OA (purity >99.9%; cat. no. 110742-200513) was purchased from the National Institutes for Food and Drug Control (Beijing, China) and was suspended in 0.6% sodium carboxymethyl cellulose for use. Crystalline SiO₂ (~95%; 1–5 µm), obtained from the National Institute of Occupational Health and Poison Control, Chinese Center for Disease Control and Prevention (Beijing, China), were subjected to grinding, and heating for at 180°C for 6 h, followed by dilution with sterile saline to a concentration of 50 mg/ml in suspension, autoclaved and stored at 4°C. Enzyme-linked immunosorbent assay (ELISA) kits, malondialdehyde (MDA), superoxide dismutase (SOD) and glutathione (GSH) test kits, and Masson's stain were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Phosphorylated (p-)AKT1 (phospho S473; cat. no. ab81283) and AKT (cat. no. ab28422) antibody were purchased from Abcam (Cambridge, MA, USA). NF-κB antibody (cat. no. sc-8008) was purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA).

A total of 96 male adult Wistar rats (aged 6–8 weeks and weighing 180–200 g) were purchased from Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). The animal experiments were reviewed and approved by the Institutional Animal Care and Use Committee at the North China University of Science and Technology (Tangshan, China). The animals received food and water according to guidelines set by the National Institutes of Health (Bethesda, MD, USA) and were housed in an air-conditioned room with a 12-h light/dark cycle at constant temperature (21°C) and 55% humidity for at least 1 week prior to the experiments.

The rats were divided into four groups according to the randomized block design, namely, a control group, model group, solvent control group and OA group, with 24 rats in each group. With the exception of the rats in the control group, the rats were induced by intratracheal instillation of SiO₂ (250 mg/kg). The rats in the OA group were intragastrically administered with OA (60 mg/kg) daily from the second day following SiO₂ administration. The rats in the solvent control group were gavaged daily with 0.6% sodium carboxymethyl cellulose (10 ml/kg) solution, whereas the rats in the control group were gavaged with physiological saline in the same conditions for 56 consecutive days. The rats (n=6/group) were sacrificed on days 7, 14, 28 and 56. Blood samples were collected into heparinized tubes via the abdominal aorta at the four distinct time points. The blood samples were immediately centrifuged at 3,000 × g for 10 min at room temperature, and the serum was frozen at −80°C for subsequent analyses. The lung tissues were immediately perfused with physiological saline solution to removed blood cells. Then right lower lobe tissues were removed and stored at −80°C for western blot analysis, and the right upper lobes were used for the determination of hydroxyproline. The left lung tissues were fixed in paraformaldehyde for the assessment of morphological changes using hematoxylin and eosin (HE) staining and Masson's staining with immunohistochemical analysis.

The oxidation/antioxidant system includes MDA, SOD and GSH peroxidase (GHS-Px). The MDA content and activities of SOD/GSH-Px in serum samples were assayed using MDA and SOD/GSH-Px test kits according to the manufacturer's protocol (Nanjing Jiancheng Bioengineering Institute). MDA, the end product of lipid peroxidation of cells, condenses with thiobarbituric acid to form a product with a maximum absorption at 532 nm, therefore, the MDA content in the samples was determined by comparing the optical density (O.D.) of the samples to the standard substance. SOD and GSH-Px, two important antioxidant enzymes, can scavenge free radicals and inhibit lipid peroxidation. The activities of SOD and GSH-Px activity are expressed in units, with 1 unit defined as 50% inhibition of nitrite formation. SOD can inhibit the hydroxylamines of superoxide radical anions oxidized to form nitrite, and appear violet with a color reagent; the change in absorbance was recorded at 550 nm. The activity of GSH-Px was determined using the disulfide-nitrobenzoic acid direct method and compared at the O.D. at 412 nm with the standard substance.

The serum contents of TNF-α and TGF-β1 were detected using ELISA, according to the manufacturer's protocol (Wuhan Boster Biological Technology, Ltd., Wuhan, China). The anti-rat TNF-α (cat. no. EK0526) or TGF-β1 (cat. no. EK0514) specific antibody was coated on the ELISA plates. The standards and samples were pipetted into the wells and the TNF-α/TGF-β1 present in the sample bound to the wells by the immobilized antibody. The liquid was removed from the wells and biotinylated anti-rat TNF-α or TGF-β1 antibody (1:100 dilution, at 37°C for 60 min) was added. Following washing away unbound biotinylated antibody, HRP-conjugated streptavidin was pipetted into the wells for 30 min at 37°C. The wells were again washed, and TMB color liquid was added to the wells for 20 min at 37°C, with color developing in proportion to the level of TNF-α (TGF-β1) bound. The stop solution alters the color from blue to yellow, and the intensity of the color was measured at 450 nm. The placental TNF-α/TGF-β1 levels were calculated as pg/ml using the standard curve.

The content of collagen in the right upper lobe lung tissue was determined using a hydroxyproline assay according to the manufacturer's protocol (Nianjing Jiancheng Bioengineering Institute.). Briefly, the lung tissue samples (80 mg) were hydrolyzed in boiling water for 5 h. Following addition of the immobilized reagents, the mixture was placed in 60°C water for 15 min and centrifuged at 3,000 × g for 15 min at room temperature after cooled down. The hydroxyproline content of the supernatant was quantified by spectrophotometry at 550 nm. The data are expressed as collagen (µg)/wet weight (mg).

The immunohistochemistry used Streptavidin-peroxidase Histostain TM-Plus kits (cat. no. SP-9000; OriGene Technologies, Inc., Beijing, China). The important stages were as follows: Paraffin-embedded sections (5 µm) were deparaffinized, rehydrated and underwent removal of endogenous peroxidase with 3% H₂O2. Following incubation with goat serum working solution, the working solution was discarded and the tissue sections were incubated with primary antibodies against AKT1-phospho S473 (1:200; Abcam), NF-κB (1:200; Santa Cruz Biotechnology, Inc.), and collagen type I (cat. no. BA0325) and III (cat. no. BA0326; 1:200; Wuhan Boster Biological Technology, Ltd.) overnight at 4°C, followed by the biotinylated secondary antibody at 37°C for 15 min and streptavidin-peroxidase at 37°C for 15 min. Immunoreactivity was visualized with DAB (Fuzhou Maixin Biotech. Co., Ltd., Fuzhou, China). Brown color staining was considered a positive result. Sections were counterstained with hematoxylin and images were captured from six separate randomly selected fields using Olympus FV1000 (magnification ×400; Olympus Inc., Center Valley, PA, USA). Quantitative analysis was performed in a blinded-manner using an automatic image analysis system at Beijing University of Aeronautics and Astronautics (Beijing, China), with the average O.D. values as quantitative indicators.

The middle lobe of the right lung (100 mg) was lysed with RIPA lysis buffer (1 ml), and then centrifuged at 10,000 × g for 15 min at 4°C. The supernatant was collected and protein content was determined using a protein assay kit (Beyotime Institute of Biotechnology, Tianjin, China). The proteins (10 µg) were separated by 10% SDS-PAGE under a constant voltage of 120 V for 2 h, and then transferred onto a polyvinylidene fluoride membrane at a constant electric current of 250 mA for 30 min (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Following blocking with 5% non-fat dry milk, the membranes were incubated at 4°C overnight with primary antibodies at the following dilution ratios: GAPDH antibody (cat. no. sc-25778, 1:2,000; Santa Cruz Biotechnology, Inc.); anti-AKT1-phospho S473 antibody (1:5,000; Abcam); anti-AKT1 antibody (1:5,000; Abcam). The membranes were then washed three times with PBST and incubated with HRP-conjugated anti-rabbit IgG antibody (cat. no. 074-1506, 1:5,000; KPL, Inc., Gaithersburg, MD, USA) for 2 h at room temperature, followed by washing with PBST. The proteins were visualized using chemiluminescence (ECL; Beyotime Institute of Biotechnology).

All data are expressed as the mean ± standard deviation. SPSS 17.0 software (SPSS, Inc., Chicago, IL, USA) was used to perform statistical analyses. Multiple group comparisons were performed using one-way analysis of variance followed by pair-wise comparison with the Student's t-test. P<0.05 was considered to indicate a statistically significant difference.

Pathological changes in the lung tissues of the rats were observed by light microscopy with HE and Masson's staining. As shown in Fig. 1Aa-d, the lungs of the rats in the control group, which received physiological saline, had a thin alveolar septum without significant inflammation and no obvious abnormalities shown by the HE stain. However, in the model group and solvent control group, at day 14 post-instillation, there was marked infiltration of inflammatory cells and alveolar septum thickening in the lungs, with occasional small numbers of cellular nodules (stage I; and Table I). At 28 days, primarily cellular nodules (stage I+) and fibrotic cellular nodules (stage II+) were observed. At 56 days, fibrous nodules were integrated with each other, there were more fibrotic cellular nodules (stages II+ and III). By contrast, OA treatment significantly reduced inflammatory cell infiltration and alveolar septum thickening at 14 days; the size and number of cellular nodules (stage I+) were decreased at 28 and 56 days. Masson's staining (Fig. 1Ba-c) showed: blue collagen fibers, red muscle fibers, cytoplasm and red blood cells, and brown nuclei. In the control group, there was a small quantity of blue collagen fiber around the bronchial and alveolar septum area during the investigation. In the model group and solvent control group, diffuse collagen fibers were increased and arranged irregularly in the nodules at 28 days; increased collagen deposition was present, which was arranged in concentric circles, at 56 days; lung fibrosis was aggravated. OA treatment significantly reduced collagen fibers, in small sections or small bundles. These results indicated that the silicosis model was successfully constructed and that OA exerted a significant protective effect.

As shown in Table II, compared with the control group, the content of MDA in sera of the model group and solvent control group increased, peaking at 14 days, followed by a marginal decrease, although significant differences were found in the statistical analysis (P<0.05). However, OA treatment significantly decreased the content of MDA, compared with the content in the model group and solvent control groups at corresponding time points (P<0.05).

The activities of SOD/GSH-Px increased in the sera from the model group and solvent control group, compared with the control group at corresponding time points (P<0.05), however, the activities of SOD/GSH-Px increased with time in the sera from the model group and solvent control group. Compared with the model group and solvent control group, the activities of SOD/GSH-Px were significantly increased in the OA group at the corresponding time points (P<0.05).

Hydroxyproline content is an important indicator of total collagen in lung fibrosis, which are predominantly composed of collagen types I and III. In the present study, we detected the content of hydroxyproline using a hydroxyproline kit, and the expression levels of collagen types I and III using immunohistochemistry (Fig. 2A-C). No significant differences in the hydroxyproline content or levels of collagen types I and III were found in the control group during the investigation. However, the total collagen/hydroxyproline content and the expression levels of collagen I and III in the model group and solvent control group were significantly higher, compared with those in the control group at corresponding time points (P<0.05). As expected, OA treatment significantly reduced these levels, compared with those in the model group and solvent control group (P<0.05), but remained higher, compared with those in the control group (P<0.05).

TNF-α and TGF-β1 are important cytokines and are involved in inflammation and pulmonary fibrosis. As shown in Fig. 3A and B, no differences in the serum contents of TNF-α and TGF-β1 were found in rats of the control group at the four time points (P>0.05). The serum contents of TNF-α in the model group and solvent control group were significantly increased, peaking at day 14 day post-instillation, with a subsequent marginal decrease, but with statistically significant differences at each time point, compared with those in the control group (P<0.05), whereas TGF-β1 increased following instillation (P<0.05). No significant differences between the model group and solvent control group were found at different time points. OA treatment had inhibitory effects on the contents of TNF-α and TGF-β1, compared with the model group and solvent group at the corresponding time points (P<0.05).

Immuno-histochemical methods were used to observe the expression of p-AKT1/NF-κB in the rat lung tissues. As shown in Fig. 4, the levels of NF-κB p65 in the nuclear fractions were significantly increased in the model group and solvent control group, compared with that in the control group (P<0.05); whereas positive staining for p-AKT1 (Fig. 5) was primarily present in the nuclei of interstitial cells in the lung tissues of the model group and solvent control group, but not in the control group. OA treatment markedly reduced this positive staining (P<0.05). The immunohistochemical results were further confirmed using western blot analysis (Fig. 6), which showed that the model group and solvent control groups exhibited increased expression levels of ph-AKT1, compared with levels in the control group at the corresponding time points (P<0.05) and that OA treatment significantly weakened the expression of p-AKT1 in the rat lungs, compared with levels in the model group and solvent control group at the corresponding time points (P<0.05).