Dysfuntion Of Releasing Adrenaline In Asthma Due To Functional Reduncdancy Primed By Nerve Growth Factor And Its Primary Proteomics Study

BackgroundBronchial asthma is a disease characterized by airway inflammation. There are complicated relationships between airway inflammation and nervous systems which innervate airways, in recent years, many studies have evidenced the concentration of NGF in serum was increased, so more and more attentions were paied to neurogenic inflammation which mediated by nerve growth factor and its role in asthma, some studies suggested that the adrenaline was decreased due to the dysfunction of synthesis and (or) release in asthma, and exogenous NGF can induce the transformation of adrenaline medullary chromaffin cells (AMCC) toward nerve cell, so we can suppose whether elevated NGF in serum would lead to the phenotype transformation of AMCC in asthma and hence the dysfunction of releasing adrenaline? Our studies aimed to investigate the possible causes of releasing adrenaline by internal and exosomatic experiments, and isolate and identify the proteins which play a critical part in this process with the tools ofproteomics.PART 1 STUDY ON NGF EXPRESSION AND PHENOTYPETRANSFORMATION OF AMCC IN ASTHMATIC RATSObjective To investigate the expression of NGF in AMCC and the morphological and functional changes in AMCC. of asthmatic rats. Methods By means of immunohistochemistry (SP) combined with the micro-image analysis to investigate the alterations of NGF immunoreactivity in asthmatic SD rats and by means of light microscopy, and electron microscopy to investigate the ultrastructural changes in AMCC, and detect the concentration of adrenaline and noradrenaline in serum by ELISA. Results The positive immunoreactivity was increased in asthmatic AMCC compared with the controls( (P＜0.05). variant extent vacuolar degeneration-like changes could be observed and there are increases in the number of lipid by the observation of light microscopy, in asthmatic AMCC, while by the observation of electron microscopy, the mitochondria is abundant, the lipid is increased in number, adrenal medullary chromaffin granula is decreased in concentration, and there are some crimples can be observed in karyolemma of the asthmatic rats AMCC. the concentration of adrenaline in serum of the asthmatic rats is decreased, while the noradrenaline is unchanged compared with the control. Conclusions The expression of NGF in AMCC is increased in asthmatic rats and the elevated NGF in serum may have resulted in the phenotypic transformation of AMCC in asthmatic rats which may have lead to the decrease of the concentration of adrenaline in serum so as to involve in the pathogenesis of asthma.Objective To investigate the possible causes of the dysfunction of adrenaline release in asthma rats and identify the role of nerve growth factor (NGF) in this process. Methods The 32 SD rats were randomly divided into four groups: control group(n=8), asthma group(n=8), NGF group (n=8) and anti-NGF group (n=8). The rats of asthma group, NGF group and anti-NGF group were sensitized and challenged with ovalbumin (OVA), and then NGF group and anti-NGF group were treated with NGF and anti-NGF, respectively. Adrenal glands were obtained for electron microscopic examination. The expression of phenylethanola- mine N-methyltransferase (PNMT) was analyzed by immunohistochemistry combined with the micro-image analysis. The concentrations of adrenaline and noradrenaline in serum were measured by ELISA. Results The data from the electron microscopy showed: (1) Compared with the control group, the number of mitochondria increased, while the concentration of the chromaffin granula decreased in asthma, NGF and anti-NGF groups. (2)There are some drumstick-like and villiform processes in the adrenaline medullary chromaffin cells (AMCC) membrane of the NGF group. The results of immunohistochemistry showed that the average gray value of PNMT of the NGF group was 218±38, which was significantly higher than those in the control group (182±24), asthma group (197±33) and anti-NGF group (195±415, t =23.42, 19.76, 17.93, all P＜0.05). Serum levels of adrenaline in the NGF group were (2.9±0.5) ng/ml. They were significantly lower than those in the control group (7.1±0.4) ng/ml, asthma group (5.9±1.7) ng/ml and anti-NGF group (5.7±0.6)ng/ml, t=7.64, 5.41, 4.96, all P±0.01). Serum levels of adrenaline in the asthma group were significantly lower than those in the control group (t=5.64, P＜0.01) however, no difference was found between the asthma group and the anti-NGF group (t=0.87, P＞0.05). Conclusions The NGF may can prime the functional reduncdancy of the adrenaline medullary chromaffin cells (AMCC), which leads to the dysfunction of adrenaline release and hence the pathogenesis of asthma.Objective To study the biologic ethology effects of NGF on primary cultured adrenaline medullary chromaffin cells (AMCC) and investigate the possible mechanism of these changes.Methods To establish primary cultured AMCC by means of enzyme digestion and purify the cells by means of isopycnic gradient centrifugation and differential plating. to observe the morphological and ultrastructural changes after the addition of NGF and detect the concentration of adrenaline and noradrenaline in serum by ELISA.Results confervaceous processes can be observed after 2 days of addition of NGF to the culture and the processes will strench longer as days go by. by the observation of electron microscopy, there are some drumstick-like and villiform processes in the cell membrane and some vesiculation can be observed near the cell membrane of the primary cultured AMCC cells after the addition of NGF. the bioblast is abundant but the structure is not clear in the intracytoplasm and the concentration of adrenaline in serum is decreased after the addition of NGF.Conclusions NGF can prime the functional reduncdancy of AMCC, which will lead to the Dysfunction of releasing adrenaline because of ohenotvoic and functional changes.Objective To compare the proteome of primarycultured adrenaline medullary chromaffin cells (AMCC) treated with NGF with that of control for identifing critical proteins in this process. Methods Tools such as 2-dimensional polyacrylamide gel electrophoresis (2-DE) and matrix associated laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS), coupled with bioinformatics, enabled us to find the differently expressed proteins in the primary cultured AMCC treated with NGF. Results The AMCC cells for 2-DE were treated with NGF, and dissolved in lysis buffer to obtain the total protein of the cells. There were about (752±234), (693±246) proteins detected by the Coomasie Briliant Blue staining in the control group and NGF treated group perspectively. Compared with the control group, there were 37 down-regulated proteins, and 48 up-regulated proteins in the NGF treated group. We randomly selected 20 proteins among the spots on the basis of the intensity and the significant difference in abundance of changes. The MALDI-TOF was used for the direct identification of these proteins. In combination with database searching, 17proteins were successfully identified by the MALDI-TOF. Among the 17proteins identified, RhoGDI alpha,HSP27 and Peripherin intermediate filament protein may have play a important part during this process. Conclusions RhoGDIα,HSP27 and Peripherin intermediate filament protein may have play a critical role in this process which would lead to the dysfunction of releasing adrenaline in asthma.