| ICS is effective to many asthma patients, but a small proportion of patients are not well controlled though using large dose of ICS. Researchers found that neutrophils are predominant in the sputum of those patients. And according to the different cell counts in sputum, four phenotypes of asthma are obtained, they are eosinophilic, neutrophilic, mixed and paucigranulocitic.It is not clear why neutrophil-predominated asthma patients are not sensitive to ICS. We don’t know whether neutrophil infiltration is an inducer or an outcome of ICS. But researchers have shown that glucocorticoid steroid can inhibit the apoptosis of neutrophils, which may be one reason for ICS resistance. Why poor is known about neutrophilic asthma.One of the reasons may be that no established neutrophilic asthma animal model is available until now. So far the most used antigen to induce asthma is OVA, and OVA-induced asthma model is dominated with eosinophils. And for the past century, researchers have made great progress in the understanding of the pathogenesis of asthma and some new drugs were made too because of OVA-induced asthma model. So we try to make a stable animal model to explore the mechanism of neutrophil-dominated asthma. Toluene diisocyanate (TDI), a commonly used industrial raw materials, has become the most important incentive for occupational asthma. With the change of lifestyles, this kind of raw materials used in the paint and foam is closely related to our life. Clinical data showed that the major inflammation cells in sputum from TDI-induced asthma patients were neutrophils. So we want to make a asthma model predominated with neutrophils.It is well known that the immunologic mechanism is involved in the pathogenesis of asthma. The increasing incidence of the development of allergic disorders seems to be associated with the modern westernized lifestyle and early life events are essential in shaping the immune answer towards the Th1-or Th2-profile, which is associated with a nonallergic or allergic phenotype, respectively. The hygiene hypothesis suggests that an early life environment rich in normal microbial flora primes the immune system in the Thl direction towards clinical balance while a’sterile’ environment promotes the development of pathological immune phenotypes. Subsequent researches revealed that the high incidence of asthma dominated with TH2inflammation and Type1diabetes dominated with TH1inflammation were correlated with the same life style. While parasitic infection could increase TH2inflammation and therefore decrease asthma incidence. All the above indicated that the balance between TH1and TH2could not illustrate the asthma mechanism. Recently, researchers showed that immune tolerance may be involved in the mechanism of asthma. Immune tolerance is dependent on CD4+Regulatory T cell, which can inhibit the function of TH1and TH2cells. And the incidence of asthma may be associated with the dysfunction of Treg.Clinical Researches demonstrated that vitamin D intake during pregnancy was inversely associated with asthma and allergic rhinitis in children. And vitamin D could decrease the dose of ICS. The data from animal and cell model showed the effect of Vitamin D on asthma may be associated with its immunological regulation. For example, Vitamin D could not only inhibit TH1and TH2inflammation, but also increase Treg proliferation and maturation,which was considered to be an important cell in the pathogenesis of asthma. In addition, some antibacterial peptide secreted by epithelial cell was assosiated with Vitamin D, which can enhance innate immunity and avoid activation of adopted immunity.Recently, researches showed that bronchial epithelial cell may play a key role in the pathogenesis of asthma. Epithelial cells play an important role in maintaining homeo stasis of the body. They display a highly regulated and impermeable barrier through the formation of tight junctions (TJs), composed of zonula occludens (ZO)1-3, occludin and claudins1-5, as well as adheren junctions (AJs), consisting of E-cadherin, β-catenin and a-catenin. E-cadherin contributes to the architecture and immunological function of airway epithelium, through the regulation of epithelial junctions, proliferation, differentiation, and production of proinflammatory mediators such as Thymus and Activation-regulated Chemokine(TARC) and thymic stromal lymphopoietin. Xiao C et al showed that E-cadherin expression was significantly lower in bronchial biopsies of asthmatic subjects than those of the nonasthmatic. Additionally, induction of E-cadherin expression can repress NF-kB activity (Solanas et al.,2008), which is critical for evoking airway inflammation. Thus, E-cadherin could be an epithelial molecular switch in mediating the immunological decision between a tolerogenic and a proinflammatory/immunogenic phenotype.Vitamin D can not only affect TH cells but also the epithelial cells. Researches on intestinal tract showed that Vitamin D could decrease the permeability of intestinal epithelial cells though altering the distribution of E-cadherin and ZO-1 and the same results was obtained from corneal epithelium, while the effect of vitamin D on bronchial epithelial is still unknown. In our previous study, we demonstrated that Vitamin D could decrease the permeability of16HBE cell lines by increasing TER and alter the distribution of E-cadherin.In our previous study we have demonstrated that TDI could disrupt E-cadherin in16HBE cell lines. So the main objective of our experiment is to made a TDI-induced asthma model and to observe the change of inflammation and the distribution of E-cadherin.content and methodsPart I:make a TDI-induced asthma model according to the method of foreign researchersPart II:observe changes in asthma inflammation and the distribution of epithelial E-cadherin after treatment with Vitamin D.ResultsAccording to the method (On days1and8, mice were dermal sensitized with toluene diisocyanate (TDI)(0.3%) or vehicle(acetone/olive oil)] and on day15they received a single challenge, via oropharyngeal aspiration,with TDI (0.01%) or vehicle.),we have made a successful asthma mice model by neutrophil airway inflammation, higher penh, raised concentration of IL-4and IFN-y, but perivascular and prebronchial inflammation did not develop in our model, which was an important feature. So we improve our mouse model by increasing the times of challenge. We investigated different times of challenge on inflammation, and the result showed a significant increase in total cell count, neutrophils, IL-4and IFN-g in auricular lymph node cells and total IgE in serum in TDI-treated mice after1,2or3TDI challenges. This response increased with increasing times of challenges. And eosinophils appeared shortly after the third challenge. After the third challenge, we found apparent inflammation in peribronchial and perivascular regions and epithelial proliferation. And a significant increase in airway reactivity was found too. To observe the duration of the inflammation, we observed three time spots (24h,72h,120h after the last challenge). The result showed that a significant decrease in total cell count, neutrophils, IL-4and IFN-g in auricular lymph node cells and total IgE in serum were found in TDI-treated mice when extending the observed time.When treated with Vitamin D, the number of total cell counts, eosinophils in BALF, IL-4and IFN-g in auricular lymph node cells and total IgE in serum were decreased. But the number of neutrophils in BALF did not decrease. Immunohistochemistry showed that E-cadherin localized especially at the lateral side and apicolateral border. The immunoreactivity of E-cadherin significantly decreased at the epithelial cell-cell contact in the TDI group. In the Vitamin D group, the immunoreactivity of E-cadherin was partly rescued.Conclusion:l.we have successfully made a TDI-induced asthma model dominated with neutrophils in BALF.2.The inflammation was inhibited by Vitamin D except for neutrophil inflammation in BALF.3.The mechanism of inhibiting the inflammation by Vitamin D may be through rescuing the distribution of E-cadherin. |
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