The Contribution Of Nerve Growth Factor Receptor NGFR To The Pathogenesis Of COPD

BackgroundChronic obstructive pulmonary disease(COPD)is the third leading cause of death in the world.In 2016,more than 3 million people died from chronic obstructive pulmonary disease(COPD),accounting for 6%of all deaths.Although most scholars believe that airway changes and pulmonary parenchymal destruction are two different aspects of COPD,some studies have found that COPD patients first develop terminal bronchioles narrowing and destruction,then central lobular emphysema occurs around them,suggesting that the factors causing airway changes may be responsible for COPD.The first histological change of airway epithelium in COPD patients is the hyperplasia of basal cells of airway epithelium.The bronchial epithelium of normal people is mostly pseudostratified ciliated columnar epithelium composed of cilia,secretory cells and basal cells.Basal cells are KRT5-positive cubic cells closely bound to the basement membrane.They have high nuclear-plasma ratio and stem/progenitor cell function.They can differentiate ciliated cells and secretory cells in vitro.Under normal physiological conditions,the adult airway epithelium is renewed about 1-4 months,and the basal cells are in a relatively dormant state.Only a small number of intermediate cells can be observed.However,in the case of injury,such as cigarette stimulation,basal cells proliferate,and intermediate cells increase.Under the influence of different factors in the microenvironment,they can regenerate normal airway epithelium,or lead to airway epithelium remodeling,which plays an important role in the pathogenesis of asthma or COPD.The number of basal progenitor cells,self-renewal ability and pluripotent differentiation potential decreased in patients with COPD,which may be one of the causes of COPD.The mechanism underlying the control of basal cell differentiation is still unclear.The neurotrophic factor family consists of several different members:nerve growth factor(NGF),brain-derived neurotrophic factor(BDNF),NT-3,and NT-4.There are two main types of receptors:one is the high affinity receptor Trk,and the other is the low affinity receptor NGFR,which belongs to the tumor necrosis factor superfamily.With the deepening of its research,it has been found that neurotrophic factors and their receptors not only exist in the nervous system,but also exist in many systems of the body including the lung and airway.It has been found that the positive rate of serum BDNF and NGFR in peripheral blood mononuclear cells of COPD patients is significantly higher.In previous experiments,we confirmed the presence of BDNF in bronchial epithelial cilia and the high expression of NGFR in bronchial epithelial basal cells by immunohistochemistry.NGFR has been found to be highly expressed in stem cells or somatic cells with stem cell function in multiple systems and to play a role in cell differentiation.We therefore speculate that NGFR may play a role in the pathogenesis of COPD by affecting the proliferation and differentiation of basal cells.ObjectiveTo research the role of NGFR in the pathogenesis of chronic obstructive pulmonary disease.1.To observe the difference of NGFR expression between chronic obstructive pulmonary disease patients and healthy smokers.2.The effect of NGFR expression on the proliferation of airway epithelial cells.3.The effect of NGFR expression on the differentiation of airway epithelial cells.Methods1.Case selectionTwenty-four smoking patients with unilateral lung lesions(lung cancer,hemoptysis,etc.)underwent bronchoscopic examination in our hospital’s bronchoscopic room(smoking index more than 200 years),and their medical history and related laboratory data were collected.Among them,12 were diagnosed as chronic obstructive pulmonary disease(COPD)according to risk factors,symptoms,physical examination,imaging manifestations,lung function,and accorded with the Global Initiative for Chronic Obstructive Pulmonary Disease(GOLD)2013 diagnostic criteria;12 were smoking non-COPD patients.2.Cell experiment(1)The expression of NGFR in bronchial epithelial cells of smoking COPD patients and smoking non-COPD smokers was detected by Western Blot method.(2)BDNF polypeptide and cigarette smoke extract were used to treat human airway basal cells and observe the effect on proliferation.(3)NGFR shRNA lentiviral particles were used to interfere with the expression of NGFR in human airway basal cells.(4)Cigarette smoke extracts were added to NGFR shRNA interference and nonsense interference airway basal cells respectively to observe whether NGFR and cigarette smoke extracts had interaction with cell proliferation.(5)TUNEL method was used to verify the apoptosis promoting effect of cigarette smoke extract on basal cells.(6)The basal cells were cultured at the gas-liquid interface to differentiate into human pseudostratified ciliated columnar epithelium in vitro.(7)Cigarette smoke extracts were used to treat human airway basal cells and observe the effect on their differentiation.(8)NGFR shRNA lentiviral particles were used to interfere with the expression of NGFR in human airway basal cells,and the effect of NGFR shRNA lentiviral particles on its differentiation into ciliated cells was observed.(9)Cigarette smoke extracts were added to NGFR shRNA interference and nonsense interference airway basal cells respectively to observe whether NGFR and cigarette smoke extracts interact with cell differentiation.Results1.NGFR,TP63 and KRT5 were expressed in all primary cultured human bronchial epithelial cells.2.The growth rate of basal airway cells was slower and the expression of NGFR was significantly higher in smoking COPD patients than that in smoking non-COPD patients(0.84±0.05,0.67±0.05,P<0.05)3.BDNF had no significant effect on the survival of basal airway cells in normal amplification stage(P>0.05).4.Interference with NGFR expression had no significant effect on the survival of human airway basal cells at normal expansion stage(P>0.05).5.Cigarette smoke extracts at concentrations of 5%and above can significantly reduce the survival rate of human airway basal cells.6.Interference with NGFR expression can attenuate the apoptosis of human airway basal cells induced by cigarette smoke extract.After treatment with 5%cigarette smoke extract for 24 hours,the survival rate of basal cells in senseless shRNA +5%cigarette smoke extract group(0.79 ± 0,02)was significantly lower than that in senseless shRNA group,while that in NGFR shRNA + 5%cigarette smoke extract group(0.92 ± 0.02)was significantly lower than that in senseless shRNA+5%cigarette smoke extract group(0.79 ± 0.02)increased significantly.7.TUNEL assay showed that 5%cigarette smoke extract group(10.0±1.1%)significantly increased the apoptosis of human airway basal cells compared with the control group(2.8 ±0.2%)(P<0.05).The apoptosis of human airway basal cells in NGFR shRNA + 5%cigarette smoke extract group(5.3 ±0.6%)was significantly lower than that in nonsense + 5%cigarette smoke extract group(10.0+ 1.1%)(P<0.05).8.Human airway basal cells were inoculated into the Transwell chamber.After 2-3 days of amplification and 4 weeks of differentiation,multiple layers of cells were observed under microscope with cilia swinging.Immunofluorescence staining with acetylated a-tubulin showed specific expression in the top layer of the cells.HE staining showed the formation of stratified ciliated columnar epithelium.Immunofluorescence staining with E cadherin,acetylated alpha tubulin,NGFR and DAPI showed that there was continuous cilia imaging near the top of the airway,almost all cells showed E-cadherin imaging,and 1-2 layers of cells near the membrane showed NGFR expression.9.The basal cells transfected with nonsense shRNA and NGFR shRNA lentiviral particles were differentiated and cultured.The number of cilia in NGFR shRNA group was significantly higher than that in nonsense shRNA group.The expression of acetylated a-tubulin protein in NGFR shRNA group(0.83 ±0.07)was significantly higher than that in control group(0.55±0.05).(P<0.05)10.Cigarette smoke extract was continuously added to the lower chamber culture medium at the beginning of the differentiation maintenance stage of the bronchial epithelial air-liquid interface culture.The proportion of cilia cells in the lower chamber culture medium was significantly reduced compared with that in the control group.Cigarette smoke extract and nonsense shRNA were quantitatively compared by Western Blot method.The expression of acetylated a-tubulin,a marker of cilia,was significantly lower in group A(0.49±0.01)than that in group A(0.62 ±0.03)(P<0.05)11.Cigarette smoke extract was continuously injected into the lower chamber at the beginning of the maintenance stage of bronchial epithelial differentiation in the air-liquid interface culture.The proportion of cilia cells in NGFR shRNA group was significantly higher than that in the control group.Western Blot assay showed that the expression of acetylated alpha tubulin protein in cigarette smoke extract ＋NGFR shRNA group(0.66 ± 0.05)was significantly higher than that in control cigarette smoke extract + nonsense shRNA group(0.49±0.01).(P<0.05)Conclusion1.NGFR pathway is one of the ways of cigarette smoke induced airway remodeling.2.High expression of NGFR may be one of the important causes of airway ciliary loss in COPD patients.3.NGFR is an important receptor in the pathogenesis of chronic obstructive pulmonary disease.