The present study was approved by the ethics committee of the Affiliated Hospital of Luzhou Medical College (Luzhou, China). A total of 20 female Wistar rats (weight, 180–200 g) were purchased from Chongqing Teng Xin Bill Experimental Animal Sales Co. Ltd. (Chongqing, China) for use in the study. The reagents used in the experiment were purchased from the following companies. Pentobarbital sodium was obtained from Jiangsu Hengrui Medicine Co. Ltd. (Lianyungang, China), while bleomycin A5 was purchased from Harbin Pharmaceutical Co. Ltd, (Shanghai, China). A rabbit anti-IL-17 immunohistochemistry kit was purchased from Beijing Boosen Biological Technology Co. Ltd. (Beijing, China), and the IL-17A ELISA kit was obtained from Bio-Rad Laboratories, Inc. (Hercules, CA, USA). A total RNA extraction kit was obtained from Tiangen Biotech Co. Ltd. (Beijing, China), while improved RPMI-1640 culture medium, 1X Dulbecco's modified Eagle's medium (DMEM) in high glucose medium and fetal bovine serum were all purchased from HyClone (GE Healthcare, Logan, UT, USA). Primers for polymerase chain reaction (PCR) were purchased from Shanghai Sangon Biological Engineering Co. Ltd. (Shanghai, China), and the RNA reverse transcription (RT) and PCR kits were purchased from Chengdu FOREGENE Biotechnology Co. Ltd. (Chengdu, China).

A total of 20 female Wistar rats were randomly divided into a control group (normal saline, NS) and a model group (BLM). Model establishment was conducted according to previously described methods (6). The rats in the BLM group were anesthetized by an intraperitoneal injection of pentobarbital sodium (40 mg/kg body weight) and the trachea was exposed according to a blunt separation method during aseptic surgery. The trachea was filled with 0.3 ml NS and BLM A5 (5 mg/kg), after which the rats were quickly erected and spun to distribute the liquid in the lung evenly. With regard to the NS group, the same methods were applied, but with an injection of the same volume of NS in the rat tracheas.

Experimental animals from the two groups were sacrificed with 10% chloral hydrate (3 ml/kg) and right ventricular blood sampling was performed following intratracheal instillation of NS or BLM at week one (day 7, n=10) and week four (day 28, n=10). Collection of the lung tissue and bronchoalveolar lavage fluid (BALF) was conducted according to previously described methods (7). A total of 40 g/l paraformaldehyde was injected into the left pulmonary tissue for fixation. Following paraffin embedding, three sections collected from different parts of the lung tissue were sliced to a thickness of 5 µm. Hematoxylin and eosin and Masson's trichrome staining were used to evaluate the pathological changes (alveolitis and pulmonary fibrosis) in the pulmonary tissue, with the specific criterion predominantly formed according to previous studies (8,9). Briefly, pathological changes were graded according to the Edmondson-Steiner grading method. Grade I, highly differentiated cancer cells, mucleocytoplasmic ratio is close to normal; grade II, moderately differentiated cancer cells, mucleocytoplasmic ratio increases with deeper nuclear staining; grade III, highly differentiated cancer cells, mucleocytoplasmic ratio is increases with marked heterogenous nuclear division; and grade IV, highest differentiation of cancer cells, reduced cytoplasm, densely stained chromatin, irregular cell shape and loose arrangement.

Lung tissue slices were subjected to dewaxing, hydration, antigen repair and serum blocking. The primary antibody used was a rabbit anti-IL-17 polyclonal antibody (dilution, 1:100; cat. no. sc-7927; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), and the secondary antibody used was a goat anti-rabbit antibody (dilution 1:100; cat. no. sc-45101; Santa Cruz Biotechnology, Inc.) with the Dako REAL™ Envision™ kit (Dako, Glostrup, Denmark). The marker used was horseradish peroxidase, while 3,3′-diaminobenzidine solution (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) was used for visualization. Other steps included hematoxylin counterstaining, dehydration using an ascending concentration of ethanol followed by treatment with xylene and mounting. The control group was treated with phosphate-buffered saline instead of the primary antibody. The image results of the immunohistochemical staining were acquired with a Leica system (Leica DMi8; Leica Microsystems, Inc., Buffalo Grove, IL, USA) and analysis was performed using Image-Pro Plus software (version 6.0; Media Cybernetics, Inc., Rockville, MD, USA). The average absorbance of the positive staining was calculated through randomly selected, stained lung tissue areas of five high power fields at two time points using ImageJ 1.48u software (National Institutes of Health, Bethesda, MD, USA).

According to previously described methods (10), BALF cells were counted and classified. A total of 10 µl BALF cell suspension liquid was inserted into a cell counting plate (Costar, Corning Life Sciences, Tewkesbury, MA, USA), and the number of cells was counted in the four large squares at the four corners at a low magnification (IX83; Olympus Corporation, Tokyo, Japan). A sedimentation smear was taken and Wright's stain (Sigma-Aldrich, St. Louis, MO, USA) cell sorting was used to select 100 white blood cells to count according to their morphological characteristics. The percentage of the various white blood cell types was calculated. In addition, the absolute value of the various white blood cell types in the BALF were determined by counting the total cell number. The formula was as follows: Cell number/ml = N/4 × 10 × C × 10⁶, where N was the total number of white blood cells in four large squares and C was the dilution ratio.

Levels of IL-17A in the BALF were detected using an ELISA kit, according to the manufacturer's instructions.

BALF was collected using a right pulmonary bronchoalveolar lavage, and the cell suspension was mixed with RPMI-1640 medium containing 100 ml/l calf serum, which was followed by incubation and purification for 3 h at 37°C in a 50 ml/l CO₂ cell culture box (Thermo Scientific 3913 incubator; Thermo Fisher Scientific, Inc., Waltham, MA, USA). Subsequently, the culture fluid was discarded and the purified and adherent AMs were cultured in DMEM. Two groups of AMs (day 7 and day 28) were cultivated at a density of 5×10⁵cells/well in six-well plates. Following culture for 12, 24 and 48 h, the cells and the culture supernatant were collected. The supernatant was used for the detection of the IL-17A concentration, while the cells were used for the detection of IL-17A mRNA expression.

Using the sequence of rat IL-17A mRNA, as listed in the PubMed database (US National Library of Medicine, National Institutes of Health), IL-17A primers were designed and synthesized by Shanghai Sangon Biological Engineering Co. Ltd. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as the internal reference. The primer sequences were as follows: IL-17A (188 bp) upstream primer, 5′-CTA CCT CAA CCG TTC CAC T-3′ and downstream primer, 5′-TTC TCA GGC TCC CTC TTC-3′; GAPDH (450 bp) upstream primer, 5′-ACC ACA GTC CAT GCC ATC AC-3′ and downstream primer, 5′-TCC ACC ACC CTG TTG CTG TA-3′. A TRIzol® kit was used to extract the total RNA from the AMs. The synthesis of cDNA by RT and the RT-PCR assay were carried out in strict accordance with the manufacturer's instructions. The PCR cycling conditions were as follows: Denaturation at 90°C for 5 min, amplification and quantification at 90°C for 30 sec, 60°C for 30 sec and 72°C for 30 sec for 30 cycles, followed by a final extension step at 72°C for 7 min. The odds ratios for the samples, where GAPDH was used as the reference gene, were used to determine the expression quantity of IL-17A mRNA.

Data are presented as the mean ±standard deviation, and were analyzed using SPSS software (version 13.0; SPSS, Inc., Chicago, IL, USA). The statistical significance of differences was assessed using one-way analysis of variance and Dixon's Q test, where P<0.05 was considered to indicate a statistically significant difference.

Inflammatory cell infiltration was observed in the alveolar cavity and pulmonary interstitium of the experimental group rats at day 7, and alveolar septal thickening and a small amount of collagen fiber organization was observed in the lung interstitium. At day 28, the alveolar exudate had decreased and the alveolar structure was destroyed. Furthermore, the interstitial gap had widened significantly and hyperplasia of the extracellular matrix was evident. Fibroblast proliferation was also observed in the pulmonary interstitium, and the accumulation of collagen fibers had formed extensive fibrosis. When compared with the NS group, the alveolitis was evident in the BLM group at day 7. By day 28, the alveolitis was relieved; however, the degree of pulmonary fibrosis remained high (Fig. 1, Table I).

Weak expression of IL-17A was observed in the lung tissues from the NS rats; the expression levels were 23.67±3.19 and 24.26±3.35 at days 7 and 28, respectively. However, the expression of IL-17A in the BLM group was significantly increased at days 7 (103.48±9.49) and 28 (87.42±7.64). When compared with the NS group at day 7, the difference was statistically significant (P<0.05), although expression levels decreased at day 28 compared with day 7 (Fig. 2).

At day 7, the total number of BALF cells in the NS group was 9.45±0.76, while the number of BALF cells in the BLM group was 35.28±2.76. When compared with the NS group, the total number of BALF cells increased significantly in the BLM group and the difference was statistically significant (P<0.05). At day 28, the total number of BALF cells in the NS group was 9.52±0.67, while the number in the BLM group was 9.71±0.80; thus, no statistically significant difference was observed (P>0.05). When compared with the NS group, the AM ratio of BALF at day 7 was smaller in the BLM group. In addition, the percentages of neutrophils and lymphocytes were significantly increased, with the differences statistically significant (P<0.05). When compared with the NS group, the classification percentage of BALF cells in the BLM group at day 28 was not significantly different (P>0.05; Table II), which demonstrated that the cell types had returned to normal.

IL-17A levels in the BALF of the NS group were 57.45±6.68 pg/ml at day 7 and 55.49±6.06 pg/ml at day 28, while the levels of IL-17A in the BLM group were 278.37±13.99 and 213.26±11.62 pg/ml at days 7 and 28, respectively. When compared with the NS group, the IL-17A levels were significantly increased in the BLM group and the difference was statistically significant (P<0.01). The level of IL-17A at day 28 was marginally lower compared with those on day 7; however, the difference was not statistically significant.

Compared with the NS group, the IL-17A concentration in the supernatant of the BLM group at days 7 and 28 increased significantly prior to AM culturing at 12, 24 and 48 h. The differences between the two groups were statistically significant (P<0.05, Table III).

Expression levels of IL-17A mRNA at days 7 and 28 were higher in the BLM group when compared with the NS group, and the differences were statistically significant (P<0.05). The difference in expression was particularly notable in the first 12 h, decreasing after 24 and 48 h (Figs. 3, 4 and 5).