Cinnamaldehyde is an effective anti-inflammatory agent for treatment of allergic rhinitis in a rat model

International Journal of Pediatric Otorhinolaryngology 84 (2016) 81–87

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Cinnamaldehyde is an effective anti-inflammatory agent for treatment of allergic rhinitis in a rat model Deniz Hancı a, Hu¨seyin Altun b, Erdem Atalay C¸etinkaya c, Nuray Bayar Muluk d,*, Betu¨l Peker Cengiz e, Cemal Cingi f a

Okmeydanı Training and Research Hospital, ENT Clinics, Istanbul, Turkey Yunus Emre Hospital, ENT Department, Uskudar, Istanbul, Turkey Antalya Ataturk State Hospital, ENT Department, Antalya, Turkey d Kırıkkale University, Medical Faculty, ENT Department, Kırıkkale, Turkey e Yunus Emre State Hospital, Pathology Clinics, Eskis¸ehir, Turkey f Eskis¸ehir Osmangazi University, Medical Faculty, ENT Department, Eskis¸ehir, Turkey b c

A R T I C L E I N F O

A B S T R A C T

Article history: Received 10 December 2015 Received in revised form 28 February 2016 Accepted 1 March 2016 Available online 7 March 2016

Objectives: The effect of cinnamaldehyde on the treatment of allergic rhinitis (AR) was investigated in rat model. Methods: Twenty-eight female Wistar albino rats were randomly divided into four groups: Group 1 (control) (C), Group 2 (AR with no treatment) (AR + NoTr), Group 3 (AR + Azelastine HCl) (AR + Aze), and Group 4 (AR + cinnamaldehyde) (AR + Cin). At day 21, AR + Aze rats were given an Azelastine HCl drop, and AR + Cin rats were given cinnamaldehyde intranasally. In all groups, allergic symptoms histopathological results were evaluated. Results: The AR + NoTr group showed the worst allergic symptoms, cilia loss and greater inflammation. In the AR + Aze and AR + Cin groups, allergic symptom scores were higher than those in the control group. However, between AR + Aze and AR + Cin groups, there were no significant differences in the allergic symptom scores Histopathological analysis revealed vascular congestion and an increase in goblet cell numbers in the AR + Cin group. However, AR + Cin rat nasal mucosa had less plasma cell infiltration compared with the AR + NoTr group. In rats from the AR + Aze group, analysis of the nasal mucosa revealed less eosinophil infiltration than that seen in the AR + NoTr group. A lower score for mast cell (MC) infiltration was observed in the nasal mucosa of rats treated with Azelastine HCl compared with cinnamaldehyde. Conclusions: In this study we observed that both Azelastine HCl and cinnamaldehyde reduced allergic symptoms in an AR rat model. Cinnamaldehyde decreased vascular congestion as well as plasma cell, eosinophil, and inflammatory cell infiltration into the lamina propria. ß 2016 Elsevier Ireland Ltd. All rights reserved.

Keywords: Allergic rhinitis (AR) Cinnamaldehyde Azelastine HCl

1. Introduction Allergic rhinitis (AR) is a major condition that occurs in individuals with asthma and is related to inflammation of the upper respiratory tract. AR is a risk factor for the development of

* Corresponding author at: Birlik Mahallesi, Zirvekent 2. Etap Sitesi, C-3 blok, No.: 62/43, 06610, C¸ankaya, Ankara, Turkey. Tel.: +90 312 4964073/535 6655718; fax: +90 312 4964073. E-mail addresses: [email protected], [email protected] (N.B. Muluk). http://dx.doi.org/10.1016/j.ijporl.2016.03.001 0165-5876/ß 2016 Elsevier Ireland Ltd. All rights reserved.

asthma, and thus children and adults with AR, especially persistent AR, should be examined for asthma [1]. When considering the role of allergies in sinus diseases, one may speculate that nasal inflammation induced by IgE-mediated mechanisms results in the development of acute and/or chronic sinus infections [2,3]. The mechanisms of allergic inflammation in the nose that predisposes an individual to the development of sinus disease are partially known [4]. Inhaled allergens may reach the sinus mucosa and initiate an allergic reaction, leading to congestion of the sinus mucosa with impaired removal of mucus. However, studies conducted in chronic sinus infection patients failed to demonstrate more severe congestion present in AR patients than in individuals without AR [4]. Allergens are not

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always responsible for severe forms of sinus infections, indicating that other factors besides allergens play a role in the etiopathogenesis of severe sinus infections [5]. AR can depreciate the quality of life by causing fatigue, headaches, cognitive impairment, and other symptoms. To maintain basal levels of inflammation in the body, the regulation of Th1 and Th2 cells is important. Dysregulation of Th responses results in the activation and accumulation of Th cells, which are causes of AR [6]. Cinnamon is a well-known spice distributed mainly in Asia, South America, and the Caribbean [7]. Cinnamon oil (usually diluted 0.5–2.5%) is more often derived from the bark than from the leaves of the tree. Its main components are cinnamaldehyde (65–80%), trans-cinnamic acid (5–10%), and eugenol (4–10%); other constituents include cinnamic alcohol, terpenes (i.e., limonene, tannins, mucilage, and the oligomer procyanidin), and traces of coumarin [8,9]. The pungent taste and smell comes from cinnamaldehyde, an antimicrobial agent [10]. Cinnamaldehyde can also be found naturally in blueberries and cranberries [11]. Other components of cinnamon and cassia include phenolic acids, proanthocyanidins [12,13], cinnamyl alcohol, terpenes, carbohydrates, coumarin, and tannins [11]. Although cassia contains much less cinnamaldehyde than cinnamon, it is much richer in coumarin [14,15]. Cinnamomum cassia is the most popular type of cinnamon. For thousands of years, cinnamomum cassia has been utilized in traditional Chinese medicine for treating various disorders including chronic gastric symptoms, circulation issues, arthritis, and the common cold [16]. Cinnamic acid is found in cinnamon oil. It can also be readily oxidized from benzaldehyde by the gentle heating of cinnamaldehyde [10]. Cinnamic acid and benzoic acid are structurally similar compounds classified as hydroxycinnamic acid and hydroxybenzoic acid, respectively [17]. The antioxidant capacity of cinnamic acids is greater than that observed in benzoic acid homologous counterparts [18]. Cinnamon bark has been reported as a successful anti-arthritic agent because of its anti-inflammatory, pain relieving, and immunoregulatory effects [19]. Its anti-inflammatory activity is attributed to procyanidin oligomers found in the bark [20]. Watersoluble cinnamon bark extract has been reported to regulate immune function in vitro and to prevent and treat inflammatory diseases [21–23]. In the present study, we investigated the efficacy of cinnamaldehyde in an AR rat model. For this study we utilized four groups of rats: (1) a healthy control group, (2) AR rats given no treatment, (3) AR rats treated with Azelastine HCl, and (4) AR rats treated with cinnamaldehyde. 2. Material and methods This study was conducted at Experimental Studies Center of Eskis¸ehir Osmangazi University. All animals were treated in compliance with the principles of the Declaration of Helsinki. 2.1. Animal subjects Twenty-eight healthy (190–220 g) female Wistar albino rats were used in this study. The experimental protocol was reviewed and approved by the Committee of Ethics of Osmangazi University, the Center of Medical and Surgical Experiments. All animal procedures were performed in accordance with the approved protocol. All rats were housed under the same conditions in a temperature- and humidity-controlled room (20  1 8C, 50  10% relative humidity) under a 14–16 h light/dark cycle. Tap water and standard feed were provided on a regular basis.

2.2. Experimental design Twenty-eight female Wistar albino rats were divided randomly into four groups (n = 7): 1. Group 1 (Control). 2. Group 2 (AR with no treatment; AR + NoTr). These rats had AR but did not receive treatment. 3. Group 3 (AR + Azelastine HCl; AR + Aze). These rats had AR and were treated with Azelastine HCl (antihistamine) for 7 days starting on day 21. 4. Group 4 (AR + cinnamaldehyde; AR + Cin), These rats had AR and were treated with cinnamaldehyde for 7 days starting on day 21.

2.3. Methods 2.3.1. Sensitization procedure for developing an AR rat model The sensitizing solution was prepared by dissolving 0.3 mg ovalbumin (OVA) (Sigma, St. Louis, MO, USA) into 1 mL saline using 30 mg aluminum hydroxide (40 mg/mL) as an adjuvant. Rats in Groups 2 (AR + NoTr), 3 (AR + Aze), and 4 (AR + Cin) were injected intraperitoneally every other day for 14 days (days 1, 3, 5, 7, 9, 11, and 13) for a total of seven injections per rat. The rats in Group 1 (Control) were given 1 mL saline plus 30 mg aluminum hydroxide intraperitoneally on these same days. On day 14 following sensitization, rats in Groups 2, 3, and 4 were treated with 25 mL 2% OVA-saline solution in the form of intranasal drops on each side of the nose, once a day for 14 days. Group 1 (control) was given intranasal drops containing saline only [24–27]. 2.3.2. Measurement of total IgE On day 28, total IgE was measured in the blood of all rats. Blood was withdrawn (1 mL) and centrifuged for 20 min at 3000 rpm. Supernatant was removed, and the sample was stored at 20 8C until further analysis. Serum IgE levels were measured using a commercially available rat IgE ELISA kit (SunReed Biotechnology Co. Ltd., China) according to the manufacturer’s instructions. Result values of this assay are expressed in KU/L. 2.3.3. Symptoms of AR Symptoms of AR including sneezing, nose rubbing, eye lacrimation, and difficulty breathing (nasal congestion) were observed and scored over a 30-min period. Sneezing was characterized by an explosive expiration immediately after a deep inhalation [28] and was scored on a scale of 0–3. Nose rubbing was characterized by external perinasal scratching with either one or both of the rat’s forelimbs [28]. Nose rubbing was scored on a scale of 0–3. Eye lacrimation was scored on the following scale: 0 (No lacrimation), 1+ (hazy eyes), 2+ (lacrimation), and 3+ (lacrimation and the onset of conjunctivitis) [29]. Nasal congestion/obstruction was evaluated on the following scale: 0 (No obstruction), 1+ (impaired inspiration), 2+ (nasal inflammation), and 3+ (severe breathing impairment) [29]. 2.3.4. Azelastine HCl treatment (Group 3) Group 3 was treated with an Azelastine HCl drop in each nostril, once a day for 7 days. Azelastine HCl treatment was given on day 21 and continued for 7 days. Intranasal Azelastine HCl treatment was administered 1 h prior to intranasal OVA application. 2.3.5. Cinnamadehyde treatment (Group 4) Group 4 was treated with 20 mL cinnamaldehyde (dissolved in distilled water (20 mg/mL)) in each nostril, twice a day for 7 days. Cinnamaldehyde treatment was given on day 21 and continued for

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Kruskal–Wallis analysis, there were statistically significant differences in IgE levels among the groups (p < 0.05). Pairwise comparisons were performed using the Mann–Whitney U test with Bonferroni adjustment, and the results revealed that the average IgE levels were significantly higher in Group 2 than in Groups 1 and 3 (padjusted < 0.0125). The allergic symptoms and histopathological changes of Groups 1–4 are listed in Table 1. Kruskal–Wallis analysis revealed statistically significant differences in allergic symptoms (sneezing, nose rubbing, eye lacrimation and nasal congestion) among the groups (p < 0.05). Pairwise comparisons were performed using the Mann–Whitney U test with Bonferroni adjustment. For sneezing, nose rubbing, and nasal congestion, Group 2 (AR + NoTr) had significantly higher scores than those observed in Groups 1 (control), 3 (AR + Aze), and 4 (AR + Cin) (padjusted < 0.0125). Furthermore, both Groups 3 (AR + Aze) and 4 (AR + Cin) had significantly higher scores for these symptoms compared with Group 1 (control) (padjusted < 0.0125). However, between Groups 3 (AR + Aze) and 4 (AR + Cin), there were no significant differences in the scores for these three symptoms (Table 2). For eye lacrimation, Group 2 (AR + NoTr) had significantly higher scores than those observed in Groups 1 (control), 3 (AR + Aze), and 4 (AR + Cin) (padjusted < 0.0125). Whereas, between Groups 3 (AR + Aze) and 4 (AR + Cin), there were no significant differences in the scores for the eye lacrimation symptom (padjusted > 0.0125) (Table 2). The differences among groups in terms of histopathology scores were analyzed using Kruskal–Wallis analysis. The following factors were assessed: vascular congestion, cilia loss, increase in goblet cell number, chondrocyte hypertrophy, and inflammatory cell infiltration (i.e., plasma cells, eosinophils, and mast cells). There was a statistically significant difference in the histopathology scores among all groups (p < 0.05). Pairwise comparisons were performed using the Mann–Whitney U test with Bonferroni correction. Specifically, the scores for vascular congestion, number of goblet cells, and plasma cell infiltration were statistically higher in Group 2 (AR + NoTr) than Groups 1 (control) and 4 (AR + Cin) (padjusted < 0.0125). Cilia loss and inflammation scores were statistically higher in Group 2 (AR + NoTr) than Group 1 (control) (padjusted < 0.0125). Chondrocyte hypertrophy was significantly higher in Group 2 (AR + NoTr) than Groups 3 (AR + Aze) and 4 (AR + Cin) (padjusted < 0.0125). Eosinophil infiltration in Group 2 (A + NoTr) was significantly higher than that observed in Groups 1

7 days. Intranasal cinnamaldehyde treatment was administered 1 h prior to intranasal OVA application. 2.3.6. Histopathology of nasal mucosa On day 28, rats were anesthetized with 1% pentobarbital sodium (50 mg/kg body weight) by intraperitoneal injection. Samples of nasal mucosa were taken [19], and 5-mm-thick sections were obtained. Tissue sections were then transferred onto adhesive slides and dried in an incubator at 37 8C overnight. The next day, samples were incubated at 60 8C for 20 min. They were deparaffinized and dehydrated by immersion in xylene twice for 10 min each. Tissue samples were removed of xylene by soaking in ascending concentrations of ethanol (70, 80, 96, and 100%). Next samples were embedded in paraffin. Tissue sections were stained with hematoxylin and eosin (H&E) and Giemsa. A minimum of 10 fields per sample were examined and evaluated for severity of changes. This was a blind study performed by a trained individual who was not aware of the treatment groups [30]. The slides were examined under light microscopy utilizing an Entella Olympus BH-2 microscope. Photos were taken with an Olympus DP-70 digital camera. In histopathological examination, the following changes were evaluated: vascular congestion, cilia loss, increase in goblet cell number, chondrocyte hypertrophy, and inflammatory cell infiltration (i.e., plasma cells, eosinophils, and mast cells). The severity of the tissue changes was evaluated using the following scale: none ( ), mild (+), moderate (++) and severe (+++) [5]. 2.4. Statistical analysis The SPSS (version 16.0) statistical package was used for statistical evaluation. Kruskal–Wallis analysis was used to analyze differences among groups. The Mann–Whitney U test with Bonferroni correction was used for pairwise comparisons to detect the specific statistically significant differences. A p-value < 0.05 was considered to indicate statistical significance. For Bonferroni corrections, a padjusted value < 0.0125 was considered statistically significant. 3. Results The average IgE levels in the blood of rats from Groups 1–4 were 1468.6, 2696.7, 1372.7 and 2192.2 KU/L, respectively. According to

Table 1 Allergic rhinitis symptoms and histopathology scores of all the groups. Group 2 (allergic rhinitis without treatment) (n = 7)

Group 1 (control) (n = 7)

Group 3 (allergic rhinitis with Azelastine HCl) (n = 7)

Group 4 (allergic rhinitis with cinnamaldehyde) (n = 7)

p*

Median

Min

Max

Median

Min

Max

Median

Min

Max

Median

Min

Max

Allergic symptoms

Sneezing Nose rubbing Eye lacrimation Nasal congestion

3.0 8.0 1.0 0.0

0.0 5.0 0.0 0.0

9.0 15.0 2.0 1.0

35.0 49.0 3.0 2.0

24.0 40.0 2.0 2.0

55.0 70.0 3.0 3.0

15.0 20.0 1.0 1.0

6.0 9.0 1.0 1.0

32.0 27.0 2.0 2.0

12.0 17.0 1.0 1.0

9.0 12.0 1.0 1.0

18.0 29.0 2.0 2.0

0.000 0.000 0.001 0.000

Histopathologic examination results

Vascular congestion Cilia loss Increase of goblet cells Inflammatory cell inflammation Plasma cell infiltration Chondrosit hypertrophy Eosinophil infiltration Mast cell infiltration

1.0 1.0 1.0 1.0

1.0 0.0 1.0 1.0

2.0 2.0 2.0 2.0

2.0 2.0 2.0 2.0

2.0 1.0 2.0 1.0

3.0 3.0 3.0 3.0

1.0 1.0 1.0 2.0

1.0 1.0 1.0 1.0

2.0 3.0 2.0 2.0

1.0 1.0 1.0 1.0

1.0 1.0 1.0 1.0

2.0 3.0 2.0 3.0

0.004 0.024 0.004 0.029

1.0 1.0 1.0 1.0

1.0 0.0 1.0 0.0

2.0 2.0 2.0 1.0

2.0 2.0 2.0 2.0

2.0 2.0 2.0 1.0

3.0 3.0 3.0 3.0

1.0 1.0 1.0 1.0

1.0 1.0 1.0 0.0

2.0 2.0 2.0 1.0

1.0 1.0 1.0 2.0

1.0 0.0 1.0 1.0

2.0 2.0 3.0 3.0

0.010 0.022 0.004 0.000

*

p value shows the results of Kruskal–Wallis variance analysis. p < 0.05 is considered statistically significant.

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Table 2 Pairwise comparisons by Mann–Whitney U test with Bonferroni adjustment. Group 1–Group2 z

padjusted

Group 1–Group3 a

z

padjusted

Group 1–Group4 a

z

padjusted

Group 2–Group3 a

z

padjusted

Group 2–Group4 a

z

padjusted

Group 3–Group4 a

padjusteda

z

Allergic symptoms

Sneezing Nose rubbing Eye lacrimation Nasal congestion

3.134 3.134 3.077 3.258

0.002 0.002 0.002 0.001

2.949 2.759 0.967 2.808

0.003 0.006 0.334 0.005

3.077 2.817 0.967 2.808

0.002 0.005 0.334 0.005

2.817 3.134 2.912 2.570

0.005 0.002 0.004 0.010

3.134 3.130 2.912 2.570

0.002 0.002 0.004 0.010

1.293 0.256 0.000 0.000

0.196 0.798 1.000 1.000

Histopathologic evaluation results

Vascular congestion Cilia loss Increase of goblet cells Inflammatory cell inflammation Plasma cell infiltration Chondrosit hypertrophy Eosinophil infiltration Mast cell infiltration

3.060 2.660 3.060 2.639

0.002 0.008 0.002 0.008

1.140 1.344 1.140 1.612

0.254 0.179 0.254 0.107

0.628 1.638 0.628 0.713

0.530 0.101 0.530 0.476

2.442 1.908 2.442 1.806

0.015 0.056 0.015 0.071

2.734 1.625 2.734 1.908

0.006 0.104 0.006 0.056

0.537 0.450 0.537 0.727

0.591 0.653 0.591 0.467

2.734 2.322 3.082 2.982

0.006 0.020 0.002 0.003

0.537 0.145 0.628 0.537

0.591 0.884 0.530 0.591

0.000 0.421 0.713 3.082

10.000 0.674 0.476 0.002

2.442 2.691 2.808 2.958

0.015 0.007 0.005 0.003

2.734 2.654 2.302 1.350

0.006 0.008 0.021 0.177

0.537 0.378 0.161 3.060

0.591 0.705 0.872 0.002

a

padjusted shows the results of Mann–Whitney U test with Bonferroni adjustment. padjusted < 0.0125 is considered statistically significant.

AR typically occurs in individuals as early as 2 years of age; however, the actual rates of prevalence in children younger than

this are unknown. AR affects 10–30% of the population, with the greatest frequency observed in children and adolescents [24]. AR is associated with reduced quality of life, sleeping disorders, emotional issues, and less productivity at the workplace. AR can occur on a scale of severity ranging from mild or moderate to more severe [31]. AR can induce additional medical complications including learning disorders and sleep-related issues (e.g., obstructive sleep apnea). Several other infections can arise from AR such as chronic and acute sinusitis, acute otitis media, serous otitis media, aggravation of the adenoidal hypertrophy, as well as asthma [31]. Children with chronic AR typically have hypernasal speech, fatigue, decreased appetite, and poor growth [32]. AR is a type I hypersensitivity allergic reaction mediated via the activation of mast cells by allergens and subsequent histamine release. Elevated levels of histamine result in increased vascular permeability, contraction of smooth muscle, increased mucus secretion, and edema. Elevated histamine levels after exposure to allergens have been observed in AR patients [33]. Cinnamtannin D-1 (CTD-1) exhibits significant immunosuppressive effects in vitro and in vivo, such as suppression of lymphocyte proliferation. This immunosuppression appears to be mediated by T cells, indicating that the immunoregulatory effect of CTD-1 is inhibition of T cell functions [16]. Cinnamon extracts (CE) inhibit histamine release and synthesis of lipid mediators during allergic reactions. In vivo studies in patients with seasonal AR demonstrated the anti-allergic properties of CE (after allergen provocation) [34]. Corren et al. [34] reported anti-allergic properties of cinnamon extract (CE) in combination with other botanical products in

Fig. 1. Normal nasal mucosa of rats in Group 1 (control) (H&E, 400).

Fig. 2. Normal nasal mucosa of rats in Group 1 (control) (Giemsa, 400).

(control) and 3 (AR + Aze) (padjusted < 0.0125). Mast cell infiltration was significantly higher in Groups 2 (A + NoTr) and 4 (AR + Cin) than in Groups 1 (control) and 3 (AR + Aze) (padjusted < 0.0125). Except mast cell infiltration, there were no significant differences for the histopathology scores of the Groups 3 (AR + Aze) and 4 (AR + Cin) (padjusted > 0.0125) (Table 2). 3.1. Histopathology results Nasal mucosa of the rats in Group 1 (control) was normal (Figs. 1 and 2). In Group 2 (AR + NoTr), we observed nasal mucosa with cilia loss (Fig. 3), goblet cell metaplasia in the epithelium, intense vascular proliferation, and increased infiltration of inflammatory cells (i.e., eosinophils, polymorphonuclear leukocytes, plasma cells, neutrophils, and lymphocytes) into the lamina propria (Figs. 3 and 4). Examination of Group 3 (AR + Aze) nasal mucosa revealed improved goblet cell metaplasia in the epithelium, decreased inflammatory cell infiltrates, and vascular proliferation in the lamina propria (Figs. 5 and 6). In Group 4 (AR + Cin), nasal mucosal showed focal goblet cell metaplasia in the epithelium, vascular proliferation, mast cells and a decrease in inflammatory cell infiltration (i.e., eosinophils, plasma cells, polymorphonuclear leukocytes, neutrophils, and lymphocytes) into the lamina propria (Figs. 7 and 8). 4. Discussion

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Fig. 3. Nasal mucosa of rats in Group 2 (allergic rhinitis with no treatment). We observed an increase in inflammatory cell infiltration (i.e., eosinophils, polymorphonuclear leukocytes, plasma cells, neutrophils, lymphocytes) in the lamina propria, and cilia loss on some epithelial cells (H&E, 400).

Fig. 6. Nasal mucosa of rats in Group 3 (allergic rhinitis with Azelastine HCl treatment). We observed improved goblet cell metaplasia and decreased inflammatory cell infiltration in the lamina propria (Giemsa, 400).

Fig. 4. Nasal mucosa of rats in Group 2 (allergic rhinitis with no treatment). We observed vascular proliferation, inflammatory cell infiltration in the lamina propria, and goblet cell metaplasia (Giemsa, 400).

Fig. 7. Nasal mucosa of rats in Group 4 (allergic rhinitis with cinnamaldehyde treatment). We observed reduced inflammatory cell infiltration (i.e., eosinophils, plasma cells, polymorphonuclear leukocytes, neutrophils, and lymphocytes) in the lamina propria (H&E, 400).

Fig. 5. Nasal mucosa of rats in Group 3 (allergic rhinitis with Azelastine HCl treatment). We observed improved goblet cell metaplasia and decreased inflammatory cell infiltration and vascular proliferation in the lamina propria (H&E, 400).

Fig. 8. Nasal mucosa of rats in Group 4 (allergic rhinitis with cinnamaldehyde treatment). We observed focal goblet cell metaplasia, vascular proliferation and mast cells (arrow) in the lamina propria (Giemsa, 400).

patients with seasonal AR [34]. It was suggested that CE acts on intracellular targets rather than on cell membrane receptors, such as the IgE receptor. CE treatment resulted in reduced activation of

mast cells (MCs) by decreasing the release of preformed b-hexosaminidase, cysLT production, chemokine and cytokine expression, as well as mast cell protease production. The

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intracellular signaling pathways ERK, JNK, p38, and Akt are involved in MC cytokine expression. These signaling pathways have also been shown to be affected by CE treatment [35]. In our study, treatment with either Azelastine HCl or cinnamaldehyde decreased nasal symptoms related to AR. Walanj et al. [36] investigated the effects of Cinnamonum zylanicum bark nasal spray in severe allergic rhinitis. AR group was given C. zylanicum bark nasal spray, 100 mg/100 mL in each nostril, twice a day for 4-weeks. The results were compared to placebo group. The efficacy outcome measure scores were obtained from Juniper rhinoconjunctivitis quality of life questionnaire (RQLQ) instrument, nasal symptom scores (NSS), total NSS (TNSS), Work Productivity and Activities Impairment – Allergy Specific (WPAI – AS) instrument. They reported that extract of C. zylanicum bark nasal spray was found to be a useful treatment in management of acute seasonal AR patients. In the AR + Cin group, the nasal mucosa exhibited lower scores for vascular congestion, goblet cell numbers, and plasma cell infiltration than those observed in the AR + NoTr group. In the AR + NoTr group, nasal mucosa showed higher scores for cilia loss and inflammatory cell inflammation compared with the control group. Chondrocyte hypertrophy was much lower in the nasal mucosa of rats from the groups treated with Azelastine HCl or cinnamaldehyde compared with the AR + NoTr group. In the Azelatine HCl-treated group, nasal mucosa had much less eosinophil infiltration compared with the AR + NoTr group. A lower score for MC infiltration was observed in the nasal mucosa of rats treated with Azelastine HCl compared with cinnamaldehyde. MCs are the main effector cells of allergic and other inflammatory reactions. To date, there are only a few anti-MC agents commercially available for therapy. Upon activation, MC exert their pro-inflammatory effects by releasing multiple inflammatory mediators including prestored histamine and proteases, as well as de novo synthesized cytokines and eicosanoids. MC is activated upon cross-linking of surface-bound IgE with antigen [37–40]. Furthermore, a subsequent challenge with the same antigen resulted in cross-linked IgE on the surface of mast cells, which resulted in histamine production [41]. MC degranulation can also lead to activation and release of other mediators (e.g., tryptase, cysteinyl leukotrienes, a-hexosaminidase, IL-4, IL-5, TNF-a, platelet-activating factor, and prostaglandins) [42]. In this study, we observed reduced MC infiltration to a greater extent in the nasal mucosa of rats treated with Azelastine HCl compared with cinnamaldehyde. Cinnamaldehyde treatment did however reduce vascular congestion, plasma cell and eosinophil infiltration, and goblet cell metaplasia in the nasal mucosa. In the Corren et al’s study [34], twenty adults with seasonal allergic rhinitis were randomizely received the combination botanical product (CBP) 2 tablets three times a day, loratadine (ClaritinTM), 10 mg once a day in the morning, or placebo. The CBP is composed of equal parts by weight Cinnamon (Cinnamomum zeylanicum) bark extract, acerola (Malphighia glabra) fruit concentrate and Spanish Needles (Bidens pilosa) leaf. During the third day of treatment, subjects underwent a nasal allergen challenge (NAC), in which nasal symptoms were assessed after each challenge dose and every 2 h for 8 h. Nasal lavage fluid was assessed for tryptase, prostaglandin D2, and leukotriene E4 concentrations and inflammatory cells. CBP prevented the increase in prostaglandin D2 release. None of the treatments significantly affected tryptase or leukotriene E4 release or inflammatory cell infiltration [34]. In our study, similarly cinnamaldehyde treatment did not reduce MC infiltration, such as Corren et al’s treatments did not affect inflammatory cell infiltration in nasal lavage fluid.

In the nasal mucosa of the AR + NoTr group, we observed a loss of cilia, goblet cell metaplasia in the epithelium, intense vascular proliferation, and increased inflammatory cell infiltration (i.e., eosinophils, polymorphonuclear leukocytes, plasma cells, neutrophils, and lymphocytes) into the lamina propria. In rats treated with Azelastine HCl, the nasal mucosa showed reduced goblet cell metaplasia, inflammatory cell infiltration, and vascular proliferation in the lamina propria. In the nasal mucosa of rats treated with cinnamaldehyde, inflammatory cell infiltration was decreased in the lamina propria, despite the presence of focal goblet cell metaplasia. In a study performed by Aswar et al. [43], the anti-allergic effects of intranasal administration of type-Aprocynidines polyphenols (TAPP) based on standardized hydroalcoholic extracts of cinnamomum zeylanicum bark (TAPP-CZ) were observed in OVAinduced AR BALB/c mice. Treatment with TAPP-CZ (10 and 30 mg/ kg in each nostril) showed a significant reduction in AR symptoms (i.e., nasal rubbing and sneezing) and decreased inflammatory mediators (i.e., serum IgE and histamine). Examination of the nasal mucosa revealed a reduction in inflammatory cells in AR mice treated with TAPP-CZ. The authors of that study concluded that TAPP-CZ can have anti-allergic effects in an AR mouse model and, thus, may be an effective drug in the treatment of AR [43]. In our study, cinnamaldehyde decreased nasal symptoms related to AR and restored basal levels of inflammation in the nasal mucosa to a greater extent than did no treatment. Azelastine and olopatadine are the only intranasal antihistamines currently available in the United States. These drugs are approved for the treatment of seasonal AR and have been shown to improve congestion, rhinorrhea, sneezing, and nasal pruritus [44,45]. Similarly, Chaaban and Corey [46] reported that intranasal antihistamines are effective for nasal congestion and some types of nonallergic rhinitis. In our study, Azelastine HCl reduced AR nasal symptoms and decreased inflammatory cell infiltration within the nasal mucosa. Hagenlocher et al. [47] analyzed the effects of cinnamon extract (CE) on human and rodent MC in vitro and in vivo. Following oral treatment with CE, cinnamon extract inhibits degranulation and de novo synthesis of inflammatory mediators in mast cells. CE also almost completely inhibited de novo synthesis of cysteinyl leukotrienes and TNFa. Cinnamon extract decreases expression of MC – specific mediators in vitro and in vivo, thus indicating its potential role as an alternative treatment for allergy-related infections. Recent studies, cinnamaldehyde is related to the transient receptor potential cation channel, member A1 (TRPA1). Proinflammatory agents trypsin and mast cell tryptase cleave and activate PAR2, which is expressed on sensory nerves to cause neurogenic inflammation. Transient receptor potential A1 (TRPA1) is an excitatory ion channel on primary sensory nerves of pain pathway [48]. Cinnamon is often added to medicines, toothpaste, oral hygiene products, chewing gum, sweets, soft drinks, chocolates, cereals, liquors, baked goods, spicy savory foods, and cosmetics, including perfumes, soaps and balms [49]. It should be kept in mind that in the literature, cinnamon-induced oral mucosal contact reaction [50]; and allergic contact stomatitis due to cinnamon-flavored chewing gum [51]. The results from our study indicated that both Azelastine HCl and cinnamaldehyde treatments were capable of reducing AR symptoms in rats. In rats treated with cinnamaldehyde, we observed nasal mucosa with decreased vascular congestion and inflammatory cell infiltration (i.e., plasma cells and eosinophils) into the lamina propria. A decrease in MC infiltration was observed primarily in the nasal mucosa of rats treated with Azelastine HCl, but not cinnamaldehyde.

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