A novel synthetic HTB derivative, BECT inhibits lipopolysaccharide-mediated inflammatory response by suppressing the p38 MAPK/JNK and NF-κB activation pathways

Article abstract of DOI:10.1016/j.pharep.2013.08.015

Activated microglia cells are well recognized as mediators of neuroinflammation, as they release nitric oxide and pro-inflammatory cytokines in various neuroinflammatory diseases. Thus, suppressing microglial activation may alleviate neuroinflammatory and neurodegenerative processes. In the present study, we synthesized and investigated the anti-neuroinflammatory effect of a novel HTB (2-hydroxy-4-trifuoromethylbenzoic acid) derivative in lipopolysaccharide (LPS)-stimulated microglial cells. Among the synthesized derivatives, the BECT [But-2-enedioic acid bis-(2-carboxy-5-trifluoromethyl- phenyl) ester] significantly decreased production of nitric oxide and other pro-inflammatory cytokines including tumor necrosis factor-α, interleukin-1β, and interleukin-6 in microglial cells. BECT also mitigated the expression of inducible nitric oxide synthase and cyclooxygenase-2 at both the mRNA and protein levels. Further mechanistic studies demonstrated that the HTB derivative inhibited phosphorylation of JNK and p38 mitogen-activated protein kinase and nuclear translocation of nuclear factor kappa-B in LPS-stimulated BV-2 microglial cells. Thus BECT, our novel synthesized compound have anti-inflammatory activity in microglial cells, and may have therapeutic potential for treating neuroinflammatory diseases.

Full text of DOI:10.1016/j.pharep.2013.08.015

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Pharmacological Reports xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Pharmacological Reports
journal homepage: www.elsevier.com/locate/pharep
Original research article
A novel synthetic HTB derivative, BECT inhibits lipopolysaccharide-mediated
inflammatory response by suppressing the p38 MAPK/JNK and NF-
activation pathways
kB
Seong-Mook Kang a, Sandeep Vasant More a, Ju-Young Park b, Byung-Wook Kim a, Park Jeong In a,
Sung-Hwa Yoon b, Dong-Kug Choi a,
*
a Department of Biotechnology, Research Institute for Biomedical and Health Science, Konkuk University, Chungju 380-701, Republic of Korea
b Department of Molecular Science and Technology, Ajou University, Suwon 443-749, Republic of Korea
A R T I C L E I N F O
A B S T R A C T
Article history:
Activated microglia cells are well recognized as mediators of neuroinflammation, as they release nitric
oxide and pro-inflammatory cytokines in various neuroinflammatory diseases. Thus, suppressing
microglial activation may alleviate neuroinflammatory and neurodegenerative processes. In the present
study, we synthesized and investigated the anti-neuroinflammatory effect of a novel HTB (2-hydroxy-4-
trifuoromethylbenzoic acid) derivative in lipopolysaccharide (LPS)-stimulated microglial cells. Among
the synthesized derivatives, the BECT [But-2-enedioic acid bis-(2-carboxy-5-trifluoromethyl-phenyl)
ester] significantly decreased production of nitric oxide and other pro-inflammatory cytokines including
Received 7 February 2013
Received in revised form 1 August 2013
Accepted 20 August 2013
Available online xxx
Keywords:
HTB derivative
tumor necrosis factor-a, interleukin-1b, and interleukin-6 in microglial cells. BECT also mitigated the
Lipopolysaccharide
Microglia
expression of inducible nitric oxide synthase and cyclooxygenase-2 at both the mRNA and protein levels.
Further mechanistic studies demonstrated that the HTB derivative inhibited phosphorylation of JNK and
p38 mitogen-activated protein kinase and nuclear translocation of nuclear factor kappa-B in LPS-
stimulated BV-2 microglial cells. Thus BECT, our novel synthesized compound have anti-inflammatory
activity in microglial cells, and may have therapeutic potential for treating neuroinflammatory diseases.
ß 2014 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp.
z o.o. All rights reserved.
Neuroinflammation
Neurodegenerative disease
Introduction
responses to inflammatory triggers [5,6]. Various studies have
reported that suppressing the exaggerated inflammatory response
Although neuroinflammation plays a critical role in brain host
defense, it also contributes to the underlying neuronal loss
occurring in various neurodegenerative disorders [1]. Uncon-
trolled activation of microglia is directly toxic to neurons, as they
release various neurotoxic substances such as nitric oxide (NO),
prostaglandin E2, superoxide, and proinflammatory cytokines
[2–4]. Inflammatory intermediates including expression of
inducible nitric oxide synthase (iNOS) and subsequent production
of NO and mitogen-activated protein kinase (MAPK) signaling
cascades such as c-Jun N-terminal kinase (JNK) and p38 in
activated glial cells play important roles in mediating glial
initiated by activated microglial cells helps to attenuate the
severity of neurodegenerative diseases [7,8]. One of the most
commonly used anti-inflammatory agents are salicylates or drugs
structurally resembling aspirin. One such derivative of aspirin is
triflusal (2-acetoxy-4-trifluoromethylbenzoic acid). Triflusal is a
fluorinated salicylate that possesses distinct pharmacologic,
pharmacokinetic, and biochemical properties from those of aspirin
[9,10]. Besides its antiplatelet action, triflusal reduces nuclear
factor kappa-B (NF-kB), activation of microglia in excitotoxic injury
and in models of Alzheimer’s disease [9,11]. Following oral
administration, triflusal is converted to its main deacetylated
active metabolite HTB (3-hydroxy-4-trifluoromethylbenzoic acid)
[12]. HTB exhibits anti-inflammatory effects by inhibiting the
cyclooxygenase-2 (COX-2) protein and NF-
synthesized HTB derivatives that may be good therapeutic agents
kB [13,14]. We have
*
Corresponding author.
E-mail address: choidk@kku.ac.kr (D.-K. Choi).
http://dx.doi.org/10.1016/j.pharep.2013.08.015
1734-1140/ß 2014 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.
Please cite this article in press as: Kang S-M, et al. A novel synthetic HTB derivative, BECT inhibits lipopolysaccharide-mediated
inflammatory response by suppressing the p38 MAPK/JNK and NF-
10.1016/j.pharep.2013.08.015
kB activation pathways. Pharmacol Rep (2014), http://dx.doi.org/

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in pathological conditions in which regulation of inflammatory
genes constitutes relevant step in the outcome of the
neurodegenerative event.
reacted with an equal volume of Griess reagent (1 part 0.1%
naphthylethylenediamine and 1 part 1% sulfanilamide in 5%
H3PO4) in 96-well plates at room temperature. Nitrite concentra-
tions were determined using standard solutions of sodium nitrite
prepared in cell culture medium. Absorbance was determined at
540 nm using a microplate reader (Tecan Trading AG, Mannedorf,
Switzerland). Results are representative of three independent
experiments.
a
Our previous report suggested that microglia are a good cellular
model for screening potential therapeutic compounds for neuroin-
flammatory disorders [15]. In the present study, we report the anti-
neuroinflammatory effect of a novel synthetic HTB derivative BECT
[But-2-enedioic acid bis-(2-carboxy-5-trifluoromethyl-phenyl)
ester] in lipopolysaccharide (LPS)-stimulated BV-2 microglial cells.
BECT inhibited the production of inflammatory markers, including
NO, COX-2, and pro-inflammatory cytokines such as tumor
Cytotoxicity assay
necrosis factor-
a
(TNF-
a
), interleukin-1
b
(IL-1
b
), and interleu-
Viability of cultured cells was determined by measuring the
reduction of MTT to formazan. Briefly, BV-2 cells (2.5 Â 104 cells/
mL) were pre-treated with different concentrations of BECT (12.5,
kin-6 (IL-6). Subsequent experiments found that pretreatment
with BECT abates, LPS-stimulated induction in phosphorylation of
JNK and p38-MAPK and decreases the nuclear translocation of the
25, and 50
mM) for 60 min in 96-well plates with 200 mL culture
NF-
k
B p65 subunit in LPS-stimulated BV-2 microglial cells. Our
B signaling
medium and then stimulated with LPS (100 ng/mL) for 24 h. Then,
0.5 mg/mL of MTT solution was added to each well. After 4 h of
incubation at 37 8C, the supernatants were removed, and the
formed formazan crystals were dissolved in DMSO. The absorbance
at 550 nm was determined using a microplate reader. Results are
representative of three independent experiments.
data indicate that inhibition JNK, p38MAPK, and NF-
k
might be one of the possible molecular mechanisms contributing
to the anti-neuroinflammatory effect of BECT in LPS-stimulated
microglial cells. These results extend our understanding of the
anti-neuroinflammatory properties of BECT and suggest a possible
new pharmacological agent for neuroinflammatory diseases.
Methodology for synthesis of BECT
Materials and methods
To a solution of 2-hydroxy-4-(trifluoromethyl)benzoic acid
(HTB) 1 (2.00 g, 9.70 mmol) in THF (150 mL) and pyridine (50 mL)
Reagents
at 0 8C was added fumaryl chloride (526
mL, 4.85 mmol). The
LPS (Escherichia coli 0111:B4, Sigma, St. Louis, MO, USA), N-(1-
naphthyl) ethylenediamine dihydrochloride, and 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT),
Tween-20, bovine serum albumin, dimethyl sulfoxide (Compound
Diluent/(1%) Vehicle control), sodium nitrite and sulfanilamide
were purchased from Sigma (St. Louis, MO, USA). Dulbecco’s
reaction mixture was stirred at room temperature for 1 h and then
poured into water and 1 N HCl solution. The solution was extracted
with ethyl acetate and the organic layer was washed with water,
dried over Na2SO4 and concentrated in vacuo. The crude product
was recrystallized with ethyl acetate/hexane (1/3) to obtain the
title compound as white solid (560 mg, 23%). Mp 177 8C; IR (KBr,
modified Eagle’s medium (DMEM), containing 4500 mg/L of
D
-
cmÀ1) 3433, 1778, 1716; 1H NMR (DMSO-d6)
7.82 (d, 2H, J = 8.0 Hz), 7.87 (s, 2H), 8.15–8.20 (d, 2H, J = 8.0 Hz); 13
NMR (DMSO-d6) 121.06, 123.40, 124.28 (JC-F = 270.7 Hz), 127.92,
d 7.23 (s, 2H), 7.78–
glucose, -arginine, 110 mg/L of sodium pyruvate, and phosphate
L
C
buffered saline (PBS) as well as other cell culture reagents were
obtained from Gibco/Invitrogen (Carlsbad, CA, USA). The protease
inhibitor cocktail tablets and phosphatase inhibitor cocktail tablets
were supplied by Roche (Indianapolis, IN, USA). Fetal bovine serum
(FBS) was purchased from PAA Laboratories Inc. (Etobicoke,
Ontario, Canada). Antibodies to iNOS, JNK, phospho-JNK, p38,
d
132.51, 133.46 (JC-F = 32.6 Hz), 133.69, 149.41, 162.47, 164.28; MS
m/z 491.02 ([M+H]+).
Isolation of total RNA and reverse transcription-polymerase chain
reaction
phospho-p38, I
Signaling Technology (Danvers, MA, USA) and antibodies to COX-2,
p65NF- B, and nucleolin were obtained from Santa Cruz
kB-a, p-IkB-a, and b-actin were supplied by Cell
BV-2 cells (50 Â 104 cells/mL) were cultured in 6-well plates,
k
and total RNA was isolated by extraction with TRIzol (Invitrogen).
Biotechnology (Santa Cruz, CA, USA).
For RT-PCR, 2.5 mg of total RNA was reverse transcribed using a
First-Strand cDNA Synthesis kit (Invitrogen). Then, cDNA was
amplified by PCR using specific primers as mentioned previously
[15,16]. The PCR was performed using the above-prepared cDNA as
a template for respective targets, as described previously [19,20].
PCR products were analyzed on 1% agarose gels. Results are
representative of three independent experiments.
Cell culture
BV-2 cells (a mouse microglial cell line), originally developed by
Dr. V. Bocchini (University of Perugia, Perugia, Italy), were used as
cited in our earlier reports [15,16]. The BV-2 cell line demonstrated
both phenotypic and functional properties of reactive microglia
cells [17]. The cells were grown and maintained in DMEM
Western blot analysis
containing 5% heat-inactivated FBS + 50
mg/mL penicillin–strep-
tomycin and maintained in a humidified incubator at 37 8C with 5%
Cells were washed twice with ice cold PBS, and total cell lysates
CO2.
were obtained by adding 50 or 100
m
L of RIPA buffer (1Â PBS, 1%
NP-40, 0.5% sodium deoxycholate, and 0.1% SDS, containing fresh
protease inhibitor cocktail) to the BV-2 cells (50 Â 104 cells/mL)
cultured in 6-well plates. Electrophoresis and immunoblotting
procedures followed those of a previous report [16]. PVDF
membranes were tagged by incubating them overnight with
NO assay
NO production was assayed by measuring the levels of nitrite in
culture medium. Accumulated levels of nitrites were measured in
the cell supernatant using the Griess reaction [18]. BV-2 cells
anti-iNOS (1:1000), anti-I
anti-p38 (1:1000), anti-phospho-p38 (1:1000), anti-JNK (1:500),
anti-phospho-JNK (1:1000) anti- -actin (1:2000), anti-COX-2
(1:1000), anti-phospho p65NF- B (1:500), and anti-nucleolin
(1:500) antibodies followed by a 1 h incubation with horseradish
kB-a (1:1000), anti-p-IkB-a (1:1000),
(2.5 Â 104 cells/mL) plated in 96-well plates in 200
mL culture
medium were pre-treated with different BECT concentrations
b
(12.5, 25, and 50
(100 ng/mL) for 24 h. Briefly, 50
m
M) for 60 min, and then stimulated with LPS
l of culture supernatant was
k
m
Please cite this article in press as: Kang S-M, et al. A novel synthetic HTB derivative, BECT inhibits lipopolysaccharide-mediated
inflammatory response by suppressing the p38 MAPK/JNK and NF-
10.1016/j.pharep.2013.08.015
kB activation pathways. Pharmacol Rep (2014), http://dx.doi.org/

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PHAREP-46; No. of Pages 9
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3
CF3
O
O
OH
O
O
OH
Cl
O
+
Cl
O
F3C
OH
O
O
HO
O
1
2
CF3
BECT
Fig. 1. Synthesis of novel 2-hydroxy-4-trifluoromethyl benzoic acid (HTB) derivative, BECT.
peroxidase-conjugated secondary antibody (1:1000–2000). The
optical densities of the antibody specific bands were analyzed with
a LAS-3000 Luminescent Image Analyzer (Fuji, Tokyo, Japan). In a
parallel experiment, nuclear proteins were extracted using the
method provided by the nuclear and cytoplasmic extraction kit
from Thermo Scientific (Rockford, IL, USA). Results are representa-
tive of three independent experiments.
10 min at room temperature, washed with PBS for 5 min, and then
treated with 10% goat serum in PBS for 60 min. The permeabilized
cells were treated with monoclonal mouse anti-human NF-kB p65
(1:200) (Santa Cruz Biotechnology) for 60 min at room tempera-
ture and washed in PBS for 5 min. The cells were then incubated in
a 1:200 dilution of Alexa Fluor 568-labeled goat anti-mouse
antibody (Invitrogen) for 60 min at room temperature and washed
in PBS for 5 min. The cells were stained with 1
mM Hoechst
Immunofluorescence assay
staining solution for 30 min at 37 8C and then washed. Finally, the
coverslips with cells were dried at 55 8C in an oven for 10–12 min
and mounted in a 1:1 mixture of xylene and malinol.
BV-2 microglia cells (5 Â 104 cells/well in 24-well plate) were
cultured on sterile cover slips in 24-well plates and treated with
50
m
M of BECT for 60 min followed by treatment with LPS (100 ng/
B p65
Statistical analyses
mL) for 30 min to detect the intracellular location of the NF-
k
sub-unit. At 30 min after LPS treatment, the cells were fixed with
methanol for 30 min at -20 8C and washed with PBS for 5 min. The
fixed cells were then permeabilized with 1% Triton X-100 in PBS for
Statistical analyses were conducted using GraphPad software
version 5 (GraphPad, La Jolla, CA, USA). Values are presented as
mean Æ standard error. Significant differences between the groups
Table 1
HTB Derivatives inhibit lipopolysaccharide (LPS)-induced nitric oxide (NO) production in BV-2 microglia cells.
Derivative name
HTB
R1
R2
H
%NO inhibition
7.53
OH
HTB-1
H
H
H
18.47
HTB-2
7.51
HTB-3
HTB-4
61.72
30.55
H
HTB: 3-hydroxy-4-trifluoromethylbenzoic acid. Among the synthetic HTB derivatives that we screened by microglia cell-based assay, HTB-4 was identified as one among the
novel synthetic compounds that strongly attenuated NO production with no significant cell toxicity observed in MTT assay in lipopolysaccharide (LPS)-stimulated BV-2
microglial cells.
Please cite this article in press as: Kang S-M, et al. A novel synthetic HTB derivative, BECT inhibits lipopolysaccharide-mediated
inflammatory response by suppressing the p38 MAPK/JNK and NF-
10.1016/j.pharep.2013.08.015
kB activation pathways. Pharmacol Rep (2014), http://dx.doi.org/

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S.-M. Kang et al. / Pharmacological Reports xxx (2014) xxx–xxx
were determined using a one-factor analysis of variance (ANOVA)
followed by Tukey’s post hoc-test. *p < 0.05 was considered statisti-
cally significant.
(Fig. 2b) with no significant toxicity seen in MTT assay (Fig. 2a) in
lipopolysaccharide (LPS)-stimulated BV-2 microglial cells. We
renamed HTB-4 as BECT in our further discourse.
Effect of BECT on NO production and cytotoxicity in LPS stimulated BV-
2 microglial cells
Results
Synthesis and purification of BECT
BV-2 microglial cells were pre-treated for 60 min with
different concentrations of BECT (12.5, 25, and 50
mM), followed
2-Hydroxy-4-(trifluoromethyl) benzoic acid (HTB) was reacted
with fumaryl chloride in a mixed solution of pyridine and THF at
room temperature to yield BECT. The chemical structure of BECT is
shown in Fig. 1. BECT was further purified by recrystallization from
ethyl acetate and methanol as a white solid and was identified by
NMR, IR and MS.
by LPS (100 ng/mL) treatment for 24 h. The accumulated nitrite
in the culture medium estimated by the Griess reaction was used
as an index of NO release. As shown in Fig. 2d; LPS treatment
significantly stimulated NO production (12.46 Æ 0.34 mM) as
compared to that in the control. The control and BECT treatments
did not induce increase in NO levels. Pre-treatment with BECT
(12.5, 25, and 50 mM) significantly reduced NO levels in LPS-
induced BV-2 microglial cells in a dose-dependent manner to
9.32 Æ 0.48 mM, 6.92 Æ 0.47 mM, and 5.43 Æ 0.19 mM, respective-
ly. The MTT assay was performed to determine whether BECT had
any cytotoxic effect on BV-2 microglia cells. Viability of control
cells was used as the 100% reference value. BV-2 cells were treated
with LPS (100 ng/mL) with or without different concentrations of
BECT (12.5, 25, and 50 mM) for 24 h. LPS (100 ng/mL) in
combination with BECT did not result in any significant cytotoxici-
ty or changes in microglia cell viability (Fig. 2c). Hence, our results
demonstrate that BECT was one of the compounds to markedly
inhibit LPS-stimulated NO release with no significant cytotoxicity
in BV-2 microglial cells.
Novel HTB derivatives as inhibitors of LPS-induced microglial NO
production
Production of NO has been used as a representative measure of
inflammatory activation of microglial cells [21]. Hence, the
suppressive effect of the synthetic HTB derivatives (Table 1) was
evaluated on NO release in LPS-stimulated microglia. BV-2
microglial cells were stimulated with LPS in the presence or
absence of 10
mM of the novel synthetic derivatives for 24 h.
Accumulated nitrite in the culture media was measured by the
Griess reaction. Among the synthetic HTB derivatives that we
screened by microglia cell-based assay, HTB-4 was identified as a
novel synthetic compound that strongly attenuated NO production
(a)
(b)
120
40
$
100
80
60
40
20
0
###
30
20
10
0
***
***
***
***
HTB-2 HTB-3 HTB-4
LPS(100ng/ml)
HTB-1
HTB-2 HTB-3 HTB-4
LPS(100ng/ml)
-
-
HTB-1
-
-
(c)
(d)
15
10
5
120
100
80
60
40
20
0
###
$
***
***
***
0
-
-
+
-
+
+
+
-
-
+
-
-
+
+
+
LPS(100ng/mL)
BECT (μM)
LPS(100ng/ml)
BECT (μM)
-
50
12.5
25
50
50
12.5
25
50
Fig. 2. The effect of HTB derivatives on nitric oxide (NO) production and cytotoxicity in lipopolysaccharide (LPS)-stimulated BV-2 microglial cells. BV-2 microglial cells were
treated with LPS (100 ng/ml) in the absence or presence of the novel synthetic HTB derivatives (10 M) for 24 h. (a) Cytotoxicity of each compound was assessed by MTT
assays and results are expressed as a percentage of surviving cells over control cells. (c) Cells were treated with varying doses of BECT (12.5, 25, and 50 M) and examined by
m
m
MTT assay. $p < 0.05 compared with the control group. Significance was determined by analysis of variance (ANOVA) followed by Tukey’s multiple comparison test. (b) Nitrite
content in the culture media was measured using the Griess reaction and the results were expressed as a percentage of released NO from LPS-stimulated BV-2 cells. Among
the compounds tested, HTB-4 was one of the compounds to significantly reduce LPS-induced NO production in BV-2 cells with no significant cytotoxicity. (d) BV-2 cells were
pre-treated with different concentrations of BECT (12.5, 25, and 50
mM) for 60 min before incubating with LPS (100 ng/mL) for 24 h. Data are expressed as a percentage of the
control. Data are mean Æ standard error (SEM) of three independent experiments. ###p < 0.001 compared with the control group and ***p < 0.001 compared with the LPS-treated
group. Significance was determined by one-way ANOVA followed by Tukey’s multiple comparison test.
Please cite this article in press as: Kang S-M, et al. A novel synthetic HTB derivative, BECT inhibits lipopolysaccharide-mediated
inflammatory response by suppressing the p38 MAPK/JNK and NF-
10.1016/j.pharep.2013.08.015
kB activation pathways. Pharmacol Rep (2014), http://dx.doi.org/

G Model
PHAREP-46; No. of Pages 9
S.-M. Kang et al. / Pharmacological Reports xxx (2014) xxx–xxx
5
(a)
(b)
iNOS
iNOS
β-Actin
GAPDH
3
2
1
0
3
2
1
0
###
###
***
***
***
***
***
-
-
+
-
-
+
+
+
LPS(100ng/mL)
BECT (μM)
-
-
+
-
-
+
5
+
+
LPS(100ng/mL)
BECT (μM)
50
12.5
25
50
20
10
20
(c)
(d)
COX-2
COX-2
GAPDH
β-Actin
2.5
2.5
###
###
2.0
1.5
1.0
0.5
2.0
1.5
1.0
0.5
**
0.0
0.0
-
-
+
-
-
+
5
+
+
LPS(100ng/mL)
-
-
+
-
-
+
+
+
LPS(100ng/mL)
BECT (μM)
20
10
20
BECT (μM)
50
12.5
25
50
Fig. 3. Effect of BECT on lipopolysaccharide (LPS)-induced inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) mRNA and protein expression in LPS-
stimulated BV-2 microglial cells. BV-2 microglial cells were pre-treated with the indicated concentrations of BECT for 60 min before incubating with LPS (100 ng/mL) for 6 h.
Total RNA was extracted and further analyzed by reverse transcription polymerase chain reaction (RT-PCR). GAPDH was used as the control gene. Quantified data are shown
in the lower panel. mRNA levels of (a) iNOS and (c) COX-2 were normalized to GAPDH levels and expressed as a relative change in comparison to the LPS treatment. BV-2 cells
were pretreated with BECT (5, 10, and 20
m
M) for 60 min and then stimulated with LPS (100 ng/ml) for 18 h. Western blot analysis was then performed to detect iNOS and
-actin antibody to confirm equal loading. Quantification of (b) iNOS and (d) COX-2 were
COX-2 using anti-iNOS/COX-2 antibodies. The blot was stripped and reprobed with
b
performed by normalization with
b
-actin and expressed as a relative change in comparison to the LPS treatment. Data are mean Æ standard error (SEM) of three independent
experiments. ###p < 0.001 compared with the control group; **p < 0.01 and ***p < 0.001 compared with the LPS-treated group. Significance was determined by one-way ANOVA
followed by Tukey’s multiple comparison test.
Effect of BECT on LPS-stimulated iNOS and COX-2 mRNA and protein
expression in LPS-stimulated BV-2 microglial cells
were pretreated with BECT (12.5, 25, and 50 mM) for 1 h in the
presence or absence of LPS (100 ng/mL) for 6 h. Our data revealed
that exposing BV-2 microglial cells to LPS (100 ng/mL) for 6 h
Experiments involving the detection of iNOS and COX-2 mRNA
and protein levels were performed by expression analysis. COX-2 is
another important inflammatory enzyme that is upregulated in
various neurodegenerative diseases [22]. Therefore, the effect of
BECT on COX-2 expression in LPS-stimulated BV-2 microglial cells
was investigated. BV-2 microglia cells were pretreated with BECT
resulted in a significant increase in the production of TNF-a, IL-1b,
and IL-6 compared to that in the control (Fig. 4a). Pretreatment
with BECT at various concentrations decreased LPS-induced mRNA
expression of TNF-
(Fig. 4b-d). We found a non-significant decrease in LPS-induced
TNF- and IL-1 mRNA expression levels at 12.5 and 25 M,
whereas 50 M resulted in a significant decrease in TNF- and IL-
1b mRNA expression (Fig. 4b and c). Our results indicate that BECT
a, IL-1b, and IL-6 in a dose-dependent manner
a
b
m
(5, 10, 12.5, 20, 25, and 50
m
M) for 1 h followed by stimulation
m
a
with LPS (100 ng/mL) for another 6 and 24 h. As shown in Fig. 3,
pre-treatment with BECT at various concentrations significantly
reduced LPS-induced iNOS mRNA (Fig. 3a) and protein expression
(Fig. 3b) in a dose-dependent manner. Similarly we found that
treating BV-2 cells with LPS (100 ng/mL) significantly increased
COX-2 mRNA (Fig. 3c) and protein expression (Fig. 3d). Pretreat-
ment with BECT at various concentrations inhibited LPS-induced
COX-2 mRNA expression and protein level in BV-2 microglial cells
in a dose-dependent manner.
suppressed production of TNF-a, IL-6, and IL-1b at the transcrip-
tional level in response to LPS.
Effect of BECT on LPS-induced p38, ERK, and JNK MAPK
phosphorylation in BV-2 microglial cells
MAPK signaling pathways control the release of pro-inflamma-
tory mediators in activated microglial cells [25,26]. Therefore, we
examined whether BECT influenced these signaling pathways in
BV-2 microglia cells. BV-2 cells were pre-treated with various
Inhibitory effect of BECT on pro-inflammatory cytokine expression in
microglial cells
concentrations of BECT (5, 10, and 20
mM) for 60 min and then
stimulated with LPS (100 ng/mL) for 30 min. We found that LPS
treatment significantly increased p38MAPK and JNK phosphory-
lation at 30 min compared to that in the control. Pretreatment with
BECT significantly inhibited LPS-stimulated upregulation of
p38MAPK and JNK phosphorylation in a dose-dependent fashion
Based on the earlier findings that proinflammatory cytokines
released from microglia are responsible for neuronal death [23,24],
we investigated the potential effects of BECT on reducing pro-
inflammatory cytokines such as TNF-a, IL-6, and IL-1b. BV-2 cells
Please cite this article in press as: Kang S-M, et al. A novel synthetic HTB derivative, BECT inhibits lipopolysaccharide-mediated
inflammatory response by suppressing the p38 MAPK/JNK and NF-
10.1016/j.pharep.2013.08.015
kB activation pathways. Pharmacol Rep (2014), http://dx.doi.org/

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2.5
(b)
(a)
###
2.0
1.5
1.0
0.5
TNF-α
IL-1β
IL-6
**
GAPDH
-
-
+
-
-
+
+
+
LPS(100ng/mL)
BECT (μM)
0.0
LPS(100ng/mL)
50
12.5
25
50
-
-
+
-
-
+
+
+
BECT (μM)
50
12.5
25
50
(c)
(d)
2.0
2.0
1.5
1.0
0.5
0.0
###
###
1.5
1.0
0.5
0.0
***
***
***
-
-
+
-
-
+
+
+
-
-
+
-
-
+
+
+
LPS(100ng/mL)
BECT (μM)
LPS(100ng/mL)
BECT (μM)
50
12.5
25
50
50
12.5
25
50
Fig. 4. Effects of BECT on pro-inflammatory cytokine gene expression in lipopolysaccharide (LPS)-simulated BV-2 microglial cells. BV-2 microglial cells were treated with LPS
(100 ng/ml) in the absence or presence of BECT (12.5, 25, and 50 M), and total RNA was isolated at 6 h after the treatment. (a) The levels of tumor necrosis factor- (TNF- ),
interleukin-1 (IL-1 ), and interleukin-6 (IL-6) mRNA were determined by reverse transcription polymerase chain reaction (RT-PCR) and then subjected to densitometric
quantification. Levels of (b) TNF- , (c) IL-1 , and (d) IL-6 mRNA were normalized to GAPDH levels and expressed as relative change in comparison to the LPS treatment.
Results are expressed as a ratio of TNF- /GAPDH, IL-1
/GAPDH, and IL-6/GAPDH. ###p < 0.001 compared with the control group; **p < 0.01 and ***p < 0.001 compared with
the LPS-treated group. Significance was determined by one-way ANOVA followed by Tukey’s multiple comparison test.
m
a
a
b
b
a
b
a
b
(Fig. 5a and b), indicating that BECT is capable of downregulating
vital signal transduction pathways involved in LPS-stimulated BV-
2 microglial cells. In addition, we did not observe any BECT-
induced change in the ERK signaling expression pattern in LPS-
stimulated BV-2 microglial cells.
Effect of BECT on LPS-induced I
kB-a phosphorylation and NF-kB
activation in microglial cells
NF-kB plays a very important role in LPS-induced expression of
iNOS, COX-2, and the production of pro-inflammatory cytokines
(a)
(b)
p-JNK
Total JNK
β-Actin
p-p38
Total p38
β-Actin
2.0
2.0
###
###
1.5
1.5
*
*
***
1.0
1.0
0.5
0.0
0.5
0.0
LPS(100ng/mL)
BECT (μM)
-
-
+
-
-
+
-
-
+
-
-
+
5
+
+
LPS(100ng/mL)
BECT (μM)
20
20
20
10
20
Fig. 5. Inhibition of lipopolysaccharide LPS-stimulated p38 and JNK phosphorylation by BECT in BV-2 microglial cells. BV-2 microglial cell lysates were obtained after a 30 min
activation with LPS (100 ng/ml) in the absence or presence of BECT (5, 10, and 20 M) and phosphorylation of (a) p38 and (b) JNK in the lysates was analyzed by Western blot.
m
Total protein was separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Levels of phospho-mitogen activated protein kinases (MAPKs)
are expressed as a relative change in comparison to the LPS treatment. Densitometric analysis of the p38 and JNK-MAPK band are shown in the lower panel. Data are
mean Æ standard error of three independent experiments. ###p < 0.001 compared with control group; *p < 0.05 and ***p < 0.001 compared with LPS-treated group. Significance
was determined by one-way ANOVA followed by Tukey’s multiple comparison test.
Please cite this article in press as: Kang S-M, et al. A novel synthetic HTB derivative, BECT inhibits lipopolysaccharide-mediated
inflammatory response by suppressing the p38 MAPK/JNK and NF-
10.1016/j.pharep.2013.08.015
kB activation pathways. Pharmacol Rep (2014), http://dx.doi.org/

G Model
PHAREP-46; No. of Pages 9
S.-M. Kang et al. / Pharmacological Reports xxx (2014) xxx–xxx
7
Fig. 6. Effect of BECT on inhibiting p-IkB-a and p65 nuclear factor NF-kB activation in lipopolysaccharide (LPS)-stimulated BV-2 microglial cells. Cells were treated with the
respective dose (20
mM) of BECT, 60 min before LPS (100 ng/mL) treatment. Total nuclear and whole cell proteins were electrophoresed followed by immunoblotting using (a)
anti-p-I B- and (b) NF-kB p65. Densitometric analysis of p-IkB-a and NF-kB p65 is shown in the lower panel. Results are expressed as a ratio of p-Ik a/b-actin and NF-kB
k
a
B-
mM) for 60 min and then stimulated
B subunit was determined by immunofluorescence assay. Data are mean Æ standard error of three
p65/nucleolin. BV-2 microglial cells were seeded at a density of 5 Â 104 cells/well on a 24-well plate. (c) Cells were treated with BECT (20
with LPS (100 ng/mL) for 30 min. Sub-cellular location of the p65NF-
independent experiments. ##p < 0.01 and ###p < 0.001 compared with the control group; **p < 0.01 and ***p < 0.001 compared with the LPS-treated group. Significance was
k
determined by one-way ANOVA followed by Tukey’s multiple comparison test.
[27]. Activation of NF-
subsequent nuclear translocation of the NF-
k
B includes I
k
B-
a
k
phosphorylation and
B p65 subunit into the
the therapeutic effects of aspirin and salicylate derivatives are far
from being established and remain open for discussion. Initial
experiments using aspirin, triflusal and HTB showed that both 4-
trifluoromethyl derivatives were more potent than aspirin; thus,
indicating that the presence of the acetyl moiety in 4-trifuor-
nucleus. Based on these reports, BV-2 microglial cells were treated
with 20 M of BECT for 1 h with or without LPS (100 ng/mL) for
30 min to examine whether BECT regulates the NF- B pathway, and
the levels of the p-I B- and NF- B p65 subunits were investigated.
As shown in Fig. 6a, exposing BV-2 cells to LPS significantly induced
B- phosphorylation. Treatment of LPS-stimulated BV-2 micro-
glial cells with BECT significantly reduced I B- phosphorylation. As
shown in Fig. 6b, stimulating BV-2 cells with LPS (100 ng/mL) for
30 min significantly increased nuclear translocation of the NF-
p65 subunit. Pretreatment of BV-2 cells with BECT (20 M)
significantly inhibited p65NF- B nuclear translocation in response
m
k
k
a
k
omethylbenzoic acid is not a key requirement for inhibiting NF-kB
activation [13]. With respect to the above structure to activity
profile we added new substitutions to HTB and synthesized novel
HTB derivatives that have inhibitory activities against microglial
activation and might also attenuate the progression of neuroin-
flammatory disease. HTB is the main deacetylated metabolite of
triflusal with structural analogy to aspirin [13]. The purpose of this
study was to address the effect of a novel HTB derivative on
inhibiting LPS-induced inflammatory response in BV-2 microglial
cells. As NO is a vital proinflammatory mediator and plays an
important role in neuroinflammatory diseases, the HTB derivatives
were screened for their ability to inhibit NO release in response to
LPS using a BV-2 microglia cell-based assay [28]. Among the
battery of HTB derivatives screened, few synthetic compounds
reduced NO release in LPS-stimulated BV-2 microglial cells. Among
these derivatives, BECT was one of the compounds exhibiting
significant inhibitory effect on NO release with least cytotoxicity in
LPS-stimulated BV-2 microglial cells. The results from our
experiment matched a similar finding in which HTB decreased
Ik a
k
a
kB
m
k
to LPS; nucleolin was used as the internal control (Fig. 6b).
Additionally, BECT treatment did not induce any significant increase
inp65NF-
by immunofluorescence assay, and we confirmed translocation of
p65 NF- B into the nucleus within 30 min of LPS stimulation, which
kBnucleartranslocation.NF-kBlevelswerealsoconfirmed
k
was inhibited by treatment with 20
mM BECT (Fig. 6c).
Discussion
One of the most popular prototype synthetic anti-inflammatory
drugs worldwide is aspirin. Nevertheless, the mechanism behind
Please cite this article in press as: Kang S-M, et al. A novel synthetic HTB derivative, BECT inhibits lipopolysaccharide-mediated
inflammatory response by suppressing the p38 MAPK/JNK and NF-
10.1016/j.pharep.2013.08.015
kB activation pathways. Pharmacol Rep (2014), http://dx.doi.org/

G Model
PHAREP-46; No. of Pages 9
8
S.-M. Kang et al. / Pharmacological Reports xxx (2014) xxx–xxx
the release of NO in response to LPS and IgG/ovalbumin [13]. Further
studies on BECT were performedbased on its strong inhibitory effect
against NO production in microglial cells. Neuroinflammation is a
process that results primarily from an abnormally high or chronic
activation of microglia. This overactive state results in increased
levels of inflammatory and oxidative stress molecules, which can
lead to neuronal damage or death. Therefore, inhibiting excess
microglial activation might be a therapeutic approach to prevent the
progression of neurodegenerative diseases [29].
against NO inhibition and also exhibited inhibition of JNK,
p38MAPK, and NF- B signaling as mechanistic pathways involved
k
in modulation of inflammatory mediators. Thus, our results
suggest that BECT is a novel HTB derivative which has good
anti-neuroinflammatory activity and hence should be further
assessed by additional mechanistic and preclinical studies for its
use as an anti-neuroinflammatory agent.
Authors’ contributions
Activated microglia produce several potentially neurotoxic
mediators such as iNOS and COX-2 [30,31]. We found that BECT
dose-dependently reduced iNOS and COX-2 mRNA and protein
expression levels in LPS-stimulated BV-2 microglial cells. Our
results were in agreement with earlier reports showing decrease in
COX-2 release by HTB [14,32]. Pro-inflammatory cytokines such as
Seong-Mook Kang, Ju-Young Park, Byung-Wook Kim, Park Jeong
In were carried out the execution of experiments. Preparation of
manuscript with figures and referencing was done by Sandeep
Vasant More. Planning and interpretation of experimental data
were done by Sung-Hwa Yoon and Dong-Kug Choi.
IL-1b, IL-6, and TNF-a are crucial mediators during neuroin-
flammation [33]. Our mRNA expression profile analysis results
indicated a dose dependent decrease in mRNA expression in TNF-
Conflict of interest
a
, IL-1
the regulation of proinflammatory cytokines [34] with iNOS [35]
and COX-2 [36]. LPS increases nuclear translocation of the NF-
p65 subunit through I B- degradation and promotes tran-
scription of target genes [37]. In parallel to these reports, HTB is
also reported to suppress the activation of I B- , but has no effect
on I B- [32]. Hence, we further studied the effects of BECT on LPS-
induced I B- phosphorylation and nuclear translocation of NF-
B by immunoblot and immunofluorescence staining. In our
experiments, we observed that BECT decreased I B- phosphor-
ylation and nuclear translocation of NF- B p65 significantly as
b, and IL-6. NF-kB is a key transcription factor implicated in
Authors have no conflict of interest.
k
B
Funding
k
a/b
This study was supported by the Basic Science Research
Program through the National Research Foundation of Korea
(NRF) funded by the Ministry of Education, Science, and
Technology (2011-0014923).
k
b
k
a
k
a
k
k
a
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k
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Please cite this article in press as: Kang S-M, et al. A novel synthetic HTB derivative, BECT inhibits lipopolysaccharide-mediated
inflammatory response by suppressing the p38 MAPK/JNK and NF-
10.1016/j.pharep.2013.08.015
kB activation pathways. Pharmacol Rep (2014), http://dx.doi.org/