The present study was performed in accordance with the guidelines of the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals, and was approved by the Animal Care and Use Committee of the Medical College of Qingdao University (Qingdao, China). A total of 72 male Wistar rats (specific pathogen-free; age, 6–8 weeks old; weight, 200±25 g) were purchased from Qingdao DaRen Fortune Animal Technology Co., Ltd. (Qingdao, Shandong, China). Animals were housed on a temperature (~22°C) and humidity (~55%) controlled environment with 12 h/12 h light cycle and free access to food and water at the Laboratory Animal Center, Medical College of Qingdao University.

A single intratracheal instillation of 5 mg/kg bleomycin (Nippon Kayaku Co., Ltd., Tokyo, Japan) dissolved in normal saline was administered to induce pulmonary fibrosis in rats on day 0 (18). The control (C) group (n=24) was intratracheally administered with an equal volume of normal saline (10 ml/kg body weight). Bleomycin-treated rats were randomly divided into the two groups (n=24 per group): The model (M) and pirfenidone (P) groups. From the second day following modeling, pirfenidone (50 mg/kg; Beijing Kangdini Pharaceutical Co., Ltd., Beijing, China) was orally administered once daily to rats in P group via a gastric tube. Prior to the present study, preliminary experiments were performed with different concentrations of pirfenidone (10, 30, 50 and 100 mg/kg) and 50 mg/kg induced the most notable inhibitory effects on bleomycin-induced pulmonary fibrosis in rats according to the histological analysis (data not shown). For rats in M group, 2 ml saline was orally administered once daily. Subsequently, at 7, 14 and 28 days following the beginning of the experiment, 8 rats were sacrificed at each time point and lungs were isolated and perfused with saline prior to histology analysis.

The left lungs of rats from each group were collected for morphologic studies. Lungs were inflated with 4% paraformaldehyde and fixed in formalin at 4°C for 24 h. After embedding by paraffin, lungs were cut into 5-µm slices and stained with hematoxylin and eosin (H&E) and Masson's trichrome kits (cat. no. MST-8003; Fuzhou Maixin Biotechnology Development Co., Ltd., Fuzhou, China) according to manufacturer's protocol. To assess alveolitis and fibrosis, H&E Masson-stained sections were evaluated via semi-quantitative histology by a pathologist who was blinded to the treatment groups, using a light microscope at ×200 and a scoring system, as described previously (19,20).

The content of hydroxyproline in lung tissues was determined using a commercial ELISA kit (cat. no. CEA621Ge; Uscn Life Science, Inc., Wuhan, China) according to the manufacturer's protocol. Briefly, lung issue samples were homogenized using a pestle and mortar in the liquid nitrogen and lysed using RIPA lysis buffer (cat. no. 20-188; Merck Millipore, Billerica, MA, USA) contained with phosphatase inhibitor cocktail (cat. no. 524627; Life Technologies; Thermo Fisher Scientific, Inc., Waltham, MA, USA) at 4°C for 30 min. Following this, the lysates were centrifuged at 10,000 x g, 4°C for 10 min, and supernatants were harvested and quantified using the BCA Protein assay kit (cat. no. 71285; Life Technologies; Thermo Fisher Scientific, Inc.). Then, lung tissue extractions were added to the 96-well ELISA plate, which was coated with rat horseradish peroxidase (HRP)-conjugated hydroxyproline antibody, provided by the ELISA kit. The hydroxyproline concentration was quantified following addition of the chromogenic substrate tetramethylbenzidine and analyzed using a microtiter plate reader (BioTek Instruments, Inc., Winooski, VT, USA) at a wavelength of 450 nm.

Immunohistochemical staining was performed to view the distribution of periostin. Primarily, lung sections were incubated with 3% H₂O₂ at room temperature for 10 min to eliminate the activity of endogenous peroxidase. Tissue sections were subsequently incubated with anti-periostin monoclonal antibody (1:65; cat. no. GTX100602; Bioworld Technology, Inc., St. Louis Park, MN, USA) at 5°C overnight and biotin-conjugated rabbit anti-rat IgG (1:300; cat. no. SP-9001; Hebei Bio-High Technology, Hebei, China) at 5°C for 1 h. Finally, sections were incubated with HRP-conjugated streptavidin at room temperature for 10 min and were visualized using 3,3′-diaminobenzidine tetrahydrochloride (Bio-High technology, Hebei, China). Yellow granules observed in tissue cells using a light microscope at ×400 and extracellular matrix were considered to indicate a positive result.

The right lung lobes were frozen in liquid nitrogen immediately following harvesting, and were subsequently used for RT-qPCR and western blotting. Total RNA was isolated using an Ultrapure RNA kit (CWbio Co., Ltd., Beijing, China) according to the manufacturer's protocol. Reverse transcription was performed using a HiFi-MMLV cDNA kit (CWbio Co., Ltd.) according to manufacture's protocol. Following this, qPCR of genes were performed on an ABI7500 system (Applied Biosystems; Thermo Fisher Scientific, Inc.) using SYBR^™ Green PCR Master Mix (Thermo Fisher Scientific, Inc.) with the following thermocycling conditions, 50°C for 3 min, 95°C for 3 min, and 40 cycles of 95°C for 10 sec and 60°C for 30 sec. Primers for periostin and GAPDH (used as control) were designed based on the mRNA sequence. Primer sequences were as follows: Periostin, forward 5′-ACTCCGTGTCTTCGTGTATC-3′ and reverse 5′-CTTTCTTCATTAGTCATTCCCT-3′; and GAPDH, forward 5′-TGGAGTCTACTGGCGTCTT-3′ and reverse 5′-TGTCATATTTCTCGTGGTTCA-3′. Relative gene expression levels were quantified using the 2^−ΔΔCq method (21).

Expression of periostin and TGF-β1 protein was determined by western blotting in lung tissue samples. Firstly, lung issue samples were homogenized using the mortar in the liquid nitrogen and lysed using RIPA lysis buffer contained with phosphatase inhibitor cocktail at 4°C for 30 min. Following this, the lysates were centrifuged at 10,000 x g, 4°C for 10 min and supernatants were harvested, quantified using the BCA Protein assay kit, and boiled with loading buffer [10% (v/v) SDS, 50% (v/v) glycerol, 0.2 M 8 tris-HCl (pH 6.8), 0.1 M dithiothreitol and bromophenol blue]. Following this, 15 µg protein for each sample was loaded into each lane of a 4% SDS-PAGE and separated by 12% SDS-PAGE. Then, proteins were electro-transferred to a polyvinylidene difluoride membrane. The membranes were blocked with 5% non-fat milk at room temperature for 1 h. Membranes were subsequently incubated with primary antibodies against TGF-β1 (1:1,000; cat. no. BZ00924) and periostin (1:1,000; cat. no. BZ1801) purchased from Bioworld Technology, Inc. (St. Louis Park, MN, USA) at 4°C overnight, and β-actin monoclonal antibody (1:1,000; cat. no. TA811000; OriGene Technologies, Inc., Beijing, China) as an internal control. β-actin was incubated with the membranes at 4°C overnight. The following day, samples were incubated with HRP-conjugated secondary antibody (1:3,000; cat. no. TA140003; OriGene Technologies, Inc.) at room temperature for 1 h. Enhanced chemiluminescent autoradiography reagent was then used to visualize the specific protein bands. Total TGF-β1, periostin and β-actin levels were determined using inbuilt software in the EC3-600 BioImaging System (UVP, LLC, Phoenix, AZ, USA).

SPSS v19.0 (IBM Corp., Armonk, NY, USA) was used for statistical analysis. Data are presented as the mean ± standard deviation. Significant differences among groups were identified via one-way analysis of variance and Tukey's tests for multiple comparisons. Correlations between the expression of periostin and TGF-β1, fibrosis score, and the content of hydroxyproline were analyzed using Spearman's rank correlation coefficient test. P<0.05 was considered to indicate a statistically significant difference.

The effects of pirfenidone on bleomycin-induced fibrogenesis were evaluated by H&E and Masson's staining and the pathology was quantified using a scoring system for alveolitis and fibrosis. As presented in Figs. 1–3, a lung phenotype of thickening of the alveolar interval, distortion of lung structure and fibrosis induced by bleomycin was observed compared with the control. Pirfenidone treatment was able to alleviate the bleomycin-induced fibrotic effects on rat lungs. Following statistical analysis, alveolitis and fibrosis scores were demonstrated to be significantly decreased following pirfenidone treatment throughout the experiment course (7, 14 and 28 days) when compared with that of the M group (P<0.05).

The effect of pirfenidone on lung hydroxyproline content at days 7, 14, and 28 is presented in Fig. 4. There was a significant increase of hydroxyproline content in the M group compared with the C group at days 7, 14 and 28, respectively (P<0.05 or P<0.01). However, pirfenidone administration significantly ameliorated this increase at days 14 (P<0.05) and 28 (P<0.01).

Representative histological features at day 14 as well as the expression of periostin during the whole course in different groups are presented in Fig. 5A-D. Periostin immunohistochemistry of the C group demonstrated weak peribronchial deposition, and periostin was rarely observed in pulmonary epithelial cells and alveolar macrophages, whereas in the M group, a strong expression of periostin was observed in the interstitia, particularly in the fibrotic foci, and periostin was also evident in the thickened alveolar walls underneath regenerating epithelial cells. In the P group, however, the expression of periostin was markedly decreased in the interstitial (Fig. 5A).

The periostin expression was detected at both the mRNA and protein level. RT-qPCR results demonstrated that periostin mRNA was significantly higher in the M group than in the C group at each time point (P<0.05 or P<0.01; Fig. 5B), whereas pirfenidone significantly inhibited the transcription of periostin when compared with the M group at days 7 (P<0.05) and 14 (P<0.01; Fig. 5B). Similar results of periostin expression at the protein level were achieved via western blotting, although there was no significant difference between the M and P groups at day 7 (Fig. 5C and D).

The expression of TGF-β1 in different groups was also measured by western blotting and the results are presented in Fig. 5E and F. The expression of TGF-β1 was significantly increased in the M group than that in the C group at days 7 (P<0.05), 14 (P<0.01), and 28 (data not shown), which was significantly ameliorated at days 14 (P<0.01) and 28 (data not shown).

As demonstrated in Fig. 6A and B, periostin expression was positively correlated with the hydroxyproline (r=0.9186, P<0.001) and TGF-β1 (r=0.8748, P<0.001) content. In addition, Fig. 6C also demonstrated that periostin expression was positively correlated with the fibrosis score (r=0.4826, P<0.001).