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R E S E A R C H A R T I C L E Open Access
Fucoxanthin, the constituent of Laminaria
japonica, triggers AMPK-mediated
cytoprotection and autophagy in
hepatocytes under oxidative stress
Eun Jeong Jang
1†
, Sang Chan Kim
1†
, Ju-Hee Lee
1,2
, Jong Rok Lee
1
, Il Kon Kim
3
, Su Youn Baek
1*
and Young Woo Kim
1,3*
Abstract
Background: Laminaria japonica has frequently been used as a food supplement and drug in traditional oriental
medicine. Among the major active constituents responsible for the bioactivities of L. japonica, fucoxanthin (FX) has
been considered as a potential antioxidant. This study was conducted to examine the effects of L. japonica extract (LJE)
or FX against oxidative stress on hepatocytes and to elucidate the overall their cellular mechanisms of the effects.
Methods: We constructed an in vitro model with the treatment of arachidonic acid (AA) + iron in HepG2 cells to
stimulate the oxidative damage. The cells were pre-treated with LJE or FX for 1 h, and incubated with AA + iron. The
effect on oxidative damage and cellular mechanisms of LJE or FX were assessed by cytological examination and several
biochemical assays under conditions with or without kinase inhibitiors.
Results: LJE or FX pretreatment effectively blocked the pathological changes caused by AA + iron treatment, such as cell
death, altered expression of apoptosis-related proteins such as procaspase-3 and poly (ADP-ribose) polymerase, and
mitochondria dysfunction. Moreover, FX induced AMPK activation and AMPK inhibitor, compound C, partially reduced the
protective effect of FX on mitochondria dysfunction. Consistent with AMPK activation, FX increased the protein levels of
autophagic markers (LC3II and beclin-1) and the number of acridine orange stained cells, and decreased the
phosphorylation of mTOR and simultaneously increased the phosphorylation of ULK1. And the inhibition of autophagy by
3-methylanine or bafilomycin A1 partially inhibited the protective effect of FX on mitochondria dysfunction.
Conclusion: These findings suggest that FX have the function of being a hepatic protectant against oxidative damages
through the AMPK pathway for the control of autophagy.
Keywords: Fucoxanthin, Oxidative stress, AMPK, Autophagy, AMPK/mTOR/ULK-1 pathway
Background
Laminaria japonica, one of the most well known brown
seaweeds, is referred to as “Dashima”in Korean,
“Kombu”in Japanese, and “Haidai”in Chinese. L. japonica
is widely used as a food supplement, as well as a drug for
treatment of various diseases [1]. L. japonica has abundant
bioactive components, including polyphenols, pigments,
polysaccharides, minerals and amino acids [1]. Among
bioactive compounds in L. japonica, fucoxanthin (FX), a
marine carotenoid, has remarkable biological properties,
including anti-cancer, obesity and inflammation [1–4]. FX
has attracted much attention as a potential antioxidant
given its unique chemical structure (Fig. 2a), which in-
cludes an allenic bond, epoxide group, and hydroxyl group
[5]. Liu et al. (2011) reported that FX significantly recov-
ered cell proliferation and increased the levels of glutathi-
one and decreased intracellular reactive oxygen species
(ROS) induced by ferric nitrilotriacetate [6]. In recently,
Seo et al. (2016) showed that FX inhibited lipid
* Correspondence: rhodeus@dhu.ac.kr;ywkim@dhu.ac.kr
†
Equal contributors
1
College of Oriental Medicine, Daegu Haany University, Gyeongsan,
Gyeongsangbuk-do 38610, South Korea
Full list of author information is available at the end of the article
© The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and
reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to
the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver
(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
Jang et al. BMC Complementary and Alternative Medicine (2018) 18:97
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accumulation and ROS formation by controlling adipo-
genic and lipogenic factors and ROS-regulating enzymes
during differentiation in 3 T3-L1 adipocytes [7]. It is indi-
cated that FX can effectively protect against hepatotoxicity
by reducing intracellular ROS, associated with the antioxi-
dant effects of FX.
ROS is a group of molecules including superoxide
anion, hydroxyl radical and hydrogen peroxide, mainly
produced in the mitochondria [8]. However, excess ROS
can be involved in oxidative stress that destroys the
structure of vital biomolecules, potentially leading to
cellular dysfunction and remodeling [9]. Oxidative stress
is known to activate the AMP-activated protein kinase
(AMPK) signaling system in neuronal, heart, muscle,
pancreatic and liver cells [10,11]. Interestingly, AMPK
is known to be involved in ROS-induced autophagy that
promotes cell survival in response to cellular stress such
as malnutrition, hypoxia or ischemia [12]. Indeed, oxy-
gen and nutrient deprivation induce the activation of
AMPK leading to autophagy by inhibition of mTORC1
and phosphorylation of ULK1 [13,14].
Autophagy is an important cell pathway for cell
homeostasis and survival by removing damaged organ-
elles and intracellular microbial pathogens [15]. Hepato-
cytes may be particularly dependent on the underlying
autophagy for normal physiological function due to their
high biosynthetic activity. In addition, autophagy plays a
crucial role in the non-alcoholic and alcoholic liver dis-
eases, drug-induced hepatic damage, viral hepatitis, fi-
brosis, liver cancer and hepatic ischemia reperfusion
injury [15–17]. In liver ischemia reperfusion injuries, au-
tophagy provides a prosurvival activity allowing the cell
to cope with nutrient starvation and anoxia [16]. During
hepatitis B or C infection, the level of autophagy is typic-
ally increased to promote viral growth [17]. In hepato-
cellular carcinoma, the level of autophagy is thought to
be involved in both tumorigenesis and tumor suppres-
sion [18,19].
In this regard, we tested whether L. japonica and FX
alleviated hepatic oxidative stress in an in vitro model,
HepG2 cells established by arachidonic acid (AA) + iron.
Specifically, we explored the abilities of FX in regulation
of autophagy and the underlying molecular mechanisms
of their effects.
Methods
Reagents
AA and Compound C (C.C) were purchased from
Calbiochem (San Diego, CA, USA). Anti-phospho-ACC,
phospho-LKB1, procaspase-3, PARP, Bcl
XL,
LC3 I/II,
beclin-1, AMPK, and phospho-AMPK antibodies were
obtained from Cell Signaling Technology (Beverly, MA,
USA). Bal-A1 was purchased from Santa Cruz Biotech-
nology (Santa Cruz, CA, USA). Horseradish peroxidase-
conjugated goat anti-rabbit, rabbit anti-goat, and goat
anti-mouse IgGs were obtained from Zymed Laboratories
(San Francisco, CA, USA). FX, acrydine orange hemi zinc
chloride salt, 3-methyladenine (3-MA), anti-ß-actin anti-
body and other reagents were purchased from Sigma-
Aldrich (St. Louis, MO, USA).
Preparation of the L. japonica extract (LJE)
L. japonica was purchased from Daewon pharmacy
(Daegu, Korea), which is standardized with a standard herb
of L. japonica in Korea Food and Drug Administrations.
The L. japonica (100 g) were extracted as previously
described [20,21]. The yield of lyophilized LJE was esti-
matedtobe1.19%basedonthedriedweight.
Cell culture
HepG2 cells, a human hepatocyte-derived cell line, were
provided by American Type Culture Collection (Rockville,
MD, USA), and cultured as previously described [20]. To
simulate oxidative stress, cells were incubated with 10 μM
AA for 12 h, followed by exposure to 5 μM iron for 1 h.
The cells were treated with FX or LJE for 1 h prior to the
incubation with AA at the indicated doses.
Cell viability assay
The cells were plated at a density of 1 × 10
5
cells per well
in a 48-well for 24 h as previously described [20]. The
media was incubated with 0.25 mg/ml MTT for 2 h, and
formazan crystals were dissolved with the addition of
200 μl DMSO.
Terminal deoxynucleotidyl transferase dUTP nick end
labeling (TUNEL) assay
The TUNEL assay was performed using the DeadEnd™
Colorimetric TUNEL System, according to the manufac-
turer’s instruction. The samples were washed and exam-
ined under light microscope.
Western blot analysis
The cells were plated at a density of 5 × 10
5
cells per well in
a 6-well plate for 24 h. After the treatment designated, cells
were lysed in RIPA buffer (Thermo Scientific, Rockford, IL,
USA) as previously described [20,21]. The protein bands
were detected using Fusion Solo scanning system (Vilber
Lourmat, Paris, France), and quantified using Image J ver
1.42 software (NIH, Bethesda, USA).
Measurement of ROS production
DCFH-DA, a cell-permeable non-fluorescent probe, has
been used as a substrate for quantitation of intracellular
oxidant production in HepG2 cells [21]. After treatment
of reagents, cells were stained with 10 μM DCFH-DA
for 30 min at 37 °C. The fluorescence intensity in the
cells was measured at an excitation/emission wavelength
Jang et al. BMC Complementary and Alternative Medicine (2018) 18:97 Page 2 of 11
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of 485/535 nm, using in the cells measured in a micro-
plate reader.
Determinant of glutathione (GSH) content
Intracellular GSH content was quantified using a commer-
cial GSH BIOXYTECH GSH-400 kit (Oxis International,
Portland, OR) according to the manufacturer’sprotocol,
and the absorbance level was measured at 405 nm.
Flow cytometric analysis of ΔΨm
ΔΨm was measured using rhodamine123 (Rh123).
Following treatment, cells were stained with 0.05 μg/ml
of Rh123 for 1 h and then harvested by trypsinization.
The change in ΔΨm was monitored using a FACS flow
cytometer (Partec, Münster, Germany). In each analysis,
a total of 10,000 events were recorded as previous
described [20].
Acridine orange (AO) staining
HepG2 cells were plated on 18-mm cover glasses and in-
cubated for 24 h to reach at approximately 70% conflu-
ence. They were then incubated either in the presence
or absence of 30 μM FX, washed twice with PBS and
fixed with ice-cold 4% paraformaldehyde for 10 min at
room temperature. Subsequently, the cells were stained
with AO (1 μg/ml) for 15 min at room temperature,
washed, and examined under a fluorescence microscope
(Nikon, Japan).
Profiling the content of fucoxanthin by ultra performance
liquid chromatography (UPLC)
Water ACQUITYTM ultraperformance LC system
(USA) was used to assess UPLC analysis as previously
described [20]. Waters ACQUITYTM BEH C18 column
(1.7 μm, 2.1 mm × 100 mm) was used as Waters
ACQUITYTM PDA and HPLC Column, and the fuco-
xanthin was analyzed at 330 nm. The standard fucoxan-
thin was melted by methanol and diluted to make
solution containing 1 μg/ml. L. japonica 1 g was also
added with methanol 10 ml, and then sonication was
perfomed for 3 h. After then, it was filtered through a
0.2 μm filter (Nalgene, NY, USA). A mobile phase was a
mixed liquid of the acetonitrile and water, and the ana-
lysis condition was as in Table 1. The sample was
injected with 2 μl, and a flow rate was 0.4 ml/min.
Statistical analysis for study
GraphPad Prism software version 5.01 (Graph Pad Software,
La Jolla, CA) was used for all statistical analyses as previ-
ously described [20,21]. Significance levels were calculated
by repeated measures of ANOVA with the Dunnett post
hoc test under 95% confidence interval. Data were presented
as the mean with standard deviation (mean ± S.D.). Within
figures, the Pvalues were displayed with asterisks
(***P < 0.001, **P < 0.01, *P < 0.05).
Results
L. japonica Extract (LJE) decreases AA + iron induced
cytotoxicity in HepG2 cells
An MTT assay for cell viability indicated that LJE pre-
treatment (3, 10, 30, and 50 μg/ml) significantly pro-
tected cells from the potential injury induced by AA +
iron. Since the maximum cell viability was achieved at
30 μg/ml of LJE, the same concentration was applied in
subsequent experiments (Fig. 1a). In western blot ana-
lysis, treatment of AA + iron markedly induced
decreases in the protein levels of procaspase-3 and
Bcl
XL
, verifying AA + iron induction of apoptosis, which
was completely blocked by LJE pretreatment (Fig. 1b).
Morphological examination by light microscopy and
TUNEL assay (Fig. 1c) confirmed the cytoprotective
effect of LJE against the synergized toxicity of AA + iron.
After treatment with LJE, positive staining located in the
nucleus by AA + iron were apparently decreased
(Fig. 1c). To further examine the antioxidative effects of
LJE, we measured the contents of GSH and ROS. The
intracellular concentration of GSH was substantially
decreased by AA + iron, but was recovered by LJE treat-
ment. In contrast, treatment with LJE alone had no ef-
fects on cellular GSH levels (Fig. 1d). The ROS
generation assay using DCFH-DA indicated that LJE
treatment effectively abrogated increases in ROS produc-
tion caused by AA + iron (Fig. 1e). The effect of LJE on
AA + iron-induced loss of mitochondrial membrane
potential (ΔΨm) monitored by FACS analysis of Rho123
staining (Fig. 1f ). Rho123 fluorescence intensity was not
significantly altered in LJE-treated cells compared to
untreated controls, though AA + iron markedly reduced
rhodamine fluorescence, indicating the loss of ΔΨm
(Fig. 1f). LJE treatment significantly restrored the loss of
ΔΨm caused by AA + iron (Fig. 1f ).
Table 1 Solvent gradient for the analysis fucoxanthin in L. japonica
Time (min) 0.1% FA/
Water (%)
0.1% FA/
Acetonitrile (%)
Flow rate
(ml/min)
0 98 2 0.40
1 98 2 0.40
2 90 10 0.40
4 70 30 0.40
7 50 50 0.40
9 30 70 0.40
10 10 90 0.40
12 0 100 0.40
14 98 2 0.40
16 98 2 0.40
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a
c
d
f
e
b
0
20
40
60
80
100
120
Vehicle - 3 10 30 50
Cell viability (%)
-actin
Procaspase-3
BclXL
Vehicle _ LJE LJE (30µg/ml)
AA+iron
(LJE, µg/ml)
0
20
40
60
80
Vehicle - LJE LJE
TUNEL-positive cells (%)
AA+iron
**
Vehicle
LJE
AA+iron
-
**
** **
NS NS
**
AA+iron
0
10000
20000
30000
40000
Vehicle - LJE LJE
ROS product
AA+iron
0
30
60
90
120
150
Vehicle - LJE LJE
GSH (nmol/mg protein)
AA+iron
** **
** **
(30µg/ml)
(30µg/ml) (30µg/ml)
Counts
Rhodamine 123
Vehicle AA+iron AA+iron+LJE(30µg/ml) LJE (30µg/ml)
0
30
60
90
120
Vehicle - LJE LJE
Loss of m (%)
AA+iron
** **
(30µg/ml)
Fig. 1 L. japonica extract (LJE) decreases AA + iron induced cytotoxicity in HepG2 cells. HepG2 cells were incubated with indicated dose of LJE
for 1 h and later treated with 10 μM AA for 12 h, being followed by exposure to 5 μM iron for 3 h. (a) Cell viability was assessed by the MTT
assay. (b) Expression of proteins associated with apoptosis was determined by western blot analysis. Equal protein loading was verified by β-actin.
(c) The levels of apoptosis in each groups examined by TUNEL assay. Representative images show apoptosis of HepG2 cells in vehicle control, LJE
treated, AA + iron treated, and AA + iron treated with LJE groups (left). Percentage of TUNEL+ cell nuclei calculated relative to total number of
cell nuclei (right). (d) Cellular GSH content was assessed in cells by using GSH assay kit. (e) Cellular reactive oxygen species production was moni-
tored by measuring intensity of dichloro fluoresce in fluorescence. (f)ΔΨm depolarization monitored by FACS analysis of Rh123 staining. Relative
proportions of low Rh-123 intensity (RN1 fractions) are expressed as the mean ±S.D. of three separated experiments. For panel from A to E, data
represent the mean ± S.D. for the four replicates. ** p< 0.01
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Fucoxanthin (FX) ameliorates AA + iron-induced
cytotoxicity
Next, we determined the effects of FX against oxidative
stress induced by AA + iron (Fig. 2a). FX treatment inhib-
ited death of cell induced by AA + iron, and this decrease
in cell viability was recovered by pre-treatment with 30 μM
of FX (Fig. 2b). In western blot analysis, cleavage of PARP
and caspase-3 were strongly observed in AA + iron-treated
cells, which were blocked by FX pretreatment (Fig. 2c). FX
treatment significantly inhibited the change in ΔΨmcaused
by AA + iron (Fig. 2d). These results indicate that FX
remarkably suppressed AA + iron-induced collapse of
ΔΨm, consequently protecting liver cells.
FX-induced the activation of AMPK alleviates cell damage
by oxidative stress
Stimulation of FX (30 μM) markedly induced the phos-
phorylation of AMPK (Fig. 3a). This compound also in-
duced the phosphorylation of LKB1, an upstream kinase
of AMPK, and ACC, a primary downstream target of
AMPK (Fig. 3a). To determine the role of AMPK in pro-
tection of HepG2 cells by FX, we measured ΔΨm levels
after treating with a chemical inhibitor of AMPK, C.C.
C.C inhibited the protective effect of FX on AA + iron-
induced the loss of ΔΨm in HepG2 cells (Fig. 3b). Col-
lectively, these results suggest that FX activates the
LKB1-AMPK signaling pathway, and that this activation
0
20
40
60
80
100
120
Vehicle - 3 10 30 60
Cell viability (%)
AA+iron
**
**
FX (µM)
Fucoxanthin
pro-PARP
cleaved form
cleaved form
pro-caspase 3
-act in
Vehicle - FX FX
AA+iron
0
0.5
1
1.5
2
2.5
0
0.5
1
1.5
2
2.5
3
3.5
4
Vehicle FX FX
AA+iron
Relative protein level
(fold of vehicle)
Cleaved
PA R P
Cleaved
caspase 3
**
**
Relative protein level
(fold of vehicle)
0
20
40
60
80
Vehicle - FX FX
AA+iron
** **
Loss of m (%)
** **
NS
(30µM)
(30µM)
(30µM)
a
c
d
b
Fig. 2 Fucoxanthin (FX) ameliorates AA + iron-induced cytotoxicity. HepG2 cells were incubated with indicated dose of FX for 1 h and later treated with
10 μM AA for 12 h, being followed by exposure to 5 μMironfor3h.(a) The chemical structure of FX. (b) The effects of FX on cell viability was assessed by
the MTT assay. (c) Expression of proteins associated with apoptosis was determined by western blot analysis. Equal protein loading was verified by β-actin.
(d)ΔΨm depolarization monitored by FACS analysis of Rh123 staining. Relative proportions of low Rh-123 intensity (RN1 fractions) are expressed as the
mean ± S.D. of three separated experiments. For panel b,cand d, data represent the mean ± S.D. for the four replicates. ** p<0.01
Jang et al. BMC Complementary and Alternative Medicine (2018) 18:97 Page 5 of 11
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of beneficial molecules is responsible for FX’s inhibition
of mitochondria damage induced by oxidative stress.
FX triggers AMPK-dependent cytoprotective autophagy
Treatment with 30 μM FX upregulated beclin-1 and
promoted the conversion of LC3 I to LC3 II as com-
pared to the control (Fig. 4a). The AVOs were clearly
observed in the HepG2 cells (red fluorescence) following
treatment with FX (Fig. 4b). As shown in Fig. 4c, inhib-
ition of AMPK activity by C.C markedly attenuated FX-
induced accumulation of beclin-1, suggesting that
AMPK is critical in the regulation of FX-induced au-
tophagy. In addition, western blotting revealed that FX
rapidly downregulated the phosphorylation of mTORC1
(Ser2448), which is known to negatively modulate au-
tophagy and be inhibited by AMPK (Fig. 5a). In contrast,
the phosphorylation levels of ULK1 (Ser555) was in-
creased during FX treatment in time-dependent manner
(Fig. 5a). Furthermore, we manipulated autophagy activ-
ity using 3-MA and Baf-A1 to suppress autophagy.
FACS analysis of ΔΨm showed that 3-MA and Baf-A1
partially blocked the effect of FX on mitochondrial pro-
tection (Fig. 5b).
Discussion
In our results, LJE or FX ameliorated oxidative damage
induced by AA + iron on hepatocytes, as confirmed by
the inhibition of cell death and the restorating the loss
of ΔΨm. We also demonstrated that these hepatoprotec-
tive effects of FX can be attributed to the function of au-
tophagy via the AMPK/mTORC1/ULK-1 axis.
To determine the capacity of LJE or FX to reduce oxi-
dative damage, we employed an in vitro model, HepG2
cells treated with AA + iron. In liver, the level of iron is
tightly regulated by the control of absorption, storage
and recycling, which is critical for the protection of liver
tissues as well as other organ tissues from iron-induced
cellular damages [21]. However, a chronic increase of
iron level in liver can result in excess ROS production
and liver injury, such as steatohepatitis, fibrosis,
a
b
FX (30µM)
Vehicle 10' 30' 1 3 6 (H)
p-ACC
p-LKB1
p-AMPK
-act in
LKB1
0
0.5
1
1.5
2
2.5
3
Relative level of p-LKB1
(fold ofvehicle)
Vehicle 10’ 30’ 1 3 6 (H)
FX
**
*
0
0.4
0.8
1.2
1.6
Relative level of p-AMPK
(foldof vehicle)
FX
Vehicle 10’ 30’ 1 3 6 (H)
*
0
20
40
60
80
100
C. C
**
*
Loss of m (%)
- + - + - +
AA+iron - - + + + +
FX (30µM) - - - - + +
Fig. 3 FX-induced the activation of AMPK alleviates cell damage by oxidative stress. (a) FX induces phosphorylation of the proteins associated with
AMPK pathway, ACC, LKB1 and AMPK. Western blot analyses were performed with the lysates of cells that had been treated with 30 μM FX for the
indicated time period. β-actin served as a loading control. Protein levels were presented as relative band intensities to control (vehicle treated) group.
Results represent the mean ±S.D. for three separate experiments. * p<0.05; **p< 0.01; *** p<0.001.(b) The effect of FX to restore ΔΨmwasrevered
by C.C. Following treatment with 10 μM C.C for 1 h, cells were incubated with FX and/or AA + iron, and ΔΨm was evaluated with Rh123 stain by FACS.
Data represent the mean± S.D. for four replicates. * p<0.05; ** p<0.01
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cirrhosis, and hepatocellular carcinoma [22]. The release
of AA can also be induced by an increase in oxidative
stress that originates from excessive levels of iron [23].
Although prostaglandins, essential components in cellu-
lar protection, are produced from AA, excessive AA can
induce extremely high levels of cellular and mitochon-
drial ROS, negatively influencing the functions of several
processes related to mitochondrial respiration [23]. The
combinatorial treatment of AA and iron can thus reduce
cell viability, and this treatment may be used to test the
potential of cytoprotective agents targeting mitochondria
against severe oxidative stress. In this study, AA + iron
successfully induced cell death, production of ROS and
damage of mitochondria in HepG2 cells. However,
pretreatment of LJE or FX significantly blocked the abil-
ity of AA + iron to induce similar detrimental effects in
HepG2 cells.
Recent studies have shown that AMPK serves as a key
regulator of hepatocytes viability under oxidative stress
[10,11,23]. Indeed, many natural compounds, such as
resveratrol, sauchinone, and isoliquiritigenin, have been
reported to protect hepatocytes by inhibiting production
of ROS and mitochondrial dysfunction through activa-
tion of AMPK [24–26]. In our study, FX upregulated the
phosphorylation of AMPK, ACC (the downstream
target) and LKB1 (the essential upstream kinase) in he-
patocytes. Furthermore, the inhibition of AMPK using
compound C reduced the beneficial effect of FX on
LC3 I
LC3 II
Beclin-1
-actin
F X (30µM) Control
Relative level of LC3 II
(fold of v e h ic le)
Re la ti ve lev e l of beclin-1
(fold of vehicle)
0
0.4
0.8
1.2
1.6
2
FX (30µM)
Vehicle 10' 30' 1 3 6 (H)
0
0.4
0.8
1.2
1.6
Vehicle 10’ 30’ 1 3 6 (H)
FX
Vehicle 10’ 30’ 1 3 6 (H)
FX
**
*
***
C.C (µM) - 10 - 10
FX (µM) - - 30 30
Beclin-1
-act in
*** ***
a
b
c
Fig. 4 The effect of FX on autophagy induction in HepG2 cells. (a) FX induces time-dependent activation of the autophagy related proteins, LC3II and
Becline-1. Western blot analyses were performed on the lysates of cells that had been treated with 30 μM FX for the indicated time period. β-actin served
as a loading control. Protein levels were presented as relative band intensities to control (vehicle treated) group. Results represent the mean ± S.D.forfour
separate experiments. ** p< 0.01; *** p< 0.001. (b) The pictures of the fluorescence micrographs show the formation of AVOs resulting from the treatment
of FX in HepG2 cells. Cells were incubated either in the presence or absence of 30 μM FX for 6 h and were stained with AO. The presence of AVOs was
indicated by the red fluorescence. (c) Inhibition of FX-induced autophagy by C.C. Western blot analysis of Beclin-1 were performed with lysates of HepG2
cells that had been pretreated with 10 μM C.C for 1 h being followed by exposure to 30 μMFXfor1h
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mitochondria. Those results partially suggest that FX
protects cells through activation of AMPK.
It is now widely accepted that AMPK induce autophagy,
in turn, serving to reduce oxidative damage [12–14]. Au-
tophagy can suppress cell death by eliminating damaged
organelles or unnecessary cellular components formed
from a variety of stresses, thereby playing adaptive roles to
protect organisms against infections, cancer, neurodegen-
eration, aging, and heart diseases [15–18,27]. The process
of autophagy involves formation of double membrane
vesicles (autophagosome) that enwrap portions of the
cytoplasm [24]. To detect the development of AVOs,
HepG2 cells treated with FX were stained with acridine
orange. Our results demonstrated that the bright red
fluorescence significantly increased after FX treatment,
indicating the development of AVOs. Western blotting
studies also demonstrated that FX induced HepG2 cell au-
tophagy, as shown by the conversion of LC3B-I in LC3B-
II and the expression of Beclin-1, indicators of autophagy
[24,28]. Furthermore, AMPK inhibition by a chemical in-
hibitor of AMPKα, C.C abolished the increased protein
level of beclin-1 by FX. Consequently, our current findings
suggest that the activation of AMPK by FX be involved in
induction of autophagy in HepG2 cells.
AMPK activation promoted autophagy through direct
activation of ULK1 and inhibition of mTORC1, a nega-
tive regulator of autophagy [14]. mTORC1 is important
in the autophagy and its activity inhibits autophagy by
ULK1 phosphorylation, which induce disassociation be-
tween ULK1 and AMPK [14]. In this study, we observed
a significant increase in the levels of ULK1 and AMPK
phosphorylation in response to FX. Thus, our findings
indicate a model in which FX induces autophagy by acti-
vating the AMPK–ULK1–mTORC1 axis. Finally, present
study showed that 3-MA and Baf-A1, the autophagy in-
hibitors, partially reversed the inhibitory effect of FX on
AA + iron-induced mitochondrial membrane potential
depolarization in HepG2 cells, suggesting the involve-
ment of AMPK-induced autophagy as the hepatoprotec-
tive activity of FX in HepG2 cells.
Although there are various component in L. japonica,
in this study, we confirmed the content of FX (mass
p-mTOR
(Ser2448)
ULK1
FX (30µM)
Vehicle 10' 30' 1 3 6 (H)
-actin
mTOR
p-ULK1
(Ser 555)
0
0.2
0.4
0.6
0.8
1
1.2
1.4
Relative levelof p-mTOR
(fo ld o fvehi cle )
*
***
FX
Vehicle 10’ 30’ 1 3 6 (H)
0
0.5
1
1.5
2
2.5
3
Relative leve l of p-ULK1
(foldofvehicle)
Vehicle 10’ 30’ 1 3 6 (H)
FX
**
*
0
20
40
60
80
100
-+-+-+
++ ++ ++
FX
(30µM)
AA+iron
Veh i c l e 3MA Baf-A1
*** *** **
*** ***
Loss of m (%)
a
b
Fig. 5 The role of AMPK activation by FX in autophagy induction. (a) Western blot analysis for the phosphorylated level of mTOR (Ser2448) and ULK1
(Ser555) in HepG2 cells treated with 30 μM FX for indicated time period. Results represent the mean ± S.D. for four separate experiments.
*p< 0.05; ** p< 0.01; *** p<0.001.(b) The effect of FX to restore ΔΨm was revered by 3-MA and Baf-A1. After 3-MA and Baf-A1 treatment (5 μMfor
1 h, respectively), cells were incubated with FX for 1 h, being followed by the addition of AA (for 12 h) + iron (1 h). Data represent the mean ± S.D. for
three replicates. ** p< 0.01; *** p<0.001
Jang et al. BMC Complementary and Alternative Medicine (2018) 18:97 Page 8 of 11
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
Fig. 6 UPLC chromatogram of fucoxanthin standard (a) and fucoxanthin in L. japonica (b). The peak represents fucoxanthin (330 nm)
Arachidonic acid
+ Iron
Oxidative
stress
Intracellular ROS
Mitochondrial
dysfunction
Fucoxanthin
LKB1
P
AMPK
P
mTOR
ULK1
AutophagyApotopsis
Laminaria japonica
Fig. 7 Schematic diagram showed that FX induces AMPK-mediated autophagy contributing to ameliorates oxidative stress in HepG2 cells
Jang et al. BMC Complementary and Alternative Medicine (2018) 18:97 Page 9 of 11
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
accuracy; 28.197 ppm) in L. japonica (Fig. 6). Recently,
some report also showed that FX is known to significantly
inhibit on the proliferation of HepG2 cells [29]. The result
showed that both the protein degradation and transcrip-
tional repression was responsible for cyclin D suppression
by FX in HepG2 cells by inducing G1 arrest as mediated
with GADD45A and MAPK pathway [29]. But, in the
present study, FX showed a significantly protective effect
on HepG2 cells against AA + iron-induced oxidative in-
jury. Therefore, the active compound in the L. japonica
and FX in the aspect of cell protection as well as their
machanism remains to be further established.
Conclusion
Our results suggested that FX could protect hepatocytes
against AA + iron-induced oxidative stress and trigger
autophagy, which is likely associated with the LKB1-
AMPKαsignaling pathway (Fig. 7). The current study
also showed that FX or Laminaria japonica likely con-
tributed to further understanding of its potential use as
a hepatic protectant and nutraceutical.
Abbreviations
3-MA: 3-methyladenine; AA: Arachidonic acid; ACC: Acetyl-CoA carboxylase;
AMPK: AMP-activated protein kinase; ATG: Autophagy-related proteins; AVO: Acidic
vesicular organelle; Bal-A1: Bafilomycin A1; Bcl-xL: B-cell lymphoma-extra large;
C.C: Compound C; DCFH-DA: 2′,7′-Dichlorofluorescein diacetate; DMSO: Dimethyl
sulphoxide; FBS: Fetal bovine serum; FX: Fucoxanthin; GSH: Glutathione;
LC3: Microtubule-associated protein 1 light chain 3; LJE: L. japonica extract;
LKB1: Liver kinase B1; mTOR: mammalian target of rapamycin; MTT: 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide; PARP: Poly (ADP-ribose)
polymerase; PBS: Phosphate-buffered saline; Rh123: Rhodamine 123; ROS: Reactive
oxygen species; TUNEL: Terminal deoxynucleotidyl transferase dUTP nick-end
labeling; ΔΨm: mitochondrial membrane potential
Funding
This work was supported by the National Research Foundation of Korea (NRF)
grant funded by the Korea Government [MSIP] (No.2015K1A3A1A59069800)
and (No. 2017R1D1A3B03027847), and the NRF grant funded by the Korea
government [MSIP] (No. 2012R1A5A2A42671316), and and also by the Grant
K18102 awarded to Korea Institute of Oriental Medicine (KIOM) from Korea
Ministry of Education, Science and Technology (MEST).
Availability of data and materials
The datasets generated and/or analyzed during this study are available from
the corresponding author on reasonable request.
Authors’contributions
E.J.J., S.C.K., J.H.L. and S.Y.B. conducted research and cell experiments of
fucoxanthin and L. japonica. J.R.L. analyzed the contents of fucoxanthin in L.
japonica. S.C.K. and S.Y.B. helped the writing the paper and the analysis of
data. E.J.J., J.H.L., S.Y.B., I.K.K. and Y.W.K designed research and wrote the
paper. S.C.K., S.Y.B. and Y.W.K. supported financial funding. All authors read
and approved the final manuscript.
Ethics approval and consent to participate
Not applicable
Consent for publication
Not applicable
Competing interests
The authors declare that they have no competing interests.
Publisher’sNote
Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations.
Author details
1
College of Oriental Medicine, Daegu Haany University, Gyeongsan,
Gyeongsangbuk-do 38610, South Korea.
2
College of Korean Medicine,
Dongguk University, Gyungju, Gyeongbuk 38066, South Korea.
3
Kyungpook
National University, Daegu 41566, South Korea.
Received: 31 August 2017 Accepted: 8 March 2018
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