| Background and ObjectivesBronchial asthma is a chronic nonspecific airway airway inflammatory disease that involves many cellular components, inflammatory cells and neurotransmitters.Asthma incidence is increasing in recent years. Effective intervention to asthma is always the hot spot of international respiratory research.Regarding the mechanisms of asthma,it was widely recognized that it is a result of immunological mechanisms in the bronchial airway.The roles of neuronal factors in asthma are receiving more and more attention in recent years. Nerve growth factor (NGF) is a member of the neurotrophic factor family. The function of NGF is to regulate neuronal development, maintenance and survival of neuronal functions. It is also involved in the regulation of complex immune-neuro interactions.Many experiments have showed that NGF is involved in the pathogenesis of many diseases, such as ulcerative colitis, rheumatoid arthritis,chronic neuritis,allergic rhinitis,bronchial asthma etc. It has been confirmed that NGF plays significate roles in asthma.But the mechanism of asthma need further investigation. GABA is the primary inhibitory neurotransmitter in the adult mammalian brain. However, recently, glutamic acid decarboxylase (GAD),a major GABA synthetic enzyme,and GABAAR were detected in a variety of human and mouse cell lines and whole mouse lung.Mucus hypersecretion is a cardinal feature of bronchial asthma and associated with goblet cell metaplasia.And it is the result of the up-regulation of mucin expressed by goblet cell of airway.MUC5AC is the major inducible mucin in the airways and its expression is controlled by GABAA receptors in airway epithelial cells.In this experiment,we used mouse model to explore the function of NGF in mucous secretion of airway. The present study was designed to observe the effects of the nerve growth factor on airway mucus secretion and GABAAR expression in a mouse model of asthma. In this report, we investigate the relationships between GABAARa and MUC5AC expression,as well as the change after the intervention of anti-NGF,and then proceeds the mechanism of mucus hypersecretion in airway epithelium in asthmatics, which provide theoretical basis for early intervention.Materials and MethodsBALB/c female mice,Six-to eight-week-old,were randomly divided into three groups(n=10mice/group):the normal control group,the asthma group,the NGF-blocking group.The mice were initially sensitized with1mg of OVA in100mg of Al(OH)3by intraperitoneal injection on day1and day8.From day15to day21,the mice were exposed to OVA aerosol,10ml of a2%solution, per day.Exposure was more than30minutes every time.The control mice were treated in the same way with saline without OVA. For the NGF-blocking group,the mice were sensitized and challenged in the same way as the asthma group, but they were treated with anti-NGF (1:50)50μl by intranasal administration3h before allergen challenge.Twenty-four hours after the last challenge,mice were anesthetized (Chloral hydrate,20mg/kg i.p.) and the thoracic cavities were carefully opened.Tracheas were exposed, cannulated and, while the left lung lobe was tied off with a silk thread, the right lobe was lavaged with saline,The BALF was centrifuged to used for cell count and classification count.Left lungs were cutted off, upper lobes of mice were immobilized by4%paraformaldehyde, paraffined slice routinely;the lower lobes of mice were fastly putted into liquid nitrogen,and then transferred to-80℃refrigerator,prepared for use. HE stain of lung tissues were used for observing inflammation in lung tissues.RT-PCR and immunohistochemistry were used to assay for GABAARa and MUC5AC mRNA and protein. Statistical analysis was performed using software(SPSS18.0).Results1Symptom:After OVA challenged,the mice of asthma group appeared symptoms of acute asthma, such as activity less,bowing the back, sneeze,nod sample breathing,urinary and fecal incontinence, and so on.These symptoms in the NGF-blocking group compared with asthma group were alleviated, The normal control group mice had no significant wheezing.2Cell count and classification count:In BALF, The number of total cells, the ratio of Neutrophils, Eosinophil Cells were obviously increased in the asthma group compared with the normal control group(P<0.05). In BALF,the ratio of Eosinophil Cells,Neutrophils and lymphocytes were conspicuously reduced in normal control groups compared with the asthma group(P<0.05).3The pathological changes of the lung tissues:In the lung tissues of asthma group, HE staining showed shedding epithelial cells,and inflammatory cells infiltrating around blood vessel wall and bronchial wall,and the broad airway alveolar interval,and the alveolar spaces to which the inflammatory substances exuded to.Those pathological changes of the NGF-blocking group were alleviated than the asthma group. In control group,the alveolar walls was integrated,and there were no inflammatory cells infiltrating around blood vessel wall and bronchial wall.4The expression of MUC5AC and GABAARa protein in lung tissue:The expression of GABAARa and MUC5AC protein were higher than that of the normal control group(P<0.05).The NGF-blocking group significantly reduced the expression of GABAARa and MUC5AC protein compared with the asthma group(P<0.05).5The expression of GABAARa and MUC5AC mRNA in lung tissue:we examined the lung expression of GABAARa and MUC5AC mRNA by RT-PCR.The expression of GABAARa and MUC5AC mRNA were higher than that of the normal control group(P<0.05).Anti-NGF inhibits the expression of GABAARa and MUC5AC mRNA compared with the asthma group(P<0.05).Conclusions1The mRNA and protein levels of GABAARa and MUC5AC in a mouse model of acute asthma are higher than that in the normal control group,which point out that GABAARa was likely linked to excessive mucus production of asthma.2Anti-NGF could inhibit the expression of GABAARa and MUC5AC,and it may play a role in the treatment of excessive mucus production through down-regulating the expression of GABAARa. |
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