Protective Effects of Ethanolic Extracts from Artichoke, an Edible Herbal Medicine, against Acute

12 Pages·2017·2.84 MB·English

nutrients
Article
Protective Effects of Ethanolic Extracts from
Artichoke, an Edible Herbal Medicine, against Acute
Alcohol-Induced Liver Injury in Mice
XuchongTang1,*,RuofanWei1,AihuaDeng2andTingpingLei3
1 CollegeofChemicalEngineering,HuaqiaoUniversity,Xiamen361021,China;[email protected]
2 CollegeofLifeandEnvironmentalScience,HunanUniversityofArtsandScience,Changde415000,China;
[email protected]
3 CollegeofMechanicalEngineeringandAutomation,HuaqiaoUniversity,Xiamen361021,China;
[email protected]
* Correspondence:[email protected];Tel.:[phone]
Received:7August2017;Accepted:6September2017;Published:11September2017
Abstract: Oxidativestressandinflammationarewell-documentedpathologicalfactorsinalcoholic
liver disease (ALD). Artichoke (Cynara scolymus L.) is a healthy food and folk medicine with
anti-oxidative and anti-inflammatory properties. This study aimed to evaluate the preventive
effects of ethanolic extract from artichoke against acute alcohol-induced liver injury in mice.
Male Institute of Cancer Research mice were treated with an ethanolic extract of artichoke (0.4,
0.8,and1.6g/kgbodyweight)bygavageoncedaily. Upto40%alcohol(12mL/kgbodyweight)
was administered orally 1 h after artichoke treatment. All mice were fed for 10 consecutive
days. Results showed that artichoke extract significantly prevented elevated levels of aspartate
aminotransferase,alanineaminotransferase,triglyceride,totalcholesterol,andmalondialdehyde.
Meanwhile,thedecreasedlevelsofsuperoxidedismutaseandglutathionewereelevatedbyartichoke
administration. Histopathological examination showed that artichoke attenuated degeneration,
inflammatory infiltration and necrosis of hepatocytes. Immunohistochemical analysis revealed
that expression levels of toll-like receptor (TLR) 4 and nuclear factor-kappa B (NF-κB) in liver
tissuesweresignificantlysuppressedbyartichoketreatment. Resultsobtaineddemonstratedthat
artichokeextractexhibitedsignificantpreventiveprotectiveeffectagainstacutealcohol-inducedliver
injury. Thisfindingismainlyattributedtoitsabilitytoattenuateoxidativestressandsuppressthe
TLR4/NF-κBinflammatorypathway. Tothebestofourknowledge,theunderlyingmechanismsof
artichokeonacuteALDhavebeenrarelyreported.
Keywords: artichoke; acute alcohol liver disease; oxidative stress; inflammation;
TLR4/NF-κBpathway
1. Introduction
Drinkingalcoholhasalwaysbeendeemedessentialinmanyareas,suchasinsocialgatherings,
statusfunctions,personalinteractions,andconformance. However,long-termexcessivealcoholintake
can result in alcoholic liver disease (ALD). ALD is the leading cause of cirrhosis and liver-related
deathworldwidefordecadesandisresponsiblefor4%ofglobalmortality[1–3]. ALDencompassesa
histologicalspectrumofliverinjurythatrangesfromsteatosis(fattyliver)toalcoholicsteatohepatitis
(ASH),andinseverecases,fibrosis,cirrhosis,andultimatelyhepatocellularcarcinoma[4,5]. Thus,the
controlofALDatanearlystage,forexample,atastagepriortotheoccurrenceofASH,couldbeof
greatsignificanceinpreventingdevelopmentofALD.
Nutrients2017,9,1000;doi:10.3390/nu9091000 www.mdpi.com/journal/nutrients
Nutrients2017,9,1000 2of12
Potentialmechanismsofacutealcohol-inducedliverinjuryareassociatedwithoxidativestress,
steaotosis,endotoxin,dysregulatedimmunity,andinflammation. However,inrecentdecades,studies
have focused on the inflammatory pathway in ALD, and increasing evidence demonstrates that
ASHiscausedbythelipopolysaccharide(LPS)bindingtotoll-likereceptor(TLR)-inducednuclear
factor-kappaB(NF-κB)activationpathway[6–8]. TLRsarepattern-recognitionreceptorsthatenable
theinnateimmunesystemtoreactimmediatelytoinfectionsbyrecognizingbothbacterialandviral
constituents. AmongtheTLRs,TLR4caninitiateactivationofNF-κBandcascaderesponsefurther
causingtheaccumulationofpro-inflammationcytokines,ultimatelyresultingintheaggravationof
inflammatoryprogress[9–12].
Although various treatments, such as nutritional therapy, pharmacological therapy,
psychotherapy,andsurgery,arecurrentlyavailableforthespectrumsofALD,nosatisfactorytherapyis
availableexceptforabstinence[13–15]. Medicationsthatactasanti-inflammatoriesandanti-oxidants,
arefrequentlyusedastherapeuticdrugsinALD.Forexample,thereissilymarin,whichalleviates
liverinjurymainlybyreducingfreeradicalactivityandlipidperoxidation,protectingthelivercell
membrane, and promoting hepatocytes regeneration, and bifendate, which alleviates liver injury
mainlybyreducingserumlevelofalanineaminotransferase(ALT),inflammatorycellinfiltrationand
liverhistologicalchanges. However,clinicalapplicationsarelimitedbecauseofsideeffectsamong
otherreasons. Forexample,silymarinhaspoororal-bioavailability,andbifendatemaycauseliver
hypertrophy[16–18]. Thus, findingconvincinglyeffectivetreatmentdrugswithfewersideeffects
withoutcompromisingtherapeuticeffectcontinuestobeanimportantgoal.
Artichoke (Cynara scolymus L.), an edible herbal medicine of the family Compositae, is a
perennialherbwidelystudiedbecauseofitspossibleantioxidativeandhepatoprotectiveeffects[19–22].
Theextractsandderivativesfromartichokecontainavarietyofdicaffeoylquinicacidsandmanykinds
of flavonoid functional compounds, such as cynarin (1,5-dicaffeoylquinic acids), chlorogenic acid
(3-caffe-oylquinicacid),luteolinglucoside,andapigeninglucoside[23,24],whichexhibitanti-microbial,
anti-allergic,anti-inflammatory,andanticancereffects. Onestudyindicatedthatartichokeextracthad
potentialinreducinghypercholesterolemiathroughpreventinglipidperoxidationandameliorating
hepatic antioxidant status [25]. Another report demonstrated that artichoke aqueous leaf extract
reducedserumtotalcholesterol(TC),triglycerides(TG),verylowdensitylipoprotein,glucoselevels,
andplasmamalondialdehyde(MDA)levelsinstreptozotocin-treateddiabeticrats[26]. Reportsalso
demonstrated that artichoke showed marked anti-inflammatory effects on tissue plasminogen
activator-inducedinflammationandantitumoractivityinaninvivotwo-stagecarcinogenesistestin
mice[27].Besides,itwasreportedthatartichokeextractwasverysafetothehumanbodyasnoobvious
sideeffectswereobservedaftercontinuousmedicationforseveralmonths[28].Therefore,artichokehas
abroadapplicationprospectsinALDtreatmentduetoitsanti-oxidantandanti-inflammatoryeffects.
Thepurposeofthisstudywastoinvestigatetheprophylacticprotectiveeffectsofethanolicextract
fromartichokeonacutealcohol-inducedinjuryinanacuteALDmicemodel. Todate,theindicatorsof
aspartateaminotransferase(AST),ALT,TG,TC,MDA,glutathione(GSH),andsuperoxidedismutase
(SOD)wereassessed. Thepossiblemechanismforacutealcohol-inducedliverinjurywasdiscussed
usingthesignalsofTLR4andNF-κB.
2. MaterialsandMethods
2.1. Materials
Artichokesinfreeze-driedpowder(8.89%caffeicacidderivatives,0.98%chlorogenicacid,0.56%
cynarin)weresuppliedbyHuimeiAgriculturalScienceandTechnologyCo.,Ltd. (Hunan,China),
andtheartichokesweredilutedinto0.04g/mL,0.08g/mL,and0.16g/mLsuspensionswithdistilled
water,respectively. EdiblealcoholwasobtainedfromBeijingRedStarCo.,Ltd. (Beijing,China),and
ediblealcoholwasdilutedwithdistilledwatertoaconcentrationof40%(w/v). Regularchowdiet
(40–43%corn,26%bran,29%beancake,1%salt,1%bonemeal,1%lysine)formicewaspurchased
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fromHuayueyangBiotechnologyCO.,Ltd. (Beijing,China). BifendatewasprovidedbyBeijingUnion
PharmaceuticalFactory(Beijing,China),andbifendatewasdilutedasa0.036g/mLsuspensionwith
distilledwater. DiagnostickitsforAST,ALT,TG,TC,SOD,MDA,andGSHwerereceivedfromthe
NanjingJianchengInstituteofBiotechnology(Nanjing,China). AntibodiesforTLR4andNF-κBp50
werepurchasedfromOriGeneTechnologies,Inc. (Rockville,MD,USA)andNovusBiologicals,Inc.
(Littleton,CO,USA),respectively. Allotherchemicalsusedwereofanalyticalreagentandobtained
fromSinopharmChemicalReagentCo.,Ltd. (Shanghai,China).
2.2. ExperimentalAnimals
Seven-week-old male Institute of Cancer Research (ICR) mice (25 ± 2 g) were supplied by
the Shanghai Laboratory Animal Center (Shanghai, China) and acclimated for one week prior to
use. These mice were kept under environmentally controlled conditions (12-h normal light/dark
cycles,22±2◦Cand50±10%relativehumidity)withchowdietandwateradlibitum. Allanimal
experiments were approved by the Animal Care and Use Committee of the Huaqiao University
(ApprovalNo. SCXK(HU)2012-0002)andfollowedtheNationalInstitutesofHealthGuidelinesfor
animalcare(ApprovalNo. HQ-ECLA-20160517).
TheICRmicewererandomlydividedinto6groupsof10micepergroup:
(1) Controlgroup: miceweregavagedwithsamevolumeof0.9%salinetwiceperday(intervaltime,
onehour).
(2) EtOH group (model group): mice were gavaged with same volume of 0.9% saline and with
12mL/kgbodyweight(BW)alcoholonehouraftersalineadministrationperday.
(3) Positivecontrolgroup(EtOH+bifendate): miceweregavagedwith0.36g/kgBWofbifendate
andwith12mL/kgBWalcoholonehourafterbifendatepretreatmenteachday.
(4) Low-dose artichoke group (EtOH + artichoke 0.4): mice were gavaged with 0.4 g/kg BW of
artichokeandwith12mL/kgBWalcoholonehourafterartichokepretreatmenteachday.
(5) Middle-doseartichokegroup(EtOH+artichoke0.8): miceweregavagedwith0.8g/kgBWof
artichokeandwith12mL/kgBWalcoholonehourafterartichokepretreatmenteachday.
(6) High-dose artichoke group (EtOH + artichoke 1.6): mice were gaveged with 1.6 g/kg BW of
artichokeandwith12mL/kgBWalcoholonehourafterartichokepretreatmenteachday.
All groups were fed for 10 consecutive days. Then, all groups were fasted for 12 h and
subsequentlyanesthetizedbypentobarbitalsolution(60mg/kgBW)beforetheexperiment.
2.3. SerumBiochemicalAssays
Blood samples were collected from the retrobulbar vessels of the mice. The samples were
centrifugedat1500rpmfor10minat4◦C(GTR16-2,BeijingEraBeiliCentrifugeCo.,Ltd,Beijing,
China) to separate the serum after standing for 1 h at room temperature. Serum ALT, AST, TG,
andTCactivitiesweresubsequentlysubjectedtodiagnostickittesting(NanjingJianchengInstitute
of Biotechnology) according to the instructions provided using spectrophotometer determination
(UV2550, Shimadzu Crop., Kyoto, Japan). Briefly, for assessment of ALT and AST, the samples
weremixedwithsubstratesorbuffersolution. Afterincubationatroomtemperaturefor5min,the
absorbance at 505 nm was measured. The final data of ALT and AST were represented as U/L.
ForassessmentofTGandTC,thesamplesweretransferredintoa96-wellplatecontainingsubstrates
orbuffersolution. Afterincubationat37◦Cfor10min,theplatewasincubatedforanadditionaltime
afteraddingcolordevelopingagentandtheabsorbanceat510nmwasmeasured. Thefinaldataare
representedasµmol/L.
2.4. HepaticAntioxidantandOxidativeStressMarkerAssays
Theliverswereweighedaccurately. Atotalof0.5glivertissuewascutandwashedwithdistilled
water, thenexcesswaterwasdriedup. Thenthelivertissuewascutintoslicesandhomogenated
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withninevolumesofphosphatebuffer(4.5mL)inanicebath(pH7.2–7.4). Theresultingsuspension
was centrifuged at 12,000 rpm for 10 min at 4 ◦C (GTR16-2, Beijing Era Beili Centrifuge Co., Ltd,
Beijing, China), and the supernatant was measured by diagnostic kits of SOD, MDA, and GSH
(NanjingJianchengInstituteofBiotechnology)accordingtothemanufacturer’sinstructions. Inbrief,
theconcentrationsofSOD,MDAandGSHwereassayedbyhydroxylaminemethod,thiobarbituric
acid-reactivemethodandmicroplatemethod,respectively.
2.5. HistologicalExaminationofLiverTissue
Liver tissues were fixed in 10% neutral formalin buffer for 24 h, and 5-µm sections were cut
andstainedwithhematoxylinandeosin(H&E),andthenobservedusingaNikonDS-Fi2fluorescent
microscope(Nikon,Tokyo,Japan). Themagnificationwas200×. Atleast10areasofeachtissueslice
wereobserved. Representativeimageswerepresented. Analysesofpathologicalchangeswerebased
onproportionofinflammation,necrosis(0point,0foci;1point,<2foci;2points,2–4foci;3points,
>4foci,per200×field)andsteatosis(0point,<5%;1point,6–33%;2points,34–66%;3points,>66%),
whichwereassessedbythreeexaminersindependently[29].
2.6. ImmunohistochemicalAnalysisofTLR4andNF-κB
Liver tissues were fixed in 10% neutral formalin buffer and embedded in paraffin.
Five-millimeter-thick paraffin sections were cut and were then heated in unmasking solution at
95 ◦Cfor15 minafterdeparaffinization. Nonspecific bindingsiteswereblockedwithgoatserum.
Sectionswerethenincubatedovernightat4◦Cinahumidifiedchamberwiththefollowingprimary
antibodies: rabbitanti-TLR4(1:50)andrabbitanti-NF-κBp50(1:250). Theexaminers,blindedtothe
experimentalgroups,countedthecellslabeledwithTLR4andNF-κBp50throughoutfiverandom
lesionregionsinthestainedareasundera200×lightmicroscope. Then,theexpressionlevelsofTLR4
andNF-κBp50wereanalyzedbymeanintegratedopticaldensity(IOD).
2.7. StatisticalAnalysis
All quantifications for assays were repeated for three times and a mean value was used
by taking mean of the triplicate plus/minus standard deviation (mean ± SD) for each group.
Statisticallysignificantdifferences(p<0.05)wereevaluatedbyone-wayanalysisofvarianceusing
SPSS18.0(SPSSInc.,Chicago,IL,USA).
3. ResultsandDiscussion
3.1. LiverIndexofICRMice
Theliverindexwascalculatedasaratio(%)oftheliverweight(g)tobodyweight(g)[30,31].
AtthefirststageofALDformation,excessivedrinkingimpliesalargenumberofcalories,resultingina
dramaticincreaseinliverweight,bodyweightandliverindex. Thus,theliverindexisaninformative
ratioforpredictingALD.Table1showstheliverindicesofmiceofsixgroups. Nosignificantdifference
wasobservedinthefinalbodyweightinbifendateorartichoketreatmentmicecomparedwiththe
EtOHgroup. However,theweightgainofmicetreatedwithalcoholdecreasedsignificantly(p<0.05)
whencomparedtocontrolgroup,whichcouldbeexplainedasasymptomofanorexiacausedbyliver
damage,indicatingthesuccessfulestablishmentofanacutealcohol-inducedmicemodel. Liverindex
wassignificantlyincreasedby8.79%(p<0.05)intheEtOHgroupincomparisontothecontrolgroup,
whereas no significant decrease was shown in the bifendate group or artichoke treatment groups
compared with the EtOH group. However, the medication groups under bifendate and artichoke
treatmentallshowedaprotectiveeffectontheliverasevidencedbycomparativelylowliverindexes
whencomparedwithEtOHgroup,suggestingthatartichokeishelpfulforattenuatingethanol-induced
liverinjury.
Nutrients2017,9,1000 5of12
Table1.Effectofartichokeonliverindexofacutealcoholicliverdisease(ALD)micea.
Dosage LiverWeight InitialBody FinalBody LiverIndex
TreatmentGroup
(g/kg) (g) Weight(g) Weight(g) (%)
Nutrients 2017, 9, 1000  5 of 12 
Control _ 1.75±0.22 26.46±0.64 36.96±4.26# 4.78±0.64#
EtOH Table 1. Effe_ct of artich1o.k5e3 o±n l0iv.1er7 index of2 a6c.u2t4e ±alc0o.h7o0lic liver 2d9is.e9a4se± (A2L.9D8) m*ice a. 5.20±0.22*
EtOH+Bifendate 0.36Dosage 1.53L±ive0r .W18eight  2In6i.t1ia7l B±ody0 .W9e5ight  3F0in.1al8 B±ody2 W.1e3igh*t  L5iv.e0r5 In±dex0 .38
Treatment Group 
Low-doseartichoke 0.4 (g/kg)  1.47±0(.g2)3  26.35±(g)0 .72 28.79±(g)3 .00* 5.1(%2) ±0.64
Middle-dosearticChoontkroel  0.8 _  1.57±1.705 .±2 00.22  26.3296.4±6 ±0 0..6542  29.0366.9±6 ± 24..206 3# * 45.7.80 ±4 0.±64 #0 .44
EtOH  _  1.53 ± 0.17  26.24 ± 0.70  29.94 ± 2.98 *  5.20 ± 0.22 * 
High-dosearEttiOcHh o+ kBeifendate  1.6 0.36  1.54±1.503 .±1 02.18  25.9246.1±7 ±0 0..9855  29.8304.18± ± 21.1.23 7* * 55.0.51 4± 0±.38 0.31
Low‐dose artichoke  0.4  1.47 ± 0.23  26.35 ± 0.72  28.79 ± 3.00 *  5.12 ± 0.64 
aDataareexpressedasmeans±standarddeviation(SD)(n=10);*p<0.05,vs.thecontrolgroup;#p<0.05,vs.the
Middle‐dose artichoke  0.8  1.57 ± 0.20  26.39 ± 0.52  29.06 ± 2.03 *  5.04 ± 0.44 
EtOHgroup. High‐dose artichoke  1.6  1.54 ± 0.12  25.94 ± 0.85  29.84 ± 1.27 *  5.14 ± 0.31 
a Data are expressed as means ± standard deviation (SD) (n = 10); * p < 0.05, vs. the control group; # p < 
3.2. SerumBiochem0i.c05a,l vMs. tahre kEetrOsH group.  
Thelevel3s.2o. fSesreumru BmiochAemLiTca,l AMSarTk,erTs GandTCareearlybiochemicalandpathologicalmarkersof
hepatocytedamagTeh.eA leLvTelsi sofa sceyrutmos AolLiTc, eAnSzTy, mTGe awndh iTcCh airse meaarliyn blyiocphreemseicnalt eadndi npatthhoelocgeilclacl ymtaorpkelarss mof ,while
ASTisamitochheopnadtorciyatle ednazmyamgee. AwLhTi cish ai scyrteolseoalisce ednzfyromme wthheichli vise mraainndly optrheseernoterdg ainn tshien cethll ecybtoopdlya.smA, LTand
while AST is a mitochondrial enzyme which is released from the liver and other organs in the body. 
ASTarereleasedintotheblood,resultinginanincreaseofserumtransaminasewhendamage(e.g.,
ALT and AST are released into the blood, resulting in an increase of serum transaminase when 
inflammationandnecrosis)occursinlivercells. Meanwhile,TGandTCarecharacterizedasindicators
damage (e.g., inflammation and necrosis) occurs in liver cells. Meanwhile, TG and TC are 
offataccumulcahtaiorancteinriztehde alsi vinedricaantodrs roefs fpaot ancsceusmtuolaatilocno ihno tlhce olinvseru amndp trieospno[n1s1es, 3t2o ]a.lcohol consumption 
Asshown[1i1n,3F2]i.g ure1,serumlevelsofALT,AST,TG,andTCintheEtOHgroupgreatlyincreased
As shown in Figure 1, serum levels of ALT, AST, TG, and TC in the EtOH group greatly 
by44.63%(p<0.05),23.69%(p<0.05),43.98%(p<0.05),and57.83%(p<0.05),respectively,compared
increased by 44.63% (p < 0.05), 23.69% (p < 0.05), 43.98% (p < 0.05), and 57.83% (p < 0.05), respectively, 
withthecontrocolmgproaruedp .wInithA tSheT caonndtroTl Ggroleuvp.e lIsn, sAiSgTn iafincda TnGt dleevcerles,a ssiegsniwficearnet odbecsreeravseesd wbeeret wobeseenrvleodw  -dose
(0.4g/kgBW)beatnwdeehn ilgowh‐-ddooses e(0(.41 .g6/kgg/ BkWg) BanWd )hiagrht‐idcohseo k(1e.6 ggr/kogu BpWs.) Nartoicthaobkley g,rtohuepsA. NLoTt,abAlyS, Tth,eT AGL,Ta, ndTC
AST, TG, and TC levels in the artichoke pretreatment groups were abated in a dose‐dependent 
levelsintheartichokepretreatmentgroupswereabatedinadose-dependentmannercomparedwith
manner compared with the EtOH group. Pretreatment with high‐dose artichoke (1.6 g/kg BW) 
theEtOHgroup. Pretreatmentwithhigh-doseartichoke(1.6g/kgBW)markedlyrecoveredserum
markedly recovered serum ALT, AST, TG, and TC levels to near those of control group mice. The 
ALT, AST, TGr,easunltds TdeCmloenvsteralstedto  thnaeta  rarttihchooskee opfrectorenattmroelntg rwoausp  almsoi ceef.fecTtihvee  rine sruelvtesrsdinegm  tohen satcruattee d that
artichokepretraelcaothmole‐inndtuwceads liavlesro deysfffuenccttiivoen. inreversingtheacutealcohol-inducedliverdysfunction.
80A & 150B &
T(U/L)4600 # * # # T(U/L)12900 # * #
L S 60
A A
20 30
0 Control EtEOtOH+HEtBiOfHen+dAarttieEcthOoHk +e(A0r.t4i)EcthOoHk+e(A0r.t8i)choke(1.6) 0 Control EtEOtOHH+EtBiOfHen+dAarttieEcthOoHk +e(A0r.t4iE)cthOoHk+e(A0r.t8i)choke(1.6)
(A)  (B) 
8C & & 16D
* *
TG(umol/L)246 # # # # TC(umol/L)1482 # # # #
0 Control EtEOtOH+HEtBiOfHen+dAarttieEcthOoHk+e(A0r.t4iE)cthOoHk+e(A0r.t8i)choke(1.6) 0 Control EtEOtOH+HEtBiOfHen+dAarttieEcthOoHk +e(A0r.t4i)EcthOoHke+(A0r.t8i)choke(1.6)
(C)  (D) 
Figure1. EffectsofartichokeonlevelsofALT(A),AST(B),TG(C),andTC(D).Valuesrepresent
means±standarddeviation(SD)(n=10);*p<0.05vs. thecontrolgroup;# p<0.05vs. theEtOH
group;&p<0.05.ALT:alanineaminotransferase;AST:aspartateaminotransferase;TG:triglycerides;
TC:totalcholesterol.
Nutrients 2017, 9, 1000  6 of 12 
Figure 1. Effects of artichoke on levels of ALT (A), AST (B), TG (C), and TC (D). Values represent 
Nutrienmtse2a0n1s7 ,±9 ,st1a0n0d0ard deviation (SD) (n = 10); * p < 0.05 vs. the control group; # p < 0.05 vs. the EtOH  6of12
group; & p < 0.05. ALT: alanine aminotransferase; AST: aspartate aminotransferase; TG: triglycerides; 
TC: total cholesterol. 
3.3. HistopathologicalAnalysis
3.3. Histopathological Analysis 
The effect of artichoke on the histopathology of the acute ALD mice is presented in Figure 2.
StainiTnhge wefiftehctH o&f Earrtiecvheoaklee donn othrme hailshtoeppaatthicolaorgcyh iotef ctthuer eacwutieth AinLDth emciocem ips lperteessetnrutecdt uinre F,isgimuriela 2r. size,
tSigtahitnainrrga wngitehm He&ntE, rreegvuelaalerdh enpoarmticalc ohredpsatwici tahrccheintetrcatulrvee iwni,tahnind tchlee acromhepplaetteic sstrinuuctsuoried, isnimcoilnart rsoilzeg,r oup
tight arrangement, regular hepatic cords with central vein, and clear hepatic sinusoid in control 
(Figure2A).Nevertheless,theEtOHgroupdemonstratedevidentpathologicalchanges,includingloose
group (Figure 2A). Nevertheless, the EtOH group demonstrated evident pathological changes, 
arrangement,disarrangementofcellcords,leukocytesinfiltration,necrosis,andhepatocytessteatosis,
including loose arrangement, disarrangement of cell cords, leukocytes infiltration, necrosis, and 
whichconfirmedtheestablishmentofliverinjurywithinflammation(Figure2B).Slightleukocyte
hepatocytes steatosis, which confirmed the establishment of liver injury with inflammation (Figure 
infiltrationandhepatocytesteatosiswasstillobservedinthelow-doseartichoke(0.4g/kg)group
2B).  Slight  leukocyte  infiltration  and  hepatocyte  steatosis  was  still  observed  in  the  low‐dose 
(Figure2D).However, pretreatmentwithbifendateandartichokesexertedaregenerativeeffectof
artichoke (0.4  g/kg)  group  (Figure 2D). However, pretreatment with bifendate and artichokes 
hepatocytesandadecreaseofnecroticandinflamedareas. Thisindicatedthatartichokepretreatment
exerted a regenerative effect of hepatocytes and a decrease of necrotic and inflamed areas. This 
promotedstructurerestorationofthelivertoacertainextent.
indicated that artichoke pretreatment promoted structure restoration of the liver to a certain extent. 
Asshowninscoreresults(Figure2G–I),thepathologicalscoresofsteatosis,inflammationand
As shown in score results (Figure 2G–I), the pathological scores of steatosis, inflammation and 
nneeccrroossiiss iinnccrreeaasseedd ssiiggnnifiificcaanntltyly (p( p< <0.00.0010)1 i)n itnhet hEetOEHtO gHrogurpo uwphewnh ceonmcpoamrepda rtoed cotontrcooln gtrroolugpr. oup.
However,comparedwithEtOHgroup,thedegreeofhepaticchangehadmadeanimprovementin
However, compared with EtOH group, the degree of hepatic change had made an improvement in 
tthhee aarrttiicchhookkee aanndd bbiiffeennddaatete ppreretrteraetamtmenetn gtrgoruopu,p s,usguggegsteinstgin tghatth aarttiacrhtoickheo hkaedh pardevpernetvivene teivffeecetf ofef ctof
aalllleevviiaattiinngg aallccoohhooll‐-iinndduucceedd lilvivere rstsetaetaotsoiss,i si,nifnlaflmammamtiaotnio annadn ndecnreocsrios.s Sisi.gnSiifgicnainfitc adniftfedriefnfecreesn wceesrew  ere
oobbsseerrvveedd bbeettwweeeenn aarrttiicchhookkee llooww‐-ddoossee (0(0.4.4 gg/k/gk gBWBW) a)nadn hdighhig‐dho-dseo s(0e.8(0 g.8/kgg/ BkWg)B gWro)ugpros uinp asliln scaollres core
rreessuullttss.. IInntteerreessttiinnggllyy,, tthhee ddeeccrreeaassee eefffefecct toof faratritcihcohkoek ewwasa isni an daodsoe‐sdee-dpeenpdenendte mntamnnaenrn. er.
A  B leukocytes   C 
infiltration  CV 
CV 
CV 
normal hepatocytes 
sinusoid  steatosis  
necrosis  
D  E  F 
leukocytes   CV 
infiltration 
CV 
CV 

s
es4G &&& ore4H &&&
r &&& c
sis sco23 *** ## tion s23 *** ##
eato1 ### ### mma1 ### ### ###
St0 Control EtEOtOH+HEtBiOfHen+dAarttieEcthOoHk +e(A0r.t4i)EcthOoHk+e(A0r.t8i)choke(1.6) Infla0 Control EtEOtOH+HEtBiOfHen+dAarttieEcthOoHk +e(A0r.t4i)EcthOoHk+e(A0r.t8i)choke(1.6)
(G)  (H) 
Figure2.Cont.
NutrieNnutstr2ie0n1t7s, 290,1170,0 90, 1000  7of127 of 12 
4I
s &&
e
r
o3
c
s ***
s ##
si2 ###
o ##
r ###
c1
e
N
0 Control EtEOtOH+HEtBiOfHen+dAarttieEcthOoHk +e(A0r.t4i)EcthOoHk+e(A0r.t8i)choke(1.6)
(I) 
FigurFeig2.uEreff e2c.t oEfffaerctti chofo kaertoinchaolkcoeh oonl- inadlcuocheodl‐hinisdtoupceadth ohliosgtoicpaaltchhoalnoggeicsailn clhivaenrgteisss uines .li(vAe)r Ctoisnstureosl. (A) 
groupC,o(nBt)roEl tgOrHougpr, o(uBp) ,E(tCO)Hp ogsriotiuvpe, c(oCn) tproolsigtirvoeu pco,n(Dtr)oll ogwro-udpo,s e(Dg)r oluowp,‐d(Eo)sem girdodulpe-, d(oEs)e mgirdodulpe‐,dose 
(F)higgrho-duops, e(Fg)r ohuigph,(‐Gdo)sset egartoousips,s c(Gor)e ss,te(Hat)oisnisfl ascmomreast, io(Hn)s cionrfelasm,(mI)anteiocrno ssicsosrceosr, e(sI,) annedcrcoesnitsr aslcovreeins, and 
(CV).cHenetmraal tvoexiynl in(CaVn)d. Heoesminatsotaxiynliinng a.nOdr iegoinsianl mstaaignninifigc. aOtiroing,in2a0l0 ×m.agVnailfuiceastrieopnr, e2s0e0n×t. mVeaaluness± reSpDresent 
(n=1m0)e;a*n**s p± <SD0. 0(n0 1= v1s0.);t h**e* cpo <n t0r.o0l0g1 rvosu. pth;e## cpon<tr0o.0l 1g,r#o#u#pp; <## 0p. 0<0 01.0v1s,. #t#h# pe <E t0O.0H01g vros.u tph;e& E&tOpH< 0g.r0o1u,p; && 
&&&pp << 00..00011, .&&& p < 0.001. 
3.4. Hepatic Antioxidant and Oxidative Stress Markers 
3.4. HepaticAntioxidantandOxidativeStressMarkers
Oxidative stress plays a central role in alcohol‐induced liver injury and ALD pathogenesis [33]. 
Oxidativestressplaysacentralroleinalcohol-inducedliverinjuryandALDpathogenesis[33].
To counterbalance oxidative stress, a number of enzymatic (e.g., SOD) and non‐enzymatic (e.g., 
Tocounterbalanceoxidativestress,anumberofenzymatic(e.g.,SOD)andnon-enzymatic(e.g.,GSH)
GSH)  mechanisms  have  evolved  to  protect  against  reactive  oxygen  species  (ROS)  caused  by 
mechanismshaveevolvedtoprotectagainstreactiveoxygenspecies(ROS)causedbyoxidativestress
oxidative stress in alcoholic liver injury. SOD reduces the generation of free radical and lipid 
in alcoholic liver injury. SOD reduces the generation of free radical and lipid peroxide and even
peroxide and even accelerates its clearance, thus reducing the damage of liver cells [34,35]. GSH 
acceleratesitsclearance,thusreducingthedamageoflivercells[34,35]. GSHservesasareservoir
serves as a reservoir for cysteine to counteract ROS [36]. SOD and GSH activities reflect the body’s 
for cysteine to counteract ROS [36]. SOD and GSH activities reflect the body’s ability of clearing
ability of clearing oxygen free radicals indirectly. Meanwhile, MDA is the main product of lipid 
oxygenfreeradicalsindirectly. Meanwhile,MDAisthemainproductoflipidperoxidationinduced
peroxidation induced by ROS; in addition, MDA content can reflex the severity of free radical 
by ROS; in addition, MDA content can reflex the severity of free radical attacks on the body cells
attacks on the body cells indirectly [37,38]. Regarding oxidative stress, excessive ROS and MDA will 
indirectly[37,38]. Regardingoxidativestress,excessiveROSandMDAwillconsumealargequantity
consume a large quantity of antioxidation factors, such as SOD and GSH. The in vivo SOD and GSH 
ofantioxidationfactors,suchasSODandGSH.TheinvivoSODandGSHwouldbeunabletofight
would be unable to fight against the excessively increasing ROS and MDA once the balance is lost. 
againsttheexcessivelyincreasingROSandMDAoncethebalanceislost. Maintainingsuitablelevels
Maintaining suitable levels of SOD, GSH, and MDA plays an important role in liver protection from 
ofSOD,GSH,andMDAplaysanimportantroleinliverprotectionfromattacksoffreeradicals.
attacks of free radicals. 
AsshowninFigure3,theSODandGSHintheEtOHgroupdecreasedby20.24%(p<0.05)and
As shown in Figure 3, the SOD and GSH in the EtOH group decreased by 20.24% (p < 0.05) and 
39.08%(p<0.05)respectivelycomparedwiththecontrolgroup. Bycontrast,hepaticMDAactivity
39.08% (p < 0.05) respectively compared with the control group. By contrast, hepatic MDA activity 
increasedsignificantlyby255.14%(p<0.001)intheEtOHgroupcomparedwiththecontrolgroup.
increased significantly by 255.14% (p < 0.001) in the EtOH group compared with the control group. 
ResultsshowedthattheactivitiesofSODandGSHinthebloodincreasedsignificantly,whereasMDA
Results showed that the activities of SOD and GSH in the blood increased significantly, whereas 
decreasedsignificantlywhenartichokewassubjectedtoanalcoholdiet. Notably,adose-dependent
MDA  decreased  significantly  when  artichoke  was  subjected  to  an  alcohol  diet.  Notably,  a 
mechanism was found for all SOD, GSH and MDA activities. High-dose artichoke (1.6 g/kg BW)
dose‐dependent mechanism was found for all SOD, GSH and MDA activities. High‐dose artichoke 
administrationssignificantlyprotectedagainsttheacutealcohol-inducedelevationofMDAactivity
(1.6 g/kg BW) administrations significantly protected against the acute alcohol‐induced elevation of 
(p<0.001)andreductionofSOD(p<0.05)andGSH(p<0.05). Thissuggestedthatpretreatmentof
MDA activity (p < 0.001) and reduction of SOD (p < 0.05) and GSH (p < 0.05). This suggested that 
artichokecouldprotecttheliverfromacutealcohol-inducedhepaticoxidativestress.
pretreatment of artichoke could protect the liver from acute alcohol‐induced hepatic oxidative stress.
Nutrients2017,9,1000 8of12
Nutrients 2017, 9, 1000  8 of 12 
30B #
250A
# # # 25 # #
g)200 * mg)20 # ##
D(U/m110500 SH(U/1105 *
O G
S
50 5
0 Control EtEOtOH+HEtBiOfHe+ndAarttieEcthOoHk +e(A0r.t4iE)cthOoHk+e(A0r.t8i)choke(1.6) 0 Control EtEOtOHH+EtBiOfHen+dAarttieEcthOoHk +e(A0r.t4iE)cthOoHk+e(A0r.t8i)choke(1.6)
(A)  (B) 
&&
6C *** &&
L )5 &&
/
l4 **
o
m
u3
DA(2 ### ### ## ###
M
1
0 Control EtEOtOH+HEtBiOfHen+dAarttieEcthOoHk+e(A0r.t4i)EcthOoHk+e(A0r.t8i)choke(1.6)
(C) 
FigurFei3g.uEreff e3c. tEsfoffecatrst icohf oakrteicohnolkeve eolsno lfeSvOelDs o(Af S),OGDS H(A(B), )G,aSnHd M(BD), Aan(Cd )M.VDalAu e(sCr)e. pVreasluenest mreeparnesse±nSt Dmeans 
(n=1±0 )S;D* p(n< =0 1.005),; *** pp << 00.0.051, ,**** p* p< <0.001.0, 0*1**v ps .<t h0.e00co1 nvtsr.o tlhger ocounpt;r#olp g<ro0u.0p5;, ## #p p< <0.00.50,1 #,# #p# #< p0.<010,. 0##0#1 pv <s .0.001 
theEtvOs.H  tghreo uEp;tO&&H pg<r0o.u01p.; SO&&D :ps up< er0o.x0i1d. eSdOisDm:u tasusep;eGroSxHid:ge ludtaistmhiountaes;eM; DGAS:Hm: algolnudtaiathldioenhey;d eM. DA: 
malondialdehyde. 
3.5. TLR4andNF-κBExpressionLevels
3.5. TLR4 and NF‐κB Expression Levels 
TLRsareafamilyofpattern-recognitionreceptorsthatenabletheinnateimmunesystemtoreact
TLRs are a family of pattern‐recognition receptors that enable the innate immune system to 
immediatelytoinfectionsbyrecognizingboththebacterialandviralconstituents[39]. Amongthese,
react immediately to infections by recognizing both the bacterial and viral constituents [39]. Among 
TLR4isresponsibleforLPS-inducedinflammatoryreaction.Acuteexcessivealcoholexposureincreases
these, TLR4 is responsible for LPS‐induced inflammatory reaction. Acute excessive alcohol exposure 
gut permeability, which allows gut-derived endotoxins (LPS) to bind with TLR4. The interaction
increases gut permeability, which allows gut‐derived endotoxins (LPS) to bind with TLR4. The 
ofTLR4andLPSresultintheactivationofNF-κB,whichiscomposedofthep50andp65subunits.
interaction of TLR4 and LPS result in the activation of NF‐κB, which is composed of the p50 and p65 
Generally,NF-κBissequesteredasaninactivecomplexincytoplasmbyinhibitorysubunit-inhibitory
subunits. Generally, NF‐κB is sequestered as an inactive complex in cytoplasm by inhibitory 
κB(IκB).Onceactivated,IκBisphosphorylatedanddegraded,whichallowsNF-κBp50totranslocateto
subunit‐inhibitory κB (IκB). Once activated, IκB is phosphorylated and degraded, which allows 
thenucleusandinducetheexpressionofitstargetgenes,henceresultinginreleaseofpro-inflammatory
NF‐κB p50 to translocate to the nucleus and induce the expression of its target genes, hence 
cytokines,suchastumornecrosisfactorα(TNF-α),theinterleukin(IL)-1receptor(IL-1β),IL-6,and
resulting in release of pro‐inflammatory cytokines, such as tumor necrosis factor α (TNF‐α), the 
IL-8.Allofthesepro-inflammatorymediatorscanaggravateinflammatoryprogress,ultimatelyleading
interleukin (IL)‐1 receptor (IL‐1β), IL‐6, and IL‐8. All of these pro‐inflammatory mediators can 
tohepaticfibrosis,cirrhosis,andhepatocellularcarcinoma[8,40,41]. Thus,theexpressionlevelsof
aggravate  inflammatory  progress,  ultimately  leading  to  hepatic  fibrosis,  cirrhosis,  and 
upstreamindexTLR4anddownstreamindexNF-κBinaninflammatorypathwayaretwopivotal
hepatocellular  carcinoma  [8,40,41].  Thus,  the  expression  levels  of  upstream  index  TLR4  and 
indicatorsimplicatedininflammation.
downstream index NF‐κB in an inflammatory pathway are two pivotal indicators implicated in 
AsshowninFigure4,thepositivecellsofTLR4werestainedasbrownoryellow,whichwere
inflammation. 
distributed mainly in the cytoplasm and cell membrane, whereas the positive cells of NF-κB p50
As shown in Figure 4, the positive cells of TLR4 were stained as brown or yellow, which were 
werestainedasbrowninthenucleus. ThemeanIODresultshowedthatexpressionlevelsofTLR4
distributed mainly in the cytoplasm and cell membrane, whereas the positive cells of NF‐κB p50 
andNF-κBp50intheEtOHgroupwereelevatedsignificantly,by147.88%(p<0.001)and103.58%
were stained as brown in the nucleus. The mean IOD result showed that expression levels of TLR4 
(p<0.001),respectively,comparedwiththecontrolgroup.However,theseincreaseswereattenuatedby
and NF‐κB p50 in the EtOH group were elevated significantly, by 147.88% (p < 0.001) and 103.58% (p 
artichokepretreatmentinadose-dependentmannercomparedwiththeEtOHgroup. InthemeanIOD
< 0.001), respectively, compared with the control group. However, these increases were attenuated 
ofTLR4expression,asignificantdifference(p<0.05)wasobservedbetweenlow-dose(0.4g/kgBW)
by artichoke pretreatment in a dose‐dependent manner compared with the EtOH group. In the mean 
IOD of TLR4 expression, a significant difference (p < 0.05) was observed between low‐dose (0.4 g/kg
Nutrients2017,9,1000 9of12
Nutrients 2017, 9, 1000  9 of 12 
aBnWd)h iagnhd- dhoisgeh(‐1d.o6sge /(k1g.6B Wg/k)ga rtBicWh)o kaertpicrhetorkeea tmpreentrtegartmouepnst .gBraosuepdso. nBtahseedre sounl tsth,teh erepsruelvtse, ntthivee  effect
opfreavretincthivoek eeffaegcat ionfs atraticcuhtoekea lacgoahionls-ti nadcuutcee adlcloivhoelr‐iinndjuurcyedw liavsera sinsojucriya twedasw aistshocTiaLtRed4 wdoitwh nTrLeRg4u lation.
Tdhoewancrteigvualtaiotinono.f  NThFe-κ Bacwtiavsattihoenr efoofr eNinFh‐κibBi tewd.asT htuhse,rtehfoerree leinahseiboitfepdr. o-Tinhfluas,m  mthae torreylecaysteo koinf eswas
lpimroi‐tiendfl.amThmeaitnoflrya mcymtoaktionreys wefafes cltimwiatesdt.h Tehne uinltfilmamatmelaytosruyp epffreecsts weda.s then ultimately suppressed. 
TLR4  NF‐κB
100A &
***
Control 
80 **
**
) # #
(% 60 #
4
LR 40 ###
T
20
0
EtOH  Control EtEtOOHH+EtBOifHe+ndArattiEecthOoH k+e(A0r.ti4Ec)thOoHk+e(A0r.ti8c)hoke(1.6)
100B
Positive 
80 ***
B(%) 60 # #* # #
F-k 40 ###
N
20
0
Low  Control EtEtOOHH+EtBOifHe+ndArattiEecthOoH k+e(A0r.ti4Ec)thOoHk+e(A0r.ti8c)hoke(1.6)
Middle 
High 

Figure 4. Immunohistochemical analysed results of mice liver in TLR4 and NF‐κB. (A) mean IOD of 
Figure4. ImmunohistochemicalanalysedresultsofmiceliverinTLR4andNF-κB.(A)meanIOD
TLR4, (B) mean IOD of NF‐κB, and (Red Arrows) positive cells. Original magnification, 200×. * p < 
of TLR4, (B) mean IOD of NF-κB, and (Red Arrows) positive cells. Original magnification, 200×.
*p<0.05,**p<0.01,***p<0.001vs. thecontrolgroup;#p<0.05,###p<0.001vs. theEtOHgroup;
&p<0.05.TLR:toll-likereceptor;NF-κB:nuclearfactor-kappaB;IOD:integratedopticaldensity.
Nutrients2017,9,1000 10of12
4. Conclusions
DatapresentedclearlyshowedthatartichokereducedlevelsofAST,ALT,TG,TC,andMDA,
whileincreasingthelevelsofSODandGSHinanacuteALDmicemodel.Degenerationandnecrosisof
hepaticcellswerealsosignificantlyattenuatedbyartichoke.Besides,TLR4andNF-κBexpressionlevels
inlivertissuewereeffectivelydownregulatedbyartichoke. Ourstudydemonstratedthat1.6g/kgBW
artichokeexhibitedsignificantpreventativepotentialityforacutealcohol-inducedliverinjury,whereas
theeffectof0.4g/kgBWartichokewasnotsignificant. Thepreventativeeffectofartichokewasmainly
duetoitsabilitytoattenuateoxidativestressandinhibittheinflammatorypathwaybysuppressingthe
expressionlevelsofTLR4andNF-κB.Overall,1.6g/kgBWartichokecouldbeaneffectiveadjuvantin
thepreventionofacuteALD.Moreover,interventionsinthepathogenesisoftheTLR4/NF-κBpathway
showeditspotentialasatherapeutictargetinacutealcohol-inducedliverinjury.
Acknowledgments: This work was supported by the Xiamen Science and Technology Program
(No.3502Z20173501),theEducationDepartmentofHunanProvince(No. 17A154),theJointFundsofHunan
ProvincialNaturalScienceFoundationofChina(No.2017JJ4043),andtheFujianProvinceYoungandMiddle-aged
TeacherEducationResearchProject(No.JAT170049).
AuthorContributions:Tangconceivedanddesignedtheexperiments;TangandWeiperformedtheexperiments;
TangandWeianalyzedthedata;Weiwrotethepaper;Dengcontributedreagents/materialstools;Leirevised
themanuscript.
ConflictsofInterest:Theauthorsdeclarethattheyhavenoconflictofinterets.
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