Cytidine‐5'‐diphosphocholine (CDP‐choline) participates as an intermediary in the synthesis of phosphatidylcholine, an essential component of cellular membranes. Citicoline treatment has shown beneficial effects in cerebral ischemia, but its potential to diminish reperfusion damage in liver has not been explored. In this work, we evaluated the hepatoprotective effect of citicoline and its possible association with inflammatory/oxidative stress and mitochondrial function because they are the main cellular features of reperfusion damage. Ischemia/reperfusion (I/R) in rat livers was performed with the Pringle's maneuver, clamping the 3 elements of the pedicle (hepatic artery, portal vein, and biliary tract) for 30 minutes and then removing the clamp to allow hepatic reperfusion for 60 minutes. The I/R + citicoline group received the compound before I/R. Liver injury was evaluated by measuring aspartate aminotransferase and alanine aminotransferase as well as lactic acid levels in serum; proinflammatory cytokines, proresolving lipid mediators, and nuclear factor kappa B content were determined as indicators of the inflammatory response. Antioxidant effects were evaluated by measuring markers of oxidative stress and antioxidant molecules. Oxygen consumption and the activities of the respiratory chain were used to monitor mitochondrial function. CDP‐choline reduced aspartate aminotransferase (AST), alanine aminotransferase (ALT), as well as lactic acid levels in blood samples from reperfused rats. Diminution in tumor necrosis factor alpha (TNF‐α) and increase in the proresolving lipid mediator resolvin D1 were also observed in the I/R+citicoline group, in comparison with the I/R group. Oxidative/nitroxidative stress in hepatic mitochondria concurred with deregulation of oxidative phosphorylation, which was associated with the loss of complex III and complex IV activities. In conclusion, CDP‐choline attenuates liver damage caused by ischemia and reperfusion by reducing oxidative stress and maintaining mitochondrial function. Liver Transplantation XX XX‐XX 2018 AASLD.

Abbreviations

ADP adenosine diphosphate AMPK adenosine monophosphate–activated protein kinase ADP/O adenosine triphosphate synthesis coupled to oxygen consumption ALT alanine aminotransferase AST aspartate aminotransferase ATP adenosine triphosphate AUF arbitrary units of fluorescence CCT cytidylyltransferase CDP‐choline cytidine‐5'‐diphosphocholine CK choline kinase CTP cytidine triphosphate CMP cytidine monophosphate DCFH‐DA dichlorofluorescein diacetate DCPIP dichlorophenolindophenol EDTA ethylene diamine tetraacetic acid ELISA enzyme‐linked immunosorbent assay GSH glutathione GSSG oxidized glutathione GST glutathione S‐transferase HO‐1 heme oxygenase 1 HEPES 4‐(2‐hydroxyethyl)‐1‐piperazine ethanesulfonic acid HIF‐α hypoxia‐inducible factor alpha IL interleukin IL‐1β interleukin 1 beta I/R ischemia/reperfusion mCB mono‐chlorobimane MDA malondialdehyde NADH reduced nicotinamide adenine dinucleotide NADPH nicotinamide adenine dinucleotide phosphate NF‐κB nuclear factor kappa B NE‐PER nuclear and cytoplasmic extraction reagents NRF2 nuclear erythroid 2 p45‐related factor 2 OPA o‐phthalaldehyde PC phosphatidylcholine PPARα peroxisome proliferators‐activated receptor alpha RC respiratory control index ROS reactive oxygen species RvD1 resolvin‐D1 SEM standard error of the mean SD standard deviation TNFα tumor necrosis factor alpha Tris‐HCl trishydroxymethylaminomethane‐chloric acid CCT cytidylyltransferase CK choline kinase CTP cytidine triphosphate PC phosphatidylcholine WM molecular weight markers

Hepatic ischemia/reperfusion (I/R) involves a series of intricate events that take place during the transient blockade of blood/oxygen flow and its return to the parenchyma, conditioning temporary or definitive dysfunction of the organ. It occurs in different clinical situations ranging from trauma surgery, resection, shock, and transplantation. Release of reactive oxygen species (ROS) and inflammatory mediators underlie I/R damage. Mitochondria are not only the main sites of ROS production, but they contribute to the perpetuation of the “activation loops” between ROS generated by other sources (ie, xanthine oxidase and nicotinamide adenine dinucleotide phosphate [NADPH] oxidase) and the inflammatory cells infiltrated in the damaged tissue.(1) Mitochondria convert molecular oxygen to the relatively stable superoxide anion ( ) as a byproduct of the electron transfer activity.(2, 3) Once is dismutated to H 2 O 2 by manganese‐superoxide dismutase, can permeate through the mitochondrial outer membrane and activate cytosolic targets, including redox‐sensitive transcription factors (such as hypoxia‐inducible factor alpha [HIF‐a] and nuclear factor kappa B [NF‐ kB]).(4) In turn, H 2 O 2 may be fully reduced to water by catalase or partially reduced by transition metals to hydroxyl radical (•OH), 1 of the strongest oxidants in nature. Also, superoxide anions interact with transition metals, intensifying hydroxyl generation and reacting with other radicals, like nitric oxide (•NO), producing the powerful oxidant peroxynitrite.(4) CDP‐choline is an endogenous intermediary product in phosphatidylcholine synthesis, produced after 2 reactions: in the first step, choline kinase (CK) catalyzes the adenosine triphosphate (ATP)–dependent phosphorylation of choline forming phosphocholine and adenosine diphosphate (ADP).(5, 6) Then phosphocholine reacts with cytidine triphosphate (CTP) via CTP‐phosphocholine cytidylyltransferase, producing CDP‐choline. Finally, CDP‐choline 1,2‐diacylglycerol choline phosphotransferase forms phosphatidylcholine (PC) and CMP from CDP‐choline and diacylglycerol.(5, 6) CDP‐choline has been extensively used in clinical trials in patients with neurological disorders, including cerebral ischemia. In comparison with many other compounds, it is the only drug that has demonstrated some neuroprotective effects.(5) Its main protective action has been related to membrane stabilization by inhibiting the activation of phospholipase A,(6) preserving sphingomyelin levels,(7) and increasing PC, a main component of biological membranes. Indirectly, it is considered a signaling mediator because the administration of some forms of PC decrease the expression of transcription factors associated with lipogenesis and increase the expression of peroxisome proliferators‐activated receptor α (PPARα) and adenosine monophosphate–activated protein kinase (AMPK) in diabetic(8) and steatosis models.(9) Few reports have evaluated the effect of CDP‐choline in the context of I/R. An initial study showed that CDP‐choline diminishes oxidative stress–induced apoptotic death in cardiomyocytes subjected to hypoxia and reoxygenation; (10) shortly after, it was demonstrated that this compound maintains the permeability transition pore in the "closed state" in mitochondria isolated from hearts that underwent I/R (11) and that it protects from reperfusion‐induced ventricular arrhythmias in a hyperthyroid rat model. (12) This evidence indicates CDP‐choline acts at several points of the I/R cascade injury and even more, that it impacts on mitochondria. Therefore, in this work we evaluated for the first time whether the antioxidant and anti‐inflammatory properties of CDP‐choline preserve mitochondrial function and confer protection against reperfusion damage in liver.

1 Material and Methods This investigation was performed in accordance with the Guide for the Care and Use of Laboratory Animals, published by the US National Institutes of Health. Male Wistar rats weighing 250‐300 g were fasted for at least 6 hours before the procedure. The rats were divided into 3 groups: control, I/R, and I/R + CDP‐choline groups. Rats from the I/R+citicoline group received the compound 15 minutes before surgery in an intraperitoneal single dose (125 mg/kg), whereas control animals received an equivalent volume of saline solution.

2 I/R PROTOCOL Rats were anaesthetized with a single dose of sodium pentobarbital (60 mg/kg intraperitoneally) and complete lack of pain response was assessed by determining the pedal withdrawal reflex. Laparotomy was performed through a bisubcostal incision approximately 2 cm from the costal ridge. The xiphoid cartilage was retracted with a hemostat, and the intestinal loops were lateralized to the left while protected with gauzes soaked in physiological solution at 37°C. Then the sickle ligament was sectioned. After that, the right and central lobes were elevated, exposing the hepatic pedicle. The gastrohepatic ligament was sectioned for identification of the bile duct, portal vein, and hepatic artery. The dissection maneuvers were performed using microsurgery instruments and wet swabs with physiological solution to minimize tissue damage. The Pringle maneuver was performed, clamping the 3 elements of the pedicle (hepatic artery, portal vein, and biliary tract) by placing 2 bulldog miniclamps in parallel for 30 minutes. Then the clamps were removed to allow hepatic reperfusion for 60 minutes. Control animals were subjected to simulated surgery but not to clamping.

3 BLOOD AND TISSUE SAMPLES At the end of the protocol, approximately 2 mL of blood from the coronary sinus was collected. Blood samples were centrifuged at 750g for 10 minutes, and the serum was obtained to measure lactic acid levels and enzymatic markers. Tissue samples from the right hepatic lobule were fixed in 30% glutaraldehyde for 18 hours, sectioned and stained with hematoxylin‐eosin. (13)

4 MEASUREMENT OF LACTIC ACID IN BLOOD Lactic acid levels were measured using coronary blood. In an insulin syringe previously loaded with heparin, 1 mL of blood was taken from the coronary sinus and samples were analyzed using an BC‐5800 Auto Hematology Blood Analyzer (Mindray, Shenzhen, China) with reference values of 0.5‐1.6 mEq/L or 0.5‐1.6 mmol/L.

5 MEASUREMENT OF LIVER DAMAGE MARKERS IN SERUM Serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels were measured with a Cholestech LDX Analyzer System (CLIAwaived, San Diego, CA).

6 DETERMINATION OF CYTOKINES AND OF ANTI‐INFLAMMATORY MEDIATORS Cytokines (interleukin [IL] 1(beta), IL6, tumor necrosis factor (alpha) [TNF‐(alpha)]) in blood serum samples were measured as reported by Pérez‐Torres et al. (14) A Resolvin D1 enzyme‐linked immunosorbent assay (ELISA) kit (Cayman Chemicals, Ann Arbor, MI) and lipoxin A4 ELISA kit (Cloud‐Clone Corp., Houston, TX) were used to determine the levels of these proresolving mediators.

7 ROS DETERMINATION WITH 2', 7’ DICHLOROFLUORESCEIN DIACETATE Intracellular ROS levels were evaluated in fresh liver homogenates using 2', 7’ dichlorofluorescein diacetate (DCFH‐DA). The assay was performed by incubating 10 µM of DCFH‐DA and 1.5 mL of the homogenates in a buffer containing 50 mM of potassium phosphate, 1 mM of ethylenediaminetetraacetic acid (EDTA) and protease/phosphatase cocktail inhibitors (pH 7.4) for 15 minutes with agitation at 37°C in a dark chamber. The increase in fluorescent signal was measured at λ ex = 488 nm and λ em = 530 nm in a Shimadzu RF5000U spectrofluorophotometer. Results were expressed in arbitrary units of fluorescence (AUF) per protein milligram. 7.1 WESTERN BLOT ANALYSIS Nuclear proteins (50 μg) and homogenates (70 µg) were separated in 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to a polyvinylidene fluoride membrane. The membranes were blocked using a casein‐based blocking solution (abcam ab126587) for 1 hour at pH 7.5. Nuclear erythroid 2 p45‐related factor 2 (NRF2) and NF‐κB p65 content was analyzed with Anti‐NRF2 (abcam ab89443) and anti‐p65 NF‐κB (cell signaling #5061). Lamin B (abcam ab133741) was used as the loading control. Heme oxygenase 1 (HO‐1) content was evaluated with anti‐HO‐1 (cell signaling #5061), and actin was used as a loading marker.

8 PREPARATION OF MITOCHONDRIAL, NUCLEAR, AND CYTOSOLIC FRACTIONS At the end of the protocols, some livers were sectioned to obtain subcellular fractions. A piece was placed in a cold isolation buffer containing 250 mM of sucrose, 10 mM of tris(hydroxymethyl)aminomethane‐chloric acid, and 1 mM of EDTA, with a pH 7.3 to obtain mitochondria as previously described. (15) Nuclear extracts were obtained using the commercial kit NE‐PER nuclear and cytoplasmic extraction reagents (Thermo Fisher Scientific, Inc., Rockford, IL) and stored at ‐70oC. Finally, a third part was homogenized in Tris‐buffered saline (100 mM Tris‐HCl, 145 mM NaCl, pH 7.3) plus EDTA (10 mM) and centrifuged at 12,000g for 30 minutes. The cytosolic fraction was recovered after centrifuging the supernatant at 100,000g for 60 minutes and immediately frozen in liquid nitrogen. (16) Protein was measured by the Lowry method. (17)

9 Caspase 3 Activity Caspase 3 activity was measured by incubating 200 µM of the colorimetric substrate Ac‐DEVD‐pNA (Enzo Life Sciences, Inc., Farmingdale, NY) in a total volume of 100 μL that contained 75 μg of the cytosolic fraction and reaction buffer (100 mM of Tris‐HCl, 145 mN of NaCl) at 37°C for 60 minutes in 96‐microwell plates. Changes in absorbance were evaluated at 405 nm in a microplate reader (BioTek Instruments, Inc.).

10 CALPAIN‐1 ACTIVITY A total of 50 μg of cytosolic fractions, 10 mM of CaCl 2 , and 10 μM of the synthetic fluorogenic substrate for calpain H‐K (FAM)‐EVY~GMMK (DABCYL)‐OH (Merck, Darmstadt, Germany) were incubated in 200 μL of reaction buffer containing 100 mM of Tris‐HCl, 145 mM of NaCl, with a pH of 7.3 at 37°C for 60 minutes in 96‐microwell plates. To measure calcium‐independent activity, CaCl 2 was replaced with a reaction buffer. An increase in fluorescence was measured in a microplate reader (BioTek Instruments, Inc., Winooski, VT) at λ em and λ ex of 518 and 490 nm, respectively.

11 MITOCHONDRIAL FUNCTION Mitochondrial oxygen consumption was measured using a Clark‐type oxygen electrode (Yellow Springs Instruments, Yellow Springs, OH). The experiments were carried out in 2 mL of basic medium containing 125 mM of KCl, 10 mM of HEPES, (4‐[2‐hydroxyethyl]‐1‐piperazineethanesulfonic acid) and 3 mM of Pi, with a pH of 7.3. State 4 respiration was evaluated in the presence of 5 mM of glutamate plus 5 mM of malate or with 10 mM of succinate plus 1 µg/mL of rotenone; recordings were initiated with 2 mg of mitochondrial protein. State 3 respiration was measured after the addition of 200 µM of ADP. The respiratory control index (RC) was calculated as the ratio between state 3 and state 4 rates. The ATP synthesis coupled to oxygen consumption (ADP/O) ratio was calculated after addition of 200 µM of ADP and measuring the amount of oxygen consumed during state 3.

12 ENZYMATIC ACTIVITY OF MITOCHONDRIAL RESPIRATORY COMPLEXES Mitochondrial respiratory chain enzymatic activities were evaluated according to Spinazzi et al. (18) with minor modifications. Reduced nicotinamide adenine dinucleotide (NADH)‐Ubiquinone Oxidoreductase (complex I) activity was measured in the presence of dichlorophenolindophenol (DCPIP) as the terminal electron receptor of NADH oxidation. DCPIP reduction was followed spectrophotometrically at 600 nm in a medium containing 10 mM of TRIS (pH 7.4), 1 μM of NADH, 0.3 mM of KCN, and 50 μg of isolated mitochondria, previously subjected to at least 3 cycles of freezing and thawing. Reaction was initiated with 100 μM of DCPIP and decrease in absorbance was registered for 5 minutes. Separate cuvettes were prepared adding 10 μM of rotenone to determine the specific activity of complex I, which was expressed as nmol/minutes/mg of protein, using an extinction coefficient for NADH = 6.2 mM‐1 × cm‐1. Succinate dehydrogenase (complex II) activity was evaluated by following DCPIP reduction at 600 nm. The incubation medium contained 10 mM of phosphate buffer (pH 7.4), 300 μM of phenazine methosulfate (PMS), and 10 mM of sodium succinate. A total of 50 μg of protein were incubated in this medium at room temperature for 30 minutes and then the reaction was initiated with 100 μM of DCPIP. (19) The extinction coefficient used for DCPIP was 20.7 mM‐1 × cm‐1, and activity was expressed as nmol DCPIP red /minute/mg of protein. Decylubiquinol cytochrome c oxidoreductase (Complex III) activity was evaluated according to Trounce et al.(20) Mitochondria (30 μg) were incubated in 1 mL of 100 mM sucrose, 5 mM of tris(hydroxymethyl)aminomethane‐ chloric acid (pH 7.4) buffer containing 1 mM of EDTA, 50 μM of cytochrome c, and 200 μM of KCN for 10 minutes at 30oC. The reaction was started with decylubiquinol, which was freshly prepared and maintained reduced as described by Spinazzi et al.(18) Ferrycytochrome c reduction was followed in a double‐beam spectrophotometer at 550‐550 nm at 30oC for 1 minute. Separate cuvettes were prepared in the presence of 1 mg/mL of antimycin A, and activity was expressed as nmol cyt c red /minute/mg of protein. The extinction coefficient for cytochrome c is 18.5 mM‐1 × cm‐1. Cytochrome c oxidase (Complex IV) activity was measured following cytochrome c oxidation in a 100 mM of potassium phosphate buffer (pH 7). Mitochondrial protein (30 μg) was used to initiate the reaction, after reduced cytochrome c (50 μM) was added to the medium and baseline recorded. The extinction coefficient for cytochrome c ox is 18.5 mM‐1 × cm‐1.

13 OXIDATIVE STRESS MARKERS IN LIVER MITOCHONDRIA Malondialdehyde (MDA) was determined as previously described by Correa et al., (15) whereas protein carbonyl content was evaluated by their reactivity with dinitrophenyl hydrazine to form protein hydrazones. (21) Tyrosine‐nitration was measured by Western blot, using anti‐3‐nitrotyrosine monoclonal antibody 39B6 (1:200; Abcam, San Francisco, CA).

14 REDUCED GLUTATHIONE AND OXIDIZED GLUTATHIONE DETERMINATION Reduced glutathione (GSH) was evaluated fluorometrically according to Galván‐Arzate et al. (22) Mitochondrial protein (15 µg) was derivatized with 100 μl of o phthalaldehyde ([OPA] 1 mg/mL) in 2‐mL final volume and incubated for 20 minutes at room temperature. Fluorescent signal was measured at λ ex = 350 and λ em = 420 in a LS50B Luminescence Spectrophotometer (Perkin‐Elmer, Waltham, MA). Twin samples were used to evaluate oxidized glutathione (GSSG). Mitochondria were incubated for 30 minutes with 100 μl of 40 mM of ethylmaleimide to alkylate the glutathione‐free SH group and to prevent its reaction with OPA. Then the samples were mixed with 4.3 mL of 0.1N NaOH (pH 12) to allow GSSG reduction. An aliquot was withdrawn, incubated with 50 μg of OPA in 2‐mL final volume for 20 minutes, and fluorescence was measured. (23) Results were normalized per mg protein and expressed as GSH/GSSG ratio. 14.1 DATA ANALYSIS Differences in a single parameter among groups were compared by using a 1‐way analysis of variance followed by a Tukey's test for multiple comparisons. Results were expressed as mean ± standard deviation (SD). P value of <0.05 was considered to be statistically significant.

15 Results The first experiments described the effect of CDP‐choline administration on liver damage induced by I/R (Fig. 1). AST was elevated in sera from the I/R group (423.5 ± 82.3 UI/L) at the end of reperfusion. Significant reduction in AST levels was found in the I/R+citicoline group (211.7 ± 5.8 UI/L) (Fig. 1A). ALT increased to 364.7 ± 164.7 UI/L during reperfusion, whereas the values measured in the I/R + citicoline were similar to those of the control groups (Fig. 1B). Lactate levels augmented from 2 ± 0.1 mg/dL in the control group to 8.8 ± 0.4 mg/dL in the I/R group; CDP‐choline administration lowered this increase to 2.8 ± 0.5 mg/dL (Fig. 1C). No significant differences were found between control animals receiving CDP‐choline and the control group (data not shown). Figure 1 Open in figure viewer PowerPoint Effect of CDP‐choline on the activities of (A) AST, (B) ALT, and (C) lactic acid content in blood serum from animals subjected to hepatic I/R. Results represent the mean ± SEM of at least 4 independent experiments. *P < 0.05 versus control and **P < 0.05 versus I/R. Histological analysis was performed to correlate the increase in liver damage markers with alterations in the organization and architecture of the tissue (Fig. 2). Hematoxylin‐eosin staining in the I/R group showed marked dilatation of centrolobullar sinusoids and hypereosinophilia of the cytoplasm, besides necrotic areas, characterized by hyperchromasia due to nucleus condensation (Fig. 2B). In the I/R + citicoline group, the cytoplasm was more homogeneous; sinusoids dilatation decreased, and a lower vacuolation of hepatocytes was observed (Fig. 2C). Figure 2 Open in figure viewer PowerPoint Representative tissue samples from the right hepatic lobule sectioned and stained with hematoxylin‐eosin. Control, I/R, I/R + citicoline. Magnification (×40). It is known that inflammatory processes modulated by cytokines and other mediators contribute to I/R damage. Fig. 3A shows TNF‐α, IL‐6, and IL‐1β levels. No significant differences were found between groups, although a slight increase in TNF‐α was observed in blood samples from the I/R group, which coincided with a significant diminution of resolvin‐D1 (RvD1). This mediator increased in the I/R+citicoline group, whereas lipoxin A4 did not change in any of the evaluated groups (Fig. 3B). To give context to the results mentioned above, we evaluated the possible activation of NF‐κB by measuring p65 subunit translocation to the nucleus. Although this transcription factor regulates the expression of genes promoting inflammation and cell survival, we did not observe any changes among the groups (Fig. 3C). Figure 3 Open in figure viewer PowerPoint Serum levels of inflammatory cytokines and proresolving mediators after CDP‐choline treatment in rats subjected to hepatic I/R. (A) Tumor necrosis factor‐alpha TNF‐α, IL‐1β, and IL‐6. (B) Resolvin D1 and lipoxin A4. Data are the mean ± SEM of at least 7 independent experiments. (C) Representative Western blot of NF‐B p65 content in homogenates from 3 independent experiments of each experimental group. Bars show the mean ± SEM. *P < 0.05 versus control and **P < 0.05 versus I/R. On the other hand, the antioxidant effect of citicoline was evaluated by measuring intracellular ROS production, NRF2 nuclear translocation, and HO‐1 content in homogenates of the experimental groups. ROS increase in samples from I/R liver was significantly reduced in the IR+citicoline group (Fig. 4A) and NRF2 translocation to the nucleus also increased after citicoline treatment (Fig. 4B). However, no differences were observed in HO‐1 content (Fig. 4C). This result indicates that although citicoline activates NRF2 signaling, the augment in the antioxidant machinery might be detected and productive at longer reperfusion times. Figure 4 Open in figure viewer PowerPoint Oxidative stress markers in reperfused liver treated with citicoline. (A) Intracellular ROS content; (B) NRF2 levels in nuclear fractions; and (C) HO‐1 levels in liver homogenates. Data are the mean ± SEM of 3 independent experiments. *P < 0.05 versus control and **P < 0.05 versus I/R. As ROS production is involved in several types of cell death, we evaluated the activities of caspase 3 and of the calcium‐dependent protease calpain‐1 as reliable markers of apoptosis and necrosis. Our results showed that both proteases increase their activity in reperfused liver and that CDP‐choline diminishes significantly caspase 3 (Fig. 5A) and to a minor extent, calpain‐1 activity (Fig. 5B). Figure 5 Open in figure viewer PowerPoint Apoptotic and necrotic cell death markers in reperfused liver treated with citicoline. (A) Caspase 3 and (B) calpain‐1 activities were measured in the cytosolic fraction of the different groups. Data are the mean ± SEM of 3 independent experiments. *P < 0.05 versus control and **P < 0.05 versus I/R. Reperfusion damage is intimately associated with mitochondrial dysfunction and with ROS generation; therefore, we measured oxidative phosphorylation, the enzymatic activity of the respiratory complexes of the electron transport chain, and oxidative/nitrosative stress markers. Table 1 shows oxygen consumption in liver mitochondrial preparations obtained from the different experimental conditions. Basal respiration (state 4) with NADH‐linked substrates was similar in all groups, whereas ADP‐stimulated respiration, RC, and ADP/O diminished significantly in mitochondria from the I/R as compared with the control and the I/R+citicoline group, ie, 17.4 ± 1.5 versus 61.4 ± 21.8 and versus 66.7 ± 6.5 ngAtO/minutes/mg, 1.2 ± 0.2 versus 3 ± 1.5 and versus 4.4 ± 1.3, and 1.3 ± 0.5 versus 3.9 ± 0.2 and versus 3.3 ± 0.3, respectively. The same effect was observed when using succinate + rotenone, which pointed out to complex I and/or to downstream‐succinate dehydrogenase complexes as main targets of I/R hepatic damage. The enzymatic activities of complex I (Fig. 6A) and complex II (Fig. 6B) did not change in any of the experimental groups. complex III activity (Fig. 6C) decreased in I/R as compared with control and I/R + citicoline (88.5 ± 14 nmol NADH reduced/minutes/mg versus 2978 ± 74 and 261 ± 36 nmol NADH reduced/minutes/mg, respectively). Also, complex IV was deregulated in I/R, diminishing its activity from 601 ± 72 in control mitochondria to 315 ± 176 nmol cytochrome c oxidized/minutes/mg. The I/R + citicoline group recovered complex IV activity by 96% (Fig. 6D). Table 1. Oxygen Consumption in Isolated Mitochondria From Control, I/R, and I/R + Citicoline Livers State 4 ngAtO/minutes/mg State 3 ngAtO/minutes/mg RC ADP/O NADH‐linked substrates Control 20.1 ± 6.1 61.4 ± 21.8 3.0 ± 1.5 3.9 ± 0.2 I/R 14.4 ± 1.5 17.4 ± 1.5* 1.2 ± 0.2* 1.3 ± 0.5* I/R + citicoline 13.6 ± 3.5 66.7 ± 6.5** 4.4 ± 1.3** 3.3 ± 0.3** Succinate + rotenone Control 19.5 ± 3.1 92.9 ± 9 4.7 ± 1.1 2.9 ± 0.4 I/R 27.7 ± 1.8* 54.6 ± 08* 1.9 ± 0.4* 1.3 ± 0.1* I/R + citicoline 25.2 ± 2.7** 151.1 ± 27** 5.8 ± 1.1** 2.6 ± 0.2** Figure 6 Open in figure viewer PowerPoint Enzymatic activities of the respiratory chain complexes of isolated mitochondria from I/R livers treated with CDP‐choline. (A) NADH dehydrogenase (complex I); (B) succinate dehydrogenase (complex II); (C) ubiquinol cytochrome c oxidoreductase (complex III); (D) cytochrome c oxidase (complex IV). Results represent the mean ± SD of at least 3 different mitochondrial preparations from independent experiments. *P < 0.05 versus control and **P < 0.05 versus I/R. Oxidative stress markers, GSH levels, and tyrosine nitration in mitochondrial fractions were measured as indicators of oxidative/nitroxidative stress. Lipid peroxidation increased significantly in I/R mitochondria (1.36 ± 0.2 nmol MDA/mg) in comparison with the control group (0.62 ± 0.14 nmol MDA/mg) and CDP‐choline mitochondria (0.87 ± 0.2 nmol/mg; Fig. 7A). Reduced GSH values in I/R mitochondria (2064.9 ± 13.7 µg/g protein) were significantly lower than in control (2390.3 ± 154.4 µg/g protein, P < 0.05) and in I/R + citicoline mitochondria (2286.7 ± 111.9 µg/g protein, P < 0.05); GSSG levels increased in I/R as compared with control group mitochondria (381.1 ± 10.6 µg/g protein versus 331.5 ± 17 µg/g protein, P < 0.05). GSH/GSSG ratio decreased in I/R mitochondria and was recovered to control levels in mitochondria from the I/R + citicoline group (Fig. 7B). Protein carbonylation increased in I/R mitochondria and was practically absent in the other groups (Fig. 7C); pro‐oxidant events depicted by 3‐nitrotyrosine labeling were higher in I/R mitochondria and diminished in the I/R+citicoline group (Fig. 7D). Figure 7 Open in figure viewer PowerPoint Oxidative/nitrosative markers in isolated mitochondria from I/R livers treated with CDP‐choline. (A) MDA levels and (B) GSH/GSSG ratio. Each value represents the mean ± SD of at least 3 separate experiments. *P < 0.05 versus control and **P < 0.05 versus I/R. (C) Carbonylation and (D) 3‐nitrotyrosine content evaluated by immunodetection (lower panels). Representative image of at least 3 different experiments along with its corresponding Coomasie blue‐stained gel presented as loading marker. In the carbonylation assay, each sample included a negative control (‐), without DNPH. Rows in which DPH was added to the assay are shown as (+).

16 Discussion Although different surgical and pharmacological strategies have been tested to inhibit hepatic reperfusion damage in experimental models, their routine use in the clinic is not recommended due to the complexity of the injury mechanisms activated during reperfusion. The benefit of citicoline administration posterior to ischemia is relevant because of the obvious clinical application in reperfused organs. However, specifically in liver transplantation, pharmacological preconditioning is used as a promissory strategy to protect the graft against I/R injury during the donation procedure, explaining in part that most of the protective published strategies against I/R in liver are evoked in a preconditioning scheme. For example, ischemic preconditioning activates mechanisms that inhibit apoptosis, (24) but might cause direct trauma to major vascular structures, not to mention that many donor livers are obtained from deceased patients. (25) On the other hand, the alternative use of remote ischemic conditioning did not show short‐term protective effects against reperfusion injury in orthotopic liver transplantation, (26, 27) whereas pharmacological interventions with methylprednisolone, trimetazidine, dextrose, and ulinastatin have not been recommended for routine use, even though it was reported that such agents decreased liver parenchymal injury. (28) Other agents such as antioxidants, adenosine agonists, and nitric oxide donors might have potential utility in hepatic transplantation. (29) Citicoline exerts hepatoprotective effects by preventing serum elevation of AST and ALT. It preserves tissue architecture and decreases the levels of lactic acid by reducing oxidative stress in a reperfused liver. It promotes the increase in proresolving lipid mediator RvD1 content, although it has no significant effect on TNF‐α, IL‐1β, and IL‐6 levels. In this sense, a recent report showed that RvD1 has similar protective effects as we found for citicoline in a model of hepatic I/R injury in rats. (25) The association between the molecular pathways activated by CDP‐choline and the formation of these resolving molecules is not known. A possible link might be related to the crucial role that these lipid mediators seem to play in ROS reduction, and recent data show that the levels of RvD1 and of its precursor docosahexaenoic acid (DHA) decrease in patients with acutely symptomatic carotid disease. (30) It is tempting to speculate that either the precursors or the enzymes involved in RvD1 biosynthesis are compromised during reperfusion in a ROS sensitive manner. The observed increase in RvD1 deserves further investigation. We would continue studying this issue using a different experimental setting, as the observed increase in RvD1 did not correlate with decrease in proinflammatory cytokines. Findings in our work are clearly related to the antioxidant properties of citicoline. The question how CDP‐choline affect ROS production in vivo, is under debate, but a hypothesis is that ROS reduction might result from both the stabilization of membranes associated to the synthesis of phosphatidylcholine (PC) and to the intrinsic ROS scavenger properties of this phospholipid. (31) It is widely accepted that complex I and complex III are the main contributors to superoxide production during I/R and that ROS‐induced damage to the electron transfer chain components potentiates the injury cascade. (32) However, the relative importance of the respiratory complexes, as well as of other potential sources in ROS production during reperfusion, are still debated. A recent report suggests that complex III might be a main ROS generator during reperfusion, providing that succinate‐driven reverse electron transfer ceases after the opening of the permeability transition pore (PTP). (33) Other reports suggest that complex IV dysfunction contributes to ROS production because of the accumulation of reduced intermediates of the electron transport chain. Another explanation is that ROS‐linked phosphorylation of complex IV subunits promotes disruption of the functional assembly of complexes known as “respirosome,” perpetuating a vicious cycle of ROS production. (34) In this work, we show that DCP‐choline maintains oxidative phosphorylation and preserves the activity of respiratory complex III and complex IV. Our observations, along with a previous demonstration that citicoline inhibits the opening of the PTP in reperfused hearts, (11) restore the transmembrane potential in hyperglycemic rats,(35) and maintain the activity of aconitase, an Fe‐S cluster enzyme considered a reliable marker of oxidant damage in mitochondria, (12) supporting our assumption that its antioxidant effect stabilizes mitochondrial function. However, we did not find changes in mitochondrial GSH levels. This result contrasts with the assumption that choline liberated from citicoline, once converted to betaine, participates in the synthesis of both S‐adenosyl‐L‐methionine and S‐adenosyl‐L‐homocysteine that are metabolized to GSH. A possible explanation is the observation that CPD‐choline induces other mechanisms for protection before it has an effect on glutathione redox status in cerebral ischemia. (36) In conclusion, the administration of CDP‐choline prior to I/R reduces liver damage, mainly due to its antioxidant properties. These findings suggest a potential use of CDP‐choline in hepatic ischemia (trauma surgery, hypovolemic shock, resections, transplantation, and so on) in order to mitigate the damage associated with the I/R process.

17 Funding: This research was partially funded by the National Science and Technology Council (CONACYT) Grant 181593 to Francisco Correa.

18 Potential conflict of interest: Nothing to report.

Acknowledgment We thank Dr. A. Hernández from CINVESTAV for generously donating the anti‐actin antibody.