| Background:Allergic rhinitis is an inflammatory disorder of the nasal mucosa characterised by nasal itching, sneezing, anterior nasal secretions, and nasal blockage. These symptoms arise from the interaction between mediators and neural, vascular, and glandular structures within the nose. Allergic rhinitis (AR) results from a complex allergen driven mucosal inflammation occurring from a vicious cycle between resident and infiltrating inflammatory cells, a number of inflammatory mediators including cytokines and neurotransmitters triggering the sensory nerve activation, leading to plasma leakage, and persistent congestion of venous sinusoids. Inhibition of cholinergic nerve by ipratropium bromide reduced the accumulation of Th2cells in lower airway in asthma patients. T-cell cholinergic receptors were found to play important role in differentiation of naive CD4Tcells toward the Th1, Th2and Th17lineages. Diverse neuropeptides are released by neuroendocrine and immune cells at the sites of allergic and inflammatory reactions. The neuropeptides and other neuromediators affect the functions of microvasculature, smooth muscle and secretory glands, and stimulate mast cell, lymphocyte and other leucocyte sharing in these reactions against immune sources. Anti-cholinergic drugs or vidian neurectomy can alleviate the symptoms of allergic rhinitis thorough there affecting the release of neurotransmitters which are affected by the action of the sympathetic and para sympathetic nervous system. Also studies made experimenting the vidian nerve and its role in allergic rhinitis it showed that the vidian nerve neurectomy was proved to be able to relive the symptoms of allergic rhinitis.Objectives:Allergic rhinitis is a common clinical disease, and the incidence has been an increasing trend in developing countries. The prevalence rate is about17%in children aged10-17years in Changsha, China. Its pathogenesis is still not fully clear, and the therapeutic methods need to be further improved. In this study, we aimed to establish the mouse model with allergic rhinitis, which treated by sympathetic inhibitor,6-hydroxydopamine, or parasympathetic inhibitor, ipratropium bromide, and to dynamic observe the expression of cytokines of Thl, Th2, Treg cells and other related inflammatory factors in nasal mucosa of allergic rhinitis, in order to further explore the effects of autonomic nervous system on nasal mucosal immunity.Methods:24mice were randomly divided into four groups:under pathogen-free conditions, mice were sensitized using OVA Ag as follows:A total of0.5mg OVA diluted to1ml by sterile normal saline with5mg aluminium hydroxide gel was administered to unanesthetized animals four times on days1,5andl4by intraperitoneal injection. Then, intranasally challenged the nasal mucosa with OVA diluted by sterile normal saline (20ul of25mg/ml OVA) were injected between days22to28. Allergic mice model was judged by symptom score at day28.Twenty-four mice were randomly divided into four groups:control group (a), model group (b), model ipratropium bromide treatment group (c), and model6-OHDA treatment group (d). In group a (n=6), mice were not sensitized and just challenged with saline. Group b (n=6), allergic mice model received intranasal ovalbumin challenge every two days (20μl of25mg/ml ovalbumin) from day29to42. Group c (n=6), allergic mice received the intranasal ovalbumin challenge every two days, but nasal administration of3.0mg/ml ipratropium bromide once a day from day29to42. The last intranasal ovalbumin was challenged30minutes after the last drop of ipratropium bromide. Group d (n=6), chemical sympathectomy was performed in allergic mice by intraperitoneally injection of6-OHDA at200mg/kg of body weight at day29and35, and mice also received intranasal ovalbumin every two days from day29to42. All mice were terminated within15minutes of the last intranasal ovalbumin challenge on day42. Evaluation of symptom scores and blood draws were performed before the termination of the mice. Control group (a), model group (b), model with ipratropium bromide treatment group (c), model with6-hydroxydopamine (6-OHDA) treatment group (d). Allergic mice model were sensitized with ovalbumin (OVA). Evaluation of allergic symptoms was recorded according to symptom score. OVA-sIgE was measured by ELISA assay. Expression of Interleukin4(IL-4), Interferon-gamma (INF-y), forkhead box P3(Foxp3), substance P (SP) and vasoactive intestinal peptides (VIP) was detected by immunohistochemistry and imaging analysis.Results:Establishment of mice allergic rhinitis:All mice were evaluated by standard symptom score of allergic rhinitis for mouse, and the results showed that17of18mice in group B, C and D were accord with the diagnosis of allergic rhinitis after ovalbumin antigen challenge. It indicated a successful establishment of allergic mouse model. One of six mice did not exhibit symptoms of allergic rhinitis in group B, and it was excluded for further experiment. The average symptom score is0.8±0.75in group A,6.4±1.14in group B,6.8±1.17in group C, and6.3±1.21in group D, respectively. After14days of different treatments in each group, the scores were changed to0.7±0.81in group A,6.2±1.09in group B,3.8±0.98in group C,7.0±1.09in group D, respectively. Ipratropium bromide treatment significantly decreased the symptom score in group C(p<0.01).1. Serum OVA-sIgE levelThe data from ELISA demonstrated that the serum OVA-sIgE concentration in group B, C, and D, was, respectively,112.5±16.3ng/ml,123.5±16.6ng/ml, and124.4±17.1ng/ml in serum after ovalbumin antigen challenge, and significantly higher than those in group A (22.1±4.33ng/ml), and the difference was statistically significant (p<0.01). There was no statistically significant difference between B, C, D group (p>0.05). After14days of different treatments for each group, the serum OVA-sIgE concentration was, respectively,26.1±4.13ng/ml in group A,120.3±16.1ng/ml in group B,74.4±9.87ng/ml in group C, and130.7±16.1ng/ml in group D. Ipratropium bromide significantly reduced serum OVA-sIgE concentration in mice allergic rhinitis (p<0.01).2. Results from Histopathological observationHistopathological examination showed that the nasal mucosa cilia were sparse, irregular arrangement, vascular dialation, gland hyperplasia, tissue edema, infiltration of eosinophils and other inflammatory cells in allergic mice compared with normal controls. Treatment of ipratropium bromide significantly reduced the infiltration of eosinophils, proliferation of glands and vascular dialation. However, treatment of6-hydroxydopamine did not change the histopathological type of mice allergic rhinitis.3. Changes of cytokines of Thl, Th2, Treg cells by different treatmentThe mice models of allergic rhinitis and control group were treated by different drugs or normal saline for14days. The percentage of IL-4positive cells in nasal mucosa was, respectively,8.2±1.45%in group A,28.2±8.41%in group B,12.4±3.12%in group C, and31.7±9.13%in group D; the percentage of INF-y positive cells was6.1±0.35%in group A,2.8±0.63%in group B,3.9±0.42%in group C, and3.6±0.51%in group D, respectively; the percentage of Foxp3positive cells was9.5±1.81%in group A,2±0.52%in group B,7.8±0.98%in group C, and2.7±0.47%in group D, respectively. The number of IL-4positive cells in nasal mucosa of model groups was higher than that of control group (p<0.01), and it was significantly decreased by treatment of ipratropium bromide in allergic mice (p<0.01). However,6-hydroxydopamine did not change the number of IL-4in nasal mucosa of allergic mice (p>0.05). On the other hand, the number of Foxp3positive cells of mice in model group was significantly lower than the control group (p<0.01), and significantly increased by treatment of ipratropium bromide (p<0.01).6-hydroxydopamine treatment did not change the expression of Foxp3in allergic mice. Although the number of INF-y positive cells in allergic rhinitis mice was significantly lower than the control group, treatments of ipratropium bromide and6-hydroxydopamine did not change its expression level (p>0.05).4. Alterations of neuropeptides (SP and VIP) by different treatmentsAfter different treatments for each group, the expression of VIP and SP in nasal mucosa was detected by immunohistochemistry and image analysis. The percentage of VIP-positive cells and fibers was respectively,5.8±0.21%in group A,26.6±6.34%in group B,16.2±1.83%in group C, and33.5±5.51%in group D. The percentage of SP-positive cells and fibers was6.9±1.53%in group A,23.5±6.02%in group B,12.6±1.31%in group C, and25.6±8.32%in group D, respectively. The expression level of SP and VIP in nasal mucosa of allergic mice was significantly higher than those in control group (p<0.01), and treatment of ipratropium bromide significantly decreased their expression levels. However,6-hydroxydopamine treatment did not change expression level in nasal mucosa of allergic mice (p>0.05).Conclusion:1. Ovalbumin antigen intraperitoneal injection combined with nasal challenge is a useful method to establish a mouse model of allergic rhinitis with high success rate;2.Cholinergic inhibitors can not only effectively relieve runny nose of mice allergic rhinitis, but also have a certain effect on sneezing, itchy nose and stuffy nose in mice allergic rhinitis.3. Inhibition of the cholinergic nerve not only alleviated symptoms of allergic rhinitis by inhibiting the impulse of the parasympathetic nerve but also modulated the T-helper type2-predominant immune reaction, and enhanced the expression of Treg cytokines. It might be a mechanism for playing a therapeutic effect in mice allergic rhinitis.4:Anti-cholinergic drugs did not alter immune responses mediated by Thl cells.5. Cholinergic inhibitors can significantly decrease the expression of SP and VIP in nasal mucosa of mice allergic rhinitis.6. The expression level of Th1/2cytokine and neuropeptides was not changed by sympathetic inhibitor,6-hydroxydopamine, in nasal mucosa of mice allergic rhinitis. |
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