A total of 100 specific pathogen-free male Wistar rats (3 weeks of age) were obtained from the Vital River Laboratory (Beijing, China). The rats were housed in a temperature-controlled facility at 22±2°C, 50±10% humidity and 0.03–0.04% CO₂, with a 12 h light/dark cycle and received food and water according to the guidelines established by the North China University of Science and Technology (16). HOPE MED 8050 exposure control apparatus was supplied by Tianjin Hope Industry and Trade Co., Ltd. (Tianjin, China). SiO₂ was obtained from Sigma Aldrich (Merck KGaA, Darmstadt, Germany). The silica content of SiO₂ was >99% and its dust particle size distribution was as follows: 97% <5 µm diameter, 80% <3 µm diameter (according to the supplier). Ac-SDKP was supplied by Bachem (Bubendorf, Switzerland). Ang II and valsartan were purchased from Sigma-Aldrich (Merck KGaA). Ac-SDKP (cat. no. CSB-E0925r) and Ang II (cat. no. CSB-E04494r) ELISA kits were obtained from CUSABIO (Wuhan, Hubei, China). The PV-6000 1-step polymer detection system for mouse, rabbit and rat antibodies were acquired from Beijing Zhongshan Jinqiao Biotechnology Co., Ltd. (Beijing, China). Enhanced chemiluminescence reagent (cat. no. RPN2232) was supplied by GE Healthcare (Chicago, IL, USA). Masson's trichrome stain was supplied by Baso Diagnostics, Inc. (Wuhan, China). Polyclonal rabbit anti-collagen (Col) I (cat. no. ab34710), anti α-smooth muscle actin (SMA; cat. no. ab32575), anti-Fibronectin (Fn) (cat. no. ab45688), rabbit monoclonal anti-AT1 (cat. no. ab124734) and mouse monoclonal anti-ACE (cat. no. ab117334) were purchased from Abcam (Cambridge, UK). Polyclonal rabbit anti-matrix metalloproteinase (MMP)-1 (cat. no. EL800296-50) and anti-tissue inhibitor of metalloproteinases-1 (TIMP-1; cat. no. EL800628-50) were obtained from Eterlife, Ltd. (Birmingham, UK). Polyclonal mouse anti-β-actin (cat. no. sc-47778) and polyclonal rabbit anti-GAPDH (cat. no. sc-25778) were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). Dulbecco's modified Eagle's medium (DMEM) was obtained from Biological Industries USA (Cromwell, CT, USA). Fetal bovine serum (FBS) was purchased from Gibco (Thermo Fisher Scientific, Inc., Waltham, MA, USA).

All animal studies were approved by the Institutional Animal Care and Use Committee of North China University of Science and Technology. HOPE MED 8050 exposure control apparatus was used to create the silicosis model. The settings were as follows: Cabinet temperature, 20–25°C; humidity, 70–75%; pressure, −50 to +50 Pa; oxygen concentration, 20%; flow rate of SiO₂: 3.0–3.5 m/min; dust mass concentration, 2,000 mg/m³. Each rat inhaled SiO₂ for 3 h per day for 0, 2, 4, 8, 12 and 16 weeks. The rats were randomly divided into 6 groups (n=10) and inhaled dust for 0, 2, 4, 8, 12 and 16 weeks to observe dynamic changes during the development of silicosis. To determine the anti-silicotic effect of Ac-SDKP, rats were randomly divided into 3 groups (n=10): i) 16 week control (treated with 0.9% saline for 16 weeks); ii) 16 week silicosis (treated with 0.9% saline 48 h before SiO₂ inhalation and thereafter for 16 weeks); and iii) Ac-SDKP post-treatment (inhaled SiO₂ and then treated with 0.9% saline for 8 weeks and Ac-SDKP for another 8 weeks). Ac-SDKP (800 mg/kg/day) or control (0.9% saline) was administered via a mini-osmotic pump implanted into the abdominal cavity as previously described (17). The rats were anesthetized, and sera and lung samples were collected and frozen at −80°C until subsequent analysis.

Rats (n=10) were anaesthetized and lung tissue was removed. Lung fibroblasts were isolated from minced tissue, as previously described (18) and plated on 25-cm² plates containing DMEM supplemented with 10% FBS and 1% penicillin-streptomycin. The cells were cultured in a humidified atmosphere containing 5% CO₂ at 37°C. Cells at 80% confluence were cultured in FBS-free DMEM for 24 h, when most cells were quiescent. The induction effect of Ang II (10⁻⁷ mol/l) on the transformation of lung fibroblasts into myofibroblasts was studied at different time points (0, 5, 15 and 30 min, and 1, 3, 6, 12, 24 and 48 h) at 37°C. Primary lung fibroblasts were divided into four groups and cultured for 24 h at 37°C: i) Control (FBS-free DMEM); ii) Ang II (10⁻⁷ mol/l); iii) Ang II + Ac-SDKP (10⁻⁸ mol/l); and iv) Ang II + valsartan (10⁻⁶ mol/l).

The right lower lungs were fixed in 4% paraformaldehyde at 4°C for 24 h, embedded in paraffin, sectioned at 5 µm and subjected to hematoxylin and eosin (H&E) and Masson trichrome staining at room temperature for pathophysiological observation. For H&E stainng, the tissues sections were stained with hematoxylin for 30 sec and with stain 1% eosin solution for 1 min. For Masson trichrome staining, the tissue sections were stained with Weigert's working hematoxylin for 10 min. Subsequently, the sections were washed in running tap water for 5 min. Biebrich scarlet staining was subsequently performed for 5 min. Phosphomolybdic acid was added for 10 min and then the solution was discarded. Tissue samples were transferred directly into Aniline blue for 5 min and rinsed in distilled water. Images in each rat were obtained from 5 randomly selected fields of view using Olympus DP80 light microscope (magnification, ×200; Olympus Corporation, Tokyo, Japan).

IHC of lung tissue sections (5 µm) and cultured fibroblasts (passage 9) was performed. Paraffin embedded sections were deparaffinized, rehydrated and underwent removal of endogenous peroxidase with 3% H₂O₂ and antigen retrieval was performed using 0.01 M citrate buffer. Following blocking with 5% bovine serum albumin (Sigma-Aldrich, Merck KGaA) in 0.1 M phosphate-buffer saline (PBS) for 30 min at room temperature to reduce nonspecific binding, samples were incubated with a primary antibody against α-SMA (1:200; cat. no. ab32575; Abcam) overnight at 4°C, followed by incubation with a goat anti-mouse/rabbit secondary antibody (cat. no. PV-6000, ready to use solution; OriGene Technologies, Inc., Beijing, China) at 37°C for 1 h. Immunoreactivity was visualized with 5% 3,3′-diaminobenzidene (cat. no. ZLI-9018; OriGene Technologies, Inc., Beijing, China) at room temperature for 5 min. Brown staining was considered positive. PBS, instead of primary antibody, was used as a negative control and α-SMA staining of vascular smooth muscle cells was used as a positive control. Images were obtained from 5 separate randomly selected fields of view using the Olympus DP80 (magnification, ×200).

Total proteins were extracted from lung tissues or cells. Lung or cultured fibroblasts were lysed with radioimmunoprecipitation assay (RIPA) buffer (1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 150 mM NaCl, 1 mM EDTA, and 50 mM Tris-HCl, pH 7.5) and centrifuged at 14,000 × g for 10 min at 4°C. Protein concentrations in supernatants were measured with a Bradford assay (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China). Protein samples (20 µg/lane) were separated by 10% SDS-PAGE and transferred onto polyvinylidene fluoride membranes. Membranes were blocked with 5% non-fat milk for 2 h at room temperature and incubated overnight at 4°C with the primary antibody against Col I, α-SMA, Fn, ACE and AT1 (all 1:500) followed by a peroxidase-labeled affinity-purified secondary antibody to rabbit/mouse IgG (1:5,000, cat. no. 074-1506, Kirkegaard & Perry Laboratories, Inc., Gaithersburg, MD, USA) for 2 h at 37°C. Target bands were visualized by the addition of an Enhanced Chemiluminescence Prime Western Blotting Detection reagent (GE Healthcare, Chicago, IL, USA). The density of specific bands was analyzed using Image-Pro Plus image processing software (version 6.0; Media Cybernetics, Inc., Rockville, MD, USA) and the results were normalized to β-actin or GAPDH (both 1:500).

Lung tissues were lysed with RIPA and the levels of Ac-SDKP and Ang II were determined by using the corresponding ELISA kits according to the manufacturer's protocol.

All experiments were performed in triplicate. The results are expressed as the mean ± standard error of the mean. Comparisons between multiple independent groups were performed using one-way analysis of variance followed by a post hoc analysis with the Bonferroni test. P<0.05 was considered to indicate a statistically significant difference.

The results of HE staining revealed that in the normal controls, the alveolar walls were thin and there was no obvious inflammatory cell infiltration (Fig. 1A). In the 2-week silicosis group, macrophages were observed in the alveoli and the alveolar wall was wider in appearance. The 4-week silicosis group exhibited isolated cell nodules that contained macrophages. The 8- and 12-week silicosis groups had significantly wider alveolar walls and a greater number of silicotic nodules. The 16-week silicosis group exhibited an increase in silicotic nodule volume, nodule fusion, interstitial fibrosis and cell fibrous nodules (Fig. 1A). Blue purple Masson trichrome staining was observed in the nodules and interstitial fibrosis area, indicating collagen deposition, which gradually increased with model preparation time and most distinct in the 16-week silicosis group (Fig. 1B).

To investigate whether ACE/Ang II/AT1 proteins are involved in the pathogenesis of silicotic fibrosis, the expression of ACE, Ang II and AT1 in rat lungs was assessed. The expression of ACE, Ang II and AT1 was increased in the 2-, 4-, 8-, 12- and 16-week silicosis groups compared with the control group (Fig. 2A and B). Ac-SDKP expression in the 2-week silicosis group was significantly higher compared with the control group, whereas expression in the 4-, 8-, 12- and 16-week silicosis groups decreased (Fig. 2C).

Western blotting revealed that, compared with the control group, α-SMA, Col I and Fn expression was gradually increased in the 4-, 8-, 12- and 16-week silicosis groups (Fig. 3A). In response to Ang II stimuli, the cultured rat lung fibroblasts underwent differentiation and produced excessive amounts of ECM in vitro. Ang II stimulated the expression of α-SMA, Col I and Fn, which increased with treatment duration (Fig. 3B). Based on these findings and results of a previous study (19), 24 h was selected as the optimal treatment time for subsequent studies.

Compared with the 16-week silicosis group, treatment with Ac-SDKP resulted in decreased AT1 expression. The expression of ACE and Ang II (Fig. 4A and B) in the Ac-SDKP treatment group decreased, but was not statistically significant. The Ang II-dependent increase in AT1 expression was also inhibited following Ac-SDKP and valsartan treatment (Fig. 4C).

IHC staining revealed that α-SMA-positive myofibroblasts were located in the nodules and distributed in the interstitial fibrotic area in the 16-week silicosis group (Fig. 5). Ac-SDKP treatment reduced the expression of α-SMA. Compared with the 16-week silicosis group, α-SMA, Col I and Fn expression levels were significantly downregulated in the Ac-SDKP post-treatment group (Fig. 6). Subsequently, α-SMA expression was assessed using IHC staining. Compared with the control group, α-SMA expression was demonstrated to be increased following Ang II induction in cultured lung fibroblasts. Compared with the Ang II group, the expression of α-SMA was decreased in the Ac-SDKP and valsartan treatment groups (Fig. 7). The Ang II-dependent increase in α-SMA, Col I and Fn expression was also inhibited following Ac-SDKP and valsartan treatment (Fig. 8).

Western blotting demonstrated that the expression of TIMP-1 was markedly increased in the silicosis group compared with the control group, whereas MMP-1 expression and the MMP-1/TIMP-1 ratio was significantly decreased (Fig. 9A). Treatment with Ac-SDKP increased the expression of MMP-1 and the MMP-1/TIMP-1 ratio. No significant differences in TIMP-1 expression were observed between the Ac-SDKP post-treatment and silicosis groups. In vitro experiments revealed that MMP-1 expression and the MMP-1/TIMP-1 ratio was reduced compared with the control and the expression of TIMP-1 was markedly increased in fibroblasts induced by Ang II (Fig. 9B). Ac-SDKP or valsartan treatment upregulated the expression of MMP-1 and enhanced the MMP-1/TIMP-1 ratio, whereas it had no significant effect on the expression of TIMP-1.