Research Article Electroacupuncture Could Regulate the NF- B Signaling Pathway to Ameliorate ...

16 Pages·2015·4.18 MB·English

HindawiPublishingCorporation
Evidence-BasedComplementaryandAlternativeMedicine
Volume2013,ArticleID924541,15pages
http://dx.doi.org/10.1155/2013/924541
Research Article
𝜅
Electroacupuncture Could Regulate the NF- B Signaling
Pathway to Ameliorate the Inflammatory Injury in Focal
Cerebral Ischemia/Reperfusion Model Rats
Wen-yiQin,1,2YongLuo,1,2LingChen,1,2TaoTao,3
YangLi,1,2Yan-liCai,1,2andYa-huiLi2,4
1DepartmentofNeurology,TheFirstAffiliatedHospitalofChongqingMedicalUniversity,Chongqing400016,China
2ChongqingKeyLaboratoryofNeurology,1YouyiRoad,YuzhongDistrict,Chongqing400016,China
3DepartmentofNeurology,AffiliatedHospitalofLuzhouMedicalCollege,Luzhou,SichuanProvince64600,China
4DepartmentofOncology,TheFirstAffiliatedHospitalofChongqingMedicalUniversity,Chongqing400016,China
CorrespondenceshouldbeaddressedtoYongLuo;[email protected]
Received23January2013;Revised10June2013;Accepted18June2013
AcademicEditor:LijunBai
Copyright©2013Wen-yiQinetal.ThisisanopenaccessarticledistributedundertheCreativeCommonsAttributionLicense,
whichpermitsunrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalworkisproperlycited.
Theactivatednuclearfactor-KappaBsignalingpathwayplaysacriticalroleininducinginflammatoryinjury.Ithasbeenreported
thatelectroacupuncturecouldbeaneffectiveanti-inflammatorytreatment.Weaimedtoexplorethecomplexmechanismbywhich
EAinhibitstheactivationoftheNF-𝜅Bsignalpathwayandameliorateinflammatoryinjuryintheshortterm;theeffectsofNEMO
BindingDomainpeptideforthispurposewerecompared.FocalcerebralI/Rwasinducedbymiddlecerebralarteryocclusionfor
2hrs.Total380maleSprague-Dawleyratsareinthestudy.Theneurobehavioralscores,infarctionvolumes,andthelevelsofIL-1𝛽
andIL-13weredetected.NF-𝜅Bp65,I𝜅B𝛼,IKK𝛼,andIKK𝛽wereanalyzedandtheabilityofNF-𝜅BbindingDNAwasinvestigated.
TheEAtreatmentandtheNBDpeptidetreatmentbothreducedinfarctsize,improvedneurologicalscores,andregulatedthelevels
ofIL-1𝛽andIL-13.ThetreatmentreducedtheexpressionofIKK𝛼andIKK𝛽andalteredtheexpressionofNF-𝜅Bp65andI𝜅B𝛼
inthecytoplasmandnucleus;theactivityofNF-𝜅Bwaseffectivelyreduced.WeconcludethatEAtreatmentmightinterferewith
theprocessofNF-𝜅Bnucleartranslocation.AnditalsocouldsuppresstheactivityofNF-𝜅Bsignalingpathwaytoamelioratethe
inflammatoryinjuryafterfocalcerebralischemia/reperfusion.
1.Introduction In Asia, especially in China, electroacupuncture (EA) is a
generalmethodforthetreatmentofcerebrovasculardiseases.
The pathological mechanism of focal cerebral ischemia/re- Many reports of basic research and clinical studies have
perfusion is very complex and involves a myriad of dis- discussedtheefficacyofEAinregulatinginflammatoryinju-
tinctmolecularsignalingandcytokinespathways[1].Asan ry [9–12]. However, the exact mechanism and functional
important and crucial pathological process following focal targets of EA treatment need to be elucidated. Significant-
cerebralischemia/reperfusion[2,3],theexcessiveactivation ly, the mechanism by which EA suppresses the activity
ofinflammationcouldinducethemoreseriousdamage,even of NF-𝜅B signaling pathway at an early stage of cerebral
thefatalinjurythanischemicinjuryitself[4].Inflammation ischemia/reperfusionneedstobediscussed.
is a pathological process that occurs via an amplification TheNF-𝜅Bfamilyoftranscriptionfactorsconsistsoffive
cascade,theNF-𝜅Bsignalingpathway[5],andthecomplex members, p50, p52, p65 (Rel A), c-Rel, and Rel B. NF-𝜅B
cytokine network is critical for the occurrence of inflam- prototypeisaheterodimercomposedoftheRelA(p65)and
matory responses [6, 7]. Furthermore, more sophisticated NF-𝜅B1(p50)subunits.Thisvariantisthemostpotentgene
approaches are required for the treatment of focal cerebral transactivator among the NF-𝜅B family and is the major
ischemia/reperfusionbecauseofthenarrowtherapeutictime NF-𝜅Bproteinfoundinthenucleus[13,14].Theinhibitory
windowandcomplexpathologicalfeaturesofthisdisease[8]. protein I𝜅B𝛼, which masks the nuclear localization signals
2 Evidence-BasedComplementaryandAlternativeMedicine
(NLS)ofNF-𝜅Bp65proteinandkeepsthemsequesteredin treatment.Theanimalswereallowedfreeaccesstostandard
aninactivestateinthecytoplasm,leadstoconstantshuttling rodent diet and tap water. Total 380 rats were randomly
of NF-𝜅B/I𝜅B𝛼 complexes between the nucleus and the dividedtofourgroups:ashamgroup(𝑛 = 95),anI/Rgroup
cytoplasm[15,16].DegradationofI𝜅Bs(especiallyI𝜅B𝛼)isa (MCAOfor2h,𝑛=95),anEAgroup(𝑛=95),andtheNBD
rapidlyinducedsignalingeventthatisinitiateduponspecific group(𝑛 = 95).Thesegroupswereassessedfrom6hto48h
phosphorylationofthemoleculesby activatedIKK [16, 17]. afterreperfusion.
The IKK complex mainly contains IKK𝛼, IKK𝛽, and a reg-
ulatorysubunitNEMO(NF-𝜅Bessentialmodulator/IKK𝛾).
2.2.InductionofFocalCerebralIschemia/Reperfusion. Tran-
IKK𝛾isnotfunctionalintheabsenceofitsinteractionswith
sient focal cerebral ischemia was induced by MCAO in
IKK𝛼 and IKK𝛽 [18]. Thus, NF-𝜅B p65/I𝜅B𝛼, IKK𝛼, and
IKK𝛽areconsideredtobesomeofthemostimportantfactors rats as previously described [21, 22]. Briefly, SD rats were
involvedinactivationoftheNF-𝜅Bsignalingpathwayduring fasted for 12h but were allowed free access to water before
surgery. Anesthetization was induced with 3.5% chloral
the early stages of focal cerebral I/R. Consequently, we will
hydrate (1mL/100g) by intraperitoneal injection. The right
observe the change of those important factors in order to
commoncarotidarteryandtherightexternalcarotidartery
explorethemechanismofEAtreatmentfortheinflammatory
injuryafterfocalcerebralI/R. were exposed through a ventral midline neck incision and
The NF-𝜅B specific inhibitor (wild-type) NBD peptide wereligatedproximallyandtemporarily.A2.0monofilament
thatdisruptstheinteractionofNEMO(IKK𝛾)withtheIKK𝛽 nylon suture (Ethicon Nylon Suture; Ethicon Inc., Osaka,
has some advantages including the following: (1) does not Japan)withitstiproundedbyheatinginaflamewasinserted
affect basal NF-𝜅B activity, (2) has well-defined molecular through an arteriectomy in the common carotid artery just
sitesofaction,(3)isspecificfortheclassicalNF-𝜅Bactivation below the carotid bifurcation and then advanced into the
pathwayanddoesnotaffecttheNF-𝜅Balternativepathway, internalcarotidarteryapproximately18mmto20mmdistal
and(4)doesnottargettheactivedomainofthekinaseand to the carotid bifurcation until a mild resistance indicated
is therefore unlikely to affect other essential kinases [19]. occlusion of the origin of the anterior cerebral artery and
ThePharmacologycharacteristicofNBDpeptidepromptsa themiddlecerebralartery.Reperfusionwasaccomplishedby
clear understanding about the mechanism of NBD peptide withdrawingthesutureafter2hofischemia.Theincisionwas
toinhibitexcessiveNF-𝜅Bactivation[20].Onthecontrary, suturedandsterilized.Theanimalsweremaintainedonthe
the mechanism by which EA inhibits the NF-𝜅B signaling ∘
37 Cthermostatictable.Theshamsurgerywasconductedin
pathway activation is not clear. Therefore, we used NBD
the ischemia/reperfusion (I/R) surgery groups by insertion
peptide as a positive intervention method control for IKKs
ofthenylonmonofilamentsutureto10mmintotheinternal
functionresearchinordertoinvestigatetheEAmechanism
carotidartery;donotinsertintothemiddlecerebralartery.
for inhibition of excessive activation of NF-𝜅B after focal
Bodytemperaturewasmonitoredinallanimals.Therec-
cerebralischemia/reperfusion.Furthermore,theeffectsofEA tal temperature was maintained at 38∘C±0.5∘C throughout
and NBD peptide treatment in this process were compared
allportionsoftheexperimentbymeansofarectalthermistor
inordertofurtherelucidatetheunderlyingmechanismand
probeandathermostaticallyregulatedheatinglampplaced
targetsofEAtreatment.
abovethebodyoftheanimal.Intrastriataltemperaturewas
According to the traditional theory of Traditional Chi-
monitored and was adjusted by manipulating the height of
neseMedicineandthebasicacupunctureresearchprinciple,
a small high-intensity lamp placed above the head. Prior
the“Baihui”(GV20)and“Siguan”acupointswerechosein
this study. The “Siguan” acupoint is composed of the two to the ischemic insult, intrastriatal temperature was held at
“Hegu”(LI4)andtwo“Taichong”(LR3)acupoints.Ouraim 36.5∘C±0.5∘Cinallgroupsduringischemia[23,24].
was to define the effective targets and mechanism of EA
treatmentthatinfluencedtheNF-𝜅Bsignalingpathwayand 2.3. Electroacupuncture Treatment. In the EA groups, the
its function in alleviating inflammatory injury after focal total SD rats (𝑛 = 95) were anesthetized with 3.5% chloral
cerebralischemia/reperfusionduringtheearlystages. hydrate(1mL/100g)byintraperitonealinjection.According
totheExperimentalAnimalsMeridiansMapping,the“Bai-
2.MaterialsandMethods hui” (GV 20)/“Siguan”acupointswere selected, which were
located at the intersection of the sagittal midline and the
2.1. Animals. The experimental protocol used in our study line linking the ears, the second metacarpal midpoint of
was approved by the Ethics Committee for Animal Exper- radialside,andthesecondtoetibialcollateralintherearof
imentation of the First Affiliated Hospital of Chongqing phalanx. These acupoints were stimulated at an intensity of
Medical University and all procedures were conducted in 1mAandfrequencyof2/20Hzfor30minusingtheG6805-
accordancewiththeNationalInstitutesofHealthGuidelines 2EAInstrument(Modelno.227033;BeijingXinshengLtd.,
∘
for Animal Research (Guide for the Care and Use of Labo- China). The SD rats were maintained on the 37 C thermo-
ratoryAnimals).MaleSDrats(280–300g)wereprovidedby static table. The EA treatment was given at once each day
theExperimentalAnimalCenteroftheChongqingMedical (from 6h to 48h after reperfusion). After ischemia for 2h,
Universityandhousedundercontrolledconditionswitha12- beganthefirstEAtreatmentwhenthenylonmonofilament
hourlight/darkcycle,atemperatureat22±2∘C,andhumidity waswithdrawn.From6hto48h,theEAtreatmentistwice,
at 60% to 70% for at least one week before operation and 3times,4times,and5timesorderly.
Evidence-BasedComplementaryandAlternativeMedicine 3
2.4. Drug Intervention Methods. NBD peptide (ENZO Life were analyzed using the Motic Med 6.0 CMIAS pathology
SciencesInc.,no.BML-P607-0500,solublein50𝜇LDMSO, imageanalysissystem(BeihangMoticInc.,Beijing,China).
final concentration 10𝜇g/𝜇L) was first time injected intrac-
erebroventricularly2hbeforesurgeryaspreviouslydescribed 2.8. Double-Immunofluorescence Labeling. Brain tissue was
[25].Briefly,thetotal72ratswereanesthetizedandattached fixed and frozen, and the 10𝜇m thick coronal brain slices
to a stereotaxic instrument (Stoelting, USA). According to werecutbeginning8mmfromtheanteriortipofthefrontal
∘
stereotacticmappingcombinedwithratsize,wedetermined lobe. Frozen sections were air dried at 50 C for 30min.
the right lateral ventricle position and drilled a cranial Sections were treated with ten mol/L sodium citrate buffer
window(cross-stitchcentered,rightshift1.3mm,backward (pH6.0)andheatedinamicrowaveovenfor20minat92–
1.5mm,downward3.8mm)forinjectionof5𝜇LNBDpeptide 98∘C. Tissues were permeated with 1% Triton X-100, and
∘
onceeachday,until48hafterreperfusion.TheNBDpeptide sections were incubated in 5% goat serum for 1h at 37 C.
∘
injection times from 6h to 48h are twice, 3 times, 4 times, Incubationswithprimaryantibodieswerecarriedoutat4 C
and5times. overnight.TheprimaryantibodieswereNF-𝜅Bp65(C22B4)
rabbitmAb(CellSignalingTechnology,1:50),I𝜅B𝛼(L35A5)
2.5. Neurobehavioral Evaluation. Neurobehavioral evalua- mouse mAb (Cell Signaling Technology, 1:50), IKK𝛼 (M-
tionwascarriedoutintheI/Rgroup,EAgroups,andNBD 110)(SantaCruz,no.sc-7183,1:25),andIKK𝛽(P-20)(Santa
peptide group. After focal cerebral ischemia/reperfusion at Cruz, no. sc-34673, 1:25). After washing, slides were incu-
differenttimepoints,theneurologicdeficitscoresofmodel batedwithfluorescentisothiocyanate-conjugatedanti-rabbit
SD rats were assessed by an investigator who was unaware (1:50), anti-mouse (1:50), and anti-goat (1:100) antibodies
∘
of animal grouping. A modified neurologic deficit score for2hat 37 C.Finally,sectionsofischemicpenumbraarea
describedbyLongaetal.wasusedforneurologicassessment. wereexaminedbylaser-scanningconfocalmicroscopyonan
0:nodeficit;1:failuretoextendleftforepawfully;2:circling OlympusIX70invertedmicroscope(Olympus,Tokyo,Japan)
to the left; 3: falling to the left; 4: no spontaneous walking equipped with Fluoview FVX confocal scan head (Leica
withadepressedlevelofconsciousness.Animalsscoring2to MicrosystemsHeidelbergGmbH,Wetzlar,Germany).
3wereincludedinexperiments.Neurobehavioralscoreswere
determinedaccordingtothemethodsdescribedbyGarciaet 2.9. Fluorescent Quantitative PCR. Total cellular RNA was
al.[26]andBedersonetal.[27]. isolatedwithTrizol(Invitrogen,Paisley,UK).cDNAwassyn-
thesizedwithSuperscriptReverseTranscriptase(Invitrogen).
2.6.InfarctVolumeAssessment. Theanimals(𝑛 = 5foreach ThepolymerasechainreactionwasperformedwiththeiQ5
group) were decapitated and 2mm thick coronal sections GradientReal-TimePCRDetectionSystem(Bio-Rad)using
from throughout the brain were stained with 2% 2,3,5- primers (Takarad) for NF-𝜅B p65, I𝜅B𝛼, IKK𝛼, and IKK𝛽
triphenyltetrazolium chloride (Sigma-Aldrich) to evaluate
(Table1).Datawereindividuallynormalizedtothemeanof
theinfarctvolume,asdescribedbyWangetal.[28].
the relative expression of GAPDH. Quantitative PCR (Q-
PCR) was performed using the Hot Start Fluorescent PCR
2.7.ImmunohistochemistryAnalysis. Immunohistochemistry Core Reagent Kits (SYBR Green I). Each reaction mixture
was performed using the avidin-biotin-peroxidase complex consisted of first-strand cDNA template, 1.25𝜇L of primer-
method.Briefly,brainswerefixedin4%phosphate-buffered probe, and 12.5𝜇L of SYBR Green in a total volume of
paraformaldehyde(PFA)andimmersedinto20%sucrosefor 25𝜇L.Thefollowingcyclingconditionswereused:95∘Cfor
3h.A4mmthickcoronalbrainslicewascut,beginning8mm ∘ ∘
10min, followed by 40 cycles of 95 C for 5s and 60 C for
awayfromtheanteriortipofthefrontallobe.Thenalongi-
30s.Subsequently,eachsampleunderwentdissociationcurve
tudinal cut (from top to bottom) was made approximately
analysis to examine primer-target specificity as previously
2mm from the midline through the ischemic hemisphere
described[29].
toremovemedialparts.Paraffin-embeddedtissuesweresec-
tionedanddewaxedforantigensretrievalbyimmersingslides
2.10. Western Blot Analysis. Rats were anesthetized with
in0.01mol/Lcitratebuffer,pH6.0,followedbyheatingina
∘ 3.5% chloral hydrate (1mL/100g) by intraperitoneal injec-
microwaveoven(temperature92–98 C)for20min.Sections
tion. The cortex of the ischemic area was homogenized in
werecooledtoroomtemperatureandwashedwithPBSfor
RIPAlysisbuffer(Beyotime,no.P0013B).Totalproteinand
5minandendogenousperoxideswereblockedin3%H2O2
cytoplasmic/nuclear proteins were extracted on ice using
for15min.Sectionswerethenincubatedfor30minwith5%
NuclearandCytoplasmicProteinExtractionKits(Beyotime,
normalgoatbloodserumtoblocknonspecificbinding.The
primaryantibody,NF-𝜅Bp65(C22B4)rabbitmAb(CellSig- no. P0027). Western blot analysis was performed with
45𝜇g protein extract separated by 10% SDS-PAGE, which
nalingTechnology,1:50),wasaddedandsectionsincubated
at 4∘C overnight. The immunostain SP Kit was used. For were then transferred to a polyvinylidene fluoride (PVDF)
negative sham group, the primary antibodies were replaced membrane (Millipore Corporation). Nonspecific epitopes
withPBS.Aslideimageofischemicpenumbraareafromeach were blocked with 5% skim milk/Tween-20-Tris-buffered
sectionwasscannedandacquiredusinganOLYMPUSPM20 saline. The following primary antibodies which were used
automaticmicroscope(Olympus,Tokyo,Japan)andaTCFY- indouble-immunofluorescencelabelinganalysis:appropriate
2050(YuanchengInc.,Beijing,China)pathologysystem.The horseradish peroxidase conjugated antibodies (anti-rabbit
visualfieldsineachsectionimage(fivesectionsineachbrain) (1:4000), anti-mouse (1:4000), and anti-goat (1:1000))
4 Evidence-BasedComplementaryandAlternativeMedicine
Table1:PrimersequencesandannealingtemperaturesemployedforQ-PCR.
Primername Sequence Annealingtemperature
󸀠 󸀠
NF-𝜅Bp65 F󸀠5-ATCGTGGAGCACTTGGTGACT-3 󸀠 55.2∘C
R5-GCCCTGGTAGGTTACTCTGTTGA-3
󸀠 󸀠
I𝜅B𝛼 F5󸀠-GTTTCCCCTCATCTTTCCCTCA-3 󸀠 56.7∘C
R5-GGGTGCGTCTTAGTGGTATCTGT-3
󸀠 󸀠
IKK𝛼 F5󸀠-GTCAGGAGAAGTTCGGTTTGA-3 54.2∘C
R5-ATTCCAGTTTCACGCTCATGGAT-3
󸀠 󸀠
IKK𝛽 F5󸀠-GTCTTGTTGATGGTTCCTGACT-3󸀠 56.0∘C
R5-AAGACAAACGAGGGCCTCACAT-3
󸀠 󸀠
F5-CTTAGCACCCCTGGCCAAG-3 ∘
GAPDH 󸀠 󸀠 54.0C
R5-GATGTTCTGGAGAGCCCCG-3
secondarydetectionantibodieswereincubatedfor2h.Bio- 5
Rad apparatus (Bio-Rad Laboratories, Richmond, CA) and
QuantityOnesoftwareversion4.6.2(Bio-RadLaboratories)
4
wereusedtoscanimmuneblotsandforanalysis.
es ∗ ∗ ∗
or # #
2.11. Electrophoretic Mobility Shift Assay (EMSA). Nuclear al sc 3
c
extracts were prepared as previously described. The NF- gi †
o
𝜅NBF-d𝜅oBubcolen-ssetrnasnudsedseqouliegnocneuc(l5e󸀠o-tAidGeTcToGrrAesGpGonGdGinAgCtToTtThe- Neurol 2
󸀠
CCCAGGC-3 ) was obtained from Santa Cruz and was 1
end-labeled with Terminal Deoxynucleotidyl Transferase
(TdT)usingBiotin-11-dUTP(Pierce).Nuclearextracts(8𝜇g) 0
were incubated at room temperature for 20min with 1𝜇L h h h h h h h h h h h h
6 2 4 8 6 2 4 8 6 2 4 8
EwMerSeAal/sGoeiln-Schuibftatebduffweirt.hFeoitrhecro1m00p-eftoitlidonexcsetussdoiefsu,nslaambeplleeds I/R I/R 1 I/R 2 I/R 4 EA EA 1 EA 2 EA 4 NBD NBD 1 NBD 2 NBD 4
(cold)oligonucleotideorunlabeledmutantoligonucleotide.
Figure 1: Neurological behavior scores from 6h to 48h after
DNA-protein complexes were resolved on a 5% nondena-
reperfusionintheratswith2hofMCAO.Theneurologicalbehavior
turing polyacrylamide gel with 20mA for 3h in 0.5× Tris- scoresinI/Rgroupwereworsefrom6hto48h.Thetreatmentwith
BorateEDTA.AfterUVcrosslinking,sampleswereblocked EAandNBDpeptidesignificantlyimprovedtheneurologicalscores,
and incubated with streptavidin-HRP conjugate (15mL). ∗𝑃<0.05, #𝑃<0.05versusI/Rgroup.Therewasmoresignificant
Hybridized samples were developed and analyzed using a improvementinNBDpeptidegroupthaninEAgroup, †𝑃<0.05.
HybridizationIncubator(WD-9403E,Beijing).
4.Results
2.12.Enzyme-LinkedImmunosorbentAssay(ELISA). Thelev-
elsofIL-1𝛽andIL-13incytosolicbrainfractionsandinserum
4.1.EAandNBDPeptideTreatmentSignificantlyDeclinedthe
wereanalyzedbyenzyme-linkedimmunosorbentassay(Ben-
Ischemia/ReperfusionDamageafterFocalCerebralIsche-
der,no.81-BMS630/96T;Abcam,no.ab100766/96T).
mia/Reperfusion
3.StatisticalAnalysis 4.1.1. Neurobehavioral Evaluation. After focal cerebral I/R,
the rats showed neurological deficit behavior at every time
The software SPSS 16.0 for Windows (SPSS Inc, Chicago,
point(Figure1).From6hto48hafterreperfusion,compared
IL) was used to conduct statistical analysis. All values are
withtheI/Rgroup,theneurobehavioralscoresofEAgroup
expressed as mean ± SD (standard deviation). Data were and NBD group were improved (𝑃 < 0.05) (Figure1). The
analyzedbyaone-wayANOVA,andintergroupdifferences effect in NBD group was more remarkable than that in EA
weredetectedbymultivariateanalysis,followedbyLSDtests. group(𝑃<0.05)(Figure1).TheseresultssuggestedthatEA
The neurological deficit scores were expressed as median treatment and NBD treatment both ameliorated the neuro-
(range)andwereanalyzedwithKruskal-Wallistest,followed logicdeficitsymptomscausedbyI/Randpromotedthemove-
by the Mann-Whitney U test with Bonferroni correction. mentrecoverydegree.ItshowedthatEAcouldalleviatethe
Valuesof𝑃<0.05wereconsideredasstatisticallysignificant. ischemiclesioninthesuperearlystageoffocalcerebralI/R.
Evidence-BasedComplementaryandAlternativeMedicine 5
50
%) 40 ∗
phere ( usion) 30 #
mis erf †
ntralateral he (24 h after rep 1200
o
C 0
Sham MCAO EA NBD Sham MCAO EA NBD
(a) (b)
Figure2:Infarctsizesat24hafterreperfusionintheratswith2hofMCAO(5ratsineachgroup,total20rats).(a)Representative2,3,5-
triphenyltetrazoliumchloridestainingofthecerebralinfarctincomparablesectionsofratbrainfrom4groupsat24hafterreperfusion.
(b)Quantificationofinfarctvolumeat24hafterreperfusion.TheEAandNBDpeptidetreatmentsignificantlyreducedtheinfarctvolume,
∗𝑃<0.05, #𝑃<0.05versusI/Rgroup.AndtheeffectofNBDpeptidewasremarkablethanEAgroup, †𝑃<0.05.
4.1.2. The Infarct Volumes Value after MCAO/R. The infarct NF-𝜅B p65 protein expression was conducted after focal
volumeinEAgroupshowedasmallerbraininfarctvolume cerebral I/R. In sham group, the NF-𝜅B p65 protein was
comparedwiththeI/Rgroup(𝑃<0.05)(Figure2).Thebrain predominantly expressed in the cytoplasm (Figures 4(a)
infarct volume of NBD group was much more significantly and4(b)).However,afterfocalcerebralI/R,expressionwas
reduced than that in EA group and I/R group (𝑃 < 0.05) observedbothinthecytoplasmandnucleusoftheneurons
(Figure2). The data suggested EA and NBD peptide treat- intheischemicpenumbraarea,althoughhigherexpression
mentcouldremarkablyreducetheinfarctsize.Moreover,the ofNF-𝜅Bp65proteinwasdetectedinthenucleusthanthatin
effectofNBDpeptideismoresignificantthanEAtreatment. thecytoplasm(𝑃<0.05)(Figures4(a)and4(b)).Significant
changes of NF-𝜅B p65 protein expression were observed in
4.2.AfterEAandNBDPeptideTreatment,theLevelofIL-1𝛽 the EA group and NBD group, which were predominantly
WasDecreasedandtheLevelofIL-13WasIncreasedinFocal detectedinthecytoplasmratherthaninthenucleus(𝑃<0.05)
CerebralIschemia/ReperfusionRats. AfterfocalcerebralI/R, (Figures 4(a) and 4(b)). These observations demonstrated
thelevelsofIL-1𝛽increasedsignificantlyfrom6hto48hboth thatthedifferentiallocalizationoftheNF-𝜅Bp65proteinin
in the brain (Figures 3(a) and 3(b)) and in serum (Figures thecytoplasmandnucleuswasalteredafterEAandNBDpep-
3(c)and3(d)).However,inEAgroupandNBDgroup,IL-1𝛽 tidetreatment,thusindicatingthatEAandNBDpeptidemay
levelsdecreasedsignificantlyandwerelowerthanthatinI/R effectivelyinhibittheprocessofNF-𝜅Bnucleartranslocation.
group(𝑃<0.05)(Figures3(a)and3(b)).TheIL-13levelswere
increasedintheI/Rgroup,especiallyat48hinbrainandat 4.4. EA and NBD Peptide Treatment Regulated the NF-𝜅B
24hinserum.IntheEAgroupandNBDgroupinthebrain p65/I𝜅B𝛼 Feedback Loop to Inhibit the NF-𝜅B Nuclear
andserum,theIL-13levelswerebothincreasedwhichwere
Translocation. The protein expression and localization of
significantlyhigherthanthoseinI/Rgroupatthesametime NF-𝜅Bp65(green)andI𝜅B𝛼(red)intheischemicpenumbra
points (𝑃 < 0.05) (Figures 3(c) and 3(d)). The IL-13 levels
areawereexaminedbydouble-immunofluorescencestaining.
in the EA group peaked at 24h in the brain and at 48h in As shown in Figure5, in sham group, low levels of NF-𝜅B
serum and peaked at 12h and 24h in NBD group (Figures p65andI𝜅B𝛼proteinexpressionwereobservedthroughout
3(c)and3(d)).ThesedatasuggestedthatEAandNBDpeptide
treatment effectively reduced IL-1𝛽 levels and increased IL- thehemisphere.AfterfocalcerebralI/R,NF-𝜅Bp65protein
expressionwasobservedbothinthenucleusandcytoplasm,
13 levels in brain and serum. Therefore, the EA and NBD
peptidetreatmenteffectivelyreducethequantityofIL-1𝛽and meanwhile there was a little expression of I𝜅B𝛼 protein
in the nucleus (Figure5). In contrast, NF-𝜅B p65 protein
restrainthedamagecausedbyinflammatoryfactorduringthe
expression was remarkably reduced in the nucleus in EA
earlystage.ButthepeaktimepointsofNBDpeptideandEA
group and NBD group; the I𝜅B𝛼 protein expression in the
treatmentarenotthesame.
cytoplasmandnucleuswassignificantlyincreased(Figure5).
4.3. EA and NBD Peptide Treatment Probably Inhibits NF- Double-immunofluorescence analysis of NF-𝜅B p65 and
𝜅BSignalingPathwayActivationthroughRegulatingtheNF- I𝜅B𝛼 protein expression demonstrated that NF-𝜅B p65
𝜅BNuclearTranslocation. Immunohistochemicalanalysisof protein was mainly localized in the cytoplasm, while I𝜅B𝛼
6 Evidence-BasedComplementaryandAlternativeMedicine
2.5 4
mL) mL)
n (ng/ 2.0 ∗ ∗ ∗ ∗ m (ng/ 3 ∗ ∗ ∗ ∗
𝛽e level of IL-1 in brai 011...505 # † # † # † 𝛽e level of IL-1 in seru 12 † # † † †
Th Th
0.0 0
6 12 24 48 6 12 24 48
(h) (h)
(a) (b)
1.5 1.5
mL) † † mL)
g/ ng/ ‡ ‡
in brain (n 1.0 ∗ ∗ ∗ # ∗ # in serum ( 1.0 ∗ ‡ ∗ ∗‡ ∗
3 3
1 1
L- L-
of I 0.5 of I 0.5
el el
v v
e e
e l e l
Th Th
0.0 0.0
6 12 24 48 6 12 24 48
(h) (h)
Sham EA Sham EA
I/R NBD I/R NBD
(c) (d)
Figure3:LevelsofIL-1𝛽andIL-13infocalischemicbraintissueandserumofratsamongsham,I/Rgroup,EAgroup,andNBDpeptidegroup
atdifferenttimepoints(5ratsineachgrouppertimepoint,total80rats).(a)IL-1𝛽levelsinthebrainoftheI/Rgroupincreasedsignificantly
from6hto48hwhichwerehigherthanthoseinEAgroupandNBDgroup, ∗𝑃<0.05.IL-1𝛽levelsintheEAgroupandNBDpeptidegroup
decreasedgradually, #𝑃<0.05, †𝑃<0.05.(b)Inserum,IL-1𝛽levelspeakedat48hintheI/Rgroupandwassignificantlyhigherthanthose
intheEAgroupandNBDgroup, ∗𝑃<0.05. †𝑃<0.05.IL-1𝛽levelsintheEAgroupdecreasedgraduallyevenat12h, #𝑃<0.05.(c)Inthe
brain,IL-13peakedintheI/Rgroupat48h,althoughtheselevelswerelowerthanthoseintheEAgroupandNBDgroup, ∗𝑃<0.05.IL-13
levelspeakedintheEAgroupat24handintheNBDgroupat12h,and24hweresignificantlyhigher, #𝑃<0.05, †𝑃<0.05versusI/Rgroup.
(d)TherewerenosignificantchangesintheserumIL-13levelsintheI/Rgroupatanyofthetimepoints,andtheselevelsweresignificantly
lowerthanthoseintheEAgroupandNBDgroup, ∗𝑃<0.05.ThelevelsinNBDgroupwerehigher, ‡𝑃<0.05versusEAgroup‡.
washighlyexpressedinnucleusandcytoplasmafterEAand and I𝜅B𝛼 protein in the EA group and NBD group was
NBDpeptidetreatment. increased (𝑃 < 0.05) (Figures 7(a), 7(c), and 7(e)). In con-
The fluorescent quantitation-PCR analysis showed that trast in the nucleus, the expression of NF-𝜅B p65 protein
the expression of NF-𝜅B p65 mRNA in I/R group was sig- was significantly increased in the I/R group and obviously
nificantlyincreasedat24hand48hwhichwerehigherthan decreased in the EA group and NBD group (𝑃 < 0.05)
thoseinEAgroupandNBDgroup(𝑃 < 0.05)(Figure6(a)). (Figures 7(b) and 7(d)). The expression of I𝜅B𝛼 protein
ExpressionofI𝜅B𝛼mRNAintheEAgroupandNBDgroup was obviously increased in the EA group and NBD group,
wasobviouslyincreasedandwashigherthanthatinI/Rgroup which were higher than that in the I/R group (𝑃 < 0.05)
(𝑃<0.05)(Figure6(b)). (Figures 7(b) and 7(f)). These data showed that EA and
The expression of NF-𝜅B p65 and I𝜅B𝛼 proteins were NBD peptide treatment effectively altered the expression
analysedbyWesternblotamongtheshamgroup,I/Rgroup, of NF-𝜅B p65 and I𝜅B𝛼 proteins in both cytoplasm and
EAgroup,andNBDgroup.IntheI/Rgroup,theexpression nucleus after focal cerebral I/R. They decreased NF-𝜅B p65
of NF-𝜅B p65 protein in the cytoplasm decreased gradu- protein expression in the nucleus and maintained it in the
ally, meanwhile low levels of I𝜅B𝛼 protein were detected. cytoplasm, while I𝜅B𝛼 protein expression was increased in
ComparedwiththeI/Rgroup,theexpressionofNF-𝜅Bp65 the nucleus and cytoplasm. Therefore, these data indicated
Evidence-BasedComplementaryandAlternativeMedicine 7
1
∗
NF-𝜅B p65 NF-𝜅B p65
alue 0.8 ∗
v
D †
O
n 0.6 #
a
e
m
5 0.4 #
6
Sham group I/R group p †
B
𝜅 0.2
NF-𝜅B p65 NF-𝜅B p65 F-
N
0
Sham group I/R group EA group NBD group
Cytoplasm
EA group NBD group Nucleus
(a) (b)
Figure4:ImmunohistochemicalanalysisofthenucleartranslocationofNF-𝜅Bp65inthesham,I/Rgroup,EAgroup,andNBDgroup(5
ratsineachgroup,total20rats).Inthetemporalneocortexofshamgroup,theimmunoreactivestainingoccurredlessinthecytoplasmand
nucleus.IntheI/Rgroup,strongimmunoreactivestainingoccurredinthecytoplasmandnucleus,especiallyinthenucleus, ∗𝑃<0.05.Inthe
EAgroup,immunoreactivestainingwaspredominantlydetectedinthecytoplasmratherthaninthenucleus, #𝑃<0.05.IntheNBDgroup,
immunoreactivestainingwasalsomaintainedinthecytoplasm, †𝑃<0.05.ComparisonofthemeanODvalueshowedthatNF-𝜅Bp65was
mainlyexpressedinthenucleusafterfocalischemia/reperfusion,andexpressionofNF-𝜅Bp65proteininthenucleusintheEAgroupand
NBDgroupwassignificantlyreduced, #𝑃<0.05, †𝑃<0.05.
thatthemechanismbywhichEAandNBDpeptidetreatment that in the EA group, especially at 12h (𝑃 < 0.05) (Figures
regulatedNF-𝜅Bnucleartranslocationwasrelatedtothehigh 10(b) and 10(d)). Expression of IKK𝛽 protein in the I/R
expressionofI𝜅B𝛼inthecytoplasmandnucleus. groupwassignificantlyincreased,especiallyat24hand48h
(𝑃 < 0.05) (Figures 10(a) and 10(d)). Compared with the
4.5.EAandNBDPeptideTreatmentRegulatedtheExpression I/R group, expression was remarkably decreased in the EA
of IKK𝛼 and IKK𝛽 mRNA and Protein after Focal Cerebral group (𝑃 < 0.05) (Figures 10(a) and 10(d)). Furthermore,
Ischemia/Reperfusion. The expression of IKK𝛼 (green) and intheNBDgroup,IKK𝛽proteinexpressionwaspersistently
IKK𝛽(red)proteinwasexaminedbydouble-immunofluores- low from 6h to 48h (𝑃 < 0.01) (Figures 10(b) and 10(d)).
cencestainingintheshamgroup,I/Rgroup,EAgroup,and ThesedatademonstratedthatEAandNBDpeptidetreatment
NBD peptide group. As shown in Figure8, little expression could effectively reduce the high levels of IKK𝛽 expression
ofIKK𝛼andIKK𝛽proteinswasobservedintheshamgroup afterfocalcerebralI/R,especiallyat24hand48h.Compared
(Figure8), and they were increased significantly after focal withtheNBDpeptidegroup,EAtreatmentcouldelsereduce
cerebral I/R (Figure8) but were obviously decreased in the IKK𝛼proteinexpression.
EA group and NBD group, especially decreased the IKK𝛽
expression(Figure8). 4.6.EAandNBDPeptideTreatmentEffectivelyRegulatedthe
The fluorescent quantitation-PCR (Q-PCR) analysis DNA Binding Capacity of NF-𝜅B. Electrophoretic mobility
showedthattheexpressionofIKK𝛼andIKK𝛽mRNAinthe shiftassays(EMSA)wereusedtofurtherinvestigatetheactiv-
I/R group was significantly increased (𝑃 < 0.05) (Figures ityofNF-𝜅BafterfocalcerebralI/Randthechangeinactivity
9(a)and9(b)).TheIKK𝛼mRNAexpressionintheEAgroup mediated by EA treatment and NBD peptide intervention.
was remarkably reduced, which was lower than that in I/R The activity of NF-𝜅B was increased after I/R, especially at
group (𝑃 < 0.05) (Figure9(a)). The peak expression in the 24h and 48h, indicating that the DNA binding capacity of
NBDgroupwashigherthanthatintheEAgroup(𝑃<0.05) NF-𝜅B was high in the I/R group (𝑃 < 0.05) (Figures 11(a)
(Figure9(a)).ComparedwiththeI/Rgroup,theexpression and11(b)).IntheEAgroupandNBDgroup,theactivityof
of IKK𝛽 mRNA in the EA group was obviously decreased NF-𝜅Bwasreducedsignificantly(𝑃 < 0.05),andtherewere
(𝑃 < 0.05) (Figure9(b)). In the NBD group, IKK𝛽 mRNA noremarkabledifferencesbetweenthose(𝑃>0.05)(Figures
expressionwasnegligiblefrom6hto48h(Figure9(b)). 11(a)and11(b)).TheresultssuggestthatEAandNBDpeptide
TheproteinexpressionofIKK𝛼andIKK𝛽wasanalysed treatmenteffectivelyreducestheactivityofNF-𝜅B.
byWesternblot.IntheI/Rgroup,IKK𝛼proteinexpression
was persistently increased from 6h to 48h (Figures 10(a) 5.Discussion
and10(c)).TheexpressionofIKK𝛼proteinwasdecreasedin
the EA group, which was lower than that in the I/R group In the present study, we investigated the anti-inflammation
(𝑃 < 0.05) (Figures 10(a) and 10(c)). In the NBD group, effectandprobablemechanismofEAontheNF-𝜅Bsignaling
expressionwasalsoreducedbutwasstillmuchhigherthan pathwayafterfocalcerebralischemia/reperfusion.Firstofall,
8 Evidence-BasedComplementaryandAlternativeMedicine
NF-𝜅B p65 I𝜅B𝛼 Merged
m
a
h
S
mmeerrggeedd
75 𝜇m 75 𝜇m 75 𝜇m
NF-𝜅B p65 I𝜅B𝛼 Merged
R
I/
75 𝜇m 75 𝜇m 75 𝜇m
NF-𝜅B p65 I𝜅B𝛼 Merged
A
E
75 𝜇m 75 𝜇m 75 𝜇m
NF-𝜅B p65 I𝜅B𝛼 Merged
D
B
N
75 𝜇m 75 𝜇m 75 𝜇m
Figure5:Double-immunofluorescentlabelingtheNF-𝜅Bp65(green)andI𝜅B𝛼(red)inthecellsintheischemiccortex(5ratsineachgroup,
total20rats).ItshowedthatNF-𝜅Bp65andI𝜅B𝛼werebothpoorlyexpressedintheshamgroup.IntheI/Rgroup,theNF-𝜅Bp65protein
wasexpressedincytoplasmandnucleus,especiallyhighlyinthenucleus;I𝜅B𝛼proteinwaspoorlyexpressedincytoplasmandnucleus.In
theEAgroupandNBDgroup,theNF-𝜅Bp65proteinwasmainlyexpressedinthecytoplasmthaninthenucleus,andtheI𝜅B𝛼proteinwas
expressedhighlyinthecytoplasmandnucleus.
wefoundthatEAtreatmenteffectivelyreducedthecerebral thefocalcerebralI/Rinjury,viaamechanismrelatedtothe
infarct volume and improved the neurobehavioral scores, suppressionoftheactivationoftheNF-𝜅Bsignalingpathway
it seemed able to alleviate the ischemic damage. Secondly, ataveryearlyphase.Moreover,theeffectsontheIKK𝛼and
the EA treatment also regulated the levels of interleukin IKK𝛽wereremarkablydifferentbetweenEAtreatmentand
(IL-1𝛽 and IL-13) in brain and serum after focal cerebral NBDpeptide.
I/R. More importantly than all of that, we observed that Theinflammatoryreactionisanimportantpathological
EA treatment reduced the expression of IKK𝛼 and IKK𝛽 stepintheprocessoffocalcerebralI/R,whichcausesrapid
protein,perhapsdeclinethefunctionofIKK𝛼andIKK𝛽,and neuronaldeathandaggravatestheischemicinjuryandbody
then regulated the NF-𝜅B p65/I𝜅B𝛼 feedback loop for the burden[30].Therefore,effectivelyrestrainingtheoccurrence
activationofNF-𝜅Bsignalingpathwayeffectivelyduringvery and development of the inflammation is considered to be
earlystageoffocalcerebralI/R,whichdemonstratedthatEA animportanttargetanddirectionofthetreatmentforfocal
hasobviouslyprotectiveandanti-inflammatoryeffectduring cerebral I/R. The cytokines that cause the inflammatory
Evidence-BasedComplementaryandAlternativeMedicine 9
8 6
†
#
A †
N ∗ A #
R ∗ N
m 6 R
65 # # m 4
p 𝛼
B B
𝜅on of NF- 4 ∗ # † † 𝜅ession of I
xpressi 2 ∗ # The expr 2
e
e
Th
0 0
6 12 24 48 6 12 24 48
(h) (h)
I/R I/R
EA EA
NBD NBD
(a) (b)
Figure6:FluorescentQuantitative-PCRanalysisoftheNF-𝜅Bp65andI𝜅B𝛼mRNAexpressionoftheshamgroup,I/Rgroup,EAgroup,
andNBDgroupintheischemicbraintissue(5ratsineachgrouppertimepoint,total80rats).(a)TheexpressionofNF-𝜅Bp65mRNAwas
significantlyincreasedintheI/RgroupcomparedwiththatintheEAandNBDgroups, ∗𝑃<0.05.At24hand48h,theNF-𝜅Bp65mRNA
intheEAgroupwashigher, #𝑃 < 0.05, †𝑃 < 0.05versusNBDgroup.(b)TheI𝜅B𝛼mRNAexpressioninEAgroupandNBDgroupwas
higher, #𝑃<0.05, †𝑃<0.05versusI/Rgroup.
response include TNF-𝛼, IL-1𝛽, cox-2, iNOS, and many of p65 and significantly reduced it in nucleus. The discov-
other types of factors [31]. IL-1𝛽 is a nerve toxin and a ery demonstrated that EA effectively inhibited p65 nuclear
majorstimulusfactoroftheinflammatoryreaction.Reducing translocationandmaintaineditinthecytoplasm,whichwe
the level of IL-1𝛽 can significantly ameliorate the ischemia also found after NBD peptide treatment. According to the
infarctionvolumeandtheoccurrenceofinflammation[32]. generalunderstandingoftheNF-𝜅Bsignalpathway,nuclear
On the contrary, the cytokines such as IL-10 and IL-13 NF-𝜅BdrivesI𝜅B𝛼expressiongeneratinganegativefeedback
exertbetteranti-inflammatoryeffect.Inflammatoryreaction loop[36,37].Inthepresentstudy,wefoundthatEAtreatment
induced by IL-1𝛽 stimulation is significantly inhibited by significantlyincreasedI𝜅B𝛼expressioninthecytoplasmand
IL-13 [33]. Focused on the inflammatory cytokines in the nucleus,especiallyexpressedinthenucleus,andalteredthe
ischemic brain and serum, we observed that the level and NF-𝜅B p65 and I𝜅B𝛼 protein expression in nucleus. The
the timing of the peak expression of IL-1𝛽 and IL-13 were resultsshowedthatEAtreatmentcouldregulateNF-𝜅B/I𝜅B𝛼
regulatedbyEAandNBDpeptidetreatment.Allofthesedata feedback loop in the nucleus and cytoplasm. Above all, it
demonstratedthatEAtreatmentcouldexertremarkableanti- revealsthatEApromotestheinactiveheterotrimergenerated
inflammatoryeffectduringtheearlystageoffocalcerebralI/R in nucleus through increasing I𝜅B𝛼 expression in the NF-
whichresemblesthefunctionofNBDpeptide.However,the 𝜅B/I𝜅B𝛼feedbackloopandthenmaintainsthehighcytosolic
effectofNBDpeptideismoreremarkableontheregulation level of p65. It can be speculated that inhibition of NF-𝜅B
ofcytokines. nucleartranslocationislikelytobeoneofthemostimportant
In order to regulate the inflammatory injury occurred mechanismsbywhichEAinhibitsNF-𝜅Bactivation.
during the early stages after reperfusion, it is necessary to The function of I𝜅B𝛼 is regulated by the I𝜅B kinases
inhibit the activation of NF-𝜅B signaling pathway which is (IKKs) in the NF-𝜅B signal pathway, and NF-𝜅B activa-
inducedbyfocalcerebralI/R.Duringtheactivationprocess tion depends on the IKK phosphorylation process [38].
ofNF-𝜅Bsignalingpathway,theinteractionofp65andI𝜅B𝛼 Both IKK𝛼 and IKK𝛽 phosphorylate I𝜅B𝛼 at Ser 32 and
onnucleartranslocationisthekeystep[34,35].Inthepresent Ser 36, although IKK𝛼 is less efficient and consequently
study,weobservedthatNF-𝜅Bp65expressedbothincyto- cannot complement IKK𝛽 knockout cells [18, 38]. In this
plasmandnucleus,theNF-𝜅Bp65predominantlyexpressed study, we observed that the expression of IKK𝛼 and IKK𝛽
innucleusafterfocalcerebralI/R.Itshowedthattheischemia was significantly increased after focal cerebral I/R, which
and anoxic pathological factors in the brain stimulated the demonstrated that the massive expression of IKK𝛼 and
p65mainlyexpressedinthenucleusandpromotedtheNF-𝜅B IKK𝛽couldberequiredforNF-𝜅Bactivation.However,EA
nucleartranslocationprocess.WeobservedthatEAandNBD treatment remarkably reduced the expression of IKK𝛼 and
peptide treatment mainly maintained high cytosolic level IKK𝛽, especially for the IKK𝛽. The results of the EA group
10 Evidence-BasedComplementaryandAlternativeMedicine
EA group I/R group NBD group Sham group
6 12 24 48 6 12 24 48 (h)
6 12 24 48 6 12 24 48(h)
NF-𝜅B p65
I𝜅B𝛼
𝛽-Actin
(a)
EA group I/R group NBD group Sham group
6 12 24 48 6 12 24 48 (h) 6 12 24 48 6 12 24 48 (h)
NF-𝜅B p65
I𝜅B𝛼
Histone
(b)
5) 4
B p6 65) 1.5
𝜅m NF- 3 # † ∗ 𝜅NF-B p ∗ ∗ ∗ ∗
cytoplas 2 # † nucleus 1.0
( # (
nisty ratio 1 # nisty ratio 0.5
nte 0 nte 0.0
I 6 12 24 48 I 6 12 24 48
(h) (h)
(c) (d)
3
∗
∗ 2.5
𝛼) ∗
𝜅B ∗ 𝛼) ∗ #
m I # # 𝜅B 2.0 ∗ #
ytoplas 2 ucleus I 1.5 ∗ #
c n
( (
o o
y rati 1 y rati 1.0 ∗ #
nist nist 0.5
nte nte
I I
0 0.0
6 12 24 48 6 12 24 48
(h) (h)
EA NBD EA NBD
I/R Sham I/R Sham
(e) (f)
Figure7:WesternblotanalysisoftheNF-𝜅Bp65andI𝜅B𝛼proteinexpressionintheshamgroup,I/Rgroup,EAgroup,andNBDgroup
inischemicbraintissue(5ratsineachgrouppertimepoint,total80rats).(a)-(b)RepresentativeWesternblotimagesshowingbandsof
NF-𝜅Bp65andI𝜅B𝛼proteinsinthecytoplasmandnucleusat6h,12h,24h,and48h.(c)–(f)Comparisonofthemeanintensityratioof
immunoblottinginthesegroupsateachtimepoint.(a),(c),(e)TheNF-𝜅Bp65andI𝜅B𝛼proteinexpressionincytoplasm.(b),(d),(f)The
NF-𝜅Bp65andI𝜅B𝛼proteinexpressioninthenucleus.