Electroacupuncture at Acupoints Reverses Plasma Glutamate, Lipid, and LDL/VLDL in an Acute ...

15 Pages·2015·2.51 MB·English

Hindawi Publishing Corporation
Evidence-Based Complementary and Alternative Medicine
Volume 2014, Article ID 659268, 14 pages
http://dx.doi.org/10.1155/2014/659268
Research Article
Electroacupuncture at Acupoints Reverses Plasma Glutamate,
1
Lipid, and LDL/VLDL in an Acute Migraine Rat Model: A H
NMR-Based Metabolomic Study
ZishanGao,1,2XuguangLiu,2ShuguangYu,2QiZhang,3QinChen,4QiaofengWu,2
JuanLiu,3BoSun,3LiFang,4JiaLin,5Bing-MeiZhu,1XianzhongYan,3andFanrongLiang2
1ClinicalAcupunctureandMoxibustionDepartment,SecondSchoolofClinicalMedicine,NanjingUniversityofChineseMedicine,
Nanjing,Jiangsu210029,China
2AcupunctureandTuinaCollege,ChengduUniversityofTCM,Chengdu,Sichuan610075,China
3NationalCenterofBiomedicalAnalysis,Beijing100850,China
4TheThirdAffiliatedHospitalofZhejiangChineseMedicalUniversity,Hangzhou,Zhejiang310006,China
5DepartmentofBiochemistryandMolecularBiology,WestChinaSchoolofPreclinicalandForensicMedicine,SichuanUniversity,
Chengdu,Sichuan610041,China
CorrespondenceshouldbeaddressedtoXianzhongYan;[email protected];[email protected]
Received28February2013;Revised16October2013;Accepted7November2013;Published28January2014
AcademicEditor:Yi-HungChen
Copyright©2014ZishanGaoetal.ThisisanopenaccessarticledistributedundertheCreativeCommonsAttributionLicense,
whichpermitsunrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalworkisproperlycited.
Background.Theobjectiveofthisstudywastoidentifypotentialbiomarkersofelectroacupuncture(EA)onrelievingacutemigraine
throughmetabolomicstudy.Methods.EAtreatmentswereperformedonbothacupointsandnonacupointsonthenitroglycerin
(NTG)-inducedmigraineratmodel.NMRexperimentsandmultivariateanalysiswereusedformetabolomicanalysis.Results.The
numberofhead-scratching,themainethologyindexofmigraineratmodel,wassignificantlyincreased(𝑃 < 0.01)afterNTG
injection.Theplasmametabolicprofileofmodelgroupwasdistinctfromthatofthecontrolgroup.Glutamatewassignificantly
increased(𝑃 < 0.01),whereaslipidsweresignificantlydecreased(𝑃 < 0.01)inmodelrats.AfterEAatacupoints,themetabolic
profile of model rats was normalized, with decreased glutamate (𝑃 < 0.05) and increased lipids (𝑃 < 0.01). In contrast, EA
at nonacupoints did not restore the metabolic profile, but with six metabolites significantly different from acupoints group.
Interestingly,thenumberofhead-scratchingandglutamatelevelweresignificantlydecreased(𝑃 < 0.05)afterreceivingEAat
bothacupointsandnonacupoints.Conclusions.EAatacupointsmayrelieveacutemigrainebyrestoringtheplasmametabolic
profileandplasmaglutamate,whileEAatnonacupointsmaymodestlyrelieveacutemigrainebydecreasingplasmaglutamate.
1.Introduction the pathophysiology of migraine is attributed to multiple
factors,andmanyoftheseaspectsarestillnotunraveled[5,
Migraineisoneofthemostcommonneurologicaldisorders 6].Drugtreatmentwithoralnonsteroidalanti-inflammatory
characterized by recurrent unilateral, throbbing headaches drugs (NSAIDs) and triptansfor relieving migraine usually
and neurological dysfunction, with or without aura [1]. have a modest effect and cause several side effects, such as
Approximately 16%–18% of women and 6%–8% of men in gastrointestinalandcardiovasculardisorders[7].
the USA suffer from migraine during the most productive Acupuncture is a procedure whereby fine needles are
years of their professional lives [2–4]. Although enhanced inserted and manipulated into related acupoints of the
excitatoryneurotransmitterssuchasglutamate(Glu),which individual,forthepurposeoftreatingthedisease.Although
facilitate spontaneous cortical spreading depression (CSD), acupuncture has been widely used for migraine prophy-
are valued as central mechanism of triggering migraine, laxis and treatment, little is understood about its biological
2 Evidence-BasedComplementaryandAlternativeMedicine
mechanism. A 2009 Cochrane meta-analysis proclaimed
One week
acupuncture as safe and effective for migraine prophy-
acclimatation
laxis compared to prophylactic drug treatment [8]. The
US Headache Consortium has also deemed acupuncture to
be an important therapy for management of migraine [9]. Rats were randomly assigned to four groups
Recently, an increasing number of studies have confirmed
that mechanistically, acupuncture analgesia works through
thereleaseofopioidpeptidesinthecentralnervoussystem
Established nitroglycerin-treated acute migraine model
(CNS),whichoccursinresponsetolong-lastingactivationof
ascendingsensorytracksstimulatedbyacupuncturemanip-
ulation[10–13].However,theprimarymechanismbywhich
acupuncturerelievesmigrainehasnotbeenwellestablished. Control and NTG EA and NA group
Further, lately a series of high quality trials have fuelled
group were restrained were restrained on the
suspicionsthatacupunctureatacupointsisnomoreeffective
on the table for 20min table and received 20min
than acupuncture at nonacupoints or sham acupuncture in
and received no treatment EA treatment
reducing migraine headaches [14–17]. Hence, two urgent
questions in the field of acupuncture for migraine have
been raised. First, are there any neurological or metabolic
2 hours after EA treatment, all the rats were sacrificed
biomarkers that provide supporting evidence for acupunc-
turetorelievemigraine?Second,aretheretrueneurological and plasma samples were collected
or metabolic differences between acupuncture at acupoints
andacupunctureatnonacupoints?
NMR experiment
To address these two questions, nitroglycerin-treated
(NTG) rats are employed in this study as acute migraine
modeltodetectmetabolicchangesofacutemigrainetreated Multivariate analysis of NMR data
byelectroacupuncture.NTGratmodelisregardedasareli-
ableanimalmodelforacutemigraine.Itreplicatesneurogenic
Result
inflammation and hyperalgesia of human migraines, and it
hasbeensuccessfullyestablishedindifferentmigrainestudies
Figure1:Trialfluidogram.
[18–22].
Metabolomics has rapidly emerged as a powerful tool
for identification of new biomarkers and for monitoring
(permitnumber:Sichuanexperimentalanimalinstitute(97)
thedynamicpathophysiologicalmetabolicchangesofwhole
6). All surgeries were performed under sodium pentobarbital
organismsindiseasestates[23].Recentadvancesinnuclear
anesthesia,andalleffortsweremadetominimizesuffering.
magnetic resonance (NMR) spectroscopy, aided by data-
reduction techniques, have facilitated the use of metabo-
2.2. Acute Migraine Model. The overall experimental
lomicsasadirectfunctionalreadoutofthepathologicalstate
approachisshown in Figure1. Fifty femaleSprague Dawley
from biofluids and tissues [24–26]. The power of metabo-
rats,3monthsoldandweighing160±20g,werepurchased
lomics lies in monitoring systemic metabolite changes and
fromtheExperimentalAnimalCenterofChengduUniversity
characterizingcompletemetabolicprofiling,whichproperly
ofTraditionalChineseMedicine.Ratswerehousedinawell-
meet the essence of Traditional Chinese Medicine (TCM)
∘
ventilated colony room at a temperature of 15–22 C and a
andacupunctureforitssensitivityandcomplexity.Therefore,
relative humidity of 50–70% on a 12h light/12h dark cycle.
there are increasing studies that employ metabolomics to
Food and tap water were provided ad libitum. Rats were
detectmetabolicevidenceforTraditionalChineseMedicine
allowed to acclimatize for one week before experiments
andacupuncture[27–30].
1 began and then were randomly divided into 4 groups
Inthecurrentstudy,weemployedadeveloped H-NMR-
using an SPSS randomization process: control (untreated,
based metabolomic technology to investigate metabolite
withoutanytreatment),NTG(nitroglycerin-treatedmodel),
changesinacutemigraineevokedinNTGratmodelandthe
EA (electroacupuncture at acupoints following NTG-
effect of electroacupuncture on treating an acute migraine
treatment), and NA (electroacupuncture at nonacupoints
attack.
following NTG-treatment). Each group has 10 rats and was
fedseparatelyintwometaboliccages.
2.Methods Allratswererestrainedonanoperatingtableforfurther
treatment. The rats in control group received no injection,
2.1. Ethics Statements. All experiments were conducted in whileotherthreegroupswereinjectedintraperitoneallywith
strictaccordancewiththeguidelinesofInternationalAssoci- NTG(10mg/kgof2mg/mLsolution[18])accordingtopre-
ationfortheStudyofPainandChinesenationalregulations viousstudy[22].Thirtyminutesafterinjection,threegroups
forexperimentalanimaluse.Thestudywasapprovedbythe ofNTGratsdisplayedredear,frequenthead-scratching,and
SichuanInstitutionalReviewBoardforAnimalExperiments climbing cage, which represent the anxiety and discomfort,
Evidence-BasedComplementaryandAlternativeMedicine 3
∘
indicating the successful establishment of migraine model at 27 C using a Carr-Purcell-Meiboom-Gill (CPMG) spin-
[18]. These typical symptoms lasted for 4 hours, which is echo pulse sequence with a total spin-spin relaxation delay
consistentwithpreviousreports.Incontrast,thecontrolrats (2n𝜏) of 320ms. Water suppression was achieved with an
didnotshowthesesymptoms. irradiation of the water peak during the recycle delay (2s)
Following NTG injection, the behavior of the rats was andmixingtime(t )of150ms.Freeinductiondecays(FIDs)
m
observedanddocumented.Thetotaltimeofethologyobser- werecollectedinto32,000datapointswithaspectralwidth
vation lasted for 4h, and each 30min of observation was of8,000Hzover64scans.Presaturatefrequencyandcentral
considered a time interval. The number of head-scratching frequencywereequallyonthewaterpeak.TheFIDswerezero
wasquantitativelymeasuredasthemainethologyindex[18– failed by a factor of two and multiplied by an exponential
22]. line-broadening factor of 0.5Hz prior to Fourier transfor-
mation.Inaddition,diffusion-editedexperimentswerealso
performedwithbipolarpulsepair-longitudinaleddycurrent
2.3.ElectroacupunctureTreatment. Onehourafterinjection,
delay(BPP-LED)pulsesequence.Thegradientamplitudewas
animals were restrained on an operating table for elec-
set at 35.0G/cm with a diffusion delay of100ms. A totalof
troacupuncture treatment. The EA group received filiform
128 transients and 16,000 data points were collected with a
needlesthatwereinsertedperpendicularlyandbilaterallyto
spectralwidthof8,000Hz.Aline-broadeningfactorof1Hz
a depth of 2-3mm at the points of SJ5 (Waiguan, located
wasappliedtoFIDsbeforeFouriertransformation.
on lateral side of forefoot, 3mm away from wrist joint and
1
in the middle of ulnar bone and radial bone) and GB34 H NMR spectra of plasma were manually phased and
(Yanglingquan,locatedonthepostlateralsideofkneejoint, baseline-correctedusingVNMR6.1Csoftware(VarianInc.).
below capitulum fibulae) along the Shao Yang meridian For CPMG spectra, each spectrum over the range of 𝛿
of Hand and Foot (see Supplementary Figure 1 available 0.4–4.4 was data-reduced into integrated regions of equal
online at http://dx.doi.org/10.1155/2014/659268). These acu- width (0.01ppm). For BPP-LED data, each spectrum over
points are most frequently used to relieve migraine attacks the range of 𝛿 0.1–6.0 was segmented into regions of equal
[31].Nonacupointswerelocated5mmabovefromeachacu- width(0.01ppm).Theregionscontainingtheresonancefrom
pointaccordingtopreviousstudies(SupplementaryFigure1) residualwater(𝛿4.6–5.1)wereexcluded.Theintegralvalues
[14]. of each spectrum were normalized to a constant sum of
Transcutaneous electroacupuncture (EA) stimulation all integrals in a spectrum to reduce any concentration
was then conducted at each acupoint for 20min [32] using plotvariationsbetweensamples.Identificationofmetabolites
a Han’s acupoint nerve stimulator (HNAS-200, Nanjing, in the spectra was accomplished based on information in
China).Stimulationfrequencywassetat14Hz,andintensity the literature and the Chenomx NMR Suite 4.5 (Chenomx,
wasincreasedfrom0.1mAtoamaximumof1.0mAuntilthe Calgary,Canada).
rats’ feet began to tremble slightly [32]. For the NA group, Based on previous studies, the HMDB website ( http://
an identical electroacupuncture procedure was conducted, www.hmdb.ca/) and the ChenomxNMR Suite software,
except that needles were placed 5mm above from each major metabolites in plasma were identified and shown
acupoint[31].AnimalsinthecontrolandNTGgroupswere in Figure2. CPMG pulse sequence was used to emphasize
alsofixedonthetablefor20min,butnoelectroacupuncture the resonances of small metabolites in plasma, while reso-
wasperformed.Allratswereconsciousduringtreatment. nancesfrommacromoleculeswereattenuated(Figure2(a)).
Figure2(b) shows diffusion-edited NMR spectra of plasma
fromeachgroup,displayingthesignalsoflipids,N-acetylgly-
2.4. Sample Collection and Pretreatment. To avoid poten-
coproteins(NAc),andO-acetylglycoproteins(OAc)groups
tial acute effects of EA, rats were anesthetized with 3%
of glycoproteins. Subtle differences of these spectra were
sodium pentobarbital (40mg/kg., i.p.) at 2 hours after elec-
observedbyvisualinspectionamongthefourgroups.Further
troacupuncturetreatment[32]andthensacrificedat4hour
analysiswasperformedusingmultivariatestatisticalanalysis
after nitroglycerin injection by exsanguination from the
femoral artery. Blood (∼3mL) from the femoral artery was todeterminethemetabolicchangesamongthefourgroups.
collected into a heparin sodium tube, placed on ice for 30
minutes,andthencentrifuged(3000×g,4∘C,15min).Plasma
2.6.PatternRecognitionandStatistics. Theresultingintegral
wascollectedandstoredat−70∘C.
datawereimportedintoSIMCA-P(version10.04;Umetrics,
Before NMR analysis, plasma samples were thawed,
Umea˚,Sweden)formultivariateanalysis.Priortoanalysis,the
centrifuged (13000×g, 4∘C, 10min), and the supernatant
CPMGdataweremean-centeredandPareto-scaled,andthe
extracted.In5mmNMRtubes,300𝜇Lofeachsupernatant
BPP-LED data were mean-centered. CPMG data and LED
sample was mixed with 250𝜇L of D2O to lock the field data were both subjected to principal component analysis
frequency and 50𝜇L of 3-trimethylsilyl-2H4-propionic acid (PCA) and partial least square discriminate analysis (PLS-
sodium salt (TSP) in D2O (1mg/mL) as a chemical shift DA) to discriminate differentiation in metabolites among
reference.Allsamplescontainedafinalvolumeof600𝜇Land
the groups. PCA was firstly performed on the normalized
werevibratedrepeatedly[33,34]. 1
H NMR dataset after Pareto scaling in this study. Data
werevisualizedbyusingtheprincipalcomponent(PC)score
1
2.5. NMR Experiments. H NMR spectra of plasma were and loading plots. Each point on the scores plot represents
acquiredwithaVarianINOVA600MHzNMRspectrometer an individual sample, and each point on the loadings plot
4 Evidence-BasedComplementaryandAlternativeMedicine
Lac
Glc Bet
Lac Gly Gln OAc NAc LipidCH2
Cho
D Cre Acet Ace Ala Val LipidCH
3
C DMG Pyr Leu/lle
U
B
Glu
A
4 3.5 3 2.5 2 1.5 1
(ppm)
(a)
Lipid Lipid(CH2)n
CH CH CO
2 2
PtdCho Lipid NAc
Lipid C=CCH2C=C LipidCH3
HC=CH OAc
D
C
B
A
5.5 5 4.5 4 3.5 3 2.5 2 1.5 1 0.5 −0
(ppm)
(b)
1
Figure2: HNMRspectraofratplasmasamples.(a)CPMGspectra;(b)LEDspectra;A,control;B,NTGgroup;C,NAgroup;D,EAgroup
(𝑛=10).
Evidence-BasedComplementaryandAlternativeMedicine 5
representsasingleNMRspectralregion.Orthogonalsignal betweenthecontrolgroupandtheNTGgroup.Basedonthe
correction(OSC)wasfurtherusedtoremovethevariations resultsofOSC-PLS-DAandmultivariateanalysisofvariance,
not correlated to group membership and to maximize the thedominantmetabolitesthatinfluencedthedifferentiation
separation,followedbyPLSanalysis.Thefinallistsofmetabo- between the control and NTG groups are displayed in the
liteswerechosenonthebasisofthevariablesofimportance corresponding loadings plots (Figure3(b)), the concentra-
parameter(VIP),whichisameasureofeachvariable’srelative tion plot (Figure6(b)), and Table1. Notably, NTG group
influenceonthemodel[35].Becauseinthisstudywefound demonstrated a significantlyhigher level of glutamate(𝑃 =
series of metabolite changes and they were correlated with 0.009), which is an important excitatory neurotransmitter
eachotheramongmetabolicsystems,thereforemultivariate fortriggeringmigraine[36],andsignificantlylowerlevelsof
analysisofvariancewasperformedtodiscriminatesignificant LDL/VDL (𝑃 = 0.001) and lipid (𝑃 = 0.008) compared
changesofmetabolitesidentifiedbySIMCA-P.Ethologydata with the controls. Moreover, PCA, PLS analysis with OSC
werecalculatedbyrepeatedmeasuresanalysisofvarianceand pretreatment were also performed on the 1H NMR LED
multivariateanalysisofvariance. dataset,whichissimilartotheCPMGprocedure.Theresults
illustrated an obvious separation between the controls and
the NTG group along PC1 (Figure3(c); 𝑅2𝑋 = 53.1%, 𝑅2𝑌
3. Results = 99%, 𝑄2 = 96.3%), as shown in Table2 and Figure6(b).
Together,weextractedanenhancedanaerobicglycolysisand
3.1. Electroacupuncture at Both Acupoints and Nonacupoints
reduced gluconeogenesis from the metabolic profiling of
Reduce Head-Scratching Number in NTG Rats. Initially, the
nitroglycerin-treatedacutemigraineratmodel,asdisplayed
quantitativeethologyobservations,forthepurposeofdetect-
bydecreasedglucose,pyruvicacidandincreasedlacticacid,
ingNTG-inducedacutemigraineratmodelandtheeffectof
alanine, and 3-hydroxybutyric acid (3-HB) (Figure6(b)).
EA,wererecordedandcalculated.FollowingNTGinjection,
Meanwhile, the enhancement of lipid metabolism was also
thenumberofhead-scratchingintheNTGgroup,EAgroup,
demonstratedintheNTGacutemigraineratmodel,asindi-
and NA group increased significantly from NTG injection
cated by decreased levels of lipid, unsaturated lipid (UFA),
to 60min compared to the control group (𝑃 < 0.01)
choline,andphosphatidylcholine(Ptdcho)(Figure6(b)).The
(Figure6(a)). Specially, the number of head-scratching in
metabolite change at 1.14ppm remains unidentified, but it
the NTG group significantly increased from NTG injection
seems unlikely that this metabolite could be a component
to 60min and from 120min to 240min, indicating that a
of the NTG solution that would have influenced any group
successfulratmodelofacutemigrainehadbeenestablished
differences.
[21].Secondly,20minofEAatacupointsandEAatnonacu-
points treatments were performed accordingly on EA and
3.3.ElectroacupunctureatAcupointsReversesPlasmaMetabo-
NA groups from 60min to 120min after NTG injection.
lite Changes in the Acute Migraine Rat Model. After estab-
Subsequently,thenumberofhead-scratchingwasobviously
decreased(𝑃<0.05)inbothEAandNAgroupfrom120min lishing the metabolic profiling of NTG acute migraine rat
model,wenextdeterminedwhetherEAreversesmetabolite
to240min(Figure6(a))comparedtoNTGgroup,suggesting
changesintheacutemigraineratmodel.WeperformedPLS
thatatypicalsymptomofacutemigrainewasrelievedbyper-
analyseswithOSCpretreatmentondatafromthreegroups:
formingEAatbothacupointsandnonacupoints.Inaddition,
control,NTG,andratswhoreceivedEAatacupoints.After
the number of head-scratching was significantly decreased
(𝑃 < 0.05) in the NA group from 210min to 240min 20min of EA at acupoints, we observed that the metabolic
profiling of the EA group was similar to that of the control
followingNTGadministration(Figure6(a))comparedtoEA
group, and both these groups were clearly separated from
group.Inconclusion,thesedatashowedthatthenumberof
the NTG group along PC1 (Figure4(a); 𝑅2𝑋 = 77.4%, 𝑅2𝑌
head-scratching in NTG group was significantly increased
(𝑃<0.01)comparedtothecontrolgroup,whilethenumber = 98.3%, and 𝑄2 = 92.6%). Interestingly, the EA group
illustratedasignificantlowerlevelofglutamate(𝑃 = 0.048)
of head-scratching in EA and NA group was significantly
decreased(𝑃 < 0.05)relativetoNTGgroupafterreceiving and significant higher levels of LDL/VDL (𝑃 = 0.002) and
lipid (𝑃 = 0.002) compared with the NTG group, which
EAatbothacupointsandnonacupoints.
is the opposite of the metabolite changes in NTG group
relative to controls (Figures 6(d) and 6(e)). Meanwhile, the
3.2.NMRMetabolicProfilingCanDifferentiateAcuteMigraine concentrationplotshowedthatthelevelsoflacticacid,OAc,
RatModel. AfterNMRexperiment,22metabolitesinplasma pyruvic acid, alanine, LDL/VLDL, glutamate, glutamine,
of animal groups were identified by using Chenomx NMR creatine,andlipidinEAgroupwererestoredtolevelssimilar
Suite software and shown in Figure2. To detect the role of tothatofthecontrolgroup(Figures6(c)–6(e)).Furthermore,
electroacupunctureonmetabolitechangesinacutemigraine the diffusion-edited NMR spectra was performed, which is
rats, we first asked whether NMR metabolic profiling can similar to the CPMG procedure. The result demonstrated a
differentiatebetweenacutemigraineratmodelanduntreated cleardifferentiationamongthecontrol,NTG,andEAgroups
controlrats.Thus,weperformedPCAandPLS-DAwithOSC along PC1 (Figure4(b); 𝑅2𝑋 = 80.9%, 𝑅2𝑌 = 99.3%, 𝑄2 =
1
pretreatment on the normalized H NMR CPMG dataset. 88.7%),asshowninFigure4(b).IntheEAgroup,wefound
A clear separation was achieved predominately along PC1 significantly higher levels of lipid (CH2), NAc and lower
(Figure3(a); 𝑅2𝑋 = 62.7%, 𝑅2𝑌 = 99.1% and 𝑄2 = 97.5%) levelsofcholine(𝑃 < 0.05)comparedwiththeNTGgroup
6 Evidence-BasedComplementaryandAlternativeMedicine
100
0.30 10
t[2] 0 0.20 7 2 1
1] 0.10 8
c[ 0
∗W −0.10 9 6 5 4
−100 −0.20
−0.30
−0.40 3
84444447777615111111111111195
−200 −100 0 100 200 4.34.24.14.03.93.83.73.53.43.33.23.02.82.52.42.32.22.12.01.91.81.71.61.51.41.31.20.0.
t[1] Var ID (Primary)
(a) (b)
2 1
0.40
2
0.30
8
1 0.20 7
c[1] 0.10 6 5 3
2] 0 ∗W
t[ 0
−1
−0.10
−0.20 4
−2
8 8 8 4 4 4 1 7 3 9 5 1 7 3
−4 −3 −2 −1 0 1 2 3 4 5.9 5.5 5.1 4.3 3.9 3.5 3. 2. 2. 1. 1. 1. 0. 0.
t[1] Var ID (Primary)
(c) (d)
Figure3:ClearseparationofCPMGandLEDspectrabetweenNTG(reddots)andcontrol(blackboxes)groups.(a)ScoresplotofOSC-PLS
analysisofCPMGdata;(b)correspondingloadingslineplot.(c)ScoresplotofOSC-PLSanalysisofLEDdata;(d)correspondingloadings
lineplot.KeystoB:(1)lipidCH3;(2)lipidCH2;(3)lactate;(4)alanine;(5)acetate;(6)glutamate;(7)OAc;(8)pyruvate;(9)glutamine;(10),
glycine.KeystoD:(1)lipidCH3;(2)lipidCH2;(3)lipidCH2CH2CO;(4)NAc;(5)lipidCH2CO;(6)PUFA;(7)PtdCho;(8)lipidCH=CH
(𝑛=10).
200
1
100
t[3] 0 t[4] 0
−100
−1
−200
−300 −200 −100 0 100 200 300 −3 −2 −1 0 1 2 3
t[1] t[1]
(a) (b)
Figure4:OSC-PLSanalysismanifestEAatacupointsreversesplasmametabolitechangesintheacutemigraineratmodel.NTGgroup(red
dots),EAgroup(bluediamonds),andcontrolgroup(blackboxes).CPMGanalyseswereconducted(a)amongthreegroups.LEDanalyses
wereconducted(b)amongthreegroups(𝑛=10).
Evidence-BasedComplementaryandAlternativeMedicine 7
Table1:ChangesofplasmametabolitesinCPMGNMRspectrainducedbyNTGandtheeffectsofEA.
Metabolites Peakregions(𝛿) NTGversus NTGversusEA NTGversusNA
untreatedcontrol
Unknown 1.14,1.18 ↑ ↓ —
Lacticacid 1.34,4.1 ↑ ↓ ↓
Glutamate 2.11,2.35 ↑ ↓ ↓
Glutamine 2.42 ↑ ↓ ↓
Glycerine 3.62,3.66 ↑ ↓ ↑
OAc 2.14,2.18 ↓ ↑ ↑
LDL/VLDL 0.86 ↓ ↑ —
Glycine 3.54 ↓ ↑ —
NAc 2.02,2.06 ↓ ↑ —
Betaine 3.26 ↓ ↑ —
Lipid 1.26,1.3 ↓ ↑ ↑
3.42,3.38,
D-Glucose 3.46,3.5,3.74, ↓ ↑ ↓
3.82,3.86,
Alanine 1.46 ↑ ↑ ↓
L-Isoleucine 0.94 ↓ ↑ —
Choline 3.22 ↓ ↑ ↓
Pyruvicacid 2.38 ↓ ↑ —
Acetoaceticacid 1.9,1.94 ↑ ↓ —
3-HB 1.22 ↑ ↓ ↑
Creatine 3.94 ↓ ↑ ↑
NAc:N-acetylglycoproteins;OAc:O-acetylglycoproteins;3-HB:3-hydroxybutyricacid.Thearrowsindicatethedirectionofthechange(↑:increase; ↓:
decrease)intheconcentrationamonguntreatedcontrolgroupofanimals,EAgroupofanimals,andNAgroupofanimalsrelativetoNTGgroupanimals
(𝑛=10).
Table2:ChangesofplasmametabolitesinLEDNMRspectrainducedbyNTGandtheeffectsofEA.
Metabolites Peakregions(𝛿) NTGversusuntreatedcontrol NTGversusEA NTGversusNA
NAc 2.02,2.06 ↑ ↓ ↑
OAc 2.14,2.18 ↑ ↓ ↑
Lipoid 1.3,1.34,1.26,2.22,2.26 ↓ ↑ ↓
Unsaturatedlipid 5.26,5.34 ↓ ↑ ↓
LDL/VDL 0.86,0.9 ↓ ↑ ↑
PUFA 2.74,2.78,2.82 ↓ ↑ —
PtdCho 3.22 ↓ ↑ ↓
FA 1.58,1.74,1.78,1.7,1.82,2.26 ↓ ↑ ↑
NAc:N-acetylglycoproteins;OAc:O-acetylglycoproteins;PtdCho:phosphatidylcholine;UFA:unsaturatedlipid;FA:fattyacid;PUFA:polyunsaturatedfatty
acid(𝑛=10).
(Figures 6(c) and 6(d)). Taken together, these data sug- acupointsandEAatnonacupoints.Toaddressthisquestion,
gest that EA reverses NTG induced changes by restoring we next executed PLS analysis with OSC pretreatment on
the metabolic profiling and reversing the levels of plasma both CPMG and LED data among the control, NTG, and
metabolites, such as lactic acid, OAc, NAc, pyruvic acid, the NA groups. After 20min of EA at nonacupoints, we
alanine, LDL/VLDL, glutamate, isoleucine, creatine, lipid observed that the metabolic profiling of the NA group was
(CH2),andlipid(CH3)intheEAgroupsrelativetotheNTG clearly separated from that of the control and the NTG
group. group (Figure5(a), 𝑅2𝑋 = 74.2%, 𝑅2𝑌 = 98.9%, and 𝑄2
= 97.6%). Meanwhile, of particular interest are data shown
in Figure5(b) (𝑅2𝑋 = 65.1%, 𝑅2𝑌 = 99.1%, 𝑄2 = 96.4%)
3.4. Clear Separation of Metabolic Profiling between EA at andFigure5(d)(𝑅2𝑋=72.7%,𝑅2𝑌=98.8%,𝑄2=88.7%),
AcupointsandEAatNonacupoints. Aremainingquestionis illustrating that the metabolic profile of the NA group was
whetherthereareanymetabolomicdifferencesbetweenEAat clearly distinct from controls, unlike EA group, which was
8 Evidence-BasedComplementaryandAlternativeMedicine
200
2
100
1
t[2] 0 t[3] 0
−100 −1
−2
−200
−200 −100 0 100 200 −5 −4 −3 −2 −1 0 1 2 3 4 5
t[1] t[1]
(a) (b)
3
3
2
2
1
1
2] 0 3] 0
t[ t[
−1
−1
−2
−2
−3
−3
−3 −2 −1 0 1 2 3 −3 −2 −1 0 1 2 3
t[1] t[1]
(c) (d)
12
10
8
6
4
2
2] 0
t[ −2
−4
−6
−8
−10
−12
−10 0 10
t[1]
(e)
Figure5:OSC-PLSanalysisdiscriminatesclearseparationbetweenEAatacupointsandEAatnonacupointsintheacutemigraineratmodel.
NTGgroup(reddots),EAgroup(bluediamonds),NAgroup(purplestars),andcontrol(blackboxes)group.CPMGanalyseswereconducted
(a)amongthreegroupsand(b)amongEA,NA,andcontrolgroup.LEDanalyseswereconducted(c)amongthreegroupsand(d)amongEA,
NA,andcontrolgroupand(e)betweenEAandNAgroup(𝑛=10).
verysimilartocontrols.Furthermore,aclearseparationwas and this was characterized by different metabolic profiling
observed between the EA group and the NA group along and a series of significant changes of plasma metabolites
PC1(Figure5(e),𝑅2𝑋=63.6%,𝑅2𝑌=99.9%,𝑄2=99.3%). in the NA groups relative to EA group. In contrast, we
Thelevelsofsixmetabolitesincludinglipid(CH2),choline, found that the level of glutamate, an important excitatory
alanine, isoleucine, LDL/VLDL, and NAc were significantly neurotransmitter that triggers migraine, was significantly
different in the EA group compared to the NA group decreased in both EA and NA groups compared with the
(Figure6(f);𝑃<0.05).Combined,thesedatasuggestthatEA NTG group. This observation indicates that glutamate may
at nonacupoints cannot reverse NTG-induced changes, but be crucial for electroacupuncture to relieve acute migraine,
there are distinct differences compared to EA at acupoints, butitisnotacupoint-dependent.
Evidence-BasedComplementaryandAlternativeMedicine 9
45 ∗∗
∗∗
40
∗∗
35
∗∗
∗∗ ∗∗
30
∗∗∗∗
25
∗∗
∗∗
20 ∗∗∗∗
∗∗ ∗∗
15
∗
∗∗ ∗
∗ ∗∗
10 ∗
5
0
30 60 90 120 150 180 210 240
−5 (min)
Control NA
NTG EA
(a)
2000 180
1800 160
1600 140
1400 120 ** **
1200
100
1000
80
800
60
600 **
400 40
200 20
0 0
La Glucose OAc Lipid CH2 Lipid CH3 Choline PtdCho Betaine Glycine Creatine Alanine NAc Gly dica civury 3-HB UFA LDL Lipid Ace Isoleucine Glu Gln
P
Control Control
NTG NTG
(b)
2500 120
∗
∗∗∗∗
100
2000
80
1500
Mean Mean 60
1000
40
∗
500 ∗∗
20
∗
0 0
La Glucose OAc Alanine NAc Gly Pyruvic acid 3-HB
Control EA Control EA
NTG NA NTG NA
(c)
Figure6:Continued.
10 Evidence-BasedComplementaryandAlternativeMedicine
1000 ∗∗ 160 ∗∗
∗ ∗∗
140
∗∗
800 ∗∗ ∗∗(cid:3) (cid:3) ∗∗ ∗∗
120
600 100
an ∗∗ an 80
e e
M ∗∗ M
400
∗ ∗ 60
40
200
20
(cid:13)
0 0
LipidCH LipidCH Choline UFA PtdCho LDL Lipid Ace
2 3
(d)
800 140
∗∗ ∗
700 120
600
100
500
80
n n
a 400 a
e e
M M 60 ∗∗
300 ∗ ∗∗ ∗
∗∗
40
200
100 20
0 0
Betaine Glycine Creatine Isoleucine Glu Gln
Control EA Control EA
NTG NA NTG NA
(e)
1000 140
900 ∗ ∗ ∗
120
800 ∗
700 100
600
80
n n
a 500 a
e e
M M 60
400
∗
300 40
200 ∗
20
100
0 0
LipidCH Choline Alanine Isoleucine LDL NAc
2
EA EA
NA NA
(f)
Figure6:Thenumberofhead-scratchingandtheconcentrationofmetabolitesfromcontrol,NTG,EA,andNAgroups.Repeatedmeasures
analysisofvarianceandmultivariateanalysisofvariancewereaccordinglyconductedamongfourgroups.Eachcolumnrepresentsthemean,
withtheSEMshownbyerrorbars.(a)Thenumberofhead-scratchingfromcontrol,NTG,EA,andNAgroupsafterNTGinjection(𝑛=6).
∗𝑃<0.05,∗∗𝑃<0.01.(b)Differencesinplasmalevelsof22metabolitesweredetectedinnitroglycerin-inducedratscomparedwithcontrols.
LDL/VLDL,lipidweredrasticallydecreasedandglutamatewassignificantlyincreasedinNTGgrouprelativetocontrolgroup(𝑛 = 10).
∗∗𝑃<0.01.(c)Themetaboliteschangesofglycometabolismamongcontrol,NTG,EA,andNAgroup.EAgroupreversedplasmametabolite
changesinNTGgroup(𝑛 = 10).∗𝑃 < 0.05,∗∗𝑃 < 0.01.(d)Themetabolitechangesoflipidmetabolismamongfourgroups(𝑛 = 10).
∗𝑃 < 0.05,∗∗𝑃 < 0.01.(e)Themetabolitechangesofaminoacidsmetabolismamongfourgroups(𝑛 = 10).∗𝑃 < 0.05,∗∗𝑃 < 0.01.(f)
SignificantdifferencesofmetabolitechangesbetweenEAandNAgroup(𝑛=10).∗𝑃<0.05.