Study Of The Mechanism Of Nerve Growth Factor Regulating Airway Neurogenic Inflammation In COPD

Tobacco smoking is the main risk factor for COPD (chronicobstructive pulmonary disease),smoke directly attack and damage theairway epithelium firstly, the damaged epithelium lesion lead to theexpose of the sensory nerve ending, the release of neuropeptides fromairway nerves leading to inflammatory effects. the abnomalinflammation include neurogenic inflammation developed then. Themolecular mechanisms underlying neurogenic inflammation areorchestrated by a large number of neuropeptides including tachykininssuch as substance P(SP) and neurokinin A, or calcitonin gene-relatedpeptide and endothelin-1,resulting in imflammation reaction, such asmucus excretion, hyperemia,increase in vasopermeability, edema,contraction of airway smooth muscle and immunocyte activation.furthermore, only a limited therapeutical effect be achieved by singleneuropeptide antagon.It is urgent and important to study the upstreamregulating factor to block up the release of the neuropeptide and theoccurrence and exacerbation of the inflammatory state.Recently,NGF(nerve growth factor NGF),a upstream cytokinepossessing high potency for regulating neuropeptide,is a study focus formany domestic and overseas scholar.many inflammatory diseases presenthigh levels of NGF.after NGF is synthetized and released by target cellshch as inflammatory cell and structure cell(including airway epithelim,vascular smooth muscle cell, airway smooth muscle cell, fibroblastetc.),NGF can be uptaked by nerve axon periphery and delivered toposterior root ganglion.then the sensitiveness of the sensory neuron inposterior root ganglion enhance,neuropetides such as SP and CGRP (calcitonin gene-related peptide) are synthetized and released withgreat quantity.we find Exogenous NGF upregulated NK-1R(neurokinin-1receptor) expression in the rat lungs,anti-NGF antibody inhibited NK-1Rupregul- ation and neurogenic inflammation in RSV-infected rat lungs inprophase study, indicating that NGF mediates the releases of neuropeptdeand is responsible for the exaggerated neurogenic inflammation in airway.Hence, our objective was to study the interaction between NGF andneuropeptide in COPD. we can hypothesize that nerve growth factor iselevated in COPD,NGF can regulate the neurogenic inflammation in theCOPD through neuropeptides up-regulation. To determine the thought,we checked the quantity of NGF in the serum in COPD patients firstly,then a COPD model and anti-NGF antibody were utilized to research itsmechanism, we completed the studies as follow.Study contents1. NGF changes and the correlation with lung function inCOPD patientsthree groups, including 31 cases in acute exacerbation stage,30 casesin stable stage and normal control group were checked by lung functionand the quantity of NGF in the serum were detected by enzyme-linkedimmunosorbent assay(ELISA).Results showed: As compared with the control group,the serumlevel of NGF in COPD patients in acute exacerbation stage and stablestage were increased (30.3±11.7ng/L vs 7.29±3.4ng/L,17.2±8.5ng/L vs7.29±3.4ng/L,P＜0.01), furthermore, the serum level of NGF in COPDpatients in acute exacerbation stage were elevated compared with instable stage(30.3±11.7ng/L vs 17.2±8.5ng/L,P＜0.05). the significantnegative correlation was found between the NGF and the FEV₁%pre,FEV₁/FVC%, in acute exacerbation stage, rs=-0.73, rs=-0.72(P＜0.01), while in stable stage, (rs=-0.65, rs=-0.68, P＜0.05)。 As a result, we can suggest that NGF play a critical role in theairflow limitation in COPD through the whole course of disease.2. COPD Model establishment.(1) 30 Wistar rats were divided into control and COPD grouprandomly, The COPD model was established by expose the rat to thecigarette and PPE intratracheal injection, lung function tests wasperformed by lung function instrument. (2) the lung tissues pathologicchanges were detected by HE staining. (3)The total cell count anddifferential cell count in the bronchoalveolar lavage fluid (BALF) wereoperated.Results showed: (1) in comparsion with the normal group, PEF(23.61±2.67 ml/s vs 30.96±2.88 ml/s),FEV_0.3 (3.49±0.42 ml vs4.86±0.36 ml) and FEV_0.3/FVC [(62.25±4.84)% vs (86.68±6.12)%]decreased significantly (P＜0.01); MLI (136.92±16.71μm vs 36.70±4.48μm) increased significantly, MAN (11.07±0.26/μm² vs 24.40±2.83/μm²) decreased significantly (P＜0.01); The BALF total cell count(10⁴/ml) (23.93±3.52 VS 15.27±2.40) and the BALF neutrophilsincreased significantly (%) (7.56±1.47 VS 1.80±0.68)(P＜0.01).In study content 2, presenting with airway inflammation andemphysema, obstructive ventilation functional impairment,we createdCOPD model successfully.3. nerver growth factor roles and its mechanism of regulatingairway neurogenic inflammation in COPD.To explore the roles and mechanisms of NGF in COPD and confirmour hypothesis,an anti-NGF group besides the COPD group and thenormal control group was established meanwhile. Lung function,HEstaining and the total cell count and differential cell count in the BALFwere also performed. NGF Quantitative analysis in the BALF weredetermined by ELISA.furthermore,a series of experimental methods were adopted.the localization and half quantitative analysis of NGF in thelungs and dorsal root ganglion(DRG) in the three groups were detectedrespectively by immunohistochemistry assay and Western-blot.NGFmRNA expression in lungs and DRG in the COPD and the control groupwere determined by reverse transcription-polymerase chain reaction(RT-PCR),the changes of SP or NK-1R were investigated byimmunohistochemistry assay or in situ hybridization in the DRG or lungsin the three groups.Results showed: (1) compared COPD group with the normal group,the levels of NGF in the BALF were significantly enhanced (ng/L)(187.18±64.76 vs 75.13±20.44, P＜0.001); the expression of NGF in thelungs and DRG increased significantly in the lungs and DRG inimmunohistochemistry average gray scale (104.23±7.74 vs153.50±8.02, 108.95±7.02 vs 171.03±8.14) (P＜0.01); NGF increasedin Western blot assay(400.4±51.6ng/L vs 198.5±46.2ng/L,318.8±52.6ng/L vs 144.2±32.7ng/L)(P＜0.01); NGF mRNA are enhancedin the lungs (82.07±7.89 vs 51.80±8.34) (P＜0.01), on the contrary, nodifference were found in the DRG (56.67±6.28 vs 51.87±7.01) (P＞0.05);NK-1R increased in the lungs in immunohistochemistry (average grayscale 101.33±6.31 vs 157.67±8.15) (P＜0.01), while SP increased in theDRG in immunohistochemistry (average gray scale 103.40±7.08 vs167.06±7.83) (P＜0.01), no SPmRNA change found in the lungs inimmunohistochemistry (165.53±7.30 vs 168.07±7.05) (P＞0.05),whereas increased in the DRG (103.58±9.63 vs 173.37±12.83)(P＜0.01). (2) compared the anti-NGF group with the COPD group:NGF wre decreased in the lungs and DRG both in immunohistochemistry(average gray scale 130.78±6.35 vs 104.23±7.74, 135.68±7.13 vs108.95±7.02) and Western blot (297.9±43.3ng/L vs 400.4±51.6ng/L,222.3±46.6ng/L vs 318.8±52.6ng/L) (P＜0.01); meanwhile NK-1R decreased significantly in the lungs in immunohistochemistry (averagegray scale 138.20±7.99 vs 101.33±6.31) (P＜0.01); also SP decreased inthe DRG in immunohistochemistry (average gray scale 138.00±9.32 vs103.40±7.08) (P＜0.01); correspondingly SPmRNA decreased in theDRG in immunohistochemistry (average gray scale 143.21±11.30 vs103.58±9.63) (P＜0.01); in BALF, the total cell count and the neutrophilratio decreased (17.13±3.94 vs 23.93±3.52,4.65±0.80 vs7.56±1.47)(P＜0.01); in lung function,PEF, FEV0.3 and FEV0.3/FVC(27.88±2.98 vs 23.61±2.67, 3.93±0.25 vs 3.49±0.42,71.48±5.38 vs62.25±4.84)were elevated significantly (P＜0.05), morphologically, MLIdecreased significantly (101.88±19.67 vs 136.92±16.71,P＜0.01). (3)the significant correlation was found between the NGFmRNA in thelungs and the SPmRNA in the DRG inthe COPD group (rs=-0.81,P＜0.001).we can conclude that NGF contribute to COPD developing.We canalso conclude that NGF may modulate the neurogenic inflammation inCOPD through down regulation the synthesis and secretion ofSP, anti-NGF can amend the pathological change and relieve the airflowlimitation.In conclusion, our current studies showed: NGF can regulateneurogenic inflammation through adjust the SP synthesis and release ofsensory neuron;NGF contribute to the airflow limitation in COPD;dealing with anti-NGF can bring SPmRNA and SP protein downregulation,palliate the airway inflammation in COPD. After the studycontents, we can clarify our hypothesis that NGF serving as an importantregulator in neurogenic inflammation in COPD.