2013 Anti-inflammatory effect of

Carbohydrate Polymers 92 (2013) 84–89

Contents lists available at SciVerse ScienceDirect

Carbohydrate

Polymers

j o u r n a l h o m e p a g e :w w w.e l s e v i e r.c o m /l o c a t e /c a r b p o

Anti-inﬂammatory effect of fucoidan extracted from Ecklonia cava in zebraﬁsh model

Seung-Hong Lee a ,1,Chang-Ik Ko b ,1,Youngheun Jee c ,Yoonhwa Jeong d ,Misook Kim d ,Jin-Soo Kim e ,You-Jin Jeon b ,f ,∗

School of Medicine,Jeju National University,Jeju 690-756,Republic of Korea

Department of Marine Life Science,Jeju National University,Jeju 690-756,Republic of Korea c

Department of Veterinary Medicine and Applied Radiological Science Institute,Jeju National University,Jeju 690-756,Republic of Korea d

Department of Food Science and Nutrition,Dankook University,Gyeonggi 448-701,Republic of Korea e

Department of Seafood Science and Technology/Institute of Marine Industry,Gyeongsang National University,Tongyeong 650-160,Republic of Korea f

Marine and Environmental Research Institute,Jeju National University,Jeju 695-814,Republic of Korea

a r t i c l e

i n f o

Article history:

Received 23August 2012Received in revised form 24September 2012

Accepted 24September 2012Available online 2 October 2012

Keywords:Fucoidan Zebraﬁsh Tail-cutting LPS-treatment

Anti-inﬂammatory

a b s t r a c t

Fucoidan extracted from Ecklonia cava had strong anti-inﬂammatory activities.However,the direct effects of fucoidan of E.cava on anti-inﬂammatory activities in vivo model remained to be determined.Therefore,the present study was designed to assess in vivo anti-inﬂammatory effect of fucoidan extracted from E.cava (ECF)using tail-cutting-induced and lipopolysaccharide (LPS)-stimulated zebraﬁsh model.Treating zebraﬁsh model with tail-cutting and LPS-treatment signiﬁcantly increased the ROS and NO level.How-ever,ECF inhibited this tail-cutting-induced and LPS-stimulated ROS and NO generation.These results show that ECF alleviated inﬂammation by inhibiting the ROS and NO generation induced by tail-cutting and LPS-treatment.In addition,ECF has a protective effect against the toxicity induced by LPS exposure in zebraﬁsh embryos.This outcome could explain the potential anti-inﬂammatory activity of ECF,which might have a beneﬁcial effect during the treatment of inﬂammatory diseases.

1.Introduction

Fucoidans are commonly found in brown seaweeds and some marine invertebrates such as sea cucumbers and sea urchins (Chevolot et al.,1999;Vieira &Mourao,1988).They mainly con-sist of fucose and sulfate with small amounts of galactose,xylose,mannose,and uronic acids (Bilan et al.,2002;Chizhov,Dell,Morris,Haslam,&McDowell,1999;Partankar,Oehninger,Barnett,Williams,&Clark,1993).Fucoidans isolated from brown algae have been extensively studied because of their diverse biological activ-ities such as anticoagulant,antitumor,immunomodulatory,and anti-inﬂammatory activities (Li,Lu,Wei,&Zhao,2008).Because of these activities,fucoidans have been investigated in the recent years to be developed as drugs and functional foods.

We extracted fucoidan in our previous study from an enzy-matic hydrolysate of the brown alga,Ecklonia cava (E.cava ),and

∗Corresponding author at:Department of Marine Life Science,Jeju National Uni-versity,Jeju 690-756,Republic of Korea.Tel.:+82647543475;fax:+82647563493.

E-mail addresses:youjinj@jejunu.ac.kr ,youjinj@gmail (Y.-J.Jeon).1

These authors contributed equally to this work.

was mainly composed of fucose,with small amounts of galac-tose,xylose,and mannose.We also demonstrated that the fucoidan extracted from E.cava had anti-inﬂammatory activities (Lee et al.,2012).However,the direct effects of fucoidan extracted from E.cava on anti-inﬂammatory activities in vivo model remained to be determined.

The vertebrate zebraﬁsh (Danio rerio )is a small tropical freshwa-ter ﬁsh,which has emerged as a useful vertebrate model organism because of its small size,large clutches,transparency,low cost maintenance,morphological and physiological similarity to mam-mals (Eisen,1996;Fishman,1999).Traditionally,zebraﬁsh has been used in the ﬁelds of molecular genetics and developmental biology (Driever,Solnica-Krezel,&Schier,1996;Kimmel,1989).However,recently the value of the zebraﬁsh as a model organism for drug dis-covery and toxicological studies has been recognized (den Hertog,2005;Pichler et al.,2003).

Zebraﬁsh have well-developed innate and acquired immune systems that very similar to the mammalian immune system (Trede,Zapata,&Zon,2001).In addition,the optical transparency of zebraﬁsh embryos allows noninvasive and dynamic imaging the inﬂammation in vivo .Because of these characteristics,zebraﬁsh are a useful and popular animal model for a variety of inﬂamma-tion studies.In in vivo anti-inﬂammation test model,zebraﬁsh is

S.-H.Lee et al./Carbohydrate Polymers92 (2013) 84–8985

Table1

Chemical composition of fucoidan extracted from E.cava.a

Component Fucoidan

Total carbohydrate(%)51.8±1.3

Sulfate content(%)20.1±0.7

Uronic acid(%)11.3±0.5

Protein(%)8.7±0.3

Proportion of monosaccharide(%)

Fucose61.1±1.6

Rhamnose 3.9±0.4

Galactose27.2±1.2

Glucose0.8±0.1

Xylose7.0±0.3

a Information from Lee et al.(2012).

Experiments were performed in triplicate and the data are expressed as mean±SE. widely accepted as the best method for effective anti-inﬂammation assay(Liao et al.,2011;Novoa,Bowman,Zon,&Figueras,2009; Park&Cho,2011).Therefore,the present study was designed to assess in vivo anti-inﬂammatory effect of fucoidan extracted from E. cava using tail-cutting-induced and lipopolysaccharide-stimulated zebraﬁsh model.

2.Materials and methods

2.1.Materials

The brown alga E.cava(Phylum Phaeophyta,Class Phaeo-phyceae,Order Laminariales,Family Alariaceae)was collected from the coast of Jeju Island,South Korea.A voucher specimen has been deposited in the author’s laboratory and taxonomic identiﬁcation of E.cava was performed by Prof.Ki-Wan Lee at Jeju National Uni-versity,Republic of Korea.Salt,sand and epiphytes were removed with tap water.The samples were then rinsed carefully with fresh water and freeze-dried.The dried alga sample was ground and sifted through a50-mesh standard testing sieve.All chemicals and reagents used were of analytical grade and obtained from commer-cial sources.

2.2.Preparation of fucoidan from E.cava

Fucoidan was extracted using the previously reported methods (Lee et al.,2012).A10g sample of the ground,dried E.cava powder was homogenized with1L of distilled water(dH2O)and mixed with 100␮L of Celluclast(Novo Nordisk,Bagsvaerd,Denmark).This reac-tion continued for24h at50◦C,and then,the digest was boiled for 10min at100◦C to inactivate the enzyme.The product was clariﬁed by centrifugation(3000×g for20min)to remove the unhydrolyzed residue.Then,the enzymatic hydrolysate was adjusted to pH7.0 and precipitated with3volumes of ethanol.After centrifugation at10,000×g for20min at4◦C,the precipitate was re-dissolved in dH2O and sequentially treated with4M CaCl2.The resulting pre-cipitate was removed by centrifugation and the supernatant was treated with cetylpyridi

nium chloride.The pyridinium salts were solubilized with3M CaCl2and reprecipitated with ethanol.The precipitate was re-dissolved in dH2O,dialyzed(M W CO,10–12kDa) against water at4◦C for72h,and then lyophilized;the lyophilized sample was used as fucoidan sample.The chemical composition of the fucoidan was shown in Table1(Lee et al.,2012).

2.3.Origin and maintenance of parental zebraﬁsh

Ten adult zebraﬁsh were obtained from a commercial dealer (Seoul aquarium,Seoul,Korea)and were kept in a3L acrylic tank at 28.5◦C with a14:10h light:dark cycle.The zebraﬁsh were fed three times a day,6days/week,with tetraminﬂake food supplemented with live brine shrimps(Artemia salina;SEWHAPET food Co.,Seoul,Korea).Embryos were obtained from natural spawning that was induced in the morning by turning on the light.The collection of embryos was completed within30min.

2.4.Preparation of inﬂammation-induced zebraﬁsh model by

tail-cutting and application of test fucoidan

The zebraﬁsh larvae were anesthetized in tricaine methane-sulfonate solution(Sigma,St.Louis,MO,USA)before tail-cutting. After anesthesia,to trigger tail-cutting-induced in

ﬂammation,tail of zebraﬁsh larvae was half-cutted,around50%of tail area was removed.To make wounds as consistently sized as possible,tail-cutting was performed using blade under a dissecting microscope. As soon as tail-cutting was completed,the zebraﬁsh larvae were transferred in the fresh embryo medium.

The zebraﬁsh larvae(n=15)were randomly divided into follow-ing four groups:(1)tail-uncutted control group(negative control);

(2)tail-cutted only group;(3)tail-cutted with fucoidan extracted from E.cava(ECF)-treated group;and(4)tail-cutted with commer-cial fucoidan(CF,Sigma,St.Louis,MO,USA)-treated group.ECF and CF-treated group was exposed to test samples at28.5◦C for3h.

2.5.Preparation of inﬂammation-induced zebraﬁsh model by LPS treatment and application of test fucoidan

Synchronized zebraﬁsh embryos were collected and arrayed by pipette,10–15embryos/well,in12-well plates containing 2mL embryo medium for7–9h post-fertilization(hpf),and then incubated without or with the test samples for1h.To induce inﬂammation,5␮g/mL LPS(ﬁnal concentration)was added to the embryo medium for15–17hpf at28.5◦C.Thereafter,the zebraﬁsh embryos were transferred in the f

resh embryo medium.

2.6.Estimation of inﬂammation-induced intracellular reactive oxygen species(ROS)generation and image analysis

The generation of ROS in inﬂammatory zebraﬁsh model was analyzed using an oxidation-sensitiveﬂuorescent probe dye,2 ,7 -dichlorodihydroﬂuorescein diacetate(DCF-DA).The DCF-DA is deacetylated intracellular by nonspeciﬁc esterase and is further oxidized to the highlyﬂuorescent compound,dichloroﬂuorescein (DCF)in the presence of cellular peroxides(Rosenkranz et al.,1992). Following tail-cutting and LPS treatment,the zebraﬁsh larvae and embryos were transferred into96-well plates,treated with DCF-DA solution(20␮g/mL),and incubated for1h in the dark at28.5◦C. After incubation,the zebraﬁsh larvae and embryos were rinsed in the fresh zebraﬁsh embryo medium and anesthetized in tricaine methanesulfonate solution before observation.Individual zebraﬁsh larvae and embryoﬂuorescence intensity was quantiﬁed using a spectroﬂuorometer(Perkin-Elmer LS-5B,Norwalk,CT,USA).The images of the stained larvae and embryos were observed using a ﬂuorescent microscope,which was equipped with a Moticam color digital camera(Motix,Xiamen,China).

2.7.Estimation of inﬂammation-induced intracellular nitric oxide (NO)generation and image analysis

Generation of NO in inﬂammatory zebraﬁsh model was analyzed using aﬂuorescent probe dye,diaminoﬂuorophore 4-amino-5-methylamino-2 ,7 -diﬂuoroﬂuorescein diacetate(DAF-FM DA).Transformation of DAF-FM DA by NO in the presence of dioxygen generates highlyﬂuorescent triazole derivatives(Itoh et al.,2000).Following tail-cutting and LPS treatment,the zebraﬁsh larvae and embryos were transferred into96-well plates,treated with DAF-FM DA solution(5␮M),and incubated for1h in the dark at28.5◦C.After incubation,the zebraﬁsh larvae and embryos were

86S.-H.Lee et al./Carbohydrate Polymers92 (2013) 84–89

rinsed in the fresh zebraﬁsh embryo medium and anesthetized in tricaine methanesulfonate solution before observation.Individual zebraﬁsh larvae and embryosﬂuorescence intensity was quanti-ﬁed using a spectroﬂuorometer(Perkin-Elmer LS-5B,Norwalk,CT, USA).The images of the stained larvae and embryos were observed using aﬂuorescent microscope,which was equipped with a Moti-cam color digital camera(Motix,Xiamen,China).

2.8.Measurement of heart-beating rate and yolk sac edema size

The heart-beating rate of both atrium and ventricle was mea-sured at48hpf after LPS-exposure(Choi et al.,2007).Counting and recording of atrial and ventricular concentration were performed for3min u

nder the microscope,and results are represented as the average heart-beating rate per min.

For size analysis of yolk sac edema at48hpf after LPS-exposure, lateral views of anesthetized embryos were imaged using a micro-scope(Na et al.,2009).The outlines of the yolk sac edema were traced,and the area within each tracing was determined by Motic-images plus V.2.0for windows(Motix,Xiamen,China).

2.9.Statistical analysis

The data are presented as means±standard error.Statistical comparisons were performed using the SPSS package for Windows (Version14).p-Values of less than0.05were considered signiﬁcant.

3.Results and discussion

典雅音乐花园3.1.Inhibitory effect of fucoidan extracted from E.cava(ECF)on

tail-cutting-induced and LPS-stimulated ROS generation in

inﬂammatory zebraﬁsh model

Inﬂammation is a complex stereotypical response of the body to cell damage and vascularize tissues.The inﬂammatory responses are controlled by cytokines,products of the plasma enzyme sys-tems,and lipid mediators(Ross&Auger,2002).However,chronic and uncontrolled inﬂammations are detrimental to the tissues, which may cause chronic inﬂammation-derived diseases,such as cardiovascular diseases and cancers(Frostegard et al.,1999;Karin, Lawrence,&Nizet,2006).We have previously reported that the fucoidan extracted from E.cava had excellent anti-inﬂammatory activities(Lee et al.,2012).However,the direct effects of fucoidan extracted from E.cava on anti-inﬂammatory activities in vivo model remained to be determined.In in vivo anti-inﬂammation test model, zebraﬁsh is widely accepted as the best method for effective anti-inﬂammation assay(Liao et al.,2011;Novoa et al.,2009;Park& Cho,2011).Therefore,the present study weﬁrst investigated in vivo anti-inﬂammatory effect of fucoidan(ECF)extracted from E.cava using zebraﬁsh model.

Inﬂammation is essential for physiological responses to tissue injury and infection.The wound-induced inﬂammation involves dynamic regulation of pro-inﬂammatory mediators(Mathias et al., 2006).To mimic acute inﬂammatory states in cells or tissues,treat-ment with lipopolysaccharide(LPS)has been widely used.LPS is a gram-negative bacterial pathogen and a potent activator of innate immune response(Park&Cho,2011).Recent studies have reported that zebr

aﬁsh model was used to rapidly and simply assess the anti-inﬂammatory activity on tail-cutting-induced and LPS-stimulated inﬂammation in vivo(Liao et al.,2011;Park&Cho,2011).

Generation of intracellular ROS can be detected using oxidation sensitive dye DCF-DA as the substrate.DCF-DA exhibits noﬂuo-rescence without ROS and becomesﬂuorescent upon interaction with ROS(Handa et al.,2006).Fluorescent probes have been widely employed to monitor oxidative activity cells.During labeling,non ﬂuorescent DCF-DA dye that freely into the cells gets hydrolyzed

by Fig.1.Inhibitory effect of ECF on tail-cutting-induced ROS generation in zebraﬁsh larvae.The zebraﬁsh larvae were pretreated to tail-cutting and treated with ECF.The ROS generation level was measured after staining with DCF-DA.(A)Fluorescence micrographs of tail-cutting-induced ROS generation,as follows:(i)control;(ii)tail-cutting only;(iii)treated with fucoidan(ECF)extracted from E.cava(100␮g/mL); and(iv)treated with commercial fucoidan(CF,100␮g/mL).(B)Aﬂuorescence spectrophotometer was used for the quantitative analysis of ROS generation.Exper-iments were performed in triplicate and the data are expressed as mean±SE. *p<0.05shows signiﬁcant difference from the only tail-cutting-treated zebraﬁsh. intracellular esterase to DCF and trapped inside the cells(Veerman et al.,2004).Therefore,in this study,the generation of ROS in tail-cutting-induced and LPS-stimulated inﬂammatory zebraﬁsh model was analyzed using an oxidation-sensitiveﬂuorescent probe dye,DCF-DA.Fig.1A demonstrates that the generation of ROS in zebraﬁsh was signiﬁcantly increased by the tail-cutting as com-pared with the negative control(zebraﬁsh without tail-cutting). Tail-cutted zebraﬁsh showed the ROS level to be signiﬁcantly increased to145.21%.However,the fucoidan extracted from E.cava (ECF)treatment reduced the level of ROS in the zebraﬁsh induced by tail-cutting.In particular,ECF slightly lowered ROS level compared to the commercial fucoidan(CF).Fig.1B is a typicalﬂuorescence micrograph of the zebraﬁsh.The negative control,which contained no fucoidan or tail-cutting,generated a clear image,whereas the positive control,which was

only tail-cutting,generated aﬂuores-cence image,which suggests that ROS took place during tail-cutting in the zebraﬁsh.However,in zebraﬁsh that were treated with ECF prior to tail-cutting,a dramatic reduction in the amount of ROS was observed.Zebraﬁsh incubated for15–17hpf with LPS showed ROS generation to be signiﬁcantly higher relative to the non-LPS treated zebraﬁsh(negative control).Pretreatment with ECF together with LPS signiﬁcantly inhibited ROS generation,indi-cating protection against ROS(Fig.2A).The zebraﬁsh treated with ECF showed ROS generation to be signiﬁcantly reduced by121.41%. Fig.2B is a typicalﬂuorescence micrograph of the zebraﬁsh.The negative control,which contained no fucoidan or LPS treatment, generated a clear image,whereas the positive control,which was only LPS treatment,generated aﬂuorescence image,which sug-gests that ROS took place during LPS treatment in the zebraﬁsh.

S.-H.Lee et al./Carbohydrate Polymers92 (2013) 84–89

Fig.2.Inhibitory effect of ECF on LPS-stimulated ROS generation in zebraﬁsh embryos.The zebraﬁsh embryos were pretreated with ECF and exposed to LPS (5␮g/mL).The ROS generation level was measured after staining with DCF-DA.(A) Fluorescence micrographs of LPS-stimulated ROS generation,as follows:(i)con-trol;(ii)LPS-treated only;(iii)pretreated with fucoidan(ECF)extracted from E.cava (100␮g/mL);and(iv)pretreated with commercial fucoidan(CF,100␮g/mL).(B) Aﬂuorescence spectrophotometer was used for the quantitative analysis of ROS generation.Experiments were performed in triplicate and the data are expressed as mean±SE.*p<0.05shows signiﬁcant difference from the only LPS-treated zebraﬁsh.

However,in zebraﬁsh that were treated with ECF prior to LPS treat-ment,a dramatic reduction in the amount of ROS was observed.ECF therefore signiﬁcantly reduced the elevated ROS level induced by LPS treatment in zebraﬁsh model.Previous studies have indicated that a high ROS level induces oxidative stress which can result in a variety of biochemical and physiological lesions.Such cellular damage frequently impairs the metabolic function and results in cell death and inﬂammation of tissues(Finkel&Holbrook,2000). Our results demonstrate that treating zebraﬁsh with tail-cutting and LPS-treatment signiﬁcantly increased the ROS level.However, ECF inhibited this tail-cutting and LPS-卢荣友

treatment-induced ROS gen-eration.These results show that ECF alleviated inﬂammation by inhibiting the ROS generation induced by tail-cutting and LPS-treatment.

3.2.Inhibitory effect of ECF on tail-cutting-induced and

LPS-stimulated NO generation in inﬂammatory zebraﬁsh model NO is an important inﬂammatory mediator that is synthesized from arginine by nitric oxide synthase(NOS).Generally,NO plays an important role as a vasodilator,neurotransmitter,and in the immunological system as a defense against tumor cells, parasites,and bacteria(Nakagawa&Yokozawa,2002).However, under pathological condition,NO production is increased by the inducible NOS(iNOS),subsequently,brings about cytotoxicity,and tissue damage(Kim,Cheon,Kim,Kim,&Kim,1999).Therefore, NO inhibitors are essential for the prevention of inﬂammatory diseases.We evaluated in this study the inhibitory effect of ECF on tail-cutting and LPS-treatment-induced NO production in zebraﬁsh by using aﬂuorescent probe dye,DAF-FM DA.Transformation of DAF-FM DA by NO in the presence of dioxygen generates highly ﬂuorescent triazole derivatives(Itoh et al.,2000).Fig.3A shows that the level of NO in zebraﬁsh was signiﬁcantly elevated by the tail-cutting as compared with the negative control(zebraﬁsh

without Fig.3.Inhibitory effect of ECF on tail-cutting induced NO generation in zebraﬁsh larvae.The zebraﬁsh larvae were pretreated to tail-cutting and treated with ECF.The NO generation level was measured after staining with DAF-FM-DA.(A)Fluorescence micrographs of tail-cutting-induced NO generation,as follows:(i)control;(ii)tail-cutting only;(iii)treated with fucoidan(ECF)extracted from E.cava(100␮g/mL); and(iv)treated with commercial fucoidan(CF,100␮g/mL).(B)Aﬂuorescence spec-trophotometer was used for the quantitative analysis of NO generation.Experiments were performed in triplicate and the data are expressed as mean±SE.*p<0.05 shows signiﬁcant difference from the only tail-cutting-treated zebraﬁsh.

tail-cutting).However,the NO level in the ECF-treated zebraﬁsh was reduced signiﬁcantly,this effect is similar to the CF.The level of NO in the zebraﬁsh treated with tail-cutting was123%,but treatment with ECF in conjunction with tail-cutting treatment resulted in a lower NO level of108%.ECF therefore reduced the elevated NO level induced by tail-cutting in inﬂammatory zebraﬁsh model.Fig.3B is a typicalﬂuorescence micrograph of the zebraﬁsh.The negative control,which contained no fucoidan or tail-cutting,generated a clear image,whereas the positive control, which was only tail-cutting,generated aﬂuorescence image,which suggests that NO took place during tail-cutting in the zebraﬁsh. However,in zebraﬁsh that were treated with ECF prior to tail-cutting,a dramat

ic reduction in the amount of NO was observed. Fig.4A shows that the level of NO in zebraﬁsh was signiﬁcantly elevated by the LPS treatment as compared with the non-LPS treated zebraﬁsh(negative control).However,the NO level in the ECF-treated zebraﬁsh was reduced signiﬁcantly.The level of NO in the zebraﬁsh treated with LPS was131.72%,but pretreatment with ECF together with LPS exposure resulted in a lower NO level of 107.87%.It is similar to the CF effect.Fig.4B is a typicalﬂuorescence micrograph of the zebraﬁsh.The negative control,which contained no fucoidan or LPS treatment,generated a clear image,whereas the positive control,which was only LPS treatment,generated a ﬂuorescence image,which suggests that NO took place during LPS treatment in the zebraﬁsh.However,in zebraﬁsh that were treated with ECF prior to LPS treatment,a dramatic reduction in the amount of NO was observed.ECF therefore signiﬁcantly reduced the elevated NO level induced by LPS treatment in zebraﬁsh model.Previous studies have indicated that fucoidan of E.cava

88S.-H.Lee et al./Carbohydrate Polymers 92 (2013) 84–

Fig. 4.Inhibitory effect of ECF on LPS-stimulated NO generation in zebraﬁsh embryos.The zebraﬁsh embryos were pretreated with ECF and exposed to LPS (5␮g/mL).The NO generation level was measured after staining with DAF-FM-DA.(A)Fluorescence micrographs of LPS-stimulated NO generation,as follows:(i)con-trol;(ii)LPS-treated only;(iii)pretreated with fucoidan (ECF)extracted from

E.cava (100␮g/mL);and (iv)pretreated with commercial fucoidan (CF,100␮g/mL).(B)A ﬂuorescence spectrophotometer was used for the quantitative analysis of NO generation.Experiments were performed in triplicate and the data are expressed as mean ±SE.*p <0.05shows signiﬁcant difference from the only LPS-treated zebraﬁsh.

suppressed NO and the expression of iNOS in murine macrophage

cells (Lee et al.,2012).ECF in this study also signiﬁcantly reduced the elevated NO level induced by tail-cutting and LPS-treatment in zebraﬁsh.Although the expression level of NO synthase was not examined,ECF may therefore inhibit NO synthase in zebraﬁsh based on previously published data.These ﬁndings indicate that ECF might confer important protection against the inﬂammation induced by physical and chemical damage.

3.3.Effects of ECF on LPS-stimulated the heart-beating rate and yolk sac edema size in zebraﬁsh

In the zebraﬁsh assay it revealed potential toxicity including swelling of the yolk,small head,tail bending,and a increase of heart-beating rate (Choi et al.,2007).Zebraﬁsh embryos respond to toxicoid sensitively,allowing us to measure heart-beating rate and yolk sac edema size.As shown in Fig.5A,the heart-beat rate in zebraﬁsh was remarkable increased by the LPS-treatment com-pared w

ith non-LPS treated zebraﬁsh (negative control).However,the heart-beat rate in the ECF-treated zebraﬁsh was decreased sig-niﬁcantly.Zebraﬁsh incubated for 48hpf without LPS-treatment observed no yolk sac edema.The size of yolk sac edema in zebraﬁsh was elevated by the LPS-treatment compared with neg-ative control group.However,ECF clearly protected zebraﬁsh against LPS-treatment (Fig.5B).Previously,Na et al.(2009)reported that antioxidant vitamin E protects 3,3 ,4,4 ,5-pentachlorobiphenyl (PCB126)induced toxicity such as pericardial sac edema,yolk sac edema,and growth retardation in zebraﬁsh embryos.In the present study,we found that ECF reduced heart-beating rate and yolk sac edema size produced by LPS exposure in zebraﬁsh embryos.The ﬁndings suggest that ECF might confer important protection against the toxicity induced by

LPS.

150

155

160

165

170

175

H e a r t b e a t i n g r a t e /m i n

Control None 100 μg/mLECF LPS

A 0.02

0.0210.0220.0230.0240.0250.026

Y o l k s a c e d e m a s i z e (μm 2)

Control None 100 μg/mLECF LPS

B Fig.5.Effects of ECF on LPS-stimulated the heart-beating rate (A)and yolk sac edema size (B)in zebraﬁsh embryos.The zebraﬁsh embryos were pretreated with ECF and exposed to LPS (5␮g/mL).The data was quantitated by image analysis.Experiments were performed in triplicate and the data are expressed as mean ±SE.*p <0.05shows signiﬁcant difference from the only LPS-treated zebraﬁsh.

Overall,the above results suggest that ECF could act as strong inhibitors of ROS and NO in tail-cutting-induced and LPS-stimulated inﬂammatory zebraﬁsh model.In addition,ECF has a protective effect against the toxicity induced by LPS exposure in zebraﬁsh embryos.This outcome could explain the potential anti-inﬂammatory activity of ECF,which might have a beneﬁcial effect during the treatment of inﬂammatory diseases.

4.Conclusion

Fucoidan extracted from E.cava had strong anti-inﬂammatory activities.However,the direct effects of fucoidan extracted from E.cava on anti-inﬂammatory activities in vivo model remained to be determined.Therefore,the present study we ﬁrst inves-tigated the anti-inﬂammatory effect of fucoidan,extracted from E.cava ,on tail-cutting-induced and LPS-stimulated inﬂammation,in in vivo zebraﬁsh model.Fucoidan from E.cava demonstrated strong anti-inﬂammatory properties against tail-cutting and LPS treatment-induced inﬂammation.The fucoidan also has a pro-tective effect against the toxicity induced by LPS exposure in zebraﬁsh embryos.The fucoidan from E.cava exhibited profound anti-inﬂammatory effect both in vitro as well as in vivo ,suggesting that the fucoidan might be a strong anti-inﬂammatory agent.

Acknowledgment

This work was supported by the National Research Foundation of Korea Grant funded by the Korean Government (Ministry of Edu-cation,Science and Technology).

S.-H.Lee et al./Carbohydrate Polymers92 (2013) 84–8989

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