Correlation between sestrin2 expression and airway remodeling in COPD

Airway tissues were obtained from lobectomy or segmentectomy in four patients with lung cancer in situ and four COPD patients with comorbidity of lung cancer in situ at the First Affiliated Hospital of Anhui Medical University, China. Serum was 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 this hospital during the same period. All patients with COPD met the diagnostic criteria of the 2018 GOLD guideline [23]. The exclusion criteria included 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, and interstitial lung disease); patients with a history of taking corticosteroids or immunosuppressants regularly; and patients with a history of participating in any health care activities before enrollment in this study.

A pulmonary function test was performed on each subject at 15 min after inhaling 400 μg of salbutamol (Ventolin, GlaxoSmithKline, London, UK) using 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.

The GE LightSpeed VCT (GE Healthcare, Milwaukee, US), GE Discovery C1750(GE Healthcare, Milwaukee, US), and Toshiba Aquilion 16-slice CT (Toshiba Medical Systems, Tokyo, Japan) were used for all scanning. All parameters were obtained by CT plain image reconstruction and were reconstructed using a 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 were as follows: GE LightSpeed VCT and GE Discovery C1750 machines used a tube voltage of 120 kV, automatic tube current regulation, a layer thickness of 5.0 mm, a reconstruction layer thickness 0.625 mm, and pitch 1.375; Toshiba Aquilion 16-layer CT instruments used a tube voltage of 120 kV, a tube current of 150 mA, a layer thickness 5.0 mm, a reconstruction layer thickness of 1.0 mm, and a pitch 0.980. Airway quantitative measurements were performed using the thoracic VCAR software supplied by GE in the apical segment of the right upper lobe (RB1). The following parameters could be automatically measured using the thoracic VCAR software: the 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(A_O), and the relative wall thickness (RWT) (expressed as the ratio of wall thickness and external diameter) [24].

Blood samples were collected from all subjects by venipuncture with a tube without anticoagulants. The serum was collected after centrifugation for 20 min at 1000×g and then stored at − 80 °C. The serum sestrin2 and MMP9 concentrations were determined using human enzyme-linked immunosorbent assay (ELISA) kits following the manufacturer’s instructions. The ELISA kits were against sestrin2(Cloud-Clone Crop, Wuhan, China) and MMP9 (CUSABIO, Wuhan, China).

Human lung tissues were fixed in 4% paraformaldehyde and then embedded in paraffin. Sections with a thickness of 5 μm were prepared for staining. Hematoxylin-eosin (HE), periodic Schiff-methenamine silver (PASM) and Masson staining kits (Solarbio, Beijing, China) were employed to test for structural changes of the human airway. The experimental methods followed the manufacturer’s instructions. Immunohistochemical staining was applied to detect the expression of sestrin2 and MMP9 in these sections. Antigens were retrieved according to primary antibody specifications. An endogenous peroxidase blocker was added and incubated for 30 min at 37 °C. Slides were then incubated with rabbit anti-sestrin2 antibody (ProteinTech#10795–1-AP, Chicago, USA; diluted 1:200) and mouse anti-MMP9 antibody (Abcam #ab58803, Cambridge, UK; diluted 1:100) at 4 °C overnight. Then, the slides were washed with PBS, followed by incubation 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 using a light microscope (Leica ICC50 W, Wetzlar, Germany). Semiquantitative assessment of MMP9 and sestrin2 protein expression was performed using Image-Pro Plus6.0 (Media Cybernetics, Inc., USA). The mean integral optical density of protein staining was the ratio of the integral optical density of protein stained-positive epithelium to the area of corresponding bronchial epithelium.

All subjects recruited in this study were divided into two groups: a COPD group(n = 62) and a control group(n = 62). There were no significant differences in age, sex, BMI, or smoking index between these two groups. However, other pulmonary function indicators (FEV1, FEV1%, and FEV1/FVC) were significantly decreased in the COPD group (P < 0.001). The demographic characteristics of all subjects are listed in Table 1.

To characterize airway remodeling at the pathological level, airway tissues were isolated and various staining methods were then used. HE staining demonstrated that the airway epithelial structure and ciliated cells were intact in the control group, while the ciliated cells were detached and lodged to different degrees in the COPD group (Fig. 1a). Masson staining showed that collagen deposition in the bronchial epithelial area of the COPD group was significantly increased compared with the control group (Fig. 1b). Continuous Rbm was clearly observed in the control group, but the reticular basement membrane was fragmentary in the COPD group based on PASM staining (Fig. 1c).

Brown staining represents positive protein expression in immunohistochemical analysis using DAB. As described previously [25, 26], sestrin2 expression was largely located in the cytoplasm of bronchial epithelial cells and was significantly increased in the COPD group (Fig. 2a). Stronger MMP9 expression was also clearly detected in the cytoplasm in the COPD group when compared with the control group (Fig. 2b). The mean integral optical densities of sestrin2 and MMP9 were significantly higher in the COPD group compared with the control group (P < 0.001; Fig. 2c-d).

Compared with the control group, the serum sestrin2 concentration was significantly increased in patients with COPD(P < 0.001) (Table 2, Fig. 2e). Similarly, the expression of MMP9 in serum was significantly higher in patients with COPD than in controls(P < 0.001) (Table 2, Fig. 2f). There was no difference between smokers and non-smokers in terms of serum senestrin2 and MMP9 concentration (see Additional file 1). There was also no difference between the different sexes in COPD patients (see Additional file 2).

Compared with the control group, chest CT imaging showed that the bronchial wall was thickened, and the bronchial lumen was rough and curved in patients with COPD(Fig. 3). We demonstrated that the values of airway parameters in chest CT (Ai8, A_O, WA%, and RWT) were all significantly increased in the COPD group(P < 0.001) (Table 2, Fig. 4).

To elucidate the relationship between sestrin2 and airway remodeling in patients with COPD, the correlation between serum sestrin2 concentration and MMP9, as well as airway parameters in chest CT, were analyzed in patients with COPD. The results showed that the serum sestrin2 concentration was positively correlated with serum MMP9 concentration (r = 0.264;P = 0.038) (Fig. 5), Ai8 (r = 0.287;P = 0.024) (Fig. 6a), A_O(r = 0.273;P = 0.032) (Fig. 6b),WA% (r = 0.294;P = 0.020) (Fig. 6c) and RWT (r = 0.304;P = 0.016) (Fig. 6d). However, there were no significant associations between serum sestrin2 concentration and parameters of lung function (Table 3).

There was no association between serum MMP9 concentration and Ai8 (r = 0.123; P = 0.340), A_O (r = 0.144; P = 0.264), WA% (r = 0.087; P = 0.500) and RWT (r = 0.128; P = 0.323), as shown in Additional file 3.