A Chinese Herbal Formula, Gengnianchun, Ameliorates 𝛽...

Research ArticleA Chinese Herbal Formula Gengnianchun Ameliorates120573-Amyloid Peptide Toxicity in a Caenorhabditis elegansModel of Alzheimerrsquos Disease

Fanhui Meng12 Jun Li2 Yanqiu Rao2 WenjunWang2 and Yan Fu1

1Department of Gynecology The First Hospital of Jilin University Changchun China2Department of Integrated Traditional Chinese Medicine and Western Medicine Obstetrical and Gynecological HospitalFudan University Shanghai China

Correspondence should be addressed to Wenjun Wang wenjunwang63163com and Yan Fu f yjlueducn

Received 26 April 2017 Revised 26 August 2017 Accepted 7 September 2017 Published 11 October 2017

Academic Editor Louise Bennett

Copyright copy 2017 Fanhui Meng et al This 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

Alzheimerrsquos disease (AD) is an age-related neurodegenerative disorder and the few drugs that are currently available only treat thesymptoms Traditional medicine or phytotherapy has been shown to protect against AD In our previous studies Gengnianchun(GNC) a traditional Chinese medicine formula with a prolongevity effect protected against A120573-induced cytotoxicity inpheochromocytoma cells (PC-12 cells) and hippocampal cells Here we investigated the effects and possible mechanisms by whichGNC protected against A120573 toxicity using transgenic Caenorhabditis elegans CL4176 Our results showed that GNC effectivelydelayed the A120573 toxicity-triggered body paralysis of CL4176 worms GNC decreased A120573 by reducing A120573 mRNA levels MoreoverGNC significantly reduced reactive oxygen species in the AD model worms compared with the controls In addition GNCupregulated the daf-16 sod-3 hsp-162 genes and enhanced DAF-16 translocation from the cytoplasm to the nuclei under oxidativestress conditions GNC treatment of C elegans strains lacking DAF-16 did not affect the paralysis phenotype Taken together thesefindings suggest that GNC could protect against A120573-induced toxicity via the DAF-16 pathway in C elegans Further studies arerequired to analyze its effectiveness in more complex animals

1 Introduction

Alzheimerrsquos disease (AD) is a chronic progressive neurode-generative disorder with a prevalence of 5 in people over 60and the risk increases with age [1] As the global populationlives longer AD has become a great burden to society partic-ularly countries in which ageing individuals represent a largeproportion of the population [2] Currently a limited numberof effective therapeutics for AD are available and only a fewdrugs have been approved by the FDA asAD treatments suchas acetylcholinesterase (AChE) inhibitors and the N-methyl-D-aspartate receptor antagonist memantine [3 4] Howeverthese drugs only alleviate symptoms for a short period andtheir side effects are unfavourable Hence additional effectivedrug candidates must be identified to combat AD

Traditional Chinese medicine and other natural herb-based therapies have been reported to be effective in pre-venting or protecting against AD for many years For

example a recombinant buckwheat trypsin inhibitor (rBTI)was shown to protect against AD by promoting the activityof the autophagy-lysosomal degradation pathway [5] Lycoriscompounds were shown to modulate inflammatory- andstress-related gene expression to alleviate 120573-amyloid inducedtoxicity in C elegans [6] Gengnianchun (GNC) a traditionalChinese herbal medicine formula has been clinically usedfor more than 20 years in China The GNC formula iscomposed of Radix Rehmanniae Rhizoma Coptidis RadixPaeoniae Alba Rhizoma Anemarrhenae Cistanche SalsaRadixMorindae Officinalis Poria EpimediumBrevicornumCortex Phellodendri Amurensis Fructus Lycii Semen Cus-cutae and Carapax et plastrum Testudinis It is currentlyused in the clinic to alleviate declining functions related toageing As shown in our previous studies GNC enhancedthe performance of ovariectomized rats in the Morris watermaze test and elevated the activities of hippocampal acetyl-choline (ACh) acetylcholinesterase (AChE) and choline

HindawiEvidence-Based Complementary and Alternative MedicineVolume 2017 Article ID 7480980 10 pageshttpsdoiorg10115520177480980

2 Evidence-Based Complementary and Alternative Medicine

acetyltransferase (ChAT) [7] In addition GNC upregulatedbrain-derived neurotrophic factor (BDNF) expression andimproved learning and memory in ovariectomized rats [8]GNC has recently been shown to attenuate A120573

25ndash35-inducedcytotoxicity in pheochromocytoma cells (PC-12 cells) andhippocampal cells [9 10] Ageing and oxidative stress areknown to have important roles in the progression of AD[11 12] GNC enhances the resistance to oxidative stress andimproves lifespan in vivo [13] Based on these results GNCmay be a promising therapeutic agent for the treatment ofAD

Although the aetiology of AD remains unclear the mostwidely accepted hypothesis is the ldquoamyloid hypothesisrdquowhich suggests that A120573 neurotoxicity plays a central role inthe pathogenesis of AD [14] The toxic beta amyloid (1ndash42)peptide is derived from the abnormal processing of theamyloid precursor protein (APP) which forms extracellularsenile plaques in the human brain and leads to downstreamneurotoxic events including neuronal dysfunction stressresponses and inflammation [15]

Furthermore oxidative stress is extensive in Alzheimerrsquosdisease brains 120573-Amyloid accumulation is associated withmarkers of oxidative stress including protein oxidation lipidperoxidation and oxidation of nucleic acids [16ndash18] A120573 hasbeen shown to induce oxidative stress in vitro and in vivo [19]In vitro cells exposed to A120573 showed high levels of hydrogenperoxide and production of reactive oxygen intermediates[20 21] Previous in vivo studies have reported an increasein H2O2levels and in the peroxidation of proteins and lipids

in AD transgenic mouse models that express mutant amyloidprecursor protein (APP) and presenilin-1 (PS-1) [22 23] InAD patients A120573 accumulates in human brain mitochondriaresulting in a significant decline in mitochondrial functionwhich can result in increased generation of ROS [24 25]

The transgenic Caenorhabditis elegans model has beenused to evaluate the efficacy of anti-AD drugs since 1995 [26]The transgenic C elegans strain CL4176 expresses humanA1205731ndash42 inmuscles under a temperature-inducible system and

the expression and subsequent aggregation of A120573 result inparalysis In transgenic CL4176 worms Ginkgo biloba extractEGb 761 [27] Liuwei Dihuang [28] cocoa peptide [29] andother compounds [5 30] were shown to significantly reduceA120573 toxicity and ameliorate AD symptoms

In the present study we used transgenicC elegansCL4176to investigate whether GNC exhibits protective effects againstA120573 toxicity in vivo and to elucidate some of the mechanismsinvolved in its protective effects

2 Materials and Methods

21 Preparation of the GNC Formula GNC formula is com-posed of 12 crude herbsThe preparation of the GNC formulahas been reported in our previously published paper [13]Thecomposition of GNC followed traditional Chinese medicinaltheory and the composition is derived fromour clinical expe-rience In this study we used amixture of thewater extracts of12 crude herbsThewater extracts were purchased fromTian-jiang Pharmaceutical (Jiangyin China) These products weremanufactured with rigid quality control protocols following

the rigid specifications of the Pharmacopeia of China Thewater extracts in this study were produced in the same batch

22 C elegans Strains and Maintenance The wild-type Celegans strain N2 (Bristol) transgenic C elegans strainCL4176 (smg-1ts [myo-3A1205731ndash42 long 31015840-untranslated region(UTR)]) TJ356 (zIs356 [daf-16 pdaf-16abGFP + rol-6(su1006)]) and E coli OP50 were obtained from theCaenorhabditis Genetics Center (CGC) at the Univer-sity of Minnesota (Minneapolis MN USA) CL4176 isa temperature-sensitive mutant when the temperature isincreased from 16∘C (permissive temperature for the smg-1ts mutation) to 25∘C (a nonpermissive temperature) theworms will express higher levels of the human A120573

1ndash42peptide in muscles CL4176 were maintained at 16∘C on solidnematode growth medium (NGM) plates seeded with E coliOP50 whereas wild-type worms N2 and TJ356 strain weremaintained at 20∘C

23 Paralysis Assays Synchronized CL4176 gravid adultswere picked onto plates to lay eggs and after 3-4 h wormswere removed and eggs were maintained at 16∘C on solidNGM plates that contained the vehicle control (H

2O) and

different concentrations of GNC (00394 0394 197 394and 788mgmL) The nematodes were maintained at 16∘Cfor 48 hours then transgene expression was induced byincreasing the temperature from 16∘C to 25∘C Each wormwas gently touchedwith a platinum loop to confirmparalysisand the nematode was scored as paralyzed if it only moved itshead or did not show a full body wave Paralyzed nematodeswere counted at 1 hour intervals from 28 to 38 h after thetemperature was increased to 25∘C

24 Western Blotting of A120573 Species The A120573 species in thetransgenic C elegans strains were identified by immunoblot-ting using a Tris-Tricine gel and a standard Western blot-ting protocol [31] Worms were harvested by washing withM9 buffer centrifuged at 12000 rpm for 15 minutes andsonicated in RIPA lysis buffer (Pierce Rockford IL USA)containing a protease inhibitor cocktail for 30 minutes at4∘C The protein concentrations were quantified using aBradford Protein Assay Kit (Beyotime Shanghai China)Before loading samples were boiled with loading buffer at100∘C for 5 minutes Equal amounts of protein were loadedin each lane of the Tricine-SDS-PAGE gel (consisting ofa 10 ldquospacer gelrdquo between a 4 stacking gel and 16separating gel) and electrophoresis was started with an initialvoltage of 30V and maintained at this voltage until thesamples have completely entered the stacking gel and thenthe samples were run at 120V for approximately 8 hoursThe gel was transferred onto polyvinylidene fluoride (PVDF)membranes PVDF membranes were blocked with 5 non-fat milk in Tris-buffered saline with Tween (TBST) (100mMTris pH 75 150mM NaCl 01 Tween-20) overnight atroom temperature Amyloid protein species were detectedwith a BAM-10 monoclonal antibody (1 1000 Sigma) and aperoxidase-conjugated secondary anti-mouse IgG (1 5000Sigma) Actin was detected with an anti-actin antibody

Evidence-Based Complementary and Alternative Medicine 3

(1 1000 Abcam) and was used as an internal control Themean optical density was analyzed using ImageJ software(US National Institutes of Health Bethesda MD US) Threeindependent experiments were conducted and consistentresults were obtained from these independent experiments

25 Measurement of Reactive Oxygen Species (ROS) Levels inC elegans Intracellular ROS levels inC elegansCL4176 weremeasured using 27-dichlorofluorescein diacetate (H

2DCF-

DA) according to a previously reported method [6] Theprocedures used for GNC administration were the same asdescribed in the paralysis assay section Exactly 50 CL4176wormswere harvested 31 hours after the temperature increaseusing phosphate-buffered saline (PBS) containing 1 Tween-20 and were washed twice with 1x PBS to remove E coliOP50 Then the worms were sonicated 4 times for 15 seceach and pipetted into the wells of a 96-well plate containing200120583l of PBS plus H

2DCF-DA (final concentration 50120583M

in PBS) The fluorescence was quantified using a SynergyHT microplate fluorescence reader (Bio-Tek InstrumentsWinooski USA) at 485 nm excitation and 530 nm emissionThe experiment was independently performed three times

26 Quantitative Real-Time RT-PCR (qRT-PCR) AnalysisWorms were collected by washing them with M9 bufferthree times and were pelleted by centrifugation at 4000 rpmfor 1min The worms were freeze-thawed and transferreddirectly into 1mL of TRIzol reagent (Thermo Fisher Sci-entific Shanghai China) After 200120583L of chloroform wasadded the worm suspension was shaken vigorously andcentrifuged at 12000timesg for 10min The total nematodeRNA in the supernatant was precipitated using isopropanolThe RNA concentration and integrity were measured usinga NanoDrop spectrophotometer The cDNAs were synthe-sized by reverse transcription using FastQuant RT Kit (withgDNase Tiangen Beijing China) according to the manufac-turerrsquos protocol The expression of the 120573-amyloid gene amy-1 (forward 51015840-CCGACATGACTCAGGATATGAAGT-31015840reverse 51015840-CACCATGAGTCCAATGATTGCA-31015840) DAF-16 (forward 51015840- GAAAGAGCTCGTGGTGGGTTATTA-31015840 reverse 51015840-TCCGCGGCGAGATTTTTC-31015840) sod-3 (for-ward 51015840-AGCATCATGCCACCTACGTGA-31015840 reverse 51015840-CACCACCATTGAATTTCAGCG-31015840) and small heat shockprotein hsp-162 (forward 51015840-CTGCAGAATCTCTCCATC-TGAGTC-31015840 reverse 51015840-AGATTCGAAGCAACTGCACC-31015840) were determined by qRT-PCR performed on a qTOWER22 Real-Time PCR System (Analytik Jena AG ThuringiaGermany) using SuperReal PreMix Plus (SYBR Green Tian-gen Beijing China) The relative levels of gene expressionwere calculated by the 2minusΔΔCT method using the gene act-4(forward 51015840-GCCACCGCTGCCTCCTCATC-31015840 reverse 51015840-CCGGCAGACTCCATACCCAAGAAG-31015840) as the internalcontrol The experiment was repeated in triplicate

27 C elegans RNA Interference (RNAi)Assay Daf-16 expres-sion was knocked down by feeding the C elegans CL4176with E coli strain HT115 (DE3) bacteria carrying daf-16

dsRNA The RNAi plates were prepared with NGM con-taining 1mM isopropyl 120573-D-1-thiogalactopyranoside (IPTG)and 50 120583gmL ampicillin C elegans was fed with E coliHT115 strains expressing dsRNA specific to the daf-16 geneAfter 3-4 h worms were removed and eggs were permittedto mature to L4 young larvae The L4 larvae were transferredto another plate containing dsRNA and allowed to lay eggsSubsequently the resultant adult worms were used for theparalysis assays Animals fed HT115 bacteria harboring theempty vector L4440 were used as negative controls

28 DAF-16 Nuclear Translocation Quantification Age-synchronized day 1 adult nematodes of the TJ356 transgenicstrain stably expressing a DAF-16GFP fusion protein asa reporter pretreated with GNC (394mgmL) or controlwere exposed to 50120583M juglone for 5min then immobilizedwith azide sodium and placed on 2 agarose pads on aglass slide The subcellular DAF-16 distribution was analyzedby a Nikon SMZ 1500 fluorescence microscope Expressionpatterns of TJ356 worms were classified into three categories(cytosolic intermediate and nuclear) At least 20 animalsfrom each group were examined and the experiment wasperformed three times independently

29 RNA-Seq Analysis of Wild-Type C elegans N2 To furtherinvestigate the transcriptional effects of GNC treatmentwe analyzed gene expression of C elegans wild-type N2treated with GNC (394mgmL) and control at the same age(old worms 22-days old) Synchronized populations werecultured in S-complete liquid medium containing GNC ora vehicle control (H

2O) and E coli OP50 were added to

the medium as a food source Total RNA was extractedusing RNAiso Plus Total RNA extraction reagent (TaKaRaChina) according to the manufacturerrsquos instructions RNAwas further purified with an RNAClean XP Kit (BeckmanCoulter Inc Kraemer Boulevard Brea CA USA) and anRNase-Free DNase Set (QIAGEN GmBH Germany) Thesamples were sent to Shanghai Biotechnology Corporationfor library construction and sequencing using IlluminaHiSeq2500 (Illumina USA) The sequence quality of the data setswas assessed with an Agilent Bioanalyzer 2100 (Agilent tech-nologies Santa Clara CA USA) Transcript expression levelswere estimated using fragments per kilobases per millionreads (FPKM) values to allow us to compare different genesor samples The up- or downregulated genes were identifiedby filtering the RNA-seq data with the following cut-offtwofold change in the expression level and a false discoveryrate (FDR) of less than 005 All analyses were performedat Shanghai Biotechnology Corporation (Shanghai China)Gene expression data determined using next-generationRNA sequencing technology has been deposited in the GeneExpressionOmnibus (GEO) database with accession numberGSE98195 Publicly available databases primarily AgriGO(httpbioinfocaueducnagriGO) for the Gene Ontology(GO) enrichment analysis andKEGG for the pathway enrich-ment analysis (httpwwwgenomejpkegg) were used toinvestigate the biological significance of the differentiallyexpressed genes


Control (CL4176) Control GNC (394 mgmL)

No shi Temperature increased to 25∘C for 29 hours

(a)

0

20

40

60

80

100

Perc

enta

ge n

ot p

aral

yzed

()

Hours aer temperature increase (h)38373635343332313029282726

Control00394mgmL-GNC0394mgmL-GNC

197mgmL-GNC394mgmL-GNC788 mgmL-GNC

(b)

Figure 1 GNC delayed A120573-induced paralysis in transgenic C elegans CL4176 at 25∘C (a) Representative image of transgenic C elegansCL4176 showing paralysis caused by A120573 expression Representative images of control worms (no temperature shift) at 16∘C and the effect ofthe temperature increase to 25∘C for 29 h to induce A120573 expression in control and GNC-treated (394mgmL) transgenicC elegansCL4176 areshown (b) Curves show the course of A120573-induced paralysis in transgenic C elegans CL4176 treated with a vehicle control (H

2O) or various

doses of GNC GNC increased the time to paralysis onset in CL4176 worms in a dose-dependent manner (at least 35 worms were tested ineach group and the experiments were performed more than 3 times)

210 Statistical Analysis Statistical analyses of differencesbetween groups in the paralysis assays were performed usingthe log-rank test Studentrsquos 119905-test was performed to comparetwo data sets Significant differences betweenmultiple groupswere evaluated using one-way analysis of variance (ANOVA)withDuncanrsquos test GraphPad Prism software 60 was used forstatistical analyses Differences were considered significant ata 119901 value less than 005

3 Results

31 GNC Delayed the Paralysis of A120573-Transgenic C elegansCL4176 Nematodes To evaluate whether GNC amelioratedthe toxicity of the 120573-amyloid peptide we used transgenic Celegans CL4176 expressing human A120573

1ndash42 in the muscles TheCL4176 strain displays a phenotype of A120573 production andaggregation when the temperature is increased to 25∘C andprogressive paralysis subsequently occursWe treatedCL4176worms with different concentrations of GNC (00394 0394

197 394 and 788mgmL) at 16∘C for 48 h and then trans-ferred them to 25∘C As shown in Figure 1 untreated controlworms exhibited almost complete paralysis within 34 h at25∘C In contrast GNC significantly increased the paralysistime of CL4176 worms We also found that GNC delayedparalysis in a dose-dependent manner and the maximumincrease of paralysis time was observed with 394mgmLThese observations suggested that GNCmay have the poten-tial to protect against 120573-amyloid peptide induced toxicity

32 GNC Reduced the Levels of A120573 in C elegans CL4176by Downregulating the Expression of the A120573 Transgene Todeterminewhether GNCdelayed onset of paralysis in CL4176by reducing the A120573 levels we examined the effect of GNC onthe expression of the A120573 transgene and performed Westernbolt analysis to monitor A120573 levels Both GNC-treated andcontrol worms were harvested 31 h after the temperatureincrease and parallel populations were processed for qRT-PCR and the Western blotting assay As shown in Figure 2


A

Actin

Control GNC Control GNC

(a)

Control GNC

lowast

00

02

04

06

08

10

12

Ratio

of A

on

imm

unob

lot

(b)Control GNC

lowastlowast

00

02

04

06

08

10

12

Rela

tive t

rans

crip

t lev

els

(c)

Figure 2 GNC reduced the A120573mRNA and protein levels in C elegans CL4176 (a) Western blot of the transgenic C elegans CL4176 treatedwithGNC (394mgmL) and the control (H

2O)A representative blot of three independent experiments is shown (b)Themeanoptical density

was calculated using ImageJ Quantitative comparisons of the A120573 levels depict a significant 35 reduction in A120573 levels (119901 = 00383) in GNC(394mgmL) pretreated worms (c) The GNC (394mgmL) treatment decreased the levels of the amy-1 transcript by 49 (119901 = 00044)120573-Actin (act-4) was used as an endogenous control and the expression levels were determined by qRT-PCR using the 2minusΔΔCT method Thedata are displayed as means plusmn SD lowast119901 le 005 lowastlowast119901 le 001

treatment with 394mgmL GNC resulted in a 49 decreasein the expression of the A120573 transgene (119901 = 00044) and asignificant 35 reduction in A120573 levels (119901 = 00383) ThusGNC potentially delayed the paralysis of C elegans CL4176by decreasing A120573 transgene expression and A120573 levels whichcontribute to the paralysis phenotype of C elegans CL4176

33 GNC Reduced Oxidative Stress in C elegans CL4176Nematodes GNC was previously shown to enhance theresistance of wild-type C elegans N2 to oxidative stress[13] Because oxidative stress is a major contributing factorto AD we investigated the ROS levels in the transgenicstrain CL4176 in vivo to determine whether GNC decreasedoxidative stress in the AD model worms At 16∘C GNC(394mgmL) reduced total ROS levels by 20 (119901 = 0033)compared with the vehicle control When the temperaturewas increased to induce A120573 expression ROS levels increasedin both GNC-treated and control worms (119901 lt 001) At 31 hafter the temperature increase to 25∘C GNC (394mgmL)also significantly decreased ROS levels by 356 (119901 =0024) compared to the vehicle control (Figure 3) Based onthese results oxidative stress is associated with the paralysis

phenotype caused by A120573 in C elegans and GNCmay protectagainst A120573 toxicity through its antioxidative stress activity

34 Effects of GNC on the Expression of Stress-Induced Genesin C elegans To further confirm that GNC increased theresistance of C elegans CL4176 to oxidative stress we exam-ined the transcript levels of daf-16 superoxide dismutasesod-3 and heat shock protein hsp-162 after the temperatureincrease Based on the qRT-PCR results the transcripts of allthree genes were expressed at significantly higher levels inworms treated with GNC than in controls at 31 h after thetemperature increase (daf-16 18-fold increase 119901 = 00017sod-3 23-fold increase 119901 = 00029 hsp-162 27-foldincrease 119901 = 00001) (Figure 4)

35 DAF-16 Is Required for the Protective Effects of GNCon Paralysis Delay DAF-16 plays an important role in theprotection against A120573 toxicity Our previous study reportedthat GNC enhances antioxidative stress ability in C elegansN2 via DAF-16 [13] To further test whether DAF-16 isrequired for the protective effect of GNC against A120573 toxicitywe performed the experiments by using RNAi knockdown



lowast

lowast

No shi

0

1

2

3

Relat

ive fl

uore

scen

ce

Shi to 25∘C

Figure 3 ROS levels in C elegans CL4176 at 16∘C or 25∘C that hadbeen pretreated with GNC (394mgmL) or the control At 16∘C(no temperature shift) GNC (394mgmL) reduced ROS levels by20 (119901 = 0033) compared with the vehicle control When thetemperature shifted to 25∘C GNC (394mgmL) decreased ROSlevels by 356 (119901 = 0024) compared to the vehicle control Thedata are displayed as means plusmn SD lowast119901 le 005

ControlGNC

daf-16 sod-3 hsp-1620

1

2

3

4

Rela

tive t

rans

crip

t lev

els

lowastlowast

lowastlowast

lowast lowast lowast

Figure 4 Quantification of mRNA expression levels in GNC-treated worms The GNC (394mgmL) treatment affected therelative levels of the daf-16 sod-3 and hsp-162 transcripts 120573-Actin(act-4) was used as an endogenous control and the expression levelswere determined by qRT-PCRusing the 2minusΔΔCT methodThedata aredisplayed as means plusmn SD lowastlowast119901 le 001 lowastlowastlowast119901 le 0001

of DAF-16 expression We found that GNC at 394mgmLsignificantly delayed A120573-induced paralysis in worms grownon bacteria containing empty vector but not onDAF-16 RNAibacteria (Figure 5) This result indicated that reducing theactivity of DAF-16 abolished the protective effect of GNCsuggesting a requirement of DAF-16 for protective effect ofGNC

0

20

40

60

80

100

Perc

enta

ge n

ot p

aral

yzed

()


Daf-16 RNAＣ + controlDaf-16 RNAＣ + NCEmpty vectoＬ + control

Empty vectoＬ + NC

Figure 5 Paralysis in CL4176 with or without Daf-16 knockdown byRNAi Curves show the course ofA120573-induced paralysis in transgenicC elegans CL4176 (at least 35 worms were tested in each group andthe experiments were performed more than 3 times)

To further clarify DAF-16 is required for GNC alleviatingA120573 toxicity we examined the translocation of DAF-16 Thesubcellular distribution of DAF-16 was classified into the cat-egories of ldquocytosolicrdquo ldquointermediaterdquo and ldquonuclearrdquo as rep-resentative images showed (Figure 6(a)) Under the juglone-induced oxidative stress conditions fractions of intermediateand nuclear localization of DAF-16 were greater in GNC-pretreated worms (Figure 6(b)) Therefore GNC enhancedthe translocation of DAF-16 from the cytoplasm to nuclei inoxidative stress

These findings indicated that DAF-16 is required for theprotective effects of GNC on A120573 toxicity

36 Genome-Wide Transcriptional Profiling of GNC-Treated CelegansN2 Ageing plays a critical role inADand remains themost dominant risk factor for AD [32] The exact pathogen-esis of AD is still unclear it may be connected with ageinggenetic factors and environmental factors [33] In additionAD shares some common signaling pathways with ageingsuch as the insulinIGF-1 signaling pathway which is theclassical and conserved signaling pathway regulated by ageing[34] Our previous study has showed that GNC extended thelifespan of wild-type C elegansN2 [13] here we analyzed theeffect of GNC on C elegans N2 gene expression using RNA-seq assays Differentially expressed genes in response to theGNC treatment were assessed A total of 1120 genes wereupregulated and 324 genes were downregulated compared tothe control treatment Two different databases GOBiologicalProcess and KEGG Pathways were used to investigate thefunctional classes of differentially expressed genes Bio-logical processes including cellular process (GO0009987)developmental process (GO0032502) andmetabolic process(GO0008152) were significantly upregulated (119901 lt 005) inGNC-treated worms (See S Figure 1 in SupplementaryMate-rial available online at httpsdoiorg10115520177480980)


Cytosolic Intermediate Nuclear

(A) (B) (C)

(a)

IntermediateCytosolic

Nuclear

Control GNC0

20

40

60

80

Wor

ms w

ith re

spec

tive D

AF-

16lo

caliz

atio

n (

)

100

(b)Figure 6 GNC promoted DAF-16 nuclear localization (a) Representative images for the localization phenotype of DAF-16GFP in TJ356adults Worms were classified into the categories of ldquocytosolicrdquo (A) ldquointermediaterdquo (B) and ldquonuclearrdquo (C) according to their localizationphenotypes (b) Quantization of the distribution of cytosolic intermediate and nuclear DAF-16GFP under oxidative stress conditions Thesubcellular distribution of DAF-16 was examined in approximately 20 animals per condition by fluorescence microscopy The phenotyperesults were presented in ratio to the whole population at each treatment condition

The metabolic pathways affected by GNC were evaluatedusing KEGG We identified 5 different KEGG pathways thatwere associated with the prolongevity effect of GNC (STable 1)

4 Discussion

To elucidate the regulatory mechanisms of ameliorating 120573-amyloid peptide toxicity by GNC we performed experiments

by using C elegans in this study We observed that GNCeffectively delayed the onset of 120573-amyloid induced paralysisin C elegans CL4176 in a dose-dependent manner Themaximum increase in the time of paralysis onset was delayedby the 394mgmL treatment GNC delayed ageing in ourprevious study and in this study we analyzed the sequencesof the transcriptomes from GNC-treated and control-treatedC elegans using the RNA-seq method The TGF-beta sig-naling pathway Wnt signaling pathway longevity regulating


pathway FOXO signaling pathway and oxidative phospho-rylation were associated with the lifespan extension effect ofGNC These findings indicated that GNC not only extendedthe lifespan but also protected against A120573-induced toxicity inC elegans

Although the exact cause of AD is still under debate theamyloid cascade hypothesis is the best accepted aetiologyA120573 deposition leads to an inflammatory response cytokinerelease microglial activation and reactive astrocytosis theseprocesses lead to neuronal dysfunction and ultimately large-scale cell death [35] In this study GNC suppressed thetranscription of amy-1 in C elegans CL4176 suggesting thatthe delayed onset of paralysis was at least partially due to areduced amy-1 level Moreover GNC significantly reducedA120573 levels compared to controls Many compounds such ascocoa peptide [29] and Liuwei Dihuang [28] exert protectiveeffects against A120573 toxicity in the same way Since A120573 is notendogenously produced in worms more research is neededto study the effect of GNC on A120573-induced toxicity

Oxidative stress is a key factor in the ageing process andhas been shown to play a vital role in the pathophysiologyof AD [22] In this study we measured the ROS levels inC elegans CL4176 and observed a significant increase inROS levels (more than 2-fold) when the temperature wasincreased to 25∘C suggesting that ROS production increasedin the process of A120573-induced paralysisThis finding is similarto those observed in AD patients Sekler et al observed aclose correlation between oxidative stress markers and thestages of AD in patients with AD [36] According to ourprevious studies GNC possesses strong antioxidant activity[13] We examined whether GNC affected ROS levels in Celegans CL4176 to determine the potential mechanism bywhichGNCdelayed the onset of paralysisThe results showedthat GNC reduced total ROS levels under both normalconditions and A120573-induced toxicity compared with controlsInC elegans ROS production is a typical outcome of amyloidaccumulation and the effect of GNC on ROS levels may bea consequence of amyloid reduction However we cannotexclude the possibility that GNC has direct scavenging effectson ROS since the antioxidant properties of GNC have alsobeen observed in our previous study [13]

C elegansDAF-16 is the sole ortholog of the FOXO familyof transcription factors and plays a central role in promotingcellular antioxidant defenses [37] DAF-16 activation stim-ulates the transcription of genes that encode antioxidantenzymes such as superoxide dismutase (sod) Small heatshock proteins (hsps) are reported to participate in protectiveresponses to the abnormal accumulation of toxic proteins[38] We evaluated the expression levels of daf-16 sod-3 andhsp-162 to further investigate the mechanism by which GNCprotects against A120573 toxicity in C elegans and observed a sig-nificant increase in the levels of these transcripts in C elegansCL4176 pretreated with GNC These findings suggest GNCmight increase the antioxidative stress ability of C elegansCL4176 via DAF-16 In TCMmanymedicinal plants increasethe resistance to oxidative stress and have been characterizedas potential treatments for mitigating AD For examplethe aqueous extract of Centella asiatica reduced oxidativestress in a rat streptozotocin-induced model of AD [39]

As shown in the study by Turgut et al Capparis spinosaL exerts a neuroprotective effect by reducing oxidativestress [40] Our results indicated that the antioxidative stressactivity of GNC may be one of the mechanisms by which itprotected C elegans from A120573 toxicity

Next we further investigated whether GNC requiresDAF-16 to protect against A120573 toxicity in C elegans by feedingDAF-16 RNAi bacteria to transgenic CL4176 worms Theresults showed that RNAi of DAF-16 significantly eliminatedthe GNC-mediated decrease of paralysis in AD wormsindicating that theDAF-16 transcription factor is required forGNCprotection against A120573 toxicityMeanwhile the paralyticphenotype was shortened in DAF-16 knockdown C eleganswith or without GNC treatment which also confirmed thatDAF-16 plays an important role in alleviating A120573 toxicity

To further validate that DAF-16 is required for theprotective effect of GNC we examined the effect of the GNCon the translocation of DAF-16 from the cytoplasm to nucleiThe results showed thatGNCenhancedDAF-16 translocationfrom the cytoplasm to nuclei under oxidative stress condi-tions In this study we found that ROS production increasedin the process of A120573-induced paralysis suggesting wormswere in the oxidative stress conditions Furthermore nuclearlocalization of DAF-16 is a prerequisite for transcriptionalactivation of its target genes such as sod-3 mtl-1 and clt-2 [41] In this study GNC caused a significant increase ofsod-3 mRNA levels in CL4176 compared with the untreatedworms Taken together we speculated that GNC amelioratedA120573 toxicity via DAF-16 pathway

5 Conclusions

In summary in this study we reported that GNC whichexhibits prolongevity activity alleviated A120573-induced paral-ysis in C elegans through lowering amy-1 and A120573 levelsreducing ROS and increasing the daf-16 sod-3 and hsp-162mRNAs all of which may have mutually exert the protectivemechanisms against A120573 toxicity Furthermore DAF-16 isessential for the protective effect of GNC Taken togetherour observations suggest that GNC could protect againstA120573-induced toxicity in a C elegans model of AD Furtherstudies need to be conducted in murine models and humansto analyze the effectiveness of GNC on AD

Conflicts of Interest

The authors declare no conflicts of interest

Acknowledgments

We thank Professor Jian Fei Dr Ping Yang andDr ZhuanbinWu for their technical assistance This work was supportedby the National Natural Science Foundation of China (no81273956) and the SpecializedResearch Fund for theDoctoralProgram of Higher Education (no 20130071110062) Thestudy was conducted at Shanghai Research Center for ModelOrganisms Shanghai China


References

[1] M Prince and J Jackson ldquoWorld Alzheimer reportrdquo Tech RepAlzheimerrsquos Disease International London UK 2009

[2] F Mangialasche M Kivipelto A Solomon and L FratiglionildquoDementia prevention current epidemiological evidence andfuture perspectiverdquoAlzheimerrsquos Research andTherapy vol 4 no1 article 6 2012

[3] N Tabet ldquoAcetylcholinesterase inhibitors for Alzheimerrsquos dis-ease anti-inflammatories in acetylcholine clothingrdquo Age andAgeing vol 35 no 4 pp 336ndash338 2006

[4] G L Wenk G Quack H-J Moebius and W Danysz ldquoNointeraction of memantine with acetylcholinesterase inhibitorsapproved for clinical userdquo Life Sciences vol 66 no 12 pp 1079ndash1083 2000

[5] J Li X Cui X Ma and Z Wang ldquorBTI reduced 120573-amyloid-induced toxicity by promoting autophagy-lysosomal degra-dation via DAF-16 in Caenorhabditis elegansrdquo ExperimentalGerontology vol 89 pp 78ndash86 2017

[6] L Xin R Yamujala Y Wang et al ldquoAcetylcholineestarase-inhibiting alkaloids from lycoris radiata delay paralysis ofamyloid beta-expressing transgenic C elegans CL4176rdquo PLoSONE vol 8 no 5 Article ID e63874 2013

[7] F G Zhao W J Wang W J Zhou and D J Li ldquoEffect ofGengnianchun Recipe on learning memory function and hip-pocampal cholinergic system in ovariectomized ratsrdquo ChineseJournal of Integrated Traditional and Western medicine vol 28no 3 pp 234ndash237 2008

[8] F G Zhao W J Wang W J Zhou and D J Li ldquoEffect of Geng-nianchun formula on hippocampal BDNF of ovariectomizedSD ratrdquo Journal of Experimental Traditional Medical Formulaevol 16 no 15 article 46 2010

[9] J Li W Wang D Li and W Zhou ldquoGengnianchun recipeinhibits apoptosis of pheochromocytoma cells from beta-amyloid 25-35 insult better than monotherapies and theircompoundsrdquo Neural Regeneration Research vol 6 no 36 pp2815ndash2821 2011

[10] Li JProteetion of decoctionGengnianehun to PC12 cells and hip-pocampal cells fromA12057325-35 insult FudanUniversity ShanghaiChina 2010

[11] S Kang Y-H Lee and J E Lee ldquoMetabolism-centric overviewof the pathogenesis of Alzheimerrsquos diseaserdquo Yonsei MedicalJournal vol 58 no 3 pp 479ndash488 2017

[12] M Luca A Luca and C Calandra ldquoThe role of oxidativedamage in the pathogenesis and progression of Alzheimerrsquosdisease and vascular dementiardquoOxidativeMedicine andCellularLongevity vol 2015 Article ID 504678 8 pages 2015

[13] FMeng J LiWWang and Y Fu ldquoGengnianchun a traditionalchinesemedicine enhances oxidative stress resistance and lifes-pan in caenorhabditis elegans by modulating daf-16FOXOrdquoEvidence-Based Complementary and Alternative Medicine vol2017 Article ID 8432306 10 pages 2017

[14] K Sambamurti N H Greig and D K Lahiri ldquoAdvances in thecellular and molecular biology of the beta-amyloid protein inAlzheimerrsquos diseaserdquo NeuroMolecular Medicine vol 1 no 1 pp1ndash31 2002

[15] J Hardy and D J Selkoe ldquoThe amyloid hypothesis ofAlzheimerrsquos disease progress and problems on the road totherapeuticsrdquo Science vol 297 no 5580 pp 353ndash356 2002

[16] M A Smith P L R Harris L M Sayre J S Beckmanand G Perry ldquoWidespread peroxynitrite-mediated damage in

Alzheimerrsquos diseaserdquo The Journal of Neuroscience vol 17 no 8pp 2653ndash2657 1997

[17] R J Mark M A Lovell W R Markesbery K Uchida andM P Mattson ldquoA role for 4-hydroxynonenal an aldehydicproduct of lipid peroxidation in disruption of ion homeostasisand neuronal death induced by amyloid 120573-peptiderdquo Journal ofNeurochemistry vol 68 no 1 pp 255ndash264 1997

[18] A Nunomura G Perry M A Pappolla et al ldquoRNA oxidationis a prominent feature of vulnerable neurons in Alzheimerrsquosdiseaserdquo The Journal of Neuroscience vol 19 no 6 pp 1959ndash1964 1999

[19] D A Butterfield and D Boyd-Kimball ldquoAmyloid 120573-peptide(1ndash42) contributes to the oxidative stress and neuro-degeneration found in Alzheimer disease brainrdquo BrainPathology vol 14 no 4 pp 426ndash432 2004

[20] C Behl J B Davis R Lesley and D Schubert ldquoHydrogenperoxide mediates amyloid 120573 protein toxicityrdquo Cell vol 77 no6 pp 817ndash827 1994

[21] M E Harris K Hensley D A Butterfield R A Leedle and JM Carney ldquoDirect evidence of oxidative injury produced by theAlzheimerrsquos120573-Amyloid peptide (1ndash40) in cultured hippocampalneuronsrdquo Experimental Neurology vol 131 no 2 pp 193ndash2021995

[22] Y Zhao and B Zhao ldquoOxidative stress and the pathogenesis ofAlzheimerrsquos diseaserdquoOxidativeMedicine andCellular Longevityvol 2013 Article ID 316523 10 pages 2013

[23] Y Matsuoka M Picciano J La Francois and K Duff ldquoFibrillar120573-amyloid evokes oxidative damage in a transgenic mousemodel of Alzheimerrsquos diseaserdquo Neuroscience vol 104 no 3 pp609ndash613 2001

[24] L Tillement L Lecanu and V Papadopoulos ldquoAlzheimerrsquosdisease effects of 120573-amyloid on mitochondriardquoMitochondrionvol 11 no 1 pp 13ndash21 2011

[25] C A Hansson Petersen N Alikhani H Behbahani et alldquoThe amyloid 120573-peptide is imported into mitochondria viathe TOM import machinery and localized to mitochondrialcristaerdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 105 no 35 pp 13145ndash13150 2008

[26] C D Link ldquoExpression of human beta-amyloid peptide intransgenic Caenorhabditis elegansrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 92 no20 pp 9368ndash9372 1995

[27] Y Wu Z Wu P Butko et al ldquoAmyloid-120573-induced pathologicalbehaviors are suppressed by Ginkgo biloba extract EGB 761 andginkgolides in transgenic Caenorhabditis elegansrdquo The Journalof Neuroscience vol 26 no 50 pp 13102ndash13113 2006

[28] J S Sangha X Sun O S D Wally et al ldquoLiuwei dihuang(LWDH) a traditional chinese medicinal formula protectsagainst 120573-amyloid toxicity in transgenic Caenorhabditis ele-gansrdquo PLoS ONE vol 7 no 8 Article ID e43990 2012

[29] PMartorell E Bataller S Llopis et al ldquoA cocoa peptide protectsCaenorhabditis elegans from oxidative stress and 120573-amyloidpeptide toxicityrdquo PLoS ONE vol 8 no 5 Article ID e632832013

[30] J Yang X Huang Q Wan et al ldquoOtophylloside B pro-tects against A120573 toxicity in Caenorhabditis elegans models ofAlzheimerrsquos Diseaserdquo Natural Products and Bioprospecting vol7 no 2 pp 207ndash214 2017

[31] H Schagger ldquoTricine-SDS-PAGErdquo Nature Protocols vol 1 no1 pp 16ndash22 2006


[32] J C Udeochu J M Shea and S A Villeda ldquoMicroglia com-munication parallels between aging and Alzheimerrsquos diseaserdquoClinical and Experimental Neuroimmunology vol 7 no 2 pp114ndash125 2016

[33] N Yang Y Wei Q Xu and B Tang ldquoProgress in epigeneticresearch on Alzheimer diseaserdquo Chinese Journal of MedicalGenetics vol 33 no 2 pp 252ndash255 2016

[34] E Cohen and A Dillin ldquoThe insulin paradox aging proteotox-icity and neurodegenerationrdquoNature Reviews Neuroscience vol9 no 10 pp 759ndash767 2008

[35] P Eikelenboom S-S Zhan W A van Gool and D AllsopldquoInflammatory mechanisms in Alzheimerrsquos diseaserdquo Trends inPharmacological Sciences vol 15 no 12 pp 447ndash450 1994

[36] MA Sekler JM Jimenez L Rojo et al ldquoCognitive impairmentand Alzheimerrsquos disease links with oxidative stress and choles-terol metabolismrdquoNeuropsychiatric Disease and Treatment vol4 no 4 pp 715ndash722 2008

[37] AMukhopadhyay SW Oh andH A Tissenbaum ldquoWormingpathways to and from DAF-16FOXOrdquo Experimental Gerontol-ogy vol 41 no 10 pp 928ndash934 2006

[38] V Fonte D R Kipp J Yerg III et al ldquoSuppression of in vivo120573-amyloid peptide toxicity by overexpression of the HSP-162small chaperone proteinrdquo Journal of Biological Chemistry vol283 no 2 pp 784ndash791 2008

[39] M H Veerendra Kumar and Y K Gupta ldquoEffect of Centellaasiatica on cognition and oxidative stress in an intracerebroven-tricular streptozotocin model of Alzheimerrsquos disease in ratsrdquoClinical and Experimental Pharmacology and Physiology vol 30no 5-6 pp 336ndash342 2003

[40] N H Turgut H Kara E Arslanbas D G Mert B Tepe and HGungor ldquoEffect of Capparis spinosa L on cognitive impairmentinduced by D-galactose in mice via inhibition of oxidativestressrdquo Turkish Journal of Medical Sciences vol 45 no 5 pp1127ndash1136 2015

[41] C T Murphy S A McCarroll C I Bargmann et al ldquoGenesthat act downstream of DAF-16 to influence the lifespan ofCaenorhabditis elegansrdquoNature vol 424 no 6946 pp 277ndash2832003

Submit your manuscripts athttpswwwhindawicom

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


acetyltransferase (ChAT) [7] In addition GNC upregulatedbrain-derived neurotrophic factor (BDNF) expression andimproved learning and memory in ovariectomized rats [8]GNC has recently been shown to attenuate A120573

25ndash35-inducedcytotoxicity in pheochromocytoma cells (PC-12 cells) andhippocampal cells [9 10] Ageing and oxidative stress areknown to have important roles in the progression of AD[11 12] GNC enhances the resistance to oxidative stress andimproves lifespan in vivo [13] Based on these results GNCmay be a promising therapeutic agent for the treatment ofAD

Although the aetiology of AD remains unclear the mostwidely accepted hypothesis is the ldquoamyloid hypothesisrdquowhich suggests that A120573 neurotoxicity plays a central role inthe pathogenesis of AD [14] The toxic beta amyloid (1ndash42)peptide is derived from the abnormal processing of theamyloid precursor protein (APP) which forms extracellularsenile plaques in the human brain and leads to downstreamneurotoxic events including neuronal dysfunction stressresponses and inflammation [15]

Furthermore oxidative stress is extensive in Alzheimerrsquosdisease brains 120573-Amyloid accumulation is associated withmarkers of oxidative stress including protein oxidation lipidperoxidation and oxidation of nucleic acids [16ndash18] A120573 hasbeen shown to induce oxidative stress in vitro and in vivo [19]In vitro cells exposed to A120573 showed high levels of hydrogenperoxide and production of reactive oxygen intermediates[20 21] Previous in vivo studies have reported an increasein H2O2levels and in the peroxidation of proteins and lipids

in AD transgenic mouse models that express mutant amyloidprecursor protein (APP) and presenilin-1 (PS-1) [22 23] InAD patients A120573 accumulates in human brain mitochondriaresulting in a significant decline in mitochondrial functionwhich can result in increased generation of ROS [24 25]

The transgenic Caenorhabditis elegans model has beenused to evaluate the efficacy of anti-AD drugs since 1995 [26]The transgenic C elegans strain CL4176 expresses humanA1205731ndash42 inmuscles under a temperature-inducible system and

the expression and subsequent aggregation of A120573 result inparalysis In transgenic CL4176 worms Ginkgo biloba extractEGb 761 [27] Liuwei Dihuang [28] cocoa peptide [29] andother compounds [5 30] were shown to significantly reduceA120573 toxicity and ameliorate AD symptoms

In the present study we used transgenicC elegansCL4176to investigate whether GNC exhibits protective effects againstA120573 toxicity in vivo and to elucidate some of the mechanismsinvolved in its protective effects

2 Materials and Methods

21 Preparation of the GNC Formula GNC formula is com-posed of 12 crude herbsThe preparation of the GNC formulahas been reported in our previously published paper [13]Thecomposition of GNC followed traditional Chinese medicinaltheory and the composition is derived fromour clinical expe-rience In this study we used amixture of thewater extracts of12 crude herbsThewater extracts were purchased fromTian-jiang Pharmaceutical (Jiangyin China) These products weremanufactured with rigid quality control protocols following

the rigid specifications of the Pharmacopeia of China Thewater extracts in this study were produced in the same batch

22 C elegans Strains and Maintenance The wild-type Celegans strain N2 (Bristol) transgenic C elegans strainCL4176 (smg-1ts [myo-3A1205731ndash42 long 31015840-untranslated region(UTR)]) TJ356 (zIs356 [daf-16 pdaf-16abGFP + rol-6(su1006)]) and E coli OP50 were obtained from theCaenorhabditis Genetics Center (CGC) at the Univer-sity of Minnesota (Minneapolis MN USA) CL4176 isa temperature-sensitive mutant when the temperature isincreased from 16∘C (permissive temperature for the smg-1ts mutation) to 25∘C (a nonpermissive temperature) theworms will express higher levels of the human A120573

1ndash42peptide in muscles CL4176 were maintained at 16∘C on solidnematode growth medium (NGM) plates seeded with E coliOP50 whereas wild-type worms N2 and TJ356 strain weremaintained at 20∘C

23 Paralysis Assays Synchronized CL4176 gravid adultswere picked onto plates to lay eggs and after 3-4 h wormswere removed and eggs were maintained at 16∘C on solidNGM plates that contained the vehicle control (H

2O) and

different concentrations of GNC (00394 0394 197 394and 788mgmL) The nematodes were maintained at 16∘Cfor 48 hours then transgene expression was induced byincreasing the temperature from 16∘C to 25∘C Each wormwas gently touchedwith a platinum loop to confirmparalysisand the nematode was scored as paralyzed if it only moved itshead or did not show a full body wave Paralyzed nematodeswere counted at 1 hour intervals from 28 to 38 h after thetemperature was increased to 25∘C

24 Western Blotting of A120573 Species The A120573 species in thetransgenic C elegans strains were identified by immunoblot-ting using a Tris-Tricine gel and a standard Western blot-ting protocol [31] Worms were harvested by washing withM9 buffer centrifuged at 12000 rpm for 15 minutes andsonicated in RIPA lysis buffer (Pierce Rockford IL USA)containing a protease inhibitor cocktail for 30 minutes at4∘C The protein concentrations were quantified using aBradford Protein Assay Kit (Beyotime Shanghai China)Before loading samples were boiled with loading buffer at100∘C for 5 minutes Equal amounts of protein were loadedin each lane of the Tricine-SDS-PAGE gel (consisting ofa 10 ldquospacer gelrdquo between a 4 stacking gel and 16separating gel) and electrophoresis was started with an initialvoltage of 30V and maintained at this voltage until thesamples have completely entered the stacking gel and thenthe samples were run at 120V for approximately 8 hoursThe gel was transferred onto polyvinylidene fluoride (PVDF)membranes PVDF membranes were blocked with 5 non-fat milk in Tris-buffered saline with Tween (TBST) (100mMTris pH 75 150mM NaCl 01 Tween-20) overnight atroom temperature Amyloid protein species were detectedwith a BAM-10 monoclonal antibody (1 1000 Sigma) and aperoxidase-conjugated secondary anti-mouse IgG (1 5000Sigma) Actin was detected with an anti-actin antibody




2DCF-














(a)

0

20

40

60

80

100

Perc

enta

ge n

ot p

aral

yzed

()




(b)


2O) or various



3 Results






A

Actin


(a)

Control GNC

lowast

00

02

04

06

08

10

12

Ratio

of A

on

imm

unob

lot

(b)Control GNC

lowastlowast

00

02

04

06

08

10

12

Rela

tive t

rans

crip

t lev

els

(c)











lowast

lowast

No shi

0

1

2

3

Relat

ive fl

uore

scen

ce

Shi to 25∘C


ControlGNC


1

2

3

4

Rela

tive t

rans

crip

t lev

els

lowastlowast

lowastlowast




0

20

40

60

80

100

Perc

enta

ge n

ot p

aral

yzed

()



Empty vectoＬ + NC







(A) (B) (C)

(a)


Nuclear

Control GNC0

20

40

60

80

Wor

ms w

ith re

spec

tive D

AF-

16lo

caliz

atio

n (

)

100



4 Discussion











5 Conclusions




Acknowledgments



References

















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















2DCF-














(a)

0

20

40

60

80

100

Perc

enta

ge n

ot p

aral

yzed

()




(b)


2O) or various



3 Results






A

Actin


(a)

Control GNC

lowast

00

02

04

06

08

10

12

Ratio

of A

on

imm

unob

lot

(b)Control GNC

lowastlowast

00

02

04

06

08

10

12

Rela

tive t

rans

crip

t lev

els

(c)











lowast

lowast

No shi

0

1

2

3

Relat

ive fl

uore

scen

ce

Shi to 25∘C


ControlGNC


1

2

3

4

Rela

tive t

rans

crip

t lev

els

lowastlowast

lowastlowast




0

20

40

60

80

100

Perc

enta

ge n

ot p

aral

yzed

()



Empty vectoＬ + NC







(A) (B) (C)

(a)


Nuclear

Control GNC0

20

40

60

80

Wor

ms w

ith re

spec

tive D

AF-

16lo

caliz

atio

n (

)

100



4 Discussion











5 Conclusions




Acknowledgments



References

















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















(a)

0

20

40

60

80

100

Perc

enta

ge n

ot p

aral

yzed

()




(b)


2O) or various



3 Results






A

Actin


(a)

Control GNC

lowast

00

02

04

06

08

10

12

Ratio

of A

on

imm

unob

lot

(b)Control GNC

lowastlowast

00

02

04

06

08

10

12

Rela

tive t

rans

crip

t lev

els

(c)











lowast

lowast

No shi

0

1

2

3

Relat

ive fl

uore

scen

ce

Shi to 25∘C


ControlGNC


1

2

3

4

Rela

tive t

rans

crip

t lev

els

lowastlowast

lowastlowast




0

20

40

60

80

100

Perc

enta

ge n

ot p

aral

yzed

()



Empty vectoＬ + NC







(A) (B) (C)

(a)


Nuclear

Control GNC0

20

40

60

80

Wor

ms w

ith re

spec

tive D

AF-

16lo

caliz

atio

n (

)

100



4 Discussion











5 Conclusions




Acknowledgments



References

















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















A

Actin


(a)

Control GNC

lowast

00

02

04

06

08

10

12

Ratio

of A

on

imm

unob

lot

(b)Control GNC

lowastlowast

00

02

04

06

08

10

12

Rela

tive t

rans

crip

t lev

els

(c)











lowast

lowast

No shi

0

1

2

3

Relat

ive fl

uore

scen

ce

Shi to 25∘C


ControlGNC


1

2

3

4

Rela

tive t

rans

crip

t lev

els

lowastlowast

lowastlowast




0

20

40

60

80

100

Perc

enta

ge n

ot p

aral

yzed

()



Empty vectoＬ + NC







(A) (B) (C)

(a)


Nuclear

Control GNC0

20

40

60

80

Wor

ms w

ith re

spec

tive D

AF-

16lo

caliz

atio

n (

)

100



4 Discussion











5 Conclusions




Acknowledgments



References

















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















lowast

lowast

No shi

0

1

2

3

Relat

ive fl

uore

scen

ce

Shi to 25∘C


ControlGNC


1

2

3

4

Rela

tive t

rans

crip

t lev

els

lowastlowast

lowastlowast




0

20

40

60

80

100

Perc

enta

ge n

ot p

aral

yzed

()



Empty vectoＬ + NC







(A) (B) (C)

(a)


Nuclear

Control GNC0

20

40

60

80

Wor

ms w

ith re

spec

tive D

AF-

16lo

caliz

atio

n (

)

100



4 Discussion











5 Conclusions




Acknowledgments



References

















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















(A) (B) (C)

(a)


Nuclear

Control GNC0

20

40

60

80

Wor

ms w

ith re

spec

tive D

AF-

16lo

caliz

atio

n (

)

100



4 Discussion











5 Conclusions




Acknowledgments



References

















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
























5 Conclusions




Acknowledgments



References

















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















References

















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















A Chinese Herbal Formula, Gengnianchun, Ameliorates 𝛽...

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Transcript of A Chinese Herbal Formula, Gengnianchun, Ameliorates 𝛽...