Research Article Catalpol Modulates Lifespan via DAF-16 ...downloads.hindawi.com/journals/ecam/2015/524878.pdfResearch Article Catalpol Modulates Lifespan via DAF-16/FOXO and SKN-1/Nrf2

Research ArticleCatalpol Modulates Lifespan via DAF-16FOXO andSKN-1Nrf2 Activation in Caenorhabditis elegans

Hyun Won Seo1 Se Myung Cheon1 Myon-Hee Lee2 Hong Jun Kim3

Hoon Jeon1 and Dong Seok Cha1

1Department of Oriental Pharmacy College of Pharmacy Woosuk University Jeonbuk 565-701 Republic of Korea2Department of Medicine HematologyOncology Division Brody School of Medicine East Carolina UniversityGreenville NC 27834 USA3Department of Korean Medical Prescription College of Korean Medicine Woosuk University Jeonbuk 565-701 Republic of Korea

Correspondence should be addressed to Dong Seok Cha chawoosukackr

Received 26 November 2014 Revised 5 January 2015 Accepted 9 January 2015

Academic Editor Chang-Gue Son

Copyright copy 2015 Hyun Won Seo et alThis is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Catalpol is an effective component of rehmannia root and known to possess various pharmacological propertiesThe present studywas aimed at investigating the potential effects of catalpol on the lifespan and stress tolerance usingC elegansmodel systemHereincatalpol showed potent lifespan extension of wild-type nematode under normal culture condition In addition survival rate ofcatalpol-fed nematodes was significantly elevated compared to untreated control under heat and oxidative stress but not underhyperosmolality conditions We also found that elevated antioxidant enzyme activities and expressions of stress resistance proteinswere attributed to catalpol-mediated increased stress tolerance of nematode We further investigated whether catalpolrsquos longevityeffect is related to aging-related factors including reproduction food intake and growth Interestingly catalpol exposure couldattenuate pharyngeal pumping rate indicating that catalpol may induce dietary restriction of nematode Moreover locomotoryability of aged nematode was significantly improved by catalpol treatment while lipofuscin levels were attenuated suggesting thatcatalpol may affect age-associated changes of nematode Our mechanistic studies revealed thatmek-1 daf-2 age-1 daf-16 and skn-1are involved in catalpol-mediated longevity These results indicate that catalpol extends lifespan and increases stress tolerance of Celegans via DAF-16FOXO and SKN-1Nrf activation dependent on insulinIGF signaling and JNK signaling

1 Introduction

Aging is a universal biological process in all living organismsthat is influenced by environmental nutritional and geneticfactors [1] Although mankind has already concerned withdelaying aging and keeping ourselves young from the ancienttime it had been believed that the development of antiagingdrug is not feasible However increasing lines of evidencedemonstrated the pharmacological intervention in the agingprocess In recent years antiaging studies on the traditionalherbal medicine have received increasing attention becausethey are promising candidates for the treatment of variousaging-associated diseases Indeedmanynatural products andtheir active compounds such as ginsenoside curcumin andresveratrol are known to protect our body and give a positive

effect against aging in mammals as well as invertebratesthrough different mechanisms [2ndash4]

Rehmannia glutinosa has been widely used for the treat-ment of aging-related diseases as a traditional herbalmedicine in Korea and China Catalpol is an importantiridoid glucoside with the molecular formula of C

15H22O10

which is prevalent in the roots of Rehmannia glutinosa(Figure 1) Previous research showed that catalpol is effectivein various age-related degenerative diseases including cancerstroke cognition deficit diabetes Alzheimerrsquos disease andParkinsonrsquos disease [5ndash9] These studies suggest that catalpolhas therapeutic potential against aging However the sci-entific evidence on the antiaging effect of catalpol awaitsidentification

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2015 Article ID 524878 10 pageshttpdxdoiorg1011552015524878

2 Evidence-Based Complementary and Alternative Medicine

OH

HOO

OH

HOH

O

O

OH

HO

H

HO

Figure 1 The structure of catalpol

Accordingly the present studywas designed to investigatethe longevity effects of catalpol using Caenorhabditis elegansmodel system C elegans has become a widely acceptedmodel for work on aging research due to their short lifespanmorphological simplicity ease of maintenance and geneticmanipulation [10] In addition many previous papers notedthat compounds which have lifespan extension propertiesin C elegans can be applicable for the treatment of cancerand neurodegenerative diseases in humans indicating thisnematodemodel provides an excellent environment for iden-tifying drugs for prolonging human lifespan [11 12] Hereinwe dissected the effects of catalpol on the lifespan and survivalrate under normal and stress conditions Furthermore toverify the underlying pharmacologicalmechanisms catalpol-mediated changes in antioxidant enzyme activities agingrelated factors and lifespan of knockout mutant strains wereemployed

2 Materials and Methods

21 Chemicals C elegans Strains and Maintenance Catalpolwas purchased from Sigma-Aldrich (St Louis MO USA) Toprepare plates supplemented with catalpol the stock solutionin dimethyl sulfoxide (DMSO) was inserted into autoclavedNGM plates (at 50∘C) A final DMSO concentration of 01(vv) was maintained under all conditions Bristol N2 (wild-type) and Escherichia coli OP50 strain were kindly providedby Dr Myon-Hee Lee (East Carolina University NC USA)All other strains were obtained from the CaenorhabditisGenetic Center (CGC University of Minnesota MinneapisMN) The transgenic strain CF1553 (muIs84) was used tovisualize SOD-3 expression Mechanistic study was per-formed using several null mutant strains including GR1307(mgDf50) VC199 (ok434) EU1 (zu67) DR1572 (e1368)TJ1052 (hx546) and FK171 (ks54) The worms were grown at22∘C on nematode growthmedium (NGM) agar plate with Ecoli OP50 as described previously [13]

22 LifespanAssay The lifespan assays were performed usingmutants as well as wild-type at least 3 times independentlyat 22∘C To obtain age-synchronized nematodes eggs weretransferred to NGM plate in the absence or presence of625 125 and 25120583M of catalpol after embryo isolation oron the 7th day of adulthood respectively Test worms wereconsidered dead when they failed to respond to proddingwith the tip of a platinum wire [14] The worms weretransferred to fresh NGM plate every 2 days

23 Assessment of Stress Resistance The age-synchronizedN2 worms were bred on NGM agar plates with or withoutvarious concentrations of catalpol For the heat toleranceassay the adult day 4 worms were transferred to fresh platesand then incubated at 36∘C The survival rate was scoredas previously described [15] Oxidative stress tolerance wasassessed as described previously with minor modification[16] Briefly the adult day 4 worms were subjected to platecontaining 60mMparaquat and then survivals were recordedover 30 h Resistance to osmotic stress was measured byplacing the adult day 4 worms to NGM agar plate containing500mMNaCl [17] Survival rate of the worms was calculatedafter 12 h incubation The survival of worms was determinedtouch-provoked movement Worms which failed to respondto gentle touch with a platinum wire were considered to bedead Each test was performed at least 3 times

24 Measurement of SOD Activities To assess enzymaticactivity the worm homogenates were prepared Briefly thewild-type worms were harvested from plate with M9 bufferon the adult day 5 and washed 3 times Then the collectedworms were resuspended in homogenization buffer (10mMTris-HCl 150mM NaCl 01mM EDTA and pH 75) andhomogenized on ice SOD activity wasmeasured spectropho-tometrically analysing the decolorization of formazan usingenzymatic reaction between xanthine and xanthine oxidaseThe reactionmixture contained 20 120583L of worm homogenates480 120583L of 16mM xanthine and 048mM nitroblue tetra-zolium (NBT) in 10mM phosphate buffer (pH 80) Afterpreincubation at room temperature for 5 minutes the reac-tion was initiated by adding 1mL of xanthine oxidase (005UmL) and incubation at 37∘C for 20min The reaction wasstopped by adding 500 120583L of 69mMSDS and the absorbanceat 570 nm was measured The SOD activity was expressed asa percentage of the scavenged amount per control

25 Analysis of Intracellular ROS Intracellular reactive oxy-gen species (ROS) in the nematodes was measured usingmolecular probe 2101584071015840-dichlorodihydrofluorescein diacetate(H2DCF-DA) Equal number of wild-type worms was incu-

bated in the absence or presence of catalpol On the 4thday of adulthood animals were exposed to NGM agarplate containing 30mM paraquat for 3 h Subsequently 5worms were transferred into the wells of a 96-well platecontaining 50 120583L of M9 buffer Immediately after additionof 50 120583L of 25 120583M H

2DCF-DA solution resulting in a final

concentration 125 120583M basal fluorescence was quantified ina microplate fluorescence reader at excitation 485 nm andemission 535 nm Plates were read every 30min for 2 h

26 Measurement of Aging-Related Factors The age-syn-chronized N2 worms were bred on NGM agar plates withor without various concentrations of catalpol On the 4thdays of adulthood single worms were transferred to freshplate and their pharynx contractions were counted under aninvertedmicroscope for 1min For the reproduction assay N2worms were raised from embryo as in the lifespan assay L4larvae were individually transferred to the fresh plate everyday to distinguish the parent from the progeny The progeny

Evidence-Based Complementary and Alternative Medicine 3

Table 1 Effects of catalpol on the lifespan of mutant C elegans

Genotype Mean lifespana Maximum lifespan Change in mean lifespanc () Log-rank testdUntreated Treatedb Untreated Treatedb

Wild-type 123 plusmn 03 158 plusmn 04 18 27 285 P lt 0001lowastlowastlowast

skn-1 (zu67) 118 plusmn 03 117 plusmn 03 20 19 minus08 P = 0595daf-16 (mgDf50) 117 plusmn 04 118 plusmn 04 19 19 09 P = 0611daf-2 (e1368) 161 plusmn 08 161 plusmn 07 27 26 00 P = 0951age-1 (hx546) 152 plusmn 05 157 plusmn 06 28 29 34 P = 0356sir-21 (ok434) 114 plusmn 06 126 plusmn 07 19 23 103 P = 0010lowast

mek-1 (ks54) 102 plusmn 03 99 plusmn 03 18 17 minus11 P = 0803aMean lifespan presented as mean plusmn SEMbCatalpol-treated concentration was 25120583McChange in mean lifespan compared with untreated group of each strain ()dStatistical significance of the difference between survival curves was determined by log-rank test using the Kaplan-Meier survival analysis Differencescompared to the control were considered significant at lowast119875 lt 005 and lowastlowastlowast119875 lt 0001

was counted at the L2 or L3 stage For the growth alterationassay photographs were taken of adult day 4 worms andthe body length of each animal was analyzed by the Nikonsoftware (Nikon Japan) All the tests were repeated at least 3times

27 Measurement of Body Movement The age-synchronizedN2wormswere bred onNGMagar plateswith orwithout var-ious concentrations of catalpol On the 7th days of adulthoodsingle worms were transferred to fresh plate and their bodymovements were recorded under an inverted microscope for20 seconds The body movements of animals were analyzedby Nikon image software and data was expressed as totaltravel distance and average speed

28 Fluorescence Microscopy and Visualization The age-synchronized transgenic nematodes including CF1553 con-taining a SOD-3GFP reporter were maintained in thepresence or absence of catalpol On the 3rd days of adulthoodnematodes were exposed to heat shock at 36∘C for 2 h andallowed to recover at 22∘C for 4 h Prior to microscopy obser-vation transgenic animals were anesthetized with sodiumazide (2) andmounted on 2 agarose padTheGFPfluores-cence of GFP-expressing populations was directly observedunder a fluorescencemicroscope (NikonEclipseNi-u Japan)To determine the protein expression levels photographs ofthe transgenic worms were taken and assayed using ImageJsoftware All experiments were done in triplicate

29 DataAnalysis Thedata from the lifespan assay and stressresistance assays were plotted using Kaplan-Meier analysisand statistical significance was analyzed by log-rank testOther data were presented as mean plusmn standard deviation orstandard error of the mean as indicated Statistical signifi-cance of differences between the control and treated groupswas analyzed by one-way analysis of variance (ANOVA)

3 Results

31 Effect of Catalpol on Lifespan-Extension and Stress Resis-tance To determine the lifespan-extension properties of

catalpol lifespan assays were performed using wild-typeworms Herein we found a concentration-dependent effectof catalpol on longevity (Figures 2(a) and 2(c)) In additionthere was a significant increase (285 at 25 120583M of catalpol119875 lt 0001) in the estimated mean life of the catalpol-treated worms compared to control worms (Table 1) Toaddress the possibility that catalpol may shift wormrsquos lifespanindependent of affecting developmental stage of wormslifespan assay was conducted again using aged infertileworms (7 days of adulthood) in the presence or absenceof catalpol Interestingly catalpol-fed aged worms displayeddose-dependent increase in lifespan though being not asdrastically as compared to catalpol exposure after embryo iso-lation (Figures 2(b) and 2(d)) Then we determined whethercatalpol has protective effects on three different kinds ofstress conditions including thermal oxidative and osmoticstress using wild-type worms As can be seen in Figure 3(a)thermotolerance was elevated as a result of catalpol treatmentand consequently increased survival rate dose-dependentlyFurthermore the results showed that catalpol-treated wormslived longer than control worms under 60mM paraquat-induced oxidative stress (Figure 3(b)) However in the caseof hypertonic stress assay catalpol failed to increase theresistance to osmotic stress (Data not shown)

32 Effect of Catalpol on Antioxidant Enzymes and Intracellu-lar ROS Levels To verify the possible mechanism of catalpol-mediated lifespan extension and elevated stress toleranceactivities and expressions of stress resistance proteins wereinvestigated In the present study we measured activities ofantioxidant enzymes such as superoxide dismutase (SOD)and catalase using prepared worm homogenates As notedin Figures 4(a) and 4(b) both of them were significantlyupregulated in the presence of catalpol 25120583M of catalpolincreased SOD and catalase activities about 501 and 464respectively (119875 lt 0001) We also quantified sod-3 geneexpressions using transgenic strain CF1553 (sod-3pgfp + rol-6) As can be seen in Figure 4(c) catalpol treatment stronglyenhanced the GFP intensity of nematode in head tailand around vulva compared to vehicle-treated control Ourresults showed that catalpol increases sod-3 gene expression

4 Evidence-Based Complementary and Alternative Medicine

0

20

40

60

80

100

120

1 5 9 13 17 21 25 29Age (days)

Surv

ival

()

0120583M625 120583M

125 120583M25120583M

(a)

0

20

40

60

80

100

120

1 5 8 11 14 17 20 23 26

Surv

ival

()

Age (days)0120583M625 120583M

125 120583M25120583M

(b)

0

2

4

6

8

10

12

14

16

18

20

Mea

n lif

espa

n (d

ays)

0

(120583M)625 125 25

lowastlowastlowastlowastlowastlowast

(c)

0

2

4

6

8

10

12

14

16

Mea

n lif

espa

n (d

ays)

0

(120583M)625 125 25

lowast

(d)

Figure 2 Effects of catalpol on the lifespan of wild-type N2 nematodes Worms were grown in the NGM agar plate at 22∘C in the absenceor presence of catalpol after embryo isolation or 7th day of adulthood respectively The number of worms used per each lifespan assayexperiment was 36ndash57 and three independent experiments were repeated (119873 = 3) The mortality of each group was determined by dailycounting of surviving and dead animals The lifespan of catalpol-treated worms after embryo isolation (a) and 7th day of adulthood (b)was plotted as a survival curve The mean lifespan of the catalpol-treated worms after embryo isolation (c) and 7th day of adulthood (d)was calculated from the survival curves in (a) and (b) respectively Statistical difference between the curves was analyzed by log-rank testError bars represent the standard error of mean (SEM) Differences compared to the control were considered significant at lowast119875 lt 005 andlowastlowastlowast119875 lt 0001 by one-way ANOVA

suggesting that catalpol is an activator of SOD Then weevaluated the influence of catalpol on the accumulationof intracellular ROS using H

2DCF-DA probe Figure 4(d)

shows diminished intracellular ROS level in the catalpol-fednematode by 4457 at 25 120583M (119875 lt 0001) compared tocontrol

33 Effect of Catalpol on Aging-Related Factors Previousstudies have suggested that longevity is closely intercon-nected with reproduction food intake and growth in manyspecies including C elegans [18ndash20] Here in this workwe showed that catalpol treatment failed to alter the totalprogeny number while egg laying of worms was delayed at

Evidence-Based Complementary and Alternative Medicine 5

0

20

40

60

80

100

120

1 3 5 7 9 11 13 15 17 19Time (h)

Surv

ival

()

0120583M625120583M

125120583M25120583M

(a)

0

20

40

60

80

100

120

1 4 7 10 13 16 19 22 25 28Time (h)

Surv

ival

()

0120583M625120583M

125120583M25120583M

(b)

Figure 3 Effects of catalpol on the stress tolerance of wild-type N2 nematodes (a) To assess thermal tolerance worms were incubated at36∘C and then their viability was scored (b) For the oxidative stress assays worms were transferred to NGM agar plate containing 60mM ofparaquat and then their viability was scored Statistical difference between the curves was analyzed by log-rank test

the maximum concentration indicating that catalpol mightaffect germline signaling results in delayed spermatogenesisor oogenesis (Figure 5(a)) In addition no significant changein body length of wormswas detected after catalpol exposuresuggesting that catalpolrsquos activity is independent of growthas well as fertility (Figure 5(b)) Then we measured thenumber of pharyngeal pumping to estimate the food intakeof worms As shown in Figure 5(c) the number of pharyngealcontractions declined gradually with increasing age andthis age-associated diminishment was further attenuated bycatalpol treatment Based on this result we could estimate theprobability of dietary restriction like effects by catalpol

34 Effect of Catalpol on Body Movements and LipofuscinAccumulation Then we evaluated whether catalpol mayaffect age-associated changes in C elegans such as bodymovements and intestinal lipofuscin levels To estimate thehealthspan of worms we recorded travel distance of agedworms (7 days of adulthood) for 20 seconds duration Ascan be seen in Figure 6(a) catalpol exposure induced dose-dependent increase in body movement of worms Surpris-ingly 25 120583M of catalpol enhanced the total travel distanceand average speed of aged worms to over 30 compared tountreated control suggesting that functional aging of wormis strongly delayed by catalpol (Figure 6(a)) Since lipofuscinis known as an endogenousmarker of cellular damage duringaging in many organisms including C elegans [21] wemeasured the autofluorescence level of lipofuscin Our resultsrevealed that fluorescence intensity from intestinal lipofuscinwas significantly attenuated in the presence of catalpol by1325 at 25120583M (119875 lt 0001 Figure 6(b))

35 Mechanistic Studies The underlying mechanism ofcatalpol-mediated longevity was dissected using loss of func-tion mutant worms relevant to aging [22] We found that themean lifespan of sir-21 mutants was significantly enhancedby catalpol exposure indicating that SIR-21 is not responsiblefor catalpolrsquos activity (Table 1) However catalpol failed toincrease the lifespan of mutants including mek-1 daf-2 age-1 daf-16 and skn-1 Thus we estimated that these genesare involved in catalpol-induced lifespan regulation (Table 1)We double checked the involvement of daf-16 using TJ356strain which carries daf-16gfp transgene As can be seenin Figure 7 heat shock triggers DAF-16 nuclear localizationand catalpol-fed worms also exhibited similar phenotypesuggesting that catalpol activates the transcriptional activityof DAF-16

4 Discussion

In the current study we investigated the antiaging activityof catalpol an active compound of Rehmannia glutinosausing C elegansmodel system We found that catalpol treat-ment significantly enhanced the lifespan of wild-type wormsunder both of normal and stress conditions In additionintriguingly lifespan of worms was prolonged even when thecatalpol exposure is started at adulthoodThis result suggeststhat catalpol may give a positive effect on the senescencea biological aging after maturation and thus should be anattractive candidate for antiaging drug discovery

Previous reports have revealed that accumulation ofoxidative stress caused ROS is a major factor in aging [2324] Our additional studies suggest that catalpol-induced

6 Evidence-Based Complementary and Alternative Medicine

0

20

40

60

80

100

120

140

160

180SO

D ac

tivity

( o

f con

trol)

lowastlowastlowast

lowastlowastlowast

0 25(120583M)

625 125

(a)

0

20

40

60

80

100

120

140

160

180

Cata

lase

activ

ity (U

mg

prot

ein)

0 25

lowastlowastlowast

lowastlowastlowastlowast

(120583M)625 125

(b)

0

1

2

3

4

5

Fluo

resc

ence

inte

nsity

lowastlowastlowast

0 25(120583M)

(c)

0

50

100

150

200

250

300

0 30 60 90 120

ROS

leve

l (

of c

ontro

l)

0120583M625120583M

125120583M25120583M

Time (h)

lowastlowast

lowastlowastlowast

(d)

Figure 4 Effects of catalpol on the antioxidant enzyme activity and intracellular ROS levels of wild-type N2 nematodes (a) The enzymaticreaction of xanthine with xanthine oxidase was used to generate ∙O

2

minus and the SOD activity was estimated spectrophotometrically throughformazan formation by NBT reduction SOD activity was expressed as a percentage of the scavenged amount per control (b) Catalase activitywas calculated from the concentration of residual H

2O2 as determined by a spectrophotometric method Catalase activity was expressed in

Umgprotein (c) Fluorescence intensity of SOD-3GFP expression in control and catalpol-treatedCF1553worms and their imagesMeanGFPintensity was represented as mean plusmn SEM of values from 19ndash24 animals per each experiment (119873 = 3) (d) Intracellular ROS accumulationwas quantified spectrometrically at excitation 485 nm and emission 535 nm Plates were read every 30min for 2 h Data are expressed as themean plusmn SEM of three independent experiments (119873 = 3) Differences compared to the control were considered significant at lowastlowast119875 lt 001and lowastlowastlowast119875 lt 0001 by one-way ANOVA

elevation of antioxidant enzyme activities that resulted inattenuated intracellular ROS might be attributed to extendedlifespan Moreover oxidative stress promotes accumulationof lipofuscin a marker of cellular damage during aging via

degeneration of cellular components in many organismsincludingC elegans [25]We demonstrated that the intestinallipofuscin levels of worms were significantly decreased bycatalpol treatment compared to control Since early studies

Evidence-Based Complementary and Alternative Medicine 7

050

100150200250300

1 2 3 4 5 6

Num

ber o

f pro

geny

wor

m

TotalDay

0120583M625120583M

125120583M25120583M

lowastlowastlowast

lowast

(a)

0

200

400

600

800

1000

1200

1400

0 25(120583M)

625 125

Body

leng

th (120583

m)

(b)

0

50

100

150

200

250

300

Pum

ping

rate

(min

)

0 25(120583M)

625 125

lowastlowast

(c)

Figure 5 Effects of catalpol on the various aging-related factors of wild-type N2 nematodes (a) Daily and total reproductive outputs werecounted The progeny was counted at the L2 or L3 stage (b) For the growth alteration assay photographs were taken of worms and the bodylength of each animal was analyzed (c) On the 4th day of adulthood the pharyngeal pumping rates Data are expressed as the mean plusmn SEMof three independent experiments (119873 = 3) Differences compared to the control were considered significant at lowast119875 lt 005 lowastlowast119875 lt 001 andlowastlowastlowast119875 lt 0001 by one-way ANOVA

have suggested that antioxidant treatment prevents lipofuscin[26] our results provide a further evidence that catalpolrsquosantioxidant potential might be contributed to delaying aging

Herein we also checked whether some alterations inaging-related factors are associated with catalpol-mediatedlongevity Reductions of age-related parameters includingreproduction food intake and growth have been known toclosely interconnectedwith longevity inmany species [18 19]We displayed that catalpol attenuated food intake of wormswhile no change was observed in other parameters such as

reproduction and growth indicating that dietary restriction(DR) like effects of catalpol might be possibly linked withlifespan-extension

Nowadays the goal of antiaging medicine has beenchanged from simply extending lifespan to increasinghealthspan Here we showed that catalpol supplementationeffectively delayed age-related deterioration of body move-ment of worms compared with untreated control indicat-ing catalpol could enhance healthspan of worms The eat-2 (ad465) mutant shows dietary restriction phenotype via

8 Evidence-Based Complementary and Alternative Medicine

0

05

1

15

2

25

3

35

4

45Tr

avel

dist

ance

(mm

)

0 25(120583M)

625 125

lowastlowast

(a)

0

10

20

30

40

Fluo

resc

ence

inte

nsity

0 25(120583M)

lowastlowastlowast

(b)

Figure 6 Effects of catalpol on the body movement and lipofuscin accumulation in wild-type N2 nematodes (a) Body movement of wormswere counted under a dissecting microscope for 1min (b) Fluorescence intensity of lipofuscin and autofluorescence image worms on the 8thday of adulthoodThe fluorescence intensity was quantified using ImageJ software by determining average pixel intensity Mean fluorescenceintensity of lipofuscin was represented as mean plusmn SEM of values from 18-19 animals per each experiment (119873 = 3) Differences compared tothe control were considered significant at lowastlowast119875 lt 001 and lowastlowastlowast119875 lt 0001 by one-way ANOVA

Control Heat-shock Catalpol (25120583M)

Figure 7 Effects of catalpol on the nuclear localization of DAF-16The translocation of DAF-16 was visualized under fluorescencemicroscopeusing TJ356 strain which carries daf-16gfp transgene To induce heat-shock worms were incubated at 36∘C for 2 h Worms were subjectedto analyze GFP expression on the 4th day of adulthood

slowing the rate of pharyngeal pumping [27] Previous studiessuggest that the locomotory ability is highly preserved toolder ages of eat-2 mutant compared to wild-type worms[28] Therefore catalpol may not be responsible for muscledeterioration in the pharynx and resulted in diminished pha-ryngeal pumping rate but rather modulates age-associatedfunctional decline via DR

To understand underlying genetic mechanisms by whichcatalpol extends lifespan we conducted lifespan assay usingseveral null mutants Previous studies have indicated thatgenetic interference by conserved transcriptional factorsincluding DAF-16 and SKN-1 increases lifespan in C elegans[29 30] DAF-16 a FOXO-family transcriptional factor hasnumerous target genes which confer an enhanced stress resis-tance and extended lifespan [31] Our experiments demon-strate that DAF-16 is required for catalpol-mediated lifespan

extension We further investigated whether catalpol acceler-ates DAF-16 activation using TJ356 strain which carries daf-16gfp transgene Importantly we confirmed that catalpolexposure induces nuclear translocation of DAF-16 Indeedas noted above catalpol increased the expression of SOD-3 a downstream target of DAF-16 In addition we foundthat another transcriptional factor SKN-1 is also involvedin catalpolrsquos longevity properties The skn-1 gene encodesa worm homolog of the Nrf2 that is critical for oxidativestress resistance and promotes longevity [32] The SKN-1is associated with inducing DR response via downstreameffector NLP-7 a neuropeptide [33] Therefore it is plausiblethat SKN-1 possibly serves as a target molecule of catalpol forDR response

Both DAF-16 and SKN-1 have been shown to be inhibitedby the insulinIGF signaling (IIS) pathway in C elegans

Evidence-Based Complementary and Alternative Medicine 9

[34 35] To test the possibility whether IIS pathway is respon-sible for catalpol-mediated activation of these transcriptionalfactors we analyzed the lifespan of daf-2 and age-1 nullmutants These genes are known to play an important rolein IIS pathway by encoding DAF-2insulin like receptorand AGE-1phosphoinositide 3-kinase (PI3K) respectivelyIn this study we observed no significant extended lifespan ofboth mutants after catalpol exposure indicating that catalpolmay activate DAF-16 and SKN-1 via inhibition of IIS pathway

Additional input into DAF-16 regulation is allowed toJNK pathway a member of the MAPK family Previousgenetic analysis suggests that the JNK pathway also par-ticipated in stress resistance and longevity as a positiveregulator of DAF-16 in C elegans [36] Our findings indicatethat catalpol failed to increase the lifespan of mek-1 mutantlacking MEK-1 (MAPKK) in the JNK pathway suggestingthat catalpol may also activate DAF-16 via regulation of JNKsignaling independent of IIS pathway

SIR-21 a family of NAD+-dependent histone deacet-ylases is another evolutionary conserved regulator oflongevity Previous studies have revealed that overexpressionof SIR-21 can increase the lifespan of C elegans througheither downregulation of IIS pathway or direct activationof DAF-16 in a parallel with IIS signaling [37 38] In thisstudy the possible involvement of SIR-21 was also inves-tigated using sir-21 null mutants Our observation showsthat catalpol significantly prolonged the lifespan of sir-21silenced worms suggesting that catalpolrsquos longevity activitiesare independent of regulation of SIR-21

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgment

This work was supported by the research grant fromWoosukUniversity (2014)

References

[1] D Harman ldquoAging a theory based on free radical and radiationchemistryrdquo Journal of Gerontology vol 11 no 3 pp 298ndash3001956

[2] J Zhu X Mu J Zeng et al ldquoGinsenoside rg1 prevents cognitiveimpairment and hippocampus senescence in a rat model of d-galactose-induced agingrdquo PLoS ONE vol 9 no 6 Article IDe101291 2014

[3] L-R Shen L D Parnell J M Ordovas and C-Q LaildquoCurcumin and agingrdquo BioFactors vol 39 no 1 pp 133ndash1402013

[4] J H Chung V Manganiello and J R B Dyck ldquoResveratrol asa calorie restriction mimetic therapeutic implicationsrdquo Trendsin Cell Biology vol 22 no 10 pp 546ndash554 2012

[5] C R Pungitore M J Ayub E J Borkowski C E Tonn andG M Ciuffo ldquoInhibition of Taq DNA polymerase by catalpolrdquoCellular andMolecular Biology vol 50 no 6 pp 767ndash772 2004

[6] Y-R Liu R-Y Lei C-E Wang et al ldquoEffects of catalpolon ATPase and amino acids in gerbils with cerebral

ischemiareperfusion injuryrdquo Neurological Sciences vol35 no 8 pp 1229ndash1233 2014

[7] J-P Shieh K-C Cheng H-H Chung Y-F Kerh C-H Yehand J-T Cheng ldquoPlasma glucose lowering mechanisms ofcatalpol an active principle from roots of rehmannia glutinosain streptozotocin-induced diabetic ratsrdquo Journal of Agriculturaland Food Chemistry vol 59 no 8 pp 3747ndash3753 2011

[8] Z Xia R Zhang P Wu Z Xia and Y Hu ldquoMemory defectinduced by beta-amyloid plus glutamate receptor agonist isalleviated by catalpol and donepezil through different mecha-nismsrdquo Brain Research vol 1441 pp 27ndash37 2012

[9] X-L Zhang L-J An Y-M Bao J-Y Wang and B Jiangldquod-galactose administration induces memory loss and energymetabolism disturbance in mice protective effects of catalpolrdquoFood and Chemical Toxicology vol 46 no 8 pp 2888ndash28942008

[10] L Guarente and C Kenyon ldquoGenetic pathways that regulateageing inmodel organismsrdquoNature vol 408 no 6809 pp 255ndash262 2000

[11] J J Collins K Evason and K Kornfeld ldquoPharmacology ofdelayed aging and extended lifespan of Caenorhabditis elegansrdquoExperimental Gerontology vol 41 no 10 pp 1032ndash1039 2006

[12] M Petrascheck X Ye and L B Buck ldquoAn antidepressant thatextends lifespan in adult Caenorhabditis elegansrdquo Nature vol450 no 7169 pp 553ndash556 2007

[13] S Brenner ldquoThe genetics of Caenorhabditis elegansrdquo Geneticsvol 77 no 1 pp 71ndash94 1974

[14] G J Lithgow T M White S Melov and T E JohnsonldquoThermotolerance and extended life-span conferred by single-gene mutations and induced by thermal stressrdquo Proceedings ofthe National Academy of Sciences of the United States of Americavol 92 no 16 pp 7540ndash7544 1995

[15] E-Y Lee Y-H Shim D J Chitwood S B Hwang J Lee andY-K Paik ldquoCholesterol-producing transgenic Caenorhabditiselegans lives longer due to newly acquired enhanced stress resis-tancerdquo Biochemical and Biophysical Research Communicationsvol 328 no 4 pp 929ndash936 2005

[16] R AMekheimer A A R Sayed and EAAhmed ldquoNovel 124-triazolo[15-a]pyridines and their fused ring systems attenuateoxidative stress and prolong lifespan ofCaenorhabiditis elegansrdquoJournal of Medicinal Chemistry vol 55 no 9 pp 4169ndash41772012

[17] M Horikawa and K Sakamoto ldquoFatty-acid metabolism isinvolved in stress-resistance mechanisms of CaenorhabditiselegansrdquoBiochemical andBiophysical ResearchCommunicationsvol 390 no 4 pp 1402ndash1407 2009

[18] L Partridge D Gems and D J Withers ldquoSex and death whatis the connectionrdquo Cell vol 120 no 4 pp 461ndash472 2005

[19] C Morck and M Pilon ldquoC elegans feeding defective mutantshave shorter body lengths and increased autophagyrdquo BMCDevelopmental Biology vol 6 article 39 2006

[20] L Bordone and L Guarente ldquoCalorie restriction SIRT1 andmetabolism understanding longevityrdquo Nature Reviews Molec-ular Cell Biology vol 6 no 4 pp 298ndash305 2005

[21] U T Brunk and A Terman ldquoLipofuscin mechanisms of age-related accumulation and influence on cell functionrdquo FreeRadical Biology amp Medicine vol 33 no 5 pp 611ndash619 2002

[22] K Pietsch N Saul S Chakrabarti S R Sturzenbaum RMenzel and C E W Steinberg ldquoHormetins antioxidants andprooxidants defining quercetin- caffeic acid- and rosmarinicacid-mediated life extension in C elegansrdquo Biogerontology vol12 no 4 pp 329ndash347 2011

10 Evidence-Based Complementary and Alternative Medicine

[23] A Bokov A Chaudhuri and A Richardson ldquoThe role ofoxidative damage and stress in agingrdquo Mechanisms of Ageingand Development vol 125 no 10-11 pp 811ndash826 2004

[24] T Finkel and N J Holbrook ldquoOxidants oxidative stress and thebiology of ageingrdquoNature vol 408 no 6809 pp 239ndash247 2000

[25] B Gerstbrein G Stamatas N Kollias and M Driscoll ldquoIn vivospectrofluorimetry reveals endogenous biomarkers that reporthealthspan and dietary restriction in Caenorhabditis elegansrdquoAging Cell vol 4 no 3 pp 127ndash137 2005

[26] A Terman and U T Brunk ldquoLipofuscin mechanisms offormation and increase with agerdquo APMIS vol 106 no 2 pp265ndash276 1998

[27] B Lakowski and S Hekimi ldquoThe genetics of caloric restrictionin Caenorhabditis elegansrdquo Proceedings of the National Academyof Sciences of the United States of America vol 95 no 22 pp13091ndash13096 1998

[28] C Huang C Xiong and K Kornfeld ldquoMeasurements of age-related changes of physiological processes that predict lifespanof Caenorhabditis elegansrdquoProceedings of theNational Academyof Sciences of the United States of America vol 101 no 21 pp8084ndash8089 2004

[29] M Hansen S Taubert D Crawford N Libina S-J Leeand C Kenyon ldquoLifespan extension by conditions that inhibittranslation in Caenorhabditis elegansrdquo Aging Cell vol 6 no 1pp 95ndash110 2007

[30] J Wang S Robida-Stubbs J M A Tullet J-F Rual M Vidaland T Keith Blackwell ldquoRNAi screening implicates a SKN-1-dependent transcriptional response in stress resistance andlongevity deriving from translation inhibitionrdquo PLoS Geneticsvol 6 no 8 2010

[31] 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

[32] S-K Park P M Tedesco and T E Johnson ldquoOxidative stressand longevity in Caenorhabditis elegans as mediated by SKN-1rdquoAging Cell vol 8 no 3 pp 258ndash269 2009

[33] J R Cypser D Kitzenberg and S-K Park ldquoDietary restrictionin C elegans recent advancesrdquo Experimental Gerontology vol48 no 10 pp 1014ndash1017 2013

[34] C J Kenyon ldquoThe genetics of ageingrdquoNature vol 464 no 7288pp 504ndash512 2010

[35] J M A Tullet M Hertweck J H An et al ldquoDirect inhibition ofthe longevity-promoting factor SKN-1 by insulin-like signalingin C elegansrdquo Cell vol 132 no 6 pp 1025ndash1038 2008

[36] E Schaffitzel and M Hertweck ldquoRecent aging research inCaenorhabditis elegansrdquo Experimental Gerontology vol 41 no6 pp 557ndash563 2006

[37] H A Tissenbaum and L Guarente ldquoIncreased dosage of a sir-2 gene extends lifespan in Caenorhabditis elegansrdquo Nature vol410 no 6825 pp 227ndash230 2001

[38] A Berdichevsky M Viswanathan H R Horvitz and LGuarente ldquoC elegans SIR-21 interacts with 14-3-3 proteins toactivate DAF-16 and extend life spanrdquo Cell vol 125 no 6 pp1165ndash1177 2006

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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MEDIATORSINFLAMMATION

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Behavioural Neurology

EndocrinologyInternational Journal of

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Oxidative Medicine and Cellular Longevity

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The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

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