Cytoprotective Effect of Makgeolli Lees on Paraquat Induced Oxidative Stress in A549 Cells via Activation of NRF2 and Antioxidant Genes

Abstract

Makgeolli lees (ML) has several physiological effects such as antioxidant, antidiabetic, and anticancer properties, but its biological functions have not been determined definitively. Here, we tested whether ML has a cytoprotective effect on paraquat (PQ)-induced oxidative stress in the human lung carcinoma cell line A549. At 0.1 mg/ml ML, viability of PQ-exposed A549 cells was restored by 12.4%, 18.5%, and 48.6% after 24, 48, and 72 h, respectively. ML also reduced production of the intracellular reactive oxygen species (ROS) that were generated by PQ treatment. Further experiments revealed that ML treatment enhanced the expression and nuclear translocation of nuclear factor erythroid 2-related factor 2 (NRF2) as well as ARE-GFP reporter activity. ML treatment also effectively increased the expression of NRF2's target genes NAD(P)H dehydrogenase quinone 1 (NQO1) and heme oxygenase 1 (HO-1). Moreover, we found that expression of cytoprotective genes, including glutathione peroxidases (GPXs), superoxide dismutase (SOD1), catalase (CAT), peroxiredoxin 3 (PRDX3), and peroxiredoxin 4 (PRDX4), was greatly enhanced by treatment with ML during PQ exposure. Taken together, the data suggest that treatment of PQ-exposed A549 cells with ML ameliorates cytotoxicity through induction of NRF2 expression and its target genes HO-1, NQO1, and other antioxidant genes. Thus, ML may serve as a functional food applicable to ROS-mediated human diseases.

Introduction

Wines and their derivatives can delay tumor onset [8], protect from coronary heart disease [28], and reduce susceptibility to LDL oxidation and aggregation [12]. For instance, unlike other alcoholic beverages, makgeolli is regarded as a functional food because of its high nutritious value and a wide range of physiological functions; its consumption has markedly increased in Korea and other countries [6].

Makgeolli is a traditional and popular rice wine in Korea. It contains a relatively low concentration of alcohol (6–8%), and is rather rich in proteins, minerals, vitamins, dietary fiber, organic acids, and uncharacterized bioactive compounds [11]. Makgeolli contains the byproduct “lees,” which is formed during fermentation and aging of alcoholic drinks such as wine, cider, and beer [30]. During the brewing of makgeolli by means of nuruk or koji (a Korean fermentation starter), a massive amount of makgeolli lees (ML) is formed [20]. At present, ML is used as a cheap animal feed and fertilizer. As an industrial waste, ML is considered a cause of environmental pollution if not properly processed [2,7]. Therefore, it would be useful to find an alternate use for ML.

ML that is used in this study was extracted from makgeolli residue with ethanol at the final yield of 6.62% and contains 1.0 ± 0.1 (mean ± SD) mg/g total flavonoids, 35.0 ± 1.1 mg/g polyphenols, 394.1 ± 17.0 mg/g total sugar, and 367.8 ± 15.0 mg/g reducing sugar [19]. Choi et al. [5] demonstrated that ML still contains considerable amounts of dietary fiber, proteins, minerals, vitamins, and organic acids. It has been also reported that ML has antioxidant, antidiabetic, antiobesity, anticancer, and antihypertensive properties [6]. In addition, it is known that ML contributes to a reduction in blood cholesterol and the incidence of atherosclerosis [15,21]. Therefore, ML may be an inexpensive nutraceutical supplement for many human diseases. Despite the high nutritious value and various useful biological effects, practical applications of ML have so far been limited.

Paraquat (1,1’-dimethyl-4,4’-bipyridinium dichloride; PQ) is a well-known potent herbicide that is banned in many countries but is still used in developing countries owing to its low cost and high effectiveness. PQ is known to exert its toxic effects via oxidative stress, although the exact molecular mechanism is still unknown [9]. On the other hand, PQ is a useful inducer of reactive oxygen species (ROS); however, chronic exposure to PQ is associated with liver damage, kidney failure, and Parkinsonian lesions [4]. Moreover, poisoning with PQ in mammals and other animals mainly causes serious lung injury such as bronchial or alveolar hemorrhage, interstitial edema, hypoxemia, and leukocyte infiltration via production of intracellular ROS [3].

Nuclear factor erythroid 2–related factor 2 (NRF2) is a pivotal transcription factor in ROS-mediated pathophysiological processes and is located in the cytoplasm, where it is degraded by Kelch-like ECH-associated protein (Keap1). Under oxidative conditions caused by one of many oxidants or electrophilic stimuli, NRF2 dissociates from inhibitory complexes and is translocated to the nucleus, where it serves as a transcription factor through binding to the antioxidant response element (ARE) in the promoter regions of target genes [34]. It has been well established that transcriptional regulation by NRF2 via binding to ARE is a crucial event in modulation of oxidative stress and cytoprotection against pro-oxidant stimuli. If oxidative stress is present, NRF2 binds to the ARE in the promoter region of phase II detoxification enzymes and cytoprotective genes and activates such enzymes and antioxidant genes, including glutathione peroxidases (GPXs), NAD(P)H dehydrogenase quinone 1 (NQO1), and heme oxygenase 1 (HO-1) [26]. In addition, many antioxidant enzymes, such as superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX), and peroxiredoxin (PRDX) have been reported to inactivate ROS directly; the activation of these enzymes is regulated by NRF2 [22,33].

The aim of this study was to test whether ML has a cytoprotective effect on PQ-induced oxidative stress, and if so, whether this effect is mediated by activation of the NRF2–ARE detoxification axis.

 

Materials and Methods

Reagents

ML was obtained from Kooksoondang Brewery Co., Ltd. (Seongnam, South Korea). Paraquat dichloride (1,1’-dimethyl-4,4’-bipyridinium dichloride; PQ), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and trypan blue dye, 2’,7’-dichlorofluorescein diacetate (DCF-DA) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Hydrogen peroxide was obtained from Junsei Chemicals Co., Ltd. (Tokyo, Japan). Lipofectamine 2000 was purchased from Invitrogen (Life Technologies, Coventry, UK). DMEM/F12 (Dulbecco’s modified Eagle’s medium supplemented with nutrient mixture F-12 [Ham]) and the Opti-MEM serum-free medium were purchased from Gibco (Grand Island, NY, USA). Fetal bovine serum was purchased from Atlas Biologicals (Fort Collins, CO, USA). A penicillin-streptomycin solution was purchased from Hyclone Laboratories, Inc. (South Logan, NY, USA). Antibodies against NRF2, HO-1, and NQO1 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA), and anti-β-actin, anti–peroxiredoxin 3, anti–peroxiredoxin 4, and anti–superoxide dismutase 1 antibodies were purchased from Abcam (Cambridge, MA, USA). Horseradish peroxidase–conjugated anti–rabbit immunoglobulin G (IgG) antibody and anti–mouse IgG antibody were also obtained from Santa Cruz Biotechnology.

Cell Culture

A human lung carcinoma cell line, A549, was purchased from ATCC (Manassas, VA, USA) and grown in DMEM/F12 containing 10% (v/v) of heat-inactivated fetal bovine serum and 1% (v/v) of the penicillin-streptomycin solution (10,000 U/ml penicillin and 10,000 μg/ml streptomycin) in a humidified atmosphere (incubator) containing 5% of CO2 at 37℃

Preparation of ML

The procedure for preparation of ML was described previously by Kim et al. [19]. Briefly, Makgeolli residue was added to 10 volumes of 95% ethanol and incubated at room temperature for 24 h; then the supernatant was collected and this extraction process was repeated three times. The resulting ethanol extract was dried at 55℃ and concentrated in an evaporator under reduced pressure (Eyela Rotary evaporator N-1000; Tokyo Rikakikai Co., Ltd., Japan) to obtain a powder; the final yield was 6.62%. The ML powder was then dissolved in dimethyl sulfoxide (DMSO) and stored at -20℃ for subsequent use.

Cell Viability Assay

A549 cells were seeded in a 96-well plate at the density of 104/well and grown overnight to ~80% confluence. The cells were then incubated with various doses of ML or PQ for indicated periods. After completion of the incubation with ML, PQ, or ML with PQ, an MTT solution was added to each well of the 96-well plate at the final concentration of 0.5 mg/ml and incubated for 4 h at 37℃ After incubation with MTT, the resulting formazan crystals were dissolved in 100 μl of DMSO, and absorbance was measured on a Victor X3 multilabel reader (Perkin Elmer, Waltham, MA, USA) at 590 nm wavelength.

Measurement of Intracellular ROS Production

This analysis was conducted by a DCF-DA assay. In brief, A549 cells were seeded in a black 96-well flat-bottom plate at a density of 5 × 104/well and grown overnight to ~90% confluence. Next, the cells were incubated with 100 μl of 25 μM DCF-DA in 1× PBS for 30 min followed by two washes with 1× PBS. After that, the cells were incubated with 0.1 mg/ml ML, 0.2 mM PQ, or in combination, and fluorescence intensity of the formed DCF was measured on the Victor X3 multi plate reader, at excitation and emission wavelengths of 485 and 535 nm, respectively, for 30 min in every 5 min.

Quantitative Real-Time Reverse-Transcriptase PCR (qRT-PCR)

A549 cells were seeded in a 6-well plate at the density of 4 × 105/well and grown overnight to ~90% confluence. Then, the cells were incubated with different doses of ML, PQ, or ML with PQ for indicated periods. After that, the cells were washed with 1× cold PBS. Total RNA was isolated with an RNA extraction kit (Qiagen, Valencia, CA, USA), and the RNA quality was analyzed using a scandrop spectrophotometer (Analytik Jena AG; Jena, Germany). A total of 1 μg of RNA was used to synthesize cDNA by means of the Maxime RT PreMix kit (Intron Biotechnology, Seoul, Korea), and the reaction was run in a Veriti 96-Well Thermal Cycler (Applied Biosystems, Singapore). Quantitative real-time PCR was performed by means of the iQ SYBR Green Supermix Kit (Bio-Rad, Singapore) on a CFX96 Real-Time PCR detection system (Bio-Rad). The primer sequences are listed in Table 1. The expression data were normalized to GAPDH.

Table 1.qRT-PCR primer sets for this study.

ARE Reporter Assay

A green fluorescent protein (GFP)-based ARE reporter assay kit, including an inducible transcription factor–responsive GFP reporter, a negative control (in which GFP expression is controlled by a minimal promoter), and a positive control containing a construct constitutively expressing GFP, was purchased from Qiagen. Briefly, cells were seeded at a density of 105/well in a 24-well plate and grown overnight to ~70–80% confluence; then, the cells were transiently transfected with various ARE-GFP reporter constructs. The construct constitutively expressing GFP was used to visually verify transfection efficiency. The transfection experiments were performed using the Lipofectamine 2000 reagent. After 24 h of the transfection, the cells were incubated with PQ, ML, or PQ with ML for 24 h. Then, fluorescence microscopy was used to measure the GFP intensity. Fluorescence intensity was analyzed by ImageJ software (National Institutes of Health, Bethesda, MD, USA).

Whole-Cell Protein Extraction

A549 cells were seeded in a 60 mm cell culture dish at the density of 4 × 105/well and incubated with various doses of ML, PQ, or ML with PQ for various periods. The cells were harvested by scraping and washed with 1×PBS; then total protein was extracted with RIPA lysis buffer containing a protease inhibitor cocktail, 2 mM phenylmethylsulfonyl fluoride, and 1 mM sodium orthovanadate (Santa Cruz Biotechnology). The protein concentration was determined using the BCA protein assay kit (Pierce, Rockford, IL, USA).

Preparation of Nuclear Extracts

Nuclear extracts were prepared according to a nuclear extraction kit (Active Motif, Carlsbad, CA, USA). Briefly, media were aspirated from the cell culture dishes, and the cells were washed twice with 1× PBS containing phosphatase inhibitors. The cells were then scraped and centrifuged at 200 ×g for 5 min at 4℃. The cell pellets were gently resuspended in a 1× hypotonic solution containing a detergent, and homogenized by pipetting up and down several times, after which the samples were incubated for 15 min on ice. The cytoplasmic fraction was separated by centrifuging the homogenized pellet at 14,000 ×g for 1 min at 4℃. After that, the remaining nuclear pellet was resuspended in lysis buffer containing 10 mM dithiothreitol and protease inhibitors, incubated for 30 min on ice on a rocking platform at 150 rpm, vortexed for 30 sec at the maximal setting, and centrifuged at 14,000 ×g for 10 min at 4℃. The nuclear extracts were collected and stored at -80℃ until analysis.

Western Blot Analysis

Equal amounts of protein (50 μg) were loaded into each well of a 4–20% sodium dodecyl sulfate polyacrylamide gradient gel (Mini-PROTEAN Precast Gel; Bio-Rad) and separated by electrophoresis and then transferred to a nitrocellulose membrane. After blocking with 5% skim milk in Tris-buffered saline (TBS) containing 0.1% Tween 20, we incubated the membrane with primary antibodies overnight at 4℃ and HRP-conjugated secondary antibodies for 1 h at room temperature. Immunoreactive proteins were visualized by means of the chemiluminescent ECL assay (Advansta, Melno Park, CA, USA). Skim milk and primary and secondary antibodies were d iluted in 1× TBS c ontaining 0.1% Tween 20. All washing steps involved 1× TBS containing 0.1% Tween 20, lasted 5 min, and were repeated three times. β-Actin served as an internal control. The western blot data were quantified in the ImageJ software.

Statistical Analysis

All data were expressed as the mean ± standard deviation (SD). Significance of the differences among the treatment groups was evaluated by Student’s t test and two-way analysis of variance, followed by the post hoc Bonferroni test. Differences with a p value < 0.05 were considered statistically significant.

 

Results

ML Protects Cytotoxicity of PQ-Exposed A549 Cells

To evaluate the effect of ML on A549 cell viability, we incubated the cells with different doses of ML (from 0.1 to 1.0 mg/ml) for 24, 48, or 72 h, and then the MTT assay was performed. Our r esults r evealed that ML d id n ot r educe the viability of A549 cells but instead enhanced cell viability at the concentration of 0.1 mg/ml even after a prolonged incubation period, such as 72 h (Fig.1 A). Because ML showed a beneficial effect on cell viability, we next assessed the cytoprotective effect of ML on PQ-exposed A549 cells. Before this assay, we incubated A549 cells with PQ at different concentrations (0.1–0.7 mM) for various periods (24, 48, or 72 h) to evaluate the cytotoxicity of PQ toward A549 cells. In line with our previous studies, PQ strongly reduced the viability of A549 cells. As shown in Fig.1 B, PQ exerted its cytotoxic effects in both dose- and time-dependent manners. Accordingly, we next cotreated the cells with 0.1–1.0 mg/ml ML and 0.2 mM PQ (the standard lethal concentration) to analyze the effects of ML on the PQ-induced cytotoxicity. As shown in Fig. 1C, treatment with 0.2 mM PQ reduced cell viability by 16%, 43.5%, and 53.6% after 24, 48, and 72 h, respectively, but cotreatment with 0.1 mg/ml ML significantly attenuated the PQ-induced cytotoxicity by 12.4%, 18.5%, and 48.6% after 24, 48, and 72 h, respectively. ML was somewhat less effective at 0.25 mg/ml: this dose attenuated the PQ-induced cytotoxicity by 11.87%, 25.5%, and 37.1% after 24, 48, and 72 h, respectively. Thus, our data suggested that 0.1 and 0.25 mg/ml were effective doses of ML for protection against PQ-induced cytotoxicity. We chose 0.1 mg/ml ML for evaluation of the cytoprotection against PQ in further experiments.

Fig. 1.Protective effect of makgeolli lees (ML) on paraquat (PQ)-induced cytotoxicity toward A549 cells. (A) Effects of ML (0.1–1.0 mg/ml) on A549 cells viability. (B) Effects of different doses of PQ (0.1–0.7 mM) on A549 cell viability. All cell treatments were performed as indicated in the figure, and viability was assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The data are presented as the mean ± SD of three independent experiments. *denotes significant differences between control and treatment groups (*p < 0.05, **p < 0.01). (C) The protective effect of ML on PQ-treated A549 cells. *Statistically significant differences between groups “PQ only” and “PQ plus ML cotreatment” (*p < 0.05).

ML Ameliorates PQ-Induced Oxidative Stress by Scavenging Intracellular ROS

Next, we tested whether ML protects A549 cells from PQ-induced oxidative stress through scavenging of intracellular ROS or via other mechanisms. To quantify the intracellular ROS production, the DCF-DA assay was performed (Fig.2). Here, 100 μM H2O2 was used as a positive control (ROS inducer). The results showed that both 0.2 mM PQ and 100 μM H2O2 greatly increased intracellular ROS production by 69.54% and 41.1%, respectively. Incubation of untreated cells with 0.1 mg/ml ML alone produced no significant change in intracellular ROS, as compared with the control. In contrast, cotreatment with ML and PQsignificantly reduced the production of intracellular ROS by 11% in comparison with PQ treatment (p < 0.05). Thus, these data suggested that ML has an intracellular ROS-scavenging effect, and this action may contribute to cytoprotection under oxidative stress.

Fig. 2.Makgeolli lees (ML) reduces the paraquat (PQ)-induced intracellular production of reactive oxygen species (ROS). A549 cells were incubated with 100 μM H2O2, 0.2 mM PQ, and 0.1 mg/ml ML alone or in combination with 0.2 mM PQ for 30 min. Then, intracellular ROS levels were measured by the 2’,7’-dichlorofluorescein diacetate (DCF-DA) assay. The data are presented as the mean ± SD of three independent experiments (***p < 0.001).

ML Induces the Expression of NRF2 and Enhances AREGFP Reporter Activity

The serious toxicity of PQ toward A549 cells and the partial restoration of cell viability by ML treatment raised the question whether NRF2 is involved in this effect. It is well known that activation of NRF2 can prevent cell death caused by various environmental toxins that can induce oxidative stress [33]. To determine whether ML can induce NRF2 signaling, expression of NRF2 and its nuclear translocation were examined in A549 cells that were exposed to ML, PQ, or the combination of both. As shown in Figs.3 A–3C, treatment of A549 cells with 0.1 mg/ml ML significantly increased both mRNA (3-fold, p < 0.05) and protein levels of NRF2 (p < 0.01). The cotreatment with 0.1 mg/ml ML and 0.2 mM PQ also effectively increased the NRF2 mRNA (2.5-fold, p < 0.05) and protein levels (p < 0.01). Next, we analyzed the distribution of NRF2 in the cytoplasm and nucleus. As shown in Fig. 3D, the protein level of NRF2 was significantly increased in the nuclear fraction in A549 cells exposed to 0.1 mg/ml ML alone (p < 0.05) or in combination with PQ (p < 0.01). These data showed that ML can enhance NRF2 expression and promote its nuclear translocation.

Fig. 3.Makgeolli lees (ML) enhances the expression of NRF2 and promotes its nuclear translocation. (A) The mRNA level of NRF2 in cells incubated with paraquat (PQ), ML, or ML with PQ was measured by quantitative real-time reversetranscriptase PCR. The data are presented as the mean ± SD of at least three independent experiments (*p < 0.05). (B) The protein level of NRF2 in the cells incubated for 24 h with the indicated doses of ML. The protein levels were measured by western blotting, and band intensity was quantified in the ImageJ software. *Statistically significant differences between the control and treatment groups (*p < 0.05). (C) Protein levels of NRF2 in the cells treated with PQ, ML, or ML plus PQ for 24 h (**p < 0.01). (D) Protein levels of NRF2 in nuclear and cytosolic fractions of the cells treated with PQ, ML, or ML plus PQ for 24 h. The protein levels were quantified by western blotting, and the data were normalized to β-actin and lamin B for the cytosolic and nuclear proteins, respectively (*p < 0.05, **p < 0.01).

To dissect the regulation of antioxidant-related gene expression that is governed by activation of NRF2 during PQ-induced oxidative stress, we performed a reporter assay that is based on an ARE-containing reporter, where the ARE reporter encodes a GFP gene under the control of a minimal (m)CMV promoter and tandem repeats of the ARE transcriptional response element (Figs.4A and 4B). Transfection of the inducible ARE-GFP and constitutively active ARE-GFP vectors into the cells caused activation of the ARE reporter activity, in contrast to the non-inducible ARE-GFP control vector. On the other hand, ML alone and the combination of ML with PQ strongly enhanced the ARE-GFP reporter activity, in comparison with cells treated with PQ only. Therefore, our results suggested that ML can effectively enhance the binding of NRF2 to the ARE reporter.

Fig. 4.Makgeolli lees (ML) effectively activates an antioxidant response element (ARE)-green fluorescent protein (GFP) reporter. Cells were seeded to attain 20–30% confluence on the following day. Next, the cells were transfected with an inducible ARE-responsive GFP reporter, a construct constitutively expressing GFP, and a GFP reporter construct in which GFP expression is controlled by a minimal promoter. Then, 6 h after the transfection, the cells were incubated with ML, paraquat (PQ), or ML plus PQ. After 24 h of the incubation, fluorescent images were acquired to analyze the GFP activity. (A) Representative fluorescent images showing the effects of PQ, ML, or PQ plus ML on the binding of NRF2 to the ARE-responsive GFP reporter. (B) Quantification of fluorescence intensity. The data are presented as the mean ± SD of at least three independent experiments (**p < 0.01).

ML Activates Antioxidant-Related Gene Expression

The binding of NRF2 to the ARE in the promoter region of antioxidant genes gives rise to the expression of several antioxidant genes and phase II detoxification enzymes [26,31].

Accordingly, we evaluated the effect of ML on the expression of genes that counteract oxidative damage. As shown in Fig. 5A, we observed an increase in the protein levels of NQO1 and HO-1 during treatment with 0.1 mg/ml ML in a time-dependent manner. Similarly, our results showed that cotreatment with 0.1 mg/ml ML and 0.2 mM PQ significantly increased both mRNA and protein levels of NQO1 and HO-1 in A549 cells as compared with the cells treated with PQ only (Figs. 5B and 5C). We also analyzed the expression of some other antioxidant genes associated with cytoprotection. ML treatment in combination with PQ significantly increased the mRNA expression of GPX3 (Fig.5D), CAT, SOD1 (Fig.5E ), PRDX4, and PRDX5 (Fig.5F) as compared with the cells treated with PQ only. Moreover, the protein levels of PRDX3, PRDX4, and SOD1 were also increased by ML alone and in cotreatment with PQ, as compared with the cells treated with PQ only (Figs.5G and 5H).

Fig. 5.Makgeolli lees (ML) enhances the expression of NRF2’s target genes and other cytoprotective genes. (A) The protein level of NQO1 and HO-1 in the cells treated with 0.1 mg/ml ML for indicated periods of time. *Statistically significant differences between the control and treatment groups (*p < 0.05). (B) The mRNA level of NQO1 and HO-1 in the cells incubated with paraquat (PQ), ML, or PQ plus ML for 24 h. (C) The protein levels of NQO1 and HO-1 in the cells treated with PQ, ML, or PQ plus ML for 24 h. (D) The mRNA level of GPXs. (E) The mRNA levels of CAT and SOD1. (F) The mRNA level of PRDXs. (G) The protein levels of PRDX3, PRDX4, and SOD1 in the cells treated with PQ, ML, or PQ plus ML for 24 h. The mRNA and protein levels were analyzed by quantitative real-time reverse-transcriptase PCR and western blotting, respectively. The data are presented as the mean ± SD of at least three independent experiments. In panels B–G, the asterisks denote statistically significant differences among the cells treated with PQ only and cells treated with ML with or without PQ (*p < 0.05).

 

Discussion

In this study, we examined the cytoprotective effect of ML on PQ-induced oxidative stress in human lung alveolar A549 cells. One of the most salient findings of this study is that ML activates the NRF2–ARE regulatory axis, which is a master regulator of the mechanisms of defense against oxidative stress.

PQ-induced cellular toxicity is solely mediated by oxidative stress, judging by the production of ROS during the redox cycle. The pulmonary toxicity pattern of PQ is in many ways similar to that of several other lung toxins such as oxygen, nitrofurantoin, and bleomycin, which mainly cause severe tissue damage followed by fibrosis after systemic or respiratory administration [3]. Therefore, PQ can be used as a model compound for studies on pulmonary toxicity and fibrosis exclusively mediated by oxidative stress in lung tissue. In the present study, PQ exposure was shown to cause oxidative stress through accumulation of intracellular ROS, which caused a dramatic reduction in the viability of lung alveolar A549 cells. Our results are also supported by several reports showing the involvement of ROS in PQ-induced damage to lung cells [13,18].

Intracellular ROS levels are tightly regulated by inducible antioxidant pathways that respond to various cellular stressors and are mainly regulated by the nuclear transcription factor NRF2 [16,26]. NRF2 is a crucial player in cellular homeostasis; this protein is sequestered in the cytoplasm in the inactive form and is translocated into the nucleus in the active form to regulate an antioxidant response upon the exposure of cells to chemical or oxidative stress [29]. It has been reported that once NRF2 is activated, it relocates to the nucleus from the cytoplasm, binds to the ARE sites in target promoter regions, and regulates the expression of its downstream genes: antioxidant and detoxification-related genes. The transcriptional regulatory antioxidant response element ARE has structural and biological features that characterize its unique responsiveness to oxidative stress. Alteration of the cellular redox status after intracellular accumulation of ROS triggers the ARE-dependent transcriptional response, which is mainly governed by NRF2 [26].

In this study, we found that ML treatment induces NRF2 expression and promotes its nuclear translocation. Furthermore, our results show that ML treatment during oxidative stress markedly increases the binding of NRF2 to an ARE-GFP reporter, suggesting that the cytoprotective mechanism of ML may involve the NRF2–ARE regulatory axis.

To control and neutralize pro-oxidative stress caused by xenobiotics, higher animals possess well-orchestrated gene regulatory systems, including phase II detoxification enzymes [31]. These enzymes include NQO1, HO-1, SOD isoforms, CAT, PRDX isoforms, GPX, GST isoforms, and glutathione reductase [17,35]. Several studies have shown that moderate upregulation of HO-1 (less than 5-fold) is associated with protection from oxidative stressors [1,10]. It has been revealed that a 3-fold overexpression of HO-1 is involved in protection from heme-mediated damage [1], and 1.8-fold overexpression of HO-1 protein contributes to the protection from oxygen toxicity [10].

We also observed a significant increase in the mRNA and protein levels of HO-1 and NQO1 as a result of ML treatment during PQ-induced oxidative stress. Therefore, we can assume that moderate upregulation of HO-1 is involved in the ML-mediated cytoprotection against PQ-induced oxidative stress. The promoter region of the NQO1 gene contains AREs, which are essential for both constitutive and xenobiotic-induced NQO1 activity [27]. In contrast, basal NQO1 expression is nearly abrogated in cells and tissues of NRF2 knockout mice [23]. Thus, the upregulation of NQO1 as a result of ML treatment during PQ-induced oxidative stress is likely associated with regulation of the NRF2–ARE axis.

Emerging evidence suggests that in addition to HO-1 and NQO1, some other antioxidant genes such as GPXs, SOD1, CAT, PRDX3, and PRDX4 are activated by the NRF2–ARE regulatory system and perform crucial functions in cellular defense against oxidative stress [18,24]. GPXs are a key group of peroxide-scavenging enzymes, and inactivation of their activity leads to excess accumulation of cytotoxic peroxide and causes cellular damage [25]. Consequently, maintenance of the GPX activity is crucial for the protection of cells from oxidative stress.

PRDXs are enzymes that exert their antioxidant action by catalyzing reduction of hydrogen peroxide [24]. It is well known that SOD plays a critical role in the reduction of excess ROS in living organisms by converting the superoxide radical to hydrogen peroxide, which is subsequently converted to water by the action of CAT and GPX [18]. CAT, another regulator of antioxidant defense mechanisms, exerts its action by degrading hydrogen peroxide and prevents formation of the hydroxyl radical by the Fenton reaction. One report showed that overexpression of CAT makes cells more resistant to the toxic effects of hydrogen peroxide and other types of oxidant-mediated injury [32]. Conversely, mice deficient in CAT are more susceptible to hyperoxia-induced lung injury [14]. Therefore, in our study, the induction of these genes or enzymes either at the transcriptional or translational level by ML alone or during cotreatment with PQ must be strongly associated with the protection of A549 cells from PQ-induced oxidative stress.

Collectively, our results demonstrated that ML enhances expression of NRF2, promotes its nuclear translocation, and facilitates its binding to an ARE in the promoter region of the target genes, HO-1 and NQO. Additionally, ML can effectively upregulate several cytoprotective genes such as SOD1, PRDX3, and PRDX4, which contribute to protection of the lung cell line A549 from PQ-induced toxicity by decreasing the intracellular ROS level. Thus, the findings of this study provide new efficacies of ML usage that might lead to the development of a promising nutraceutical agent for the prevention or treatment of oxidative-stress-related diseases.