Research ArticleProtective Effects of an Ancient ChineseKidney-Tonifying Formula against H2O2-InducedOxidative Damage to MES235 Cells

Yihui Xu1 Wei Lin2 Shuifen Ye3 HuajinWang4 TingtingWang5 Youyan Su5

LiangningWu6 YuanwangWang7 Qian Xu5 Chuanshan Xu8 and Jing Cai5

1Second Peoplersquos Hospital Fujian University of Traditional Chinese Medicine Fuzhou Fujian Province 350003 China2Academy of Integrative Medicine Fujian University of Traditional Chinese Medicine Fuzhou Fujian Province 350122 China3Longyan First Hospital Affiliated to Fujian Medical University Longyan Fujian Province 364000 China4Tibet Autonomous Region Peoplersquos Hospital Lhasa Tibet Autonomous Region 850000 China5College of Integrative Medicine Fujian University of Traditional Chinese Medicine Fuzhou Fujian Province 350122 China6Graduate School Fujian University of Traditional Chinese Medicine Fuzhou Fujian Province 350122 China7Quanzhou Orthopedic-Traumatological Hospital Fujian University of Traditional Chinese MedicineQuanzhou Fujian Province 362000 China8School of Chinese Medicine Faculty of Medicine The Chinese University of Hong Kong Shatin Hong Kong

Correspondence should be addressed to Jing Cai caij1163com

Received 11 August 2016 Accepted 27 December 2016 Published 12 March 2017

Academic Editor Jin-Tai Yu

Copyright copy 2017 Yihui Xu et alThis is an open access article distributed under the Creative Commons Attribution License whichpermits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Oxidative damage plays a critical role in the etiology of neurodegenerative disorders including Parkinsonrsquos disease (PD) Inour study an ancient Chinese kidney-tonifying formula which consists of Cistanche Epimedii and Polygonatum cirrhifoliumwas investigated to protect MES235 dopaminergic neurons against hydrogen peroxide- (H

2O2-) induced oxidative damage The

damage effects of H2O2on MES235 cells and the protective effects of KTF against oxidative stress were evaluated using MTT

assay transmission electronmicroscopy (TEM) immunocytochemistry (ICC) enzyme-linked immunosorbent assay (ELISA) andimmunoblotting The results showed that cell viability was dramatically decreased after a 12 h exposure to 150120583M H

2O2 TEM

observation found that the H2O2-treated MES235 cells presented cellular organelle damage However when cells were incubated

with KTF (3125 625 and 125120583gml) for 24 h after H2O2exposure a significant protective effect against H

2O2-induced damage

was observed in MES235 cells Using ICC we found that KTF inhibited the reduction of the tyrosine hydroxylase (TH) inducedby H2O2 upregulated the mRNA and protein expression of HO-1 CAT and GPx-1 and downregulated the expression of caspase 3

These results indicated that KTFmay provide neuron protection against H2O2-induced cell damage through ameliorating oxidative

stress and our findings provide a new potential strategy for the prevention and treatment of Parkinsonrsquos disease

1 Introduction

Neurodegenerative diseases have recently become an impor-tant public health problem Parkinsonrsquos disease (PD) is a neu-rodegenerative disease characterized by the progressive lossof dopaminergic neurons in the substantia nigra Even tho-ugh the cause of PD remains to be clarified several linesof evidence strongly suggest that oxidative damage andmitochondrial dysfunction play an important role in the

pathological mechanisms of PD [1ndash3] Increased oxidativestress results from imbalance between oxidative productssuch as reactive oxygen species (ROS) and antioxidantmolecules in the cell [4] Enhancing antioxidative capacityof brain cells has become an effective strategy in protectingnervous cells against oxidative stress damage

PD is a difficult-to-treat disease threatening the elderlyworldwide The current therapeutic drugs have limited effi-cacy with severe side-effects Traditional Chinese herbs and

HindawiParkinsonrsquos DiseaseVolume 2017 Article ID 2879495 9 pageshttpsdoiorg10115520172879495

2 Parkinsonrsquos Disease

formula have been widely used in folk medicine to treatneurodegenerative diseases including PD for a long timeAccording to Chinese medicine theory the deficiency ofkidney is the main pathological mechanism of PD Cistanchedeserticola Y C Ma a species of Cistanche which belongs totheOrobanchaceae family is awell-knownherb in traditionalChinese medicine with so-called kidney-tonifying efficacyand is used for the treatment of kidney deficiency femaleinfertility morbid leucorrhea neurasthenia and senile con-stipation [5] It has been reported that Cistanche total glyco-sides exerted protective effects on substantia nigra dopamin-ergic neuron in mice model and cells of PD [6 7] Moreoverthe active ingredients of Cistanche have been documentedto possess neuroprotective effect through enhancing antiox-idant capacity and inhibiting apoptosis of neurons [8ndash15]Herba Epimedii is also a traditional kidney-tonifying Chineseherb with significantly antioxidative activity which is widelyutilized in treating osteoporosis and cardiovascular diseasesand improving sexual and neurological functions [16] Polyg-onatum sibiricum is often employed as an assistant to improvethe effectiveness of the monarch drug and minister drugin the traditional Chinese formula Recent report showedthat the water extracts of Polygonatum sibiricum are naturalantioxidants

Based on these benefits from the ancient Chinesekidney-tonifying formula (KTF) which consists of CistancheEpimedii and Polygonatum cirrhifolium we hypothesizedthat KTF might protect MES235 dopaminergic neuronsagainst hydrogen peroxide- (H

2O2-) induced oxidative dam-

age Herein as proof-of-concept we conducted a series ofexperiments to examine the effects of KTF on cell deathand antioxidant enzymes expression in the H

2O2-treated

MES235 cells

2 Materials and Methods

21 Chemicals and Reagents HO-1 CAT caspase 3 and 120573-actin antibodies were obtained from Cell Signaling Tech-nology Inc (Boston USA) GPx-1 antibody was fromAbcam (Cambridge Science Park UK) Rabbit Anti-TyrosineHydroxylase was from Boster (BA1454 China) Goat anti-rabbit IgG1 conjugated to a horseradish peroxidase label wasfrom Santa Cruz Biotechnology Inc (California USA) Dul-beccorsquos modified Eaglersquos medium (DMEM) was purchasedfrom GIBCO BRL Co Ltd (Gaithersburg MD USA) Allthe other reagents unless otherwise stated were from SigmaChemical Co (St Louis MO USA) RevertAid First StrandcDNA Synthesis Kit was from Fermentas Inc (BurlingtonUSA) DreamTaq Green PCR Master Mix (2x) was fromFermentas Inc (Burlington USA) Bio DL100 was fromBioFlux (Tokyo Japan) Polink-2 plus Polymer HRP Detec-tion System for Rabbit Primary Antibody (PV-9001) andDAB (3 31015840-diaminoobenzidine tetrahydrochloride) (ZLI-9032) were from ZSGB-BIO (Beijing China) ELISA kitsfor measuring CAT GPx-1 and GSH were from HufengBiotechnology Inc (Shanghai China)Ginkgo biloba extracts(GE) were from Shanghai Sine Promod Pharmaceutical CoLtd (Shanghai China)

22 Preparation and Standardization of Kidney-TonifyingFormula The components of kidney-tonifying formula Cis-tanche deserticola Epimedium brevicornum and Polygona-tum sibiricum were supplied by Second Affiliated Peo-plersquos Hospital Fujian University of Traditional ChineseMedicine and carefully authenticated according to the Chi-nese pharmacopoeia (The Pharmacopoeia Commission ofChina 2005) To prepare KTF samples Cistanche deserticolaEpimedium brevicornum and Polygonatum sibiricum at theweight ratio of 1 1 12 were mixed and ground into thepowder and then immersed in a total volume of 10 times (wv)distilled water for 1 h and boiled for 2 h After the solutionwas filtered the filtrate was collected The entire residue wascollected and further boiled with a total volume of 8 times(wv) distilled water for 2 h The solution was filtered and thetwo filtrates were combined concentrated and freeze-driedThe yield of the final dried extract was 25 (ww) of thestarting raw herbal materials and was stored at minus20∘C untilused The stock solution KTF (10mgml) was prepared bydissolving KTF in phosphate-buffer solution (PBS) followedby sonication sterilization at 100∘C and filtration

23 Cell Culture and 11986721198742Treatment MES235 cells were

cultured and exposed to H2O2as described by our previous

paper [10] Briefly MES235 cells were grown in DMEMF12(GIBCO BRL Co Ltd Gaithersburg MD USA) mediumcontaining 5 (volvol) FBS (GIBCOBRLCo Ltd Gaithers-burg MD USA) 100U penicillinstreptomycin 2mM L-glutamine (Sigma Chemical Co St Louis MO USA) andSatorsquos (50 times Satorsquos insulin 25mg transferring 25mg pyruvicacid 243mg putrescine 20mg 1mgml sodium selenate25 120583l 0315mgml progesterone) at 37∘C with 5 CO

2

Overnight grown MES235 cells were further incubated with150 120583MH

2O2for 12 h

24 KTF Treatment MES235 cells were randomly dividedinto control group (fresh culture medium) H

2O2group

(H2O2culture medium) Ginkgo biloba extract (GE) group

(H2O2and GE culture medium) and KTF groups including

low dose of KTF treatment middle dose of FTF treatmentand high dose of KTF treatmentMES235 cells at logarithmicphase were seeded in culture plate coated by poly-L-lysine(PLL) for 24 hThe cells in the control groupwere treatedwiththe fresh culture medium and those in all other groups weretreated with the culturemedium containing 150 120583MH

2O2for

12 h The cells in the GE group were treated with the culturemedium containing 125 120583gml GE and those in the low doseKTF group middle dose KTF group and high dose KTFgroup were treated with KTF at the concentrations of 3125625 and 125 120583gml for 24 h respectively

25 Cell Viability After KTF treatment for 20 h the cellswere incubated with MTT (05mgml) for 4 h at 37∘C andthe media were carefully removed and 150120583L of DMSO wasadded to each well The formazan crystals were dissolvedfor 10min and absorbance was measured at 490 nm Opticaldensity (OD) of each well was measured by spectrophotome-ter (BIO-TEK ELX 800 BioTek Instruments Inc Vermont

Parkinsonrsquos Disease 3

USA) Freshly cell culture medium was used as a negativecontrol

26 Ultrastructural Morphology Observation After KTFtreatment for 24 h the cells were collected suspended andfixed with 3 glutaraldehyde in 15 paraformaldehydesolution (pH 73) at 4∘C for 24 h Cell suspensions were thenrinsed twice with PBS and postfixed with 1 osmic acidand 15 potassium hexacyanoferrate (II) solution (pH 73)and incubated at 4∘C for 2 h then cell suspensions weredehydrated in a graded series of alcohol for 5min eachThe dehydrated pellets were embedded three times withpropylene oxide for 1 h and infiltrated with a resinpropyleneoxidemixture at 1 1 ratio for 2 h and thenwith resin for 12 h atroom temperature The inclusion was made with epoxy resin618 and Araldite and polymerization was performed at 60∘Cfor 48 h Finally ultrathin sections (80 nm) were stained withuranyl acetate and counterstained with lead citrate for 5minThe stained cells were examinedwith theH7650 transmissionelectron microscope (Hitachi Japan)

27 Immunocytochemical Assay of Tyrosine Hydroxylase (TH)After KTF treatment for 24 h the cells were washed twicefor 2min with PBS and fixed with 4 paraformaldehyde for15min The fixed cells were washed 3 times and then treatedwith 05 Triton X-100 for 20min Afterward the cells werewashed 3 times and treated with 3 H

2O2for 10min The

H2O2-treated cells were washed 3 times and then blotted

with goat serum for 20min After being blotted rabbit anti-TH (1 400) was added to each sample at 4∘C overnightThe samples were then washed 3 times and treated withPolymer Helper for 20min After Polymer treatment the cellswere washed 3 times and treated with poly-HRP anti-rabbitIgG for 20min The cells were washed 3 times and treatedwith DAB for 3min Finally the samples were washed withrunning water and treated with hematoxylin counterstainingfor 1min and then washed with running water and sealedwith permount mounting medium

28 Measurement of Antioxidative Enzymes by ELISA AfterKTF treatment for 24 h the activity level of intracellularCAT GPx-1 and GSH was measured using an ELISA kit(Hufeng Biotechnology Inc Shanghai China) according tothe manufacturerrsquos instruction Briefly 100 120583L homogenatesupernatant of the lysate of the treated cells was added tothe ELISA wells and then incubated at 37∘C for 30min Afterbeing washed five times and dried 50 120583L enzyme-labeledantibody working solution was added to each well (expectedcontrol tube)The resultant solutionwas incubated for 30minat 37∘C and washed five times and dried Substrate workingsolution (150 120583L) was added to each well and incubatedfor 15min at 37∘C Finally stop solution (50 120583L) was addedto each well and the absorbance value was determined at450 nm using an automatic ELISA reader (XL800 BioTexIncorporated Houston TX USA)

29 Western Blot Analysis After KTF treatment for 24 hthe cells were washed with ice-cold PBS and proteins wereextracted in a lysis buffer containing 50mM Tris-HCl (pH

74) 1mM EDTA 150mM NaCl 1 Nonidet P-40 1mMphenylmethylsulfonyl fluoride and 10 120583gmL aprotinin Pro-tein concentration was determined using BCA Protein AssayKit (Thermo Scientific Pierce Rockford Illinois USA)Protein were separated by SDS-PAGE electrophoresis andtransferred to PVDF (Bio-RadHercules CAUSA) Blockingwas performed with 5 nonfat dried milk the membranewas incubated with the of rabbit anti-rat HO-1 CAT caspase3 120573-actin antibody (1 1000 Cell Signaling) and rabbit anti-rat GPx-1 antibody (1 1000 Abcam) overnight at 4∘C Thetreated membrane was washed and then exposed to goatanti-rabbit IgG1 conjugated to a horseradish peroxidase label(1 5000 Santa Cruz USA) for 2 h at room temperatureAfter five more washes cross-reactivity was visualized usingElectrochemiluminescence (ECL)Western blotting detectionreagents and analyzed using scanning densitometry in anImage System (Bio-Rad Hercules CA USA)

210 RT-PCR Analysis PCR primer of HO-1 CAT GPx-1 and 120573-actin were purchased from Sangon Biotech CoLtd (Shanghai China) After KTF treatment for 24 h totalRNA of cells was isolated using Trizol reagent according tothe manufacturerrsquos instruction Reverse transcription (RT)was performed using the AMV reverse transcription system(Fermentas Burlington USA) cDNA fragment and 120573-actinwere amplified usingGreen PCRmasterMIXKit (FermentasBurlington USA) DNA was amplified immediately with asingle cycle at 94∘C for 5min and 30 cycles at 94∘C for30 s and 58∘C for 30 s and 72∘C for 30 s for 120573-actin and afinal extension step was taken at 72∘C for 10min Ethidiumbromide stained gels were scanned and qualified using TanonImage Software The intensity of each band was normalizedagainst the intensity of 120573-actin

211 Statistical Analysis Statistical analysis was done withSPSS 180 (SPSS Inc Chicago IL) All data were shown asmean plusmn SD (standard deviation) and statistical significanceof difference was performed by one-way ANOVA and LSD(linear standard deviation) A difference was considered tobe significant at 119875 lt 005

3 Results

31 The Damage Effects of H2O2on MES235 Cells and the

Protective Effects of KTF against Oxidative Stress As shownin Figure 1(a) H

2O2exhibited a dose- and time-dependent

inhibition in the viability ofMES235 dopaminergic neuronalcells After 12 h exposure to 50120583MH

2O2 a significant loss of

cell viability was noted and the cell viability loss graduallyincreased with H

2O2dose increasing to 150120583M However

further increasing H2O2dose from 200 to 500 120583M did not

result in significant higher loss of cell viability ExtendingMES235 cells exposure to H

2O2from 12 h to 24 h and 48 h

increased the loss of cell viability at lower H2O2concentra-

tion A significant loss of MES235 cell viability was notedafter 24 h exposure to 25120583M H

2O2and 48 h exposure to

125 120583MH2O2 A time-dependentmanner of cell viability loss

was observed in all tested concentration of H2O2

4 Parkinsonrsquos Disease

Cel

l via

bilit

y (

)120

100

80

60

40

20

12 h24 h

48 h

0

H2O2 (휇molL)0 125 25 50 100 150 200 300 400 500

lowastlowast

lowast

lowastlowast

lowast

lowastlowast

lowast

lowast

lowast

lowast

lowast

lowast

lowast

lowast

lowast

lowast

lowast

lowast

lowast

lowast

lowast

(a)

Cel

l via

bilit

y (

)

120

100

80

60

40

20

0Control H2O2 GE KTF-L KTF-M KTF-H

lowastlowastlowast

lowast

(b)

Figure 1 The damage effects of H2O2on MES235 cells and the protective effects of KTF against oxidative stress were evaluated using MTT

assay (a) Effects of different concentrations of H2O2treatment on cell damage MES235 cells were treated with 125 25 50 100 150 200

300 400 and 500 120583MolL H2O2for 12 24 and 48 h (b) Protective effects of different concentrations of KTF treatment on H

2O2-induced

cell damage Treatment of KTF resulted in increase of MES235 cells viability after H2O2addition MES235 cells were incubated with GE

(125 120583gml) or KTF (3125 625 125 120583gml) for 24 h after the addition of H2O2The cell viability was measured withMTT Data are expressed

as mean plusmn SD (standard deviation) in three independent experiments 119875 lt 005 compared to the control group lowast119875 lt 005 compared to theH2O2-treat group

Exposing MES235 cells to KTF alone did not havesignificant cytotoxic effect even whenKTF concentrationwasup to 100 120583gml MES235 cells cocultured with 150 120583MH

2O2

for 12 h caused cell viability loss of 4676 Cytotoxic effectof H2O2on MES235 cells was significantly attenuated upon

KTF treatment for 24 h (Figure 1(b)) Augmented viabilityof H2O2treated MES235 cells was restored to 9586 plusmn

718 9649 plusmn 1507 and 9904 plusmn 1057 by a treatmentwith KTF at concentrations of 3125 625 and 125 120583gmlrespectively

TEM showed that normal cells in control group exhibitedoval shape with intact cell membrane which has manycorrugations and microvilli-like structures Cell nucleus waselliptic in the center with a single nucleolus and had normaleuchromatin and scattered heterochromatin Mitochondriaexhibited rod-like or elliptic scattered and with normalcristae mitochondriate Rough endoplasmic reticulum waswell developed and had no obvious degranulation (Fig-ure 2(a)) However the cells treated with H

2O2presented

typical oxidative damage with smaller irregular nucleusand swelledmitochondria Obvious degranulation was foundin rough endoplasmic reticulum (Figure 2(b)) In the cellstreated by KTF ameliorated H

2O2-induced oxidative dam-

age mitochondria exhibited rod-like or elliptic scatteredand with more cristae mitochondriate Rough endoplasmicreticulum had no obvious degranulation (Figures 2(d) 2(e)and 2(f))

32 Increasing Expression of TH in MES235 Cells after KTFTreatment TH is a critical catalyzing enzyme in the con-version of the amino acid L-tyrosine to L-34-dihydroxy-phenylalanine (L-DOPA) which is the precursor to theneurotransmitters dopamine The normal expression of TH

is the mark of active neuron Therefore we compared theTH expression in the H

2O2-incubated MES235 cells with or

without following KTF treatment by immunohistochemistryThe result showed that H

2O2decreased TH expression in the

MES235 cells and following KTF treatment could improveTH expression significantly (Figure 3) This indicates thatKTF treatment was able to restore the function of the H

2O2-

treated MES235 cells

33 KTF Suppressed H2O2-Induced Caspase 3 Activation

in MES235 Cells For further investigating the mechanismof MES235 cell damage we evaluated the expression ofproapoptosis factor caspase 3 by immunoblotting Resultshowed that H

2O2treatment markedly downregulated the

expression of procaspase 3 and upregulated the expression ofcaspase 3 In contrast KTF treatment restored the procaspase3 and caspase 3 balance by increasing the expressions ofprocaspase 3 and decreasing the expression of caspase 3(Figure 4) These findings demonstrate that KTF treatmenteffectively inhibited caspase 3 activation

34 Increased Expression of Antioxidative Enzymes inMES235 Cells by KTF Treatment ELISA assay revealed thatintracellular CAT GPx-1 and GSH were downregulated after150 120583M H

2O2treatment in MES235 cells Of note follow-

ing treatment of KTF restored intracellular CAT GPx-1and GSH expression RT-PCR analysis indicated that H

2O2

treatment downregulated CAT andGPx-1mRNA expressionhowever KTF treatment restored the expressions of CATand GPx-1 mRNA in MES235 cells Immunoblot with speci-fic antibodies also confirmed that CAT and GPx-1 proteinexpression in MES235 cells were downregulated by H

2O2

treatment and KTF treatment was able to restore CAT and

Parkinsonrsquos Disease 5

(a) Control (b) H2O2 (c) GE

(d) KTF-L (e) KTF-M (f) KTF-H

Figure 2 Effect of KTF on cellular ultrastructure in H2O2-inducedMES235 cells Cellular ultrastructure was observed under TEM Normal

morphology of cytoplasm cell organelles in control group ((a) times15000) characteristic ultrastructural morphology of oxidative damage inH2O2-treated group ((b) times15000) treatment with KTF group (3125 625 and 125120583gml respectively) ((d) (e) (f) times15000) or Ginkgo

extract (125 120583gml) ((c) times15000)

GPx-1 protein expression Moreover mRNA expression ofoxidative stress responding protein HO-1 was also inhibitedafter H

2O2treatment and then was restored by following

KTF treatment Immunoblotting also proved that HO-1protein expression was markedly downregulated by H

2O2

but KTF treatment was able to restore HO-1 proteinexpression As expected the highest KTF concentrationused in our investigation has the best effect on restoringCAT GPx-1 GSH and HO-1 expression (Figures 5 and 6)Taken together the obtained results demonstrate that KTFtreatment significantly upregulated antioxidant substrates inthe H

2O2-treated MES233 cells

4 Discussion

As an important component of redox system ROS playimportant roles in regulating cell function Moderate levelsof ROS may function as signals to promote cell proliferationand survival Under physiologic conditions the balancebetween generation and elimination of ROS maintains theproper function of redox-sensitive signaling system Redoxhomeostasis can be disrupted by increase of ROS productionor decrease of ROS-scavenging capacity leading to an overall

increase in oxidative stress [17 18] subsequently causing anirreversible damage of intracellular structure and inducingcell death

High consumption of O2in brain leads to accumulation

of ROS thus neurons are particularly vulnerable to the attackof oxidative stress Postmortem investigations have shownlipid peroxidation and oxidative damage to brain cells in PDpatients [19] Growing studies confirm that oxidative damageplays a critical role in the pathogenesis of neurondegenerativediseases such as AD PDHD andALS [18 20] In the presentstudy a significant loss of cell viability was observed inMES235 dopaminergic neuronal cell after exposure to 50120583MH2O2for 12 h 25 120583M H

2O2for 24 h or 125 120583M H

2O2for

48 h As shown bymorphology observation the cells exposed12 h to 150 120583M H

2O2exhibited a typical oxidative injury

including multiple membranous vacuoles in the cytoplasmdisruption of endoplasmic reticulum and mitochondrionand condensing euchromatin in cell nucleus

According to Chinese medicine theory the deficiency ofkidney is the main pathological mechanism of PD Invigo-rating the kidney is an important strategy in the treatmentof PD In the present study we found that traditional kidney-tonifying formula KTF which consists ofCistanche Epimedii

6 Parkinsonrsquos Disease

Control GE

KTF-L KTF-M KTF-H

H2O2

(a)

Negativecontrol

(b)

0

20

40

60

80

100

120

140

160

Aver

age d

egre

e

Control H2O2 GE KTF-L KTF-M KTF-H

lowastlowast

lowastlowast

(c)

Figure 3 KTF ameliorated H2O2-induced reduction of TH inMES235 cells Cells were fixed with 4 paraformaldehyde and the expression

of TH was detected by immunocytochemistry (a) Tan represents the TH positive expression (times400) (b) Negative control (c) Results areexpressed as average grey degree Data are mean plusmn SD in three independent experiments 119875 lt 005 compared to the control group lowast119875 lt 005compared to the H

2O2-treat group

andPolygonatum sibiricum significantly enhanced cell viabil-ity and the expression of TH of H

2O2-treated MES235 cells

Cell apoptosis is a mode of programmed cell death uponvarious stimuli which activate either extrinsic or intrinsicpathways Cysteinyl aspartate-specific proteases (caspases)are highly conserved in multicellular organisms and functionas central regulators of apoptosis Activation of caspases isthe main axis of all apoptosis events Among those caspasescaspase 3 has been identified as a key mediator and indicatorof cell apoptosis Like other caspases caspase 3 usually existsas an inactive proenzyme (precaspase 3) After proteolyticprocessing it will produce two subunits which can dimerizeto form active caspase 3 subsequently evoking many of thebiochemical and biophysical changes to induce cell apoptosis[21] Our present study revealed that H

2O2treatment signif-

icantly upregulated caspase 3 but this trend was reversed byKTF treatment inMES235 cells indicating that KTF reducedcell apoptosis of H

2O2-treated MES235 cells

Antioxidative enzymes such as superoxide dismutase(SOD) CAT GPx-1 GSH and HO-1 are important defense

mechanism of cells against the oxidative stress As an antiox-idative enzyme SOD can transform superoxide radicals tohydrogen peroxide which was subsequently converted toH2O by CAT and GPx [22] GSH is also an important

antioxidant Catalytic conversion of reduced glutathione(GSH) to oxidized glutathione (GSSG) by GPx is helpfulto convert H

2O2to O2 It has been reported that the

activity of the antioxidant was decreased in PD modelmice [23ndash25] Therefore drugs that regulate expression ofenzymatic antioxidants may serve as potential candidates forthe treatment of PD Our data showed that H

2O2treatment

suppressed CAT GPx GSH and HO-1 in MES235 cellswhile KTF treatment at different concentration effectivelyrestored the expression of CAT GPx GSH and HO-1

In conclusion our findings demonstrate that traditionalChinese herb kidney-tonifying formulation which consistsofCistancheEpimedii and Polygonatum sibiricum has strongantioxidative capacity and significant neuroprotective effectsThis study provides a clue for developing the kidney-tonifyingformulation as a potential therapeutic agent to prevent andtreat Parkinsonrsquos disease

Parkinsonrsquos Disease 7

Procaspase 3

Caspase 3

훽-actin

35 KD

17 KD

45 KD

Control H2O2 GE KTF-L KTF-M KTF-H

(a)

lowast

lowastlowast

lowast

lowast

Control H2O2 GE KTF-L KTF-M KTF-H

Ratio

of p

roca

spas

e 3 an

d ca

spas

e 3훽

-act

in p

rote

in le

vel

250

200

150

100

050

000

Procaspase 3Caspase 3

(b)

Figure 4 Effect of KTF on the protein expression of caspase 3 in H2O2-induced MES235 cells (a) The protein expression of caspase 3 was

determined with western blot and quantified in (b) Data are expressed as mean plusmn SD in three independent experiments 119875 lt 005 comparedto the control group lowast119875 lt 005 compared to the H

2O2-treat group

00

01

02

03

04

05

06

CAT

conc

entr

atio

n (U

L)

Control H2O2 GE KTF-L KTF-M KTF-H

lowastlowastlowast

lowast

(a)

00

05

10

15

20

25

30

35

40

GPx

-1 co

ncen

trat

ion

(pm

olm

l)

Control H2O2 GE KTF-L KTF-M KTF-H

lowastlowast

lowast

(b)

0

20

40

60

80

100

120

GSH

conc

entr

atio

n (n

gm

l)

Control H2O2 GE KTF-L KTF-M KTF-H

lowast lowast lowast lowast

(c)

Figure 5 Effect of KTF on the level of intracellular CAT GPx-1 and GSH in H2O2-induced MES235 cells The activity level of intracellular

CAT GPx-1 and GSH was measured using an ELISA kit Data are expressed as mean plusmn SD in three independent experiments 119875 lt 005compared to the control group lowast119875 lt 005 compared to the H

2O2-treat group

8 Parkinsonrsquos Disease

HO-1

훽-actin

CAT

훽-actin

GPx-1

훽-actin

HO-1

훽-actin

CAT

훽-actin

GPx-1

훽-actin

(a) (c)

(d)(b)

00

02

04

06

08

10

12

GPx-1CATHO-1

Prot

ein

leve

l nor

mal

ized

with

훽-a

ctin

00

05

10

15

20

25m

RNA

leve

l nor

mal

ized

with

훽-a

ctin

Control

GE

KTF-LKTF-MKTF-HC

ontro

l

H2O

2

H2O2

GPx-1CATHO-1

Control

GE

KTF-LKTF-MKTF-H

H2O2

GE

KTF-

L

KTF-

M

KTF-

H

Con

trol

H2O

2

GE

KTF-

L

KTF-

M

KTF-

H

lowastlowastlowast

lowast

lowast

lowastlowast

lowast

lowastlowast

lowast

lowastlowastlowast

lowastlowastlowastlowast

lowast

lowast

lowastlowast

lowast

lowast

Figure 6 KTF increased the protein and mRNA expression of antioxidant enzymes in H2O2-induced MES235 cells (a) The protein

expressions of HO-1 CAT andGPx-1 were determined withWestern blot and quantified in (c) (b)ThemRNA expressions of HO-1 CAT andGPx-1 were determined with RT-PCR and quantified in (d) Data are expressed as mean plusmn SD in three independent experiments 119875 lt 005compared to the control group lowast119875 lt 005 compared to the H

2O2-treat group

Competing Interests

The authors declare that the research was conducted in theabsence of any commercial or financial relationships thatcould be construed as a potential conflict of interests

Authorsrsquo Contributions

Yihui Xu and Wei Lin share the same contribution to thearticle Chuanshan Xu is the cocorresponding author

Acknowledgments

This work was supported by Scientific Fund of NationalHealth and Family Planning Commission of the PeoplersquosRepublic of China (WKJ-FJ-38) Chen Keji DevelopmentFund Of Integrative Medicine (CKJ2014012) and Fund Of

KeyDisciplinary (X2014010-xueke)The authors express theirsincere thanks to Miss Xin Pan for her helpful assistance

References

[1] S Fahn andG Cohen ldquoThe oxidant stress hypothesis in Parkin-sonrsquos disease evidence supporting itrdquo Annals of Neurology vol32 no 6 pp 804ndash812 1992

[2] P Jenner andCWOlanow ldquoOxidative stress and the pathogen-esis of Parkinsonrsquos diseaserdquoNeurology vol 47 supplement 3 ppS161ndashS170 1996

[3] Y Akaneya M Takahashi and H Hatanaka ldquoInvolvement offree radicals in MPP+ neurotoxicity against rat dopaminergicneurons in culturerdquo Neuroscience Letters vol 193 no 1 pp 53ndash56 1995

[4] J Emerit M Edeas and F Bricaire ldquoNeurodegenerative dis-eases and oxidative stressrdquo Biomedicine amp Pharmacotherapyvol 58 no 1 pp 39ndash46 2004

Parkinsonrsquos Disease 9

[5] Chinese Pharmacopoeial Commission Pharmacopoeia of thePeoples Republic of China vol 1 Peoplersquos Medical PublishingHouse Beijing China 2010

[6] J Cai Y Tian R Lin X Chen Z Liu and J Xie ldquoProtectiveeffects of kidney-tonifying Chinese herbal preparation on sub-stantia nigra neurons in a mouse model of Parkinsonrsquos diseaserdquoNeural Regeneration Research vol 7 no 6 pp 413ndash420 2012

[7] WW Li R Yang and D F Cai ldquoProtective effects of Cistanchetotal glycosides on dopaminergic neuron in substantia nigra ofmodelmice of Parkinsonrsquos diseaserdquoZhongguo Zhong Xi Yi Jie HeZa Zhi vol 28 no 3 pp 248ndash251 2008

[8] W Wei and C Jing ldquoAn analysis of TCM pattern and formulafor parkinsonrsquos diseaserdquo Journal of Traditional ChineseMedicinevol 54 no 20 pp 1778ndash1782 2013

[9] Y Tian J Cai and X Chen ldquoProtection of kidney-tonifyingChinese herb on the nigra striatal neurons mice with Parkin-sonrsquos diseaserdquo Chinese Journal of Gerontology vol 31 no 3 pp440ndash443 2011

[10] S G Lin S F Ye JMHuang et al ldquoHow doChinesemedicinesthat tonify the kidney inhibit dopaminergic neuron apoptosisrdquoNeural Regeneration Research vol 8 no 30 pp 2820ndash28262013

[11] H Wang W-W Li D-F Cai and R Yang ldquoProtecting effect ofCistanche extracts on MPP+-induced injury of the Parkinsonrsquosdisease cell modelrdquo Journal of Chinese Integrative Medicine vol5 no 4 pp 407ndash411 2007

[12] X Geng X Tian P Tu and X Pu ldquoNeuroprotective effects ofechinacoside in themouseMPTPmodel of Parkinsonrsquos diseaserdquoEuropean Journal of Pharmacology vol 564 no 1ndash3 pp 66ndash742007

[13] X-F Tian and X-P Pu ldquoPhenylethanoid glycosides from Cis-tanches salsa inhibit apoptosis induced by 1-methyl-4-phe-nylpyridinium ion in neuronsrdquo Journal of Ethnopharmacologyvol 97 no 1 pp 59ndash63 2005

[14] X Geng L Song X Pu and P Tu ldquoNeuroprotective effectsof phenylethanoid glycosides from Cistanches salsa against 1-methyl-4-phenyl-1236-tetrahydropyridine (MPTP)-induceddopaminergic toxicity in C57 micerdquo Biological and Pharmaceu-tical Bulletin vol 27 no 6 pp 797ndash801 2004

[15] H SWong and KM Ko ldquoHerba Cistanches stimulates cellularglutathione redox cycling by reactive oxygen species generatedfrommitochondrial respiration inH9c2 cardiomyocytesrdquo Phar-maceutical Biology vol 51 no 1 pp 64ndash73 2013

[16] S C Wing Sze Y Tong T B Ng C L Yin Cheng and HP Cheung ldquoHerba Epimedii anti-oxidative properties and itsmedical implicationsrdquo Molecules vol 15 no 11 pp 7861ndash78702010

[17] D Trachootham W Lu M A Ogasawara N R-D Valle andP Huang ldquoRedox regulation of cell survivalrdquo Antioxidants andRedox Signaling vol 10 no 8 pp 1343ndash1374 2008

[18] D J Moore A B West V L Dawson and T M DawsonldquoMolecular pathophysiology of Parkinsonrsquos diseaserdquo AnnualReview of Neuroscience vol 28 pp 57ndash87 2005

[19] J Sian D T Dexter A J Lees et al ldquoAlterations in glutathionelevels in Parkinsonrsquos disease and other neurodegenerative disor-ders affecting basal gangliardquo Annals of Neurology vol 36 no 3pp 348ndash355 1994

[20] BHalliwell and JMCGutteridge ldquoRole of free radic in humandiseases an overviewrdquo Methods in Enzymology vol 186 pp 1ndash85 1990

[21] KVenderova andD S Park ldquoProgrammed cell death in Parkin-sonrsquos diseaserdquo Cold Spring Harbor Perspectives in Medicine vol2 no 8 Article ID a009365 2012

[22] I Fridovich ldquoFundamental aspects of reactive oxygen speciesor whatrsquos the matter with oxygenrdquo Annals of the New YorkAcademy of Sciences vol 893 pp 13ndash18 1999

[23] Q Xiong S Kadota T Tani and T Namba ldquoAntioxidativeeffects of phenylethanoids from Cistanche deserticolardquo Biolog-ical and Pharmaceutical Bulletin vol 19 no 12 pp 1580ndash15851996

[24] S Koppula H Kumar S V More H-W Lim S-M Hong andD-K Choi ldquoRecent updates in redox regulation and free radicalscavenging effects by herbal products in experimental modelsof Parkinsonrsquos diseaserdquoMolecules vol 17 no 10 pp 11391ndash114202012

[25] Z Fang and Y Wang ldquoEffect of isatin on the activities of anti-oxidase in PDmodel micerdquo China Pharmacy vol 19 no 22 pp1696ndash1697 2008

Submit your manuscripts athttpswwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Parkinsonrsquos Disease 3

USA) Freshly cell culture medium was used as a negativecontrol

26 Ultrastructural Morphology Observation After KTFtreatment for 24 h the cells were collected suspended andfixed with 3 glutaraldehyde in 15 paraformaldehydesolution (pH 73) at 4∘C for 24 h Cell suspensions were thenrinsed twice with PBS and postfixed with 1 osmic acidand 15 potassium hexacyanoferrate (II) solution (pH 73)and incubated at 4∘C for 2 h then cell suspensions weredehydrated in a graded series of alcohol for 5min eachThe dehydrated pellets were embedded three times withpropylene oxide for 1 h and infiltrated with a resinpropyleneoxidemixture at 1 1 ratio for 2 h and thenwith resin for 12 h atroom temperature The inclusion was made with epoxy resin618 and Araldite and polymerization was performed at 60∘Cfor 48 h Finally ultrathin sections (80 nm) were stained withuranyl acetate and counterstained with lead citrate for 5minThe stained cells were examinedwith theH7650 transmissionelectron microscope (Hitachi Japan)

27 Immunocytochemical Assay of Tyrosine Hydroxylase (TH)After KTF treatment for 24 h the cells were washed twicefor 2min with PBS and fixed with 4 paraformaldehyde for15min The fixed cells were washed 3 times and then treatedwith 05 Triton X-100 for 20min Afterward the cells werewashed 3 times and treated with 3 H

2O2for 10min The

H2O2-treated cells were washed 3 times and then blotted

with goat serum for 20min After being blotted rabbit anti-TH (1 400) was added to each sample at 4∘C overnightThe samples were then washed 3 times and treated withPolymer Helper for 20min After Polymer treatment the cellswere washed 3 times and treated with poly-HRP anti-rabbitIgG for 20min The cells were washed 3 times and treatedwith DAB for 3min Finally the samples were washed withrunning water and treated with hematoxylin counterstainingfor 1min and then washed with running water and sealedwith permount mounting medium

28 Measurement of Antioxidative Enzymes by ELISA AfterKTF treatment for 24 h the activity level of intracellularCAT GPx-1 and GSH was measured using an ELISA kit(Hufeng Biotechnology Inc Shanghai China) according tothe manufacturerrsquos instruction Briefly 100 120583L homogenatesupernatant of the lysate of the treated cells was added tothe ELISA wells and then incubated at 37∘C for 30min Afterbeing washed five times and dried 50 120583L enzyme-labeledantibody working solution was added to each well (expectedcontrol tube)The resultant solutionwas incubated for 30minat 37∘C and washed five times and dried Substrate workingsolution (150 120583L) was added to each well and incubatedfor 15min at 37∘C Finally stop solution (50 120583L) was addedto each well and the absorbance value was determined at450 nm using an automatic ELISA reader (XL800 BioTexIncorporated Houston TX USA)

29 Western Blot Analysis After KTF treatment for 24 hthe cells were washed with ice-cold PBS and proteins wereextracted in a lysis buffer containing 50mM Tris-HCl (pH

74) 1mM EDTA 150mM NaCl 1 Nonidet P-40 1mMphenylmethylsulfonyl fluoride and 10 120583gmL aprotinin Pro-tein concentration was determined using BCA Protein AssayKit (Thermo Scientific Pierce Rockford Illinois USA)Protein were separated by SDS-PAGE electrophoresis andtransferred to PVDF (Bio-RadHercules CAUSA) Blockingwas performed with 5 nonfat dried milk the membranewas incubated with the of rabbit anti-rat HO-1 CAT caspase3 120573-actin antibody (1 1000 Cell Signaling) and rabbit anti-rat GPx-1 antibody (1 1000 Abcam) overnight at 4∘C Thetreated membrane was washed and then exposed to goatanti-rabbit IgG1 conjugated to a horseradish peroxidase label(1 5000 Santa Cruz USA) for 2 h at room temperatureAfter five more washes cross-reactivity was visualized usingElectrochemiluminescence (ECL)Western blotting detectionreagents and analyzed using scanning densitometry in anImage System (Bio-Rad Hercules CA USA)

210 RT-PCR Analysis PCR primer of HO-1 CAT GPx-1 and 120573-actin were purchased from Sangon Biotech CoLtd (Shanghai China) After KTF treatment for 24 h totalRNA of cells was isolated using Trizol reagent according tothe manufacturerrsquos instruction Reverse transcription (RT)was performed using the AMV reverse transcription system(Fermentas Burlington USA) cDNA fragment and 120573-actinwere amplified usingGreen PCRmasterMIXKit (FermentasBurlington USA) DNA was amplified immediately with asingle cycle at 94∘C for 5min and 30 cycles at 94∘C for30 s and 58∘C for 30 s and 72∘C for 30 s for 120573-actin and afinal extension step was taken at 72∘C for 10min Ethidiumbromide stained gels were scanned and qualified using TanonImage Software The intensity of each band was normalizedagainst the intensity of 120573-actin

211 Statistical Analysis Statistical analysis was done withSPSS 180 (SPSS Inc Chicago IL) All data were shown asmean plusmn SD (standard deviation) and statistical significanceof difference was performed by one-way ANOVA and LSD(linear standard deviation) A difference was considered tobe significant at 119875 lt 005

3 Results

31 The Damage Effects of H2O2on MES235 Cells and the

Protective Effects of KTF against Oxidative Stress As shownin Figure 1(a) H

2O2exhibited a dose- and time-dependent

inhibition in the viability ofMES235 dopaminergic neuronalcells After 12 h exposure to 50120583MH

2O2 a significant loss of

cell viability was noted and the cell viability loss graduallyincreased with H

2O2dose increasing to 150120583M However

further increasing H2O2dose from 200 to 500 120583M did not

result in significant higher loss of cell viability ExtendingMES235 cells exposure to H

2O2from 12 h to 24 h and 48 h

increased the loss of cell viability at lower H2O2concentra-

tion A significant loss of MES235 cell viability was notedafter 24 h exposure to 25120583M H

2O2and 48 h exposure to

125 120583MH2O2 A time-dependentmanner of cell viability loss

was observed in all tested concentration of H2O2

4 Parkinsonrsquos Disease

Cel

l via

bilit

y (

)120

100

80

60

40

20

12 h24 h

48 h

0

H2O2 (휇molL)0 125 25 50 100 150 200 300 400 500

lowastlowast

lowast

lowastlowast

lowast

lowastlowast

lowast

lowast

lowast

lowast

lowast

lowast

lowast

lowast

lowast

lowast

lowast

lowast

lowast

lowast

lowast

(a)

Cel

l via

bilit

y (

)

120

100

80

60

40

20

0Control H2O2 GE KTF-L KTF-M KTF-H

lowastlowastlowast

lowast

(b)

Figure 1 The damage effects of H2O2on MES235 cells and the protective effects of KTF against oxidative stress were evaluated using MTT

assay (a) Effects of different concentrations of H2O2treatment on cell damage MES235 cells were treated with 125 25 50 100 150 200

300 400 and 500 120583MolL H2O2for 12 24 and 48 h (b) Protective effects of different concentrations of KTF treatment on H

2O2-induced

cell damage Treatment of KTF resulted in increase of MES235 cells viability after H2O2addition MES235 cells were incubated with GE

(125 120583gml) or KTF (3125 625 125 120583gml) for 24 h after the addition of H2O2The cell viability was measured withMTT Data are expressed

as mean plusmn SD (standard deviation) in three independent experiments 119875 lt 005 compared to the control group lowast119875 lt 005 compared to theH2O2-treat group

Exposing MES235 cells to KTF alone did not havesignificant cytotoxic effect even whenKTF concentrationwasup to 100 120583gml MES235 cells cocultured with 150 120583MH

2O2

for 12 h caused cell viability loss of 4676 Cytotoxic effectof H2O2on MES235 cells was significantly attenuated upon

KTF treatment for 24 h (Figure 1(b)) Augmented viabilityof H2O2treated MES235 cells was restored to 9586 plusmn

718 9649 plusmn 1507 and 9904 plusmn 1057 by a treatmentwith KTF at concentrations of 3125 625 and 125 120583gmlrespectively

TEM showed that normal cells in control group exhibitedoval shape with intact cell membrane which has manycorrugations and microvilli-like structures Cell nucleus waselliptic in the center with a single nucleolus and had normaleuchromatin and scattered heterochromatin Mitochondriaexhibited rod-like or elliptic scattered and with normalcristae mitochondriate Rough endoplasmic reticulum waswell developed and had no obvious degranulation (Fig-ure 2(a)) However the cells treated with H

2O2presented

typical oxidative damage with smaller irregular nucleusand swelledmitochondria Obvious degranulation was foundin rough endoplasmic reticulum (Figure 2(b)) In the cellstreated by KTF ameliorated H

2O2-induced oxidative dam-

age mitochondria exhibited rod-like or elliptic scatteredand with more cristae mitochondriate Rough endoplasmicreticulum had no obvious degranulation (Figures 2(d) 2(e)and 2(f))

32 Increasing Expression of TH in MES235 Cells after KTFTreatment TH is a critical catalyzing enzyme in the con-version of the amino acid L-tyrosine to L-34-dihydroxy-phenylalanine (L-DOPA) which is the precursor to theneurotransmitters dopamine The normal expression of TH

is the mark of active neuron Therefore we compared theTH expression in the H

2O2-incubated MES235 cells with or

without following KTF treatment by immunohistochemistryThe result showed that H

2O2decreased TH expression in the

MES235 cells and following KTF treatment could improveTH expression significantly (Figure 3) This indicates thatKTF treatment was able to restore the function of the H

2O2-

treated MES235 cells

33 KTF Suppressed H2O2-Induced Caspase 3 Activation

in MES235 Cells For further investigating the mechanismof MES235 cell damage we evaluated the expression ofproapoptosis factor caspase 3 by immunoblotting Resultshowed that H

2O2treatment markedly downregulated the

expression of procaspase 3 and upregulated the expression ofcaspase 3 In contrast KTF treatment restored the procaspase3 and caspase 3 balance by increasing the expressions ofprocaspase 3 and decreasing the expression of caspase 3(Figure 4) These findings demonstrate that KTF treatmenteffectively inhibited caspase 3 activation

34 Increased Expression of Antioxidative Enzymes inMES235 Cells by KTF Treatment ELISA assay revealed thatintracellular CAT GPx-1 and GSH were downregulated after150 120583M H

2O2treatment in MES235 cells Of note follow-

ing treatment of KTF restored intracellular CAT GPx-1and GSH expression RT-PCR analysis indicated that H

2O2

treatment downregulated CAT andGPx-1mRNA expressionhowever KTF treatment restored the expressions of CATand GPx-1 mRNA in MES235 cells Immunoblot with speci-fic antibodies also confirmed that CAT and GPx-1 proteinexpression in MES235 cells were downregulated by H

2O2

treatment and KTF treatment was able to restore CAT and

Parkinsonrsquos Disease 5

(a) Control (b) H2O2 (c) GE

(d) KTF-L (e) KTF-M (f) KTF-H

Figure 2 Effect of KTF on cellular ultrastructure in H2O2-inducedMES235 cells Cellular ultrastructure was observed under TEM Normal

morphology of cytoplasm cell organelles in control group ((a) times15000) characteristic ultrastructural morphology of oxidative damage inH2O2-treated group ((b) times15000) treatment with KTF group (3125 625 and 125120583gml respectively) ((d) (e) (f) times15000) or Ginkgo

extract (125 120583gml) ((c) times15000)

GPx-1 protein expression Moreover mRNA expression ofoxidative stress responding protein HO-1 was also inhibitedafter H

2O2treatment and then was restored by following

KTF treatment Immunoblotting also proved that HO-1protein expression was markedly downregulated by H

2O2

but KTF treatment was able to restore HO-1 proteinexpression As expected the highest KTF concentrationused in our investigation has the best effect on restoringCAT GPx-1 GSH and HO-1 expression (Figures 5 and 6)Taken together the obtained results demonstrate that KTFtreatment significantly upregulated antioxidant substrates inthe H

2O2-treated MES233 cells

4 Discussion

As an important component of redox system ROS playimportant roles in regulating cell function Moderate levelsof ROS may function as signals to promote cell proliferationand survival Under physiologic conditions the balancebetween generation and elimination of ROS maintains theproper function of redox-sensitive signaling system Redoxhomeostasis can be disrupted by increase of ROS productionor decrease of ROS-scavenging capacity leading to an overall

increase in oxidative stress [17 18] subsequently causing anirreversible damage of intracellular structure and inducingcell death

High consumption of O2in brain leads to accumulation

of ROS thus neurons are particularly vulnerable to the attackof oxidative stress Postmortem investigations have shownlipid peroxidation and oxidative damage to brain cells in PDpatients [19] Growing studies confirm that oxidative damageplays a critical role in the pathogenesis of neurondegenerativediseases such as AD PDHD andALS [18 20] In the presentstudy a significant loss of cell viability was observed inMES235 dopaminergic neuronal cell after exposure to 50120583MH2O2for 12 h 25 120583M H

2O2for 24 h or 125 120583M H

2O2for

48 h As shown bymorphology observation the cells exposed12 h to 150 120583M H

2O2exhibited a typical oxidative injury

including multiple membranous vacuoles in the cytoplasmdisruption of endoplasmic reticulum and mitochondrionand condensing euchromatin in cell nucleus

According to Chinese medicine theory the deficiency ofkidney is the main pathological mechanism of PD Invigo-rating the kidney is an important strategy in the treatmentof PD In the present study we found that traditional kidney-tonifying formula KTF which consists ofCistanche Epimedii

6 Parkinsonrsquos Disease

Control GE

KTF-L KTF-M KTF-H

H2O2

(a)

Negativecontrol

(b)

0

20

40

60

80

100

120

140

160

Aver

age d

egre

e

Control H2O2 GE KTF-L KTF-M KTF-H

lowastlowast

lowastlowast

(c)

Figure 3 KTF ameliorated H2O2-induced reduction of TH inMES235 cells Cells were fixed with 4 paraformaldehyde and the expression

of TH was detected by immunocytochemistry (a) Tan represents the TH positive expression (times400) (b) Negative control (c) Results areexpressed as average grey degree Data are mean plusmn SD in three independent experiments 119875 lt 005 compared to the control group lowast119875 lt 005compared to the H

2O2-treat group

andPolygonatum sibiricum significantly enhanced cell viabil-ity and the expression of TH of H

2O2-treated MES235 cells

Cell apoptosis is a mode of programmed cell death uponvarious stimuli which activate either extrinsic or intrinsicpathways Cysteinyl aspartate-specific proteases (caspases)are highly conserved in multicellular organisms and functionas central regulators of apoptosis Activation of caspases isthe main axis of all apoptosis events Among those caspasescaspase 3 has been identified as a key mediator and indicatorof cell apoptosis Like other caspases caspase 3 usually existsas an inactive proenzyme (precaspase 3) After proteolyticprocessing it will produce two subunits which can dimerizeto form active caspase 3 subsequently evoking many of thebiochemical and biophysical changes to induce cell apoptosis[21] Our present study revealed that H

2O2treatment signif-

icantly upregulated caspase 3 but this trend was reversed byKTF treatment inMES235 cells indicating that KTF reducedcell apoptosis of H

2O2-treated MES235 cells

Antioxidative enzymes such as superoxide dismutase(SOD) CAT GPx-1 GSH and HO-1 are important defense

mechanism of cells against the oxidative stress As an antiox-idative enzyme SOD can transform superoxide radicals tohydrogen peroxide which was subsequently converted toH2O by CAT and GPx [22] GSH is also an important

antioxidant Catalytic conversion of reduced glutathione(GSH) to oxidized glutathione (GSSG) by GPx is helpfulto convert H

2O2to O2 It has been reported that the

activity of the antioxidant was decreased in PD modelmice [23ndash25] Therefore drugs that regulate expression ofenzymatic antioxidants may serve as potential candidates forthe treatment of PD Our data showed that H

2O2treatment

suppressed CAT GPx GSH and HO-1 in MES235 cellswhile KTF treatment at different concentration effectivelyrestored the expression of CAT GPx GSH and HO-1

In conclusion our findings demonstrate that traditionalChinese herb kidney-tonifying formulation which consistsofCistancheEpimedii and Polygonatum sibiricum has strongantioxidative capacity and significant neuroprotective effectsThis study provides a clue for developing the kidney-tonifyingformulation as a potential therapeutic agent to prevent andtreat Parkinsonrsquos disease

Parkinsonrsquos Disease 7

Procaspase 3

Caspase 3

훽-actin

35 KD

17 KD

45 KD

Control H2O2 GE KTF-L KTF-M KTF-H

(a)

lowast

lowastlowast

lowast

lowast

Control H2O2 GE KTF-L KTF-M KTF-H

Ratio

of p

roca

spas

e 3 an

d ca

spas

e 3훽

-act

in p

rote

in le

vel

250

200

150

100

050

000

Procaspase 3Caspase 3

(b)

Figure 4 Effect of KTF on the protein expression of caspase 3 in H2O2-induced MES235 cells (a) The protein expression of caspase 3 was

determined with western blot and quantified in (b) Data are expressed as mean plusmn SD in three independent experiments 119875 lt 005 comparedto the control group lowast119875 lt 005 compared to the H

2O2-treat group

00

01

02

03

04

05

06

CAT

conc

entr

atio

n (U

L)

Control H2O2 GE KTF-L KTF-M KTF-H

lowastlowastlowast

lowast

(a)

00

05

10

15

20

25

30

35

40

GPx

-1 co

ncen

trat

ion

(pm

olm

l)

Control H2O2 GE KTF-L KTF-M KTF-H

lowastlowast

lowast

(b)

0

20

40

60

80

100

120

GSH

conc

entr

atio

n (n

gm

l)

Control H2O2 GE KTF-L KTF-M KTF-H

lowast lowast lowast lowast

(c)

Figure 5 Effect of KTF on the level of intracellular CAT GPx-1 and GSH in H2O2-induced MES235 cells The activity level of intracellular

CAT GPx-1 and GSH was measured using an ELISA kit Data are expressed as mean plusmn SD in three independent experiments 119875 lt 005compared to the control group lowast119875 lt 005 compared to the H

2O2-treat group

8 Parkinsonrsquos Disease

HO-1

훽-actin

CAT

훽-actin

GPx-1

훽-actin

HO-1

훽-actin

CAT

훽-actin

GPx-1

훽-actin

(a) (c)

(d)(b)

00

02

04

06

08

10

12

GPx-1CATHO-1

Prot

ein

leve

l nor

mal

ized

with

훽-a

ctin

00

05

10

15

20

25m

RNA

leve

l nor

mal

ized

with

훽-a

ctin

Control

GE

KTF-LKTF-MKTF-HC

ontro

l

H2O

2

H2O2

GPx-1CATHO-1

Control

GE

KTF-LKTF-MKTF-H

H2O2

GE

KTF-

L

KTF-

M

KTF-

H

Con

trol

H2O

2

GE

KTF-

L

KTF-

M

KTF-

H

lowastlowastlowast

lowast

lowast

lowastlowast

lowast

lowastlowast

lowast

lowastlowastlowast

lowastlowastlowastlowast

lowast

lowast

lowastlowast

lowast

lowast

Figure 6 KTF increased the protein and mRNA expression of antioxidant enzymes in H2O2-induced MES235 cells (a) The protein

expressions of HO-1 CAT andGPx-1 were determined withWestern blot and quantified in (c) (b)ThemRNA expressions of HO-1 CAT andGPx-1 were determined with RT-PCR and quantified in (d) Data are expressed as mean plusmn SD in three independent experiments 119875 lt 005compared to the control group lowast119875 lt 005 compared to the H

2O2-treat group

Competing Interests

The authors declare that the research was conducted in theabsence of any commercial or financial relationships thatcould be construed as a potential conflict of interests

Authorsrsquo Contributions

Yihui Xu and Wei Lin share the same contribution to thearticle Chuanshan Xu is the cocorresponding author

Acknowledgments

This work was supported by Scientific Fund of NationalHealth and Family Planning Commission of the PeoplersquosRepublic of China (WKJ-FJ-38) Chen Keji DevelopmentFund Of Integrative Medicine (CKJ2014012) and Fund Of

KeyDisciplinary (X2014010-xueke)The authors express theirsincere thanks to Miss Xin Pan for her helpful assistance

References

[1] S Fahn andG Cohen ldquoThe oxidant stress hypothesis in Parkin-sonrsquos disease evidence supporting itrdquo Annals of Neurology vol32 no 6 pp 804ndash812 1992

[2] P Jenner andCWOlanow ldquoOxidative stress and the pathogen-esis of Parkinsonrsquos diseaserdquoNeurology vol 47 supplement 3 ppS161ndashS170 1996

[3] Y Akaneya M Takahashi and H Hatanaka ldquoInvolvement offree radicals in MPP+ neurotoxicity against rat dopaminergicneurons in culturerdquo Neuroscience Letters vol 193 no 1 pp 53ndash56 1995

[4] J Emerit M Edeas and F Bricaire ldquoNeurodegenerative dis-eases and oxidative stressrdquo Biomedicine amp Pharmacotherapyvol 58 no 1 pp 39ndash46 2004

Parkinsonrsquos Disease 9

[5] Chinese Pharmacopoeial Commission Pharmacopoeia of thePeoples Republic of China vol 1 Peoplersquos Medical PublishingHouse Beijing China 2010

[6] J Cai Y Tian R Lin X Chen Z Liu and J Xie ldquoProtectiveeffects of kidney-tonifying Chinese herbal preparation on sub-stantia nigra neurons in a mouse model of Parkinsonrsquos diseaserdquoNeural Regeneration Research vol 7 no 6 pp 413ndash420 2012

[7] WW Li R Yang and D F Cai ldquoProtective effects of Cistanchetotal glycosides on dopaminergic neuron in substantia nigra ofmodelmice of Parkinsonrsquos diseaserdquoZhongguo Zhong Xi Yi Jie HeZa Zhi vol 28 no 3 pp 248ndash251 2008

[8] W Wei and C Jing ldquoAn analysis of TCM pattern and formulafor parkinsonrsquos diseaserdquo Journal of Traditional ChineseMedicinevol 54 no 20 pp 1778ndash1782 2013

[9] Y Tian J Cai and X Chen ldquoProtection of kidney-tonifyingChinese herb on the nigra striatal neurons mice with Parkin-sonrsquos diseaserdquo Chinese Journal of Gerontology vol 31 no 3 pp440ndash443 2011

[10] S G Lin S F Ye JMHuang et al ldquoHow doChinesemedicinesthat tonify the kidney inhibit dopaminergic neuron apoptosisrdquoNeural Regeneration Research vol 8 no 30 pp 2820ndash28262013

[11] H Wang W-W Li D-F Cai and R Yang ldquoProtecting effect ofCistanche extracts on MPP+-induced injury of the Parkinsonrsquosdisease cell modelrdquo Journal of Chinese Integrative Medicine vol5 no 4 pp 407ndash411 2007

[12] X Geng X Tian P Tu and X Pu ldquoNeuroprotective effects ofechinacoside in themouseMPTPmodel of Parkinsonrsquos diseaserdquoEuropean Journal of Pharmacology vol 564 no 1ndash3 pp 66ndash742007

[13] X-F Tian and X-P Pu ldquoPhenylethanoid glycosides from Cis-tanches salsa inhibit apoptosis induced by 1-methyl-4-phe-nylpyridinium ion in neuronsrdquo Journal of Ethnopharmacologyvol 97 no 1 pp 59ndash63 2005

[14] X Geng L Song X Pu and P Tu ldquoNeuroprotective effectsof phenylethanoid glycosides from Cistanches salsa against 1-methyl-4-phenyl-1236-tetrahydropyridine (MPTP)-induceddopaminergic toxicity in C57 micerdquo Biological and Pharmaceu-tical Bulletin vol 27 no 6 pp 797ndash801 2004

[15] H SWong and KM Ko ldquoHerba Cistanches stimulates cellularglutathione redox cycling by reactive oxygen species generatedfrommitochondrial respiration inH9c2 cardiomyocytesrdquo Phar-maceutical Biology vol 51 no 1 pp 64ndash73 2013

[16] S C Wing Sze Y Tong T B Ng C L Yin Cheng and HP Cheung ldquoHerba Epimedii anti-oxidative properties and itsmedical implicationsrdquo Molecules vol 15 no 11 pp 7861ndash78702010

[17] D Trachootham W Lu M A Ogasawara N R-D Valle andP Huang ldquoRedox regulation of cell survivalrdquo Antioxidants andRedox Signaling vol 10 no 8 pp 1343ndash1374 2008

[18] D J Moore A B West V L Dawson and T M DawsonldquoMolecular pathophysiology of Parkinsonrsquos diseaserdquo AnnualReview of Neuroscience vol 28 pp 57ndash87 2005

[19] J Sian D T Dexter A J Lees et al ldquoAlterations in glutathionelevels in Parkinsonrsquos disease and other neurodegenerative disor-ders affecting basal gangliardquo Annals of Neurology vol 36 no 3pp 348ndash355 1994

[20] BHalliwell and JMCGutteridge ldquoRole of free radic in humandiseases an overviewrdquo Methods in Enzymology vol 186 pp 1ndash85 1990

[21] KVenderova andD S Park ldquoProgrammed cell death in Parkin-sonrsquos diseaserdquo Cold Spring Harbor Perspectives in Medicine vol2 no 8 Article ID a009365 2012

[22] I Fridovich ldquoFundamental aspects of reactive oxygen speciesor whatrsquos the matter with oxygenrdquo Annals of the New YorkAcademy of Sciences vol 893 pp 13ndash18 1999

[23] Q Xiong S Kadota T Tani and T Namba ldquoAntioxidativeeffects of phenylethanoids from Cistanche deserticolardquo Biolog-ical and Pharmaceutical Bulletin vol 19 no 12 pp 1580ndash15851996

[24] S Koppula H Kumar S V More H-W Lim S-M Hong andD-K Choi ldquoRecent updates in redox regulation and free radicalscavenging effects by herbal products in experimental modelsof Parkinsonrsquos diseaserdquoMolecules vol 17 no 10 pp 11391ndash114202012

[25] Z Fang and Y Wang ldquoEffect of isatin on the activities of anti-oxidase in PDmodel micerdquo China Pharmacy vol 19 no 22 pp1696ndash1697 2008

Submit your manuscripts athttpswwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

4 Parkinsonrsquos Disease

Cel

l via

bilit

y (

)120

100

80

60

40

20

12 h24 h

48 h

0

H2O2 (휇molL)0 125 25 50 100 150 200 300 400 500

lowastlowast

lowast

lowastlowast

lowast

lowastlowast

lowast

lowast

lowast

lowast

lowast

lowast

lowast

lowast

lowast

lowast

lowast

lowast

lowast

lowast

lowast

(a)

Cel

l via

bilit

y (

)

120

100

80

60

40

20

0Control H2O2 GE KTF-L KTF-M KTF-H

lowastlowastlowast

lowast

(b)

Figure 1 The damage effects of H2O2on MES235 cells and the protective effects of KTF against oxidative stress were evaluated using MTT

assay (a) Effects of different concentrations of H2O2treatment on cell damage MES235 cells were treated with 125 25 50 100 150 200

300 400 and 500 120583MolL H2O2for 12 24 and 48 h (b) Protective effects of different concentrations of KTF treatment on H

2O2-induced

cell damage Treatment of KTF resulted in increase of MES235 cells viability after H2O2addition MES235 cells were incubated with GE

(125 120583gml) or KTF (3125 625 125 120583gml) for 24 h after the addition of H2O2The cell viability was measured withMTT Data are expressed

as mean plusmn SD (standard deviation) in three independent experiments 119875 lt 005 compared to the control group lowast119875 lt 005 compared to theH2O2-treat group

Exposing MES235 cells to KTF alone did not havesignificant cytotoxic effect even whenKTF concentrationwasup to 100 120583gml MES235 cells cocultured with 150 120583MH

2O2

for 12 h caused cell viability loss of 4676 Cytotoxic effectof H2O2on MES235 cells was significantly attenuated upon

KTF treatment for 24 h (Figure 1(b)) Augmented viabilityof H2O2treated MES235 cells was restored to 9586 plusmn

718 9649 plusmn 1507 and 9904 plusmn 1057 by a treatmentwith KTF at concentrations of 3125 625 and 125 120583gmlrespectively

TEM showed that normal cells in control group exhibitedoval shape with intact cell membrane which has manycorrugations and microvilli-like structures Cell nucleus waselliptic in the center with a single nucleolus and had normaleuchromatin and scattered heterochromatin Mitochondriaexhibited rod-like or elliptic scattered and with normalcristae mitochondriate Rough endoplasmic reticulum waswell developed and had no obvious degranulation (Fig-ure 2(a)) However the cells treated with H

2O2presented

typical oxidative damage with smaller irregular nucleusand swelledmitochondria Obvious degranulation was foundin rough endoplasmic reticulum (Figure 2(b)) In the cellstreated by KTF ameliorated H

2O2-induced oxidative dam-

age mitochondria exhibited rod-like or elliptic scatteredand with more cristae mitochondriate Rough endoplasmicreticulum had no obvious degranulation (Figures 2(d) 2(e)and 2(f))

32 Increasing Expression of TH in MES235 Cells after KTFTreatment TH is a critical catalyzing enzyme in the con-version of the amino acid L-tyrosine to L-34-dihydroxy-phenylalanine (L-DOPA) which is the precursor to theneurotransmitters dopamine The normal expression of TH

is the mark of active neuron Therefore we compared theTH expression in the H

2O2-incubated MES235 cells with or

without following KTF treatment by immunohistochemistryThe result showed that H

2O2decreased TH expression in the

MES235 cells and following KTF treatment could improveTH expression significantly (Figure 3) This indicates thatKTF treatment was able to restore the function of the H

2O2-

treated MES235 cells

33 KTF Suppressed H2O2-Induced Caspase 3 Activation

in MES235 Cells For further investigating the mechanismof MES235 cell damage we evaluated the expression ofproapoptosis factor caspase 3 by immunoblotting Resultshowed that H

2O2treatment markedly downregulated the

expression of procaspase 3 and upregulated the expression ofcaspase 3 In contrast KTF treatment restored the procaspase3 and caspase 3 balance by increasing the expressions ofprocaspase 3 and decreasing the expression of caspase 3(Figure 4) These findings demonstrate that KTF treatmenteffectively inhibited caspase 3 activation

34 Increased Expression of Antioxidative Enzymes inMES235 Cells by KTF Treatment ELISA assay revealed thatintracellular CAT GPx-1 and GSH were downregulated after150 120583M H

2O2treatment in MES235 cells Of note follow-

ing treatment of KTF restored intracellular CAT GPx-1and GSH expression RT-PCR analysis indicated that H

2O2

treatment downregulated CAT andGPx-1mRNA expressionhowever KTF treatment restored the expressions of CATand GPx-1 mRNA in MES235 cells Immunoblot with speci-fic antibodies also confirmed that CAT and GPx-1 proteinexpression in MES235 cells were downregulated by H

2O2

treatment and KTF treatment was able to restore CAT and

Parkinsonrsquos Disease 5

(a) Control (b) H2O2 (c) GE

(d) KTF-L (e) KTF-M (f) KTF-H

Figure 2 Effect of KTF on cellular ultrastructure in H2O2-inducedMES235 cells Cellular ultrastructure was observed under TEM Normal

morphology of cytoplasm cell organelles in control group ((a) times15000) characteristic ultrastructural morphology of oxidative damage inH2O2-treated group ((b) times15000) treatment with KTF group (3125 625 and 125120583gml respectively) ((d) (e) (f) times15000) or Ginkgo

extract (125 120583gml) ((c) times15000)

GPx-1 protein expression Moreover mRNA expression ofoxidative stress responding protein HO-1 was also inhibitedafter H

2O2treatment and then was restored by following

KTF treatment Immunoblotting also proved that HO-1protein expression was markedly downregulated by H

2O2

but KTF treatment was able to restore HO-1 proteinexpression As expected the highest KTF concentrationused in our investigation has the best effect on restoringCAT GPx-1 GSH and HO-1 expression (Figures 5 and 6)Taken together the obtained results demonstrate that KTFtreatment significantly upregulated antioxidant substrates inthe H

2O2-treated MES233 cells

4 Discussion

As an important component of redox system ROS playimportant roles in regulating cell function Moderate levelsof ROS may function as signals to promote cell proliferationand survival Under physiologic conditions the balancebetween generation and elimination of ROS maintains theproper function of redox-sensitive signaling system Redoxhomeostasis can be disrupted by increase of ROS productionor decrease of ROS-scavenging capacity leading to an overall

increase in oxidative stress [17 18] subsequently causing anirreversible damage of intracellular structure and inducingcell death

High consumption of O2in brain leads to accumulation

of ROS thus neurons are particularly vulnerable to the attackof oxidative stress Postmortem investigations have shownlipid peroxidation and oxidative damage to brain cells in PDpatients [19] Growing studies confirm that oxidative damageplays a critical role in the pathogenesis of neurondegenerativediseases such as AD PDHD andALS [18 20] In the presentstudy a significant loss of cell viability was observed inMES235 dopaminergic neuronal cell after exposure to 50120583MH2O2for 12 h 25 120583M H

2O2for 24 h or 125 120583M H

2O2for

48 h As shown bymorphology observation the cells exposed12 h to 150 120583M H

2O2exhibited a typical oxidative injury

including multiple membranous vacuoles in the cytoplasmdisruption of endoplasmic reticulum and mitochondrionand condensing euchromatin in cell nucleus

According to Chinese medicine theory the deficiency ofkidney is the main pathological mechanism of PD Invigo-rating the kidney is an important strategy in the treatmentof PD In the present study we found that traditional kidney-tonifying formula KTF which consists ofCistanche Epimedii

6 Parkinsonrsquos Disease

Control GE

KTF-L KTF-M KTF-H

H2O2

(a)

Negativecontrol

(b)

0

20

40

60

80

100

120

140

160

Aver

age d

egre

e

Control H2O2 GE KTF-L KTF-M KTF-H

lowastlowast

lowastlowast

(c)

Figure 3 KTF ameliorated H2O2-induced reduction of TH inMES235 cells Cells were fixed with 4 paraformaldehyde and the expression

of TH was detected by immunocytochemistry (a) Tan represents the TH positive expression (times400) (b) Negative control (c) Results areexpressed as average grey degree Data are mean plusmn SD in three independent experiments 119875 lt 005 compared to the control group lowast119875 lt 005compared to the H

2O2-treat group

andPolygonatum sibiricum significantly enhanced cell viabil-ity and the expression of TH of H

2O2-treated MES235 cells

Cell apoptosis is a mode of programmed cell death uponvarious stimuli which activate either extrinsic or intrinsicpathways Cysteinyl aspartate-specific proteases (caspases)are highly conserved in multicellular organisms and functionas central regulators of apoptosis Activation of caspases isthe main axis of all apoptosis events Among those caspasescaspase 3 has been identified as a key mediator and indicatorof cell apoptosis Like other caspases caspase 3 usually existsas an inactive proenzyme (precaspase 3) After proteolyticprocessing it will produce two subunits which can dimerizeto form active caspase 3 subsequently evoking many of thebiochemical and biophysical changes to induce cell apoptosis[21] Our present study revealed that H

2O2treatment signif-

icantly upregulated caspase 3 but this trend was reversed byKTF treatment inMES235 cells indicating that KTF reducedcell apoptosis of H

2O2-treated MES235 cells

Antioxidative enzymes such as superoxide dismutase(SOD) CAT GPx-1 GSH and HO-1 are important defense

mechanism of cells against the oxidative stress As an antiox-idative enzyme SOD can transform superoxide radicals tohydrogen peroxide which was subsequently converted toH2O by CAT and GPx [22] GSH is also an important

antioxidant Catalytic conversion of reduced glutathione(GSH) to oxidized glutathione (GSSG) by GPx is helpfulto convert H

2O2to O2 It has been reported that the

activity of the antioxidant was decreased in PD modelmice [23ndash25] Therefore drugs that regulate expression ofenzymatic antioxidants may serve as potential candidates forthe treatment of PD Our data showed that H

2O2treatment

suppressed CAT GPx GSH and HO-1 in MES235 cellswhile KTF treatment at different concentration effectivelyrestored the expression of CAT GPx GSH and HO-1

In conclusion our findings demonstrate that traditionalChinese herb kidney-tonifying formulation which consistsofCistancheEpimedii and Polygonatum sibiricum has strongantioxidative capacity and significant neuroprotective effectsThis study provides a clue for developing the kidney-tonifyingformulation as a potential therapeutic agent to prevent andtreat Parkinsonrsquos disease

Parkinsonrsquos Disease 7

Procaspase 3

Caspase 3

훽-actin

35 KD

17 KD

45 KD

Control H2O2 GE KTF-L KTF-M KTF-H

(a)

lowast

lowastlowast

lowast

lowast

Control H2O2 GE KTF-L KTF-M KTF-H

Ratio

of p

roca

spas

e 3 an

d ca

spas

e 3훽

-act

in p

rote

in le

vel

250

200

150

100

050

000

Procaspase 3Caspase 3

(b)

Figure 4 Effect of KTF on the protein expression of caspase 3 in H2O2-induced MES235 cells (a) The protein expression of caspase 3 was

determined with western blot and quantified in (b) Data are expressed as mean plusmn SD in three independent experiments 119875 lt 005 comparedto the control group lowast119875 lt 005 compared to the H

2O2-treat group

00

01

02

03

04

05

06

CAT

conc

entr

atio

n (U

L)

Control H2O2 GE KTF-L KTF-M KTF-H

lowastlowastlowast

lowast

(a)

00

05

10

15

20

25

30

35

40

GPx

-1 co

ncen

trat

ion

(pm

olm

l)

Control H2O2 GE KTF-L KTF-M KTF-H

lowastlowast

lowast

(b)

0

20

40

60

80

100

120

GSH

conc

entr

atio

n (n

gm

l)

Control H2O2 GE KTF-L KTF-M KTF-H

lowast lowast lowast lowast

(c)

Figure 5 Effect of KTF on the level of intracellular CAT GPx-1 and GSH in H2O2-induced MES235 cells The activity level of intracellular

CAT GPx-1 and GSH was measured using an ELISA kit Data are expressed as mean plusmn SD in three independent experiments 119875 lt 005compared to the control group lowast119875 lt 005 compared to the H

2O2-treat group

8 Parkinsonrsquos Disease

HO-1

훽-actin

CAT

훽-actin

GPx-1

훽-actin

HO-1

훽-actin

CAT

훽-actin

GPx-1

훽-actin

(a) (c)

(d)(b)

00

02

04

06

08

10

12

GPx-1CATHO-1

Prot

ein

leve

l nor

mal

ized

with

훽-a

ctin

00

05

10

15

20

25m

RNA

leve

l nor

mal

ized

with

훽-a

ctin

Control

GE

KTF-LKTF-MKTF-HC

ontro

l

H2O

2

H2O2

GPx-1CATHO-1

Control

GE

KTF-LKTF-MKTF-H

H2O2

GE

KTF-

L

KTF-

M

KTF-

H

Con

trol

H2O

2

GE

KTF-

L

KTF-

M

KTF-

H

lowastlowastlowast

lowast

lowast

lowastlowast

lowast

lowastlowast

lowast

lowastlowastlowast

lowastlowastlowastlowast

lowast

lowast

lowastlowast

lowast

lowast

Figure 6 KTF increased the protein and mRNA expression of antioxidant enzymes in H2O2-induced MES235 cells (a) The protein

expressions of HO-1 CAT andGPx-1 were determined withWestern blot and quantified in (c) (b)ThemRNA expressions of HO-1 CAT andGPx-1 were determined with RT-PCR and quantified in (d) Data are expressed as mean plusmn SD in three independent experiments 119875 lt 005compared to the control group lowast119875 lt 005 compared to the H

2O2-treat group

Competing Interests

The authors declare that the research was conducted in theabsence of any commercial or financial relationships thatcould be construed as a potential conflict of interests

Authorsrsquo Contributions

Yihui Xu and Wei Lin share the same contribution to thearticle Chuanshan Xu is the cocorresponding author

Acknowledgments

This work was supported by Scientific Fund of NationalHealth and Family Planning Commission of the PeoplersquosRepublic of China (WKJ-FJ-38) Chen Keji DevelopmentFund Of Integrative Medicine (CKJ2014012) and Fund Of

KeyDisciplinary (X2014010-xueke)The authors express theirsincere thanks to Miss Xin Pan for her helpful assistance

References

[1] S Fahn andG Cohen ldquoThe oxidant stress hypothesis in Parkin-sonrsquos disease evidence supporting itrdquo Annals of Neurology vol32 no 6 pp 804ndash812 1992

[2] P Jenner andCWOlanow ldquoOxidative stress and the pathogen-esis of Parkinsonrsquos diseaserdquoNeurology vol 47 supplement 3 ppS161ndashS170 1996

[3] Y Akaneya M Takahashi and H Hatanaka ldquoInvolvement offree radicals in MPP+ neurotoxicity against rat dopaminergicneurons in culturerdquo Neuroscience Letters vol 193 no 1 pp 53ndash56 1995

[4] J Emerit M Edeas and F Bricaire ldquoNeurodegenerative dis-eases and oxidative stressrdquo Biomedicine amp Pharmacotherapyvol 58 no 1 pp 39ndash46 2004

Parkinsonrsquos Disease 9

[5] Chinese Pharmacopoeial Commission Pharmacopoeia of thePeoples Republic of China vol 1 Peoplersquos Medical PublishingHouse Beijing China 2010

[6] J Cai Y Tian R Lin X Chen Z Liu and J Xie ldquoProtectiveeffects of kidney-tonifying Chinese herbal preparation on sub-stantia nigra neurons in a mouse model of Parkinsonrsquos diseaserdquoNeural Regeneration Research vol 7 no 6 pp 413ndash420 2012

[7] WW Li R Yang and D F Cai ldquoProtective effects of Cistanchetotal glycosides on dopaminergic neuron in substantia nigra ofmodelmice of Parkinsonrsquos diseaserdquoZhongguo Zhong Xi Yi Jie HeZa Zhi vol 28 no 3 pp 248ndash251 2008

[8] W Wei and C Jing ldquoAn analysis of TCM pattern and formulafor parkinsonrsquos diseaserdquo Journal of Traditional ChineseMedicinevol 54 no 20 pp 1778ndash1782 2013

[9] Y Tian J Cai and X Chen ldquoProtection of kidney-tonifyingChinese herb on the nigra striatal neurons mice with Parkin-sonrsquos diseaserdquo Chinese Journal of Gerontology vol 31 no 3 pp440ndash443 2011

[10] S G Lin S F Ye JMHuang et al ldquoHow doChinesemedicinesthat tonify the kidney inhibit dopaminergic neuron apoptosisrdquoNeural Regeneration Research vol 8 no 30 pp 2820ndash28262013

[11] H Wang W-W Li D-F Cai and R Yang ldquoProtecting effect ofCistanche extracts on MPP+-induced injury of the Parkinsonrsquosdisease cell modelrdquo Journal of Chinese Integrative Medicine vol5 no 4 pp 407ndash411 2007

[12] X Geng X Tian P Tu and X Pu ldquoNeuroprotective effects ofechinacoside in themouseMPTPmodel of Parkinsonrsquos diseaserdquoEuropean Journal of Pharmacology vol 564 no 1ndash3 pp 66ndash742007

[13] X-F Tian and X-P Pu ldquoPhenylethanoid glycosides from Cis-tanches salsa inhibit apoptosis induced by 1-methyl-4-phe-nylpyridinium ion in neuronsrdquo Journal of Ethnopharmacologyvol 97 no 1 pp 59ndash63 2005

[14] X Geng L Song X Pu and P Tu ldquoNeuroprotective effectsof phenylethanoid glycosides from Cistanches salsa against 1-methyl-4-phenyl-1236-tetrahydropyridine (MPTP)-induceddopaminergic toxicity in C57 micerdquo Biological and Pharmaceu-tical Bulletin vol 27 no 6 pp 797ndash801 2004

[15] H SWong and KM Ko ldquoHerba Cistanches stimulates cellularglutathione redox cycling by reactive oxygen species generatedfrommitochondrial respiration inH9c2 cardiomyocytesrdquo Phar-maceutical Biology vol 51 no 1 pp 64ndash73 2013

[16] S C Wing Sze Y Tong T B Ng C L Yin Cheng and HP Cheung ldquoHerba Epimedii anti-oxidative properties and itsmedical implicationsrdquo Molecules vol 15 no 11 pp 7861ndash78702010

[17] D Trachootham W Lu M A Ogasawara N R-D Valle andP Huang ldquoRedox regulation of cell survivalrdquo Antioxidants andRedox Signaling vol 10 no 8 pp 1343ndash1374 2008

[18] D J Moore A B West V L Dawson and T M DawsonldquoMolecular pathophysiology of Parkinsonrsquos diseaserdquo AnnualReview of Neuroscience vol 28 pp 57ndash87 2005

[19] J Sian D T Dexter A J Lees et al ldquoAlterations in glutathionelevels in Parkinsonrsquos disease and other neurodegenerative disor-ders affecting basal gangliardquo Annals of Neurology vol 36 no 3pp 348ndash355 1994

[20] BHalliwell and JMCGutteridge ldquoRole of free radic in humandiseases an overviewrdquo Methods in Enzymology vol 186 pp 1ndash85 1990

[21] KVenderova andD S Park ldquoProgrammed cell death in Parkin-sonrsquos diseaserdquo Cold Spring Harbor Perspectives in Medicine vol2 no 8 Article ID a009365 2012

[22] I Fridovich ldquoFundamental aspects of reactive oxygen speciesor whatrsquos the matter with oxygenrdquo Annals of the New YorkAcademy of Sciences vol 893 pp 13ndash18 1999

[23] Q Xiong S Kadota T Tani and T Namba ldquoAntioxidativeeffects of phenylethanoids from Cistanche deserticolardquo Biolog-ical and Pharmaceutical Bulletin vol 19 no 12 pp 1580ndash15851996

[24] S Koppula H Kumar S V More H-W Lim S-M Hong andD-K Choi ldquoRecent updates in redox regulation and free radicalscavenging effects by herbal products in experimental modelsof Parkinsonrsquos diseaserdquoMolecules vol 17 no 10 pp 11391ndash114202012

[25] Z Fang and Y Wang ldquoEffect of isatin on the activities of anti-oxidase in PDmodel micerdquo China Pharmacy vol 19 no 22 pp1696ndash1697 2008

Submit your manuscripts athttpswwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Parkinsonrsquos Disease 5

(a) Control (b) H2O2 (c) GE

(d) KTF-L (e) KTF-M (f) KTF-H

Figure 2 Effect of KTF on cellular ultrastructure in H2O2-inducedMES235 cells Cellular ultrastructure was observed under TEM Normal

morphology of cytoplasm cell organelles in control group ((a) times15000) characteristic ultrastructural morphology of oxidative damage inH2O2-treated group ((b) times15000) treatment with KTF group (3125 625 and 125120583gml respectively) ((d) (e) (f) times15000) or Ginkgo

extract (125 120583gml) ((c) times15000)

GPx-1 protein expression Moreover mRNA expression ofoxidative stress responding protein HO-1 was also inhibitedafter H

2O2treatment and then was restored by following

KTF treatment Immunoblotting also proved that HO-1protein expression was markedly downregulated by H

2O2

but KTF treatment was able to restore HO-1 proteinexpression As expected the highest KTF concentrationused in our investigation has the best effect on restoringCAT GPx-1 GSH and HO-1 expression (Figures 5 and 6)Taken together the obtained results demonstrate that KTFtreatment significantly upregulated antioxidant substrates inthe H

2O2-treated MES233 cells

4 Discussion

As an important component of redox system ROS playimportant roles in regulating cell function Moderate levelsof ROS may function as signals to promote cell proliferationand survival Under physiologic conditions the balancebetween generation and elimination of ROS maintains theproper function of redox-sensitive signaling system Redoxhomeostasis can be disrupted by increase of ROS productionor decrease of ROS-scavenging capacity leading to an overall

increase in oxidative stress [17 18] subsequently causing anirreversible damage of intracellular structure and inducingcell death

High consumption of O2in brain leads to accumulation

of ROS thus neurons are particularly vulnerable to the attackof oxidative stress Postmortem investigations have shownlipid peroxidation and oxidative damage to brain cells in PDpatients [19] Growing studies confirm that oxidative damageplays a critical role in the pathogenesis of neurondegenerativediseases such as AD PDHD andALS [18 20] In the presentstudy a significant loss of cell viability was observed inMES235 dopaminergic neuronal cell after exposure to 50120583MH2O2for 12 h 25 120583M H

2O2for 24 h or 125 120583M H

2O2for

48 h As shown bymorphology observation the cells exposed12 h to 150 120583M H

2O2exhibited a typical oxidative injury

including multiple membranous vacuoles in the cytoplasmdisruption of endoplasmic reticulum and mitochondrionand condensing euchromatin in cell nucleus

According to Chinese medicine theory the deficiency ofkidney is the main pathological mechanism of PD Invigo-rating the kidney is an important strategy in the treatmentof PD In the present study we found that traditional kidney-tonifying formula KTF which consists ofCistanche Epimedii

6 Parkinsonrsquos Disease

Control GE

KTF-L KTF-M KTF-H

H2O2

(a)

Negativecontrol

(b)

0

20

40

60

80

100

120

140

160

Aver

age d

egre

e

Control H2O2 GE KTF-L KTF-M KTF-H

lowastlowast

lowastlowast

(c)

Figure 3 KTF ameliorated H2O2-induced reduction of TH inMES235 cells Cells were fixed with 4 paraformaldehyde and the expression

of TH was detected by immunocytochemistry (a) Tan represents the TH positive expression (times400) (b) Negative control (c) Results areexpressed as average grey degree Data are mean plusmn SD in three independent experiments 119875 lt 005 compared to the control group lowast119875 lt 005compared to the H

2O2-treat group

andPolygonatum sibiricum significantly enhanced cell viabil-ity and the expression of TH of H

2O2-treated MES235 cells

Cell apoptosis is a mode of programmed cell death uponvarious stimuli which activate either extrinsic or intrinsicpathways Cysteinyl aspartate-specific proteases (caspases)are highly conserved in multicellular organisms and functionas central regulators of apoptosis Activation of caspases isthe main axis of all apoptosis events Among those caspasescaspase 3 has been identified as a key mediator and indicatorof cell apoptosis Like other caspases caspase 3 usually existsas an inactive proenzyme (precaspase 3) After proteolyticprocessing it will produce two subunits which can dimerizeto form active caspase 3 subsequently evoking many of thebiochemical and biophysical changes to induce cell apoptosis[21] Our present study revealed that H

2O2treatment signif-

icantly upregulated caspase 3 but this trend was reversed byKTF treatment inMES235 cells indicating that KTF reducedcell apoptosis of H

2O2-treated MES235 cells

Antioxidative enzymes such as superoxide dismutase(SOD) CAT GPx-1 GSH and HO-1 are important defense

mechanism of cells against the oxidative stress As an antiox-idative enzyme SOD can transform superoxide radicals tohydrogen peroxide which was subsequently converted toH2O by CAT and GPx [22] GSH is also an important

antioxidant Catalytic conversion of reduced glutathione(GSH) to oxidized glutathione (GSSG) by GPx is helpfulto convert H

2O2to O2 It has been reported that the

activity of the antioxidant was decreased in PD modelmice [23ndash25] Therefore drugs that regulate expression ofenzymatic antioxidants may serve as potential candidates forthe treatment of PD Our data showed that H

2O2treatment

suppressed CAT GPx GSH and HO-1 in MES235 cellswhile KTF treatment at different concentration effectivelyrestored the expression of CAT GPx GSH and HO-1

In conclusion our findings demonstrate that traditionalChinese herb kidney-tonifying formulation which consistsofCistancheEpimedii and Polygonatum sibiricum has strongantioxidative capacity and significant neuroprotective effectsThis study provides a clue for developing the kidney-tonifyingformulation as a potential therapeutic agent to prevent andtreat Parkinsonrsquos disease

Parkinsonrsquos Disease 7

Procaspase 3

Caspase 3

훽-actin

35 KD

17 KD

45 KD

Control H2O2 GE KTF-L KTF-M KTF-H

(a)

lowast

lowastlowast

lowast

lowast

Control H2O2 GE KTF-L KTF-M KTF-H

Ratio

of p

roca

spas

e 3 an

d ca

spas

e 3훽

-act

in p

rote

in le

vel

250

200

150

100

050

000

Procaspase 3Caspase 3

(b)

Figure 4 Effect of KTF on the protein expression of caspase 3 in H2O2-induced MES235 cells (a) The protein expression of caspase 3 was

determined with western blot and quantified in (b) Data are expressed as mean plusmn SD in three independent experiments 119875 lt 005 comparedto the control group lowast119875 lt 005 compared to the H

2O2-treat group

00

01

02

03

04

05

06

CAT

conc

entr

atio

n (U

L)

Control H2O2 GE KTF-L KTF-M KTF-H

lowastlowastlowast

lowast

(a)

00

05

10

15

20

25

30

35

40

GPx

-1 co

ncen

trat

ion

(pm

olm

l)

Control H2O2 GE KTF-L KTF-M KTF-H

lowastlowast

lowast

(b)

0

20

40

60

80

100

120

GSH

conc

entr

atio

n (n

gm

l)

Control H2O2 GE KTF-L KTF-M KTF-H

lowast lowast lowast lowast

(c)

Figure 5 Effect of KTF on the level of intracellular CAT GPx-1 and GSH in H2O2-induced MES235 cells The activity level of intracellular

CAT GPx-1 and GSH was measured using an ELISA kit Data are expressed as mean plusmn SD in three independent experiments 119875 lt 005compared to the control group lowast119875 lt 005 compared to the H

2O2-treat group

8 Parkinsonrsquos Disease

HO-1

훽-actin

CAT

훽-actin

GPx-1

훽-actin

HO-1

훽-actin

CAT

훽-actin

GPx-1

훽-actin

(a) (c)

(d)(b)

00

02

04

06

08

10

12

GPx-1CATHO-1

Prot

ein

leve

l nor

mal

ized

with

훽-a

ctin

00

05

10

15

20

25m

RNA

leve

l nor

mal

ized

with

훽-a

ctin

Control

GE

KTF-LKTF-MKTF-HC

ontro

l

H2O

2

H2O2

GPx-1CATHO-1

Control

GE

KTF-LKTF-MKTF-H

H2O2

GE

KTF-

L

KTF-

M

KTF-

H

Con

trol

H2O

2

GE

KTF-

L

KTF-

M

KTF-

H

lowastlowastlowast

lowast

lowast

lowastlowast

lowast

lowastlowast

lowast

lowastlowastlowast

lowastlowastlowastlowast

lowast

lowast

lowastlowast

lowast

lowast

Figure 6 KTF increased the protein and mRNA expression of antioxidant enzymes in H2O2-induced MES235 cells (a) The protein

expressions of HO-1 CAT andGPx-1 were determined withWestern blot and quantified in (c) (b)ThemRNA expressions of HO-1 CAT andGPx-1 were determined with RT-PCR and quantified in (d) Data are expressed as mean plusmn SD in three independent experiments 119875 lt 005compared to the control group lowast119875 lt 005 compared to the H

2O2-treat group

Competing Interests

The authors declare that the research was conducted in theabsence of any commercial or financial relationships thatcould be construed as a potential conflict of interests

Authorsrsquo Contributions

Yihui Xu and Wei Lin share the same contribution to thearticle Chuanshan Xu is the cocorresponding author

Acknowledgments

This work was supported by Scientific Fund of NationalHealth and Family Planning Commission of the PeoplersquosRepublic of China (WKJ-FJ-38) Chen Keji DevelopmentFund Of Integrative Medicine (CKJ2014012) and Fund Of

KeyDisciplinary (X2014010-xueke)The authors express theirsincere thanks to Miss Xin Pan for her helpful assistance

References

[1] S Fahn andG Cohen ldquoThe oxidant stress hypothesis in Parkin-sonrsquos disease evidence supporting itrdquo Annals of Neurology vol32 no 6 pp 804ndash812 1992

[2] P Jenner andCWOlanow ldquoOxidative stress and the pathogen-esis of Parkinsonrsquos diseaserdquoNeurology vol 47 supplement 3 ppS161ndashS170 1996

[3] Y Akaneya M Takahashi and H Hatanaka ldquoInvolvement offree radicals in MPP+ neurotoxicity against rat dopaminergicneurons in culturerdquo Neuroscience Letters vol 193 no 1 pp 53ndash56 1995

[4] J Emerit M Edeas and F Bricaire ldquoNeurodegenerative dis-eases and oxidative stressrdquo Biomedicine amp Pharmacotherapyvol 58 no 1 pp 39ndash46 2004

Parkinsonrsquos Disease 9

[5] Chinese Pharmacopoeial Commission Pharmacopoeia of thePeoples Republic of China vol 1 Peoplersquos Medical PublishingHouse Beijing China 2010

[6] J Cai Y Tian R Lin X Chen Z Liu and J Xie ldquoProtectiveeffects of kidney-tonifying Chinese herbal preparation on sub-stantia nigra neurons in a mouse model of Parkinsonrsquos diseaserdquoNeural Regeneration Research vol 7 no 6 pp 413ndash420 2012

[7] WW Li R Yang and D F Cai ldquoProtective effects of Cistanchetotal glycosides on dopaminergic neuron in substantia nigra ofmodelmice of Parkinsonrsquos diseaserdquoZhongguo Zhong Xi Yi Jie HeZa Zhi vol 28 no 3 pp 248ndash251 2008

[8] W Wei and C Jing ldquoAn analysis of TCM pattern and formulafor parkinsonrsquos diseaserdquo Journal of Traditional ChineseMedicinevol 54 no 20 pp 1778ndash1782 2013

[9] Y Tian J Cai and X Chen ldquoProtection of kidney-tonifyingChinese herb on the nigra striatal neurons mice with Parkin-sonrsquos diseaserdquo Chinese Journal of Gerontology vol 31 no 3 pp440ndash443 2011

[10] S G Lin S F Ye JMHuang et al ldquoHow doChinesemedicinesthat tonify the kidney inhibit dopaminergic neuron apoptosisrdquoNeural Regeneration Research vol 8 no 30 pp 2820ndash28262013

[11] H Wang W-W Li D-F Cai and R Yang ldquoProtecting effect ofCistanche extracts on MPP+-induced injury of the Parkinsonrsquosdisease cell modelrdquo Journal of Chinese Integrative Medicine vol5 no 4 pp 407ndash411 2007

[12] X Geng X Tian P Tu and X Pu ldquoNeuroprotective effects ofechinacoside in themouseMPTPmodel of Parkinsonrsquos diseaserdquoEuropean Journal of Pharmacology vol 564 no 1ndash3 pp 66ndash742007

[13] X-F Tian and X-P Pu ldquoPhenylethanoid glycosides from Cis-tanches salsa inhibit apoptosis induced by 1-methyl-4-phe-nylpyridinium ion in neuronsrdquo Journal of Ethnopharmacologyvol 97 no 1 pp 59ndash63 2005

[14] X Geng L Song X Pu and P Tu ldquoNeuroprotective effectsof phenylethanoid glycosides from Cistanches salsa against 1-methyl-4-phenyl-1236-tetrahydropyridine (MPTP)-induceddopaminergic toxicity in C57 micerdquo Biological and Pharmaceu-tical Bulletin vol 27 no 6 pp 797ndash801 2004

[15] H SWong and KM Ko ldquoHerba Cistanches stimulates cellularglutathione redox cycling by reactive oxygen species generatedfrommitochondrial respiration inH9c2 cardiomyocytesrdquo Phar-maceutical Biology vol 51 no 1 pp 64ndash73 2013

[16] S C Wing Sze Y Tong T B Ng C L Yin Cheng and HP Cheung ldquoHerba Epimedii anti-oxidative properties and itsmedical implicationsrdquo Molecules vol 15 no 11 pp 7861ndash78702010

[17] D Trachootham W Lu M A Ogasawara N R-D Valle andP Huang ldquoRedox regulation of cell survivalrdquo Antioxidants andRedox Signaling vol 10 no 8 pp 1343ndash1374 2008

[18] D J Moore A B West V L Dawson and T M DawsonldquoMolecular pathophysiology of Parkinsonrsquos diseaserdquo AnnualReview of Neuroscience vol 28 pp 57ndash87 2005

[19] J Sian D T Dexter A J Lees et al ldquoAlterations in glutathionelevels in Parkinsonrsquos disease and other neurodegenerative disor-ders affecting basal gangliardquo Annals of Neurology vol 36 no 3pp 348ndash355 1994

[20] BHalliwell and JMCGutteridge ldquoRole of free radic in humandiseases an overviewrdquo Methods in Enzymology vol 186 pp 1ndash85 1990

[21] KVenderova andD S Park ldquoProgrammed cell death in Parkin-sonrsquos diseaserdquo Cold Spring Harbor Perspectives in Medicine vol2 no 8 Article ID a009365 2012

[22] I Fridovich ldquoFundamental aspects of reactive oxygen speciesor whatrsquos the matter with oxygenrdquo Annals of the New YorkAcademy of Sciences vol 893 pp 13ndash18 1999

[23] Q Xiong S Kadota T Tani and T Namba ldquoAntioxidativeeffects of phenylethanoids from Cistanche deserticolardquo Biolog-ical and Pharmaceutical Bulletin vol 19 no 12 pp 1580ndash15851996

[24] S Koppula H Kumar S V More H-W Lim S-M Hong andD-K Choi ldquoRecent updates in redox regulation and free radicalscavenging effects by herbal products in experimental modelsof Parkinsonrsquos diseaserdquoMolecules vol 17 no 10 pp 11391ndash114202012

[25] Z Fang and Y Wang ldquoEffect of isatin on the activities of anti-oxidase in PDmodel micerdquo China Pharmacy vol 19 no 22 pp1696ndash1697 2008

Submit your manuscripts athttpswwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

6 Parkinsonrsquos Disease

Control GE

KTF-L KTF-M KTF-H

H2O2

(a)

Negativecontrol

(b)

0

20

40

60

80

100

120

140

160

Aver

age d

egre

e

Control H2O2 GE KTF-L KTF-M KTF-H

lowastlowast

lowastlowast

(c)

Figure 3 KTF ameliorated H2O2-induced reduction of TH inMES235 cells Cells were fixed with 4 paraformaldehyde and the expression

of TH was detected by immunocytochemistry (a) Tan represents the TH positive expression (times400) (b) Negative control (c) Results areexpressed as average grey degree Data are mean plusmn SD in three independent experiments 119875 lt 005 compared to the control group lowast119875 lt 005compared to the H

2O2-treat group

andPolygonatum sibiricum significantly enhanced cell viabil-ity and the expression of TH of H

2O2-treated MES235 cells

Cell apoptosis is a mode of programmed cell death uponvarious stimuli which activate either extrinsic or intrinsicpathways Cysteinyl aspartate-specific proteases (caspases)are highly conserved in multicellular organisms and functionas central regulators of apoptosis Activation of caspases isthe main axis of all apoptosis events Among those caspasescaspase 3 has been identified as a key mediator and indicatorof cell apoptosis Like other caspases caspase 3 usually existsas an inactive proenzyme (precaspase 3) After proteolyticprocessing it will produce two subunits which can dimerizeto form active caspase 3 subsequently evoking many of thebiochemical and biophysical changes to induce cell apoptosis[21] Our present study revealed that H

2O2treatment signif-

icantly upregulated caspase 3 but this trend was reversed byKTF treatment inMES235 cells indicating that KTF reducedcell apoptosis of H

2O2-treated MES235 cells

Antioxidative enzymes such as superoxide dismutase(SOD) CAT GPx-1 GSH and HO-1 are important defense

mechanism of cells against the oxidative stress As an antiox-idative enzyme SOD can transform superoxide radicals tohydrogen peroxide which was subsequently converted toH2O by CAT and GPx [22] GSH is also an important

antioxidant Catalytic conversion of reduced glutathione(GSH) to oxidized glutathione (GSSG) by GPx is helpfulto convert H

2O2to O2 It has been reported that the

activity of the antioxidant was decreased in PD modelmice [23ndash25] Therefore drugs that regulate expression ofenzymatic antioxidants may serve as potential candidates forthe treatment of PD Our data showed that H

2O2treatment

suppressed CAT GPx GSH and HO-1 in MES235 cellswhile KTF treatment at different concentration effectivelyrestored the expression of CAT GPx GSH and HO-1

In conclusion our findings demonstrate that traditionalChinese herb kidney-tonifying formulation which consistsofCistancheEpimedii and Polygonatum sibiricum has strongantioxidative capacity and significant neuroprotective effectsThis study provides a clue for developing the kidney-tonifyingformulation as a potential therapeutic agent to prevent andtreat Parkinsonrsquos disease

Parkinsonrsquos Disease 7

Procaspase 3

Caspase 3

훽-actin

35 KD

17 KD

45 KD

Control H2O2 GE KTF-L KTF-M KTF-H

(a)

lowast

lowastlowast

lowast

lowast

Control H2O2 GE KTF-L KTF-M KTF-H

Ratio

of p

roca

spas

e 3 an

d ca

spas

e 3훽

-act

in p

rote

in le

vel

250

200

150

100

050

000

Procaspase 3Caspase 3

(b)

Figure 4 Effect of KTF on the protein expression of caspase 3 in H2O2-induced MES235 cells (a) The protein expression of caspase 3 was

determined with western blot and quantified in (b) Data are expressed as mean plusmn SD in three independent experiments 119875 lt 005 comparedto the control group lowast119875 lt 005 compared to the H

2O2-treat group

00

01

02

03

04

05

06

CAT

conc

entr

atio

n (U

L)

Control H2O2 GE KTF-L KTF-M KTF-H

lowastlowastlowast

lowast

(a)

00

05

10

15

20

25

30

35

40

GPx

-1 co

ncen

trat

ion

(pm

olm

l)

Control H2O2 GE KTF-L KTF-M KTF-H

lowastlowast

lowast

(b)

0

20

40

60

80

100

120

GSH

conc

entr

atio

n (n

gm

l)

Control H2O2 GE KTF-L KTF-M KTF-H

lowast lowast lowast lowast

(c)

Figure 5 Effect of KTF on the level of intracellular CAT GPx-1 and GSH in H2O2-induced MES235 cells The activity level of intracellular

CAT GPx-1 and GSH was measured using an ELISA kit Data are expressed as mean plusmn SD in three independent experiments 119875 lt 005compared to the control group lowast119875 lt 005 compared to the H

2O2-treat group

8 Parkinsonrsquos Disease

HO-1

훽-actin

CAT

훽-actin

GPx-1

훽-actin

HO-1

훽-actin

CAT

훽-actin

GPx-1

훽-actin

(a) (c)

(d)(b)

00

02

04

06

08

10

12

GPx-1CATHO-1

Prot

ein

leve

l nor

mal

ized

with

훽-a

ctin

00

05

10

15

20

25m

RNA

leve

l nor

mal

ized

with

훽-a

ctin

Control

GE

KTF-LKTF-MKTF-HC

ontro

l

H2O

2

H2O2

GPx-1CATHO-1

Control

GE

KTF-LKTF-MKTF-H

H2O2

GE

KTF-

L

KTF-

M

KTF-

H

Con

trol

H2O

2

GE

KTF-

L

KTF-

M

KTF-

H

lowastlowastlowast

lowast

lowast

lowastlowast

lowast

lowastlowast

lowast

lowastlowastlowast

lowastlowastlowastlowast

lowast

lowast

lowastlowast

lowast

lowast

Figure 6 KTF increased the protein and mRNA expression of antioxidant enzymes in H2O2-induced MES235 cells (a) The protein

expressions of HO-1 CAT andGPx-1 were determined withWestern blot and quantified in (c) (b)ThemRNA expressions of HO-1 CAT andGPx-1 were determined with RT-PCR and quantified in (d) Data are expressed as mean plusmn SD in three independent experiments 119875 lt 005compared to the control group lowast119875 lt 005 compared to the H

2O2-treat group

Competing Interests

The authors declare that the research was conducted in theabsence of any commercial or financial relationships thatcould be construed as a potential conflict of interests

Authorsrsquo Contributions

Yihui Xu and Wei Lin share the same contribution to thearticle Chuanshan Xu is the cocorresponding author

Acknowledgments

This work was supported by Scientific Fund of NationalHealth and Family Planning Commission of the PeoplersquosRepublic of China (WKJ-FJ-38) Chen Keji DevelopmentFund Of Integrative Medicine (CKJ2014012) and Fund Of

KeyDisciplinary (X2014010-xueke)The authors express theirsincere thanks to Miss Xin Pan for her helpful assistance

References

[1] S Fahn andG Cohen ldquoThe oxidant stress hypothesis in Parkin-sonrsquos disease evidence supporting itrdquo Annals of Neurology vol32 no 6 pp 804ndash812 1992

[2] P Jenner andCWOlanow ldquoOxidative stress and the pathogen-esis of Parkinsonrsquos diseaserdquoNeurology vol 47 supplement 3 ppS161ndashS170 1996

[3] Y Akaneya M Takahashi and H Hatanaka ldquoInvolvement offree radicals in MPP+ neurotoxicity against rat dopaminergicneurons in culturerdquo Neuroscience Letters vol 193 no 1 pp 53ndash56 1995

[4] J Emerit M Edeas and F Bricaire ldquoNeurodegenerative dis-eases and oxidative stressrdquo Biomedicine amp Pharmacotherapyvol 58 no 1 pp 39ndash46 2004

Parkinsonrsquos Disease 9

[5] Chinese Pharmacopoeial Commission Pharmacopoeia of thePeoples Republic of China vol 1 Peoplersquos Medical PublishingHouse Beijing China 2010

[6] J Cai Y Tian R Lin X Chen Z Liu and J Xie ldquoProtectiveeffects of kidney-tonifying Chinese herbal preparation on sub-stantia nigra neurons in a mouse model of Parkinsonrsquos diseaserdquoNeural Regeneration Research vol 7 no 6 pp 413ndash420 2012

[7] WW Li R Yang and D F Cai ldquoProtective effects of Cistanchetotal glycosides on dopaminergic neuron in substantia nigra ofmodelmice of Parkinsonrsquos diseaserdquoZhongguo Zhong Xi Yi Jie HeZa Zhi vol 28 no 3 pp 248ndash251 2008

[8] W Wei and C Jing ldquoAn analysis of TCM pattern and formulafor parkinsonrsquos diseaserdquo Journal of Traditional ChineseMedicinevol 54 no 20 pp 1778ndash1782 2013

[9] Y Tian J Cai and X Chen ldquoProtection of kidney-tonifyingChinese herb on the nigra striatal neurons mice with Parkin-sonrsquos diseaserdquo Chinese Journal of Gerontology vol 31 no 3 pp440ndash443 2011

[10] S G Lin S F Ye JMHuang et al ldquoHow doChinesemedicinesthat tonify the kidney inhibit dopaminergic neuron apoptosisrdquoNeural Regeneration Research vol 8 no 30 pp 2820ndash28262013

[11] H Wang W-W Li D-F Cai and R Yang ldquoProtecting effect ofCistanche extracts on MPP+-induced injury of the Parkinsonrsquosdisease cell modelrdquo Journal of Chinese Integrative Medicine vol5 no 4 pp 407ndash411 2007

[12] X Geng X Tian P Tu and X Pu ldquoNeuroprotective effects ofechinacoside in themouseMPTPmodel of Parkinsonrsquos diseaserdquoEuropean Journal of Pharmacology vol 564 no 1ndash3 pp 66ndash742007

[13] X-F Tian and X-P Pu ldquoPhenylethanoid glycosides from Cis-tanches salsa inhibit apoptosis induced by 1-methyl-4-phe-nylpyridinium ion in neuronsrdquo Journal of Ethnopharmacologyvol 97 no 1 pp 59ndash63 2005

[14] X Geng L Song X Pu and P Tu ldquoNeuroprotective effectsof phenylethanoid glycosides from Cistanches salsa against 1-methyl-4-phenyl-1236-tetrahydropyridine (MPTP)-induceddopaminergic toxicity in C57 micerdquo Biological and Pharmaceu-tical Bulletin vol 27 no 6 pp 797ndash801 2004

[15] H SWong and KM Ko ldquoHerba Cistanches stimulates cellularglutathione redox cycling by reactive oxygen species generatedfrommitochondrial respiration inH9c2 cardiomyocytesrdquo Phar-maceutical Biology vol 51 no 1 pp 64ndash73 2013

[16] S C Wing Sze Y Tong T B Ng C L Yin Cheng and HP Cheung ldquoHerba Epimedii anti-oxidative properties and itsmedical implicationsrdquo Molecules vol 15 no 11 pp 7861ndash78702010

[17] D Trachootham W Lu M A Ogasawara N R-D Valle andP Huang ldquoRedox regulation of cell survivalrdquo Antioxidants andRedox Signaling vol 10 no 8 pp 1343ndash1374 2008

[18] D J Moore A B West V L Dawson and T M DawsonldquoMolecular pathophysiology of Parkinsonrsquos diseaserdquo AnnualReview of Neuroscience vol 28 pp 57ndash87 2005

[19] J Sian D T Dexter A J Lees et al ldquoAlterations in glutathionelevels in Parkinsonrsquos disease and other neurodegenerative disor-ders affecting basal gangliardquo Annals of Neurology vol 36 no 3pp 348ndash355 1994

[20] BHalliwell and JMCGutteridge ldquoRole of free radic in humandiseases an overviewrdquo Methods in Enzymology vol 186 pp 1ndash85 1990

[21] KVenderova andD S Park ldquoProgrammed cell death in Parkin-sonrsquos diseaserdquo Cold Spring Harbor Perspectives in Medicine vol2 no 8 Article ID a009365 2012

[22] I Fridovich ldquoFundamental aspects of reactive oxygen speciesor whatrsquos the matter with oxygenrdquo Annals of the New YorkAcademy of Sciences vol 893 pp 13ndash18 1999

[23] Q Xiong S Kadota T Tani and T Namba ldquoAntioxidativeeffects of phenylethanoids from Cistanche deserticolardquo Biolog-ical and Pharmaceutical Bulletin vol 19 no 12 pp 1580ndash15851996

[24] S Koppula H Kumar S V More H-W Lim S-M Hong andD-K Choi ldquoRecent updates in redox regulation and free radicalscavenging effects by herbal products in experimental modelsof Parkinsonrsquos diseaserdquoMolecules vol 17 no 10 pp 11391ndash114202012

[25] Z Fang and Y Wang ldquoEffect of isatin on the activities of anti-oxidase in PDmodel micerdquo China Pharmacy vol 19 no 22 pp1696ndash1697 2008

Submit your manuscripts athttpswwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Parkinsonrsquos Disease 7

Procaspase 3

Caspase 3

훽-actin

35 KD

17 KD

45 KD

Control H2O2 GE KTF-L KTF-M KTF-H

(a)

lowast

lowastlowast

lowast

lowast

Control H2O2 GE KTF-L KTF-M KTF-H

Ratio

of p

roca

spas

e 3 an

d ca

spas

e 3훽

-act

in p

rote

in le

vel

250

200

150

100

050

000

Procaspase 3Caspase 3

(b)

Figure 4 Effect of KTF on the protein expression of caspase 3 in H2O2-induced MES235 cells (a) The protein expression of caspase 3 was

determined with western blot and quantified in (b) Data are expressed as mean plusmn SD in three independent experiments 119875 lt 005 comparedto the control group lowast119875 lt 005 compared to the H

2O2-treat group

00

01

02

03

04

05

06

CAT

conc

entr

atio

n (U

L)

Control H2O2 GE KTF-L KTF-M KTF-H

lowastlowastlowast

lowast

(a)

00

05

10

15

20

25

30

35

40

GPx

-1 co

ncen

trat

ion

(pm

olm

l)

Control H2O2 GE KTF-L KTF-M KTF-H

lowastlowast

lowast

(b)

0

20

40

60

80

100

120

GSH

conc

entr

atio

n (n

gm

l)

Control H2O2 GE KTF-L KTF-M KTF-H

lowast lowast lowast lowast

(c)

Figure 5 Effect of KTF on the level of intracellular CAT GPx-1 and GSH in H2O2-induced MES235 cells The activity level of intracellular

CAT GPx-1 and GSH was measured using an ELISA kit Data are expressed as mean plusmn SD in three independent experiments 119875 lt 005compared to the control group lowast119875 lt 005 compared to the H

2O2-treat group

8 Parkinsonrsquos Disease

HO-1

훽-actin

CAT

훽-actin

GPx-1

훽-actin

HO-1

훽-actin

CAT

훽-actin

GPx-1

훽-actin

(a) (c)

(d)(b)

00

02

04

06

08

10

12

GPx-1CATHO-1

Prot

ein

leve

l nor

mal

ized

with

훽-a

ctin

00

05

10

15

20

25m

RNA

leve

l nor

mal

ized

with

훽-a

ctin

Control

GE

KTF-LKTF-MKTF-HC

ontro

l

H2O

2

H2O2

GPx-1CATHO-1

Control

GE

KTF-LKTF-MKTF-H

H2O2

GE

KTF-

L

KTF-

M

KTF-

H

Con

trol

H2O

2

GE

KTF-

L

KTF-

M

KTF-

H

lowastlowastlowast

lowast

lowast

lowastlowast

lowast

lowastlowast

lowast

lowastlowastlowast

lowastlowastlowastlowast

lowast

lowast

lowastlowast

lowast

lowast

Figure 6 KTF increased the protein and mRNA expression of antioxidant enzymes in H2O2-induced MES235 cells (a) The protein

expressions of HO-1 CAT andGPx-1 were determined withWestern blot and quantified in (c) (b)ThemRNA expressions of HO-1 CAT andGPx-1 were determined with RT-PCR and quantified in (d) Data are expressed as mean plusmn SD in three independent experiments 119875 lt 005compared to the control group lowast119875 lt 005 compared to the H

2O2-treat group

Competing Interests

The authors declare that the research was conducted in theabsence of any commercial or financial relationships thatcould be construed as a potential conflict of interests

Authorsrsquo Contributions

Yihui Xu and Wei Lin share the same contribution to thearticle Chuanshan Xu is the cocorresponding author

Acknowledgments

This work was supported by Scientific Fund of NationalHealth and Family Planning Commission of the PeoplersquosRepublic of China (WKJ-FJ-38) Chen Keji DevelopmentFund Of Integrative Medicine (CKJ2014012) and Fund Of

KeyDisciplinary (X2014010-xueke)The authors express theirsincere thanks to Miss Xin Pan for her helpful assistance

References

[1] S Fahn andG Cohen ldquoThe oxidant stress hypothesis in Parkin-sonrsquos disease evidence supporting itrdquo Annals of Neurology vol32 no 6 pp 804ndash812 1992

[2] P Jenner andCWOlanow ldquoOxidative stress and the pathogen-esis of Parkinsonrsquos diseaserdquoNeurology vol 47 supplement 3 ppS161ndashS170 1996

[3] Y Akaneya M Takahashi and H Hatanaka ldquoInvolvement offree radicals in MPP+ neurotoxicity against rat dopaminergicneurons in culturerdquo Neuroscience Letters vol 193 no 1 pp 53ndash56 1995

[4] J Emerit M Edeas and F Bricaire ldquoNeurodegenerative dis-eases and oxidative stressrdquo Biomedicine amp Pharmacotherapyvol 58 no 1 pp 39ndash46 2004

Parkinsonrsquos Disease 9

[5] Chinese Pharmacopoeial Commission Pharmacopoeia of thePeoples Republic of China vol 1 Peoplersquos Medical PublishingHouse Beijing China 2010

[6] J Cai Y Tian R Lin X Chen Z Liu and J Xie ldquoProtectiveeffects of kidney-tonifying Chinese herbal preparation on sub-stantia nigra neurons in a mouse model of Parkinsonrsquos diseaserdquoNeural Regeneration Research vol 7 no 6 pp 413ndash420 2012

[7] WW Li R Yang and D F Cai ldquoProtective effects of Cistanchetotal glycosides on dopaminergic neuron in substantia nigra ofmodelmice of Parkinsonrsquos diseaserdquoZhongguo Zhong Xi Yi Jie HeZa Zhi vol 28 no 3 pp 248ndash251 2008

[8] W Wei and C Jing ldquoAn analysis of TCM pattern and formulafor parkinsonrsquos diseaserdquo Journal of Traditional ChineseMedicinevol 54 no 20 pp 1778ndash1782 2013

[9] Y Tian J Cai and X Chen ldquoProtection of kidney-tonifyingChinese herb on the nigra striatal neurons mice with Parkin-sonrsquos diseaserdquo Chinese Journal of Gerontology vol 31 no 3 pp440ndash443 2011

[10] S G Lin S F Ye JMHuang et al ldquoHow doChinesemedicinesthat tonify the kidney inhibit dopaminergic neuron apoptosisrdquoNeural Regeneration Research vol 8 no 30 pp 2820ndash28262013

[11] H Wang W-W Li D-F Cai and R Yang ldquoProtecting effect ofCistanche extracts on MPP+-induced injury of the Parkinsonrsquosdisease cell modelrdquo Journal of Chinese Integrative Medicine vol5 no 4 pp 407ndash411 2007

[12] X Geng X Tian P Tu and X Pu ldquoNeuroprotective effects ofechinacoside in themouseMPTPmodel of Parkinsonrsquos diseaserdquoEuropean Journal of Pharmacology vol 564 no 1ndash3 pp 66ndash742007

[13] X-F Tian and X-P Pu ldquoPhenylethanoid glycosides from Cis-tanches salsa inhibit apoptosis induced by 1-methyl-4-phe-nylpyridinium ion in neuronsrdquo Journal of Ethnopharmacologyvol 97 no 1 pp 59ndash63 2005

[14] X Geng L Song X Pu and P Tu ldquoNeuroprotective effectsof phenylethanoid glycosides from Cistanches salsa against 1-methyl-4-phenyl-1236-tetrahydropyridine (MPTP)-induceddopaminergic toxicity in C57 micerdquo Biological and Pharmaceu-tical Bulletin vol 27 no 6 pp 797ndash801 2004

[15] H SWong and KM Ko ldquoHerba Cistanches stimulates cellularglutathione redox cycling by reactive oxygen species generatedfrommitochondrial respiration inH9c2 cardiomyocytesrdquo Phar-maceutical Biology vol 51 no 1 pp 64ndash73 2013

[16] S C Wing Sze Y Tong T B Ng C L Yin Cheng and HP Cheung ldquoHerba Epimedii anti-oxidative properties and itsmedical implicationsrdquo Molecules vol 15 no 11 pp 7861ndash78702010

[17] D Trachootham W Lu M A Ogasawara N R-D Valle andP Huang ldquoRedox regulation of cell survivalrdquo Antioxidants andRedox Signaling vol 10 no 8 pp 1343ndash1374 2008

[18] D J Moore A B West V L Dawson and T M DawsonldquoMolecular pathophysiology of Parkinsonrsquos diseaserdquo AnnualReview of Neuroscience vol 28 pp 57ndash87 2005

[19] J Sian D T Dexter A J Lees et al ldquoAlterations in glutathionelevels in Parkinsonrsquos disease and other neurodegenerative disor-ders affecting basal gangliardquo Annals of Neurology vol 36 no 3pp 348ndash355 1994

[20] BHalliwell and JMCGutteridge ldquoRole of free radic in humandiseases an overviewrdquo Methods in Enzymology vol 186 pp 1ndash85 1990

[21] KVenderova andD S Park ldquoProgrammed cell death in Parkin-sonrsquos diseaserdquo Cold Spring Harbor Perspectives in Medicine vol2 no 8 Article ID a009365 2012

[22] I Fridovich ldquoFundamental aspects of reactive oxygen speciesor whatrsquos the matter with oxygenrdquo Annals of the New YorkAcademy of Sciences vol 893 pp 13ndash18 1999

[23] Q Xiong S Kadota T Tani and T Namba ldquoAntioxidativeeffects of phenylethanoids from Cistanche deserticolardquo Biolog-ical and Pharmaceutical Bulletin vol 19 no 12 pp 1580ndash15851996

[24] S Koppula H Kumar S V More H-W Lim S-M Hong andD-K Choi ldquoRecent updates in redox regulation and free radicalscavenging effects by herbal products in experimental modelsof Parkinsonrsquos diseaserdquoMolecules vol 17 no 10 pp 11391ndash114202012

[25] Z Fang and Y Wang ldquoEffect of isatin on the activities of anti-oxidase in PDmodel micerdquo China Pharmacy vol 19 no 22 pp1696ndash1697 2008

Submit your manuscripts athttpswwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

8 Parkinsonrsquos Disease

HO-1

훽-actin

CAT

훽-actin

GPx-1

훽-actin

HO-1

훽-actin

CAT

훽-actin

GPx-1

훽-actin

(a) (c)

(d)(b)

00

02

04

06

08

10

12

GPx-1CATHO-1

Prot

ein

leve

l nor

mal

ized

with

훽-a

ctin

00

05

10

15

20

25m

RNA

leve

l nor

mal

ized

with

훽-a

ctin

Control

GE

KTF-LKTF-MKTF-HC

ontro

l

H2O

2

H2O2

GPx-1CATHO-1

Control

GE

KTF-LKTF-MKTF-H

H2O2

GE

KTF-

L

KTF-

M

KTF-

H

Con

trol

H2O

2

GE

KTF-

L

KTF-

M

KTF-

H

lowastlowastlowast

lowast

lowast

lowastlowast

lowast

lowastlowast

lowast

lowastlowastlowast

lowastlowastlowastlowast

lowast

lowast

lowastlowast

lowast

lowast

Figure 6 KTF increased the protein and mRNA expression of antioxidant enzymes in H2O2-induced MES235 cells (a) The protein

expressions of HO-1 CAT andGPx-1 were determined withWestern blot and quantified in (c) (b)ThemRNA expressions of HO-1 CAT andGPx-1 were determined with RT-PCR and quantified in (d) Data are expressed as mean plusmn SD in three independent experiments 119875 lt 005compared to the control group lowast119875 lt 005 compared to the H

2O2-treat group

Competing Interests

The authors declare that the research was conducted in theabsence of any commercial or financial relationships thatcould be construed as a potential conflict of interests

Authorsrsquo Contributions

Yihui Xu and Wei Lin share the same contribution to thearticle Chuanshan Xu is the cocorresponding author

Acknowledgments

This work was supported by Scientific Fund of NationalHealth and Family Planning Commission of the PeoplersquosRepublic of China (WKJ-FJ-38) Chen Keji DevelopmentFund Of Integrative Medicine (CKJ2014012) and Fund Of

KeyDisciplinary (X2014010-xueke)The authors express theirsincere thanks to Miss Xin Pan for her helpful assistance

References

[1] S Fahn andG Cohen ldquoThe oxidant stress hypothesis in Parkin-sonrsquos disease evidence supporting itrdquo Annals of Neurology vol32 no 6 pp 804ndash812 1992

[2] P Jenner andCWOlanow ldquoOxidative stress and the pathogen-esis of Parkinsonrsquos diseaserdquoNeurology vol 47 supplement 3 ppS161ndashS170 1996

[3] Y Akaneya M Takahashi and H Hatanaka ldquoInvolvement offree radicals in MPP+ neurotoxicity against rat dopaminergicneurons in culturerdquo Neuroscience Letters vol 193 no 1 pp 53ndash56 1995

[4] J Emerit M Edeas and F Bricaire ldquoNeurodegenerative dis-eases and oxidative stressrdquo Biomedicine amp Pharmacotherapyvol 58 no 1 pp 39ndash46 2004

Parkinsonrsquos Disease 9

[5] Chinese Pharmacopoeial Commission Pharmacopoeia of thePeoples Republic of China vol 1 Peoplersquos Medical PublishingHouse Beijing China 2010

[6] J Cai Y Tian R Lin X Chen Z Liu and J Xie ldquoProtectiveeffects of kidney-tonifying Chinese herbal preparation on sub-stantia nigra neurons in a mouse model of Parkinsonrsquos diseaserdquoNeural Regeneration Research vol 7 no 6 pp 413ndash420 2012

[7] WW Li R Yang and D F Cai ldquoProtective effects of Cistanchetotal glycosides on dopaminergic neuron in substantia nigra ofmodelmice of Parkinsonrsquos diseaserdquoZhongguo Zhong Xi Yi Jie HeZa Zhi vol 28 no 3 pp 248ndash251 2008

[8] W Wei and C Jing ldquoAn analysis of TCM pattern and formulafor parkinsonrsquos diseaserdquo Journal of Traditional ChineseMedicinevol 54 no 20 pp 1778ndash1782 2013

[9] Y Tian J Cai and X Chen ldquoProtection of kidney-tonifyingChinese herb on the nigra striatal neurons mice with Parkin-sonrsquos diseaserdquo Chinese Journal of Gerontology vol 31 no 3 pp440ndash443 2011

[10] S G Lin S F Ye JMHuang et al ldquoHow doChinesemedicinesthat tonify the kidney inhibit dopaminergic neuron apoptosisrdquoNeural Regeneration Research vol 8 no 30 pp 2820ndash28262013

[11] H Wang W-W Li D-F Cai and R Yang ldquoProtecting effect ofCistanche extracts on MPP+-induced injury of the Parkinsonrsquosdisease cell modelrdquo Journal of Chinese Integrative Medicine vol5 no 4 pp 407ndash411 2007

[12] X Geng X Tian P Tu and X Pu ldquoNeuroprotective effects ofechinacoside in themouseMPTPmodel of Parkinsonrsquos diseaserdquoEuropean Journal of Pharmacology vol 564 no 1ndash3 pp 66ndash742007

[13] X-F Tian and X-P Pu ldquoPhenylethanoid glycosides from Cis-tanches salsa inhibit apoptosis induced by 1-methyl-4-phe-nylpyridinium ion in neuronsrdquo Journal of Ethnopharmacologyvol 97 no 1 pp 59ndash63 2005

[14] X Geng L Song X Pu and P Tu ldquoNeuroprotective effectsof phenylethanoid glycosides from Cistanches salsa against 1-methyl-4-phenyl-1236-tetrahydropyridine (MPTP)-induceddopaminergic toxicity in C57 micerdquo Biological and Pharmaceu-tical Bulletin vol 27 no 6 pp 797ndash801 2004

[15] H SWong and KM Ko ldquoHerba Cistanches stimulates cellularglutathione redox cycling by reactive oxygen species generatedfrommitochondrial respiration inH9c2 cardiomyocytesrdquo Phar-maceutical Biology vol 51 no 1 pp 64ndash73 2013

[16] S C Wing Sze Y Tong T B Ng C L Yin Cheng and HP Cheung ldquoHerba Epimedii anti-oxidative properties and itsmedical implicationsrdquo Molecules vol 15 no 11 pp 7861ndash78702010

[17] D Trachootham W Lu M A Ogasawara N R-D Valle andP Huang ldquoRedox regulation of cell survivalrdquo Antioxidants andRedox Signaling vol 10 no 8 pp 1343ndash1374 2008

[18] D J Moore A B West V L Dawson and T M DawsonldquoMolecular pathophysiology of Parkinsonrsquos diseaserdquo AnnualReview of Neuroscience vol 28 pp 57ndash87 2005

[19] J Sian D T Dexter A J Lees et al ldquoAlterations in glutathionelevels in Parkinsonrsquos disease and other neurodegenerative disor-ders affecting basal gangliardquo Annals of Neurology vol 36 no 3pp 348ndash355 1994

[20] BHalliwell and JMCGutteridge ldquoRole of free radic in humandiseases an overviewrdquo Methods in Enzymology vol 186 pp 1ndash85 1990

[21] KVenderova andD S Park ldquoProgrammed cell death in Parkin-sonrsquos diseaserdquo Cold Spring Harbor Perspectives in Medicine vol2 no 8 Article ID a009365 2012

[22] I Fridovich ldquoFundamental aspects of reactive oxygen speciesor whatrsquos the matter with oxygenrdquo Annals of the New YorkAcademy of Sciences vol 893 pp 13ndash18 1999

[23] Q Xiong S Kadota T Tani and T Namba ldquoAntioxidativeeffects of phenylethanoids from Cistanche deserticolardquo Biolog-ical and Pharmaceutical Bulletin vol 19 no 12 pp 1580ndash15851996

[24] S Koppula H Kumar S V More H-W Lim S-M Hong andD-K Choi ldquoRecent updates in redox regulation and free radicalscavenging effects by herbal products in experimental modelsof Parkinsonrsquos diseaserdquoMolecules vol 17 no 10 pp 11391ndash114202012

[25] Z Fang and Y Wang ldquoEffect of isatin on the activities of anti-oxidase in PDmodel micerdquo China Pharmacy vol 19 no 22 pp1696ndash1697 2008

Submit your manuscripts athttpswwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Parkinsonrsquos Disease 9

[5] Chinese Pharmacopoeial Commission Pharmacopoeia of thePeoples Republic of China vol 1 Peoplersquos Medical PublishingHouse Beijing China 2010

[6] J Cai Y Tian R Lin X Chen Z Liu and J Xie ldquoProtectiveeffects of kidney-tonifying Chinese herbal preparation on sub-stantia nigra neurons in a mouse model of Parkinsonrsquos diseaserdquoNeural Regeneration Research vol 7 no 6 pp 413ndash420 2012

[7] WW Li R Yang and D F Cai ldquoProtective effects of Cistanchetotal glycosides on dopaminergic neuron in substantia nigra ofmodelmice of Parkinsonrsquos diseaserdquoZhongguo Zhong Xi Yi Jie HeZa Zhi vol 28 no 3 pp 248ndash251 2008

[8] W Wei and C Jing ldquoAn analysis of TCM pattern and formulafor parkinsonrsquos diseaserdquo Journal of Traditional ChineseMedicinevol 54 no 20 pp 1778ndash1782 2013

[9] Y Tian J Cai and X Chen ldquoProtection of kidney-tonifyingChinese herb on the nigra striatal neurons mice with Parkin-sonrsquos diseaserdquo Chinese Journal of Gerontology vol 31 no 3 pp440ndash443 2011

[10] S G Lin S F Ye JMHuang et al ldquoHow doChinesemedicinesthat tonify the kidney inhibit dopaminergic neuron apoptosisrdquoNeural Regeneration Research vol 8 no 30 pp 2820ndash28262013

[11] H Wang W-W Li D-F Cai and R Yang ldquoProtecting effect ofCistanche extracts on MPP+-induced injury of the Parkinsonrsquosdisease cell modelrdquo Journal of Chinese Integrative Medicine vol5 no 4 pp 407ndash411 2007

[12] X Geng X Tian P Tu and X Pu ldquoNeuroprotective effects ofechinacoside in themouseMPTPmodel of Parkinsonrsquos diseaserdquoEuropean Journal of Pharmacology vol 564 no 1ndash3 pp 66ndash742007

[13] X-F Tian and X-P Pu ldquoPhenylethanoid glycosides from Cis-tanches salsa inhibit apoptosis induced by 1-methyl-4-phe-nylpyridinium ion in neuronsrdquo Journal of Ethnopharmacologyvol 97 no 1 pp 59ndash63 2005

[14] X Geng L Song X Pu and P Tu ldquoNeuroprotective effectsof phenylethanoid glycosides from Cistanches salsa against 1-methyl-4-phenyl-1236-tetrahydropyridine (MPTP)-induceddopaminergic toxicity in C57 micerdquo Biological and Pharmaceu-tical Bulletin vol 27 no 6 pp 797ndash801 2004

[15] H SWong and KM Ko ldquoHerba Cistanches stimulates cellularglutathione redox cycling by reactive oxygen species generatedfrommitochondrial respiration inH9c2 cardiomyocytesrdquo Phar-maceutical Biology vol 51 no 1 pp 64ndash73 2013

[16] S C Wing Sze Y Tong T B Ng C L Yin Cheng and HP Cheung ldquoHerba Epimedii anti-oxidative properties and itsmedical implicationsrdquo Molecules vol 15 no 11 pp 7861ndash78702010

[17] D Trachootham W Lu M A Ogasawara N R-D Valle andP Huang ldquoRedox regulation of cell survivalrdquo Antioxidants andRedox Signaling vol 10 no 8 pp 1343ndash1374 2008

[18] D J Moore A B West V L Dawson and T M DawsonldquoMolecular pathophysiology of Parkinsonrsquos diseaserdquo AnnualReview of Neuroscience vol 28 pp 57ndash87 2005

[19] J Sian D T Dexter A J Lees et al ldquoAlterations in glutathionelevels in Parkinsonrsquos disease and other neurodegenerative disor-ders affecting basal gangliardquo Annals of Neurology vol 36 no 3pp 348ndash355 1994

[20] BHalliwell and JMCGutteridge ldquoRole of free radic in humandiseases an overviewrdquo Methods in Enzymology vol 186 pp 1ndash85 1990

[21] KVenderova andD S Park ldquoProgrammed cell death in Parkin-sonrsquos diseaserdquo Cold Spring Harbor Perspectives in Medicine vol2 no 8 Article ID a009365 2012

[22] I Fridovich ldquoFundamental aspects of reactive oxygen speciesor whatrsquos the matter with oxygenrdquo Annals of the New YorkAcademy of Sciences vol 893 pp 13ndash18 1999

[23] Q Xiong S Kadota T Tani and T Namba ldquoAntioxidativeeffects of phenylethanoids from Cistanche deserticolardquo Biolog-ical and Pharmaceutical Bulletin vol 19 no 12 pp 1580ndash15851996

[24] S Koppula H Kumar S V More H-W Lim S-M Hong andD-K Choi ldquoRecent updates in redox regulation and free radicalscavenging effects by herbal products in experimental modelsof Parkinsonrsquos diseaserdquoMolecules vol 17 no 10 pp 11391ndash114202012

[25] Z Fang and Y Wang ldquoEffect of isatin on the activities of anti-oxidase in PDmodel micerdquo China Pharmacy vol 19 no 22 pp1696ndash1697 2008

Submit your manuscripts athttpswwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Submit your manuscripts athttpswwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom