Distinct Effects of Imperatorin on Allergic Rhinitis ...jpet.aspetjournals.org/content/jpet/early/2011/07/05/jpet.111... · Caspase-1 is characterized by its ability to activate the

JPET #184275

1

Distinct Effects of Imperatorin on Allergic Rhinitis: Imperatorin Inhibits

Caspase-1 Activity In Vivo and In Vitro System.

Hyun-A Oh, Hyung-Min Kim*, Hyun-Ja Jeong*

Department of Pharmacology, College of Oriental Medicine, Kyung Hee University,

Seoul,130-701, Republic of Korea (H.A.O., H.M.K); and Biochip Research Center, Hoseo

University 165, Sechul-ri, Baebang-myun, Asan, Chungnam, 336-795, Republic of Korea

(H.J.J)

JPET Fast Forward. Published on July 5, 2011 as DOI:10.1124/jpet.111.184275

Copyright 2011 by the American Society for Pharmacology and Experimental Therapeutics.

This article has not been copyedited and formatted. The final version may differ from this version.JPET Fast Forward. Published on July 5, 2011 as DOI: 10.1124/jpet.111.184275

at ASPE

T Journals on Septem

ber 9, 2018jpet.aspetjournals.org

Dow

nloaded from

http://jpet.aspetjournals.org/

JPET #184275

2

Running title: Effect of imperatorin on allergic rhinitis

Address correspondence to: Dr. HJ Jeong ( ) and HM Kim

HJ Jeong, Biochip Research Center, Hoseo University 165, Sechul-ri, Baebang-myun, Asan,

Chungnam, 336-795, Republic of Korea, Tel.: +82 41 540 9681, Fax: +82 41 542 9681, e-

mail: [email protected].; HM Kim, Department of Pharmacology, College of Oriental

Medicine, Kyung Hee University, Seoul,130-701, Republic of Korea, e-mail:

[email protected].

The number of text page is 31

The number of figure is 8

The number of reference is 34

The number of words in the abstract is 249

The number of words in the introduction is 705

The number of words in the discussion is 634

ABBREVIATIONS: AR, allergic rhinitis; IPT, imperatorin; DEX, dexamethasone; OVA,

ovalbumin; HMC-1 cell, human mast cell line; IMDM, iscove’s modified dulbecco’s

medium; PBS, phosphate-buffered saline; PBST, 1 × PBS containing 0.05% tween-20;

PMACI, PMA and calcium ionophore A23187; ANOVA, analysis of variance.

A recommended section: Inflammation, Immunopharmacology, and Asthma


at ASPE



Dow

nloaded from


JPET #184275

3

ABSTRACT

Because imperatorin (IPT), the furanocoumarins exhibits anti-inflammatory activity, we

reasoned that IPT might modulate the allergic rhinitis (AR). The aim of this study was to

analyze the regulation of AR by IPT. Here, we show the effect and mechanism of IPT in an

ovalbumin (OVA)-induced AR model. The number of rubs after the OVA challenge in the

OVA-sensitized mice was significantly higher than that in the OVA-unsensitized mice. The

increased number of rubs was inhibited by the oral administration of IPT. The increased

levels of IgE and histamine in the OVA-sensitized mice were reduced by IPT administration.

The levels of interferon-γ were enhanced while the levels of interleukin (IL)-4 were reduced

on the spleen tissue of the IPT-administered AR mice. Protein levels of IL-1β, macrophage

inflammatory protein-2, intercellular adhesion molecule -1, and cyclooxygenase-2 were

reduced by IPT administration in the nasal mucosa of the OVA-sensitized mice. In the IPT-

administered mice, the number of eosinophils and mast cells infiltration increased by OVA-

sensitization were also decreased. In addition, IPT inhibited caspase-1 activity in the same

nasal mucosa tissue. In activated human mast cells, the receptor interacting protein (RIP)2,

IκB kinase (IKK)-β, nuclear factor (NF)-κB/Rel A, and caspase-1 activation were increased,

but increased RIP2, IKK-β, NF-κB/Rel A, and caspase-1 activation were inhibited by the

treatment of IPT. In addition, IPT inhibited caspase-1 activity and IL-1β production in IgE-

stimulated bone marrow-derived mast cells. We can conclude that IPT exerts significant

effects by regulating of caspase-1 activation in AR animal and in vitro models.


at ASPE



Dow

nloaded from


JPET #184275

4

Introduction

Allergic rhinitis (AR) is a global health problem that causes major illness and disability

and common manifestation of allergic diseases, affecting approximately 500 million people

worldwide (Bousquet et al., 2008). Many of the symptoms of patients with AR, including

sneezing, itching, and respiratory obstruction cause a lot of pain. However, the symptoms of

AR do not end here. If prolonged, AR, can cause problems in the nasal voice box, and can

cause very severe eye and ear symptoms. These symptoms are due to the release of histamine

and other active substances by mast cells, which stimulate the dilation of blood vessels,

irritate nerve endings and increase the secretion of tears (Whitcup, 2006).

Since the discovery by Coffman and colleagues of two distinct types of Th in mice

(Mosmann et al., 1986), mutual regulation between Th1 cells and Th2 cells has been

considered important for homeostatic maintenance of the immune system in the whole body.

Dysregulated Th1 and Th2 responses lead to excessive Th1 cell or Th2 cell activation,

resulting in the development of autoimmune diseases associated with the accumulation of

Th1 cells or in an induction of allergic diseases due to the accumulation of Th2 cells,

respectively (Bach, 2002). In response to exposure to allergens, patients with AR present an

inflammatory Immunoglobulin (Ig)E-mediated response characterized by a Th2 immunologic

pattern with mast cells and eosinophils activation and the release of inflammatory mediators,

interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α (Johansson et al., 2001).

Leukotrienes and prostanoids produced by the 5-lipoxygenase and cyclooxygenase (COX)-2

pathways have potent pro-inflammatory and vascular actions that implicate them in the

allergic and inflammatory reactions (Montuschi et al., 2007). Eosinophils are innate effector

cells that are important in immune responses against helminth parasitic infections and

contribute to the pathology associated with allergic inflammatory conditions. Mast cells


at ASPE



Dow

nloaded from


JPET #184275

5

contribute to the induction and/or maintenance of eosinophilic inflammation by a variety of

mechanisms, including IgE-dependent and IgE-independent processes (Pawankar et al., 2007).

The recruitment of these mast cells to inflammatory sites occurs in response to chemotactic

and activation signals (Bournazou et al, 2010). Minimal persistent inflammation is a

physiopathological phenomenon referring to the presence of an inflammatory cell infiltrate

(eosinophils and neutrophils) associated with the expression of intercellular adhesion

molecule-1 (ICAM-1) in the epithelial cells of the mucosa exposed to the allergen, in the

absence of clinical symptoms. ICAM-1 is still only expressed in the mucosal epithelial cells

of allergic patients. ICAM-1 was considered a marker of allergic inflammation (Montoro et al,

2007). Macrophage-inflammatory protein 2 (MIP-2) is a potent chemoattractant for immune

cells (Gupta et al., 1996).

Caspase-1 is a member of the cystein-aspartic acid protease (caspase) family (Stutz et al.,

2009). Caspase-1 is characterized by its ability to activate the inactive precursors of IL-1�

and IL-18 that are involved in inflammation. Caspase-1 contains an N-terminal caspase

recruitment domain (CARD). This CARD promotes the proteolytic activation of the recruited

caspase-1 in inflammation (Stutz et al., 2009). Caspase-1 is activated within inflammasome, a

large cytosolic protein complex that is induced by a growing number of endogenous,

microbial, chemical or environmental stimuli (Stutz et al., 2009). Specific adaptor molecules

of the receptor interacting protein-2 (RIP2, CARD containing kinase) regulate the activation

of caspase-1 through the CARD-CARD interaction (Kobayashi et al., 2002). RIP2 then

recruits the IκB kinase (IKK) complex through the direct interaction of its intermediate

domain with IKK-β, leading to the activation of the nuclear factor (NF)-κB (Inohara et al,

2000).

Imperatorin (IPT, Fig. 1A) is one of the furanocoumarins. Furanocoumarins are depicted


at ASPE



Dow

nloaded from


JPET #184275

6

with miscellaneous biological functions including vasorelaxation of corpus cavernosum

(Chen et al., 2000), increased cell differentiation in osteoblasts (Kuo et al., 2005), anti-

convulsant (Luszczki et al., 2009), anti-diabetic (Liang et al., 2009), vascular vasodilation

(He et al., 2007), and reduction in liver steatosis (Ogawa et al., 2007). Lin et al.,

demonstrated that glutamate release was facilitated by IPT in rat hippocampal nerve terminals

(Lin et al., 2010). In addition, Adebajo et al., reported that the IPT had the main anti-

trichomonal activity (Adebajo et al., 2009).

This present study was designed to investigate the possibility of applying this IPT for the

regulation of AR. Furthermore, we aimed to validate a possible mechanism in the ovalbumin

(OVA)-induced AR models and activated mast cells.


at ASPE



Dow

nloaded from


JPET #184275

7

Material and methods

Materials. IPT, dexamethasone (DEX), OVA, phorbol 12-myristate 13-acetate (PMA),

A23187, O-phthaldialdehyde (OPA), avidin peroxidase (AP), 2'-azino-bis (3-

ethylbenzithiazoline-6-sulphonic acid) tablets substrate (ABTS), bicinchoninic acid (BCA),

anti-dinitrophenyl (DNP) IgE, DNP-human serum albumin (HSA), and other reagents were

purchased from Sigma (St. Louis, MO, USA). Fetal bovine serum (FBS), iscove’s modified

dulbecco’s medium (IMDM), and streptomycin were purchased from Gibco BRL (Grand

Island, NY, USA). Anti-mouse IgE/IL-1β (mature form detection Ab)/ΙL-4/IFN-

γ antibody (Ab), biotinylated anti-mouse IgE/IL-1β/IL-4/IFN-γ Ab, recombinant mouse (rm)

IgE/IL-1β/IL-4/IFN-γ, anti-human IL-1β Ab, biotinylated anti-human IL-1β Ab, and

recombinant human (rh) IL-1β were purchased from Pharmingen (Sandiego, CA, USA). Ab

for IKK-β, RIP2, caspase-1, COX-2, NF-κB/Rel A, IκBα, histon, and actin were obtained

from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The mouse OVA-specific IgE kit

was purchased from DS Pharma Biomedical Co. Ltd. (Osaka, Japan) The caspase-1 assay kit

was supplied by R&D Systems Inc. (Minneapolis, MN, USA).

OVA-induced AR animal model. We maintained 6-week-old female BALB/c (Charles

River Technology) mice under pathogen-free conditions. Mouse care and experimental

procedures were performed under approval from the animal care committee of Kyung Hee

University [KHUASP (SE)-10-016]. The mice were sensitized on days 1, 5, and 14 by

intraperitoneal (i.p) injection of 100 μg OVA emulsified and 20 mg aluminum hydroxide

(Sigma) in a 100 μl phosphate-buffered saline (PBS) and challenged intranasally with 1.5 mg

OVA in 2 μl PBS or PBS. The mice were challenged intranasally with PBS in a similar

manner for the negative control. IPT (0.1 and 1 mg/kg), DEX (5 mg/ml), or a control vehicle

(distilled water.) was administrated orally for 10 days before the intranasal (i.n.) OVA


at ASPE



Dow

nloaded from


JPET #184275

8

challenge (Fig. 1B). Bain et al., reported that OVA primed mice displayed a significant

increase in sneezing behavior when challenged intranasally (i.n.) with OVA (2011). In our

study, although the frequency of sneezing was increased by the OVA challenge, there were no

appreciable differences in the appearance of the mice between the OVA-unsensitized and

OVA-sensitized groups (data not shown). Nasal symptoms were only evaluated by counting

the number of nasal rubs that occurred in the 10 min after OVA i.n. provocation at the 10 day

mark after the challenge. We measured OVA-specific IgE as relevant end point for AR. The

numbers of mice in each group was 5.

Culture of HMC-1 cells. The human mast cell line (HMC-1) was generously provided by

Eichi Morri (Osaka University, Japan). HMC-1 cells were grown in IMDM supplemented

with 100 unit/ml penicillin, 100 mg/ml streptomycin and 10% heat-inactivated FBS at 37°C

5% CO2 and 95% humidity. HMC-1 cells (3 × 105 cells/ml) were treated with IPT (1 and 10

μg/ml) for 1 hr prior to stimulation with PMA and calcium ionophore A23187 (PMACI)

incubated for 2 hrs or 8 hrs.

Histamine assay. The histamine was measured from HMC-1 cells and serum according to

the manufacturer’s specifications using the histamine assay kit supplied by Oxford

Biomedical Research (Oxford, MI, USA).

Enzyme-linked immunosorbent assay (ELISA). HMC-1 cells (3 × 105) were treated

with IPT (1 and 10 μg/ml) for 1 h prior to stimulation with PMACI incubated for 8 h.

Cytokines of serum, mucosa, and spleen tissue, and supernatant were measured by an ELISA.

The ELISA was performed by coating 96-well plates with 1 μg/well of capture Ab. Before

the subsequent steps in the assay, the coated plates were washed twice with 1 × PBS

containing 0.05% tween-20 (PBST). All reagents and coated wells used in this assay were

incubated for 2 h at room temperature. The standard curve was generated from the known


at ASPE



Dow

nloaded from


JPET #184275

9

concentrations of cytokine, as provided by the manufacturer. After exposure to the medium,

the assay plates were exposed sequentially to each of the biotin-conjugated secondary

antibodies, and an AP and ABTS substrate solution containing 30% H2O2. The plates were

read at 405 nm. Appropriate specificity controls were included, and all samples were run in

duplicate. The OVA-specific IgE was measured from the serum according to the

manufacturer’s specifications using an OVA-specific IgE kit. Cytokine levels in the spleen

and nasal mucosa were divided according to the total protein. The protein was estimated

using the BCA method with the BCA protein assay kit (Pierce, Rockford, IL, USA). This

method combines the reduction of cupric ions to cuprous ions by the protein in an alkaline

medium and then the subsequent reaction of the cuprous ions with two molecules of BCA to

give an intense purple color read at 560 nm.

Reverse transcription-polymerase chain reaction (RT-PCR). HMC-1 cells (3 × 106)

were treated with IPT (1 and 10 μg/ml) for 1 h prior to stimulation with PMACI incubated for

6 h. The total RNA was isolated from the cells and nasal mucosa according to the

manufacturer’s specification using an easy-BLUETM RNA extraction kit (iNtRON Biotech,

Korea). The concentration of total RNA in the final elutes was determined by

spectrophotometry. Total RNA (2.5 μg) was heated at 65°C for 10 min and then chilled on ice.

Each sample was reverse-transcribed to cDNA for 90 min at 37°C using a cDNA synthesis kit

(Amersham Pharmacia Biotech, Piscataway, NJ, USA). The PCR was performed with the

following primers for the mouse IL-1β (5’ AGG CCA CAG GTA TTT TGT CG 3’; 5’ GCC

CAT CCT CTG TGA CTC AT 3’), mouse GAPDH (5’TTC ACC ACC ATG GAG AAG GC

3’; 5’GGC ATG GAC TGT GGT CAT GA 3’), human IL-1β (5` GGG GTA CCT TAG GAA

GAC ACA AAT TG 3`; 5` CCG GAT CCA TGG CAC CTG TAC GAT CA 3`), and human

GAPDH (5` CCT GCT TCA CCA CCT TCT TG 3`; 5` CAA AAG GGT CAT CAT CTC TG


at ASPE



Dow

nloaded from


JPET #184275

10

3`). GAPDH was used to verify whether equal amounts of RNA were used for reverse

transcription and PCR amplification from different experimental conditions. Saturation

curves for PCR were obtained from various experimental conditions (RNA concentrations,

annealing temperatures, and PCR cycle numbers). We determined the optimal amplification

conditions (annealing temperature and PCR cycle number) of primers for the PCR. The

annealing temperature was 50°C for mouse and human IL-1β and 60°C for mouse and human

GAPDH respectively. Products were electrophoresed on a 1.5% agarose gel and visualized by

staining with ethidium bromide.

Western blot analysis. HMC-1 cells (3 × 106) were treated with IPT (1 and 10 μg/ml) for

1 h prior to stimulation with PMACI incubated for 2 h. Western blot analysis was used for

nasal mucosa tissue extracts and cell extracts were prepared by a detergent lysis procedure.

Samples were heated at 95°C for 5 min, and briefly cooled on ice. Following the

centrifugation at 15,000 × g for 5 min, 50 μg aliquots were resolved by 10% SDS-PAGE. The

resolved proteins were electrotransferred overnight to nitrocellulose membranes in 25 mM

Tris, pH 8.5, 200 mM glycerin, 20% methanol at 25 V. Blots were blocked for at least 2 h

with PBST containing 5% nonfat dry milk and then incubated with primary antibodies for 1 h

at room temperature. Blots were developed by peroxidase-conjugated secondary antibodies,

and proteins were visualized by enhanced chemiluminescence procedures (Amersham

Bioseciences, Piscataway, NJ, USA) according to the manufacturer's instructions.

Histological examination. Tissue samples were immediately fixed with 10%

formaldehyde and embedded in paraffin. The sections of the nasal mucosa sample were 4 μm

thick. Each section was stained with hematoxylin and eosin (H&E, for eosinophils), alcian

blue and safranine O (A&S, for mast cells), CD4 (for T cells, Abbiotec, San diego, CA, USA),

F4/80 (for macrophages, eBioscience, San diego, CA, USA) or immunohistochemical stain


at ASPE



Dow

nloaded from


JPET #184275

11

(for IL-1β) before dewaxing and dehydration. The numbers of eosinophils, mast cells, T cells,

macrophages, and IL-1β on both sides of the septal mucosa were counted. Sections were

coded and randomly analyzed by two blinded observers.

Myeloperoxidase (MPO) assay. To evaluate the effect of IPT on neutrophils infiltration,

the activity of tissue MPO was assessed. A biopsy was placed in 0.75 ml of 80 mM PBS, pH

5.4 containing 0.5% hexadecyltrimethylammonium bromide and homogenized (45 s at 0°C)

in a motor-driven homogenizer. The homogenate was decanted into a microfuge tube, and the

vessel was washed with a second 0.75 ml aliquot of hexadecyltrimethylammonium bromide

in a buffer. The wash was added to the tube, and the 1.5 ml sample was centrifuged at 12000

× g at 4°C for 15min. Samples of the resulting supernatant were added to 96-well microlitre

plates in triplicate at a volume of 30 μl. For the MPO assay, 200 μl of a mixture containing

100 μl of 80 mM PBS pH 5.4, 85 μl of 0.22 M PBS pH 5.4 and 15 μl of 0.017% hydrogen

peroxide were added to the wells. The reaction was started by the addition of 20 μl of 10 mM

O-dianisidine dihydrochloride in 80 mM PBS (pH 5.4). The plates were incubated at 37°C for

3 min and then placed on ice. The reaction was stopped by the addition of 30 μl of 1.46 M

sodium acetate, pH 3.0. Enzyme activity was determined colorimetrically using a late reader

set to measure absorbance at 460 nm and is expressed as OD mg per tissue.

Caspase-1 assay. HMC-1 cells (3 × 106) were treated with IPT (1 and 10 μg/ml) for 1 h

prior to stimulation with PMACI incubated for 2 h. The caspase-1 assay used nasal mucosa

tissue and cell extracts. Caspase-1 activity was measured according to the manufacturer’s

specifications using a caspase assay kit (R & D system). Equal amounts of the total protein

were quantified by a BCA protein quantification kit (Sigma) in each lysate. Catalytic activity

of caspase-1 from the cell lysate was measured by the proteolytic cleavage of WEHD-pNA or

YVAD-pNA (Biovision, Inc. California, USA) for 4 h or various times at 37°C. The plates


at ASPE



Dow

nloaded from


JPET #184275

12

were read at 405 nm.

Transient transfection and luciferase assay. For the transfection, we seeded the HMC-1

cells (1 × 107) in a 100 mm culture dish. We then used Lipofectamine™ 2000 (Invitrogen,

Carlsbad, CA, USA) to transiently transfect reporter gene constructs into HMC-1 cells.

HMC-1 cells (3 × 106) were treated with IPT (1 and 10 μg/ml) for 1 h prior to stimulation

with PMACI incubated for 24 h. We mixed 20 μl of cell extract and 100 μl of the luciferase

assay reagent at room temperature. To measure the luciferase activity, we used a luminometer

(1420 luminescence counter, Perkin Elmer) in accordance with the manufacturer's protocol.

The transfection experiments were performed in at least three different experiments, all with

similar results. The relative luciferase activity was defined as the ratio of firefly luciferase

activity to renilla luciferase activity.

IKK-β assay. An IKK-β kinetic assay was performed according to the manufacturer’s

specifications using an IKK-β activity assay kit (EMD chemical, Gibbstown, NJ, USA).

Preparation of mouse bone marrow-derived mast cells (BMMCs). BMMCs were

generated from the femoral bone marrow cells of mice as described previously. Cells were

incubated in RPMI 1640 supplemented with 10% heat-inactivated FBS, 100 U/ml penicillin,

100 µg/ml streptomycin, 100 μM 2-mercaptoethanol, 10 mM sodium pyruvate, 10 μM MEM

nonessential amino acid solution (Invitrogen), 100 U/m murine IL-3 (R&D Systems), and 0.5

U/ml murine stem cell factor (SCF; R&D Systems) at 37°C in a humidified atmosphere in the

presence of 5% CO2. After 4 weeks of being cultured, more than 96% of the cells were

identifiable as mast cells as determined by toluidine blue staining. BMMCs were treated with

anti-DNP IgE (10 μg/ml) and HSA (50 ng/ml).

Statistical analysis. The experiments shown are a summary of the data from at least three

experiments and statistical analyses were performed using SPSS statistical software (SPSS


at ASPE



Dow

nloaded from


JPET #184275

13

11.5, USA). Treatment effects were analyzed by one-way ANOVA, offered by Tukey’s

multiple range tests, and P < 0.05 was used to indicate significance.


at ASPE



Dow

nloaded from


JPET #184275

14

Results

Effects of IPT on clinical symptoms and histamine, IgE, IL-4, IFN-γ, and IL-1β levels

in the AR model. To investigate the inhibitory effects of IPT in the AR model, we sensitized

mice on days 1, 5, and 14 by i.p. injections of 100 μg OVA emulsified in 20 mg aluminum

hydroxide and the challenged mice with 1.5 mg OVA. DEX (5 mg/kg) used as a positive

control. The numbers of nasal and ear rubs after the OVA challenge in the OVA-sensitized

mice were significantly higher than those in the OVA-unsensitized mice. Increased rub scores

were inhibited by treatment with IPT (Fig. 2A). The spleen weights after the OVA challenge

in the OVA-sensitized mice were significantly higher than those in the OVA-unsensitized

mice. Increased spleen weights were reduced by IPT administration (Fig. 2B). Histamine

levels in the serum were reduced by IPT (Fig. 2C). Levels of IgE in the AR mice were

significantly higher than those in the serum, spleen, and nasal mucosa tissues of the OVA-

unsensitized mice (Fig. 2D and E). Total IgE and OVA-specific IgE levels increased by the

OVA in the serum were reduced by IPT. To identify the Th1/Th2 immune reaction in IPT-

administered mice, we measured IL-4 and IFN-γ levels in the spleen. As shown in Fig. 2F

and G, the levels of IL-4 in the AR mice significantly increased compared to those in the

normal mice. IL-4 levels significantly decreased in the IPT-administered AR mice. However,

IFN-γ levels increased by OVA were not changed in the IPT-administered AR mice. The

protein levels of IL-1β in the serum and spleen tissue were increased in the AR mice

compared to those levels in the control mice (Fig. 2H). However, protein levels of IL-1β were

significantly reduced by IPT administration. IPT did not affect various allergic factors by

itself.

Effects of IPT on IL-1β, MIP-2, ICAM-1, and COX-2 levels in the nasal mucosa tissue

of the AR model. To evaluate the regulatory effect of IPT on IL-1β expression, we measured


at ASPE



Dow

nloaded from


JPET #184275

15

the protein and mRNA levels of IL-1β in the AR model. The protein and mRNA levels of IL-

1β in the nasal mucosa tissue were increased in the AR mice compared to those levels in the

control mice (Fig. 3A and B). However, protein and mRNA levels of IL-1β were reduced by

IPT administration. Levels of MIP-2, ICAM-1, and COX-2 in the AR mice were significantly

higher than those in the nasal mucosa tissues of the OVA-unsensitized mice (Fig. 3C-F).

Increased levels of MIP-2, ICAM-1, and COX-2 were inhibited by IPT (Fig. 3C-F).

Effects of IPT on inflammation of nasal mucosa tissue. The respective numbers of

inflammatory cells (eosinophil, mast cell, neutrophils, macrophages, and T cells) in the nasal

mucosa in the AR mice were significantly higher than those in the control mice. In the IPT-

administered mice, the increase in eosinophil, mast cells, macrophages, and T cells

infiltration (MPO activation) by OVA sensitization was decreased. But IPT did not affect

neutrophil infiltration (Fig. 4B). Immunohistochemical analysis of the nasal mucosa sections

in the AR mice revealed that IL-1β is highly expressed; whereas in the IPT-administered mice,

it is decreased (Fig. 4A).

Effects of IPT on caspase-1 activation in the nasal mucosa tissues. Caspase-1 plays a

key role in inflammatory responses by cleaving pro-IL-1β into secreted pro-inflammatory

cytokines. To investigate the effect of IPT on caspase-1 activation, a caspase-1 assay was

performed with the nasal mucosa tissues. As shown in Fig. 5A, IPT (1 mg/kg) and DEX (5

mg/kg) inhibited OVA-induced caspase-1 activation. IPT also reduced the expression of

caspase-1 in the nasal mucosa tissue (Fig. 5B and C).

Effects of IPT on PMACI-induced IL-1β expression and histamine release in HMC-1

cells. Mast cells play a major role in the inflammatory reaction of AR (Pawankar et al., 2007).

Since IL-1β is a major cytokine released from mast cells after allergic responses, we

examined the effect of IPT on the expression of IL-1β in human mast cells. The protein and


at ASPE



Dow

nloaded from


JPET #184275

16

mRNA levels of IL-1β were significantly inhibited by IPT (Fig. 6A and B). Also, the

inhibitory effects of IPT on PMACI-induced histamine release from HMC-1 cells are shown

in Fig. 6C. IPT did not affect IL-1β and histamine by itself. We examined cell viability using

a MTT assay and found that IPT had no effect on cell viability (Fig. 6D).

Effects of IPT on PMACI-induced NF-κB activation in HMC-1 cells. To assess the

regulatory mechanism of IPT on allergic inflammation in the in vitro model, we examined the

effect of IPT on PMACI-induced NF-κB activation, which is known to be important for

cytokine expression in HMC-1 cells. Because the suppression of NF-κB is linked with anti-

inflammation, we postulated that IPT mediates its effects at least partly through the

suppression of NF-κB activation. The pretreatment with IPT (1 and 10 μg/ml) inhibited the

PMACI-induced NF-κB/Rel A levels in the nuclear extract (Fig. 7A). As a marker of NF-κB

activation, the degradation of IκB-α in cell lysates was detected. We showed that IPT (1 and

10 μg/ml) inhibited the PMACI-induced IκB-α degradation (Fig. 7A). Histone and actin

expression levels were not changed by any treatment in the nuclear and cytoplasmic extracts.

We next examined whether IPT could modulate the luciferase expression specifically via the

NF-κB. The NF-κB luciferase reporter gene constructs (pNF-κB-LUC, plasmid containing

NF-κB binding site; STRATAGENE, La Jolla, CA, USA) were transiently transfected into

HMC-1 cells, which was treated by IPT and then stimulated by PMACI. As shown in Fig. 7B,

PMACI increased reporter gene activity. However, the increased activity was decreased by

the treatment of IPT (P < 0.05). IPT did not affect NF-κB activation by itself.

Effects of IPT on PMACI-induced RIP2/IKK-β/caspase-1 activation in HMC-1 cells.

RIP2 is a CARD-containing kinase that interacts with caspase-1 and plays an important role

in NF-κB activation (Kelsall, 2005). Apoptosis-associated speck-like protein containing a

CARD (ASC) is a PYRIN and CARD-containing molecule, important in the induction of


at ASPE



Dow

nloaded from


JPET #184275

17

apoptosis and caspase-1 activation (Sarkar et al., 2006). Expressions of RIP2 and IKK-β were

inhibited by IPT in HMC-1 cells (Fig. 8A). In the IKK-β assay, IKK-β activity was also

inhibited by IPT (Fig. 8B). To evaluate whether IPT regulated the caspase-1 activation, we

performed a Western blot analysis and caspase-1 assay. As shown in Fig. 8C and D, caspase-1

activity increased by PMACI was inhibited by IPT. A caspase-1 kinetic assay was used to

evaluate the binding affinity of IPT for the caspase-1 catalytic domain. Additionally,

recombinant caspase-1 (Biovision, Inc. California, USA) was also used to confirm the effect

of IPT in the kinetic assay. As shown in Fig. 8E, caspase-1 activity was increased after a

treatment with YVAD-pNA (caspase-1 substrate), but the IPT or caspase-1 inhibitor inhibited

the binding of YVAD-pNA with the caspase-1 catalytic domain. Interestingly, IPT (10 μg/ml)

exhibited a more potent binding affinity than did the caspase-1 inhibitor. In addition, caspase-

1 activity and IL-1β production increased by anti-DNP IgE and HSA were inhibited by IPT in

BMMCs (Fig. 8F and G). IPT did not affect the expression of RIP2, activation of IKK-β and

caspase-1, and production of IL-1β by itself.


at ASPE



Dow

nloaded from


JPET #184275

18

Discussion

In this study, IPT reduced the allergic inflammatory reaction in the AR model. AR is

characterized by a two-phase allergic reaction. In the early-phase inflammatory response

allergen-IgE dependent activation of mast cells and basophils results in the production of

pharmacologically active mediators such as histamine, prostaglandins, leukotrienes, and

cytokines which produce sneezing, rhinorrhea, and itching (Jeong et al., 2009). The late-

phase of AR show accumulations of mast cells, eosinophils, and basophils in the epithelium

and an accumulation of eosinophils in the deeper lamina propria (Fuentes-Beltrán et al, 2009).

Recruitment of inflammatory cells, including eosinophils, mast cells, basophils, and T cells,

results in the further release of histamine and leukotrienes, as well as in the release of other

compounds including proinflammatory cytokines, COX-2, and chemokines, which sustain the

allergic response and promote the late phase response (Fuentes-Beltrán et al., 2009; Fukui et

al., 2009). In previous studies, polyphenolic phytochemicals including rosmarinic acid have

been shown to inhibit the IgE response (Makino et al., 2001) and inflammation characterized

by polymorphonuclear leukocytes (eosinophils, and neutrophils) infiltration (Osakabe et al.,

2002). Glucocorticosteroid (GC) is the most effective drug for AR. GC inhibits the function

of infiltrating inflammatory cells and their recruitment into the nasal mucosa. GC inhibits the

maturation, cytokine production, COX-2 expression, FcεRI expression, and mediator release

of mast cells (Takano et al., 2004; Smith et al., 2002). We observed that IPT inhibited the IgE

production, inflammatory cytokine production, chemokine production, and COX-2

expression in the mice AR model. DEX also reduced the allergic and inflammatory reaction.

Therefore, our results suggest that the effect of IPT is similar to the mechanism of rosmarinic

acid or GC. We can also deduce that IPT has an anti-allergic effect.

Inflammasomes are multiprotein cytoplasmic complexes that mediate the activation of


at ASPE



Dow

nloaded from


JPET #184275

19

inflammatory caspase-1 (Stutz et al., 2009). Inflammasome regulates the activation and

secretion of caspase-1-regulated IL-1β and IL-18. Caspase-1–/– mice show the decreased

production of IL-6 after stimulation with lipopolysaccharide (Martinon, 2005). Grzegorczyk

et al., have reported a significant increase in caspase-1 level in serum from allergic asthmatic

patients as compared to a control group (Grzegorczyk et al., 2002). Caspase-1 activity was

increased in patients with the Netherton syndrome (Hosomi et al., 2008). In this study, we

observed that caspase-1 was activated in the AR mice. IPT inhibited caspase-1 activity and

IL-1β production. Therefore, we postulate that the inhibitory effect of IPT on inflammatory

cytokine production might be derived from the regulation of caspase-1 activation.

Mast cells arise from pluripotential stem cells and mature in the tissue. They have the

ability to generate immune reactions following exposure to a variety of receptor-mediated

signals initiated by both innate and acquired immune response mechanisms. Activated mast

cells release a broad spectrum of mediators including cytokines such as IL-1β (Johansson et

al., 2001; Howarth, 2003). Suppression of NF-κB activation has been linked with the

inhibition of inflammatory cytokine (IL-1β, IL-6, and TNF-α) expression (Inohara et al.,

2000). Activation by RIP2 induces caspase-1 oligomerization and promotes caspase-1

activation, with the latter inducing cytokine stimulation (Inohara et al., 2000). RIP2 and IKK

complexes may play an important role for NF-κB activation (Inohara et al., 2000). In this

study, IPT inhibited the IL-1β expression and production. We also confirmed that IPT

suppressed the RIP2/IKK-β/caspase-1 activation in mast cells. This result suggested that the

inhibitory effect of IPT on the allergic inflammatory reaction might be derived through the

regulation of RIP2/IKK-β/caspase-1 activation.

IPT has not been elucidated for the effect and mechanism on inflammatory reactions. We

first time observed that IPT can regulate the reduction of inflammatory cytokine expression


at ASPE



Dow

nloaded from


JPET #184275

20

and the inhibition of caspase-1 activation, which causes the symptoms of AR, nasal itching

and eye's mucous membrane inflammation were found to alleviate symptoms. Therefore, the

author sees the potential as the drugs of IPT have on inhibitory action on AR.


at ASPE



Dow

nloaded from


JPET #184275

21

Authorship Contributions

Participated in research design: Kim HM and Jeong HJ.

Conducted experiments: Oh HA.

Performed data analysis: Jeong HJ and Oh HA.

Wrote or contributed to the writing of the manuscript: Kim HM, Jeong HJ, and Oh HA.

The authors have no conflicting financial interests.

`


at ASPE



Dow

nloaded from


JPET #184275

22

References

Adebajo AC, Iwalewa EO, Obuotor EM, Ibikunle GF, Omisore NO, Adewunmi CO,

Obaparusi OO, Klaes M, Adetogun GE, Schmidt TJ, Verspohl EJ (2009) Pharmacological

properties of the extract and some isolated compounds of Clausena lansium stem bark:

anti-trichomonal, antidiabetic, anti-inflammatory, hepatoprotective and antioxidant effects.

J Ethnopharmacol 122:10-19.

Bach JF (2002) The effect of infections on susceptibility to autoimmune and allergic diseases.

N Engl J Med 347:911-920.

Bain G, Lorrain DS, Stebbins K, Broadhead A, Santini A, Prodanovich P, Darlington J, King

C, Lee C, Baccei C, Stearns B, Truong Y, Hutchinson J, Prasit P, Evans J (2011)

Pharmacology of AM211, a potent and selective DP2 receptor antagonist that is active in

animal models of allergic inflammation. J Pharmacol Exp Ther. Epub ahead of print.

Bournazou I, Mackenzie KJ, Duffin R, Rossi AG, and Gregory CD (2010) Inhibition of

eosinophil migration by lactoferrin. Immunol Cell Biol 88:220-223.

Bousquet J, Khaltaev N, Cruz AA, Denburg J, Fokkens WJ, Togias A, Zuberbier T, Baena-

Cagnani CE, Canonica GW, van Weel C, et al. (2008) World Health Organization; GA (2)

LEN; Allergen. Allergic Rhinitis and its Impact on Asthma (ARIA) 2008 update (in

collaboration with the World Health Organization, GA (2) LEN and Allergen). Allergy

86:8-160.

Chen J, Chiou WF, Chen CC, and Chen CF (2000) Effect of the plant-extract osthole on the

relaxation of rabbit corpus cavernosum tissue in vitro. J Urol 163:1975-1980.

Fuentes-Beltrán A, Montes-Vizuet R, Valencia-Maqueda E, Negrete-García MC, García-Cruz

Mde L, and Teran LM (2009) Chemokine CC-ligand 5 production and eosinophil

activation into the upper airways of aspirin-sensitive patients. Clin Exp Allergy 39:491-499.


at ASPE



Dow

nloaded from


JPET #184275

23

Fukui N, Honda K, Ito E, and Ishikawa K (2009) Peroxisome proliferator-activated receptor

gamma negatively regulates allergic rhinitis in mice. Allergol Int 58:247-253.

Grzegorczyk J, Kowalski ML, Pilat A, and Iwaszkiewicz J (2002) Increased apoptosis of

peripheral blood mononuclear cells in patients with perennial allergic asthma/rhinitis:

relation to serum markers of apoptosis. Mediators Inflamm 11:225-233.

Gupta S, Feng L, Yoshimura T, Redick J, Fu SM, and Rose CE (1996) Intra-alveolar

macrophage-inflammation peptide 2 induces rapid neutrophil localization in the lung. Am J

Respir Cell Mol Biol 15:656-663.

He JY, Zhang W, He LC, and Cao YX (2007) Imperatorin induces vasodilatation possibly via

inhibiting voltage dependent calcium channel and receptor-mediated Ca2+ influx and

release. Eur J Pharmacol 573:170-175.

Hosomi N, Fukai K, Nakanishi T, Funaki S, and Ishii M (2008) Caspase-1 activity of stratum

corneum and serum interleukin-18 level are increased in patients with Netherton syndrome.

Br J Dermatol 159:744-746.

Howarth PH (2003) Allergic and nonallergic rhinitis. In: Middleton E, Reed C, Ellis E,

Adkinson N, Yunginger J, Busse W et al. Allergy, Principles and Practice, vol. 2, Allergy,

Principles and Practice. 6th edn. Philadelphia: Mosby 1391-410.

Inohara N, Koseki T, Lin J, del Peso L, Lucas PC, Chen FF, Ogura Y, and Núñez G (2000) An

induced proximity model for NF-kappa B activation in the Nod1/RICK and RIP signaling

pathways. J Biol Chem 275:27823-27831.

Jeong HJ, Moon PD, Kim SJ, Seo JU, Kang TH, Kim JJ, Kang IC, Um JY, Kim HM, Hong

SH (2009) Activation of hypoxia-inducible factor-1 regulates human histidine

decarboxylase expression. Cell Mol Life Sci 66:1309-1319.


at ASPE



Dow

nloaded from


JPET #184275

24

Johansson SG, Hiurihane JO, Bousquet J, Bruijnzeel-Koomen C, Dreborg S, Haahtela T,

Kowalski ML, Mygind N, Ring J, van Cauwenberge P et al. (2001) A revised nomenclature

for allergy: an EAACI position statement from the EAACI nomenclature task force.

Allergy 56:813-824.

Kobayashi K, Inohara N, Hernandez LD, Galán JE, Núñez G, Janeway CA, Medzhitov R,

Flavell RA (2002) RICK/Rip2/CARDIAK mediates signaling for receptors of the innate

and adaptive immune systems. Nature 416:194-199.

Kuo PL, Hsu YL, Chang CH, and Chang JK (2005) Osthole-mediated cell differentiation

through bone morphogenetic protein-2/p38 and extracellular signal-regulated kinase 1/2

pathway in human osteoblast cells. J Pharmacol Exp Ther 314:1290-1299.

Liang HJ, Suk FM., Wang CK, Hung LF, Liu DZ, Chen NQ, Chen YC, Chang CC, and Liang

YC (2009) Osthole, a potential anti-diabetic agent, alleviates hyperglycemia in db/db mice.

Chem Biol Interact 181:309-315.

Lin TY, Lu CW, Huang WJ, and Wang SJ (2010) Osthole or imperatorin-mediated facilitation

of glutamate release is associated with a synaptic vesicle mobilization in rat ippocampal

glutamatergic nerve endings. Synapse 64:390-396.

Luszczki JJ, Wojda E, Andres-MachM, Cisowski W, Glensk M, Glowniak K, and Czuczwar

SJ (2009) Anticonvulsant and acute neurotoxic effects of imperatorin, osthole and

valproate in the maximal electroshock seizure and chimney tests in mice: a comparative

study. Epilepsy Res 85:293-299.

Makino T, Furuta A, Fujii H, Nakagawa T, Wakushima H, Saito K, and Kano Y (2001) Effect

of oral treatment of Perilla frutescens and its constituents on type-I allergy in mice. Biol

Pharm Bull 24:1206-1209.


at ASPE



Dow

nloaded from


JPET #184275

25

Martinon F (2005) NLRs join TLRs as innate sensors of pathogens. Trends Immunol 26:447-

454.

Montuschi P, Sala A, Dahlen SE, and Folco G (2007) Pharmacological modulation of the

leukotriene pathway in allergic airway disease. Drug Discov Today 12:404-412.

Montoro J, Sastre J, Jáuregui I, Bartra J, Dávila I, A del Cuvillo, Ferrer M, Mullol J, and

Valero A (2007) Allergic rhinitis: Continuous or on demand antihistamine therapy? J

Investig Allergol Clin Immunol 17:21-27.

Mosmann TR, Cherwinski H, Bond MW, Giedin MA, and Coffman RL (1986) Two types of

murine helper T cell clone. I. Definition according to profiles of lymphokine activities and

secreted proteins. J Immunol 136:2348-2357.

Ogawa H, Sasai N, Kamisako T, and Baba K (2007) Effects of osthol on blood pressure and

lipid metabolism in stroke-prone spontaneously hypertensive rats. J Ethnopharmacol

112:26-31.

Osakabe N, Yasuda A, Natsume M, Sanbongi C, Kato Y, Osawa T, and Yoshikawa T (2002)

Rosmarinic acid, a major polyphenolic component of Perilla frutescens, reduces

lipopolysaccharide (LPS)-induced liver injury in D-galactosamine (D-GalN)-sensitized

mice. Free Radic Biol Med 33:798-806.

Pawankar R, Lee KH, Nonaka M, and Takizawa R (2007) Role of mast cells and basophils in

chronic rhinosinusitis. Clin Allergy Immunol 20:93-101.

Sarkar A, Duncan M, Hart J, Hertlein E, Guttridge DC, and Wewers MD (2006) ASC directs

NF-κB activation by regulating receptor interaction protein-2 (RIP2) caspase-1 interactions.

J immunol 176:4979-4986.


at ASPE



Dow

nloaded from


JPET #184275

26

Smith SJ, Piliponsky AM, Rosenhead F, Elchalal U, Nagler A, and Levi-Schaffer F (2002)

DEX inhibits maturation, cytokine production and Fc epsilon RI expression of human cord

blood-derived mast cells. Clin Exp Allergy 32:906-913.

Stutz A, Golenbock DT, and Latz E Inflammasomes: too big to miss. J Clin Invest 119:3502-

3511.

Takano H, Osakabe N, Sanbongi C, Yanagisawa R, Inoue K, Yasuda A, Natsume M, Baba S,

Ichiishi E, and Yoshikawa T (2004) Extract of Perilla frutescens enriched for rosmarinic

acid, a polyphenolic phytochemical, inhibits seasonal allergic rhinoconjunctivitis in

humans. Exp Biol Med 229:247-254.

Whitcup SM (2006) Recent advances in ocular therapeutics. Int Ophthalmol Clin 46:1-6.


at ASPE



Dow

nloaded from


JPET #184275

27

Footnotes

This research was supported by Basic Science Research Program through the National

Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and

Technology [2009-0090401].

*Kim HM and Jeong HJ contributed equally to this work.


at ASPE



Dow

nloaded from


JPET #184275

28

Legends for figure

Fig. 1. Structure of IPT (A) and Schematic protocols of AR animal model (B).

Fig. 2. Effects of IPT on clinical symptoms, spleen weight, and histamine, IgE, IL-4, IFN-γ,

and IL-1β levels in the AR model. We sensitized mice on days 1, 5, and 14 by intraperitoneal

injections of 100 μg OVA emulsified in 20 mg of aluminum hydroxide and we challenged

mice with 1.5 mg OVA. Mice received IPT before the intranasal OVA challenge for 10 days.

(A) The number of the nasal and ear rubs that occurred in the 10 min after the OVA intranasal

provocation. (B) Spleen weight, (C) Serum was isolated from blood and then assayed about

histamine. (D and E) IgE or OVA-specific IgE, (F) IL-4, (G) IFN-γ, and (H) IL-1β were

measured by the ELISA method. All parameters measured in the tissue homogenate were

presented as a ratio to the total protein levels in the tissue. .#P < 0.05; significantly different

from the OVA-unsensitized mice. *P < 0.05; significantly different from the OVA-sensitized

mice. N=5. DEX, dexamethasone.

Fig. 3. Effects of IPT on IL-1β, ΜIP-2, ICAM-1, and COX-2 expression in the nasal mucosa

of the AR mice. We sensitized mice on days 1, 5, and 14 by intraperitoneal injections of 100

μg OVA emulsified in 20 mg of aluminum hydroxide and we challenged the mice with 1.5

mg OVA. The mice received IPT for 10 days before the intranasal OVA challenge. (A) IL-1β,

(C) MIP-2, and (D) ICAM-1 were measured by the ELISA method in the nasal mucosa tissue.

(B) Messenger RNA was measured using the RT-PCR method. (E) COX-2 protein expression

was evaluated by using Western blot analysis. (F) The protein levels of COX-2 were


at ASPE



Dow

nloaded from


JPET #184275

29

quantified by densitometry. #P < 0.05; significantly different from the OVA-unsensitized mice.

*P < 0.05; significantly different from the OVA-sensitized mice. N=5. DEX, dexamethasone.

Fig. 4. Effects of IPT on eosinophil and mast cell infiltration, and IL-1β expression in the AR

nasal mucosa tissue. (A) Nasal mucosa stained with H&E (for eosinophils = arrow head),

A&S (for mast cells = black arrow) and immunohistochemical DAB stain (for IL-1β = red

arrow). (B) Eosinophil, mast cells, neutrophil, macrophages, and T cells (MPO activity) were

counted by two individuals. After five randomly selected tissue sections per mouse were

counted. The absolute number of cells was counted as the mean ± standard error of the mean

(S.E.M.). #P < 0.05; significantly different from the OVA-unsensitized mice. *P < 0.05;

significantly different from the OVA-sensitized mice. DEX, dexamethasone. (Original

magnification × 400, scale bar=100 μm).

Fig. 5. Effects of IPT on caspase-1 activation in the nasal mucosa of the AR mice. We

sensitized mice on days 1, 5, and 14 by intraperitoneal injections of 100 μg OVA emulsified

in 20 mg of aluminum hydroxide and we challenged the mice with 1.5 mg OVA. Mice

received IPT for 10 days before the intranasal OVA challenge. (A) Protein was assayed about

caspse-1. (B) Caspase-1 protein expression was evaluated by using Western blot analysis. (C)

The protein levels were quantified by densitometry. Results are representative of three

independent experiments. #P < 0.05; significantly different from the OVA-unsensitized mice.

*P < 0.05; significantly different from the OVA-sensitized mice. N=5. DEX, dexamethasone.

Fig. 6. Effects of IPT on PMACI-induced IL-1β expression and histamine release in HMC-1

cells. (A) HMC-1 cells were treated with IPT (1 and10 μg/ml) for 1 h and then stimulated


at ASPE



Dow

nloaded from


JPET #184275

30

with PMACI for 8 h. IL-1β was measured by the ELISA method. (B) Messenger RNA was

measured using the RT-PCR method. (C) Secreted histamine was assayed by a histamine

assay. (D) Cell viability was evaluated by a MTT assay. #P < 0.05; significantly different

from the unstimulated cells. *P < 0.05; significantly different from the PMACI-stimulated

cells. M, marker.

Fig. 7. Effects of IPT on PMACI-induced NF-κB activation in HMC-1 cells. (A) HMC-1

cells were treated with IPT (1 and 10 μg/ml) for 1 h and then stimulated with PMACI for 2 h.

Nuclear protein and cytoplasmic protein were prepared and analyzed for NF-κB and IκB-

α by Western blotting as described in the experimental procedures. Results are

representative of three independent experiments. (B) The NF-κB activity was assayed by a

luciferase assay. Values are the mean ± S.E.M. of duplicate determinations from three

separate experiments. #P < 0.05: significantly different from the unstimulated cells. * P <

0.05: significantly different from the PMACI-stimulated cells.

Fig. 8. Effects of IPT on PMACI-induced RIP2/IKK-β/caspase-1 activation in HMC-1 cells.

HMC-1 cells were pretreated with IPT for 1 h prior to PMACI stimulation for 2 hr. (A) RIP2

and IKK-β protein expressions were evaluated by using Western blot analysis. (B) An IKK-

β assay was performed. (C) The levels of caspase-1 were assayed by Western blot analysis.

(D) The enzymatic activity of caspase-1 was tested by a caspase-1 colorimetric assay. (E)

Catalytic activity of recombinant caspase-1 (8 units) was measured by YVAD-pNA

(caspase-1 substrate), IPT (1 and 10 μg/ml), or caspase-1 inhibitor (10 µM) for various

times. (F) The effect of IPT (10 μg/ml) on caspase-1 activity in IgE-stimulated BMMCs. (G)

The effect of IPT (10 μg/ml) on IL-1β production in IgE-stimulated BMMCs. Data represent


at ASPE



Dow

nloaded from


JPET #184275

31

the mean ± S.E.M. of the three independent experiments. #P < 0.05: significantly different

from the unstimulated cells. * P < 0.05: significantly different from the stimulated cells.


at ASPE



Dow

nloaded from



at ASPE



Dow

nloaded from



at ASPE



Dow

nloaded from



at ASPE



Dow

nloaded from



at ASPE



Dow

nloaded from



at ASPE



Dow

nloaded from


This article has not been copyedited and form

atted. The final version m

ay differ from this version.

JPET

Fast Forward. Published on July 5, 2011 as D

OI: 10.1124/jpet.111.184275

at ASPET Journals on September 9, 2018 jpet.aspetjournals.org Downloaded from



at ASPE



Dow

nloaded from



at ASPE



Dow

nloaded from


Documents

Distinct Effects of Imperatorin on Allergic Rhinitis ...jpet.aspetjournals.org/content/jpet/early/2011/07/05/jpet.111... · Caspase-1 is characterized by its ability to activate the