and a range of concentrations of test compounds in binding buffer (50 mM Tris pH 7.4, 5 mM MgCl₂, 1 mM EDTA) with 0.5% (w/v) bovine serum albumin (BSA) (ICP Bio, New Zealand), at 30°C for 60 min. Stock solutions of putative cannabinoid ligands were prepared in DMSO to a concentration of 10 mM. Six different final concentrations of compounds were used ranging from 0.1 nM to 50 µM. Non-specific binding was determined in the presence of 1 µM non-radioactive CP 55,940 (Tocris Cookson). Assays were terminated by addition of 3 mL ice-cold binding buffer and filtration through GF/C filters (Whatman) pre-soaked in cold binding buffer, followed by two washes in the same buffer.

Radioactivity was determined by incubation of filters with Irgasafe scintillation fluid (PerkinElmer) and scintillation counting in a Wallac Trilux using Microbeta Trilux software. Data was analysed using the Prism 4.02 program (GraphPad Software, San Deigo, CA, USA).

cAMP assay

CHO-CB₁ and CHO-CB₂ cells were seeded at a density of 10⁴ cells per well in poly-L-lysine treated 96-well culture plates (BD Biosciences). The following day wells were incubated with 40 µL DMEM/F12 containing 0.5% (w/v) BSA and 0.5 mM 3-isobutyl-1-methylxanthine (Sigma-Aldrich) for 30 min prior to 15 min stimulation with 50 µM forskolin (Tocris Cookson) and varying concentrations of indicated compounds at 37°C, 5% CO₂. Assays were stopped by removal of media and addition of 100% ice-cold ethanol. Plates were then frozen for a minimum of 2 h before complete evaporation of ethanol. The well contents were then reconstituted in 50 µL cAMP assay buffer (20 mM HEPES pH 7.5 and 5 mM EDTA). Half of the reconstituted sample was transferred to round bottom 96-well plates (Greiner Bio-One GmbH) with 50 µL 0.01% w/v cAMP dependent protein kinase A [PKA (Sigma-Aldrich) in 1 mM sodium citrate pH 6.5 with 2 mM dithiothreitol] and 25 µL [³H]-cAMP (at 22 nM in cAMP assay buffer) (GE Healthcare, Life Sciences). Samples were then allowed to equilibrate for 3–18 h. Following this, a charcoal slurry [5% (w/v) activated charcoal and 0.2% (w/v) BSA in cAMP assay buffer] was added to the samples and the plates centrifuged at 3000×g, 4°C for 5 min. A sample of the supernatant was then transferred to 96-well flexible microplates (PerkinElmer) and 200 µL Irgasafe scintillation fluid (PerkinElmer) added. Plates were sealed, vigourously agitated and scintillation counting performed by a Wallac Trilux using Microbeta Trilux software.

[³⁵S]GTPγS binding assay

Human CB₂ receptor expressing CHO-K1 membranes (5 µg per incubation mixture) were diluted in 50 mM Tris-HCl (pH 7.5) and 0.5 mM EDTA and added to the indicated compounds in a pre-mixed incubation cocktail. Final incubation concentrations were 55 mM Tris-HCl (pH 7.4), 1 mM EDTA, 100 mM NaCl, 5 mM MgCl₂, 0.5% BSA, 50 µM GDP, 0.2 nM [³⁵S]GTPγS (PerkinElmer) with varied concentrations of compounds (0.1 nM–10 µM) and 5 µg membrane. Incubations were continued for 60 min at 30°C in a shaking water bath. Assays were terminated by addition of 2 mL ice-cold wash buffer (50 mM Tris-HCl, pH 7.5 and 5 mM MgCl₂) and filtration through pre-soaked GF/C filters (Whatman), followed by two further washes. Radioactivity was determined as described for competition binding assays.

Hepatic ischaemia-reperfusion

All animal care and experimental procedures complied with the National Institutes of Health (NIH) guidelines for the care and use of laboratory animals and were approved by the Institutional Animal Care and Use Committees of the National Institute on Drug Abuse (NIAAA). Male C57BL/6J mice (25–30 g; Jackson Laboratories, Bar Harbor, ME, USA) were anesthetized with pentobarbital sodium (65 mg·kg⁻¹ i.p.). We used the model of segmental (70%) hepatic ischaemia, as described by Mukhopadhyay et al., (2011b). Briefly, the liver was exposed by midline laparotomy and the hepatic artery and the portal vein were clamped using an atraumatic micro-serrefine. This method of partial ischaemia prevents mesenteric venous congestion by allowing portal decompression throughout the right and caudate lobes of the liver. The duration of hepatic ischaemia was 60 min, after which the vascular clips were removed and liver was reperfused for 2, 6 or 24 h, as indicated. Sham surgeries were identical except that hepatic blood vessels were not clamped with a micro-serrefine. The liver was kept moist at 37°C with gauze soaked in 0.9% saline. Body temperature was monitored with a rectal temperature probe and was maintained at 37°C by a heating blanket. Treatments with HU-910 0.3, 1, 3 and 10 mg·kg⁻¹or vehicle (i.p.), started 2 h before I/R or were given after ischaemia at the moment of reperfusion, as indicated in the text. CB₁ and CB₂ receptor antagonists were given 2 h before ischaemia or HU-910 treatment. At the experimental end points, blood was collected and liver samples were removed and snap-frozen in liquid nitrogen for determining biochemical parameters or fixed in 4% buffered formalin for histopathological evaluation (Moon et al., 2008).

Serum aspartate amino-transferase (AST) and alanine amino-transferase (ALT) levels

The activities of AST and ALT, indicators of liver cellular damage (necrosis), were measured in serum samples using a clinical chemistry analyser system (VetTest 8008, IDEXX laboratories, Westbrook, ME, USA) (Moon et al., 2008).

Histological examination of liver sections

Liver samples were fixed in 4% buffered formalin. After embedding and cutting 5 µm slices, all sections were stained with haematoxylin/eosin. Neutrophils were stained for myeloperoxidase (MPO) using anti-MPO antibody, according to the manufacturer’s protocol (Biocare Medical, Concord, CA, USA), and samples were counter-stained with nuclear fast red as described (Mukhopadhyay et al., 2011b). Histological evaluation was performed without knowledge of the treatments.

Real-time PCR analyses of mRNA

Total RNA was isolated from liver homogenate using TRIzol reagents (Invitrogen, Carlsbad, CA, USA) according to manufacturer’s instructions. The isolated RNA was treated with RNase-free DNase (Ambion, Austin, TX, USA) to remove traces of genomic DNA contamination. Samples (1µg) of total RNA were reverse-transcribed to cDNA using the SuperScript II (Invitrogen, Carlsbad, CA, USA). The target gene expression was quantified with Power SYBER Green PCR Master Mix using an ABI HT7900 real-time PCR instrument (Applied Biosystems, Foster City, CA, USA). Each amplified sample in all wells was analysed for homogeneity using dissociation curve analysis. After denaturation at 95°C for 2 min, 40 cycles were performed at 95°C for 10 s and at 60°C for 30 s. Relative quantification was calculated using the comparative CT method (2-ΔΔCt method: ΔΔCt =ΔCt sample −ΔCt reference). Lower ΔCT values and lower ΔΔCT reflect a relatively higher amount of gene transcript.

Primers used were as follows:

CCL3, 5′-TGCCCTTGCTGTTCTTCTCTG-3′ and 5′-CAACGATGAATTGGCGTGG-3′; CXCL2, 5′-AGTGAACTGCGCTGTCAATGC-3′ and 5′-AGGCAAACTTTTTGACCGCC-3′; TNF-α, 5′-AAGCCTGTAGCCCACGTCGTA-3′ and 5′-AGGTACAACCCATCGGCTGG-3′; ICAM-1, 5′-AACTTTTCAGCTCCGGTCCTG-3′ and 5′-TCAGTGTGAATTGGACCTGCG-3′; CB₁, 5′-ATGAAGTCGATCCTAGATGGCCTTGCAGA-3′ and 5′TCACAGAGCCTCGGCAGACGTG-3′; CB₂, 5′-GACCTTCACAGCCTCTGTGGGTA-3 and 5′-GATTTTCCCATCAGCCTCTGTCT-3; and actin, 5′-TGCACCACCAACTGCTTAG-3′ and 5′-GGATGCAGGGATGATGTTC-3′.

Hepatic 4-hydroxynonenal (HNE) content

Lipid peroxides are unstable indicators of oxidative stress in cells that decompose to form more complex and reactive compounds such as HNE, which has been shown to be capable of binding to proteins and forming stable HNE adducts. HNE in the hepatic tissues was determined using a kit (Cell Biolabs, CA, USA). In brief, BSA or hepatic tissue extracts (10 µg·mL⁻¹) are adsorbed onto a 96-well plate for 12 h at 4°C. HNE adducts present in the sample or standard were probed with anti-HNE antibody, followed by an horseradish peroxidase (HRP)-conjugated secondary antibody. The content of HNE-protein adducts in an experimental sample was determined by comparing with a standard curve (Mukhopadhyay et al., 2010).

Detection of hepatic carbonyl adducts

Carbonyl content in liver tissues was determined by OxiSelect Protein Carbonyl elisa Kit (Cell Biolabs, CA, USA) (Mukhopadhyay et al., 2011a). In brief, BSA standards or protein samples (10 µg·mL⁻¹) were adsorbed onto a 96-well plate for 2 h at 37°C. The protein carbonyls present in the sample or standard were derivatized to DNP hydrazone and probed with an anti-DNP antibody, followed by an HRP-conjugated secondary antibody. The protein carbonyl content in unknown sample was determined by comparing with a standard curve, prepared from reduced and oxidized BSA standards.

Detection of apoptosis by caspase 3/7 activity assays

Caspase 3/7 activity in hepatic tissue lysate was measured using the Apo-One Homogenous Caspase-3/7 assay kit (Promega Corp., Madison, WI, USA). An aliquot of caspase reagent was added to each well and mixed on a plate shaker for 1 h at room temperature shielded from light, and the fluorescence was measured (Rajesh et al., 2009; 2010).

Hepatic DNA fragmentation elisa

The quantitative determinations of cytoplasmic histone-associated-DNA-fragmentation (mono and oligonucleosomes) due to cell death in liver homogenizates were measured using elisa kit (Roche Diagnostics GmbH) (Mukhopadhyay et al., 2009; Rajesh et al., 2010).

Isolation and stimulation of hepatic Kupffer cells

Livers were perfused in deeply anaesthetized mice and the livers were excised for isolation of Kupffer cells. Hepatic cellular contents were released from the stroma by digestion with the perfusion medium (hepatocyte wash media) containing collagenase type 1 (Sigma, St. Louis, MO, USA) and 2% Penicillin and Streptomycin (Invitrogen, Carlsbad, CA, USA). Kupffer cells were isolated using Optiprep gradient. Kupffer cell fraction was aspirated and washed twice with RPMI 1640 medium. Homogenous Kupffer cells population was obtained by using negative selection (anti-CD146) LS column according to the manufacturer’s instruction (Miltenyi Biotec, Auburn, CA, USA) as described earlier (Mukhopadhyay et al., 2011b). After additional washes, Kupffer cells were plated on to 96-well or six-well plates in RPMI 1640 media, containing 10% FBS and penicillin-streptomycin, and incubated overnight in a CO₂ incubator at 37°C and 5% CO₂. Cells were maintained for 2 h with RPMI 1640 medium containing FBS (2%). HU-910 or HU-308 or the CB₂ receptor antagonists (SR144528 or AM-630) at indicated concentrations were added 1 h prior to LPS treatment. Cells were treated for 6 h with LPS (Escherichia coliO127:BB catalogue # L3129, Sigma Chemicals, St Louis, MO, USA). After the end of treatments, culture supernatants were removed and snap frozen in liquid nitrogen and assayed for the TNF-α concentrations with the use of elisa kit (Mouse TNF-α; catalogue # SMTA00; R&D Systems, Minneapolis, MN, USA) as described by Mukhopadhyay et al., (2011b).

Cell surface inter-cellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1) expression assay

Cell surface expression of ICAM-1 and VCAM-1 in the HLSEC was measured by in situelisaas described (Rajesh et al., 2007). In brief, HLSEC cells were grown in 96-well plates. After treatments as described in Figure 14, in situelisa was performed with anti-mouse ICAM-1 or VCAM-1 monoclonal antibodies (1:1500 dilution; R&D Systems) and by measuring the absorbance colorimetrically at 450 nm using the HRP-3,3′,5,5′-tetramethylbenzidine developing system (Sigma, St. Louis, MO, USA). Each treatment was performed in triplicate, and the experiments were repeated three times.

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 …

Analysis of data

For binding data the K_i was determined from IC₅₀ values derived from competition binding data fitted with one site competition non-linear regression analysis by Prism 4.02 using the K_d values shown in Table 1. pIC₅₀ values were determined from cAMP assays by fitting a sigmoidal concentration response curve. Results shown were generated by averaging at least three independently determined pIC₅₀ values. Data shown is mean IC₅₀(95% confidence interval). Emax values were calculated as a percentage of the maximal response detected in parallel cAMP assays with HU-210 or HU-308 for CB₁ and CB₂receptor expressing cells respectively. Data are displayed as the mean ± SEM.

Table 1

HU-910 activates CB₂ receptors in vitro

For all experiments presented in Figures 2–14, values have been expressed as means and variability as SEM. Statistical significance among groups was determined by one-way anovafollowed by Newman–Keuls post hoc analysis using GraphPad Prism 5 software (San Diego, CA, USA). Probability values of P < 0.05 were considered significant.

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 …

Materials

The chemical structure of HU-910 is shown in Figure 1. The synthesis of HU-910 has been presented at a meeting of the International Cannabinoid Research Society (Magid et al., 2010a) and in a patent (Magid et al., 2010b). HU-910 has a unique bicyclic structure, in the non-aromatic portion of the molecule. Its synthesis follows a number of steps starting with a Suzuki cross-coupling reaction using (+)-camphor-10-sulphonyl chloride (Sigma-Aldrich) and 2,6-dimethoxy-4-(2-methyloctan-2-yl)benzene (Dominianni et al., 1977).

The CB₁ and CB₂ receptor antagonists/inverse agonists SR141716A and SR144528 were obtained from the National Institute on Drug Abuse (NIDA) Drug Supply Program. For in vivo administration, all drugs were dissolved in vehicle solution (one drop of Tween-80 in 3 mL 2.5% dimethyl sulphoxide in saline) as previously described (Batkai et al., 2007). Vehicle solution was used in control experiments.

HU-910 attenuates markers of hepatic I/R injury (ALT, AST)

For assessments of hepatocellular damage of the post-ischaemic liver, the serum transaminase activities (AST and ALT), markers of necrosis, were measured. After 1 h of ischaemia and a subsequent 2 or 6 h of reperfusion (I/R 2 h and I/R 6 h respectively), a dramatic increase in liver enzyme activities were observed in vehicle-treated C57Bl6/J mice as compared with sham-operated controls, which almost returned to baseline at 24 h of reperfusion (I/R 24 h) (Figure 2). HU-910 was able to attenuate these increases in the markers of hepatic I/R injury at all measured time points, while HU-910 alone had no effects on ALT and AST levels in the sham animals compared with the vehicle-treated group (Figure 2).

Because the peak elevation of ALT and AST is known to occur around 6 h of reperfusion (Abe et al., 2009; Mukhopadhyay et al., 2011b), as also demonstrated in Figure 2, this time point was chosen to evaluate the maximal hepatocellular injury (necrosis) in the subsequent experiments.

Pretreatment with HU-910 (0.3, 1, 3 or 10 mg·kg⁻¹ i.p.) 2 h before the induction of the ischaemia dose-dependently attenuated the serum transaminase elevations (ALT and AST) at 6 h of reperfusion compared with vehicle (Figure 3). Furthermore, pretreatment with the CB₂ receptor antagonist SR144528 (3 mg·kg⁻¹ i.p.) significantly attenuated the effect of 3 or 10 mg·kg⁻¹ HU-910 at the time of the peak serum ALT and AST elevations, implying a role for CB₂ receptors in the beneficial effects of HU-910 (Figure 3). Because the protective effect of 3 and 10 mg·kg⁻¹ of HU-910 were similar (Figure 3), and the protection afforded by the lower dose could almost completely be prevented by pretreatment with SR144528 (3 mg·kg⁻¹ i.p.), 3 mg·kg⁻¹ HU-910 was used in the subsequent experiments. Given alone, SR144528 (3 mg·kg⁻¹) did not affect the I/R-induced increase in liver enzymes, while pretreatment with the CB₁ receptor antagonist SR141716A (3 mg·kg⁻¹ and 10 mg·kg⁻¹) alone attenuated the markers of hepatic I/R injury and potentiated the effect of HU-910, when given in combination (Figure 3). At higher doses (above 10 mg·kg⁻¹), SR144528 alone did attenuate the hepatic I/R injury (data not shown).

Figure 3

HU-910 dose-dependently attenuates hepatic ischaemia/reperfusion (I/R) injury: role of CB_1/2receptors. (A,B) Serum transaminase alanine amino-transferase (ALT) (A) and aspartate amino-transferase (AST) (B) levels in sham operated mice treated with vehicle …

HU-910 treatment given immediately after the induction of the ischaemia (Figure 4A) or 1 h following the reperfusion (Figure 4B) still attenuated, although to a lesser extent than in the pretreatment experiments, the hepatic injury measured at 6 h of reperfusion.

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 …

HU-910 improves I/R-induced histological damage

Haematoxylin and eosin staining of representative liver sections after 24 h of reperfusion showed (Figure 5) that I/R induced marked coagulation necrosis (lighter areas, with marked inflammatory cell infiltration), which was dramatically reduced and became more focal in mice treated with HU-910 (3 mg·kg⁻¹ i.p.). Pretreatment with the CB₂ receptor antagonist/inverse agonist SR144528 (3 mg·kg⁻¹ i.p.) almost completely prevented the protective effect of HU-910, as seen on liver histology. Vehicle or HU-910 treatment in the sham animals had no effect on liver histopathology. A similar histological profile was seen throughout the group (n = 3–5 mice).

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 …

HU-910 attenuates the marked neutrophil infiltration induced by I/R

Neutrophils are important mediators of the delayed tissue injury following I/R. Most of the hepatic neutrophil infiltration is known to occur between 6 and 24 h of reperfusion (Abe et al., 2009; Mukhopadhyay et al., 2011b). An indicator of neutrophil infiltration is the tissue MPO activity. In sham-treated mice, MPO staining was barely detectable (Figure 6). In contrast, there was a marked increase in infiltrating MPO-positive immune cells (brown staining) after 24 h of reperfusion in vehicle treated animals, which was significantly attenuated by HU-910 3 mg·kg⁻¹ i.p. (Figure 6). Pretreatment with the CB₂ receptor antagonist SR144528 (3 mg·kg⁻¹ i.p.) almost completely prevented the effect of HU-910 seen on hepatic neutrophil infiltration. Vehicle or HU-910 treatment in the sham animals had no effect on liver MPO staining. A similar histological profile was seen throughout the group (n = 3–5).

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 …

HU-910 attenuates the hepatic pro-inflammatory chemokine, cytokine and adhesion molecule expression induced by I/R

I/R greatly increased the expression of mRNA of pro-inflammatory chemokines CCL3, CXCL2 and CCL2 (Figure 7A–C), the pro-inflammatory cytokine TNF-α (Figure 8A) and the adhesion molecule ICAM-1 (Figure 8B) in liver tissue as documented by real-time PCR, which was attenuated by HU-910 (3 mg·kg⁻¹ i.p.) given before the ischaemia (Figures 7and ​and8).8). Hepatic TNF-α, CCL3, CXCL2 and CCL2 and ICAM-1 mRNA (part of the acute inflammatory response orchestrated by activated Kupffer and endothelial cells) were maximal at 2 h of reperfusion (Figures 7 and ​and8)8) and decreased by 24 h of reperfusion almost to control levels. HU-910 given before ischaemia significantly decreased the peak values of CCL3, CXCL2 and CCL2 (Figure 7A–C), TNF-α (Figure 8A) and ICAM-1 (Figure 8B) mRNA in liver. These effects could be largely attenuated by pretreatment with the CB₂ receptor antagonist SR144528 (3 mg·kg⁻¹ i.p.) (Figures 7 and ​and88).

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 …

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 …

HU-910 decreases the increased oxidative stress and hepatocyte cell death induced by I/R

The rate of oxidative posttranslational modification of proteins and lipid peroxidation was negligible in sham livers, as indicated by the low level of carbonyl adducts and HNE. We found a time-dependent increase in HNE peaking at 24 h of reperfusion, which was significantly attenuated by HU-910 (3 mg·kg⁻¹ i.p.) (Figure 9A). Hepatic content of carbonyl adducts also peaked at 24 h of reperfusion, which was significantly attenuated by HU-910 (3 mg·kg⁻¹ i.p.) (Figure 9B).

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) …

Apoptotic cell death in the liver following I/R was evaluated by monitoring caspase 3/7 activity and DNA fragmentation (Figure 9C,D). All markers of cell death in the liver were markedly increased at 24 h of reperfusion following 60 min of ischaemia. HU-910 (3 mg·kg⁻¹ i.p.) significantly attenuated the increase of both markers (Figure 9C,D).

All effects of HU-910 on oxidative and cell death markers were largely prevented by the CB₂ receptor antagonist SR144528 (3 mg·kg⁻¹ i.p.) (Figure 9A–D).

HU-910 treatment (administered after ischaemia) attenuates histological damage, oxidative stress, cell death and acute pro-inflammatory response following hepatic ischaemia-reperfusion injury

I/R induced marked coagulation necrosis (lighter areas, with marked inflammatory cell infiltration), which was dramatically reduced and became more focal in mice treated with HU-910 (3 mg·kg⁻¹ given immediately after the ischaemic period) (Figure 10A). HU-910 also attenuated the observed increase in infiltrating MPO-positive immune cells (brown staining) after 24 h of reperfusion in vehicle-treated animals (Figure 10B), and also the markers of oxidative stress (protein carbonyl, HNE) and apoptotic cell death (caspase 3/7 activity and DNA fragmentation) (Figure 11A–D).

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 …

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 …

Furthermore, HU-910 (3 mg·kg⁻¹; administered immediately after the ischaemic period) was also able to attenuate the increased expression of mRNA of CCL3, CXCL2 and CCL2 (Figure 12A–C), of the adhesion molecule ICAM-1 (Figure 12D) and of TNF-α (Figure 12E), after 6 h of reperfusion.

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), …

Only the higher dose of HU-910 (10 mg·kg⁻¹) was able to improve the histopathological injury and attenuate the neutrophil infiltration when it was given 1 h after the ischaemic period (data not shown).

HU-910 attenuates the TNF-α production in mouse Kupffer cells induced by LPS

Because the acute inflammatory response in the liver is orchestrated mainly by activated Kupffer and endothelial cells, which express CB₂ receptors (Rajesh et al., 2007; Hall et al., 2010; Pacher and Mechoulam, 2011), we also tested the concentration-dependent effect of HU-910 on inflammatory responses of these cell types. Kupffer cells were isolated from mice as described in the Methods section, and then treated with bacterial LPS (100 ng·mL⁻¹) in the presence or absence of HU-910 (10 nM–10 µM), and the CB₂ receptor antagonist SR144528 (1–6 µM). As shown in Figure 13, LPS treatment drastically enhanced TNF-α production in Kupffer cells, which was attenuated in a concentration-dependent manner by HU-910. The effect of 3 µM HU-910 was largely prevented by pretreatment with the CB₂ receptor antagonist SR144528 at 1 µM. Interestingly, higher concentrations of SR144528 by themselves attenuated the TNF-α production induced by LPS, indicating a non-specific effect at these concentrations. Addition of vehicle, HU-910 (10 µM) or SR144528 (1 µM) had no effect on the negligible baseline TNF-α levels in unstimulated Kupffer cells.

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 …

HU-910 attenuates the adhesion molecules expression in HLSEC induced by TNF-α

Treatment of HLSEC cells with TNF-α (50 ng·mL⁻¹) for 6 h markedly enhanced the production of the adhesion molecules, ICAM-1 and VCAM-1 (Figure 14). This was inhibited when cells were treated with HU-910 (10 nM–3 µM). The effect of HU-910 (3 µM) was largely attenuated by pretreatment with the CB₂ receptor antagonist SR144528 at 1 µM. Vehicle, HU-910 (3 µM) or SR144528 (1 µM) had no effect on baseline TNF-α levels in unstimulated HLSEC.

This study was supported by the Intramural Research Program of NIH/NIAAA (to P. P.) and by NIDA grant #9789 (to R. M.). Dr Béla Horváth is a recipient of a Hungarian Research Council Scientific Research Fund Fellowship (OTKA-NKTH-EU MB08-80238). The authors are indebted to Drs George Kunos and Bin Gao for providing key resources and support.

4-HNE

4-hydroxy-2-nonenal (marker of lipid peroxidation)

CB₂ or CB₁ receptor

cannabinoid 1 or 2 receptor

HU-910

((1S,4R)-2-(2,6-dimethoxy-4-(2-methyloctan-2-yl)phenyl)-7,7-dimethylbicyclo[2.2.1]hept-2-en-1-yl)methanol

I/R

ischaemia/reperfusion

ICAM-1

inter-cellular adhesion molecule 1, CD54;CCL2, monocyte chemotactic protein-1

CCL3

macrophage inflammatory protein-1α

CXCL2

macrophage inflammatory protein-2α

VCAM-1

vascular cell adhesion molecule 1

• Abe Y, Hines IN, Zibari G, Pavlick K, Gray L, Kitagawa Y, et al. Mouse model of liver ischemia and reperfusion injury: method for studying reactive oxygen and nitrogen metabolites in vivo. Free Radic Biol Med. 2009;46:1–7. [PMC free article][PubMed]
• Alexander SPH, Mathie A, Peters JA. Guide to Receptors and Channels (GRAC), 5th Edition. Br J Pharmacol. 2011;164(Suppl. 1):S1–S324. [PMC free article][PubMed]
• Batkai S, Osei-Hyiaman D, Pan H, El-Assal O, Rajesh M, Mukhopadhyay P, et al. Cannabinoid-2 receptor mediates protection against hepatic ischemia/reperfusion injury. FASEB J. 2007;21:1788–1800. [PMC free article] [PubMed]
• Caraceni P, Pertosa AM, Giannone F, Domenicali M, Grattagliano I, Principe A, et al. Antagonism of the cannabinoid CB-1 receptor protects rat liver against ischaemia-reperfusion injury complicated by endotoxaemia. Gut. 2009;58:1135–1143.[PubMed]
• Di Marzo V. Targeting the endocannabinoid system: to enhance or reduce? Nat Rev Drug Discov. 2008;7:438–455. [PubMed]
• Dominianni SJ, Ryan CW, DeArmitt CW. Synthesis of 5-(tert-alkyl)resorcinols. J Org Chem. 1977;42:344–346. [PubMed]
• Engerson TD, McKelvey TG, Rhyne DB, Boggio EB, Snyder SJ, Jones HP. Conversion of xanthine dehydrogenase to oxidase in ischemic rat tissues. J Clin Invest.1987;79:1564–1570. [PMC free article] [PubMed]
• Ferdinandy P, Schulz R. Nitric oxide, superoxide, and peroxynitrite in myocardial ischaemia-reperfusion injury and preconditioning. Br J Pharmacol.2003;138:532–543. [PMC free article] [PubMed]
• Gero D, Szabo C. Role of the peroxynitrite-poly (ADP-ribose) polymerase pathway in the pathogenesis of liver injury. Curr Pharm Des. 2006;12:2903–2910. [PubMed]
• Hall D, Poussin C, Velagapudi VR, Empsen C, Joffraud M, Beckmann J, et al. Peroxisomal and microsomal lipid pathways associated with resistance to hepatic steatosis and reduced pro-inflammatory state. J Biol Chem. 2010;285:31011–31023. [PMC free article] [PubMed]
• Hanus L, Breuer A, Tchilibon S, Shiloah S, Goldenberg D, Horowitz M, et al. HU-308: a specific agonist for CB₂, a peripheral cannabinoid receptor. Proc Natl Acad Sci U S A. 1999;96:14228–14233. [PMC free article] [PubMed]
• Huffman JW. CB₂ receptor ligands. Mini Rev Med Chem. 2005;5:641–649.[PubMed]
• Jaeschke H. Role of reactive oxygen species in hepatic ischemia-reperfusion injury and preconditioning. J Invest Surg. 2003;16:127–140. [PubMed]
• Jaeschke H. Mechanisms of Liver Injury. II. Mechanisms of neutrophil-induced liver cell injury during hepatic ischemia-reperfusion and other acute inflammatory conditions. Am J Physiol Gastrointest Liver Physiol. 2006;290:G1083–G1088.[PubMed]
• Julien B, Grenard P, Teixeira-Clerc F, Van Nhieu JT, Li L, Karsak M, et al. Antifibrogenic role of the cannabinoid receptor CB₂ in the liver. Gastroenterology.2005;128:742–755. [PubMed]
• Liaudet L, Szabo G, Szabo C. Oxidative stress and regional ischemia-reperfusion injury: the peroxynitrite-poly(ADP-ribose) polymerase connection. Coron Artery Dis. 2003;14:115–122. [PubMed]
• Lotersztajn S, Teixeira-Clerc F, Julien B, Deveaux V, Ichigotani Y, Manin S, et al. CB₂receptors as new therapeutic targets for liver diseases. Br J Pharmacol.2008;153:286–289. [PMC free article] [PubMed]
• Magid L, Goodfellow CE, Srebnik M, Glass M, Alexandrovich A, Hanuš L, et al. Design, synthesis and neuroprotrective potential of novel CB2 receptor selective agonists. 20th Annual Symposium on the Cannabinoids; International Cannabinoid Research Society, Research Triangle Park, NC, USA. 2010a. Abstracts, p 42.
• Magid L, Mechoulam R, Shohami E, Bab I. Novel arylated camphenes, processes for their preparation and uses thereof. 2010b. Israeli Patent PCT/IL2010/000970.
• Montecucco F, Lenglet S, Braunersreuther V, Burger F, Pelli G, Bertolotto M, et al. CB₂ cannabinoid receptor activation is cardioprotective in a mouse model of ischemia/reperfusion. J Mol Cell Cardiol. 2009;46:612–620. [PubMed]
• Moon KH, Hood BL, Mukhopadhyay P, Rajesh M, Abdelmegeed MA, Kwon YI, et al. Oxidative inactivation of key mitochondrial proteins leads to dysfunction and injury in hepatic ischemia reperfusion. Gastroenterology. 2008;135:1344–1357.[PMC free article] [PubMed]
• Mukhopadhyay P, Rajesh M, Batkai S, Kashiwaya Y, Hasko G, Liaudet L, et al. Role of superoxide, nitric oxide, and peroxynitrite in doxorubicin-induced cell death in vivo and in vitro. Am J Physiol Heart Circ Physiol. 2009;296:H1466–H1483.[PMC free article] [PubMed]
• Mukhopadhyay P, Rajesh M, Batkai S, Patel V, Kashiwaya Y, Liaudet L, et al. CB₁cannabinoid receptors promote oxidative stress and cell death in murine models of doxorubicin-induced cardiomyopathy and in human cardiomyocytes. Cardiovasc Res. 2010;85:773–784. [PMC free article] [PubMed]
• Mukhopadhyay P, Horvath B, Rajesh M, Matsumoto S, Saito K, Batkai S, et al. Fatty acid amide hydrolase is a key regulator of endocannabinoid-induced myocardial tissue injury. Free Radic Biol Med. 2011a;50:179–195. [PMC free article][PubMed]
• Mukhopadhyay P, Rajesh M, Horvath B, Batkai S, Park O, Tanchian G, et al. Cannabidiol protects against hepatic ischemia/reperfusion injury by attenuating inflammatory signaling and response, oxidative/nitrative stress, and cell death.Free Radic Biol Med. 2011b;50:1368–1381. [PMC free article] [PubMed]
• Murikinati S, Jüttler E, Keinert T, Ridder DA, Muhammad S, Waibler Z, et al. Activation of cannabinoid 2 receptors protects against cerebral ischemia by inhibiting neutrophil recruitment. FASEB J. 2010;24:788–798. [PubMed]
• Pacher P, Gao B. Endocannabinoids and liver disease. III. Endocannabinoid effects on immune cells: implications for inflammatory liver diseases. Am J Physiol Gastrointest Liver Physiol. 2008;294:G850–G854. [PMC free article] [PubMed]
• Pacher P, Hasko G. Endocannabinoids and cannabinoid receptors in ischaemia-reperfusion injury and preconditioning. Br J Pharmacol. 2008;153:252–262.[PMC free article] [PubMed]
• Pacher P, Mechoulam R. Is lipid signaling through cannabinoid 2 receptors part of a protective system? Prog Lipid Res. 2011;50:193–211. [PMC free article] [PubMed]
• Pacher P, Batkai S, Kunos G. The endocannabinoid system as an emerging target of pharmacotherapy. Pharmacol Rev. 2006;58:389–462. [PMC free article][PubMed]
• Pacher P, Beckman JS, Liaudet L. Nitric oxide and peroxynitrite in health and disease. Physiol Rev. 2007;87:315–424. [PMC free article] [PubMed]
• Pertwee RG. Pharmacological actions of cannabinoids. Handb Exp Pharmacol.2005;168:1–51. [PubMed]
• Rajesh M, Pan H, Mukhopadhyay P, Batkai S, Osei-Hyiaman D, Hasko G, et al. Cannabinoid-2 receptor agonist HU-308 protects against hepatic ischemia/reperfusion injury by attenuating oxidative stress, inflammatory response, and apoptosis. J Leukoc Biol. 2007;82:1382–1389. [PMC free article][PubMed]
• Rajesh M, Mukhopadhyay P, Batkai S, Mukhopadhyay B, Patel V, Hasko G, et al. Xanthine oxidase inhibitor allopurinol attenuates the development of diabetic cardiomyopathy. J Cell Mol Med. 2009;13:2330–2341. [PMC free article][PubMed]
• Rajesh M, Mukhopadhyay P, Bátkai S, Patel V, Saito K, Matsumoto S, et al. Cannabidiol attenuates cardiac dysfunction, oxidative stress, fibrosis, and inflammatory and cell death signaling pathways in diabetic cardiomyopathy. J Am Coll Cardiol. 2010;56:2115–2125. [PMC free article] [PubMed]
• Teixeira-Clerc F, Belot MP, Manin S, Deveaux V, Cadoudal T, Chobert MN, et al. Beneficial paracrine effects of cannabinoid receptor 2 on liver injury and regeneration. Hepatology. 2010;52:1046–1059. [PMC free article] [PubMed]
• Zhang M, Martin BR, Adler MW, Razdan RK, Jallo JI, Tuma RF. Cannabinoid CB₂receptor activation decreases cerebral infarction in a mouse focal ischemia/reperfusion model. J Cereb Blood Flow Metab. 2007;27:1387–1396.[PMC free article] [PubMed]
• Zhang M, Adler MW, Abood ME, Ganea D, Jallo J, Tuma RF. CB₂ receptor activation attenuates microcirculatory dysfunction during cerebral ischemic/reperfusion injury. Microvasc Res. 2009a;78:86–94. [PMC free article] [PubMed]
• Zhang M, Martin BR, Adler MW, Razdan RJ, Kong W, Ganea D, et al. Modulation of cannabinoid receptor activation as a neuroprotective strategy for EAE and stroke.J Neuroimmune Pharmacol. 2009b;4:249–259. [PMC free article] [PubMed]