Hepatic I/R injury is a major clinical problem implicated in the liver failure associated with liver transplantation, hepatic surgery and circulatory shock, with unfortunately only limited therapeutic options. Cannabidiol, is a nonpsychotropic component of marijuana, which has been shown to exert antioxidant and anti-inflammatory effects both in vitro
and in various preclinical models of neurodegeneration and inflammatory disorders, independent from conventional CB1
receptors (reviewed in [13
]). In the present study, we demonstrate that CBD exerts protective effect against liver I/R reperfusion damage by attenuating major pro-inflammatory and stress signaling pathways, as well as oxidative/nitrative stress and cell death.
Initially, the destructive effects of I/R is inflicted by the generation of superoxide and other forms of reactive oxygen species (ROS) following reoxygenation during reperfusion from the activation of various sources (e.g. xathine oxidoreductases [49
]). This also leads to early impairment of the activities of the enzymes of the mitochondrial respiratory chain, mitochondrial dysfunction, allowing more ROS to leak out of the respiratory chain [29
]. Consistently with previous studies demonstrating mitochondrial dysfunction in various forms of I/R [29
], we found marked depression of mitochondrial complex I activity in the livers exposed to ischemia followed by reperfusion. This decreased complex I activity was most pronounced 2 hours following ischemia, but also persisted at 6 and 24 hours of reperfusion. In agreement with previous studies establishing that NADPH oxidase-derived superoxide (particularly gp91phox/NOX2-derived (gp91phox is abundantly present in various inflammatory cells including neutrophil granulocytes)), plays an important role in the development of hepatic I/R-induced injury [4
], we also found significantly increased expression of mRNA of gp91phox at 6 and 24 hours of reperfusion. The peak increase in gp91phox mRNA expression occurred 24 hours following the ischemic insult coinciding with the marked inflammatory cell infiltration in damaged livers. However, the absence of significantly increased gp91phox mRNA expression 2 hours following ischemia, coupled with markedly depressed mitochondrial complex I activity, support the view that at early stage of reperfusion injury mitochondria play important role in reactive oxygen species (ROS) generation.
The increased superoxide and NO generation during early hepatic reperfusion (the latter being most likely derived from increased iNOS induction) favors the formation of the potent oxidant peroxynitrite [53
] via a diffusion limited reaction of superoxide with nitric oxide (NO) [54
], further impairing mitochondrial [55
] and cellular functions and increasing ROS generation [7
]. Consistently with several recent studies demonstrating markedly enhanced iNOS gene/protein expressions during hepatic I/R [56
], we found significant time-dependent increases in the liver iNOS mRNA expression at 2, 6 and 24 hours of reperfusion, peaking at 24 hours when the massive inflammatory cell infiltration occurred. There was also significant increase in hepatic nitrotyrosine (NT) content as early as at 2 hours of reperfusion, with further increase at 6 hours and dramatic enhancement at 24 hours of reperfusion. While nitrotyrosine has been considered a marker of peroxynitrite formation previously, there is some evidence that heme-protein peroxidase activity, in particular neutrophil-derived myeloperoxidase (MPO), may significantly contribute to nitrotyrosine formation in vivo via the oxidation of nitrite to nitrogen dioxide under certain inflammatory conditions [46
], therefore it is rather used as a collective index of nitrative sress [3
]. Since the neutrophil recruitment is known to occur in this hepatic I/R model predominantly from 6 hours of reperfusion (peaking between 12–24 hours), it is not very likely that the latter mechanism was significantly involved in the increased hepatic nitrotyrosine content/staining observed at 2 and 6 hours of reperfusion compared to sham operated animals, however it may contribute to the additional elevation observed at 24 hours, at a time when there is a profound increase in the MPO positive infiltrating neutrophils. This is also supported by our results clearly demonstrating absence of infiltrating inflammatory cells (including MPO positive cells) at 2 and 6 hours of reperfusion in liver tissues. Furthermore, at earlier time points of reperfusion (2 and 6 hours) the NT staining is clearly localized in endothelial cells and perivascular hepatocytes which are the primary targets of I/R-induced injury (the staining being stronger at 6 hours of reperfusion when gp91phox and iNOS mRNA expressions are also elevated). At 24 hours of reperfusion we observed further dramatic enhancement of hepatic NT content/staining in endothelial cells and perivascular hepatocytes in the damaged areas, as well as in infiltrating (largely MPO positive) inflammatory cells in the necrotic areas. Importantly, the time-dependent increases in hepatic NT content/staining paralleled with increased expressions of superoxide generating gp91phox and iNOS, as well as with increased hepatic HNE adduct levels (marker of lipid peroxidation/oxidative stress [41
]). Thus, it is very likely that the above described increased NT content/staining during reperfusion originates, at least in part, from increased peroxynitrite generation. Importantly, NO which is readily diffusible at a distance of several cells, irrespective of its source can rapidly react with superoxide (even produced in neighboring cells) to form peroxynitrite, resulting in decreased NO bioavailability with consequent loss of its protective effects [3
]. It should also be noted that under various pro-inflammatory conditions both eNOS and iNOS may also be uncoupled and generate additional ROS aggravating the tissue damage [3
]. Increasing evidence suggests that peroxynitrite during I/R injury may modulates/trigger various key stress signaling (e.g. p38 MAPK, JNK), pro-inflammatory (e.g. nuclear factor kappa B (NF-KB)) and cell death signaling pathways [3
], promoting cell death (both apoptotic and necrotic).
Indeed, sustained ROS/RNS generation during hepatic reperfusion activates important stress signaling (e.g. p38MAPK, JNK) [65
] and pro-inflammatory pathways (e.g. NF-KB [4
], COX-2 [68
]) in various cell types, in turn rmodulating/regulating important inflammatory and cell death processes. In agreement with these observations, we demonstrate time-dependent activation of these pathways at 2, 6 and/or 24 hours of reperfusion in the liver. After a more prolonged period of reperfusion, increased amounts of pro-inflammatory chemokines and cytokines are produced from activated Kupffer cells (resident macrophages of the liver) and endothelium leading to the priming and recruitment of neutrophils and other inflammatory cells, into the liver vasculature upon reperfusion, attachment to the activated endothelium and consequent activation resulting in further ROS/RNS generation and release of pro-inflammatory mediators leading to endothelial damage and dysfunction [4
]. Subsequently, adherent inflammatory cells transmigrate through the injured endothelium, attach to hepatocytes, and become fully activated to release oxidants and proteolytic enzymes, which in turn triggers the intracellular oxidative/nitrative stress and mitochondrial dysfunction in hepatocytes, eventually culminating in cell death (both apoptotic and necrotic), as also demonstrated in our report. Hepatic I/R also leads to significant reduction of endothelial NO synthase activity in sinusoidal endothelial cells during I/R [70
] resulting in an imbalance between sinusoidal vasoconstrictors (e.g., endothelins) and vasodilators (NO) in the liver, creating a situation favoring sinusoidal vasoconstriction and injury during reperfusion [4
]. In agreement with our recent results, and as already mentioned above, hepatic I/R also activates Kupffer cells, which in concert with activated inflammatory cells then produce proinflammatory cytokines, free radicals, oxidants, and large amounts of NO due to iNOS expression [4
], leading to more sustained formation of peroxynitrite and decreased NO bioavailability. These events lead to further activation of endothelial cells, neutrophils, and hepatocytes, resulting in amplified ROS/RNS generation in the delayed phase of hepatic I/R aggravating the cell death of hepatocytes and consequent organ injury.
Consistent with the above mentioned sequel of pathological events and previous reports using the same or very similar models [26
], we have found increased serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities (markers of liver injury), hepatic oxidative/nitrative stress (HNE, NT content/staining, gp91phox and iNOS mRNA), mitochondrial dysfunction (decreased complex I activity), enhanced acute inflammatory response (TNF-α, COX-2, MIP-1α/CCL3, MIP-2/CXCL2, ICAM-1/CD54, mRNA levels, NF-KB), and stress signaling (p38 MAPK and JNK) activation at 2 and/or 6 hours of reperfusion. This was followed by tissue neutrophil infiltration and cell death (DNA fragmentation, PARP activity, and TUNEL) at 24 hours of reperfusion. Indeed, both oxidative and nitrative stress appeared to peak at 24 hours of reperfusion, as well as apoptotic cell death (DNA fragmentation and deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)), while the poly(ADP-ribose) polymerase (PARP) activity was already at peak at 2–6 hours, indicating that the predominant type of cell death at the earlier time points of reperfusion is necrotic. The latter is also supported by peak observable levels of serum ALT and AST (marker of hepatocyte necrosis) at 6 hours of reperfusion in our model, and gradual return close to normal levels thereafter by 24 hours.
We found that CBD, given prior to the induction of I/R, significantly attenuated the elevations of serum liver transaminases (ALT/AST), decreased tissue oxidative and nitrative stress (HNE, NT content/staining, gp91phox and iNOS expressions), attenuated acute and chronic hepatic inflammatory response (TNF-α, MIP-1α/CCL3, MIP-2/CXCL2, ICAM-1/CD54, COX-2 mRNA levels, NF-KB activation, and tissue neutrophil infiltration), stress signaling (p38 MAPK, JNK), and cell death (DNA fragmentation, PARP activity and TUNEL). CBD also exerted similar protective effects in CB2
knockout mice against I/R injury, indicating that its protective effects were not mediated by CB2
receptors. It is also unlikely that the in vitro described inverse agonistic property of CBD at CB2
receptors contributed to its beneficial effect observed in the current in vivo study, because CB2
inverse agonists by themselves are not attenuating the I/R-induced tissue (including hepatic) injury, but are able to prevent the protective effect of pure CB2
agonist in the same models [35
]. Importantly from a clinical point of view, the protective effects of CBD against liver damage were also preserved when it was given right after the ischemic episode up to 90 minutes of reperfusion.
The beneficial effects of CBD against I/R injury can only be explained in part by its direct antioxidant properties [24
]. In fact, in the first study demonstrating its direct and indirect antioxidant effects [24
], CBD was more protective against glutamate-induced neurotoxicity than any of the well-know antioxidants (e.g., ascorbate or α-tocopherol), indicating additional cytoprotective effects of CBD beyond its antioxidant properties. Moreover, pro-oxidant effects of CBD were also recently described in various immune cells (e.g. lymphocytes) in vitro [77
], which may contribute to apoptosis induction in these cells, an overall anti-inflammatory response. CBD has also been reported to exert potent anti-inflammatory effects in numerous inflammatory disease models in which conventional antioxidants are not very effective (e.g., in autoimmune arthritis [13
]), and was recently shown to attenuate key pro-inflammatory signaling processes (e.g. NF-KB activation) and/or its consequences (e.g. adhesion molecules, COX-2, iNOS expressions, etc.) in kidneys with nephropathy [38
], diabetic hearts [25
], human cardiomyocytes exposed to high glucose [25
], and in bacterial lipopolysaccharide/endotoxin (LPS)-activated microglia cells [80
], likewise in livers exposed to I/R injury in the current report. Notably, the most prominent effects of CBD in our in vivo liver I/R injury model were the suppression of the acute inflammatory response (orchestrated mostly by Kupffer cells and activated endothelium), as well as the marked attenuation of the delayed inflammatory cell infiltration. In further support of the anti-inflammatory effects of this natural constituent of marijuana, we also provide in vitro evidence that CBD attenuates LPS-triggered NF-KB activation and TNF-α production in isolated Kupffer cells, as well as the adhesion molecules (ICAM-1 and VCAM-1) expression in primary human liver sinusoidal endothelial cells stimulated with TNF-α, likewise the attachment of human neutrophils to the activated endothelium. These protective effects of CBD were preserved in CB2
knockout mice and were not prevented by CB1/2
antagonists in vitro.
Although following intraperitoneal injection numerous biotransformation products of CBD has been reported in mouse livers, and over 50 metabolites were identified in the urine with considerable variations among rat, dog and man, their biological activities and significance are largely elusive [81
]. On the basis of numerous in vitro studies (both in cell free and cellular systems; even though the metabolism of CBD can not be excluded in some cells in vitro), it is very likely CBD exerts direct anti-inflammatory and antioxidant effects. However, the indirect protective effect of its certain metabolites in in vivo models can not be excluded, which deserves further exploratory studies.
Collectively, our results indicate that CBD may represent a novel protective strategy against I/R-induced injury and inflammatory diseases by attenuating the acute and chronic pro-inflammatory response, oxidative/nitrative stress, cell death and interrelated signaling. Furthermore, our results also underline the importance of the careful evaluation of the markers of oxidative/nitrative stress, inflammation, cell death and signaling processes at various time points of reperfusion, since these may show considerable time-dependent differences.