research article effects of electroacupuncture on chronic

Research ArticleEffects of Electroacupuncture on Chronic UnpredictableMild Stress Rats Depression-Like Behavior and Expression ofp-ERKERK and p-P38P38

Jian Xu1 Yanling She2 Ning Su2 Ruixin Zhang3 Lixing Lao34 and Shifen Xu1

1Shanghai Municipal Hospital of Traditional Chinese Medicine Shanghai University of Traditional Chinese MedicineShanghai 200071 China2Guangzhou University of Traditional Chinese Medicine Guangzhou 510405 China3University of Maryland School of Medicine Baltimore MD 21201 USA4School of Chinese Medicine The University of Hong Kong Pokfulam Hong Kong

Correspondence should be addressed to Shifen Xu xu teacher2006126com

Received 20 December 2014 Revised 7 April 2015 Accepted 12 May 2015

Academic Editor Antonella Di Sotto

Copyright copy 2015 Jian Xu et al This is an open access article distributed under the Creative Commons Attribution License whichpermits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

We investigate the antidepressant-like effect and mechanism of electroacupuncture (EA) on a chronic unpredictable mild stressrats depression-like behavior In our study depression in rats was induced by unpredictable chronic mild stress (UCMS) andisolation for four weeks Male Sprague-Dawley rats were randomly divided into four groups Normal Model EA and Sham EAEA treatment was administered for two weeks once a day for five days a week Two acupoints Yintang (EX-HN3) and Baihui(GV20) were selected For sham EA acupuncture needles were inserted shallowly into the acupoints EX-HN3 and GV20 Noelectrostimulatorwas connectedThe antidepressant-like effect of the electroacupuncture treatmentwasmeasured by sucrose intaketest open field test and forced swimming test in rats The protein levels of phosphorylated extracellular regulated protein kinases(p-ERK12)ERK12 and p-P38P38 in the hippocampus (HP) were examined by Western blot analysis Our data demonstrate thatEA treatment decreased the immobility time of forced swimming test and improved the sucrose solution intake in comparison tounpredictable chronic mild stress and placebo sham control Electroacupuncture may act on depression by enhancing p-ERK12and p-p38 in the hippocampus

1 Introduction

Depression a debilitating illness that exerts a large emotionaland monetary cost on society [1] involves emotion cogni-tion and physical symptoms Clinical depression is charac-terized in the DSM-IV-R (APA2004 DSM IV-R MassonAPA) by low mood (sadness loss of motivation feelings ofworthlessnessguilt and suicidal ideas) reduced cognition(concentrationattention deficit) low or impaired psychomo-tor activity fatigue loss of appetite and sleeplessness Theillness is characterized by loss of confidence in oneselfthe world and the future and it is often preceded by aperiod of acute or chronic stress [2] Depression is a seriouscondition that often requires medical intervention Althoughclinically it is treated with antidepressants however not all

patients respond to antidepressant treatment Moreover thetherapeutic effect takes several weeks to manifest and is oftenaccompanied by unwanted side effects [3] For these reasonsnew strategies for treating depression are urgently needed

Acupuncture is a major therapy that has been used inChina for thousands of years to treat various medical andpsychiatric conditions [4] and some studies substantiate itsbenefits for depression Duan et al reported that somaticsymptoms of depression were alleviated faster and with feweradverse effects when electroacupuncture (EA) with Baihui(GV20) and Yintang (EX-HN3) was combined with theantidepressant fluoxetine [5] Manber et al recently reportedthat acupuncture was more effective than nonspecific shamacupuncture control on depression in pregnant women andthat the response rate was comparable to that observed for

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2015 Article ID 650729 8 pageshttpdxdoiorg1011552015650729

2 Evidence-Based Complementary and Alternative Medicine

conventional treatments of similar length [6] A review showsthat acupuncture therapy is safe and effective in depressivedisorder and poststroke depression and may be consideredan alternative option for treating both disorders [7]

It has been reported that acupuncture treatment couldactivate ERK-CREB pathway and alleviate depressive-like behavior [8] The mitogen-activated protein kinases(MAPKs) in mammals include c-Jun NH2-terminal kinase(JNK) p38MAPK and extracellular signal-regulated kinase(ERK) These enzymes are serine-threonine protein kinasesthat regulate various cellular activities including prolifer-ation differentiation apoptosis or survival inflammationand innate immunity The compromised MAPK signalingpathways contribute to the pathology of diverse humandiseases including neurodegenerative disorders and cognitivedisorder [9] ERK12 and p38MAPKs are members of theMAPK family that are activated by a variety of environmentalstresses and inflammatory cytokines Currently availabledata suggest that ERK- and p38-responsive MAPKs functionduring mitogenic and stressful conditions respectively [10]

In the present study we used sucrose intake test openfield test and forced swimming test to determine whetherEA affects UCMS-induced depression-like behavior Toexamine the mechanism underlying the effect of EA onUCMS-induced depressive-like hippocampal nerve systemresponses we used Western blot analysis to examine theprotein levels of p-ERK12ERK12 and p-P38P38 in thehippocampus

2 Materials and Methods

21 Animal Preparation Male Sprague-Dawley rats weighingbetween 180 and 200 grams were obtained from the MedicalSchool of Shanghai Jiaotong University Shanghai ChinaThey were kept under controlled environmental conditions(22 plusmn 1∘C relative humidity 40ndash60 alternate dark-lightcycles from 900 am to 900 pm and water and food adlibitum) The rats were housed separately in standard cagesand given one week to adapt to the environment beforeexperimentation The animal protocols were approved bythe Ethical Committee of Shanghai Jiaotong University ofMedical Sciences

22 Experiment Design The rats were randomly divided intofour groups of 7 rats in each group Normal group Modelgroup EA group and Sham EA group The Normal groupwas given no stress except general handling for 4 weeksThe rats in the Model group EA group and Sham groupwere exposed to CUMS twice a day for 2 weeks Afterwardsthe Model group was sequentially exposed to CUMS twicea day for 2 weeks the EA group received EA treatment inthe morning and CUMS for two weeks once a day in theafternoon the Sham EA group received EA treatment in themorning and CUMS once a day in the afternoon for twoweeks We employed the three commonly used behavioraltests for depression the sucrose intake test forced swim tests(FST) and open field tests (eg (a) total distance traveled(b) distance traveled within a central area (c) time spent in

central area and (d) rearing and grooming) The tests wereconducted for four weeks after the initial intervention Afterthe behavioral tests had been completed rats were sacrificedby decapitation for Western blot analysis

23 Chronic Unpredictable Stress The CUS paradigm wasperformed as previously described [11] with minor modifi-cations Rats that are 8 weeks old were randomly exposedto one of the following stressors food deprivation waterdeprivation cage tilt 45 swimming in 10∘C ice water restraintstress cage shaking and light on or light off twice daily fortwo weeks Subsequently according to different groups theywere exposed twice or once daily for another two weeksStressors were given randomly at different times of the dayto establish unpredictability The schedule of stressors isshown in Table 1 The rats in the Normal control group werehoused undisturbedly except for necessary procedures suchas routine cage cleaning

24 Acupuncture Treatment Procedures We used two acu-points Yintang (EX-HN3) and Baihui (GV20) commonlyemployed in the clinic for the treatment of depression [1213] In humans EX-HN3 is located midway between themedial ends of the two eyebrows and GV20 on the midlineat the apex of the skull Anatomical equivalents of thesepoints were located in the animals [10] After the skin hadbeen cleaned with alcohol swabs disposable acupunctureneedles (025 times 13mm Suzhou Medical Appliance FactorySuzhou China) were inserted about 5mm deep into theacupoints An electrostimulator Huatuorsquos Acupoint NerveStimulator (SDZ-V Huatuo Medical Technology Co LtdSuzhou China) was connected and the electrical currentwas delivered to the needles The anode was inserted intoEX-HN3 the cathode was inserted into GV20 EA frequencywas held constant at 2Hz pulse width 03ms and intensitywas adjusted slowly over the period of approximately twominutes to the designated level (ie 1 2 or 3mA) Mildmuscle twitching was observedThe needles were retained forfifteen minutes As previously reported by Lao et al each ratwas placed in an inverted clear 510158401015840times 810158401015840times 1110158401015840 plastic chamberduring the treatment and was neither restrained nor givenanesthetic [14] The animals remained awake and still duringtreatment and no signs of distress were observed Treatmentwas given for two weeks once a day at approximately 1000am fromMonday to Friday

25 Sham Control Procedure For control after the skin wascleaned with alcohol swabs disposable acupuncture needles(025 times 13mm Suzhou Medical Appliance Factory SuzhouChina) were inserted about 2mm deep into the acupointsEX-HN3 and GV20 A piece of adhesive tape was secured atthe surface No electrostimulator was connectedThe needleswere retained for fifteen minutes Each rat was placed inan inverted clear 510158401015840times 810158401015840times 1110158401015840 plastic chamber during thetreatment and was neither restrained nor given anesthetictoo Control and treatment animals were treated on the sameschedule to make the procedures comparable

Evidence-Based Complementary and Alternative Medicine 3

Table 1 Unpredictable stressors for 14 days

Day Time of 1st stressor 1st stressor Time of 2nd stressor 2nd stressor1 1000 am 10∘C swim stress (4min) 400 pm Lights off (2 h)2 1230 pm Lights off (3 h) 700 pm Lights on overnight3 1000 am 4∘C cold isolation (20min) 400 pm Cage shaking (5min)4 1100 am 4∘C cold isolation (1 h) 200 pm Restraint stress (40min)5 300 pm Lights off (2 h) 500 pm Cage tilting 45∘ (2 h)6 1000 am Food deprivation (24 h)7 1000 am Water deprivation (24 h)8 330 pm 10∘C swim stress (15min) 700 pm Lights on overnight9 400 pm Restraint stress (1 h) 500 pm Lights off (1 h)10 1000 am Cage shaking (5min) 330 pm 4∘C cold isolation (15min)11 1200 pm Lights off (2 h) 700 pm Lights on (2 h)12 1000 am Restraint stress (1 h) 200 pm 10∘C swim stress (5min)13 1000 am Food deprivation (24 h)14 1000 am Water deprivation (24 h)

CUMS twice a day CUMS once a dayEA once a day

29th day

Sucroseintake test

30th day

OPT

31st day 32nd day

FSTFSThabituation

Behavioral tests

1stndash14th day 15thndash28th day

Figure 1 Timeline for all procedures

26 Behavioral Tests Behavioral tests were performed fourweeks after the initial intervening They were conductedbetween 4 pm and 9 pm and videotaped in a random orderfor later scoring by two observers blinded to the treatmentassignment Interrater reliability correlation was gt090 for alltests Timeline for all procedures can be seen in Figure 1

261 FST [15] The FST was conducted over three days andconsisted of two pretests and a test During the pretestswhich were for training purposes each rat was placed for15 minutes in a clear Plexiglas cylinder (25 cm in diameter65 cm high) filled with 50 cm of 25 plusmn 1∘C water The resultsof the pretests were not recorded Twenty-four hours afterthe second pretest each rat was returned to the apparatusfor five minutes Upon completion of the trial the animalwas dried thoroughly and placed back in its home cageThe apparatus was drained and cleaned after each useBehavioral changes were scored for analysis following themethods previously reported [16] immobility was defined asmaking only thosemovements necessary to stay afloat longerimmobility indicated more serious depression Immobilitylatency was operationally defined as at least ten consecutiveseconds of immobility

262 OFT [17] The open field test (OFT) is a commonlyused qualitative and quantitative measure of general loco-motor activity and willingness to explore in rodents [18]

The open field is a table that may have surrounding wallsto prevent escape Usually the field is marked in a grid andsquare crossings rearing and time spent moving are used toassess the activity of the rodent In modern open field appa-ratus infrared beams can be used to automate the assessmentprocess The open field test was designed to measure anxietyand depression as well as behavioral responses such aslocomotive activity and exploratory behaviors Some studiesused the open field test to measure depression [19 20] Ratsin the open field were videomonitored with an automatedactivity monitoring system (TRU Scan CoulBourn Instru-ments USA) The rat was placed in a randomized startingcorner of a square black apparatus (50 cm long times 50 cmwide) with walls 100 cm high Its behavior including totaldistance traveled total movement time distance traveledwithin the central area and time spent in the central areawas recorded automatically for five minutes and analyzedwith computer software Incidents of rearing and grooming(face washing body and genital grooming body and pawlicking and scratching)were counted by an investigator Totaldistance traveled indicates an animalrsquos spontaneous activityTime spent and distance traveled within the central areashow the animalrsquos ability to explore Rearing and groomingincidents indicate the animalrsquos curiosity All activity wasrecorded by a video camera mounted above the open fieldand scored in real time (or digitized and scored later) by anadvanced motion recognition software package that detectsand analyzes movements


263 Sucrose Intake Test The sucrose intake test is a behav-ioral task used to assess the degree of anhedonia in rats [21]The sucrose intake test was performed on the first day of thetest Before the test the rats were habituated for 24 hoursto two bottles one with 1 sucrose (Sigma) and the otherwith tap water On the last day of EA treatment all rats weredeprived of water for 24 h Then the rats were given 24 hexposure to the two identical bottles again to test for fluidconsumption Two-bottle tests for each cage were adoptedthroughout the procedure Sucrose solution consumptionwas recorded by calculating the volume of test solution

264 Western Blot Analysis After the behavioral tests hadbeen completed rats were sacrificed by decapitation forWestern blot analysisThen theHPwas dissected Brain tissuewas homogenized in an extraction buffer containing SDS lysisbuffer protease inhibitor mixture and phosphatase inhibitormixtureThe homogenates were centrifuged at 13000 rpm for15min at 4∘C The same level of total proteins was loadedon the gels and separated on 10 SDS-PAGE and transferredonto PVDF membranes Blots were blocked in blockingbuffer (1lowastTBS 01 Tween-20 with 5 nonfat dry milk) for1 hour at room temperature Following washing blots wereincubated overnight at 4∘C with primary antibodies againstERK12 (1 1000) ERK12 phosphorylated onThr202Tyr204(1 1000) p38MAPK (1 1000) and p38MAPK phosphory-lated on Thr180Tyr182 (1 1000) After washing 2 times inTBS-T and 3 times in 1milk-TBST for 5min each blots wereincubated with goat anti-mouse or anti-rabbit secondaryantibody for 1 h at room temperature and thenwashed 2 timesin 1 milk-TBST and 3 times in TBS-T for 5 minutes eachBand intensity was detected by LI-COROdyssey infrared flu-orescence scanning imaging system and quantified by ImageJThe relative level of each signal protein was calculated as theratio between phosphorylated and total protein [22ndash24]

27 Statistical Analysis Data were analyzed using SPSS150for Windows XP All data were expressed as mean plusmn SD Ananalysis was performed byANOVA with Bonferronirsquos test formultiple comparisons and by t-test a 119875 value of lt005 wasconsidered statistically significant

3 Results

31 FST The forced swimming results are shown in Figure 2There are significant differences observed among groups[F(324)

=2693119875 lt 001]TheModel rats showed a significantincrease in the immobile time compared to Normal rat (119875 lt001) EA treatment had a significant effect on the decreasedimmobile time compared to sham control (119875 lt 001)suggesting that EA may decrease immobile time of FST indepressed rat but Sham EA had no effect on it

32 Open Field Tests The results of the open field testsare shown in Figure 3 In the total distance there was asignificant difference among groups [F

(324)= 3965 119875 lt

005] The Model rats showed a significant decrease of thetotal distance compared to Normal rat (119875 lt 005) However

0

10

20

30

40

50

Imm

obile

tim

e (s)

lowastlowast lowastlowast

Normal Model EA Sham EA

Figure 2 Immobility time (in seconds) of forced swimming test inthe following groups (119899 = 7 per group) Normal Model EA andSham EA lowastlowast119875 lt 001

there was no significant difference between EA and shamcontrol (119875 gt 005) In the central distance there was nosignificant difference among groups [F

(324)= 2442 119875 gt

005] For time in the central area there was a significantdifference among groups [F

(324)= 4812 119875 lt 001] The

Model rats showed a significant decrease of time in the centralarea compared toNormal rat (119875 lt 005) EA treatment hadnosignificant effect on the time in the central area compared tosham control (119875 gt 005) There was no significant differenceobserved among groups in rearing and grooming incidents[F(324)

= 271119875 gt 005] suggesting that EAhadno significanteffect on open field tests compared to sham control

33 Sucrose Intake Test The results of the sucrose intaketest are shown in Figure 4 The sucrose solution intakesignificantly differed among groups [F

(324)= 1019 119875 lt 001]

The sucrose intake was significantly reduced in model ratcompared to Normal group (119875 lt 001) It was improvedafter EA treatment compared to sham control (119875 lt 005)suggesting that EA may improve sucrose solution intake indepression rat However Sham EA had no effect on it

34 The Effects of Western Blot Analysis

341 The Effect of p-ERK12 Western Blot Analysis Westernblot analysis revealed that there was no significant differencein the ERK12 protein level among groups in the HP [F

(320)=

1499 119875 gt 005] However a p-ERK12 level significantlydiffered among groups in the HP [F

(320)= 8408 119875 lt 001]

Chronic stress significantly decreased p-ERK12 in the HP(119875 lt 005) compared to the Normal group EA treatmentsignificantly increased p-ERK12 level in HP compared toSham EA (119875 lt 005) At the same time from the ratiobetween p-ERK12 andERK therewas a significant differenceamong groups [F

(320)= 8284 119875 lt 001] Model group

significantly decreased compared to Normal group (119875 lt005) EA treatment increased the ratio between p-ERK12and ERK compared to sham control (119875 lt 001) suggestingthat EA may reverse the deficits in p-ERK in the HP ondepression rats However sham control had no effect on it(see Figures 5 and 6)


Normal Model0

500

1000

1500

Tota

l dist

ance

(cm

)

EA Sham EA

lowast

(a)

0

200

400

600

Dist

ance

in ce

ntra

l are

a (cm

)


(b)

0

100

200

300

400lowast

Tim

e in

cent

ral a

rea (

s)


(c)

0

5

10

15

Rear

ing

and

groo

min

g in

cide

nts


(d)

Figure 3 Open field test scores in the following groups (119899 = 7 per group) Normal Model EA and Sham EA (a) Total distance (b) distancein the central area (c) time in central area and (d) rearing and grooming incidents lowast119875 lt 005 There was no significant difference betweenEA and Sham EA in all parameters of OFT

0

50

100

150lowastlowast

lowast

Sucr

ose s

olut

ion

inta

ke (m

L)


Figure 4 Sucrose solution intake in the following groups (119899 = 7 pergroup) Normal Model EA and Sham EA lowast119875 lt 005 lowastlowast119875 lt 001

342 The Effect of p-p38 Western Blot Analysis Westernblot analysis revealed that there was no significant differencein the p38 protein level among groups in the HP [F

(320)

= 3104 119875 gt 005] However a p-p38 level significantly

differed among groups in the HP [F(320)

= 5895 119875 lt 001]Unpredictable chronic mild stress decreased p-p38 in theHP compared to the Normal rat EA treatment significantlyincreased p-p38 level in HP compared to sham control(119875 lt 001) At the same time from the ratio between p-p38 and p38 there were significant differences among groups[F(320)

= 8102 119875 lt 001] Model group decreased the ratiosignificantly compared to Normal group (119875 lt 001) EAtreatment had a significant effect on depression model ratscompared to sham control (119875 lt 001) suggesting that EAmay reverse the deficits in p-p38 in theHP on depression ratsHowever Sham EA had no effect on it (see Figures 7 and 8)

4 Discussion

The present study showed that EA treatment decreased theimmobility time of forced swimming test and improved thesucrose solution intake in comparison to placebo sham con-trol (119875 lt 005 119875 lt 001) However for the open field test EAhad no significant effect compared to placebo sham controlThese results suggest that EA stimulation may alleviate some


0

50

100

150lowast lowast

p-ERKGAPDH p-ERKERKERKGAPDH

Prot

ein

leve

l (

of c

ontro

l) lowast lowastlowast

NormalModel

EASham EA

Figure 5 ERK and p-ERK protein expression in the hippocampusin the following groups (119899 = 6 per group) Normal Model EA andSham EA lowast119875 lt 005 lowastlowast119875 lt 001

p-ERK

ERK

GAPDH

Normal ModelEA Sham EA

Figure 6 Representative Western blots showing levels of p-ERKERK in the hippocampus of the following groups (119899 = 6 per group)Normal Model EA and Sham EA

symptoms in this animal model of depression Some otherstudies that researched antidepressant drugs showed thatthe results in OFT also had no significant difference [2526] For example fluoxetine decreased immobility of forcedswimming test but did not affect activities in an open field[25] Baicalein reduced the immobility time in the forcedswimming test and tail suspending test of mice but therewas no effect on open field test [27] Our results are similarto those reported by others [28ndash30] Lee et al [28] observedthe effect of acupuncture in a rat model of depression Theyfound that acupuncture treatment showed significantly lessimmobility time compared to control Neither open fieldtests nor MWMs were performed Dos Santos Jr et al [29]used rats to investigate the effects of EA on depressive-likesymptoms with a learned helplessness test and the FST TheEA group showed significantly enhanced active avoidance inthe learned helplessness test and less immobility in the FST(119875 lt 001) compared to sham control but with EA there wasno effect on number of squares crossed in an open field testWe suppose that maybe open field test is not sensitive enoughto depression

We also found that chronic stress exposure caused deficitsin p-ERK and p-p38 in the HP which could be reversedby electroacupuncture treatment There was a significantdifference between EA treatment and sham control (119875 lt001) ERK and p38 belong to MARK family ERK pathway

0

50

100

150

200lowastlowast

lowastlowastlowast

p-P38GAPDH p-P38P38P38GAPDH

Prot

ein

leve

l (

of c

ontro

l)

NormalModel

EASham EA

Figure 7 P38 and p-P38 protein expression in the hippocampus inthe following groups (119899 = 6 per group) Normal Model EA andSham EA lowast119875 lt 005 lowastlowast119875 lt 001

p-P38

P38

GAPDH


Figure 8 RepresentativeWestern blots showing levels of p-P38 P38in the hippocampus of the following groups (119899 = 6 per group)Normal Model EA and Sham EA

in an intracellular signaling cascade is implicated in severalforms of learning memory and neuroplasticity [31] Stresscaused a reduction in ERK phosphorylation in both thehippocampus and the prefrontal cortex whereas it led toa nonsignificant decrease in BDNF levels only in the HPThe involvement of ERK activation in the stress responseand antidepressant therapy has also been shown in anotherstudy [32]The p38MAPKmainly functioned as mediators ofcellular stresses since increasing evidence implicates stress asan important factor in the vulnerability to depression [33]

The mechanisms of acupuncture on depression havebeen exploredWe previously reported that acupuncturemayact on depression by mediating the regulation of centralmonoamine neurotransmitters including norepinephrine(NE) 5-hydroxytryptamine (5-HT) and dopamine (DA)[34] It has been reported that acupuncturersquos antidepressiveeffects may involve neuropeptide Y (NPY) activation in thehypothalamus [28] One research has reported that themech-anism underlying the antidepressant-like effects of EA mayassociate with the enhancement of amplifying neural progen-itors (ANPs) proliferation and preserving quiescent neuralprogenitors (QNPs) from apoptosis [35] In our study wefound that EAmay enhance p-ERK12 andp-p38 in theHPon


depression rats Lu et al [8] have reported that acupuncturetreatment could alleviate depressive-like behaviors includingOFT and sucrose intake and its mechanism could activateERK-CREBpathwayWe got the same result that acupunctureimproves the p-ERK12 in the hippocampus despite thedifferent results onOFTWe indicated that acupuncture couldrelieve some symptoms in this animal model of depressionincluding forced swimming test and sucrose solution intakeIn that article EA was compared to Paroxetine and bothof them were equally effective However in our study EAwas compared to placebo control in order to make the effectof EA clear Although EA could increase p-ERK12 in thehippocampus which has been reported by some studies [836] we found that EA could enhance the p-p38 The MAPKfamily is subdivided into three main classes ERK Jun N-terminal kinases (JNK) and the p38 kinase and they allare involved in differentiation survival and structural andfunctional plasticity of neurons So far only ERK pathwaywas examined in animal model of depression and effect ofacupuncture However the levels of JNK and p38 kinasescan be also altered in depression [37] In order to determinewhether EA could play the role on p-p38 in hippocampus wedetected the level of p-p38 kinases and found that EA couldimprove the p-p38 and p-ERK12 in the hippocampus at thesame time

5 Conclusion

In conclusion we found that EA could alleviate unpredictablechronic mild stress-induced depression-like behavior EAcould improve p-ERK12 and p-p38 in the HP in the ratsexposed to chronic unpredicted mild stress Our resultssuggest that the antidepressant-like effect of acupuncturemight be mediated by modulating the p-ERK12 and p-p38MAPK pathway in the hippocampus Further studiesto investigate EArsquos mechanisms of action on depression areneeded

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Authorsrsquo Contribution

Jian Xu and Yanling She made the same work for this paper

Acknowledgments

The study was partly supported by the National NaturalScience Foundation of China (81102636) Shanghai Edu-cation Bureau of Scientific Research Innovation Project(2014ZZ116) and ShanghaiMunicipal Commission of Healthand Family Planning (ZY3-CCCX-3-3022 ZYSNXD-CC-HPGC-JD-004)

References

[1] J A Mauskopf G E Simon A Kalsekar C Nimsch EDunayevich and A Cameron ldquoNonresponse partial responseand failure to achieve remission humanistic and cost burden inmajor depressive disorderrdquo Depression and Anxiety vol 26 no1 pp 83ndash97 2009

[2] F Lavergne and T M Jay ldquoA new strategy for antidepressantprescriptionrdquo Frontiers in Neuroscience vol 4 article 192 2010

[3] C Taylor A D Fricker L A Devi and I Gomes ldquoMechanismsof action of antidepressants from neurotransmitter systems tosignaling pathwaysrdquo Cellular Signalling vol 17 no 5 pp 549ndash557 2005

[4] A Yeung R Schnyer R Ma and DMischoulon ldquoAcupuncturefor the treatment of psychiatric disordersrdquo in Natural Medica-tion for Psychiatric Disorders Considering the Alternatives pp283ndash302 Lippincott Williams ampWilins Philadelphia Pa USA2nd edition 2008

[5] D-M Duan Y Tu L-P Chen and Z-J Wu ldquoEfficacyevaluation for depression with somatic symptoms treated byelectroacupuncture combined with fluoxetinerdquo Journal of Tra-ditional Chinese Medicine vol 29 no 3 pp 167ndash173 2009

[6] R Manber R N Schnyer D Lyell et al ldquoAcupuncture fordepression during pregnancy a randomized controlled trialrdquoObstetrics and Gynecology vol 115 no 3 pp 511ndash520 2010

[7] Z-J Zhang H-Y Chen K-C Yip R Ng and V TWong ldquoTheeffectiveness and safety of acupuncture therapy in depressivedisorders systematic review and meta-analysisrdquo Journal ofAffective Disorders vol 124 no 1-2 pp 9ndash21 2010

[8] J Lu J Liang J-RWang L Hu Y Tu and J-Y Guo ldquoAcupunc-ture activates ERK-CREB pathway in rats exposed to chronicunpredictable mild stressrdquo Evidence-based Complementary andAlternativeMedicine vol 2013 Article ID 469765 7 pages 2013

[9] P P Roux and J Blenis ldquoERK and p38MAPK-activated proteinkinases a family of protein kinases with diverse biologicalfunctionsrdquoMicrobiology andMolecular Biology Reviews vol 68no 2 pp 320ndash344 2004

[10] E K Kim and E-J Choi ldquoCompromised MAPK signaling inhuman diseases an updaterdquo Archives of Toxicology vol 89 no6 pp 867ndash882 2015

[11] P Willner ldquoValidity reliability and utility of the chronic mildstress model of depression a 10-year review and evaluationrdquoPsychopharmacology vol 134 no 4 pp 319ndash329 1997

[12] X Cheng and L Deng Chinese Acupuncture and MoxibustionForeign Language Press Beijing China 1st edition 1987

[13] X M Shi Clinical Acupuncture amp Moxibustion Therapy ThePeoplersquos Medical Publishing House Beijing China 2004

[14] L Lao R-X Zhang G Zhang X Wang B M Berman and KRen ldquoA parametric study of electroacupuncture on persistenthyperalgesia and Fos protein expression in ratsrdquo Brain Researchvol 1020 no 1-2 pp 18ndash29 2004

[15] Y Kitada T Miyauchi A Satoh and S Satoh ldquoEffects ofantidepressants in the rat forced swimming testrdquo EuropeanJournal of Pharmacology vol 72 no 2-3 pp 145ndash152 1981

[16] P J Allen K E DrsquoAnci R B Kanarek and P F Ren-shaw ldquoChronic creatine supplementation alters depression-likebehavior in rodents in a sex-dependent mannerrdquo Neuropsy-chopharmacology vol 35 no 2 pp 534ndash546 2010

[17] J A Gray R F Drewett and B Lalljee ldquoEffects of neona-tal castration and testosterone injection on adult open-fieldbehaviour in rats with atypical sex difference in defecationrdquoAnimal Behaviour vol 23 no 4 pp 773ndash778 1975


[18] S C Stanford ldquoThe Open Field Test reinventing the wheelrdquoJournal of Psychopharmacology vol 21 no 2 pp 134ndash135 2007

[19] R R Parekar K S Jadhav P A Marathe and N N RegeldquoEffect of Saraswatarishta in animalmodels of behavior despairrdquoJournal of Ayurveda and Integrative Medicine vol 5 no 3 pp141ndash147 2014

[20] L SGodlevsky TNMuratovaNVKresyunG vanLuijtelaarand A M L Coenen ldquoAnxiolytic and antidepressive effects ofelectric stimulation of the paleocerebellar cortex in pentylenete-trazol kindled ratsrdquoActa Neurobiologiae Experimentalis vol 74no 4 pp 456ndash464 2014

[21] Y-H Lin A-H Liu Y Xu L Tie H-M Yu and X-J Li ldquoEffectof chronic unpredictable mild stress on brain-pancreas relativeprotein in rat brain and pancreasrdquo Behavioural Brain Researchvol 165 no 1 pp 63ndash71 2005

[22] K Yang Y Song Y B Tang et al ldquomAChRs activationinduces epithelial-mesenchymal transition on lung epithelialcellsrdquo BMC Pulmonary Medicine vol 14 no 1 p 53 2014

[23] G-N Xu K Yang Z-P Xu et al ldquoProtective effects of aniso-damine on cigarette smoke extract-induced airway smoothmuscle cell proliferation and tracheal contractilityrdquo Toxicologyand Applied Pharmacology vol 262 no 1 pp 70ndash79 2012

[24] Y Song H-Z Lu J-R Xu et al ldquoCarbocysteine restores steroidsensitivity by targeting histone deacetylase 2 in a thiolGSH-dependent mannerrdquo Pharmacological Research vol 91 pp 88ndash98 2015

[25] R M Santiago T Zaminelli T B Bassani et al ldquoThe mecha-nism of antidepressant-like effects of piroxicam in ratsrdquo Journalof Pharmacology and Pharmacotherapeutics vol 6 no 1 pp 7ndash12 2015

[26] D T Chau P V Rada K Kim R A Kosloff and B G HoebelldquoFluoxetine alleviates behavioral depression while decreasingacetylcholine release in the nucleus accumbens shellrdquoNeuropsy-chopharmacology vol 36 no 8 pp 1729ndash1737 2011

[27] Z Xiong B Jiang P-F Wu et al ldquoAntidepressant effects of aplant-derived flavonoid baicalein involving extracellular signal-regulated kinases cascaderdquo Biological and Pharmaceutical Bul-letin vol 34 no 2 pp 253ndash259 2011

[28] B Lee I Shim H-J Lee Y Yang and D-H Hahm ldquoEffectsof acupuncture on chronic corticosterone-induced depression-like behavior and expression of neuropeptide Y in the ratsrdquoNeuroscience Letters vol 453 no 3 pp 151ndash156 2009

[29] J G Dos Santos Jr F Kawano M M Nishida Y YamamuraL E Mello and A Tabosa ldquoAntidepressive-like effects ofelectroacupuncture in ratsrdquo Physiology amp Behavior vol 93 no1-2 pp 155ndash159 2008

[30] H Kim H-J Park H S Shim et al ldquoThe effects of acupuncture(PC6) on chronic mild stress-induced memory lossrdquo Neuro-science Letters vol 488 no 3 pp 225ndash228 2011

[31] S L Gourley F J Wu D D Kiraly et al ldquoRegionally specificregulation of ERK MAP kinase in a model of antidepressant-sensitive chronic depressionrdquo Biological Psychiatry vol 63 no4 pp 353ndash359 2008

[32] X Qi W Lin J Li et al ldquoFluoxetine increases the activityof the ERK-CREB signal system and alleviates the depressive-like behavior in rats exposed to chronic forced swim stressrdquoNeurobiology of Disease vol 31 no 2 pp 278ndash285 2008

[33] K S Kendler L MThornton and C O Gardner ldquoGenetic risknumber of previous depressive episodes and stressful life eventsin predicting onset of major depressionrdquoThe American Journalof Psychiatry vol 158 no 4 pp 582ndash586 2001

[34] S-F Xu L-X Zhuang C-Z Tang and J-J Yang ldquoEffects ofacupuncture and embedding thread on central monoamineneurotransmitters in the depression model ratrdquo Chineseacupuncture ampmoxibustion vol 27 no 6 pp 435ndash437 2007

[35] L Yang N Yue X Zhu et al ldquoElectroacupuncture pro-motes proliferation of amplifying neural progenitors and pre-serves quiescent neural progenitors from apoptosis to alleviatedepressive-like and anxiety-like behavioursrdquo Evidence-BasedComplementary and Alternative Medicine vol 2014 Article ID872568 13 pages 2014

[36] L Yang N Yue X Zhu et al ldquoElectroacupuncture upregulatesERK signaling pathways and promotes adult hippocampalneural progenitors proliferation in a rat model of depressionrdquoBMC Complementary and Alternative Medicine vol 13 article288 2013

[37] B Budziszewska M Szymanska M Leskiewicz et al ldquoThedecrease in JNK- and p38-MAP kinase activity is accompaniedby the enhancement of PP2A phosphatase level in the brain ofprenatally stressed ratsrdquo Journal of Physiology and Pharmacol-ogy vol 61 no 2 pp 207ndash215 2010

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


conventional treatments of similar length [6] A review showsthat acupuncture therapy is safe and effective in depressivedisorder and poststroke depression and may be consideredan alternative option for treating both disorders [7]

It has been reported that acupuncture treatment couldactivate ERK-CREB pathway and alleviate depressive-like behavior [8] The mitogen-activated protein kinases(MAPKs) in mammals include c-Jun NH2-terminal kinase(JNK) p38MAPK and extracellular signal-regulated kinase(ERK) These enzymes are serine-threonine protein kinasesthat regulate various cellular activities including prolifer-ation differentiation apoptosis or survival inflammationand innate immunity The compromised MAPK signalingpathways contribute to the pathology of diverse humandiseases including neurodegenerative disorders and cognitivedisorder [9] ERK12 and p38MAPKs are members of theMAPK family that are activated by a variety of environmentalstresses and inflammatory cytokines Currently availabledata suggest that ERK- and p38-responsive MAPKs functionduring mitogenic and stressful conditions respectively [10]

In the present study we used sucrose intake test openfield test and forced swimming test to determine whetherEA affects UCMS-induced depression-like behavior Toexamine the mechanism underlying the effect of EA onUCMS-induced depressive-like hippocampal nerve systemresponses we used Western blot analysis to examine theprotein levels of p-ERK12ERK12 and p-P38P38 in thehippocampus

2 Materials and Methods

21 Animal Preparation Male Sprague-Dawley rats weighingbetween 180 and 200 grams were obtained from the MedicalSchool of Shanghai Jiaotong University Shanghai ChinaThey were kept under controlled environmental conditions(22 plusmn 1∘C relative humidity 40ndash60 alternate dark-lightcycles from 900 am to 900 pm and water and food adlibitum) The rats were housed separately in standard cagesand given one week to adapt to the environment beforeexperimentation The animal protocols were approved bythe Ethical Committee of Shanghai Jiaotong University ofMedical Sciences

22 Experiment Design The rats were randomly divided intofour groups of 7 rats in each group Normal group Modelgroup EA group and Sham EA group The Normal groupwas given no stress except general handling for 4 weeksThe rats in the Model group EA group and Sham groupwere exposed to CUMS twice a day for 2 weeks Afterwardsthe Model group was sequentially exposed to CUMS twicea day for 2 weeks the EA group received EA treatment inthe morning and CUMS for two weeks once a day in theafternoon the Sham EA group received EA treatment in themorning and CUMS once a day in the afternoon for twoweeks We employed the three commonly used behavioraltests for depression the sucrose intake test forced swim tests(FST) and open field tests (eg (a) total distance traveled(b) distance traveled within a central area (c) time spent in

central area and (d) rearing and grooming) The tests wereconducted for four weeks after the initial intervention Afterthe behavioral tests had been completed rats were sacrificedby decapitation for Western blot analysis

23 Chronic Unpredictable Stress The CUS paradigm wasperformed as previously described [11] with minor modifi-cations Rats that are 8 weeks old were randomly exposedto one of the following stressors food deprivation waterdeprivation cage tilt 45 swimming in 10∘C ice water restraintstress cage shaking and light on or light off twice daily fortwo weeks Subsequently according to different groups theywere exposed twice or once daily for another two weeksStressors were given randomly at different times of the dayto establish unpredictability The schedule of stressors isshown in Table 1 The rats in the Normal control group werehoused undisturbedly except for necessary procedures suchas routine cage cleaning

24 Acupuncture Treatment Procedures We used two acu-points Yintang (EX-HN3) and Baihui (GV20) commonlyemployed in the clinic for the treatment of depression [1213] In humans EX-HN3 is located midway between themedial ends of the two eyebrows and GV20 on the midlineat the apex of the skull Anatomical equivalents of thesepoints were located in the animals [10] After the skin hadbeen cleaned with alcohol swabs disposable acupunctureneedles (025 times 13mm Suzhou Medical Appliance FactorySuzhou China) were inserted about 5mm deep into theacupoints An electrostimulator Huatuorsquos Acupoint NerveStimulator (SDZ-V Huatuo Medical Technology Co LtdSuzhou China) was connected and the electrical currentwas delivered to the needles The anode was inserted intoEX-HN3 the cathode was inserted into GV20 EA frequencywas held constant at 2Hz pulse width 03ms and intensitywas adjusted slowly over the period of approximately twominutes to the designated level (ie 1 2 or 3mA) Mildmuscle twitching was observedThe needles were retained forfifteen minutes As previously reported by Lao et al each ratwas placed in an inverted clear 510158401015840times 810158401015840times 1110158401015840 plastic chamberduring the treatment and was neither restrained nor givenanesthetic [14] The animals remained awake and still duringtreatment and no signs of distress were observed Treatmentwas given for two weeks once a day at approximately 1000am fromMonday to Friday

25 Sham Control Procedure For control after the skin wascleaned with alcohol swabs disposable acupuncture needles(025 times 13mm Suzhou Medical Appliance Factory SuzhouChina) were inserted about 2mm deep into the acupointsEX-HN3 and GV20 A piece of adhesive tape was secured atthe surface No electrostimulator was connectedThe needleswere retained for fifteen minutes Each rat was placed inan inverted clear 510158401015840times 810158401015840times 1110158401015840 plastic chamber during thetreatment and was neither restrained nor given anesthetictoo Control and treatment animals were treated on the sameschedule to make the procedures comparable





29th day

Sucroseintake test

30th day

OPT

31st day 32nd day

FSTFSThabituation

Behavioral tests











3 Results




(324)= 3965 119875 lt


0

10

20

30

40

50

Imm

obile

tim

e (s)





(324)= 2442 119875 gt


(324)= 4812 119875 lt 001] The




(324)= 1019 119875 lt 001]




(320)=


(320)= 8408 119875 lt 001]


(320)= 8284 119875 lt 001] Model group



Normal Model0

500

1000

1500

Tota

l dist

ance

(cm

)

EA Sham EA

lowast

(a)

0

200

400

600

Dist

ance

in ce

ntra

l are

a (cm

)


(b)

0

100

200

300

400lowast

Tim

e in

cent

ral a

rea (

s)


(c)

0

5

10

15

Rear

ing

and

groo

min

g in

cide

nts


(d)


0

50

100

150lowastlowast

lowast

Sucr

ose s

olut

ion

inta

ke (m

L)




(320)





4 Discussion



0

50

100

150lowast lowast


Prot

ein

leve

l (

of c

ontro


NormalModel

EASham EA


p-ERK

ERK

GAPDH





0

50

100

150

200lowastlowast

lowastlowastlowast


Prot

ein

leve

l (

of c

ontro

l)

NormalModel

EASham EA


p-P38

P38

GAPDH







5 Conclusion






Acknowledgments


References












































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




















29th day

Sucroseintake test

30th day

OPT

31st day 32nd day

FSTFSThabituation

Behavioral tests











3 Results




(324)= 3965 119875 lt


0

10

20

30

40

50

Imm

obile

tim

e (s)





(324)= 2442 119875 gt


(324)= 4812 119875 lt 001] The




(324)= 1019 119875 lt 001]




(320)=


(320)= 8408 119875 lt 001]


(320)= 8284 119875 lt 001] Model group



Normal Model0

500

1000

1500

Tota

l dist

ance

(cm

)

EA Sham EA

lowast

(a)

0

200

400

600

Dist

ance

in ce

ntra

l are

a (cm

)


(b)

0

100

200

300

400lowast

Tim

e in

cent

ral a

rea (

s)


(c)

0

5

10

15

Rear

ing

and

groo

min

g in

cide

nts


(d)


0

50

100

150lowastlowast

lowast

Sucr

ose s

olut

ion

inta

ke (m

L)




(320)





4 Discussion



0

50

100

150lowast lowast


Prot

ein

leve

l (

of c

ontro


NormalModel

EASham EA


p-ERK

ERK

GAPDH





0

50

100

150

200lowastlowast

lowastlowastlowast


Prot

ein

leve

l (

of c

ontro

l)

NormalModel

EASham EA


p-P38

P38

GAPDH







5 Conclusion






Acknowledgments


References












































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




















3 Results




(324)= 3965 119875 lt


0

10

20

30

40

50

Imm

obile

tim

e (s)





(324)= 2442 119875 gt


(324)= 4812 119875 lt 001] The




(324)= 1019 119875 lt 001]




(320)=


(320)= 8408 119875 lt 001]


(320)= 8284 119875 lt 001] Model group



Normal Model0

500

1000

1500

Tota

l dist

ance

(cm

)

EA Sham EA

lowast

(a)

0

200

400

600

Dist

ance

in ce

ntra

l are

a (cm

)


(b)

0

100

200

300

400lowast

Tim

e in

cent

ral a

rea (

s)


(c)

0

5

10

15

Rear

ing

and

groo

min

g in

cide

nts


(d)


0

50

100

150lowastlowast

lowast

Sucr

ose s

olut

ion

inta

ke (m

L)




(320)





4 Discussion



0

50

100

150lowast lowast


Prot

ein

leve

l (

of c

ontro


NormalModel

EASham EA


p-ERK

ERK

GAPDH





0

50

100

150

200lowastlowast

lowastlowastlowast


Prot

ein

leve

l (

of c

ontro

l)

NormalModel

EASham EA


p-P38

P38

GAPDH







5 Conclusion






Acknowledgments


References












































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Normal Model0

500

1000

1500

Tota

l dist

ance

(cm

)

EA Sham EA

lowast

(a)

0

200

400

600

Dist

ance

in ce

ntra

l are

a (cm

)


(b)

0

100

200

300

400lowast

Tim

e in

cent

ral a

rea (

s)


(c)

0

5

10

15

Rear

ing

and

groo

min

g in

cide

nts


(d)


0

50

100

150lowastlowast

lowast

Sucr

ose s

olut

ion

inta

ke (m

L)




(320)





4 Discussion



0

50

100

150lowast lowast


Prot

ein

leve

l (

of c

ontro


NormalModel

EASham EA


p-ERK

ERK

GAPDH





0

50

100

150

200lowastlowast

lowastlowastlowast


Prot

ein

leve

l (

of c

ontro

l)

NormalModel

EASham EA


p-P38

P38

GAPDH







5 Conclusion






Acknowledgments


References












































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

50

100

150lowast lowast


Prot

ein

leve

l (

of c

ontro


NormalModel

EASham EA


p-ERK

ERK

GAPDH





0

50

100

150

200lowastlowast

lowastlowastlowast


Prot

ein

leve

l (

of c

ontro

l)

NormalModel

EASham EA


p-P38

P38

GAPDH







5 Conclusion






Acknowledgments


References












































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















5 Conclusion






Acknowledgments


References












































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of










































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















research article effects of electroacupuncture on chronic

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