research article endogenous n-3 fatty acids alleviate...

Research ArticleEndogenous n-3 Fatty Acids AlleviateCarbon-Tetrachloride-Induced Acute Liver Injury inFat-1 Transgenic Mice

Ruibing Feng1 Meng Wang1 Chunyan Yan2 Peng Li1 Meiwan Chen1

Chengwei He1 and Jian-Bo Wan1

1State Key Laboratory of Quality Research in Chinese Medicine Institute of Chinese Medical Sciences University of Macau Macau2College of Pharmacy Guangdong Pharmaceutical University Guangzhou 510006 China

Correspondence should be addressed to Chengwei He chengweiheumacmo and Jian-Bo Wan jbwanumacmo

Received 6 September 2016 Accepted 11 October 2016

Academic Editor Ravirajsinh Jadeja

Copyright copy 2016 Ruibing Feng et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

n-3 polyunsaturated fatty acids (PUFAs) are beneficial for numerous models of liver diseasesThe probable protective effects of n-3PUFA against carbon-tetrachloride- (CCl4-) induced acute liver injury were evaluated in a fat-1 transgenic mouse that synthesizesendogenous n-3 from n-6 PUFA Fat-1 mice and their WT littermates were fed a modified AIN93 diet containing 10 corn oiland were injected intraperitoneally with a single dose of CCl4 or vehicle CCl4 challenge caused severe liver injury in WT miceas indicated by serum parameters and histopathological changes which were remarkably ameliorated in fat-1 mice Endogenousn-3 PUFA decreased the elevation of oxidative stress induced by CCl4 challenge which might be attributed to the activation ofNrf2keap1 pathway Additionally endogenous n-3 PUFA reduces hepatocyte apoptosis via suppressing MAPK pathway Thesefindings indicate that n-3 PUFA has potent protective effects against acute liver injury induced by CCl4 in mice suggesting that n-3PUFA can be used for the prevention and treatment of liver injury

1 Introduction

Liver is a vital organ that has extensive synthetic metabolicand detoxifying functions [1] This tissue is also a maintarget that is subject to acute or chronic injury induced by avariety of drugs or xenobiotics such as alcohol heavymetalsand carbon tetrachloride (CCl4) [2] CCl4 an analogue ofhuman hepatotoxin has been widely used in in vitro and invivo models to induce liver injury [3] CCl4 is oxidized bycytochrome P4502E1 (CYP2E1) in the liver to generate thehighly reactive species such as trichloromethyl (∙CCl

3) and

peroxy trichloromethyl (∙OOCCl3) radicals [4] which can

trigger oxidative stress lipid peroxidation and hepatocyteapoptosis leading ultimately to hepatotoxicity [5]

Oxidative stress is mainly responsible for the pathogene-sis of CCl4-induced liver injury which can disturb the redoxhomeostasis and elevate the excessive production of reac-tive oxygen species (ROS) [6] Antioxidant defense system

including nonenzymatic antioxidants and enzymatic antiox-idants contributes to protecting the liver against oxidativestress in living organismsThe expressions of these antioxida-tive enzymes are regulated by a redox-sensitive transcriptionfactor nuclear factor-erythroid 2-related factor-2 (Nrf2) andits downstream proteins including heme oxygenase-1 (HO-1) glutamate cysteine ligase (GCL) and NAD(P)Hquinoneoxidoreductase-1 (NQO1) [7 8] Oxidative stress also elevatescytochrome C in the cytoplasm which is released fromthe mitochondria which induces the activation of caspasecascades eventually leading to hepatocyte apoptosis

Growing evidence indicates that n-3 polyunsaturatedfatty acids (PUFAs) mainly 120572-linolenic acid (ALA) eicos-apentaenoic acid (EPA) and docosahexaenoic acid (DHA)exhibit profoundly therapeutic efficacy in several modelsof liver disease including nonalcoholic liver disease [9]parenteral nutrition-associated liver disease [10] alcohol-induced liver injury [11] hepatic steatosis [12 13] and

Hindawi Publishing CorporationOxidative Medicine and Cellular LongevityVolume 2016 Article ID 7962948 12 pageshttpdxdoiorg10115520167962948

2 Oxidative Medicine and Cellular Longevity

D-galactosaminelipopolysaccharide-induced hepatitis [14]However the impacts of n-3 PUFA on CCl4-induced liverinjury have not been sufficiently addressed The fat-1 trans-genic mouse was genetically modified to express a fat-1 genethat encodes n-3 PUFA desaturase [15 16] This enzyme canendogenously convert n-6 PUFA to n-3 PUFA in mammalsleading to higher n-3 PUFA level in tissues from fat-1 micecompared to the wild-type (WT) littermates when fed thesame diet rich in n-6 PUFA Thus fat-1 mice are a well-established animal model to investigate the role of n-3 PUFAin CCl4-induced liver injury Therefore the aims of currentstudy are to evaluate the probable effects of n-3 PUFA againstCCl4-induced acute liver injury and to elucidate the potentialmolecular mechanisms underlying this action

2 Materials and Methods

21 Animals and Treatments Fat-1 transgenic mice with agenetic background of C57BL6 were provided by Dr JingX Kangrsquos lab at Massachusetts General Hospital (BostonMA USA) Male heterozygous fat-1 mice were crossed withC57BL6 female mice to yield heterozygous fat-1 and WToffspringThe fat-1 phenotype of each offspringwas identifiedby the analysis of total lipids from mouse tail by using gaschromatography-mass spectrometry (GC-MS) The femalefat-1-positive and WT littermates were maintained in a spe-cific pathogen-free room at the Experimental Animal CenterGuangdong Pharmaceutical University Mice were fed an n-6 PUFA-rich but n-3 PUFA-deficient diet (a modified AIN93containing 10 corn oil) which contains 20 protein 58carbohydrate and 22 fat (TROPHIC Animal Feed High-Tech Co Ltd Nantong China) Mice (10ndash12 weeks old) weredivided into three groups (119899 = 10) that is WT controlWTCCl4 and fat-1CCl4 groupsThemice in CCl4 challengegroups were injected intraperitoneally with 10mLkg CCl4(02 dissolved in olive oil) [17] whileWT controls receivedan equal volume olive oil (ip) After 24 h of CCl4 or vehicletreatment all mice were anesthetized by an injection (ip100mgkg) with sodium pentobarbital Blood samples andliver tissues were immediately collected The animal exper-imental protocols (ICMS-AEC-2015-028) were performedaccording to the Guide to Animal Use and Care of the Uni-versity of Macau and were approved by the ethics committee

22 Analysis of Fatty Acid Composition in the Liver Fattyacid profile was analyzed according to a simplified methodby using GC-MS as described previously [18 19] In briefapproximately 10mgof liver tissuewas ground in liquid nitro-gen and methylated with 15mL of 14 boron trifluoride-methanol reagent (Sigma-Aldrich) and 15mL of hexane at100∘C for 1 h After cooling fatty acid methyl esters (FAME)were extracted in upper hexane layer GC-MS analysis wasconducted on a Thermo Fisher Scientific ISQ Series Sin-gle Quadrupole GC-MS system equipped with a TriPlusRSH autosampler (Thermo Fisher Waltham MA USA)Separation of FAME was achieved on an Omegawax 250fused silica capillary column (30m times 025mm id 025 120583mfilm thickness Supelco Bellefonte PA) The optimum oven

temperature program was as follows it was initially set at180∘C for 3min ramped to 206∘C at 2∘Cmin held at 206∘Cfor 25min ramped to 240∘C at 10∘Cmin and held at 240∘Cfor 5min Peaks in the chromatogram were identified bycomparing their retention times and mass spectrums withGLC-461 reference standard (Nu-Chek Prep Elysian MNUSA) containing 32 FAME

23 Measurement of Serum Aminotransferase Levels Activi-ties of serum aspartate aminotransferase (AST) and alanineaminotransferase (ALT) were colorimetrically examined byusing their commercial kits (Nanjing Jiancheng Bioengineer-ing Institute Nanjing China)

24 Determination of Oxidative Stress Parameters in the LiverPartial liver tissues were weighed and homogenized with coldradioimmunoprecipitation assay (RIPA) buffer (BeyotimeInstitute of Biotechnology Nanjing China) to prepare 10liver homogenate After centrifugation the supernatant wassubjected to measure the levels of malondialdehyde (MDA)reduced glutathione (GSH) and oxidized glutathione (GSSG)and the activities of catalase (CAT) glutathione peroxidase(GSH-Px) glutathione reductase (GR) and superoxide dis-mutase (SOD) in the liver by the corresponding kits (NanjingJiancheng Bioengineering Institute) Total proteins in thehomogenate were quantified using PierceBCAKit (ThermoFisher) Values were normalized against hepatic total proteincontent

25 Histopathological Analysis Histopathological changes ofthe liver were observed by hematoxylin and eosin (HampE)staining [20] The liver tissue from the same lobe in eachmouse was fixed in 10 formalin overnight dehydratedin alcohol with different concentrations and embedded inparaffin The liver sections (5120583m thickness) were stainedwith HampE using a standard protocol The histopathologicalchanges of each mouse were examined and photographed byan Olympus CX-31 light microscope (Olympus Corp TokyoJapan)

26 TUNEL Assay To evaluate apoptotic cells in the livertissue a terminal deoxynucleotidyl transferase-mediateddeoxyuridine 5-triphosphate (dUTP) nick end labeling(TUNEL) assay was conducted by using ApopTag Plus InSitu Apoptosis Fluorescein Detection Kit (S7111 EMD Milli-pore Corporation BillericaMAUSA) In brief liver cryostatsection (8 120583M)was fixed in 1 paraformaldehyde andwashedwith PBS three times Then the section was incubated withgreen fluorescein labeled dUTP solution at 37∘C for 1 h Thesection was counterstained with DAPI and examined usingan Olympus BX63 fluorescence microscope (Tokyo Japan)

27 Immunofluorescence Assay Immunofluorescence analy-sis of hepatic Nrf2 was conducted as described previously[15] Briefly the cryostat section of liver tissue (8120583M) wasfixed in cooled acetone for 10min at 4∘C and then washedwith PBS After blocking the endogenous peroxidase with 5goat serum for 20min the liver section was incubated with

Oxidative Medicine and Cellular Longevity 3

Table 1 The primer sequences used in quantitative reverse transcription PCR analysis

Gene Full name GenBank accession number Primer sequences (forwardreverse)

HO-1 Heme oxygenase-1 NM 010442 51015840-AAGCCGAGAATGCTGAGTTCA-31015840

51015840-GCCGTGTAGATATGGTACAAGGA-31015840

GCLC Glutamate cysteine ligasecatalytic subunit NM 010295 51015840-GGGGTGACGAGGTGGAGTA-31015840

51015840-GTTGGGGTTTGTCCTCTCCC-31015840

GCLM Glutamate cysteine ligasemodifier subunit NM 008129 51015840-AGGAGCTTCGGGACTGTATCC-31015840

51015840-GGGACATGGTGCATTCCAAAA-31015840

NQO1 Quinone oxidoreductase-1 NM 008706 51015840-AGGATGGGAGGTACTCGAATC-31015840

51015840-AGGCGTCCTTCCTTATATGCTA-31015840

GADPH Glyceraldehyde-3-phosphatedehydrogenase NM 008085 51015840-TGGATTTGGACGCATTGGTC-31015840

51015840-TTTGCACTGGTACGTGTTGAT-31015840

1 100 rabbit anti-mouse Nrf2 antibody (Santa Cruz Biotech-nology Dallas USA) at 4∘C overnight and then incubatedwith 1 1000 Alexa Fluor 568-labeled secondary antibody(Life Technologies Carlsbad CA USA) in the dark at roomtemperature for 1 h Nuclei were counterstained with DAPIfor 10minThe fluorescence was observed and photographedby an Olympus BX63 fluorescent microscope (Olympus)

28 RT-PCRAssay Total RNA from the same lobe of liver tis-sue was extracted by a commercial RNAiso Plus kit accordingto the manufacturerrsquos protocol (Takara Tokyo Japan) cDNAwas synthesized by reverse transcription and amplified byPCR with the primers shown in Table 1 using PrimeScript RTReagent kit (Takara) The sequence of primers was designedfrom the PrimerBank and synthesized by Invitrogen LifeTechnologies (Shanghai China) PCR products were sepa-rated by agarose gel electrophoresis stained with ethidiumbromide and visualized under UV light

29 Western Blotting Assay Total protein cytosolic protein(exclusively for Nrf2) and nuclear protein (exclusively forNrf2) from liver tissues were prepared Total proteins wereisolated from liver tissue by RIPA buffer with 1 phos-phatase and protease inhibitors (Beyotime) The extractionand isolation of nuclear and cytosolic proteins were con-ducted by a Nuclear and Cytoplasmic Protein Extraction Kit(Beyotime) Protein concentration was quantified by using aPierce BCA Kit (Thermo Fisher) An aliquot of 20120583g totalprotein was loaded and separated on 10ndash15 SDS-PAGEand then subsequently electrophoretically transferred onto apolyvinylidene fluoride (PVDF) membrane The transferredmembrane was incubated with primary antibodies at 4∘Covernight (Table 2) and then incubated with the correspond-ing secondary antibodies (1 1000) at room temperature for1 h The band was visualized by ECL Detection Reagent (GEHealthcare BioSciences NJ USA) in a FluorChem Imagingsystem (Cell Biosciences Santa Clara CA USA)

210 Statistical Analysis Data are presented as mean plusmn stan-dard deviation (SD) To test the difference between groupsone-way analysis of variance (ANOVA) followed by Tukeyrsquospost hoc test was performed by using GraphPad Prism 60

software (San Diego CA USA) Statistical significance wasaccepted at the level of 119901 lt 005

3 Results

31 Fatty Acid Composition in Liver Tissues To measure theeffect of fat-1 expression on hepatic fatty acid profile livertissues from fat-1 and WT mice were determined by GC-MS Because fat-1 gene can encode n-3 PUFA desaturasethat allows converting n-6 PUFA to n-3 PUFA in fat-1mice compared with WTCCl4 group liver tissues fromfat-1CCl4 group exhibited higher amounts of n-3 PUFAincluding ALA (183n-3) EPA (205n-3) and DHA (226n-3) and lower levels of n-6 PUFA mainly linoleic acid (LA182n-6) and arachidonic acid (AA 204n-6) leading to aremarkable increase in total n-3 PUFA and decreases in totaln-6 PUFA and n-6n-3 ratio (Table 3) The level of totalsaturated fatty acids (SFA) in fat-1CCl4micewas significantlyhigher and the level of total monounsaturated fatty acids(MUFAs) tended to be lowerThese results demonstrated thatthe expression of fat-1 gene greatly elevated n-3 PUFA levelsin the liver although both groups were fed the identical diet

CCl4 exposure also greatly altered the fatty acid compo-sition in the liver Compared to WT control the WTCCl4group showed decreased levels in SFA mainly 140 160 and180 and increased levels in MUFA mainly 161 and 181leading to increased ratios of 161160 and 181180 the fattyacid desaturation indexThese findings also suggest that CCl4challenge may increase the activity or expression of stearoyl-CoA desaturase-1 (SCD-1)

32 Endogenous n-3 PUFA Ameliorates the Features ofAcute CCl4-Induced Liver Injury Liver injury was evaluatedby serum enzyme activities and hepatic histopathologicalchanges ALT and AST are released into the blood once thestructural integrity of the hepatocyte was damaged their lev-els are the most commonly used markers of liver injury [21]As shown in Figure 1(a) after acute CCl4 challenge the serumlevels of ALT andAST inWTCCl4 group increased 63 and 71times respectively over those in WT group However theseelevations were significantly blunted in fat-1CCl4 groupThehistological changes in the liver were evaluated byHampE stain-ing (Figure 1(b)) WT group exhibited normal architecture


Table 2 Primary antibodies used in immunoblot analysis

Primary antibody Full name Source Dilution CompanyBax BCL2-associated X protein Rabbit 1 1000 Cell Signaling TechnologyBcl-2 B-cell lymphoma-2 Rabbit 1 1000 Cell Signaling TechnologyCaspase-3 Cysteinyl aspartate specific proteinase-3 Rabbit 1 1000 Cell Signaling TechnologyCaspase-9 Cysteinyl aspartate specific proteinase-9 Rabbit 1 1000 Cell Signaling TechnologyCYP2E1 Cytochrome P4502E1 Rabbit 1 500 AbcamCyto-C Cytochrome C Rabbit 1 1000 Cell Signaling TechnologyERK Extracellular signal-regulated protein kinase Rabbit 1 1000 Cell Signaling TechnologyGADPH Glyceraldehyde-3-phosphate dehydrogenase Rabbit 1 1000 Cell Signaling TechnologyGCLC Glutamate cysteine ligase catalytic subunit Rabbit 1 1000 AbcamGCLM Glutamate cysteine ligase modifier subunit Rabbit 1 500 Santa Cruz BiotechnologyHO-1 Heme oxygenase-1 Rabbit 1 1000 AbcamJNK c-Jun N-terminal kinase Rabbit 1 1000 Cell Signaling TechnologyKeap1 Kelch-like ECH-associated protein-1 Rabbit 1 1000 Cell Signaling TechnologyLamin B Lamin B Rabbit 1 500 Cell Signaling TechnologyNrf2 Nuclear factor erythroid 2-related factor-2 Rabbit 1 1000 Santa Cruz BiotechnologyNQO1 NAD(P)Hquinone oxidoreductase-1 Rabbit 1 1000 Santa Cruz Biotechnologyp38 p38 mitogen-activated protein kinase Rabbit 1 1000 Cell Signaling Technologyp62 Nucleoporin p62 Rabbit 1 1000 Cell Signaling Technologyp-ERK Phosphorylated extracellular signal-regulated protein kinase Rabbit 1 1000 Cell Signaling Technologyp-JNK Phosphorylated c-Jun N-terminal kinase Rabbit 1 1000 Cell Signaling Technologyp-p38 Phosphorylated p38 mitogen-activated protein kinase Rabbit 1 1000 Cell Signaling Technology

with clear nuclear distribution CCl4 induced histologicalchanges including severely disrupted hepatic architecture andextensive hepatocellular necrosis around the blood vessels inWTCCl4 group which was reduced in fat-1CCl4 group

33 Endogenous n-3 PUFA Reduces CCl4-Induced OxidativeStress in the Liver Oxidative stress is characterized as a redoximbalance between prooxidants and endogenous antioxi-dants including nonenzymatic antioxidants (eg GSH) andenzymatic antioxidants (eg SOD CAT and GSH-Px) [22]MDA is an end product of lipid peroxidation (LPO) andhas been widely used as a marker of oxidative stress [22]As shown in Table 4 CCl4 exposure induced a remarkableincrease of hepatic MDA production by 877 (252 plusmn 034versus 473 plusmn 052 119901 lt 001) and a remarkable decreasein hepatic lipid peroxidation was observed in fat-1CCl4group Conversely CCl4 challenge depleted endogenousenzymatic and nonenzymatic antioxidants which can protecthepatocytes against oxidative stress as it is indicated thatthe activities of SOD CAT and GSH-Px and GSH levelin WTCCl4 group were significantly reduced to 705557 590 and 684 of those of WT group respectivelyThis depletion of endogenous antioxidants was markedlyameliorated in fat-1CCl4 group As a radical scavenger GSHcan be oxidized to GSSG under oxidative stress GSSG is alsoreduced back to GSH by glutathione reductase (GR) HenceGSHGSSG has been also used as a marker of oxidative stress[23] CCl4 exposure significantly increased the GSSG leveland decreased GR activity in the liver leading to a greatdecrease in GSHGSSG ratio these changes were remarkablyameliorated in fat-1mice

34 Endogenous n-3 PUFA Upregulates Antioxidant Enzymesvia Nuclear Translocation of Nrf2 To understand the under-lying molecular mechanisms for the protective effects ofendogenous n-3 PUFA against oxidative stress triggered byCCl4 the activation of Nrf2 a main transcription factorregulating antioxidant responses in the liver was examinedby immunofluorescence assay and immunoblot analysis Asshown in Figure 2(a) the significant nuclear translocationof Nrf2 was detected in fat-1CCl4 group compared toWTCCl4 group which was in accordance with the resultsof immunoblot analysis Endogenous n-3 PUFA in fat-1mice greatly decreased the protein expression of Nrf2 inthe cytoplasm but increased Nrf2 expression in the nucleuswithout changing the level of total Nrf2 expression in the liver(Figure 2(b)) In addition the Kelch-like ECH-associatedprotein-1 (Keap1) a repressor protein and p62 a substrateadaptor sequestosome-1 protein that competes with Nrf2 forbinding to Keap1 were examined in the liver by westernblot As shown in Figure 2(c) the lower protein expressionof Keap1 and higher expression of p62 were detected in fat-1CCl4 mice compared to WTCCl4 group

Nrf2-regulated genes such as HO-1 GCLC GCLMand NQO1 were also examined As shown in Figure 3both mRNA and protein levels of HO-1 GCLC GCLMand NQO1 in the liver were obviously upregulated in fat-1CCl4 group compared to WTCCl4 group InterestinglyCCl4 challenge notably promoted nuclear translocation ofNrf2 elevated Nrf2 expression in the nucleus and increasedthe expression of its downstream genes (Figures 2 and 3)which were consistent with the previous studies [3 4] Amost plausible explanation is the adaptive cytoprotective


Table 3 Fatty acid composition () of liver tissues

Fatty acids of total fatty acidsCommon name Symbol WT WTCCl4 Fat-1CCl4Lauric acid 120 027 plusmn 022 023 plusmn 015 026 plusmn 022Myristic acid 140 084 plusmn 028 060 plusmn 013 076 plusmn 011lowastlowast

Palmitic acid 160 285 plusmn 43 244 plusmn 16 267 plusmn 38Palmitoleic acid 161 116 plusmn 043 159 plusmn 042 132 plusmn 038lowastlowast

Stearic acid 180 230 plusmn 74 145 plusmn 29 195 plusmn 54lowast

Oleic acid 181 151 plusmn 50 208 plusmn 23 177 plusmn 31lowast

Linolenic acid 1826 171 plusmn 56 246 plusmn 23 213 plusmn 42lowast

120574-Linolenic acid 1836 041 plusmn 020 035 plusmn 010 028 plusmn 016120572-Linolenic acid 1833 027 plusmn 005 022 plusmn 005 036 plusmn 007lowastlowast

Arachidic acid 200 045 plusmn 018 023 plusmn 008 041 plusmn 012lowastlowast

Eicosenoic acid 201 026 plusmn 006 026 plusmn 003 025 plusmn 005Dihomo-120574-linoleic acid 2036 043 plusmn 014 060 plusmn 010 043 plusmn 008lowastlowast

Arachidonic acid 2046 908 plusmn 148 821 plusmn 186 473 plusmn 129lowastlowast

Eicosapentaenoic acid 2053 003 plusmn 002 004 plusmn 003 042 plusmn 016lowastlowast

Behenic acid 220 017 plusmn 005 015 plusmn 005 017 plusmn 001Erucic acid 221 020 plusmn 006 018 plusmn 011 029 plusmn 008lowast

Docosadienoic acid 2226 025 plusmn 010 010 plusmn 011 002 plusmn 001lowast

Docosatetraenoic acid 2246 028 plusmn 016 045 plusmn 012 022 plusmn 010lowastlowast

Docosapentaenoic acid 2253 004 plusmn 003 007 plusmn 006 015 plusmn 021Lignoceric acid 240 002 plusmn 001 002 plusmn 002 001 plusmn 001Docosahexaenoic acid 2263 167 plusmn 039 194 plusmn 033 416 plusmn 117lowastlowast

Nervonic acid 241 009 plusmn 004 020 plusmn 009 013 plusmn 009SFAs 535 plusmn 119 404 plusmn 41 481 plusmn 91lowastlowast

MUFAs 169 plusmn 55 231 plusmn 27 198 plusmn 36lowast

n-3 PUFAs 204 plusmn 038 230 plusmn 033 51 plusmn 14lowastlowast

n-6 PUFAs 275 plusmn 65 343 plusmn 28 270 plusmn 56lowast

Total PUFAs 296 plusmn 68 366 plusmn 30 322 plusmn 68n-6n-3 PUFAs 134 plusmn 15 151 plusmn 17 540 plusmn 075lowastlowast

161160 004 plusmn 002 007 plusmn 002 005 plusmn 002181180 080 plusmn 052 151 plusmn 042 101 plusmn 041lowastlowast

Values are expressed as the means plusmn SD (119899 = 10) 119901 lt 005 and 119901 lt 001 versus WT group lowast119901 lt 005 and lowastlowast119901 lt 001 versus WTCCl4 group SFAsaturated fatty acid MUFA monounsaturated fatty acid PUFA polyunsaturated fatty acid

reaction of organisms in response to oxidative stimuli Theseresults demonstrate that the protection of endogenous n-3PUFA against CCl4-induced liver damage is correlated withameliorating oxidative stress in the liver via activating Nrf2and upregulating its downstream genes

35 Endogenous n-3 PUFA Reduces Hepatocyte Apoptosis viaRegulating MAPK Signal Pathway As cell apoptosis directlyreflects the extent of liver injury caused by CCl4 a TUNELassay was conducted to estimate the regulation of cell apop-tosis by endogenous n-3 PUFA As shown in Figure 4(a) after24 h of CCl4 challenge the number of TUNEL-positive cellsin the liver section was significantly increased over the WTcontrol group In fat-1CCl4 group this increase in apoptoticcells was significantly decreased In addition during theCCl4-induced liver injury there was a cascade of apoptosis-related molecular events [24 25] After CCl4 challenge theprotein expressions of the proapoptotic proteins including

cytochrome C caspase-3 caspase-9 and Bax were obviouslyincreased in liver tissues from WT mice while the levelsof the antiapoptotic factor Bcl-2 was significantly decreasedIn agreement with the reduction of TUNEL-positive cellscompared with WTCCl4 mice the fat-1CCl4 mice showeddecreased levels of the aforementioned proapoptotic proteinsand increased level of Bax in the liver as indicated bywestern blot analyses (Figure 4(b)) These results indicatethat endogenous n-3 PUFA effectively prevents CCl4-inducedDNA fragmentation

To understand the underlying molecular mechanisms forthe inhibitory effects of endogenous n-3 PUFA on CCl4-induced hepatocyte apoptosis ERK JNK and p38 the majorcomponents in mitogen-activated protein kinase (MAPK)pathways which are critical regulators of cell proliferationand death in response to diverse stresses were examined byimmunoblottingOxidative stress in the liver activatesMAPKafter CCl4 challenge and results in activation of JNK p38 and


WTCCl4WT WTCCl4WT0

200

400

600

800

ALT

(UL

)

0

200

400

600

800

1000

AST

(UL

)

lowastlowastlowastlowast

Fat-1CCl4 Fat-1CCl4(a)

WTCCl4 Fat-1CCl4WT

(b)

Figure 1 Endogenous n-3 PUFA alleviates CCl4-induced acute liver injury in fat-1mice (a) Plasma levels of alanine aspartate transaminase(AST) and aminotransferase (ALT) (b) Representative hematoxylin and eosin (HampE) staining of liver tissue sections (magnification 400x)Values represent the means plusmn SD (119899 = 10) 119901 lt 0001 versus WT group lowastlowast119901 lt 001 versus WTCCl4 group

Table 4 Effects of endogenous omega-3 fatty acids on oxidative stress parameters in the liver

Parameters WT WTCCl4 Fat-1CCl4MDA (nmolmg protein) 252 plusmn 034 473 plusmn 052 399 plusmn 054lowastlowast

SOD (Umg protein) 3038 plusmn 338 2143 plusmn 328 2618 plusmn 442lowastlowast

CAT (Umg protein) 135 plusmn 19 752 plusmn 226 997 plusmn 210lowast

GSH-Px (Umg protein) 9756 plusmn 3172 5654 plusmn 1991 7701 plusmn 2834lowastlowast

GR (Ug protein) 106 plusmn 17 622 plusmn 177 792 plusmn 172lowast

GSH (mgg protein) 116 plusmn 18 796 plusmn 089 927 plusmn 193lowastlowast

GSSG (mgg protein) 126 plusmn 036 229 plusmn 056 172 plusmn 040lowast

GSHGSSG (fold) 972 plusmn 227 362 plusmn 089 546 plusmn 094lowastlowast

Data are expressed as mean plusmn SD (119899 = 10) 119901 lt 005 and 119901 lt 001 versus WT group lowast119901 lt 005 and lowastlowast119901 lt 001 versus WTCCl4 group

ERK [25] As shown in Figure 4(c) CCl4 exposure obviouslyincreased the phosphorylated protein levels of JNK p38 andERK12 without changing their total expressions in livertissues from WT mice These increases in phosphorylatedkinases were all downregulated by endogenous n-3 PUFA infat-1 mice Thus protective effects of endogenous n-3 PUFAagainst CCl4-induced hepatocyte apoptosis are associatedwith suppressing MAPK pathways

4 Discussion

In this study we used fat-1 transgenic mice to investigatethe role of endogenous n-3 PUFA in CCl4-caused acuteliver damage We demonstrate that CCl4 challenge causedsevere liver injury in WT mice as illustrated by markedlyelevated serum activities of AST and ALT oxidative stressand hepatocyte apoptosis Those pathological alterations


WT

DAPI Nrf2 Merge

WT

CCl 4

Fat-1

CCl

4

(a)

Nrf2

Lamin B

GADPH

Nrf2

Lamin B

GADPH

Cytoplasm

Nucleus

WT WTCCl4 Fat-1CCl4

(b)

Total Nrf2

GADPH

p62

Keap1

WT WTCCl4 Fat-1CCl4

(c)

Figure 2 Endogenous n-3 PUFA induces nuclear translocation of Nrf2 (a) Immunofluorescence staining of Nrf2 (b) Western blot analysisof Nrf2 in the nucleus and cytoplasm (c) Western blot analysis of total Nrf2 keap1 and p62 in liver tissue


GCLM

GCLC

GADPH

NQO1

HO-1

WT WTCCl4 Fat-1CCl4

(a)

GCLM

GCLC

GADPH

NQO1

HO-1

WT WTCCl4 Fat-1CCl4

(b)

Figure 3 Endogenous n-3 PUFA upregulates mRNA (a) and protein (b) expressions of Nrf2 target genes including HO-1 GCLC GCLMand NQO1

were remarkably relieved in fat-1 mice after CCl4 challengewhich was associated with activating Nrf2 and regulatingMAPK signal pathway

The fat-1 transgenicmice have beenwidely used as a noveltool for investigating the benefits of long chain n-3 PUFAsand the mechanisms underlying their actions [26] Fat-1transgenic mice carrying a fat-1 gene fromC elegans encodea desaturase that can convert n-3 to n-6 PUFA resulting inabundant n-3 PUFA without changing total PUFA in theirorgans and tissues (Table 3) Fat-1 mice and WT littermatesendogenously generate distinct fatty acid profiles in the liverwhile feeding them the same diet rich in n-6 PUFA Thusseveral variables arising from different diets such as flavoroxidation degree and unwanted components of fat usedmay be well avoided [15] As expected in this study livertissues from fat-1CCl4 group exhibited higher amounts ofn-3 PUFA particularly EPA and DHA and lower level ofn-6 PUFA leading to a remarkable increase in total n-3PUFA in the liver compared to WTCCl4 group As a well-characterized animal model the fat-1 mice were studied toexamine the impacts of endogenous n-3 PUFA on CCl4-induced acute liver injury

Oxidative stress is critical during the pathogenesis ofCCl4-induced acute liver injury [27] CCl4 challenge pro-duces highly reactive species and increases cellular pro-duction of ROS and MDA leading to oxidative stress intissues especially the liver where it is primarily metabolized[6] Additionally CCl4-induced oxidative stress also depletesantioxidant defense system including endogenous nonenzy-matic (eg GSH) and enzymatic (eg SOD CAT GSH-Pxand GR) antioxidants GSH has been considered to be thefirst line of defense against free radicals It was documentedthat GSH is an important antioxidant in eliminating toxicfree radicals and reactive toxic CCl4 metabolites [28 29]The sulfhydryl residues of GSH molecule are easily oxidizedto GSSG which can be reduced back to GSH by GR [30]Thus GSHGSSG ratio serves as a reliable marker to evaluatethe redox status and potential of oxidative stress [30] In ourstudy CCl4challenge increased hepatic MDA and GSSG and

the ratio of GSHGSSG and depleted GSH SOD CAT GSH-Px and GR in livers of WT mice which was ameliorated infat-1mice after CCl4 treatment (Table 4) To examine how n-3PUFA improves the antioxidant defense system the nucleartranslocation of Nrf2 and the expressions of Nrf2 targetgenes in the liver were evaluated Nrf2 acts as a transcriptionfactor which plays a key role in regulating the expressionof antioxidant proteins in response to oxidative stress [31]Under physiological condition Nrf2 is attached to Keap1 aspecific repressor in the cytoplasm which promotes Nrf2degradation by the ubiquitin proteasome pathway [32] Inthe presence of ROS Nrf2 degradation ceases while stabi-lized Nrf2 translocates into the nucleus which triggers theexpression of a series of antioxidants includingHO-1 GCLCGCLM and NQO1 through antioxidant response element(ARE) HO-1 is a strong antioxidant with antiapoptotic andanti-inflammatory effects in the liver GCLC and GCLM arekey rate-limiting enzymes in GSH biosynthesis [33] p62 isa substrate adaptor sequestosome-1 protein that modulatesthe Nrf2-Keap1 signaling pathway by competing with Nrf2for binding to Keap1 In this study the significant nucleartranslocation of Nrf2 was observed in fat-1CCl4 group asevidenced by immunofluorescence assay and immunoblotanalysis (Figure 2) Additionally fat-1CCl4 mice showedlower protein expression of Keap1 and higher expressionsof p62 HO-1 GCLC GCLM and NQO1 in the liver whencompared toWTCCl4 mice (Figure 3) These results suggestthat the protective effects of endogenous n-3 PUFA againstCCl4-caused acute liver damage might be attributable toreducing oxidative stress via the activation of Nrf2-keap1pathway

The induction of hepatocyte apoptosis has been wellstudied in acute liver injury induced by CCl4 exposure[34] Our data demonstrated that CCl4 challenge markedlyinduced hepatocyte apoptosis in WT mice (Figure 4(a))which was significantly reduced in fat-1mice after CCl4 expo-sure The mitochondrial apoptotic pathway was consideredto be involved in various types of cellular stress [35] Oxida-tive stress causes elevated cytochrome C in the cytoplasm


DAPI TUNEL MergeW

TW

TCC

l 4Fat-1

CCl

4

(a)

Bcl-2

Caspase-9

Caspase-3

GADPH

Bax

Cyto-C

WT WTCCl4 Fat-1CCl4

(b)

GADPH

p-ERK12

p-p38

Total JNK

Total p38

Total ERK12

p-JNK

WT WTCCl4 Fat-1CCl4

(c)

Figure 4 Endogenous n-3 PUFA protects against CCl4-induced hepatocyte apoptosis in fat-1mice via regulating MAPK signaling pathway(a) Representative images of TUNEL stained liver sections (magnification 200x) green fluorescence indicates the positive cells and cellularnucleus is labeled by staining DAPI with blue fluorescence (b) Western blot analysis of apoptosis-related proteins including cytochrome Ccaspase-3 caspase-9 Bcl-2 and Bax (c) Western blot analysis of total and phosphorylated protein expression of JNK p38 and ERK


Trichloromethyl radicalsOxidative

stress

ROS

Nrf2

Fat-1

Nrf2

Nrf2 HO-1 uarrGCLC uarrGCLM uarrNQO1 uarr

Keap1

Keap1

ROS

Caspase-3

Caspase-9

Apoptosis

Cytochrome C

MAPKs

JNK p38 ERK p

CCl4

120596-3

120596-6

darr Bcl-2Bax uarr

ARE

Figure 5 A schematic diagram of the potential mechanisms that underlie the protective effects of n-3 PUFA against CCl4-induced acute liverinjury

which is released from themitochondria which consequentlyinduces the activation of caspase cascades including caspase-3 and caspase-9 The mitochondrial apoptotic pathway isalso regulated by several apoptosis-related factors such asBax and Bcl-2 [36] The ratio of proapoptotic protein Bax toantiapoptotic protein Bcl-2 is critical for cell death or survivalIncreased BaxBcl-2 ratio leads to cytochrome C releasecaspase-3 activation and eventually apoptosis [37] Our datarevealed that endogenous n-3 PUFA significantly inhibitedthe upregulation of cytochromeC caspase-3 caspase-9 andBax and normalized the downregulation of Bcl-2 expressioninduced byCCl4 (Figure 4(b)) whichwaswell consistentwithTUNEL staining results

The MAPK family members including JNK p38 andJNK are crucial for the regulation of cell proliferationdifferentiation apoptosis and cellular responses to oxidativestress [38 39] Activated MAPKs can inactivate Bcl-2 byphosphorylation activate caspase-9 and regulate the releaseof cytochrome C from the mitochondria [40] Previousstudies revealed that suppressing protein expressions of thephosphorylated MAPK members contributed to the inhibi-tion of CCl4-induced apoptosis [38 39] In this study theupregulation of phosphorylated JNK p38 and JNK inducedby CCl4 challenge was attenuated in the livers of fat-1 miceThese findings suggest that reducing hepatocyte apoptosisvia suppressing MAPK pathway might also contribute to

the inhibitory function of n-3 PUFA on CCl4-induced liverinjury

In conclusion endogenous n-3 PUFA effectively ame-liorated CCl4-induced acute liver injury and this protectiveeffect might be associated with ameliorating oxidative stressvia Nrf2 activation and reducing apoptosis via suppressionof MAPK pathway as illustrated in Figure 5 Our findingssuggest that dietary supplement with n-3 PUFA may bebeneficial for the prevention of liver injury

Abbreviations

ALT Alanine transaminaseARE Antioxidant response elementAST Aspartate transaminaseBax Bcl-2-associated X proteinBcl-2 B-cell lymphoma-2CAT CatalaseCaspase-3 Cysteinyl aspartate specific proteinase-3Caspase-9 Cysteinyl aspartate specific proteinase-9CCl4 Carbon tetrachlorideCYP2E1 Cytochrome P4502E1Cyto-C Cytochrome CDAPI 46-Diamidino-2-phenylindoleERK Extracellular signal-regulated kinaseFAME Fatty acid methyl esters


GADPH Glyceraldehyde-3-phosphatedehydrogenase

GCLC Glutamate cysteine ligase catalytic subunitGCLM Glutamate cysteine ligase modifier subunitGSH Reduced glutathioneGSH-Px Glutathione peroxidaseGSSG Oxidized glutathioneHampE Hematoxylin and eosinHO-1 Heme oxygenase-1JNK c-Jun N-terminal kinaseKeap1 Kelch-like ECH-associated protein-1MAPK Mitogen-activated protein kinaseMDA MalondialdehydeMUFA(s) Monounsaturated fatty acid(s)Nrf2 Nuclear factor-erythroid 2-related factor-2NQO1 NAD(P)Hquinone oxidoreductase-1PUFA(s) Polyunsaturated fatty acid(s)p62 Nucleoporin p62ROS Reactive oxygen speciesSFA(s) Saturated fatty acid(s)SOD Superoxide dismutaseTUNEL Terminal deoxynucleotidyl transferase-

mediated deoxyuridine 5-triphosphatenick end labeling

WT Wild-type

Competing Interests

The authors declare that there are no competing interests

Acknowledgments

Thepresent study was supported by grants from the ResearchCommittee of the University of Macau (MYRG111-ICMS13to Jian-Bo Wan) and from Macao Science and TechnologyDevelopment Fund (0102013A1 to Jian-Bo Wan)

References

[1] L Qu H Xin G Zheng Y Su and C Ling ldquoHepatoprotectiveactivity of the total saponins from Actinidia valvata dunn rootagainst carbon tetrachloride-induced liver damage in micerdquoEvidence-Based Complementary and Alternative Medicine vol2012 Article ID 216061 13 pages 2012

[2] R N Jadeja N H Urrunaga S Dash S Khurana and NK Saxena ldquoWithaferin-a reduces acetaminophen-induced liverinjury in micerdquo Biochemical Pharmacology vol 97 no 1 pp122ndash132 2015

[3] D Dong L Xu L Yin Y Qi and J Peng ldquoNaringin preventscarbon tetrachloride-induced acute liver injury in micerdquo Jour-nal of Functional Foods vol 12 pp 179ndash191 2015

[4] C Su X Xia Q Shi et al ldquoNeohesperidin dihydrochalcone ver-sus CCl4-induced hepatic injury through differentmechanismsthe implication of free radical scavenging and Nrf2 ActivationrdquoJournal of Agricultural and Food Chemistry vol 63 no 22 pp5468ndash5475 2015

[5] A F Ismail A A Salem andMM Eassawy ldquoHepatoprotectiveeffect of grape seed oil against carbon tetrachloride inducedoxidative stress in liver of 120574-irradiated ratrdquo Journal of Photo-chemistry and Photobiology B Biology vol 160 pp 1ndash10 2016

[6] G M Campo A Avenoso S Campo et al ldquoHyaluronicacid and chondroitin-4-sulphate treatment reduces damagein carbon tetrachloride-induced acute rat liver injuryrdquo LifeSciences vol 74 no 10 pp 1289ndash1305 2004

[7] B O Cho H W Ryu C H Jin et al ldquoBlackberry extractattenuates oxidative stress through up-regulation of Nrf2-dependent antioxidant enzymes in carbon tetrachloride-treatedratsrdquo Journal of Agricultural and Food Chemistry vol 59 no 21pp 11442ndash11448 2011

[8] Y-F Lu J Liu K C Wu Q Qu F Fan and C D KlaassenldquoOverexpression of Nrf2 protects against microcystin-inducedhepatotoxicity in micerdquo PLoS ONE vol 9 no 3 Article IDe93013 2014

[9] E Scorletti L Bhatia K GMccormick et al ldquoEffects of purifiedeicosapentaenoic and docosahexaenoic acids in nonalcoholicfatty liver disease Results from the WELCOMElowast studyrdquo Hep-atology vol 60 no 4 pp 1211ndash1221 2014

[10] E M Tillman and R A Helms ldquoOmega-3 long chain polyun-saturated Fatty acids for treatment of parenteral nutrition-associated liver disease a review of the literaturerdquo The Journalof Pediatric Pharmacology and Therapeutics vol 16 pp 31ndash382011

[11] M Wang X Zhang C Yan et al ldquoPreventive effect of 120572-linolenic acid-rich flaxseed oil against ethanol-induced liverinjury is associated with ameliorating gut-derived endotoxin-mediated inflammation in micerdquo Journal of Functional Foodsvol 23 pp 532ndash541 2016

[12] M Wang X Zhang K Feng et al ldquoDietary 120572-linolenic acid-rich flaxseed oil prevents against alcoholic hepatic steatosis viaameliorating lipid homeostasis at adipose tissue-liver axis inmicerdquo Scientific Reports vol 6 p 26826 2016

[13] L L Huang J B Wan B Wang et al ldquoSuppression of acuteethanol-induced hepatic steatosis by docosahexaenoic acid isassociated with downregulation of stearoyl-CoA desaturase 1and inflammatory cytokinesrdquo Prostaglandins Leukotrienes andEssential Fatty Acids vol 88 no 5 pp 347ndash353 2013

[14] C Schmocker K H Weylandt L Kahlke et al ldquoOmega-3 fatty acids alleviate chemically induced acute hepatitis bysuppression of cytokinesrdquo Hepatology vol 45 no 4 pp 864ndash869 2007

[15] J-B Wan L-L Huang R Rong R Tan J Wang and J XKang ldquoEndogenously decreasing tissue n-6n-3 fatty acid ratioreduces atherosclerotic lesions in apolipoprotein E-deficientmice by inhibiting systemic and vascular inflammationrdquo Arte-riosclerosisThrombosis and Vascular Biology vol 30 no 12 pp2487ndash2494 2010

[16] J X Kang J Wang L Wu and Z B Kang ldquoTransgenic micefat-1 mice convert n-6 to n-3 fatty acidsrdquo Nature vol 427 no6974 p 504 2004

[17] H Xu T Guo Y-F Guo et al ldquoCharacterization and protectionon acute liver injury of a polysaccharide MP-I from Mytiluscoruscusrdquo Glycobiology vol 18 no 1 pp 97ndash103 2008

[18] X-J Zhang L-L Huang X-J Cai P Li Y-T Wang and J-BWan ldquoFatty acid variability in three medicinal herbs of Panaxspeciesrdquo Chemistry Central Journal vol 7 no 1 article 12 2013

[19] X-J Zhang L-L Huang H Su et al ldquoCharacterizing plasmaphospholipid fatty acid profiles of polycystic ovary syndromepatients with and without insulin resistance using GC-MSand chemometrics approachrdquo Journal of Pharmaceutical andBiomedical Analysis vol 95 pp 85ndash92 2014

[20] G-J Huang J-S Deng S-S Huang Y-Y Shao C-CChen and Y-H Kuo ldquoProtective effect of antrosterol from


Antrodia camphorata submerged whole broth against carbontetrachloride-induced acute liver injury in micerdquo Food Chem-istry vol 132 no 2 pp 709ndash716 2012

[21] R-B Ding K Tian L-L Huang et al ldquoHerbal medicines forthe prevention of alcoholic liver disease a reviewrdquo Journal ofEthnopharmacology vol 144 no 3 pp 457ndash465 2012

[22] R-B Ding K Tian Y-W Cao et al ldquoProtective effect of Panaxnotoginseng saponins on acute ethanol-induced liver injuryis associated with ameliorating hepatic lipid accumulationand reducing ethanol-mediated oxidative stressrdquo Journal ofAgricultural and Food Chemistry vol 63 no 9 pp 2413ndash24222015

[23] Y-T Tung J-H Wu C-C Huang et al ldquoProtective effect ofAcacia confusa bark extract and its active compound gallicacid against carbon tetrachloride-induced chronic liver injuryin ratsrdquo Food and Chemical Toxicology vol 47 no 6 pp 1385ndash1392 2009

[24] X-J Zhang C He P Li H Su and J-B Wan ldquoGin-senoside Rg1 a potential JNK inhibitor protects againstischemiareperfusion-induced liver damagerdquo Journal of Func-tional Foods vol 15 pp 580ndash592 2015

[25] H-Y Kim J Park K-H Lee et al ldquoFerulic acid protects againstcarbon tetrachloride-induced liver injury in micerdquo Toxicologyvol 282 no 3 pp 104ndash111 2011

[26] J X Kang ldquoFat-1 transgenic mice a new model for omega-3researchrdquo Prostaglandins Leukotrienes and Essential Fatty Acidsvol 77 no 5-6 pp 263ndash267 2007

[27] J-C Lin Y-J Peng S-Y Wang et al ldquoSympathetic nervoussystem control of carbon tetrachloride-induced oxidative stressin liver through 120572-adrenergic signalingrdquo Oxidative Medicineand Cellular Longevity vol 2016 Article ID 3190617 11 pages2016

[28] G Ray and S A Husain ldquoOxidants antioxidants and carcino-genesisrdquo Indian Journal of Experimental Biology vol 40 no 11pp 1213ndash1232 2002

[29] M Boll L W Weber E Becker and A Stampfl ldquoMecha-nism of carbon tetrachloride-induced hepatotoxicity Hepato-cellular damage by reactive carbon tetrachloride metabolitesrdquoZeitschrift fur Naturforschung C vol 56 pp 649ndash659 2001

[30] K Aoyama and T Nakaki ldquoGlutathione in cellular redoxhomeostasis association with the excitatory amino acid carrier1 (EAAC1)rdquoMolecules vol 20 no 5 pp 8742ndash8758 2015

[31] Y-W Cao Y Jiang D-Y Zhang et al ldquoProtective effects ofPenthorum chinense Pursh against chronic ethanol-inducedliver injury in micerdquo Journal of Ethnopharmacology vol 161 pp92ndash98 2015

[32] K Taguchi H Motohashi and M Yamamoto ldquoMolecularmechanisms of the Keap1-Nrf2 pathway in stress response andcancer evolutionrdquoGenes to Cells vol 16 no 2 pp 123ndash140 2011

[33] Y Chen H G Shertzer S N Schneider D W Nebert and TP Dalton ldquoGlutamate cysteine ligase catalysis dependence onATP and modifier subunit for regulation of tissue glutathionelevelsrdquoThe Journal of Biological Chemistry vol 280 no 40 pp33766ndash33774 2005

[34] E Karakus A Karadeniz N Simsek et al ldquoProtective effectof Panax ginseng against serum biochemical changes andapoptosis in liver of rats treated with carbon tetrachloride(CCl4)rdquo Journal of Hazardous Materials vol 195 pp 208ndash2132011

[35] B-Y Yang X-Y Zhang S-W Guan and Z-C Hua ldquoProtectiveeffect of procyanidin B2 against CCl4-induced acute liver injuryin micerdquoMolecules vol 20 no 7 pp 12250ndash12265 2015

[36] KKimN Jung K Lee et al ldquoDietary omega-3 polyunsaturatedfatty acids attenuate hepatic ischemiareperfusion injury in ratsby modulating toll-like receptor recruitment into lipid raftsrdquoClinical Nutrition vol 32 no 5 pp 855ndash862 2013

[37] DM Finucane E Bossy-Wetzel N J Waterhouse T G CotterandD R Green ldquoBax-induced caspase activation and apoptosisvia cytochrome c release from mitochondria is inhibitable byBcl-xLrdquoThe Journal of Biological Chemistry vol 274 no 4 pp2225ndash2233 1999

[38] J-Q Ma J Ding L Zhang and C-M Liu ldquoHepatoprotectiveproperties of sesamin against CCl4 induced oxidative stress-mediated apoptosis in mice via JNK pathwayrdquo Food andChemical Toxicology vol 64 pp 41ndash48 2014

[39] D N Dhanasekaran and E P Reddy ldquoJNK signaling inapoptosisrdquo Oncogene vol 27 no 48 pp 6245ndash6251 2008

[40] D-I Kim S-J Lee S-B Lee K Park W-J Kim and S-KMoon ldquoRequirement for RasRafERK pathway in naringin-induced G1 -cell-cycle arrest via p21WAF1 expressionrdquo Carcino-genesis vol 29 no 9 pp 1701ndash1709 2008

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


D-galactosaminelipopolysaccharide-induced hepatitis [14]However the impacts of n-3 PUFA on CCl4-induced liverinjury have not been sufficiently addressed The fat-1 trans-genic mouse was genetically modified to express a fat-1 genethat encodes n-3 PUFA desaturase [15 16] This enzyme canendogenously convert n-6 PUFA to n-3 PUFA in mammalsleading to higher n-3 PUFA level in tissues from fat-1 micecompared to the wild-type (WT) littermates when fed thesame diet rich in n-6 PUFA Thus fat-1 mice are a well-established animal model to investigate the role of n-3 PUFAin CCl4-induced liver injury Therefore the aims of currentstudy are to evaluate the probable effects of n-3 PUFA againstCCl4-induced acute liver injury and to elucidate the potentialmolecular mechanisms underlying this action

2 Materials and Methods

21 Animals and Treatments Fat-1 transgenic mice with agenetic background of C57BL6 were provided by Dr JingX Kangrsquos lab at Massachusetts General Hospital (BostonMA USA) Male heterozygous fat-1 mice were crossed withC57BL6 female mice to yield heterozygous fat-1 and WToffspringThe fat-1 phenotype of each offspringwas identifiedby the analysis of total lipids from mouse tail by using gaschromatography-mass spectrometry (GC-MS) The femalefat-1-positive and WT littermates were maintained in a spe-cific pathogen-free room at the Experimental Animal CenterGuangdong Pharmaceutical University Mice were fed an n-6 PUFA-rich but n-3 PUFA-deficient diet (a modified AIN93containing 10 corn oil) which contains 20 protein 58carbohydrate and 22 fat (TROPHIC Animal Feed High-Tech Co Ltd Nantong China) Mice (10ndash12 weeks old) weredivided into three groups (119899 = 10) that is WT controlWTCCl4 and fat-1CCl4 groupsThemice in CCl4 challengegroups were injected intraperitoneally with 10mLkg CCl4(02 dissolved in olive oil) [17] whileWT controls receivedan equal volume olive oil (ip) After 24 h of CCl4 or vehicletreatment all mice were anesthetized by an injection (ip100mgkg) with sodium pentobarbital Blood samples andliver tissues were immediately collected The animal exper-imental protocols (ICMS-AEC-2015-028) were performedaccording to the Guide to Animal Use and Care of the Uni-versity of Macau and were approved by the ethics committee

22 Analysis of Fatty Acid Composition in the Liver Fattyacid profile was analyzed according to a simplified methodby using GC-MS as described previously [18 19] In briefapproximately 10mgof liver tissuewas ground in liquid nitro-gen and methylated with 15mL of 14 boron trifluoride-methanol reagent (Sigma-Aldrich) and 15mL of hexane at100∘C for 1 h After cooling fatty acid methyl esters (FAME)were extracted in upper hexane layer GC-MS analysis wasconducted on a Thermo Fisher Scientific ISQ Series Sin-gle Quadrupole GC-MS system equipped with a TriPlusRSH autosampler (Thermo Fisher Waltham MA USA)Separation of FAME was achieved on an Omegawax 250fused silica capillary column (30m times 025mm id 025 120583mfilm thickness Supelco Bellefonte PA) The optimum oven

temperature program was as follows it was initially set at180∘C for 3min ramped to 206∘C at 2∘Cmin held at 206∘Cfor 25min ramped to 240∘C at 10∘Cmin and held at 240∘Cfor 5min Peaks in the chromatogram were identified bycomparing their retention times and mass spectrums withGLC-461 reference standard (Nu-Chek Prep Elysian MNUSA) containing 32 FAME

23 Measurement of Serum Aminotransferase Levels Activi-ties of serum aspartate aminotransferase (AST) and alanineaminotransferase (ALT) were colorimetrically examined byusing their commercial kits (Nanjing Jiancheng Bioengineer-ing Institute Nanjing China)

24 Determination of Oxidative Stress Parameters in the LiverPartial liver tissues were weighed and homogenized with coldradioimmunoprecipitation assay (RIPA) buffer (BeyotimeInstitute of Biotechnology Nanjing China) to prepare 10liver homogenate After centrifugation the supernatant wassubjected to measure the levels of malondialdehyde (MDA)reduced glutathione (GSH) and oxidized glutathione (GSSG)and the activities of catalase (CAT) glutathione peroxidase(GSH-Px) glutathione reductase (GR) and superoxide dis-mutase (SOD) in the liver by the corresponding kits (NanjingJiancheng Bioengineering Institute) Total proteins in thehomogenate were quantified using PierceBCAKit (ThermoFisher) Values were normalized against hepatic total proteincontent

25 Histopathological Analysis Histopathological changes ofthe liver were observed by hematoxylin and eosin (HampE)staining [20] The liver tissue from the same lobe in eachmouse was fixed in 10 formalin overnight dehydratedin alcohol with different concentrations and embedded inparaffin The liver sections (5120583m thickness) were stainedwith HampE using a standard protocol The histopathologicalchanges of each mouse were examined and photographed byan Olympus CX-31 light microscope (Olympus Corp TokyoJapan)

26 TUNEL Assay To evaluate apoptotic cells in the livertissue a terminal deoxynucleotidyl transferase-mediateddeoxyuridine 5-triphosphate (dUTP) nick end labeling(TUNEL) assay was conducted by using ApopTag Plus InSitu Apoptosis Fluorescein Detection Kit (S7111 EMD Milli-pore Corporation BillericaMAUSA) In brief liver cryostatsection (8 120583M)was fixed in 1 paraformaldehyde andwashedwith PBS three times Then the section was incubated withgreen fluorescein labeled dUTP solution at 37∘C for 1 h Thesection was counterstained with DAPI and examined usingan Olympus BX63 fluorescence microscope (Tokyo Japan)

27 Immunofluorescence Assay Immunofluorescence analy-sis of hepatic Nrf2 was conducted as described previously[15] Briefly the cryostat section of liver tissue (8120583M) wasfixed in cooled acetone for 10min at 4∘C and then washedwith PBS After blocking the endogenous peroxidase with 5goat serum for 20min the liver section was incubated with



















3 Results







































WTCCl4WT WTCCl4WT0

200

400

600

800

ALT

(UL

)

0

200

400

600

800

1000

AST

(UL

)



WTCCl4 Fat-1CCl4WT

(b)













4 Discussion



WT

DAPI Nrf2 Merge

WT

CCl 4

Fat-1

CCl

4

(a)

Nrf2

Lamin B

GADPH

Nrf2

Lamin B

GADPH

Cytoplasm

Nucleus

WT WTCCl4 Fat-1CCl4

(b)

Total Nrf2

GADPH

p62

Keap1

WT WTCCl4 Fat-1CCl4

(c)



GCLM

GCLC

GADPH

NQO1

HO-1

WT WTCCl4 Fat-1CCl4

(a)

GCLM

GCLC

GADPH

NQO1

HO-1

WT WTCCl4 Fat-1CCl4

(b)








DAPI TUNEL MergeW

TW

TCC

l 4Fat-1

CCl

4

(a)

Bcl-2

Caspase-9

Caspase-3

GADPH

Bax

Cyto-C

WT WTCCl4 Fat-1CCl4

(b)

GADPH

p-ERK12

p-p38

Total JNK

Total p38

Total ERK12

p-JNK

WT WTCCl4 Fat-1CCl4

(c)




stress

ROS

Nrf2

Fat-1

Nrf2


Keap1

Keap1

ROS

Caspase-3

Caspase-9

Apoptosis

Cytochrome C

MAPKs

JNK p38 ERK p

CCl4

120596-3

120596-6

darr Bcl-2Bax uarr

ARE






Abbreviations






WT Wild-type

Competing Interests


Acknowledgments


References
















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


































3 Results







































WTCCl4WT WTCCl4WT0

200

400

600

800

ALT

(UL

)

0

200

400

600

800

1000

AST

(UL

)



WTCCl4 Fat-1CCl4WT

(b)













4 Discussion



WT

DAPI Nrf2 Merge

WT

CCl 4

Fat-1

CCl

4

(a)

Nrf2

Lamin B

GADPH

Nrf2

Lamin B

GADPH

Cytoplasm

Nucleus

WT WTCCl4 Fat-1CCl4

(b)

Total Nrf2

GADPH

p62

Keap1

WT WTCCl4 Fat-1CCl4

(c)



GCLM

GCLC

GADPH

NQO1

HO-1

WT WTCCl4 Fat-1CCl4

(a)

GCLM

GCLC

GADPH

NQO1

HO-1

WT WTCCl4 Fat-1CCl4

(b)








DAPI TUNEL MergeW

TW

TCC

l 4Fat-1

CCl

4

(a)

Bcl-2

Caspase-9

Caspase-3

GADPH

Bax

Cyto-C

WT WTCCl4 Fat-1CCl4

(b)

GADPH

p-ERK12

p-p38

Total JNK

Total p38

Total ERK12

p-JNK

WT WTCCl4 Fat-1CCl4

(c)




stress

ROS

Nrf2

Fat-1

Nrf2


Keap1

Keap1

ROS

Caspase-3

Caspase-9

Apoptosis

Cytochrome C

MAPKs

JNK p38 ERK p

CCl4

120596-3

120596-6

darr Bcl-2Bax uarr

ARE






Abbreviations






WT Wild-type

Competing Interests


Acknowledgments


References
















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















































WTCCl4WT WTCCl4WT0

200

400

600

800

ALT

(UL

)

0

200

400

600

800

1000

AST

(UL

)



WTCCl4 Fat-1CCl4WT

(b)













4 Discussion



WT

DAPI Nrf2 Merge

WT

CCl 4

Fat-1

CCl

4

(a)

Nrf2

Lamin B

GADPH

Nrf2

Lamin B

GADPH

Cytoplasm

Nucleus

WT WTCCl4 Fat-1CCl4

(b)

Total Nrf2

GADPH

p62

Keap1

WT WTCCl4 Fat-1CCl4

(c)



GCLM

GCLC

GADPH

NQO1

HO-1

WT WTCCl4 Fat-1CCl4

(a)

GCLM

GCLC

GADPH

NQO1

HO-1

WT WTCCl4 Fat-1CCl4

(b)








DAPI TUNEL MergeW

TW

TCC

l 4Fat-1

CCl

4

(a)

Bcl-2

Caspase-9

Caspase-3

GADPH

Bax

Cyto-C

WT WTCCl4 Fat-1CCl4

(b)

GADPH

p-ERK12

p-p38

Total JNK

Total p38

Total ERK12

p-JNK

WT WTCCl4 Fat-1CCl4

(c)




stress

ROS

Nrf2

Fat-1

Nrf2


Keap1

Keap1

ROS

Caspase-3

Caspase-9

Apoptosis

Cytochrome C

MAPKs

JNK p38 ERK p

CCl4

120596-3

120596-6

darr Bcl-2Bax uarr

ARE






Abbreviations






WT Wild-type

Competing Interests


Acknowledgments


References
















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















WT

DAPI Nrf2 Merge

WT

CCl 4

Fat-1

CCl

4

(a)

Nrf2

Lamin B

GADPH

Nrf2

Lamin B

GADPH

Cytoplasm

Nucleus

WT WTCCl4 Fat-1CCl4

(b)

Total Nrf2

GADPH

p62

Keap1

WT WTCCl4 Fat-1CCl4

(c)



GCLM

GCLC

GADPH

NQO1

HO-1

WT WTCCl4 Fat-1CCl4

(a)

GCLM

GCLC

GADPH

NQO1

HO-1

WT WTCCl4 Fat-1CCl4

(b)








DAPI TUNEL MergeW

TW

TCC

l 4Fat-1

CCl

4

(a)

Bcl-2

Caspase-9

Caspase-3

GADPH

Bax

Cyto-C

WT WTCCl4 Fat-1CCl4

(b)

GADPH

p-ERK12

p-p38

Total JNK

Total p38

Total ERK12

p-JNK

WT WTCCl4 Fat-1CCl4

(c)




stress

ROS

Nrf2

Fat-1

Nrf2


Keap1

Keap1

ROS

Caspase-3

Caspase-9

Apoptosis

Cytochrome C

MAPKs

JNK p38 ERK p

CCl4

120596-3

120596-6

darr Bcl-2Bax uarr

ARE






Abbreviations






WT Wild-type

Competing Interests


Acknowledgments


References
















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















GCLM

GCLC

GADPH

NQO1

HO-1

WT WTCCl4 Fat-1CCl4

(a)

GCLM

GCLC

GADPH

NQO1

HO-1

WT WTCCl4 Fat-1CCl4

(b)








DAPI TUNEL MergeW

TW

TCC

l 4Fat-1

CCl

4

(a)

Bcl-2

Caspase-9

Caspase-3

GADPH

Bax

Cyto-C

WT WTCCl4 Fat-1CCl4

(b)

GADPH

p-ERK12

p-p38

Total JNK

Total p38

Total ERK12

p-JNK

WT WTCCl4 Fat-1CCl4

(c)




stress

ROS

Nrf2

Fat-1

Nrf2


Keap1

Keap1

ROS

Caspase-3

Caspase-9

Apoptosis

Cytochrome C

MAPKs

JNK p38 ERK p

CCl4

120596-3

120596-6

darr Bcl-2Bax uarr

ARE






Abbreviations






WT Wild-type

Competing Interests


Acknowledgments


References
















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















DAPI TUNEL MergeW

TW

TCC

l 4Fat-1

CCl

4

(a)

Bcl-2

Caspase-9

Caspase-3

GADPH

Bax

Cyto-C

WT WTCCl4 Fat-1CCl4

(b)

GADPH

p-ERK12

p-p38

Total JNK

Total p38

Total ERK12

p-JNK

WT WTCCl4 Fat-1CCl4

(c)




stress

ROS

Nrf2

Fat-1

Nrf2


Keap1

Keap1

ROS

Caspase-3

Caspase-9

Apoptosis

Cytochrome C

MAPKs

JNK p38 ERK p

CCl4

120596-3

120596-6

darr Bcl-2Bax uarr

ARE






Abbreviations






WT Wild-type

Competing Interests


Acknowledgments


References
















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















stress

ROS

Nrf2

Fat-1

Nrf2


Keap1

Keap1

ROS

Caspase-3

Caspase-9

Apoptosis

Cytochrome C

MAPKs

JNK p38 ERK p

CCl4

120596-3

120596-6

darr Bcl-2Bax uarr

ARE






Abbreviations






WT Wild-type

Competing Interests


Acknowledgments


References
















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




















WT Wild-type

Competing Interests


Acknowledgments


References
















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of











































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















research article endogenous n-3 fatty acids alleviate...

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