Study On Pathogenic Mechanism For Respiratory Tract Inflammation

Allergic rhinitis (AR) is a kind of worldwide common ailment of the nasal allergy, and its prevalence was up to 10%~25%, and even in some regions can be disturbed up to 50%. As its incidence has been increasing annually, AR affects human life quality seriously. Furthermore, AR can give rise to many complications, including bronchial asthma (BA), sinusitis, nasal polyps, otitis media, among which the most serious complication was the bronchial asthma. Epidemiological studies showed that the B A incidence strongly correlated to the severing degree of the AR, this was mainly because BA and AR have similarities in many aspects, such as anatomical location, physiology and etiological factors. In 1997, Grossman proposed the concept of "one airway, one disease", which highlights the integrity of airway inflammation.The pathogenesis of AR and BA has not been quite clearly, it may be involved in the interaction of many inflammatory mediators, cytokines and transcriptional factors. Eosinophil (EOS) was the main effector, which may cause inflammatory damage to the respiratory tract mucosa. There was a strong correlation between the severe degree of the airway inflammation and the infiltration of eosinophils. Eosinophils originate from the hemopoietic precursor cell in the bone marrow, then flow into blood circulation. When it was accumulated in the respiratory mucosa, eosinophils may play important biological effect. Th2 type cytokines (as IL-5, IL-13), EOS chemokine eotaxin took part in that process. The allergic reaction that induced by allergen may lead to the imbalance between Th1 and Th. Th2 type cytokines (IL-5, IL-13 ) may accelerate the formation of the EOS, and accumulate them in the airway mucosa, soas to inhibit their apoptosis. In addition, the eotaxin may play very important role in the process of chemotaxis from the marrow in the respiratory tract. The eotaxin receptor, so called CCR3 can be mainly expressed on the surface of EOS and partially on the Th2 cells. The amount of the eotaxin could increase and induce EOS accumulating when the organism were stimulated by allergen.Recently, people have recognized the occurrence and development of respiratory inflammation from the point of view of genetic control level. NF- k B can be considered as one of transcriptional factors, it has had certain functions and existed in various kinds of cells, so as to accelerate to the transcription of genes, such as cytokines and chemokines. NF- k B can also accelerate the expression of the Th2 type cytokines, being positive control effect to inflammation.Nasal polyps (NP) is a kind of inflammatory mass, which may affect the local microenvironment of nasal cavity. It may has closed relationship with AR in its pathogenesis, chronic inflammations characteristic and selectively EOS etc. infiltration. With regard to the function, Th2 type cytokine IL-6 and inducible nitric oxide synthetase (iNOS) both can subjecte to the control of NF- k B, and also have close relationship to the formation of NP.The incidence of respiratory inflammation was very high in Xinjiang, however till now, there have not been effective therapeutics to cure this ailment. So it was very important to investigate thoroughly, the pathogenic mechanism of respiratory inflammation. Experimental animal model can be considered as the main tools and basic methods in the research of the human diseases.The majow procedures in this study were listed as follows:(D Establishment of AR model in rats; (2) By means of in situ hybridization and immunohistochemistry technique, the activation of NF-kB in the respiratory inflammatory tissue of AR rat was measured. Atthe same time the EOS infiltration in the respiratory mucosa was observed, and the pathogenesis of the inflammatory reaction investigated in respiratory tract; ?Monitored the pathophysiologic variation of the NF-kB, IL-5, IL-13, eotaxin and EOS in the process of respiratory inflammation of AR rat pre and post futicasone propionce to therapy to investigate the mechanism of glucocorticoid therapy; (D Evaluation of the consistency of the upper and lower respiratory tract inflammation; (5) Through examination of NF-kB, IL-6 and iNOS in the resected specimen of nasal polyps, their expression and significance were investigated and analysed in NP.Part One— -. Establishment of the model of allergic rhinitis in rats.Objective: Establish stable experimental the model of allergic rhinitis in rats. Methods: NIH mouse and SD rat randomly divided into two groups, that the experimental group (1, 2) and control group (1, 2). In each group these are 60 rats, each group subdivided into two small groups. In the experimental group, ovalbumin(OVA) was injected intraperitonea-lly once in each other day for 7 times, after this OVA challenged intranasally once in every day for 7 times; In the control group the OVA was replaced by physiological saline. Results: In the experimental group, all the rats presented the same signs such as scratching their nose, sneezing, copious nasal discharge and asthmatoid breathing. The nasal mucosa became edemata and the glands proliferated, great deal of EOS was found in the nasal mucosa and lung tissue; In the control group there was no such manifestations. Conclusion: This experiment of the AR model was successfully established in rats, it was many advantages, such as simple and repeatable, it provides a very effective method and histomorphological evidence to diagnosis, treatment and further investigation of Allergic rhinitis.~> Monitoring the regulation of NF-KBmRNA to EOS infiltration Th2 cytokines- IL-S, IL-13 and eotaxin in the respiratory tract in rats with allergic rhinitis modelObjective: Investigate the regulation effect of NF-KBmRNA to EOS infiltration, Th2 cytokines- IL-5, IL-13 and eotaxin in the respiratory tract of AR rats. Methods: Select 6~8 weeks old SD rats. They were randomly divided into a sensitized group (A) and a control group (B), in each group there were 60 rats. The rats were sensitized by OVA. Then according to the time that they were killed, each group randomly was subdivided again into one hour group (Al, Bl), 24 hour group (A2, B2), 48 hour group (A3, B3), 72hour group (A4, B4) and 10 day group (A5, B5) in each small group there were 10 rats. Obtained the nasal mucosa and broncho-lung tissue from each group rats: ? The expression and pathomorphologic changes of EOS were observed by HE staining; (2) Through in situ hybridization method, the expression of NF-kB in nasal mucosa and broucho-lung tissue was examined in different time; (3) Through immunohistochemiscal method, the expression of IL-5, IL-13 and eotaxin in nasal mucosa and broncho-lung tissue in different time was measured. Results: ?In the sensitized group, there were inflammation in the nasal mucosa and broncho-lung tissue in all groups, the degree of inflammation correlates with the sensitized time. The EOS infiltration, epithelial destruction, tissue edema, vessel dilation were significant in the groups of A2, A3 and A4, but comparatively slight in the groups of Al and A5; In the A5 group we found that the smooth muscle proliferation, and the epithelium began to repair. ?In the sensitized group, we found various amount of positive expression of the NF-KBmRNA, IL-5, IL-13 and eotaxin in the nasal mucosa, broncho-lung tissue epithelium, inflammatory cells and the endothelium of the blood vessels, compared tothe control group it was statistically significant (P<0.05). (3)In groups of A1-A5, continuous overexpression of NF-KBmRNA in the nasal mucosa was revealed, but in the A5 group its expression lowered obviously in the broncho-lung tissue; the expression of IL-5 reached its peak in the nasal and broncho-lung tissue in the group of A2, but lowered in A4-A5; In nasal mucosa, continuous over expression of IL-13 was reveald in A1-A4 and began to lower in A5, but in the broncho-lung tissue, continuous over expression was revealed in A1-A4 and reached its peak in A5; the overexpression of eotaxin was revealed in both nasal and broncho-lung tissue. @In the various sensitized group, there was a strong correlation between the eosinophil infiltration in the nasal, bronchial and lung tissue and the positive expression of the NF-KBmRNA, IL-5, IL-13 and eotoxin in the corresponding site. ?In the various sensitized groups, there were also positive correlation between the NF-KBmRNA in the nasal, bronchial and lung tissue and the expression of the IL-5, IL-13 and eotaxin. Conclusion: ?EOS is the main effecting cell in the process of respiratory inflammation in the experimental AR rat model ; All of the IL-5, IL-13 and eotaxin probably directly or indirectly take part in the process of the chemiotaxis, gathering and activiation of EOS in the respiratory tract; ?NF-kB probably take part in the EOS infiltration and the regulation of the IL-5, IL-13,eotaxin in the respiratory tract inflammation.Part twoExperimental research of the fluticasone propionate treatment of the respiratory allergic diseaseObjective Investigate the pathophysiological variation and its significance of the EOS, NF-KBmRAN, IL-5,IL-13 and eotaxin in therespiratory tissue of AR rats that treated by fluticasone propionate(FP). Methods: ?There were 30 SD rats that aged from 6 to 8 weeks, male to female ratio was 1:1, they were randomly divided into 3 groups, that the control group ( I ), the sensitized group (II) and the treatment group (III). In each group, there were 10 rats. In the group II and group III, the rats were sensitized and challenged by ovalbumin (OVA). In the group I , the ovalbumin was replaced by physiological saline. After the rats models were established, each rat in the group I and group II was given lOOul physiological saline intranasal drops (50ul in each side), rats in group III were given lOOul futicasone propionate nasal drops (50ul each side), once in a day and continued for 7 days. After that group II and group III were challenged by OVA, in group I , the OVA was replaced by physiological saline.;?The expression of NF-KBmRNA in upper and lower respiratory tract tissue was examined by the method of in situ hybridization. (3)The expression of IL-5, IL-13 and eotaxin in upper and lower respiratory tract tissue was detected by the method of immunohistochemistry. ?EOS infiltration and inflammatory reaction in upper and lower respiratory tract were observed by the method of HE staining. Results: ?In group I , there was no AR findings; In group II, there were obvious symptoms and signs of AR; In group III the findings were slighter than group II. ?In group II, there were great deal of EOS infiltrated and epithelial damage in the respiratory tract; In group III, there were few infiltrated EOS and the epithelial damage was also significantly slight than group II; In group I , there were no EOS infiltration and epithelial damage in respiratory tract. (3)In group II, the expression of NF-KBmRNA, IL-5JL-13 and eotaxin were significantly increased in upper and lower respiratory tract, and compared to group I and group III, the difference was statistically significant (P<0.001); there was no significant statistical difference between group I and group III (P>0.005). Conclusion: ?FP can inhibit the activiation of NF-KBmRNAin upper and lower respiratory tract, it may decrease the expression of the inflammatory factors like IL-5, IL-13 and eotaxin in the respiratory tract; ?Intranasal steroid treatment may also alleviate the inflammation in the lower respiratory tract.Part ThreeEvaluation of the consistency of the upper and lower airway inflammation and its significanceObjective: To evaluate the consistency of the upper and lower airway inflammation and its significance. Methods: Methods are same to the methods of the second experiment in part one and the methods of part two. Results: ?In the experimental group, the rats had asthmatoid breathing as well as nasal symptoms; In the treatment group, the breathing symptoms subsided after alleviation of the nasal symptoms. ? Histomorphological variations: There were significant EOS infiltration and destruction in the nasal mucosa and broncho-lung tissue of the rats in the sensitized group, the inflammation of both uppe* and lower respiratory tract had been improved after treatment. (3) In the experimental group, the expression of IL-5, IL-13 and eotaxin had been elevated obviously in the nasal, bronchial and lung tissue, at the same time, NF-kB had been activated; In the treatment group the expression of those factors had been lowered obviously. Conclusion: The upper and lower respiratory tract inflammation were identical in the sensitized AR rats that were challenged intranasally. The allergic reactions in upper and lower respiratory tract can be improved after treated with corticosteroids, such as fluticasone propionate.Part FourExpression of NF-KBmRNA> iNOS and IL-6 in nasal polyp and initial analysis of its roleObjective: To evaluate the expression and significance of NF-kB mRNA, iNOS and IL-6 in the pathophysiology of nasal polyp(NP). Methods: The specimens of 59 patients with nasal polyps and 20 patients with nasal inferior turbinates (IT) were studied by in situ hybridization for NF-KBmRNA, and by immunohistochemistry for iNOS and IL-6. Tissue sections were observed under optical microscope. Results: ? NF-KBmRNA, iNOS and IL-6 overexpression were localized mainly to the nasal polyps epithelium, the serious inflammatory cells, in submucosal endothelial cells around the vessel and in submucosal glands. ? The positive frequency of activation of NF-KBmRNA was higher in NP (54.24%) than in IT (P<0.05), whereas the positive frequency of iNOS and IL-6 overexpression were higher in NP (62.71% and 64.41%) than in IT (/)<0.05).(3)Eosinophils and neutrophils in NP were higher than in IT (P<0.05). Conclusion: These results suggested that NF-kB ,iNOS and IL-6 may correlate the formation of NP. NF-kB ,iNOS , and IL-6 may act as anti-apoptotic molecules in eosinophils and neutrophils with may be responsible for the recruitment of inflammatory cells, particularly eosinophils, through the initiation of the transcriptional pathway of the related cytokines. NF-kB may also play a role in the pathogenesis process, and a potential target for new therapeutic intervention for human NP.