Article pubs.acs.org/JAFC
Monascin Attenuates Oxidative Stress-Mediated Lung Inflammation via Peroxisome Proliferator-Activated Receptor-Gamma (PPAR-γ) and Nuclear Factor-Erythroid 2 Related Factor 2 (Nrf-2) Modulation Wei-Hsuan Hsu, Bao-Hong Lee, and Tzu-Ming Pan* Department of Biochemical Science and Technology, College of Life Science, National Taiwan University, No. 1, Sec. 4, Roosevelt Road, Taipei 10617, Taiwan S Supporting Information *
ABSTRACT: We speculated that peroxisome proliferator-activated receptor (PPAR)-γ agonists may modulate the oxidative stress pathway to ameliorate the development of airway inflammation. The effect of Monascus-fermented metabolite monascin (MS) and rosiglitazone (Rosi) on oxidative stress-induced lung inflammation was evaluated. Luciferase assay and DNA binding activity assay were used to point out that MS may be a novel PPAR-γ agonist and nuclear factor-erythroid 2 related factor 2 (Nrf2) activator. We used hydrogen peroxide (H2O2) to induce inflammation in lung epithelial cells. MS and Rosi prevented H2O2induced ROS generation in A549 epithelial cells through PPAR-γ translocation, avoiding inflammatory mediator expression via inhibiting nuclear factor (NF)-κB translocation. The regulatory ability of MS was abolished by siRNA against PPAR-γ. MS also elevated antioxidant enzyme expression via Nrf-2 activation. Both PPAR-γ and Nrf-2 might have benefits against lung inflammation. MS regulated PPAR-γ and Nrf-2 to improve lung oxidative inflammation. KEYWORDS: hydrogen peroxide, lung inflammation, monascin, Nrf-2, oxidative stress, PPAR-γ
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INTRODUCTION Airway inflammation of the lung is a main feature of respiratory diseases that involves many different types of inflammatory and epithelial cells of the respiratory tract. The airway epithelial cells, which are the point of first contact between the stimuli and the respiratory system, play a key role in the pathogenous process by releasing inflammatory mediators.1 Oxidative stress has been shown to play an important role between inflammation and lung damage.2−4 The lung tissue has an antioxidant system to protect itself against exposure to endogenous and/or exogenous oxidants;5 however, excessively produced reactive oxygen species (ROS) can still cause inflammation in the lungs. Exposure to different stimuli causes the production of ROS in the lung epithelial cells and generates inflammatory cytokines and/or chemokines; lung epithelial cells also express adhesion molecules on the cell surface and result in lung inflammation, including narrowing of airways, secretion of large amounts of mucus, and infiltration of inflammatory cells.4,6,7 The lungs combat oxidative injury in part by the transcription factor nuclear factor-erythroid 2 related factor 2 (Nrf2). After translocation to the nucleus, this factor binds to antioxidant response elements (AREs), which locate in promoter regions of relevant genes, thereby transactivating several dozen antioxidant and cellular defense genes.8 One of the major ligand-activated transcription factors up-regulated by Nrf-2 is peroxisome proliferator-activated receptor-γ (PPARγ),9 which is a member of the nuclear hormone receptor family that not only is prominently involved in adipogenesis and metabolic regulation but also exerts pleiotropic anti-inflammatory effects in the lung and other organs.10 PPAR-γ enhances the transcription of anti-inflammatory and antioxidant genes, © 2014 American Chemical Society
several of which are also up-regulated by Nrf-2, and PPAR-γ may in turn regulate the expression of Nrf-2.11 Cho and colleagues have showed that PPAR-γ agonist reduced oxidantinduced lung injury in wild-type but not Nrf-2-deficient mice.12 Fermentation products of Monascus have been used as dietary colorants for thousands of years. Monascus-fermented rice, known as red mold rice (RMR), is a common food material and traditional health remedy in Asian countries. Several chemical compounds of RMR have been purified and identified, such as monacolins, pigments, γ-aminobutyric acid (GABA), and dimerumic acid. Yellow pigment monascin (MS), isolated from Monascus-fermented products, is a secondary metabolite with an azaphilonoid structure and has been reported to have cytotoxic and anti-inflammatory activities.13,14 MS also reduces tumor necrosis factor (TNF)-α, resulting in endothelial adhesiveness as well as down-regulating generation of intracellular ROS, activation of nuclear factor (NF)-κB, and expression of vascular cell adhesion molecule-1 (VCAM-1) in aortic endothelial cells.15 Our research group previously found that MS has antiobesity potential via prevention of adipogenesis and modulation of lipolysis activity,16 and MS also has hypolipidemic and high-density lipoprotein cholesterol-raising abilities.17 We showed that MS negatively regulates the expression of endothelial adhesion molecules and positively elevates expression of endothelial NO synthase (eNOS) in human umbilical vein endothelial cells (HUVECs) induced by TNF-α.18 With its antioxidant and anti-inflammatory properReceived: Revised: Accepted: Published: 5337
April 7, 2014 May 12, 2014 May 27, 2014 May 27, 2014 dx.doi.org/10.1021/jf501373a | J. Agric. Food Chem. 2014, 62, 5337−5344
Journal of Agricultural and Food Chemistry
Article
Figure 1. MS attenuated H2O2-induced lung oxidative damage in airway epithelial cells. A549 cells were treated with H2O2 and different concentrations of MS for 12 h. (A) Effect of H2O2 and/or MS on intracellular ROS levels in A549 cells. MS significantly attenuated H2O2-mediated ROS generation. (B−D) Western blot analysis of protein extracts obtained from A549 cells. Cells were lysed and immunoblotted with antibodies against (B) phosphorylated-PKC, (C) phosphorylated-JNK, (D) cytosol NF-κB, and nuclear NF-κB. Each protein was normalized with total (B) PKC, (C) JNK, (D) GAPDH, and Lamin B. MS reduced PKC and JNK phosphorylation as well as NF-κB translocation caused by H2O2. (E) Realtime PCR analysis of adhesion molecules expression in cells. GAPDH was used as a control. MS decreased ICAM-1, VCAM-1, and eotaxin mRNA expression induced by H2O2. Results are expressed as the mean ± SD (n = 3). (∗) Significantly different from the H2O2 treatment alone group at p < 0.05. H2O2, hydrogen peroxide (400 μM); MS, monascin; NAC, N-acetylcysteine (1 mM); ROS, reactive oxygen species; PKC, protein kinase C; JNK, c-Jun NH2-terminal kinase; NF-κB, nuclear factor-κB; ICAM-1, intercellular adhesion molecule-1; VCAM-1, vascular cell adhesion molecule-1. (BCRC 60430; Food Industry Research and Development Institute, Hsinchu, Taiwan). Cells were maintained in Ham’s F-12K medium supplemented with 10% FBS, streptomycin (100 mg/mL), and penicillin (100 U/mL) in a 5% CO2 incubator at 37 °C. All experiments were performed when cells were 80−90% confluent. Determination of ROS Level. The assay for oxidative stress was monitored by the measurement of ROS.10 A549 cells were collected and stained with 10 μM DCFH-DA prior to incubation for 30 min at 37 °C. ROS level was analyzed by FACS (Becton-Dickinson Lmmunocytometery Systems, San Jose, CA, USA). Immunoblot Analysis. A549 cells were lysed, and the cell lysates were centrifuged (10000g for 10 min) to recover the supernatant. The supernatant was taken as the cell extract. The protein concentration in the cell extract was 50 μg and was determined using a Bio-Rad protein assay kit. The samples were subjected to 10% SDS−polyacrylamide gel electrophoresis (PAGE). The protein spots were electrotransferred to a polyvinyldiene difluoride (PVDF) membrane. The membrane was incubated with block buffer and then probed with primary antibody overnight at 4 °C. The membrane was washed and shaken in a solution of HRP-linked anti-rabbit IgG secondary antibody. The expressions of proteins were detected by enhanced chemiluminescent (ECL) reagent (Millipore, Billerica, MA, USA). Nuclear and Cytosol Protein Extraction. Cells were collected for nuclear protein extraction according to the fractionation kit protocol supplied from BioVision (Mountain View, CA, USA). Briefly,
ties, MS may have potential to improve lung inflammation by regulating the oxidative stress pathway.
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MATERIALS AND METHODS
Chemicals and Reagents. 2,7-Dichlorodihydrofluorescein diacetate (DCFH-DA), D-glucose, hydrogen peroxide (H2O2), Nacetylcysteine (NAC), tert-butylhydroquinone (tBHQ), and trypsin were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Fetal bovine serum (FBS) was purchased from Life Technologies (Carlsbad, CA, USA). Dimethyl sulfoxide (DMSO) was purchased from Wako Pure Chemical Industries (Saitama, Japan). Ham’s F-12K medium was purchased from HyClone Laboratories (Logan, UT, USA). Sodium dodecyl sulfate (SDS) was purchased from Merck (Darmstadt, Germany). The Bio-Rad protein assay dye was from Bio-Rad Laboratories (Hercules, CA, USA). PPAR-γ antagonist GW9662 was purchased from Cayman Chemical Co. (Ann Arbor, MI, USA). Monascin Purification. MS (Supporting Information Supplemental Figure 1) was obtained as previously described,14 which was identified by nuclear magnetic resonance (NMR, Varian Gemini, 200 MHz, FT-NMR, Varian Inc., Palo Alto, CA, USA) and electrospray ionization-mass spectrometry (ESI-MS, FinniganMAT LCQ, Thermo Electron Co., Waltham, MA, USA) analysis. Cell Culture. Human lung adenocarcinoma epithelial cell line A549 was obtained from the Bioresource Collection and Research Center 5338
dx.doi.org/10.1021/jf501373a | J. Agric. Food Chem. 2014, 62, 5337−5344
Journal of Agricultural and Food Chemistry
Article
Figure 2. MS ameliorated lung oxidative and inflammatory injury through PPAR-γ regulation in airway epithelial cells. A549 cells were treated with H2O2 and different concentrations of MS for 12 h. (A, B) Western blot analysis of protein extracts obtained from A549 cells. Each protein was normalized with GAPDH or Lamin B. MS elevated PPAR-γ expression and promoted PPAR-γ translocation from cytoplasm to the nucleus. (C) Intracellular ROS levels in A549 cells. MS and Rosi significantly reduced H2O2-mediated ROS generation, whereas ROS level was restored by siPPAR-γ. (D) Real-time PCR analysis of adhesion molecules expression in cells. GAPDH was used as a control. The inhibitory effect of MS on adhesion molecules expression was abolished by siPPAR-γ. Results are expressed as the mean ± SD (n = 3). (∗) Significantly different from the H2O2 treatment alone group at p < 0.05. H2O2, hydrogen peroxide (400 μM); MS, monascin (20 μM); Rosi, rosiglitazone (20 μM); siPPAR-γ, siRNA against PPAR-γ (50 nM); ICAM-1, intercellular adhesion molecule-1; VCAM-1, vascular cell adhesion molecule-1. cells were harvested and added to 200 μL of cytosol extraction buffer containing dithiothreitol (DTT) and protease inhibitors. After vortexing and 10 min of incubation on ice, the extracts were centrifuged at 16000g for 5 min at 4 °C, and the supernatant was removed to separate the cytoplasmic fraction from nuclei. The nuclei pellets were then further added to 100 μL of nuclei extraction buffer, vortexed, and set on ice every 10 min for 40 min. After centrifuging, the supernatant nuclear extracts were stored at −80 °C for future use. The protein concentration of the cell extract was determined using a Bio-Rad protein assay kit. RNA Preparation and Real-Time PCR. Total RNA was isolated using Trizol (Life Technologies) according to the manufacturer’s instructions. cDNA from 3 μg of RNA was generated using a SuperScript III First-Strand Synthesis System for RT-PCR (Life Technologies) according to the manufacturer’s instructions. The reverse transcription product was diluted in water, and a volume corresponding to 30 ng of original RNA was used for real-time PCR. Real-time PCR amplification and detection were performed using the SYBR Green qPCR SuperMix-UDG with ROX (Life Technologies) in a fluorescence thermal cycler (StepOne Real-Time PCR System, Life Technologies) according to the manufacturer’s protocol. All amplifications were conducted within the linear range of the assay, normalized to respective GAPDH levels using SPSS version 17.0 (SPSS Institute, Inc., Chicago, IL, USA). Transfection of Cells with Small Interfering RNA (siRNA). PPAR-γ and Nrf-2 siRNA were purchased from Santa Cruz Biotechnology Inc. (Burlingame, CA, USA), and specific siRNA was transfected into A549 cells by lipofectamine RNAiMAX reagent (Invitrogen, Carlsbad, CA, USA). PPAR-γ and Nrf-2 Luciferase Assay. The specific PPAR-γ reporter assay was carried out in A549 cells according to the previous study.19 The plasmid was designed by Blossom Biotechnologies Inc.
(Taipei, Taiwan). In addition, we used a DNA fragment containing three copies of the ARE4 elements from the GCL gene (5′-CCC GTG ACT CAG CGC TCC GTG ACT CAG CGC TCC GTG ACT CAG CGC T-3′), which was subcloned into a pGL3-promoter vector to construct pGL3-ARE4-Luc. A549 cells were transiently transfected with a DNA mixture containing 2 μg of pGL3-ARE4-Luc and 0.5 μg of control plasmid pRL-TK (Promega, Madison, WI, USA) using the lipofectamine-2000 transfection reagent in serum-free medium (Invitrogen). After treatment with samples for various times, luciferase activity was conducted utilizing the Dual-Luciferase Reporter Assay System (Promega). Luciferase activity of pRL-TK was used to normalize the transfection efficiency. PPAR-γ DNA Binding Assay. Nuclear extracts were prepared from A549 cells by means of a commercially available kit and performed according to the instructions of the manufacturer. The binding activities of the PPAR-γ were measured by using specific transcription factor assay kits (Panomics, Redwood City, CA, USA). Oligonucleotides encoding for the human PPAR-γ consensus binding site were bound to microtiter plates. The binding of activated PPAR-γheterodimers, in the prepared nuclear extracts, to the immobilized DNA was revealed by incubation with PPAR-γ specific antibody using ELISA technology. Optical density was determined at 450 nm. Statistical Analysis. The statistical significance was determined by one-way analysis of variance (ANOVA) using the general linear model procedure of SPSS software (SPSS Institute, Inc.), followed by ANOVA with Duncan’s test. The results were considered to be statistically significant if the p value was
