A novel synthetic compound MCAP suppresses LPS-induced murine microglial activation in vitro via inhibiting NF-kB and p38 MAPK pathways

Article abstract of DOI:10.1038/aps.2015.138

Aim: To investigate the anti-neuroinflammatory activity of a novel synthetic compound, 7-methylchroman-2-carboxylic acid N-(2-trifluoromethyl) phenylamide (MCAP) against LPS-induced microglial activation in vitro.Methods:Primary mouse microglia and BV2 microglia cells were exposed to LPS (50 or 100 ng/mL). The expression of iNOS and COX-2, proinflammatory cytokines, NF-κB and p38 MAPK signaling molecules were analyzed by RT-PCR, Western blot and ELISA. The morphological changes of microglia and nuclear translocation of NF-κB were visualized using phase contrast and fluorescence microscopy, respectively. Results: Pretreatment with MCAP (0.1, 1, 10 μmol/L) dose-dependently inhibited LPS-induced expression of iNOS and COX-2 in BV2 microglia cells. Similar results were obtained in primary microglia pretreated with MCAP (0.1, 0.5 μmol/L). MCAP dose-dependently abated LPS-induced release of TNF-α, IL-6 and IL-1β, and mitigated LPS-induced activation of NF-κB by reducing the phosphorylation of IκBα in BV2 microglia cells. Moreover, MCAP attenuated LPS-induced phosphorylation of p38 MAPK, whereas SB203580, a p38 MAPK inhibitor, significantly potentiated MCAP-caused inhibition on the expression of MEF-2 (a transcription factor downstream of p38 MAPK). Conclusion: MCAP exerts anti-inflammatory effects in murine microglia in vitro by inhibiting the p38 MAPK and NF-κB signaling pathways and proinflammatory responses. MCAP may be developed as a novel agent for treating diseases involving activated microglial cells.

Full text of DOI:10.1038/aps.2015.138

Acta Pharmacologica Sinica (2016) 37: 1–10
© 2016 CPS and SIMM All rights reserved 1671-4083/16
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Original Article
A novel synthetic compound MCAP suppresses LPS-
induced murine microglial activation in vitro via
inhibiting NF-kB and p38 MAPK pathways
Byung-Wook KIM1, 2, Sandeep Vasant MORE1, Yo-Sep YUN1, Hyun-Myung KO1, Jae-Hwan KWAK3, Heesoon LEE4, Kyoungho
1,
SUK2, In-Su KIM1, , Dong-Kug CHOI
*
*
1Department of Biotechnology, College of Biomedical and Health Science, Konkuk University, ChungJu 27478, Republic of Korea;
2Department of Pharmacology, Brain Science and Engineering Institute, Kyungpook National University School of Medicine, Daegu
41944, Republic of Korea; 3College of Pharmacy, Kyungsung University, Busan 48434, Republic of Korea; 4College of Pharmacy,
Chungbuk National University, Cheongju 28644, Republic of Korea
Aim: To investigate the anti-neuroinflammatory activity of a novel synthetic compound, 7-methylchroman-2-carboxylic acid N-(2-
trifluoromethyl) phenylamide (MCAP) against LPS-induced microglial activation in vitro.
Methods: Primary mouse microglia and BV2 microglia cells were exposed to LPS (50 or 100 ng/mL). The expression of iNOS and
COX-2, proinflammatory cytokines, NF-κB and p38 MAPK signaling molecules were analyzed by RT-PCR, Western blot and ELISA.
The morphological changes of microglia and nuclear translocation of NF-ĸB were visualized using phase contrast and fluorescence
microscopy, respectively.
Results: Pretreatment with MCAP (0.1, 1, 10 μmol/L) dose-dependently inhibited LPS-induced expression of iNOS and COX-2 in BV2
microglia cells. Similar results were obtained in primary microglia pretreated with MCAP (0.1, 0.5 μmol/L). MCAP dose-dependently
abated LPS-induced release of TNF-α, IL-6 and IL-1β, and mitigated LPS-induced activation of NF-κB by reducing the phosphorylation
of IκBα in BV2 microglia cells. Moreover, MCAP attenuated LPS-induced phosphorylation of p38 MAPK, whereas SB203580, a p38
MAPK inhibitor, significantly potentiated MCAP-caused inhibition on the expression of MEF-2 (a transcription factor downstream of p38
MAPK).
Conclusion: MCAP exerts anti-inflammatory effects in murine microglia in vitro by inhibiting the p38 MAPK and NF-κB signaling
pathways and proinflammatory responses. MCAP may be developed as a novel agent for treating diseases involving activated
microglial cells.
Keywords: 7-methylchroman-2-carboxylic acid N-(2-trifluoromethyl) phenylamide (MCAP); microglia; NO; NF-κB; MAPKs; LPS; SB203580;
neuroinflammation
Acta Pharmacologica Sinica advance online publication, 1 Feb 2016; doi: 10.1038/aps.2015.138
process[1-3]. In addition to lipopolysaccharides (LPS), BV2
Introduction
Microglial cells are the prime effector cells that participate in microglia cells also respond to other inflammatory triggers,
immune and inflammatory responses of host defense and tis- such as nitric oxide (NO), prostaglandins and proinflamma-
sue repair in the central nervous system (CNS). The over-acti- tory cytokines. These microglial products are thought to be
vation of microglia in various neuropathological conditions, responsible for neuroglia-mediated neurotoxicity[4]. Thus,
such as Alzheimer’s disease (AD), Parkinson’s disease (PD), the suppression of microglial activation could be an effec-
multiple sclerosis and cerebral ischemia, leads to increased tive therapeutic approach for alleviating the progression of
neural cell death and further deteriorates the on-going disease neurodegenerative diseases involving excessive microglial
activation[5, 6]. Despite the fact that many of the leading anti-
inflammatory compounds are analogues of natural products
*
To whom correspondence should be addressed.
that are not readily amenable to high throughput chemical
screening and that the inhibition of multiple protein kinases is
involved in the regulation of interleukin-1β (IL-1β) and induc-
E-mail choidk@kku.ac.kr (Dong- Kug CHOI);
kis5497@kku.ac.kr (In-Su KIM)
Received 2015-09-23 Accepted 2015-12-17

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Kim BW et al
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ible nitric oxide synthase (iNOS) induction[7], there is a need Cell culture, morphological examination, NO release assay and
for developing a new chemical class of compounds that are cell viability
cell-permeable inhibitors of glial activation and are more spe- Primary microglial were obtained from a 2 to 3-d old mice by
cific inhibitors of the protein kinases involved in the regulation chopping and mechanically disrupting the whole brain using
of proinflammatory mediators.
a 70 μmol/L nylon mesh. The cells obtained were seeded in
Nuclear factor kappa B (NF-κB) is a transcription factor culture flasks and grown at 37°C in a 5% CO2 atmosphere in
that regulates the expression of numerous proinflammatory DMEM supplemented with 10% FBS and 100 U/mL P–S. The
genes[8-11] and has also been demonstrated to play an essential culture medium was changed initially after 5 days and then
role in the LPS-induced expression of iNOS, cyclooxygenase-2 afterward every 3 days, and the cells were used after being in
(COX-2) and proinflammatory cytokines. Therefore, it has culture for 21 days. Primary microglial cells were obtained
been suggested that inhibition of NF-κB activation might help by shaking the mixed glial cultures at 200 rounds/min for 4 h.
to mitigate on-going inflammation.
The culture medium was then centrifuged at 1200 rounds/min
In the present study, we synthesized a novel MCAP [7-meth- for 10 min. Finally, the primary microglia cultures were
ylchroman-2-carboxylic acid N-(2-trifluoromethyl) phenyl- grown and maintained in DMEM supplemented with 10% FBS
amide] and then investigated its possible anti-inflammatory and P–S. The BV2 microglia cells were acquired as described
effects to an LPS-induced inflammatory response in primary previously[15]. The BV2 cells were cultured in DMEM supple-
microglia and BV2 microglia cells. MCAP significantly mented with 5% FBS and 100 U/mL P–S and maintained in a
inhibited the release of NO in LPS-induced primary microg- humidified incubator in an atmosphere of 5% CO2.
lia and BV2 microglia cells. Moreover, MCAP significantly
In all of the experiments, the cells were seeded at a density
suppressed the release of inflammatory mediators, such as of 5×105 cells/mL and pretreated for 1 h with the indicated
iNOS, COX-2 and cytokines, in LPS-stimulated BV2 cells. In concentrations of MCAP before incubation in a medium
addition, we also demonstrated that MCAP reduced the phos- containing LPS (50 or 100 ng/mL). In the cell morphology
phorylation of p38 mitogen-activated protein kinase (MAPK) experiment, the primary microglial were pretreated with 0.5
when it was accompanied by the inhibition of myocyte- μmol/L of MCAP followed by LPS (50 ng/mL) induction for
enhancer factor (MEF-2), a transcription factor downstream 24 h, and the cellular morphology images were observed by
of p38 MAPK[12-14]. Furthermore, MCAP inhibited the nuclear using phase contrast microscopy (Axio; Carl Zeiss, Germany).
translocation of NF-ĸB, which ultimately contributes to the The LPS-induced release of NO in the culture supernatants
inflammatory milieu. In conclusion, our findings suggest that was determined by measuring nitrite, a major stable prod-
MCAP is a therapeutic candidate for the treatment of vari- uct of NO, using the Griess reagent (1% sulfanilamide/0.1%
ous neurodegenerative diseases wherein microglial activation N-(1-naphthyl)-ethylenediamine dihydrochloride/2.5%
plays an important role in the pathogenesis of the disease.
H3PO4). The cell viability of the cultured cells was determined
by measuring the reduction of MTT to formazan. Briefly, the
cells (5×104 cells/well) were seeded and treated with different
concentrations of MCAP (0.1 and 0.5 or 0.1, 1 and 10 μmol/L)
Materials and methods
Reagents
MCAP (chemical structure and synthesis are shown in Fig- followed by an incubation with LPS for 24 h. Following the
ure 1), lipopolysaccharide (LPS, E. coli 0111:B4), dimethyl 24-h LPS incubation, 50 μL of each culture medium was mixed
sulfoxide (DMSO), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl- with an equal volume of Griess reagent. Nitrite levels were
tetrazolium bromide (MTT), sulfanilamide, N-(1-naphthyl)- determined using a microplate reader at 540 nm (Tecan Trad-
ethylenediamine dihydrochloride and Tween-20 were pur- ing AG, Basel, Switzerland), and nitrite concentrations were
chased from Sigma–Aldrich (St Louis, MO, USA). Dulbecco's calculated by reference to a standard curve generated by
Modified Eagle Medium (DMEM), fetal bovine serum (FBS), known concentrations of sodium nitrite. Cell viability was
penicillin–streptomycin (P–S), trypsin/EDTA (TE) and TRIzol, measured by adding 0.5 mg/mL of MTT to each well. After
as well as other cell culture plates, were obtained from Gibco- incubation for another 4 h at 37 °C in 5% CO2, the superna-
BRL (Rockville, MD, USA). The protease inhibitor cocktail tants were removed from each well and the formazan crystals
tablets and the phosphatase inhibitor cocktail tablets were formed were dissolved in DMSO. The absorbance was deter-
supplied by Roche (Mannheim, Germany). The antibodies for mined at 540 nm using a microplate reader (Tecan Trading
COX-2, NF-κB p65, MEF-2 and nucleolin were obtained from AG).
Santa Cruz (Santa Cruz, CA, USA). iNOS, IκBα, phosphor(p)-
IκBα, p38, p-p38, SB203580 (p38 specific inhibitor) and β-actin Reverse transcription–polymerase chain reaction (RT-PCR)
were procured from Cell Signaling (Danvers, MA, USA). The The BV2 microglia cells were plated overnight in 6-well cul-
nuclear extract kit was purchased from Thermo Scientific ture plates at a density of 5×105 cells/mL and pretreated for
(Rockford, IL, USA). The nitric oxide synthase 2, inducible 1 h with various concentrations of MCAP, including 0.1, 1
(NOS2) enzyme-linked immunosorbent assay (ELISA) kit was and 10 μmol/L, before incubation in a medium containing
from USCNLIFETM (Wuhan, China), and the prostaglandin LPS (100 ng/mL). Total RNA was extracted using TRIZOL
E2 (PGE2) ELISA kit was purchased from Cayman Chemical (Invitrogen Life Technologies, Carlsbad, CA, USA). RNA (2.5
Company (Ann Arbor, MI, USA).
μg) was reverse-transcribed using the Superscript™ III kit
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(Invitrogen) according to the manufacturer’s instructions. The to the BV2 cells (1×106 cells/mL) cultured in 6-well plates. The
iNOS, COX-2, tumor necrosis factor-α (TNF-α), IL-1β, IL-6 cells were scraped, incubated for 10 min on ice and centrifuged
and GAPDH genes were amplified from the cDNA by poly- at 14000 rounds/min for 10 min at 4ºC. The protein concen-
merase chain reaction (PCR). The cDNA was amplified by tration was determined by the DC protein assay from Bio-Rad
PCR using the specific primers presented in Table 1. The PCR (Hercules, CA, USA), and 10 μg of the whole cell lysate was
was performed using an initial step of denaturation (5 min at used for inducible (NOS2) ELISA kit. The iNOS enzyme activ-
94°C), 20–28 cycles of amplification (94°C for 30 s, 54−58°C ity in lysate was determined spectrophotometrically (Tecan
for 1 min and 72 °C for 1 min) and an extension (72 °C for 5 Trading AG) at 450 nm. Each experiment was performed in
min). The PCR products were analyzed on 1% agarose gels. triplicate.
The GAPDH mRNA served as the internal control for sample
loading and mRNA integrity. The band intensity was quanti- Prostaglandin E2 (PGE2) measurement
fied by densitometry analysis using Multi-gauge software V3.1 The BV2 microglia cells cultured in 24-well plates were pre-
(Fujifilm, Tokyo, Japan).
treated for 1 h with the indicated doses of MCAP followed
by an incubation with LPS for 24 h. The PGE2 level in the
culture supernatant was measured by a commercial kit (Cay-
Western blot analysis
The BV2 cells were plated overnight in 6-well culture plates at man Chemicals) according to the manufacturer’s instructions.
a density of 5×105 cells/mL and further incubated in serum- Briefly, 50 μL of the PGE2 acetylcholinesterase conjugate (as
free medium for at least 3 h before treatment. The cells were tracer), 50 μL of the PGE2 monoclonal antibody and 50 μL of
lysed using RIPA buffer (1× PBS, 1% NP-40, 0.5% sodium the culture supernatant were combined in each well of a plate
deoxycholate and 0.1% SDS, containing fresh protease inhibi- pre-coated with a goat polyclonal mouse immunoglobulin G
tor cocktail). The electrophoresis and immunoblotting proce- (IgG) and was incubated for 18 h at 4°C. The wells were emp-
dures were performed according to a previous report[16]. The tied and rinsed five times with wash buffer. Ellman’s reagent
membranes were probed with primary antibodies to iNOS, (200 mL) was added to each well to develop in the dark for
IκBα, p-IκBα, p38, p-p38 (1:1000; Cell Signaling), β-actin 60–90 min at room temperature. The PGE2 concentrations
(1:2000; Cell Signaling), COX-2, p65, MEF-2 (1:1000; Santa were determined using a PGE2 standard. The absorbance
Cruz) and nucleolin (1:500; Santa Cruz). After incubation with at 420 nm was determined using a microplate reader (Tecan
the appropriate primary antibody, the membranes were incu- Trading AG).
bated for 1 h at room temperature with a secondary antibody
conjugated to horseradish peroxidase (1:2000 dilution; rabbit, Immunofluorescence (IF) assay
Cell Signaling, goat; Santa Cruz). The blots were visualized by For the detection of the intracellular location of the p65 sub-
a PowerOpti-ECL (Animal Genetics Inc, Tallahassee, FL, USA) unit of NF-κB, the BV2 cells (1×105 cells/well in 12-well plate)
detection system according to the recommended procedure. were cultured on sterile cover slips placed in 24-well plates
In a parallel experiment, the nuclear protein was prepared and treated with the compound followed by LPS. Sixty min
using a nuclear extraction kit (Thermo Scientific, Rockford, IL, after the LPS treatment, the cells were fixed with methanol for
USA) according to the manufacturer’s protocol.
20 min at −20°C and washed with PBS for 5 min. The fixed
cells were then permeabilized with 1% Triton X-100 in PBS for
10 min at room temperature and washed with 0.02% Tween-
Inducible NOS activity assay
To obtain the total cell lysate, 70 μL of RIPA buffer was added 20 in PBS for 5 min. The permeabilized cells were then treated
with 1:200 dilution of NF-κB (p65) (Santa Cruz) overnight at
4°C and washed with 0.02% Tween-20 in PBS for 5 min. The
cells were then incubated in a 1:100 dilution of an Alexa Fluor
Table 1. PCR primers used in this study.
568-labeled antibody (Invitrogen) for 1 h at room temperature
Gene target Accession
Primer sequence
Size
(bp)
and washed with 0.02% Tween-20 in PBS for 5 min. The cells
were then stained with 1 μmol/L of a Hoechst (Invitrogen)
staining solution for 5 min at room temperature and then
washed. Finally, all of the images were captured with a fluo-
rescence microscope (Axio; Carl Zeiss)
iNOS
COX-2
TNF-α
IL-1β
NM_010927 F 5′-CTTGCAAGTCCAAGTCTTGC-3′
R 5′-GTATGTGTCTGCAGATGTGCTG-3′
NM_011198 F 5′-ACATCCCTGAGAACCTGCAGT-3′
R 5′-CCAGGAGGATGGAGTTGTTGT-3′
369
414
390
385
435
378
NM_01369
F 5′-TTCGAGTGACAAGCCTGTAGC-3′
R 5′-AGATTGACCTCAGCGCTGAGT-3′
Statistical analysis
The values given are the means±SEM of at least three separate
experiments conducted in triplicate. Comparisons between
groups were analyzed using a One-way analysis of variance
(ANOVA) followed by Tukey’s multiple comparison test using
the software Graphpad Prism V5.01 (GraphPad Software Inc,
San Diego, CA, USA).
NM_008361 F 5′-CATATGAGCTGAAAGCTCTCCA-3′
R 5′-GACACAGATTCCATGGTGAAGTC-3′
IL-6
NM_031168 F 5′-CATATGAGCTGAAAGCTCTCCA-3′
R 5′-GACACAGATTCCATGGTGAAGTC-3′
GAPDH
GU214026
F 5′-CCAGTATGACTCCACTCACG-3′
R 5′-CCTTCCACAATGCCAAAGTT-3′
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LPS group in Figure 2A; P<0.01 vs the LPS group in Figure
2B). We observed a significant inhibition of NO after treat-
Results
Synthesis of MCAP
The synthetic strategy used to prepare the target compound, ment with 0.5 μmol/L and 10 μmol/L of MCAP. To further
MCAP, is outlined in Figure 1. Initially, 2’-hydroxy-4’-methyl- evaluate the cytotoxic effects of MCAP and/or LPS in primary
acetophenone was treated with diethyl oxalate in sodium microglia and BV2 microglia cells, cell viability was deter-
ethoxide and then with a hydrochloride solution to give the mined by an MTT assay. MCAP- and LPS-treated microglial
cyclized intermediate ethyl 7-methyl-4-oxo-4H-chromene- cells individually did not elicit any signs of toxicity at the
2-carboxylate in a 98% yield. The catalytic hydrogenation of selected concentrations. We also found that MCAP alone at
this ester was carried out with 10% Pd/C in ethanol in the doses of 0.5 μmol/L and 10 μmol/L had no toxic effects on
presence of a catalytic amount of acetic acid under a H2 gas primary microglia and BV2 microglia cells, respectively (Fig-
atmosphere to give the ethyl 7-methylchroman-2-carboxylate ure 2C−D). In addition, we examined the cell morphology of
in a 99% yield. Treatment of the intermediate with potassium the primary microglial cells that were incubated with MCAP
hydroxide in water afforded 7-methylchroman-2-carboxylic (0.5 μmol/L) in the presence or absence of LPS (50 ng/mL).
acid in a 96% yield. The coupling reaction of the acid with Bright field images were obtained after 24 h using the inverted
2-(trifluoromethyl) aniline using N,N-carbonyldiimidazole microscope. The shape of the LPS-treated microglial cells was
(CDI) in anhydrous THF provided the MCAP. The oil product ramified compared to the control group, indicating activation
was obtained in a 25% yield after flash column chromatogra- of the microglial cells. This morphological change induced by
1
phy[17]. IR (KBr): 3421, 1710, 1535, 634 cm-1; H NMR (CDCl3, LPS treatment was successfully inhibited by pretreatment with
300 MHz): δ8.39 (d, 1H, Ј=8.1 Hz, Ar-H), 7.63 (d, 1H, Ј=8.1 Hz, 0.5 μmol/L of MCAP (Figure 2E).
Ar-H), 7.58 (t, 1H, Ј=8.1 Hz, Ar-H), 7.24 (t, 1H, Ј=8.1 Hz,
MCAP regulates LPS-induced iNOS and COX-2 production in BV2
microglia cells
Ar-H), 6.98 (d, 1H, Ј=7.7 Hz, Ar-H), 6.78 (s, 1H, Ar-H), 6.76
(d, 1H, Ј=7.7 Hz, Ar-H), 4.65 (dd, 1H, Ј=9.8, 2.9 Hz, OCH),
2.95-2.75 (m, 2H, CH2), 2.52-2.06 (m, 2H, CH2), 2.32 (s, 3H, Because MCAP, at the indicated concentrations (0.1, 1 and 10
Ar-CH3). MCAP (purity>99%) was dissolved in DMSO.
μmol/L), attenuated NO production, we further examined
the effect of MCAP on the mRNA and protein expressions
Effect of MCAP on NO production in LPS-stimulated primary of iNOS and COX-2 in the BV2 cells. The inhibitory effects
microglia and BV2 microglia cells of MCAP on the mRNA and protein expressions of iNOS
Initially, we aimed to evaluate the effect of MCAP on NO and COX-2 were determined by RT-PCR and Western blot
production in LPS-stimulated primary microglia and BV2 analysis, respectively. The levels of iNOS and COX-2 mRNA
microglia cells. For this, the primary microglia were pre- were markedly increased after 24 h of LPS (100 ng/mL) treat-
treated with MCAP (0.1 or 0.5 μmol/L), while the BV2 cells ment, and MCAP significantly inhibited iNOS and COX-2
were treated with 0.1, 1 or 10 μmol/L of MCAP for 1 h before mRNA expression in the LPS-stimulated BV2 cells in a con-
the addition of LPS (50 and 100 ng/mL, respectively), Incuba- centration-dependent manner (Figure 3A−B; P<0.05, P<0.01
tion with LPS alone markedly increased NO production in vs LPS group). LPS-stimulated BV2 cells showed a significant
the primary microglia (14.1±1.2 μmol/L; Figure 2A; P<0.01 vs increase in iNOS and COX-2 protein levels when compared
the control group) and BV2 cells (25.6±2.9 μmol/L; Figure 2B; to the controls (P<0.01 vs the control group). Pre-treatment
P<0.01 vs the control group) compared to the control. How- with MCAP at various concentrations (0.1, 1 and 10 μmol/L)
ever, pretreatment with MCAP prevented the increased NO significantly attenuated the LPS-stimulated increase in iNOS
production levels in both LPS-stimulated microglial cells in a and COX-2 levels (Figure 3C−D; P<0.01 vs the LPS group).
concentration-dependent manner (P<0.05 and P<0.01 vs the A Western blot analysis showed that the reduction in iNOS
Figure 1. The structure and synthesis of MCAP. NaOEt, sodium ethoxide; EtOH, ethanol; CDI, N,N-carbonyldiimidazole.
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Figure 2. Effect of MCAP on cell viability and NO production in LPS-stimulated microglia. Mouse primary microglia (A, C) and BV2 microglia (B, D) cells
were pretreated with various concentrations of MCAP (0.1 and 0.5 µmol/L for the primary microglia and 0.1, 1 and 10 µmol/L for the BV2 cells) for 1 h
before incubation with LPS (50 and 100 ng/mL, respectively) for 24 h. The nitrite content was measured using the Griess reaction (A, B). The viability
in MCAP-treated cells was evaluated using an MTT assay (C, D). The results are displayed as a percentage of the control samples. The morphological
changes are represented in the primary microglia cells (E). Scale bar, 50 µmol/L. The data represent the mean±SEM from three independent
experiments. cP<0.01 vs the control group; eP<0.05, fP<0.01 vs the LPS alone group by a one-way ANOVA followed by Tukey’s multiple comparison test.
and COX-2 protein levels was correlated with the reduction or 10 μmol/L) concentration-dependently decreased the PGE2
in their corresponding mRNA levels. In addition, MCAP synthesis (Figure 3F; P<0.01 vs the LPS group).
reduced the LPS-stimulated iNOS enzyme activity in the BV2
cells in a dose-dependent manner. The data showed a signifi- MCAP inhibited the production of proinflammatory cytokines in
cant reduction in the enzyme activity by MCAP treatment at the LPS-induced BV2 microglia cells
a 10 μmol/L concentration in the LPS-treated BV2 cells (Fig- Proinflammatory cytokines, such as TNF-α, IL-1β and IL-6,
ure 3E; P<0.01 vs the LPS group). PGE2 represents the most play central roles in microglia-mediated inflammation. There-
important inflammatory product of COX-2 activity; therefore, fore, we analyzed the effects of MCAP on proinflammatory
we quantified the PGE2 levels present in the supernatant of cytokines (TNF-α, IL-1β and IL-6). The BV2 cells were incu-
the LPS-exposed BV2 cells. To assess whether MCAP inhib- bated with MCAP (0.1, 1 and 10 μmol/L) in the presence or
its LPS-induced PGE2 production in the BV2 cells, the cells absence of LPS (100 ng/mL). The RT-PCR results demon-
were pretreated with MCAP for 1 h and then stimulated with strated that the mRNA levels of these cytokines were elevated
LPS (100 ng/mL). After incubation for 24 h, the cell culture 6 h after the LPS treatment. Pre-treatment with MCAP signifi-
medium was harvested and the production of PGE2 was mea- cantly inhibited LPS-induced TNF-α, IL-1β and IL-6 produc-
sured using an ELISA. As shown in Figure 2F, the amount tion, compared to the LPS-treated cells (Figure 4A). The rep-
of PGE2 present in the culture medium increased to approxi- resentative quantification data revealed that LPS-stimulated
mately 221.8±4.3 pg/mL after a 24-h exposure to LPS alone proinflammatory cytokine mRNA levels (TNF-α, IL-1β and
(P<0.01 vs the control group). Pretreatment with MCAP (0.1, 1 IL-6) were significantly decreased following MCAP treatment
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Figure 3. MCAP attenuates expression of iNOS and COX-2 levels in LPS-stimulated BV2 microglia cells. The BV2 cells were pre-treated with the
indicated concentrations of MCAP for 1 h before incubating them with LPS (100 ng/mL) for 6 h (RT-PCR) and 18 h (immunoblotting). Total RNA was
prepared and analyzed for iNOS (A) and COX-2 (B) gene expression by RT-PCR. The lysates were analyzed by immunoblotting with iNOS (C) and COX-2
(D) antibodies. The quantification of the data are shown in the lower panel. The BV2 cells were pre-treated with the indicated concentrations of MCAP
for 1 h before incubating with LPS (100 ng/mL) for 24 h. The lysates were analyzed by an inducible (NOS2) ELISA kit. The absorbance was determined
spectrophotometrically at 450 nm (E). The PGE2 concentration in the culture medium was measured using a commercial ELISA kit. The absorbance
was measured at 420 nm spectrophotometrically (F). The data represent the mean±SEM from three independent experiments. cP<0.01 vs the control
group; eP<0.05, fP<0.01 vs the LPS alone group by a one-way ANOVA followed by Tukey’s multiple comparison test.
(Figure 4B−D; P<0.05 and P<0.01 vs the LPS group). Thus, our expression, the effect of MCAP on the blockade of NF-κB
results indicate that MCAP inhibited the expression of cyto- activity was determined by an immunocytochemistry-immu-
kines involved in the inflammatory process.
nofluorescence (ICC-IF) assay. LPS treatment caused a signifi-
cant increase in the translocation of the NF-κB p65 subunit into
MCAP suppressed NF-κB activity in the LPS-stimulated BV2 the nucleus within 30 min after LPS stimulation. In contrast,
microglia cells
treatment with MCAP markedly suppressed the translocation
NF-κB is one of the principal factors for iNOS and COX-2 expres- of NF-κB p65 induced by LPS (Figure 5A). We also investi-
sion mediated by LPS or proinflammatory mediators[9, 18]
To gated the effect of MCAP on LPS-induced NF-κB p65 nuclear
.
elucidate the molecular mechanism by which MCAP inhibits translocation as measured by a Western blot analysis. LPS-
iNOS and COX-2 expression and proinflammatory cytokine stimulated BV2 cells were pre-treated with MCAP (0.1, 1 or
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Figure 4. MCAP decreases the production of proinflammatory cytokines. (A) The cells were pre-treated with the indicated doses of MCAP for 1 h before
LPS (100 ng/mL) treatment. The levels of TNF-α, IL-1β, IL-6 and GAPDH mRNA were determined by RT–PCR. The representative densitometry analyses
of TNF-α (B), IL-1β (C) and IL-6 (D) compared with GAPDH mRNA. The data represent the mean±SEM from three independent experiments. cP<0.01 vs
the control group; eP<0.05, fP<0.01 vs the LPS alone group by a one-way ANOVA followed by Tukey’s multiple comparison test.
10 μmol/L) for 1 h, and NF-κB activity was evaluated accord-
Discussion
ing to the nuclear translocation of the p65 subunit of NF-κB In the present study, we demonstrated that a synthetic com-
(Figure 5B; P<0.05 and P<0.01 vs LPS group). Furthermore, pound, named 7-methylchroman-2-carboxylic acid N-(2-
the phosphorylation of IκBα was also significantly inhibited trifluoromethyl) phenylamide (MCAP), exhibits a significant
by MCAP treatment of the BV2 cells (Figure 5C; P<0.05 and inhibitory action by blocking the synthesis of proinflammatory
P<0.01 vs LPS group). Taken together, these results indicate mediators in LPS-stimulated BV2 microglia cells. The func-
that inhibition of NF-κB activation by MCAP may be the tional role of microglia in the diseased brain is controversial
mechanism responsible for the suppression of iNOS, COX-2 because it has been observed to protect as well as to destroy
and other proinflammatory cytokine expression in the LPS- or exacerbate the neurotoxicity[24]. Activated microglia pro-
stimulated BV2 cells.
duces several neurotoxic molecules, such as iNOS, COX-2
and cytokines, such as TNF-α, IL-1β and IL-6[25-27]. Based on
MCAP inhibited the phosphorylation of p38 MAPK and the the strong inhibitory action of MCAP on NO, iNOS activity
expression of MEF-2 in the LPS-induced BV2 cells
and PGE2 production, MCAP was further investigated for its
The phosphorylation of p38 MAPK induces the expression of effect on iNOS and COX-2 expression along with its effect on
proinflammatory molecules and is one of the important signal- the release of proinflammatory cytokines. Additionally, we
ing pathways during inflammation[19, 20]. MEF-2 is a transcrip- only included the primary microglia results concerning cell
tion factor that is activated by p38 MAPK during inflamma- viability, cell morphology and NO production because they
tion[12, 21, 22]. Therefore, we examined the effect of MCAP on the also might reflect the result of our compound in an animal
LPS-induced phosphorylation of p38 MAPK in the BV2 cells study. However, due to time constraints and cell availability,
by a Western blot analysis. Pretreatment with MCAP at the we decided to carry out the rest of the experiments in the BV2
indicated concentrations (0.1, 1 and 10 μmol/L) significantly cells[15, 28]. We observed that MCAP satisfactorily inhibited the
inhibited the LPS-stimulated p38 phosphorylation in the BV2 mRNA expressions of TNF-α, IL-1β and IL-6, with the most
cells (Figure 6A; P<0.05 and P<0.01 vs the LPS group). Fur- significant inhibition at 1 and 10 μmol/L for TNF-α and IL-6
thermore, the effect of MCAP on the inhibition of MEF-2 acti- secretion. MCAP was also observed to dose-dependently
vation was consistent with the effect of a specific p38 inhibitor reduce the mRNA and protein expressions of iNOS and COX-
SB203580[23] when examined simultaneously in LPS-stimulated 2. In addition, 0.5 μmol/L of MCAP restored the morphologi-
BV2 cells (Figure 6B; P<0.05 and P<0.01 vs LPS group). These cal changes in LPS- (50 ng/mL) stimulated primary microglia
results suggest that MCAP is indeed capable of disrupting p38 cells. Our results are consistent with previous reports on LPS-
MAPK, the key signal transduction pathway elicited by LPS in induced morphological changes in microglia cells[29, 30]
.
the BV2 cells, and subsequently preventing the production of
proinflammatory mediators.
The MAPK signaling pathways fine-tune a variety of cel-
lular activities, such as survival, differentiation, proliferation
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Figure 5. Inhibition of LPS-induced NF-κB activity by MCAP. (A) The BV2 cells were treated with LPS (100 ng/mL) in the absence or presence of MCAP
for the indicated times. The nuclear translocation of the NF-κB p65 subunit was determined by an immunofluorescence assay. Scale bar, 20 µmol/L.
(B) Nuclear extracts were prepared and analyzed by a Western blot to asses NF-κB p65 and nucleolin. (C) The cytosolic extracts were prepared and
analyzed by a Western blot using IκBα, p-IκBα and β-actin antibodies. The densitometry analysis of NF-κB p65 and p-IκBα is presented in the lower
panel. The data represent the mean±SEM from three independent experiments. cP<0.01 vs the control group; eP<0.05 and fP<0.01 vs the LPS alone
group by a one-way ANOVA followed by Tukey’s multiple comparison test.
and neuronal death. Deviation from the normal MAPK sig- gated the effect of MCAP on the protein expression of MEF-2
naling pathways has been implicated in the development of in the nuclear extracts of the LPS-stimulated microglial cells.
many neurodegenerative diseases, such as AD, PD, and amyo- MCAP significantly inhibited the expression of MEF-2. We
trophic lateral sclerosis (ALS)[31, 32]. Hyper-phosphorylation also assessed the synergistic effect of MCAP and SB203580,
of MAPK molecules ultimately activates the transcription a specific inhibitor of p38 MAPK. Our primary goal in per-
factor NF-κB and the subsequent production of inflammatory forming this experiment was to observe the synergistic effect
molecules[33]. Furthermore, several others studies[34, 35] have of MCAP and SB203580 in combination with LPS to inhibit
reported that the NF-κB and p38 MAPK pathways are impor- MEF-2 levels. Additionally, some evidence[37] shows that
tant targets for therapeutic drugs designed to control inflam- SB203580 does not affect MEF-2 expression per se. Further-
mation in the CNS. We also found that MCAP significantly more, we found a significant decrease in MEF-2 expression in
attenuated the LPS-stimulated phosphorylation of p38 MAPK the BV2 cells treated with SB203580 and MCAP.
in a dose-dependent manner. However, there was no effect of
In addition, several others studies have reported that the
MCAP on LPS-induced ERK and JNK phosphorylation (data NF-κB and p38 MAPK pathways are important targets for
not shown). MEF-2 is the downstream signaling transcription therapeutic drugs (involved in the control of proinflamma-
factor involved in mediating the inflammatory effects of p38 tory gene expression) designed to control inflammation in the
MAPK signaling[12, 14, 36]. Therefore, in our study, we investi- CNS[34, 38]. In our study, MCAP decreased the phosphorylation
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9
Figure 6. Inhibitory effects of MCAP on the LPS-induced activation of p38 MAPK and the expression of MEF-2 in the BV2 cells. (A) The BV2 cells were
treated with the indicated doses of MCAP for 1 h before LPS treatment (100 ng/mL) for 30 min. The densitometry analysis of the p38 and p-p38 bands
is presented in the lower panel. (B) The cells were pre-treated with SB203580 (10 µmol/L) and MCAP (10 µmol/L) for 1 h before LPS (100 ng/mL)
treatment. The level of MEF-2 protein was determined by immunoblotting. The quantification data are shown in the lower panel. The data represent
e
f
the mean±SEM from three independent experiments. cP<0.01 vs the control group; P<0.05, P<0.01 vs the LPS alone group by a one-way ANOVA
followed by Tukey’s multiple comparison test.
neuroinflammation on the pathogenesis of Parkinson's disease. BMB
Rep 2010; 43: 225−32.
of IκBα in a dose-dependent manner, and this effect was also
correlated to a parallel decrease in the nuclear translocation of
2
More SV, Kumar H, Kim IS, Koppulla S, Kim BW, Choi DK. Strategic
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the p65 subunit of NF-κB as confirmed by immunocytochemis-
try and a Western blot analysis. Our results are in accordance
with previous reports demonstrating that the inhibition of
IκBα phosphorylation leads to the diminished nuclear translo-
3
4
cation of the p65 subunit of NF-κB[16, 39]
.
In conclusion, the results of this study suggest that pre-treat-
ment with MCAP mitigates LPS-induced iNOS, COX-2 and
cytokine production in activated microglia. Apart from the
inhibitory effects on microglial p38 MAPK and NF-κB, further
research is needed to affirm the current findings that MCAP
may have therapeutic potential for ameliorating microglia-
mediated neuronal damage in many neuroinflammatory con-
ditions.
5
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Acknowledgements
This work was supported by the National Research Founda-
tion of Korea (NRF) grant funded by the Korea government
(MEST) (NRF-2014R1A2A2A04007791) and supported by the
Basic Science Research Program through the NRF funded by
the Ministry of Education, Science and Technology (NRF-2011-
0024940).
8
9
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Author contribution
Byung-Wook KIM, In-Su KIM and Dong-Kug CHOI designed
the experiments; Byung-Wook KIM, Sandeep Vasant MORE,
Jae-Hwan KWAK and Yo-Sep YUN performed parts of the
experiments; Byung-Wook KIM, Hyun-Myung KO, Heesoon
LEE, Kyoungho SUK and In-Su KIM analyzed the data;
Byung-Wook KIM, Sandeep Vasant MORE, In-Su KIM and
Dong-Kug CHOI wrote the manuscript.
12 Han J, Jiang Y, Li Z, Kravchenko VV, Ulevitch RJ. Activation of the
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Nature 1997; 386: 296−9.
13 Wang BW, Chang H, Kuan P, Shyu KG. Angiotensin II activates
myostatin expression in cultured rat neonatal cardiomyocytes via p38
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