HO-1 Antioxidant Pathway in Human

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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

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Journal of Agricultural and Food Chemistry

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

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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*

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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

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

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315211, China

16 17

&

These authors contributed equally to this work.

18 19

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

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

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

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ACS Paragon Plus Environment


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Abstract

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Astaxanthin is a powerful antioxidant that possesses potent protective effects against

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various human diseases and physiological disorders. However, the mechanisms

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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

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

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

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

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

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

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

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

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

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antioxidant response element-driven luciferase activity upon astaxanthin treatment.

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

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

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

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generating small amounts of ROS.

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

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

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Introduction

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

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many vascular functions including cell adhesion, inflammatory responses, regulation

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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effects7. Astaxanthin

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

is

present

in

most

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

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than

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

vitamin

E

(VE)8.

The

biological functions of astaxanthin


include


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

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

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

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astaxanthin include directly scavenging cellular ROS that are trapped inside the

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

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

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

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

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

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

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precise effective utilization ratio and actual concentration of astaxanthin that can

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

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

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

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

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reported.

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

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astaxanthin in HUVECs, and investigated the mechanisms underlying stimulation of

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the Nrf-2/ARE signaling pathway.

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Materials and Methods

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2.1 Chemicals

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


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

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

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(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).

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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

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

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

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

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

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Chemical Industries Ltd. (Osaka, Japan).

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2.2 Cell culture and astaxanthin preparation

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

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

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

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



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different concentrations for different time courses. To prepare different concentrations

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

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

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

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according to the working concentrations.

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2.3 Kinetic uptake assay

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

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

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

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

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

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

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leupeptin. The homogenates containing astaxanthin were extracted with 1 ml

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

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

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

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

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

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

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

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

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


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75% A, 25% B.

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2.4 Cell viability assay

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

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

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

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

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

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

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done in triplicate.

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2.5 Antioxidant activity assays

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

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

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

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

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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

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

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

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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

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

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2.6 Intracellular ROS assay



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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

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

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

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

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

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

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

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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

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

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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).

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2.7 Real-time quantitative PCR

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

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

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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

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

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


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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

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

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TGAGCCAGGAACAGAGTG ;

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NQO1-R:

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CAGTGGTGGATGGTTGTG,γ-GCL-R:

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β-actin-F:

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

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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

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

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normalized to that of β-actin.

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2.8 Western blot analysis

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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

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

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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,


and

β-actin-R:


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

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transferred to polyvinylidene fluoride membranes. The membranes were blocked with

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

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

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

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(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

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

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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

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

216

represent as the % of control.

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2.9 Transient transfection and luciferase reporter assays

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

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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


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

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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.

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2.10 Nrf-2 RNA interference assay

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

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

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used:

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

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

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reduced serum medium. Transient transfection of siRNAs was conducted using

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

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

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

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

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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.

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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



244

Kit.

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2.11 Statistical analysis

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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.

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3. Results

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3.1 Astaxanthin uptake

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

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

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

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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.

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3.2 Astaxanthin increased intracellular ROS but did not cause cytotoxicity

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

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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

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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).

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


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

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

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

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

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

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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

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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

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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

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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

HO-1 Antioxidant Pathway in Human

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