FSTL1 Aggravates Airway Inflammation And Remodeling In COPD By Promoting Autophagy

Chronic obstructive pulmonary disease(COPD)is one of the recognized life-threatening respiratory diseases.In the 21st century,it will still be a major issue that endangers human health.With an ageing population worldwide,the impact of COPD on human health will be more serious.Nowadays,more than 100 million people over 40 have been affected by COPD in our country.Chronic obstructive pulmonary disease is characterized by persistent airflow limitation and emphysema associated with airway remodeling and inflammation.Airway remodeling in COPD involves airway epithelial cell proliferation,reticular basement membrane(RBM)thickening,collagen deposition,peribronchial fibrosis,airway epithelial-mesenchymal transition and bronchial smooth muscle cell proliferation.Meanwhile,COPD is a complex inflammatory disease involving multiple types of inflammatory cells and mediators.During the emergence and progression of the disease,complex congenital factors such as host factors and extrinsic factors such as smoking and pollution promote the disease jointly.Although chronic obstructive pulmonary disease is a preventable and treatable disease,there is still no accurate treatment method to effectively delay or reverse the progress of the disease.In summary,more in-depth studies are needed to answer and solve some key problems.Follistatin-like protein 1(FSTL1)known as Follistatin related protein(FRP)。It is engaged in the modulation of various physiological processes,including cell apoptosis,metaboly,immunoreaction and endocrine.FSTL1 is known to be associated with a variety of diseases,including cardiovascular disease,cancer and autoimmune diseases.FSTL1 is engaged in lung growth and maturation.Studies have shown that FSTL11 deficiency leads to postnatal death as a result of respiratory failure due to multiple defects in lung development.FSTL1 also participated in the pathogenesis and development of diseases including bronchial asthma,pulmonary fibrosis,and pulmonary infection.FSTL1 is a novel inflammatory mediator,which plays an essential role in the regulation of inflammatory cells.Inflammation is a characteristic change of many chronic respiratory diseases,especially in chronic obstructive pulmonary disease.Levels of FSTL1 in COPD patients with pulmonary hypertension are elevated in recent studies.Autophagy is a mechanism that relies on lysosomes to degrade organelles or proteins to achieve self-renewal and self-protection of cells.Therefore,dysfunction of autophagy can cause various diseases.Researches proved that autophagy-related protein ATG 8/microtubule-related protein-ILC3 is a candidate molecular target for chronic obstructive pulmonary disease.Further studies have confirmed that autophagy-related proteins may exert an important influence in the pathological formation of emphysema in COPD.Clinical study found that increased levels of autophagy(LC3Ⅱ/Ⅰ and Beclin-1)in patients with COPD were negatively correlated with the predicted Forced Expiratory Volume in 1 second(FEV1),and positively correlated with the circulating proinflammatory cytokines IL-6,IL-8 and TNF-α.These studies indicate that autophagy is an important indicator to evaluate the incidence and severity of COPD.The above researches show that the FSTL1 and autophagy may generate vital influences in COPD airway inflammation and remodeling.However,what is the relationship between FSTL1 and autophagy and how do they mediate airway inflammation and remodeling,there is no report.In view of the above problems,this topic mainly studies the mechanism of airway inflammation and remodeling mediated by FSTL1 and autophagy so to find a therapeutic target for COPD.ObjectivesTo explore the specific mechanisms of FSTL1 and autophagy mediated airway inflammation and remodeling in COPD,and to find an effective target for reducing airway inflammation and remodeling in COPD.Methods1.Expression of FSTL1 and autophagy in COPD patients.1.1 PatientsSerum samples from patients with stable COPD,the patients were treated at Qilu Hospital of Shandong University between January 2017 to January 2018(n=25).The control group(n=28)were obtained from non-COPD patients.Lung samples from patients with COPD(n=7)were collected from patients who underwent pneumonectomy for suspected or confirmed lung cancer.The lung tissue specimens of the control group(n=6)were collected from the body donation center of Qilu Hospital.The cadavers were collected without a history of chronic obstructive pulmonary disease before life,and the causes of death were not respiratory diseases.All the subjects had no other respiratory diseases,such as bronchial asthma,interstitial pulmonary disease,silicosis,or pulmonary infectious diseases.This research was ratified by the Ethics Committee of Qilu Hospital of Shandong University and has obtained written informed consent.1.1.1 Inclusion criteria of COPD group:(1)Diagnosis According to GOLD 2016 and the lung function were classified.1.1.2 Exclusion criteria:(1)Unstable angina or myocardial infarction,pulmonary embolism and cerebral infarction occurred in recent 1 month.(2)Severe hypertension,systolic blood pressure>180mmHg,diastolic blood pressure>120mmHg.(3)Malignant arrhythmia.(4)Extremely weak body.(5)Patients with mental diseases and rheumatoid arthritis.1.2 Hematoxylin&Eosin(H&E)staining was used to observe the pathological manifestations of lung tissue.1.3 Immunohistochemical staining was employed to test the expression of FSTL1 and autophagy marker LC3B in lung samples.1.4 The level of FSTL1 in serum was determined by enzyme-linked immunosorbent assay.2.Effects of FSTL1 and autophagy on airway inflammation and remodeling in COPD.2.1 Animals.Age,weight,and sex-matched(Eight-week-old,20±3g,female)wild-type C57BL/6 mice(6 mice in each group)were recruited from Animal Experimental Center of Shandong University(Jinan).The mice lived at room temperature in a pathogen free environment,controlled circadian rhythm,and had free access to drinking water and standard laboratory feed.The Fstl1flox/+ mice were generously presented by Prof.Xiang Gao of Nanjing University and Prof.Xu Zhang of Institute of Neuroscience,Shanghai Institute of Biological Sciences.Cmv-Cre mice were purchased from the Animal Experiment Center of Nanjing University.FSTL1+/-mice were obtained by mating FSTL1flox/+ mice with CMV-Cre mice,which were determined by genotype detection.2.2 Animal2.2.1 group:Randomly divided into three groups,6 in each group,include CON-WT,CS-WT,CS+3MA-WT,CON-FSTL1flox/+,CS-FSTL1flox/+,CON-FSTL 1+/-CS-FSTL1+/-.2.2.2 CS inductionCS induction was performed for 12 weeks,6 days a week,2 times a day,with 4 Harden filter cigarettes(10 mg of tar,1.0 mg of nicotine,12 mg of carbon monoxide),using a homemade smoke cage for CS induction.Mice in the control group were exposed to indoor air under the same conditions.2.2.3 3-methyladenine(3-MA)treatment3-methyladenine(Sigma,USA)was intraperitoneally injected for 12 weeks at an injection dose of 20mg/kg/day.Abdominal injection was completed 2 h before CS induction.2.3 HE(hematoxylin&eosin)staining was used to observe the pathological changes of lung tissue in experimental mice.2.4 Immunohistochemical staining was used to detect the expression of FSTL1,LC3B,P62,collagen 1 and α-smooth muscle actin(α-SMA).2.5 The levels of IL-8,TNF-α and LTB4 in bronchoalveolar lavage fluid were determined by enzyme-linked immunosorbent assay(ELISA).2.6 Lung function was measured for total lung volume and lung compliance.2.7 The expression of autophagosomes in airway specimens was observed by transmission electron microscopy.Results1.There was no statistical difference in gender,age and BMI between COPD patients and control group.HE staining of respiratory tract specimens of COPD patients showed inflammatory infiltration,RBM fragmentation,loss of airway epithelial integrity and ciliary lodging.2.Immunohistochemical staining showed that the positive areas of FSTL1 and LC3B in airway epithelial cells and interstitium were significantly higher than those in the control group.3.The level of FSTL1 in serum of COPD patients was obviously higher than that in control group by ELISA.4.FSTL1 was extremely expressed in the airway epithelial cells and lung interstitium of CS-induced mice.The results of western blot and immunohistochemical staining revealed that FSTL1 was visibly expressed in the airway epithelial cells and lung interstitium of CS-induced mice than in the control group.5.Increased activity of autophagy in CS-WT mice was accompanied by increased airway inflammation and remodeling.Compared with CON-WT mice,CS-WT mice displayed the following changes:Immunohistochemical staining and western blot presented enhancive expression of LC3B,P62,Collagen I and a-SMA.The formation of autophagosomes increased in airway epithelial cells under transmission electron microscope.The concentrations of TNF-α,IL-8 and LTB4 in bronchoalveolar lavage fluid were significantly elevated by ELISA.6.Inhibition of autophagy with 3-methyladenine alleviated airway remodeling and inflammation in CS-induced wild-type mice.Compared with CS-induced mice,3-MA pretreated mice showed a lower level of Collagen I,α-SMA and the inflammatory mediators mentioned above.7.Conditional knockout of FSTL1 gene can reduce the activation of autophagy after CS induction.Conversion of LC3B and formation of autophagosome increased after CS-induction,however these changes were weaker in FSTL1+/-mice than in FSTL1Flox/+ mice under the same CS induction.8.Conditional knockout of FSTL1 gene alleviates airway remodeling and inflammation after CS exposure.Immunohistochemical staining and Western blotting analysis manifested that collagen I and a-SMA were increased more lighter in FSTLL+/-mice after CS induction than in FSTLlflox/+ mice.Meanwhile,the concentrations of IL-8,LTB4 and TNF-α in FSTLL+/-mice were lower than those in FSTL1flox/+ mice.9.Both conditional knockout of FSTL1 gene and inhibitors of autophagy can improve the reduction of lung function in mice induced by CS.After CS induction,The total lung volume and lung compliance of CS-WT mice and CS-FSTL1flox/+ mice increased.The above changes in CS+3MA-WT mice were weaker than those in CS-WT mice.The changes in CS-FSTL1+/-mice were weaker than those in CS-FSTL1flox/+mice.The above experimental results showed that the level of FSTL1 and autophagy in COPD patients were increased.Both conditional knockout of FSTL1 and autophagy inhibitor can reduce CS-induced airway inflammation and airway remodeling.FSTL1 promotes airway inflammation and airway remodeling in chronic obstructive pulmonary disease by enhancing autophagy.Conclusion1.CS induction enhanced autophagy in COPD.2.Conditional knockout of FSTL1 and autophagy inhibitors can improve CS-induced airway inflammation and airway remodeling in COPD.3.FSTL1 promotes cigarette-induced airway inflammation and airway remodeling in COPD by enhancing autophagy.4.FSTL1 and autophagy may be potential targets for the treatment of COPD.