In this study we explored the therapeutic potential of mitochondrially-targeted antioxidants in a well-established mouse model of segmental hepatic ischemia-reperfusion (I/R) injury [13
]. Previous in vitro, ex vivo, and in vivo studies using this and other models of hepatic I/R have implicated an important role of oxidative/nitrative stress, mitochondrial dysfunction and inflammation in hepatic I/R injury, and demonstrated beneficial effects of various global antioxidants [5
]. However, the exact role of mitochondrial ROS generation during liver I/R injury is still largely elusive, likewise the spatial-temporal relationship of the oxidative/nitrative stress and inflammatory cell infiltration. We hypothesized that the I/R-induced mitochondrial oxidative stress results in mitochondrial injury and dysfunction, which may trigger a cascade of deleterious cellular consequences continuously fueling oxidative/nitrative stress, endothelial and hepatocyte cell demise, local inflammation, eventually culminating in liver dysfunction/failure. To test this hypothesis, we administered membrane-permeable small molecule antioxidants that target mitochondria 1.5 hr before I/R challenge and observed marked improvements in each of these pathogenic events including, most importantly, hepatocellular damage. These findings indicate that mitochondrial ROS is a crucial mediator of hepatic I/R injury, which can be selectively targeted by novel antioxidants for therapeutic gain.
The early damaging effect of I/R is caused by the generation of superoxide and other forms of ROS during reperfusion which may involve activation of xathine oxidoreductases [15
], impairment of the activities of the enzymes of the mitochondrial respiratory chain, mitochondrial dysfunction, allowing more ROS to leak out of the respiratory chain [6
]. Superoxide may also readily react with nitric oxide (NO) during early hepatic reperfusion (the latter can be derived from nitric oxide synthases, most likely iNOS [39
]) via a diffusion limited reaction to form a more potent oxidant peroxynitrite [40
], further impairing mitochondrial [12
] and cellular functions and increasing ROS generation [3
]. Hepatic I/R also attenuates endothelial NO synthase activity in sinusoidal endothelial cells during I/R leading to endothelial dysfunction, favoring sinusoidal vasoconstriction. The initial oxidant-induced injury also leads to the activation of endothelial cells and the resident macrophages of the liver, the Kupffer cells [2
], which together orchestrate the acute and delayed pro-inflammatory response leading to attraction of neutrophils and other inflammatory cells into the damaged tissue upon reperfusion [2
]. These inflammatory cells further release oxidants and proteolytic enzymes enhancing intracellular oxidative/nitrative stress and mitochondrial dysfunction in hepatocytes, thereby promoting apoptotic and/or necrotic, or other forms of cell demise [6
Increased oxidative/nitrative stress was one of the earliest features we observed during the initial phase of hepatic I/R (I/R 2h) both in the liver, as well as in the isolated liver mitochondria. The above mentioned changes were also accompanied by structural mitochondrial injury in the liver, decreased mitochondrial complex I activity, and hepatocellular necrosis. The early mitochondrial and hepatocellular injury and dysfunction were coupled with an acute pro-inflammatory cytokine/chemokine response (originating from activated Kupffer and endothelial cells) without significant inflammatory cell infiltration. The above mentioned pathological changes were followed by profound delayed neutrophil infiltration and a secondary wave of ROS/RNS generation from 6 hours following ischemia (IR 6 h), peaking at 24 hours of reperfusion (I/R 24h). The later response, consistently with other reports [20
], most likely involved the phagocyte NAD(P)H oxidase isoform gp91phox/NOX2 [16
], increased iNOS and cyclooxygenase 2 expression[20
], and coincided with enhanced leukocyte infiltration in the injured livers. The I/R-induced ROS/RNS generation may also induce expression of adhesion molecules through the activation of NF-κB[18
]. Indeed, we found increased expression of adhesion molecule ICAM-1 in the liver exposed to I/R. This, coupled with the pro-inflammatory cytokines/chemokines released by activated Kupffer and endothelial cells, and possibly certain subpopulations of T cells, may facilitate migration, adhesion and activation of neutrophils to the site of injury, which may further amplify oxidative and nitrative stress, and these processes are interrelated leading to a concerted activation of various mitochondrial and other (e.g. PARP-1-dependent) cell death pathways.
A single dose of mitochondrially-targeted antioxidants, MitoQ/Mito-CP, not only attenuated the I/R-induced liver dysfunction, mitochondrial and hepatic early and delayed oxidative and nitrative stress (HNE/carbonyl adducts, malondialdehyde, 8-OHdG, and nitrotyrosine), mitochondrial and histopathological injury/dysfunction, but also blunted the early and the secondary wave of pro-inflammatory responses and associated cell death. Our results are also in agreement with a recent study demonstrating protective effects of MitoQ against alcohol-induced liver injury, where MitoQ successfully attenuated the alcohol-induced hepatic mitochondrial complex I dysfunction and associated oxidative/nitrative damage [14
]. Even though the exact mechanisms of the direct protective effects of MitoQ/Mito-CP are not known, our study supports the view that these antioxidants can attenuate mitochondrial dysfunction and ROS/RNS generation in vivo under pathological conditions, most likely by attenuating lipid peroxidation and/or by quenching peroxynitrite. The most likely primary cellular targets of the protective effects of these compounds against liver I/R injury are the hepatocytes and endothelial cells; however in light of the emerging view that mitochondrial ROS/RNS formation may directly or indirectly control expression of NAD(P)H oxidases [42
] and other key processes in various cell types, a direct effect of MitoQ/Mito-CP on early Kupffer cell activation cannot be excluded either. On the other hand, compelling evidence suggests that various molecules released from necrotic hepatocytes during early hepatic I/R (e.g. high mobility group 1 proteins (HMGB1) and DNA fragments), as well as lipid peroxidation products such as hydroxynonenal (HNE), are key triggers of Kupffer cells activation via toll like receptors (TLRs) [2
]. Thus, attenuation of early mitochondrial dysfunction, oxidative/nitrative stress, and subsequent necrotic cell death in hepatocytes and endothelial cells during early I/R can also lead indirectly to attenuation of the acute pro-inflammatory response orchestrated by activated Kupffer cells and consequent delayed inflammatory cell infiltration. summarizes the key pathological processes during hepatic I/R injury.
Simplified mechanisms of the interplay of mitochondrial dysfunction and oxidative stress with inflammatory responses and cell death during hepatic ischemia-reperfusion (I/R) injury
Collectively, these observations strongly indicate that early mitochondrial ROS generation triggers the deleterious cascade of inflammation and tissue injury associated with hepatic I/R (). Thus, mitochondrially-targeted antioxidants such as MitoQ, which appear to be safe in humans and are recently being evaluated for potential therapeutic use [45
] (see ClinicalTrials.gov), may represent a promising approach to attenuate the I/R-inflicted liver and most likely other forms of tissue injury, and inflammation.