Epigallocatechin-3-gallate rescues LPS-impaired adult hippocampal neurogenesis through suppressing the TLR4-NF-κB signaling pathway in mice

Abstract

Adult hippocampal dentate granule neurons are generated from neural stem cells (NSCs) in the mammalian brain, and the fate specification of adult NSCs is precisely controlled by the local niches and environment, such as the subventricular zone (SVZ), dentate gyrus (DG), and Toll-like receptors (TLRs). Epigallocatechin-3-gallate (EGCG) is the main polyphenolic flavonoid in green tea that has neuroprotective activities, but there is no clear understanding of the role of EGCG in adult neurogenesis in the DG after neuroinflammation. Here, we investigate the effect and the mechanism of EGCG on adult neurogenesis impaired by lipopolysaccharides (LPS). LPS-induced neuroinflammation inhibited adult neurogenesis by suppressing the proliferation and differentiation of neural stem cells in the DG, which was indicated by the decreased number of Bromodeoxyuridine (BrdU)-, Doublecortin (DCX)- and Neuronal Nuclei (NeuN)-positive cells. In addition, microglia were recruited with activatingTLR4-NF-κB signaling in the adult hippocampus by LPS injection. Treating LPS-injured mice with EGCG restored the proliferation and differentiation of NSCs in the DG, which were decreased by LPS, and EGCG treatment also ameliorated the apoptosis of NSCs. Moreover, pro-inflammatory cytokine production induced by LPS was attenuated by EGCG treatment through modulating the TLR4-NF-κB pathway. These results illustrate that EGCG has a beneficial effect on impaired adult neurogenesis caused by LPSinduced neuroinflammation, and it may be applicable as a therapeutic agent against neurodegenerative disorders caused by inflammation.

Full text

www.kjpp.net Korean J Physiol Pharmacol 2016;20(1):41-51
41
Korean J Physiol Pharmacol 2016;20(1):41-51
http://dx.doi.org/10.4196/kjpp.2016.20.1.41
Author contributions: K.J.S., H.G.L., J.Y.J. and W.J.K. designed the experi-
ments. Cell cultures, immunohistochemistry and molecular works was
performed by S.H.C., H.G.L., H.M.K. and M.S.K. K.J.S. and H.G.L. took care
all animals. K.J.S., H.G.L., J.Y.J. and W.J.K. analyzed the data and wrote the
manuscript.
This is an Open Access article distributed under the terms
of the Creative Commons Attribution Non-Commercial
License, which permits unrestricted non-commercial use, distribution, and
reproduction in any medium, provided the original work is properly cited.
Copyright © Korean J Physiol Pharmacol, pISSN 1226-4512, eISSN 2093-3827
INTRODUCTION
Neuroinflammation with microglial activation plays a role
in neurodegenerative diseases, such as Alzheimer's disease and
multiple sclerosis [1], and microglia involved in the inflammation
of the central nervous system have neuroprotective effects [2-
4]. Recent
in vivo
and
in vitro
studies have suggested that
microglia are essential in neural development, synapse formation,
and neuroinflammation involved in the impairment of adult
neurogenesis [5-7]. This evidence was supported by the Gram-
negative bacterial immune stimulation lipopolysaccharide (LPS)-
induced neuroinflammatory model, which showed that LPS-
induced activation of the Toll-like receptor 4 (TLR4) in microglia
led to the production of the pro-inflammatory cytokines, IL-
1
b
, IL-6, and TNF-
a
through the MAPK and NF
k
B pathways
in BV2 cell lines [8,9]. In addition to the immune response by
activated microglia, the neuroprotective cytokines, IL-4 and IL-
25, are also produced [10,11]. Moreover, other studies showed that
Original Article
Epigallocatechin3gallate rescues LPSimpaired adult
hippocampal neurogenesis through suppressing the TLR4NF
k
B
signaling pathway in mice
Kyung-Joo Seong
1,2,3,#
, Hyun-Gwan Lee
1,2,3,#
, Min Suk Kook
1,2,4
, Hyun-Mi Ko
5
, Ji-Yeon Jung
1,2,3,
*, and Won-Jae Kim
1,2,3,
*
1
Dental Science Research Institute,
2
Medical Research Center for Biomineralization Disorders,
3
Department of Oral Physiology,
4
Department of Oral and
Maxillofacial Surgery, School of Dentistry, Chonnam National University, Gwangju 61186,
5
Department of Microbiology, Collage of Medicine, Seonam
Universtity, Namwon 55724, Korea
ARTICLE INFO
Received July 9, 2015
Revised
August 21, 2015
Accepted
September 1, 2015
*Correspondence
Won-Jae Kim
E-mail: wjkim@jnu.ac.kr
Ji-Yeon Jung
E-mail: jjy@jnu.ac.kr
Key Words
Adult Neurogenesis
Epigallocatechin-3-gallate
Neural stem cells
Neuronal Inammation
NF-
k
B signaling
TLR4
#
These authors contributed equally to
this work.
ABSTRACT Adult hippocampal dentate granule neurons are generated from
neural stem cells (NSCs) in the mammalian brain, and the fate specification of
adult NSCs is precisely controlled by the local niches and environment, such as
the subventricular zone (SVZ), dentate gyrus (DG), and Toll-like receptors (TLRs).
Epigallocatechin-3-gallate (EGCG) is the main polyphenolic flavonoid in green
tea that has neuroprotective activities, but there is no clear understanding of the
role of EGCG in adult neurogenesis in the DG after neuroinflammation. Here, we
investigate the eect and the mechanism of EGCG on adult neurogenesis impaired
by lipopolysaccharides (LPS). LPS-induced neuroinflammation inhibited adult
neurogenesis by suppressing the proliferation and dierentiation of neural stem cells
in the DG, which was indicated by the decreased number of Bromodeoxyuridine
(BrdU)-, Doublecortin (DCX)- and Neuronal Nuclei (NeuN)-positive cells. In
addition, microglia were recruited with activatingTLR4-NF-
k
B signaling in the
adult hippocampus by LPS injection. Treating LPS-injured mice with EGCG restored
the proliferation and differentiation of NSCs in the DG, which were decreased
by LPS, and EGCG treatment also ameliorated the apoptosis of NSCs. Moreover,
pro-inflammatory cytokine production induced by LPS was attenuated by EGCG
treatment through modulating the TLR4-NF-
k
B pathway. These results illustrate
that EGCG has a beneficial effect on impaired adult neurogenesis caused by LPS-
induced neuroinammation, and it may be applicable as a therapeutic agent against
neurodegenerative disorders caused by inammation.

42
http://dx.doi.org/10.4196/kjpp.2016.20.1.41Korean J Physiol Pharmacol 2016;20(1):41-51
Seong KJ et al
adult neurogenesis in the hippocampus was affected through the
TLR and NF-
k
B pathway in microglia [12 ,13].
Adult neurogenesis is a process in which new neurons are
continuously generated throughout adult life from the neural
stem cells (NSCs) in the mammalian brain. Small populations
of neurons continue to be born in the adult subventricular
zone, the olfactory system, and the DG of the hippocampus.
The neurogenic niches are important for the NSCs to have
self-renewal and multipotent properties that can give rise
to neurons, astrocytes, and oligodendrocytes [14-16]. The
neuronal fate specification (neurogenesis) and glial fate
specification (gliogenesis) of the NSCs is determined by cellular
communication within the microenvironment [17]. The newborn
neurons are critical to cognitive functions including learning
and memory. For instance, physical activity and an enriched
environment, which are associated with improved memory
function and synaptic plasticity, enhance adult neurogenesis
in the DG [18,19]. Furthermore, adult neurogenesis in the DG
declines with aging and neurodegenerative diseases, which in
turn causes cognitive deficits [20-22].
Green tea has been shown to function as an anticancer
reagent that inhibits the development and progression of skin,
lung, mammary gland, and gastrointestinal tract tumors
in animal models [23,24]. Among the various polyphenols
in green tea including epigallocatechin-3-gallate (EGCG),
(–)-epigallocatechin, (–)-epicatechin, (+)-gallocatechin, and other
catechins, EGCG is the most abundant and the most biologically
active component [25-27]. However, the recovery effects of EGCG
on adult neurogenesis following neuroinflammation at the DG
in the hippocampus are still unknown. In the present study, we
investigate the effect of EGCG on impaired adult neurogenesis in
a mouse model of LPS-induced neuroinflammation.
Here, we present the effects of EGCG on impaired adult
hippocampal neurogenesis
in vivo
caused by neuroinflammation.
We induced inflammation by administering LPS in a
mouse model and explored the positive effects of EGCG
in NSC proliferation and neural differentiation using an
immunohistochemical approach to detect bromodeoxyuridine
(BrdU, an S-phase marker of proliferating cells), doublecortin
(DCX, a marker of immature newborn neurons), neuronal nuclei
(NeuN, a marker of mature neurons), ionized calcium binding
adapter molecule 1 (Iba-1, a marker of microglia), and the levels
of cytokines.
METHODS
Animals & chemicals
7-week-old male C57BL/6 mice were purchased from Daehan
Biolink (Chungbuk, Korea). The animals were kept under
standard conditions of 22
o
C and a humidity of 55% with a
12 hour light-12 hour dark cycle, and allowed free access to
food and water. Mice were divided into the following three
groups: saline-treated group (sham control), saline-treated
with lipopolysaccharide (LPS,
E
.
coli
, serotype 055:B5, St. Louis,
MO, USA) intracerebroventricular (I.C.V.) injection group,
and epigallocatechin-3-gallate (EGCG)-treated with LPS I.C.V.
injection group (n=5 mice/ each group). Supplementary Figure 1
demonstrated the schematic experimental design of time courses
with LPS, EGCG and BrdU injection followed by sampling the
brain for immunohistochemistry. All experiments were approved
by the Animal Care and Use Committee of Chonnam National
University. All chemicals used in the experiments were supplied
by Sigma (St. Louis, MO, USA).
LPS-induced neuroinflammation in the brain
Experimental mice were anesthetized with zoletil (VIRBAC,
Milperra, Australia) (25 mg/kg) and Rompun (Bayer HealthCare,
Mississauga, Canada) (10 mg/kg). LPS for a single unilateral
stereotaxic injection was prepared in phosphate-buffered saline
(PBS, pH 7.4) at a final concentration of 1.0 mg/ml and was
injected into the brain (0.6 mm caudal to the bregma, 1.5 mm to
the right of the bregma, 2.0 mm ventral to the bregma). Mice were
injected with vehicle (sterile saline) or LPS (3
m
g in 3
m
l sterile
saline) following brain surgery at a dose of 1
m
g/min.
EGCG treatment
EGCG (0.5 mg/kg) prepared in physiological saline (PBS
containing 0.9% NaCl) was injected intraperitoneally (i.p.) for
three times with 6 hours interval at 3 hours after recovered from
anesthesia for LPS-injection.
BrdU injection
BrdU (Sigma, St. Louis, MO, USA) was dissolved in
physiological saline and injected i.p. (50 mg/kg). To examine
the proliferation of NSCs in the DG, BrdU was injected into the
animals once, 3 hours prior to sacrifice. To study the survival rate
of newborn cells generated from NSCs and the differentiation rate
of NSCs, BrdU was injected once daily for 5 consecutive days and
the mice were sacrificed on 28
th
day after the final BrdU injection.
Tissue preparation
Animals were perfused transcardially with 0.1 M PBS, followed
by fresh cold 4% paraformaldehyde (PFA) in 0.1 M PBS. The
brains were removed and fixed overnight in 4% PFA in 0.1 M
PBS at 4
o
C, and then washed for 6 hours in PBS at 4
o
C. Sucrose-
saturated brains were then embedded in freezing media (O.C.T
compound, Leica Biosystems, Richmond, USA), frozen in chilled
isopentane (–25
o
C), and stored at –80
o
C until sectioning. Brains

EGCG in adult neurogenesis impaired neuroinflammation in mouse
Korean J Physiol Pharmacol 2016;20(1):41-51www.kjpp.net
43
were cryocut coronally at a thickness of 40
m
m using a Cryostat
(Model CM3050; Leica Microsystems, Richmond, USA) and
stored in cryoprotectant solution (25% ethylene glycol, 25%
glycerol, and 0.05 M sodium phosphate buffer, Na-PB) at –20
o
C
until immunohistochemical (IHC) processing.
Fluorescent immunohistochemical staining (IHC)
Sections were washed in Na-PB and mounted on charged
slide glasses for IHC. The sections immunostained for BrdU
were pretreated with 2 N HCl for 30 minutes at 37
o
C and
neutralized with PBS before incubation with primary antibodies.
Sections were incubated for 60 minutes with 5% normal horse
serum in 0.4% Triton X-100 in PBS (PBST). The sections were
incubated overnight at 4
o
C with primary antibodies in the same
buffer solution. Primary antibodies were used at the following
concentrations: rat anti-BrdU (1:200; Abcam, Cambridge, UK),
goat anti-doublecortin (DCX, 1:100; Cell Signaling, Danvers,
MA), mouse anti-neuronal nuclear antigen (NeuN, 1:100;
Chemicon, Temecula, CA), and rabbit anti-ionized calcium-
binding adapter molecule 1 (Iba-1, 1:100; Wako Chemicals USA,
Inc., Richmond, VA). Sections were washed three times with
PBST for 10 minutes at room temperature and blocked in PBST
containing 5% horse serum for 30 minutes. Sections were then
incubated for 2 hours with secondary antibodies conjugated
to FITC (Jackson Immuno-Research, West Grove, PA) or CY3
(Jackson Immuno-Research, West Grove, PA). The sections
were washed three times with PBST, and stained with 10 mg/ml
4’6’-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich, St. Louis,
USA) for 30 minutes before mounted.
Quantification and image analysis
IHC images were acquired with a confocal microscope (Zeiss,
Thornwood, NY) equipped with an argon/krypton laser (488
nm), two helium/neon lasers (543 and 633 nm), and a coherent
laser (Santa Clara, CA), using the 20x objective lens. Images
were analyzed with the Image J (Ver.1.4, NIH, USA). Cells were
counted in a defined frame size of 20~50 with 35
m
m×35
m
m
in every selected section. Colocalizations were investigated
by performing z-stack acquisitions and three-dimensional
reconstructions with the LSM software (Zeiss, Thornwood, NY).
Adobe Photoshop (Version CS6, Adobe Systems, San Jose, CA)
was used to adjust the contrast and brightness.
Western blot analysis
Brain was homogenized in RIPA buffer (150 mM NaCl, 1.0%
IGEPAL
Ⓡ
CA-630, 0.5% sodium deoxycholate and 0.1% SDS in
50 mM Tris, Sigma, St. Louis, MO, USA). Proteins were separated
by SDS-PAGE and transferred to a nitrocellulose membrane. The
membranes were incubated with primary anti-TLR4, anti-RelA,
anti-pRelA, and anti-Actin antibodies (Cell Signaling, Danvers,
MA or Abcam, Cambridge, UK). The expression levels of protein
were visualized and analyzed with Fusion FX (Vilber Lourmat,
Eberhardzell, Germany).
Cytokine measurements
The levels of IL-1
b
, IL-6, and TNF-
a
in the DG were measured
using a commercial ELISA kit (R&D Systems, Minneapolis,
MN, USA) as per the manufacturer's instructions, and using
quantitative real-time PCR (Bio-Rad Lab. Inc., CA, USA). In
detail, the total RNAs were prepared using Trizol Reagent (Gibco
BRL) and reverse transcription was performed with a RT system
containing Moloney Murine Leukemia Virus reverse transcriptase
(Promega, Madison, WI) in accordance with the manufacturer’s
instructions. PCR was performed in a Palm-Cycler thermocycler
(Corbett Life Science, Sydney, Australia) and the product was
resolved in a 1.2% agarose gel. Real time amplification of cDNA
was conducted in a Rotor-Gene 3000 System (Corbett Research,
Morklake, Australia) using the SYBR Green PCR Master Mix
Reagent Kit (Qiagen, Valencia, CA). The PCR conditions were as
follows: incubation for 5 minutes at 95
o
C, followed by 30 cycles of
denaturation for 15 seconds at 95
o
C, annealing for 15 seconds at
62
o
C and extension for 15 seconds at 72
o
C. The primers were as
follows: mouse IL-1
b
; 5'-AGG AGA ACC AAG CAA CGA CA-3'
and 5'-CTT GGG ATC CAC ACT CTC CAG-3', mouse IL-6; 5'-
GCC TTC TTG GGA CTG ATG CT-3' and 5'-GCC TTC TTG
GGA CTG ATG CT-3', mouse TNF-
a
; 5'-ATG GCC TCC CTC
TCA TCA GT-3' and 5'-CTT GGT GGT TTG CTA CGA CG-
3' and
b
-actin; 5'-GAT CTG GCA CCA CAC CTT CT-3' and 5'-
GGG GTG TTG AAG GTC TCA AA-3'. The relative levels of
mRNA were calculated using the standard curve generated from
the cDNA dilutions. The mean cycle threshold (Ct) values from
quadruplicate measurements were employed in the calculation
of gene expression, with normalization to β-actin as an internal
control. Calculation of the relative levels of gene expression was
performed using Corbett Robotics Rotor-Gene software (Rotor-
Gene 6 version 6.1, Build 90).
Data analysis
A complete series of 1 in 10 sections of the DG was analyzed.
For each experiment, 9 sections per mouse were selected for
analysis. The immunolabeled cells were counted and multiplied
by 10 to obtain the total number of labeled cells throughout
the DG. The data are expressed as the mean±SEM. Statistical
analyses were performed using a one-way analysis of variance
(ANOVA) followed by a Student-Newman-Keuls test for multiple
comparisons. Results were considered statistically significant
when the p value was less than 0.05.

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Seong KJ et al
RESULTS
EGCG recovered the proliferation of adult NSCs in the
LPS-injured hippocampal DG
To study the effect of EGCG in the DG related to neurogenesis,
the proliferation of adult NSCs in the DG was initially examined
in an LPS-induced neuroinflammation mouse model by
administering LPS (3
m
g/animal with 1 mg/ml stock) via
intracerebroventricular (I.C.V.) injection (n=5 each). The number
of BrdU (S-phase marker)-positive cells were compared at 3 hours
following a single BrdU injection (i.p. 50 mg/kg), which indicates
proliferating cells, and were found to be significantly decreased
at 1 day (0.7066±0.04408×10
3
, 38% of decreased compared to
sham control,
F
(4.4)=1.076, p=0.0002, two-tailed unpaired one-
way ANOVA) and day 3 (0.8292±0.03464×10
3
, 29% of decreased
compared to sham control,
F
(4.4)=1.577, p=0.0003, two-tailed
unpaired one-way ANOVA) post-LPS injection in mice treated
with either vehicle or EGCG compared with the sham controls.
However, EGCG significantly improved the number of BrdU-
positive cells affected by LPS in the hippocampal DG (day1;
0.8242±0.01520×10
3
, 16% of increased compared with LPS only,
F
(4.4)=8.406, p=0.0357, day 3; 0.9462±0.03309×10
3
, 14% of
increased compared with LPS only,
F
(4.4)=1.096, p=0.0404, two-
tailed unpaired one-way ANOVA) (Fig. 1). These results show
that treatment with EGCG recovered NSC proliferation, which
was impaired by LPS-induced neuroinflammation.
EGCG ameliorated the immature neuronal
differentiation of adult NSCs in the LPS-injured
hippocampal DG
DCX (immature neuronal marker) is transiently expressed
in the soma and dendrites of immature newborn neurons.
The newborn NSCs are differentiated into neurons to serve
as functional units in the nervous system following their
proliferation. Thus, we studied whether EGCG ameliorated
the differentiation of adult NSCs after LPS-induced injury by
counting the number of BrdU- and DCX double-positive cells
and the ratio of BrdU
+
DCX
+
cells/BrdU
+
cells in the hippocampal
DG 5 days after injection with BrdU (n=5 each). The number of
double positive cells was decreased post LPS injection compared
to that of sham controls (1.070±0.06641×10
3
, 32% of decreased
compared to sham control,
F
(4.4)=1.287, p=0.0013), but it was
significantly improved after injection with EGCG in the LPS-
injured group compared with the LPS-injured group with no
treatment (1.398±0.07632×10
3
, 30% of increased compared to LPS
only,
F
(4.4)=1.321, p=0.0118) (Fig. 2A and B). Similarly, the ratio
of the total number of BrdU- and DCX-double positive cells to
the total number of BrdU-positive cells was also decreased in the
LPS-treated group compared with the sham controls (43.60±1.887
×10
3
, 11.4% of decreased compared to sham control,
F
(4.4)=1.017,
p=0.0026), but the ratio was recovered in the EGCG-treated
LPS-injured group compared with the LPS-injured group with
no treatment (50.80±1.985×10
3
, 7.2% of increased compared to
LPS only,
F
(4.4)=1.107, p=0.0302, two-tailed unpaired one-way
A NOVA) (Fig. 2A and C). These results suggest that EGCG had
a recovery effect on the neuronal differentiation of adult NSCs,
which was impaired by LPS in the early stage.
Fig. 1. The effect of EGCG on the prolife-
ration of adult NSCs in the DG impaired
by LPS-induced neuroinflammation.
(A) Representative images show BrdU-
positive cells in the adult dentate gyrus
(DG) on day 1 and day 3 after BrdU in-
jection following LPS injection. Newly
proliferated cells in the subgranular zone
(SGZ) of the DG were immunolabeled
using anti-BrdU (green). (B, C) Quantitative
analysis of the number of BrdU-positive
cells in the DG of the hippocampus
on day 1 and day 3 after LPS injection.
(n=5 each). BrdU-positive cells were
decreased in the LPS-injured group at
1
st
, and 3
rd
day post brain inflammation,
which was improved significantly
by EGCG (p=0.0357 and p=0.0404
respectively, one-way ANOVA) (*p<0.05,
***p<0.001). Data are expressed as the
mean±SEM. Scale bar, 200
m
m.

EGCG in adult neurogenesis impaired neuroinflammation in mouse
Korean J Physiol Pharmacol 2016;20(1):41-51www.kjpp.net
45
EGCG amended the mature neuronal differentiation
of adult NSCs in the LPS-injured hippocampal DG
To investigate the effect of EGCG on the maturation of
newborn neurons following LPS-induced neuroinflammation,
immunohistochemical analysis was performed using antibodies
against the mature neuronal marker, NeuN, and BrdU. The
results show that the number of cells co-localized with NeuN and
BrdU are decreased in the LPS-injured group (0.6280±0.03967×
10
3
, 27% of decreased compared to sham control,
F
(4.4)=1.623,
p=0.0021), but the number of these cells are increased in the
EGCG-treated LPS-injured group compared with the vehicle-
treated LPS-injured group (0.7476±0.02307×10
3
, 19% of increased
compared to LPS only,
F
(4.4)=2.958, p=0.0313) (Fig. 3A and B).
Furthermore, the ratio of NeuN/BrdU double-positive cells to
the total BrdU-positive cells in the EGCG-treated LPS-injured
group was significantly increased compared with that of the
vehicle-treated LPS-injured group (52.80±1.158, 5.6% of increased
compared to LPS only, F(4.4)=2.791, p=0.0378, two-tailed
unpaired one-way ANOVA), which was decreased compared
with the sham control group (Fig. 3C). These results support
the notion of a beneficial effect of EGCG on mature neuronal
differentiation of adult NSCs, which was impaired by LPS-
induced neuroinflammation.
EGCG improved adult NSC survival in the LPS-injured
hippocampal DG
Following NSC proliferation in the DG, the newborn NSCs
survive and differentiate into neurons. To determine the survival
rate of newborn cells derived from NSCs in the DG, the number
of BrdU-positive cells was quantified 3 hours and 28 days after
the last injection of BrdU, which was injected once a day for 5
consecutive days. The total number of BrdU-positive cells was
decreased in the LPS-injured group, and was recovered in the
EGCG-treated LPS-injured group up to the level of the sham
controls (n=5 each). Subsequently, we quantified the survival rate
of newborn cells by counting the total BrdU-positive cells, 28
days after the final of 5 consecutive days of BrdU injections, to
BrdU-positive cells, 3 hours after the final of 5 consecutive days
of BrdU injections. The rate was decreased in all groups, but the
EGCG-treated LPS-injured group showed an improved survival
rate of newborn cells compared with the LPS-injured group
with no treatment (43.20±0.8602, 3.8% of increased compared
to LPS only,
F
(4.4)=1.162, p=0.0170, two-tailed unpaired one-
way ANOVA) (Fig. 4A and C). Further, to examine the effect of
Fig. 2. The effect of EGCG on the im-
mature neuronal differentiation in
the DG after LPS-induced neuroin-
flammation. (A) Representative images
show BrdU- and DCX-positive cells in the
DG 5 days after BrdU injection following
LPS injection. BrdU (green) and DCX
(red) double stained cells (yellow)
represent newly generated immature
neurons (arrows). (B, C) Quantitative
analysis of the number of BrdU-po-
sitive cells and/or DCX-positive cells
(n=5 each). The differentiation of the
NSCs in the hippocampal DG affected
by LPS-induced neuroinflammation
was recovered by EGCG (0.5 mg/kg)
(p=0.0302, one-way ANOVA) (*p<0.05,
**p<0.01). Data are expressed as the
mean±SEM. Scale bar, 200
m
m.

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Seong KJ et al
EGCG on newborn cell survival related to apoptosis, the number
of cleaved caspase 3-positive cells was counted. The total number
of cleaved caspase 3-positive cells was significantly decreased in
the EGCG-treated LPS-injured group compared with the vehicle-
treated LPS-injured group (0.4880±0.01393×10
2
, 12% of decreased
compared to LPS only, F (4.4)=1.062, p=0.0109, two-tailed
unpaired one-way ANOVA) (Fig. 4B and D). The results show
that neuroinflammation decreased the survival of dividing cells,
whereas EGCG recovered the survival of dividing cells, which
was impaired by LPS-induced neuroinflammation.
EGCG downregulated the expression of
proinflammatory cytokines through the TLR4/NF-
k
B
pathway in LPS-induced neuroinflammation
To study the effect of EGCG on neuroinflammation in
detail, the activity of microglia following LPS I.C.V. injection
was quantified by the immunoreactivity of Iba-1, a specific
marker of microglia (n=5 each). The number of Iba-1-positive
cells in the DG was increased (day 1; 4.876±0.2238×10
3
, 54% of
increased compared to sham control,
F
(4.4)=2.181, p=0.0002,
day 3; 4.528±0.2567×10
3
, 39% of increased compared to sham
control,
F
(4.4)=2.917, p=0.0025, two-tailed unpaired one-way
ANOVA ) by LPS injection, which was suppressed at day 1
and day 3 of EGCG treatment (day 1; 3.393±0.2497×10
3
, 7% of
decreased compared to LPS only,
F
(4.4)=1.245, p=0.0022, day
3; 3.402±0.1609×10
3
, 4% of decreased compared to LPS only,
F
(4.4)=2.544, p=0.0059, two-tailed unpaired one-way ANOVA )
(Fig. 5A and B). Since LPS acted as a TLR4 agonist, the expression
of TLR4, NF-
k
B, and cytokines was examined as molecular
targets of EGCG in adult neurogenesis impaired by LPS-induced
neuroinflammation. The expression of TLR4 and phospho-
RelA (activated form) was upregulated in the LPS-injured group
compared with the sham controls, but the expression of TLR4
and phospho-RelA was suppressed in the EGCG-treated LPS-
injured group compared with the LPS-injured group. Therefore,
LPS activated the expression of TLR4 and stimulated NF-
k
B, but
EGCG attenuated the LPS-induced TLR4 and NF-
k
B (Fig. 5C).
In addition, LPS upregulated the production of IL-1
b
, IL-6, and
TNF-
a
by microglia, however, the increased mRNA and protein
levels of cytokines induced by LPS injection were compromised
by EGCG up to 53.75%, 46.67%, and 30.71% for IL-1
b
, IL-6 and
TNF-
a
transcripts, respectively, and up to 72.61%, 47.59% and
59.57% for proteins, respectively, compared with the expression
levels of the LPS-injured group (p<0.05) (Fig. 5D~G). The results
demonstrate that EGCG suppressed the inflammatory activities
Fig. 3. The effect of EGCG on the mature
neuronal differentiation in the DG after
LPS- induced neuroinflammation. (A)
Representative images show BrdU and
NeuN double immunostained cells in
the DG 28 days after BrdU injection
following LPS injection. BrdU (green)
and NeuN (red) double immunostained
cells (yellow) represent newly generated
mature neurons (arrows). (B, C) Quantitative
analysis of the number of BrdU- and/or
NeuN-positive cells (n=5 each). BrdU and
NeuN double positive cells represent
matured neurons from adult NSCs that
are affected by neuroinflammation, and
the damage was rescued by EGCG (0.5
mg/kg) (p=0.0378, one-way ANOVA) (*p
<0.05, **p<0.01). Data are expressed as
the mean±SEM. Scale bar, 200 µm.

EGCG in adult neurogenesis impaired neuroinflammation in mouse
Korean J Physiol Pharmacol 2016;20(1):41-51www.kjpp.net
47
of microglia, resulting in the attenuation of proinflammatory
cytokine production via the TLR4/NF-
k
B signaling pathway.
DISCUSSION
Adult NSCs in the hippocampal DG have the ability to generate
self-renewing neural stem cells possessing multipotency [28].
Adult neurogenesis occurs in three discrete stages, proliferation
of NSCs, cell survival, and neuronal differentiation, which are
affected by various factors such as hormones, growth factors, enriched
environment, stress, drugs, and pathological stimulation [29,30].
The process in the DG affected by these factors is associated
with cognitive functions including learning and memory [31].
Neuroinflammation in the brain following injuries causes neural
development-related diseases, which modulate adult hippocampal
neurogenesis [32,33]. During the inflammatory response
following injury to the central nervous system (CNS), microglial
cells and a population of glial cells of the CNS as immune cells
produce proinflammatory cytokines. In particular, IL-6 and NO
affect adult hippocampal neurogenesis, which are involved in the
pathogenesis of neurological diseases such as Alzheimer's disease
(AD) and Parkinson's disease (PD) [34,35].
EGCG, among the catechins, a major subgroup of poly-
phenolic flavonoids in green tea, is well-known for its anti-
carcinogenic effects via the suppression of the proliferation and
angiogenesis of cancer cells [36]. Even though the instability
of catechins, previous studies were showing that EGCG was
effectively delivered over blood brain barrier and was founded
physiologically activated forms in the brain [37,38]. In addition,
it may be used for the prevention and treatment of AD and PD
based on the evidence that EGCG promotes adult hippocampal
neurogenesis [39-41]. Moreover, recent studies have demonstrated
that EGCG had a neuroprotective effect through the reduction of
neuroinflammation [42-45]. Oxidative stress has been implicated
in the pathophysiology of the majority of neurodegenerative
diseases, and most of the researches in this field have focused on
EGCG as a natural pharmacological compound [46]. However,
there have been no studies assessing the effect of EGCG on
neurogenesis in the DG of the adult hippocampus impaired by
neuroinflammation. Therefore, here, we investigated whether
EGCG improves adult neurogenesis via the modulation of
neuroinflammation
in vivo
.
In the present study, the effect of EGCG on the proliferation of
adult NSCs in the DG following LPS injection was investigated.
Proliferation of adult NSCs in the DG was inhibited by LPS
Fig. 4. The effect of EGCG on the survival
of newborn cells in the DG after LPS-
induced neuroinflammation. (A) Re-
presentative images show BrdU-positive
cells (green) in the DG at 3 hours (upper)
or 28 days (lower) after consecutive
BrdU injection for 5 days, following LPS
injection. (B) Representative images
show cleaved caspase-3 immunoreactive
cells (red) in the DG 28 days after
consecutive BrdU injection for 5 days.
(C) The survival rate of newborn cells
in LPS-induced injures was improved
significantly by EGCG (0.5 mg/kg) which
was analyzed by counting BrdU-positive
cells in the DG of the hippocampus at 5
days over 28 days (n =5 each, p=0.0170,
one-way ANOVA) (*p<0.05, **p<0.01).
(D) Quantitative analysis of the number
of cleaved caspase-3-positive cells in the
DG of the hippocampus (n=5 each). The
number of cleaved caspase-3-positive
cells in the DG of the hippocampus
was decreased in the EGCG-treated
LPS-injured group (p=0.0109, one-way
ANOVA) (*p<0.05). Data are expressed as
the mean±SEM; Scale bar in A and B, 200
m
m.

48
http://dx.doi.org/10.4196/kjpp.2016.20.1.41Korean J Physiol Pharmacol 2016;20(1):41-51
Seong KJ et al
injection, however, the number of BrdU-positive cells indicating
the rate of adult NSC proliferation was improved within one day
in the EGCG-treated LPS-injured group (Fig. 1A~C). Moreover,
the survival rate of newborn cells, represented by the ratio of
the total number of BrdU-positive cells 28 days after the final
BrdU injection following LPS injection, was amended by EGCG
treatment (Fig. 2A~C). These results suggest that EGCG rescued
the proliferation and survival of adult NSCs in the DG impaired
by LPS-induced neuroinflammation, which is consistent with
that of previous reports showing the effective role of EGCG in
increasing cell proliferation in mouse hippocampal DG and
neural progenitor cell proliferation during adult hippocampal
neurogenesis [47, 4 8].
To investigate the effect of EGCG on immature neuronal
differentiation in the DG following LPS-induced neuroin-
flammation, immunohistochemical analysis was performed using
a marker of immature neural differentiation, DCX, and BrdU, 5
days after BrdU injection following LPS with or without EGCG
injection. The number of double positive cells (DCX
+
and BrdU
+
)
was decreased post LPS injection compared with that of the sham
controls, however, it was significantly improved in the EGCG-
treated LPS-injured group compared with the LPS-injured group
with no treatment (Fig. 3A~C). The results indicate that EGCG
ameliorated the neuronal differentiation of NSCs impaired by LPS
in the adult hippocampal DG at an early stage. In addition, NeuN
(a mature neuronal marker) and BrdU- positive cells at 28 days
were decreased in the LPS-injured group, whereas the number
of double positive cells was increased in the EGCG-treated LPS-
Fig. 5. The effect of EGCG on microglia and proinflammatory cytokines after LPS- induced neuroinflammation. (A) Representative images show
Iba-1-positive cells (green) in the DG on day 1 (upper) and day 3 (lower) after LPS injection. (B) The positive effect of EGCG was quantified by counting
the number of Iba-1-positive cells that was significantly reduced by EGCG (0.5 mg/kg) compared with LPS only (n=5 each, day 1; p=0.0002, day 3;
p=0.0025 respectively, one-way ANOVA). (C) Western blotting analysis was performed to quantify the TLR4, Rel A, active form of Rel A (pRel A), and
b
-actin as a control in the hippocampal DG. The TLR4-NF
k
B pathway was increased by LPS, which was rescued by EGCG (0.5 mg/kg) (n=4 each). (D~G)
Quantitative real-time PCR and ELISA was performed to measure IL-1β, IL-6, and TNF-α in the hippocampus after LPS-induced neuroinflammation (n=4
each). The levels of IL-1
b
, IL-6, and TNF-
a
were suppressed by EGCG treatment in LPS-induced (n=4 each) p<0.05, **p<0.01 compared with the control,
★
p<0.05 and
★★
p< 0.01 compared with the LPS-injured group, ***p<0.001). Data are expressed as the mean±SEM. Scale bar in A, 200
m
m (upper) and
in an inset, 100
m
m (lower).

EGCG in adult neurogenesis impaired neuroinflammation in mouse
Korean J Physiol Pharmacol 2016;20(1):41-51www.kjpp.net
49
injured group compared with the vehicle-treated LPS-injured
group (Fig. 4A~C). It is indicating that EGCG had a recovery
effect on the neuronal differentiation of adult NSCs at a late
stage, which was impaired by LPS induced neuroinflammation.
These results demonstrate that the neuronal differentiation of
adult NSCs in the DG is rescued by the injection of EGCG into
mice with LPS-induced neuroinflammation. Our data also was
supported the beneficial effect of EGCG on adult neurogenesis
by showing improved proliferation and differentiation of adult
mouse hippocampal NSCs in the DG under normal conditions
[48].
TLRs are expressed in immune and non-immune cells and
are crucial for innate immune responses. For instance, TLR4 is
activated by LPS-mediated inflammation in immune cells [49],
and is also found in the central nervous system for regulating
neurogenesis [50]. A previous report showed that EGCG has
anti-inflammatory activity via the downregulation of TLR4
expression [51]. Thus, the recruitment of activated microglia
was evaluated in LPS-induced neuroinflammation by showing
the number of Iba-1-positive microglial cells. LPS activated the
concentrating microglia in the brain, however, the activated
microglia were suppressed by EGCG treatment, which modified
the TLR4-mediated NF-
k
B pathway and proinflammatory
cytokines (Fig. 5A~G). The results suggest a potential role of
EGCG in significantly improving adult neurogenesis impaired
by neuroinflammation, and show that the beneficial effect
was associated with the downregulation of proinflammatory
cytokines through the modulation of microglial activity.
It is still unclear whether EGCG decreases the level of oxidative
stress in neuroinflammation for its improvement of adult
neurogenesis. Published reports have demonstrated that microglia
produce reactive oxygen species (ROS) in PD [52,53], and
in vivo
and
in vitro
models of AD have shown that EGCG modulates the
cellular mechanisms of neuroprotection and neurorestoration
through the activation of the protein kinase C pathway for
improving cell survival and apoptosis and antioxidant function
against ROS [54,55]. These works support that EGCG may be
involved in the regulation of ROS for rescuing adult neurogenesis
affected by neuroinflammation. To effectively address these
questions, studies regarding the molecular mechanisms of EGCG
in relation to ROS using primary cultures of adult NSCs are
required in the future.
In conclusion, we suggest that the administration of EGCG
was beneficial for improving the proliferation, survival rate, and
neuronal differentiation of adult NSCs in the DG by suppressing
the activity of microglia and the TLR4-mediated NF-
k
B path-
way in impaired neurogenesis caused by neuroinflammation.
Therefore, EGCG may be a potential therapeutic agent for
neuroinflammatory diseases.
ACKNOWLEDGEMENTS
This work was supported by the National Research Foundation
of Korea (NRF) grant funded by the Korea government (MSIP)
(2011-0030121), (2014R1A2A2A01007582) and by a grant
(CRI 12052-22) from Chonnam National University Hospital
Biomedical Research Institute.
SUPPLEMENTARY MATERIALS
Supplementary data including one figure can be found with
this article online at http://pdf.medrang.co.kr/paper/pdf/Kjpp/
Kjpp020-01-06-s001.pdf.
REFERENCES
1. Ekdahl CT, Kokaia Z, Lindvall O. Brain inflammation and
adult neurogenesis: the dual role of microglia.
Neuroscience.
2009;158:1021-1029.
2. Polazzi E, Monti B. Microglia and neuroprotection: from in vitro
studies to therapeutic applications.
Prog Neurobiol.
2010;92:293-
315.
3. Dheen ST, Kaur C, Ling EA. Microglial activation and its
implications in the brain diseases.
Curr Med Chem.
2007;14:1189-
1197.
4. Lourbopoulos A, Ertürk A, Hellal F. Microglia in action: how aging
and injury can change the brain's guardians.
Front Cell Neurosci.
2015;9:54.
5. Casano AM, Peri F. Microglia: multitasking specialists of the
brain.
Dev Cell.
2015;32:4 69- 477.
6. Delpech JC, Madore C, Nadjar A, Joffre C, Wohleb ES, La yé
S. Microglia in neuronal plasticity: Influence of stress.
Neu-
ropharmacology.
2015;96:19-28.
7. Bilimoria PM, Stevens B. Microglia function during brain de-
velopment: New insights from animal models.
Brain Res.
2015;
1617:7-17.
8. Jeong JW, Lee HH, Han MH, Kim GY, Kim WJ, Choi YH. Anti-
inflammatory effects of genistein via suppression of the toll-like
receptor 4-mediated signaling pathway in lipopolysaccharide-
stimulated BV2 microglia.
Chem Biol Interact.
2014;212:30-39.
9. Su X, Chen Q, Chen W, Chen T, Li W, Li Y, Dou X, Zhang Y, Shen Y,
Wu H, Yu C. Mycoepoxydiene inhibits activation of BV2 microglia
stimulated by lipopolysaccharide through suppressing NF-κB,
ERK 1/2 and toll-like receptor pathways.
Int Immunopharmacol.
2014;19:88-93.
10. Park KW, Lee DY, Joe EH, Kim SU, Jin BK. Neuroprotective role
of microglia expressing interleukin-4.
J Neurosci Res.
2005;81:397-
402.
11. Zhang Q, Yuan L, Liu D, Wang J, Wang S, Zhang Q, Gong Y, Liu
H, Hao A, Wang Z. Hydrogen sulfide attenuates hypoxia-induced
neurotoxicity through inhibiting microglial activation.
Pharmacol
Res.
2014;84:32-44.
12. Lehnardt S. Innate immunity and neuroinflammation in the

50
http://dx.doi.org/10.4196/kjpp.2016.20.1.41Korean J Physiol Pharmacol 2016;20(1):41-51
Seong KJ et al
CNS: the role of microglia in Toll-like receptor-mediated neuronal
injury.
Glia.
2010;58:253-263.
13. Matsuda T, Murao N, Katano Y, Juliandi B, Kohyama J, Akira
S, Kawai T, Nakashima K. TLR9 signalling in microglia atte-
nuates seizure-induced aberrant neurogenesis in the adult hip-
pocampus.
Nat Commun.
2015;6:6514.
14. Suhonen JO, Peterson DA, Ray J, Gage FH. Differentiation of
adult hippocampus-derived progenitors into olfactory neurons in
vivo.
Nature.
1996;383:624-627.
15. Gage FH. Mammalian neural stem cells.
Science.
2000;287:1433-
1438.
16. Suh H, Deng W, Gage FH. Signaling in adult neurogenesis.
Annu
Rev Cell Dev Biol.
2009;25:253-275.
17. Wen S, Li H, Liu J. Dynamic signaling for neural stem cell fate
determination.
Cell Adh Migr.
20 09;3:107-117.
18. Brown J, Cooper-Kuhn CM, Kempermann G, Van Praag H, Winkler
J, Gage FH, Kuhn HG. Enriched environment and physical activity
stimulate hippocampal but not olfactory bulb neurogenesis.
Eur J
Neurosci.
2003;17:2042-2046.
19. van Praag H, Kempermann G, Gage FH. Running increases
cell proliferation and neurogenesis in the adult mouse dentate
gyrus.
Nat Neurosci.
1999;2:266-270.
20. Drapeau E, Mayo W, Aurousseau C, Le Moal M, Piazza PV, Abrous
DN. Spatial memory performances of aged rats in the water maze
predict levels of hippocampal neurogenesis.
Proc Natl Acad Sci U S
A.
2003;100:14385-14390.
21. Tatebayashi Y, Lee MH, Li L, Iqbal K, Grundke-Iqbal I. The
dentate gyrus neurogenesis: a therapeutic target for Alzheimer's
disease.
Acta Neuropathol.
2003;105:225-232.
22. Abrous DN, Koehl M, Le Moal M. Adult neurogenesis: from
precursors to network and physiology.
Physiol Rev.
2005;85:523-569.
23. Rogers AE, Hafer LJ, Iskander YS, Yang S. Black tea and mammary
gland carcinogenesis by 7,12-dimethylbenz[a]anthracene in rats fed
control or high fat diets.
Carcinogenesis.
1998;19:1269-1273.
24. Zhou H, Chen JX, Yang CS, Yang MQ, Deng Y, Wang H. Gene
regulation mediated by microRNAs in response to green tea
polyphenol EGCG in mouse lung cancer.
BMC Genomics.
2014;15
Suppl 11:S3.
25. Singh BN, Shankar S, Srivastava RK. Green tea catechin, epigallo-
catechin-3-gallate (EGCG): mechanisms, perspectives and clinical
applications.
Biochem Pharmacol.
2011;82:1807-1821.
26. Lecumberri E, Dupertuis YM, Miralbell R, Pichard C. Green tea
polyphenol epigallocatechin-3-gallate (EGCG) as adjuvant in cancer
therapy.
Clin Nutr.
2013;32:894-903.
27. Steinmann J, Buer J, Pietschmann T, Steinmann E. Anti-infective
properties of epigallocatechin-3-gallate (EGCG), a component of
green tea.
Br J Pharmacol.
2013;168:1059-1073.
28. Gould E. How widespread is adult neurogenesis in mammals?
Nat
Rev Neurosci.
2007;8:481-488.
29. Lledo PM, Alonso M, Grubb MS. Adult neurogenesis and functional
plasticity in neuronal circuits.
Nat Rev Neurosci.
2006;7:179-193.
30. Kriegstein A, Alvarez-Buylla A. The glial nature of embryonic and
adult neural stem cells.
Annu Rev Neurosci.
2009;32:149-184.
31. Drew LJ, Fusi S, Hen R. Adult neurogenesis in the mammalian
hippocampus: why the dentate gyrus?
Learn Mem.
2013;20:710-729.
32. Singhal G, Jaehne EJ, Corrigan F, Toben C, Baune BT. Inflam-
masomes in neuroinflammation and changes in brain function: a
focused review.
Front Neurosci.
2014;8:315.
33. Monje ML, Toda H, Palmer TD. Inflammatory blockade restores
adult hippocampal neurogenesis.
Science.
2003;302:1760-1765.
34. Filiou MD, Arefin AS, Moscato P, Graeber MB. 'Neuroinflammation'
differs categorically from inflammation: transcriptomes of Al-
zheimer's disease, Parkinson's disease, schizophrenia and in-
flammatory diseases compared.
Neurogenetics.
2014;15:201-212.
35. Song C, Wang H. Cytokines mediated inflammation and
decreased neurogenesis in animal models of depression.
Prog
Neuropsychopharmacol Biol Psychiatry.
2011;35:760-768.
36. Roy AM, Baliga MS, Katiyar SK. Epigallocatechin-3-gallate induces
apoptosis in estrogen receptor-negative human breast carcinoma
cells via modulation in protein expression of p53 and Bax and
caspase-3 activation.
Mol Cancer Ther.
2005;4:81-90.
37. Abd El Mohsen MM, Kuhnle G, Rechner AR, Schroeter H, Rose S,
Jenner P, Rice-Evans CA. Uptake and metabolism of epicatechin
and its access to the brain after oral ingestion.
Free Radic Biol Med.
2002;33:1693-1702.
38. Lin LC, Wang MN, Tseng TY, Sung JS, Tsai TH. Pharmacokinetics
of (-)-epigallocatechin-3-gallate in conscious and freely moving
rats and its brain regional distribution.
J Agric Food Chem.
2007;55:1517-1524.
39. Li J, Ye L, Wang X, Liu J, Wang Y, Zhou Y, Ho W. (-)-Epigallocatechin
gallate inhibits endotoxin-induced expression of inflammatory
cytokines in human cerebral microvascular endothelial cells.
J
Neuroinflammation.
2012;9:161.
40. Mandel SA, Amit T, Weinreb O, Reznichenko L, Youdim MB.
Simultaneous manipulation of multiple brain targets by green tea
catechins: a potential neuroprotective strategy for Alzheimer and
Parkinson diseases.
CNS Neurosci Ther.
2008;14:352-365.
41. Avramovich-Tirosh Y, Reznichenko L, Mit T, Zheng H, Fridkin
M, Weinreb O, Mandel S, Youdim MB. Neurorescue activity, APP
regulation and amyloid-beta peptide reduction by novel multi-
functional brain permeable iron- chelating- antioxidants, M-30 and
green tea polyphenol, EGCG.
Curr Alzheimer Res.
2007;4:403-411.
42. Herges K, Millward JM, Hentschel N, Infante-Duarte C, Aktas O,
Zipp F. Neuroprotective effect of combination therapy of glatiramer
acetate and epigallocatechin-3-gallate in neuroinflammation.
PLoS
One.
2011;6:e25456.
43. Wu KJ, Hsieh MT, Wu CR, Wood WG, Chen YF. Green tea extract
ameliorates learning and memory deficits in ischemic rats via
its active component polyphenol epigallocatechin-3-gallate by
modulation of oxidative stress and neuroinflammation.
Evid Based
Complement Alternat Med.
2012;2012:163106.
44. Weinreb O, Amit T, Youdim MB. A novel approach of proteomics
and transcriptomics to study the mechanism of action of the
antioxidant-iron chelator green tea polyphenol (-)-epigallocatechin-
3-gallate.
Free Radic Biol Med.
2007;43:546-556.
45. Weinreb O, Mandel S, Amit T, Youdim MB. Neurological me-
chanisms of green tea polyphenols in Alzheimer's and Parkinson's
diseases.
J Nutr Biochem.
2004;15:506-516.
46. Gan L, Meng ZJ, Xiong RB, Guo JQ, Lu XC, Zheng ZW, Deng YP,
Luo BD, Zou F, Li H. Green tea polyphenol epigallocatechin-3-
gallate ameliorates insulin resistance in non-alcoholic fatty liver
disease mice.
Acta Pharmacol Sin.
2015;36:597-605.
47. Wang Y, Li M, Xu X, Song M, Tao H, Bai Y. Green tea epigal-
locatechin-3-gallate (EGCG) promotes neural progenitor cell pro-

EGCG in adult neurogenesis impaired neuroinflammation in mouse
Korean J Physiol Pharmacol 2016;20(1):41-51www.kjpp.net
51
liferation and sonic hedgehog pathway activation during adult
hippocampal neurogenesis.
Mol Nutr Food Res.
2012;56:1292-1303.
48. Yoo KY, Choi JH, Hwang IK, Lee CH, Lee SO, Han SM, Shin HC,
Kang IJ, Won MH. (-)-Epigallocatechin-3-gallate increases cell
proliferation and neuroblasts in the subgranular zone of the dentate
gyrus in adult mice.
Phytother Res.
2010;24:1065-1070.
49. Wang W, Deng M, Liu X, Ai W, Tang Q, Hu J. TLR4 activation
induces nontolerant inflammatory response in endothelial
cells.
Inflammation.
2011;34:509-518.
50. Rolls A, Shechter R, London A, Ziv Y, Ronen A, Levy R, Schwartz M.
Toll-like receptors modulate adult hippocampal neurogenesis.
Nat
Cell Biol.
2007;9:1081-1088.
51. Hong Byun E, Fujimura Y, Yamada K, Tachibana H. TLR4 sig-
naling inhibitory pathway induced by green tea polyphenol epi-
gallocatechin-3-gallate through 67-kDa laminin receptor.
J Im-
munol.
2010;185:33-45.
52. Kumar P, Kumar A. Protective effects of epigallocatechin gal-
late following 3-nitropropionic acid-induced brain damage:
possible nitric oxide mechanisms.
Psychopharmacology (Berl).
2009;207:257-270.
53. Mao H, Fang X, Floyd KM, Polcz JE, Zhang P, Liu B. Induction
of microglial reactive oxygen species production by the
organochlorinated pesticide dieldrin.
Brain Res.
2007;1186:267-274.
54. Levites Y, Amit T, Youdim MB, Mandel S. Involvement of protein
kinase C activation and cell survival/ cell cycle genes in green tea
polyphenol (-)-epigallocatechin 3-gallate neuroprotective action.
J
Biol Chem.
2002;277:30574-30580.
55. Weinreb O, Amit T, Mandel S, Youdim MB. Neuroprotective
molecular mechanisms of (-)-epigallocatechin-3-gallate: a reflective
outcome of its antioxidant, iron chelating and neuritogenic
properties.
Genes Nutr.
2009;4:283-296.

Supplementary figure 1. The schematic representation of experimental research design. (A) LPS (black arrow), EGCG (red arrows, 3
times with 6 hours interval), and BrdU (gray arrow) injection were done in the time course and the animals were sacrificed on day 1, 3, and 5
for immunohistochemical analysis to the quantification of NSC proliferation and immature differentiation. (B) LPS (black arrow), EGCG (red
arrows), and 5 consecutive BrdU (gray arrows) injection daily were done in the time course and the animals were sacrificed on day 5 and 28 for
immunohistochemical analysis to the quantification of NSC cell survival and mature differentiation. LPS, lipopolysaccharide, EGCG, epigallocatechin-
3-gallate, BrdU, bromodeoxyuridine, injt, injection, and Sac, scarified.