Δ8-Tetrahydrocannabivarin prevents hepatic ischaemia/reperfusion injury by decreasing oxidative stress and inflammatory responses through cannabinoid CB2 receptors
Abstract
BACKGROUND AND PURPOSE
Activation of cannabinoid CB2 receptors protects against various forms of ischaemia-reperfusion (I/R) injury. Δ8-Tetrahydrocannabivarin (Δ8-THCV) is a synthetic analogue of the plant cannabinoid Δ9-tetrahydrocannabivarin, which exhibits anti-inflammatory effects in rodents involving activation of CB2receptors. Here, we assessed effects of Δ8-THCV and its metabolite 11-OH-Δ8-THCV on CB2 receptors and against hepatic I/R injury.
EXPERIMENTAL APPROACH
Effects in vitro were measured with human CB2 receptors expressed in CHO cells. Hepatic I/R injury was assessed in mice with 1h ischaemia and 2, 6 or 24h reperfusion in vivo.
KEY RESULTS
Displacement of [3H]CP55940 by Δ8-THCV or 11-OH-Δ8-THCV from specific binding sites in CHO cell membranes transfected with human CB2 receptors (hCB2) yielded Ki values of 68.4 and 59.95 nM respectively. Δ8-THCV or 11-OH-Δ8-THCV inhibited forskolin-stimulated cAMP production by hCB2CHO cells (EC50= 12.95 and 14.3 nM respectively). Δ8-THCV, given before induction of I/R, attenuated hepatic injury (measured by serum alanine aminotransferase and aspartate aminotransferase levels), decreased tissue protein carbonyl adducts, 4-hydroxy-2-nonenal, the chemokines CCL3 and CXCL2,TNF-α, intercellular adhesion molecule 1 (CD54) mRNA levels, tissue neutrophil infiltration, caspase 3/7 activity and DNA fragmentation. Protective effects of Δ8-THCV against liver damage were still present when the compound was given at the beginning of reperfusion. Pretreatment with a CB2 receptor antagonist attenuated the protective effects of Δ8-THCV, while a CB1 antagonist tended to enhance it.
CONCLUSIONS AND IMPLICATIONS
Δ8-THCV activated CB2 receptors in vitro, and decreased tissue injury and inflammation in vivo, associated with I/R partly via CB2 receptor activation.
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
Introduction
Transient ischaemia followed by reperfusion (I/R) is a principal mechanism of organ injury in common pathological conditions, such as myocardial infarction and stroke, and may accompany surgical interventions involving vascular occlusion. In particular, solid organ transplantation inherently exposes the graft to some degree of I/R injury, which considerably affects outcome. The deleterious effects of I/R arise from the acute generation of reactive oxygen (ROS) and nitrogen species (RNS) (Ferdinandy and Schulz, 2003; Pacher et al., 2007) subsequent to re-oxygenation upon vascular re-opening. These compounds cause direct tissue damage and initiate a chain of deleterious cellular responses leading to inflammation and cell death, and eventually to target organ failure (Liaudet et al., 2003; Pacher and Hasko, 2008).
Δ8-Tetrahydrocannabivarin (O-4395, Δ8-THCV) is a synthetic analogue of Δ9-tetrahydrocannabivarin (Δ9-THCV), a plant cannabinoid, which is a propyl homologue of Δ9-tetrahydrocannabinol, the main psychoactive ingredient of marijuana (Gill et al., 1970). Initial pharmacological experiments have already suggested that the plant-derived Δ9-THCV was a possible ligand for cannabinoid (CB) receptors (seePertwee, 2008). It has been shown that Δ9-THCV may act as a CB1 receptor antagonist (Thomas et al., 2005; Pertwee et al., 2007; Ma et al., 2008; Pertwee, 2008; receptor nomenclature follows Alexander et al., 2011); however, it can also activate CB2 receptors (Bolognini et al., 2010), as well as non-CB1, non-CB2 receptor targets (see Pertwee, 2008). Studies using Δ8-THCV have shown that it has a similar pharmacological profile to Δ9-THCV. Thus, both compounds behave as CB1 receptor antagonists in vitroand in vivo at low doses. However, Δ8-THCV may also exhibit agonistic activity in vivo at higher doses (Pertwee et al., 2007). Regarding CB2 receptors, evidence from a very recent study implies that Δ9-THCV may behave as partial agonists at CB2 receptors, not only in vitro, but also in vivo, as shown by reduced inflammation and inflammatory pain in mouse models (Bolognini et al., 2010). The rationale behind the development of Δ8-THCV is that it is more stable than the plant-derived analogue Δ9-THCV, and it is easier to synthesize.
The protective role of CB2 receptor activation in a well-characterized mouse model of hepatic I/R injury has previously been demonstrated (Batkai et al., 2007; Rajesh et al., 2007; Moon et al., 2008). In the current study, we have investigated the effects of Δ8-THCV on the course of liver damage, oxidative stress, and acute and chronic inflammatory response and cell death. In our model, tissue damage is induced by the generation of reactive oxygen species (ROS) that is followed by a cascade of acute and chronic inflammatory response. Because a recent study has found that Δ9-THCV displayed high selectivity and potency as a CB2 receptor agonist (Bolognini et al., 2010), and CB2 receptors on endothelial, inflammatory and perhaps some parenchyma cells mediate protective effects in hepatic (Batkai et al., 2007; Rajesh et al., 2007), cardiac (Pacher and Hasko, 2008; Defer et al., 2009; Montecucco et al., 2009) and cerebral (Zhang et al., 2007; 2009a,b; Murikinati et al., 2010) I/R injury (Pacher and Mechoulam, 2011), we also explored the plausible role of CB2 receptors in the effects of Δ8-THCV on hepatic I/R injury. Our findings reinforce the potential of Δ8-THCV for prevention/treatment of hepatic, and perhaps other forms of, I/R injury.
Discussion
In the present study, we have demonstrated that Δ8-THCV exerted protective effects against liver I/R reperfusion damage by attenuating tissue injury, oxidative stress and inflammatory response. Hepatic I/R injury may occur in a number of clinical settings, such as those associated with low flow states, surgical procedures and transplantation, affecting morbidity and mortality. The damage to the liver caused by I/R represents a continuum of processes that culminate in tissue injury. These processes are triggered when the liver is transiently deprived of oxygen and subsequently re-oxygenated. The destructive effects of I/R are induced by the rapid generation of ROS from the activation of various cellular sources, such as xanthine oxido-reductases (Engerson et al., 1987; Pacher et al., 2006) and impairment of the mitochondrial respiratory chain (Jaeschke, 2003; Moon et al., 2008), resulting in increased lipid peroxidation, oxidative modification of proteins and DNA damage, impairing pivotal cellular functions. The increased ROS during early reperfusion also leads to activation of Kupffer cells (the resident inflammatory cells of the liver) and endothelial cells with consequent increased production of pro-inflammatory chemokines (this response peaks 2 h following reperfusion) and cytokines in these cells, leading to the priming and recruitment of neutrophils and other inflammatory cells into the liver vasculature upon reperfusion. These inflammatory cells attach to the activated endothelium and generate ROS and RNS, and pro-inflammatory mediators leading to endothelial dysfunction, followed by transmigration through the injured endothelium, attachment to hepatocytes and further activation to release oxidants and proteolytic enzymes. These changes in turn trigger intracellular oxidative/nitrative stress and mitochondrial dysfunction in hepatocytes (the oxidative/nitrative stress peaks at 24 h of reperfusion), culminating in cell death (both apoptotic and necrotic) (Jaeschke, 2006; Pacher and Hasko, 2008).
In agreement with this cascade of pathological events and previous reports using the same model (Batkaiet al., 2007; Rajesh et al., 2007; Moon et al., 2008), we have found increased hepatic carbonyl and 4-HNE adduct levels (markers of lipid peroxidation and oxidative stress), and increased serum ALT and AST activities (markers of liver injury/necrosis) at 2 h of reperfusion, accompanied by peak increases in markers of acute inflammatory response and endothelial activation [mRNA for TNF-α, CCL3, CXCL2, ICAM-1 (CD54)] in the liver. The peak levels of serum ALT and AST could be detected at 6 h of reperfusion in our model, which gradually returned to almost normal levels by 24 h of reperfusion, indicating that the predominant type of cell death at the earlier time points of reperfusion is necrotic. As the inflammatory reactions progress, the histological picture of post-ischaemic liver morphology at 24 h of reperfusion is characterized by marked coagulation necrosis (lighter areas) with massive inflammatory cell infiltration (Figures 4 and ?and5).5). In this model of I/R, MPO-positive neutrophil recruitment starts from 6 h of reperfusion and peaks during 12–24 h of reperfusion (Moon et al., 2008) (Figure 5), which coincides with the peak of oxidative stress and apoptotic cell death (caspase 3/7 activity and DNA fragmentation) 24 h following reperfusion (Figures 7 and ?and88).
Δ8-THCV is a synthetic analog of Δ9-THCV, a natural component of marijuana. The latter has been shown to exert protective effects against seizure activity (Hill et al., 2010); has also hypophagic properties; and is in phase I development as a potential treatment for obesity, diabetes and related metabolic disorders (Riedel et al., 2009). On the other hand, Δ9-THCV also exhibits significant potency and efficacy as a CB2 receptor agonist in vivo, and exerts anti-oedema and anti-hyperalgesic activity in a model of local inflammation (Bolognini et al., 2010). Based on these data, we hypothesized that Δ8-THCV may also activate CB2 receptors and mediate beneficial effects in hepatic I/R.
Indeed, we found that Δ8-THCV and one of its metabolites, 11-OH-Δ8-THCV (Harvey and Brown, 1988), behaved as potent CB2 receptor agonists in vitro. In our binding assay, Δ8-THCV caused agonist displacement in hCB2-transfected CHO cells and also inhibited forskolin-induced stimulation of cAMP production (with an EC50 of 13 nM and Emax of 52%) in these cells, which were not significantly different from the recently reported values for Δ9-THCV (EC50= 38 nM and Emax= 40%; Bolognini et al., 2010).
Various cannabinoid ligands may also interact with the synthetic and metabolic enzymes of endocannabinoids, eliciting indirect effects mediated by changes of the levels of these bioactive substances, with potential relevance to tissue injury (Pacher and Mechoulam, 2011). However, we found that Δ8-THCV in relevant concentrations had no effect on the activities of the major enzymes involved in endocannabinoid synthesis (NAPE-PLD and DAGL) or degradation (FAAH and MAGL). Although in this study we have not tested the effect of Δ8-THCV on the putative endocannabinoid transporter(s), on the basis of the data described earlier, it is unlikely that Δ8-THCV would directly lead to major changes in tissue endocannabinoid levels in normal livers. However, it is very likely that by decreasing inflammation and oxidative stress during I/R, which are involved in modulating endocannabinoid production/degradation (Pacher and Hasko, 2008), Δ8-THCV may indirectly affect tissue endocannabinoid levels in damaged tissues, an interesting issue which should be explored in future studies.
In our in vivo model of liver I/R injury, Δ8-THCV, given prior to the induction of I/R, significantly attenuated the elevations of serum liver transaminases (ALT/AST), decreased tissue oxidative stress (carbonyl adducts and HNE), attenuated acute and chronic hepatic inflammatory response (TNF-α, CCL3, CXCL2, ICAM-1/CD54 mRNA levels and tissue neutrophil infiltration), and necrotic (ALT/AST levels and coagulation necrosis) and apoptotic (caspase 3/7 activity, DNA fragmentation) cell death. The protective effects of Δ8-THCV were largely abolished by pretreatment with the CB2 receptor antagonist SR144528, indicating that these were, at least in part, mediated by CB2 receptor activation (CB2antagonist in tested dose had no significant effect on I/R injury by itself). Pretreatment with the CB1receptor antagonist SR141716 did not prevent the beneficial effect of Δ8-THCV, in fact it showed protective effects by itself, which tended to be more pronounced when Δ8-THCV was given in combination with SR141716. The protective effect of the CB1 receptor antagonist observed in our study is consistent with the protection observed in rat liver I/R complicated with endotoxemia recently (Caraceniet al., 2009) and in other models of I/R injury (Pacher and Hasko, 2008; Zhang et al., 2009b), likewise with the proposal of combining CB2 receptor agonists with CB1 receptor antagonists for the treatment of reperfusion damage (Pacher and Hasko, 2008; Zhang et al., 2009b). From this perspective, it would be interesting to see in future studies if Δ8-THCV could inhibit CB1 receptors in in vitro and in vivo models of injury. Importantly, the protective effects of Δ8-THCV against liver damage were also preserved when it was given after the ischaemia, at the moment of reperfusion, which is very important from a clinical point of view.
In summary, our results indicate that Δ8-THCV, a more stable and more readily synthesized analogue of the naturally occurring Δ9-THCV, may represent a novel protective strategy against I/R injury by attenuating oxidative stress, acute and chronic inflammatory response, and cell death.
