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International Journal of
Molecular Sciences
Article
Antarctic Krill Oil Diet Protects against
Lipopolysaccharide-Induced Oxidative Stress,
Neuroinflammation and Cognitive Impairment
Ji Yeon Choi, Jun Sung Jang, Dong Ju Son, Hyung-Sik Im, Ji Yeong Kim, Joung Eun Park,
Won Rak Choi, Sang-Bae Han and Jin Tae Hong * ID
College of Pharmacy and Medical Research Center, Chungbuk National University,
194-31 Osongsaemgmyeong 1-ro, Osong-eup, Heungdeok-gu, Cheongju 28160, Chungbuk, Korea;
cjy8316@hanmail.net (J.Y.C.); chvictory07@hanmail.net (J.S.J.); sondj1@hotmail.com (D.J.S.);
ihs311@naver.com (H.-S.I.); ronevans@naver.com (J.Y.K.); wlwl83@naver.com (J.E.P.);
cwonr@sk.com (W.R.C.); shan@chungbuk.ac.kr (S.-B.H.)
*Correspondence: jinthong@chungbuk.ac.kr; Tel.: +82-43-261-2813; Fax: +82-43-268-2732
Received: 30 September 2017; Accepted: 22 November 2017; Published: 28 November 2017
Abstract:
Oxidative stress and neuroinflammation are implicated in the development and pathogenesis
of Alzheimer’s disease (AD). Here, we investigated the anti-inflammatory and antioxidative effects
of krill oil. Oil from Euphausia superba (Antarctic krill), an Antarctic marine species, is rich in
eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). We examined whether krill oil diet
(80 mg/kg/day for one month) prevents amyloidogenesis and cognitive impairment induced by
intraperitoneal lipopolysaccharide (LPS) (250
µ
g/kg, seven times daily) injections in AD mice model
and found that krill oil treatment inhibited the LPS-induced memory loss. We also found that krill
oil treatment inhibited the LPS-induced expression of inducible nitric oxide synthase (iNOS) and
cyclooxygenase-2 (COX-2) and decreased reactive oxygen species (ROS) and malondialdehyde levels.
Krill oil also suppresses I
κ
B degradation as well as p50 and p65 translocation into the nuclei of
LPS-injected mice brain cells. In association with the inhibitory effect on neuroinflammation and
oxidative stress, krill oil suppressed amyloid beta (1–42) peptide generation by the down-regulating
APP and BACE1 expression
in vivo
. We found that eicosapentaenoic acid (EPA) and docosahexaenoic
acid (DHA) (50 and 100
µ
M) dose-dependently decreased LPS-induced nitric oxide and ROS generation,
and COX-2 and iNOS expression as well as nuclear factor-
κ
B activity in cultured microglial BV-2
cells. These results suggest that krill oil ameliorated impairment via anti-inflammatory, antioxidative,
and anti-amyloidogenic mechanisms.
Keywords: neuroinflammation; amyloidogenesis; oxidation; nuclear factor-κB; krill oil
1. Introduction
Dietary intervention with marine products, including marine-derived oils, has been widely
used during the past decades. Furthermore, within the previous three decades, natural marine
products have shown many promising activities against inflammation, cancer, infectious diseases and
neurological disorders [
1
]. The consumption of marine products prevents neurodegenerative processes
and maintains cognitive capacities in the elderly [
2
]. Of these products, krill oil can regulate lipid
metabolism, inflammation, and oxidative stress [
3
]. The shrimp-like Euphausia superba (Antarctic krill)
is one of the most important Antarctic marine species [
4
]. Previous studies have demonstrated that
krill oil has anti-inflammatory and antioxidative effects due to its eicosapentaenoic acid (EPA) and
docosahexaenoic acid (DHA) contents, which can be absorbed very quickly and cross the blood–brain
barrier (BBB) [
5
]. It was also reported that EPA and DHA, which are found in animal-based sources
Int. J. Mol. Sci. 2017,18, 2554; doi:10.3390/ijms18122554 www.mdpi.com/journal/ijms
Int. J. Mol. Sci. 2017,18, 2554 2 of 15
of omega-3 fats, play a significant role in lowering tumor necrosis alpha (TNF-
α
), interleukin 1 beta
(IL-1
β
), and prostaglandin E
2
levels [
6
]. Additionally, krill oil is rich in vitamin A and E, and the
carotenoid astaxanthin, which is likely stable and resistant to oxidation [
7
]. Therefore, high levels of
these components make krill oil more superior than fish oil in terms of its biological effects [8].
Oxidative stress and inflammation are the two major processes in the development of Alzheimer’s
disease (AD). Oxidative stress is a condition in which oxidant generation overwhelms antioxidant
defenses and is largely implicated in the pathogenesis of many neurologic and psychiatric, diseases
including AD [
2
]. Increased oxidative stress leads to damage to lipids, DNA, and proteins, and thus causes
a functional decline in neurons [
9
]. Oxidative stress has been proposed to upregulate amyloid beta (A
β
)
peptide generation via induction of
β
- and
γ
-secretase activity [
10
]. Hydrogen peroxide (H
2
O
2
) in human
neuroblastoma cells reportedly enhances BACE1 expression and A
β
accumulation, eventually causing
significant cell damage [
11
,
12
]. Additionally, AD brain exhibit oxidative stress-mediated injury since A
β
peptides increase superoxide anion production in the brain [
13
]. Thus, synaptic loss and increased number
of extracellular A
β
peptides could be associated with oxidative brain damage [
14
]. Brain inflammation is
also a pathological hallmark of the AD. The activated microglial cells produce inflammatory mediators and
accumulate around amyloid plaques in the brains of individuals with the AD, and have been implicated in
promoting neurodegeneration [
15
]. Chronically activated glia can kill adjacent neurons by releasing highly
toxic products such as reactive oxygen species (ROS), nitric oxide (NO), and complement factors, thereby
enhancing APP production and amyloidogenic processing [
16
]. Exposure of lipopolysaccharide (LPS) has
cognitive-behavioral consequences due to A
β
aggregation in the hippocampus and pro-inflammatory
reactions in response to oxidative damages [
17
]. Therefore, the study of protective compounds that
inhibit oxidative pathways and inflammatory responses is an aspect of further research for treating
neurodegenerative diseases.
Nuclear factor-kappa B (NF-
κ
B) is a redox transcription factor that influences the levels of
oxidative stress in neurons [
18
,
19
]. Expression of several inflammatory genes such as inducible nitric
oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), as well as inflammatory cytokines, can be
regulated by NF-
κ
B activation [
20
]. It is also known that oxidative stress can activate NF-
κ
B in
several disease statuses. Moreover, the promoter of neuronal BACE1, a limiting enzyme producing
A
β
, has NF-
κ
B DNA consensus sequences [
21
]. Epidemiologic studies have demonstrated that the
anti-inflammatory and antioxidative therapies could decrease the risk of the AD by reducing NF-
κ
B
activity [
22
]. Thus, blocking NF-
κ
B can facilitate AD management by reducing neuroinflammation,
oxidative stress, and amyloidogenesis [23].
In the present study, we investigate whether Antarctic krill oil has antioxidative and anti-
inflammatory properties as well as anti-amyloidogenic property against LPS-induced memory
dysfunction in cultured neuronal macrophages and in vivo mice models.
2. Results
2.1. Krill Oil Treatment Attenuates LPS-Induced Cognitive Impairment
Effect of krill oil on cognitive and memory improvement was estimated using the water maze
and passive avoidance tests. We investigated the ability of mice to learn locations and perform spatial
memory recall through escape latency and measuring the distance in the water maze. The LPS-injected
mice learned more slowly than control mice and krill oil-treated mice. Krill oil-treated mice exhibited
a reduction in escape latency over the training period (Figure 1A). Krill oil-treated mice also showed
a shorter escape distance (Figure 1B) compared with LPS-injected mice. After the final day of the
water maze test, we performed a probe test to calculate the time spent in the target quadrant zone,
thereby testing for the maintenance of memory. Krill oil-treated mice spent much more time in
the quadrant zone than LPS-injected mice (Figure 1C). Then, through the passive avoidance test,
we tested the mice to assess for how long they can remember the locations. Although there was no
Int. J. Mol. Sci. 2017,18, 2554 3 of 15
significant difference in the learning pattern, krill oil-treated mice recorded increased step-through
latency compared with the LPS-injected mice group (Figure 1D).
Int. J. Mol. Sci. 2017, 18, 2554 3 of 15
Figure 1. Effect of Krill oil on memory impairment. To investigate the effect of Krill oil in LPS-induced
memory impairment, we performed: a water maze test (A,B); a probe test (C); and a step-through type
passive avoidance test (D). Memory function was determined by the escape latencies (A, s) and distance
(B, cm) for 5 days, and time spent in the target quadrant (C, %) in the probe test after administration of
LPS. Each value is mean ± Standard deviation (SD) from eight mice. Group differences were analyzed
by one-way ANOVA followed by Bonferroni’s post-hoc analysis.
#
Significantly different from control
group (p < 0.05). * Significantly different from LPS-treated group (p < 0.05).
2.2. Krill Oil Treatment Inhibits the Accumulation of Aβ Peptides, Amyloidogenesis, and NF-κB Activation
To investigate whether Aβ deposition by immunohistochemical analysis was paralleled with Aβ
level in the brain, quantitative analyses of Aβ level was performed using ELISA. Aβ
level in the brains
of LPS-injected mice was significantly higher than that of control mice, but it was reduced in the krill
oil-treated mice brains (Figure 2A). Because Aβs are produced by activated β-secretase, we measured
the activity of β-secretase in the hippocampus. The activity of β-secretase was increased in the brains
of LPS-injected mice, while the activity was significantly decreased in krill oil-treated mice brains
(Figure 2B). To confirm whether krill oil influenced amyloidogenesis inhibition in the brain, we
performed the Western blot assay. LPS-elevated expression of APP, BACE1, and C99 was
significantly decreased by krill oil treatment (Figure 2C). NF-κB activity is implicated in
amyloidogenesis and neuroinflammation. Thus, due to p50, p65, and IκB phosphorylation, NF-κB
activation was confirmed. Phosphorylation of IκB and translocation of p50 and p65 were significantly
decreased by the treatment of krill oil (Figure 2D).
Figure 1.
Effect of Krill oil on memory impairment. To investigate the effect of Krill oil in LPS-induced
memory impairment, we performed: a water maze test (
A
,
B
); a probe test (
C
); and a step-through type
passive avoidance test (
D
). Memory function was determined by the escape latencies (
A
, s) and distance
(
B
, cm) for 5 days, and time spent in the target quadrant (
C
, %) in the probe test after administration of
LPS. Each value is mean
±
Standard deviation (SD) from eight mice. Group differences were analyzed
by one-way ANOVA followed by Bonferroni’s post-hoc analysis.
#
Significantly different from control
group (p< 0.05). * Significantly different from LPS-treated group (p< 0.05).
2.2. Krill Oil Treatment Inhibits the Accumulation of AβPeptides, Amyloidogenesis, and NF-κB Activation
To investigate whether A
β
deposition by immunohistochemical analysis was paralleled with
A
β
level in the brain, quantitative analyses of A
β
level was performed using ELISA. A
β
level
in the brains of LPS-injected mice was significantly higher than that of control mice, but it was
reduced in the krill oil-treated mice brains (Figure 2A). Because A
β
s are produced by activated
β
-secretase, we measured the activity of
β
-secretase in the hippocampus. The activity of
β
-secretase
was increased in the brains of LPS-injected mice, while the activity was significantly decreased in
krill oil-treated mice brains (Figure 2B). To confirm whether krill oil influenced amyloidogenesis
inhibition in the brain, we performed the Western blot assay. LPS-elevated expression of APP, BACE1,
and C99 was significantly decreased by krill oil treatment (Figure 2C). NF-
κ
B activity is implicated
in amyloidogenesis and neuroinflammation. Thus, due to p50, p65, and I
κ
B phosphorylation, NF-
κ
B
activation was confirmed. Phosphorylation of I
κ
B and translocation of p50 and p65 were significantly
decreased by the treatment of krill oil (Figure 2D).
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Int. J. Mol. Sci. 2017, 18, 2554 4 of 15
Figure 2. Effect of Krill oil on the LPS-induced amyloidogenesis and NF-κB activity in mice brain. The
levels of Aβ
1–42
in mice brain (n = 5) were measured by ELISA (A). The activity of β-secretase in mice
brain (n = 5) was investigated using assay kit (B). The expression of APP, BACE1 and C99 were
detected by Western blotting using specific antibodies in the mouse brain (C). Phosphorylation of IκB,
and p50 and p65 translocation were detected by Western blotting using specific antibodies in mice
brain; β-actin and Histone H1 protein were used as an internal control (D). For the cropped images,
samples were run in the same gels under same experimental conditions and processed in parallel. The
graphs under Western blotting are the relative protein expression of three bands. Each band is
representative of three experiments. Group differences were analyzed by one-way ANOVA followed
by Bonferroni’s post-hoc analysis.
#
Significantly different from control group (p < 0.05). * Significantly
different from LPS-treated group (p < 0.05).
2.3. Krill Oil Treatment Inhibits Neuroinflammation
We performed immunohistochemistry and Western blotting to detect the expression of GFAP
(an astrocyte activation marker), Iba-1 (a microglial cell activation marker), and inflammatory
proteins (iNOS and COX-2) in the brain, which consequently indicates the activation of astrocytes
and microglia as well as the occurrence of neuroinflammation. The GFAP-reactive cell number and
Iba-1-reactive cell number were reduced in krill oil-treated mice as opposed to LPS-injected mice,
which showed the much higher number of cells reactive for these marker proteins compared with
control mice (Figure 3A). The expression of iNOS and COX-2 was also significantly decreased in the
brains of krill oil-treated mice than LPS-injected mice brains (Figure 3B). We also investigated the
inhibitory effect of krill oil on neuroinflammation through Western blotting; treatment with LPS
elevated the expression of inflammatory proteins (iNOS and COX-2), GFAP, and Iba-1, but the
expression was significantly reduced by krill oil treatment (Figure 4A). We investigated expression
levels of the pro-inflammatory cytokines-related factors IL-6, IL-1β and TNF-α in brain tissues. Our
results suggested that krill oil treatment decreased LPS-induced mRNA levels of IL-6 (Figure 4B), IL-
1β (Figure 4C) and TNF-α (Figure 4D) in brain tissues.
Figure 2.
Effect of Krill oil on the LPS-induced amyloidogenesis and NF-
κ
B activity in mice brain.
The levels of A
β1–42
in mice brain (n= 5) were measured by ELISA (
A
). The activity of
β
-secretase in
mice brain (n= 5) was investigated using assay kit (
B
). The expression of APP, BACE1 and C99 were
detected by Western blotting using specific antibodies in the mouse brain (
C
). Phosphorylation of I
κ
B,
and p50 and p65 translocation were detected by Western blotting using specific antibodies in mice
brain;
β
-actin and Histone H1 protein were used as an internal control (
D
). For the cropped images,
samples were run in the same gels under same experimental conditions and processed in parallel.
The graphs under Western blotting are the relative protein expression of three bands. Each band is
representative of three experiments. Group differences were analyzed by one-way ANOVA followed
by Bonferroni’s post-hoc analysis.
#
Significantly different from control group (p< 0.05). * Significantly
different from LPS-treated group (p< 0.05).
2.3. Krill Oil Treatment Inhibits Neuroinflammation
We performed immunohistochemistry and Western blotting to detect the expression of GFAP
(an astrocyte activation marker), Iba-1 (a microglial cell activation marker), and inflammatory proteins
(iNOS and COX-2) in the brain, which consequently indicates the activation of astrocytes and microglia
as well as the occurrence of neuroinflammation. The GFAP-reactive cell number and Iba-1-reactive
cell number were reduced in krill oil-treated mice as opposed to LPS-injected mice, which showed
the much higher number of cells reactive for these marker proteins compared with control mice
(Figure 3A). The expression of iNOS and COX-2 was also significantly decreased in the brains of krill
oil-treated mice than LPS-injected mice brains (Figure 3B). We also investigated the inhibitory effect of
krill oil on neuroinflammation through Western blotting; treatment with LPS elevated the expression
of inflammatory proteins (iNOS and COX-2), GFAP, and Iba-1, but the expression was significantly
reduced by krill oil treatment (Figure 4A). We investigated expression levels of the pro-inflammatory
cytokines-related factors IL-6, IL-1
β
and TNF-
α
in brain tissues. Our results suggested that krill oil
treatment decreased LPS-induced mRNA levels of IL-6 (Figure 4B), IL-1
β
(Figure 4C) and TNF-
α
(Figure 4D) in brain tissues.
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Figure 3. Effect of Krill oil on LPS-induced neuroinflammation in the mouse brain. Immunostaining
of GFAP, Iba-1, iNOS, and COX-2 proteins in the hippocampus were performed in 20 µm-thick
sections of mice brain with specific primary antibodies and the biotinylated secondary antibodies
(A,B). Similar patterns were observed in five mice brain. Group differences were analyzed by one-
way ANOVA followed by Bonferroni’s post-hoc analysis.
#
Significantly different from control group
(p < 0.05). * Significantly different from LPS-treated group (p < 0.05).
2.4. Krill Oil Inhibits LPS-Induced Oxidative Stress
Krill oil decreased superoxide anion production in the mice brain. Intracellular superoxide
radical production was measured by dihydroethidium in the brain. Furthermore, another study has
shown that local LPS administration contributes to the activation of astroglial or microglial cells in
place of this toxin administration. Additionally, it was reported that brain damage can be caused by
inflammation and oxidative stress after prolonged exposure to LPS for ≥7 days. The accumulation of
excessive intracellular ROS with increased enzymatic sources characterizes oxidative stress.
Although the intensity of oxidative stress varies because oxidation usually occurs only for a short
time, systemic LPS administration for a prolonged period will damage the brain due to exposure to
oxidative stress. The brain sections were double stained with DHE (red) and DAPI (blue) staining.
The krill oil-treated mice showed a significant decrease in DHE signal intensity compared with the
LPS-injected mice (Figure 4E,F). We also evaluated malondialdehyde (MDA) and H
2
O
2
levels, which
are indicators of oxidative stress. MDA was significantly increased in the brains of LPS-injected mice
compared with control mice. However, in contrast to LPS-injected mice, krill oil-treated mice showed
lower MDA (Figure 4G) and H
2
O
2
levels (Figure 4H).
Figure 3.
Effect of Krill oil on LPS-induced neuroinflammation in the mouse brain. Immunostaining
of GFAP, Iba-1, iNOS, and COX-2 proteins in the hippocampus were performed in 20
µ
m-thick
sections of mice brain with specific primary antibodies and the biotinylated secondary antibodies
(
A
,
B
). Similar patterns were observed in five mice brain. Group differences were analyzed by one-way
ANOVA followed by Bonferroni’s post-hoc analysis.
#
Significantly different from control group
(p< 0.05). * Significantly different from LPS-treated group (p< 0.05).
2.4. Krill Oil Inhibits LPS-Induced Oxidative Stress
Krill oil decreased superoxide anion production in the mice brain. Intracellular superoxide
radical production was measured by dihydroethidium in the brain. Furthermore, another study has
shown that local LPS administration contributes to the activation of astroglial or microglial cells in
place of this toxin administration. Additionally, it was reported that brain damage can be caused by
inflammation and oxidative stress after prolonged exposure to LPS for
≥
7 days. The accumulation of
excessive intracellular ROS with increased enzymatic sources characterizes oxidative stress. Although
the intensity of oxidative stress varies because oxidation usually occurs only for a short time, systemic
LPS administration for a prolonged period will damage the brain due to exposure to oxidative stress.
The brain sections were double stained with DHE (red) and DAPI (blue) staining. The krill oil-treated
mice showed a significant decrease in DHE signal intensity compared with the LPS-injected mice
(Figure 4E,F). We also evaluated malondialdehyde (MDA) and H
2
O
2
levels, which are indicators of
oxidative stress. MDA was significantly increased in the brains of LPS-injected mice compared with
control mice. However, in contrast to LPS-injected mice, krill oil-treated mice showed lower MDA
(Figure 4G) and H2O2levels (Figure 4H).
Int. J. Mol. Sci. 2017,18, 2554 6 of 15
Int. J. Mol. Sci. 2017, 18, 2554 6 of 15
Figure 4. Effect of Krill oil on the LPS-induced neuroinflammation and oxidative stress in the mouse
brain. The expression of GFAP, Iba-1, iNOS, and COX-2, were detected by Western blotting using
specific antibodies in the mice brain. Each blot is representative of three experiments (A). For the
cropped images, samples were run in the same gels under same experimental conditions and
processed in parallel. The graphs under Western blotting are the relative protein expression of three
ban ds. Each band is repr esentative of t hree exp erime nts. mRNA levels o f IL-6 (B); IL-1β (C); and TNF-
α (D) were detected by qRT-PCR in brain tissues (n = 5). Intracellular superoxide radical production
was measured by dihydroethidium in the brain. The brain sections were double stained with DHE
(red) and DAPI staining (blue) (E). Similar patterns were observed in five mice brain. The graph is a
quantification of the DHA fluorescent signal in the brain tissues (F). MDA (G); and hydrogen peroxide
level (H) were assessed by using a specific detection kit as described in Methods (n = 5). Values
measured from each group of mice were calibrated by the amount of protein and expressed as mean
± SD. Group differences were analyzed by one-way ANOVA followed by Bonferroni’s post-hoc
analysis.
#
Significantly different from control group (p < 0.05). * Significantly different from LPS-
treated group (p < 0.05).
2.5. EPA and DHA Prevent LPS-Stimulated Nuclear Translocation of the NF-κB Complex
We investigated the effects of EPA and DHA, the components of krill oil, on NF-κB nuclear
translocation in microglial BV-2 cells using immunofluorescence imaging. LPS induced nuclear
translocation of p65, the NF-κB protein, in 30 min. In contrast, EPA and DHA pretreatment prevented
the dose-dependent nuclear translocation of p65 (Figures 5A and 6A). To clarify whether EPA and
DHA influenced the inhibition of p65 translocation, we performed Western blotting. We determined
NF-κB activation through the detection of p50, p65, and IκB phosphorylation. Phosphorylation of IκB
and translocation of p50 and p65 were significantly decreased by the treatment of EPA and DHA
(Figures 5B and 6B).
Figure 4.
Effect of Krill oil on the LPS-induced neuroinflammation and oxidative stress in the mouse
brain. The expression of GFAP, Iba-1, iNOS, and COX-2, were detected by Western blotting using
specific antibodies in the mice brain. Each blot is representative of three experiments (
A
). For the
cropped images, samples were run in the same gels under same experimental conditions and processed
in parallel. The graphs under Western blotting are the relative protein expression of three bands.
Each band is representative of three experiments. mRNA levels of IL-6 (
B
); IL-1
β
(
C
); and TNF-
α
(
D
) were detected by qRT-PCR in brain tissues (n= 5). Intracellular superoxide radical production
was measured by dihydroethidium in the brain. The brain sections were double stained with DHE
(red) and DAPI staining (blue) (
E
). Similar patterns were observed in five mice brain. The graph
is a quantification of the DHA fluorescent signal in the brain tissues (
F
). MDA (
G
); and hydrogen
peroxide level (
H
) were assessed by using a specific detection kit as described in Methods (n= 5).
Values measured from each group of mice were calibrated by the amount of protein and expressed as
mean
±
SD. Group differences were analyzed by one-way ANOVA followed by Bonferroni’s post-hoc
analysis.
#
Significantly different from control group (p< 0.05). * Significantly different from LPS-treated
group (p< 0.05).
2.5. EPA and DHA Prevent LPS-Stimulated Nuclear Translocation of the NF-κB Complex
We investigated the effects of EPA and DHA, the components of krill oil, on NF-
κ
B nuclear
translocation in microglial BV-2 cells using immunofluorescence imaging. LPS induced nuclear
translocation of p65, the NF-
κ
B protein, in 30 min. In contrast, EPA and DHA pretreatment prevented
the dose-dependent nuclear translocation of p65 (Figures 5A and 6A). To clarify whether EPA and
DHA influenced the inhibition of p65 translocation, we performed Western blotting. We determined
NF-
κ
B activation through the detection of p50, p65, and I
κ
B phosphorylation. Phosphorylation of
I
κ
B and translocation of p50 and p65 were significantly decreased by the treatment of EPA and DHA
(Figures 5B and 6B).
Int. J. Mol. Sci. 2017,18, 2554 7 of 15
Int. J. Mol. Sci. 2017, 18, x FOR PEER REVIEW 7 of 15
Figure 5. Inhibitory effect of Krill oil on neuroinflammatory responses in microglia cells. To find the
effect of anti-oxidative stress, microglial BV-2 cells were treated with 1 µg/mL of LPS and 50, 100 µM
of EPA. The cultured microglial BV-2 cells were incubated with anti-p65 (green) and DAPI staining
(blue) (A). Quantification of p65 subcellular distribution in one representative of three independent
experiments (B). Fluorescence was developed using Alexa 488-conjugated anti-mouse secondary
antibodies. Phosphorylation of IκB, and p50 and p65 translocation were detected by Western blotting
using specific antibodies in microglial BV-2 cells. β-actin and Histone H1 protein were used as an
internal control (C). Hydrogen peroxide level was assessed using a specific detection kit, as described
in Methods (D) (n = 5). NO level was measured in EPA treated microglial BV-2 cells (E) (n = 5). COX-
2 and iNOS proteins were detected by Western blotting using specific antibodies in EPA treated
microglial BV-2 cells (F). The graphs under Western blotting are the relative protein expression of
bands. Each band is representative of three experiments. Group differences were analyzed by one-
way ANOVA followed by Bonferroni’s post-hoc analysis.
#
Significantly different from control group
(p < 0.05). * Significantly different from LPS-treated group (p < 0.05).
2.6. EPA and DHA Prevent LPS-Stimulated Oxidative Stress and Neuroinflammation
We evaluated the H
2
O
2
level as an indicator of oxidative stress. To elucidate the effect of
antioxidative stress, microglial BV-2 cells were treated with 1 µg/mL LPS and 50 and 100 µM of EPA
or DHA. The microglial BV-2 cells treated with EPA/DHA showed lower H
2
O
2
levels (Figures 5C and
6C). Furthermore, it was detected that the NO level was decreased dose-dependently in microglial
BV-2 cells (Figures 5D and 6D). Then, we detected the expression of inflammatory proteins (iNOS
and COX-2) by Western blotting. The levels of iNOS and COX-2 protein were increased in LPS-
treated cells, whereas the expressions were dose-dependently reduced by EPA and DHA treatment
(Figures 5E and 6E).
Figure 5.
Inhibitory effect of Krill oil on neuroinflammatory responses in microglia cells. To find the
effect of anti-oxidative stress, microglial BV-2 cells were treated with 1
µ
g/mL of LPS and 50, 100
µ
M
of EPA. The cultured microglial BV-2 cells were incubated with anti-p65 (green) and DAPI staining
(blue) (
A
). Quantification of p65 subcellular distribution in one representative of three independent
experiments (
B
). Fluorescence was developed using Alexa 488-conjugated anti-mouse secondary
antibodies. Phosphorylation of I
κ
B, and p50 and p65 translocation were detected by Western blotting
using specific antibodies in microglial BV-2 cells.
β
-actin and Histone H1 protein were used as an internal
control (
C
). Hydrogen peroxide level was assessed using a specific detection kit, as described in Methods
(
D
) (n= 5). NO level was measured in EPA treated microglial BV-2 cells (
E
) (n= 5). COX-2 and iNOS
proteins were detected by Western blotting using specific antibodies in EPA treated microglial BV-2
cells (
F
). The graphs under Western blotting are the relative protein expression of bands. Each band is
representative of three experiments. Group differences were analyzed by one-way ANOVA followed by
Bonferroni’s post-hoc analysis.
#
Significantly different from control group (p< 0.05). * Significantly
different from LPS-treated group (p< 0.05).
2.6. EPA and DHA Prevent LPS-Stimulated Oxidative Stress and Neuroinflammation
We evaluated the H
2
O
2
level asan indicator of oxidativestress. To elucidate the effect of antioxidative
stress, microglial BV-2 cells were treated with 1
µ
g/mL LPS and 50 and 100
µ
M of EPA or DHA.
The microglial BV-2 cells treated with EPA/DHA showed lower H
2
O
2
levels (Figures 5C and 6C).
Furthermore, it was detected that the NO level was decreased dose-dependently in microglial BV-2 cells
(Figures 5D and 6D). Then, we detected the expression of inflammatory proteins (iNOS and COX-2) by
Western blotting. The levels of iNOS and COX-2 protein were increased in LPS-treated cells, whereas the
expressions were dose-dependently reduced by EPA and DHA treatment (Figures 5E and 6E).
Int. J. Mol. Sci. 2017,18, 2554 8 of 15
Int. J. Mol. Sci. 2017, 18, 2554 8 of 15
Figure 6. Inhibitory effect of Krill oil on neuroinflammatory responses in microglia cells. To find the
effect of anti-oxidative stress, microglial BV-2 cells were treated with 1 µg/mL of LPS and 50, 100 µM
of DHA. The cultured microglial BV-2 cells were incubated with anti-p65 (green) and DAPI staining
(blue) (A). Quantification of p65 subcellular distribution in one representative of three independent
experiments (B). Fluorescence was developed using Alexa 488-conjugated anti-mouse secondary
antibodies. Phosphorylation of IκB, and p50 and p65 translocation were detected by Western blotting
using specific antibodies in microglial BV-2 cells. β-actin and Histone H1 protein were used as an
internal control (C). Hydrogen peroxide level was assessed by using a specific detection kit as
described in Methods (D) (n = 5). NO level was measured in DHA treated microglial BV-2 cells (E) (n
= 5). COX-2 and iNOS proteins were detected by Western blotting using specific antibodies in DHA
treated microglial BV-2 cells (F). The graphs under Western blotting are the relative protein expression
of bands. Each band is representative of three experiments. Group differences were analyzed by one-
way ANOVA followed by Bonferroni’s post-hoc analysis.
#
Significantly different from control group
(p < 0.05). * Significantly different from LPS-treated group (p < 0.05).
3. Discussion
The data from the present study revealed that krill oil supplementation in diet could suppress
neuroinflammation, oxidative stress, and amyloidogenesis in LPS-induced AD model. Oxidative
stress and neuroinflammatory cascades can lead to neurodegenerative diseases, including AD; thus,
the administration of anti-inflammatory and anti-oxidative agents reduces the risk of or delays the
neuropathologic features of AD [24,25]. There are different mechanisms related with AD progression.
Recently, a series of studies proved that systemic administration of LPS contributes to increased
neuroinflammation and oxidative stress along with direct damage of the BBB, thereby causing
amyloidogenesis and memory deficiency [26,27]. Furthermore, LPS-induced brain inflammation is
accompanied by neuronal and glial cell activation resulting in the release of neurotoxic factors such
as inflammatory cytokines or free radicals [28,29]. The chronic administration of LPS can cause spatial
memory and learning impairment analogous to cognitive decline during AD, which is associated
with inflammation and amyloidogenesis due to increased Aβ deposition [30–32]. In the present study,
Figure 6.
Inhibitory effect of Krill oil on neuroinflammatory responses in microglia cells. To find the
effect of anti-oxidative stress, microglial BV-2 cells were treated with 1
µ
g/mL of LPS and 50, 100
µ
M
of DHA. The cultured microglial BV-2 cells were incubated with anti-p65 (green) and DAPI staining
(blue) (
A
). Quantification of p65 subcellular distribution in one representative of three independent
experiments (
B
). Fluorescence was developed using Alexa 488-conjugated anti-mouse secondary
antibodies. Phosphorylation of I
κ
B, and p50 and p65 translocation were detected by Western blotting
using specific antibodies in microglial BV-2 cells.
β
-actin and Histone H1 protein were used as an internal
control (
C
). Hydrogen peroxide level was assessed by using a specific detection kit as described in
Methods (
D
) (n= 5). NO level was measured in DHA treated microglial BV-2 cells (
E
) (n= 5). COX-2 and
iNOS proteins were detected by Western blotting using specific antibodies in DHA treated microglial
BV-2 cells (
F
). The graphs under Western blotting are the relative protein expression of bands. Each band
is representative of three experiments. Group differences were analyzed by one-way ANOVA followed
by Bonferroni’s post-hoc analysis.
#
Significantly different from control group (p< 0.05). * Significantly
different from LPS-treated group (p< 0.05).
3. Discussion
The data from the present study revealed that krill oil supplementation in diet could
suppress neuroinflammation, oxidative stress, and amyloidogenesis in LPS-induced AD model.
Oxidative stress and neuroinflammatory cascades can lead to neurodegenerative diseases, including
AD; thus, the administration of anti-inflammatory and anti-oxidative agents reduces the risk of or
delays the neuropathologic features of AD [
24
,
25
]. There are different mechanisms related with AD
progression. Recently, a series of studies proved that systemic administration of LPS contributes to
increased neuroinflammation and oxidative stress along with direct damage of the BBB, thereby causing
amyloidogenesis and memory deficiency [
26
,
27
]. Furthermore, LPS-induced brain inflammation is
accompanied by neuronal and glial cell activation resulting in the release of neurotoxic factors such as
inflammatory cytokines or free radicals [
28
,
29
]. The chronic administration of LPS can cause spatial
memory and learning impairment analogous to cognitive decline during AD, which is associated
with inflammation and amyloidogenesis due to increased A
β
deposition [
30
–
32
]. In the present study,
Int. J. Mol. Sci. 2017,18, 2554 9 of 15
we found that krill oil decreased amyloidogenesis and memory deficiency via the prevention of brain
damage by oxidative stress and neuroinflammation. We also found that the krill oil components EPA
and DHA reduced LPS-induced oxidative stress and inflammatory response in BV-2 cells.
Recent studies revealed that A
β
production plays a major role in regulating microglial ROS
generation in during AD [
33
,
34
]. Hence, oxidative stress leading to attack by free radicals on neural cells
increases lipid peroxidation, subsequently causing neurodegenerative conditions such as AD [
25
]. In our
study, krill oil inhibited LPS-induced lipid peroxidation as well as H
2
O
2
generation, and these inhibitory
effects were associated with reduced A
β
accumulation level. The promoters of APP and BACE1 contain
NF-
κ
B consensus sequences, which control the transcription of these genes [
21
]. NF-
κ
B is activated
by inflammatory mediators and oxidative stress [
35
]. Thus, the inhibitory effect of krill oil on NF-
κ
B
could be associated with its overall anti-amyloidogenic property owing to its anti-inflammatory and
anti-oxidative effects. In our previous study, L-theanine, EGCG, and punicalagin, which are antioxidant
compounds, showed anti-neuroinflammatory responses and anti-amyloidogenic activity through
antioxidative mechanisms [
36
–
38
]. The findings of several studies suggest that patients with mild
Alzheimer’s deterioration could benefit from taking dietary supplement formulation containing both
the omega-3 fatty acids, EPA and DHA [
39
]. The high content of the two biologically active components
EPA and DHA are responsible for the majority of physiological effects of krill oil [
40
]. EPA or DHA
intake resulted in an increased incorporation of omega-3 fatty acids in membrane phospholipids
of immune cells; they can be absorbed quickly, cross the BBB, and reduce inflammatory responses
as well as the activation of microglia in the brain [
41
]. EPA and DHA can modulate the expression
of several inflammatory genes such as COX-2 and iNOS by significantly reducing NF-
κ
B activity,
which subsequently lowers the induction of inflammation and oxidative stress in cells [
42
,
43
]. In the
present study, LPS-induced phosphorylation of I
κ
B and translocation of p50 and p65 were significantly
decreased by treatment with EPA or DHA. Furthermore, EPA and DHA reduced the increased level
of LPS-induced oxidative stress and neuroinflammatory gene expression. Thus, antioxidative and
anti-inflammatory properties of krill oil could be significant for anti-amyloidogenesis through reducing
NF-κB activation, and this effect could be associated with the effects of EPA and DHA.
Krill consumption by humans can potentially help healthy nutrition strategy to protect
against progressive cognitive loss [
2
]. Taken together, these data indicate that antioxidative,
anti-neuroinflammatory, and anti-amyloidogenic effects of krill oil could enhance memory function.
Hence, krill oil can be employed for the development of functional food or drug for treating AD.
4. Methods
4.1. Ethical Approval
The experimental protocols (27 March 2017) were carried out according to the guidelines for
animal experiments of the Institutional Animal Care and Use Committee (IACUC) of Laboratory
Animal Research Center at Chungbuk National University, Korea (CBNUA-1073-17-01). All efforts
were made to minimize animal suffering and to reduce the number of animals used. All mice were
housed in three mice per cage with automatic temperature control (21–25
◦
C), relative humidity
(45–65%), and 12 h light-dark cycle illuminating from 08:00 a.m. to 08:00 p.m. Food and water were
available ad libitum. They were fed pellet diet consisting of crude protein 20.5%, crude fat 3.5%,
crude fiber 8.0%, crude ash 8.0%, calcium 0.5%, phosphorus 0.5% per 100 g of the diet (obtained from
Daehan Biolink, Chungcheongbuk-do, Korea). During this study, all mice were especially observed
for the normal body posture, piloerection, ataxia, urination, etc. twice daily to minimize their pain
and discomfort.
Int. J. Mol. Sci. 2017,18, 2554 10 of 15
4.2. Materials
4.2.1. Preparation of Enzymatically Decomposed Krill Oil
The Antarctic krill oil was supplied from Alpha B&H (Eumseong-gun, Chungcheongbuk-do,
Korea) and we feed the rodent chow supplemented with 5 wt % of krill oil (2018 Teklad Rodent Diet,
Envigo Bioproducts) ad libitum. Enzymatically decomposed Krill oil was prepared as reported
previously and stored at room temperature until use. Briefly, frozen or freeze-dried krill were thawed.
Salt was removed from the krill by washing with tap water, and then they were pulverized using
a pin-type mill. Pulverized krill (62.29%, w/w) were mixed with alcalase enzyme (0.19%, w/w),
a non-specific subtilisin-related serine protease separated from Bacillus licheniformis, and water
(37.52%, w/w), then stirred for 30–60 min at room temperature. Before performing the enzyme reaction,
the pH of the krill was adjusted to be 7.5–9.0. The enzyme reaction was performed at 57
±
3
◦
C for
3.5
±
0.5 h until liquefied. After performing the enzyme reaction, the pH of the reactant was adjusted
to 4.5
±
0.5 by adding 1.84 part by weight citric acid and/or ascorbic acid per 100 parts by weight
liquefied krill and letting stand for 30 min. The enzymes were inactivated by heating at 94
±
5
◦
C.
The sludge including shell and head of krill was removed by decanter centrifugation (3.0 t/h) at >70
◦
C.
The lipids and phospholipids of filtrate were extracted by centrifugation at 5000 rpm (1.0–2.0 t/h).
The extract was sterilized and concentrated under reduced pressure on a rotary evaporator at 80–90
◦
C
until the water content has dropped below 3%. The sterilized concentrate was filtered using 50 mesh
sieve and was stored at room temperature until use.
The main components in krill oil are about 7% docosapentaenoic acid (C22:6, DHA) and 12%
eicosapentaenoic acid (C20:5, EPA). Furthermore, we purchased EPA and DHA from TOCRIS. The EPA
and DHA (final concentration of 100 mM) were dissolved in 100% dimethyl sulfoxide (DMSO),
and aliquots were stored at
−
20
◦
C until use
in vitro
. The LPS was purchased from Sigma (serotype
O55:B5, Sigma, St. Louis, MO, USA). The LPS (final concentration of 1 mg/mL) was dissolved in PBS,
and aliquots in PBS were stored at −20 ◦C until use.
4.2.2. Animal Experiment
Eight- to ten-week-old male imprinting control region (ICR) mice (Daehan Biolink,
Chungcheongbuk-do, Korea) were maintained and handled in accordance with the humane animal
care and use guidelines of Korean FDA. ICR mice were randomly divided into three groups: (I) Control
group; (II) LPS group; and (III) Krill oil + LPS group. Each group was assigned 10 mice. The Krill
oil diet (80 mg/kg) was given to (III) group daily for 4 weeks. Intraperitoneal (i.p.) injection of LPS
(250
µ
g/kg) was administered except for control group on the 4th week for 7 days. Control mice were
given an equal volume of vehicle instead. The behavioral tests of learning and memory capacity were
assessed using water maze, probe and passive avoidance test. Mice were sacrificed after behavioral
tests by CO2asphyxiation (Figure 7).
Int. J. Mol. Sci. 2017, 18, 2554 10 of 15
4.2. Materials
4.2.1. Preparation of Enzymatically Decomposed Krill Oil
The Antarctic krill oil was supplied from Alpha B&H (Eumseong-gun, Chungcheongbuk-do,
Korea) and we feed the rodent chow supplemented with 5 wt % of krill oil (2018 Teklad Rodent Diet,
Envigo Bioproducts) ad libitum. Enzymatically decomposed Krill oil was prepared as reported
previously and stored at room temperature until use. Briefly, frozen or freeze-dried krill were
thawed. Salt was removed from the krill by washing with tap water, and then they were pulverized
using a pin-type mill. Pulverized krill (62.29%, w/w) were mixed with alcalase enzyme (0.19%, w/w),
a non-specific subtilisin-related serine protease separated from Bacillus licheniformis, and water
(37.52%, w/w), then stirred for 30–60 min at room temperature. Before performing the enzyme
reaction, the pH of the krill was adjusted to be 7.5–9.0. The enzyme reaction was performed at 57 ± 3
°C for 3.5 ± 0.5 h until liquefied. After performing the enzyme reaction, the pH of the reactant was
adjusted to 4.5 ± 0.5 by adding 1.84 part by weight citric acid and/or ascorbic acid per 100 parts by
weight liquefied krill and letting stand for 30 min. The enzymes were inactivated by heating at 94 ±
5 °C. The sludge including shell and head of krill was removed by decanter centrifugation (3.0 t/h) at
>70 °C. The lipids and phospholipids of filtrate were extracted by centrifugation at 5000 rpm (1.0–2.0
t/h). The extract was sterilized and concentrated under reduced pressure on a rotary evaporator at
80–90 °C until the water content has dropped below 3%. The sterilized concentrate was filtered using
50 mesh sieve and was stored at room temperature until use.
The main components in krill oil are about 7% docosapentaenoic acid (C22:6, DHA) and 12%
eicosapentaenoic acid (C20:5, EPA). Furthermore, we purchased EPA and DHA from TOCRIS. The
EPA and DHA (final concentration of 100 mM) were dissolved in 100% dimethyl sulfoxide (DMSO),
and aliquots were stored at −20 °C until use in vitro. The LPS was purchased from Sigma (serotype
O55:B5, Sigma, St. Louis, MO, USA). The LPS (final concentration of 1 mg/mL) was dissolved in PBS,
and aliquots in PBS were stored at −20 °C until use.
4.2.2. Animal Experiment
Eight- to ten-week-old male imprinting control region (ICR) mice (Daehan Biolink,
Chungcheongbuk-do, Korea) were maintained and handled in accordance with the humane animal
care and use guidelines of Korean FDA. ICR mice were randomly divided into three groups: (I)
Control group; (II) LPS group; and (III) Krill oil + LPS group. Each group was assigned 10 mice. The
Krill oil diet (80 mg/kg) was given to (III) group daily for 4 weeks. Intraperitoneal (i.p.) injection of
LPS (250 µg/kg) was administered except for control group on the 4th week for 7 days. Control mice
were given an equal volume of vehicle instead. The behavioral tests of learning and memory capacity
were assessed using water maze, probe and passive avoidance test. Mice were sacrificed after
behavioral tests by CO
2
asphyxiation (Figure 7).
Figure 7. Timeline depicts the treatment of Krill oil and assessments of cognitive functions of mice.
Figure 7. Timeline depicts the treatment of Krill oil and assessments of cognitive functions of mice.
Int. J. Mol. Sci. 2017,18, 2554 11 of 15
4.2.3. Behavior Tests
Memory test was performed by the Morris’s water maze test as described elsewhere with
SMART-CS (Panlab, Barcelona, Spain) program and equipment [
44
]. The platform was removed
from the pool which was used in the water maze test, and the mice were allowed to swim freely.
The swimming pattern of each mouse was monitored and recorded for 60 s using the SMART-LD
program (Panlab). Retained spatial memory was estimated by the time spent in the target quadrant area.
The passive avoidance response was determined using a “step-through” apparatus (Med Associates,
Georgia, VT, USA). All three behavior test were done as described elsewhere [44].
4.2.4. Brain Collection and Preservation
After behavioral tests, mice were perfused with phosphate-buffered saline (PBS) with heparin
under inhaled CO
2
anesthetization. The brains were immediately removed from the skulls and
divided into left and right hemisphere. One was stored at
−
80
◦
C, while the other was fixed in 4%
paraformaldehyde for 72 h at 4 ◦C and transferred to 30% sucrose solutions.
4.2.5. Immunohistochemical Staining
Immunohistochemical staining was performed as described previously [
45
]. The sections
were incubated overnight with a rabbit/mouse polyclonal antibody against GFAP; SC-33673
(1:300, Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), IBA-1; NB100-1028, iNOS; NB300-605
(1:300; Novus Biologicals, Inc., Littleton, CO, USA), COX-2; #12282 (1:300; Cell Signaling Technology,
Inc., Beverly, MA, USA). To prevent nonspecific staining, a blocking step was included. Sections were
incubated at room temperature for 2 h with 5% bovine serum albumin [
46
] (in PBS), and incubated for
overnight at 4
◦
C with the primary antibody in blocking solution (5% BSA). Immunohistochemical
staining was performed on 8 mice per group (3 sections per each mouse).
4.2.6. Immunofluorescence Staining
The microglial BV-2 cells were incubated for 2 h at room temperature with a goat polyclonal
antibody against p65 (1:500, Santa Cruz Biotechnologies, Inc., Santa Cruz, CA, USA). After washing
with PBS, the brain sections were incubated with an anti-rabbit or anti-mouse secondary antibody
labeled with Alexa-Fluor 488 for 2 h at room temperature. Sections were then dehydrated in ethanol,
cleared in xylene and covered with Permount. Final images were acquired using a confocal laser
scanning microscope (TCS SP2, Leica Microsystems AG, Wetzlar, Germany).
4.2.7. Western Blot Analysis
We extracted total protein by total lysis buffer (iNtRON Biotechnology, 17081). Furthermore,
we used nuclear extraction kit (Abcam, ab113474, Cambridge, MA, USA) for obtaining nuclear protein.
In the
in vivo
study, to compare the expression of protein levels through Western blotting, we selected
and used 3 of 10 mice brain from each group. An equal amount of total protein (20
µ
g) was resolved on
8–15% sodium dodecyl sulfate-polyacrylamide gel and then transferred to a nitrocellulose membrane
(Hybond ECL; Amersham Pharmacia Biotech, Piscataway, NJ, USA). To detect target proteins,
specific antibodies against APP; NB110-55461, IBA-1; NB100-1028, iNOS; NB300-605 (1:1000, Novus
Biologicals, Inc., Littleton, CO, USA), BACE1; #5606, COX-2; #12282 (1:1000, Cell Signaling Technology,
Inc., Beverly, MA, USA), GFAP; SC-33673 and
β
-actin; SC-47778 were used. The blots were then
incubated with the corresponding conjugated goat anti-rabbit; SC-2004 or goat anti-mouse; SC-2005 or
donkey anti-goat; SC-2020 IgG-horseradish peroxidase (HRP) (1:5000; Santa Cruz Biotechnology Inc.,
Santa Cruz, CA, USA) secondary antibodies. Immunoreactive proteins were detected with an enhanced
chemiluminescence Western blotting detection system. The relative density of the protein bands was
scanned by densitometry using MyImage (SLB, Seoul, Korea) and quantified by Labworks 4.0 software
(UVP Inc., Upland, CA, USA).
Int. J. Mol. Sci. 2017,18, 2554 12 of 15
4.2.8. Nitrate Assay
Microglial BV-2 cells were plated at a density of 5
×
10
5
cells/well in 6-well plates per 2 mL
medium for 24 h. After removing the culture medium, the cells were then treated with LPS (1
µ
g/mL)
and EPA, DHA (50, 100
µ
M) per 2 mL medium for 24 h. The nitrite in the supernatant was assessed
using a NO detection kit (iNtRON Biotechnology, Seongnam, Korea), according to the manufacturer’s
instructions. Finally, the resulting color was assayed at 520 nm using a microplate absorbance reader
(VersaMax ELISA, Molecular Devices, Sunnyvale, CA, USA).
4.2.9. RNA Isolation and Quantitative Real-Time RT-PCR
Tissue RNA was isolated from homogenized hippocampus using RiboEX (Gene All, Seoul,
Korea), and total RNA (0.2
µ
g) was reverse-transcribed into cDNA according to the manufacturer’s
instructions using Applied Biosystems (Foster City, CA, USA). For the quantitative, real-time,
reverse transcriptase polymerase chain reaction (PCR) assays, the linearity of the amplification
of IL-6, IL-1
β
, TNF-
α
and
β
-actin cDNAs was established in preliminary experiments. All signal
mRNAs were normalized to
β
-actin mRNA. cDNAs were amplified by real-time PCR in
duplicate with QuantiNova SYBR green PCR kit (Qiagen, Valencia, CA, USA). Each sample
was run with the following primer sets: IL-6, 5
0
-GAGGATACCACTCCCAACAGACC-3
0
(sense),
5
0
-AAGTGCATCATCGTTGTTCATACA-3
0
(antisense); IL-1
β
, 5
0
-GTGGCTAAGGACCAAGACCA-3
0
(sense), 5
0
-TACCAGTTGGGGAACTCTGC-3
0
(antisense); TNF-
α
, 5
0
-GATCTCAAAGACAACCAACAT
GTG-3
0
(sense), 5
0
-CTCCAGCTGGAAGACTCCTCCCAG-3
0
(antisense);
β
-actin: 5
0
-TGGAATCC
TGTGGCATCCATGAAAC-30(sense), 50-TAAAACGCAGCTCAGTAACAGTCCG-30(antisense).
4.2.10. Measurement of Aβ1–42
Lysates of brain tissue were obtained through protein extraction buffer containing a protease
inhibitor. A
β1–42
levels were determined using each specific ELISA Kit (CUSABIO, College Park,
MD, USA). Protein was extracted from brain tissues using a protein extraction buffer (PRO-PREPTM,
Intron Biotechnology, Korea), incubated on ice for 1 h and centrifuged at 13,000
×
gfor 15 min at
4
◦
C. In brief, 100
µ
L of sample was added into a pre-coated plate and incubated for 2 h at 37
◦
C.
After removing any unbound substances, a biotin-conjugated antibody specific for A
β1–42
was added
to the wells. After washing, avidin conjugated Horseradish Peroxidase (HRP) was added to the wells.
Following a wash to remove any unbound avidin-enzyme reagent, a substrate solution was added
to the wells and color developed in proportion to the amount of A
β1–42
bound in the initial step.
The color development was stopped and the intensity of the color was measured using a microplate
absorbance reader (Sunrise™, TECAN, Männedorf, Switzerland).
4.2.11. Oxidative Stress Assay
Hydrogen peroxides were measured according to the manufacturer’s instructions (Cell Biolabs,
San Diego, CA, USA). Malondialdehyde (MDA) and hydrogen peroxide were measured according
to the manufacturer’s instructions (Cayman Chemical, Ann Arbor, MI, USA). To perform the assay,
the brain tissues were homogenized, then normalized to protein concentration. Superoxide production
in the brain was detected by dihydroethidium staining (Sigma-Aldrich). Brains were incubated with
5
µ
M DHE for 30 min at 37
◦
C in a humidified chamber protected from light. The average fluorescence
intensity of the nuclei was then analyzed using Image Pro-Plus software (Media Cybernetics, Inc.,
Rockville, MD, USA).
4.2.12. Assay of β-Secretase Activities
β
-secretase activity in the mice brains was determined using a commercially available
β
-secretase
activity kit (Abcam, Inc., Cambridge, MA, USA) using a fluorescence spectrometer (Gemini EM,
Molecular Devices, CA, USA) as described elsewhere [47].
Int. J. Mol. Sci. 2017,18, 2554 13 of 15
4.2.13. Microglial BV-2 Cell Culture
Microglial BV-2 cells were maintained with serum-supplemented culture media of DMEM
supplemented with FBS (10%) and penicillin (100 units/mL). The microglial BV-2 cells were incubated
in the culture medium in a humidified incubator at 37
◦
C and 5% CO
2
. The cultured cells were
treated simultaneously with LPS (1
µ
g/mL) and several concentrations (50, 100
µ
M) of EPA and DHA
dissolved in DMSO.
4.2.14. Statistical Analysis
For the measurement of the image data, ImageJ (Wayne Rasband, National Institutes of Health,
Bethesda, MD, USA) was used. For the measurement of the image data, ImageJ (Wayne Rasband,
National Institutes of Health, Bethesda, MD, USA) was used. Group differences were analyzed
by one-way ANOVA followed by Bonferroni’s post-hoc analysis using GraphPad Prism 5 software
(Version 5.02, GraphPad Software, Inc., La Jolla, CA, USA).
Acknowledgments:
This work is financially supported by the National Research Foundation of Korea [NRF]
Grant funded by the Korea government (MSIP) (No. MRC, 2017R1A5A2015541), and by the Functional Districts
of the Science Belt support program, Ministry of Science, ICT and Future Planning.
Author Contributions:
Ji Yeon Choi, Dong Ju Son, Jun Sung Jang and Jin Tae Hong designed the experiments
and wrote the manuscript. Ji Yeon Choi, Dong Ju Son, Jun Sung Jang performed the experiments. Ji Yeon Choi,
Dong Ju Son, Jun Sung Jang, Hyung-Sik Im, Ji Yeong Kim, Joung Eun Park, Won Rak Choi, Sang-Bae Han
and Jin Tae Hong analyzed the data. Jin Tae Hong supervised the whole paper. All authors have reviewed
the manuscript.
Conflicts of Interest: The authors declare no conflict of interest.
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