Neuroprotective Effects of Gagam-Sipjeondaebo-Tang, a

Research ArticleNeuroprotective Effects of Gagam-Sipjeondaebo-Tanga Novel Herbal Formula against MPTP-Induced ParkinsonianMice and MPP+-Induced Cell Death in SH-SY5Y Cells

Jade Heejae Ko1 Ju-Hee Lee 1 Bobin Choi1 Ju-Yeon Park1 Young-Won Kwon 1

Songhee Jeon2 Sun-Dong Park 1 and Seung-NamKim 1

1College of KoreanMedicine Dongguk University Goyang 10326 Republic of Korea2Department of Biomedical Sciences Center for Creative Biomedical Scientists Chonnam National UniversityGwangju 61469 Republic of Korea

Correspondence should be addressed to Seung-Nam Kim snkimdonggukedu

Received 24 August 2018 Revised 19 October 2018 Accepted 5 November 2018 Published 4 December 2018

Academic Editor Yoshiji Ohta

Copyright copy 2018 Jade Heejae Ko et alThis is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Parkinsonrsquos disease is a neurodegenerative disease characterized by progressive cell death of dopaminergic neuron and followingneurological disorders Gagam-Sipjeondaebo-Tang (GST) is a novel herbal formula made of twelve medicinal herbs derivedfrom Sipjeondaebo-Tang which has been broadly used in a traditional herbal medicine In the present study we investigatedthe effects of GST against 1-methyl-4-phenyl-1236-tetrahydropyridine (MPTP)-induced motor abnormalities in mice and 1-methyl-4-phenylpyridinium (MPP+)-inducedneurotoxicity in SH-SY5Y cell First we found thatGST alleviatedmotor dysfunctioninduced by MPTP and the result showed dopaminergic neurons recovery in substantia nigra In the cell experiment pretreatmentwith GST increased the cell viability and attenuated apoptotic cell death in MPP+-treated SH-SY5Y cells GST also inhibitedreactive oxygen species production and restored the mitochondrial membrane potential loss which were induced by MPP+Furthermore GST extract significantly activated ERK and Akt cell survival-related proteins in SH-SY5Y cells The effect of GSTpreventing mitochondrial dysfunction was antagonized by pretreatment of PD98059 and LY294002 selective inhibitors of ERKand Akt respectively Taken together GST alleviated abnormal motor functions and recovered neuronal cell death mitochondrialdysfunction possibly via ERK and Akt activation Therefore we suggest that GST may be a candidate for the treatment andprevention of Parkinsonrsquos disease

1 Introduction

Parkinsonrsquos disease (PD) is characterized by extensive celldeath of dopaminergic neuron in the substantia nigra andsubsequently develops into other neurological disorders [1]It has been known that familial and sporadic pathologicalfactors induce neuronal deficits by reactive oxygen stressand following mitochondrial dysfunction [2] Consideringhow rapidly population of PD grows there is comparativelyless development of conventional methods so that alternativetreatments for PD has been drawing attention [3]

Sipjeondaebo-Tang (SDT Shi-Quan-Da-Bu-Tang in Chi-nese and Juzen-Taiho-To in Japanese) is broadly used totreat neurological patients in traditional East Asian herbal

medicine [4] It has been shown that SDT has anti-inflammatory effect in LPS-stimulated RAW cells [5] reduc-tion of toxicity and regulation of immune system in hepatictumor [6] It was also revealed that SDT recovered DNAfragmentation and mitochondrial dysfunction in antitumordrug given mice [7] Gagam-Sipjeondaebo-Tang (GST) is anovel herbal formula of twelve medicinal herbs derived fromSDT GST is modified formula of SDT with two additionalherbs which are Uncaria rhynchophylla and Gastrodia elataBlume The two herbs were reported to have positive effecton PD [8 9] and have been used for patients who havemotor dysfunctions in East Asia countries FurthermoreUncaria rhynchophylla extract has shown neuroprotectiveeffect in MPP+-induced SH-SY5Y cells and MPTP-induced

HindawiEvidence-Based Complementary and Alternative MedicineVolume 2018 Article ID 2420809 9 pageshttpsdoiorg10115520182420809

2 Evidence-Based Complementary and Alternative Medicine

Table 1 The compositions of GST

Latin Name Scientific name (Family name) RatioGinseng Radix Alba Panax ginseng C A Meyer (Araliaceae) 3Atractylodis Rhizoma Alba Atractylodes macrocephala Koidzumi (Compositae) 2Hoelen Poria cocosWolf (Polyporaceae) 2Glycyrrhizae Radix et Rhizoma Glycyrrhiza uralensis Fischer (Leguminosae) 2Rehmanniae Radix Preparata Rehmannia glutinosa Liboschitz ex Steudel (Scrophulariaceae) 2Angelicae Gigantis Radix Angelica gigas Nakai (Umbelliferae) 2Cnidii Rhizoma Cnidium officinaleMakino (Umbelliferae) 2Paeoniae Radix Paeonia lactiflora Pallas (Paeoniaceae) 2Astragali Radix Astragalus membranaceus Bunge (Leguminosae) 3Bupleuri Radix Bupleurum falcatum Linne (Umbelliferae) 2Uncariae Ramulus et Uncus Uncaria rhynchophylla (Rubiaceae) 2Gastrodiae Rhizoma Gastrodia elata Blume (Orchidaceae) 2

mice by increasing cell viability and ameliorating behavioralimpairment through dopamine and tyrosine hydroxylase(TH) expression elevation [10] Anti-apoptotic and antiox-idative aspects of Gastrodia elata Blume in MPP+-inducedcytotoxicity dopaminergic cells were also suggested [11] Theearlier studies suggest that GST potentially has neuropro-tective effects of dopaminergic neuron and its followingmotor dysfunctions of PD Therefore in this study weinvestigated effects of GST on PD and its underlying mech-anism using 1-methyl-4-phenyl-1236-tetrahydropyridine(MPTP)-induced Parkinsonian mice model and 1-methyl-4-phenylpyridinium (MPP+)-induced cell death

2 Materials and Methods

21 Animals Male C57BL6 mice (Orient-Bio Co Seong-nam Republic of Korea) 9 weeks of age and weighing20ndash25 g each were used The animals were housed under a1212-h lightdark cycle with free access to food and waterAll experiments were approved by the Dongguk UniversityAnimal Care Committee for animal welfare (2016-DGU-46) and maintained in strict accordance with guidelines Todevelop PD animal model we used acute MPTP regimen onmice Briefly intraperitoneal MPTP injection (20 mgkg ofbody weight Sigma-Aldrich St Louis USA) was performedevery 2 hours four times a day in total One day afterMPTP injection rotarod test was done and only mice thatshowed motor function deficits more than 30 comparedto control group were divided into experimental groups forthis study control group MPTP group and MPTP+GSTgroup 300 mgkg of GST was given to MPTP+GST grouponce a day for 5 days by oral administration There were 6mice in each group The same volume of physiological salinewas administered to mice in the control group After thebehavioral tests the mice were sacrificed and the substantianigra in the brain was removed Detailed timeline of theanimal study is shown in Figure 1(a)

22 Behavioral Test We have conducted rotarod test andpole test to examine the motor function of the mice For therotarod test mice were located on a rotating rod accelerating

from 5 to 20 rpm for a 240 seconds time period Each mousewas scored for time they sustained on the rod The mice thatcompleted the task over 240 seconds scored as 240 whichwas the maximum value To examine pole test mice werelocated head toward up close to top of the pole We recordedthe time when themicersquos heads completely rotated downward(P1) and when they reached the floor (P2) Falling latencywas calculated by subtracting P1 fromP2Themaximum timelimit was determined as 120 seconds

23 Plant Materials and Preparation of GST Extract TheGST was formulated from twelve medicinal herbs Panaxginseng C A Meyer Atractylodes macrocephala KoidzumiPoria cocos Wolf Glycyrrhiza uralensis Fischer Rehmanniaglutinosa Liboschitz ex Steudel Angelica gigas Nakai Cnid-ium officinale Makino Paeonia lactiflora Pallas Astragalusmembranaceus Bunge Bupleurum falcatum Linne Uncariarhynchophylla and Gastrodia elata Blume All herbs werepurchased as dried herbs from Omniherb (Seoul Republicof Korea) Briefly a mixture of the twelve dried components(100 g weight ratios shown inTable 1) was extracted in 800mlof 30 ethanol by heating at 80∘C for 4 h and extracts werefiltered twice through a filter paper (40 120583mWhatman Buck-inghamshireUK) Filtrateswere concentrated under reducedpressure using a rotary vacuum evaporator (EYELA TokyoJapan) at 40sim45∘C and the concentrate was lyophilized usinga freeze dryer (EYELA Tokyo Japan) The yield of the GSTextract (dried powder) was 227 of the weight of the driedstarting materials

24 Cell Culture SH-SY5Y cells a human neuroblastomacell line were purchased from the Korean Cell Line Bank(Seoul Republic of Korea) Cells were cultured in Dulbeccorsquosmodified Eaglersquos medium (DMEM Welgene GyeongsanRepublic of Korea) supplemented with 10 fetal bovineserum (FBS Thermo Scientific Waltham USA) and 1penicillinstreptomycin Cultures were maintained at 37∘C ina CO2incubator with a controlled humidified atmosphere

composed of 95 air and 5 CO2 FBS cell culture media

penicillinstreptomycin and all other reagents used for cell

Evidence-Based Complementary and Alternative Medicine 3

Day 1Day 0 3 4 51 week

AdaptationMPTP

Behavioral test

GST

Behavioral test

2

(a)

Tim

e on

rod

(sec

)

0

50

100

150

200

250

Control MPTP MPTP+GST

lowast

(b)

0

5

10

15

20

25

Late

ncy

to fa

ll (s

ec)


lowastlowast

(c)

00

05

10

15

TH - 60kDa

- 42kDa

(rel

ativ

e exp

ress

ion)

TH

-act

in

lowast

MPTP+GSTMPTPControl

-actin

(d)

Figure 1 Effects of GST extract on motor functions in MPTP-induced Parkinsonian mice (a) Timeline of the animal test in this studyFilled arrowheads indicate MPTP injections to induce Parkinsonian mice Blank arrowheads indicate GST treatment Dashed arrow showsthe day of behavioral test (b) Effects of GST on the duration of MPTP and control mice stayed on the rotating rod (c) The result of timespent in the pole testThe time spent before mice arrived on the ground from top of the pole wasmeasured (d)Western blot shows differenceof TH level between control group MPTP group and GST administered MPTP group p lt 001 and p lt 0001 compared to controlgroup lowast p lt 005 and lowastlowast p lt 001 compared to MPTP group analyzed by one-way ANOVA followed by Bonferronirsquos post hoc test

culture studies were purchased from HyClone Laboratories(Logan USA)

25 Cell Viability Assay Cell viabilities were evaluated usinga 3-(45-dimethylthiazol-2-yl)-25-diphenyltetrazolium bro-mide (MTT Sigma-Aldrich St Louis USA) assay SH-SY5Y

cells were plated at a density of 3-5 times 103 cellswell andpretreated with either dimethyl sulfoxide (DMSO JunseiChemical Co Tokyo Japan) or GST (10-300 120583gml) for 1h and then incubated in the presence or absence of 1 mMMPP+ (Sigma-Aldrich St Louis USA) for 24 h Viable cellswere then stained with MTT solution (02 mgml 3 h) and


formazan crystals were dissolved in 100 120583l of 01 DMSOAbsorbance was measured at 540 nm using a multimodemicroplate reader (Tecan Morrisville USA)

26 Measurement of Mitochondrial Membrane PotentialsMitochondrial membrane potentials were measured by flowcytometry using rhodamine 123 (Sigma-Aldrich St LouisUSA) a membrane-permeable cationic fluorescent dye SH-SY5Y cells were stained with 005 120583gml of rhodamine 123for 1 h and harvested by trypsinization After washing withphosphate-buffered saline (PBS) containing 1 FBS changesin mitochondrial membrane potentials were examined bymeasuring fluorescence intensities using a CytoFLEX flowcytometer (Beckman Coulter Inc Brea USA)

27 Measurement of ROS Production SH-SY5Y cells wereplated at a density of 2 times 105 cellswell for 24 h Cellswere incubated with 100 120583gml of GST for 1 h and latertreated with 1 mM MPP+ for 18 h After the treatment cellswere stained with 5 120583M of 21015840 71015840-dichlorodihydrofluoresceindiacetate (H

2DCF-DA Sigma-Aldrich St Louis USA) for 30

min at 37∘C For each analysis a total of 10000 events wererecorded Fluorescence intensity in the cells was measuredusing a CytoFLEX flow cytometer

28 Western Blot Analysis Samples were lysed with RIPAbuffer (Thermo Scientific Waltham USA) containing phos-phatase and protease inhibitor cocktail (Gen-DEPOT KatyUSA) and then protein concentrations were assessed usinga BCA protein assay kit (Thermo Scientific Waltham USA)Equal amount of proteins were separated by 10 SDS-PAGEtransferred onto PVDF membranes (Millipore BurlingtonUSA) and blocked with 5 skim milk Membranes wereincubated with primary antibodies overnight at 4∘C washed3 times with PBS containing 01 Tween 20 and incu-bated with HRP-conjugated secondary antibodies (NovusLittleton USA) for 1 h at room temperature Detection wasperformed using an enhanced chemiluminescence system(Amersham Biosciences Little Chalfont UK) The primaryantibodies used in this study were as follows 120573-actin waspurchased from Santa Cruz (Santa Cruz USA) TH wasobtained from Novus (Littleton USA) procaspase-3 Bcl-2 phospho-Akt Akt phospho-ERK12 and ERK12 wereobtained from Cell Signaling Technologies (Danvers USA)The band intensity of the detected proteins were measured byImage J version 18

29 Statistical Analysis All the data are shown as means plusmnSEM One-way ANOVA followed by Bonferronirsquos multiplecomparison tests was performed to determine the signifi-cance of differences using GraphPad Prism software (Graph-Pad Software Inc La Jolla USA) Statistical significance wasset at p lt 005 for all analyses

3 Result

31 GST Improves Motor Function Impairment in Parkin-sonian Mice We performed the pole test and rotarod test

to examine the effect of GST on improvement of motorfunction First of all mice in the MPTP group spent 50less time on the rotarod compared to the mice in thecontrol group This shows that MPTP resulted in motordysfunction in mice consequently (p lt 001 Figure 1(b))Compared to MPTP group motor function of mice wasrecovered to normal level in MPTP+GST group (p lt 005Figure 1(b)) Similarly pole test showed that administrationof GST reduced total time spent until reaching the floorcompared with mice in MPTP group which means that GSTtreatment ameliorated motor complications caused byMPTP(p lt 0001 control versus MPTP and p lt 001 MPTP versusMPTP+GST Figure 1(c)) Next we performed western blotanalysis for all the experimental groups of mice Comparedwith the control group the level of TH was about 60 lowerin the MPTP group Importantly we could find increasedlevel of TH in the GST administered MPTP group comparedwith the MPTP group (p lt 001 Control versus MPTP and plt 005 MPTP versus MPTP+GST Figure 1(d))

32 Effects of GST on MPP+-Induced Cell Death in SH-SY5YCells To evaluate cytotoxicity of GST SH-SY5Y cells weretreated with various concentrations of GST extract (10 3050 100 300 or 500 120583gml) for 24 h and then the cell viabilitywas measured using MTT assay GST concentration rangedfrom 10 to 300 120583gml and did not affect viability of SH-SY5Y cells whereas 500 120583gml of GST treatment reducedthe cell viability (p lt 0001 Figure 2(a)) Next to examinethe effect of GST against MPP+-induced neurotoxicity SH-SY5Y cells were treated with GST (10 100 200 or 300120583gml) for 1 h and then further exposed to 1 mM MPP+ for24 h As a result 100 120583gml of GST pretreatment showedsignificant increase in cell viability against MPP+-inducedcell death According to these results 100 120583gml of GSTwas selected as an effective dose for further experiments (plt 0001 Figure 2(b)) And the effect of GST on apoptosisinduced by MPP+ was investigated by western blot analysisof apoptosis-related proteins expression As a result MPP+reduced expression level of procaspase-3 and Bcl-2 whereascleaved caspase-3 was increased (Figure 2(c)) And theywere altered by GST pretreatment (Figure 2(c)) To test cellsignaling proteins related to cell proliferation we examinedAkt and ERK phosphorylation levels by western blot analysisIn the result both phosphorylation levels of Akt and ERKwere decreased by MPP+ and these were altered by GSTpretreatment (Figure 2(d)) Overall these results suggest thatGST recovered cell viability against MPP+-toxicity possiblyvia change of apoptosis- and cell proliferation-related pro-teins expression level

33 Effects of GST on MPP+-Induced Oxidative Stress in SH-SY5Y Cells To further investigate the protective effects ofGST against oxidative stress intracellular reactive oxygenspecies (ROS) levels were assessed by the flow cytometricassay using H

2DCF-DA SH-SY5Y cells were pretreated with

100 120583gml of GST for 1 h and then stimulated with or without1 mM MPP+ for 18 h After staining with 5 120583M H

2DCF-

DA ROS production was monitored by measuring intensity


0

20

40

60

80

100

120C

ell v

iabi

lity

()

10 30 50 100 300 5000GST (gml)

(a)

GST (gml)

0

20

40

60

80

100

120

Cel

l via

bilit

y (

)

- 10 100 200 300Con

－００+ (1 mM)

lowastlowastlowast

(b)

-32kDa

-19kDa-17kDa

-27kDa

-42kDa

GST (100 gml) minus minus

Bcl-2

Procaspase-3

Cleaved caspase-3

-actin

+ +MPP+

(c)

-60kDa

-60kDa

-44kDa-42kDa

-44kDa-42kDa

p-Akt

Akt

p-ERK

ERK

GST (100 gml) minus minus + +MPP+

(d)

Figure 2 Effects of GST extract on MPP+-induced cell death in SH-SY5Y cells (a) Effects of GST extract on SH-SY5Y cell viability Cellswere treated with various concentrations of GST extract (10 30 50 100 300 or 500 120583gml) for 24 h Cell viability was determined by MTTassay Values were expressed as percentages of the control (b) Effects of GST extract against MPP+-induced neurotoxicity in SH-SY5Y cellsSH-SY5Y cells were pretreated with GST (10 100 200 or 300 120583gml) for 1 h and then exposed to 1 mM MPP+ for 24 h The cell viabilitywas measured using an MTT assay p lt 0001 compared to control group lowastlowastlowastp lt 0001 compared to MPP+ treatment analyzed byone-way ANOVA followed by Bonferronirsquos post hoc test (c) Effects of GST on MPP+-induced apoptosis in SH-SY5Y cells The expressionof apoptosis-related proteins were measured by western blot in cells treated with 100 120583gml GST in the absence or presence of 1 mM MPP+for 24 h (d) Effects of GST on MPP+-induced changes of cell signaling proteins in SH-SY5Y cellsThe expression of cell proliferation-relatedproteins was assayed by western blot in cells treated with 100 120583gml GST in the absence or presence of 1 mMMPP+ for 24 h

of DCF fluorescence in cells using the flow cytometer Theresults showed that 1 mM MPP+ treatment induced about30 ROS generation (p lt 0001 Control versus MPP+)whereas pretreatment of GST significantly inhibited MPP+-induced ROS increment (p lt 0001MPP+ versusMPP++GSTFigure 3)

34 Effects of GST on MPP+-Induced Mitochondrial Dys-function in SH-SY5Y Cells To examine whether GST hasprotective effect on mitochondrial dysfunction by MPP+in SH-SY5Y cells mitochondrial membrane potentials weremeasured using the flow cytometry SH-SY5Y cells weretreated with 100 120583gml of GST for 1 h and then stimulatedwith or without 1 mM MPP+ for 18 h After staining withrhodamine 123 cells were loaded on the flow cytometerMPP+ treatment increased the proportion of cells with lowrhodamine 123 fluorescence intensity (431 plusmn 04 p lt 0001

control versus MPP+) as indicated bymitochondrial damageand dysfunction (Figure 4) In contrast GST pretreatmentsignificantly reduced the proportion of cells with low Rh123fluorescence intensity (185 plusmn 09 p lt 0001 MPP+ versusMPP++GST) These results show that GST alleviated MPP+-induced mitochondrial dysfunction

35 Effects of GST on the Akt and ERK Signaling in SH-SY5Y Cells To elucidate the cellular mechanism linkedwith the cytoprotective effects of GST we examined cellsurvival-related signaling pathways by western blot analy-sis According to the result we found that expressions ofphosphorylated-Akt and -ERK were significantly increasedby treatment of 100 120583gml of GST (Figure 5(a))The phospho-rylation of ERK was strongly expressed in 10 min after GSTtreatment and sustained for 3 h Meanwhile Akt phosphory-lation started 30 min after GST treatment peaked at 3 h andlasted for 6 h


(100 gml)

0

10

20

30

40

DCF

fluo

resc

ence

()

- GST GSTCon

lowastlowastlowast

－００+ (1 mM)

Figure 3 Effects of GST extract onMPP+-inducedROS generation in SH-SY5Y cellsCells were pretreatedwith 100 120583gml GST for 1 h andthen stimulated with or without 1 mM MPP+ for 18 h After staining with 5 120583MH

2DCF-DA ROS production was monitored by measuring

intensity of DCF fluorescence in cells using a flow cytometer p lt 0001 compared to control group lowastlowastlowastp lt 0001 compared to MPP+treatment analyzed by one-way ANOVA followed by Bonferronirsquos post hoc test

0

200

400

Control

Cou

nt

FITC-H

Multi-sample P1

GST

103 104 105

－００++GST－００+

(a)

Rh1

23+

cells

()

0

20

40

60

80

100

Con GST GST

MPP+ (1 mM)

-

lowastlowastlowast

(b)

Figure 4 Effects of GST extract on MPP+-induced mitochondrial dysfunction in SH-SY5Y cells Cells were pretreated with 100 120583gmlGST for 1 h and then stimulated with or without 1 mM MPP+ for 18 h After staining with rhodamine 123 cells were subjected on flowcytometer p lt 0001 compared to control group lowastlowastlowast p lt 0001 compared to MPP+ treatment analyzed by one-way ANOVA followedby Bonferronirsquos post hoc test

To investigate the role of ERK and Akt activation inthe cytoprotective effect of GST mitochondrial membranepotentials were measured after treating SH-SY5Y cells withspecific inhibitors of each protein As shown in Figure 5(b)the protective effect of GST against the toxicity of MPP+was partially blocked by PD98059 and LY294002 selectiveinhibitors of ERK and Akt respectively These observationssuggest that the neuroprotective effects of GST extract wereassociated at least in part with ERK and Akt activation

4 Discussion

It has been thought that loss of dopaminergic neurons in thesubstantia nigra as a result of oxidative stress is one of the

potential causes of PD [12] For the treatment of PD a lot ofdrugs including dopaminergic drugs and nondopaminergicdrugs have been widely used in last few decades howeverdetrimentalmotor complications of the current PD treatmentmedications are still considerable [13] There are a numberof on-going researches aiming for discovering alternative PDtreatments with fewer side effects Herbal medicines are oneof the alternative treatments of PD and many other diseases

GST is modified formula of SDT with two additionalherbs Uncaria rhynchophylla and Gastrodia elata BlumeSDT has been used for patients diagnosed with deficiencyof qi and blood in East Asian medicine and Uncaria rhyn-chophylla and Gastrodia elata Blume have been known tocontrol tremor in the traditional herbal medicine [5 8 9 14]


Time (h) 30 1 1263

-actin

p-ERK

ERK

p-Akt

Akt

GST (100 gml)

-44kDa

-44kDa

-42kDa

-42kDa

-60kDa

-60kDa

-42kDa

100

(a)

Rh1

23+

cells

()

0

20

40

60

80

100

ampampamp

ampampamp

----

-+--

++

--

++

+

-

+

+

+

-

GST

LY294002PD98059

MPP+

lowastlowastlowast

(b)

Figure 5 Effects of GST on the Akt and ERK signaling in SH-SY5Y cells (a) Akt and ERK activation by GST SH-SY5Y cells were treatedwith 100 120583gml GST for the indicated times The protein levels of Akt and ERK and their phosphorylated forms were analyzed by westernblot (b) Role of Akt and ERK activation by GST in mitochondrial function After LY294002 and PD98059 pretreatment (10 120583M 1 h) cellswere incubated with GST for 1 h and then treated with 1 mMMPP+ for 18 h After staining with rhodamine 123 cells were subjected on flowcytometer p lt 0001 compared to control group lowastlowastlowast p lt 0001 compared to MPP+ treatment ampampamp p lt 0001 compared to MPP++GSTtreatment analyzed by one-way ANOVA followed by Bonferronirsquos post hoc test

Because patients diagnosed with PD are generally consideredto exhibit unbalanced qi and blood with tremor in orientalmedicine GST has been used in order to alter the symptomsof PD under clinical conditions

In the present study we examined the effect of GST invivo and in vitro by using MPTP-induced PD mice modelandMPP+-induced cell death in SH-SY5Ycells In the animalstudy improvement in motor function of PD mice modelwas evident in rotarod test and pole test after GST treatmentNeuroprotective effect of GST was suggested by the in vitrostudy MTT assay results showed that GST increased viabilityof cells and inhibited apoptosis against MPP+ toxicity Also

decrease in proportion of low fluorescence intensity wasdetected after GST pretreatment indicating that mitochon-drial dysfunctionwas attenuated byGSTpretreatmentMPP+toxicity caused increment of ROS and resulted in oxidativestress in SH-SY5Y cells By using H

2DCF-DA staining it was

shown that GST inhibited MPP+-induced increase of ROSWe could also see that protective effect of GST was

attenuated when selective inhibitors antagonized ERK orAktAkt andERKaremitogen activated protein kinases which areknown to regulate cell proliferation and cell differentiationby activating downstream CRE gene pathway Akt- and ERK-initiated transcription of CRE-binding protein is known to


induce upregulation of Bcl-2 which is antiapoptotic andprosurvival [15 16] Upregulation of Bcl-2 controlsmitochon-drial dysfunction by regulating activation of BAX and BAKwhich are mitochondrial membrane proteins [17] Thus ourresults possibly indicate that GST recovered mitochondrialdysfunctions via Akt and ERK signaling pathways

Still the main component of GST which shows theseeffects on PD remains unclear but previous studies havedrawn possible candidates One candidate chemical ofRehmannia catalpol is an ingredient of SDT Catalpol isknown to have neuroprotective effect in neurotoxicity byattenuating mitochondrial dysfunction and MAO-B activity[18] Furthermore in Alzheimers disease mouse model SDTattenuates 120573-amyloid deposits [19] and catalpol has shownits neuroprotective effect from 120573-amyloid induced neuroninjury and following cognitive functions in aged rats [20]Further researches are needed to find a main component ofherbal formula for development of optimal drug for PD

5 Conclusion

We examined membrane potential ROS apoptosis- and cellproliferation-related proteins in order to see the effect of GSTon PD by using both animal model and in vitromethod As aresult it was revealed that specific dose of GST increased cellviability and ameliorated MPTP-inducedmotor dysfunctionMPP+-induced mitochondrial dysfunction was prevented inSH-SY5Y cells and dopaminergic neurons were protected inPD mouse model Our data suggests that GST may be apotential candidate for alternative treatment of PD

Data Availability

All the data used to support the findings of this study areincluded within the article

Conflicts of Interest

The authors declare that there are no conflicts of interest

Authorsrsquo Contributions

Jade Heejae Ko and Ju-Hee Lee contributed equally to thiswork

Acknowledgments

This researchwas supported by a grant of Comprehensive andIntegrative Medicine RampD project through Comprehensiveand Integrative Medicine Institute (CIMI) funded by theMinistry of Health amp Welfare (MOHW) Republic of Korea(Grant Number 090-091-3000-3038-301-320-01)

References

[1] S S Rao L A Hofmann and A Shakil ldquoParkinsonrsquos diseasediagnosis and treatmentrdquoAmerican family physician vol 74 no12 pp 2046ndash2054 2006

[2] S R Subramaniam andM Chesselet ldquoMitochondrial dysfunc-tion and oxidative stress in Parkinsonrsquos diseaserdquo Progress inNeurobiology vol 106-107 pp 17ndash32 2013

[3] X-Z Li S-N Zhang S-M Liu and F Lu ldquoRecent advances inherbal medicines treating Parkinsonrsquos diseaserdquo Fitoterapia vol84 no 1 pp 273ndash285 2013

[4] H Liu J Wang A Sekiyama and T Tabira ldquoJuzen-taiho-toan herbal medicine activates and enhances phagocytosis inmicrogliamacrophagesrdquo The Tohoku Journal of ExperimentalMedicine vol 215 no 1 pp 43ndash54 2008

[5] Y Oh W Cho Y H Jeong G Y Im M C Yang andJ Y Ma ldquoFermentation Improves Anti-Inflammatory Effectof Sipjeondaebotang on LPS-Stimulated RAW 2647 CellsrdquoAmerican Journal of Chinese Medicine vol 40 no 04 pp 813ndash831 2012

[6] X Du L Liu and Y H Zhang ldquoEffect of association ofShiquandabu Tang with Hetaokun on liver and immunity oftumour mouserdquo China journal of Chinese materia medica vol29 pp 449ndash451 2004

[7] D K Kim J S Eun H Jeon and J M Song ldquoEffect of Sipjeon-Daebo-Tang on Thymocytes of Anti-tumor Drugs Adminis-teredMicerdquoTheKorea Journal of Herbology vol 13 no 2 p 1291998

[8] ADoo S KimDHahmet al ldquoGastrodia elata Blume alleviatesL-DOPA-induced dyskinesia by normalizing FosB and ERKactivation in a 6-OHDA-lesioned Parkinsonrsquos disease mousemodelrdquo BMC Complementary and Alternative Medicine vol 14no 1 2014

[9] J S Shim H G Kim M S Ju J G Choi S Y Jeong andM S Oh ldquoEffects of the hook of Uncaria rhynchophylla onneurotoxicity in the 6-hydroxydopamine model of Parkinsonrsquosdiseaserdquo Journal of Ethnopharmacology vol 126 no 2 pp 361ndash365 2009

[10] Y Lan J Zhou J Liu et al ldquoUncaria rhynchophylla Ame-liorates Parkinsonrsquos Disease by Inhibiting HSP90 ExpressionInsights fromQuantitative ProteomicsrdquoCellular Physiology andBiochemistry vol 47 no 4 pp 1453ndash1464 2018

[11] H An I S Kim S Koppula et al ldquoProtective effects ofGastrodia elata BlumeonMPP+-induced cytotoxicity in humandopaminergic SH-SY5Y cellsrdquo Journal of Ethnopharmacologyvol 130 no 2 pp 290ndash298 2010

[12] P J Houghton and M J Howes ldquoNatural products andderivatives affecting neurotransmission relevant to Alzheimerrsquosand Parkinsonrsquos diseaserdquo Neurosignals vol 14 no 1-2 pp 6ndash222005

[13] P Maiti J Manna and G L Dunbar ldquoCurrent understandingof the molecular mechanisms in Parkinsonrsquos disease Targets forpotential treatmentsrdquo Translational Neurodegeneration vol 6no 1 2017

[14] W-Y Jeon I-S Shin H-K Shin andM-Y Lee ldquoGastroprotec-tive effect of the traditional herbalmedicine Sipjeondaebo-tangwater extract against ethanol-induced gastric mucosal injuryrdquoBMC Complementary and Alternative Medicine vol 14 2014

[15] J E Chipuk J C Fisher C P Dillon R W Kriwacki TKuwana and D R Green ldquoMechanism of apoptosis inductionby inhibition of the anti-apoptotic BCL-2 proteinsrdquo Proceedingsof the National Acadamy of Sciences of the United States ofAmerica vol 105 no 51 pp 20327ndash20332 2008

[16] L Singh N Pushker N Saini S Sen A Sharma S Bakhshiet al ldquoExpression of pro-apoptotic Bax and anti-apoptotic Bcl-2 proteins in human retinoblastomardquo Clinical amp experimentalophthalmology vol 43 no 3 pp 259ndash267 2015


[17] C Orskov T Buhl L Rabenhoj H Kofod and J J HolstldquoCarboxypeptidase-B-like processing of the C-terminus ofglucagon-like peptide-2 in pig and human small intestinerdquoFEBS Letters vol 247 no 2 pp 193ndash196 1989

[18] J Bi X BWang LChen SHao L J An B Jiang et al ldquoCatalpolprotects mesencephalic neurons against MPTP induced neu-rotoxicity via attenuation of mitochondrial dysfunction andMAO-B activityrdquo Toxicology in vitro An International JournalPublished in Association with BIBRA vol 22 no 8 pp 1883ndash1889 2008

[19] H Hara S Kataoka M Anan A Ueda TMutoh and T TabiraldquoThe Therapeutic Effects of the Herbal Medicine Juzen-taiho-to on Amyloid-120573 Burden in a Mouse Model of AlzheimerrsquosDiseaserdquo Journal of Alzheimerrsquos Disease vol 20 no 2 pp 427ndash439 2010

[20] Z Xia F Wang S Zhou et al ldquoCatalpol protects synaptic pro-teins from beta-amyloid induced neuron injury and improvescognitive functions in aged ratsrdquoOncotarget vol 8 no 41 2017

Stem Cells International

Hindawiwwwhindawicom Volume 2018


MEDIATORSINFLAMMATION

of

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Immunology ResearchHindawiwwwhindawicom Volume 2018

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine


Behavioural Neurology

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary andAlternative Medicine

Volume 2018Hindawiwwwhindawicom

Submit your manuscripts atwwwhindawicom


Table 1 The compositions of GST

Latin Name Scientific name (Family name) RatioGinseng Radix Alba Panax ginseng C A Meyer (Araliaceae) 3Atractylodis Rhizoma Alba Atractylodes macrocephala Koidzumi (Compositae) 2Hoelen Poria cocosWolf (Polyporaceae) 2Glycyrrhizae Radix et Rhizoma Glycyrrhiza uralensis Fischer (Leguminosae) 2Rehmanniae Radix Preparata Rehmannia glutinosa Liboschitz ex Steudel (Scrophulariaceae) 2Angelicae Gigantis Radix Angelica gigas Nakai (Umbelliferae) 2Cnidii Rhizoma Cnidium officinaleMakino (Umbelliferae) 2Paeoniae Radix Paeonia lactiflora Pallas (Paeoniaceae) 2Astragali Radix Astragalus membranaceus Bunge (Leguminosae) 3Bupleuri Radix Bupleurum falcatum Linne (Umbelliferae) 2Uncariae Ramulus et Uncus Uncaria rhynchophylla (Rubiaceae) 2Gastrodiae Rhizoma Gastrodia elata Blume (Orchidaceae) 2

mice by increasing cell viability and ameliorating behavioralimpairment through dopamine and tyrosine hydroxylase(TH) expression elevation [10] Anti-apoptotic and antiox-idative aspects of Gastrodia elata Blume in MPP+-inducedcytotoxicity dopaminergic cells were also suggested [11] Theearlier studies suggest that GST potentially has neuropro-tective effects of dopaminergic neuron and its followingmotor dysfunctions of PD Therefore in this study weinvestigated effects of GST on PD and its underlying mech-anism using 1-methyl-4-phenyl-1236-tetrahydropyridine(MPTP)-induced Parkinsonian mice model and 1-methyl-4-phenylpyridinium (MPP+)-induced cell death

2 Materials and Methods

21 Animals Male C57BL6 mice (Orient-Bio Co Seong-nam Republic of Korea) 9 weeks of age and weighing20ndash25 g each were used The animals were housed under a1212-h lightdark cycle with free access to food and waterAll experiments were approved by the Dongguk UniversityAnimal Care Committee for animal welfare (2016-DGU-46) and maintained in strict accordance with guidelines Todevelop PD animal model we used acute MPTP regimen onmice Briefly intraperitoneal MPTP injection (20 mgkg ofbody weight Sigma-Aldrich St Louis USA) was performedevery 2 hours four times a day in total One day afterMPTP injection rotarod test was done and only mice thatshowed motor function deficits more than 30 comparedto control group were divided into experimental groups forthis study control group MPTP group and MPTP+GSTgroup 300 mgkg of GST was given to MPTP+GST grouponce a day for 5 days by oral administration There were 6mice in each group The same volume of physiological salinewas administered to mice in the control group After thebehavioral tests the mice were sacrificed and the substantianigra in the brain was removed Detailed timeline of theanimal study is shown in Figure 1(a)

22 Behavioral Test We have conducted rotarod test andpole test to examine the motor function of the mice For therotarod test mice were located on a rotating rod accelerating

from 5 to 20 rpm for a 240 seconds time period Each mousewas scored for time they sustained on the rod The mice thatcompleted the task over 240 seconds scored as 240 whichwas the maximum value To examine pole test mice werelocated head toward up close to top of the pole We recordedthe time when themicersquos heads completely rotated downward(P1) and when they reached the floor (P2) Falling latencywas calculated by subtracting P1 fromP2Themaximum timelimit was determined as 120 seconds

23 Plant Materials and Preparation of GST Extract TheGST was formulated from twelve medicinal herbs Panaxginseng C A Meyer Atractylodes macrocephala KoidzumiPoria cocos Wolf Glycyrrhiza uralensis Fischer Rehmanniaglutinosa Liboschitz ex Steudel Angelica gigas Nakai Cnid-ium officinale Makino Paeonia lactiflora Pallas Astragalusmembranaceus Bunge Bupleurum falcatum Linne Uncariarhynchophylla and Gastrodia elata Blume All herbs werepurchased as dried herbs from Omniherb (Seoul Republicof Korea) Briefly a mixture of the twelve dried components(100 g weight ratios shown inTable 1) was extracted in 800mlof 30 ethanol by heating at 80∘C for 4 h and extracts werefiltered twice through a filter paper (40 120583mWhatman Buck-inghamshireUK) Filtrateswere concentrated under reducedpressure using a rotary vacuum evaporator (EYELA TokyoJapan) at 40sim45∘C and the concentrate was lyophilized usinga freeze dryer (EYELA Tokyo Japan) The yield of the GSTextract (dried powder) was 227 of the weight of the driedstarting materials

24 Cell Culture SH-SY5Y cells a human neuroblastomacell line were purchased from the Korean Cell Line Bank(Seoul Republic of Korea) Cells were cultured in Dulbeccorsquosmodified Eaglersquos medium (DMEM Welgene GyeongsanRepublic of Korea) supplemented with 10 fetal bovineserum (FBS Thermo Scientific Waltham USA) and 1penicillinstreptomycin Cultures were maintained at 37∘C ina CO2incubator with a controlled humidified atmosphere

composed of 95 air and 5 CO2 FBS cell culture media

penicillinstreptomycin and all other reagents used for cell



AdaptationMPTP

Behavioral test

GST

Behavioral test

2

(a)

Tim

e on

rod

(sec

)

0

50

100

150

200

250


lowast

(b)

0

5

10

15

20

25

Late

ncy

to fa

ll (s

ec)


lowastlowast

(c)

00

05

10

15

TH - 60kDa

- 42kDa

(rel

ativ

e exp

ress

ion)

TH

-act

in

lowast

MPTP+GSTMPTPControl

-actin

(d)













3 Result







2DCF-



0

20

40

60

80

100

120C

ell v

iabi

lity

()

10 30 50 100 300 5000GST (gml)

(a)

GST (gml)

0

20

40

60

80

100

120

Cel

l via

bilit

y (

)

- 10 100 200 300Con

－００+ (1 mM)

lowastlowastlowast

(b)

-32kDa

-19kDa-17kDa

-27kDa

-42kDa


Bcl-2

Procaspase-3

Cleaved caspase-3

-actin

+ +MPP+

(c)

-60kDa

-60kDa

-44kDa-42kDa

-44kDa-42kDa

p-Akt

Akt

p-ERK

ERK


(d)







(100 gml)

0

10

20

30

40

DCF

fluo

resc

ence

()

- GST GSTCon

lowastlowastlowast

－００+ (1 mM)




0

200

400

Control

Cou

nt

FITC-H

Multi-sample P1

GST

103 104 105

－００++GST－００+

(a)

Rh1

23+

cells

()

0

20

40

60

80

100

Con GST GST

MPP+ (1 mM)

-

lowastlowastlowast

(b)



4 Discussion





Time (h) 30 1 1263

-actin

p-ERK

ERK

p-Akt

Akt

GST (100 gml)

-44kDa

-44kDa

-42kDa

-42kDa

-60kDa

-60kDa

-42kDa

100

(a)

Rh1

23+

cells

()

0

20

40

60

80

100

ampampamp

ampampamp

----

-+--

++

--

++

+

-

+

+

+

-

GST

LY294002PD98059

MPP+

lowastlowastlowast

(b)











5 Conclusion


Data Availability






Acknowledgments


References


























of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of





















AdaptationMPTP

Behavioral test

GST

Behavioral test

2

(a)

Tim

e on

rod

(sec

)

0

50

100

150

200

250


lowast

(b)

0

5

10

15

20

25

Late

ncy

to fa

ll (s

ec)


lowastlowast

(c)

00

05

10

15

TH - 60kDa

- 42kDa

(rel

ativ

e exp

ress

ion)

TH

-act

in

lowast

MPTP+GSTMPTPControl

-actin

(d)













3 Result







2DCF-



0

20

40

60

80

100

120C

ell v

iabi

lity

()

10 30 50 100 300 5000GST (gml)

(a)

GST (gml)

0

20

40

60

80

100

120

Cel

l via

bilit

y (

)

- 10 100 200 300Con

－００+ (1 mM)

lowastlowastlowast

(b)

-32kDa

-19kDa-17kDa

-27kDa

-42kDa


Bcl-2

Procaspase-3

Cleaved caspase-3

-actin

+ +MPP+

(c)

-60kDa

-60kDa

-44kDa-42kDa

-44kDa-42kDa

p-Akt

Akt

p-ERK

ERK


(d)







(100 gml)

0

10

20

30

40

DCF

fluo

resc

ence

()

- GST GSTCon

lowastlowastlowast

－００+ (1 mM)




0

200

400

Control

Cou

nt

FITC-H

Multi-sample P1

GST

103 104 105

－００++GST－００+

(a)

Rh1

23+

cells

()

0

20

40

60

80

100

Con GST GST

MPP+ (1 mM)

-

lowastlowastlowast

(b)



4 Discussion





Time (h) 30 1 1263

-actin

p-ERK

ERK

p-Akt

Akt

GST (100 gml)

-44kDa

-44kDa

-42kDa

-42kDa

-60kDa

-60kDa

-42kDa

100

(a)

Rh1

23+

cells

()

0

20

40

60

80

100

ampampamp

ampampamp

----

-+--

++

--

++

+

-

+

+

+

-

GST

LY294002PD98059

MPP+

lowastlowastlowast

(b)











5 Conclusion


Data Availability






Acknowledgments


References


























of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of



























3 Result







2DCF-



0

20

40

60

80

100

120C

ell v

iabi

lity

()

10 30 50 100 300 5000GST (gml)

(a)

GST (gml)

0

20

40

60

80

100

120

Cel

l via

bilit

y (

)

- 10 100 200 300Con

－００+ (1 mM)

lowastlowastlowast

(b)

-32kDa

-19kDa-17kDa

-27kDa

-42kDa


Bcl-2

Procaspase-3

Cleaved caspase-3

-actin

+ +MPP+

(c)

-60kDa

-60kDa

-44kDa-42kDa

-44kDa-42kDa

p-Akt

Akt

p-ERK

ERK


(d)







(100 gml)

0

10

20

30

40

DCF

fluo

resc

ence

()

- GST GSTCon

lowastlowastlowast

－００+ (1 mM)




0

200

400

Control

Cou

nt

FITC-H

Multi-sample P1

GST

103 104 105

－００++GST－００+

(a)

Rh1

23+

cells

()

0

20

40

60

80

100

Con GST GST

MPP+ (1 mM)

-

lowastlowastlowast

(b)



4 Discussion





Time (h) 30 1 1263

-actin

p-ERK

ERK

p-Akt

Akt

GST (100 gml)

-44kDa

-44kDa

-42kDa

-42kDa

-60kDa

-60kDa

-42kDa

100

(a)

Rh1

23+

cells

()

0

20

40

60

80

100

ampampamp

ampampamp

----

-+--

++

--

++

+

-

+

+

+

-

GST

LY294002PD98059

MPP+

lowastlowastlowast

(b)











5 Conclusion


Data Availability






Acknowledgments


References


























of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




















0

20

40

60

80

100

120C

ell v

iabi

lity

()

10 30 50 100 300 5000GST (gml)

(a)

GST (gml)

0

20

40

60

80

100

120

Cel

l via

bilit

y (

)

- 10 100 200 300Con

－００+ (1 mM)

lowastlowastlowast

(b)

-32kDa

-19kDa-17kDa

-27kDa

-42kDa


Bcl-2

Procaspase-3

Cleaved caspase-3

-actin

+ +MPP+

(c)

-60kDa

-60kDa

-44kDa-42kDa

-44kDa-42kDa

p-Akt

Akt

p-ERK

ERK


(d)







(100 gml)

0

10

20

30

40

DCF

fluo

resc

ence

()

- GST GSTCon

lowastlowastlowast

－００+ (1 mM)




0

200

400

Control

Cou

nt

FITC-H

Multi-sample P1

GST

103 104 105

－００++GST－００+

(a)

Rh1

23+

cells

()

0

20

40

60

80

100

Con GST GST

MPP+ (1 mM)

-

lowastlowastlowast

(b)



4 Discussion





Time (h) 30 1 1263

-actin

p-ERK

ERK

p-Akt

Akt

GST (100 gml)

-44kDa

-44kDa

-42kDa

-42kDa

-60kDa

-60kDa

-42kDa

100

(a)

Rh1

23+

cells

()

0

20

40

60

80

100

ampampamp

ampampamp

----

-+--

++

--

++

+

-

+

+

+

-

GST

LY294002PD98059

MPP+

lowastlowastlowast

(b)











5 Conclusion


Data Availability






Acknowledgments


References


























of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




















(100 gml)

0

10

20

30

40

DCF

fluo

resc

ence

()

- GST GSTCon

lowastlowastlowast

－００+ (1 mM)




0

200

400

Control

Cou

nt

FITC-H

Multi-sample P1

GST

103 104 105

－００++GST－００+

(a)

Rh1

23+

cells

()

0

20

40

60

80

100

Con GST GST

MPP+ (1 mM)

-

lowastlowastlowast

(b)



4 Discussion





Time (h) 30 1 1263

-actin

p-ERK

ERK

p-Akt

Akt

GST (100 gml)

-44kDa

-44kDa

-42kDa

-42kDa

-60kDa

-60kDa

-42kDa

100

(a)

Rh1

23+

cells

()

0

20

40

60

80

100

ampampamp

ampampamp

----

-+--

++

--

++

+

-

+

+

+

-

GST

LY294002PD98059

MPP+

lowastlowastlowast

(b)











5 Conclusion


Data Availability






Acknowledgments


References


























of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




















Time (h) 30 1 1263

-actin

p-ERK

ERK

p-Akt

Akt

GST (100 gml)

-44kDa

-44kDa

-42kDa

-42kDa

-60kDa

-60kDa

-42kDa

100

(a)

Rh1

23+

cells

()

0

20

40

60

80

100

ampampamp

ampampamp

----

-+--

++

--

++

+

-

+

+

+

-

GST

LY294002PD98059

MPP+

lowastlowastlowast

(b)











5 Conclusion


Data Availability






Acknowledgments


References


























of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of






















5 Conclusion


Data Availability






Acknowledgments


References


























of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




























of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of























of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of



















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