Male C57BL/6 mice (weighing 20–22 g, 8–10 weeks-old) were purchased from the Experimental Animal Center of Lanzhou University. All mice were housed in cages (humidity 45–50%, sizes 20×30 cm², bedding-wood shavings, 6 animals/cage) with free access to tap water and food in a room, which was maintained at 22±2°C in a 12-h light (8:00 a.m.)/dark (8:00 p.m.) cycle. All animal protocols in the present study were approved by the Ethics Committee of Lanzhou University (approval no. SYXK Gan 2009-0005).

The animal model of pulmonary fibrosis in the present study was established according to the protocols previously described by Tian et al (16), Luque et al (17) and Wei et al (18). Briefly, mice were anesthetized by an intraperitoneal (i.p.) injection of 70 mg/kg pentobarbital sodium (Sigma-Aldrich; Merck KGaA), and were intratracheally administered 4 mg/kg BLM (Sigma-Aldrich; Merck KGaA) to induce fibrosis. A total of 54 mice were randomly assigned to six groups of 9 mice each as follows: i) Control group, instilled with saline alone; ii) BLM group, instilled with BLM alone; iii) BLM+KP-13 group, instilled with BLM and treated with KP-13 [1 mg/kg, i.p.]; iv) BLM+KP-234+KP-13 group, instilled with BLM and treated with KP-234 (1 mg/kg, i.p.) and KP-13 (i.p.); and v) BLM+Cetrorelix+KP-13group, instilled with BLM and treated with Cetrorelix and KP-13 (i.p.). vi) KP-13 group were only treated with KP-13 (i.p.). After intratracheal instillation of BLM, the mice received all the injections daily from the third day up to 28 days. KP-13 and KP-234 were provided by Dr Min Chang (Lanzhou University). KP-13 and KP-234 were first dissolved in 2% DMSO and diluted in saline immediately prior to injection, as previously described (9). Cetrorelix (Sigma-Aldrich; Merck KGaA) was dissolved in saline and injected i.p. (1 mg/kg/10 ml) 30 min prior to KP-13 injection. The mice were sacrificed on day 28 after injection of BLM. All experiments were performed between 9:00 a.m. and 6:00 p.m. In addition, the dose selection was based on previous studies by Jiang et al (8,9).

The histopathological assay was performed, according to previous studies. Briefly, whole left lungs were fixed in 4% paraformaldehyde at 4°C overnight and embedded in paraffin. The 8-µm sections were stained with hematoxylin and eosin (H&E), Masson's trichrome stain and Picro-Sirius Red (PSR), using standard methods. The images were performed by an ordinary optical microscope (H&E and Masson) and polarized light microscope (PSR; Zeiss AG).

RT-qPCR was conducted according to the manufacturer's protocol (Takara Bio, Inc.). Total RNA was extracted using TRIzol^® reagent (Thermo Fisher Scientific, Inc.) following the manufacturer's protocol and 1 µg RNA sample was reverse transcribed into cDNA with the 5X PrimeScript RT Master Mix (Takara Bio, Inc.), the following conditions: 37°C for 15 min, 85°C for 5 sec, 4°C for 3 min. Amplification was conducted in a 25 µl reaction compound composed of 12.5 µl 2X SYBR Premis Ex TaqII (Takara Bio, Inc.), 8.5 µl ddH₂O, 2 µl cDNA, 1 µl forward primer and 1 µl reverse primer, and was carried out under the following conditions: 95°C for 30 sec, followed by 40 cycles at 95°C for 5 sec, 60°C for 30 sec and 72°C for 30 sec. Gene expression was evaluated using the 2^−∆∆Cq method (19) where ∆Cq=Cq_{target gene}-Cq_GAPDH and ∆∆Cq=Cq_Drug-Cq_Control. The primers are presented in Table I and GAPDH was used as the reference gene.

Western blotting was performed following the manufacturer's protocol (Bio-Rad Laboratories, Inc). Protein was extracted with RIPA buffer containing protease inhibitor (Gibco; Thermo Fisher Scientific, Inc.). The protein concentration was determined using a BCA protein assay kit (Pierce; Thermo Fisher Scientific, Inc.). The protein samples (40 µg per lane) were separated by SDS-PAGE on 10% gels and then transferred onto PVDF membranes. The membranes were blocked in 5% fat-free milk in TBST (0.1% Tween-20) at room temperature for 2 h, and incubated with specific antibodies overnight at 4°C as follows: Anti-α-smooth muscle actin (α-SMA; cat. no. 19245; 1:1,000; Cell Signaling Technology, Inc.), anti-TGF-β (cat. no. 3711; 1:1,000; Cell Signaling Technology, Inc.), anti-phosphorylated (p)-Smad2/3 (cat. no. 8828; 1:1,000; Cell Signaling Technology, Inc.) or anti-total (t)-Smad2/3 (cat. no. 3102; 1:1,000; Cell Signaling Technology, Inc.), anti-Bcl-2 (cat. no. D198628-0100; 1:500; BBI Life Sciences), anti-Bax (cat. no. D190756-0100; 1:500; BBI Life Sciences), anti-active caspase-3 (cat. no. D195315-0100; 1:500; BBI Life Sciences) and anti-GAPDH (cat. no. 5174; 1:2,000; Cell Signaling Technology, Inc.). After washing three times with PBS, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (cat. no. A0208, 1:5,000; Beyotime Institute of Biotechnology) at room temperature for 1 h. The result was visualized with chemiluminescence reagents using an ECL kit (Thermo Fisher Scientific, Inc.) and exposed to a film. The intensity of the blots was quantified with densitometry (Image J 1.49v; National Institutes of Health).

Total protein from the lungs of mice in each group were extracted with RIPA lysis buffer containing protease inhibitor (Gibco; Thermo Fisher Scientific, Inc.). Total proteins were determined using a bicinchoninic acid protein assay kit (Sangon Biotech Co., Ltd.). Interleukin (IL)-1β, IL-6 and tumor necrosis factor-α (TNF-α) in the lung tissue were measured using ELISA kits (cat. no. E-EL-M0049 for TNF-α, cat.no. E-EL-M0044 for IL-6, cat. no. E-EL-M0037 for IL-1β, Elabscience), according to the manufacturer's protocol.

All data are presented as the mean ± SEM for two repeats twice of each experiment. Overall survival was defined as the time period from the first day of BLM-induced pulmonary fibrosis to the date of succumbing, or until the 28th day of BLM-induced pulmonary fibrosis. Comparisons of mortality were made by analyzing Kaplan-Meier survival curves, and then log-rank tests to assess for differences in survival. The statistical analysis was conducted by two-way ANOVA followed by Dunnett's post-hoc test using SPSS 19.0 (IBM Corp.). P<0.05 was considered to indicate a statistically significant difference.

A comparison of 28-day survival curves among the four groups of mice with pulmonary fibrosis revealed that KP-13 improved the survival of mice with BLM-induced (4 mg/kg) pulmonary fibrosis (Fig. 1A). Additionally, mice in the BLM group had lost an amount of body weight between days 2 and 28 compared with the control mice, and it reached a significant difference at 28 days (P<0.001; Fig. 1B). Treatment with KP-13markedly inhibited these changes compared with the BLM group. The increased spleen/body weight ratio reflected the progression of inflammation. As shown in Fig. 1C, BLM treatment increased this ratio, while KP-13 treatment inhibited the increase in the ratio (P<0.05; BLM group compared with BLM+KP-13 group), suggesting that KP-13 suppresses inflammation.

Pulmonary injury and fibrosis induced by BLM in mice were evaluated by histopathological examination. The findings included severe edema, alveolar collapse, alveolar membrane thickening and inflammatory cell infiltration (Fig. 2). H&E, Masson's trichrome and PSR staining revealed severe collagen deposition induced by BLM in the lungs of the mice. However, treatment with KP-13 markedly reversed these changes and alleviated collagen deposition.

It was recently demonstrated that BLM-induced pulmonary fibrosis is correlated with alterations in inflammatory cytokines (TNF-α, IL-1β and TGF-β) and fibrosis-related factors [collagen type I α 1 (Colla1), actin α 2 (Acta2), tissue inhibitor of metalloproteinase 1 (Timp1) and matrix metalloproteinase 2 (MMP2)] (16). In the present study, the levels of these factors were increased in response to intratracheal BLM instillation compared with control mice. However, these increases were markedly inhibited by KP-13 application.

As shown in Fig. 3, the mRNA levels of Colla1, Acta2 and MMP2 were significantly increased following intratracheal BLM treatment (P<0.01 for Colla1; P<0.001 for MMP2 and Acta2; Fig. 3). Administration of KP-13 to the mice inhibited the expression of these genes induced by BLM administration (P<0.01 for Colla1; P<0.05 for MMP2 and Acta2; Fig. 3). TIMP1 is a collagenase inhibitory protein in pulmonary fibrosis. Thus, its expression level is inversely related to the level of fibrosis (20). Fig. 3C showed that BLM treated decreased the expression of TIMP1, whereas the decrease was markedly changed by KP-13 application (P<0.05, Fig. 3C). These results reflect the induction of fibrosis by BLM and the beneficial effect of KP-13 on IPF. As IPF is associated with inflammation, the expression levels of related inflammatory cytokines were also evaluated. The protein levels of IL-1β, TNF-α and IL-6, and the mRNA level of TGF-β were statistically significantly increased in the lung following intratracheal BLM treatment (P<0.001 for IL-1β and TGF-β; P<0.01 for TNF-α and IL-6; Fig. 4), while KP-13 injection significantly decreased the expression of those factors (P<0.05).

A main characteristic of pulmonary fibrosis is the over-proliferation of α-SMA-positive fibroblasts in the whole lung (21,22). Therefore, the ability of KP-13 to modulate the expression of α-SMA, which is a key marker of myofibroblasts, was further evaluated. The western blotting results demonstrated that BLM treatment upregulated the expression of α-SMA in lung tissues compared with the control group (P<0.001; Fig. 5), whereas the levels of α-SMA were reduced following KP-13 treatment compared with the BLM group (P<0.01; Fig. 5).

A number of previous studies have demonstrated the physiological and pathological roles of the KP/GPR54 signaling pathway in the regulation of the reproductive system, diabetes, adiposity, inhibition of cancer metastasis, and atherosclerotic plaque progression and instability (11,23–25). Recent studies have also reported that KP is a potent stimulator of GnRH secretion, and GnRH was reported to be associated with inflammation and fibrosis (14,15).

Therefore, KP-234, an antagonist of GPR54, and Cetrorelix, an antagonist of GnRHR, were used to determine whether they could block the anti-fibrotic effects of KP-13. The results revealed that KP-234, but not Cetrorelix, significantly attenuated the effects of KP-13 on BLM-induced pulmonary injury and fibrosis (P<0.05 for BLM+KP-13 group and BLM+KP-234+KP-13 group; Figs. 1, 2 and 5).

TGF-β is a key mediator of pulmonary fibrosis, as it regulates the synthesis of extracellular matrix proteins via the TGF-β/Smad2/3 signaling pathway (26). To elucidate the possible mechanisms through which KP-13 ameliorates BLM-induced pulmonary injury and fibrosis, the effects of KP-13 on the TGF-β/Smad2/3 signaling pathway were examined. As demonstrated by the western blot analysis results (Fig. 6A-C), the expression of TGF-β1, as well as the phosphorylation of Smad2/3, were significantly increased after treatment with BLM (P<0.01 for TGF-β1 and Smad2/3), which was downregulated following KP-13 application (P<0.05 for TGF-β1; P<0.001 for Smad2/3). Furthermore, the levels of pro-apoptosis related proteins, such as Bax and caspase-3 (P<0.05 for between Bax and caspase-3, Fig. 6A, D-F), were increased in the BLM group compared with the control. However, these pro-apoptosis proteins were significantly downregulated after KP-13 application (P<0.05, Fig. 6A, E and F). Meanwhile, anti-apoptosis related protein (Bcl-2) was markedly decreased by BLM, whereas KP-13 upregulated its expression level (Fig. 6A).