{'fraction_non_alphanumeric': 0.04494275711987889, 'fraction_numerical': 0.02403254801778787, 'mean_word_length': 4.828232699200441, 'pattern_counts': {'":': 0, '<': 7, '<?xml version=': 0, '>': 0, 'https://': 0, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 0, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
Treatment with dimethyl fumarate ameliorates liver ischemia/reperfusion injury

AIM To investigate the hypothesis that treatment with dimethyl fumarate (DMF) may ameliorate liver ischemia/reperfusion injury (I/RI). METHODS Rats were divided into 3 groups: sham, control (CTL), and DMF. DMF (25 mg/kg, twice/d) was orally administered for 2 d before the procedure. The CTL and DMF rats were subjected to ischemia for 1 h and reperfusion for 2 h. The serum alanine aminotransferase (ALT) and malondialdehyde (MDA) levels, adenosine triphosphate (ATP), NO × metabolites, anti-oxidant enzyme expression level, anti-inflammatory effect, and anti-apoptotic effect were determined. RESULTS Histological tissue damage was significantly reduced in the DMF group (Suzuki scores: sham: 0 ± 0; CTL: 9.3 ± 0.5; DMF: 2.5 ± 1.2; sham vs CTL, P < 0.0001; CTL vs DMF, P < 0.0001). This effect was associated with significantly lower serum ALT (DMF 5026 ± 2305 U/L vs CTL 10592 ± 1152 U/L, P = 0.04) and MDA (DMF 18.2 ± 1.4 μmol/L vs CTL 26.0 ± 1.0 μmol/L, P = 0.0009). DMF effectively improved the ATP content (DMF 20.3 ± 0.4 nmol/mg vs CTL 18.3 ± 0.6 nmol/mg, P = 0.02), myeloperoxidase activity (DMF 7.8 ± 0.4 mU/mL vs CTL 6.0 ± 0.5 mU/mL, P = 0.01) and level of endothelial nitric oxide synthase expression (DMF 0.38 ± 0.05-fold vs 0.17 ± 0.06-fold, P = 0.02). The higher expression levels of anti-oxidant enzymes (catalase and glutamate-cysteine ligase modifier subunit and lower levels of key inflammatory mediators (nuclear factor-kappa B and cyclooxygenase-2 were confirmed in the DMF group. CONCLUSION DMF improved the liver function and the anti-oxidant and inflammation status following I/RI. Treatment with DMF could be a promising strategy in patients with liver I/RI.


INTRODUCTION
Liver ischemia/reperfusion injury (I/RI) is a common pathologic process caused by many clinical settings, such as liver resection, liver transplantation, hypo volemic shock, and trauma [1] . Temporary clamping of the portal triad, which is a common strategy during liver surgery, produces liver I/RI [2] that may result in postoperative liver dysfunction [3] . I/R generates reactive oxygen species (ROS), which can damage lipids, proteins and tissues and lead to local inflammatory responses, endothelial and Kupffer cell activation, cytokine/chemokine release, and cell apoptosis [4] . The I/Rinduced oxidative stress and tissue damage involve multiple cell signaling pathways that result in liver failure [5] . BG12 has been approved by the Food and Drug Administration for the treatment of patients with multiple sclerosis, in whom it reduces disease activity and the progression of relapsingremitting multiple sclerosis [6] . Dimethyl fumarate (DMF), which has a mild side effect profile, has been available for medical use for over twenty years, despite an unclear mechanism of action. Methyl fumarates were first investigated for their antiproliferative and antioxidant effects; however, they quickly became repurposed for use as potent antipsoriatic drugs in Europe [7] . We have investigated the usefulness of DMF and reported its effects on acute [8] and chronic pancreatitis [9] . However, to our knowledge, the effects of DMF on liver I/RI have not been investigated. The aim of the present study was to test the hypothesis that treatment with the potent antioxidant DMF may attenuate the severity of liver I/RI in experimental animals by upregulating cellular anti-oxidant and anti-inflammatory machinery in liver tissue.

Animals
SpragueDawley male rats weighing 230250 g were purchased from Charles River (Wilmington, MA, United States). The rats were free of all pathogens and housed under standard conditions (room tempera ture: 22 ℃: humidity: 50% ± 5%, 12:12 h light/dark cycle). The study was approved by the Institutional Animal Care and Use Committee of the University of California, Irvine.

Experimental design
The rats were randomly divided into 3 experimental groups: (1) the sham group (n = 6) was subjected to exposure of the hepatic artery, portal vein, and bile duct region but no I/RI; (2) the control (CTL) group (n = 7) was subjected to 1 hour of ischemia followed by 2 h of reperfusion. Finally; and (3) the DMF group (n = 6) received DMF (25 mg/kg, twice/d) by oral gavage 2 d prior to the operation.

Surgical procedure
Under general anesthesia using isoflurane, a midline incision was made. The left and median portal triads were occluded by a microvascular clamp to achieve 70% liver ischemia. After 1 h of clamping, the clamp was removed, and restored hepatic blood flow was confirmed visually prior to wound closure. During the surgery, the body temperature was maintained at approximately 37.5 ℃ with a heating blanket. Two hours post clamp removal (reperfusion time), blood samples and ischemic liver lobes were collected under deep general anesthesia for analysis, and the animals were sacrificed [10] .

Histopathological analysis
Histopathological analysis was performed as previously described [11] . The severity of I/RI was blindly graded using the modified Suzuki criteria [12] .

Assessment of hepatic apoptosis
Apoptosis was quantified by the terminal de oxynucleotidyl transferase (TdT)mediated dUTP nick end labeling (TUNEL) technique (Millipore, Bedford, MA, United States). For each section, 20 random fields were examined by confocal microscopy (20 × objective). An apoptotic index (i.e., the number of nuclei labeled by the TUNEL method/the total number of nuclei) was calculated [13] .

Measurement of serum alanine aminotransferase and malondialdehyde
Serum alanine aminotransferase (ALT) levels were determined to assess the liver function using commer cial kits (BioVision, Milpitas, CA, United States) according to the manufacturer's instructions. The serum malondialdehyde (MDA) formation assay was performed using a thiobarbituric acid reactive substances (TBARS) assay kit (Cayman, Ann Arbor, MI, United States) according to the manufacturer's instructions.

Adenosine triphosphate determination
The adenosine triphosphate (ATP) levels of liver tissue were determined using a colorimetric/fluorometric assay kit (Bio Vision, Milpitas, CA, United States) according to the manufacturer's instructions. We used 20 mg of liver tissue for the assay and calculated the ATP content. Protein concentration was performed with a BioRad assay kit (BioRad, Richmond, CA, United States) based on the Lowry method [14] .

Myeloperoxidase assay
The presence of myeloperoxidase (MPO) was used as an index of neutrophil accumulation in the liver [15] and was determined using an MPO colorimetric assay kit (BioVision, Milpitas, CA, United States) according to the manufacturer's instructions.

Protein extraction and Western blots
Western blots were performed as previously des cribed [11] .

Measurement of serum inflammatory mediators
The levels of serum inflammatory mediators were determined using a standard rat cytokine kit (Ray Biotech, Norcross, GA, United States) according to the manufacturer's instructions. Fifteen rat cytokines/ chemokines were analyzed.

Statistical analyses
All the results are presented as the mean ± SD. Comparisons between two groups were performed with Student's ttest or MannWhitney's U test, as appropriate, using Stat ViewJ 5.0 software (SAS, Cary, NC, United States). Statistical significance was defined as a P value less than 0.05.

Histopathological changes
The histopathological findings are given in Figure 1A and B. The liver tissue was histologically normal in the sham group ( Figure 1A). In contrast, substantial intracellular vacuolization, sinusoidal dilatation, congestion, and focal necrosis of the liver parenchyma were observed in the CTL group ( Figure 1A). These changes were notably reduced in the DMF treatment group ( Figure 1A). The Suzuki scores of the groups differed significantly (CTL 9.3 ± 0.5 vs DMF 2.5 ± 1.2, P < 0.0001; Figure 1B), indicating that pretreatment with DMF ameliorated the I/RIinduced histological changes.

Effect of DMF on hepatocellular apoptosis
Representative confocal microscopy images of hepatocytelabeling TUNEL (green fluorescence) are cellular ATP levels. DMFtreated livers demonstrated significantly higher ATP contents than those in the CTL group (20.3 ± 0.4 nmol/mg vs 18.3 ± 0.6 nmol/mg, respectively, P = 0.02; Figure 2C). Neutrophil infiltration in the DMF-treated group, as analyzed by MPO activity, was significantly lower (7.8 ± 0.4 mU/mL) than that in the CTL group (6.0 ± 0.5 mU/mL) ( Figure 2D, P = 0.01), indicating that DMF treatment decreased neutrophil migration into the hepatocytes. Figure 3BD shows the protein expression of nuclear factor erythroid 2related factor 1 (Nrf2) pathway target antioxidant enzymes. CAT and GCLM expression levels in the liver were significantly enhanced by DMF treatment compared with those in the CTL group (CAT: 1.50 ± 0.18fold vs 0.93 ± 0.13fold, respectively, P = 0.03, Figure 3B; GCLM: 0.42 ± 0.07fold vs 0.22 ± 0.05fold, respectively, P = 0.04, Figure 3C). Additionally, GCLC protein expression was higher in the DMF group than in the CTL group (CTL: 0.48 ± 0.08fold vs DMF 0.77 ± 0.13fold, P = 0.10; Figure 3D). The protein expression levels of GPx, HO-1, SOD and NQO-1 did not differ significantly between the two groups (GPx: P = 0.22, HO1: P = shown in Figure 1C and D. No TUNELpositive apoptotic cells were found in the sham group. Although the number of TUNELpositive apoptotic cells increased dramatically in the CTL group, only a few positive cells were detected in the DMF group. The apoptotic index in the DMF group (1.3 ± 0.9) was significantly lower than that of the CTL group (17.2 ± 3.6) ( Figure 1D; P = 0.002). These results suggest that DMF protected the liver from hepatocellular apoptosis.

Effect of DMF on serum ALT and MDA
To further confirm the protective effect of DMF on hepatic I/R injury, the level of ALT, a hepatocyte damage marker, was measured. The ALT levels were significantly lower in the DMF group than in the CTL group (5026 ± 2305 U/L vs 10592 ± 1152 U/L, respectively, P = 0.04; Figure 2A). To determine the cellular damage under oxidative stress after I/RI, the serum MDA level, which is a valid biochemical marker of lipid peroxidation, was measured. The serum MDA levels were significantly lower in the DMF group (DMF 18.2 ± 1.4 µmol/L vs CTL 26.0 ± 1.0 µmol/L, P = 0.0009; Figure 2B).

Effect of DMF on ATP content and MPO activity in hepatocytes
To assess the ability of hepatocytes, we studied the

Effect of DMF on iNOS and eNOS expression
To determine whether DMF exerts its protective role through NOmediated production, we detected the levels of NO synthases (eNOS and iNOS). The protein expression of eNOS was enhanced by DMF administration relative to that in the CTL group (0.38 ± 0.05fold vs 0.17 ± 0.06fold, respectively, P = 0.02; Figure 3F). However, no significant difference in iNOS expression was found between the two groups (CTL: 0.027 ± 0.12fold vs DMF 0.027 ± 0.009fold, P = 0.97).

Effect of DMF on NF-κ B and COX-2 expression
To elucidate the effect of DMF on inflammation, The levels of serum alanine aminotransferase (ALT) (A) and malondialdehyde (MDA) (B) and liver adenosine triphosphate (ATP) (C) and myeloperoxidase (MPO) (C) in the liver tissue were determined. Serum ALT and MDA in the DMF group were significantly lower than in the CTL group (ALT: P < 0.05, MDA: P < 0.001; data represent the mean ± SD). Liver ATP in the DMF group was significantly higher than in the CTL group (P < 0.05; data represent the mean ± SD). Liver MPO in the DMF group was significantly lower than in the CTL group (P < 0.05; data represent the mean ± SD). DMF: Dimethyl fumarate; CTL: Control.  Figure 3B; GCLM: P = 0.04, Figure 3C). The protein expression of eNOS was also enhanced by DMF administration compared to that of the CTL group (P = 0.02, Figure 3F). Furthermore, DMF decreased the levels of NF-κB and COX-2 expression in the liver (NF-κB, P = 0.01, Figure 3G; COX-2, P = 0.007, Figure 3H). The protein expression levels of western blot analysis of NFκB and COX2 proteins were performed. Both the expression levels of both NF κB and COX2 decreased significantly in DMFtreated animals' liver tissues (NFκB: CTL: 0.75 ± 0.08fold vs DMF 0.44 ± 0.04fold, P = 0.01, Figure 3G; COX2: CTL: 1.64 ± 0.26fold vs DMF 0.48 ± 0.19fold, P = 0.007; Figure 3H) compared to those of the CTL group.

Effect of DMF on the production of cytokines and chemokines
We analyzed direct changes in serum cytokine and chemokine production in response to I/RI. DMF administration significantly decreased the serum levels of many inflammatory mediators relative to those in the CTL group ( Figure 4). Significant reductions of the following were observed in the DMFtreated group: cluster of differentiation 86 (CD86), cytokine induced neutrophil chemoattractant2α (CINC2α), CINC3, ciliary neurotrophic factor (CNTF), Fas ligand, interleukin-6 (IL-6), IL-10, macrophage inflammatory protein3α (MIP3α), plateletderived growth factor AA (PDGFAA), tissue inhibitor of metalloproteinase 1 (TIMP1), tumor necrosis factor α (TNFα) and vascular endothelial growth factor (VEGF).

DISCUSSION
In this study, we evaluated the effects of DMF, a potent antioxidant, on a rodent hepatic I/RI model. Our data demonstrated that the pharmacological supplementation of DMF resulted in a significant increase of antioxidant enzymes and a substantial reduction of inflammatory mediators, leading to significant amelioration of the liver damage caused by I/R. Many studies have shown that oxidative stress plays a crucial role in the pathogenesis of I/RI [16] . MDA is an indicator of lipid peroxidation and cellular damage under oxidative stress, and the elevation of MDA is associated with liver I/RI [17] . We observed that the MDA levels were significantly decreased in the DMF treated rats, suggesting lower I/RIinduced oxidative stress in the DMFtreated liver. ROS can be contained by endogenous antioxidant enzymes, such as SOD, CAT and GPx [1] . ROSinduced lipid peroxidation can be blocked by these antioxidative enzymes. CAT is an antioxidant enzyme that converts H2O2 to water and thereby prevents the transformation of H2O2 into highly toxic hydroxyl radicals. Previous reports showed that CAT has beneficial effects toward the end of the ischemic period and that its activities are decreased after I/RI [18] . GSH protects cells from oxidative injury by scavenging radicals and reducing lipid peroxidation products. GSH is synthesized by GSH synthetase and glutamatecysteine ligase (GCL), which is a heterodimer composed of GCLM and GCLC subunits. Both GCLM and GCLC are upregulated for GSH synthesis by oxidants, providing a protective mechanism against oxidative stress [19] . We observed that DMF increased CAT, GCLM and GCLC expression, which may explain the observed decrease in ROS related liver damage.
The accumulation of neutrophils, as confirmed by measuring the MPO activity, was significantly reduced by the administration of DMF. Neutrophils are believed to mediate hepatic damage caused by the production of ROS and reactive halogen species. These toxic products can directly damage hepatocytes and endothelial cells and induce other inflammatory mediators [20] . The initial phase of injury, which occurs within 2 h after reperfusion, is characterized by Kupffer cellinduced oxidant stress. Activated Kupffer cells produce and secrete proinflammatory cytokines, including TNFα, IL6, COX2, and iNOS [3] . In this study, we observed significantly decreased TNF-α, IL6 and COX2 protein expression, indicating that DMF may inhibit the activation of Kupffer cells by enhancing antioxidant effects. We also found a significant decrease in the expression level of NFκB 2 h after reperfusion in the DMFpretreated rats. A previous report showed that DMF inhibited proinflammatory cytokine production and NFκB signaling by inhibiting nuclear translocation [21] . Previous reports also demonstrated that DMF has a cardioprotective effect during I/RI through the Nrf2 and NFκB pathway [22] , strongly supporting our results. Moreover, recent studies have demonstrated that NFκB is involved in the regulation of COX2 and iNOS expression [3] . Our data showed that DMF can inhibit NFκB expression and suppress COX2 expression but does not affect iNOS expression.
We found that twelve plasma cytokines and chemokines were decreased by DMF administration. CINC and MIP are members of the CXC chemokine family, are potent chemotactic factors for neutrophils, and contribute to neutrophil recruitment in in flammation [23] . MIP3α is expressed mainly in the liver and is produced by dendritic cells and macrophages after an inflammatory response, resulting in the recruitment of activated T cells into the liver [24] . CD86 was also reported to be involved as the costimulatory signaling molecule for antigenpresenting cells (APCs) in Tcell activation [25] . The upregulation of CD86 in sinusoidal endothelial cells contributes to liver injury after warm I/R [26] . VEGF is a wellestablished angiogenesis factor and has been recently found to have potent pro-inflammatory properties in the early period after transplant [27] . PDGFAA is an important mediator of connective tissue expansion during liver fibrosis [28] . TIMP-1 is significantly increased in patients with fulminant hepatitis, reflecting severe hepatic inflammation [29] . Fas ligand is expressed on infiltrating immune cells and induces apoptosis of hepatocytes, leading to hepatocyte injury [30] . CNTF, a member of the IL6 superfamily, is involved in fever induction and a hepatic acutephase protein response [31] . Taken together, DMF suppressed plasma cytokines and chemokines related to apoptosis, inflammation and fibrosis. Further studies will be conducted to explore this Nrf2 anti-inflammatory effect.
The histological investigation suggested that I/RI caused severe pathological alterations in the liver. Pretreatment with DMF ameliorated I/RIinduced histological changes, and this effect was attributed to its antioxidant efficacy. The observed endothelial dysfunction could be a consequence of decreased synthesis or bioavailability of NO [32] . A previous report showed that the observed endothelial dysfunction is characterized by a marked reduction in NO availability and decreased expression and phosphorylation of eNOS [17] . Our results showed that DMF enhanced eNOS protein expression, suggesting that DMF pretreatment may protect endothelial function from I/RI. Furthermore, cellular apoptosis consumes large amounts of nicotinamide adenine dinucleotide (NAD + ), and the process to resynthesize NAD + decreases the level of cellular ATP [33] . Our data showed that the ATP content was increased in the DMF group compared to that in the CTL group, suggesting that DMF has an antiapoptotic effect. In fact, the apoptotic index in the DMF group was significantly lower than that in the CTL group. A previous report showed that liver I/RI mainly occurred in the form of apoptosis [13] . Excessive oxygen free radicals could directly react with unsaturated fatty acids on the surfaces of mitochondrial membranes, leading to the destruction of their structure and function. Large amounts of cytochrome C and other apoptosispromoting substances were released, triggering cell apoptosis. In this study, we showed that DMF may have inhibited the apoptotic pathway mediated by mitochondria.
In conclusion, our biochemical and histological findings indicated that DMF pretreatment was very effective in preventing tissue damage in liver I/RI. First, the burden of oxidative stress, which increases during I/RI, was alleviated by DMF, which increased the expression of antioxidant enzymes and decreased the level of NFκB. Second, DMF improved the recovery of endothelial dysfunction and the production of ATP in hepatocytes. Other protective effects against I/RI on hepatocytes were clearly demonstrated by DMF administration, including improvements in the ALT level and the histopathological features. Future studies are needed to explore the efficacy of DMF in the management of patients with liver interventions.

Background
Liver ischemia/reperfusion injury (I/RI). generates reactive oxygen species, which can damage lipids, proteins and tissues and lead to local inflammatory responses, endothelial and Kupffer cell activation, cytokine/chemokine release, and cell apoptosis. The I/R-induced oxidative stress and tissue damage involve multiple cell signaling pathways that result in liver failure.

Research frontiers
To knowledge, the effects of dimethyl fumarate (DMF) on liver I/RI have not been investigated.

Innovations and breakthroughs
DMF treatment could be a promising strategy to improve clinical outcome in patients with liver I/RI.

Applications
First, the burden of oxidative stress, which increases during I/RI, was alleviated by DMF, which increased the expression of anti-oxidant enzymes and decreased the level of NF-κB. Second, DMF improved the recovery of endothelial dysfunction and the production of adenosine triphosphate in hepatocytes.