Anti-Allergic Asthma Properties of Brazilin through Inhibition of TH2 Responses in T
Cells and in a Murine Model of Asthma
Chen-Chen Lee 1,2*, Chien-Neng Wang2, Jaw-Jou Kang3, Jiunn-Wang Liao4,
Bor-Luen Chiang5, Hui-Chen Chen2, Chia-Der Lin6, Shih-Hsuan Huang3, Yu-Ting
Lai7
1
Department of Microbiology, School of Medicine, 2Graduate Institute of Basic
Medical Science, 2College of Medicine, China Medical University, Taichung,
Taiwan.3 Institute of Toxicology, College of Medicine, National Taiwan University,
Taipei, Taiwan, 4 Graduate Institute of Veterinary Pathology, College of Veterinary
Medicine, National Chung Hsing University, Taichung, Taiwan, ROC. 5 Department
of Clinical Medicine, College of Medicine, National Taiwan University, Taipei,
Taiwan. 6Department of Otolaryngology, School of Medicine, 7Graduate Institute of
Immunology, China Medical University, Taichung, Taiwan.
*Correspondence should be addressed to this author at
Department of Microbiology and Immunology,
School of Medicine
College of Medicine,
China Medical University
No.91 Hsueh-Shih Road,Taichung, Taiwan 40402, R.O.C
Tel: 886-4-22052121 ext 7706
Fax: 886-4-22333641
Email: leechenchen@mail.cmu.edu.tw
One-sentence summary: Brazilin showed a good potential drug for allergic asthma
through inhibition of TH2 responses.
Running title: brazilin inhibit airway inflammation
1
Abstract
2
The aim of the study was to determine whether brazilin exhibits any
3
anti-inflammatory effects that inhibit T helper cell type II (TH2) responses and
4
whether it suppresses allergic inflammation reactions in a murine model of asthma.
1
5
We found that brazilin inhibited phorbol myristate acetate (PMA) + cyclic adenosine
6
monophosphate (cAMP)-induced IL-4 and IL-5 expression in EL-4 T cells with
7
respect to both RNA and protein levels in a dose-dependent manner. After
8
intratracheal instillation of brazilin in ovalbumin (OVA)-immunized mice, we found
9
that brazilin-treated mice exhibited decreases in the release of IL-4, IL-5, IL-13,
10
eotaxin, and TNF-α in bronchoalveolar lavage fluid (BALF), inhibited TH2
11
functioning by a decrease in IL-4 production, and the attenuation of the OVA-induced
12
lung eosinophilia, and airway hyperresponsiveness and remodeling. These results
13
suggest that brazilin exhibits an anti-TH2 reaction both in vitro and in vivo. Brazilin as
14
demonstrates therapeutic potential for allergic diseases.
15
Keywords: brazilin, airway remodeling, allergic inflammation, GATA-3
2
16
Introduction
17
Asthma is a chronic inflammatory disease that affects about 300 million people
18
worldwide; in 2005, 255,000 people died from this disease (World Health
19
Organization). Allergic asthma is clinically characterized by the hypersecretion of
20
mucus, chronic inflammation of the airways, and airway hyperresponsiveness (AHR).
21
According to studies of patients and animal models of asthma, it is suggested that in
22
allergic asthma the CD4+ T helper (TH2) 2 lymphocytes induce an inflammatory
23
cascade via the production of cytokines that is comprised of eosinophil action, IgE
24
production, and mast cell activation; all of these in turn produce the necessary
25
mediators causing AHR (1). TH2 cells are the major effector cells in airway
26
inflammation and are associated with lung dysfunction via their recruitment and
27
activation of eosinophils (2). The pathological role of TH2 cells is mediated by the
28
release of TH2 cytokines such as IL-4, IL-5, and IL-13. IL-4 and IL-13 induce IgE
29
isotype switching and are implicated in the stimulation of VCAM-1 expression (3)
30
and the enhancement of eosinophil recruitment to the lungs (4).Caesalpinia sappan L.
31
is used in traditional Chinese medicine as an analgesic and anti-inflammatory agent,
32
and is used to treat emmeniopathy, sprains, and convulsions (3). Brazilin
33
[7,11b-dihydrobenz[b]indeno[1,2-d]-pyran-3,
34
component of C. sappan L. (4) is a natural red pigment that is usually used for
3
6a,9,10(6H)-
tetrol],
the
major
35
histological staining. Brazilin induces hypoglycemic action in experimental diabetic
36
animals (5) and vasorelaxation via nitric oxide synthase activation in endothelial cells
37
(6). In addition, it also exerts many biological effects, including anti-platelet
38
aggregation (7), inhibition of protein kinase C and insulin receptor kinase in rat livers
39
(8), as well as protection of cultured hepatocytes from BrCCl3-induced toxicity (9). It
40
also exhibits anti-inflammation potential, including inhibiting the induction of
41
immunological tolerance caused by sheep red blood cells in vivo (10), the production
42
of anti-proinflammatory cytokines (11), anti-complementary activity (12), and the
43
proliferation of mitogen-stimulated T and B cells (13). However, no study has
44
investigated the therapeutic effects of brazilin in allergic diseases. Therefore, in this
45
study, we used mitogen-induced TH2 cytokine-producing cells to investigate the
46
ability of brazilin to regulate TH2 responses in T cells. Materials and methods
47
Drugs and chemicals
48
Brazilin was purchased from ICN Pharmaceuticals (Irvine, CA, USA). Its chemical
49
structure is shown in Fig 1A. Phorbol myristate acetate (PMA), dibutyryl-cyclic
50
adenosine monophosphate (cAMP), and OVA (grade V) were purchased from Sigma
51
Chemical (St. Louis, MO, USA).
52
EL-4 murine T-lymphoma cells and Jurkat human T-lymphoma cells were
53
purchased from ATCC (Manassas,VA, USA). EL-4 cells were cultured in DMEM
4
54
supplemented with 10% heat-inactivated FBS
Confluent cells were subcultured at a
55
ratio of 1:3, and media were changed twice a week.
56
Cytotoxicity assay
57
EL-4 T cells were pretreated with various concentrations of brazilin for 10 min
58
and cultured with or without PMA (5 ng·mL−1) plus cAMP (250 μM) for 24 h. At this
59
point, the number of viable cells was determined using trypan blue staining (16).
60
Cytokine assay
61
5x105 EL-4 cells were cultured in 24-wells dish and treated with different
62
concentration of brazilin for 10 min and then in the presence or absence of 5ng.ml-1
63
PMA combined 250μM cAMP for 24h. Cell culture supernatants were collected 24 h
64
after different drug treatments and stored at −20°C before analysis by ELISA
65
according to the manufacturer's instructions. Standards were prepared from
66
recombinant mouse IFN-γ, IL-4, IL-5, TNF-, IL-13, and eotaxin, (R&D Systems,
67
Minneapolis, MN, USA).
68
Quantitative real-time PCR
69
RNA was converted into cDNA and subsequently quantified by quantitative
70
real-time PCR using an ABI PRISM 7900 Sequence Detector (Applied Biosystems,
71
Foster City, CA, USA). The partial cycles that resulted in a statistically significant
72
increase in IL-4, IL-5, GATA-3, c-Maf, and T-bet products were determined
5
73
(threshold cycle; Ct) and normalized to the Ct for -actin. IL-4, IL-5, GATA-3, c-Maf,
74
T-bet and -actin were amplified using a SYBR Green kit (Applied Biosystems). The
75
primer sequences used were as follows and all the primers were designed using ABI
76
primer 3 software and identified the specificity using BLAST (the basic local
77
alignment search tool) :
78
IL-4, sense 5′-CTCATGGAGCTGCAGAGACTCTT-3′,
79
antisense 5′-CATTCATGGTGCAGCTTATCGA-3′;
80
IL-5, sense 5′-TGACCGCCAAAAAGAGAAGTG-3′,
81
antisense 5′-GAACTCTTGCAGGTAATCCAGGAA-3′;
82
GATA-3, sense 5′- CAGAACCGGCCCCTTATCA-3′ ,
83
antisense 5′- ACAGTTCGCGCAGGATGTC-3′;
84
c-Maf, sense 5′-AGAGGCGGACCCTGAAAAA-3′ ,
85
antisense 5′-GTGTCTCTGCTGCACCCTCTT-3′;
86
T-bet, sense 5′-CTGGATGCGCCAGGAAGT-3′,
87
antisense 5′-TGTTGGAAGCCCCCTTGTT-3′;
88
-actin, sense 5′-ACTGCCGCATCCTCTTCCT-3′,
89
antisense 5′-ACCGCTCGTTGCCAATAGTG-3′.
90
91
Animals and experimental protocol
Female BALB/c mice aged 6–8 weeks were obtained from the Animal Center of
6
92
the College of Medicine, National Taiwan University. The Animal Care and Handling
93
protocols are approved by the Animal Committee of China Medical University. mice
94
were intraperitoneally sensitized by a 50 g injection of OVA emulsified in 2 mg
95
aluminum hydroxide (AlumImuject; Pierce Chemical, Rockford, IL, USA)(18) in a
96
total volume of 200 L; the injections were boosted 2 times with 25 g OVA
97
emulsified in 2 mg of aluminum hydroxide and challenged 3 times with OVA (100 g
98
in a total volume of 40L) by intranasal administration on consecutive days (Fig.
99
2A). To confirm that the OVA immunization procedure was successful, OVA-specific
100
IgE was detected in mouse serums on day 30 after injection. There were 8–10 mice
101
per group. Mice were euthanized using CO2.
102
Measurement of airway resistance in anesthetized mice
103
Airway resistance was assessed as an increase in pulmonary resistance after
104
challenge with aerosolized MCh in anesthetized mice using a modification of the
105
techniques described by Glaab et al.(19) and Kang et al.(20). Mice were anesthetized
106
with 70–90 mg·kg–1 pentobarbital sodium (Sigma) and tracheostomized; mice were
107
mechanically ventilated using a computer-controlled small animal ventilator () at 150
108
breaths·min−1 with a tidal volume of 0.3 mL and a positive end-expiratory pressure of
109
3–4 cm H2O. Flow was measured by the electronic differentiation of the volume
110
signal. Pressure, flow, and volume change were recorded. Pulmonary resistance was
7
111
calculated using a software program (). MCh aerosol was generated with an in-line
112
nebulizer and administrated directly through the ventilator. Data are expressed as the
113
pulmonary resistance (RL) and represent 3 independent experiments.
114
Bronchoalveolar lavage and lung histology
115
Bronchoalveolar lavage using 1 mL HBSS instilled by syringe was harvested by
116
gentle aspiration 3 times, and subsequently centrifuged (21). An aliquot of BALF cells
117
was used for differential cell count with Liu stained cytospins. In total, 300 cells were
118
counted at least four area of the slide under a light microscope. BAL fluid
119
supernatants were assayed by ELISA. Lungs were fixed with 10% neutral
120
phosphate-buffered formalin, and sections were prepared and stained with
121
hematoxylin/eosin (H&E), periodic acid-Schiff (PAS), and Masson’s trichrome in
122
order to quantify the number of infiltrating inflammatory cells, mucous production,
123
and collagen fibril deposition by microscopy. Airway inflammation was quantified as
124
the number of inflammatory cells per mm2 of subepithelial and subendothelial areas.
125
The number of PAS-positive and PAS-negative bronchial epithelial cells was
126
determined in individual airways. Results are expressed as the percentage of
127
PAS-positive cells per bronchiole. The scores for collagen fibril deposition were
128
determined in individual peribronchiolar and perivascular areas. Each set of 3 sections
129
was given a score from 0–4 for collagen fibril deposition as follows: 0 = normal lung;
8
130
1 = sparse fibrosis with fine fibrils involving <25% of the peribronchiolar area; 2 = mild
131
fibrosis with fine fibrils throughout the peribronchiolar and perivascular areas; 3 =
132
moderate fibrosis with fibrils throughout the peribronchiolar and perivascular areas with
133
fibrils increasing in the submucosal area; 4 = severe fibrosis with fibrils involving the
134
whole peribronchiolar area throughout the inflammatory infiltrate. Means were calculated
135
from the scores of the 3 sections for each mouse.
136
OVA-specific IgE antibody assay
137
ELISA was used to determine anti-OVA immunoglobulin (Ig)E antibody titer. In
138
brief, 96-well microtiter plates were coated with OVA (1 μg·well−1) or predetermined
139
concentrations of anti-mouse IgE (Pharmingen, San Diego, CA, USA) in NaHCO3
140
buffer (pH 9.6). After incubation overnight at 4°C, the plates were washed and
141
blocked with 3% bovine serum albumin (BSA) in PBS for 2 h at room temperature.
142
Serum samples were diluted 10X and added to each well and were kept overnight at
143
4°C. The plates were then washed. Either biotin-conjugated anti-mouse IgE (0.5
144
mg·mL−1; Pharmingen) diluted in 3% BSA-PBS buffer (1:500) was added for 45 min
145
at room temperature. Next, avidin-conjugated horseradish peroxidase (1:5000; Pierce
146
Biotechnology, Rockford, IL, USA) was added for an additional 30 min at room
147
temperature. The reaction was developed by a peroxidase substrate,
148
3,3′,5,5′-tetramethylbenzidine (KPL, Gaithersburg, MD, USA), and halted by 2N
9
149
H2SO4. Absorbance was determined at 450 nm using a microplate reader. Antibody
150
levels were compared to standard serum ; the IgE concentration in standard serum
151
were arbitrarily designated as 1 ELISA unit (1 EU). The standard serum is the
152
previous serum from immunized mice.
153
Lymph node preparation and Lung mononucleocyte preparation
154
Mediastinal lymph nodes were harvested and pooled from each group at the time
155
of sacrifice (day 43). Single-cell suspensions were obtained by mechanical disruption.
156
Cells were stimulated in vitro with CD3 and CD28 (0.5 µg·mL−1 each) for 72 h. The
157
cell media were collected for cytokine analysis. Lungs were perfused with 10 mL PBS
158
through the right ventricle. Lungs were subsequently harvested and pooled from each
159
group at the time of euthanasia (day 43). The lungs were cut into small pieces after
160
their removal from animals, and single-cell suspensions were obtained using a
161
stainless-steel cell dissociation sieve. Debris was removed by using a cell strainer
162
(100 µm; BD Biosciences). Then, mononucleocytes were isolated using Ficoll-Plaque
163
Plus according to the manufacturer's instructions (GE Healthcare, Sweden). Cells
164
were stimulated in vitro with CD3 and CD28 (0.5 µg·mL−1 each) for 72 h. The cell
165
media were collected for cytokine analysis.
166
Statistical analysis
167
All experimental data are expressed as mean (S.E.M.) using one-way ANOVA
10
168
followed by the Newman–Keuls post-hoc test. Statistical significance was set at p <
169
0.05.
170
Results
171
Brazilin inhibits the mitogen-induced expression of TH2 cytokines in EL-4 T cells
172
First, we evaluated the possible cytotoxic effects of brazilin on EL-4 cells. After
173
treatments with 3, 10, or 30 µM brazilin for 24 h, EL-4 cells did not exhibit any
174
cytotoxicity as shown by the trypan blue exclusion assay (Fig. 1B). Next, we
175
investigated the production of IL-4 and IL-5. EL-4 cells treated with different
176
concentrations of brazilin did not exhibit any apparent changes with respect to IL-4
177
and IL-5 production (Fig. 1C). Because PMA activates protein kinase C (PKC) and
178
cAMP activates protein kinase A (PKA), all of which are involved in EL-4 T cell
179
activation and the release of IL-4 and IL-5 (25), we used a mixture of PMA and
180
cAMP to drive EL-4 T cells to behave like TH2 cells. Brazilin (3, 10, and 30 µM)
181
inhibited the PMA + cAMP-induced IL-4 and IL-5 proteins (Fig. 1C) as well as
182
mRNA (Fig. 1D) expression in a dose-dependent manner. Compared with the control
183
group only treated with PMA + cAMP, IL-4 production was reduced by 20.2% ± 4.2
184
and 31.8% ± 2.4 by 10 and 30 M brazilin respectively; IL-5 production was reduced
185
by 43.1% ± 10.6 and 59.3% ± 16.7 by 10 and 30 M brazilin respectively. Compared
186
with the control group only treated with PMA + cAMP, IL-4 mRNA production was
11
187
reduced by 31.1% ± 0.5 and 44.5% ± 0.3 by 10 and 30 M brazilin respectively; IL-5
188
mRNA was reduced by 29% ± 1.0 and 49.9% ± 15.6 by 10 and 30 M respectively. In
189
addition, we analyzed the TH2- and TH1-related transcription factors and found that
190
brazilin inhibited PMA + cAMP-induced GATA-3 and c-Maf mRNA expression in a
191
dose-dependent manner. However, T-bet was not induced by PMA + cAMP treatment,
192
and treatment with brazilin did result in an obvious change in T-bet mRNA expression
193
(Fig. 1E).
194
Inhibition of allergen-induced airway inflammation by brazilin
195
We examined the effects of brazilin in a murine model of asthma. First, we found
196
that i.t. of brazilin did not affect cell profile in BALF in PBS sensitized and
197
challenged mice (Fig. 2A). Next, mice were sensitized to OVA with 3 intraperitoneal
198
injections and challenged with intranasal OVA droplet aspiration (Figure 2B). Mice
199
were treated once a day with brazilin on days 35–39. To investigate the effects of
200
brazilin on airway inflammation, we analyzed the cellular composition of BALF 24 h
201
after the last of 3 sequential OVA challenges (given on days 40–42) on day 43. No
202
obvious infiltration of inflammatory cells was noted in the BALF from mice
203
sensitized and challenged with PBS (PBS group). However, after sensitization and
204
challenging with OVA (OVA group), the numbers of macrophages and eosinophils in
205
BALF were significantly increased compared with PBS group (Fig. 2B). After the
12
206
intratracheal instillation of brazilin, challenging of the OVA group induced lower
207
numbers of eosinophils in the BALF, although the number of macrophages was not
208
affected.
209
Histological examination of lung sections from the OVA group exhibited a large
210
number of infiltrating inflammatory cells (Figure ) and increased mucus formation
211
(Fig) around the airways compared to the PBS group. After treatment with brazilin,
212
challenging of the OVA group exhibited substantially less inflammatory cell
213
infiltration and mucus formation than in OVA mice without brazilin treatment.
214
Intratracheal instillation of brazilin decreases BALF TH2 cytokines levels and lung
215
TH2 transcription factors mRNA expression in a murine model of asthma
216
For further analysis the mechanisms of brazilin-induced inhibition of airway
217
inflammation, one day after the final challenge (Fig.2B), we measured cytokine
218
contents in the BALF. OVA groups exhibited increases in IL-4, IL-5, IL-13, eotaxin,
219
and TNF-α release in BALF compared to the control group (PBS groups) (Fig. 3).
220
After treatment with brazilin, the levels of IL-4, IL-5, eotaxin, and TNF-α in BALF
221
decreased in a dose-dependent manner. Compared with the OVA group, BALF
222
cytokine production was reduced as follows at 43.9 and 439 µg mouse–1 of the
223
indicated protein respectively: IL-4, 31.2% ± 9.73 and 59.7% ± 4.1; IL-5, 20.6% ± 7.3
224
and 41.5% ± 6.5; IL-13, 38.5% ± 7.3 and 60.7% ± 8.7; eotaxin, 43.8% ± 6.9 and
13
225
64.5% ± 3.7; and TNF-α 44.4 ± 14 and 52.2% ± 9.3 (Fig). However, the level of
226
IFN- in the BALF was below the detection limit of the kit.
227
Next, analysis of TH2 transcription factors expression in lungs, we found
228
OVA-immunized showed significantly increase in GATA-3 and c-Maf but not T-bet
229
mRNA expression (FigB). After treatment of brazilin, the levels of GATA-3 and
230
c-Maf mRNA expression in lungs decreased in a dose-dependent manner.
231
Intratracheal instillation of brazilin decreases TH2-cell activation in the lungs in a
232
murine model of asthma
233
Next, we investigated whether brazilin inhibits T-lymphocyte activation in the
234
lungs and mediastinal lymph nodes. As shown in Figure 5, treatment with a mixture of
235
CD3 and CD28 (CD3 + CD28) induced the production of IL-4 and IFN-γ in lung
236
(Fig. ) and mediastinal lymph node cells (Fig. 4B) isolated from OVA-immunized
237
mice. Brazilin inhibited the CD3 + CD28-induced IL-4 production in a
238
dose-dependent manner, whereas IFN-γ did not. As CD3 + CD28 induced both naïve
239
and effector T cells activation, PBS group also can induced IL-4 and IFN-γ production.
240
As our amimal model activated TH2 response, the OVA-immunized group induced
241
higher IL-4 production than PBS group.
242
Intratracheal
243
hyperresponsiveness and remodeling
delivery
of
brazilin suppresses
14
the development
of
airway
244
We investigated whether brazilin affects the development of airway
245
hyperresponsiveness and remodeling in a murine model of asthma. One day after the
246
final challenge, airway responsiveness was assessed by pulmonary resistance using
247
invasive body plethysmography. The baseline value of lung resistance is 1.25 ± 0.13.
248
(cmH2O/L/sec). BALB/c mice sensitized and challenged with OVA exhibited an increase
249
in airway resistance to methacholine (MCh) inhalation compared to mice sensitized
250
and challenged with PBS (FigA; OVA and PBS groups). After treatment with brazilin,
251
airway resistance was significantly diminished in comparison with that of the
252
untreated OVA group. Histological examination of lung sections from the OVA group
253
showed greater fibrosis and the formation of collagen fibers around the airway
254
compared to that in the PBS group (FigB). After treatment with brazilin, challenging
255
of the OVA group showed substantially less fibroblast deposition and collagen fiber
256
formation than in OVA mice without brazilin treatment. Further identification of the
257
expressed extracellular matrix deposition-related genes revealed that collagen,
258
metalloproteinase (MMP)-2, and MMP9 mRNA were increased in the OVA group
259
without brazilin (FigC). After treatment with brazilin, collagen, MMP2, and MMP9
260
mRNA expression decreased compared with the OVA group without brazilin
261
Discussion
262
In this study, we used PMA + cAMP to activate EL-4 T cells to IL-4- and
15
263
IL-5-producing cells. Previous studies found that antigens can be mimicked by the use
264
of agents such as PMA and calcium ionophores, or by anti-CD3 and anti-CD28
265
antibodies(22). However, under such conditions, T cells produce INF- in greater
266
amounts than either IL-4 or IL-5, which drives the cells to behave more like TH1 than
267
TH2 cells. Lee et al. (15) found PMA + cAMP induces EL-4 T cells to produce IL-4
268
and IL-5. In the present study, we also found that IFN-γ production was below the
269
detection level (data not shown) and did not induce T-bet (TH1 cell specific
270
transcription factor) expression in PMA + cAMP-stimulated EL-4 T cells. In addition,
271
GATA-3 and c-Maf, which are master Th2 transcription factors, were highly induced.
272
Hayakawa et al. (27) found that PMA + cAMP induced GATA-3 gene transcription
273
and translation. Because EL-4 T cells can induce IL-4, IL-5, and IL-13 at the
274
transcriptional and translational levels (28-30) in addition to Th2 transcriptional
275
factors upon mitogen stimulation (27), we used PMA + cAMP-treated EL-4 T cells as
276
a new screening platform for allergic diseases.
277
We found that brazilin possesses therapeutic potential for treating allergic asthma
278
via several mechanisms. First, brazilin inhibited PMA + cAMP-induced TH2 cell
279
activation in a dose-dependent manner by decreasing IL-4 and IL-5 expressions with
280
respect to mRNA and protein levels in addition to the expression of GATA-3 and
281
c-Maf mRNA. The signaling pathways that involve PMA + cAMP inducing the
16
282
GATA-3-dependent production of IL-4 and IL-5 in T cells include the pathways
283
regulated by phosphoinositide-dependent kinase 1 and PKA (23-24) as well as a
284
soluble secreted form (e.g., protein product of the ST2 gene) (25), and the p38
285
mitogen-activated protein kinase pathway (26). However, whether these signaling
286
pathways are involved in the brazilin-induced inhibition of the PMA +
287
cAMP-induced, GATA-3-dependent production of IL-4 and IL-5 in EL-4 T cells
288
requires further investigation. In addition, previous study also found that GATA-3
289
signaling involved IκB activation (phosphorylation and then degradation) induced
290
NF-κB1/p50 activation in TH2 cell activation in murine spenocyte (29-30).
291
Second, brazilin possesses therapeutic potential in vivo by reducing the release of
292
TH2 cytokine in BALF, by decrease TH2 transcription factors mRNA expression in
293
lung tissues, and by inhibiting airway inflammation and hyperresponsiveness in a
294
murine model of asthma. We found that the level of peribronchial airway
295
inflammation was more serious than that of the perivascular areas in the lung sections
296
of OVA-immunized mice. Brazilin inhibited the antigen- induced peribronchial and
297
perivascular inflammation in a similar manner. In order to identify whether brazilin
298
inhibits TH2 activation in vivo, we isolated mononuclear lung and mediastinal lymph
299
node cells, and treated them with anti-CD3 and anti-CD28 antibodies to activate the T
300
cells. We found that brazilin also specifically inhibited TH2 activation in vivo by
17
301
reducing IL-4 production but not the release of IFN-γ in mononuclear lung and
302
mediastinal lymph node cells.
303
Airway remodeling is an important feature of asthma—especially in severe
304
cases. Airway remodeling tends to be milder and reversible in acute and mild asthma
305
models (27). In this study, we observed features of airway remodeling in
306
OVA-immunized mice was inhibited by brazilin treatment, such as subepithelial
307
fibrosis, goblet and mucous gland hyperplasia, as well as the inhibition of
308
extracellular matrix deposition. Subepithelial fibrosis is a process that includes
309
increased matrix deposition in the subepithelial lamina reticularis of the basement
310
membrane, disruption of elastin filaments, and the deposition of type 1, 3, 5, and 6
311
collagens and proteoglycans throughout the airway wall (including the smooth
312
muscle), causing the thickening of the airways (28). and
313
In conclusion, we demonstrated the mechanisms of brazilin –induced inhibitory
314
effect of allergic asthma which through suppresses TH2 cells activation including TH2
315
cytokines and transcription factors in vitro and in vivo. This study provides a novel
316
platform for the screening of allergic diseases. Brazilin has the potentially to be used
317
as drug for treating allergic asthma.
18
318
319
320
Acknowledgements
This study was supported by the National Science Council in Taiwan (NSC
97-2320-B-039-007-MY3) and China Medical University (-S-).
321
322
19
323
References
324
1. Wills-karp, M. Immunologic basis of antigen-induced airway hyperresponsiveness
325
326
327
Annu. Rev. Immunol. 1999, 17, 255.
2. Watanabe, A.; Mishima, H.; Renzi, P. M.; Xu, L. J.; Hamid, Q.Martin, J. G. Transfer
of allergic airway responses with antigen-primed CD4+ but not CD8+ T cells in
328
329
330
brown Norway rats J. Clin. Invest. 1995, 96, 1303-1310.
3. Baek, N. I.; Jeon, S. G.; Ahn, E. M.; Hahn, J. T.; Bahn, J. H.; Jang, J. S.; Cho, S. W.;
Park, J. K.Choi, S. Y. Anticonvulsant compounds from the wood of Caesalpinia
331
332
sappan L. Arch. Pharma. Res. 2000, 23, 344- 348.
4. Hikino, H.; Taguchi, T.; Fujimura, H.Hiramatsu, Y. Antiinflammatory principles of
333
Caesalpinia sappan wood and of Haematoxylon campechianum wood. Planta Medica
334
335
336
1977, 31, 214- 220.
5. Moon, C. K.; Lee, S. H.; Lee, M. O.Kim, S. G. Effects of brazilin on glucose
oxidation, lipogenesis and therein involved enzymes in adipose tissues from diabetic
337
338
339
KK-mice Life Science 1993, 53, 1291- 1297.
6. Hu, C. M.; Kang, J. J.; Lee, C. C.; Li, C. H.; Liao, J. W.Cheng, Y. W. Induction of
vasorelaxation through activation of nitric oxide synthase in endothelial cells by
340
341
342
brazilin Eur. J. Pharmacol. 2003, 468, 37-45.
7. Hwang, G. S.; Kim, J. Y.; Chang, T. S.; Jeon, S. D.; So, D. S.Moon, C. K. Effects of
brazilin on the phospholipase A2 activity and changes of intracellular free calcium
343
344
345
concentration in rat platelets. Arch. Pharma. Res. 1998, 21, 774-778.
8. Kim, S. G.; Kim, Y. M.; Khil, L. Y.; Jeon, S. D.; So, D. S.; Moon, C. H.Moon, C. K.
Brazilin inhibits activities of protein kinase C and insulin receptor kinase in rat liver.
346
347
Arch. Pharma. Res. 1998, 21, 140- 146.
9. Moon, C. K.; Park, K. S.; Kim, S. G.; Won, H. S.Chung, J. H. Brazilin protects
348
349
350
351
cultured rat hepatocytes from BrCCl3-induced toxicity Drug Chem.Toxicol. 1992, 15,
81- 91.
10. Mok, M. S.; Jeon, S. D.; Yang, K. M.; So, D. S.Moon, C. K. Effects ofbrazilin on
induction of immunological tolerance by sheep red blood cells in C57BL/6 female
352
353
354
mice. Arch. Pharm. Res. 1998, 21, 769- 773.
11. Bae, I. K.; Min, H. Y.; Han, A. R.; Seo, E. K.Lee, S. K. Suppression of
lipopolysaccharide-induced expression of inducible nitric oxide synthase by brazilin
355
356
357
in RAW 264.7 macrophage cells Eur. J. Pharmacol. 2005, 513, 237-242.
12. Oh, S. R.; Kim, D. S.; Lee, I. S.; Jung, K. Y.; Lee, J. J.Lee, H. K.
Anticomplementary activity of constituents from the heartwood of Caesalpinia sappan
358
359
Planta Med. 1998, 64, 456- 458.
13. Ye, M.; Xie, W. D.; Lei, F.; Meng, Z.; Zhao, Y. N.; Su, H.Du, L. J. Brazilein, an
20
360
important immunosuppressive component from Caesalpinia sappan L. Inter.
361
362
363
Immunolpharmacol. 2006, 6, 426-432.
14. Oh, S., R,, ; Kim, D. S.; Lee, I. S.; Jung, K. Y.; Lee, J. J.Lee, H. K.
Anticomplementary activity of constituents from the heartwood of Caesalpinia sappan
364
365
366
367
Planta Medica 1998, 64, 456-458.
15. Lee, H. J.; Koyano-Nakagawa, N.; Naito, Y.; Nishida, J.; Arai, N.; Arai, K.Yokota,
T. cAMP activates the IL-5 promoter synergistically with phorbol ester through the
signaling pathway involving protein kinase A in mouse thymoma line EL-4 J.
368
369
370
Immunol. 1993, 151, 6135-6142.
16. Sugiura, H.; Liu, X.; Duan, F.; Kawasaki, S.; Togo, S.; Kamio, K.; Wang, X. Q.;
Mao, L.; Ahn, Y.; Ertl, R. F.; Bargar, T. W.; Berro, A.; Casale, T. B.Rennard, S. I.
371
Cultured lung fibroblasts from ovalbumin-challenged "asthmatic" mice differ
372
373
374
375
functionally from normal Am. J. Respir. Cell Mol. Biol. 2007, 37, 424-430.
17. Lee, C. C.; Wang, C. N.; Lai, Y. T.; Kang, J. J.; Liao, J. W.; Chiang, B. L.; Chen, H.
C.Cheng, Y. W. Shikonin inhibits maturation of bone marrow-derived dendritic cells
and suppresses allergic airway inflammation in a murine model of asthma Brit. J.
376
377
378
379
Pharmacol. 2010, 161, 1496-1511.
18. Kool, M.; Soullie, T.; van Nimwegen, M.; Willart, M. A.; Muskens, F.; Jung, S.;
Hoogsteden, H. C.; Hammad, H.Lambrecht, B. N. Alum adjuvant boosts adaptive
immunity by inducing uric acid and activating inflammatory dendritic cells J Exp Med
380
381
382
2008, 205, 869-882.
19. Glaab, T.; Mitzner, W.; Braun, A.; Ernst, H.; Hohllfeld, J. M.; Krug, N.Hoymann,
H. G. Repetitive measurements of pulmonary mechanics to inhaled cholinergic
383
384
385
386
challenge in spontaneously breathing mice J App. Physiol. 2004, 97, 1104-1111.
20. Wagers, S. S.; Haverkamp, H. C.; Bates, J. H. T.; Norton, R. J.;
Thompson-Figueroa, J. A.; Sullivan, M. J.Irvin, C. G. Intrinsic and antigen-induced
airway hyperresponsiveness are the result of diverse physiological mechanisms J.
387
388
389
Appl. Physiol. 2007, 102, 221-230.
21. Lee, C. C.; Huang, H. Y.Chiang, B. L. Lentiviral-mediated interleukin-4 and
interleukin-13 RNA interference decrease airway inflammation and
390
391
392
393
hyperresponsiveness Human Gene Therapy 2011, 22, 577-586.
22. Verhoef, C. M.; Van Roon, J. A.; Vianen, M. E.; Glaudemans, C. A.; Lafeber, F.
P.Bijlsma, J. W. Lymphocyte stimulation by CD3-CD28 enables detection of low T
cell interferon-gamma and interleukin-4 production in rheumatoid arthritis Scand J
394
395
396
Immunol 1999, 50, 427-432.
23. Siegel, M. D.; Zhang, D. H.; Ray, P.Ray, A. Activation of the interleukin-5
promoter by cAMP in murine EL-4 cells requires the GATA-3 and CLE0 elements J
397
Biol.Chem. 1995, 270, 24548-24555.
21
398
24. Lacour, M.; Arrighi, J. F.; Muller, K. M.; Carlberg, C.; Saurat, J. H.Hauser, C.
399
cAMP up-regulates IL-4 and IL-5 production from activated CD4+ T cells while
400
401
402
decreasing IL-2 release and NF-AT induction Inter. Immunol. 1994, 6, 1333-1343.
25. Hayakawa, M.; Yanagisawa, K.; Aoki, S.; Hayakawa, H.; Takezako, N.Tominaga,
S. T-helper type 2 cell-specific expression of the ST2 gene is regulated by
403
404
405
transcription factor GATA-3 BBA 2005, 1728, 53-64.
26. Chen, C. H.; Zhang, D. H.; LaPorte, J. M.Ray, A. Cyclic AMP activates p38
mitogen-activated protein kinase in Th2 cells: phosphorylation of GATA-3 and
406
407
stimulation of Th2 cytokine gene expression J. Immunol. 2000, 165 5597- 5605.
27. Bergeron, C.; Al-Ramli, W.Hamid, Q. Remodeling in asthma Proc. Am. Thor. Soc.
408
409
410
2009, 6, 301-305.
28. Holgate, S. T.; Holloway, J.; Wilson, S.; Bucchiere, F.; Davies, D. E.Puddicombe,
S. Epithelial-mesenchymal communication in the pathogenesis of chronic asthma
411
412
413
Proc. Am. Thor. Soc. 2004, 1, 93-98.
29. Gueders, M. M.; Foidart, J. M.; Noel, A.Cataldo, D. D. Matrix metalloproteinases
(MMPs) and tissue inhibitors of MMPs in the respiratory tract: potential implications
414
415
416
in asthma and other lung diseases Eur J Pharmacol 2006, 533, 133-144.
30. Mautino, G.; Oliver, N.; Chanez, P.; Bousquet, J.Capony, F. Increased release of
matrix metalloproteinase-9 in bronchoalveolar lavage fluid and by alveolar
417
418
macrophages of asthmatics Am J Respir Cell Mol Biol 1997, 17, 583–591.
31. Cataldo, D.; Munaut, C.; Noel, A.; Frankenne, F.; Bartsch, P.; Foidart, J. M.Louis,
419
R. MMP-2-and MMP-9-linked gelatinolytic activity in the sputum from patients with
420
421
422
423
424
asthma and chronic obstructive pulmonary disease Int Arch Allergy Immunol 2000,
123, 259-267.
32. Bosse, M.; Chakir, J.; Rouabhia, M.; Boulet, L. P.; Audette, M.Laviolette, M.
Serum matrix metalloproteinase-9: tissue inhibitor of metalloproteinase-1 ratio
correlates with steroid responsiveness in moderate to severe asthma Am J Respir Crit
425
426
427
Care Med 1999, 159, 596-602.
33. Zhang, D. L.; Bar-Eli, M.; Meloche, S.Brodt, P. Dual regulation of MMP-2
expression by the type 1 insulin-like growth factor receptor - the phosphatidylinositol
428
429
430
431
3-kinase/Akt and Raf/ERK pathways transmit opposing signals J Biol Chem 2004,
279, 19683-19690.
34. Zheng, T.; Zhu, Z.; Wang, Z. D.; Homer, R. J.; Ma, B.; Riese, R. J.; Chapman, H.
A.; Shapiro, S. D.Elias, J. A. Inducible targeting of IL-13 to the adult lung causes
432
433
434
435
matrix metalloproteinase- and cathepsin-dependent emphysema J Clin Invest 2000,
106 1081-1093.
35. Chang, C. J.; Yang, Y. H.; Liang, Y. C.; Chiu, C. J.; Chu, K. H.; Chou, H.
N.Chiang, B. L. A novel phycobiliprotein alleviates allergic airway inflammation by
22
436
437
438
modulating immune responses Am J Respir Crit Care Med 2011, 183, 15-25.
36. Li, C. Y.; Suen, J. L.; Chiang, B. L.; Lee Chao, P. D.Fang, S. H. Morin promotes
the production of Th2 cytokine by modulating bone marrow-derived dendritic cells
439
440
441
Am J Chin Med 2006, 34, 667-684.
37. Lin, Y. L.; Lee, S. S.; Hou, S. M.Chiang, B. L. Polysaccharide purified from
Ganoderma lucidum induces gene expression changes in human dendritic cells and
442
443
444
445
446
promotes T helper 1 immune response in BALB/c mice Mol Pharmacol 2006, 70,
637-644.
38. Lu, Y.; Yang, J. H.; Li, X.; Hwangbo, K.; Hwang, S. L.; Taketomi, Y.; Murakami,
M.; Chang, Y. C.; Kim, C. H.; Son, J. K.Chang, H. W. Emodin, a naturally occurring
anthraquinone derivative, suppresses IgE-mediated anaphylactic reaction and mast
447
448
449
cell activation Biochem Pharmacol 2011, 82, 1700-1708.
39. Karlberg, M.; Ekoff, M.; Huang, D. C.; Mustonen, P.; Harvima, I. T.Nilsson, G.
The BH3-mimetic ABT-737 induces mast cell apoptosis in vitro and in vivo: potential
450
451
452
for therapeutics J Immunol 2010, 185, 2555-2562.
40. Kim, S. H.; Lee, S.; Kim, I. K.; Kwon, T. K.; Moon, J. Y.; Park, W. H.Shin, T. Y.
Suppression of mast cell-mediated allergic reaction by Amomum xanthiodes Food
453
454
Chem Toxicol 2007, 45, 2138-2144.
41. Nagai, H. Recent research and developmental strategy of anti-asthma drugs
455
456
Pharmacol Ther 2012, 133, 70-78.
457
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Figure 1. Brazilin inhibited mitogens-induced TH2 cytokines expression in murine
459
EL-4 T cells. (A). Chemical structure of brazilin. (B) Brazilin did not induced
460
cytotoxicity effects in EL-4 T cells. Cytotoxicity effect detected by trypan blue
461
exclusion and data was expressed as Mean ± SEM (n=5). (C) IL-4 and IL-5
462
production detected by ELISA and data was expressed as Mean ± SEM (n=6). (D)
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IL-4 and IL-5 mRNA expression and (E) GATA-3 and c-Maf mRNA expression
464
detected by real-time PCR and data was expressed as Mean ± SEM (n=6). #p<0.001,
465
compared to the control, without PMA +cAMP group. *p<0.05; **p<0.01;
466
***p<0.001, compared to the control, with PMA combined cAMP group. C
467
represented control group which means cells cultured in medium contained 0.1%
468
DMSO. #p<0.001, compared to the control, without PMA + ionomycin group.
469
*p<0.05; **p<0.01; ***p<0.001, compared to the C group, with PMA combined
470
ionomycin group. C represented control group which means cells cultured in medium
471
contained 0.1% DMSO.
472
Figure 2. Airway inflammation inhibited by brazilin. Profile of cell in BALF and
473
histological analysis of lung tissue in mice after brazilin treatment, shown as the daily
474
dose (μg) given i.t. for 5 days. (A) Brief scheme of animal sensitization and challenge
475
of PBS and (B) brief scheme of animal sensitization and challenge of OVA. Total cell
476
counts were determined on 3 ml BALF and differential cell counts were assessed by
24
477
Liu staining. Data were expressed as mean ± SEM. (n=10). #p<0.001, compared with
478
the PBS group;*p<0.05; **p<0.01, versus the OVA group. Lung sections were stained
479
with haematoxylin/eosin (H&E) for measurement of inflammatory cells (C) and
480
periodic acid-Schiff (PAS) for measurement of mucus production around the airways
481
(D). Data revealed a different extent of cellular infiltration of the peri-airway region.
482
Original magnification: ×200. Quantification of infiltration was by counting the
483
number of inflammatory cells per mm2 of subepithelial and subendothelial area.
484
Mucus-producing cells were quantified as the percentage of PAS-positive cells per
485
bronchiole. Data were expressed as mean ± SEM. (n=5) #p<0.001, compared with the
486
PBS group;***p<0.001 versus the OVA group. i.p., intraperitoneal; i.n., intranasal; i.t.,
487
intratracheal. PBS group represented that mice were sensitized and challenged with
488
PBS and given i.t. of PBS contained 1%DMSO. OVA group represented that mice
489
were sensitized and challenged with OVA combined alum and given i.t. of PBS
490
contained 1%DMSO.
491
Figure 3.Suppression of cytokine levels in BALF and transcription factors in lung
492
tissues after administration of brazilin in a murine model of asthma. (A) Cytokine
493
levels in BALF were analyzed by enzyme-linked immunosorbent assay. Data were
494
expressed as mean ± SEM. (n=10). (B)GATA-3, c-Maf and T-bet mRNA expression
495
in lung tissues was detected by real-time PCR and data was expressed as Mean ±
25
496
SEM (n=6). #p<0.001, compared with the respective PBS group; *p<0.05; **p<0.01;
497
***p<0.001 different from the OVA group without brazilin.
498
Figure 4. Brazilin suppressed TH2 cells function in lung cells and mediastinal lymph
499
node cells in a murine model of asthma. Lung cells and mediastinal lymph node cells
500
were isolated from naïve or OVA-immunized mice. Lung cells (A) and mediastinal
501
lymph node cells (B) were treated with 1μg.mL-1 CD3 + 1μg.mL-1 CD28 for 72h,
502
and medium was collected for investigation of cytokine production by enzyme-linked
503
immunosorbent assay (ELISA). Data were expressed as mean ± SEM. (n=6)
504
#p<0.001, compared with the respective PBS group; ***p<0.001, versus the OVA
505
group without brazilin.
506
Figure 5. Airway hyperresponsiveness and remodeling inhibited by brazilin in
507
BALB/c mice. (A). Airway resistance as measured by invasive body plethysmography.
508
Data were expressed as the mean ± SEM. of the ratio of airway resisitance over the
509
baseline (n=5). #p<0.001, compared with the PBS group. ***p<0.001 compared with
510
the OVA group without brazilin. (B). Lung sections were stained with masson’s
511
trichrome for measurement of collagen fibers deposition around the airways and
512
vascular area. Data revealed a different extent of collagen fibers deposition of the
513
peri-airway region. Original magnification: ×200. Each set of three sections was given
514
a score of 0-4 for collagen fibers deposition and a mean derived from the three
26
515
sections scores for each individual mouse (0 = normal lung; 1 = sparse fibrosis with
516
fine fibrils involving <25% of peribronchiolar area; 2 = mild fibrosis with fine fibrils
517
throughout the peribronchiolar and perivascular areas; 3 = moderate fibrosis with
518
fibrils throughout the peribronchiolar and perivascular areas with fibrils increasing
519
submucosal area; 4 = severe fibrosis with fibrils involving the whole peribronciolar
520
area, throughout the inflammatory infiltrate). In lung section of the OVA group, the
521
fibrosis is indicated with arrow. (C) Collagen, MMP2, and MMP9 mRNA expressions
522
of lung tissues detected by real-time PCR and data was expressed as Mean ± SEM
523
(n=6). #p<0.05, compared to the PBS control. *p<0.05, compared to the OVA control,
524
without brazilin.
525
27
526
Primers sequences for real-time PCR
Gene
Name
Sense sequence
Anti-sense sequence
527
528
28