| Background Allergic rhinitis(AR)is an inflammatory reactive disease of nasal mucosa,which is dominated by immune response of Th2 mediated by Ig E.The main clinical symptoms are itchy nose,sneeze,rhinorrhea and nasal obstruction.Local allergic rhinitis(LAR)refers to the type I allergic reaction with mast cell activation and eosinophil infiltration in the nasal mucosa of patients with non-atopy after exposure to inhaled allergens,and at the same time,it also presents typical symptoms similar to AR.Different from the classic AR,the SPT and serum s Ig E were negative.SPT and s Ig E in blood mainly reflected atopy,and could not accurately reflect the local state of the target organ.For LAR patients,the nasal allergen provocation test can induce typical AR symptoms,suggesting that there may be non-Ig E mediated inflammatory mechanism involved in the pathogenesis of LAR,which may be different from the classic AR pathogenesis.The classical pathogenesis of AR is the imbalance of Th1/Th2 cells.Th1 cytokines are inhibited and Th2 cytokines are dominant.IL-4 is the main cytokine secreted by Th2 cells,the production of Ig E depends on IL-4,but s Ig E is not detected in the serum of LAR patients,suggesting that the immune response of Th2 cells does not play a leading role in LAR.At present,it is known that Th17 cells and Th1 cells have an interactive regulatory effect,and the cytokine IL-17 A secreted by Th17 cells is positively related to the symptoms of AR,and IL-17 A may be involved in the pathogenesis of LAR.Mast cells and eosinophils are the primary and secondary effector cells of nasal allergy.When activated,mast cells can release tryptase and other the inflammatory mediators.Tryptase can combine with PAR2,wchich result in mast cells can produce cytokines and chemokines,and then eosinophils were selectively collected to migrate,infiltrate in the nasal mucosa,and release ECP toxic protein,causing nasal mucosa tissue damage.Taking PAR2 as the entry point,the change of Th1/Th2/Th17 cytokines in HDM after activation of PAR2 was explored to elucidate the possible pathogenesis of LAR.Part I Establishment of LAR mouse modelObjective To construct and evaluate the LAR mouse model by nasal sensitization of HDM.Methods The mice were divided into four groups: nasal sensitization group,nasal sensitization control group,intraperitoneal sensitization group and intraperitoneal sensitization control group.The nasal sensitization group was sensitized by nasal drip of HDM,the sensitization period was 14 days to establish LAR mice model,PBS was the blank control.In the intraperitoneal sensitization group,the traditional AR mice model established by intraperitoneal injection of HDM was used as the positive control.HE staining and methylene blue staining were used to observe the infiltration of eosinophils and mast cells in each group,as well as the pathological changes of the nasal mucosa.The success of LAR animal model was judged by EISA and the determination of histamine in nasal lavage fluid and the specific Ig E in serum of HDM.Results After the challenge of HDM,the mice in the nasal sensitization group and the intraperitoneal sensitization group showed obvious sneezing,scratching nose and runny nose.The total score was more than 5 points,which was statistically significant compared with the control group.There were obvious inflammatory reactions in the nasal mucosa of the mice in the nasal sensitization group and the mice in the intraperitoneal sensitization group.There were eosinophils and lymphocytes infiltration in different degrees,submucosal vasodilation,gland hyperplasia,and the number of mast cells in methylene blue staining.The nasal mucosa of the control group had no above manifestations.The concentration of histamine in nasal lavage fluid in nasal sensitization group and intraperitoneal sensitization group was significantly higher than that in control group;the serum total Ig E and HDM s Ig E in intraperitoneal sensitization group were increased with statistical significance;while the total Ig E and HDM s Ig E in nasal sensitization group had no significant change.Conclusion Mice intranasal sensitized with HDM can be successfully established LAR model.And the nasal mucosa shows allergic inflammatory reaction,the histamine in the nasal lavage fluid increased after nasal stimulation,while the serum HDM s Ig E is negative.Part II Study on the characteristics of immune response to different patterns of exposure to HDM in LAR.Objective To compare the change of Th1/Th2/Th17 cytokines expression level in LAR and make sure that there is non Ig E mediated inflammation in LAR.Methods Different sensitization courses were used to establish the mouse model:HDM exposure mice sensitized for 14 days and 28 days,and PBS was used as the blank controlgroup.The changes of Th1/Th2/Th17 cytokines in nasal mucosa were detected by flow cytometry CBA technology,the changes of serum total Ig E,HDM s Ig E and histamine in nasal lavage fluid were detected by ELISA,and the changes of the above indexes were compared under different allergen exposure patterns.Results The total scores of sneezing,nose running and nose rubbing in mice sensitized by short-term and long-term nasal passages were more than 5 points after the last nasal provocation,and allergic inflammatory reactions occurred in nasal mucosa.The serum total Ig E and HDM s Ig E in the short-term nasal sensitization group and the control group did not change.With the prolongation of sensitization time,the serum total Ig E and HDM s Ig E increased and LAR developed into AR model.Th17 cytokines(IL-17A),Th1 cytokines(IFN-γ,IL-2)and Th2 cytokines(IL-4,IL-10)in the nasal mucosa for a short period of sensitization significantly increased,while there was no significant change in Th2 cytokines(IL-4,IL-10).Conclusion With the prolongation of nasal allergen exposure,LAR model gradually evolves into AR model.Th1/Th2/Th17 cytokines are all involved in the allergic inflammatory reaction.In the pathogenesis of LAR,Th1/Th17 reaction plays a important role,and the production of IL-17 A indirectly inhibits the production of s Ig E.Part Ⅲ PAR2 activation participates in allergic sensitization to HDM in LAR modelObjective To investigate the role of PAR2 in allergic sensitization to HDM in LAR model.Methods The experimental mice were divided into 4 groups: LAR group,agonist group,inhibitor group and normal control group.The expression of PAR2 in nasal mucosa was detected by immunohistochemistry,the m RNA of PAR2,tryptase and ECP in nasal mucosa was detected by q RT-PCR,and the change of Th1/Th2/Th17 cytokines in serum was detected by flow cytometry CBA.Results Immunohistochemistry showed that PAR2 presented brownish yellow granules in the cytoplasm of submucous tissue,which was increased by agonists,but not inhibitors.The agonists could further aggravate the damage of the nasal mucosa,and the epithelial layer of the mucosa was exfoliated and the structure was disordered.PAR2 agonists increased the expression of tryptase m RNA and ECP m RNA in nasal mucosa,while the inhibitors were just the opposite.PAR2 activator induced the serum IL-17 A and IL-10 to further increase,IFN-γ to further decrease,but had no significant effect on IL-4.The inhibitor reduced the increase of IL-17 A,IL-10 and the decrease of IFN-γ.Conclusion HDM can induce the immune inflammatory response of nasal mucosa partly depending on the activation of PAR2 in LAR,revealing that PAR2 can be used as the target of LAR treatment. |
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