# Effects of Radix Adenophorae and Cyclosporine  A on an OVA-Induced Murine Model of Asthma by

Authors: Seong-Soo Roh; Seung-Hyung Kim; Young-Cheol Lee; Young-Bae Seo
Journal: Mediators of Inflammation
 (2008-01-10)

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## Abstract

The present study is performed to investigate the inhibitory effects of Radix Adenophorae extract (RAE) on ovalbumin-induced asthma murine model. To study the anti-inflammatory and antiasthmatic effects of RAE, we examined the development of pulmonary eosinophilic inflammation and inhibitory effects of T cells in murine by RAE and cyclosporine A (CsA). We examined determination of airway hyperresponsiveness, flow cytometric analysis (FACS), enzyme-linked immunosorbent assay (ELISA), quantitative real time (PCR),  hematoxylin-eosin staining, and Masson trichrome staining in lung tissue, lung weight, total cells, and eosinophil numbers in lung tissue. We demonstrated how RAE suppressed development on inflammation and decreased airway damage.


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## Body

## 1. Introduction

Radix Adenophorae is a dry root of Umbelliferae adenophora triphilla var. japonica Hara. Radix Adenophorae has the actions of reinforcing phagocytosis of giant cell, rising leukocyte, regulating humoral and cellular immunity, antimutation,  restraining adenocarcinoma cell, strengthening cardiac function, allaying a fever, and easing pain and cough [1].
It has researched that Radix Adenophorae has
the chemicals such as cycloartenyl acetate, lupenone, β-sitosterol, taraxerone, octacosanoic acid, and praeruptorin
A [2].
These chemicals are known to possess anti-inflammatory and immunomodulatory activity [3–7].In modern clinical medicine, it can cure atrophic gastritis, pneumonia, thrush, lung cancer, pharyngolaryngitis, infective inflammation of trachea and lungs kinds of eosinophilic inflammation, and bronchial hyperresponsiveness
[8].
And asthma is a chronic inflammatory disease of the mucosa and is associated with excess production of Th2 cytokines (IL-4, lL-5, IL-13), eosinophilic infiltration in lung.Th2
cells play a critical role in the pathogenesis of allergy and asthma [9]
and eosinophils
have a crucial role in the pathogenesis of allergic diseases and produce
factors such as CCR3, CCR5, EOTAXIN 2, TGF-β1, and TARC.
The inflammatory aspects of the disease are complex with mast cells, dendritic
cells, and T
and B lymphocytes, and eosinophils playing important roles. Increased
eosinophils and T lymphocytes in the bronchial mucosa and bronchoalveolar
lavage fluid (BALF)
are
distinctive features of the inflammatory response in patients with asthma and
appear to correlate with the severity of the disease [10–12].Clinically
Radix
Adenophorae
has been used
for treatment of asthma in Korea.
But a research for this has not been accomplished yet. So to clarify the anti-inflammatory and antiasthmatic
effects of Radix
Adenophorae (RAE) and
cyclosporine
A(CsA),
we examined the influence of RAE and CsA on the development of pulmonary
eosinophilic inflammation in murine model of asthma.Therefore,
we decided to investigate the effects of Radix Adenophorae
on total pulmonary airflow in mice, eosinophil influx,
total leukocytes number, cell surface markers by FACS, and cytokines
production in BALF by ELISA were determined.


## 2. Materials and Methods

Plant
material and Radix Adenophorae of extracts. The
sample of the Radix
Adenophorae
was
purchased from Oriental Medical Hospital (Daejeon, South Korea)
in 2007. The plant was identified by Professor Young-Bae Seo and avoucher specimens (Adenophorae)
are deposited in our laboratory.
Plant material (200 g) was extracted three times with distilled H2O
(or distilled water).
Then, the extract was filtered and evaporated on a rotary evaporator (BUCHI,
Switzerland),
dried by a freeze drier (EYELA FDU-540, Tokyo, Japan)
to yield the extract of Adenophorae
Radix (20 g). The yield (w/w) of the extract was about 10%.Animals
Seven-eight-week-old female
C57bl/6 mice
were obtained at Daehan Biolink Co. Ltd.  (Eumsung, South Korea).
All animal procedures were conducted in accordance with the guidelines of the Institutional
Animal Care and Use Committee, Korea Research Institute of Bioscience and Biotechnology
(Daejeon, South Korea).
We were divided
into five groups and each group had six mice.Isolation CD4+ T cells
Splenocytes
were isolated from naive C57bl/6 mice. Cells were enriched for CD4+ cell
populations by first staining the cells with anti-CD4 (BD PharMingen, Calif,
USA). CD25-cells were isolated from this population by first staining with fluorescein isothiocyanate
(FITC)—conjugated
anti-CD25 mAb (BD PharMingen) followed by incubation with magnetic-activated
cell sorting anti-FITC beads (Miltenyi Biotec, Auburn, Calif, USA).
CD4+ T cells were selected on a (CS) column, and the flow-through was collected as CD4+ T cells. Isolated cells
were activated by overnight incubation on 24-well plates coated with 1 μg/mL anti-CD3 (datasheet:
MCA500GA; description: Rat
antimouse CD3; specificity:
CD3; format: purified; product type: monoclonal antibody; clone:
KT3; isotype:
IgG2a; quantity: 0.25 mg, AbD Serotec.), 1 μg/mL anti-CD28 (datasheet: MCA1363; description:
Hamster antimouse CD28; specificity:
CD28; format: purified; product type: monoclonal antibody; clone: 37.51.1; isotype: IgG; quantity: 0.2 mg) and
with
RAE  (100, 50, 10 μg/mL)
added to RPMI medium supplemented with 1 unit/mL penicillin, 1 μg/mL streptomycin,
20 mM L-glutamine,
50 mM-mercaptoethanol, and 5% FBS for 48 hours after stimulation.Cytokine measurements
After
48-hours cultures, samples
were centrifuged at 2 000 rpm for 10 minutes,
and the supernatants were stored at−80°C.
Mouse IL-4, IL-5, IL-13, and IFN-γ
were quantified using ELISA kits (BioSource International, Camarillo, Calif, USA)
according to the manufacturer’s
protocols.Digestion of pulmonary tissue and cells preparations
Single-cell
suspensions from lung tissues and BALF were isolated by mechanical disruption
in RPMI 1640 medium supplemented with 2 mM L-glutamine, 100 U/mL penicillin,
100 mL streptomycin, 50 mM 2-mercaptoethanol, 20 mM HEPES, and 2% heat-inactivated
fetal bovine serum (FBS, GIBCO, Grand
Island, NY, USA).
Briefly, lungs were subsequently removed from thoracic cavity. After mincing
using sterile scalpels, tissue was incubated in PBS containing 1μg/mL collagenase
IV and 2 μg/mL dispase
II for 40 minutes at 37°C in a sterile polypropylene tube. After incubation, lung tissue was
vigorously pipetted up and down to further dissolve remaining tissue clumps and
then filtered using 70 μm cellstrainer
(Falcon, Le Pont de Claix, France). Total cells were counted manually in a hemocytometer
chamber (Fisher). A number of 2−4×103
cells were
spun onto glass slides  (Cytospin centrifuge, Cellspin, Hanil, South Korea)
(400 xg for
4 minutes).
Differential count was made according to standard morphologic criteria.OVA sensitization and inhalation
By
modified protocol as previously described [13, 14],
OVA (500 μg/mL)
in PBS was mixed with equal volumes of 10% (w/v)  aluminum
potassium sulfate (alum; Sigma-Aldrich)
in distilled water. Then incubated for 60 minutes at RT after adjustment to pH 6.5 using 10 N NaOH, and centrifuged at 750 Xg for
5 minutes.
OVA/alum pellet was resuspened to the original volume in distilled water. The
experimental animals were sensitized by intraperitoneal
(i.p) injection of OVA/alum {500⁢μg/ml,0. 2μg alum-precipitated Ag containing 100 μg of OVA  (Sigma-Aldrich, Seoul, South Korea) bound to 4 mg of
aluminum hydroxide (Sigma-Aldrich)
in PBS} with the amount of 200 μg on day 0, and 100 mL on days 7 and 14. And the mice were challenged with an intratracheal
(i.t) injection of 100 mL OVA/alum
(500 μg/mL)
on day 21, and underwent an aerosol challenge with OVA/alum for 30 min/day on 5
days/week for 8 weeks (at a flow rate of 250 L/min,
2.5% ovalbumin in normal saline). Seven days after the second sensitization,
mice were exposed to aerosolized ovalbumin for 30 min/day on 5 days/week for 8
weeks (at a flow rate of 250 L/min,
2.5% ovalbumin in normal saline). The
RAE
(450, 45 mg/kg)
in solution form were orally administered five
times a week
and CsA (10 mg/kg)
injection three times a week
for the last 8 weeks once 0.1 mL.
One day after the last of the OVA exposures, samples (BALF, lung cells, and
blood) were collected.BALF
To
evaluate airway inflammation, we experimented the accumulation of eosinophils
in BALF. Mice were sacrificed with an intraperitoneal injection of sodium pentoparbitone
(100 mg/kg).
The trachea was cannulated and the left bronchi were tied for histological
experiment.Immediately
after sacrifice, cells in the lungs were recovered by flushing 1 mL of BALF
(1 mM EDTA, 10% FBS, PBS) into the lungs via the trachea. Total cell counts
were determined and 100 mL of fluid were cytospin onto glass slides using a cytospin
centrifuge (Cellspin, Hanil, South Korea)
(400⁢×g for 4 minutes).
Differential cell counts were performed after staining with Diff-Quik Stain Set
(Baxter Healthcare Corporation,
Miami, Fla, USA).
The supernatant of BALF was stored at –25°C for determination of cytokines.Determination of airway hyperresponsiveness
Total
pulmonary airflow in mice was estimated in a modification of the method [15, 16].
Buxco system (Biosystem XA; Buxco Electronics Inc.,
Troy, Conn, USA)
was used to evaluate the extent of airway constriction in different groups of
mice following the protocol described previously.
Penh
is equal to Pausex PIF/PEF, where Pause=(Te−Tr)/Tr (PIF: peak inspiratory flow; PEF: peak expiratory flow; Te: expiratory time; Tr: relaxation time). In this experiment, the mice were aerosolized ovalbumin for 30 min/day on 3 days/week for 12 weeks. Twenty four hours after final inhalation, the mice were given aerosolized normal saline, 50 μg/mL methacholine
(Sigma-Aldrich).
And then airway reactivity was monitored for 1, 3, 10, and 30
minutes, serially. Differences of Penh value between groups were evaluated by means of student’s
t-test.Antibodies and flow cytometric analysis
All
antibodies (CD3, CD4, CD8, CD69, CCR3, CD11b, Gr-1, IgE, and B220) for flow
cytometric analysis were purchased from Becton Dickinson (BD) PharMingen (San Diego, Calif, USA).
Cells from lung tissues and BALF were stained with the indicated antibodies in
staining buffer (PBS containing 1% FBS and 0.01% NaN3)
for 10 minutes on ice, and analyzed by two-color flow cytometry on a FACScan using CellQuest
software (BD Biosciences, Mountain View, Calif, USA).Enzyme-linked immimosorbent assay
Interleukin
(IL-4, IL-5, IL-13, IL-I0), IgE production from BALF and histamine release in
serum of the indicated mice(n=6) was measured by ELISA according
to the manufacturer's instruction on a monoclonal antibody-based mouse ELISA
kit.
All
data represent the standard deviation of at least three different determinants
and were compared using Student's t-test.Quantitative real-time PCR
To study antiasthmatic
effects of Radix Adenophorae extract on cytokine gene expression from lung
cells, quantitative real-time PCR was performed after quantitative
normalization for each gene by a densitometry usingβ-actin
gene expression. Briefly, total cellular RNA was extracted from the lung by the
phenol-chloroform-based
method (RNAsolB: Tel-Test Co. Inc., Friendswood, Tex, USA)
according to the manufacturer's instructions. cDNA synthesis from 3 μg of total RNA was carried out using a ReverTraAce-a-cDNA Synthesis kit (Toyobo
Co., Ltd., Osaka, Japan)
according to the manufacturer’s instructions. Real-time
quantitative PCR was performed using the Applied Biosystems 7500 Fast
Real-Time
PCR system (Applied
Biosystems, Foster City, Calif, USA)
with the following primers.
The primer sequences are as follows: mouse eotaxin
2, 5′-CCTGGACCAAAAACTCCAAA-3′
and 5′-GCGACTGGTGCTGATATTC-3′ CCR3, 5′-CCCGAACTGTGACITTTGCT-3′ and
5′-CCTCTGTATAGCGAGGACTG-3′ IL-13,
5′-ATGCCCAACAAAGCAGAGAC-3′ and 5′-TGAGAGAACCAGGGAGCTGT-3′ TNF-α, 5′-TGGGAGGAAGGGGTCTAAG-3′ and 5′-ACCTACGACGTGGGCTACAG-3′ IL-10,
5′-AAGCAGCCTTGCAGAAAAGA-3′ and 5′-TGGGAAGTGGGTGCAGTTAT-3′ TGF-β1,
5′-TGGAGCAACATGTGGAACTC-3′ and 5′-CTGCCGTACAACTCCAGTGA-3′ TARC,
5′-CAGGGATGCCATCGTGTTTCT-3′ and 5′-GGTCACAGGCCGCTTTATGTT-3′β-actin, 5′-TGGAATCCTGGTCCATGAAAC-3′ and 5′-GTCACAGTCAGCTGTATAGGG-3′.Proinflammatory
gene expression was analyzed with SYBR Green PCR Mastermix (ABl) and a final
concentration of 200 nM primers, usingβ-actin
as the internal standard. The following PCR profile was used: 2 minutes at 50°C, 10 minutes at 94°C, and 40 cycles of 1 minute at 94°C, and 1 minute at 60°C. The amount of SYBR Green was measured at the end of each cycle. The
cycle number at which the emission intensity of the sample rises above the
baseline is referred as to the relative quantitative (RQ)
and is proportional to the target concentration. Real-time
PCR was performed in duplicate and analyzed by  applied Biosystems 7500 Fast Real-Time PCR system manual (threshold: 0.05; baseline: 6–15 cycles). To
generate the standard curves for Proinflammatory
cytokine and β-actin,
serially diluted
cDNA (1/1-1/16)
was prepared and real-time
PCR was performed as above. Relative quantitative (RQ) evaluations by RQ-PCR were
expressed at various samples.H&E and M-T staining in murine OVA-induced asthma lung tissue
Three
experimental groups were treated with different concentrations of RAE five
times a week and
CsA three times a week
for the later 8 weeks. At the end of the experiment, the mice lung sections
were stained with H&E and M-T and analyzed histologically by modified
protocol as previously described [17, 18].Statiscal analysis
For
statistical analysis of data,P values were analyzed
using a student’s t-test software program (Startview 5.1; Abacus Concepts, Berkeley, Calif, USA). Results were
considered statistically significant when P values
were *P<.05, **P<.01, and
***P<.001.


## 3. Results and Discussion

First,
we examined how RAE made an effect toward CD4+ T cells in
vitro.
Splenocytes
were isolated from naive C57bl/6 mice. CD4+ T cells were selected on a CS
column, and the flow-through was collected as CD4+ T cells. Isolated cells were
activated by overnight incubation on 24-well plates coated with 1 μg/mL anti-CD3, 1 μg/mL anti-CD28, and with RAE (100,
50, 10 μg/mL)
and CsA (Cyclosporine A 10 μg/mL)
added to RPMI medium supplemented with 1 unit/mL penicillin, 1 μg/mL streptomycin, 20 mM L-glutamine,
50 mM-mercaptoethanol,
and 5% FBS for 48 hours after stimulation. After 48-hour culture, samples were centrifuged at 2 000 rpm for 10 minutes.
Mouse IL-4, IL-5, IL-13, and IFN-γ.
The
levels of cytokines
were
quantified using ELISA kits according to the manufacturer’s
protocols. As
you see Table 1, RAE decreased IL-4, IL-5, and IL-13 cytokines, It has been suggested that RAE may be has suppressing
activities of CD4+ T cells, eosinophils inducing asthmatic inflammations.Table 1
Effects of RAE on cytokines in
CD4+ t cells isolated from
Naive C57bl/6 Mice (NM: Normal C57BL/6 mice; CT: a-CD3/CD28+
CD4+ T cells; CsA: a-CD3/CD28+
CD4+ T cells + CsA;  RAE 100: a-CD3/CD28+ CD4+ T cells + RAE (100 μg/mL); RAE 50 : a-CD3/CD28+ CD4+ T cells + RAE (50 μg/mL); RAE 10 : a-CD3/CD28+ CD4+ T cells + RAE (10 μg/mL)).
IFN-gamma (pg/mL)IL-4 (pg/mL)IL-5 (pg/mL)IL-13 (pg/mL)NM1023.8± 39.3693.8± 109.91.0± 0.215.2± 1.0CT1452.5± 62.44681± 303.554.5± 5.1515.5± 18.7CsA809±  32.2***537±  84.3***3.9±  0.4***15.4±  0.7***RAE 1002028.9±  192.9*2859.5±  198.7**21.7±  1.4***316.2±  32.6***RAE 501529.1± 159.93114.1±  76.2**28.3±  2.7**394.4±  21.7**RAE 101335.4± 55.63597.2± 434.756.7± 4.6496.5± 7.2The results expressed the mean± S.E
(N=6). Statistically significant value compared with control group data by t-test
(***P<.001,**P<.01,*P<.05).To evaluate the effects of RAE and CsA on
airway hyperresponsiveness, total pulmonary airflow in mice was estimated in
murine model of asthma. Penh was measured by Buxco system on day 1 after final
inhalation and then immediately samples were collected. Exposure of animals to
aerosolized OVA resulted in increased airway hyperresponsiveness (AHR) compared with that of animals receiving PBS only (Figure1).
As shown in Figure 1,
relative to animals sensitized with OVA (control group), RAE-(450 mg/kg)
and CsA-(10 mg/kg)
treated groups showed a significant (**p<.01,***p<.001) decrease in methacholine-induced AHR. But RAE (45 mg/kg)
group did not show significant decrease in Penh value.
This was accompanied by changes in the lung and BAL total cells counts
(Figure 4). Therefore,
above results indicate that RAE (450 mg/kg)
and CsA have inhibitory effects on AHR.Statistically significant,
value compared with control byt-test
(*P<.05,**P<.01).To
clarify the efficacy of RAE on lung cells of murine asthma model, the left
lungs were histologically examined 24 hours
after the final antigen challenge. Histological analyses of lungs from
PBS-exposed sensitized mice showed normal lung histology (Figures2(a),
3(a)).
In contrast, similar to the BALF study, histological sections of lung tissue
from OVA-exposed mice exhibited airway inflammation and infiltrating
eosinophils were chiefly observed in the peribronchial regions of the lung (Figures 2(b), 2(c), 3(b), 3(c)).
While on the other hand,
exhibition of airway inflammation was decreased in histological sections of
lung tissue from RAE and CsA (Figures 2(d), 3(d)),
RAE 450 mg/kg (Figures 2(e), 3(e)), and RAE 45 mg/kg-treated
mice (Figures 2(f), 3(f)).
The lung tissue of CsA and RAE
treatments
on mice
group showed much less eosinophils, leukocytes, and collagen accumulating compared with that of OVA-induced mice group.Figure 1
Effects of RAE and CsA on methacholine-induced AHR in the sensitization (NM: normal C57BL/6 mice; CT: OVA-induced asthma mice (control); CsA: OVA-induced asthma mice treated with cyclosporine A (10 mg/kg); RAE 450 mg/kg: OVA-induced asthma mice treated with RAE (450 mg/kg); RAE 45 mg/kg: OVA-induced asthma mice treated with RAE (45 mg/kg)).Figure 2
Effect of RAE on histology of lung tissue (H&E and Masson trichrome staining) in lung tissues of OVA-induced asthma murine ((a): normal C57BL/6 mice; (b): OVA-induced asthma mice (Control 1); (c): OVA-induced asthma mice (Control 2); (d): OVA-induced asthma mice treated with cyclosporine A (10 mg/kg); (e): OVA-induced asthma mice treated with RAE (450 mg/kg); (f): OVA-induced asthma mice treated with RAE (45 mg/kg)).Effect of RAE on immunohistochemistry of lung tissue (Congo red stain) in lung tissues of OVA-induced asthma murine ((a): normal C57bl/6 mice; (b) and (c): OVA-induced asthma mice (control); (d): OVA-induced asthma mice treated with cyclosporine A (10 mg/kg); (e): OVA-induced asthma mice treated with RAE (450 mg/kg); (f): OVA-induced asthma mice treated with RAE (45 mg/kg)).
(a)(b)(c)(d)(e)(f)Effects of RAE on lung weights and total lung cells in OVA-induced asthma murine (NM: normal C57BL/6 mice; CT: OVA-induced asthma mice (control); CsA: OVA-induced asthma mice treated with cyclosporine A (10 mg/kg); RAE 450 mg/kg: OVA-induced asthma mice treated with RAE (450 mg/kg); RAE 45 mg/kg: OVA-induced asthma mice treated with RAE (45 mg/kg)).
(a)(b)(c)(d)The
number of BAL eosinophils was sequentially increased after two or three
consecutive days of OVA exposure. To evaluate the effect of RAE and CsA on the
recruitment of cells to the airway, we investigated the lung weight, total lung
cell, total leukocytes, and eosinophils in BALF. In RAE and CsA-treated
groups, total lung weight was significantly reduced compared with OVA-exposed control
group, and the number of total leukocytes in BALF, total lung cells, and eosinophils were also significantly reduced compared to those in the control
group (Figure4).C57BL/6
mice were injected, inhaled, and sprayed with ovalbumin (OVA) for 8
weeks (five times a week) for asthma induction. Two experimental groups were treated with
different concentrations of Radix Adenophorae extract
(RAE) for the later 8 weeks (five times a week). At the end of the experiment,
the mice lungs were removed and weighted.The cell surface
expression of CCR3 was analyzed by flow cytometry. To detect CCR3 expression on
lung, lymph node, BALF, and spleen cells, two-color immune-staining was performed using FITC-conjugated
anti-CCR3 with PE-conjugated anti-CD3 mAb as described in materials and
methods.To
evaluate the efficacy of RAE and CsA treatments on granulocytes population and CCR3 expression, we compared the effects of RAE
and CsA on intracellular CCR3 expression in OVA-induced asthma lung cells by
using flow cytometry. The percentages of CCR3-positive cells were decreased
when compared with control group (**P<.01, Figure 5). Results
obtained with FACS were also confirmed by real-time PCR, as the relative
quantitiveness RQ of
mRNA expression in lung cells expressing CCR3 was significantly decreased in
cells treated with RAE and CsA when compared with control group (Table 3).
Moreover, RAE and CsA treated group with OVA resulted in significant reductions
Gr-1+/CD11b+ cells in lung (**P<.01) (Figure 6).Figure 5
Effect of RAE on CD3e−/CCR3+ population in lung tissues of OVA-induced asthma murine (NM: normal C57BL/6 mice; CT: OVA-induced asthma mice (control); CsA: OVA-induced asthma mice treated with cyclosporine A (10 mg/kg); RAE 450 mg/kg: OVA-induced asthma mice treated with RAE (450 mg/kg); RAE 45 mg/kg: OVA-induced asthma mice treated with RAE (45 mg/kg)).Effects of RAE on CD4+/CD8−, GR-1+/CD11b+, IgE+/B220+, and CD3e+/CD69+ cells absolute number in lung and spleen tissues of OVA-induced asthma murine (NM: normal C57BL/6 mice; CT: OVA-induced asthma mice (control); CsA: OVA-induced asthma mice treated with cyclosporine A (10 mg/kg); RAE 450 mg/kg: OVA-induced asthma mice treated with RAE (450 mg/kg); RAE 45 mg/kg: OVA-induced asthma mice treated with RAE (45 mg/kg)).
(a)(b)(c)(d)C57BL/6
mice were injected, inhaled, and sprayed with OVA for 8
weeks (five
times a week) for asthma induction.
Two experimental groups were treated with
different concentrations of RAE for the later 8 weeks (five times a week). At
the end of the experiment, the mice lungs were removed and analyzed by flow
cytometer.Effects of RAE on leukocyte subsets
in lungs and BALF were marked with change in numbers of CD4+/CD8− T cells (Th2 cells).
CD3  +/CD69+ (early activated T cells) IgE+/B220+ B cells in a murine
model of asthma compared to control group, and the deficits in CD4+/CD8− T
cells were accompanied by concurrent decreases in absolute number of
CD3+/CD69+, IgE+/B220+ B cells (Table 2, Figure 6). RAE and CsA groups treated with OVA resulted in further significant reductions in CD4+/CD8− T cells
(**P<.01)
and
IgE+/B220+ B cells (**P<.01) in BALF and lung
tissues significantly
were decreased and
CD4+/CD8−
T cells
and
CD3+/CD69+ cells
(**P<.01)
in lung tissues significantly
were also decreased (Table 2,
Figure 6).Table 2
Effects of RAE on CD4+/CD8−, GR-1+/CD11b+, IgE+/B220+, and CD3e+/CD69+ cells absolute number in lymph nodes and BAL fluid of OVA-induced asthma murine.
Cell phenotypeOrganNormal C57BL/6OVA-induced asthma mice (Absolute no.)ControlCsARAE (450)RAE (45)CD4+/CD8− (×104 cells)LN1221.9± 34.9232.7± 12.8150.3± 41.1**193.95± 10.1*244.7± 32.2BA23.9± 0.633.6± 0.813.2± 3.1**11.3± 0.1**27.7± 1.5*Gr-1+/CD11b+ (×104 cells)LN125.6± 0.573.6± 13.845.0± 11.9**33.7± 1.0**45.6± 8.6*IgE+/B220+ (×104 cells)LN13.6± 0.16.6± 1.23.6± 1.12.1± 0.3*3.0± 0.9BA20.3± 0.012.6± 0.070.5± 0.03**0.5± 0.02**1.1± 0.05*CD3+/CD69+ (×104 cells)LN132.5± 3.054.2± 1.435.6± 8.136.6± 1.947.6± 7.1Table 3
Effect of RAE on Eotaxin2, CCR3, IL-13, IL-10, TNF-α, and TARC mRNA expression in lung tissue of OVA-induced murine model of asthma (NM: normal C57BL/6 mice; CT: OVA-induced asthma mice (control); CsA: OVA-induced asthma mice treated with cyclosporine A  (10 mg/kg); RAE 45 mg/kg: OVA-induced asthma mice treated with RAE (450 mg/kg); RAE 450 mg/kg: OVA-induced asthma mice treated with RAE (450 mg/kg)).
OVA/alumGroupExotaxin2CCR3IL-10IL-13TARCTNF-α−NM0.020.030.050.0160.6560.152+CT111111+CsA0.2020.4040.6210.3860.120.255+RAE (450 mg/kg)0.4620.4990.5770.2840.4870.401+RAE (45 mg/kg)0.620.7250.5040.7630.9440.625Each point represents the mean ± S.E of 6 mice. Statistically significant, value compared with control byt-test (*P<.05,**P<.01) (LN1: Lymph node; BA2: Bronchoalveolar Lavage).To
study whether RAE was related to inflammatory cytokine production, BALF
supernatants were collected, and the productions of IL-4, IL-5, IL-10, IL-13, IgE, histamine
were analyzed by ELISA.
Asshown
in Figure7,
IL-4, IL-5, IL-13, IgE productions in
BALF and histamine production
in
serum were suppressed by RAE and CsA. Both IL-4
and IL-13
levels were significantly reduced in RAE- and CsA-treated
groups of mice.Effect of RAE on cytokines, immunoglobulin E levels in BALF and histamine release in serum (NM: normal C57BL/6 mice; CT: OVA-induced asthma mice (control); CsA: OVA-induced asthma mice treated with cyclosporine A (10 mg/kg); RAE 450 mg/kg: OVA-induced asthma mice treated with RAE (450 mg/kg); RAE 450 mg/kg: OVA-induced asthma mice treated with RAE (450 mg/kg)).
(a)(b)Because
IgE levels in BALF are dependent upon IL-4, IL-5, IL-13 and may be considered
an additional index of Th2 cytokine secretion, we measured IgE in BALF from mice in all groups. We found
that IgE levels in BALF from OVA-induced murine model of asthma were
significantly increased compared with normal groups. RAE and CsA treatments of these mice significantly inhibited the production of IgE. These results
support the conclusion that RAE and CsA suppressed the generation of a Th2-type
immune response and activity of mast cells in this animal model of asthma.To
investigate the effect of RAE on mRNA expression in lung tissue, total cellular
RNA was extracted from lung cells treated with or without RAE in the presence
or absence of OVA sensitization and inhalation. As shown in Table3, the mRNA for
eotaxin2, CCR3, IL-13, IL-10, TARC, and TNF-α was detectable in
the lung cells treated with PBS only (NM), OVA (CT), CsA (10 mg/kg), and
RAE
(450, 45 mg/kg), respectively (Table 3).C57BL/6
mice were injected, inhaled and, sprayed with OVA for 8
weeks (five
times a week) for asthma induction. Two experimental groups were treated with
different concentrations of Radix Adenophorae extract
(RAE) for the later 8 weeks (five times a week). At the end of the experiment,
the mice lungs were removed and analyzed by real-time PCR.PCR
products for eotaxin2, CCR3, IL-13, TARC, and TNF-α amplified from lung
cell RNA preparations were decreased in
the RAE-treated groups
compared with control groups. Furthermore, the RAE had little effects on IL-10 mRNA expression in lung cells (Table 3).
The results demonstrated that RAE and CsA significantly affected eotaxin2,
CCR3, IL-13, TARC, and TNF-α mRNAs expression
but not other cytokines in lung cells. This was accompanied by changes in the
eosinophil influx (Figure 5), BAL cytokines
(IL-13) production (Figure 7) in some degree.C57BL/6 mice were injected, inhaled, and sprayed with OVA for 8
weeks (five
times a week) for asthma induction. Two experimental groups were treated with
different concentrations of Radix Adenophorae extract (RAE) for
the later 8 weeks (five times a week). At the end of the experiment, the BALF
were collected and cytokine productions were analyzed by ELISA.Bronchial asthma is
a chronic inflammatory disorder of the airways characterized by variable airway
obstruction, airway eosinophilic inflammation, and AHR [19]. Persistent
inflammation of bronchial mucosa, mainly characterized by eosinophilic
infiltration, is considered as an essential process in the pathogenesis of asthma [20, 21].Asthma
is a chronic lung disease characterized by allergen-induced airway inflammation
and orchestrated by Th2 cells [22]. RAE is one of the well known herbs used in oriental medicine for treatment of anti-inflammatory and many allergic
diseases.Anti-inflammatory
effects of RAE on the development of OVA-induced eosinophilia and hyperresponsiveness in murine
model of asthma have not been fully investigated in vivo.Immunomodulators
such as cyclosporine A (CsA) inhibit single allergen-induced allergic
inflammation such as eosinophilic, lymphocytic infiltration, and mRNA expression for IL-4 and IL-5 [23]. In this study, the RAE could
modulate the immune responses of BALF cells and lung cells in OVA-induced
murine model of asthma. Hence, the suppressive activity of RAE and CsA on
inflammatory cytokine productions might have important implications with regard
to RAE therapeutic activity in asthma and pulmonary inflammation. The main
question to be addressed is whether RAE is involved in the pathology of airway
inflammation and asthma. In the present study, we examined lung cells
inflammation and BALF analyses.Airway
allergen challenge causes cellular inflammation in the airways, which is
dominated by eosinophils in both humans and mice [24].
It has been suggested that eosinophils contribute to several of the clinical
features of allergic asthma, including tissue damage and AHR
[25].We
measured the effects of RAE on methacholine-induced
AHR in the sensitization. As shown in Figure1, relative to animals
sensitized with OVA (control group), RAE-(450 mg/kg) and
CsA-(10 mg/kg)
treated groups showed a significant decrease in methacholine-induced
AHR. This was accompanied by changes in the lung and BAL total cells counts
(Figure 4). Therefore,
above results indicate that RAE (450 mg/kg)
and CsA have inhibitory effects on airway hyperresponsiveness.In aspect of
histological analyses of sections from OVA-induced asthma model mice, the
lung tissue of CsA and RAE treatments of mice
group showed much less eosinophils, leukocytes, and collagen accumulating compared with that of OVA-induced mice group (Figures2, 3).To
evaluate the effect of RAE and CsA on the recruitment of cells to the airway,
we investigated the lung weight, total lung cell, total leukocytes, and eosinophils in BALF. In groups
treated with RAE
and CsA,
total lung weight was significantly reduced compared with OVA-exposed control
group, and the number of total leukocytes in BALF, total lung cells, and eosinophils was also significantly reduced compared to those in the control group (Figure4). These
results
indicate that RAE (450 mg/kg)
and
CsA have inhibitory effects on eosinophils and leukocytes infiltration into
the lungs.Antigen-activated
CD4+ T cells have been shown to induce many of the characteristic features of
asthma, including the secretion of cytokines such as IL-4, IL-5, and IL-13,
which regulate mucus production, inflammation, and adhesion molecules [26–28].High
levels of interleukin-4 and interleukin-5 are related to clinical measures of
disease severity [29, 30]; IL-5 has been shown to mediate the recruitment of eosinophils [31]. The reduction in BAL IL-13 levels would
suggest a beneficial therapeutic effect via decreased mucus production [32].To
evaluate the efficacy of
RAE and CsA treatments on granulocytes population and CCR3 expression, we compared the effects of RAE
and CsA on intracellular CCR3 expression in OVA-induced asthma lung cells by
using flow cytometry. The percentages of CCR3-positive cells were decreased
when compared with control group (Figure5). Results
obtained with FACS were also confirmed by real-time PCR, as the relative
quantitiveness RQ of
mRNA gene expression in lung cells expressing CCR3 was significantly decreased
in cells treated with RAE and CsA when compared with control group (Table 3).
Our results indicate that RAE could decrease CCR3 expression of BALF cells
(especially eosinophils), data of which are compatible with airway eosinophil
influx and hyperresponsiveness.CD11b
expression on the surface of circulating eosino-phils is significantly elevated
in various allergic disorders, including atopic dermatitis and bronchial asthma
[33]. We observed that inhalation challenge with RAE and CsA administration
resulted in a decrease in airway CD11b+ macrophage, Gr-1 granulocytes when
compared with that observed after OVA challenge only. RAE and CsA groups treated with OVA resulted in significant
reductions Gr-1+/CD11b cells in lung and lymph nodes (Table 2, Figure 6). Our results show that RAE and CsA downregulate CCR3 expression and
CD4+CD8− T cells, CD3+ CD69+, IgE+ B220+ B cells in BALF and lung cells (Table 2, Figure 6).In
study, various cytokines production of BALF cells from OVA-induced control
subjects have been enhanced, but RAE- and CsA-treated subjects
significantly reduced. We suggest that the inhibitory mechanism of RAE and CsA
on IL-4, IL-5, IL-10, and IL-13 production may have involved reduced production of IL-4, IL-5, IL-13,
IgE and this would suppress the eosinophils influx in airway and then more
factors synthesized by RAE and CsA treated in BALF such as IgE were decreased
(Figure7). This was
accompanied by changes in the BAL eosinophils counts (Figure 4) and mRNA
expression in lung tissue (Table 3).RAE decreased IL-4,
IL-5, and IL-13 levels in BALF and histamine release in serum significantly
(Figure7), and mRNA
expression of IL-13 in the lung as well (Table 3).
The lung histology in our study (Figures 2, 3) showed that
RAE reduced the eosinophils in lung tissue, and this result may be due to the
decreased IL-4 level which caused reduction in rolling and adhesion of
eosinophils. Based on these results, it is inferred that RAE may have an antiallergic effect on
allergic bronchial asthma via suppressing IL-4 secretion, and in consequence,
inhibiting IgE secretion from B cells, and reduced IL-4 may reduce eosinophils
infiltration into the lungs via reducing the rolling on and adhesion of
circulating eosinophils to the endothelial cells.IL-5
is a Th2-type cytokine that promotes the recruitment and activation of
eosinophils. IL-5 stimulates the release of chemical mediators from the
eosinophils [34]. Therefore, IL-5 has been implicated in the pathogenesis of
allergic diseases [35], and many studies have focused IL-5 to be a viable
target for the treatment of asthma and allergic diseases [36].In
our study, RAE significantly reduced IL-5 levels in BALF (Figure7) as well as mRNA expression of IL-13 in lung (Table 3).
Like this, RAE reduced eosinophils infiltration and activation in OVA-induced
asthmatic mice. We considered that this action may be due to inhibiting IL-13,
IL-5 secretion from Th2 cells.These
results indicate that RAE may suppress the excess activity of T cells and Th2
cytokines, which are implicated in the pathogenesis of allergic asthma, and consequently
regulate the Th1/Th2 imbalance of the immune system in allergic asthma. From
these results, we hypothesize that RAE has an immunoregulatory function
between Th1 and Th2, and suppressive effect on excessive Th2 cytokines.Th2
cytokines as well as elevated IgE levels are associated with the development of
airway hyperreactivity. Especially, IL-13 has been
shown to mediate the airway hyperresponsiveness in an allergic lung in
several studies [37].Furthermore,
mRNA expressions of these cytokines in lung were reduced remarkably by RAE
(Table2). From these results, we presume that RAE may suppress the airway hyperresponsiveness by
way of reducing these cytokines.Cytokines
and chemokines produced by Bronchial epithelial cells include IL-6, IL-8,
G-CSF, GM-CSF, RANTES, eotaxin, and TARC. Proinflammatory
cytokines IL-1 and TNF-alpha generally up-regulate
expression and release these cytokines/chemokines [38].The
bronchial epithelium of asthmatics and normal subjects expressed TARC protein,
and the asthmatics showed more intense expression than the normal subjects. And
combination of TNF-alpha and IL-4 induced expression of TARC protein.
Furthermore, expressions of TARC protein and mRNA were almost completely
inhibited by glucocorticoids. The airway epithelium represents an important
source of TARC, which potentially plays a role via a paracrine mechanism in the
development of allergic respiratory diseases [39].Our
study shows that eotaxin2, TNF-α, and
TARC mDNA gene expression in lung cells were significantly decreased in cells
treated with RAE and CsA when compared with control group (Table 3).
Our results indicate that RAE may have effects of antibronchial
injury, data of which are compatible with airway eosinophils influx and
hyperresponsiveness. So histological
analyses of sections from OVA-induced asthma model mice, the
lung tissue of CsA and RAE treatments
of mice
group showed much less eosinophils, leukocytes, and collagen accumulating compared with that of OVA-induced mice group (Figures 2, 3).In
summary, RAE and CsA have profound effects on airway inflammation and AHR in a murine model
of asthma through suppression of IL-13, IL-5, IgE, eosinophils CCR3 expression, and CD11b
cells expression. The therapeutic activity of RAE on asthma in oriental
medicine may be partly related tothe following. (a) Immunomodulatory
agents
contained in RAE reduce Th2 cells immune responses. (b)
Reduced production
of IgE will make a marked decrease for asthma attack occurring in individuals. (c) RAE may have an
effect of suppressing eosinophils activity and decrease airway damage.



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