Research ArticleNeuroprotective Effect of Echinacoside inSubacute Mouse Model of Parkinsonrsquos Disease

Yan Liang 12 Chang Chen 2 Baomei Xia 3 Wei Wu2 Juanjuan Tang4 Qing Chen5

Lili Tang2 Hui Yang2 Zhennian Zhang2 Yan Lu2 Ye Yang 6 and Yang Zhao 2

1Nanjing University of Chinese Medicine Nanjing Jiangsu China2Department of Neurology The Third Affiliated Hospital of Nanjing University of Chinese Medicine Nanjing Jiangsu China3Faculty of Rehabilitation Science Nanjing Normal University of Special Education Nanjing Jiangsu China4Physiology Research Section School of Medicine and Life Sciences Nanjing University of Chinese Medicine Nanjing Jiangsu China5Department of Neurology Nanjing Pukou Hospital of Traditional Chinese Medicine Nanjing Jiangsu China6Center forModernization of ChineseMedicine andDatabaseTheThird AffiliatedHospital of NanjingUniversity of Chinese MedicineNanjing Jiangsu China

Correspondence should be addressed to Ye Yang yangye876sinacom and Yang Zhao yangzhaotcm163com

Yan Liang Chang Chen and Baomei Xia contributed equally to this work

Received 9 July 2018 Revised 16 December 2018 Accepted 15 January 2019 Published 30 January 2019

Academic Editor Stephen H Safe

Copyright copy 2019 Yan Liang et alThis is an open access article distributed under theCreative CommonsAttribution License whichpermits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Objective To study the protective effect of Echinacoside for 1-methyl-4-phenyl-1236-tetrahydropyridine (MPTP) induceddopaminergic (DA) neurons injury in the subacute mouse model of Parkinsonrsquos disease (PD) and to explore its mechanism ofaction Methods We chose 10 weeks of healthy wild type C57BL6 male mice hypodermic MPTP 30 mgkgday five days toprepare PD subacute mouse model Behavior indexes of open field test and pole test were applied to examine the function ofECH to PD subacute mice model of PD sample actionThe effects of ECH on dopaminergic neurons and astrocyte were examinedusing Immunohistochemistry including tyrosine hydroxylase (TH) and glial fibrillary acidic protein (GFAP) expression The totalnumbers of TH-positive neurons and GFAP-positive cells in the substantia nigra pars compacts (SNpc) and ventral tegmentalarea (VTA) were obtained stereologically using the optical fractionator method Enzyme-linked immunosorbent assay (ELISA)method was used to detect the inflammatory cytokines in the serum including TNF-120572 (Ttumor necrosis factor alpha) and IFN-120574(interferon gamma) Protein expressions of ionized calcium binding adaptor molecule 1 (IBA-1) TNF-120572 Cleaved caspase-3 glialderived neurotrophic factor (GDNF) and phosphorylated and total extracellular signal-regulated kinase (p-ERK and ERK) inthe anatomical region of substantia nigra (SN) were tested by protein immunoblot method (ie Western blotting) Results ECHreversed the reduction of total distance in open field test in MPTP-induced PD model mice (P lt 001) shortened the return timeand total time of PD subacute model mice in pole test (P lt 001 P lt 005) significantly reversed the reduction of TH positiveneurons induced by MPTP (P lt 005) and reduced the activation of astrocytes (P lt 005) Meanwhile ECH significantly inhibitedthe expression of IBA-1 Cleaved caspase-3 and TNF-120572 in midbrain of MPTP model mice (P lt 005 P lt 005 and P lt 005) andupregulated the expression of GDNF (P lt 005) And ECH lowered the level of TNF-120572 and IFN-120574 in serum (P lt 005 P lt 005)Conclusion ECH has protective effects on the MPTP subacute model mice its mechanism may be through inhibiting activationof microglia and astrocytes reducing inflammatory reaction and promoting the secretion of neurotrophic factors and eventuallyresulting in the reduction of the DA neurons apoptosis

1 Introduction

Parkinsonrsquos disease (PD) is the second most common age-related neurodegenerative disease which is characterizedby progressive loss of dopaminergic (DA) neurons and the

appearance of Lewy body in the cytoplasm in the midbrainsubstantia nigra compacta Chronic neuroinflammation isalso one of the signs of PD pathophysiology The post-mortem analysis of human PD patients and experimentalanimals showed that activation of glial cells and increased

HindawiBioMed Research InternationalVolume 2019 Article ID 4379639 8 pageshttpsdoiorg10115520194379639

2 BioMed Research International

levels of proinflammatory cytokines are common featuresof PD brain [1 2] Previous study showed that activationof astrocytes and microglia due to the release of promotinginflammatory cell factor aggravated the degeneration of DAneurons in the SNpc [3]Therefore the inflammatory processis considered to be a promising intervention target for PDand other neurodegenerative diseases Echinacoside (ECH)is derived from the fleshy stems of Cistanche deserticolaIts pharmacological effects involve skeleton protection liverprotection antioxidation anti-inflammation antiaging [4]With the deepening and development of pharmacology agrowing number of studies have been focused on the role ofECH in the treatment of neurological diseases in recent years[5ndash7] The purpose of this study was to establish a subacutePDmodel of MPTP in vivo by using C57BL6mice in whichSelegiline was used as the positive drug control to study theneuroprotective effect of ECH on PD model mice and itsunderlying mechanism of action

2 Materials and Methods

21 Animals and Groups Experiments were conducted usingmale C57BL6J mice at 10 weeks of age and weighing 25 to30g The animals were maintained in standard conditions(1212h lightdark cycle 22plusmn2∘C and relative humidity of55plusmn5) and allowed access to food and water ad libitumAll animal procedures conformed to the Guide for theCare and Use of Laboratory Animals and were approvedby the Institutional Animal Care and Use Committee atNanjing University of Chinese medicine The experimenterswere blinded to the assignments of the mice Before theexperiment the animals were housed in our facilities for twoweeks to acclimate then the animals were randomly dividedinto five groups (10mice in each group) Saline control groupmodel group (MPTP) MPTP+ECH group positive drugcontrol group (MPTP+Selegiline) and ECH group

22 Drugs and Reagents Echinacoside (HPLCge9882854-37-3) was purchased from Chengdu Research Institute ofBiology of the Chinese Academy of Sciences China Selegi-line was purchased fromOrion Finland 1-Methyl-4-phenyl-1236-tetrahydropyridine hydrochloride (MPTP M0896)and anti-tyrosine hydroxylase (TH) antibody (T1299) werepurchased from Sigma America Antiglial fibrillary acidicprotein (GFAP) antibody (MAB360) were purchased fromMillipore America Diaminobenzidine (DAB AR1000) waspurchased from Boster Biological Technology Co LtdAmerica ELISA Kits (SBJ-M0030 SBJ-M0038) were pur-chased from Nanjing SenBeiJia Biological Technology CoLtd The primary antibodies used in the study were asfollows Anti-ionized calcium binding adaptor molecule 1(IBA-1) antibody (016-20001Wako Japan) anti-TNF-120572 anti-body (bs-0078R Beijing Biosynthesis BiotechnologyCo LtdChina) anti-Cleaved Caspase-3 antibody (9664s Cell Sig-naling Technology America) antiglial derived neurotrophicfactor (GDNF) antibody (sc-328 Santa cruz America)antiphosphorylated and total extracellular signal-regulatedkinase (p-ERK andERK) antibody (4370 4695 Cell Signaling

Technology America) and anti-120573-Tublin antibody (10094-1-AP Proteintech Group America) The secondary antibodiesused in the study were as follows HRP-labeled Goat anti-Rabbit IgG and HRP-labeled Goat anti-mouse IgG (ZB-2301ZB-2305 Beijing Zhongshan Jinqiao Biotechnology Co LtdChina)

23 Administration of Drugs The animals were given ECH30mgkg Selegiline 1mgkg or the same volume of salinegavage at 1000am per day for 7 days before the beginningof model-making process MPTP (30mgkg) was injectedsubcutaneously to themodel-making animals at 900am fromday 8d to day 12d and ECH or Selegiline was given gavageadministration after 1h while non-model-making animalswere given subcutaneously or gavage treatment with salineFrom 13d to 16d ECH or Selegiline was still given to themodel-making animal at 1000am daily while non-mold-making animals were given saline gavage At 1500pm onday 16 behavioral tests were conducted on all the groupsof animals At 1000am on day 17 all the animals weresacrificed after orbital blood extraction 4 mice of each groupwere perfused via intracardial infusion with saline (09)followed by 4 paraformaldehyde (PFA) and the brains werecollected In each group another 6 animalsrsquo tissues of ventralmidbrain and striatum were dissected rapidly on ice frozen inliquid nitrogen and stored at -80∘C until used

24 Locomotor Activity The open-field test can be used toobserve and evaluate the spontaneous activity of mice Thetest device consists of two parts the open-field universalsound insulation box and the automatic data collection andprocessing system The open-field reaction box is an organicglass box (40cm long 40cm wide and 15cm high) and thetest area is well illuminated The experiment was carried outin a quiet environment Put the animal into the center of thebottom of the box and turn on the camera to observe for 5minThe automatic data acquisition device sends out infraredray to track all the trajectories of the random movementin the mouse box which is total distance Before the test75 ethanol was used to thoroughly wipe the inner wall andbottom surface of the box so as not to affect the results of thenext test Replace the animals and continue the experiment

25 Pole Test Pole test can be used to assess the coordinationand balance ability of mice and it was conducted at 1500pmon day 16 According to Chenrsquos protocol [8] the PV tube witha length of 55cm and a diameter of 1cm was selected Theoutside of the PV tube was tightly wrapped with white tape toprevent slip The PV tube was fixed on the plastic foam baseand placed in the cage and the basewas coveredwith dressingThe upper end of the tube was covered with a sphericalprotruding point with a diameter of 2cm which was usedas the attachment point of the mouse during the experimentThemouse was placed on the spherical protruding point withits head upward during the test and the time from the top ofthe placed bar to the head to turn down (t-turn) and the totaltime from the top of the placed bar to climb to the bottomof the tube to land (t-total) were recorded Each mouse was

BioMed Research International 3

tested 3 times with an interval of 2min between each test andan average value of the 3 times was taken as the data

26 Immunohistochemical Staining [8] After perfusion fromthe left ventricle the brains ofmice were rapidly removed andplaced in 4paraformaldehyde to be fixed overnight Sucrosesolution was used for gradient dehydration and OCT gel wasused for embedding We prepared brain tissue sections (30microns) with frozen slicer and chose SN-VTA slices in linewith the atlas of Paxinos and Franklin [9] Sections wereblocked for 1h and incubated with anti-TH primary antibody(1 1000) and anti-GFAP primary antibody (1 1000) for 12hAfter washing there times in 001M PBS the slices were incu-bated with the secondary antibody of goat anti-mouse IgGHampL (HRP) (12000) for 2h After washing the brain slices amodification of the ABC system (Kit Vectastain ABC VectorLaboratory Inc) was used to detect the antibody complexImage-Pro Express 6 (Media Cybernetics) was applied tostereologically quantify the number of TH positive neuronsand GFAP positive astrocytes and the mean number of themin each brain was picked up through the definite quantity offour alternating sections Olympus camera (DP72 Olympus)was used to take images at an original magnification of 100times

27WesternBlotting Tissues of substantia nigrawere lysed inRIPA buffer containing protease inhibitors and phosphataseinhibitors Protein concentration was determined colorimet-rically by NANODROP 2000 (Thermo Scientific) Proteinlysates were separated by 10 SDS-PAGE electrophoresisand were transferred onto polyvinylidenedifluoride (PVDF)membranes After blocking with 5BSA for 1h the followingantibodies were used Anti-IBA-1 antibody (1300) anti-TNF-120572 antibody (1200) anti-Cleaved Caspase-3 antibody(1200) anti-GDNF antibody (1200) anti-phosphorylated-ERK (1500) antibody and anti-ERK antibody (1500) anti-120573-Tublin antibody (12000) After the blots were incubatedwith antibodies overnight at 4∘C they were incubated withhorseradish peroxidase-conjugated secondary antibodies for1h The blots were visualized using the Super Signal WestPico Chemiluminescent Substrate (Thermo Fisher ScientificInc) The resulting data was analyzed statistically using SPSSStatistics 20 All experiments were performed in triplicateThe final data are expressed as a ratio of the relative opticaldensity (ROD) of the protein of interest to the ROD of 120573-tubulin A p value lt 005 was considered significant

28 Enzyme-Linked Immunosorbent Assay Theblood of eachmouse was taken out 15ml to be centrifuged under thecondition of 4∘C 3000 rmin and 10 cm centrifugal radiusfor 10 min and then the blood was stored in 20∘C Thecontents of inflammatory factors such as TNF-120572 and IFN-120574 were detected by enzyme-linked immunoassay kit All theoperations are carried out in accordance with the instructionof kits strictly

29 Statistical Analysis SPSS Version 200 (IBM) was usedfor all statistical analyses All experiments were performedat least three times Comparisons between two groups were

Saline

MPTP

MPTP+ECH

MPTP+Selegilin

eECH

0

10000

20000

30000

tota

l dis

tanc

e (m

m)

lowast

Figure 1 Total distance of open field test Saline means salinecontrol group MPTP means model group MPTP+ECH meansMPTP+ECH group MPTP+Selegiline means positive drug controlgroup and ECHmeans ECH grouplowastplt005 comparedwith Salineplt005 and plt001 compared with MPTP

performed using a two-sample Studentrsquos t-test and compar-isons between multiple groups were performed using a one-way ANOVA followed by post hoc analysis of Tukeyrsquos HSDand LSD multiple comparisons test All data are presented asmean plusmn SEM and statistical significance was accepted at the5 level unless otherwise indicated

3 Result

31 Effect of ECH on Behavior Test in Subacute PDModelMice

311 Locomotor Activity In the open field test the totaldistances of activity in MPTP model group mice was signif-icantly shortened and the difference was statistically signif-icant compared with that in saline control group (Plt005)While compared with the model group the total distanceof activity in MPTP+ECH group was significantly longer(Plt001) (Figure 1)

312 Equilibrium Coordination In the pole test the returntime of mice in the MPTP model group was significantlylonger than that of saline control group (Plt001) while com-pared with the model group the time of MPTP+ECH groupwas shorter and the difference was statistically significant (Plt 001) As to the total time it was significantly increased inthe model group compared to saline control group (P lt 001)meanwhile it was significantly shortened in the MPTP+ECHgroup compared to the model group (P lt 005) (Figure 2)

32 Effects of ECH on Dopaminergic Neurons in the Midbrainof Subacute PD Model Mice Tyrosine hydroxylase is thespecific markers for dopaminergic neurons the number ofTH positive cells can be used as the number of DA neuronsThe stereo counting showed that theTHpositive cells numberof themidbrain in theMPTPmodel group decreased by 631compared with the saline control group in which the number

4 BioMed Research International

Saline

MPTP

MPTP+ECH

MPTP+Selegilin

eECH

00

05

10

15

20

T-tu

rn (s

ec) lowastlowast

(a)

Saline

MPTP

MPTP+ECH

MPTP+Selegilin

eECH

0

5

10

15

T-to

tal (

sec) lowastlowast

(b)

Figure 2 Pole test (a) Time of return (b) Time of total Saline means saline control group MPTP means model group MPTP+ECHmeansMPTP+ECH group MPTP+Selegiline means positive drug control group and ECH means ECH group lowastlowastplt001 compared with salineplt005 and plt001 compared with MPTP

Saline MPTP+Selegiline

MPTP ECH

MPTP+ECH

Saline

MPTP

MPTP+ECH

MPTP+Selegilin

eECH

0

2000

4000

6000

8000

10000

TH+ co

unts

SN

pc

(a)

Saline

MPTP

MPTP+ECH

MPTP+Selegilin

eECH

(b)

0

2000

4000

6000

8000

TH+ co

unts

VTA

lowast

lowastlowastlowast

Figure 3 Representativemicrophotographs of dopaminergic neurons stained for TH and the quantification of TH positive cell in each groupThe pictures were taken at an original magnification of 10times Scale bars 100120583m Saline means saline control groupMPTPmeans model groupMPTP+ECH means MPTP+ECH group MPTP+Selegiline means positive drug control group and ECH means ECH group lowastplt005 lowastlowastlowastplt0001 compared with saline plt001 and plt0001 compared with MPTP

in SNpc area decreased by 599 and the number in the VTAarea decreased by 446 Both differences were significantcompared with the control group (Plt0001 Plt005) Whilein the ECH treatment group the number of TH positivecells in both SNpc and VTA increased observably which was

statistically significant compared with the model group (P lt0001 P lt 0001) (Figure 3)

33 Effects of ECH on Astrocytes in the Midbrain of SubacutePD Model Mice As a marker of activation in astrocyte

BioMed Research International 5

Saline MPTP+Selegiline

MPTP ECH

MPTP+ECH

(a)

Saline

MPTP

MPTP+ECH

MPTP+Selegilin

eECH

0

500

1000

1500

GFA

P-IR

coun

ts V

TA(b)

Saline

MPTP

MPTP+ECH

MPTP+Selegilin

eECH

0

2000

4000

6000

GFA

P-IR

coun

ts S

Npc

lowastlowastlowast

lowastlowastlowast

Figure 4 Representative microphotographs of dopaminergic neurons stained for GFAP and the quantification of GFAP positive cell in eachgroup The pictures were taken at an original magnification of 10times Scale bars 100120583m Saline means saline control group MPTP meansmodel group MPTP+ECHmeans MPTP+ECH group MPTP+Selegiline means positive drug control group and ECHmeans ECH group lowastlowastlowastplt0001 compared with saline plt0001 compared with MPTP

GFAP can be used to detect the number of active astro-cytes According to the results of immunohistochemistry theastrocytes in model group mice were activated in a patternof hypertrophy and the number of active astrocytes wassignificantly increased in both SNpc area and VTA areacompared with saline control group (P lt 0001 P lt 0001)After the ECH treatment the morphology of astrocytes wererestored to the saline control group and the number ofGFAP positive cells in SNpc and VTA area was significantlydecreased (P lt 0001 P lt 0001) (Figure 4)

34 Effects of ECH on Serum Inflammatory Factors in Suba-cute PD Model Mice Enzyme-linked immunosorbent assayshowed that the concentrations of inflammatory factorsincluding TNF-120572 and IFN-120574 in the serum of model groupmice were significantly higher than that of saline controlgroup (P lt 005 P lt 001) After the treatment of ECH theconcentrations of the two factors were significantly reduced(P lt 001 P lt 005) (Figure 5)

35 Effects of ECH on the Expression of Midbrain ProteinsIncluding IBA-1 Cleaved Caspase-3 GDNF TNF-120572 p-ERKand ERK in Subacute PD Mice Western blotting analy-sis showed that MPTP induced a marked IBA-1 CleavedCaspase-3 and TNF-120572protein increase (Plt001 Plt0001 and

Plt0001) while the expression of GDNF and ERK phospho-rylation level was significantly decreased (Plt005 Plt005)in midbrain tissue compared to saline control group Afterthe ECH administration the expressions of IBA-1 CleavedCaspase-3 and TNF-120572were significantly decreased (P lt 001Plt 0001 and Plt 001) and the expressions of GDNF and thephosphorylation level of ERK were significantly increased (Plt 0001 P lt 001) (Figure 6)

4 Discussion

Parkinsonrsquos disease is a common central nervous systemdegeneration disease in the elderly Its main clinical fea-tures are not only bradykinesia static tremor myotoniaand abnormal posture but also olfactory disorder con-stipation insomnia fatigue depression and anxiety andother nonmotor symptoms [10 11] The pathogenesis of PDis complex which may be caused by aging genetic andenvironmental factors The pathological change of SNpc DAneuron degeneration lost and appearance of Lewy body maybe connected with cell autophagy proteasome dysfunctionmitochondrial function defect oxidative stress and immuneresponse inflammation excitatory neurotoxicity and othercomplex mechanisms [10 12] Currently dopaminergic drugtherapy is the major treatment of PD Neither supplementing

6 BioMed Research International

Saline

MPTP

MPTP+ECH

MPTP+Selegilin

eECH

0

50

100

150

200

250

TNF-

o

f ser

um (p

gm

l)lowast

(a)

Saline

MPTP

MPTP+ECH

MPTP+Selegilin

eECH

0

50

100

150

200

IFN

- o

f ser

um (p

gm

l)

lowastlowast

(b)

Figure 5 Alteration of TNF-120572 and IFN-120574 in serum of mice Saline means saline control group MPTP means model group MPTP+ECHmeans MPTP+ECH group MPTP+Selegiline means positive drug control group and ECH means ECH group lowastplt005 and lowastlowastplt001compared with saline plt005 and plt001 compared with MPTP

the DA neurotransmitter nor activating DA receptors canreverse the loss of DA neurons and the appearance of Lewybody That means the current PD treatment plan is mainlyfor the symptoms without affecting the progress of the disease[13]Therefore there has been increasing interests in studyingagents like ECH that affect the mechanistic processes ofPD as novel treatment This study specifically found that(1) ECH could improve the PD-like behavior of subacutemodel mice induced by MPTP and the loss of DA neuronsin the substantia nigra of model mice was saved which issimilar to the reports of Qing Zhao [14] (2) ECH couldalso reverse against the astrocyte overactivation microgliaactivation serum and mesencephalic inflammatory factorsin midbrain induced by MPTP in PD subacute model mice(3) ECH was able to increase the expression of GDNF andameliorate the overexpression of MPTP-induced IBA-1 andCleaved Caspase-3 and the decrease of phosphorylated ERK

MPTP is the most commonly used environmental toxinfor the preparation of PD animal model which is highlyfat-soluble and extremely easy to pass through the bloodbrain barrier MPTP is metabolized by monoamine oxidasein astrocytes and turned into ionized 1-methyl-4-phenylpyridine (MPP+) transferred by the Dopamine transporter(DAT) to the DA neurons MPTP produces obvious patho-logical and biochemical damage of DA neurons in C57BL6Jmice so it has a great advantage over other PD models interms of efficacy evaluation We prepared subacute PDmod-els in C57BL6J mice by MPTP (subcutaneous injection) forfive consecutive days and observe thatMPTP can successfullyinduce animal PD behavior cause the loss of DA neurons byimmune histochemical and behavior testing

Echinacoside is the key components in the identificationand assaying of herba cistanche in Chinese pharmacopoeiaIn recent years it has been found that ECH can cross theblood brain barrier [15] and has a good protective effect onthe central nervous system According to Zhaorsquos research[14] we chose the optimal therapeutic does of 30mgkg

ECH as an intervention the behavior of MPTP-inducedanimal models improved markedly the DA neurons deathsdropped significantly the expression of Cleaved Caspase-3decreased obviously and all the above indicated ECH couldprotect DA neurons of PD model mice by antiapoptoticeffect This is similar to Zhaorsquos report [14] The differencebetween two experiments lies in Zhao detected apoptosisrelated protein directly but Zhao has not studied howMPTPcauses apoptosis of DA neurons and how ECH works againstapoptosis Therefore this study explored the mechanism ofMPTP-induced apoptosis of DA neurons and the role of ECHin neurorescue based on glial cells

Microglia is an immune cell in the brain when activatedit produces and releases proinflammatory cytokines thatcause damage to DA neurons Inflammatory factors such asIFN-120574 IL-1120573 TNF-120572 IL-2 and IL-6 [1] are founded in the SNstriatum and cerebrospinal fluids of autopsy in patients withPDmeanwhile the expressions of TNF-120572 receptors R1 (p55)bcl-2 soluble Fas caspase-1 and caspase-3 are increased [16]Therefore proinflammatory cytokines activated and releasedby microglia-mediated factor may be the one reason of deathof DA neurons Astrocytes secrete a variety of neurotrophicfactors of which GDNF is particularly important becauseit can specifically promote the repair of DA neurons ininjured midbrain and it is considered as the most promisingnutrient factor in PD treatment According to reports inthe literature [17] significant improvement can be observedafter astrocytes which are carrying the high expression ofGDNF gene were implanted into the SN region of animalmodels of PD The treatment of GDNF depends on theERK signaling pathway which promotes cell survival [18] Ithas been reported that application of phosphatidylinositol-related D1-like receptor agonists in rat astrocytes can enhancethe expression of phosphorylated ERK and promote themigration of astrocytes [19] GDNF can activate ERK andinduce the upregulation of its own expression in SH-SY5Ycells and rat VTA region [20 21] At the same time ERK

BioMed Research International 7

IBA-1

Cleaved-Cas3

GDNF

TNF-

-tubulin

p-ERK

ERK

Saline

MPTPMPTP+ECH

MPTP+Selegilin

e

ECH

(a)

Saline

MPTP

MPTP+ECH

MPTP+Selegilin

eECH

0

1

2

3

4

IBA-1

IBA-

1

-tubu

lin

lowastlowast

(b)

Saline

MPTP

MPTP+ECH

MPTP+Selegilin

eECH

0

1

2

3

4

Cleaved-Caspase-3

Cle

aved

-Cas

3

-tubu

lin lowastlowastlowast

(c)

Saline

MPTP

MPTP+ECH

MPTP+Selegilin

eECH

00

05

10

15

GDNF

GD

NF

-tu

bulin

lowast

(d)

Saline

MPTP

MPTP+ECH

MPTP+Selegilin

eECH

00

05

10

15

20

TNF-

TNF-

-tubu

lin

lowastlowastlowast

(e)

Saline

MPTP

MPTP+ECH

MPTP+Selegilin

eECH

00

05

10

15

20

p-Erk Erk

p-Er

k E

rk

lowast

(f)

Figure 6 Protein expression of IBA-1 Cleaved-Caspase-3GDNF TNF-120572 p-ERK and ERK Salinemeans saline control groupMPTPmeansmodel group MPTP+ECH means MPTP+ECH group MPTP+Selegiline means positive drug control group and ECH means ECH grouplowastplt005 lowastlowastplt001 lowastlowastlowastplt0001 compared with saline plt005 plt001 and plt0001 compared with MPTP

signaling pathway also plays a key role in the inflammatorypathological changes of PD and their coeffects may affect thepathogenesis of PD and the progression of the disease In theresting state ERKMAPK are located in the cytoplasm of thecells When activated ERK is rapidly phosphorylated Thenactivated ERK enters the nucleus to regulate the activities ofother transcription factors which make the change of genetranscription impact the ascension or decline of the expres-sion of respective protein gene and finally cause the changesin cellrsquos energy metabolism physiological and biochemicalfunction [22] This study found that ECH can upregulate thephosphorylation of ERK increase the expression of GDNFand significantly downregulate the activation of microglia-IBA-1 Therefore we believe that ECH exerts neurotrophiceffects by upregulating the phosphorylation level of ERKincreasing the expression of GDNF and at the same timeinhibiting the activation of microglia and astrocytes and

further reducing the content of inflammatory factor in serumand midbrain which restrains DA neuronal apoptosis

The antiapoptotic effect of ECH has been confirmedTo our knowledge it is the first time to explore the signalpathways of anti-inflammatory and antiapoptosis and anti-inflammatory effects of ECH The expression of GDNFmight be related to the regulation of ERK signaling pathwayThe results of this experiment enriched and extended theprotective mechanisms of ECH in PD providing usefultargets for PD therapeutics development and also the relatedbasic research In the past ten years there have been alarge number of experimental studies on traditional Chinesemedicine compound andmonomer that have been confirmedwith the protective effects on DA neurons in PD On thisbasis exploring and confirming the targets of these drugs andtheir neuroprotective mechanisms can provide a theoreticalbasis for the clinical treatment of PD and drug screening

8 BioMed Research International

Data Availability

The data used to support the findings of this study areavailable from the corresponding author upon request

Conflicts of Interest

The authors declare no conflicts of interest

Authorsrsquo Contributions

Yan Liang Chang Chen Baomei Xia Juanjuan Tang andYang Zhao conceived the idea for the study Chang Chen YanLiang Ye Yang andWei Wuwrote the main manuscript textQing Chen Baomei Xia Yan Liang Lili Tang and Hui Yangprepared Figures 1ndash4 Qing Chen Chang Chen Baomei XiaZhennian Zhang and Yan Lu prepared Figures 5 and 6 Allauthors reviewed the manuscript Yan Liang Chang Chenand Baomei Xia contributed equally to this work

Acknowledgments

The study was supported by the National natural sciencefoundation training project of Construction fund for thekey subject of ldquoChinese medicine brain diseaserdquo (201705201707 and 201708) the Nanjing medical science and tech-nology development project (YKK17149) and the Trans-lational medicine project of Nanjing TCM translationalmedicine base (ZHCX201806)The study was also supportedby the Natural Science Foundation of Jiangsu Province(BK20170769 BK20161044) Natural Science Fund for Col-leges and Universities of Jiangsu Province (17KJD360001)and the National Natural Science Foundation of China(81703483 81603089)

References

[1] A L de Lella Ezcurra M Chertoff C Ferrari M Gracia-rena and F Pitossi ldquoChronic expression of low levels oftumor necrosis factor-alpha in the substantia nigra elicitsprogressive neurodegeneration delayed motor symptoms andmicrogliamacrophage activationrdquoNeurobiology of Disease vol37 no 3 pp 630ndash640 2010

[2] H D E Booth W D Hirst and R Wade-Martins ldquoThe Roleof Astrocyte Dysfunction in Parkinsonrsquos Disease PathogenesisrdquoTrends in Neurosciences vol 40 no 6 pp 358ndash370 2017

[3] Q Wang Y Liu and J Zhou ldquoNeuroinflammation in Parkin-sonrsquos disease and its potential as therapeutic targetrdquo in Transla-tional Neurodegeneration vol 4 article no 19 2015

[4] L Lei F Q Yang T Y Zhang P Tu L Wu and Y ItoldquoPreparative isolation and purification of acteoside and 21015840-acetyl acteoside from Cistanches salsa (CA Mey) G Beckby high-speed counter-current chromatographyrdquo Journal ofChromatography A vol 912 no 1 pp 181ndash185 2001

[5] W Chen H-R Lin C-M Wei et al ldquoEchinacoside aphenylethanoid glycoside from Cistanche deserticola extendslifespan of Caenorhabditis elegans and protects from A120573-induced toxicityrdquo Biogerontology vol 19 no 1 pp 47ndash65 2017

[6] Y-J Shiao M-H Su H-C Lin and C-R Wu ldquoEchinacosideameliorates the memory impairment and cholinergic deficit

induced by amyloid beta peptides via the inhibition of amyloiddeposition and toxicologyrdquo Food amp Function vol 8 no 6 pp2283ndash2294 2017

[7] Y Zhang H Long F Zhou et al ldquoEchinacosidersquos nigrostriataldopaminergic protection against 6-OHDA-Induced endoplas-mic reticulum stress through reducing the accumulation ofSeipinrdquo Journal of Cellular and Molecular Medicine vol 21 no12 pp 3761ndash3775 2017

[8] C Chen B Xia L Tang et al ldquoEchinacoside protects againstMPTPMPP+-induced neurotoxicity via regulating autophagypathway mediated by Sirt1rdquoMetabolic Brain Disease 2018

[9] G Paxinos and B J F Keith The mouse brain in stereotaxiccoordinates Gulf professional publishing Houston USA 2004

[10] L V Kalia and A E Lang ldquoParkinsonrsquos diseaserdquoThe Lancet vol386 no 9996 pp 896ndash912 2015

[11] R B Postuma D Berg C H Adler et al ldquoThe new definitionand diagnostic criteria of Parkinsonrsquos diseaserdquo The LancetNeurology vol 15 no 6 pp 546ndash548 2016

[12] P Calabresi and M Di Filippo ldquoMultitarget disease-modifyingtherapy in Parkinsonrsquos diseaserdquo The Lancet Neurology vol 14no 10 article no 159 pp 975-976 2015

[13] E Valera and E Masliah ldquoTherapeutic approaches in Parkin-sonrsquos disease and related disordersrdquo Journal of Neurochemistryvol 139 Supplement 1 pp 346ndash352 2016

[14] Q Zhao J Gao W Li andD Cai ldquoNeurotrophic and neurores-cue effects of Echinacoside in the subacuteMPTPmousemodelof Parkinsonrsquos diseaserdquo Brain Research vol 1346 pp 224ndash2362010

[15] M Zhu C Lu and W Li ldquoTransient exposure to echinacosideis sufficient to activate Trk signaling and protect neuronal cellsfrom rotenonerdquo Journal of Neurochemistry vol 124 no 4 pp571ndash580 2013

[16] R Gordon V Anantharam and A G Kanthasamy ldquoProte-olytic activation of proapoptotic kinase protein kinase Cdeltaby tumor necrosis factor alpha death receptor signaling indopaminergic neurons during neuroinflammationrdquo Journal ofNeuroinflammation vol 9 article 82 2012

[17] A Drinkut Y Tereshchenko J B Schulz M Bahr and SKugler ldquoEfficient gene therapy for Parkinsonrsquos disease usingastrocytes as hosts for localized neurotrophic factor deliveryrdquoMolecularTherapy vol 20 no 3 pp 534ndash543 2012

[18] E Knapska and L Kaczmarek ldquoA gene for neuronal plas-ticity in the mammalian brain Zif268Egr-1NGFI-A Krox-24TIS8ZENKrdquo Progress in Neurobiology vol 74 no 4 pp183ndash211 2004

[19] C Huang J Wu R Liao andW Zhang ldquoSKF83959 an Agonistof Phosphatidylinositol-Linked D1-Like Receptors PromotesERK12 Activation and Cell Migration in Cultured Rat Astro-cytesrdquo PLoS ONE vol 7 no 11 Article ID e49954 2012

[20] D-Y He and D Ron ldquoAutoregulation of glial cell line-derivedneurotrophic factor expression implications for the long-lasting actions of the anti-addiction drug IbogainerdquoThe FASEBjournal official publication of the Federation of AmericanSocieties for Experimental Biology vol 20 no 13 pp 2420ndash24222006

[21] C Barcia ldquoGlial-Mediated Inflammation Underlying Parkin-sonismrdquo Scientifica vol 2013 Article ID 357805 15 pages 2013

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

Stem Cells International

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

MEDIATORSINFLAMMATION

of

EndocrinologyInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Disease Markers

Hindawiwwwhindawicom Volume 2018

BioMed Research International

OncologyJournal of

Hindawiwwwhindawicom Volume 2013

Hindawiwwwhindawicom Volume 2018

Oxidative Medicine and Cellular Longevity

Hindawiwwwhindawicom Volume 2018

PPAR Research

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Immunology ResearchHindawiwwwhindawicom Volume 2018

Journal of

ObesityJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Computational and Mathematical Methods in Medicine

Hindawiwwwhindawicom Volume 2018

Behavioural Neurology

OphthalmologyJournal of

Hindawiwwwhindawicom Volume 2018

Diabetes ResearchJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Research and TreatmentAIDS

Hindawiwwwhindawicom Volume 2018

Gastroenterology Research and Practice

Hindawiwwwhindawicom Volume 2018

Parkinsonrsquos Disease

Evidence-Based Complementary andAlternative Medicine

Volume 2018Hindawiwwwhindawicom

Submit your manuscripts atwwwhindawicom

BioMed Research International 3

tested 3 times with an interval of 2min between each test andan average value of the 3 times was taken as the data

26 Immunohistochemical Staining [8] After perfusion fromthe left ventricle the brains ofmice were rapidly removed andplaced in 4paraformaldehyde to be fixed overnight Sucrosesolution was used for gradient dehydration and OCT gel wasused for embedding We prepared brain tissue sections (30microns) with frozen slicer and chose SN-VTA slices in linewith the atlas of Paxinos and Franklin [9] Sections wereblocked for 1h and incubated with anti-TH primary antibody(1 1000) and anti-GFAP primary antibody (1 1000) for 12hAfter washing there times in 001M PBS the slices were incu-bated with the secondary antibody of goat anti-mouse IgGHampL (HRP) (12000) for 2h After washing the brain slices amodification of the ABC system (Kit Vectastain ABC VectorLaboratory Inc) was used to detect the antibody complexImage-Pro Express 6 (Media Cybernetics) was applied tostereologically quantify the number of TH positive neuronsand GFAP positive astrocytes and the mean number of themin each brain was picked up through the definite quantity offour alternating sections Olympus camera (DP72 Olympus)was used to take images at an original magnification of 100times

27WesternBlotting Tissues of substantia nigrawere lysed inRIPA buffer containing protease inhibitors and phosphataseinhibitors Protein concentration was determined colorimet-rically by NANODROP 2000 (Thermo Scientific) Proteinlysates were separated by 10 SDS-PAGE electrophoresisand were transferred onto polyvinylidenedifluoride (PVDF)membranes After blocking with 5BSA for 1h the followingantibodies were used Anti-IBA-1 antibody (1300) anti-TNF-120572 antibody (1200) anti-Cleaved Caspase-3 antibody(1200) anti-GDNF antibody (1200) anti-phosphorylated-ERK (1500) antibody and anti-ERK antibody (1500) anti-120573-Tublin antibody (12000) After the blots were incubatedwith antibodies overnight at 4∘C they were incubated withhorseradish peroxidase-conjugated secondary antibodies for1h The blots were visualized using the Super Signal WestPico Chemiluminescent Substrate (Thermo Fisher ScientificInc) The resulting data was analyzed statistically using SPSSStatistics 20 All experiments were performed in triplicateThe final data are expressed as a ratio of the relative opticaldensity (ROD) of the protein of interest to the ROD of 120573-tubulin A p value lt 005 was considered significant

28 Enzyme-Linked Immunosorbent Assay Theblood of eachmouse was taken out 15ml to be centrifuged under thecondition of 4∘C 3000 rmin and 10 cm centrifugal radiusfor 10 min and then the blood was stored in 20∘C Thecontents of inflammatory factors such as TNF-120572 and IFN-120574 were detected by enzyme-linked immunoassay kit All theoperations are carried out in accordance with the instructionof kits strictly

29 Statistical Analysis SPSS Version 200 (IBM) was usedfor all statistical analyses All experiments were performedat least three times Comparisons between two groups were

Saline

MPTP

MPTP+ECH

MPTP+Selegilin

eECH

0

10000

20000

30000

tota

l dis

tanc

e (m

m)

lowast

Figure 1 Total distance of open field test Saline means salinecontrol group MPTP means model group MPTP+ECH meansMPTP+ECH group MPTP+Selegiline means positive drug controlgroup and ECHmeans ECH grouplowastplt005 comparedwith Salineplt005 and plt001 compared with MPTP

performed using a two-sample Studentrsquos t-test and compar-isons between multiple groups were performed using a one-way ANOVA followed by post hoc analysis of Tukeyrsquos HSDand LSD multiple comparisons test All data are presented asmean plusmn SEM and statistical significance was accepted at the5 level unless otherwise indicated

3 Result

31 Effect of ECH on Behavior Test in Subacute PDModelMice

311 Locomotor Activity In the open field test the totaldistances of activity in MPTP model group mice was signif-icantly shortened and the difference was statistically signif-icant compared with that in saline control group (Plt005)While compared with the model group the total distanceof activity in MPTP+ECH group was significantly longer(Plt001) (Figure 1)

312 Equilibrium Coordination In the pole test the returntime of mice in the MPTP model group was significantlylonger than that of saline control group (Plt001) while com-pared with the model group the time of MPTP+ECH groupwas shorter and the difference was statistically significant (Plt 001) As to the total time it was significantly increased inthe model group compared to saline control group (P lt 001)meanwhile it was significantly shortened in the MPTP+ECHgroup compared to the model group (P lt 005) (Figure 2)

32 Effects of ECH on Dopaminergic Neurons in the Midbrainof Subacute PD Model Mice Tyrosine hydroxylase is thespecific markers for dopaminergic neurons the number ofTH positive cells can be used as the number of DA neuronsThe stereo counting showed that theTHpositive cells numberof themidbrain in theMPTPmodel group decreased by 631compared with the saline control group in which the number

4 BioMed Research International

Saline

MPTP

MPTP+ECH

MPTP+Selegilin

eECH

00

05

10

15

20

T-tu

rn (s

ec) lowastlowast

(a)

Saline

MPTP

MPTP+ECH

MPTP+Selegilin

eECH

0

5

10

15

T-to

tal (

sec) lowastlowast

(b)

Figure 2 Pole test (a) Time of return (b) Time of total Saline means saline control group MPTP means model group MPTP+ECHmeansMPTP+ECH group MPTP+Selegiline means positive drug control group and ECH means ECH group lowastlowastplt001 compared with salineplt005 and plt001 compared with MPTP

Saline MPTP+Selegiline

MPTP ECH

MPTP+ECH

Saline

MPTP

MPTP+ECH

MPTP+Selegilin

eECH

0

2000

4000

6000

8000

10000

TH+ co

unts

SN

pc

(a)

Saline

MPTP

MPTP+ECH

MPTP+Selegilin

eECH

(b)

0

2000

4000

6000

8000

TH+ co

unts

VTA

lowast

lowastlowastlowast

Figure 3 Representativemicrophotographs of dopaminergic neurons stained for TH and the quantification of TH positive cell in each groupThe pictures were taken at an original magnification of 10times Scale bars 100120583m Saline means saline control groupMPTPmeans model groupMPTP+ECH means MPTP+ECH group MPTP+Selegiline means positive drug control group and ECH means ECH group lowastplt005 lowastlowastlowastplt0001 compared with saline plt001 and plt0001 compared with MPTP

in SNpc area decreased by 599 and the number in the VTAarea decreased by 446 Both differences were significantcompared with the control group (Plt0001 Plt005) Whilein the ECH treatment group the number of TH positivecells in both SNpc and VTA increased observably which was

statistically significant compared with the model group (P lt0001 P lt 0001) (Figure 3)

33 Effects of ECH on Astrocytes in the Midbrain of SubacutePD Model Mice As a marker of activation in astrocyte

BioMed Research International 5

Saline MPTP+Selegiline

MPTP ECH

MPTP+ECH

(a)

Saline

MPTP

MPTP+ECH

MPTP+Selegilin

eECH

0

500

1000

1500

GFA

P-IR

coun

ts V

TA(b)

Saline

MPTP

MPTP+ECH

MPTP+Selegilin

eECH

0

2000

4000

6000

GFA

P-IR

coun

ts S

Npc

lowastlowastlowast

lowastlowastlowast

Figure 4 Representative microphotographs of dopaminergic neurons stained for GFAP and the quantification of GFAP positive cell in eachgroup The pictures were taken at an original magnification of 10times Scale bars 100120583m Saline means saline control group MPTP meansmodel group MPTP+ECHmeans MPTP+ECH group MPTP+Selegiline means positive drug control group and ECHmeans ECH group lowastlowastlowastplt0001 compared with saline plt0001 compared with MPTP

GFAP can be used to detect the number of active astro-cytes According to the results of immunohistochemistry theastrocytes in model group mice were activated in a patternof hypertrophy and the number of active astrocytes wassignificantly increased in both SNpc area and VTA areacompared with saline control group (P lt 0001 P lt 0001)After the ECH treatment the morphology of astrocytes wererestored to the saline control group and the number ofGFAP positive cells in SNpc and VTA area was significantlydecreased (P lt 0001 P lt 0001) (Figure 4)

34 Effects of ECH on Serum Inflammatory Factors in Suba-cute PD Model Mice Enzyme-linked immunosorbent assayshowed that the concentrations of inflammatory factorsincluding TNF-120572 and IFN-120574 in the serum of model groupmice were significantly higher than that of saline controlgroup (P lt 005 P lt 001) After the treatment of ECH theconcentrations of the two factors were significantly reduced(P lt 001 P lt 005) (Figure 5)

35 Effects of ECH on the Expression of Midbrain ProteinsIncluding IBA-1 Cleaved Caspase-3 GDNF TNF-120572 p-ERKand ERK in Subacute PD Mice Western blotting analy-sis showed that MPTP induced a marked IBA-1 CleavedCaspase-3 and TNF-120572protein increase (Plt001 Plt0001 and

Plt0001) while the expression of GDNF and ERK phospho-rylation level was significantly decreased (Plt005 Plt005)in midbrain tissue compared to saline control group Afterthe ECH administration the expressions of IBA-1 CleavedCaspase-3 and TNF-120572were significantly decreased (P lt 001Plt 0001 and Plt 001) and the expressions of GDNF and thephosphorylation level of ERK were significantly increased (Plt 0001 P lt 001) (Figure 6)

4 Discussion

Parkinsonrsquos disease is a common central nervous systemdegeneration disease in the elderly Its main clinical fea-tures are not only bradykinesia static tremor myotoniaand abnormal posture but also olfactory disorder con-stipation insomnia fatigue depression and anxiety andother nonmotor symptoms [10 11] The pathogenesis of PDis complex which may be caused by aging genetic andenvironmental factors The pathological change of SNpc DAneuron degeneration lost and appearance of Lewy body maybe connected with cell autophagy proteasome dysfunctionmitochondrial function defect oxidative stress and immuneresponse inflammation excitatory neurotoxicity and othercomplex mechanisms [10 12] Currently dopaminergic drugtherapy is the major treatment of PD Neither supplementing

6 BioMed Research International

Saline

MPTP

MPTP+ECH

MPTP+Selegilin

eECH

0

50

100

150

200

250

TNF-

o

f ser

um (p

gm

l)lowast

(a)

Saline

MPTP

MPTP+ECH

MPTP+Selegilin

eECH

0

50

100

150

200

IFN

- o

f ser

um (p

gm

l)

lowastlowast

(b)

Figure 5 Alteration of TNF-120572 and IFN-120574 in serum of mice Saline means saline control group MPTP means model group MPTP+ECHmeans MPTP+ECH group MPTP+Selegiline means positive drug control group and ECH means ECH group lowastplt005 and lowastlowastplt001compared with saline plt005 and plt001 compared with MPTP

the DA neurotransmitter nor activating DA receptors canreverse the loss of DA neurons and the appearance of Lewybody That means the current PD treatment plan is mainlyfor the symptoms without affecting the progress of the disease[13]Therefore there has been increasing interests in studyingagents like ECH that affect the mechanistic processes ofPD as novel treatment This study specifically found that(1) ECH could improve the PD-like behavior of subacutemodel mice induced by MPTP and the loss of DA neuronsin the substantia nigra of model mice was saved which issimilar to the reports of Qing Zhao [14] (2) ECH couldalso reverse against the astrocyte overactivation microgliaactivation serum and mesencephalic inflammatory factorsin midbrain induced by MPTP in PD subacute model mice(3) ECH was able to increase the expression of GDNF andameliorate the overexpression of MPTP-induced IBA-1 andCleaved Caspase-3 and the decrease of phosphorylated ERK

MPTP is the most commonly used environmental toxinfor the preparation of PD animal model which is highlyfat-soluble and extremely easy to pass through the bloodbrain barrier MPTP is metabolized by monoamine oxidasein astrocytes and turned into ionized 1-methyl-4-phenylpyridine (MPP+) transferred by the Dopamine transporter(DAT) to the DA neurons MPTP produces obvious patho-logical and biochemical damage of DA neurons in C57BL6Jmice so it has a great advantage over other PD models interms of efficacy evaluation We prepared subacute PDmod-els in C57BL6J mice by MPTP (subcutaneous injection) forfive consecutive days and observe thatMPTP can successfullyinduce animal PD behavior cause the loss of DA neurons byimmune histochemical and behavior testing

Echinacoside is the key components in the identificationand assaying of herba cistanche in Chinese pharmacopoeiaIn recent years it has been found that ECH can cross theblood brain barrier [15] and has a good protective effect onthe central nervous system According to Zhaorsquos research[14] we chose the optimal therapeutic does of 30mgkg

ECH as an intervention the behavior of MPTP-inducedanimal models improved markedly the DA neurons deathsdropped significantly the expression of Cleaved Caspase-3decreased obviously and all the above indicated ECH couldprotect DA neurons of PD model mice by antiapoptoticeffect This is similar to Zhaorsquos report [14] The differencebetween two experiments lies in Zhao detected apoptosisrelated protein directly but Zhao has not studied howMPTPcauses apoptosis of DA neurons and how ECH works againstapoptosis Therefore this study explored the mechanism ofMPTP-induced apoptosis of DA neurons and the role of ECHin neurorescue based on glial cells

Microglia is an immune cell in the brain when activatedit produces and releases proinflammatory cytokines thatcause damage to DA neurons Inflammatory factors such asIFN-120574 IL-1120573 TNF-120572 IL-2 and IL-6 [1] are founded in the SNstriatum and cerebrospinal fluids of autopsy in patients withPDmeanwhile the expressions of TNF-120572 receptors R1 (p55)bcl-2 soluble Fas caspase-1 and caspase-3 are increased [16]Therefore proinflammatory cytokines activated and releasedby microglia-mediated factor may be the one reason of deathof DA neurons Astrocytes secrete a variety of neurotrophicfactors of which GDNF is particularly important becauseit can specifically promote the repair of DA neurons ininjured midbrain and it is considered as the most promisingnutrient factor in PD treatment According to reports inthe literature [17] significant improvement can be observedafter astrocytes which are carrying the high expression ofGDNF gene were implanted into the SN region of animalmodels of PD The treatment of GDNF depends on theERK signaling pathway which promotes cell survival [18] Ithas been reported that application of phosphatidylinositol-related D1-like receptor agonists in rat astrocytes can enhancethe expression of phosphorylated ERK and promote themigration of astrocytes [19] GDNF can activate ERK andinduce the upregulation of its own expression in SH-SY5Ycells and rat VTA region [20 21] At the same time ERK

BioMed Research International 7

IBA-1

Cleaved-Cas3

GDNF

TNF-

-tubulin

p-ERK

ERK

Saline

MPTPMPTP+ECH

MPTP+Selegilin

e

ECH

(a)

Saline

MPTP

MPTP+ECH

MPTP+Selegilin

eECH

0

1

2

3

4

IBA-1

IBA-

1

-tubu

lin

lowastlowast

(b)

Saline

MPTP

MPTP+ECH

MPTP+Selegilin

eECH

0

1

2

3

4

Cleaved-Caspase-3

Cle

aved

-Cas

3

-tubu

lin lowastlowastlowast

(c)

Saline

MPTP

MPTP+ECH

MPTP+Selegilin

eECH

00

05

10

15

GDNF

GD

NF

-tu

bulin

lowast

(d)

Saline

MPTP

MPTP+ECH

MPTP+Selegilin

eECH

00

05

10

15

20

TNF-

TNF-

-tubu

lin

lowastlowastlowast

(e)

Saline

MPTP

MPTP+ECH

MPTP+Selegilin

eECH

00

05

10

15

20

p-Erk Erk

p-Er

k E

rk

lowast

(f)

Figure 6 Protein expression of IBA-1 Cleaved-Caspase-3GDNF TNF-120572 p-ERK and ERK Salinemeans saline control groupMPTPmeansmodel group MPTP+ECH means MPTP+ECH group MPTP+Selegiline means positive drug control group and ECH means ECH grouplowastplt005 lowastlowastplt001 lowastlowastlowastplt0001 compared with saline plt005 plt001 and plt0001 compared with MPTP

signaling pathway also plays a key role in the inflammatorypathological changes of PD and their coeffects may affect thepathogenesis of PD and the progression of the disease In theresting state ERKMAPK are located in the cytoplasm of thecells When activated ERK is rapidly phosphorylated Thenactivated ERK enters the nucleus to regulate the activities ofother transcription factors which make the change of genetranscription impact the ascension or decline of the expres-sion of respective protein gene and finally cause the changesin cellrsquos energy metabolism physiological and biochemicalfunction [22] This study found that ECH can upregulate thephosphorylation of ERK increase the expression of GDNFand significantly downregulate the activation of microglia-IBA-1 Therefore we believe that ECH exerts neurotrophiceffects by upregulating the phosphorylation level of ERKincreasing the expression of GDNF and at the same timeinhibiting the activation of microglia and astrocytes and

further reducing the content of inflammatory factor in serumand midbrain which restrains DA neuronal apoptosis

The antiapoptotic effect of ECH has been confirmedTo our knowledge it is the first time to explore the signalpathways of anti-inflammatory and antiapoptosis and anti-inflammatory effects of ECH The expression of GDNFmight be related to the regulation of ERK signaling pathwayThe results of this experiment enriched and extended theprotective mechanisms of ECH in PD providing usefultargets for PD therapeutics development and also the relatedbasic research In the past ten years there have been alarge number of experimental studies on traditional Chinesemedicine compound andmonomer that have been confirmedwith the protective effects on DA neurons in PD On thisbasis exploring and confirming the targets of these drugs andtheir neuroprotective mechanisms can provide a theoreticalbasis for the clinical treatment of PD and drug screening

8 BioMed Research International

Data Availability

The data used to support the findings of this study areavailable from the corresponding author upon request

Conflicts of Interest

The authors declare no conflicts of interest

Authorsrsquo Contributions

Yan Liang Chang Chen Baomei Xia Juanjuan Tang andYang Zhao conceived the idea for the study Chang Chen YanLiang Ye Yang andWei Wuwrote the main manuscript textQing Chen Baomei Xia Yan Liang Lili Tang and Hui Yangprepared Figures 1ndash4 Qing Chen Chang Chen Baomei XiaZhennian Zhang and Yan Lu prepared Figures 5 and 6 Allauthors reviewed the manuscript Yan Liang Chang Chenand Baomei Xia contributed equally to this work

Acknowledgments

The study was supported by the National natural sciencefoundation training project of Construction fund for thekey subject of ldquoChinese medicine brain diseaserdquo (201705201707 and 201708) the Nanjing medical science and tech-nology development project (YKK17149) and the Trans-lational medicine project of Nanjing TCM translationalmedicine base (ZHCX201806)The study was also supportedby the Natural Science Foundation of Jiangsu Province(BK20170769 BK20161044) Natural Science Fund for Col-leges and Universities of Jiangsu Province (17KJD360001)and the National Natural Science Foundation of China(81703483 81603089)

References

[1] A L de Lella Ezcurra M Chertoff C Ferrari M Gracia-rena and F Pitossi ldquoChronic expression of low levels oftumor necrosis factor-alpha in the substantia nigra elicitsprogressive neurodegeneration delayed motor symptoms andmicrogliamacrophage activationrdquoNeurobiology of Disease vol37 no 3 pp 630ndash640 2010

[2] H D E Booth W D Hirst and R Wade-Martins ldquoThe Roleof Astrocyte Dysfunction in Parkinsonrsquos Disease PathogenesisrdquoTrends in Neurosciences vol 40 no 6 pp 358ndash370 2017

[3] Q Wang Y Liu and J Zhou ldquoNeuroinflammation in Parkin-sonrsquos disease and its potential as therapeutic targetrdquo in Transla-tional Neurodegeneration vol 4 article no 19 2015

[4] L Lei F Q Yang T Y Zhang P Tu L Wu and Y ItoldquoPreparative isolation and purification of acteoside and 21015840-acetyl acteoside from Cistanches salsa (CA Mey) G Beckby high-speed counter-current chromatographyrdquo Journal ofChromatography A vol 912 no 1 pp 181ndash185 2001

[5] W Chen H-R Lin C-M Wei et al ldquoEchinacoside aphenylethanoid glycoside from Cistanche deserticola extendslifespan of Caenorhabditis elegans and protects from A120573-induced toxicityrdquo Biogerontology vol 19 no 1 pp 47ndash65 2017

[6] Y-J Shiao M-H Su H-C Lin and C-R Wu ldquoEchinacosideameliorates the memory impairment and cholinergic deficit

induced by amyloid beta peptides via the inhibition of amyloiddeposition and toxicologyrdquo Food amp Function vol 8 no 6 pp2283ndash2294 2017

[7] Y Zhang H Long F Zhou et al ldquoEchinacosidersquos nigrostriataldopaminergic protection against 6-OHDA-Induced endoplas-mic reticulum stress through reducing the accumulation ofSeipinrdquo Journal of Cellular and Molecular Medicine vol 21 no12 pp 3761ndash3775 2017

[8] C Chen B Xia L Tang et al ldquoEchinacoside protects againstMPTPMPP+-induced neurotoxicity via regulating autophagypathway mediated by Sirt1rdquoMetabolic Brain Disease 2018

[9] G Paxinos and B J F Keith The mouse brain in stereotaxiccoordinates Gulf professional publishing Houston USA 2004

[10] L V Kalia and A E Lang ldquoParkinsonrsquos diseaserdquoThe Lancet vol386 no 9996 pp 896ndash912 2015

[11] R B Postuma D Berg C H Adler et al ldquoThe new definitionand diagnostic criteria of Parkinsonrsquos diseaserdquo The LancetNeurology vol 15 no 6 pp 546ndash548 2016

[12] P Calabresi and M Di Filippo ldquoMultitarget disease-modifyingtherapy in Parkinsonrsquos diseaserdquo The Lancet Neurology vol 14no 10 article no 159 pp 975-976 2015

[13] E Valera and E Masliah ldquoTherapeutic approaches in Parkin-sonrsquos disease and related disordersrdquo Journal of Neurochemistryvol 139 Supplement 1 pp 346ndash352 2016

[14] Q Zhao J Gao W Li andD Cai ldquoNeurotrophic and neurores-cue effects of Echinacoside in the subacuteMPTPmousemodelof Parkinsonrsquos diseaserdquo Brain Research vol 1346 pp 224ndash2362010

[15] M Zhu C Lu and W Li ldquoTransient exposure to echinacosideis sufficient to activate Trk signaling and protect neuronal cellsfrom rotenonerdquo Journal of Neurochemistry vol 124 no 4 pp571ndash580 2013

[16] R Gordon V Anantharam and A G Kanthasamy ldquoProte-olytic activation of proapoptotic kinase protein kinase Cdeltaby tumor necrosis factor alpha death receptor signaling indopaminergic neurons during neuroinflammationrdquo Journal ofNeuroinflammation vol 9 article 82 2012

[17] A Drinkut Y Tereshchenko J B Schulz M Bahr and SKugler ldquoEfficient gene therapy for Parkinsonrsquos disease usingastrocytes as hosts for localized neurotrophic factor deliveryrdquoMolecularTherapy vol 20 no 3 pp 534ndash543 2012

[18] E Knapska and L Kaczmarek ldquoA gene for neuronal plas-ticity in the mammalian brain Zif268Egr-1NGFI-A Krox-24TIS8ZENKrdquo Progress in Neurobiology vol 74 no 4 pp183ndash211 2004

[19] C Huang J Wu R Liao andW Zhang ldquoSKF83959 an Agonistof Phosphatidylinositol-Linked D1-Like Receptors PromotesERK12 Activation and Cell Migration in Cultured Rat Astro-cytesrdquo PLoS ONE vol 7 no 11 Article ID e49954 2012

[20] D-Y He and D Ron ldquoAutoregulation of glial cell line-derivedneurotrophic factor expression implications for the long-lasting actions of the anti-addiction drug IbogainerdquoThe FASEBjournal official publication of the Federation of AmericanSocieties for Experimental Biology vol 20 no 13 pp 2420ndash24222006

[21] C Barcia ldquoGlial-Mediated Inflammation Underlying Parkin-sonismrdquo Scientifica vol 2013 Article ID 357805 15 pages 2013

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

Stem Cells International

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

MEDIATORSINFLAMMATION

of

EndocrinologyInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Disease Markers

Hindawiwwwhindawicom Volume 2018

BioMed Research International

OncologyJournal of

Hindawiwwwhindawicom Volume 2013

Hindawiwwwhindawicom Volume 2018

Oxidative Medicine and Cellular Longevity

Hindawiwwwhindawicom Volume 2018

PPAR Research

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Immunology ResearchHindawiwwwhindawicom Volume 2018

Journal of

ObesityJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Computational and Mathematical Methods in Medicine

Hindawiwwwhindawicom Volume 2018

Behavioural Neurology

OphthalmologyJournal of

Hindawiwwwhindawicom Volume 2018

Diabetes ResearchJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Research and TreatmentAIDS

Hindawiwwwhindawicom Volume 2018

Gastroenterology Research and Practice

Hindawiwwwhindawicom Volume 2018

Parkinsonrsquos Disease

Evidence-Based Complementary andAlternative Medicine

Volume 2018Hindawiwwwhindawicom

Submit your manuscripts atwwwhindawicom

4 BioMed Research International

Saline

MPTP

MPTP+ECH

MPTP+Selegilin

eECH

00

05

10

15

20

T-tu

rn (s

ec) lowastlowast

(a)

Saline

MPTP

MPTP+ECH

MPTP+Selegilin

eECH

0

5

10

15

T-to

tal (

sec) lowastlowast

(b)

Figure 2 Pole test (a) Time of return (b) Time of total Saline means saline control group MPTP means model group MPTP+ECHmeansMPTP+ECH group MPTP+Selegiline means positive drug control group and ECH means ECH group lowastlowastplt001 compared with salineplt005 and plt001 compared with MPTP

Saline MPTP+Selegiline

MPTP ECH

MPTP+ECH

Saline

MPTP

MPTP+ECH

MPTP+Selegilin

eECH

0

2000

4000

6000

8000

10000

TH+ co

unts

SN

pc

(a)

Saline

MPTP

MPTP+ECH

MPTP+Selegilin

eECH

(b)

0

2000

4000

6000

8000

TH+ co

unts

VTA

lowast

lowastlowastlowast

Figure 3 Representativemicrophotographs of dopaminergic neurons stained for TH and the quantification of TH positive cell in each groupThe pictures were taken at an original magnification of 10times Scale bars 100120583m Saline means saline control groupMPTPmeans model groupMPTP+ECH means MPTP+ECH group MPTP+Selegiline means positive drug control group and ECH means ECH group lowastplt005 lowastlowastlowastplt0001 compared with saline plt001 and plt0001 compared with MPTP

in SNpc area decreased by 599 and the number in the VTAarea decreased by 446 Both differences were significantcompared with the control group (Plt0001 Plt005) Whilein the ECH treatment group the number of TH positivecells in both SNpc and VTA increased observably which was

statistically significant compared with the model group (P lt0001 P lt 0001) (Figure 3)

33 Effects of ECH on Astrocytes in the Midbrain of SubacutePD Model Mice As a marker of activation in astrocyte

BioMed Research International 5

Saline MPTP+Selegiline

MPTP ECH

MPTP+ECH

(a)

Saline

MPTP

MPTP+ECH

MPTP+Selegilin

eECH

0

500

1000

1500

GFA

P-IR

coun

ts V

TA(b)

Saline

MPTP

MPTP+ECH

MPTP+Selegilin

eECH

0

2000

4000

6000

GFA

P-IR

coun

ts S

Npc

lowastlowastlowast

lowastlowastlowast

Figure 4 Representative microphotographs of dopaminergic neurons stained for GFAP and the quantification of GFAP positive cell in eachgroup The pictures were taken at an original magnification of 10times Scale bars 100120583m Saline means saline control group MPTP meansmodel group MPTP+ECHmeans MPTP+ECH group MPTP+Selegiline means positive drug control group and ECHmeans ECH group lowastlowastlowastplt0001 compared with saline plt0001 compared with MPTP

GFAP can be used to detect the number of active astro-cytes According to the results of immunohistochemistry theastrocytes in model group mice were activated in a patternof hypertrophy and the number of active astrocytes wassignificantly increased in both SNpc area and VTA areacompared with saline control group (P lt 0001 P lt 0001)After the ECH treatment the morphology of astrocytes wererestored to the saline control group and the number ofGFAP positive cells in SNpc and VTA area was significantlydecreased (P lt 0001 P lt 0001) (Figure 4)

34 Effects of ECH on Serum Inflammatory Factors in Suba-cute PD Model Mice Enzyme-linked immunosorbent assayshowed that the concentrations of inflammatory factorsincluding TNF-120572 and IFN-120574 in the serum of model groupmice were significantly higher than that of saline controlgroup (P lt 005 P lt 001) After the treatment of ECH theconcentrations of the two factors were significantly reduced(P lt 001 P lt 005) (Figure 5)

35 Effects of ECH on the Expression of Midbrain ProteinsIncluding IBA-1 Cleaved Caspase-3 GDNF TNF-120572 p-ERKand ERK in Subacute PD Mice Western blotting analy-sis showed that MPTP induced a marked IBA-1 CleavedCaspase-3 and TNF-120572protein increase (Plt001 Plt0001 and

Plt0001) while the expression of GDNF and ERK phospho-rylation level was significantly decreased (Plt005 Plt005)in midbrain tissue compared to saline control group Afterthe ECH administration the expressions of IBA-1 CleavedCaspase-3 and TNF-120572were significantly decreased (P lt 001Plt 0001 and Plt 001) and the expressions of GDNF and thephosphorylation level of ERK were significantly increased (Plt 0001 P lt 001) (Figure 6)

4 Discussion

Parkinsonrsquos disease is a common central nervous systemdegeneration disease in the elderly Its main clinical fea-tures are not only bradykinesia static tremor myotoniaand abnormal posture but also olfactory disorder con-stipation insomnia fatigue depression and anxiety andother nonmotor symptoms [10 11] The pathogenesis of PDis complex which may be caused by aging genetic andenvironmental factors The pathological change of SNpc DAneuron degeneration lost and appearance of Lewy body maybe connected with cell autophagy proteasome dysfunctionmitochondrial function defect oxidative stress and immuneresponse inflammation excitatory neurotoxicity and othercomplex mechanisms [10 12] Currently dopaminergic drugtherapy is the major treatment of PD Neither supplementing

6 BioMed Research International

Saline

MPTP

MPTP+ECH

MPTP+Selegilin

eECH

0

50

100

150

200

250

TNF-

o

f ser

um (p

gm

l)lowast

(a)

Saline

MPTP

MPTP+ECH

MPTP+Selegilin

eECH

0

50

100

150

200

IFN

- o

f ser

um (p

gm

l)

lowastlowast

(b)

Figure 5 Alteration of TNF-120572 and IFN-120574 in serum of mice Saline means saline control group MPTP means model group MPTP+ECHmeans MPTP+ECH group MPTP+Selegiline means positive drug control group and ECH means ECH group lowastplt005 and lowastlowastplt001compared with saline plt005 and plt001 compared with MPTP

the DA neurotransmitter nor activating DA receptors canreverse the loss of DA neurons and the appearance of Lewybody That means the current PD treatment plan is mainlyfor the symptoms without affecting the progress of the disease[13]Therefore there has been increasing interests in studyingagents like ECH that affect the mechanistic processes ofPD as novel treatment This study specifically found that(1) ECH could improve the PD-like behavior of subacutemodel mice induced by MPTP and the loss of DA neuronsin the substantia nigra of model mice was saved which issimilar to the reports of Qing Zhao [14] (2) ECH couldalso reverse against the astrocyte overactivation microgliaactivation serum and mesencephalic inflammatory factorsin midbrain induced by MPTP in PD subacute model mice(3) ECH was able to increase the expression of GDNF andameliorate the overexpression of MPTP-induced IBA-1 andCleaved Caspase-3 and the decrease of phosphorylated ERK

MPTP is the most commonly used environmental toxinfor the preparation of PD animal model which is highlyfat-soluble and extremely easy to pass through the bloodbrain barrier MPTP is metabolized by monoamine oxidasein astrocytes and turned into ionized 1-methyl-4-phenylpyridine (MPP+) transferred by the Dopamine transporter(DAT) to the DA neurons MPTP produces obvious patho-logical and biochemical damage of DA neurons in C57BL6Jmice so it has a great advantage over other PD models interms of efficacy evaluation We prepared subacute PDmod-els in C57BL6J mice by MPTP (subcutaneous injection) forfive consecutive days and observe thatMPTP can successfullyinduce animal PD behavior cause the loss of DA neurons byimmune histochemical and behavior testing

Echinacoside is the key components in the identificationand assaying of herba cistanche in Chinese pharmacopoeiaIn recent years it has been found that ECH can cross theblood brain barrier [15] and has a good protective effect onthe central nervous system According to Zhaorsquos research[14] we chose the optimal therapeutic does of 30mgkg

ECH as an intervention the behavior of MPTP-inducedanimal models improved markedly the DA neurons deathsdropped significantly the expression of Cleaved Caspase-3decreased obviously and all the above indicated ECH couldprotect DA neurons of PD model mice by antiapoptoticeffect This is similar to Zhaorsquos report [14] The differencebetween two experiments lies in Zhao detected apoptosisrelated protein directly but Zhao has not studied howMPTPcauses apoptosis of DA neurons and how ECH works againstapoptosis Therefore this study explored the mechanism ofMPTP-induced apoptosis of DA neurons and the role of ECHin neurorescue based on glial cells

Microglia is an immune cell in the brain when activatedit produces and releases proinflammatory cytokines thatcause damage to DA neurons Inflammatory factors such asIFN-120574 IL-1120573 TNF-120572 IL-2 and IL-6 [1] are founded in the SNstriatum and cerebrospinal fluids of autopsy in patients withPDmeanwhile the expressions of TNF-120572 receptors R1 (p55)bcl-2 soluble Fas caspase-1 and caspase-3 are increased [16]Therefore proinflammatory cytokines activated and releasedby microglia-mediated factor may be the one reason of deathof DA neurons Astrocytes secrete a variety of neurotrophicfactors of which GDNF is particularly important becauseit can specifically promote the repair of DA neurons ininjured midbrain and it is considered as the most promisingnutrient factor in PD treatment According to reports inthe literature [17] significant improvement can be observedafter astrocytes which are carrying the high expression ofGDNF gene were implanted into the SN region of animalmodels of PD The treatment of GDNF depends on theERK signaling pathway which promotes cell survival [18] Ithas been reported that application of phosphatidylinositol-related D1-like receptor agonists in rat astrocytes can enhancethe expression of phosphorylated ERK and promote themigration of astrocytes [19] GDNF can activate ERK andinduce the upregulation of its own expression in SH-SY5Ycells and rat VTA region [20 21] At the same time ERK

BioMed Research International 7

IBA-1

Cleaved-Cas3

GDNF

TNF-

-tubulin

p-ERK

ERK

Saline

MPTPMPTP+ECH

MPTP+Selegilin

e

ECH

(a)

Saline

MPTP

MPTP+ECH

MPTP+Selegilin

eECH

0

1

2

3

4

IBA-1

IBA-

1

-tubu

lin

lowastlowast

(b)

Saline

MPTP

MPTP+ECH

MPTP+Selegilin

eECH

0

1

2

3

4

Cleaved-Caspase-3

Cle

aved

-Cas

3

-tubu

lin lowastlowastlowast

(c)

Saline

MPTP

MPTP+ECH

MPTP+Selegilin

eECH

00

05

10

15

GDNF

GD

NF

-tu

bulin

lowast

(d)

Saline

MPTP

MPTP+ECH

MPTP+Selegilin

eECH

00

05

10

15

20

TNF-

TNF-

-tubu

lin

lowastlowastlowast

(e)

Saline

MPTP

MPTP+ECH

MPTP+Selegilin

eECH

00

05

10

15

20

p-Erk Erk

p-Er

k E

rk

lowast

(f)

Figure 6 Protein expression of IBA-1 Cleaved-Caspase-3GDNF TNF-120572 p-ERK and ERK Salinemeans saline control groupMPTPmeansmodel group MPTP+ECH means MPTP+ECH group MPTP+Selegiline means positive drug control group and ECH means ECH grouplowastplt005 lowastlowastplt001 lowastlowastlowastplt0001 compared with saline plt005 plt001 and plt0001 compared with MPTP

signaling pathway also plays a key role in the inflammatorypathological changes of PD and their coeffects may affect thepathogenesis of PD and the progression of the disease In theresting state ERKMAPK are located in the cytoplasm of thecells When activated ERK is rapidly phosphorylated Thenactivated ERK enters the nucleus to regulate the activities ofother transcription factors which make the change of genetranscription impact the ascension or decline of the expres-sion of respective protein gene and finally cause the changesin cellrsquos energy metabolism physiological and biochemicalfunction [22] This study found that ECH can upregulate thephosphorylation of ERK increase the expression of GDNFand significantly downregulate the activation of microglia-IBA-1 Therefore we believe that ECH exerts neurotrophiceffects by upregulating the phosphorylation level of ERKincreasing the expression of GDNF and at the same timeinhibiting the activation of microglia and astrocytes and

further reducing the content of inflammatory factor in serumand midbrain which restrains DA neuronal apoptosis

The antiapoptotic effect of ECH has been confirmedTo our knowledge it is the first time to explore the signalpathways of anti-inflammatory and antiapoptosis and anti-inflammatory effects of ECH The expression of GDNFmight be related to the regulation of ERK signaling pathwayThe results of this experiment enriched and extended theprotective mechanisms of ECH in PD providing usefultargets for PD therapeutics development and also the relatedbasic research In the past ten years there have been alarge number of experimental studies on traditional Chinesemedicine compound andmonomer that have been confirmedwith the protective effects on DA neurons in PD On thisbasis exploring and confirming the targets of these drugs andtheir neuroprotective mechanisms can provide a theoreticalbasis for the clinical treatment of PD and drug screening

8 BioMed Research International

Data Availability

The data used to support the findings of this study areavailable from the corresponding author upon request

Conflicts of Interest

The authors declare no conflicts of interest

Authorsrsquo Contributions

Yan Liang Chang Chen Baomei Xia Juanjuan Tang andYang Zhao conceived the idea for the study Chang Chen YanLiang Ye Yang andWei Wuwrote the main manuscript textQing Chen Baomei Xia Yan Liang Lili Tang and Hui Yangprepared Figures 1ndash4 Qing Chen Chang Chen Baomei XiaZhennian Zhang and Yan Lu prepared Figures 5 and 6 Allauthors reviewed the manuscript Yan Liang Chang Chenand Baomei Xia contributed equally to this work

Acknowledgments

The study was supported by the National natural sciencefoundation training project of Construction fund for thekey subject of ldquoChinese medicine brain diseaserdquo (201705201707 and 201708) the Nanjing medical science and tech-nology development project (YKK17149) and the Trans-lational medicine project of Nanjing TCM translationalmedicine base (ZHCX201806)The study was also supportedby the Natural Science Foundation of Jiangsu Province(BK20170769 BK20161044) Natural Science Fund for Col-leges and Universities of Jiangsu Province (17KJD360001)and the National Natural Science Foundation of China(81703483 81603089)

References

[1] A L de Lella Ezcurra M Chertoff C Ferrari M Gracia-rena and F Pitossi ldquoChronic expression of low levels oftumor necrosis factor-alpha in the substantia nigra elicitsprogressive neurodegeneration delayed motor symptoms andmicrogliamacrophage activationrdquoNeurobiology of Disease vol37 no 3 pp 630ndash640 2010

[2] H D E Booth W D Hirst and R Wade-Martins ldquoThe Roleof Astrocyte Dysfunction in Parkinsonrsquos Disease PathogenesisrdquoTrends in Neurosciences vol 40 no 6 pp 358ndash370 2017

[3] Q Wang Y Liu and J Zhou ldquoNeuroinflammation in Parkin-sonrsquos disease and its potential as therapeutic targetrdquo in Transla-tional Neurodegeneration vol 4 article no 19 2015

[4] L Lei F Q Yang T Y Zhang P Tu L Wu and Y ItoldquoPreparative isolation and purification of acteoside and 21015840-acetyl acteoside from Cistanches salsa (CA Mey) G Beckby high-speed counter-current chromatographyrdquo Journal ofChromatography A vol 912 no 1 pp 181ndash185 2001

[5] W Chen H-R Lin C-M Wei et al ldquoEchinacoside aphenylethanoid glycoside from Cistanche deserticola extendslifespan of Caenorhabditis elegans and protects from A120573-induced toxicityrdquo Biogerontology vol 19 no 1 pp 47ndash65 2017

[6] Y-J Shiao M-H Su H-C Lin and C-R Wu ldquoEchinacosideameliorates the memory impairment and cholinergic deficit

induced by amyloid beta peptides via the inhibition of amyloiddeposition and toxicologyrdquo Food amp Function vol 8 no 6 pp2283ndash2294 2017

[7] Y Zhang H Long F Zhou et al ldquoEchinacosidersquos nigrostriataldopaminergic protection against 6-OHDA-Induced endoplas-mic reticulum stress through reducing the accumulation ofSeipinrdquo Journal of Cellular and Molecular Medicine vol 21 no12 pp 3761ndash3775 2017

[8] C Chen B Xia L Tang et al ldquoEchinacoside protects againstMPTPMPP+-induced neurotoxicity via regulating autophagypathway mediated by Sirt1rdquoMetabolic Brain Disease 2018

[9] G Paxinos and B J F Keith The mouse brain in stereotaxiccoordinates Gulf professional publishing Houston USA 2004

[10] L V Kalia and A E Lang ldquoParkinsonrsquos diseaserdquoThe Lancet vol386 no 9996 pp 896ndash912 2015

[11] R B Postuma D Berg C H Adler et al ldquoThe new definitionand diagnostic criteria of Parkinsonrsquos diseaserdquo The LancetNeurology vol 15 no 6 pp 546ndash548 2016

[12] P Calabresi and M Di Filippo ldquoMultitarget disease-modifyingtherapy in Parkinsonrsquos diseaserdquo The Lancet Neurology vol 14no 10 article no 159 pp 975-976 2015

[13] E Valera and E Masliah ldquoTherapeutic approaches in Parkin-sonrsquos disease and related disordersrdquo Journal of Neurochemistryvol 139 Supplement 1 pp 346ndash352 2016

[14] Q Zhao J Gao W Li andD Cai ldquoNeurotrophic and neurores-cue effects of Echinacoside in the subacuteMPTPmousemodelof Parkinsonrsquos diseaserdquo Brain Research vol 1346 pp 224ndash2362010

[15] M Zhu C Lu and W Li ldquoTransient exposure to echinacosideis sufficient to activate Trk signaling and protect neuronal cellsfrom rotenonerdquo Journal of Neurochemistry vol 124 no 4 pp571ndash580 2013

[16] R Gordon V Anantharam and A G Kanthasamy ldquoProte-olytic activation of proapoptotic kinase protein kinase Cdeltaby tumor necrosis factor alpha death receptor signaling indopaminergic neurons during neuroinflammationrdquo Journal ofNeuroinflammation vol 9 article 82 2012

[17] A Drinkut Y Tereshchenko J B Schulz M Bahr and SKugler ldquoEfficient gene therapy for Parkinsonrsquos disease usingastrocytes as hosts for localized neurotrophic factor deliveryrdquoMolecularTherapy vol 20 no 3 pp 534ndash543 2012

[18] E Knapska and L Kaczmarek ldquoA gene for neuronal plas-ticity in the mammalian brain Zif268Egr-1NGFI-A Krox-24TIS8ZENKrdquo Progress in Neurobiology vol 74 no 4 pp183ndash211 2004

[19] C Huang J Wu R Liao andW Zhang ldquoSKF83959 an Agonistof Phosphatidylinositol-Linked D1-Like Receptors PromotesERK12 Activation and Cell Migration in Cultured Rat Astro-cytesrdquo PLoS ONE vol 7 no 11 Article ID e49954 2012

[20] D-Y He and D Ron ldquoAutoregulation of glial cell line-derivedneurotrophic factor expression implications for the long-lasting actions of the anti-addiction drug IbogainerdquoThe FASEBjournal official publication of the Federation of AmericanSocieties for Experimental Biology vol 20 no 13 pp 2420ndash24222006

[21] C Barcia ldquoGlial-Mediated Inflammation Underlying Parkin-sonismrdquo Scientifica vol 2013 Article ID 357805 15 pages 2013

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

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of

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Disease Markers

Hindawiwwwhindawicom Volume 2018

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OncologyJournal of

Hindawiwwwhindawicom Volume 2013

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Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

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Hindawiwwwhindawicom Volume 2018

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Parkinsonrsquos Disease

Evidence-Based Complementary andAlternative Medicine

Volume 2018Hindawiwwwhindawicom

Submit your manuscripts atwwwhindawicom

BioMed Research International 5

Saline MPTP+Selegiline

MPTP ECH

MPTP+ECH

(a)

Saline

MPTP

MPTP+ECH

MPTP+Selegilin

eECH

0

500

1000

1500

GFA

P-IR

coun

ts V

TA(b)

Saline

MPTP

MPTP+ECH

MPTP+Selegilin

eECH

0

2000

4000

6000

GFA

P-IR

coun

ts S

Npc

lowastlowastlowast

lowastlowastlowast

Figure 4 Representative microphotographs of dopaminergic neurons stained for GFAP and the quantification of GFAP positive cell in eachgroup The pictures were taken at an original magnification of 10times Scale bars 100120583m Saline means saline control group MPTP meansmodel group MPTP+ECHmeans MPTP+ECH group MPTP+Selegiline means positive drug control group and ECHmeans ECH group lowastlowastlowastplt0001 compared with saline plt0001 compared with MPTP

GFAP can be used to detect the number of active astro-cytes According to the results of immunohistochemistry theastrocytes in model group mice were activated in a patternof hypertrophy and the number of active astrocytes wassignificantly increased in both SNpc area and VTA areacompared with saline control group (P lt 0001 P lt 0001)After the ECH treatment the morphology of astrocytes wererestored to the saline control group and the number ofGFAP positive cells in SNpc and VTA area was significantlydecreased (P lt 0001 P lt 0001) (Figure 4)

34 Effects of ECH on Serum Inflammatory Factors in Suba-cute PD Model Mice Enzyme-linked immunosorbent assayshowed that the concentrations of inflammatory factorsincluding TNF-120572 and IFN-120574 in the serum of model groupmice were significantly higher than that of saline controlgroup (P lt 005 P lt 001) After the treatment of ECH theconcentrations of the two factors were significantly reduced(P lt 001 P lt 005) (Figure 5)

35 Effects of ECH on the Expression of Midbrain ProteinsIncluding IBA-1 Cleaved Caspase-3 GDNF TNF-120572 p-ERKand ERK in Subacute PD Mice Western blotting analy-sis showed that MPTP induced a marked IBA-1 CleavedCaspase-3 and TNF-120572protein increase (Plt001 Plt0001 and

Plt0001) while the expression of GDNF and ERK phospho-rylation level was significantly decreased (Plt005 Plt005)in midbrain tissue compared to saline control group Afterthe ECH administration the expressions of IBA-1 CleavedCaspase-3 and TNF-120572were significantly decreased (P lt 001Plt 0001 and Plt 001) and the expressions of GDNF and thephosphorylation level of ERK were significantly increased (Plt 0001 P lt 001) (Figure 6)

4 Discussion

Parkinsonrsquos disease is a common central nervous systemdegeneration disease in the elderly Its main clinical fea-tures are not only bradykinesia static tremor myotoniaand abnormal posture but also olfactory disorder con-stipation insomnia fatigue depression and anxiety andother nonmotor symptoms [10 11] The pathogenesis of PDis complex which may be caused by aging genetic andenvironmental factors The pathological change of SNpc DAneuron degeneration lost and appearance of Lewy body maybe connected with cell autophagy proteasome dysfunctionmitochondrial function defect oxidative stress and immuneresponse inflammation excitatory neurotoxicity and othercomplex mechanisms [10 12] Currently dopaminergic drugtherapy is the major treatment of PD Neither supplementing

6 BioMed Research International

Saline

MPTP

MPTP+ECH

MPTP+Selegilin

eECH

0

50

100

150

200

250

TNF-

o

f ser

um (p

gm

l)lowast

(a)

Saline

MPTP

MPTP+ECH

MPTP+Selegilin

eECH

0

50

100

150

200

IFN

- o

f ser

um (p

gm

l)

lowastlowast

(b)

Figure 5 Alteration of TNF-120572 and IFN-120574 in serum of mice Saline means saline control group MPTP means model group MPTP+ECHmeans MPTP+ECH group MPTP+Selegiline means positive drug control group and ECH means ECH group lowastplt005 and lowastlowastplt001compared with saline plt005 and plt001 compared with MPTP

the DA neurotransmitter nor activating DA receptors canreverse the loss of DA neurons and the appearance of Lewybody That means the current PD treatment plan is mainlyfor the symptoms without affecting the progress of the disease[13]Therefore there has been increasing interests in studyingagents like ECH that affect the mechanistic processes ofPD as novel treatment This study specifically found that(1) ECH could improve the PD-like behavior of subacutemodel mice induced by MPTP and the loss of DA neuronsin the substantia nigra of model mice was saved which issimilar to the reports of Qing Zhao [14] (2) ECH couldalso reverse against the astrocyte overactivation microgliaactivation serum and mesencephalic inflammatory factorsin midbrain induced by MPTP in PD subacute model mice(3) ECH was able to increase the expression of GDNF andameliorate the overexpression of MPTP-induced IBA-1 andCleaved Caspase-3 and the decrease of phosphorylated ERK

MPTP is the most commonly used environmental toxinfor the preparation of PD animal model which is highlyfat-soluble and extremely easy to pass through the bloodbrain barrier MPTP is metabolized by monoamine oxidasein astrocytes and turned into ionized 1-methyl-4-phenylpyridine (MPP+) transferred by the Dopamine transporter(DAT) to the DA neurons MPTP produces obvious patho-logical and biochemical damage of DA neurons in C57BL6Jmice so it has a great advantage over other PD models interms of efficacy evaluation We prepared subacute PDmod-els in C57BL6J mice by MPTP (subcutaneous injection) forfive consecutive days and observe thatMPTP can successfullyinduce animal PD behavior cause the loss of DA neurons byimmune histochemical and behavior testing

Echinacoside is the key components in the identificationand assaying of herba cistanche in Chinese pharmacopoeiaIn recent years it has been found that ECH can cross theblood brain barrier [15] and has a good protective effect onthe central nervous system According to Zhaorsquos research[14] we chose the optimal therapeutic does of 30mgkg

ECH as an intervention the behavior of MPTP-inducedanimal models improved markedly the DA neurons deathsdropped significantly the expression of Cleaved Caspase-3decreased obviously and all the above indicated ECH couldprotect DA neurons of PD model mice by antiapoptoticeffect This is similar to Zhaorsquos report [14] The differencebetween two experiments lies in Zhao detected apoptosisrelated protein directly but Zhao has not studied howMPTPcauses apoptosis of DA neurons and how ECH works againstapoptosis Therefore this study explored the mechanism ofMPTP-induced apoptosis of DA neurons and the role of ECHin neurorescue based on glial cells

Microglia is an immune cell in the brain when activatedit produces and releases proinflammatory cytokines thatcause damage to DA neurons Inflammatory factors such asIFN-120574 IL-1120573 TNF-120572 IL-2 and IL-6 [1] are founded in the SNstriatum and cerebrospinal fluids of autopsy in patients withPDmeanwhile the expressions of TNF-120572 receptors R1 (p55)bcl-2 soluble Fas caspase-1 and caspase-3 are increased [16]Therefore proinflammatory cytokines activated and releasedby microglia-mediated factor may be the one reason of deathof DA neurons Astrocytes secrete a variety of neurotrophicfactors of which GDNF is particularly important becauseit can specifically promote the repair of DA neurons ininjured midbrain and it is considered as the most promisingnutrient factor in PD treatment According to reports inthe literature [17] significant improvement can be observedafter astrocytes which are carrying the high expression ofGDNF gene were implanted into the SN region of animalmodels of PD The treatment of GDNF depends on theERK signaling pathway which promotes cell survival [18] Ithas been reported that application of phosphatidylinositol-related D1-like receptor agonists in rat astrocytes can enhancethe expression of phosphorylated ERK and promote themigration of astrocytes [19] GDNF can activate ERK andinduce the upregulation of its own expression in SH-SY5Ycells and rat VTA region [20 21] At the same time ERK

BioMed Research International 7

IBA-1

Cleaved-Cas3

GDNF

TNF-

-tubulin

p-ERK

ERK

Saline

MPTPMPTP+ECH

MPTP+Selegilin

e

ECH

(a)

Saline

MPTP

MPTP+ECH

MPTP+Selegilin

eECH

0

1

2

3

4

IBA-1

IBA-

1

-tubu

lin

lowastlowast

(b)

Saline

MPTP

MPTP+ECH

MPTP+Selegilin

eECH

0

1

2

3

4

Cleaved-Caspase-3

Cle

aved

-Cas

3

-tubu

lin lowastlowastlowast

(c)

Saline

MPTP

MPTP+ECH

MPTP+Selegilin

eECH

00

05

10

15

GDNF

GD

NF

-tu

bulin

lowast

(d)

Saline

MPTP

MPTP+ECH

MPTP+Selegilin

eECH

00

05

10

15

20

TNF-

TNF-

-tubu

lin

lowastlowastlowast

(e)

Saline

MPTP

MPTP+ECH

MPTP+Selegilin

eECH

00

05

10

15

20

p-Erk Erk

p-Er

k E

rk

lowast

(f)

Figure 6 Protein expression of IBA-1 Cleaved-Caspase-3GDNF TNF-120572 p-ERK and ERK Salinemeans saline control groupMPTPmeansmodel group MPTP+ECH means MPTP+ECH group MPTP+Selegiline means positive drug control group and ECH means ECH grouplowastplt005 lowastlowastplt001 lowastlowastlowastplt0001 compared with saline plt005 plt001 and plt0001 compared with MPTP

signaling pathway also plays a key role in the inflammatorypathological changes of PD and their coeffects may affect thepathogenesis of PD and the progression of the disease In theresting state ERKMAPK are located in the cytoplasm of thecells When activated ERK is rapidly phosphorylated Thenactivated ERK enters the nucleus to regulate the activities ofother transcription factors which make the change of genetranscription impact the ascension or decline of the expres-sion of respective protein gene and finally cause the changesin cellrsquos energy metabolism physiological and biochemicalfunction [22] This study found that ECH can upregulate thephosphorylation of ERK increase the expression of GDNFand significantly downregulate the activation of microglia-IBA-1 Therefore we believe that ECH exerts neurotrophiceffects by upregulating the phosphorylation level of ERKincreasing the expression of GDNF and at the same timeinhibiting the activation of microglia and astrocytes and

further reducing the content of inflammatory factor in serumand midbrain which restrains DA neuronal apoptosis

The antiapoptotic effect of ECH has been confirmedTo our knowledge it is the first time to explore the signalpathways of anti-inflammatory and antiapoptosis and anti-inflammatory effects of ECH The expression of GDNFmight be related to the regulation of ERK signaling pathwayThe results of this experiment enriched and extended theprotective mechanisms of ECH in PD providing usefultargets for PD therapeutics development and also the relatedbasic research In the past ten years there have been alarge number of experimental studies on traditional Chinesemedicine compound andmonomer that have been confirmedwith the protective effects on DA neurons in PD On thisbasis exploring and confirming the targets of these drugs andtheir neuroprotective mechanisms can provide a theoreticalbasis for the clinical treatment of PD and drug screening

8 BioMed Research International

Data Availability

The data used to support the findings of this study areavailable from the corresponding author upon request

Conflicts of Interest

The authors declare no conflicts of interest

Authorsrsquo Contributions

Yan Liang Chang Chen Baomei Xia Juanjuan Tang andYang Zhao conceived the idea for the study Chang Chen YanLiang Ye Yang andWei Wuwrote the main manuscript textQing Chen Baomei Xia Yan Liang Lili Tang and Hui Yangprepared Figures 1ndash4 Qing Chen Chang Chen Baomei XiaZhennian Zhang and Yan Lu prepared Figures 5 and 6 Allauthors reviewed the manuscript Yan Liang Chang Chenand Baomei Xia contributed equally to this work

Acknowledgments

The study was supported by the National natural sciencefoundation training project of Construction fund for thekey subject of ldquoChinese medicine brain diseaserdquo (201705201707 and 201708) the Nanjing medical science and tech-nology development project (YKK17149) and the Trans-lational medicine project of Nanjing TCM translationalmedicine base (ZHCX201806)The study was also supportedby the Natural Science Foundation of Jiangsu Province(BK20170769 BK20161044) Natural Science Fund for Col-leges and Universities of Jiangsu Province (17KJD360001)and the National Natural Science Foundation of China(81703483 81603089)

References

[1] A L de Lella Ezcurra M Chertoff C Ferrari M Gracia-rena and F Pitossi ldquoChronic expression of low levels oftumor necrosis factor-alpha in the substantia nigra elicitsprogressive neurodegeneration delayed motor symptoms andmicrogliamacrophage activationrdquoNeurobiology of Disease vol37 no 3 pp 630ndash640 2010

[2] H D E Booth W D Hirst and R Wade-Martins ldquoThe Roleof Astrocyte Dysfunction in Parkinsonrsquos Disease PathogenesisrdquoTrends in Neurosciences vol 40 no 6 pp 358ndash370 2017

[3] Q Wang Y Liu and J Zhou ldquoNeuroinflammation in Parkin-sonrsquos disease and its potential as therapeutic targetrdquo in Transla-tional Neurodegeneration vol 4 article no 19 2015

[4] L Lei F Q Yang T Y Zhang P Tu L Wu and Y ItoldquoPreparative isolation and purification of acteoside and 21015840-acetyl acteoside from Cistanches salsa (CA Mey) G Beckby high-speed counter-current chromatographyrdquo Journal ofChromatography A vol 912 no 1 pp 181ndash185 2001

[5] W Chen H-R Lin C-M Wei et al ldquoEchinacoside aphenylethanoid glycoside from Cistanche deserticola extendslifespan of Caenorhabditis elegans and protects from A120573-induced toxicityrdquo Biogerontology vol 19 no 1 pp 47ndash65 2017

[6] Y-J Shiao M-H Su H-C Lin and C-R Wu ldquoEchinacosideameliorates the memory impairment and cholinergic deficit

induced by amyloid beta peptides via the inhibition of amyloiddeposition and toxicologyrdquo Food amp Function vol 8 no 6 pp2283ndash2294 2017

[7] Y Zhang H Long F Zhou et al ldquoEchinacosidersquos nigrostriataldopaminergic protection against 6-OHDA-Induced endoplas-mic reticulum stress through reducing the accumulation ofSeipinrdquo Journal of Cellular and Molecular Medicine vol 21 no12 pp 3761ndash3775 2017

[8] C Chen B Xia L Tang et al ldquoEchinacoside protects againstMPTPMPP+-induced neurotoxicity via regulating autophagypathway mediated by Sirt1rdquoMetabolic Brain Disease 2018

[9] G Paxinos and B J F Keith The mouse brain in stereotaxiccoordinates Gulf professional publishing Houston USA 2004

[10] L V Kalia and A E Lang ldquoParkinsonrsquos diseaserdquoThe Lancet vol386 no 9996 pp 896ndash912 2015

[11] R B Postuma D Berg C H Adler et al ldquoThe new definitionand diagnostic criteria of Parkinsonrsquos diseaserdquo The LancetNeurology vol 15 no 6 pp 546ndash548 2016

[12] P Calabresi and M Di Filippo ldquoMultitarget disease-modifyingtherapy in Parkinsonrsquos diseaserdquo The Lancet Neurology vol 14no 10 article no 159 pp 975-976 2015

[13] E Valera and E Masliah ldquoTherapeutic approaches in Parkin-sonrsquos disease and related disordersrdquo Journal of Neurochemistryvol 139 Supplement 1 pp 346ndash352 2016

[14] Q Zhao J Gao W Li andD Cai ldquoNeurotrophic and neurores-cue effects of Echinacoside in the subacuteMPTPmousemodelof Parkinsonrsquos diseaserdquo Brain Research vol 1346 pp 224ndash2362010

[15] M Zhu C Lu and W Li ldquoTransient exposure to echinacosideis sufficient to activate Trk signaling and protect neuronal cellsfrom rotenonerdquo Journal of Neurochemistry vol 124 no 4 pp571ndash580 2013

[16] R Gordon V Anantharam and A G Kanthasamy ldquoProte-olytic activation of proapoptotic kinase protein kinase Cdeltaby tumor necrosis factor alpha death receptor signaling indopaminergic neurons during neuroinflammationrdquo Journal ofNeuroinflammation vol 9 article 82 2012

[17] A Drinkut Y Tereshchenko J B Schulz M Bahr and SKugler ldquoEfficient gene therapy for Parkinsonrsquos disease usingastrocytes as hosts for localized neurotrophic factor deliveryrdquoMolecularTherapy vol 20 no 3 pp 534ndash543 2012

[18] E Knapska and L Kaczmarek ldquoA gene for neuronal plas-ticity in the mammalian brain Zif268Egr-1NGFI-A Krox-24TIS8ZENKrdquo Progress in Neurobiology vol 74 no 4 pp183ndash211 2004

[19] C Huang J Wu R Liao andW Zhang ldquoSKF83959 an Agonistof Phosphatidylinositol-Linked D1-Like Receptors PromotesERK12 Activation and Cell Migration in Cultured Rat Astro-cytesrdquo PLoS ONE vol 7 no 11 Article ID e49954 2012

[20] D-Y He and D Ron ldquoAutoregulation of glial cell line-derivedneurotrophic factor expression implications for the long-lasting actions of the anti-addiction drug IbogainerdquoThe FASEBjournal official publication of the Federation of AmericanSocieties for Experimental Biology vol 20 no 13 pp 2420ndash24222006

[21] C Barcia ldquoGlial-Mediated Inflammation Underlying Parkin-sonismrdquo Scientifica vol 2013 Article ID 357805 15 pages 2013

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

Stem Cells International

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

MEDIATORSINFLAMMATION

of

EndocrinologyInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Disease Markers

Hindawiwwwhindawicom Volume 2018

BioMed Research International

OncologyJournal of

Hindawiwwwhindawicom Volume 2013

Hindawiwwwhindawicom Volume 2018

Oxidative Medicine and Cellular Longevity

Hindawiwwwhindawicom Volume 2018

PPAR Research

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Immunology ResearchHindawiwwwhindawicom Volume 2018

Journal of

ObesityJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Computational and Mathematical Methods in Medicine

Hindawiwwwhindawicom Volume 2018

Behavioural Neurology

OphthalmologyJournal of

Hindawiwwwhindawicom Volume 2018

Diabetes ResearchJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Research and TreatmentAIDS

Hindawiwwwhindawicom Volume 2018

Gastroenterology Research and Practice

Hindawiwwwhindawicom Volume 2018

Parkinsonrsquos Disease

Evidence-Based Complementary andAlternative Medicine

Volume 2018Hindawiwwwhindawicom

Submit your manuscripts atwwwhindawicom

6 BioMed Research International

Saline

MPTP

MPTP+ECH

MPTP+Selegilin

eECH

0

50

100

150

200

250

TNF-

o

f ser

um (p

gm

l)lowast

(a)

Saline

MPTP

MPTP+ECH

MPTP+Selegilin

eECH

0

50

100

150

200

IFN

- o

f ser

um (p

gm

l)

lowastlowast

(b)

Figure 5 Alteration of TNF-120572 and IFN-120574 in serum of mice Saline means saline control group MPTP means model group MPTP+ECHmeans MPTP+ECH group MPTP+Selegiline means positive drug control group and ECH means ECH group lowastplt005 and lowastlowastplt001compared with saline plt005 and plt001 compared with MPTP

the DA neurotransmitter nor activating DA receptors canreverse the loss of DA neurons and the appearance of Lewybody That means the current PD treatment plan is mainlyfor the symptoms without affecting the progress of the disease[13]Therefore there has been increasing interests in studyingagents like ECH that affect the mechanistic processes ofPD as novel treatment This study specifically found that(1) ECH could improve the PD-like behavior of subacutemodel mice induced by MPTP and the loss of DA neuronsin the substantia nigra of model mice was saved which issimilar to the reports of Qing Zhao [14] (2) ECH couldalso reverse against the astrocyte overactivation microgliaactivation serum and mesencephalic inflammatory factorsin midbrain induced by MPTP in PD subacute model mice(3) ECH was able to increase the expression of GDNF andameliorate the overexpression of MPTP-induced IBA-1 andCleaved Caspase-3 and the decrease of phosphorylated ERK

MPTP is the most commonly used environmental toxinfor the preparation of PD animal model which is highlyfat-soluble and extremely easy to pass through the bloodbrain barrier MPTP is metabolized by monoamine oxidasein astrocytes and turned into ionized 1-methyl-4-phenylpyridine (MPP+) transferred by the Dopamine transporter(DAT) to the DA neurons MPTP produces obvious patho-logical and biochemical damage of DA neurons in C57BL6Jmice so it has a great advantage over other PD models interms of efficacy evaluation We prepared subacute PDmod-els in C57BL6J mice by MPTP (subcutaneous injection) forfive consecutive days and observe thatMPTP can successfullyinduce animal PD behavior cause the loss of DA neurons byimmune histochemical and behavior testing

Echinacoside is the key components in the identificationand assaying of herba cistanche in Chinese pharmacopoeiaIn recent years it has been found that ECH can cross theblood brain barrier [15] and has a good protective effect onthe central nervous system According to Zhaorsquos research[14] we chose the optimal therapeutic does of 30mgkg

ECH as an intervention the behavior of MPTP-inducedanimal models improved markedly the DA neurons deathsdropped significantly the expression of Cleaved Caspase-3decreased obviously and all the above indicated ECH couldprotect DA neurons of PD model mice by antiapoptoticeffect This is similar to Zhaorsquos report [14] The differencebetween two experiments lies in Zhao detected apoptosisrelated protein directly but Zhao has not studied howMPTPcauses apoptosis of DA neurons and how ECH works againstapoptosis Therefore this study explored the mechanism ofMPTP-induced apoptosis of DA neurons and the role of ECHin neurorescue based on glial cells

Microglia is an immune cell in the brain when activatedit produces and releases proinflammatory cytokines thatcause damage to DA neurons Inflammatory factors such asIFN-120574 IL-1120573 TNF-120572 IL-2 and IL-6 [1] are founded in the SNstriatum and cerebrospinal fluids of autopsy in patients withPDmeanwhile the expressions of TNF-120572 receptors R1 (p55)bcl-2 soluble Fas caspase-1 and caspase-3 are increased [16]Therefore proinflammatory cytokines activated and releasedby microglia-mediated factor may be the one reason of deathof DA neurons Astrocytes secrete a variety of neurotrophicfactors of which GDNF is particularly important becauseit can specifically promote the repair of DA neurons ininjured midbrain and it is considered as the most promisingnutrient factor in PD treatment According to reports inthe literature [17] significant improvement can be observedafter astrocytes which are carrying the high expression ofGDNF gene were implanted into the SN region of animalmodels of PD The treatment of GDNF depends on theERK signaling pathway which promotes cell survival [18] Ithas been reported that application of phosphatidylinositol-related D1-like receptor agonists in rat astrocytes can enhancethe expression of phosphorylated ERK and promote themigration of astrocytes [19] GDNF can activate ERK andinduce the upregulation of its own expression in SH-SY5Ycells and rat VTA region [20 21] At the same time ERK

BioMed Research International 7

IBA-1

Cleaved-Cas3

GDNF

TNF-

-tubulin

p-ERK

ERK

Saline

MPTPMPTP+ECH

MPTP+Selegilin

e

ECH

(a)

Saline

MPTP

MPTP+ECH

MPTP+Selegilin

eECH

0

1

2

3

4

IBA-1

IBA-

1

-tubu

lin

lowastlowast

(b)

Saline

MPTP

MPTP+ECH

MPTP+Selegilin

eECH

0

1

2

3

4

Cleaved-Caspase-3

Cle

aved

-Cas

3

-tubu

lin lowastlowastlowast

(c)

Saline

MPTP

MPTP+ECH

MPTP+Selegilin

eECH

00

05

10

15

GDNF

GD

NF

-tu

bulin

lowast

(d)

Saline

MPTP

MPTP+ECH

MPTP+Selegilin

eECH

00

05

10

15

20

TNF-

TNF-

-tubu

lin

lowastlowastlowast

(e)

Saline

MPTP

MPTP+ECH

MPTP+Selegilin

eECH

00

05

10

15

20

p-Erk Erk

p-Er

k E

rk

lowast

(f)

Figure 6 Protein expression of IBA-1 Cleaved-Caspase-3GDNF TNF-120572 p-ERK and ERK Salinemeans saline control groupMPTPmeansmodel group MPTP+ECH means MPTP+ECH group MPTP+Selegiline means positive drug control group and ECH means ECH grouplowastplt005 lowastlowastplt001 lowastlowastlowastplt0001 compared with saline plt005 plt001 and plt0001 compared with MPTP

signaling pathway also plays a key role in the inflammatorypathological changes of PD and their coeffects may affect thepathogenesis of PD and the progression of the disease In theresting state ERKMAPK are located in the cytoplasm of thecells When activated ERK is rapidly phosphorylated Thenactivated ERK enters the nucleus to regulate the activities ofother transcription factors which make the change of genetranscription impact the ascension or decline of the expres-sion of respective protein gene and finally cause the changesin cellrsquos energy metabolism physiological and biochemicalfunction [22] This study found that ECH can upregulate thephosphorylation of ERK increase the expression of GDNFand significantly downregulate the activation of microglia-IBA-1 Therefore we believe that ECH exerts neurotrophiceffects by upregulating the phosphorylation level of ERKincreasing the expression of GDNF and at the same timeinhibiting the activation of microglia and astrocytes and

further reducing the content of inflammatory factor in serumand midbrain which restrains DA neuronal apoptosis

The antiapoptotic effect of ECH has been confirmedTo our knowledge it is the first time to explore the signalpathways of anti-inflammatory and antiapoptosis and anti-inflammatory effects of ECH The expression of GDNFmight be related to the regulation of ERK signaling pathwayThe results of this experiment enriched and extended theprotective mechanisms of ECH in PD providing usefultargets for PD therapeutics development and also the relatedbasic research In the past ten years there have been alarge number of experimental studies on traditional Chinesemedicine compound andmonomer that have been confirmedwith the protective effects on DA neurons in PD On thisbasis exploring and confirming the targets of these drugs andtheir neuroprotective mechanisms can provide a theoreticalbasis for the clinical treatment of PD and drug screening

8 BioMed Research International

Data Availability

The data used to support the findings of this study areavailable from the corresponding author upon request

Conflicts of Interest

The authors declare no conflicts of interest

Authorsrsquo Contributions

Yan Liang Chang Chen Baomei Xia Juanjuan Tang andYang Zhao conceived the idea for the study Chang Chen YanLiang Ye Yang andWei Wuwrote the main manuscript textQing Chen Baomei Xia Yan Liang Lili Tang and Hui Yangprepared Figures 1ndash4 Qing Chen Chang Chen Baomei XiaZhennian Zhang and Yan Lu prepared Figures 5 and 6 Allauthors reviewed the manuscript Yan Liang Chang Chenand Baomei Xia contributed equally to this work

Acknowledgments

The study was supported by the National natural sciencefoundation training project of Construction fund for thekey subject of ldquoChinese medicine brain diseaserdquo (201705201707 and 201708) the Nanjing medical science and tech-nology development project (YKK17149) and the Trans-lational medicine project of Nanjing TCM translationalmedicine base (ZHCX201806)The study was also supportedby the Natural Science Foundation of Jiangsu Province(BK20170769 BK20161044) Natural Science Fund for Col-leges and Universities of Jiangsu Province (17KJD360001)and the National Natural Science Foundation of China(81703483 81603089)

References

[1] A L de Lella Ezcurra M Chertoff C Ferrari M Gracia-rena and F Pitossi ldquoChronic expression of low levels oftumor necrosis factor-alpha in the substantia nigra elicitsprogressive neurodegeneration delayed motor symptoms andmicrogliamacrophage activationrdquoNeurobiology of Disease vol37 no 3 pp 630ndash640 2010

[2] H D E Booth W D Hirst and R Wade-Martins ldquoThe Roleof Astrocyte Dysfunction in Parkinsonrsquos Disease PathogenesisrdquoTrends in Neurosciences vol 40 no 6 pp 358ndash370 2017

[3] Q Wang Y Liu and J Zhou ldquoNeuroinflammation in Parkin-sonrsquos disease and its potential as therapeutic targetrdquo in Transla-tional Neurodegeneration vol 4 article no 19 2015

[4] L Lei F Q Yang T Y Zhang P Tu L Wu and Y ItoldquoPreparative isolation and purification of acteoside and 21015840-acetyl acteoside from Cistanches salsa (CA Mey) G Beckby high-speed counter-current chromatographyrdquo Journal ofChromatography A vol 912 no 1 pp 181ndash185 2001

[5] W Chen H-R Lin C-M Wei et al ldquoEchinacoside aphenylethanoid glycoside from Cistanche deserticola extendslifespan of Caenorhabditis elegans and protects from A120573-induced toxicityrdquo Biogerontology vol 19 no 1 pp 47ndash65 2017

[6] Y-J Shiao M-H Su H-C Lin and C-R Wu ldquoEchinacosideameliorates the memory impairment and cholinergic deficit

induced by amyloid beta peptides via the inhibition of amyloiddeposition and toxicologyrdquo Food amp Function vol 8 no 6 pp2283ndash2294 2017

[7] Y Zhang H Long F Zhou et al ldquoEchinacosidersquos nigrostriataldopaminergic protection against 6-OHDA-Induced endoplas-mic reticulum stress through reducing the accumulation ofSeipinrdquo Journal of Cellular and Molecular Medicine vol 21 no12 pp 3761ndash3775 2017

[8] C Chen B Xia L Tang et al ldquoEchinacoside protects againstMPTPMPP+-induced neurotoxicity via regulating autophagypathway mediated by Sirt1rdquoMetabolic Brain Disease 2018

[9] G Paxinos and B J F Keith The mouse brain in stereotaxiccoordinates Gulf professional publishing Houston USA 2004

[10] L V Kalia and A E Lang ldquoParkinsonrsquos diseaserdquoThe Lancet vol386 no 9996 pp 896ndash912 2015

[11] R B Postuma D Berg C H Adler et al ldquoThe new definitionand diagnostic criteria of Parkinsonrsquos diseaserdquo The LancetNeurology vol 15 no 6 pp 546ndash548 2016

[12] P Calabresi and M Di Filippo ldquoMultitarget disease-modifyingtherapy in Parkinsonrsquos diseaserdquo The Lancet Neurology vol 14no 10 article no 159 pp 975-976 2015

[13] E Valera and E Masliah ldquoTherapeutic approaches in Parkin-sonrsquos disease and related disordersrdquo Journal of Neurochemistryvol 139 Supplement 1 pp 346ndash352 2016

[14] Q Zhao J Gao W Li andD Cai ldquoNeurotrophic and neurores-cue effects of Echinacoside in the subacuteMPTPmousemodelof Parkinsonrsquos diseaserdquo Brain Research vol 1346 pp 224ndash2362010

[15] M Zhu C Lu and W Li ldquoTransient exposure to echinacosideis sufficient to activate Trk signaling and protect neuronal cellsfrom rotenonerdquo Journal of Neurochemistry vol 124 no 4 pp571ndash580 2013

[16] R Gordon V Anantharam and A G Kanthasamy ldquoProte-olytic activation of proapoptotic kinase protein kinase Cdeltaby tumor necrosis factor alpha death receptor signaling indopaminergic neurons during neuroinflammationrdquo Journal ofNeuroinflammation vol 9 article 82 2012

[17] A Drinkut Y Tereshchenko J B Schulz M Bahr and SKugler ldquoEfficient gene therapy for Parkinsonrsquos disease usingastrocytes as hosts for localized neurotrophic factor deliveryrdquoMolecularTherapy vol 20 no 3 pp 534ndash543 2012

[18] E Knapska and L Kaczmarek ldquoA gene for neuronal plas-ticity in the mammalian brain Zif268Egr-1NGFI-A Krox-24TIS8ZENKrdquo Progress in Neurobiology vol 74 no 4 pp183ndash211 2004

[19] C Huang J Wu R Liao andW Zhang ldquoSKF83959 an Agonistof Phosphatidylinositol-Linked D1-Like Receptors PromotesERK12 Activation and Cell Migration in Cultured Rat Astro-cytesrdquo PLoS ONE vol 7 no 11 Article ID e49954 2012

[20] D-Y He and D Ron ldquoAutoregulation of glial cell line-derivedneurotrophic factor expression implications for the long-lasting actions of the anti-addiction drug IbogainerdquoThe FASEBjournal official publication of the Federation of AmericanSocieties for Experimental Biology vol 20 no 13 pp 2420ndash24222006

[21] C Barcia ldquoGlial-Mediated Inflammation Underlying Parkin-sonismrdquo Scientifica vol 2013 Article ID 357805 15 pages 2013

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

Stem Cells International

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

MEDIATORSINFLAMMATION

of

EndocrinologyInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Disease Markers

Hindawiwwwhindawicom Volume 2018

BioMed Research International

OncologyJournal of

Hindawiwwwhindawicom Volume 2013

Hindawiwwwhindawicom Volume 2018

Oxidative Medicine and Cellular Longevity

Hindawiwwwhindawicom Volume 2018

PPAR Research

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Immunology ResearchHindawiwwwhindawicom Volume 2018

Journal of

ObesityJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Computational and Mathematical Methods in Medicine

Hindawiwwwhindawicom Volume 2018

Behavioural Neurology

OphthalmologyJournal of

Hindawiwwwhindawicom Volume 2018

Diabetes ResearchJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Research and TreatmentAIDS

Hindawiwwwhindawicom Volume 2018

Gastroenterology Research and Practice

Hindawiwwwhindawicom Volume 2018

Parkinsonrsquos Disease

Evidence-Based Complementary andAlternative Medicine

Volume 2018Hindawiwwwhindawicom

Submit your manuscripts atwwwhindawicom

BioMed Research International 7

IBA-1

Cleaved-Cas3

GDNF

TNF-

-tubulin

p-ERK

ERK

Saline

MPTPMPTP+ECH

MPTP+Selegilin

e

ECH

(a)

Saline

MPTP

MPTP+ECH

MPTP+Selegilin

eECH

0

1

2

3

4

IBA-1

IBA-

1

-tubu

lin

lowastlowast

(b)

Saline

MPTP

MPTP+ECH

MPTP+Selegilin

eECH

0

1

2

3

4

Cleaved-Caspase-3

Cle

aved

-Cas

3

-tubu

lin lowastlowastlowast

(c)

Saline

MPTP

MPTP+ECH

MPTP+Selegilin

eECH

00

05

10

15

GDNF

GD

NF

-tu

bulin

lowast

(d)

Saline

MPTP

MPTP+ECH

MPTP+Selegilin

eECH

00

05

10

15

20

TNF-

TNF-

-tubu

lin

lowastlowastlowast

(e)

Saline

MPTP

MPTP+ECH

MPTP+Selegilin

eECH

00

05

10

15

20

p-Erk Erk

p-Er

k E

rk

lowast

(f)

Figure 6 Protein expression of IBA-1 Cleaved-Caspase-3GDNF TNF-120572 p-ERK and ERK Salinemeans saline control groupMPTPmeansmodel group MPTP+ECH means MPTP+ECH group MPTP+Selegiline means positive drug control group and ECH means ECH grouplowastplt005 lowastlowastplt001 lowastlowastlowastplt0001 compared with saline plt005 plt001 and plt0001 compared with MPTP

signaling pathway also plays a key role in the inflammatorypathological changes of PD and their coeffects may affect thepathogenesis of PD and the progression of the disease In theresting state ERKMAPK are located in the cytoplasm of thecells When activated ERK is rapidly phosphorylated Thenactivated ERK enters the nucleus to regulate the activities ofother transcription factors which make the change of genetranscription impact the ascension or decline of the expres-sion of respective protein gene and finally cause the changesin cellrsquos energy metabolism physiological and biochemicalfunction [22] This study found that ECH can upregulate thephosphorylation of ERK increase the expression of GDNFand significantly downregulate the activation of microglia-IBA-1 Therefore we believe that ECH exerts neurotrophiceffects by upregulating the phosphorylation level of ERKincreasing the expression of GDNF and at the same timeinhibiting the activation of microglia and astrocytes and

further reducing the content of inflammatory factor in serumand midbrain which restrains DA neuronal apoptosis

The antiapoptotic effect of ECH has been confirmedTo our knowledge it is the first time to explore the signalpathways of anti-inflammatory and antiapoptosis and anti-inflammatory effects of ECH The expression of GDNFmight be related to the regulation of ERK signaling pathwayThe results of this experiment enriched and extended theprotective mechanisms of ECH in PD providing usefultargets for PD therapeutics development and also the relatedbasic research In the past ten years there have been alarge number of experimental studies on traditional Chinesemedicine compound andmonomer that have been confirmedwith the protective effects on DA neurons in PD On thisbasis exploring and confirming the targets of these drugs andtheir neuroprotective mechanisms can provide a theoreticalbasis for the clinical treatment of PD and drug screening

8 BioMed Research International

Data Availability

The data used to support the findings of this study areavailable from the corresponding author upon request

Conflicts of Interest

The authors declare no conflicts of interest

Authorsrsquo Contributions

Yan Liang Chang Chen Baomei Xia Juanjuan Tang andYang Zhao conceived the idea for the study Chang Chen YanLiang Ye Yang andWei Wuwrote the main manuscript textQing Chen Baomei Xia Yan Liang Lili Tang and Hui Yangprepared Figures 1ndash4 Qing Chen Chang Chen Baomei XiaZhennian Zhang and Yan Lu prepared Figures 5 and 6 Allauthors reviewed the manuscript Yan Liang Chang Chenand Baomei Xia contributed equally to this work

Acknowledgments

The study was supported by the National natural sciencefoundation training project of Construction fund for thekey subject of ldquoChinese medicine brain diseaserdquo (201705201707 and 201708) the Nanjing medical science and tech-nology development project (YKK17149) and the Trans-lational medicine project of Nanjing TCM translationalmedicine base (ZHCX201806)The study was also supportedby the Natural Science Foundation of Jiangsu Province(BK20170769 BK20161044) Natural Science Fund for Col-leges and Universities of Jiangsu Province (17KJD360001)and the National Natural Science Foundation of China(81703483 81603089)

References

[1] A L de Lella Ezcurra M Chertoff C Ferrari M Gracia-rena and F Pitossi ldquoChronic expression of low levels oftumor necrosis factor-alpha in the substantia nigra elicitsprogressive neurodegeneration delayed motor symptoms andmicrogliamacrophage activationrdquoNeurobiology of Disease vol37 no 3 pp 630ndash640 2010

[2] H D E Booth W D Hirst and R Wade-Martins ldquoThe Roleof Astrocyte Dysfunction in Parkinsonrsquos Disease PathogenesisrdquoTrends in Neurosciences vol 40 no 6 pp 358ndash370 2017

[3] Q Wang Y Liu and J Zhou ldquoNeuroinflammation in Parkin-sonrsquos disease and its potential as therapeutic targetrdquo in Transla-tional Neurodegeneration vol 4 article no 19 2015

[4] L Lei F Q Yang T Y Zhang P Tu L Wu and Y ItoldquoPreparative isolation and purification of acteoside and 21015840-acetyl acteoside from Cistanches salsa (CA Mey) G Beckby high-speed counter-current chromatographyrdquo Journal ofChromatography A vol 912 no 1 pp 181ndash185 2001

[5] W Chen H-R Lin C-M Wei et al ldquoEchinacoside aphenylethanoid glycoside from Cistanche deserticola extendslifespan of Caenorhabditis elegans and protects from A120573-induced toxicityrdquo Biogerontology vol 19 no 1 pp 47ndash65 2017

[6] Y-J Shiao M-H Su H-C Lin and C-R Wu ldquoEchinacosideameliorates the memory impairment and cholinergic deficit

induced by amyloid beta peptides via the inhibition of amyloiddeposition and toxicologyrdquo Food amp Function vol 8 no 6 pp2283ndash2294 2017

[7] Y Zhang H Long F Zhou et al ldquoEchinacosidersquos nigrostriataldopaminergic protection against 6-OHDA-Induced endoplas-mic reticulum stress through reducing the accumulation ofSeipinrdquo Journal of Cellular and Molecular Medicine vol 21 no12 pp 3761ndash3775 2017

[8] C Chen B Xia L Tang et al ldquoEchinacoside protects againstMPTPMPP+-induced neurotoxicity via regulating autophagypathway mediated by Sirt1rdquoMetabolic Brain Disease 2018

[9] G Paxinos and B J F Keith The mouse brain in stereotaxiccoordinates Gulf professional publishing Houston USA 2004

[10] L V Kalia and A E Lang ldquoParkinsonrsquos diseaserdquoThe Lancet vol386 no 9996 pp 896ndash912 2015

[11] R B Postuma D Berg C H Adler et al ldquoThe new definitionand diagnostic criteria of Parkinsonrsquos diseaserdquo The LancetNeurology vol 15 no 6 pp 546ndash548 2016

[12] P Calabresi and M Di Filippo ldquoMultitarget disease-modifyingtherapy in Parkinsonrsquos diseaserdquo The Lancet Neurology vol 14no 10 article no 159 pp 975-976 2015

[13] E Valera and E Masliah ldquoTherapeutic approaches in Parkin-sonrsquos disease and related disordersrdquo Journal of Neurochemistryvol 139 Supplement 1 pp 346ndash352 2016

[14] Q Zhao J Gao W Li andD Cai ldquoNeurotrophic and neurores-cue effects of Echinacoside in the subacuteMPTPmousemodelof Parkinsonrsquos diseaserdquo Brain Research vol 1346 pp 224ndash2362010

[15] M Zhu C Lu and W Li ldquoTransient exposure to echinacosideis sufficient to activate Trk signaling and protect neuronal cellsfrom rotenonerdquo Journal of Neurochemistry vol 124 no 4 pp571ndash580 2013

[16] R Gordon V Anantharam and A G Kanthasamy ldquoProte-olytic activation of proapoptotic kinase protein kinase Cdeltaby tumor necrosis factor alpha death receptor signaling indopaminergic neurons during neuroinflammationrdquo Journal ofNeuroinflammation vol 9 article 82 2012

[17] A Drinkut Y Tereshchenko J B Schulz M Bahr and SKugler ldquoEfficient gene therapy for Parkinsonrsquos disease usingastrocytes as hosts for localized neurotrophic factor deliveryrdquoMolecularTherapy vol 20 no 3 pp 534ndash543 2012

[18] E Knapska and L Kaczmarek ldquoA gene for neuronal plas-ticity in the mammalian brain Zif268Egr-1NGFI-A Krox-24TIS8ZENKrdquo Progress in Neurobiology vol 74 no 4 pp183ndash211 2004

[19] C Huang J Wu R Liao andW Zhang ldquoSKF83959 an Agonistof Phosphatidylinositol-Linked D1-Like Receptors PromotesERK12 Activation and Cell Migration in Cultured Rat Astro-cytesrdquo PLoS ONE vol 7 no 11 Article ID e49954 2012

[20] D-Y He and D Ron ldquoAutoregulation of glial cell line-derivedneurotrophic factor expression implications for the long-lasting actions of the anti-addiction drug IbogainerdquoThe FASEBjournal official publication of the Federation of AmericanSocieties for Experimental Biology vol 20 no 13 pp 2420ndash24222006

[21] C Barcia ldquoGlial-Mediated Inflammation Underlying Parkin-sonismrdquo Scientifica vol 2013 Article ID 357805 15 pages 2013

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

Stem Cells International

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

MEDIATORSINFLAMMATION

of

EndocrinologyInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Disease Markers

Hindawiwwwhindawicom Volume 2018

BioMed Research International

OncologyJournal of

Hindawiwwwhindawicom Volume 2013

Hindawiwwwhindawicom Volume 2018

Oxidative Medicine and Cellular Longevity

Hindawiwwwhindawicom Volume 2018

PPAR Research

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Immunology ResearchHindawiwwwhindawicom Volume 2018

Journal of

ObesityJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Computational and Mathematical Methods in Medicine

Hindawiwwwhindawicom Volume 2018

Behavioural Neurology

OphthalmologyJournal of

Hindawiwwwhindawicom Volume 2018

Diabetes ResearchJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Research and TreatmentAIDS

Hindawiwwwhindawicom Volume 2018

Gastroenterology Research and Practice

Hindawiwwwhindawicom Volume 2018

Parkinsonrsquos Disease

Evidence-Based Complementary andAlternative Medicine

Volume 2018Hindawiwwwhindawicom

Submit your manuscripts atwwwhindawicom

8 BioMed Research International

Data Availability

The data used to support the findings of this study areavailable from the corresponding author upon request

Conflicts of Interest

The authors declare no conflicts of interest

Authorsrsquo Contributions

Yan Liang Chang Chen Baomei Xia Juanjuan Tang andYang Zhao conceived the idea for the study Chang Chen YanLiang Ye Yang andWei Wuwrote the main manuscript textQing Chen Baomei Xia Yan Liang Lili Tang and Hui Yangprepared Figures 1ndash4 Qing Chen Chang Chen Baomei XiaZhennian Zhang and Yan Lu prepared Figures 5 and 6 Allauthors reviewed the manuscript Yan Liang Chang Chenand Baomei Xia contributed equally to this work

Acknowledgments

The study was supported by the National natural sciencefoundation training project of Construction fund for thekey subject of ldquoChinese medicine brain diseaserdquo (201705201707 and 201708) the Nanjing medical science and tech-nology development project (YKK17149) and the Trans-lational medicine project of Nanjing TCM translationalmedicine base (ZHCX201806)The study was also supportedby the Natural Science Foundation of Jiangsu Province(BK20170769 BK20161044) Natural Science Fund for Col-leges and Universities of Jiangsu Province (17KJD360001)and the National Natural Science Foundation of China(81703483 81603089)

References

[1] A L de Lella Ezcurra M Chertoff C Ferrari M Gracia-rena and F Pitossi ldquoChronic expression of low levels oftumor necrosis factor-alpha in the substantia nigra elicitsprogressive neurodegeneration delayed motor symptoms andmicrogliamacrophage activationrdquoNeurobiology of Disease vol37 no 3 pp 630ndash640 2010

[2] H D E Booth W D Hirst and R Wade-Martins ldquoThe Roleof Astrocyte Dysfunction in Parkinsonrsquos Disease PathogenesisrdquoTrends in Neurosciences vol 40 no 6 pp 358ndash370 2017

[3] Q Wang Y Liu and J Zhou ldquoNeuroinflammation in Parkin-sonrsquos disease and its potential as therapeutic targetrdquo in Transla-tional Neurodegeneration vol 4 article no 19 2015

[4] L Lei F Q Yang T Y Zhang P Tu L Wu and Y ItoldquoPreparative isolation and purification of acteoside and 21015840-acetyl acteoside from Cistanches salsa (CA Mey) G Beckby high-speed counter-current chromatographyrdquo Journal ofChromatography A vol 912 no 1 pp 181ndash185 2001

[5] W Chen H-R Lin C-M Wei et al ldquoEchinacoside aphenylethanoid glycoside from Cistanche deserticola extendslifespan of Caenorhabditis elegans and protects from A120573-induced toxicityrdquo Biogerontology vol 19 no 1 pp 47ndash65 2017

[6] Y-J Shiao M-H Su H-C Lin and C-R Wu ldquoEchinacosideameliorates the memory impairment and cholinergic deficit

induced by amyloid beta peptides via the inhibition of amyloiddeposition and toxicologyrdquo Food amp Function vol 8 no 6 pp2283ndash2294 2017

[7] Y Zhang H Long F Zhou et al ldquoEchinacosidersquos nigrostriataldopaminergic protection against 6-OHDA-Induced endoplas-mic reticulum stress through reducing the accumulation ofSeipinrdquo Journal of Cellular and Molecular Medicine vol 21 no12 pp 3761ndash3775 2017

[8] C Chen B Xia L Tang et al ldquoEchinacoside protects againstMPTPMPP+-induced neurotoxicity via regulating autophagypathway mediated by Sirt1rdquoMetabolic Brain Disease 2018

[9] G Paxinos and B J F Keith The mouse brain in stereotaxiccoordinates Gulf professional publishing Houston USA 2004

[10] L V Kalia and A E Lang ldquoParkinsonrsquos diseaserdquoThe Lancet vol386 no 9996 pp 896ndash912 2015

[11] R B Postuma D Berg C H Adler et al ldquoThe new definitionand diagnostic criteria of Parkinsonrsquos diseaserdquo The LancetNeurology vol 15 no 6 pp 546ndash548 2016

[12] P Calabresi and M Di Filippo ldquoMultitarget disease-modifyingtherapy in Parkinsonrsquos diseaserdquo The Lancet Neurology vol 14no 10 article no 159 pp 975-976 2015

[13] E Valera and E Masliah ldquoTherapeutic approaches in Parkin-sonrsquos disease and related disordersrdquo Journal of Neurochemistryvol 139 Supplement 1 pp 346ndash352 2016

[14] Q Zhao J Gao W Li andD Cai ldquoNeurotrophic and neurores-cue effects of Echinacoside in the subacuteMPTPmousemodelof Parkinsonrsquos diseaserdquo Brain Research vol 1346 pp 224ndash2362010

[15] M Zhu C Lu and W Li ldquoTransient exposure to echinacosideis sufficient to activate Trk signaling and protect neuronal cellsfrom rotenonerdquo Journal of Neurochemistry vol 124 no 4 pp571ndash580 2013

[16] R Gordon V Anantharam and A G Kanthasamy ldquoProte-olytic activation of proapoptotic kinase protein kinase Cdeltaby tumor necrosis factor alpha death receptor signaling indopaminergic neurons during neuroinflammationrdquo Journal ofNeuroinflammation vol 9 article 82 2012

[17] A Drinkut Y Tereshchenko J B Schulz M Bahr and SKugler ldquoEfficient gene therapy for Parkinsonrsquos disease usingastrocytes as hosts for localized neurotrophic factor deliveryrdquoMolecularTherapy vol 20 no 3 pp 534ndash543 2012

[18] E Knapska and L Kaczmarek ldquoA gene for neuronal plas-ticity in the mammalian brain Zif268Egr-1NGFI-A Krox-24TIS8ZENKrdquo Progress in Neurobiology vol 74 no 4 pp183ndash211 2004

[19] C Huang J Wu R Liao andW Zhang ldquoSKF83959 an Agonistof Phosphatidylinositol-Linked D1-Like Receptors PromotesERK12 Activation and Cell Migration in Cultured Rat Astro-cytesrdquo PLoS ONE vol 7 no 11 Article ID e49954 2012

[20] D-Y He and D Ron ldquoAutoregulation of glial cell line-derivedneurotrophic factor expression implications for the long-lasting actions of the anti-addiction drug IbogainerdquoThe FASEBjournal official publication of the Federation of AmericanSocieties for Experimental Biology vol 20 no 13 pp 2420ndash24222006

[21] C Barcia ldquoGlial-Mediated Inflammation Underlying Parkin-sonismrdquo Scientifica vol 2013 Article ID 357805 15 pages 2013

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

Stem Cells International

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

MEDIATORSINFLAMMATION

of

EndocrinologyInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Disease Markers

Hindawiwwwhindawicom Volume 2018

BioMed Research International

OncologyJournal of

Hindawiwwwhindawicom Volume 2013

Hindawiwwwhindawicom Volume 2018

Oxidative Medicine and Cellular Longevity

Hindawiwwwhindawicom Volume 2018

PPAR Research

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Immunology ResearchHindawiwwwhindawicom Volume 2018

Journal of

ObesityJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Computational and Mathematical Methods in Medicine

Hindawiwwwhindawicom Volume 2018

Behavioural Neurology

OphthalmologyJournal of

Hindawiwwwhindawicom Volume 2018

Diabetes ResearchJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Research and TreatmentAIDS

Hindawiwwwhindawicom Volume 2018

Gastroenterology Research and Practice

Hindawiwwwhindawicom Volume 2018

Parkinsonrsquos Disease

Evidence-Based Complementary andAlternative Medicine

Volume 2018Hindawiwwwhindawicom

Submit your manuscripts atwwwhindawicom

Stem Cells International

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

MEDIATORSINFLAMMATION

of

EndocrinologyInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Disease Markers

Hindawiwwwhindawicom Volume 2018

BioMed Research International

OncologyJournal of

Hindawiwwwhindawicom Volume 2013

Hindawiwwwhindawicom Volume 2018

Oxidative Medicine and Cellular Longevity

Hindawiwwwhindawicom Volume 2018

PPAR Research

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Immunology ResearchHindawiwwwhindawicom Volume 2018

Journal of

ObesityJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Computational and Mathematical Methods in Medicine

Hindawiwwwhindawicom Volume 2018

Behavioural Neurology

OphthalmologyJournal of

Hindawiwwwhindawicom Volume 2018

Diabetes ResearchJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Research and TreatmentAIDS

Hindawiwwwhindawicom Volume 2018

Gastroenterology Research and Practice

Hindawiwwwhindawicom Volume 2018

Parkinsonrsquos Disease

Evidence-Based Complementary andAlternative Medicine

Volume 2018Hindawiwwwhindawicom

Submit your manuscripts atwwwhindawicom