| Bronchial asthma (bronchial asthma, referred to as asthma), is a chronic airway inflammation which many inflammatory cells, especially macrophages, mast cells, eosinophils and T lymphocytes take part in. The death rate and prevalence of bronchial asthma increase gradually at home and abroad. Bronchial asthma invalid some 100 million people around the world, becoming a serious threat to people's health a major chronic diseases. China's asthma incidence is 1%, children up to 3%.Nerve growth factor (NGF) has been specifically implicated in asthma pathogenesis due to its ability to promote sensory neuron hyperreactivity and induce airway inflammation. Blocking NGF can effectively relieve asthmatic pathological changes and reduce a variety of cytokines. While the mechanism of NGF involved in the pathogenesis of asthma not fully understood. In addition, NGF regulates PC 12 cells in part by stimulating phosphorylation of Src homology 2β(SH2-Bβ) on serine/threonine kinase (Akt/PKB). SH2-Bβis a member of a widely expressed, highly conserved family of adaptor proteins and can bind to Janus kinase 2 (JAK2) and the activated receptor tyrosine kinases for insulin, insulin-like growth factor I, NGF, platelet-derived growth factor, and fibroblast growth factor. Moreover, SH2-Bβis critical for both NGF-dependent neurite outgrowth and neurite maintenance.Additionally, SH2-Bβenhances and prolongs of NGF-induced Akt phosphorylation and of Akt enzymatic activity in PC 12 cells. Li has also characterized SH2-Bβexpression in alveolar macrophages in the bronchoalveolar lavage fluid (BALF) of asthmatic guinea pigs and its role in NGF-TrkA-mediated asthma. Together, these studies suggest that SH2-Bβplays a fundamental role in receptor tyrosine kinase-mediated cellular functions. Together, these studies suggest that SH2-Bβplays a fundamental role in receptor tyrosine kinase-mediated cellular functions. Based on the above information, we hypothesized that upregulation of Akt phosphorylation by SH2-Bβmay contribute to the pathogenesis of NGF-mediated asthma.By the study, using the mouse model of asthma, we observed expression of Akt at asthmatic lung tissue, observed the relationship of between NGF and SH2-B(3 with airway hyperresponsiveness, observed lung tissue pathological changes and changes in the number of inflammatory cells after blocking NGF and SH2-Bβ, observed effects of NGF and SH2-B(3 on the activity of Akt in the pathogenesis of asthma using several methods. These findings may pave the way for exploring the NGF-mediated asthma pathogenesis and the development of new therapeutic approaches to allergic airway diseases.Methods1,AnimalsFifty-five normal female BALB/c mice (6 to 8 weeks old) weighing 22±2.5 g were obtained from the Laboratory Animal Research Center in Beijing, China, they were randomly divided into four groups:control group, ovalbumin group, R555E group, and anti-NGF group.2,OVA-treated asthma mice model and DrugsOn days (d) 0,7,14, and 21, all mice except the control group were actively sensitized with an intra-abdominal injection of 1 mg OVA (Sigma) and 100 mg aluminum hydroxide in 0.5 ml phosphate buffered saline (PBS), for the control group only with 0.5 ml PBS. The control group mice were then exposed to aerosolized PBS for 30 min per day from d 22 to d 30. Meanwhile, the OVA-sensitized mice were exposed to 10 mg/ml aerosolized OVA (1 g OVA in 100 ml sterile PBS in a nebulizer) for 30 min per day from d 22 to d 33. The anti-NGF group and R555E group mice were anesthetized and respectively administered an intravenous injection of 50μl polyclonal rabbit anti-mouse NGF antibody (Sigma, St Louis, MO, USA) diluted 1:10 in PBS and 50μl R555E. Intravenous treatment was administered 3h before allergen aerosol challenge on d 31,32, and 33.3,Determination of Airway responsiveness Airway responsiveness to muscarinic acetylcholine(mAch) was measured in AniRes2005 lung function system. Inspiratory resistance (IR) and expiratory resistance(ER) are detected.4,Preparing the histological slicesThe animals were perfused transcardially with 4% paraformaldehyde in PBS(0.1M, pH 7.4). The lungs were removed and postfixed. The lung slices at same positions of four groups.5,Detecting the expressions of Akt at lungs by using immunohisto-chemical technique.6,Total BALF cell counts were performed using hemacytometer. A differential cell count was carried out by Wright-Giemsa staining on the basis of morphologic criteria.7,Determinating p-Akt of expression in lungs of 4 groups by Immunofluorescence8,The expressions of p-Akt in lungs were detected by Western blotting.9,The expressions of Akt mRNA in lungs were detected by Western blotting.10,Hematoxylin-Eosin(HE) staining was employed to detect the histological changes in different groups.The frozen lungs slices from different groups at 24 h reperfusion were stained with HE solution, and the histopathologic changes of hippocampuses were observed under the microscope.11,StatisticsAll values are expressed as the mean±SD. One-way analysis of variance (ANOVA) was used to compare group differences in Akt levels, while two-way ANOVA was used to assess differences in airway resistance. 1,Respiratory frequencys have no significant differences in each group2,Pathological changes in the bronchi and lung tissueInflammatory cells, mucous edema, and epithelial lesions were significantly more prevalent in asthmatic mice than in those treated with anti-NGF or R555E. Many inflammatory cells, including mononuclear cells, macrophages, lymphocytes, and eosinophils, were observed in asthmatic pulmonary alveoli and the surrounding bronchi, in contrast with control mice. However, these finds reduced anti-NGF-and R555E-treated mice.3,BALF cell counting and differential countingThe total number of cells in the BALF was significantly higher in OVA-sensitized mice than in control mice. The numbers of macrophages, eosinophils, and lymphocytes were also significantly increased in OVA-sensitized mice but reduced in anti-NGF-and R555E-treated mice.4,Airway resistance measurementThe airway response to MCH was increased in the OVA group compared with the control group (P<0.01). Treatment of OVA-sensitized mice with intravenous anti-NGF and R555E prior to OVA challenge prevented this increase in airway reactivity.5,Immunohistochemistry observed expression of AktAkt levels significantly increased in OVA group, but reduced in NGF group.6,Immunofluorescent analysis of the effects of NGF and SH2-Bβon Akt activationImmunofluorescent staining showed that p-Akt levels significantly increased in macrophages, eosinophils, and lymphocytes and bronchial tissue of the OVA group's lung tissue (P< 0.01), as compared with the control group. Meanwhile, the overexpression of p-Akt at the above sites was reduced in both anti-NGF-and R555E-treated groups. 7,Western blot analysis revealed p-Akt protein expressionIn the lung tissue of the control group, p-Akt was expressed at low levels, but these levels increased markedly in the OVA group. However, anti-NGF and R555E greatly decreased the p-Akt upregulation caused by OVA.8,Effects of anti-NGF and R555E on Akt mRNAAkt mRNA expression was significantly higher in the OVA group than in the control group, but that anti-NGF and R555E treatment significantly decreased Akt mRNA expression in these OVA-sensitized mice.Conclusions1,The airway response was increased in the OVA group, NGF participates in airway hyperresponsiveness and inflammatory response of in the lungs of asthmatic mice. Blocking NGF significantly reduced airway hyperresponsiveness and inflammatory response in the lungs of asthmatic mice2,Akt may be involved in pathogenesis of asthma, NGF regulates activity of AKT may be one of the mechanisms of NGF involved in asthma.3,SH2-Bβinvolved in airway hyper-responsiveness and inflammatory response of lung of asthmatic mice, and blocking SH2-Bβcould alleviate airway hyperresponsiveness and inflammatory response in the lungs of asthmatic mice.4,NGF mediated SH2-Bβ/Akt signaling pathway may be one of the pathways NGF-mediated asthma.
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