Article pubs.acs.org/JAFC
Sulforaphane Attenuates Homocysteine-Induced Endoplasmic Reticulum Stress through Nrf-2-Driven Enzymes in Immortalized Human Hepatocytes Canxia He,†,∥ Baolong Li,‡,∥ Wei Song,† Zhongqing Ding,† Shuran Wang,*,§ and Yujuan Shan*,† †
School of Food Science and Engineering, Harbin Institute of Technology, No. 73 Huanghe Road, Harbin 150090, China Center of Safety Evaluation of Drugs, Heilongjiang University of Chinese Medicine, No. 24 Heping Road, Harbin 150040, China § School of Public Health, Jilin Medical College, No. 5 Jilin Street, Jilin 132013, China ‡
ABSTRACT: In the present study, we investigated the potential efficacy of cruciferous vegetable-derived sulforaphane (SFN) in improving homocysteine (HCY)-stressed cells. After human hepatocyte line HHL-5 was preincubated with SFN and subsequently with 10 mmol/L HCY, SFN improved the pathologic changes which are caused by HCY, including cell morphological abnormality, endoplasmic reticulum (ER) swelling, excessive generation of reactive oxygen species (ROS), the increased malondialdehyde (MDA) levels, as well as the increased activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST). Phase II enzymes, thioredoxin reductase-1 (TrxR-1) and NAD(P)H:quinone oxidoreductase 1 (NQO1), were involved in the protective effect of SFN against injury by HCY. The ER stress-specific proteins, such as glucoseregulated protein-78 (GRP78) and protein kinase RNA (PKR)-like ER kinase (PERK), were strikingly abolished by SFN. Furthermore, Nrf-2 translocation was enhanced by SFN, which lead to the induction of TrxR-1and NQO1. KEYWORDS: sulforaphane, endoplasmic reticulum stress, phase II enzyme, oxidative stress
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INTRODUCTION Homocysteine (HCY) is a sulfhydryl amino acid derived from dietary methionine as a byproduct of cellular methylation reactions. HCY is perhaps the most reactive amino acid in the pathology of hyper-homocysteinemia (HHCY) through transmethylation to methionine or trans-sulfuration to cysteine, as well as conversion to other metabolites including HCY-thiolactone, CY-containing disulfides, homocysteic acid or S-nitroso-HCY.1 HHCY, a condition with elevated blood HCY levels (>15 μmol/L), has been widely recognized as an independent risk factor for cardiovascular diseases.2,3 HHCY is also a risk factor for neurodegenerative disorders, such as dementia and Alzheimer’s disease.4 Moreover, HHCY and the following endoplasmic reticulum (ER) stress induced by alcohol have emerged as a novel mechanism for alcoholic liver disease.5,6 Patients with liver cirrhosis or chronic alcohol-induced liver injury were accompanied by elevated levels of HCY.7 Also in Sprague−Dawley rats, HCY-induced hepatic lipid accumulation is mediated by SREBP2 and CREB, two downstream ER-reactive proteins mediating ER stress.8,9 The highly reactive thiol group of HCY may be responsible for the change of redox state, which leads to the accumulation of various misfolding proteins and subsequently to ER stress. At present, ER stress has been closely related to a variety of diseases such as liver or neurodegenerative disorders, and so on. Up to now, limited treatments were publicly accepted to attenuate the levels of HCY in serum or plasma. Although folic acid supplementation combined with multivitamins has been widely adopted, some drawbacks still prevail. For instance, the potential of folate to increase methylation could have profound effects on cellular function and pro-atherogenic molecule expression.10 In addition, a systematic review which included © 2014 American Chemical Society
12 randomized clinical trials involving 47,429 participants found no evidence to suggest that HCY-lowering interventions with folic acid-based multivitamin therapy at any dosage prevent cardiovascular events.11 Furthermore, a few studies demonstrated that the improved endothelial function in coronary artery diseases is largely independent of the decreased HCY.12 All these results suggested that lowering HCY without targeting its potential mechanism is not effective to overcome HHCY-related disorders. Nowadays, plant-based antioxidants such as carotenoids, polyphenols, and isothiocyanates (ITC) have been widely accepted by human populations for enhancing endogenous cellular defense.13 Sulforaphane (SFN), an indirect antioxidant exclusively existing in cruciferous vegetables such as broccoli and brussel sprouts, has attracted a lot of attention due to its remarkable ability to activate multiple endogenous enzymes via the up-regulation of nuclear factor erythroid-2-related factor 2 (Nrf-2).14 Numerous evidence suggested that a variety of beneficial functions owned by SFN contributed to the induction of Nrf-2 driven proteins. For instance, the aortic protection from diabetes by SFN was associated with the up-regulation of Nrf-2 and its downstream antioxidants.15 Also, it has been proposed that the cardiovascular protective effects of broccoli sprouts may be due to the induction of phase II proteins by SFN.16,17 In the aortic smooth muscle cells, SFN at physiologically relevant concentrations dramatically enhanced these cells’ resistance to oxidative and electrophilic stress through inducing a series of Received: Revised: Accepted: Published: 7477
April 26, 2014 June 26, 2014 June 27, 2014 June 27, 2014 dx.doi.org/10.1021/jf501944u | J. Agric. Food Chem. 2014, 62, 7477−7485
Journal of Agricultural and Food Chemistry
Article
Figure 1. Dual-growth control and morphological improvement in HHL-5 cells by SFN. (A) Cells (104/well) were seeded in a 96-well plate and incubated with 1−140 μmol/L SFN for 24 and 48 h. (B) HHL-5 cells were preincubated with 1, 5, 10, and 20 μmol/L SFN for 24 h and then incubated with 10 mmol/L HCY for 24 h. The absorbance was measured by a microplate reader at 490 nm. Data are presented as the mean ± SD (n = 6). (C) Cells were grown in DMEM/F12 medium, 10 mmol/L HCY, or 10 μmol/L SFN plus 10 mmol/L HCY for 24 h. Cell morphological changes were observed under an inverted microscope (×100). Bars with no letters in common are significantly different (p < 0.05). 100 U/mL penicillin, and 100 μg/mL streptomycin in a humidified atmosphere of 5% CO2 at 37 °C. SFN was dissolved in dimethyl sulfoxide (DMSO), with a final concentration in the medium was ⩽0.1% (v/v).20 DL-HCY was freshly prepared in serum-free medium and sterilized by 0.22 μm filtration. The range of DL-HCY concentrations was selected on the basis of other research21 and our preliminary tests. HHL-5 cells were pretreated with 1−20 μmol/L SFN for 24 h (for dose course) or 20 μmol/L SFN for 6−48 h (for time course) followed by 10 mmol/L HCY for 6 h. Cell Survival Assay. MTT assay was performed according to the manufacturer’s protocol (Sigma) and our previous method.22 Briefly, cells (1 × 104/well) were seeded in 96-well plates and grown to 80% confluence. Various concentrations of SFN or HCY were exposed to HHL-5 cells or vehicle for the desired time. Afterward, MTT (5 mg/mL in PBS) was added to each well and incubated at 37 °C for 4 h. Formazan crystals formed were dissolved by DMSO, and its optical density was measured with a microplate reader (Bio-Tek Instruments, Inc., USA) at 490 nm. Cell viability was calculated from the optical density readings, when vehicle-treated cells were taken as 100% viable. The experiment was repeated three times, and the IC50 value was calculated with the SPSS13.0 software. Morphological Changes. HHL-5 cells were seeded in a 6-well plate and attached overnight. Then, the cells were grown in DMEM/ F12 medium, 10 mmol/L HCY, or 10 μmol/L SFN with 10 mmol/L HCY for 24 h. Cell morphological changes were observed under an inverted microscope and photographed (Olympus Optical Co., Ltd., Japan). Determination of ROS Generation. The membrane-permeable probe 2′,7′-dichlorofluorescin diacetate (H2DCFDA) enters the cells and produces fluorescence after intracellular oxidation by ROS such as hydrogen peroxide (H2O2) and hydroxyl radical. Intracellular oxidant stress was monitored by changes in fluorescence intensity resulting from intracellular probe oxidation.23 Following treatment, cells were incubated with 5 μmol/L DCFH-DA at 37 °C for 30 min in the dark and then rinsed with PBS three times. Intracellular ROS was imaged using an Olympus X70 fluorescence microscope with ultraviolet (UV)
antioxidants and phase II enzymes.18 Accordingly, impaired Nrf2 signaling heightens ER stress response in the lungs of patients with chronic obstructive pulmonary disease.19 Given the potent antioxidant effects of SFN, it is of keen interest to determine whether SFN can protect against the HCY-induced injuries of which HCY is responsible for the mechanisms of numerous disorders. Our present results demonstrate that SFN improves both the ER swelling and reactive oxygen species (ROS) production by HCY. The underlining mechanism may be involved with enhancing phase II enzymes and reversing ER stress, all of which may be related to the activation of Nrf-2.
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MATERIALS AND METHODS
Chemicals and Reagents. Sulforaphane (1-isothiocyanato-4(methylsulfinyl) butane, SFN, purity ⩾98%) was purchased from LKT laboratories (St. Paul, MN), dissolved in dimethyl sulfoxide (DMSO) and stored as a stock solution of 100 mmol/L at −20 °C.20 DL-homocysteine (purity ⩾ 95%), 2′,7′-dichlorofluorescin diacetate (H2DCFDA), and 3-[4,5-dimethylthiazol-2-yl]-2, 5-diphenyl tetrazolium bromide (MTT) were obtained from Sigma (St. Louis, MO). Dulbecco’s modified Eagle’s medium−Ham’s F-12 medium (DMEM-F12) and fetal bovine serum (FBS) were from Hyclone (Logan, UT). Antibodies against glucose-regulated protein-78 (GRP78), protein kinase RNA (PKR)-like ER kinase (PERK), phospho (p)-PERK, thioredoxin reductase-1 (TrxR-1), NAD(P)H:quinone oxidoreductase 1 (NQO1), Nrf-2, LaminB1, and β-actin were from Santa Cruz Biotechnology (Santa, CA). Other chemicals and solvents used were of the highest analytic grade. The nuclear extraction kit was purchased from Chemicon International (UK). Cell Culture and Treatments. Immortalized human hepatocytes (defined as HHL-5) were kindly supplied by Professor Duncan McGeoch, Medical Research Council (MRC) Virology Unit, UK. Immortalized human hepatocytes HHL-5 were maintained in DMEM/ F12 medium supplemented with 10% FBS, 2 mmol/L L-glutamine, 7478
dx.doi.org/10.1021/jf501944u | J. Agric. Food Chem. 2014, 62, 7477−7485
Journal of Agricultural and Food Chemistry
Article
Preparation of Cell Lysates and Western Blotting. After various treatments, cells were washed twice with ice-cold PBS and subjected to whole cell lysis buffer [50 mmol/L Tris-HCl (pH 7.4), 150 mmol/L NaCl, 2 mmol/L EDTA, 10% glycerol, 1% Nonidet P-40 containing 0.1 mol/L PMSF, and 1× protease inhibitor cocktail (Sigma)] for 30 min on ice. After centrifugation at 16,000g for 10 min at 4 °C, the supernatants were collected for Western blot analysis. Equal amounts of protein (40−60 μg) were separated on 8% to 10% SDS−PAGE and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Bedford, MA). The membrane was blocked with 5% nonfat milk for 30 min at room temperature and were then incubated at 4 °C overnight with the following primary antibodies: NQO1, TrxR-1, GRP78, PERK, P-PERK, Nrf-2, LaminB1, and β-actin. The membrane was then incubated with the secondary alkaline phosphatase-conjugated IgG and detected with Western Blue Stabilized Substrate for alkaline phosphatase (Promega). The relative densities of the individual bands were analyzed densitometrically using the ChemiImager 4000 instrument (Alpha Innotech, USA). Nuclear Extraction. Nuclear protein extraction was regularly performed as previously described.28 After SFN treatments, cells were harvested and washed with phosphate-buffered saline (PBS). Nuclear and cytoplasmic extractions were isolated using Chemicon Nuclear Extraction Kit. Then, each pellet was suspended in cell lysis buffer containing 1% Nonidet P-40, 50 mmol/L Tris (pH 7.4), 150 mM NaCl, 1 mmol/L PMSF, and 1× protease inhibitor cocktail, then incubated on ice for 30 min. Lysates were centrifuged at 10,000g for 10 min at 4 °C, and the supernatant was used for Western blot analysis. Statistical Analysis. All results shown are representative of at least three independent experiments. Data are presented as the mean ± SD. Statistical analysis was performed by one-way ANOVA, followed by Dunnett’s test and SNK test using the SPSS 13.0 software. Probability values of
