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Mitochondrial reactive oxygen species generation has been implicated in the pathophysiology of ischemia-reperfusion (I/R) injury, however its exact role and its spatial-temporal relationship with inflammation are elusive. Herein we explored the spatial-temporal relationship of oxidative/nitrative stress and inflammatory response during the course of hepatic I/R and the possible therapeutic potential of mitochondrial-targeted antioxidants, using a mouse model of segmental hepatic ischemia-reperfusion injury. Hepatic I/R was characterized by early (at 2 hours of reperfusion) mitochondrial injury, decreased complex I activity, increased oxidant generation in the liver or liver mitochondria, and profound hepatocellular injury/dysfunction with acute pro-inflammatory response (TNF-α, MIP-1αCCL3, MIP-2/CXCL2) without inflammatory cell infiltration, followed by marked neutrophil infiltration and more pronounced secondary wave of oxidative/nitrative stress in the liver (starting from 6 hours of reperfusion and peaking at 24 hours). Mitochondrially-targeted antioxidants, MitoQ or Mito-CP, dose-dependently attenuated I/R-induced liver dysfunction, the early and delayed oxidative and nitrative stress response (HNE/carbonyl adducts, malondialdehyde, 8-OHdG, and 3-nitrotyrosine formation), mitochondrial and histopathological injury/dysfunction, as well as delayed inflammatory cell infiltration and cell death.
Mitochondrially generated oxidants play a central role in triggering the deleterious cascade of events associated with hepatic I/R, which may be targeted by novel antioxidants for therapeutic advantage.
Ischemia-reperfusion (I/R) inflicted by transient ischemia followed by reperfusion is a pivotal mechanism of organ injury during various pathological conditions (e.g. myocardial infarction, stroke, vascular surgeries, circulatory shock, and organ transplantation). Surgical interventions in the liver, transplantation, inflammatory and vascular diseases, circulatory shock or various toxic insults may lead to hepatic I/R injury resulting in organ dysfunction or failure, depending on the duration of the insult [1–3]. Early generation of reactive oxygen (e.g. superoxide and hydrogen peroxide) and nitrogen (e.g. peroxynitrite) species (ROS/RNS) during reperfusion is considered playing a pivotal role in initiating a chain of deleterious cellular responses leading to inflammation and cell death, which eventually culminate in target organ dysfunction/failure [3–11]. Mitochondrial proteins are particularly sensitive targets for ROS/RNS , and their inactivation by oxidants may contribute to dysfunction and injury during hepatic  and other forms of liver injury . Dysfunctional mitochondria may also generate significant amount of ROS during hepatic I/R in addition to activated xanthine oxidoreductases, NADPH oxidases in various key cell types of the liver (e.g. hepatocytes, endothelial, Kupffer, and infiltrating immune cells) [15–17].
Despite numerous reports on the role of oxidative stress and mitochondrial dysfunction in hepatic I/R injury, and on the beneficial effects of global antioxidants, the exact role of mitochondrial ROS generation in liver I/R injury is still largely elusive. Furthermore, there is very limited information on the spatial-temporal relationship of the oxidative and nitrative stress and inflammatory response during hepatic I/R, and on the main cellular sources/targets of ROS/RNS generation. In this study, we used a well-established mouse model of in vivo segmental hepatic I/R injury [13, 18–20] and two well-characterized mitochondrially-targeted small-molecule antioxidants, MitoQ and Mito-CP [21–24], to investigate the role of oxidative stress and mitochondrial oxidant generation during the course of hepatic I/R.
The study has been approved by the Institutional Animal Care and Use Committees of NIAAA, and has been carried out in line with the National Institutes of Health (NIH) guidelines for the care and use of laboratory animals. Male C57BL/6J mice (25–30g; Jackson Laboratories, Bar Harbor, ME) were anesthetized with pentobarbital sodium (65 mg/kg i.p.). In our study, the model of segmental (70%) hepatic ischemia-reperfusion has been used, as described [13, 18–20, 25, 26]. Two mitochondrially-targeted antioxidants, which most likely inhibit lipid peroxidation and also quench peroxynitrite, Mito-Q and Mito-CP were synthesized as described [21, 27]. MitoQ and Mito-CP were stored in ethanol at 50 mg/ml, further diluted in saline and administered at 0.3–3 mg/kg or as described, i.p. once, 1.5 hour before I/R. Mito-Q and Mito-CP at 3 mg/kg or vehicle were also given right after the ischemia at the moment of reperfusion (post I (R 0h)) or 3h after ischemia (post I (R 3h)), as indicated in the text. At the experimental end points at 2, 6 and 24 hours following ischemia (I/R 2, 6 and 24h), 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.
The activities of aspartate amino-transferase (AST) and alanine amino-transferase (ALT), indicators of liver cellular damage, were measured in serum samples using a clinical chemistry analyzer system (VetTest 8008, IDEXX laboratories, Westbrook, ME) [13, 19].
Liver samples were fixed in 4% buffered formalin. After embedding and cutting 5 μm slices, all sections were stained with hematoxylin/eosin (HE). Myeloperoxidase staining of neutrophils was done by using anti-myeloperoxidase antibody according to the manufacturer’s protocol (Biocare Medical, Concord, CA), and samples were counter-stained with nuclear fast red as described . Liver sections were also stained for malondialdehyde (Genox Corporation, Baltimore, MD), nitrotyrosine (Cayman Chemical, Ann Arbor, MI) and 8-hydroxy-2′-deoxyguanosine (8-OHdG) (Genox Corporation, Baltimore, MD) according to the manufacturer’s protocol and/or as described [20, 28]. Both malondialdehyde and nitrotyrosine stained sections were counter stained with nuclear fast red. The specific staining was visualized and images were acquired using microscope IX-81 with 20X, 40X and 100x objectives (Olympus, Center Valley, PA) [20, 28]. Histological evaluation was performed in a blinded manner.
Livers of anesthetized animals were perfusion fixed with 1.25 % glutaraldehyde in 0.1 M cacodylate buffer. Livers were harvested and processed in standard fashion (Epon embedded) for transmission electron microscopy using a JEOL 1011 Electron Microscope as described .
Total RNA was isolated from liver homogenate using TRIzol reagents (Invitrogen, Carlsbad, CA) according to manufacturer’s instructions. The isolated RNA was treated with RNase-free DNase (Ambion, Austin, TX) to remove traces of genomic DNA contamination. One microgram of total RNA was reverse-transcribed to cDNA using the SuperScript II (Invitrogen, Carlsbad, CA). 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). Each amplified sample in all wells was analyzed for homogeneity using dissociation curve analysis. Relative quantification was calculated using the comparative CT method.
TNF-α, 5′-AAGCCTGTAGCCCACGTCGTA-3′ and 5′-AGGTACAACCCATCGGCTGG-3′; MIP-1α, 5′-TGCCCTTGCTGTTCTTCTCTG-3′ and 5′-CAACGATGAATTGGCGTGG-3′; MIP-2, 5′-AGTGAACTGCGCTGTCAATGC-3′ and 5′-AGGCAAACTTTTTGACCGCC-3′; gp91Phox (NOX2), 5′-GACCATTGCAAGTGAACACCC-3′ and 5′-AAATGAAGTGGACTCCACGCG-3′; and actin, 5′-TGCACCACCAACTGCTTAG-3′ and 5′-GGATGCAGGGATGATGTTC-3′.
Liver tissues were homogenized in mammalian tissue protein extraction reagent (Pierce, Rockford, IL, USA) supplemented with protease and phosphatase inhibitors (Roche Diagnostics) Blots were probed with gp91phox (BioLegend Inc, San Diego, CA) used at 1:1000 dilution and incubated overnight at 4 C. After subsequent washing with PBS–Tween, the membranes were probed with appropriate secondary antibodies conjugated with HRP (Pierce, Rockford, IL) and incubated 1h at room temperature. Then the membranes were developed using a SuperSignal–West Pico substrate chemiluminescence detection kit (Pierce, Rockford, IL). To confirm uniform loading, membranes were stripped and re-probed with β-actin (Chemicon, Ramona, CA, USA).
Mitochondria were isolated from the livers of mice treated with vehicle, or Mito-CP/MitoQ with or without exposure to I/R using tissue mitochondrial isolation kit (Pierce Biotechnology, Rockford, IL) as decribed .
Microplate assay kits (MitoSciences, Inc., Eugene, OR, USA) were used to determine the activity of Mitochondrial complex I, complex II and complex IV according to the manufacturer’s instructions. The complex enzymes were immunocaptured within the wells of the microplate and activities were determined colorimetrically. Complex activities were expressed as percentage activity compared to the liver samples of the vehicle-treated mice as described .
Lipid peroxides are unstable indicators of oxidative stress in cells that decompose to form more complex and reactive compounds such as 4-hydroxynonenal (HNE), which has been shown to be capable of binding to proteins and forming stable HNE adducts. HNE adducts in the hepatic tissues or from mitochondrial fractions were determined using a kit (Cell Biolabs, San Diego, CA). In brief, BSA or hepatic tissue extracts (10 μg/mL) were adsorbed onto a 96-well plate. HNE adducts present in the sample or standard were probed with anti-HNE antibody, followed by an HRP conjugated secondary antibody. The HNE-protein adducts content in an unknown sample was determined by comparing with a standard curve.
The protein oxidation was also determined in isolated mitochondrial fractions by OxyBlot Protein Detection Kit from EMD Millipore (Billerica, MA) according to manufacturer instructions. This method is based on the immunoblot detection of carbonyl groups introduced into proteins by oxidative reactions with ozone or oxides of nitrogen or by metal catalyzed oxidation.
Mitochondria for oxyblot analyses were purified by magnetic beads method (Mitochondria Isolation Kit, Miltenyi Biotec, Auburn, CA) as described earlier . The isolation of mitochondria was carried out according to manufacturer’s instruction. Briefly, fresh harvested liver was homogenized by gentleMACS dissociator (Miltenyi Biotec, Auburn, CA). To magnetically label mitochondria, the homogenate was incubated with 50 μl of monoclonal anti-TOM22-conjugated microBeads for 1 hour at 4°C. Then, the suspension of labeled mitochondria was loaded onto a pre-equilibrated MACS Column (Miltenyi Biotec, Auburn, CA), previously placed in the magnetic field of a MACS Separator (Miltenyi Biotec, Auburn, CA). Due to the magnetic field generated by the MACS Separator, labeled-mitochondria were retained into the column. The column was washed three times with 3 ml suspension buffer provided with the kit. Subsequently, the column was removed from the magnetic field and the retained mitochondria were eluted in 1 ml suspension buffer. The mitochondrial lysates were prepared in RIPA buffer and the purity of mitochondrial fraction was analyzed by western blot with cytochrome c oxidase (COX IV) antibody (Abcam, Cambridge, MA).
Protein oxidation is defined as the covalent modification of the proteins induced by reactive oxygen species. Oxidative modification of proteins can be induced by wide array of pro-oxidant stimulus, and elevated levels were demonstrated in several pathological conditions. An ELISA-based assay (Cell Biolabs Inc., San Diego, CA) was used to measure the protein carbonyl levels in isolated liver mitochondrial fractions. In brief, BSA standards or protein samples were adsorbed on to 96-well plates for 2 h at 37°C. The protein carbonyls present in the samples or standards were derivatized to DNP hydrazones with dinitrophenylhydrazine (DNPH) and incubated with anti-DNP antibody, followed with HRP conjugated secondary antibody. Then the absorbance was measured at 450 nm using a plate reader (Spectramax Plus, Molecular Device, Sunnyvale, CA) as described [32, 33]..
NT content was measured by the NT ELISA kit from Hycult Biotechnology (Cell Sciences, Canton, MA) from tissue homogenates as described [20, 33]. Levels were presented as fold changes compared to vehicle-treated control sample.
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, Indianapolis, IN). Hepatic PARP activity was assayed using a colorimetric kit according to the manufacturer’s protocol (Trevigen, Gaithersburg, MD) as described .
Results are expressed as means ± SEM. Statistical significance among groups was determined by one-way ANOVA followed by Newman-Keuls post hoc analysis using GraphPad Prism 5 software (San Diego, CA). Probability values of P<0.05 were considered significant.
After 1 hour of ischemia and a subsequent 2 or 6 hours of reperfusion (I/R 2 h or I/R 6h), a dramatic increase in the serum transaminase enzyme activities (AST and ALT; markers of hepatocellular damage/necrosis) were observed in vehicle-treated C57BL/6J mice (peaking at 6h of reperfusion), as compared with sham-operated controls (Figure 1B–D). At 24 hours of reperfusion (I/R 24h) the transaminase activities declined compared I/R 6h, but were still elevated compared to sham-operated controls (Figure 1D). Pretreatment with MitoQ or Mito-CP (Figure 1A) (0.3–3 mg/kg i.p.) 1.5 hours before the induction of the ischemia dose-dependently attenuated the serum transaminase elevations at 6 hours of reperfusion compared to vehicle (Figure 1B–C). MitoQ or Mito-CP (3 mg/kg i.p.) 1.5 hours before the induction of the ischemia significantly attenuated the serum transaminase elevations at 2, 6, and 24 hours of reperfusion compared to vehicle (Figure 1D). MitoQ and Mito-CP (3 mg/kg i.p.) were also effective in attenuating the I/R-induced peak ALT/AST elevations at 6 h of reperfusion when administered right after the 1 h ischemic period before the start of the reperfusion (Figure 2A; post I (R 0h)), however they lost their protective effects on liver dysfunction when administered at 3 hours of reperfusion (Figure 2B; post I (R 3h)).
We hypothesized that the mitochondrial dysfunction/ROS generation triggers the deleterious cascade of events during I/R (e.g. secondary inflammatory response and ROS/RNS generation, etc.), therefore we studied mitochondrial structure early at 2 h of reperfusion. The electron microscopy of the non-ischemic control livers of mice undergoing sham surgery (sham) revealed normal hepatic tissue with good preservation of hepatocytes and lining cells of sinusoidal venules, as well as normal mitochondrial structure (Figure 3A, left panels), which were similar in sham mice treated with MitoQ or Mito-CP (3 mg/kg i.p.) (Figure 3A middle and right panels).
Post-ischemic liver tissue at 2 hours of reperfusion (I/R 2h) exhibited marked disintegration of ultrastructure (e.g., swelling of mitochondria, vacuolization, nuclear and cytoplasmic degeneration) in most hepatocytes (Figure 3B, left panels). The majority of mitochondria exhibited alterations in size, shape, and matrix. In some mitochondria the matrix totally disappeared and only the outer membrane remained, while in others, the cristae appeared disorganized because of edema in the matrix, and many of them lost the spherical or ellipsoid shape (Figure 3B, left panels). Mito-CP and MitoQ pretreatment (3 mg/kg i.p.) markedly attenuated the morphological and mitochondrial injury induced by 1 h of ischemia followed by 2 hours of reperfusion (I/R 2h) to a similar extent (Figure 3B, middle and right panels).
Representative liver sections of sham mice, mice exposed to 1 hour of ischemia followed by 2, 6 and 24 hours of reperfusion (I/R) with vehicle or Mito-CP/MitoQ pretreatment were stained with hematoxylin and eosin (Figure 4). The I/R-induced marked coagulation necrosis was histologically clearly separated by 6 and 24h of I/R (lighter areas, with marked inflammatory cell infiltration at 24 hours of reperfusion), which was dramatically reduced and became more focal in Mito-CP/MitoQ treated mice (Figure 4). Mito-CP/MitoQ treatment alone or vehicle had no effect on the liver histopathology. Figure 4 depicts 200× magnifications.
The rate of hepatic lipid peroxidation (HNE; Figure 5A) was negligible in sham-operated mouse liver as indicated by the low levels of HNE adducts. Hepatic HNE adducts time-dependently increased at 2, 6 and 24 hours of reperfusion, and were markedly attenuated by mitochondrial antioxidants (Figure 5A). Consistently with early mitochondrial injury (Figure 3) and ROS/RNS formation (Figure 5A–D), the level of hepatic and mitochondrial HNE adducts (Figure 5A and B) and oxidized proteins (protein carbonyl adducts; Figure 5C and D) were elevated at 2 h of reperfusion. Marked attenuation of mitochondrial complex I activity was also observed at 2h of reperfusion, without significant changes in complex II and IV activities (Figure 5E).
Mitochondrial antioxidants almost completely prevented the I/R-induced early elevation of HNE and/or protein oxidation in the mitochondria (Figure 5A–D), as well as attenuated the impaired complex I activity measured from isolated liver mitochondria (Figure 5E, left panel). These data indicate that mitochondria represent the primary source of ROS generation during early reperfusion injury and Mito-CP/MitoQ is able to selectively target ROS in these organelles.
24 hours of I/R also induced marked increases in liver malondialdehyde formation (another index of lipid peroxidation; brown staining), which was predominantly localized to endothelial cells, perivascular hepatocytes, and infiltrating (or attached to the endothelium) inflammatory cells (Figure 6A, second pair of images from the left). Mitochondrial antioxidants markedly attenuated the I/R-induced malondialdehyde formation in the liver (Figure 6A, ,22 pairs of images from the right). Similar pattern but weaker staining (without infiltrating immune cells) was observed at earlier time points (I/R 2 and 6h; data not shown). Minimal staining could be seen in the livers of control mice exposed to sham surgery (Figure 6A, left panels).
24 hours of I/R was also associated with increased 8-OHdG formation (a marker of oxidative DNA injury; strong dark blue nuclear staining) in endothelial cells, perivascular hepatocytes and infiltrating inflammatory cells (Figure 6B, second pair of images from the left). Mitochondrial antioxidants markedly attenuated the I/R-induced 8-OHdG formation in the liver (Figure 6B, ,22 pairs of images from the right). Minimal nuclear 8-OHdG staining could be seen in the livers of control mice exposed to sham surgery (Figure 6B, left panels). The upper row of images (Figures 6A and B) depicts 400× magnification, the lower 1000×.
Consistently with previous studies [16, 20] from 6 h of reperfusion there was an increased expression (mRNA and/or protein) of ROS generating NADPH oxidase isoform NOX2/gp91phox in the liver, coinciding with the inflammatory cell infiltration; which were also attenuated by Mito-CP/MitoQ pretreatments (Figure 7A,B). In contrast, when Mito-CP/MitoQ were administered 3 hours after the ischemia they were not able to attenuate the I/R-induced NOX2 protein expression determined at 24 hours of reperfusion (Figure 7C).
I/R was also associated with time-dependent marked increases of hepatic and/or mitochondrial 3-nitrotyrosine formation (Figures 8A–C, and and9),9), a marker of peroxynitrite formation and more broadly nitrative stress ), showing similar cellular localization to malondialdehyde and 8-OHdG in endothelial cells, perivascular hepatocytes, and infiltrating (or attached to the endothelium) inflammatory cells (Figures 6, ,88 and and9).9). Mito-CP/MitoQ markedly attenuated the hepatic/mitochondrial nitrative damage (Figures 8A–C and and9).9). Figure 8C depicts 400× magnification, Figure 9 depicts 1000×.
I/R induced time-dependent changes in the mRNA expression of the pro-inflammatory cytokine tumor necrosis factor α (TNF-α), chemokines macrophage inflammatory proteins 1α and 2 (MIP-1α/CCL3 and MIP2/CXCL2), intercellular adhesion molecule 1 (ICAM-1/CD54) (Figures 10, and 11A), and hepatic myeloperoxidase activity and staining (an indicator of leukocyte infiltration; Figure 11B-D). The I/R-induced acute hepatic pro-inflammatory response (TNF-α, MIP-1α/2, ICAM-1; most likely orchestrated by activated Kupffer and endothelial cells [20, 35] peaked at 2 hours of reperfusion and was largely declined thereafter by 24 hours of reperfusion (Figures 10 and 11A). In contrast, the delayed inflammatory cell (predominantly myeloperoxidase positive neutrophil (brown staining)) infiltration started only from 6 hours of reperfusion peaking at 24 hours of reperfusion (Figures 11C and D). These changes in inflammatory responses were largely attenuated by pre-treatment with Mito-CP/MitoQ (3 mg/kg i.p., Figures 10, and 11A–D). Figure 11C depicts 200× magnification, Figure 11D depicts 400×.
I/R markedly increased the poly (ADP-ribose) polymerase-dependent (mostly necrotic) and apoptotic (DNA fragmentation, caspase 3/7activity) cell death (Figure 12A–C) in the liver, which were prevented by Mito-CP/MitoQ. Notably, while PARP activity was already increased early following reperfusion (Figure 12A), the apoptotic cell death was evident mainly at 24 h of I/R only (Figure 12B and C).
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, 18–20, 26]. 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, 7, 13, 15, 17, 36]. 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, 37, 38], 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, 13, 20]. 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 ) via a diffusion limited reaction to form a more potent oxidant peroxynitrite , further impairing mitochondrial  and cellular functions and increasing ROS generation [3, 4]. 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 , 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, 20]. 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, 9, 41].
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 , most likely involved the phagocyte NAD(P)H oxidase isoform gp91phox/NOX2 , increased iNOS and cyclooxygenase 2 expression, 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, 20]. 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 . 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  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, 43, 44]. 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. Figure 13 summarizes the key pathological processes during hepatic 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 (Figure 13). Thus, mitochondrially-targeted antioxidants such as MitoQ, which appear to be safe in humans and are recently being evaluated for potential therapeutic use [45, 46] (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.
This study was supported by the Intramural Research Program of NIH/NIAAA (to P.P.). 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 Dr. George Kunos, the Scientific Director of NIAAA/NIH for providing key resources and support.
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