Background
Allergic rhinitis (AR) is accepted to be an IgE-mediated inflammatory disease in the nasal mucosa and characterized by intense infiltration and activation of inflammatory cells such as mast cells, and eosinophils, among others [
1]. Although AR is not life-threatening disease, it can deteriorate the quality of life and an economic burden through the clinical symptoms such as sneezing, rhinorrhea, itching and nasal congestion [
1,
2]. These clinical conditions are well known to be primarily induced by chemical mediators such as histamine and leukotrienes, which are secreted by mast cells, basophils and eosinophils [
1]. In addition to the secretion of chemical mediators, these cells also release several types of cytokines and chemokines, which are responsible for the amplification and the persistence of allergic inflammatory responses in nasal mucosa [
1]. On the other hand, there is much evidence that nasal mucosa is innervated by sensory, sympathetic and parasympathetic nerves. After stimulation with aeroallergen, sensory nerves transmit signals generating sensations such as itching and motor reflexes, including sneezing [
3‐
6]. Antigenic stimulation on sensory nerves also causes axonal reflex to produce neuropeptides, substance P (SP) and calcitonin gene-related peptides (CGRP), which are responsible for vasodilation, edema and activation of inflammatory cells in nasal mucosa [
3‐
6].
Quercetin is one of the well-characterized flavonoids and found in onion, red wine and mulberry, among others [
7]. It is reported that quercetin exerts many beneficial activities on human health, including anti-oxidative, anti-diabetic and anti-cancer activities [
8‐
10]. In regard to allergic diseases, quercetin is reported to attenuate clinical conditions of allergic diseases such as AR through its suppressive effects on the release of inflammatory cytokines and chemical mediators from mast cells and eosinophils after immunological stimulation [
11,
12]. Therapeutic potential of quercetin on allergic airway diseases is also observed in animal experimental models of asthma, in which oral administration of quercetin showed the inhibitory action on bronchial hyper-reactivity to specific allergen [
13,
14]. It is also reported that quercetin could effectively block the development of anaphylactic responses against peanuts in the experimental mouse model and in vitro cell lines [
15,
16]. Although these reports strongly suggest that quercetin will be a good candidate as a potential drug to allergic diseases, the influence of quercetin on neuropeptide productions is poorly understood. In the present study, therefore, we examined the influence of quercetin on neuropeptide production by using AR model rat.
Methods
Animals
Specific pathogen free, male Sprague-Dawley (SD) rats, 5 weeks of age were purchased from CLEA JAPAN Co., Ltd. (Tokyo, Japan). These animals were maintained in our animal facilities at 25 ± 2 °C with 55 ± 10 % humidity under a 12-h light/dark cycle and were allowed free access to tap water and standard laboratory rodent chow (Oriental Yeast Co., Ltd., Tokyo, Japan) throughout the experiments. Each control and each experimental group consisted of five rats. All animal experiments were approved by the Ethics Committee for Animal Experiments of Showa University (Approved No. 05112).
Sensitization and challenge procedures
SD rats were sensitized with toluene 2,4-diisocyanate (TDI) according to the method described previously [
17]. Briefly, 5 μl of a 10 % TDI (Wako Pure Chemicals Co., Ltd., Osaka, Japan) solution in ethyl acetate (Wako Pure Chemicals Co., Ltd.) was instilled bilaterally into the nasal vestibule once a day for 5 consecutive days. This sensitization procedure was repeated after a 2-day interval. To induce nasal allergy-like symptoms, 5 μl of 10 % TDI solution in ethyl acetate was applied bilaterally on the nasal vestibule of sensitized rats. Control rats were treated with ethyl acetate only by the same procedure.
Treatment of rats with agents
Quercetin purchased from Sigma-Aldrich Co., Ltd. (St. Louis, MO, USA) was well mixed with 5 % tragacanth gum solution at a concentration of 7.5 mg/ml. Rats were orally administered with either 10, 20, 25 or 30 mg/kg of quercetin once a day for 2 to 7 days via a stomach tube in a volume not exceeding 1.0 ml. Olopatadine hydrochloride (OH), a second generation histamine H
1 receptor antagonist, was used for treatment as a positive control. OH for human use was purchased from Kyowa Hakko Kirin Co., Ltd. (Tokyo, Japan). This was dissolved in distilled water at a concentration of 10 mg/ml and administered orally into rats at a dose of 10 mg/kg once a day for 5 days via a stomach tube [
19]. Treatment was started 5 days after the final second sensitization with TDI.
Assay for nasal symptoms
Nasal allergy-like symptoms were assessed by counting numbers of sneezing and nasal rubbing movements for 10 min just after TDI nasal challenge. The experimental rats were placed into the plastic animal cage (35 × 20 × 30 cm) for about 10 min for acclimation. After the nasal instillation of 10 % TDI solution in ethyl acetate in a volume of 5 μl, rats were placed into the plastic cage (one animal/cage) and the number of sneezes and nasal rubbing movements for 10 min were counted [
19,
20].
Preparation of nasal lavage fluids
The rats were killed by intraperitoneal injection with 100 mg/kg sodium pentobarbital (Kyoritsu Seiyaku Co., Ltd., Tokyo, Japan) 6 h after TDI nasal challenge. The trachea was exposed and cannulated to introduce 1.0 ml phosphate buffered saline. The lavage fluid exiting the nares was collected and centrifuged at 3000 rpm for 15 min at 4 °C. After measuring IgA by ELISA (Bethyl Lab., Inc., Montgomery, TX, USA), the fluids were stored at -40 °C until used.
Assay for neuropeptides
Substance P (SP), calcitonin gene-related peptide (CGRP), and nerve growth factor (NGF) in nasal lavage fluids were examined by commercially available ELISA test kits according to the manufacturer's recommendations. SP ELISA test kits were obtained from ENZO Life Science Inc. (Farmingdale, NY, USA), CGRP from Phoenix Pharmaceuticals, Inc. (Burlingame, CA, USA) and NGF from Chemicon International Inc. (Temecula, CA, USA), and the minimum detectable levels of these ELISA test kits was 8.04 pg/ml for SP, 0.01 ng/ml for CGRP and 10.0 pg/ml for NGF.
Statistical analysis
The results are shown as the mean ± standard errors of the means (SEM) of five rats/group. Statistical analyses were performed with analysis of variance (ANOVA) followed by Bonferroni correction. A value of P < 0.05 was considered statistically significant.
Discussion
AR is a global health problem that affects patients of all ages and characterized mainly by the three cardinal symptoms such as sneezing, nasal obstruction and watery rhinorrhea [
1,
2]. These clinical symptoms are considered to be caused by the chemical mediators such as histamine, prostaglandins and leukotriens secreted from inflammatory cells especially mast cells [
1,
2]. In addition to the development of clinical symptoms, these mediators stimulate sensory nerves to release several types of neuropeptides which in turn produce itching, nasal congestion and sneezing [
3‐
6]. From these established concepts, histamine H
1 receptor antagonists are recommended as a first choice of the agents for AR treatment as well as topical corticosteroids [
2]. On the other hand, there is much evidence that oral administration of quercetin and its derivative, isoquercetin, into allergic patients could modify favorably the clinical conditions of the diseases and the therapeutic mode of quercetin may be owing, at least in part, to its suppressive effects on inflammatory cell (e.g. mast cells and eosinophils) activation [
11,
12]. However, the influence of quercetin on the production of neuropeptides, which are responsible for the development of clinical symptoms of AR, is not fully understood. The present study, therefore, was undertaken to examine the influence of quercetin on neuropeptide production using TDI-sensitized rats.
The present results clearly showed that oral administration of quercetin at more than 25 mg/kg for 5 days into TDI-sensitized rats significantly inhibited the development of nasal allergy-like symptoms such as sneezing and nasal rubbing movements, which are induced by nasal antigenic challenge. The clinical nasal allergic reaction is divided into the early (or immediate)- and the late-phase response [
2]. The early phase-allergic response is occurred within minutes of allergen exposure and characterized by sneezing, nasal itching and watery rhinorrhea, which are mainly caused by the chemical mediators secreted from mast cells and basophils [
2]. It is also reported that the chemical mediators stimulate nasal sensory neurons to cause sneezing reflax via the central nervous system [
3,
4,
6]. There is also a local effect, so called axonal reflax, in which sensory nerves release several types of neuropeptides such as SP and CGRP, leading to vasodilation and plasma exudation [
3,
4,
6]. These responses originated in sensory nerve activation are called neurogenic inflammation [
4]. Intranasal instillation of TDI into rats is well accepted to induce nasal neurogenic inflammation and used frequently for analysis the mechanisms of nasal neurogenic inflammation [
17‐
19]. Together with these reports, the present results may be interpreted that oral administration of quercetin into TDI-sensitized rats attenuates the development of nasal neurogenic inflammation and results in inhibition of the development of nasal allergy-like symptoms induced by TDI nasal provocation in rats.
The second part of experiments was undertaken to evaluate the influence of quercetin on the development of nasal neurogenic inflammation by examining the contents of neuropeptides in nasal lavage fluids. The present data clearly showed that oral administration of quercetin at more than 25 mg/kg for 5 days could significantly inhibit the appearance of neuropeptides, SP, CGRP and NGF in response to TDI nasal challenge. SP and CGRP act synergistically and increase each other mast cell degranulation to produce chemical mediators responsible for development of the early-phase allergic response [
6,
21]. These two neuropeptides also activate macrophages to produce pro-inflammatory cytokines, including interleukin (IL)-1β, IL-3 and TNF-α, which are functioned in additional stimulus to allergic inflammation [
22,
23]. Furthermore, SP increases the ability of sensory nerves and nasal epithelial cells to produce NGF [
24,
25], which contributes to amplifying the early-phase allergic response through the promotion of both survival and degranulation of mast cells [
6,
25]. NGF also activates tyrosine kinase A (TrkA) receptor which in turn initiates signaling via the phosphatidylinositol 3 kinase/phosphatidylinositol phosphate 3 (PI3K/PIP3) pathway to increase expression and sensitivity of transient receptor potential vallinoid (TRPV1) receptor, which is responsible for the production and release of SP and CGRP from sensory neurons [
6]. These reports may suggest that oral administration of quercetin at more than 25 mg/kg for 5 days into TDI-sensitized rats inhibit neuropeptide productions from sensory neurons and results in attenuation of the appearance of nasal allergy-like symptoms induced by TDI nasal provocation.
Although the present results strongly suggest that quercetin attenuates nasal allergy-like symptoms through the suppression of neuropeptide release after antigenic challenge, the precise mechanisms by which quercetin inhibits neuropeptide production from sensory nerves after antigenic challenge. It is reported that the activation of the extracellular signal-regulated kinase 1/2 (ERK1/2) pathway is essential for the production of both SP and CGRP in dorsal root ganglions and peripheral nerve fibers [
26]. The activation of ERK1/2 and nuclear factor-κB (NF-κB) pathway is also reported to enhance SP production and is responsible for the development of neurogenic inflammation [
26,
27]. Furthermore, NGF production in keratinocytes requires the activation of mitogen-activated protein kinase (MAPK), especially ERK1/2, and phosphorylation of NF-κB after stimulation with SP, CGRP and other inflammatory mediators [
28]. Neuropeptides synthetized in the neuron cell body are stored in vesicles and the vesicles are transported to the release sites such as the axon terminals [
29]. When an action potential reaches the axon terminals, Ca
2+ is influx into the axon terminals through Ca
2+ channels and increases the intracellular Ca
2+ concentrations. And then neuropeptides are released by exocytosis to the extracellular space [
28,
29]. It is reported that Ca
2+ is an essential molecule for activation of both MAPK, including ERK1/2 and NF-κB after stimulation of cells with inflammatory molecules such as cytokines and neuropeptides [
30]. Quercetin is reported to increase in intracellular Ca
2+ contents after compound 48/80 stimulation in human mast cell line in vitro [
31]. From these reports, there is possibility that oral administration of quercetin into TDI-sensitized rats inhibits changes in Ca
2+ contents in the axon terminals after TDI nasal challenge and results in suppression of neuropeptide appearance in nasal lavage fluids.
Allergic diseases such as AR, asthma and atopic dermatitis (AD) are characterized by intense infiltration and activation of Th2 T-cells, mast cells and eosinophils into the inflamed tissues. Th2 T-cells are believed to orchestrate the development and maintenance of allergic inflammatory responses through the secretion of inflammatory cytokines such as IL-3, IL-5 and thymus and activation-regulated chemokine (TARC), which are responsible for promotion of migration and activation of inflammatory cells, including mast cells and eosinophils [
1]. It is reported that quercetin at 5.0 μM significantly suppressed TARC production from human keratinocytes, HaCat cell after TNF-α stimulation in vitro [
32]. Oral administration of 2.5 mM quercetin three times a week for 3 weeks into AD experimental model mouse, Nc/Nga is also reported to decrease serum TARC levels along with attenuation of clinical symptoms [
32]. Furthermore, there is evidence that quercetin at 5.0 μM significantly suppresses eosinophil degranulation and the ability of eosinophil to produce chemokines such as eotaxin, RANTES and MIP-1β, which are implicated in development and persistence of eosinophilic inflammatory responses [
1], after immunological and no-immunological stimulations in vitro [
11,
12,
33‐
35]. In regard to mast cells functions, quercetin inhibits the release of histamine and several types of inflammatory cytokines responsible for the development of allergic responses [
8,
35‐
37]. Together with these reports and the present results, it is strongly suggest that quercetin modulates the activation of inflammatory cells and neuropeptide productions and results in improvement of clinical conditions of allergic diseases, especially AR.