A total of 20 male Sprague-Dawley rats (age, 3 months; body weight, 250 g) were purchased from Hunan Slake Jingda Laboratory Animal Co., Ltd. [license no. SCXK (Hunan) 2016-0002]. All animals were raised in non-toxic plastic boxes, stainless steel wire cages and metal cages, and housed in a specific pathogen-free environment that was automatically maintained at a temperature of 22±2°C and a relative humidity of 45–65%, under a controlled 12-h light/dark cycle with access to food and water ad libitum. All animal experiments were approved by the Ethics Committee of Guizhou College of Traditional Chinese Medicine (Guiyang, China).

Following anesthesia with sodium pentobarbital (40 mg/kg, intraperitoneal), rat skins were disinfected with iodophor. The epidermis and muscles along the middle of the neck were cut using surgical scissors to expose the trachea. Papain solution (2 mg/ml) was injected into the trachea at a dose of 1 ml/kg from top to bottom to reach the lungs. Subsequently, the animals were picked up and rotated to ensure that the drug had fully entered the trachea. Thereafter, the wound was sutured, and the animals were placed on an electric blanket. The animals were anesthetized with isoflurane (1% in oxygen) and decapitated 8 days after remodeling. Airway tissues from 3 animals were collected on ice and fixed in 4% paraformaldehyde overnight at 4°C. The tissues were sectioned into 5-µm thick sections and mounted on slides, followed by hematoxylin and eosin staining for 5 min at room temperature. The stained sections were observed under a light microscope (magnification: ×200; BX51; Olympus Corporation).

A total of 12 rats were sacrificed and thoracotomized under aseptic conditions to collect airway fibroblasts as previously described (21). A sterile scalpel was used to collect the tissue surrounding the airway; freshly obtained airway tissue from COPD rats was washed five times with phosphate-buffered saline (PBS) on an aseptic worktable. The tissue was then cut into 2×2-mm pieces using sterilized ophthalmic scissors. The tissue was pasted into the culture plate, and placed in a 5% CO₂ incubator at 37°C for 4 h. After cell adherence, freshly prepared medium containing DMEM (Gibco; Thermo Fisher Scientific, Inc.) and 10% fetal bovine serum (FBS; cat. no. 04-007-1A; Biological Industries) was added to the culture plate, and the cells were further cultured in a 5% CO₂ incubator at 37°C. Cell growth was monitored, the cell supernatant was periodically discarded, and newly prepared DMEM +10% FBS culture medium was added into the culture dish.

The cultured dishes (3×10³/ml) were rinsed three times in PBS (3 min/wash), fixed in 4% paraformaldehyde for 15 min at room temperature, and permeated with 0.5% Triton X-100 PBS for 20 min at room temperature. Bovine serum albumin (5%; Gibco; Thermo Fisher Scientific, Inc.) was added to the culture dish, which was subsequently sealed at 37°C for 30 min. Anti-vimentin antibody (1:250; cat. no. ab92547; Abcam) was added to the dish and incubated at 37°C for 3 h. The diluted Cy3-conjugated secondary antibody (1:200, cat. no. BA1032, Wuhan Boster Biological Technology, Ltd.) was added and incubated at 37°C for 30 min. Subsequently, DAPI was used to stain the nuclei for 3 min at room temperature, and the images were observed under a fluorescence microscope (magnification: ×200) as previously described (22).

Airway fibroblasts (3×10³ cells/well) were seeded in 6-well plates. Following transfection and/or treatment, total mRNA was extracted using a TRIzol^® assay kit (Baosheng Science & Technology Innovation Co. Ltd.). RNA quality was evaluated based on the optical density (260/280 nm). mRNA was transcribed into cDNA using a SMART^® MMLV Reverse Transcriptase kit (cat. no. 639522; Takara Biotechnology Co., Ltd.) at 37°C using the following thermocycling conditions: 25°C for 10 min, 37°C for 120 min and 85°C for 5 min, which was subsequently used as the template for fluorescent qPCR. The reaction mixture included the following components: Deionized water (9.5 µl), cDNA (1 µl), primers (2 µl) and 2X UltraSYBR Mixture (12.5 µl; HY-K0501; MedChemExpress LLC) under the following cycling conditions: 35 cycles of 94°C denaturation for 45 sec, 59°C annealing for 45 sec and 72°C extension for 60 sec. The relative mRNA expression of HSG, β-catenin and Ras homology family member A (RhoA) was calculated using GAPDH as the reference using the 2^−ΔΔCq method (23). The primers used in the study are presented in Table I.

Airway fibroblasts were divided into nine groups: Control, vector, HSG overexpression (HSG), lithium chloride (LiCl), vector + LiCl, HSG + LiCl, 4-ethyl-5,6-dihydro-5-methyl-(1,3)dioxolo(4,5-j)phenanthridine (HLY78), vector + HLY78, and HSG + HLY78 (Fig. 1A). Wnt signaling pathway agonists, LiCl (cat. no. B6083) and HLY78 (cat. no. C5433) were purchased from Apexbio Technology, LLC. The cells were treated with either 10 mM LiCl or 10 µM HLY78 for 24 h, at 48 h at 37°C following transfection with the HSG vector.

The HSG gene sequence (NM_130894) was obtained from the National Centre for Biotechnology Information database, and a biosynthetic gene fragment was cloned into the pCDNA3.1 (+) vector (Shanghai GenePharma Co. Ltd.) at the digestion site (Hind III/Xho I). Cells at 60% confluence were transfected with empty vector or HSG (1 µg/ml) using Lipofectamine^® 3000 (Invitrogen; Thermo Fisher Scientific, Inc.). After 6 h, the medium was replaced with fresh DMEM containing 10% FBS and cultured in a 5% CO₂ incubator at 37°C for 48 h. The cells were then used in the following experiments.

After discarding the culture medium, the cells (3×10⁵/ml) were washed with PBS and digested with trypsin for 2–3 min. Next, the cells were resuspended, inoculated into 96-well plates and placed in a 5% CO₂ incubator at 37°C for 24 h. The assay was performed as previously described (24). The optical density was determined using a microplate reader (Bio-Tek Instruments, Inc.) at 570 nm to determine cell viability.

The cells (3×10⁵/ml) were incubated in the dark with Annexin V-fluorescein isothiocyanate and propidium iodide (cat. no. AP101-100; Multisciences Biotech Co., Ltd.) for 30 min at room temperature. Subsequently, apoptosis was detected using a flow cytometer (BD Biosciences) within 1 h. Totally, four quadrants were divided based upon the fluorescence intensity and analyzed using FlowJo v10 (FlowJo, LLC). Quadrant Q2-2 was added to box quadrant Q2-4 to obtain the percentage of apoptotic cells.

The cells were collected and total protein was isolated using a triple prep kit (cat. no. 28-9425-44, ReadyPrep; GE Healthcare Life Sciences). Protein concentration was measured using a bicinchoninic acid assay kit (cat. no. P0009: Beyotime Institute of Biotechnology). Volumes were adjusted to normalize the protein content, and then aliquots were processed for the ELISA using rat TGF-β1 (cat. no. MM-0181R1), rat MMP-9 (cat. no. MM-20918R1) and rat PDGF assay kits (cat. no. MM-0076R1; all MlBio; Shanghai Enzyme Biotechnology Co., Ltd.).

Following transfection and/or treatment as aforementioned, protein was extracted from each group using a protein isolation kit (cat. no. 28-9425-44; GE Healthcare Life Sciences). Protein levels were quantified with a bicinchoninic acid protein assay kit (cat. no. P0009; Beyotime Institute of Biotechnology). Proteins (25 µg/lane; 0.5 µg/µl) were separated via 12% SDS-PAGE and transferred onto polyvinylidene fluoride membranes. The membranes were blocked in 5% skimmed milk for 2 h at room temperature and incubated with the following primary antibodies overnight at 4°C: Mouse monoclonal anti-GAPDH (1:2,000; cat. no. TA-08; OriGene Technologies, Inc.); rabbit polyclonal anti-HSG (1:5,000; cat. no. ab124773; Abcam); rabbit polyclonal anti-β-catenin (1:300; cat. no. ab32572; Abcam) and rabbit polyclonal anti-RhoA (1:2,000; cat. no. ab187026; Abcam). The IgG horseradish peroxidase-conjugated secondary antibody (mouse: 1:100; cat. no. ab131368; Abcam; rabbit: 1:100; ZB-2301; OriGene Technologies, Inc.) was added and co-incubated for 2 h at room temperature. An enhanced chemiluminescence exposure liquid droplet (cat. no. RJ239676; Thermo Fisher Scientific, Inc.) was added to the membranes. The membranes were visualized using a gel imaging system (Bio-Rad Laboratories, Inc.). Densitometry was performed using Quantity One version 1.4.6 (Bio-Rad Laboratories, Inc.).

Data were presented as the mean ± standard error of the mean with 6 independent repeats and analyzed using SPSS version 17.0 (SPSS, Inc.). Significant differences were determined using one-way analysis of variance, followed by the Student-Newman-Keuls post hoc test. P<0.05 was considered to indicate a statistically significant difference.

The airway tissue of the control group exhibited regular alveolar structure (Fig. 1B). Pathological expansion and fusion of alveolar cavities were not observed. The wall of the bronchial tube was normal, the epithelium of the airway mucosa was smooth, cilia were neatly arranged, and obvious inflammatory exudation was not found in the tracheal cavity. In contrast, the airway tissue of the COPD group showed evidence of emphysema, alveolar dilatation, an alveolar wall that disintegrated and fused to form lung ulcers, and markedly decreased numbers of alveoli (Fig. 1B). The goblet cells of the bronchial epithelium showed proliferation, and a number of inflammatory cells such as neutrophils and giant cells infiltrated the lumen, accompanied by the proliferation of fibrous connective tissue.

The identification of airway fibroblasts is shown in Fig. 1C, wherein red indicates vimentin expression, and nuclear staining by DAPI is observed in blue. All cells showed vimentin expression, indicating successful preparation of the airway fibroblasts. Compared with the vector group, the HSG vector upregulated HSG expression at both the mRNA and protein levels (both P<0.05; Fig. 2A and B), whereas the control vector did not influence HSG expression. Thereafter, the effects of HSG overexpression on cell viability were measured. The cell viability of the HSG group was decreased (vs. vector, P<0.05), which was inhibited by HLY78 and LiCl (vs. HSG, P<0.05; Fig. 3). Of note, LiCl and HLY78 alone increased cell viability (vs. control, P<0.05).

To further examine the effects of HSG overexpression on apoptosis, flow cytometry was used to detect the cell distribution following double staining. The apoptosis rate in the HSG group was significantly higher compared with that observed in the vector group (P<0.05; Fig. 4). LiCl and HLY78 reduced the effects of HSG overexpression on apoptosis (vs. HSG, P<0.05). The rates of apoptosis in the HSG + LiCl and HSG + HLY78 groups were significantly lower compared with that detected in the HSG group (P<0.05). However, LiCl and HLY78 alone did not affect apoptosis (vs. control, P>0.05; Fig. 4).

The protein levels of TGF-β1, MMP-9, and PDGF in each group are shown in Fig. 5. The levels of TGF-β1 (Fig. 5A), MMP-9 (Fig. 5B), and PDGF (Fig. 5C) in the HSG group were decreased compared with in the control group. In contrast, these levels were higher in the HSG + LiCl and HSG + HLY78 groups compared with the HSG group (P<0.05). LiCl and HLY78 alone also increased the levels of TGF-β1, MMP-9, and PDGF (vs. control; P<0.05).

The expression of β-catenin and RhoA in each group was normalized to GAPDH and is presented in Fig. 6. The mRNA levels of β-catenin (Fig. 6A) and RhoA (Fig. 6B) were higher in the HSG group compared with the vector group (both P<0.05). However, the expression of β-catenin and RhoA was reduced by LiCl and HLY78, respectively (vs. HSG, P<0.05). The protein expression of β-catenin (Fig. 6C) and RhoA (Fig. 6D) was also detected using western blot analysis. Consistent with the mRNA expression, the protein levels of β-catenin and RhoA (normalized to GAPDH) in the HSG group were higher (vs. control, P<0.05), and the expression of β-catenin and RhoA was reduced by LiCl and HLY78, respectively (vs. HSG, P<0.05). The expression of β-catenin and RhoA was promoted by LiCl and HLY78 alone, respectively (vs. control, P<0.05).