Study On Neurogenic Inflatmmation Signal Conduct Pathway Caused By Nerve Growth Factor

Asthma is an airway inflammation disease characterized by recurrent reversible airway obstruction and development of airway hyperreactivity, participated by many kinds of immunity inflammation cells (such as adipose cells, acidophilia granular cells, lymphocyte, and so on) and the cell components. At present, it is believed that the neurogenic inflammation is one of the most important mechanisms of asthma. Recent studies discovered that NGF as representative neurotrophin factor possibly induced the release of tachykinins, caused airway hyperreactivity and airway reconstruction, and contributed to pathology of asthma.Some researchers reported that NGF was elevated in patients' blood serum with the allergy disease and asthma. Usually, it was obviously higher (P＜0.01) than in the healthy control group's; NGF inpatients' blood serum with allergy asthma is obviously higher than in patients' with the non-allergy asthma, and the more serious asthma, the higher airway hyperreactivity, and showed higher level of NGF in blood serum. NGF is close with airway hyperreactivity. In a research, injecting 8ng/kg NGF into a guinea pig's intravenous induced the airway hyperreactivity to the histamine, and the airway resistance obviously after half hour, and in 3 hours, it eliminated the airway functional change caused by NGF with anti-NGF while it could suppress the airway hyperreactivity conducted by NGF with neurokinin acceptor-1 inhibitor SR140333. It was prompted that airway hyperreactivity caused by neurokinin. NGF is involed in neurogenic inflammation through neurokinins, such as SP and neurokinin A, and so on. The neurokinin is the powerful inflammation factor affected the airway function: it can cause smooth muscle contraction, enhancement of airway hyperreactivity, promotion vasodilatation, increasment of the capillaries insight increase and mucus hypesecretion, and can aggravates the condition of asthma.NGF can lure sense neuron of airway inflammation to increase SP expression. Our former findings also showed its NK-1R expression was upregulated in the lung organization after rats was injected with NGF, and it reduced neurogenic inflammation of lung organiation infected by RSV with the anti-NGF, and its NK-1R expression was declined. It prompts that NGF has the direct relationship to produce tachykinin. In recent years, the signal conduct mechanism became a study hot spot more and more at asthma function. With which signal conduct pathway does NGF upregulate neurokinin SP and anticipate asthma neurogenic inflammation actually? At present, the most findings related NGF receptor and the signal conduct pathway were obtained by studying on the nerve cell's model and the vitro tumor cells. However, studies on the lung's and the airway's structure and infiltrating inflammatory cells are few. Recently, a research discovered that after being given vitro NGF intervention, trkA and SP expression of protein was strengthened in respiratory nerve fiber's cholinergic neuron under the controlling mouse. It prompts that NGF receptor trkA still plays an important role in neurogenic inflammation with SR After NGF and trkA union, the receptor appears two polymerization, and it causes phosphorylation of the acceptor's interior tyrosine, as well as following activation downriver signal conduct enzyme, including phospho-lipin enzyme C (PLC-γ), adjustment protein Shc, Phospho-lipin acid radical myokinase-3 (PI3). It participates the physiological change in the cells differentiation, activates Ras/MAPK related pathway through PLC-γand Shc's downriver signal conduct pathway. Ras/MAPK way is an important way in the cells signal conduct pathway. Generally, the Ras-MAPK cascade includes Ras/Raf/MAPKK/MAPK/C-fos. Ras, the G protein, existing by GTP form, unifies with Raf, and is activated. Raf returns to the cells membrane through Ras, and is phosphorylized and acidificated. Raf kinase phosphorylats MAPK kinase (MAPKK, also called MEK), and MAPKK activates MAPK (MAPK also called ERK). Instant inchoate original cancer gene c-fos is the downriver member to activate the MAPK function, and the activated MAPK transfers to the cells nucleus and activates c-fos and the c-jun gene. Its product c-Fos forms the different source dimer with c-Jun, named the activated protein-1 (AP-1). The latter unifying the TPA response part adjusts the activated MAPK by gene expression related to the cells growing and cells differentiation, and cause some important cytoblastema proteins and the membrane protein phosphorylation, or after MAPK nucleus indexing, activates the intranuclear transferring factor and the nucleoalbumin (such as c-myc, C-jun, C-fos), and regulates gene transfer and expression, thus separately, and transmit biology effect in the level from of protein kinase and transfer. MAPK also promotes the smooth muscle to multiply, the transferring factor and the cytoblastema protein to phosphorylize, and participates hormone to cause resistant asthma. Obviously, to study the result of asthma neurogenic inflammation function mechanism caused by NGF will be a new treating target since the Ras-MAPK signal transmitting way plays the vital role in asthma pathogenesis.1. Study of nerve growth factor regulating asthma rats' Ras-MAPK signal conduct pathwayFirstly, SD rats were divided into 3 groups randomly: Asthma group, control group, anti-NGF group, each group of 12. The asthmatic model was established by inhalation and injection of ovalbumin (OVA). Lung tissue of the three groups were detected the pathologic changes by HE staining to observe the model of airway inflammations. Quantitative analysis and localization of the lung tissues, dosal root ganglion pan-Ras, pERK, c-fos protein were determined.Results showed: in the lung tissues, pan-Ras, pERK, c-fos positive reaction matter assumes yellow-brown color, mainly locating in the cytoplasma. Asthma group assumes streng positive reaction, the control group assumes weak positive reaction. The average gray value of pan-Ras, pERK, c-fos in asthma group (152.65±15.95, 152.89±13.52, 130.62±10.46) respectively is lower than that in the control groups (174.74±14.61,179.55±13.83,165.04±11.57) (P<0.01). Comparing the anti-NGF group with the asthma group in rats' lung tissues, the immune reaction masculine cells expression reduced obviously, the value of pan-Ras, pERK, c-fos respectively is 174.08±14.94, 177.80±15.08 and 165.02±11.33. In the dosal root ganglion, panRas, pERK, c-fos positive reaction matter assumes yellow-brown color, located mainly in the cytoplasma, but pERK is located in the cytoplasma and the cells nucleus. The control group assumes the weak positive reaction while the asthma group strong positive reaction. The anti-NGF group assumes the weak positive reaction. Comparing to the value of the control group pan-Ras, pERK, c-fos (171.44±16.43, 175.34±13.24, 175.51±7.76), that of the asthma groups (151.40±15.89, 154.84±13.22, 137.21±13.05)obviously reduced, P<0.01. After the anti-NGF intervened, pan-Ras, pERK, c-fos the expression reduced, which (168.41±17.27, 170.78±13.34 and 163.89±8.92). Comparing to the asthma group, it has statistics difference, P<0.05.2. Study of NGF inducing the NHBEC Ras-MAPK signal conduct pathwayThe NHBEC was cultured in the DMEM medium containing 10% calf blood serum. 8 groups were divided: (1) A group: the control group; (2) B group: NGF group; (3) C group: NGF+PD98059 group; (4) D group: PD98059 group; (5) E group: NGF+PMA group; (6) F group: PMA group; (7) G group: NGF+AG490 group; (8) H group: AG490 group. The cells intervention was carried on separately. After intervened, immunity signed semi-definite quantity was taken to detect the protein content in cells c-fos, p-ERK and total-ERK while the RT-PCR method to each group of cell NK-1R content. And immune cells chemical qualitative applied to observe each group of cell express NK-1R.Result showed that comparing with the control group, the level of NHBEC c-fos protein and phosphorylized ERK1/2 protein affected in 30min by NGF rose to the peak value remarkably. The total-ERK protein level didn't influenced by NGF. But, in the NHBEC of the PD98059 and the AG490 groups, the level didn't be detected nearly; the level rose to the peak value remarkably in the NGF group affected in 30min by NGF, and c-fos/GAPDH and p-erk/GAPDH light density ratio was 0.8354±0.1012, 1.3187±0.2078 respectively, comparing with the control, P<0.01. PD98059 might suppress NGF to induce the NHBEC to express the level completely, and c-fos/GAPDH and pERK/GAPDH light density ratio were 0.3012±0.1034 and 0.3146±0.0957 respectively, comparing with the NGF group, P<0.01. PMA might stimulate NGF to induce the NHBEC to express c-los and the phosphorylized ERK1/2 protein. But STAT3 conduct way specific inhibitor AG490 could not suppress NGF to do that. The immune cell chemistry findings showed that NK-1R expression assumed negative reaction in the control and the PD98059 group; strong positive in the NGF and the NGF+PMA groups; positive in the PMA group; and weak positive in the NGF+PD98059 group. The RT-PCR result showed that, the NK-1RmRNA level expressed few in the PD98059 and the AG490 group, comparing with the control group; but in the NGF group, affected 1h by NGF, the NK-1RmRNA level rose to 1.0682±0.1597 remarkably, comparing with the control group (0.3753+0.0642), P<0.01. The PD98059 might suppress NGF to induce the NHBEC to produce NK-1RmRNA (0.4787±0.0981), comparing with the NGF group, P<0.01. PMA might stimulate NGF to induce the NHBEC to express NK-1RmRNA.3.Study of applying the c-fos RNAi technology on Ras-MAPK signal conduct pathway induced by the NGFNHBEC was cultured, the process was designed that c-los siRNA trans-dyes into cells through the positive ion fat plastid, and the NGF intervention was carried on. The NGF+fos1 group, The NGF+fos2 group, The NGF+fos3 group, the NGF+irrelevant siRNA group, the NGF+HK siRNA group, the NGF group and the control group were decided. Then, the total protein immunity signed semi-definite quantity was extracted to determine each group of c-los protein expression separately; the total RNA RT-PCR semi-definite quantity was extracted to determine NK-1RmRNA, and application immune cell chemical definite quality was applied to determine each group of NK-1R expression.Result showed: stimulated by NGF, the c-los protein expression increased obviously comparing with the control group, c-fos/GAPDH extinction ratio 1.1861±0.1427 comparing 0.3122±0.0914,P<0.01;c-fos shRNA could suppress NGF stimulating the NHBEC to express the c-fos protein, the value of c-fos/GAPDH extinction ratio is 1.0965±0.2591, 0.6574±0.1373, 0.3773±0.136 respectively; comparing with the NGF group, P<0.01; Irrelevant shRNA, the spatial matter did not have this function. Compares with the control group, the NHBEC affected by NGF in 1 hour, NK-1R mRNA increased remarkably, NK-1R/beta-actin extinction ratio (1.0047±0.1106) comparing with the control group (NK-1R/beta-actin extinction ratio, 0.4131±0.0473), P<0.01. C-fos shRNA could suppresse NGF to induce the NHBEC express NK-1RmRNA,its NK-1R/beta-actin extinction ratio (0.6214±0.0971) comparing with the NGF group, P<0.01.Irrelevant shRNA, the spatial matter did not have this function. The immune cell chemical result showed that the NK-1R positive product is brown, by NHBEC cytoblastema coloration primarily. The NK-1R expression assumed negative reaction in control group; the NGF and the NGF+HK siRNA assumes strong positive reaction; NGF+the irrelevant siRNA group assumed positive reaction; but the NGF+siRNA assumed weak.The above three parts prompted: NGF might participate asthma neurogenic inflammation; Its functional mechanism might be upregulating expression of neurogenic inflammation medium SP receptor-NK1R through Ras-MAPK signal conduct pathway; siRNA could cut off Ras-MAPK signal conduct pathway, suppress SP receptor -NK1R expression, and become a new way treating asthma hopefully.