The Effect Of Adiponectin Expressed By Bronchial Epithelial Cells In The Inflammation Of Acute Exacerbation Of Chronic Obstructive Pulmonary Disease

Background:Chronic obstructive pulmonary disease(COPD)is a common global disease that endangers human health.The incidence of COPD is increasing,and it has become the fourth leading cause of hunan death in the world.The exacerbation of COPD(AECOPD)is important in the process of the clinical events.The occurrence of repeated exacerbations will accelerate the deterioration of lung function,seriously impact on the patient's quality of life,increase the risk of death and the economic burden.Therefore,the further study of the pathogenesis of AECOPD will provide a better theoretical basis for the prevention and treatment of AECOPD.Current studies suggest that COPD is a chronic inflammatory disease that involves respiratory epithelial cells,inflammatory cells,chemokines and cytokines.Many cytokines are involved in airway inflammation in COPD,and this cytokine-mediated airway inflammation is one of the important pathogenic features of COPD.The acute exacerbation of the disease is the further aggravation of its chronic inflammation.Adiponectin(APN)is a cytokine that is mainly secreted by adipocytes and has important in vivo biological activities,such as regulation of glycolipid metabolism and anti-atherosclerotic,anti-apoptotic,antiinflammatory and anti-oxidative effects.In recent years,it has been found that APN regulates COPD inflammation through autocrine or paracrine pathways.Daniele et al found that serum APN in COPD patients inhibited the release of pro-inflammatory factors such as tumour necrosis factor-alpha(TNF-?),which indicates that APN plays a protective role in the pathogenesis of COPD as an anti-inflammatory factor.While Tomoda et al found that the serum APN levels of COPD patients were significantly higher than those of normal people.Kirdar et al showed that serum levels of APN were significantly higher in patients with acute exacerbation of COPD(AECOPD)compared to those of the stable group and suggested that APN might be a marker of a systemic inflammatory response in COPD.Sood et al indicated that APN promotes inflammation in some cases.APN plays a dual role in the occurrence and development of COPD,but the role of APN in COPD,especially in exacerbation phase has not been specifically explored.Therefore,further study on the role of APN in the pathogenesis of AECOPD may contribute to the early prevention and treatment of AECOPD,which has strong application value and research prospects.Objective:1.This study prospectively analysed serum adiponectin(both HMW and globular isoforms)levels in patients with AECOPD caused by bacterial infection and tried to explore the changes of serum adiponectin levels before and after anti-infection treatment.2.We also used lipopolysaccharide(LPS),apoptotic cells and inflammatory factors to stimulate bronchial epithelial cells to explore the role of adiponectin and its possible mechanism,so as to lay a foundation for regulating or controlling the mechanism of adiponectin on AECOPD.3.We used adiponectin and LPS to stimulate bronchial epithelial cells,to investigate the role of adiponectin in LPS induced inflammation.Methods:1.Between January 2016 and December 2016,40 Chinese patients with AECOPD induced by bacterial infection at Shanxi Dayi Hospital affiliated to Shanxi Medical University were enrolled in the current study.Diagnosis of AECOPD was based on FEV1/FVC<70% according to the Global Initiative for Chronic Obstructive Lung Disease(GOLD)criteria that was published in 2016.Exclusion criteria included severe hepatic,renal or endocrine disorders and cardiac failure.During hospitalisation,all patients received positive anti-infection treatment and other therapeutic regimens according to the GOLD criteria.This study adhered to the guidelines of the Declaration of Helsinki and was approved by the Ethics Committees of Shanxi Medical University(reference number 2011061 from March 2011).All patients provided written informed consent to participate in the study and granted permission to use their samples.All enrolled patients underwent pulmonary function tests before hospital discharge.Physiological assessment included measurements of forced expiratory volume in 1 second(FEV1)and forced vital capacity(FVC).Lung function measurements were performed according to American Thoracic Society(ATS)recommendations.2.Fasting blood samples were taken from 40 AECOPD patients induced with bacterial infection twice at the beginning and end of the antiinfection treatment.After incubation at room temperature for 2 hours,the samples were centrifuged at 1500 g for 10 minutes to obtain serum.Serum adiponectin(HMW and globular isoforms)and IL-6 levels were measured using ELISA kits.3.The immortalised human bronchial epithelial cells(16HBECs)were purchased from Bena Culture Collection.The cells were cultured in high glucose Dulbecco's Modified Eagle Medium with 10% certified FBS,100 U/mL penicillin and 100?g/mL streptomycin at 37 C in a humidified 5% CO2 incubator.Growth-arrested HBECs(in a low concentration of serum for 24 hours after reaching 70% confluence)were then stimulated for 6,12 or 24 hours with recombinant human TNF-?(0,50,100 or 150 ng/mL),IL-6(0,50,100 or 150 ng/mL),LPS(0,5,10 or 20 ng/mL),apoptotic cells(0,1×106,5×106 or 10×106)or anti-TNF-?(50,100 or 150ng/mL)for 6 hours and then 10 ng/mL LPS.Then,the expression of adiponectin was detected by qRT-PCR and western blotting,the secretion of adiponectin was dectected by ELISA.4.Growth-arrested HBECs(in a low concentration of serum for 24 hours after reaching70% confluence)were then stimulated for 24 hours with adiponectin(0,1,2,5,10,20ug/ml),LPS(100ng/ml)or adiponectin(10ug/ml)combined with LPS(100ng/ml).The levels of pro-and anti-inflammatory factors(TNF-??IL-1??IL-6?IL-8?IL-4 and IL-10)were detected by ELISA.5.And then completed the statistical analysis.Results:1.The concentration of both APN isoforms(HMW and gAd)and in the serum of patients with COPD in the acute exacerbation phase was significantly lower than that in the remission stage(t or z = 2.084 or-5.511 both P<0.05),but the difference in IL-6 was not significant(t =0.919 P>0.1).2.Although there was no significant correlation between the serum levels of either APN isoform and the parameters of pulmonary function tests or hospital stays(All P>0.05),we found that the concentration of gAd divided by the level of HMW was negatively correlated with the number of days of hospital stay(r=-0.494 P=0.001).3.In the ROC curve analysis of the multivariable logistic regression model to predict AECOPD induced by bacterial infection,the results showed that the sensitivity and specificity of HMW were better than that of gAd or gAd/HMW,and the AUC of HMW was the largest(76.3%)and indicated potential clinical usefulness.4.Stimulating HBECs with TNF-? showed that TNF-? promoted the mRNA expression of APN in a dosedependent but not time-dependent manner,and significant upregulation began at a concentration of 100 ng/mL.But 50 ng/mL TNF-? had been able to significantly promote the protein expression of APN.Strangely,although TNF-? promoted APN secretion in a dose-dependent manner,the difference was not significant.5.IL-6(100 ng/mL)also promoted the expression of APN but significantly inhibited its expression after 24 hours of stimulation.Conversely,150 ng/mL IL-6 significantly inhibited the expression of APN.However,the trend in the APN mRNA and protein expression was not entirely consistent,and IL-6 did not affect the secretion of APN,indicating that APN transcription,translation and secretion are affected by other factors.6.Lipopolysaccharide(LPS)is the main factor that induces inflammation after bacterial infection.LPS stimulation of HBECs inhibited the expression of APN in a dose-dependent manner,and 15 ng/mL LPS had a significant inhibitory effect.Similarly,15 ng/mL LPS significantly inhibited the protein expression of APN.LPS inhibited the secretion of APN,but the difference was not obvious.7.In addition,when apoptotic cells were co-cultured with HBECs,apoptotic cells promoted the expression of APN in a dose-dependent manner,and 5 × 106 cells significantly promoted the expression of APN.Apoptotic cells enhanced the protein expression of APN,but the change level was not significant.Apoptotic cells also promoted the secretion of APN in a dose-dependent manner,but only the group with 10 × 106 cells was significantly different.8.Although anti-TNF-? did not affect the expression or secretion of APN in HBECs,when HBECs were pretreated with anti-TNF-? and then LPS was added,the expression and secretion of APN increased significantly with increasing anti-TNF-? concentrations,and anti-TNF-? at100 ng/mL significantly antagonised LPS-mediated inhibition of APN expression.9.When HBECs were pretreated with anti-TNF-? and then apoptotic cells were added for 6hours,we could not found that the role of apoptotic cells in promoting the expression and secretion of adiponectin in HBECs.10.APN significantly promote the secretion of pro-inflammatory factors(TNF-?,IL-1?,IL-6 and IL-8),but the effect was not dose dependent.11.APN increased the secretion of anti-inflammatory factors(IL-4 and IL-10)in a dose dependent manner.12.APN(10ug/ml)significantly blocked the effect of LPS on pro-inflammatory factor secretion and enhanced the effect of LPS on the secretion of anti-inflammatory factors.Conclusions:1.APN expression was increased in AECOPD patients after treatment,suggesting that the increased expression of APN promotes the rehabilitation of AECOPD patients.2.HBECs secrete APN and many factors regulate the expression and secretion of APN.3.APN plays a role in regulating the dynamic balance of inflammation during the development of COPD and inhibits further expansion of inflammation.