A total of 60 patients with lung cancer undergoing a unilateral lobectomy were recruited from Tianjin Chest Hospital (Tianjin, China) between October 2012 and April 2013 for the present study. Patient characteristics are presented in Table I. According to their lung function, all individuals were divided into a non-COPD group (n=20) and a COPD group (n=40). According to a random number table, the COPD group was further randomly divided into a COPD control group (n=20) and a COPD treatment group (n=20). The COPD treatment group were given a 5-day lung protection treatment (atomized inhalation of 1,000 µg ipratropium bromide, 30 µg ambroxol and 2 mg budesonide 3 times/day; intravenous injection of 150 mg ambroxol hydrochloride dissolved in 100 ml saline and 0.2 g doxofylline dissolved in 100 ml saline once/day) once they were hospitalized. The non-COPD group and COPD control group received no intervention.

The present study was approved by the ethics committee of Tianjin Chest Hospital and written consent was obtained from all patients and their families prior to enrollment. The diagnosis of patients with COPD was performed in accordance with the Guidelines for Diagnosis and Treatment of Chronic Obstructive Pulmonary Disease (2013 revision) (19). The standard method of confirming COPD is the examination of lung function: Following treatment with a bronchial dilation agent, the patients were defined as incomplete reversible airflow limitation with forced expiratory volume in 1 sec (FEV1) <80%, and FEV1/forced vital capacity (FVC) <70%. The exclusion criteria were as follows: i) Patients with a lung infection or COPD acute attack in the short term (one month prior); ii) patients with other chronic diseases (for example, bronchiectasis, asthma, pulmonary interstitial fibrosis or silicosis); iii) patients with chronic diseases of the heart, brain, liver, kidney or other systems; and iv) patients taking medication, including antibiotics, cortical hormone, bronchodilators or expectorants.

EBC was obtained following the 5-day lung protection treatment described above using an RTube condenser (Model Austin TX 78720; RTube starter kit; Respiratory Research Inc., Austin, TX, USA). Patients were required to gargle to clean up oral secretions and wear a nasal splint prior to EBC collection. Patients breathed through a disposable biting mouthpart. A one-way valve in the collecting tube allowed the exhaled air to enter the collecting tube and be condensed into liquid through a cooling tube. The procedure lasted 10–15 min and 1–3 ml condensate was collected. Patients were required to ingest the saliva during process of collection. Collection was stopped if the patient coughed. The condensate was removed into cryovial and stored at −80°C for subsequent use.

Pneumonectomy was performed to obtain lung tissue specimens under aseptic conditions. Lung tissue samples were obtained during lobectomy through resection of the adjacent lung tissues, located >5 cm from the nodule. Partial samples were fixed in 10% formalin at room temperature for 12 h and cut into 5-µm paraffin sections, which were used for immunohistochemistry experiments. At the time of sampling, other lung tissues were immediately frozen in liquid nitrogen and stored at −80°C; these were used for reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis.

Levels of SP-A and SP-D in EBC were measured using ELISA assay kits (cat nos. RGP011R and RGP011R; BioVendor Laboratorni Medicina, a.s., Brno, Czech Republic), according to the manufacturer's protocol. A total of 10 µl each sample was analyzed on the same ELISA plate, including a negative control. Detection was performed at 450 nm using a multiscan spectrum microplate reader (model ELx808; BioTek Instruments, Inc., Winooski, VT, USA). The concentrations of SP-A and SP-D were calculated from the calibration curve drawn from the data for standard albumin.

Total RNA from lung tissue was extracted using aUNlQ-10 Column total RNA purification kit (Sangon Biotech Co., Ltd., Shanghai, China). cDNA was generated following the manufacturer's protocol of a Transcriptor First Strand cDNA Synthesis kit (Roche Diagnostics GmbH, Mannheim, Germany). The expression levels of SP-A and SP-D were analyzed using RT-qPCR with FastStart Universal SYBR-Green Master (Roche Diagnostics GmbH) and a Bio-Rad CFX96 qPCR system (Bio-Rad Laboratories, Inc., Hercules, CA, USA), according to the manufacturers' protocol. The primers used were as follows: SP-A forward, 5′-ATGTCACTGTGTCTTTGGCTTTC-3′ and reverse, 5′-TCAAAATTCACAAACAGCCAGCC-3′; SP-D forward, 5′-AAAGTGTCGGGGAGAAGA-3′ and reverse, 5′-TCAGTCATGCTCAGGAAA-3′. qPCR analysis was carried out in a total volume of 20 µl, containing 9 µl 2X SYBR-Green qPCR Mix (Takara Bio, Inc., Otsu, Japan), 0.1 µM each specific primer and 100 ng template cDNA. The reaction mixtures were heated to 94°C for 30 sec, followed by 40 cycles at 94°C for 10 sec, 60°C for 45 sec, and 72°C for 45 sec. The housekeeping gene GAPDH was used as a reference gene, with primers as follows: GAPDH forward, 5′-CGGAGTCAACGGATTGGTCGTAT-3′ and reverse, 5′-AGCCTTCTCCATGGTGGTGAAGAC-3′. The primers for the RT-qPCR experiments were designed using Primer Premier version 5.0 (Premier Biosoft International, Palo Alto, CA, USA) and synthesized by Sangon Biotech Co., Ltd. All RT-qPCR analysis was performed in triplicate and quantified using the 2^−ΔΔCq method (20).

The lung tissue slides were deparaffinized in xylene, hydrated through a graded series of ethanols (anhydrous ethanol, 95% ethanol and 75% ethanol for 5 min, respectively), and rehydrated in deionized water. Inactivation of endogenous peroxidase was performed using 3% H₂O₂ in methanol for 10 min. Antigen retrieval was performed by heating with citric acid solution (pH 6.0) for 30 sec at 125°C and subsequently for 25 min at 94°C, followed by cooling for 15 min. Tissues were blocked with 1% bovine serum albumin (Shenhang Biotechnology Co., Ltd., Shanghai, China) at room temperature for 20 min, and subsequently incubated with diluted primary mouse anti-human SP-A antibody (cat no. sc-7700; 1:200) and anti-thyroid transcription factor-1 (TTF-1) antibody (cat no. sc-12524; 1:200) (both from Abcam) overnight at 4°C. Following washing with PBS, sections were incubated with secondary anti-mouse SP-A antibody (cat no. sc-7701; 1:500) and anti-mouse TTF-1 antibody (cat no. sc-12525; 1:500) (both from Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA) at 37°C for 30 min and visualized with diaminobenzidine and hematoxylin-eosin staining at room temperature for 15 min. The proportions of SP-A⁺ alveolar type II (ATII) cells were evaluated under a light microscope (BX50; Olympus Corporation, Tokyo, Japan) at ×400 magnification.

All data are expressed as the mean ± standard deviation. Statistical analysis was performed using SPSS software (version 19.0; IBM Corp., Armonk, NY, USA). Paired sample t-test was conducted when comparing the expression levels of SP-A and SP-D in EBC in the COPD treatment group prior to and following lung protection treatment. Multiple comparisons between the groups were performed using one-way analysis of variance followed by Student-Newman-Keuls test. Correlation analysis was conducted using Spearman's rank correlation test. P<0.05 was considered to indicate a statistically significant difference.

As presented in Table I, the clinical data of patients, including sex, age, smoking history and FEV1, was analyzed in the three groups, including the non-COPD group, COPD control group and COPD treatment group. No statistically significant difference was observed in sex, age and smoking between patients of the three groups. FEV1 in patients with COPD was significantly decreased compared with patients in the non-COPD group (P<0.05). There was no significant difference in FEV1 between the COPD control group and COPD treatment group (P>0.05).

The mRNA expression levels of SP-A and SP-D in lung tissue were measured using RT-qPCR (Fig. 1A). Significant differences in SP-A and SP-D expression were observed among the three groups (P<0.05). SP-A in the non-COPD group (3.21±0.52) exhibited a significantly increased mRNA expression compared with the COPD control group (2.25±0.95) and COPD treatment group (2.75±0.15; P<0.05). SP-D expression exhibited a similar profile, with the highest levels (2.59±0.44) in the non-COPD group and lowest expression levels (2.00±0.13) in the COPD control group (P>0.05).

In addition, the expression levels of SP-A and SP-D in EBC were measured using ELISA analysis (Fig. 1B). The non-COPD group exhibited the highest SP-A expression of 8.57±0.73 pg/ml and SP-D expression of 4.55±0.99 ng/ml, which were significantly increased compared with the other two groups (P<0.05). The COPD control group exhibited the lowest expression levels of SP-A and SP-D (P<0.05). In the COPD treatment group, lung protection treatment for 5 days promoted SP-A (5.82±0.27 vs. 4.98±0.96 pg/ml; P<0.05) and SP-D expression (4.01±0.8 vs. 3.66±0.79 ng/ml; P<0.05) in EBC.

As presented in Fig. 2, SP-A and TTF-1 immunostaining was performed to measure the proportion of SP-A⁺ ATII in patient lung tissues. Areas of ATII cells, club cells and alveolar macrophages were observed to be positive for SP-A immunostaining. In addition, ATII cells, bronchial epithelial cells and club cells were positive for TTF-1 immunostaining. The proportions of SP-A⁺ ATII cells in the non-COPD group, COPD control group and COPD treatment group was 62.00±5.86, 44.00±5.89 and 52.55±10.46%, respectively.

The correlation between the expression levels of SP-A, SP-D and lung function was analyzed in all three patient groups, as exhibited Fig. 3. The results of the present study demonstrated that FEV1 was positively correlated with the expression levels of SP-A in EBC (r=0.494, P<0.05) and SP-D (r=0.253, P<0.05). Additionally, FEV1 in lung tissue was positively correlated with the mRNA expression of SP-A (r=0.264, P<0.05) and SP-D (r=0.281, P<0.05).

The correlation between the proportion of SP-A⁺ ATII and lung function was analyzed. A positive correlation between SP-A⁺ AII and FEV1 was observed (r=0.795, P<0.05).

The correlation between the content of SP-A and SP-D in EBC, and the corresponding mRNA in lung tissue, was analyzed (Fig. 4). There was a positive correlation between SP-A in EBC and SP-A mRNA in lung tissue (r=0.542, P<0.05). A positive correlation additionally existed between the content of SP-D in EBC and the mRNA expression of SP-D in lung tissue (r=0.478, P<0.05). In addition, the proportion of SP-A⁺ ATII exhibited a positive correlation with SP-A content in EBC (r=0.522, P<0.05) and mRNA expression levels of SP-A in lung tissue (r=0.299, P<0.05). Notably, no correlation was observed between SP-A⁺ ATII and SP-D contents in EBC (r=0.145, P>0.05). No correlation was observed between SP-A⁺ ATII and the mRNA expression levels of SP-D in lung tissue (r=0.200, P>0.05).