| In recent years, with the rapid advance of industrialization, the rapid increase in the number of city vehicles, city to speed up the process, as well as coal, oil and other energy using rapid growth, air pollution is becoming more and more serious in China, which causing a serious threat to the health of residents. At present, the air pollution in our country presents the new compound pollution of soot and motor vehicle pollution coexist, haze and photochemical smog frequent and high status of NO2. NO2 is one of the main components of air pollution, has caused widespread concern around the world. NO2 is a primary pollutant that is found both indoors and in the outdoor atmosphere.The monitoring data of NO2 shows that outdoor NO2 levels usually do not exceed 0.5 ppm with heavy traffic, while indoor levels can reach up to 2 ppm in power plants, refineries, and ice-skating rinks. In place of occupation exposure, it may even reach up to 4ppm. Respiratory system is the main toxicity target organ of NO2. After inhalation, NO2 can rech the lower respiratory tract and even the deep lung and it mainly cause injury of respiratory bronchioles and alveolar. Therefore, NO2-related health risks have become the global focal point.Asthma is a disease characterized by variable airway obstruction and airway allergic inflammation associated with several inflammatory cells and mediators. Imbalance between Thl and Th2 and the excessive differentiation of Th2 cells is prominent in the pathogenesis of asthma. At present, there are about 300,000,000 patients with bronchial asthma in the world, and in China the incidence of asthma was 1% and it can be up to 3% in children. Recently, a growing number of epidemiologic studies linked NO2 pollution and increased asthma morbidity and mortality of residents, particularly in children. More recent evidence suggests that exposure to pollutants may also contribute to the development of the sensitization of atopic individuals to aeroallergens. So far, the study on relationship between NO2 and asthma development mainly concentrated in the field of epidemiology, and the results are not consistent as well as lacks direct evidence. The related experimental studys on mechanism of NO2 are few. So the objective of this study is to investigate the impact of NO2 on change of asthma susceptibility and its mechanism of transcription regulatory in the pathogenesis of asthma.Firstly, in order to investigate the effects of NO2 exposure on inflammation in rat lung. Adult wistar rats were exposed to NO2 (5 mg/m3,5 h/day, for 7 days). The centent of IL-4 in rat lung was examined by ELISA; and the mRNA expression of IFN-y> Muc5ac^ T-bet and GATA3 in rat lung were measured by real-time RT-PCR analysis. The results demonstrate that acute exposure to NO2 increased IL-4 content in rat lung. Furthermore, Muc5ac and GATA3 mRNA expression in rat lung were increased significantly after acute NO2 exposure, while T-bet and IFN-γ mRNA expression were decreased. The results implied that acute NO2 inhalation could cause airway inflammation and imbalance between Th1 and Th2, which suggestted that the molecular mechanisms might be involved in over-expression of transcription factor GATA3 and repressing the expression of T-betSecondly, people are often low-level chronic exposed to NO2 in daily life. Asthma is a chronic airway inflammation. However, in the case of low dose chronic exposure, wether NO2 could eventually lead to airway inflammation and airway mucus hypersecretion is not clear, through regulating the expression of T-bet/GATA3 and Thl/Th2 imbalance. The molecular mechanism is not well understood. In the present study, pathological features, cytokines, transcription factor and JAK/STAT6 signaling pathway were investigated following chronic inhalation exposure of rats to NO2(2 and 5 mg/m3,5 h/d, for 28 d). The histopathologic change in rat lung was observed by Hematoxylin and eosin (HE) staining. The level of IL-4 in rat lung was examined by ELISA. The mRNA and protein expressions of some transcription factors in rat lung were measured by real-time RT-PCR analysis and Western blotting technique. The results show that chronic inhalation of NO2 could cause airway epithelial cell damage, wall surrounding a small amount of inflammatory cell infiltration and visible mucus secretion in airway. By electron microscopy, collagen fibers deposition and caducous cilia was observed in the lung of the NO2-treated rats. Consistently with the results from acute exposure, chronic inhalation of N02 could up-regulate IL-1β、ICAM-1、Muc5ac and GATA3 expression and reduce IFN-y and T-bet expression in a concentration-dependent manner in rat lung. In addition, JAK/STAT6 signaling pathway and some signal regulation factors (ERK1/2、Lck and NF-kB) were activated by NO2 expourse. Through NO2 chronic inhalation exposure model, this experiment further prove that NO2 exposure can induce Thl/Th2 imbalance and Th2-dominant responses, and JAK/STAT6 signal transduction pathway and signal regulation factors (NF-kB, ERK1/2 and Lck) participated in the regulation. It implied that the above pathway might be an important mechanism of airway inflammation induced by NO2 exposure.Thirdly, in order to futher explore the possible molecular mechanism of increase of asthma susceptibility induced by NO2, the asthmatic rat model was established. Adult male wistar rats (42 days old) were divided randomly into 3 equal groups of 6 animals each:control group, ovalbumin (OVA) group (asthma group) and NO2+OVA group. The rats in NO2+OVA group were exposed to NO2 (5 mg/m3) for 5 h/d for 42 d, while control group and OVA group were exposed to filtered air for the same period of time. The rats in OVA group and in NO2+OVA group were sensitized by administering OVA (100 mg, grade V) with A1(OH)3 (100 mg) in 1 mL of total volume via intraperitoneal (i.p.) injection on days 22 and 29, and were then challenged using aerosolized 1% OVA in saline for 20 min on days 36-42. As controls, rats were administered saline. The pathological change was measured by HE methods. The levels of OVA-specific immunoglobulin E (IgE) in serum and IL-4 in lung were measured by ELISA. The mRNA and protein expressions of some transcription factors in rat lung were measured by real-time RT-PCR analysis and Western blotting technique. The results showed that, NO2 inhalation exposure aggravated the inflammatory cell infiltration, airway mucus hypersecretion, collagen fibers deposition and caducous cilia in lung tissues of asthmatic rats, accompanied by promoted increase of IL-4, IL-1(3, ICAM-1, Muc5ac, GATA3 expression and serum OVA specific IgE content, and decrease of IFN-y and T-bet expression. Furthermore, NO2 inhalation exposure promoted the protein activation levels of JAK/STAT6 signal transduction pathway and regulation factors (NF-kB, ERK1/2 and Lck) in lung tissue of asthmatic rats. The above results show that, NO2 exposed induced the increase of susceptibility to asthma in adult rats. NO2 may promote T-bet/GATA3 imbalance, increased Th1/Th2 offset, and adjusting the secretion of Thl/Th2 cytokines, as well as induce the activation levels of JAK/STAT6 signal transduction pathway and regulation factors (NF-kB, ERK1/2 and Lck), which thereby aggravate the inflammation and allergic reactions of asthma. This may be one of the important mechanism, that people exposed to NO2 are more sensitive, more prone to asthma, the reaction of asthma are more serious, also confirmed that NO2 pollution is an important cause of increased incidence of asthma.Forthly, epidemiological studies showed that asthmatic symptoms induced by NO2 and degree of asthma attack are also different in different age groups. Because the incidence of children is higher than adults, the authors suppose that children may be more sensitive, more prone to asthma or asthma attack is more severe than adults. So in this study, the asthmatic model was established with young rats. Infant male wistar rats (21 days old) were divided randomly into 3 equal groups of 6 animals each:control group, ovalbumin (OVA) group (asthma group) and NO2+OVA group. Experimental methods were the same as the third part. Through camparing the above indexes between infant and adult asthmatic rat after NO2 exposure, we found that the variation of the indexs of infant rats were larger than that of adult rats. Infant rats were more sensitive than adults to NO2, and damage is more serious. This result explains the author’s previous supposition and this may be an important reason why incidences of asthma for children are higher than that of adults in NO2 polluted area.Fifthly, recent research has shown that lymphocyte DNA damage level increases in children with asthma bronchiale. Elevated DNA damage may be related to increased oxidative stress. Whether NO2 itself can cause DNA damage to mammalian, and then influence the development of asthma, has not been reported in the literature. In the present study, comet, micronucleus (MN) and DNA-protein crosslinks (DPC) assays were used to investigate the genotoxicity following in vivo inhalation exposure of rats to NO2. The results show that both acute and chronic inhalation exposure of rats to NO2 induced DNA strand breakage and the formation of DPC in the cells from lung, as well as resulted in obvious increase of MN frequency in the bone marrow cells of rats. Furthermore, above genotoxic responses showed significant linear dose-dependent manners. These results implicate that NO2 can induce DNA damage and the reason may be related to increased oxidative stress, which might be an important cause of increased susceptibility to asthma.In conclusion, this study displayed the inflammatory injury effect of NO2 inhalation on the lung, demonstrated an association between NO2 inhalation and asthma, elucidated the related molecular mechanisms for increased asthma susceptibility (especially children asthma) and its possible signal transduction pathway, which will provide a theoretical basis for searching for new therapeutic targets of patients in NO2 polluted areas. |
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