HO-1 Antioxidant Pathway in Human

Jan 30, 2018 - PROYA Companies, Hangzhou, Zhejiang 310012, China. ∥ Chenghai Baoer Bio-Ltd, Lijiang, Yunnan 674202, China. ⊥Collaborative Innovati...
0 downloads 6 Views 2MB Size
Subscriber access provided by University of Florida | Smathers Libraries

Article

Astaxanthin induces the Nrf2/HO-1 antioxidant pathway in human umbilical vein endothelial cells by generating trace amounts of ROS Tingting Niu, Rongrong Xuan, Ligang Jiang, Wei Wu, Zhanghe Zhen, Yuling Song, Lili Hong, Kaiqin Zheng, Jiaxing Zhang, Qingshan Xu, Yinghong Tan, Xiaojun Yan, and Haimin Chen J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b05493 • Publication Date (Web): 30 Jan 2018 Downloaded from http://pubs.acs.org on January 31, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 40

Journal of Agricultural and Food Chemistry

1

Astaxanthin induces the Nrf2/HO-1 antioxidant pathway in human umbilical

2

vein endothelial cells by generating trace amounts of ROS

3 4

Tingting Niu1,5,&, Rongrong Xuan2,&, Ligang Jiang3, Wei Wu1, Zhanghe Zhen1, Yuling

5

Song1, Lili Hong1,Kaiqin, Zheng1,Jiaxing Zhang1,Qingshan Xu4, Yinghong Tan4,

6

Xiaojun Yan1 and Haimin Chen1*

7

1. Key Laboratory of Marine Biotechnology of Zhejiang Province, Ningbo University, Ningbo, Zhejiang 315211, China

8 9

2. Department of Gynecology and Obstetrics, The Affiliated Hospital of Medical College of Ningbo University, Ningbo, Zhejiang 315211, China

10 11

3. PROYA Companies, Hangzhou, Zhejiang 310012, China.

12

4. Chenghai Baoer Bio-Ltd, Lijiang, Yunnan 674202, China

13

5. Collaborative Innovation Center for Zhejiang Marine High-efficiency and Healthy

14

Aquaculture School of Marine Sciences, Ningbo University, Ningbo, Zhejiang

15

315211, China

16 17

&

These authors contributed equally to this work.

18 19

Corresponding author: Haimin Chen, Key Laboratory of Marine Biotechnology of

20

Zhejiang Province, Ningbo University, Post Box 71, Ningbo, Zhejiang, China 315211,

21

Tel: +86-574-87609574; Fax: +86-574-87609570; E-mail: [email protected]

22 23

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

24

Abstract

25

Astaxanthin is a powerful antioxidant that possesses potent protective effects against

26

various human diseases and physiological disorders. However, the mechanisms

27

underlying its antioxidant functions in cells are not fully understood. In the present

28

study, the effects of astaxanthin on reactive oxygen species (ROS) production and

29

antioxidant enzyme activity, as well as mitogen-activated protein kinases (MAPKs),

30

phosphatidylinositol 3-kinase (PI3K)/Akt, and the nuclear factor erythroid 2-related

31

factor 2 (Nrf-2)/heme oxygenase-1 (HO-1) pathways in human umbilical vein

32

endothelial cells (HUVECs) were examined. It was shown that astaxanthin (0.1, 1, 10

33

µM) induced ROS production by 9.35%, 14.8% and 18.06% compared to control

34

respectively in HUVECs. In addition, astaxanthin increased the mRNA levels of

35

phase II enzymes HO-1 and also promoted GSH-Px enzyme activity. Furthermore, we

36

observed ERK phosphorylation, nuclear translocation of Nrf-2, and activation of

37

antioxidant response element-driven luciferase activity upon astaxanthin treatment.

38

Knockdown of Nrf-2 by small interfering RNA inhibited HO-1 mRNA expression by

39

60%, indicating that the Nrf-2/ARE signaling pathway is activated by astaxanthin.

40

Our results suggest that astaxanthin activates the Nrf-2/HO-1 antioxidant pathway by

41

generating small amounts of ROS.

42 43

Keywords: astaxanthin, human umbilical vein endothelial cells, reactive oxygen

44

species, Nrf-2/HO-1 pathway, antioxidant

45

ACS Paragon Plus Environment

Page 2 of 40

Page 3 of 40

Journal of Agricultural and Food Chemistry

46

Introduction

47

Endothelial cells form the inner lining of blood vessels and play a crucial role in

48

many vascular functions including cell adhesion, inflammatory responses, regulation

49

of permeability, and vasoactive1. These cells, however, are highly sensitive to injury

50

caused by oxidative stress, an imbalance between oxidants and antioxidants in favor

51

of the oxidants, leading to a disruption of redox signaling and control and/or

52

molecular damage2. Previous studies have indicated that oxidative stress plays a

53

pivotal role in endothelial dysfunction that is closely associated with diabetes,

54

cardiovascular disease, hypertension, and preeclampsia3-4. To avoid the injury caused

55

by oxidative stress, cells have evolved strategies to overcome this challenge. A major

56

strategy is to activate nuclear factor erythroid2-related factor 2 (Nrf-2)/heme

57

oxygenase-1 (HO-1) signaling pathway5, which controls the expression of a number

58

of cytoprotective genes that are able to combat the harmful effects of oxidative

59

response. As a result, Nrf-2/HO-1 signaling pathway has become a therapeutic target

60

of many antioxidants. For example, epigallocatechin-3-gallate (EGCG) derived from

61

green tea induces reactive oxygen species (ROS), leading to the induction of Nrf-2

62

phosphorylation6. In addition, Nrf-2 is a critical regulator of flavonoid-mediated

63

effects7. Astaxanthin

64

(3,3’-dihydroxy-β-carotene-4,4’-dione)

is

present

in

most

65

red-colored aquatic organisms and is a potent antioxidant with 550-fold more potency

66

than

67

anti-inflammatory, antiapoptotic, neuroprotective, and cardioprotective effects9-10.

vitamin

E

(VE)8.

The

biological functions of astaxanthin

ACS Paragon Plus Environment

include

Journal of Agricultural and Food Chemistry

68

However, its hydrophilic polyene structure, which has low polarity, makes it difficult

69

to permeate the cell; thus, few studies have been conducted to study its antioxidant

70

effects at the cellular level. It has been shown that the antioxidant mechanisms of

71

astaxanthin include directly scavenging cellular ROS that are trapped inside the

72

phospholipid membrane and at the surface, protecting the mitochondrial redox state

73

and functional integrity, and activating antioxidant-related signaling pathways11-14.

74

However, few of these in vitro studies have reported the effective astaxanthin

75

concentration or the ideal incubation time that can trigger responses in cells. Instead,

76

varied astaxanthin concentrations ranging from 0.025 to 5 mM have been reported15-16,

77

in addition to the reported incubation periods ranging between 2 to 96 h17-18. The

78

precise effective utilization ratio and actual concentration of astaxanthin that can

79

permeate the cell membrane remain unclear. For example, Saw et al. demonstrated

80

that astaxanthin protects against oxidative stress via the Nrf-2/antioxidant response

81

element (ARE) pathway in human hepatoma HepG2-C8 cells19, but the amount of

82

astaxanthin that was taken up by the cells to activate this pathway in vitro was not

83

reported.

84

In this study, we determined the effective concentration and utilization ratio of

85

astaxanthin in HUVECs, and investigated the mechanisms underlying stimulation of

86

the Nrf-2/ARE signaling pathway.

87

Materials and Methods

88

2.1 Chemicals

89

Dulbecco’s Modified Eagle’s Medium (DMEM), fetal bovine serum (FBS) and

ACS Paragon Plus Environment

Page 4 of 40

Page 5 of 40

Journal of Agricultural and Food Chemistry

90

trypsin were purchased from Gibco BRL (Grand Island, NY, USA). HPLC grade

91

acetonitrile, N-hexane, ethylacetate, DMSO, 2,7-dichloroflurescenin diacetate

92

(DCFH-DA) probe, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide

93

(MTT), N-acetylcysteine (NAC) were purchased from Sigma (St. Louis, MO, USA).

94

Lysis buffer, Superoxide dismutase (SOD) Activity Assay Kit, Glutathione peroxidase

95

(GSH-Px) Activity Assay Kit were purchased from Beyotime (Shanghai, China).

96

NE-PER Nuclear and Cytoplasmic Extraction Kit was purchased from Pierce

97

(Rockford, IL, USA). Bio-Rad DC Protein Assay was purchased from Bio-Rad

98

Laboratories (Hercules, CA, USA). Anti-HO-1, phospho-JNK (Thr 183 and Tyr 185),

99

NQO1, Histone antibodies were purchased from Cell Signaling Technology (Danvers,

100

MA, USA). Phospho-extracellular signal-regulated protein kinase 1/2 (ERK1/2)

101

(Thr202/Tyr204), ERK, JNK, phospho-p38 (Thr 180/Tyr 182), p38, phospho-Akt

102

(Ser473), Akt, Nrf-2, and β-actin, horseradish peroxidase (HRP)-conjugated mouse

103

anti-rabbit IgG, HRP-conjugated goat anti-mouse IgG secondary antibodies were

104

purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Astaxanthin

105

(≥97%) isolated from Haematococcus pluvialis was purchased from Wako Pure

106

Chemical Industries Ltd. (Osaka, Japan).

107

2.2 Cell culture and astaxanthin preparation

108

HUVECs were obtained from China Center for Type Culture Collection (Wuhan,

109

China) and maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) with 10%

110

(v/v) fetal bovine serum (FBS) in a humidified incubator at 37°C with 5% CO2 and 95%

111

air. Cells were cultured until 50–70% confluence, and then treated with astaxanthin at

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

112

different concentrations for different time courses. To prepare different concentrations

113

of astaxanthin, 10 mg astaxanthin was solubilized in 500 µL dimethylsulfoxide

114

(DMSO, final concentration of 0.03%). Then it was slowly added to FBS and

115

completely mixed, after which the mixture was added to the cell culture medium

116

according to the working concentrations.

117

2.3 Kinetic uptake assay

118

Cells were treated with a serial dilution of astaxanthin (0.1, 1, and 10 µM) for

119

different time courses (6, 12, 18, 24, 36 or 48 h). At the end of each incubation period,

120

cells were harvested by trypsin treatment and counted by blood counting chamber.

121

Cells were washed three times with ice-cold phosphate-buffered saline (PBS), and

122

lysed with 200 µL lysis buffer containing 20 mM Tris (pH7.5), 150 mM NaCl, 1%

123

Triton X-100 and sodium pyrophosphate, β-glycerophosphate, EDTA, Na3VO4,

124

leupeptin. The homogenates containing astaxanthin were extracted with 1 ml

125

N-hexane:ethylacetate (1:2, v:v), followed by ultrasonic decomposition for 15 min in

126

an ice-cold container and centrifugation at 13,000 ×g for 10 min at 4°C. After

127

centrifugation, the organic layer was collected and dried under nitrogen gas, and

128

re-dissolved in methanol. Samples were analyzed by high-performance liquid

129

chromatography-mass spectrometry (Thermo Fisher Scientific, Waltham, MA, USA).

130

Analyses were performed at 25°C using a Hypersil Gold C18 column (100 mm×2.1

131

mm, 3 µm, Thermo Fisher Scientific). Acetonitrile (A) and deionized water (B) were

132

used for gradient elution. The elution protocol was as follows: 0 min, 75% A, 25% B;

133

4 min, 85% A, 15% B; 12 min, 98% A, 2% B; 13 min, 75% A, 25% B; and 16.5 min,

ACS Paragon Plus Environment

Page 6 of 40

Page 7 of 40

Journal of Agricultural and Food Chemistry

134

75% A, 25% B.

135

2.4 Cell viability assay

136

HUVECs grown in 96 well plate and incubated with different concentrations of

137

astaxanthin for 18 and 48 h, respectively. The cells were incubated with 20 µL

138

3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) for 4 h at 37°C.

139

After removing the MTT, 150 µL DMSO was added to each well. The

140

spectrophotometric absorbance of the samples was measured at a wavelength of 492

141

nm. The data are expressed as the percentage of control, and the experiments were

142

done in triplicate.

143

2.5 Antioxidant activity assays

144

HUVECs were seeded into 6-well culture plates for 24 h until 60–70% confluence,

145

after which cells were treated with astaxanthin for 18 h. Then cells were washed twice

146

with ice-cold PBS and lysed with 200 µL lysis buffer containing 1 mM

147

phenylmethylsulfonyl fluoride for 30 min on ice. The homogenates were centrifuged

148

at 13,000 ×g for 10 min at 4°C. Superoxide dismutase (SOD) was measured using a

149

SOD Activity Assay Kit according to the manufacturer’s instructions. One unit of

150

SOD activity was defined as the amount of enzyme needed to exhibit 50%

151

dismutation of superoxide radical. Glutathione peroxidase (GSH-Px) activity was

152

measured using a GSH-Px Activity Assay Kit according to the manufacturer’s

153

instructions. One unit of enzyme activity was defined as the amount of enzyme that

154

caused the oxidation of 1 µmoL NADPH to NADP per min at 25°C.

155

2.6 Intracellular ROS assay

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

156

The redox-sensitive fluorescent 2,7-dichloroflurescenin diacetate (DCFH-DA) probe

157

was used to assess the intracellular levels of ROS. Briefly, HUVECs were seeded into

158

6-well cell culture plates for 18 h until 50–60% confluence. After that, cells were

159

incubated with astaxanthin (1 µM) for either different periods of time, or with

160

different concentrations astaxanthin for 18 h. In another experiment, cells were

161

pretreated with VE (30 µM) or N-acetylcysteine (NAC, 10 mM) for 1 h, and then

162

incubated with 1 µM astaxanthin for 18 h. Additionally, we also investigated the ROS

163

production of cells induced by hydrogen peroxide (1 mM, 30min), fucoxanthin (5 µM,

164

18 h), astaxanthin (1 µM, 18 h), or NAC (10 mM, 1 h). After treatment, cell culture

165

medium was changed with fresh serum-free DMEM. DCFH-DA (15 µM) was then

166

added to the cells and incubated at 37°C for 45 min, after which cells were trypsinized

167

and washed twice with PBS. ROS measurement was conducted using a Beckman

168

Gallios Flow Cytometer (Beckman Counter, Inc., Brea, CA, USA) and the data are

169

expressed as mean DCF fluorescence intensity (sum of fluorescence intensities of all

170

cells/the number of cells).

171

2.7 Real-time quantitative PCR

172

HUVECs were cultured with astaxanthin (1 µM) for different time courses or treated

173

with different concentrations of astaxanthin for 18, 48 h. After treatment, cells were

174

harvested and total RNA was isolated with TaKaLa RNAiso Plus Reagent (TaKaLa,

175

Dalian, China) according to the manufacturer`s protocol. Total RNA (2 µg) was used

176

as template to synthesize the first strand of cDNA in a 20 µL reverse transcription (RT)

177

reaction, and 2 µL of RT product was used for PCR amplification using the

ACS Paragon Plus Environment

Page 8 of 40

Page 9 of 40

Journal of Agricultural and Food Chemistry

178

LightCycler 96 real-time PCR system (Roche, Basel, Switzerland) and SYER-Green I

179

monitoring method. Four pairs of specific primers were used for amplification as

180

previously described20, namely HO-1-F: AAGTATCCTTGTTGACACG, HO-1-R :

181

TGAGCCAGGAACAGAGTG ;

182

NQO1-R:

183

CAGTGGTGGATGGTTGTG,γ-GCL-R:

184

β-actin-F:

185

TGTGTGGACTTGGGAGAGG. β-actin was served as the internal control for the

186

real-time quantitative PCR (qPCR) analysis. The concentration of cDNA in each

187

sample was reflected by the threshold cycle (Ct) value, which was compared using the

188

relative quantification method. The relative mRNA expression of each target gene was

189

normalized to that of β-actin.

190

2.8 Western blot analysis

191

HUVECs were treated with either astaxanthin (1 µM) for different time courses, or

192

with different concentrations of astaxanthin for 18 h. In other experiment, HUVECs

193

were pretreated with VE (30 mM) or NAC (10 mM) for 1 h, and then stimulated with

194

1 µM astaxanthin for 18 h. Nuclear extracts were prepared by using the NE-PER

195

Nuclear and Cytoplasmic Extraction Kit according to the manufacturer’s protocol. For

196

total protein, cells were washed twice with ice-cold PBS and lysed with 200 µL lysis

197

buffer containing 1 mM phenylmethylsulfonyl fluoride for 30 min on ice. The

198

homogenates were centrifuged at 13,000 × g for 10 min at 4°C. Protein concentration

199

was determined with the Bio-Rad DC Protein Assay according to the manufacturer’s

NQO1-F:

AGACCTTGTGATATTCCAGTTC,

GGCAGCGTAAGTGTAAGC ;

γ-GCL-F:

ATTGATGATGGTGTCTATGC ;

CGGTGAAGGTGACAGCAG,

ACS Paragon Plus Environment

and

β-actin-R:

Journal of Agricultural and Food Chemistry

200

instructions. For western blot analysis, 30µg proteins (nuclear extracts or whole cell

201

lysates) were resolved on 10% sodium dodecyl sulfate polyacrylamide gels and then

202

transferred to polyvinylidene fluoride membranes. The membranes were blocked with

203

5% skim milk in Tris-buffered saline with Tween20 (TBST) for 2 h at room

204

temperature, and washed with TBST for three times. Then the membranes were

205

incubated overnight at 4°C with antibodies against HO-1 (1:2000), phospho-JNK

206

(1:1000), NQO1 (1:1000), Histone (1:1000), phospho-extracellular signal-regulated

207

protein kinase 1/2 (ERK1/2) (1:500), ERK (1:500), JNK (1:500), phospho-p38

208

(1:500), p38 (1:500), phospho-Akt (1:1000), Akt (1:1000), Nrf-2 (1:1000), or β-actin

209

(1:1000). The membranes were washed with TBST for three times followed by

210

incubation for 1 h at room temperature with horseradish peroxidase (HRP)-conjugated

211

mouse anti-rabbit IgG (1:2000) or HRP-conjugated goat anti-mouse IgG (1:8000)

212

secondary antibodies. After three times washing with TBST, immunoreactive proteins

213

were detected with WesternBright ECL (Advansta Inc., Menlo Park, CA, USA). The

214

results were quantified by measuring the band intensity and comparing it to that of

215

β-actin using AlphaView™ Software (Alpha Innotech, San Leandro, CA, USA). Data

216

represent as the % of control.

217

2.9 Transient transfection and luciferase reporter assays

218

To examine the effects of astaxanthin on Nrf-2 activation, HUVECs were transiently

219

co-transfected with 1 µg firefly luciferase reporter plasmid p-ARE-Luc (Clontech

220

Laboratories, Palo Alto, CA, USA) and 0.1 µg p-RL by using X-tremeGENE HP DNA

221

Transfection Reagent (Roche) according to the manufacturer’s instructions. 24h after

ACS Paragon Plus Environment

Page 10 of 40

Page 11 of 40

Journal of Agricultural and Food Chemistry

222

transfection, cells were treated with astaxanthin for 18 h. Firefly and Renilla

223

luciferase activities were measured in cell lysates using the Dual-Glo Luciferase

224

Assay System (Promega, Madison, WI, USA). All of the experiments were repeated

225

three times and the luciferase activity was calculated and normalized to renilla

226

luciferase activity.

227

2.10 Nrf-2 RNA interference assay

228

Short interfering RNA (siRNA) duplexes were synthesized by GenePharma (Shanghai,

229

China). The Nrf-2 siRNA duplex with the following sense and antisense strands was

230

used:

231

5’-AGAUCUAUAUCUUGCCUCCTT-3’ (antisense). HUVECs were cultured in

232

6-well plates. At 50–60% confluence, the media was replaced with OPTI-MEM

233

reduced serum medium. Transient transfection of siRNAs was conducted using

234

X-tremeGENE siRNA Transfection Reagent (Roche). Briefly, 2 µg siRNA and 10 µL

235

X-tremeGENE siRNA Transfection Reagent was diluted in 100 µL OPTI-MEM

236

reduced serum medium, and incubated for 5 min at room temperature. Diluted

237

X-tremeGENE siRNA Transfection Reagent was added to the siRNA dilution,

238

incubated for 20 min at room temperature, after which the transfection compound was

239

directly added to the cells. 8 h after transfection, cell culture medium was replaced

240

with fresh media containing different concentrations of astaxanthin and incubated for

241

another 48 h.

242

western blot analysis, and the mRNA level of HO-1 gene was determined by

243

qRT-PCR. The GSH-Px enzyme activity was examined by GSH-Px Activity Assay

5’-GGAGGCAAGAUAUAGAUCUTT-3’

(sense)

and

The protein expression of HO-1 and Nrf-2 were determined by

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

244

Kit.

245

2.11 Statistical analysis

246

Statistical analyses were performed using SPSS software, version 16.0 (SPSS Inc.,

247

Chicago, IL, USA). The results are expressed as mean ± standard deviation (SD) and

248

the statistical significance was analyzed by one-way ANOVA with the Tukey multiple

249

comparison test. P values less than 0.05 were considered statistically significant.

250

3. Results

251

3.1 Astaxanthin uptake

252

As shown in Figure 1, the intracellular level of astaxanthin was gradually increased

253

within 18 h, and the peak level was reached at 18 h and remained unchanged from 24

254

to 48 h. In addition, it was shown that the maximum concentration of intracellular

255

astaxanthin was 4.622 nmol/106 cells when cells were incubated with 10 µM

256

astaxanthin for 18 h, indicating an uptake rate of 0.0462%. As a control, astaxanthin

257

was undetectable in parallel untreated cell cultures.

258

3.2 Astaxanthin increased intracellular ROS but did not cause cytotoxicity

259

Treatment of cells with astaxanthin (1 µM) for different time courses caused the

260

production of small amount of ROS. As shown in Fig. 2A, incubation of cells with 1

261

µM astaxanthin for 12, 18, 24, and 48 h resulted in the increase of intracellular ROS

262

levels by 7.87%, 13.28%, 8.79%, and 1.58%, respectively, compared with control

263

cells. We also used various concentrations of astaxanthin to treat HUVECs for 18 h,

264

and low levels of ROS were observed in a concentration-dependent manner (Fig. 2B).

265

Specifically, intracellular ROS level was increased by 18.2% after treatment with 10

ACS Paragon Plus Environment

Page 12 of 40

Page 13 of 40

Journal of Agricultural and Food Chemistry

266

µM astaxanthin for 18 h. In addition, it was shown that pretreatment of cells with

267

antioxidants VE or NAC significantly inhibited astaxanthin-induced ROS production.

268

Pretreatment of cells with VE reduced 93.94% of ROS that induced by astaxanthin

269

treatment, while treatment of cells with NAC not only eliminated the

270

astaxanthin-induced ROS production, but also caused a reduction of endogenous ROS

271

level by 2.06% compared with control cells (Fig. 2C). Furthermore, previously study

272

showed that treatment of BNL CL.2 cells with fucoxanthin can cause a low level of

273

increase for intracellular ROS, which leads to the activation of intracellular

274

antioxidant pathway21. Our result also indicated that fucoxanthin caused 28.33% of

275

increase of ROS compared with control cells. To determine whether the amount of

276

ROS produced upon astaxanthin treatment can cause any cellular toxicity, we

277

evaluated cell viability with MTT method. As shown in Figure 3, treatment of cells

278

with astaxanthin did not lead to cytotoxic effect, however, which increased cell

279

survival in terms of concentration and time. Either treatment of cells with 10 µM

280

astaxanthin for 18 h, or treatment with 1 or 10 µM astaxanthin for 48 h significantly

281

increased cell survival (p