6
14
E.-J. CHOI ET AL.
Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). Jurkat cells Animals
human T lymphocytes) were obtained from the ATCC (Manassas,
(
Eight-week-old female BALB/c mice were purchased from Samtako
Osan, Republic of Korea) and housed under specific-pathogen-
free conditions. All the experiments were approved by the
Institutional Animal Care and Use Committee (IACUC) of Konkuk
University, Korea (Protocol no. KU14012).
VA, USA) and cultured in RPMI-1640 Medium (Sigma-Aldrich, St.
Louis, MO, USA) supplemented with 10% fetal bovine serum
(
ꢁ
(Gibco, Thermo Fisher Scientific, Waltham, MA, USA) at 37 C in a
5
% CO atmosphere. All other reagents were of the highest grade
2
commercially available at the time of the study.
Induction of AD lesions in the ear
General procedure and characterisation of amide derivatives
AD was induced in the mice by repeated local exposure of the
2
0
p-Coumaric acid (1.0 g, 61.09 mmol, 1.0 equiv.) was dissolved in ears to DFE and DNCB, as previously described . For the induc-
ꢁ
dimethylformamide (20 mL) and stirred at 0 C for 5 min. tion of AD, the mice were divided into six groups (control, AD-
Triethylamine (0.85 mL, 6.09 mmol, 1.0 equiv.) was added drop-wise only, NCT-only, NCPA-only, AD þ NCT, and AD þ NCPA). Structures
of NCT and NCPA and experimental design are shown in
Figure 1(a,b), respectively. To induce AD, the surfaces of both ear-
lobes were stripped five times using surgical tape (Nichiban,
Tokyo, Japan), and 20 mL 1% DNCB was applied to each ear, fol-
lowed 4 days later by 20 mL of DFE (10 mg/mL). DFE and DNCB
treatments were administered alternately once per week for
and stirred for 5 min before the corresponding amines (6.09 mmol,
1
.0 equiv.) were added, followed by a solution of benzotriazol-1-
yloxy-tris-(dimethylamino)-phosphonium hexafluorophosphate
, 20 mL). The mixture was stirred
(BOP) in dichloromethane (CH
2
Cl
2
ꢁ
at 0 C for 30 min and then at room temperature overnight. The
reaction mixture was evaporated under reduced pressure; ethyl
4
weeks. The animals in NCT-only, NCPA-only, AD þ NCT, and
acetate (30 mL) and water (H O, 20 mL) were added, and the
2
AD þ NCPA groups were orally administrated NCT or NCPA
organic layer was washed with 1 N hydrochloric acid (HCl, aq.,
(50 mg/kg/day) throughout the 4-week AD induction period.
2
3
0 mL) and sodium bicarbonate (NaHCO , aq., saturated, 20 mL).
The ear thickness was measured 24 h after DNCB or DFE appli-
The organic layer was separated and dried over anhydrous magne-
cations using a dial thickness gauge (ID-C1012XBS, Mitutoyo Co.,
Kawasaki, Japan). On days 14 and 28, blood samples were col-
lected by orbital puncture; plasma samples were prepared, and
sium sulfate (MgSO ). The solvent was evaporated under reduced
4
pressure, and the residue was further purified using an open-
bed silica gel column (4 ꢂ 30 cm, ethyl acetate:n-hexane:methanol,
ꢁ
stored at –70 C for further analysis. After blood collection on day
6
:2:1, v/v/v). Fractions containing the product were combined and
2
8, mice ears were removed and histopathologically analysed.
evaporated under reduced pressure to obtain the correspond-
ing amides.
Serum IgE and IgG2a levels were measured on days 14 and 28
after the first induction using an IgE enzyme-linked immunoassay
kit (Bethyl Laboratories, Inc., Montgomery, TX, USA) according to
the manufacturer’s instructions.
Nct
The synthesised NCT had the following characteristics: yellowish
Histological observations
ꢁ
þ
powder; melting point (mp), 143–146 C; EIMS, m/z 267 [M] ;
HREIMS, m/z 267.1259 (calcd for C17
H
17NO
2
267.1259); IR (KBr) The excised mouse ears were fixed in 4% paraformaldehyde for
6 h and embedded in paraffin, and thin (6 mm) sections were cut
3
423, 3140, 3015, 2951, 1655 cm ; H-NMR (CD OD, 300 MHz) d:
ꢃ1
1
1
3
7
7
and stained with hematoxylin and eosin (H&E). The thicknesses of
the epidermis and dermis were measured under a microscope. For
determining mast cell infiltration, skin sections were stained with
toluidine blue, and the mast cells were counted in five randomly
chosen fields of view.
.48 (1H, d, J ¼ 15.7 Hz), 7.42 (2H, d, J ¼ 8.6 Hz), 7.24 (2H, m),
.04 (2H, m), 6.99 (1H, br), 6.81 (2H, d, J ¼ 8.6 Hz), 6.41 (1H, d,
1
3
J ¼ 15.7 Hz), 3.51 (2H, t, J ¼ 7.3 Hz), 2.84 (2H, t, J ¼ 7.3 Hz). C-
0
NMR (75 MHz) d: 167.8 (C-9), 159.1 (C-4), 140.4 (C-7), 135.1 (C-1 ),
0
0
0
0
0
1
(
(
30.1 (C-3 , C-5 ), 129.1 (C-2 , C-6 ), 126.2 (C-4 ), 116.9 (C-8), 115.3
0
C-3, C-5), 114.7 (C-2, C-6), 114.5 (C-1), 40.7 (C-8 ), 34.3
C-7 ). NCT was dissolved in DMSO for in vitro and in vivo
0
þ
CD4 T-cell preparation
experiments.
þ
Naïve BALB/c mice were sacrificed and CD4 T cells were isolated
from the LNs by magnetic-activated cell sorting (MACS) separation
(
Miltenyi Biotec, Bergisch Gladbach, Germany).
Ncpa
Jurkat cells were stimulated with phorbol myristate acetate
PMA)/calcium ionophore A23187 for 24 h. The supernatants were
The synthesised NCPA had the following characteristics: white
powder; melting point (mp), 255–257 C; electron impact mass
spectrometry (EIMS), m/z 283 [M] ; high-resolution (HR) EIMS, m/z
(
ꢁ
collected, and IL-2 mRNA levels were determined.
þ
2
3
83.1209 (calcd for C H O 283.1208); IR (KBr) 3432, 3300, 3175,
17 17 3
023, 2940, 1660 cm ; proton nuclear magnetic resonance ( H-
ꢃ1
1
Analysis of mRNA expression
NMR, CD
3
OD, 500 MHz) d: 7.36 (1H, d, J ¼ 15.7 Hz), 7.30 (2H, d,
For the reverse transcription-polymerase chain reaction (RT-PCR),
the total cellular RNA was isolated from the ear tissue and the
CD4 T cell in draining lymph nodes (dLNs) and non-dLNs using
J ¼ 8.6 Hz), 6.96 (2H, d, J ¼ 8.4 Hz), 6.69 (2H, d, J ¼ 8.6 Hz), 6.62 (2H,
d, J ¼ 8.4 Hz) 6.30 (1H, d, J ¼ 15.7 Hz), 3.37 (2H, t, J ¼ 7.4 Hz), 2.66
þ
1
3
(
1
6
(
2H, t, J ¼ 7.5 Hz). C-NMR (125 MHz) d: 169.7 (C-9), 160.9 (C-4),
TRIzol according to the manufacturer’s protocol [20]. The first-
0
0
0
57.4 (C-4 ), 142.2 (C-7), 131.8 (C-1 ), 131.1(C-2, C-6), 130.9 (C-2 , C- strand complementary DNA (cDNA) was synthesised using
0
0
0
), 128.2 (C-1), 118.9 (C-8), 117.1 (C-3, C-5), 116.7 (C-3 , C-5 ), 42.9 Superscript II reverse transcriptase (Invitrogen). The conditions for
0
0
C-8 ), 36.2 (C-7 ). NCPA was dissolved in DMSO for in vitro and in RT-PCR were s1imilar to those employed in previously
2
vivo experiments.
described studies .
Choi, Eun-Ju
