1.Subjects
Airway tissues were obtained from lobectomy or segmentectomy in 4 patients with lung cancer in situ and 4 COPD patients with comorbidity of lung cancer in situ at the First Affiliated Hospital of Anhui Medical University. Serum were obtained from a total of 124 participants (62 COPD patients and 62 controls) recruited from March 2018 to September 2019. The control group was collected from the physical examination center of our hospital in the same period. All patients with COPD met the diagnostic criteria of the 2018 GOLD guideline. The exclusion criteria include patients with severe cardiovascular, hepatic and renal dysfunction, hematological system diseases, diabetes, obesity and malignant tumor; patients with mental illness; patients with other lung diseases (such as asthma, acute exacerbation of COPD, pneumonia, cystic fibrosis, active pulmonary tuberculosis, interstitial lung disease, etc.); patients with a history of taking corticosteroids or immunosuppressants regularly; patients with a history of participating in any health care activities before attending this study.
2. Pulmonary function tests
Pulmonary function test was performed on each subject at 15 minutes after inhaling salbutamol 400 µg (Ventolin, GlaxoSmithKline, London, UK) by a dry spirometer device (Erich Jaeger GmbH, Hoechberg, Germany). Pulmonary function results were reported by experienced respiratory and critical care physicians. The following parameters were recorded: forced vital capacity (FVC), forced expiratory volume in one second (FEV1), FEV1% predicted, and the FEV1/FVC ratio. FEV1/FVC༜0.7 was classified as COPD.
3.Chest computed tomography scan and analysis
The GE LightSpeed VCT, GE Discovery C1750, and Toshiba Aquilion 16-slice CT were used for scanning. All parameters were obtained by CT plain image reconstruction and were reconstructed by the standard algorithm. The lung window (window width 1500 HU, window level − 500 HU) and mediastinum window (window width 400 HU, window level 40 HU) were observed. Scanning parameters: GE LightSpeed VCT and GE Discovery CTI SO CT machines adopt tube voltage 120 kV, tube current automatic regulation, layer thickness 5.0 mm, reconstruction layer thickness 0.625 mm, pitch 1.375; Toshiba Aquilion 16-layer CT machine adopts tube voltage 120 kV, tube current 150 mA, layer thickness 5.0 mm, reconstruction layer thickness 1.0 mm, pitch 0.980. Airway quantitative measurement was performed using the thoracic VCAR software supplied by GE in the apical segment of the right upper lobe (RB1). The following parameters can be automatically measured using the thoracic VCAR software: square root of the wall area at an internal airway area of 8 mm2 (Ai8), the percentage of bronchial wall area (WA %), total airway area(AO) and relative wall thickness (RWT) (expressed as the ratio of wall thickness and external diameter)(20).
4.Measurement of serum sestrin2 and MMP9 concentrations
The blood samples were collected from all subjects by venipuncture with a tube without anticoagulants. The serum was collected after centrifuged for 20 min at 1000 × g and stored at − 80 °C. The serum sestrin2 and MMP9 concentration were detected using human enzyme-linked immunosorbent assay kits following the manufacturer's instruction. The ELISA kits were against sestrin2(Cloud-Clone Crop, Wuhan, China) and MMP9 (CUSABIO, Wuhan, China).
5. Histological staining of airway tissues
Human lung tissues were fixed in 4% paraformaldehyde and then embedded in paraffin.5-µm-thick sections were prepared before staining. HE staining, PASM staining and Masson staining kits (Solarbio, Beijing, China) were employed to test structure changes of the human airway. The experimental methods followed the manufacturer’s instructions. Immunohistochemical staining was applied to detect the expressions of sestrin2 and MMP9 of the sections. Antigens were repaired according to primary antibody specifications. The endogenous peroxidase blocker was added and incubated for 30 minutes at 37℃. The slides were incubated with rabbit anti-sestrin2 antibody (ProteinTech#10795-1-AP, Chicago, USA, diluted with 1:200) and mouse anti-MMP9 antibody (Abcam #ab58803, Cambridge, UK, diluted with 1:100) at 4℃ overnights. Then, the slides were washed with PBS, followed by incubated with biotinylated goat anti-rabbit IgG and then incubated with streptavidin-peroxidase. Diaminobenzidine (DAB) solution (ZSGB-Bio, Beijing, China) was used for staining. Finally, all slides were counterstained with hematoxylin. Staining was imaged under a light microscope (Leica ICC50 W, Wetzlar, Germany).
6.Statistical analysis
GraphPad Prism 5 (San Diego, California, USA) and SPSS 22.0 (IBM, Armonk, NY, USA) software was used to analyze the data. Categorical variables were expressed as count (%). The chi-square test examined differences between sex ratios and smoking status. Normally distributed data were compared using an unpaired t-test and expressed as mean (standard deviation). All non-normally distributed were compared by the Mann-Whitney U test and expressed as median (interquartile range). The correlation between serum sestrin2 concentration and other measurement indexes in the COPD group was analyzed by Pearson’s correlation test. A P-value of less than 0.05 was regarded as statistically significant.