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Cytoprotective Effect of Makgeolli Lees on Paraquat Induced Oxidative Stress

in A549 Cells via Activation of NRF2 and Antioxidant Genes

Article  in  Journal of Microbiology and Biotechnology · November 2015

DOI: 10.4014/jmb.1510.10093

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J. Microbiol. Biotechnol. (2016), 26(2), 277–286
http://dx.doi.org/10.4014/jmb.1510.10093 Research Article

Review

Cytoprotective Effect of Makgeolli Lees on Paraquat Induced Oxidative

Stress in A549 Cells via Activation of NRF2 and Antioxidant Genes
Miso Jeon1, Naimur Rahman1,2, and Yong-Sik Kim1,2*
1
Department of Microbiology, College of Medicine, Soonchunhyang University, Cheonan 31151, Republic of Korea
2
Institute of Tissue Engineering, College of Medicine, Soonchunhyang University, Cheonan 31151, Republic of Korea

Received: October 29, 2015

Revised: November 11, 2015
Accepted: November 12, 2015
First published online
November 24, 2015
*Corresponding author
Phone: +82-41-570-2413;
Fax: +82-41-575-2412;
E-mail: yongsikkim@sch.ac.kr
pISSN 1017-7825, eISSN 1738-8872
Copyright © 2016 by
The Korean Society for Microbiology
and Biotechnology
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.
Keywords: Makgeolli lees, paraquat, oxydative stress, NRF2, antioxidant response element, A549

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,

February 2016 ⎪ Vol. 26 ⎪ No. 2

278 Jeon et al.
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.
J. Microbiol. Biotechnol.
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°C.
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 55oC 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 -20oC 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°C.
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.
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
Table 1. qRT-PCR primer sets for this study.
Gene Sequences
Nrf2 (F) 5’-GCGACGGAAAGAGTATGAC-3’
Nrf2 (R) 5’-GTTGGCAGATCCACTGGTTT-3’
HO-1 (F) 5’-GCAACCCGACAGCATGC-3’
HO-1 (R) 5’-TGCGGTCGAGCTCTTCTG-3’
NQO1(F) 5’-CGCAGACCTTGTGATATTCCAG-3’
NQO1 (R) 5’-CGTTTCTTCCATCCTTCCAGG-3’
GAPDH (F) 5’-TCCCATCACCATCTTCCA-3’
GAPDH (R) 5’-CATCACGCCACAGTTTCC-3’
ML Protects PQ-Exposed A549 Cells 279
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 4oC. 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 4oC.
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 4oC. The nuclear extracts were collected
Amplicon sizes (bp)
99
245
249
380
February 2016 ⎪ Vol. 26 ⎪ No. 2
280 Jeon et al.

and stored at -80oC until analysis. value < 0.05 were considered statistically significant.

Western Blot Analysis Results

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°C 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 diluted in 1× TBS containing 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
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 results revealed that ML did not reduce
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. 1A).
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. 1B, 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

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).

J. Microbiol. Biotechnol.

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.
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 PQ
significantly 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.
ML Induces the Expression of NRF2 and Enhances ARE-
GFP 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. 3A–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
ML Protects PQ-Exposed A549 Cells 281
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).
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.
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.
ML Activates Antioxidant-Related Gene Expression
The binding of NRF2 to the ARE in the promoter region
February 2016 ⎪ Vol. 26 ⎪ No. 2
282 Jeon et al.

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 reverse-
transcriptase 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).

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).

J. Microbiol. Biotechnol.
 ML Protects PQ-Exposed A549 Cells 283

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).

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).
Discussion
In this study, we examined the cytoprotective effect of

February 2016 ⎪ Vol. 26 ⎪ No. 2

284 Jeon et al.
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
J. Microbiol. Biotechnol.
[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.
Acknowledgments
This study was supported by a research fund of
Soonchunhyang University and by the Basic Science
Research Program through the National Research Foundation
of Korea (NRF) funded by the Ministry of Education
(2015R1A6A1A03032522).
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