Study Of The Roles Of Nerve Growth Factor In The Development Of Bronchial Asthma

Bronchial asthma is a chronic airway inflammatory disease that affects public health of world-wide population. This nonspecific airway inflammation involves many inflammatory cells, cellular components and neurotransmitters. The pathological features are shedding of bronchial epithelial cells, infiltration of the airways by lymphocytes and eosinophils, thickening of the basement layer and an enhanced collagen deposition. Many inflammatory cells and cytokine, including mast cells, and eosinophils are involved in this inflammatory process. Many cytokines play role in the induction of asthma. One of them is TNF-α(Tumor necrosis factor-α). TNF-αis a proinflammatory cytokine that is produced by many cells and have many biological activities, and it is also an important factor in asthma pathogenesis. Regarding the induction of airway inflammation, it was widely recognized that it is a result of immune response in the bronchial airway. However, the underlying mechanism is not clear. Many believe that imbalance of two types of T-helper cells, Th1 and Th2, is the underlying cause for asthma. Specifically, the increased activity of Th2 cells is a key event in asthma induction. Th1 and Th2 are differentiated from a common precursor cells, Th0. Transcription factor GATA-3 regulates Th0 differentiation in a specific manner. Such regulation is critical in controlling Th1 and Th2. IFN-γand IL-4 are typical cytokines from Th1 and Th2.In addition to immune response, the roles of neuronal factors in airway inflammation are receiving increasing attention in recent years. Nerve growth factor (NGF) is an important member of the neurotrophic factor family and is the most important biological active substance in the nerve system. The main function of NGF is in the regulation of neuronal development, survival and maintenance of neuronal functions. It also participates in regulation of immune response and complex neuro-immune interactions. It is believed that NGF plays dual roles in regulating neuronal plasticity and immune plasticity. Much experimental evidence has demonstrated that NGF is involved in pathogenesis of many inflammatory diseases, such as bronchial asthma, ulcerative colitis, allergic rhinitis, chronic neuritis, rheumatoid arthritis, etc. It has been shown that NGF plays significant pathophysiological roles in asthma induction. However, the underlying mechanism requires further investigation. Substance P (SP) is a sensory nerve peptide and a neurotransmitter. It exerts its effect on the release of inflammatory cytokines from the inflammatory cells. SP is involved in contraction of bronchial smooth muscle, and therefore participates in asthma inflammation and induction of airway hypersensitivity. In this study, we used mouse asthma model to study the function of NGF in inflammation of asthmatic airway. The goal of this study is to examine the expression of NGF and neurotransmitter SP in bronchial tissues of asthmatic mice, and the effect of glucocorticoid (Budesonide) on their expressions. Furthermore, we studied the effects of NGF on pathological changes in the asthmatic mouse bronchial tissues, bronchial eosinophils infiltration, expressions of lung SP and TNF-α, as well as the roles NGF in dys-regulation of the balance of T1/Th2 lymphocytes in the pathological process of asthma. Through this study, we hope to open new avenues and provide further experimental evidence for therapeutic treatment of asthma.Method:To examine the expression of NGF and SP in the lung of the asthma mouse model and the regulation by glucocorticoid, we randomly divided thirty female BALB/c mice into three groups at: the normal control group, the asthma group, and the hormone-treated group and there were ten mice in each group. Asthmatic mice were sensitized on days 1 and 14 by intraperitoneal injection of 20μg ovalbumin , and on days 21-27, after the initial sensitization, the mice were challenged for 30 min with an aerosol of 3% (w/v) OVA in saline (or with saline as a control) using an ultrasonic nebulizer. The hormone-treated group was treated for 30 min with glucocorticoid (Budesonide) before each allergen challenge. Using ELISA, RT-PCR, and radioimmunoassay, we measured and compared the expression level of NGF and SP before and after administering glucocorticoid. In addition, we selected the female BALB/c mice, and divided them at random into normal control group, the asthma group, and the NGF-blocking group. The NGF-blocking group was treated 3 h before each allergen challenge with 50μl anti-NGF (1 : 50). We then monitored the effects of NGF withdrawal on the number of eosinophil in BALF. We also observed bronchial pathological changes in the asthma mice after the passive NGF withdrawal. Expression level of lung SP, as well as serum and lung TNF-αwere determined by ELISA, RT-PCR, and radioimmunoassay. To determine the role of NGF in Th0 differentiation, IFN-γ,IL-4 and GATA-3 were selected as the markers of imbalance of Thl/Th2. Using ELISA and RT-PCR, we measured changes of expression level of IFN-γ,IL-4 and GATA-3 in the asthma mice after applying exogenous anti-NGF antibody.Results: The mRNA and protein levels of NGF and protein level of SP in the asthma group and the hormone-treated group were significantly higher than those in the normal control group. The levels of NGF and SP in asthmatic lung were higher than those in the hormone-treated group. The asthmatic manifestation of the NGF-blocking group was less pronounced than that of the asthma group,and the score of asthmatic attack of NGF-blocking group was lower than that of the asthma group .The number of eosinophils in bronchoalveolar lavage fluid of the OVA sensitized and allergen-challenged mice was higher than that of the normal control group, and the number of eosinophils in bronchoalveolar lavage fluid of the mice treated with anti-NGF was lower than that of the asthma group. Histologic analyses revealed typical pathologic features of asthma in the mice that OVA sensitized and allergen-challenged. There were more inflammatory cells, including eosinophils, infiltrated around the bronchioles, and mucus and debris had accumulated in the lumen of bronchioles of the asthma group compared with that of the control. Mice in the NGF-blocking group, which were treated with anti-NGF antibody showed marked reductions in the infiltration of inflammatory cells in the peribronchiolar region, in the number of inflammatory cells, and in the amount of debris in the airway lumen. Compared to the NGF-blocking group, the number of inflammatory cells, lung SP, sera TNF-αand lung TNF-αmRNA levels were significantly higher in the asthma group. These parameters in the asthma group and NGF-blocking group were higher than those in the normal control group. The result of the study on the effect of NGF in the Th1/Th2 imbalance showed that serum IL-4 levels in the asthma group and NGF-blocking group were higher than those in the normal control group; IL-4 level in the asthma group was higher than that in the NGF-blocking group, the results of IFN-γwere opposite. Compared to the normal control group, the GATA-3 mRNA level was elevated in the asthmatic mice, which also increased than those in the NGF-blocking group.Conclusion: These findings demonstrate that the mRNA and protein levels of NGF and protein level of SP in the asthma group are higher and NGF takes part in the development of asthma. The therapy of glucocorticoid may inhibit the expression of NGF and SP in the lung of asthma mouse model. Blocking endogenous NGF can reduce the airway inflammation in the asthmatic mice, alleviate lung eosinophil infiltration, and decease the serum TNF-αand mRNA expression of lung TNF-αin the asthmatic mice. Blocking NGF can also lower SP level in the lung. All these results suggest that NGF participates in pathogenesis of asthmatic airway inflammation, specifically by regulating TNF-αand SP expressions. Blocking endogenous NGF can effectively inhibit the cytokine IL-4 expression in the Th2 cells, and increase the cytokine INF-γexpression in the Th1 cells. Blocking NGF can also inhibit GATA-3 expression, suggesting that NGF is involved in Th1/Th2 cytokine immune-imbalance by regulating GATA-3 expression.