fucoidan attenuates 6-hydroxydopamine-induced...

pISSN 2466-1384 eISSN 2466-1392

大韓獸醫學會誌 (2017) 第 57 卷第 1 號Korean J Vet Res(2017) 57(1) : 1~7https://doi.org/10.14405/kjvr.2017.57.1.1

1

<Original Article>

Fucoidan attenuates 6-hydroxydopamine-induced neurotoxicity

by exerting anti-oxidative and anti-apoptotic actions in SH-SY5Y cells

Myung-Hwan Kim1, Hoon Namgoong1, Bae-Dong Jung1, Myung-Sang Kwon1, Yeon-Shik Choi2, Taekyun Shin3,

Hyoung-Chun Kim4, Myung-Bok Wie1,*

1College of Veterinary Medicine and Institute of Veterinary Science, Kangwon National University, Chuncheon 24341, Korea2Department of Laboratory Animal Science, Korea Biopolytechnic College, Nonsan 32943, Korea

3College of Veterinary Medicine, Jeju National University, Jeju 63243, Korea4College of Pharmacy, Kangwon National University, Chuncheon 24341, Korea

(Received: October 11, 2016; Revised: November 25, 2016; Accepted: December 14, 2016)

Abstract: Parkinson’s disease (PD) is an irreversible neurological disorder with related locomotor dysfunction and ischaracterized by the selective loss of nigral neurons. PD can be experimentally induced by 6-hydroxydopamine (6-OHDA). It has been reported that reactive oxygen species, which deplete endogenous glutathione (GSH) levels, mayplay important roles in the dopaminergic cell death characteristic of PD. Fucoidan, a sulfated algal polysaccharide,exhibits anti-inflammatory and anti-oxidant actions. In this study, we investigated whether fucoidan can protect against6-OHDA-mediated cytotoxicity in SH-SY5Y cells. Cytotoxicity was evaluated by using MTT and LDH assays.Fucoidan alleviated cell damage evoked by 6-OHDA dose-dependently. Fucoidan reduced the number of apoptoticnuclei and the extent of annexin-V-associated apoptosis, as revealed by DAPI staining and flow cytometry. Elevationof lipid peroxidation and caspase-3/7 activities induced by 6-OHDA was attenuated by fucoidan, which also protectedagainst cytotoxicity evoked by buthionine-sulfoximine-mediated GSH depletion. Reduction in the glutathione/glutathionedisulfide ratio induced by 6-OHDA was reversed by fucoidan, which also inhibited 6-OHDA-induced disruption ofmitochondrial membrane potential. The results indicate that fucoidan may have protective action against 6-OHDA-mediated neurotoxicity by modulating oxidative injury and apoptosis through GSH depletion.

Keywords: apoptosis, caspase-3/7, fucoidan, glutathione/glutathione disulfide ratio, 6-hydroxydopamine

Introduction

PD is known as one of chronic neurological disorders asso-

ciated with oxidative damage to dopamine-producing neu-

rons in human midbrain [17]. The neuronal death of this area

is connected with various motor dysfunction, such as stiff-

ness or slowness [19]. Although the precise etiology of neu-

ronal injury remains unknown, accumulating evidence suggests

that impairment in the ability of mitochondria to regulate the

levels of lipid peroxides made by oxidative stress and inflam-

matory processes may be critical [6, 14]. Glutathione (GSH)

is an endogenous antioxidant that protects against various

oxidative stressors by regulating reactive oxygen species (ROS)

levels in PD patients [17]. GSH concentration is decreased in

the nigral regions of PD patients [12, 20]. The antioxidant

and neuroprotective characteristics of GSH have been amply

demonstrated in various models of oxidative injury, includ-

ing models in which GSH is depleted by the addition of L-

buthionine-(S,R)-sulfoximine (BSO) [10, 20].

Fucoidan is a fucose-rich, sulfated polysaccharide derived

from brown seaweeds and has been used as a component of

foods, cosmetics, and dietary supplements for many centu-

ries in Asian countries. The pharmacological actions of

fucoidan include anti-inflammatory, anti-oxidant, and anti-

thrombotic effects [9, 21]. However, fucoidan has not yet

been shown to be neuroprotective.

6-hydroxydopamine (6-OHDA) acts as a dopamine deplet-

ing agent which can occur toxicity to dopaminergic neurons.

Thus, it can be applicable to make an experimental form of

PD [1, 16]. Recently, fucoidan has been shown to inhibit the

toxicity of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)

by exerting anti-oxidant activities [11]. However, no report has

yet proposed the neuroprotective effect of fucoidan on 6-

OHDA-mediated toxicity in dopaminergic SH-SY5Y neuro-

blastoma cells. Previously, we found that fucoidan protected

against β-amyloid-mediated neuronal damage in rat neuronal

cultures; fucoidan exerted potent anti-oxidant effects and inhibi-

tory action of apoptosis [9]. In this study, we explored whether

*Corresponding author

Tel: +82-33-250-8676, Fax: +82-33-259-5625

E-mail: [email protected]

2 Myung-Hwan Kim, Hoon Namgoong, Bae-Dong Jung, Myung-Sang Kwon, Yeon-Shik Choi, Taekyun Shin, Hyoung-Chun Kim, Myung-Bok Wie

fucoidan protected against various oxidative injuries (includ-

ing GSH deprivation) attributable to BSO, and mitochon-

drial membrane dysfunction evoked by 6-OHDA.

Materials and Methods

Chemicals

Dulbecco’s modified Eagle’s medium (DMEM) and fetal

bovine serum (FBS) were obtained from Gibco-BRL (USA).

Penicillin-streptomycin mixture antibiotic was obtained from

Lonza (USA). Fucoidan (from Fucus vesiculosus), 6-OHDA,

the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bro-

mide (MTT), NADH, and demethy sulfoxide (DMSO) were

obtained from Sigma-Aldrich (USA).

Cell culture

The human dopaminergic SH-SY5Y cells were cultured in

DMEM supplemented with 10% heat-inactivated FBS, 100

µg/mL of streptomycin, and 100 U/mL of penicillin. Cells

were maintained at 37oC under an atmosphere of 95% air and

5% CO2. All experiments used cell monolayers at 70% con-

fluence.

MTT assay

Cells were plated into 96-well culture plates (20,000 cells/

well) at 37oC and grown in an incubator under 5% CO2.

After 24 h, cells were treated with 60 µM 6-OHDA in a vol-

ume of 200 µL; incubation continued for 15 to 17 h. Fucoidan

was pretreated 2 h prior to the exposure of 6-OHDA. The

cells were incubated with MTT solution (final concentration,

5 mg/mL) for 3 h. Then, the media were removed, 200 µL of

DMSO was added, and incubation continued for extra 30

min. The absorbance was measured at 570 nm (Molecular

Devices, USA).

Lactate dehydrogenase (LDH) measurement

Cells were plated (2 × 105 cells/well) into 24-well plates,

and treated with 60 µM 6-OHDA either alone or in combina-

tion with fucoidan for 24 h. The cytotoxicity was analyzed

by assessing the level of LDH released from destructed

membrane into the surrounding media 20-24 h after expo-

sure to 6-OHDA or fucoidan. LDH activities were measured

at an absorbance of 340 nm (Molecular Devices).

Thiobarbituric acid-reactive substances (TBARS) assay

Cellular levels of lipid peroxidation were analyzed using

OXItek TBARS Assay Kits (Enzo Life Sciences, USA). Briefly,

cultured cells were lysed with 2% (w/v) sodium dodecyl sul-

fate. Harvested cells were sonicated in PBS for 5 sec at 40 V

in an ice bath, and mixed with 100 µL of sodium dodecyl

sulfate solution and 2.5 mL of TBS/Buffer reagent. The sam-

ple mixtures were incubated at 95oC for 60 min, and cooled

in an ice bath. The supernatants were obtained by centrifuga-

tion (1,500 × g) for 30 min. The levels of TBARS were mea-

sured at an absorbance of 532 nm. The results were calculated

by standard curve which made according to difference of

malondialdehyde (MDA) concentration.

Glutathione (GSH) assay

GSH was analyzed according to the method of Chang et

al. [3]. Cultured cells were detached using trypsin and har-

vested with a cell scraper into 15-mL conical tubes, centri-

fuged briefly. Cell washing with cold PBS was repeated

twice. The cells were suspended in 5% (w/v) metaphospho-

ric acid and sonicated in an ice bath. The cells were next cen-

trifuged at 12,000 × g for 5 min at 4oC. Total GSH and

glutathione disulfide (GSSG) levels were estimated by absor-

bance at 405 nm using a GSH assay kit (Enzo Life Sciences)

and expressed as GSH/GSSG (picomolar) ratios.

DAPI staining

Cells were seeded on coverglasses into bottom-culture

dishes. After incubation for 24 h, the cells were treated with

6-OHDA or fucoidan, fixed in 70% (v/v) ethanol after wash-

ing with PBS. Cells were stained with DAPI (1 µg/mL). Mor-

phological changes in DNA were observed under an inverted

fluorescence microscope (Carl Zeiss, Germany).

Caspase-3/7 assay

To assess caspase-3/7 activities, cells were plated at a den-

sity of 0.5 × 106cells/well. After treatment of cells with 60

µM 6-OHDA, either alone or with fucoidan for 12 h, caspase-3/

7 activity was evaluated by commercial kit (Millipore, USA).

The caspase-3/7 reagent was mixed with cell suspension.

After incubation, 7-amino-actinomycin D (7-AAD) reagent

was added, and data were collected by Muse Cell Analyzer

(Millipore).

Apoptosis assay

Flow cytometric assay on apoptosis quantitatively mea-

sured using Annexin-V and 7-AAD kits (Millipore). Annexin

V binds phosphatidylserine which translocated to the outer

cell membrane during apoptotic process. 7-AAD is known as

a fluorescent, DNA-intercalating agent. All assays were car-

ried out conformity with the assay protocol. Briefly, after

exposure to 6-OHDA alone or 6-OHDA with fucoidan for 24

h, the cells were collected and resuspended in assay buffer.

Stained cells were evaluated by the Muse Cell Analyzer.

Assay of mitochondrial membrane potential (MMP)

The diminution of cellular MMP is known as a disruption

of energy production and sign of early apoptosis. After treat-

ment with 60 µM 6-OHDA, either alone or with fucoidan for

12 h, MMP was analyzed using the Muse MitoPotential Kit

(Millipore). Briefly, harvested cell suspension was mixed

with the Muse MitoPotential reagent followed by incubation

at 37oC for 20 min. Finally, the 7-AAD reagent was added,

and data were collected using the Muse Cell Analyzer.

Fucoidan attenuates 6-hydroxydopamine neurotoxicity 3

Statistics

Results were assessed by one-way ANOVA with Dunnett’s

test. A value of p < 0.05 or p < 0.01 was considered statisti-

cally significant. All data were expressed as means ± SEM.

Results

Fucoidan protects SH-SY5Y cells against 6-OHDA-

mediated neurotoxicity

Treatment of cells with 6-OHDA (60 µM) triggered 50 to

60% neurotoxicity, as revealed by the MTT assay. Thus, we

used 60 µM 6-OHDA as the control level in all experiments.

Fucoidan exerted significant protective effects at concentra-

tions of 4, 20, 40, and 60 µg/mL (Fig. 1).

Phase contrast photomicrography and DAPI staining

The protective effects of fucoidan were reflected morpho-

logically, as shown in Figure 2A–C. In the control (vehicle-

treated) group, most neurites and cell bodies were intact (Fig.

2A). However, the majority of cell bodies and neurites were

damaged after exposure to 6-OHDA for 24 h (Fig. 2B).

Fucoidan mitigated 6-OHDA-induced neurotoxicity (Fig. 2C).

DAPI staining showed that 6-OHDA induced nuclear break-

down and fragmentation (Fig. 2E), to a greater extent than

occurred in control samples (Fig. 2D). Fucoidan prevented

such cellular injuries (Fig. 2F).

Effects of fucoidan on TBARS levels, the glutathione

redox system, and neurotoxicity mediated by glutathione

deprivation

We used the TBARS assay to explore the effects of

fucoidan on lipid peroxide formation. Likewise in Figure 3A,

treatment of cells with 6-OHDA increased the TBARS lev-

els by about two-fold (compared to control values). Fucoidan

pretreatment significantly inhibited lipid peroxidation. We

evaluated the action of fucoidan on GSH redox reaction, and

markers of oxidative stress, by determining GSH/GSSG ratios.

Treatment of cells with 6-OHDA markedly diminished in

GSH/GSSG ratio (Fig. 3B). Under such circumstances, fucoidan

significantly inhibited the decline of the GSH/GSSG ratio

induced by 6-OHDA, such that the values remained normal.

Prolonged exposure (48 h) of cells to a high concentration (3

mM) of L-buthionine-(S,R)-sulfoximine (BSO; an inhibitor

Fig. 1. Protective effects of fucoidan on 6-hydroxydopamine (6-

OHDA)-induced neurotoxicity in SH-SY5Y cells. Cultures were

pretreated with 2–60 µg/mL fucoidan for 2 h prior to exposure

to 60 µM 6-OHDA for 24 h. Cell viability was determined

using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium

bromide (MTT) assay. Optical density was measured at 570 nm.

Values are means ± SEM of three independent experiments,

each performed in triplicate. *p < 0.05, **p < 0.01 compared to

control, and #p < 0.05 compared to vehicle.

Fig. 2. Morphological evidence of protection afforded by fucoidan to cells exposed to 6-OHDA-induced neurotoxicity. (A-C) Phase-

contrast photomicrographs of cultured SH-SY5Y cells exposed to vehicle (A), 60 µM 6-OHDA (the control agent) (B), and 40 µg/mL

fucoidan and 6-OHDA (C). (D-F). Each group contains examples of cells exposed to vehicle (D), 60 µM 6-OHDA (E), and 60 µM

6-OHDA+40 µg/mL fucoidan (F). Red arrows indicate apoptotic cells (E). DAPI stain (D-F). 200× (A-C), 400× (D-F).


Fig. 3. Fucoidan inhibits 6-OHDA-induced reactive oxygen species (ROS) production in, and L-buthionine-(S,R)-sulfoximine (BSO)-

induced neuronal injury to, SH-SY5Y cells. Fucoidan significantly inhibited the extent of lipid peroxidation, as measured by the

thiobarbituric acid-reactive substances (TBARS) assay (A), and accelerated recovery as assessed by evaluation of the glutathione/glu-

tathione disulfide (GSH/GSSG) ratio (B). Fucoidan significantly attenuated neuronal injury induced by glutathione deprivation of SH-

SY5Y cells (C). All values are means ± SEM of three independent experiments, each performed in triplicate. *p < 0.05, **p < 0.01

compared to control, and #p < 0.05 compared to vehicle.

Fig. 4. Changes in cumulative apoptotic cell ratio (live, early apoptotic, apoptotic/dead, and dead) as determined by assay of apoptotic

status based on caspase-3/7 activation of SH-SY5Y cells exposed to various concentrations of fucoidan and 6-OHDA, measured by

flow cytometry (A). Representative flow cytometric data are shown in (B). Apoptotic cell ratio by caspase-3/7 activation was observed

24 h after exposure to 6-OHDA. All values are means ± SEM of three independent experiments, each performed in triplicate.

*p < 0.05, **p < 0.01 compared to control, and #p < 0.05 compared to vehicle.


of GSH biosynthesis) increased the extent of neuronal injury.

Fucoidan dose-dependently protected against BSO-induced

neurotoxicity (Fig. 3C).

Effects of fucoidan on caspase-3/7 activities

We evaluated the anti-apoptotic activities of fucoidan by

measuring caspase-3/7 activities using flow cytometry. Treat-

ment of cells with 6-OHDA elevated apoptotic status (apop-

totic/dead and dead cells) based on caspase-3/7 activation.

Among cumulative cell counts, the percentage of apoptotic/

dead and dead cells increased two- and five-folds compared

to vehicle group, respectively. Fucoidan pretreatment signifi-

cantly inhibited such elevations in both apoptotic/dead and

dead cells induced by 6-OHDA (Fig. 4A). Representative

flow cytometric profiles on apoptotic cell counts are shown

in Figure 4B.

Effects of fucoidan on apoptosis

Treatment of cells with 6-OHDA increased the percent-

age of early and late apoptosis by 13% and 12%, respec-

tively. Fucoidan pretreatment (40 µg/mL) significantly decreased

by 5 and 2% (Fig. 5A). Representative apoptotic flow cyto-

metric profiles on apoptotic cell counts are shown in Fig-

ure 5B.

Effects of fucoidan on MMP

The 6-OHDA reduced the proportion of SH-SY5Y cells

exhibiting a decrease in mitochondrial depolarization. The

average percentage of depolarized dead cells in 6-OHDA-

treated group increased from 9.6% to 38%. Pretreatment with

fucoidan (40 µg/mL) significantly inhibited by 16.7% com-

pared to 6-OHDA (control) group (Fig. 6A). Representative

flow cytometric profiles of cumulative cell counts (Depolar-

ized/live and depolarized/dead cells except live and dead

cells were shown) are shown in Figure 6B.

Discussion

Although many researchers have sought to define the

pathogenesis of PD, the mechanism underlying neurodegen-

eration in the substantia nigra remains unclear. However, oxi-

dative stress, including elevation of iron concentrations and

reduced GSH levels, may be key in this context [15]. In

accordance with these ideas, the elevation of TBARS and

reduction of GSH/GSSG ratio evoked by 6-OHDA were well

observed in same cell line [3]. Especially, reduction of glu-

tathione level in nigral regions of brain is considered as a

critical factor in PD pathogenesis and secondary to complex I

inhibition [12]. In in vivo studies, fucoidan treatment has

Fig. 5. Changes in cumulative apoptotic cell ratio (live, early

apoptotic, late apoptotic, and dead) determined using the Muse

Annexin-V and dead cell assays after exposure to 40 µg/mL

fucoidan and 6-OHDA, as measured by flow cytometry (A).

Representative flow cytometric data are shown in (B). All val-

ues are expressed as means ± SEM of three independent exper-

iments, each performed in triplicate. **p < 0.01 compared to

control, and ##p < 0.01 compared to vehicle.

Fig. 6. Changes in cumulative cell ratio (live, deplolarized/live,

depolarized/dead, and dead) by changes of mitochondrial mem-

brane potential (MMP) caused by 6-OHDA alone or 6-OHDA

and fucoidan (40 µg/mL) in combination (A). Representative

flow cytometric data are shown in (B). MMPs were measured

using the Muse Mitopotential kit. All values are means ± SEM

of three independent experiments, each performed in triplicate.

**p < 0.01 compared to control, #p < 0.05, and ##p < 0.01 com-

pared to vehicle.


showed a distinct amelioration of neurodegenerative symp-

toms in experimental PD models induced by MPTP and 6-

OHDA, respectively [11, 22]. Hence, our results suggest that

fucoidan may be one of the potential neuroprotective candi-

dates derived from various natural antioxidants. Recently,

fucoidan has been reported to suppress the microglial activa-

tion via increase in ROS-generating enzyme, NADPH oxi-

dase-1 (Nox 1) expression in dopaminergic neurons of rat

substntia nigra region after 6-OHDA injection [22]. These

results mean that fucoidan could be contributable to neu-

ronal survival through modulatory action of nitric oxide,

ROS and a key proinflammatory cytokine, such as tumor

necrosis factor-α [4, 5]. Here, Cui et al. [5] have demon-

strated that fucoidan remarkably inhibited an elevation of

inducible nitric oxide synthase protein and microglial activa-

tion induced by lipopolysaccharide at similar concentrations

like us. Mitochondrial dysfunction caused by inhibition of

complex I activity may correlate with the extent of injury to

dopaminergic neurons in PD patients. Unfortunately, there

have been few pharmacological reports on the ability of

fucoidan to prevent PD. Previously, we screened some natu-

ral medicines using an in vitro PD model (human dopaminer-

gic SH-SY5Y neuroblastoma cells) [3].

In the present study, we found that fucoidan inhibited both

lipid peroxidation, and the disturbance of GSH metabolism,

induced by 6-OHDA in SH-SY5Y cells. These results sug-

gest that fucoidan may serve as a useful, natural GSH-spar-

ing agent that partially alleviates PD symptoms. Fucoidan

inhibits the neuronal damage triggered by deprivation of

endogenous GSH. Although source of fucoidan is somewhat

different, the neuroprotective basis seems to be mainly attrib-

utable to the anti-oxidant and anti-inflammatory action [4,

11, 13, 21, 22]. Previously, we showed that fucoidan pro-

tected neuronal cells by inhibiting oxidative effects, as well

as the stimulation of protein kinase C and caspase 3/9, which is

required for β-amyloid-induced neurotoxicity [9]. Similarly,

we now show that fucoidan inhibits the elevation of caspase

3/7 activity evoked by 6-OHDA and consequent apoptotic

death. An earlier report showed that fucoidan inhibited the

elevation of caspase-3 activity in a model of H2O2-induced

neurotoxicity [7], and protected against MPTP-induced lipid

peroxidation of, and 1-methyl-4-phenylpyridinium (MPP+)-

induced neuronal injury to, MN9D cells [11]. Thus, fucoidan

acts a neuroprotectant in experimental models of PD, such as

MPTP or 6-OHDA. However, fucoidan has not yet been

shown the capability to permeate the blood-brain barrier. The

MMP decreases when the mitochondrial membrane is dam-

aged or collapses after exposure to a 6-OHDA-induced auto-

oxidation cascade [2, 8]. Fucoidan increased the number of

cells in which depolarized mitochondria was apparent after

6-OHDA treatment. Recently, fucoidan has been shown to

alleviate the mitochondrial dysfunction that develops after

traumatic injury [18]. However, the exact pharmacological

properties of fucoidan are difficult to conclude easily because

sulfated polysaccharide is complex and possess broad spec-

trum therapeutic properties. Overall, our results suggest that

fucoidan pretreatment improves neuronal survival by exert-

ing anti-oxidative and anti-apoptotic effects when 6-OHDA

induces neuronal damage to SH-SY5Y cells.

Acknowledgments

This study was supported by 2014 Research Grant from

Kangwon National University (No. 120140291).

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