Research Article Neuroprotective Effects of Jitai Tablet ...downloads.hindawi.com/journals/ecam/2014/542383.pdfEvidence-Based Complementaryand AlternativeMedicine 9 days 15 days 0.E+00

Research ArticleNeuroprotective Effects of Jitai Tablet a Traditional ChineseMedicine on the MPTP-Induced Acute Model of ParkinsonrsquosDisease Involvement of the Dopamine System

Jia Liu1 Jinlong Gao1 Shaoang Tu1 Shasha Xu1 Ying Liu1

Weihu Shang1 Chenxin Gu1 Yiyun Huang2 and Mei Han13

1 College of Chemistry Beijing Normal University Beijing 100875 China2 Yale PET Center Department of Diagnostic Radiology Yale University School of Medicine New Haven CT 06510 USA3Key Laboratory of Radiopharmaceuticals Ministry of Education College of Chemistry Beijing Normal UniversityBeijing 100875 China

Correspondence should be addressed to Mei Han hanmeibnueducn

Received 25 November 2013 Revised 22 January 2014 Accepted 29 January 2014 Published 2 April 2014

Academic Editor Jen-Hwey Chiu

Copyright copy 2014 Jia Liu 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

Jitai tablet (JTT) is a traditional Chinese medicine used to treat neuropsychiatric disorders We previously demonstrated that JTTtreatment led to increased level of dopamine transporter (DAT) in the striatum thus indicating that JTT might have therapeuticpotential for Parkinsonrsquos disease (PD) which is characterized by dysregulated dopamine (DA) transmission and decreased striatalDAT expression The aim of this study was to investigate the neuroprotective effect of JTT on MPTP-induced PD mice Usinglocomotor activity test and rotarod test we evaluated the effects of JTT (050 015 or 005 gkg) on MPTP-induced behavioralimpairments Tyrosine hydroxylase TH-positive neurons in the substantia nigra and DAT and dopamine D

2receptor (D

2R) levels

in the striatum were detected by immunohistochemical staining andor autoradiography Levels of DA and its metabolites weredetermined by HPLC In MPTP-treated mice behavioral impairments were alleviated by JTT treatment Moreover JTT protectedagainst impairment of TH-positive neurons and attenuated theMPTP-induced decreases inDAT andD

2R Finally high dose of JTT

(050 gkg) inhibited the MPTP-induced increase in DA metabolism rate Taken together results from our present study provideevidence that JTT offers neuroprotective effects against the neurotoxicity of MPTP and thus might be a potential treatment for PD

1 Introduction

Parkinsonrsquos disease (PD) is a debilitating chronic and pro-gressive neurodegenerative disease Between 05 and 1 ofthe population aged 65ndash69 years and 1ndash3 of those over 80years old suffer from PD worldwide [1] The pathophysiologyof PD results from the progressive and selective loss ofdopaminergic neurons which leads to the depletion ofstriatal dopamine (DA) dopamine transporter (DAT) andDA receptors levels PD patients exhibit several major clinicalsymptoms such as bradykinesia muscle rigidity and restingtremor [2] It has been reported that clinical symptomsappeared when approximately 80 of the striatal DA is lost[3] Therefore a major treatment strategy for PD focuses

on DA supplementation through the use of DA precursorssuch as levodopa (L-DOPA) [4] DA receptor agonists andmonoamine oxidase B (MAO-B) inhibitors [1 5] Madoparwhich is composed of the DA precursor L-DOPA and thedecarboxylase inhibitor benserazide is commonly used totreat PD by increasingDA concentrations Although it is usedfor the relief of PD motor symptoms it does not preventor slow the progressive degeneration of neuronal cells andits long-term use may induce various side-effects includingfluctuations and abnormalities in involuntary movements[6] DA receptor agonists and MAO-B inhibitors are bothassociated with the same issues which greatly limit theirclinical utility [1] Thus there is still an urgent need forefficacious therapies that not only act on themotor symptoms

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2014 Article ID 542383 9 pageshttpdxdoiorg1011552014542383

2 Evidence-Based Complementary and Alternative Medicine

of PD but also protect neuronal cells and present low side-effect profiles

Traditional Chinese medicine (TCM) may offer thera-pies with better efficacy and fewer side-effects [7] TCMpreparations usually contain more than one componentand target multiple body systems for maximal therapeu-tic effects [7 8] As such TCM may prove superior toother pharmacotherapies in the treatment of chronic anddegenerative diseases such as PD [8] The Jitai tablet (JTT)is a traditional herbal formula consisting of the followingingredients Papaveraceae Corydalis (yan hu suo) 1020Solanaceae Datura metel (yang jin hua) 218 LamiaceaeSalvia Miltiorrhizae (Dan shen) 1687 Araliaceae Panaxginseng (Ren shen) 218 Apiaceae Angelica sinensis (Danggui) 1020 Apiaceae Ligusticum chuanxiong (Chuan xiong)571 Asteraceae Carthamus tinctorius (Hong hua) 1020Ranunculaceae Aconitum (Fu zi) 218Myristicaceae Myris-tica cagayanensis (Dou kou) 218 Asteraceae Aucklandia(Mu xiang) 571 Thymelaeaceae Aquilaria (Chen xiang)435 Zingiberaceae Zingiber (Gan jiang) 218 LauraceaeCinnamomum (Rou gui) 218 Semen Persicae (Tao ren)1020 Pearl powder (Zhen zhu fen) 1347 [9 10] JTT haslong been used clinically to treat neuropsychiatric disordersMoreover prior studies in our laboratory have demonstratedthat JTT could upregulate DAT in patients [11] It is wellknown that DAT level is a standard for diagnosis of PDand can be used for evaluating therapeutic efficacy in PDtreatment Therefore it is very interesting to investigate theeffects of JTT on PD

The neurotoxin 1-methyl-4-phenyl-1236-tetrahydro-pyridine (MPTP) causes damage to dopaminergic neuronsand has been widely used for generation of animal models ofPD [12] In the present study we examined the effects of JTTon behavioral changes and characteristics of the DA systemin an MPTP-induced PD mouse model Madopar was usedas a positive control drug for comparison

2 Materials and Methods

21 Animals and Treatment Procedures Male C57BL6 miceaged 6ndash8 weeks (20ndash24 g) were randomly divided intothe following groups (1) control (2) vehicle (distilledwater) (3) high dose of JTT (050 gkgday JTT-H) (4)middle dose of JTT (015 gkgday JTT-M) (5) low doseof JTT (005 gkgday JTT-L) and (6) Madopar (Roche)(012 gkgday) [13] All groups except for the control groupwere given MPTP (Sigma) intraperitoneally four times at2-hour interval at the dosage of 20mgkg on the 8th dayof the 15-day experiment period [14] JTT was dissolved indeionisedwaterTheprepared suspensionwas intragastricallygiven to mice at dose of 050 gkgday 015 gkgday or005 gkgday for 15 days (01mL10 g body weight) TheMadopar group was givenMadopar at a dose of 012 gkgdayintraperitoneally for 7 days after injection of MPTP Allanimals were maintained according to the internationalguidelines for care and use of laboratory animals Experimen-tal procedures involving animals were approved by the EthicsCommittee of Beijing Normal University (BNUEC012011)

22 Behavioral Analyses and Brain Tissue Preparation Sixmice were randomly selected from each group and a numberof behavioral tests were performed on the 9th day (1 day afterMPTP) and the 15th day (7 days after MPTP)

221 Locomotor Activity Test Locomotor activity wasassessed in an automated activity chamber (30 cm diameter15 cm high) connected to an infrared tracking analyzer thattransmitted the animalsrsquo movement distance and velocityto a computer The test was started by placing the mouse atthe center of the chamber for two min for acclimatizationand then the total horizontal ambulatory route within thefollowing 30min was recorded by a computer analyzer

222 Rotarod Test To determine fore limb and hind limbmotor coordination and balance a rotarod test as describedpreviously was used [15] Mouse was placed on the rotatingbar of the rotarod unit (DXP-3 Institute of Materia MedicaChinese Academy of Medical Sciences) set to a rotationspeed of up to 18 rpm during the test session The timespent on the rotating bar known as the latent period wasrecorded Performance was recorded as 120 s if the latentperiod exceeded 120 s

223 Brain Tissue Preparation Mice were anesthetized withsodium pentobarbital (50mgkg ip) after the behavior teston the 15th day and rapidly perfusion fixed with salinefollowed by 4 paraformaldehyde in 01M phosphate buffer(pH 74) Then the brains were removed and stored at minus80∘Cuntil use The frozen brains were cut into 18 120583m coronalsections using a cryostat microtome (CM3000 Leica Ger-many) Frozen sections of the striatum and substantia nigrawere used for immunohistochemistry and autoradiographyexperiments

23 DAT and Tyrosine Hydroxylase (TH) Immunohistochemi-cal Staining Analyses of DAT immunoreactivity in the stria-tum and TH immunoreactivity in the substantia nigra (SN)were conducted on brain sections as previously described[16] Briefly sections were incubated with either rat anti-DAT (Abcam) at 1 1000 dilutions or mouse anti-TH (Sigma)at 1 10000 dilutions at 4∘C overnight Then the slices wereincubated with peroxidase-conjugated secondary antibody(ZSGB-BIO) for DAT and the VECTASTAINABC kit (VEC-TOR) for TH All staining sections were visualized with 331015840-diaminobenzidine tertrahydrochloride (DAB) The sectionswere counted using a bright-field microscope (M165FCLeica) and optical densities (OD) of DAT and TH werecalculated by Image-ProPlus software TH-neurons weremanually counted by researchers blinded to the treatmentgroups

24 Autoradiography Experiments The brains were dividedinto two hemispheres one used for in vitro autoradiographyand the other for analysis of striatal levels of DA and itsmetabolites [17] Autoradiography was conducted on frozencryostat-cut 18 120583m thick brain slices to measure DAT and D

2

binding according to the procedures reported previously [18]

Evidence-Based Complementary and Alternative Medicine 3

9days 15days0E+00

1E + 04

2E + 04

3E + 04

4E + 04

5E + 04

6E + 04

Tota

l dist

ance

(cm

30

min

)

lowastlowast

lowastlowast

Number of days

(a)

0

5

10

15

20

25

30

35

9days 15days

Velo

city

(cm

s)

ControlVehicle

JTT-L

JTT-M

JTT-HMadopar

lowast

lowast

lowastlowast

Number of days

(b)

Figure 1 JTT improved MPTP-induced locomotor activity deficitsin PD mice These tests were used to assess mouse behaviors on the9th day (1 day after injection of MPTP) and 15th day (7 days afterinjection of MPTP) All the data total movement distance (a) andmean velocity (b) 119899 = 4ndash6 were shown asmean plusmn SEM lowast119875 lt 005lowastlowast119875 lt 001 versus vehicle group

Briefly brain sections were incubated for 60min with 50 pMof [125I]-IBZM for D

2receptor (D

2R) binding or with 50 pM

of [125I]-120573-CIT and in the presence of 1mM fluoxetinefor DAT binding Nonspecific binding was determined inthe adjacent slices in the presence of 10 120583M sulpiride (D

2R

antagonist Sigma-Aldrich Co USA) for D2R or 100mM

nomifensine (DAT antagonist Sigma-Aldrich Co USA)and 100mM fluoxetine for DAT Sections were then exposedto a super sensitive phosphor screen (PerkinElmer) for 2 h

0

20

40

60

80

100

120

9days 15days

Late

ncy

to fa

ll (s

)

ControlVehicleJTT-L

JTT-MJTT-HMadopar

lowastlowastlowastlowastlowastlowast

Number of days

Figure 2 JTT improved MPTP-induced latent period deficit in PDmice Test was done on the 9th day (1 day after injection of MPTP)and 15th day (7 days after injection ofMPTP)The data latent period119899 = 8ndash10 were shown asmeanplusmn SEM lowast119875 lt 005 lowastlowast119875 lt 001 versusvehicle group

Densitometry analysis of autoradiographs was carriedout with OptiQuant software (PerkinElmer) [125I]-IBZMand [125I]-120573-CIT were prepared according to proceduresdescribed previously [17 19]

25 Measurement of DA and Its Metabolites in the StriatumLevels of DA and its metabolites 34-dihydroxyphenylaceticacid (DOPAC) and homovanillic acid (HVA) were measuredby high performance liquid chromatography with an electro-chemical detector (HPLC-ECD) as previously described [20]Briefly the striatum was dissected weighed and homog-enized in 02M ice-cold perchloric acid The sample wasthen centrifuged twice at 14000 g for 15min at 4∘C Thesupernatant was collected and filtered through a 022120583mMillipore filter and injected into the HPLC system foranalysis

26 Statistical Analyses Statistical analyses included one-wayanalysis of variance (ANOVA) followed by LSD for behavioraltests and Tukeyrsquos HSD post hoc test for all other tests Theresults of immunohistochemical staining and autoradiogra-phy were indicated as a ratio compared to the control group

3 Results

31 Behavioral Analyses

311 Locomotor Activity Locomotor activity testing is acommonly used technique for behavioral monitoring inPD animal models In this study it was used to evaluatemotor impairment in the MPTP-treated animals As shown


Control Vehicle JTT-L

JTT-M JTT-H MadoparSN

(a)

0

20

40

60

80

100

120

140

Control Vehicle JTT-L JTT-M JTT-H Madopar

SN T

H p

ositi

ve ce

lls

( o

f con

trol)

lowast lowastlowast

lowastlowast

(b)

Figure 3 JTT protected against MPTP-induced reductions in TH expression in the mouse SN C57BL6 mice were treated with JTT for 7days and then MPTP (20mgkg 4 times at 2-hour interval ip) was injected on the 8th day Mice were sacrificed on the 7th day after MPTPinjection (15th day of the experiment period) and after behavioral tests Photomicrographs of representative SN (a) sections stained with anantibody against TH Reduced activity of TH-neurons was observed in MPTP treated mice which was partially prevented by treatment withJTT The number of TH-positive neurons in the SN (b) was expressed as the mean plusmn SD 119899 = 5 lowast119875 lt 005 lowastlowast119875 lt 001 versus vehicle group119875 lt 001 versus JTT-H

in Figures 1(a) and 1(b) on the 9th day all animals inMPTP-treated groups exhibited a significant reduction intotal movement distance and mean velocity compared tothose in the control group (119875 lt 001) On the 15th daythere was a marked improvement in locomotor activity inmice treated with high dose JTT (JTT-H) compared to thosetreated with the vehicle including both movement distanceand mean velocity (119875 lt 005) The group of mice treatedwith Madopar also demonstrated significant improvementscompared with the vehicle group in movement distance andmean velocity (119875 lt 001)

312 Rotarod Test The restorative effects of JTT on MPTP-induced motor coordination deficiencies were tested usingthe rotarod test As shown in Figure 2 on the 9th day all miceinjected with MPTP had a significant reduction in rotarodperformance compared to control mice (119875 lt 001) Onthe 15th day a significant difference was observed betweencontrol and vehicle (119875 lt 001) mice The latent periodsignificantly increased in the JTT-H and JTT-M groups (119875 lt001) and the Madopar group (119875 lt 001) when compared

with mice in the vehicle group JTT effectively counteredthe MPTP-induced behavioral coordination deficiencies in adose-dependent manner

32 Effects of JTT on MPTP-Induced Decreases in TH PositiveNeurons in the SN Immunohistochemical staining was usedto measure the levels of TH in the SN As shown in Figures3(a) and 3(b) MPTP exposure led to a remarkable loss ofTH positive neurons in the SN (119875 lt 001) compared tocontrol mice Treatment with JTT-H and JTT-M significantlyhalted TH-neuron reductions (JTT-H 119875 lt 001 JTT-M119875 lt 005) while treatment with Madopar had no effect Thisresult showed that JTT significantly protected againstMPTP-induced reduction in TH-neurons in the mouse SN whileMadopar did not

33 DAT in the Striatum Measured by ImmunohistochemicalStaining and Autoradiography DAT is a key protein used inthe clinical diagnosis of PD In this study immunochemicalstaining and autoradiography were used to detect DAT levels


Control Vehicle JTT-LSt

riatu

m

JTT-M JTT-H Madopar

500120583m

(a)

0

20

40

60

80

100

120


Stria

tum

DAT

OD

( o

f con

trol)

lowastlowastlowast

lowastlowast

lowastlowast

(b)


(c)

0

20

40

60

80

100

120


DAT

auto

radi

ogra

phy

( o

f con

trol)

lowastlowast

lowast

(d)

Figure 4 JTT protected against MPTP-induced reductions in DAT levels of the mouse striatum as measured by immunohistochemistryand autoradiography MPTP (20mgkg 4 times at 2-hour interval ip) was injected on the 8th day of the 15-day experiment period Theanimals were sacrificed on the 7th day after MPTP injections for immunohistochemistry and autoradiography studies Photomicrographsof representative striatum immunohistochemistry sections (a) stained with a DAT antibody Autoradiography of DAT binding using [125I]-120573-CIT (c) A decrease in DAT levels was observed in MPTP mice Treatment with JTT partially prevented this decrease Optical density ofDAT-positive fibers in the striatum (b) radioactivity binding of DAT in striatum (d) are expressed as the mean plusmn SD 119899 = 5 lowast119875 lt 005lowastlowast119875 lt 001 versus vehicle group 119875 lt 005 versus JTT-H

in the striatum As shown in Figures 4(a) and 4(b) MPTPtreatment elicited a significant reduction in DAT levels in thestriatum (119875 lt 001) Treatment with JTT-H (119875 lt 001) andJTT-M (119875 lt 005) significantly protected against this reduc-tion A comparison between JTT and Madopar found thatJTT-H was significantly superior in its protectiverestorativeeffect (119875 lt 005) DAT level increased by 18 in the JTT-Hgroup but only by 9 in theMadopar group when comparedto the vehicle group

Figures 4(c) and 4(d) illustrate the range of DAT levelsafter drug treatments via autoradiography using [125I]-120573-CIT MPTP treatment led to a significant reduction in DATbinding in the striatum compared with control mice Thisreduction was attenuated in mice treated with JTT whiletreatment with Madopar showed no significant effect whencompared to vehicle mice These results indicate that JTTcould significantly protect against MPTP-induced reductionsin DAT levels in the mouse striatum and that JTT-H is moreeffective than Madopar

34 Autoradiography Detected D2R in Striatum DA recep-

tors gradually decrease in the progression of PD over timeand the D

2R is one of the main receptors of the DA system

Autoradiography with [125I]-IBZM was used to detect levelsof D2R expression in the striatum As shown in Figure 5

MPTP treatment elicited a significant reduction in D2bind-

ing with [125I]-IBZM in striatum compared with that ofcontrol mice (119875 lt 001) This reduction in striatal D

2R

levels was attenuated in mice treated with JTT-H and JTT-M in a dose-dependent manner (JTT-H 119875 lt 001 JTT-M119875 lt 005) Although Madopar also had a significant effect toincrease D

2levels (119875 lt 005) in MPTP-treated animals JTT-

H had a stronger effect JTT-H reversed 26 of the MPTP-induced reductions in D

2binding compared with 17 by

Madopar

35 Levels of DA and Its Metabolites in the Striatum Thelevels of DA and its metabolites in the striatum were mea-sured by HPLC-ECD As shown in Figure 6(a) mice in



(a)

0

20

40

60

80

100

120

140


( o

f con

trol)

D2

auto

radi

ogra

phy lowastlowast

lowastlowast

lowast lowast

(b)

Figure 5 JTT protected against MPTP-induced reductions in D2R

expression in the mouse striatum as measured by autoradiographyMPTP (20mgkg 4 times at 2-hour interval ip) was injected onthe 8th day of the 15-day experiment period Mice were sacrificedfor the autoradiography study on the 7th day after MPTP injections[125I]-IBZMD

2binding is shown in autoradiograms (a) A decrease

inD2R expression was observed inMPTPmice Treatment with JTT

partially prevented this decrease Radioactivity binding of D2in the

striatum (b) is expressed as the mean plusmn SD 119899 = 5 lowast119875 lt 005 lowastlowast119875 lt001 versus vehicle group

the MPTP vehicle group showed a remarkable depletion ofDA DOPAC and HVA in the striatum compared with thosein the control group (119875 lt 001) The ratio of DOPAC andHVA to DA (Figure 6(b)) was significantly higher (119875 lt 001)in the vehicle group than the control which is indicative ofincreased DA metabolism in the vehicle group Treatmentwith JTT effectively inhibited the increase in the DOPAC andHVA to DA ratio (JTT-H 119875 lt 001) in a dose-dependentfashion despite the fact that the three dosages of JTT didnot significantly alter striatal levels of DA and its metabolitesThe effect of inhibiting this increased ratio was stronger withJTT-H and JTT-M than with Madopar (119875 lt 005) HoweverMadopar appeared to increase the levels of DA DOPAC andHVA in the striatum (119875 lt 005)

4 Discussion

The present study investigated the ability of JTT to offerprotection against the neurotoxicity of MPTP Our findingsindicated that high dose of JTT (JTT-H 050 gkgday) effec-tively protected against the MPTP-induced deficiencies inbehavioral activities in an efficacy equal to that of MadoparMoreover we found that JTT could protect against the loss ofTH-neurons in the SN JTT also attenuated the reduction inDAT levels in the striatum induced by MPTP FurthermoreJTT protected against the reduction in striatum D

2levels

typically induced by MPTP in a dose-dependent manner

We also found that JTT could attenuate the increase in DAmetabolism rates in MPTP-treated mice In general theseprotectiverestorative effects were stronger in animal treatedwith JTT-H than Madopar

Movement deficits in PD result from dopaminergicdysfunction in the brain In our study JTT treatment clearlydid improve MPTP-induced motor deficits Notably wefound that JTT could inhibit the rate of DAmetabolismwhilefailing to significantly increase the concentrations of DAand its metabolites One explanation for these findings maybe that behavioral changes are not solely directly related tostriatal DA levels and multiple factors are likely to be at play[21] As is well known long-term treatment with Madoparinducesmany side-effects and these side-effects coincidewiththe rise and fall of DA levels in the brain that occur inthe course of L-DOPA therapy [22] As for JTT preclinicaltoxicological studies have found no apparent toxicity even athigh doses (eg 98 gkgday) in SD rats during long-termuse(6months) [23] In the clinical setting JTT also demonstratedno obvious adverse effects [24ndash26] In the present study JTTwas found to improve striatal DA levels only appreciably buteffectively attenuate the increase in DA turnover rate Thismay suggest that long-term treatment with JTT is effectiveand will not induce the same side-effects as Madopar

PD is characterized by degeneration of DA neurons andcorresponds to loss of DAT a membrane protein importantfor DA reuptake Decreasing DAT levels is the gold standardby which clinical diagnoses of PD are made using medicalimaging methods [27 28] In the present study we foundthat JTT protected against MPTP-induced DAT reductionin a dose-dependent manner Relatively speaking Madopartreatment has little effect on DAT levels It has been reportedthat ginseng which is an active ingredient found in JTTcan significantly preserve DAT levels in the striatum [29]Changes observed in DAT levels during JTT treatment maymainly be due to its protection of DA turnover but may alsoresult from the direct action of JTT on DAT expression

The D2R is one of the key receptors associated with PD

but remains controversial because levels of D2R expression

may decrease in PD patients or not Antonini et alfound a significant reduction in putamen D

2binding to

[11C]raclopride in PD patients who had carried a diagnosisfor 3ndash5 years [30] Rinne et al found that striatal D

2increased

in early PD patients [31] In this study we found significantdecline in striatalD

2binding to [125I]-IBZM inMPTP-treated

mice Meanwhile JTT significantly inhibited this reductionsuggesting that JTT may play a role in reversing D

2R decline

which may be beneficial to the treatment of PDIn order to investigate the neuroprotective effect of JTT in

MPTP-induced PD model we treated mice with JTT beforeMPTP in a paradigm similar to that used in other studiesreviewed by More et al [7] JTT was found to inhibit thereduction in positive TH-neurons in SN typically observedin vehicle MPTP mice This suggests that JTT might offerprotection against dopaminergic neurodegeneration inducedby the neurotoxicity of MPTPMoreover JTT upregulates thelevels of DAT and D

2R in the striatum Some components in

the JTT prescription have been reported to have protective


0

100

200

300

400

500

600

DA DOPAC HVA

(ng

g)

ControlVehicle JTT-M

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

JTT-L JTT-HMadopar

(a)

0

05

1

15

2

25

3


(DO

PAC+

HVA

)D

A

lowast

lowastlowast

lowastlowast

(b)

Figure 6 JTT inhibited the increase in the ratio of DOPAC and HVA to DA in the striatum of MPTP mice MPTP (20mgkg 4 timesat 2-hour interval ip) was injected on the 8th day of 15-day experiment period The contents of DA DOPAC and HVA were detected byHPLC-ECD and the ratio of (DOPAC +HVA) to DAwas calculated Data regarding DA DOPAC HVA (a) and (DOPAC +HVA)DA ratios(b) are expressed as the mean plusmn SD 119899 = 4-5 lowast119875 lt 005 lowastlowast119875 lt 001 versus vehicle group

effects on neurons and upregulate DA tone For exampleRadix salvia and Radix ginseng which are components inJTT have been reported to be effective in protecting neuronsfromneurotoxicity [32ndash34] Some bioactive compounds fromherbal medicine have been reported to provide a significantneuroprotective effect in PD models and JTT does containmany constituents with neuroprotective effect such as gin-senoside Rg1 [35] Scopolamine which is an active ingredientin JTT is effective at enhancing DAT availability throughthe inhibition of muscarinic cholinergic neuronal activity[36] The mechanism underlying the ability of the JTT totreatMPTP-induced PDmice even though not entirely clearis likely to derive from the combined effects of its activeingredients JTT may act on multiple targets which reflectsthe advantage of TCM

5 Conclusions

Evidence from the present study appears to indicate thatJTT can play an important role in behavioral and motorfunction improvement in MPTP-induced PD model miceThis is related to the DA system including DAT D

2R

and TH Therefore JTT may be considered as a potentialcomplementary therapeutic agent for PD treatment because

it not only provides relief of motor symptoms but alsoprotects neurons and involves the DA system to restore levelsof DAT and D

2R

Conflict of Interests

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

Acknowledgments

This study was supported by the National Key TechnologyRampD Program (2008BAI49B04) the China National ScienceFoundation (81173139) and theMajor Research Plan of NSFC(21233003) The authors thank the National Engineering andResearch Center for TCM for providing the JTT used in thisstudy

References

[1] A Toulouse and A M Sullivan ldquoProgress in Parkinsonrsquosdiseasemdashwhere do we standrdquo Progress in Neurobiology vol 85no 4 pp 376ndash392 2008


[2] S Hunot F Boissiere B Faucheux et al ldquoNitric oxide synthaseand neuronal vulnerability in Parkinsonrsquos diseaserdquo Neuro-science vol 72 no 2 pp 355ndash363 1996

[3] J D Elsworth and R H Roth ldquoDopamine synthesis uptakemetabolism and receptors relevance to gene therapy of Parkin-sonrsquos diseaserdquo Experimental Neurology vol 144 no 1 pp 4ndash91997

[4] T Nagatsua and M Sawadab ldquoL-DOPA therapy for Parkinsonrsquosdisease past present and futurerdquo Parkinsonism and RelatedDisorders vol 15 no 1 pp S3ndashS8 2009

[5] C A Davie ldquoA review of Parkinsonrsquos diseaserdquo British MedicalBulletin vol 86 no 1 pp 109ndash127 2008

[6] S Fahn ldquoLevodopa and the progression of Parkinsonrsquos diseaserdquoTheNew England Journal of Medicine vol 351 no 24 pp 2498ndash2566 2004

[7] S V More H Kumar S M Kang et al ldquoAdvances in neu-roprotective ingredients of medicinal herbs by using cellularand animal models of Parkinsonrsquos diseaserdquo Evidence-BasedComplementary and Alternative Medicine vol 2013 Article ID957875 15 pages 2013

[8] X Z Li S N Zhang S M Liu et al ldquoRecent advances in herbalmedicines treating Parkinsonrsquos diseaserdquo Fitoterapia vol 84 pp273ndash285 2013

[9] S-P Wang L Liu L-L Wang et al ldquoScreening and analysis ofthe multiple absorbed bioactive components and metabolitesin rat plasma after oral administration of Jitai tablets byhigh-performance liquid chromatographydiode-array detec-tion coupled with electrospray ionization tandem mass spec-trometryrdquoRapid Communications inMass Spectrometry vol 24no 11 pp 1641ndash1652 2010

[10] S P Wang L Liu L L Wang et al ldquoStructural characterizationand identification of major constituents in Jitai tablets byhigh-performance liquid chromatographydiode-array detec-tion coupled with electrospray ionization tandem mass spec-trometryrdquoMolecules vol 17 no 9 pp 10470ndash10493 2012

[11] Y Liu M Han X D Liu et al ldquoDopamine transporteravailability in heroin-dependent subjects and controls longitu-dinal changes during abstinence and the effects of Jitai tabletstreatmentrdquo Psychopharmacology vol 230 no 2 pp 235ndash2442013

[12] S Przedborski andM Vila ldquoMPTP a review of its mechanismsof neurotoxicityrdquoClinical Neuroscience Research vol 1 no 6 pp407ndash418 2001

[13] X M Li H B Ma Z Q Ma et al ldquoAmeliorative andneuroprotective effect in MPTP model of Parkinsonrsquos diseaseby Zhen-Wu-Tang (ZWT) a traditional Chinese medicinerdquoJournal of Ethnopharmacology vol 130 no 1 pp 19ndash27 2010

[14] L V Jackson and S Przedborski ldquoProtocol for theMPTPmousemodel of Parkinsonrsquos diseaserdquo Nature Protocols vol 2 no 1 pp141ndash151 2007

[15] H N Zhang C N An H N Zhang and X P Pu ldquoProtocat-echuic acid inhibits neurotoxicity induced by MPTP in vivordquoNeuroscience Letters vol 474 no 2 pp 99ndash103 2010

[16] Y C Chung S R Kim J-Y Park et al ldquoFluoxetine preventsMPTP-induced loss of dopaminergic neurons by inhibitingmicroglial activationrdquo Neuropharmacology vol 60 no 6 pp963ndash974 2011

[17] Z Chen X Zhou C YWu et al ldquoSynthesis and iodine labellingof 120573-CITrsquo as a dopamine transporter imaging agentrdquo Journal ofIsotopes vol 13 no 1 pp 7ndash11 2000

[18] Z Xiong G Xu Z P Chen et al ldquoRegulation of a sapogeninfrom Rhizoma Anemarrhenae ZMR on dopaminergic systemin a chronic mdole of Parkinsonrsquos diseaserdquo Chinese Journal ofNuclear Medicine vol 29 no 5 pp 331ndash334 2009

[19] M P Kung B L Liu Y Y Yang J J Billings and H F KungldquoA kit formulation for preparation of iodine-123-IBZM a newCNS D-2 dopamine receptor imaging agentrdquo Journal of NuclearMedicine vol 32 no 2 pp 339ndash342 1991

[20] D Luo Q Zhang H Wang et al ldquoFucoidan protects againstdopaminergic neuron death in vivo and in vitrordquo EuropeanJournal of Pharmacology vol 617 no 1ndash3 pp 33ndash40 2009

[21] E Rousselet C Joubert J Callebert et al ldquoBehavioral changesare not directly related to striatal monoamine levels number ofnigral neurons or dose of parkinsonian toxin MPTP in micerdquoNeurobiology of Disease vol 14 no 2 pp 218ndash228 2003

[22] H Iderberg V Francardo and E Y Pioli ldquoAnimal models of L-DOPA-induced dyskinesia an update on the current optionsrdquoNeuroscience vol 211 pp 13ndash27 2012

[23] Y L Chen B Y Yan and Y P Deng ldquoPharmacology ofJitai tablets a traditional Chinese medicine for detoxificationrdquoChinese Journal of New Drug vol 19 no 14 pp 1229ndash1233 2010

[24] Z Li Y Tang S Hua et al ldquoEfficacy of Jitai for protractedwithdrawal symptoms in heroin dependent patientsrdquo ChineseJournal of Drug Dependence vol 16 no 4 pp 284ndash287 2007

[25] G Xu L Duan Z Wang et al ldquoA double-blind clinical trial ofJitai tablets vs Lofexidine in the treatment of heroin withdrawalsymptomsrdquo Chinese Journal of Drug Dependence vol 9 no 4pp 273ndash277 2000

[26] S Q Hao M Zhao R W Zhang et al ldquoThe effectivenesscomparison of Jitai tablets versus methadone in community-based drug treatment a 1 year follow-up studyrdquo AddictiveBehaviors vol 38 no 10 pp 2596ndash2600 2013

[27] A Winogrodzka P Bergmans J Booij E A Van Royen JC Stoof and E C Wolters ldquo[123I]120573-CIT SPECT is a usefulmethod for monitoring dopaminergic degeneration in earlystage Parkinsonrsquos diseaserdquo Journal of Neurology Neurosurgeryand Psychiatry vol 74 no 3 pp 294ndash298 2003

[28] B Ravina K Marek S Eberly et al ldquoDopamine transporterimaging is associated with long-term outcomes in ParkinsonrsquosdiseaserdquoMovement Disorders vol 27 no 11 pp 1392ndash1397 2012

[29] L Jia Y Zhao and X-J Liang ldquoCurrent evaluation of themillennium phytomedicinemdashginseng (II) Collected chemi-cal entities modern pharmacology and clinical applicationsemanated from traditional Chinese medicinerdquo Current Medici-nal Chemistry vol 16 no 22 pp 2924ndash2942 2009

[30] A Antonini J Schwarz W H Oertel O Pogarell and KL Leenders ldquoLong-term changes of striatal dopamine D

2

receptors in patients with Parkinsonrsquos disease a study withpositron emission tomography and [11C]raclopriderdquoMovementDisorders vol 12 no 1 pp 33ndash38 1997

[31] U K Rinne A Laihinen J O Rinne K Nagren J Bergmanand U Ruotsalainen ldquoPositron emission tomography demon-strates dopamine D

2receptor supersensitivity in the striatum

of patients with early Parkinsonrsquos diseaserdquoMovement Disordersvol 5 no 1 pp 55ndash59 1990

[32] HKim ldquoNeuroprotective herbs for stroke therapy in traditionaleastern medicinerdquoNeurological Research vol 27 no 3 pp 287ndash301 2005

[33] D O Kennedy and A B Scholey ldquoGinseng potential for theenhancement of cognitive performance andmoodrdquo Pharmacol-ogy Biochemistry and Behavior vol 75 no 3 pp 687ndash700 2003


[34] B G Park H J Jung Y W Cho et al ldquoPotentiation ofantioxidative and anti-inflammatory properties of culturedwildginseng root extract through probiotic fermentationrdquo Journal ofPharmacy and Pharmacology vol 65 no 3 pp 457ndash464 2013

[35] JWangH-MXuH-D Yang X-XDuH Jiang and J-X XieldquoRg1 reduces nigral iron levels of MPTP-treated C57BL6 miceby regulating certain iron transport proteinsrdquo NeurochemistryInternational vol 54 no 1 pp 43ndash48 2009

[36] H TsukadaNHarada SNishiyamaHOhba andTKakiuchildquoCholinergic neuronalmodulation alters dopamineD

2receptor

availability in vivo by regulating receptor affinity inducedby facilitated synaptic dopamine turnover positron emissiontomography studies with microdialysis in the conscious mon-key brainrdquo Journal of Neuroscience vol 20 no 18 pp 7067ndash7073 2000

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


of PD but also protect neuronal cells and present low side-effect profiles

Traditional Chinese medicine (TCM) may offer thera-pies with better efficacy and fewer side-effects [7] TCMpreparations usually contain more than one componentand target multiple body systems for maximal therapeu-tic effects [7 8] As such TCM may prove superior toother pharmacotherapies in the treatment of chronic anddegenerative diseases such as PD [8] The Jitai tablet (JTT)is a traditional herbal formula consisting of the followingingredients Papaveraceae Corydalis (yan hu suo) 1020Solanaceae Datura metel (yang jin hua) 218 LamiaceaeSalvia Miltiorrhizae (Dan shen) 1687 Araliaceae Panaxginseng (Ren shen) 218 Apiaceae Angelica sinensis (Danggui) 1020 Apiaceae Ligusticum chuanxiong (Chuan xiong)571 Asteraceae Carthamus tinctorius (Hong hua) 1020Ranunculaceae Aconitum (Fu zi) 218Myristicaceae Myris-tica cagayanensis (Dou kou) 218 Asteraceae Aucklandia(Mu xiang) 571 Thymelaeaceae Aquilaria (Chen xiang)435 Zingiberaceae Zingiber (Gan jiang) 218 LauraceaeCinnamomum (Rou gui) 218 Semen Persicae (Tao ren)1020 Pearl powder (Zhen zhu fen) 1347 [9 10] JTT haslong been used clinically to treat neuropsychiatric disordersMoreover prior studies in our laboratory have demonstratedthat JTT could upregulate DAT in patients [11] It is wellknown that DAT level is a standard for diagnosis of PDand can be used for evaluating therapeutic efficacy in PDtreatment Therefore it is very interesting to investigate theeffects of JTT on PD

The neurotoxin 1-methyl-4-phenyl-1236-tetrahydro-pyridine (MPTP) causes damage to dopaminergic neuronsand has been widely used for generation of animal models ofPD [12] In the present study we examined the effects of JTTon behavioral changes and characteristics of the DA systemin an MPTP-induced PD mouse model Madopar was usedas a positive control drug for comparison

2 Materials and Methods

21 Animals and Treatment Procedures Male C57BL6 miceaged 6ndash8 weeks (20ndash24 g) were randomly divided intothe following groups (1) control (2) vehicle (distilledwater) (3) high dose of JTT (050 gkgday JTT-H) (4)middle dose of JTT (015 gkgday JTT-M) (5) low doseof JTT (005 gkgday JTT-L) and (6) Madopar (Roche)(012 gkgday) [13] All groups except for the control groupwere given MPTP (Sigma) intraperitoneally four times at2-hour interval at the dosage of 20mgkg on the 8th dayof the 15-day experiment period [14] JTT was dissolved indeionisedwaterTheprepared suspensionwas intragastricallygiven to mice at dose of 050 gkgday 015 gkgday or005 gkgday for 15 days (01mL10 g body weight) TheMadopar group was givenMadopar at a dose of 012 gkgdayintraperitoneally for 7 days after injection of MPTP Allanimals were maintained according to the internationalguidelines for care and use of laboratory animals Experimen-tal procedures involving animals were approved by the EthicsCommittee of Beijing Normal University (BNUEC012011)

22 Behavioral Analyses and Brain Tissue Preparation Sixmice were randomly selected from each group and a numberof behavioral tests were performed on the 9th day (1 day afterMPTP) and the 15th day (7 days after MPTP)

221 Locomotor Activity Test Locomotor activity wasassessed in an automated activity chamber (30 cm diameter15 cm high) connected to an infrared tracking analyzer thattransmitted the animalsrsquo movement distance and velocityto a computer The test was started by placing the mouse atthe center of the chamber for two min for acclimatizationand then the total horizontal ambulatory route within thefollowing 30min was recorded by a computer analyzer

222 Rotarod Test To determine fore limb and hind limbmotor coordination and balance a rotarod test as describedpreviously was used [15] Mouse was placed on the rotatingbar of the rotarod unit (DXP-3 Institute of Materia MedicaChinese Academy of Medical Sciences) set to a rotationspeed of up to 18 rpm during the test session The timespent on the rotating bar known as the latent period wasrecorded Performance was recorded as 120 s if the latentperiod exceeded 120 s

223 Brain Tissue Preparation Mice were anesthetized withsodium pentobarbital (50mgkg ip) after the behavior teston the 15th day and rapidly perfusion fixed with salinefollowed by 4 paraformaldehyde in 01M phosphate buffer(pH 74) Then the brains were removed and stored at minus80∘Cuntil use The frozen brains were cut into 18 120583m coronalsections using a cryostat microtome (CM3000 Leica Ger-many) Frozen sections of the striatum and substantia nigrawere used for immunohistochemistry and autoradiographyexperiments

23 DAT and Tyrosine Hydroxylase (TH) Immunohistochemi-cal Staining Analyses of DAT immunoreactivity in the stria-tum and TH immunoreactivity in the substantia nigra (SN)were conducted on brain sections as previously described[16] Briefly sections were incubated with either rat anti-DAT (Abcam) at 1 1000 dilutions or mouse anti-TH (Sigma)at 1 10000 dilutions at 4∘C overnight Then the slices wereincubated with peroxidase-conjugated secondary antibody(ZSGB-BIO) for DAT and the VECTASTAINABC kit (VEC-TOR) for TH All staining sections were visualized with 331015840-diaminobenzidine tertrahydrochloride (DAB) The sectionswere counted using a bright-field microscope (M165FCLeica) and optical densities (OD) of DAT and TH werecalculated by Image-ProPlus software TH-neurons weremanually counted by researchers blinded to the treatmentgroups

24 Autoradiography Experiments The brains were dividedinto two hemispheres one used for in vitro autoradiographyand the other for analysis of striatal levels of DA and itsmetabolites [17] Autoradiography was conducted on frozencryostat-cut 18 120583m thick brain slices to measure DAT and D

2

binding according to the procedures reported previously [18]


9days 15days0E+00

1E + 04

2E + 04

3E + 04

4E + 04

5E + 04

6E + 04

Tota

l dist

ance

(cm

30

min

)

lowastlowast

lowastlowast

Number of days

(a)

0

5

10

15

20

25

30

35

9days 15days

Velo

city

(cm

s)

ControlVehicle

JTT-L

JTT-M

JTT-HMadopar

lowast

lowast

lowastlowast

Number of days

(b)



2receptor (D



2R



0

20

40

60

80

100

120

9days 15days

Late

ncy

to fa

ll (s

)

ControlVehicleJTT-L

JTT-MJTT-HMadopar


Number of days





3 Results






(a)

0

20

40

60

80

100

120

140


SN T

H p

ositi

ve ce

lls

( o

f con

trol)

lowast lowastlowast

lowastlowast

(b)









riatu

m

JTT-M JTT-H Madopar

500120583m

(a)

0

20

40

60

80

100

120


Stria

tum

DAT

OD

( o

f con

trol)

lowastlowastlowast

lowastlowast

lowastlowast

(b)


(c)

0

20

40

60

80

100

120


DAT

auto

radi

ogra

phy

( o

f con

trol)

lowastlowast

lowast

(d)










2R





Madopar




(a)

0

20

40

60

80

100

120

140


( o

f con

trol)

D2

auto

radi

ogra

phy lowastlowast

lowastlowast

lowast lowast

(b)








4 Discussion


2levels








2binding to


2increased




2R decline






0

100

200

300

400

500

600

DA DOPAC HVA

(ng

g)


lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

JTT-L JTT-HMadopar

(a)

0

05

1

15

2

25

3


(DO

PAC+

HVA

)D

A

lowast

lowastlowast

lowastlowast

(b)



5 Conclusions


2R



2R



Acknowledgments


References
































2











2receptor







of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















9days 15days0E+00

1E + 04

2E + 04

3E + 04

4E + 04

5E + 04

6E + 04

Tota

l dist

ance

(cm

30

min

)

lowastlowast

lowastlowast

Number of days

(a)

0

5

10

15

20

25

30

35

9days 15days

Velo

city

(cm

s)

ControlVehicle

JTT-L

JTT-M

JTT-HMadopar

lowast

lowast

lowastlowast

Number of days

(b)



2receptor (D



2R



0

20

40

60

80

100

120

9days 15days

Late

ncy

to fa

ll (s

)

ControlVehicleJTT-L

JTT-MJTT-HMadopar


Number of days





3 Results






(a)

0

20

40

60

80

100

120

140


SN T

H p

ositi

ve ce

lls

( o

f con

trol)

lowast lowastlowast

lowastlowast

(b)









riatu

m

JTT-M JTT-H Madopar

500120583m

(a)

0

20

40

60

80

100

120


Stria

tum

DAT

OD

( o

f con

trol)

lowastlowastlowast

lowastlowast

lowastlowast

(b)


(c)

0

20

40

60

80

100

120


DAT

auto

radi

ogra

phy

( o

f con

trol)

lowastlowast

lowast

(d)










2R





Madopar




(a)

0

20

40

60

80

100

120

140


( o

f con

trol)

D2

auto

radi

ogra

phy lowastlowast

lowastlowast

lowast lowast

(b)








4 Discussion


2levels








2binding to


2increased




2R decline






0

100

200

300

400

500

600

DA DOPAC HVA

(ng

g)


lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

JTT-L JTT-HMadopar

(a)

0

05

1

15

2

25

3


(DO

PAC+

HVA

)D

A

lowast

lowastlowast

lowastlowast

(b)



5 Conclusions


2R



2R



Acknowledgments


References
































2











2receptor







of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















(a)

0

20

40

60

80

100

120

140


SN T

H p

ositi

ve ce

lls

( o

f con

trol)

lowast lowastlowast

lowastlowast

(b)









riatu

m

JTT-M JTT-H Madopar

500120583m

(a)

0

20

40

60

80

100

120


Stria

tum

DAT

OD

( o

f con

trol)

lowastlowastlowast

lowastlowast

lowastlowast

(b)


(c)

0

20

40

60

80

100

120


DAT

auto

radi

ogra

phy

( o

f con

trol)

lowastlowast

lowast

(d)










2R





Madopar




(a)

0

20

40

60

80

100

120

140


( o

f con

trol)

D2

auto

radi

ogra

phy lowastlowast

lowastlowast

lowast lowast

(b)








4 Discussion


2levels








2binding to


2increased




2R decline






0

100

200

300

400

500

600

DA DOPAC HVA

(ng

g)


lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

JTT-L JTT-HMadopar

(a)

0

05

1

15

2

25

3


(DO

PAC+

HVA

)D

A

lowast

lowastlowast

lowastlowast

(b)



5 Conclusions


2R



2R



Acknowledgments


References
































2











2receptor







of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















riatu

m

JTT-M JTT-H Madopar

500120583m

(a)

0

20

40

60

80

100

120


Stria

tum

DAT

OD

( o

f con

trol)

lowastlowastlowast

lowastlowast

lowastlowast

(b)


(c)

0

20

40

60

80

100

120


DAT

auto

radi

ogra

phy

( o

f con

trol)

lowastlowast

lowast

(d)










2R





Madopar




(a)

0

20

40

60

80

100

120

140


( o

f con

trol)

D2

auto

radi

ogra

phy lowastlowast

lowastlowast

lowast lowast

(b)








4 Discussion


2levels








2binding to


2increased




2R decline






0

100

200

300

400

500

600

DA DOPAC HVA

(ng

g)


lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

JTT-L JTT-HMadopar

(a)

0

05

1

15

2

25

3


(DO

PAC+

HVA

)D

A

lowast

lowastlowast

lowastlowast

(b)



5 Conclusions


2R



2R



Acknowledgments


References
































2











2receptor







of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















(a)

0

20

40

60

80

100

120

140


( o

f con

trol)

D2

auto

radi

ogra

phy lowastlowast

lowastlowast

lowast lowast

(b)








4 Discussion


2levels








2binding to


2increased




2R decline






0

100

200

300

400

500

600

DA DOPAC HVA

(ng

g)


lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

JTT-L JTT-HMadopar

(a)

0

05

1

15

2

25

3


(DO

PAC+

HVA

)D

A

lowast

lowastlowast

lowastlowast

(b)



5 Conclusions


2R



2R



Acknowledgments


References
































2











2receptor







of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

100

200

300

400

500

600

DA DOPAC HVA

(ng

g)


lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

JTT-L JTT-HMadopar

(a)

0

05

1

15

2

25

3


(DO

PAC+

HVA

)D

A

lowast

lowastlowast

lowastlowast

(b)



5 Conclusions


2R



2R



Acknowledgments


References
































2











2receptor







of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of














































2











2receptor







of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




















2receptor







of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Documents

Research Article Neuroprotective Effects of Jitai Tablet ...downloads.hindawi.com/journals/ecam/2014/542383.pdfEvidence-Based Complementaryand AlternativeMedicine 9 days 15 days 0.E+00