research article aqueous extract of paeonia lactiflora and...

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2013 Article ID 471659 11 pageshttpdxdoiorg1011552013471659

Research ArticleAqueous Extract of Paeonia lactiflora and Paeoniflorin asAggregation Reducers Targeting Chaperones in Cell Modelsof Spinocerebellar Ataxia 3

Kuo-Hsuan Chang1 Wan-Ling Chen1 Li-Ching Lee2 Chih-Hsin Lin1

Pin-Jui Kung2 Te-Hsien Lin2 Yi-Ci Wu2 Yih-Ru Wu1 Yi-Chun Chen1

Guey-Jen Lee-Chen2 and Chiung-Mei Chen1

1 Department of Neurology Chang Gung Memorial Hospital Chang Gung University College of Medicine Taipei 10507 Taiwan2Department of Life Science National Taiwan Normal University Taipei 11677 Taiwan

Correspondence should be addressed to Guey-Jen Lee-Chen t43019ntnuedutwand Chiung-Mei Chen cmchenadmcgmhorgtw

Received 29 October 2012 Accepted 19 January 2013

Academic Editor Carlo Ventura

Copyright copy 2013 Kuo-Hsuan Chang et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Spinocerebellar ataxia (SCA) types 1 2 3 6 7 and 17 as well as Huntingtonrsquos disease are a group of neurodegenerative disorderscaused by expanded CAG repeats encoding a long polyglutamine (polyQ) tract in the respective proteins Evidence has shown thatthe accumulation of intranuclear and cytoplasmic misfolded polyQ proteins leads to apoptosis and cell death Thus suppressionof aggregate formation is expected to inhibit a wide range of downstream pathogenic events in polyQ diseases In this study weestablished a high-throughput aggregation screening system using 293 ATXN3Q

75-GFP cells and applied this system to test the

aqueous extract of Paeonia lactiflora (P lactiflora) and its constituentsWe found that the aggregation can be significantly prohibitedby P lactiflora and its active compound paeoniflorin Meanwhile P lactiflora and paeoniflorin upregulated HSF1 and HSP70chaperones in the same cellmodels Both of them further reduced the aggregation in neuronal differentiated SH-SY5YATXN3Q

75-

GFP cells Our results demonstrate how P lactiflora and paeoniflorin are likely to work on polyQ-aggregation reduction and provideinsight into the possible working mechanism of P lactiflora in SCA3 We anticipate our paper to be a starting point for screeningmore potential herbs for the treatment of SCA3 and other polyQ diseases

1 Introduction

Spinocerebellar ataxias (SCAs) are a large complex groupof heterogeneous autosomal dominant neurodegenerativedisorders characterized by cerebellar dysfunction aloneor in combination with other neurological abnormalities[1] Among them the expansions of CAG trinucleotiderepeats encoding a polyglutamine (polyQ) stretch havebeen shown to cause dominantly inherited SCA1 SCA2SCA3 SCA6 SCA7 SCA17 and dentatorubropallidoluy-sianatrophy (DRPLA) [2ndash8] These polyQ-mediated geneticdisorders in SCAs have shown selective progressive degen-eration of the cerebellum brainstem and spinal tractwith prominent pathological hallmark of intranuclear and

cytoplasmic accumulation of aggregated polyQ proteinsinside degenerated neurons [9] Different polyQ tract-containing proteins ultimately lead to the dysfunction anddegeneration of specific neuronal subpopulations [10] Theaggregated polyQ proteins may cause dysfunction of mito-chondria chaperone and ubiquitin proteasome system lead-ing to apoptosis and cell death [11ndash13] As misfolding of thepolyQ protein is likely the initial event in the pathogeniccascade suppression of protein misfolding is expected toinhibit a wide range of downstream detrimental events andto rescue neuronal dysfunction

Increasing evidence suggests that some herbs may poten-tially attenuate the deterioration of neurodegenerative dis-eases Paeonia lactiflora (P lactiflora) belonging to the

2 Evidence-Based Complementary and Alternative Medicine

Paeoniaceae family is a perennial herb frequently used asan important ingredient in many traditional prescriptions Ithas been commonly used for nourishing blood alleviatingpain reducing irritability as well as treating liver diseaseand cancer [14] Paeoniflorin one of the main compoundsextracted from P lactiflora has been reported to ameliorateneurodegenerative process in Parkinsonrsquos disease (PD) andAlzheimerrsquos disease (AD)models [15ndash17] However the effectof P lactiflora herb extract and paeoniflorin in treating SCAremains unraveled

In the present study we firstly built up an aggrega-tion screening cell model by overexpressing CAG-expandedATXN3 the causative mutation in SCA3 [2] in 293 cellsand then examined the anti-aggregation effect of P lactifloraaqueous extract and paeoniflorin We further demonstratedthat the anti-aggregation activity of P lactiflora extract andpaeoniflorin was contributed by the enhancement of heatshock transcription factor 1 (HSF1)-heat shock protein (HSP)70 chaperone system These findings provide evidence thatP lactiflora and paeoniflorin may be a novel alternativetherapeutic agent for the treatment of SCAs

2 Materials and Methods

21 P lactiflora Extract Preparation and HPLC AnalysisAqueous extract from P lactiflora was provided by Sun-Ten Pharmaceutical Company (Taipei Taiwan) Briefly 100 gof dried P lactiflora was boiled with 1500mL of water at100∘C for 30min and was sieved using a 100-mesh sieve Theextract was concentrated to 100mL and filtered through a200-mesh sieveThe extract was then dried by speed vacuumconcentration and then stored at minus20∘C until used

High pressure liquid chromatography (HPLC) was per-formed using a LaChrom Elite HPLC system (Hitachi) con-sisting of a photo diode array detector The chromatographicseparation ofP lactiflora extract (50120583L 1mgmL)was carriedout on a Hypersil ODS (C18) column (250 times 46mm 5 120583m)eluted with the mixture of 01 formic acid in water (A)or acetonitrile (B) The linear gradient elution program forA B (vv) was set as follows 95 5 (0ndash10min) 95 5ndash70 30(10ndash40min) 70 30ndash15 85 (40ndash55min) 15 85ndash95 5 (55ndash60min) 95 5 (60ndash75min) with a flow rate of 1mLminAbsorbance was monitored at 230 250 270 nm and the scanrange for photo diode array was 190sim400 nm Paeonifloringallic acid and albiflorin (2sim10120583L 20mM) were used asreference compounds for P lactiflora [18 19]

22 Cell Culture and Cell Proliferation Assay Human embry-onic kidney HEK-293 cells (ATCC No CRL-1573) werecultivated in Dulbeccorsquos modified Eaglersquos medium (DMEM)containing 10 fetal bovine serum (FBS) Human neuroblas-toma SH-SY5Y cells (ATCCNo CRL-2266) were maintainedin DMEM F12 supplemented with 10FBS Cells werecultivated at 37∘C incubator containing 5 CO

2and cell

proliferation was measured based upon the reduction of thetetrazolium salt 3[45-dimethylthiazol-2-yL]-25-diphenyl-tetrazolium bromide (MTT) Cells were plated into 48-well (5 times 104well) dishes grown for 20 hr and treated

with different concentrations of the P lactiflora extract (5sim30mgmL) or pure compound (100 nMsim1mM) After oneday 20120583L MTT (5mgmL in PBS Sigma) was added tocells and incubated for 2 hr The absorbance of the purpleformazan dye was measured at 570 nm by a Bio-Tek 120583QuantUniversal Microplate Spectrophotometer

23 Flp-In-293 Triple Fluorescent Reporter Cells and Flu-orescent Assay A triple fluorescent reporter plasmid withmCherry ZsYellow1 and AmCyan1 was first constructedin pAmCyan1-N1 A proximal promoter (minus360sim+2 withthe translation initiation site as +1) from HSF1 gene wasused as 331 bp upstream of the murine HSF1 transla-tion start site is required for maximal basal expression[20] Promoter fragments from HSF1 (enhancing chaperoneexpression) heat shock cognate protein (HSPA8 minus1140sim+38 driving constitutively expressed HSP70) [21] and heat-inducible HSP70 chaperone (HSPA1A minus273sim+215 drivingheat-inducible HSP70) [22] are placed upstream of the threefluorescent reporters The fragment containing the HSF1HSPA8 and HSPA1A driven reporters was excised withAseI and NotI restriction enzymes and used to replace anAseI-NotI fragment in pcDNA5FRTTO plasmid (Invitro-gen) The resulting triple fluorescent reporter plasmid wasused to generate Flp-In triple fluorescent reporter cells andmaintained according to the supplierrsquos instructions (Invitro-gen) Geranylgeranylacetone (GGA Sigma) or paeoniflorin(100 nMsim100120583M) was added to the medium for 24 hr Thethree fluorescence colors were analyzed simultaneously usinghigh-content analysis (HCA) system (ImageXpressMICROMolecular Devices) with excitationemission wavelengthsat 453486 (mCherry) 531540 (ZsYellow1) and 587610 nm(AmCyan1)

24 ATXN3 and HSF1 cDNA Constructs PolyadenylatedRNA (200 ng) isolated from neuroblastoma SK-N-SH cellswas reverse transcribed using the SuperScript III reversetranscriptase (Invitrogen) The sense and antisense primersused for ATXN3Q

14cDNA (+826sim+1152 NM 004993)

amplification were 51015840-ATTCAGCTAAGTATGCAA-GGTAGTTCCA (codon for Met257 underlined) and51015840-CATGCCATGGCATGTTTTTTTCCTTCTGTT (NcoIsite underlined) The amplified 31015840 polyQ-containing cDNAfragment (translated into amino acids 257sim361) wascloned into pGEM-T Easy (Promega) and sequenced TheATXN3Q

14cDNAwas excised with EcoRI (in pGEM-T Easy

vector) and NcoI and subcloned into pEGFP-N1 (Clontech)Then DNA fragment containing in-frame ATXN3Q

14-

EGFP was excised with HindIII-NotI and subcloned intothe pcDNA5FRTTO The ATXN3Q

75cDNA was made

by replacing an 88 bp ATXN3Q14

BsmBI-BsmFI fragmentwith a 271 bp ATXN3Q

75fragment from the cDNA clone

of a SCA3 patient The HSF1 cDNA (BC014638) in pOTB7was obtained from Bioresource Collection and ResearchCenter (BCRC) Food Industry Research and DevelopmentInstitute Taiwan The cDNA was excised with EcoRI andXhoI and subcloned into pcDNA3 (Invitrogen)

Evidence-Based Complementary and Alternative Medicine 3

(kD

a)

170 130 100

70

55

40

35

25

M Dox

GFP antibody

ATXN311987614 ATXN311987675

minus + minus +

ATXN311987614-GFP

ATXN311987675-GFP

(a)

10

20

30

Dox

ATXN311987614 ATXN311987675

ATXN

3H

PRT1

minus + minus +

(b)

100120583m

ATXN311987614-GFP ATXN311987675-GFP

(c)

Figure 1 Flp-In 293 cells with ATXN3Q14sim75

-GFP expression in an inducible fashion (a) Western blot analysis of ATXN3Q14sim75

-GFPprotein expression using GFP antibody after two days of induction (+Dox) (b) Real-time PCR quantification of ATXN3Q

14sim75-GFP RNA

expression relatively to HPRT after two days of induction (+Dox) (c) Fluorescence microscopy images of ATXN3Q14sim75

-GFP expressionafter six days of induction The scale bar = 100 120583m

25 Isogenic 293 and SH-SY5Y Cell Lines Human 293-derived Flp-In-293 cells (Invitrogen) were cultivated inDMEM containing 10FBS as described The clonedpcDNA5FRTTO-ATXN3Q

14and Q

75plasmids were

used to generate the isogenic ATXN3Q14sim75

cell lines bytargeting insertion into Flp-In-293 cells according to thesupplierrsquos instructions The repeats in these ATXN3 celllines were examined by PCR and sequencing These celllines were grown in medium containing 5120583gmL blasticidinand 100 120583gmL hygromycin (InvivoGen) Human SH-SY5Y-derived Flp-In host cell line was constructed as described[23] The SH-SY5Y host cells were used to generate isogenicATXN3Q

14sim75lines and maintained as described above

26 ATXN3Q75

Aggregation Assay 293ATXN3Q75-GFP

cells were plated into 96-well (2 times 104well) dishes grownfor 24 hr and treated with different concentrations of the

P lactiflora extract (2sim200120583gmL) or suberoylanilide hydrox-amic acid (SAHA Cayman Chemical) paeoniflorin (Sigma)gallic acid and albiflorin (Chromadex) (100 nMsim5 120583M) for8 hr Then doxycycline (10120583gmL BD) was added to themedium to induce ATXN3Q

75-GFP expression for 6 days

Oxaliplatin (5120583M Sigma) was also added for aggregateaccumulation through inhibition of cell division [24] Thencells were stained withHochest 33342 (01 120583gmL Sigma) andaggregation percentage was assessed by HCA system withexcitationemission wavelengths at 482536 (GFP)

SH-SY5Y ATXN3Q75-GFP cells were seeded in 6-well

(2times105well) plate with all trans retinoic acid (10 120583M Sigma)added at seeding time At day 2 cells were treated withpaeoniflorin (100 nM) or the P lactiflora extract (10 120583gmL)for 8 hr and then doxycycline (5120583gmL) was added to induceATXN3Q

75-GFP expression The cells were kept in the

medium containing 10 120583M trans retinoic acid doxycyclineand paeoniflorinP lactiflora extract for 7 days After that


Inte

nsity

(AU

) 2

15

1

05

0

Retention time (min)

Paeoniflorin(2903min)

Gallic acid(588min)

Albiflorin(2746min)

230nmHerb paeonia lactiflora

0 10 20 30 40 50 60 70

(a)

Paeoniflorin

Gallic acid

Albiflorin

Content in 1gmL

227 4733mM

03 1806mM

073 1516mM

(b)

Cel

l via

bilit

y (

) 120

80

40

Cel

l via

bilit

y (

) 120

80

40

51015

20

30

Untreated

SAHA Paeoniflorin Gallic acid AlbiflorinHEK-293 IC50 cytotoxicity

038mM 058mM

P lactiflora

SAHA Paeoniflorin Gallic acid Albiflorin P lactiflora

gt1mM gt1mM gt30mgmL

057mMgt1mM gt1mM gt30mgmL

50

50

002mMSH-SY5Y IC50 cytotoxicity

1120583M

10120583M

1mM100120583M 25mgmL

mgmL

mgmLmgmL

mgmL

mgmL100nM

(c)

Figure 2 Chemical profile and cytotoxicity of the aqueous extract of P lactiflora (a) Chromatographic patterns from HPLC analysis(230 nm) showed peaks compatible with paeoniflorin albiflorin and garlic acid (b)The relative amount of above molecules in the extract (c)Cytotoxicity of the aqueous extract of P lactiflora paeoniflorin garlic acid albiflorin and SAHA against HEK-293 and SH-SY5Y cells usingMTT viability assay The IC

50of each herbcompound was shown under the columns To normalize the relative viability in untreated cells is

set as 100 The red line represents 50 viability

cells were stained with Hochest 33342 (01120583gmL) and aggre-gation percentage was assessed as described

27 Real-Time PCR Total RNA from 293 ATXN3 lineswas extracted using Trizol reagent (Invitrogen) The RNAwas DNase (Stratagene) treated quantified and reverse-transcribed to cDNA as described Real-time quantitativePCR experiments were performed in the ABI PRISM7000 Sequence Detection System (Applied Biosystems)Amplification was performed on 100 ng cDNA with gene-specific TaqMan fluorogenic probes Hs0024525 ml forATXN3 and 4326321E for HPRT1 (endogenous control)(Applied Biosystems) Fold change was calculated using theformula 2ΔC119905 ΔC

119905= C119905(control) minus C

119905(target) in which C

119905

indicates cycle threshold

28 Western Blot Analysis Total proteins were preparedusing lysis buffer containing 50mM Tris-HCl 150mMNaCl

1mM EDTA 1mM EGTA 01 SDS and 05 sodiumdeoxycholate 1 Triton X-100 protease inhibitor cocktail(Calbiochem) Proteins (25120583g) were separated on 10 SDS-polyacrylamide gel electrophoresis and blotted on to nitrocel-lulose membranes by reverse electrophoresis After blockingthe membrane was probed with HSF1 (1 1000 dilutionAbnova) HSPA8 (1 500 dilution Santa Cruz) HSPA1A(1 500 dilution Santa Cruz) H3F3B (1 3000 dilution Gene-Tex) GFP (1 500 dilution Santa Cruz) 120573-actin (1 5000dilution Millipore) or GAPDH (1 1000 dilution MDBio) at4∘C overnight Then the immune complexes were detectedby horseradish peroxidase-conjugated goat anti-mouse orgoat anti-rabbit IgG antibody (1 5000 dilutionGeneTex) andchemiluminescent substrate (Millipore)

29 ATXN3Q75

and HSF1 cDNA Co-Transfection Humanembryonic kidney HEK-293T cells (ATCC No CRL-11268)were cultivated in DMEM containing 10 FBS as described


Cellseeding

Herbcompounds

HCA

Doxycycline (10120583M)Oxaliplatin (5120583M)

8hr1day 6days

(a)

Untreated Paeoniflorin P lactiflora

(b)

Untreated

120

80

40

Relat

ive a

ggre

gatio

n (

)

85

SAHA 3800

Paeoniflorin Gallic acid Albiflorin

100


lowast lowast lowast lowast lowastlowast

21050

100nM500nM

1120583M5120583M 200120583gmL

120583gmL

120583gmL120583gmL120583gmL

IC50 cytotoxicityeffective dose ratio gt10000 gt200

P lactifloragt15000

(c)

Figure 3 High-content compound screen using Flp-In 293 cells with inducible ATXN3Q75-GFP expression (a) Experiment flow chart

ATXN3Q75-GFP 293 cells were plated into 96-well dishes grown for 24 hr and treated with different concentrations of the herb or compound

for 8 hr Then doxycycline and oxaliplatin was added to the medium to for 6 days and aggregation percentage was assessed by HCA system(b) Representative fluorescencemicroscopy images of ATXN3Q

75-GFP cells untreated or treated with P lactiflora (10120583gmL) or paeoniflorin

(100 nM) for 6 days (c)Aggregation analysis ofATXN3Q75-GFP cells untreated or treatedwith aqueous extract ofP lactiflora (2sim200 120583gmL)

paeoniflorin garlic acid albiflorin and SAHA (100 nMsim5120583M) To normalize the relative aggregation level in untreated cells is set as 100The red line represents 85 aggregation with SAHA treatment (100 nM)

For transient overexpression cells were plated into 12-well (1 times 105well) dishes grown for 20 hr and trans-fected using T-Pro reagent (JF Biotechnology Taiwan)with pcDNA5FRTTO-ATXN3Q

75and pcDNA3-HSF1 or

pcDNA3 vector plasmids (15120583g each) The cells were grownfor 48 hr for ATXN3Q

75aggregation assay as described

210 Statistical Analysis For each set of values data wereexpressed as the means plusmn standard deviation (SD) Threeindependent experiments were performed and non-cate-gorical variables were compared using the Studentrsquos 119905-test All119875 values were two-tailed with values of 119875 lt 005 consideredsignificant

3 Results

31 Construction of 293 Cells Expressing ATXN3Q75

Aggre-gates For therapies toward the polyQ diseases we aimed to

screen herbscompounds potentially inhibiting polyQ aggre-gation As removal of the N-terminus of polyQ-expandedATXN3 is required for aggregation in vitro and in vivo [25]we clonedGFP-taggedATXN3C-terminalQ

14sim75-containing

fragment to establish Flp-In 293 cells with ATXN3Q14sim75

-GFP expression in an inducible fashion As shown inFigure 1(a) the GFP antibody detected 40 kDa ATXN3Q

14-

GFP and 57 kDa ATXN3Q75-GFP proteins in doxycycline

(Dox) induced ATXN3 cells ATXN3-RNA levels were thenexamined by real-time PCR using ATXN3-specific probe andprimers As shown in Figure 1(b) in the presence of Dox thetwo ATXN3 lines expressed sim20 times more ATXN3 RNAthan in the absence of Dox While the expressed ATXN3Q

14

was mainly diffused the expressed ATXN3Q75-GFP formed

aggregates (Figure 1(c))

32 Aqueous Extract of P lactiflori and Constituents Toexamine the potential active compounds in P lactiflori


(a)

mCherry Yellow1 Amcyan

(b)

125

100

75

50

25Fluo

resc

ence

()

GGA Paeoniflorin GGA Paeoniflorin GGA Paeoniflorin

HSF1-mCherry HSPA8-ZsYellow1 HSPA1A-AmCyan 1

100nM100nM1120583M1120583M

10120583M10120583M100120583M100120583M

119875 = 0028sim0001

119875 = 0010sim0002 119875 = 0024sim0000

119875 = 0044 sim0000

119875 = 0019sim0001

119875 = 0025sim0001

(c)

Paeoniflorin 200

150

100

50

(100nM)

H3F3B

minus + minus + minus +

+minus

()

HSF1

HSF1

HSPA8

HSPA8

HSPA1A

HSPA1A

(d)

Figure 4 Enhancement of chaperone expression by paeoniflorin in 293 cells (a) Triple fluorescent reporter plasmid with HSF1 HSPA8 andHSPA1A promoter fragments upstream of mCherry ZsYellow1 and AmCyan1 fluorescent reporters respectively (b) Microscopic imagesof the triple fluorescent reporter cells (c) Effect of GGA and paeoniflorin (100 nMsim100120583M) on HSF1 HSPA8 and HSPA1A reporters Tonormalize the fluorescence level in untreated cells is set as 100 Three independent experiments were performed with 119875 lt 005 consideredsignificant (d) Representative western blot image of paeoniflorin-(100 nM) treated 293 cells for two days using HSF1 HSPA8 HSPA1A andH3F3B antibodies Levels of HSF1 HSPA8 HSPA1A were normalized with a loading control (H3F3B) Data are expressed as the mean plusmn SEMvalues from three independent experiments


Dox

Paeoniflorin

Dox

Paeoniflorin

Relat

ive r

atio

()

Relat

ive r

atio

()

Relat

ive r

atio

() 140

100

60

20

140

100

60

20

140

100

60

20

P lactifloraP lactiflora

120573-actin

120573-actin

120573-actin

119875 = 0005119875 = 0001

119875 = 0018

119875 = 0009 119875 = 0046

HSF1

HSPA8

HSPA1A

119875 = 0005

HSPA1A

HSPA8

HSF1

minus

minus

minus

minus minus

minus

minusminus minus

minus minusminus minus

minus

minus

minus+

+ +

+ +

++

+

++ +

+ ++ +

+

Figure 5 Enhancement of HSF1 and chaperone expression by paeoniflorin and the aqueous extract of P lactiflora in ATXN3Q75-GFP 293

cells Cells were pre-treated with paeoniflorin (100 nM) or herb (10120583gmL) for 8 hours and ATXN3Q75-GFP expression induced for 6 days

Relative HSF1 HSPA8 and HSPA1A expressions were analyzed by western blot analysis using 120573-actin as a loading control

the chemical profile of aqueous extract was analyzed andquantified by full-spectrum analytic HPLC Chromato-graphic patterns showed peaks in 230 nm corresponding tothe retention time compatible with paeoniflorin garlic acidand albiflorin (Figure 2(a)) The amounts of paeonifloringarlic acid and albiflorin in aqueous extract of P lactifloriwere 227 030 and 073 respectively correspondingto 4733mM 1806mM and 1516mM respectively in 1 gmLaqueous extract (Figure 2(b))

MTT assays were performed with human embryonickidney 293 and human neuroblastoma SH-SY5Y cells aftertreatment with extract of P lactiflora and the its threeconstituents respectively for 24 hr The histone deacetylaseinhibitor suberoylanilide suberoylanilide hydroxamic acid(SAHA) known to reduce SDS-insoluble polyQ aggregate[26] was included for comparison The IC

50of the herb and

compounds were calculated using the interpolation methodBoth P lactiflora extract and its constituents paeoniflorin andalbiflorin had an IC

50higher than the highest concentration

tested (gt30mgmL for P lactiflora and gt1mM for paeoni-florin and albiflorin) suggesting their very low cytotoxicity(Figure 2(c))

33 P lactiflori Extract and Paeoniflorin Reduce ATXN3Q75

Aggregation on 293 Cell Model To screen if herbcompoundspotentially inhibit aggregation we used ATXN3Q

75-GFP

cells to examine aqueous extract of P lactiflora and its con-stituents for their potentials to reduce the ATXN3Q

75aggre-

gation The experiment flow chart is shown in Figure 3(a)and representative fluorescence microscopy images of aggre-gation after treatment with paeoniflorin and the aqueousextract of P lactiflora are shown in Figure 3(b) As a positivecontrol HDAC inhibitor SAHA reduced the ATXN3Q

75

aggregation to 85 (at 100 nM) as compared to untreatedcells (Figure 3(c)) While garlic acid did not display goodaggregation-inhibitory potential (90sim95 at 100 nMsim1120583M)P lactiflora (81sim82 at 2sim50120583gmL) paeoniflorin (73 at100 nM) and albiflorin (78 at 5 120583M) had greater aggrega-tion reduction potential than SAHA (Figure 3(c)) The IC

50

cytotoxicityeffective (reduced the ATXN3Q75

aggregationto 85 or lower) dose ratio of SAHA paeoniflorin albiflorinand extract of P lactiflora were 3800 gt10000 gt200 andgt15000 respectively Considering 2 120583gml of P lactifloraextract contained 95 nM paeoniflorin and 30 nM albiflorinand tested greatest aggregation reduction potential of 100 nMfor paeoniflorin and 5 120583M for albiflorin paeoniflorin wasregarded as a major active component for the aggregationinhibition in P lactiflora

34 Paeoniflorin Enhanced HSF1 and HSP70 ChaperoneExpression on 293 Cells To screen the potential of herbcompounds to enhance HSF1 and HSP70 chaperone expres-sion we established a triple fluorescent reporter 293 cell

8 Evidence-Based Complementary and Alternative MedicineA

ggre

gatio

n (

)

30

20

10

119875 = 0024

minusHSF1 +HSF1

50120583m

Figure 6 HSF1 overexpression in ATXN3Q75-GFP transient cell

models HEK-293T cells were co-transfected with plasmids encod-ingATXN3Q

75-GFP andplasmidwith (+HSF1) orwithout (minusHSF1)

HSF1 cDNA After 2 days aggregation percentage was assessed byHCA system (The scale bar = 50 120583m) The percentage of aggregateformation counted among three random fields

model with mCherry ZsYellow1 and AmCyan1 reportersdownstream of HSF1 HSPA8 and HSPA1A promoters(Figure 4(a)) The cloned promoters effectively drove theexpression of red yellow and blue fluorescent reporters(Figure 4(b)) As shown in Figure 4(c) treatment of GGA(100 nMsim100120583M) a potent HSP inducer for one day signifi-cantly increasedHSF1 (110sim112119875 = 0010sim0002) HSPA8(106sim116 119875 = 0024sim0000) and HSPA1A (108sim118119875 = 0019sim0001) promoter activity This was also true forpaeoniflorin (100 nMsim100 120583M) treatment with 108sim120HSF1 (119875 = 0028sim0001) 112 sim120 HSPA8 (119875 = 0044sim0000) and 110sim119 HSPA1A (119875 = 0025sim0001) promoteractivities compared to no treament The enhancement ofpaeoniflorin (100 nM) on HSF1 (158 119875 = 0027) HSPA8(140 119875 = 0011) and HSPA1A (137 119875 = 0007) expressionwas confirmed by theWestern blot inHEK-293 cells after twodays treatment (Figure 4(d))

35 P lactiflori Extract and Paeoniflorin Enhanced HSF1and HSP70 Chaperone Expression on 293 ATXN3Q

75Cell

Model To examine if paeoniflorin and P lactiflora extractalso up-regulated HSF1 and HSP70 chaperone expression inATXN3Q

75293 cells we compared the expression levels

of HSF1 HSPA8 and HSPA1A between with and withoutpaeoniflorinP lactiflora andor Dox treatment As shownin Figure 5 induced expression of ATXN3Q

75for 6 days

attenuated the expression ofHSF1 (78sim67) HSPA8 (86)and HSPA1A (82) This reduction can be rescued by theaddition of paeoniflorin (100 nM) or P lactiflora (10120583gmL)with significantly increased HSF1 (113sim126 119875 = 0005sim0001) HSPA8 (123sim124 119875 = 0018sim0005) and HSPA1A(118sim119 119875 = 0046sim0009) expression These findingssuggested that P lactiflora and paeoniflorin up-regulated

HSF1 and HSP70 chaperon expression to reduce ATXN3Q75

aggregation in this cell model

36 HSF1 Overexpression to Reduce ATXN3Q75Aggregation

To determine whether HSF1 could suppress aggregationof mutant ATXN3 we transiently co-expressed HSF1 withATXN3Q

75in HEK-293T cells As shown in Figure 6

with HSF1 co-transfection visible aggregates significantlydecreased in ATXN3Q

75cells (138 versus 240 119875 =

0024)

37 P lactiflori Extract and Paeoniflorin Reduced ATXN3Q75

Aggregation on SH-SY5Y Cell Model To test the aggregationreduction potential of P lactiflori extract and paeoniflorinin neuronal cells we constructed Flp-In SH-SY5Y cells withN-terminal truncated ATXN3Q

14sim75-GFP expression in an

inducible fashion GFP-tagged 40sim57 kDa ATXN3Q14sim75

protein in Dox-induced SH-SY5Y cells can be seen in West-ern blot (Figure 7(a)) Then we differentiated ATXN3Q

14sim75

SH-SY5Y cells using retinoic acid and found that theinduced ATXN3Q

75formed aggregates in sim1 neuronal

cells (Figure 7(b)) The treatment of paeoniflorin or P lacti-flora leaded to 21sim16 of aggregation reduction (119875= 0013sim0035) in ATXN3Q

75expressed neuronal cells (Figure 7(c))

These results confirmed the aggregation-inhibitory effect ofpaeoniflorin and P lactiflora in differentiated neurons

4 Discussion

Although Chinese herbs have been reported to reducepneumonia risk in elderly patients with dementia [27] andregarded as a potential treatment of Huntingtonrsquos disease(HD) [28] the attempts to apply this alternative treatmentin SCA are still few Okabe et al (2007) reported a patientwith SCA6 was treated with a mixture of 18 medical herbs(modified Zhengan Xifeng Tang) and then the patientrsquos ataxiawas remarkably reduced [29] However the therapeuticallyeffective compound(s) in this remedy remains unknown Inthis studywe identified the aqueous extract of P lactiflora thatreduced ATXN3-aggregates mainly via its active compoundpaeoniflorin (Figures 2 and 3) The reporter gene assay andWestern blotting further indicated the aggregation-reductioneffect of paeoniflorin was modulated by the up-regulationof HSF1 and its targets HSPA8 and HSPA1A chaperoneexpressions (Figure 4)

P lactiflora with immunomodulatory and anti-inflam-matory effects [30] has been widely used as a componentof traditional Chinese prescriptions to relieve pain and totreat rheumatoid arthritis systemic lupus erythromatosusdysmenorrhea hepatitis muscle spasm and fever with a longhistory Its main bioactive component paeoniflorin pos-sesses wide pharmacological effects in the nervous systemIt has been reported to decrease the death of rat corticalcells while exposed to H

2O2-induced oxidative stress [31]

Subcutaneous injection of paeoniflorin has shown functionalprotection in the 6-OHDA lesion rodent model of PD [16]


(kD

a)

70

55

40

35

Dox

GAPDH

-GFP

-GFP

ATXN311987614

ATXN311987675


minusminus ++

(a)

ATXN311987614

ATXN311987675

20120583m

(b)

Rela

tive a

ggre

gatio

n (

)

10080604020

119875 = 0013 0035


(c)

Figure 7 Reduction of aggregation by paeoniflorin and the aqueous extract of P lactiflora in ATXN3Q75-GFP SH-SY5Y cells (a) Western

blot analysis using GFP and GAPDH antibodies after two days induction (+Dox) (b) Microscopic images of differentiated SH-SY5Y cellsexpressing ATXN3Q

1475-GFP for 6 days (the scale bar = 20 120583m) (c) Relative aggregation after treatment with paeoniflorin (100 nM) or the

aqueous extract of P lactiflora (10120583gmL) for 6 days To normalize the relative aggregation level in untreated cells is set as 100

and reduce the MPTP-induced toxicity by activation of theadenosine A1 receptor to inhibit neuroinflammation [15]In A120573

(1ndash42)-injected AD rat model paeoniflorin attenuatesthe neurotoxicity and cognitive decline by regulating cal-cium homeostasis and ameliorating oxidative stress [17]Our results demonstrate both extract of P lactiflora andpaeoniflorin up-regulating HSF1 and HSP70 expressionsto inhibit aggregate formation in ATXN3Q

75293 cells

(Figure 5) This aggregation-inhibitory effect can also beseen in neuronal differentiated SH-SY5Y cells expressingATXN3Q

75(Figure 7) providing a novel mechanism of P

lactiflora and paeoniflorin to slow down the neurodegen-erative process by inducing the expression of heat shockproteins

Peoniflorin was the first reported active component ofherbal medicines to induce expression of heat shock proteinsin HeLa IMR-32 and normal rat kidney cells [32] Uponstress HSF1 is released from the chaperone complex self-trimerizes and then is transported into the nucleus as atranscription factor It activates chaperones which play animportant role in preventing unwanted protein aggregationOverexpression of HSF1 significantly improved the life spanof R62 Huntingtonrsquos disease mouse [33] Up-regulation ofchaperone expression by HSF1 and its activating compounds

17-(allylamino)-17-demethoxygeldanamycin (17-AAG) dem-onstrate a strong inhibitory effect on HD aggregate for-mation [34] We also showed aggregation-inhibitory effectof HSF1 overexpression in 293 cells expressing ATXN3Q

75

(Figure 6) Overexpression of its downstream target geneHSPA1A suppresses polyQ-mediated neurodegeneration inDrosophila and mouse models [35 36] Reduced expressionof HSPA8 another target genes of HSF1 [37] has been shownin the 293 cells overexpressing expanded TBP-Q

61[38] and

SCA17 lymphoblastoid cells [39] The therapeutic potentialof P lactiflora and paeoniflorin in the treatment of SCA isstrongly supported by the up-regulated HSF1 and HSP70expressions

In conclusion our in vitro study provides strong evidencethat P lactiflora and paeoniflorin could be novel therapeuticsfor SCA3 and other polyQ diseases Future application ofP lactiflora and paeoniflorin to SCA animal models wouldsolidify their effects on aggregation reduction and diseaseimprovement Since the pathogenesis of the polyQ diseases isnot completely clear and effective treatment is not availableour cell models are extremely valuable for identifying poten-tial therapeutic targets in polyQ diseases A systemic high-throughput screening of herbal and chemical compoundsusing ATXN3Q

75293 cell model is undergoing


Conflict of Interests

The authors declare that there is no conflict of interests

Acknowledgments

This work was supported by Grants NSC100-2325-B-003-001 and NSC101-2325-B-003 -002 from the National ScienceCouncil Executive Yuan NTNU100-D-02 from NationalTaiwan Normal University Taipei and Aim for the Top Uni-versity ProjectndashNTNU Taiwan K-H Chang W-L Chenand L-C Lee equally contributed to this work

References

[1] A Matilla-Duenas M Corral-Juan V Volpini and I SanchezldquoThe spinocerebellar ataxias clinical aspects and moleculargeneticsrdquo Advances in Experimental Medicine and Biology vol724 pp 351ndash374 2012

[2] YKawaguchi TOkamotoMTaniwaki et al ldquoCAGexpansionsin a novel gene for Machado-Joseph disease at chromosome14q321rdquo Nature Genetics vol 8 no 3 pp 221ndash228 1994

[3] H T Orr M Y Chung S Banfi et al ldquoExpansion of anunstable trinucleotide CAG repeat in spinocerebellar ataxiatype 1rdquo Nature Genetics vol 4 no 3 pp 221ndash226 1993

[4] S M Pulst A Nechiporuk T Nechiporuk et al ldquoModerateexpansion of a normally biallelic trinucleotide repeat in spinoo-erebellar ataxia typerdquoNatureGenetics vol 14 no 3 pp 269ndash2761996

[5] O Zhuchenko J Bailey P Bonnen et al ldquoAutosomal dominantcerebellar ataxia (SCA6) associated with small polyglutamineexpansions in the 120572

(1119860)-voltage-dependent calcium channelrdquo

Nature Genetics vol 15 no 1 pp 62ndash69 1997[6] G David N Abbas G Stevanin et al ldquoCloning of the SCA7

gene reveals a highly unstable CAG repeat expansionrdquo NatureGenetics vol 17 no 1 pp 65ndash70 1997

[7] R Koide S Kobayashi T Shimohata et al ldquoA neurologicaldisease caused by an expanded CAG trinucleotide repeat inthe TATA-binding protein gene a new polyglutamine diseaserdquoHuman Molecular Genetics vol 8 no 11 pp 2047ndash2053 1999

[8] R Koide T Ikeuchi O Onodera et al ldquoUnstable expansion ofCAG repeat in hereditary dentatorubral-pallidoluysian atrophy(DRPLA)rdquo Nature Genetics vol 6 no 1 pp 9ndash13 1994

[9] H Y Zoghbi and H T Orr ldquoPolyglutamine diseases proteincleavage and aggregationrdquoCurrentOpinion inNeurobiology vol9 no 5 pp 566ndash570 1999

[10] J R Gatchel and H Y Zoghbi ldquoDiseases of unstable repeatexpansion mechanisms and common principlesrdquo NatureReviews Genetics vol 6 no 10 pp 743ndash755 2005

[11] A Solans A Zambrano M Rodrıguez and A BarrientosldquoCytotoxicity of a mutant huntingtin fragment in yeast involvesearly alterations in mitochondrial OXPHOS complexes II andIIIrdquo Human Molecular Genetics vol 15 no 20 pp 3063ndash30812006

[12] E J Bennett T A Shaler BWoodman et al ldquoGlobal changes tothe ubiquitin system in Huntingtonrsquos diseaserdquo Nature vol 448no 7154 pp 704ndash708 2007

[13] S M Chafekar and M L Duennwald ldquoImpaired heatshock response in cells expressing full-length polyglutamine-expanded Huntingtinrdquo PLoS ONE vol 7 no 5 Article IDe37929 2012

[14] T T Ou C H Wu J D Hsu C C Chyau H J Lee and CJ Wang ldquoPaeonia lactiflora Pall inhibits bladder cancer growthinvolving phosphorylation of Chk2 in vitro and in vivordquo Journalof Ethnopharmacology vol 135 no 1 pp 162ndash172 2011

[15] H Q Liu W Y Zhang X T Luo Y Ye and X Z ZhuldquoPaeoniflorin attenuates neuroinflammation and dopaminergicneurodegeneration in the MPTP model of Parkinsonrsquos diseaseby activation of adenosine A1 receptorrdquo British Journal ofPharmacology vol 148 no 3 pp 314ndash325 2006

[16] D Z Liu J Zhu D Z Jin et al ldquoBehavioral recovery follow-ing sub-chronic paeoniflorin administration in the striatal 6-OHDA lesion rodent model of Parkinsonrsquos diseaserdquo Journal ofEthnopharmacology vol 112 no 2 pp 327ndash332 2007

[17] S Z Zhong Q H Ge Q Li R Qu and S P Ma ldquoPeoni-florin attentuates A120573

(1minus42)-mediated neurotoxicity by regulating

calcium homeostasis and ameliorating oxidative stress in hip-pocampus of ratsrdquo Journal of the Neurological Sciences vol 280no 1-2 pp 71ndash78 2009

[18] M H Jeon H J Kwon J S Jeong Y M Lee and S P HongldquoDetection of albiflorin and paeoniflorin in Paeoniae Radix byreversed-phase high-performance liquid chromatography withpulsed amperometric detectionrdquo Journal of Chromatography Avol 1216 no 21 pp 4568ndash4573 2009

[19] S L Li J Z Song F F K Choi et al ldquoChemical profil-ing of Radix Paeoniae evaluated by ultra-performance liquidchromatographyphoto-diode-arrayquadrupole time-of-flightmass spectrometryrdquo Journal of Pharmaceutical and BiomedicalAnalysis vol 49 no 2 pp 253ndash266 2009

[20] Y Zhang S Koushik R Dai and N F Mivechi ldquoStructuralorganization and promoter analysis of murine heat shocktranscription factor-1 generdquo Journal of Biological Chemistry vol273 no 49 pp 32514ndash32521 1998

[21] M He H Guo X Yang et al ldquoGenetic variations in HSPA8gene associated with coronary heart disease risk in a Chinesepopulationrdquo PloS One vol 5 no 3 p e9684 2010

[22] Y-R Wu C-K Wang C-M Chen et al ldquoAnalysis of heat-shock protein 70 gene polymorphisms and the risk of Parkin-sonrsquos diseaserdquoHumanGenetics vol 114 no 3 pp 236ndash241 2004

[23] L-C Lee C-M Chen H-C Wang et al ldquoRole of the CCAAT-binding protein NFY in SCA17 pathogenesisrdquo PLoS One vol 7no 4 Article ID e35302 2012

[24] S Flis and J Spławinski ldquoInhibitory effects of 5-fluorouracil andoxaliplatin on human colorectal cancer cell survival are syn-ergistically enhanced by sulindac sulfiderdquo Anticancer Researchvol 29 no 1 pp 435ndash441 2009

[25] A Haacke S A Broadley R Boteva N Tzvetkov F U Hartland P Breuer ldquoProteolytic cleavage of polyglutamine-expandedataxin-3 is critical for aggregation and sequestration of non-expanded ataxin-3rdquo Human Molecular Genetics vol 15 no 4pp 555ndash568 2006

[26] M Mielcarek C L Benn S A Franklin et al ldquoSAHAdecreases HDAC 2 and 4 levels in vivo and improves molecularphenotypes in the R62 mouse model of Huntingtonrsquos diseaserdquoPLoS One vol 6 no 11 Article ID e27746 2011

[27] K Iwasaki S Kato Y Monma et al ldquoA pilot study of BanxiaHoupu Tang a traditional Chinese medicine for reducingpneumonia risk in older adults with dementiardquo Journal of theAmerican Geriatrics Society vol 55 no 12 pp 2035ndash2040 2007

[28] T Satoh T Takahashi K Iwasaki et al ldquoTraditional Chinesemedicine on four patients with Huntingtonrsquos diseaserdquo Move-ment Disorders vol 24 no 3 pp 453ndash455 2009


[29] T Okabe M Fujisawa T Sekiya Y Ichikawa and J GotoldquoSuccessful treatment of spinocerebellar ataxia 6withmedicinalherbsrdquo Geriatrics amp Gerontology International vol 7 no 2 pp195ndash197 2007

[30] D-Y He and S-M Dai ldquoAnti-inflammatory and immunomod-ulatory effects of Paeonia lactiflora Pall a traditional Chineseherbal medicinerdquo Frontiers in Pharmacology vol 2 article 102011

[31] S H Kim M K Lee K Y Lee S H Sung J Kim and Y CKim ldquoChemical constituents isolated from Paeonia lactifloraroots and their neuroprotective activity against oxidative stressin vitrordquo Journal of Enzyme Inhibition andMedicinal Chemistryvol 24 no 5 pp 1138ndash1140 2009

[32] D Yan K Saito Y Ohmi N Fujie and K Ohtsuka ldquoPaeoni-florin a novel heat shock protein-inducing compoundrdquo CellStress and Chaperones vol 9 no 4 pp 378ndash389 2004

[33] M Fujimoto E Takaki T Hayashi et al ldquoActive HSF1 signifi-cantly suppresses polyglutamine aggregate formation in cellularandmousemodelsrdquo Journal of Biological Chemistry vol 280 no41 pp 34908ndash34916 2005

[34] N Fujikake Y Nagai H A Popiel Y Okamoto M Yamaguchiand T Toda ldquoHeat shock transcription factor 1-activating com-pounds suppress polyglutamine-induced neurodegenerationthrough induction of multiple molecular chaperonesrdquo Journalof Biological Chemistry vol 283 no 38 pp 26188ndash26197 2008

[35] J M Warrick H Y E Chan G L Gray-Board Y Chai HL Paulson and N M Bonini ldquoSuppression of polyglutamine-mediated neurodegeneration in Drosophila by the molecularchaperone HSP70rdquo Nature Genetics vol 23 no 4 pp 425ndash4281999

[36] C J Cummings Y Sun P Opal et al ldquoOver-expression ofinducible HSP70 chaperone suppresses neuropathology andimproves motor function in SCA1 micerdquo Human MolecularGenetics vol 10 no 14 pp 1511ndash1518 2001

[37] S A Kim J H Yoon D K Kim S G Kim and S G AhnldquoCHIP interacts with heat shock factor 1 during heat stressrdquoFEBS Letters vol 579 no 29 pp 6559ndash6563 2005

[38] L C Lee C M Chen F L Chen et al ldquoAltered expressionof HSPA5 HSPA8 and PARK7 in spinocerebellar ataxia type17 identified by 2-dimensional fluorescence difference in gelelectrophoresisrdquo Clinica Chimica Acta vol 400 no 1-2 pp 56ndash62 2009

[39] C M Chen L C Lee B W Soong et al ldquoSCA17 repeat expan-sion mildly expanded CAGCAA repeat alleles in neurologicaldisorders and the functional implicationsrdquo Clinica ChimicaActa vol 411 no 5-6 pp 375ndash380 2010

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


Paeoniaceae family is a perennial herb frequently used asan important ingredient in many traditional prescriptions Ithas been commonly used for nourishing blood alleviatingpain reducing irritability as well as treating liver diseaseand cancer [14] Paeoniflorin one of the main compoundsextracted from P lactiflora has been reported to ameliorateneurodegenerative process in Parkinsonrsquos disease (PD) andAlzheimerrsquos disease (AD)models [15ndash17] However the effectof P lactiflora herb extract and paeoniflorin in treating SCAremains unraveled

In the present study we firstly built up an aggrega-tion screening cell model by overexpressing CAG-expandedATXN3 the causative mutation in SCA3 [2] in 293 cellsand then examined the anti-aggregation effect of P lactifloraaqueous extract and paeoniflorin We further demonstratedthat the anti-aggregation activity of P lactiflora extract andpaeoniflorin was contributed by the enhancement of heatshock transcription factor 1 (HSF1)-heat shock protein (HSP)70 chaperone system These findings provide evidence thatP lactiflora and paeoniflorin may be a novel alternativetherapeutic agent for the treatment of SCAs

2 Materials and Methods

21 P lactiflora Extract Preparation and HPLC AnalysisAqueous extract from P lactiflora was provided by Sun-Ten Pharmaceutical Company (Taipei Taiwan) Briefly 100 gof dried P lactiflora was boiled with 1500mL of water at100∘C for 30min and was sieved using a 100-mesh sieve Theextract was concentrated to 100mL and filtered through a200-mesh sieveThe extract was then dried by speed vacuumconcentration and then stored at minus20∘C until used

High pressure liquid chromatography (HPLC) was per-formed using a LaChrom Elite HPLC system (Hitachi) con-sisting of a photo diode array detector The chromatographicseparation ofP lactiflora extract (50120583L 1mgmL)was carriedout on a Hypersil ODS (C18) column (250 times 46mm 5 120583m)eluted with the mixture of 01 formic acid in water (A)or acetonitrile (B) The linear gradient elution program forA B (vv) was set as follows 95 5 (0ndash10min) 95 5ndash70 30(10ndash40min) 70 30ndash15 85 (40ndash55min) 15 85ndash95 5 (55ndash60min) 95 5 (60ndash75min) with a flow rate of 1mLminAbsorbance was monitored at 230 250 270 nm and the scanrange for photo diode array was 190sim400 nm Paeonifloringallic acid and albiflorin (2sim10120583L 20mM) were used asreference compounds for P lactiflora [18 19]

22 Cell Culture and Cell Proliferation Assay Human embry-onic kidney HEK-293 cells (ATCC No CRL-1573) werecultivated in Dulbeccorsquos modified Eaglersquos medium (DMEM)containing 10 fetal bovine serum (FBS) Human neuroblas-toma SH-SY5Y cells (ATCCNo CRL-2266) were maintainedin DMEM F12 supplemented with 10FBS Cells werecultivated at 37∘C incubator containing 5 CO

2and cell

proliferation was measured based upon the reduction of thetetrazolium salt 3[45-dimethylthiazol-2-yL]-25-diphenyl-tetrazolium bromide (MTT) Cells were plated into 48-well (5 times 104well) dishes grown for 20 hr and treated

with different concentrations of the P lactiflora extract (5sim30mgmL) or pure compound (100 nMsim1mM) After oneday 20120583L MTT (5mgmL in PBS Sigma) was added tocells and incubated for 2 hr The absorbance of the purpleformazan dye was measured at 570 nm by a Bio-Tek 120583QuantUniversal Microplate Spectrophotometer

23 Flp-In-293 Triple Fluorescent Reporter Cells and Flu-orescent Assay A triple fluorescent reporter plasmid withmCherry ZsYellow1 and AmCyan1 was first constructedin pAmCyan1-N1 A proximal promoter (minus360sim+2 withthe translation initiation site as +1) from HSF1 gene wasused as 331 bp upstream of the murine HSF1 transla-tion start site is required for maximal basal expression[20] Promoter fragments from HSF1 (enhancing chaperoneexpression) heat shock cognate protein (HSPA8 minus1140sim+38 driving constitutively expressed HSP70) [21] and heat-inducible HSP70 chaperone (HSPA1A minus273sim+215 drivingheat-inducible HSP70) [22] are placed upstream of the threefluorescent reporters The fragment containing the HSF1HSPA8 and HSPA1A driven reporters was excised withAseI and NotI restriction enzymes and used to replace anAseI-NotI fragment in pcDNA5FRTTO plasmid (Invitro-gen) The resulting triple fluorescent reporter plasmid wasused to generate Flp-In triple fluorescent reporter cells andmaintained according to the supplierrsquos instructions (Invitro-gen) Geranylgeranylacetone (GGA Sigma) or paeoniflorin(100 nMsim100120583M) was added to the medium for 24 hr Thethree fluorescence colors were analyzed simultaneously usinghigh-content analysis (HCA) system (ImageXpressMICROMolecular Devices) with excitationemission wavelengthsat 453486 (mCherry) 531540 (ZsYellow1) and 587610 nm(AmCyan1)

24 ATXN3 and HSF1 cDNA Constructs PolyadenylatedRNA (200 ng) isolated from neuroblastoma SK-N-SH cellswas reverse transcribed using the SuperScript III reversetranscriptase (Invitrogen) The sense and antisense primersused for ATXN3Q

14cDNA (+826sim+1152 NM 004993)

amplification were 51015840-ATTCAGCTAAGTATGCAA-GGTAGTTCCA (codon for Met257 underlined) and51015840-CATGCCATGGCATGTTTTTTTCCTTCTGTT (NcoIsite underlined) The amplified 31015840 polyQ-containing cDNAfragment (translated into amino acids 257sim361) wascloned into pGEM-T Easy (Promega) and sequenced TheATXN3Q

14cDNAwas excised with EcoRI (in pGEM-T Easy

vector) and NcoI and subcloned into pEGFP-N1 (Clontech)Then DNA fragment containing in-frame ATXN3Q

14-

EGFP was excised with HindIII-NotI and subcloned intothe pcDNA5FRTTO The ATXN3Q

75cDNA was made

by replacing an 88 bp ATXN3Q14

BsmBI-BsmFI fragmentwith a 271 bp ATXN3Q

75fragment from the cDNA clone

of a SCA3 patient The HSF1 cDNA (BC014638) in pOTB7was obtained from Bioresource Collection and ResearchCenter (BCRC) Food Industry Research and DevelopmentInstitute Taiwan The cDNA was excised with EcoRI andXhoI and subcloned into pcDNA3 (Invitrogen)


(kD

a)

170 130 100

70

55

40

35

25

M Dox

GFP antibody

ATXN311987614 ATXN311987675

minus + minus +

ATXN311987614-GFP

ATXN311987675-GFP

(a)

10

20

30

Dox

ATXN311987614 ATXN311987675

ATXN

3H

PRT1

minus + minus +

(b)

100120583m


(c)




14sim75-GFP RNA




14and Q

75plasmids were




26 ATXN3Q75











Inte

nsity

(AU

) 2

15

1

05

0



Gallic acid(588min)

Albiflorin(2746min)


0 10 20 30 40 50 60 70

(a)

Paeoniflorin

Gallic acid

Albiflorin

Content in 1gmL

227 4733mM

03 1806mM

073 1516mM

(b)

Cel

l via

bilit

y (

) 120

80

40

Cel

l via

bilit

y (

) 120

80

40

51015

20

30

Untreated


038mM 058mM

P lactiflora




50

50


1120583M

10120583M

1mM100120583M 25mgmL

mgmL

mgmLmgmL

mgmL

mgmL100nM

(c)








119905




29 ATXN3Q75



Cellseeding

Herbcompounds

HCA


8hr1day 6days

(a)


(b)

Untreated

120

80

40

Relat

ive a

ggre

gatio

n (

)

85

SAHA 3800


100



21050

100nM500nM

1120583M5120583M 200120583gmL

120583gmL

120583gmL120583gmL120583gmL


P lactifloragt15000

(c)












3 Results




14sim75-containing



14-



14





(a)


(b)

125

100

75

50

25Fluo

resc

ence

()



100nM100nM1120583M1120583M

10120583M10120583M100120583M100120583M

119875 = 0028sim0001

119875 = 0010sim0002 119875 = 0024sim0000

119875 = 0044 sim0000

119875 = 0019sim0001

119875 = 0025sim0001

(c)

Paeoniflorin 200

150

100

50

(100nM)

H3F3B


+minus

()

HSF1

HSF1

HSPA8

HSPA8

HSPA1A

HSPA1A

(d)



Dox

Paeoniflorin

Dox

Paeoniflorin

Relat

ive r

atio

()

Relat

ive r

atio

()

Relat

ive r

atio

() 140

100

60

20

140

100

60

20

140

100

60

20


120573-actin

120573-actin

120573-actin

119875 = 0005119875 = 0001

119875 = 0018

119875 = 0009 119875 = 0046

HSF1

HSPA8

HSPA1A

119875 = 0005

HSPA1A

HSPA8

HSF1

minus

minus

minus

minus minus

minus

minusminus minus


minus

minus

minus+

+ +

+ +

++

+

++ +

+ ++ +

+






50of the herb and






75-GFP


75aggre-


75


50





ggre

gatio

n (

)

30

20

10

119875 = 0024

minusHSF1 +HSF1

50120583m







75Cell




75for 6 days








75cells (138 versus 240 119875 =

0024)






14sim75






4 Discussion






(kD

a)

70

55

40

35

Dox

GAPDH

-GFP

-GFP

ATXN311987614

ATXN311987675


minusminus ++

(a)

ATXN311987614

ATXN311987675

20120583m

(b)

Rela

tive a

ggre

gatio

n (

)

10080604020

119875 = 0013 0035


(c)







75293 cells






75


61[38] and







Acknowledgments


References


















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















(kD

a)

170 130 100

70

55

40

35

25

M Dox

GFP antibody

ATXN311987614 ATXN311987675

minus + minus +

ATXN311987614-GFP

ATXN311987675-GFP

(a)

10

20

30

Dox

ATXN311987614 ATXN311987675

ATXN

3H

PRT1

minus + minus +

(b)

100120583m


(c)




14sim75-GFP RNA




14and Q

75plasmids were




26 ATXN3Q75











Inte

nsity

(AU

) 2

15

1

05

0



Gallic acid(588min)

Albiflorin(2746min)


0 10 20 30 40 50 60 70

(a)

Paeoniflorin

Gallic acid

Albiflorin

Content in 1gmL

227 4733mM

03 1806mM

073 1516mM

(b)

Cel

l via

bilit

y (

) 120

80

40

Cel

l via

bilit

y (

) 120

80

40

51015

20

30

Untreated


038mM 058mM

P lactiflora




50

50


1120583M

10120583M

1mM100120583M 25mgmL

mgmL

mgmLmgmL

mgmL

mgmL100nM

(c)








119905




29 ATXN3Q75



Cellseeding

Herbcompounds

HCA


8hr1day 6days

(a)


(b)

Untreated

120

80

40

Relat

ive a

ggre

gatio

n (

)

85

SAHA 3800


100



21050

100nM500nM

1120583M5120583M 200120583gmL

120583gmL

120583gmL120583gmL120583gmL


P lactifloragt15000

(c)












3 Results




14sim75-containing



14-



14





(a)


(b)

125

100

75

50

25Fluo

resc

ence

()



100nM100nM1120583M1120583M

10120583M10120583M100120583M100120583M

119875 = 0028sim0001

119875 = 0010sim0002 119875 = 0024sim0000

119875 = 0044 sim0000

119875 = 0019sim0001

119875 = 0025sim0001

(c)

Paeoniflorin 200

150

100

50

(100nM)

H3F3B


+minus

()

HSF1

HSF1

HSPA8

HSPA8

HSPA1A

HSPA1A

(d)



Dox

Paeoniflorin

Dox

Paeoniflorin

Relat

ive r

atio

()

Relat

ive r

atio

()

Relat

ive r

atio

() 140

100

60

20

140

100

60

20

140

100

60

20


120573-actin

120573-actin

120573-actin

119875 = 0005119875 = 0001

119875 = 0018

119875 = 0009 119875 = 0046

HSF1

HSPA8

HSPA1A

119875 = 0005

HSPA1A

HSPA8

HSF1

minus

minus

minus

minus minus

minus

minusminus minus


minus

minus

minus+

+ +

+ +

++

+

++ +

+ ++ +

+






50of the herb and






75-GFP


75aggre-


75


50





ggre

gatio

n (

)

30

20

10

119875 = 0024

minusHSF1 +HSF1

50120583m







75Cell




75for 6 days








75cells (138 versus 240 119875 =

0024)






14sim75






4 Discussion






(kD

a)

70

55

40

35

Dox

GAPDH

-GFP

-GFP

ATXN311987614

ATXN311987675


minusminus ++

(a)

ATXN311987614

ATXN311987675

20120583m

(b)

Rela

tive a

ggre

gatio

n (

)

10080604020

119875 = 0013 0035


(c)







75293 cells






75


61[38] and







Acknowledgments


References


















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Inte

nsity

(AU

) 2

15

1

05

0



Gallic acid(588min)

Albiflorin(2746min)


0 10 20 30 40 50 60 70

(a)

Paeoniflorin

Gallic acid

Albiflorin

Content in 1gmL

227 4733mM

03 1806mM

073 1516mM

(b)

Cel

l via

bilit

y (

) 120

80

40

Cel

l via

bilit

y (

) 120

80

40

51015

20

30

Untreated


038mM 058mM

P lactiflora




50

50


1120583M

10120583M

1mM100120583M 25mgmL

mgmL

mgmLmgmL

mgmL

mgmL100nM

(c)








119905




29 ATXN3Q75



Cellseeding

Herbcompounds

HCA


8hr1day 6days

(a)


(b)

Untreated

120

80

40

Relat

ive a

ggre

gatio

n (

)

85

SAHA 3800


100



21050

100nM500nM

1120583M5120583M 200120583gmL

120583gmL

120583gmL120583gmL120583gmL


P lactifloragt15000

(c)












3 Results




14sim75-containing



14-



14





(a)


(b)

125

100

75

50

25Fluo

resc

ence

()



100nM100nM1120583M1120583M

10120583M10120583M100120583M100120583M

119875 = 0028sim0001

119875 = 0010sim0002 119875 = 0024sim0000

119875 = 0044 sim0000

119875 = 0019sim0001

119875 = 0025sim0001

(c)

Paeoniflorin 200

150

100

50

(100nM)

H3F3B


+minus

()

HSF1

HSF1

HSPA8

HSPA8

HSPA1A

HSPA1A

(d)



Dox

Paeoniflorin

Dox

Paeoniflorin

Relat

ive r

atio

()

Relat

ive r

atio

()

Relat

ive r

atio

() 140

100

60

20

140

100

60

20

140

100

60

20


120573-actin

120573-actin

120573-actin

119875 = 0005119875 = 0001

119875 = 0018

119875 = 0009 119875 = 0046

HSF1

HSPA8

HSPA1A

119875 = 0005

HSPA1A

HSPA8

HSF1

minus

minus

minus

minus minus

minus

minusminus minus


minus

minus

minus+

+ +

+ +

++

+

++ +

+ ++ +

+






50of the herb and






75-GFP


75aggre-


75


50





ggre

gatio

n (

)

30

20

10

119875 = 0024

minusHSF1 +HSF1

50120583m







75Cell




75for 6 days








75cells (138 versus 240 119875 =

0024)






14sim75






4 Discussion






(kD

a)

70

55

40

35

Dox

GAPDH

-GFP

-GFP

ATXN311987614

ATXN311987675


minusminus ++

(a)

ATXN311987614

ATXN311987675

20120583m

(b)

Rela

tive a

ggre

gatio

n (

)

10080604020

119875 = 0013 0035


(c)







75293 cells






75


61[38] and







Acknowledgments


References


















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Cellseeding

Herbcompounds

HCA


8hr1day 6days

(a)


(b)

Untreated

120

80

40

Relat

ive a

ggre

gatio

n (

)

85

SAHA 3800


100



21050

100nM500nM

1120583M5120583M 200120583gmL

120583gmL

120583gmL120583gmL120583gmL


P lactifloragt15000

(c)












3 Results




14sim75-containing



14-



14





(a)


(b)

125

100

75

50

25Fluo

resc

ence

()



100nM100nM1120583M1120583M

10120583M10120583M100120583M100120583M

119875 = 0028sim0001

119875 = 0010sim0002 119875 = 0024sim0000

119875 = 0044 sim0000

119875 = 0019sim0001

119875 = 0025sim0001

(c)

Paeoniflorin 200

150

100

50

(100nM)

H3F3B


+minus

()

HSF1

HSF1

HSPA8

HSPA8

HSPA1A

HSPA1A

(d)



Dox

Paeoniflorin

Dox

Paeoniflorin

Relat

ive r

atio

()

Relat

ive r

atio

()

Relat

ive r

atio

() 140

100

60

20

140

100

60

20

140

100

60

20


120573-actin

120573-actin

120573-actin

119875 = 0005119875 = 0001

119875 = 0018

119875 = 0009 119875 = 0046

HSF1

HSPA8

HSPA1A

119875 = 0005

HSPA1A

HSPA8

HSF1

minus

minus

minus

minus minus

minus

minusminus minus


minus

minus

minus+

+ +

+ +

++

+

++ +

+ ++ +

+






50of the herb and






75-GFP


75aggre-


75


50





ggre

gatio

n (

)

30

20

10

119875 = 0024

minusHSF1 +HSF1

50120583m







75Cell




75for 6 days








75cells (138 versus 240 119875 =

0024)






14sim75






4 Discussion






(kD

a)

70

55

40

35

Dox

GAPDH

-GFP

-GFP

ATXN311987614

ATXN311987675


minusminus ++

(a)

ATXN311987614

ATXN311987675

20120583m

(b)

Rela

tive a

ggre

gatio

n (

)

10080604020

119875 = 0013 0035


(c)







75293 cells






75


61[38] and







Acknowledgments


References


















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















(a)


(b)

125

100

75

50

25Fluo

resc

ence

()



100nM100nM1120583M1120583M

10120583M10120583M100120583M100120583M

119875 = 0028sim0001

119875 = 0010sim0002 119875 = 0024sim0000

119875 = 0044 sim0000

119875 = 0019sim0001

119875 = 0025sim0001

(c)

Paeoniflorin 200

150

100

50

(100nM)

H3F3B


+minus

()

HSF1

HSF1

HSPA8

HSPA8

HSPA1A

HSPA1A

(d)



Dox

Paeoniflorin

Dox

Paeoniflorin

Relat

ive r

atio

()

Relat

ive r

atio

()

Relat

ive r

atio

() 140

100

60

20

140

100

60

20

140

100

60

20


120573-actin

120573-actin

120573-actin

119875 = 0005119875 = 0001

119875 = 0018

119875 = 0009 119875 = 0046

HSF1

HSPA8

HSPA1A

119875 = 0005

HSPA1A

HSPA8

HSF1

minus

minus

minus

minus minus

minus

minusminus minus


minus

minus

minus+

+ +

+ +

++

+

++ +

+ ++ +

+






50of the herb and






75-GFP


75aggre-


75


50





ggre

gatio

n (

)

30

20

10

119875 = 0024

minusHSF1 +HSF1

50120583m







75Cell




75for 6 days








75cells (138 versus 240 119875 =

0024)






14sim75






4 Discussion






(kD

a)

70

55

40

35

Dox

GAPDH

-GFP

-GFP

ATXN311987614

ATXN311987675


minusminus ++

(a)

ATXN311987614

ATXN311987675

20120583m

(b)

Rela

tive a

ggre

gatio

n (

)

10080604020

119875 = 0013 0035


(c)







75293 cells






75


61[38] and







Acknowledgments


References


















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Dox

Paeoniflorin

Dox

Paeoniflorin

Relat

ive r

atio

()

Relat

ive r

atio

()

Relat

ive r

atio

() 140

100

60

20

140

100

60

20

140

100

60

20


120573-actin

120573-actin

120573-actin

119875 = 0005119875 = 0001

119875 = 0018

119875 = 0009 119875 = 0046

HSF1

HSPA8

HSPA1A

119875 = 0005

HSPA1A

HSPA8

HSF1

minus

minus

minus

minus minus

minus

minusminus minus


minus

minus

minus+

+ +

+ +

++

+

++ +

+ ++ +

+






50of the herb and






75-GFP


75aggre-


75


50





ggre

gatio

n (

)

30

20

10

119875 = 0024

minusHSF1 +HSF1

50120583m







75Cell




75for 6 days








75cells (138 versus 240 119875 =

0024)






14sim75






4 Discussion






(kD

a)

70

55

40

35

Dox

GAPDH

-GFP

-GFP

ATXN311987614

ATXN311987675


minusminus ++

(a)

ATXN311987614

ATXN311987675

20120583m

(b)

Rela

tive a

ggre

gatio

n (

)

10080604020

119875 = 0013 0035


(c)







75293 cells






75


61[38] and







Acknowledgments


References


















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















ggre

gatio

n (

)

30

20

10

119875 = 0024

minusHSF1 +HSF1

50120583m







75Cell




75for 6 days








75cells (138 versus 240 119875 =

0024)






14sim75






4 Discussion






(kD

a)

70

55

40

35

Dox

GAPDH

-GFP

-GFP

ATXN311987614

ATXN311987675


minusminus ++

(a)

ATXN311987614

ATXN311987675

20120583m

(b)

Rela

tive a

ggre

gatio

n (

)

10080604020

119875 = 0013 0035


(c)







75293 cells






75


61[38] and







Acknowledgments


References


















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















(kD

a)

70

55

40

35

Dox

GAPDH

-GFP

-GFP

ATXN311987614

ATXN311987675


minusminus ++

(a)

ATXN311987614

ATXN311987675

20120583m

(b)

Rela

tive a

ggre

gatio

n (

)

10080604020

119875 = 0013 0035


(c)







75293 cells






75


61[38] and







Acknowledgments


References


















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















Acknowledgments


References


















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















research article aqueous extract of paeonia lactiflora and...

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