Animal experiments were approved by the Animal Ethical Committee of Suleyman Demirel University (B.30.2.SDÜ.0.05.06.00–196, 2012; Isparta, Turkey), and the study was conducted in accordance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals (8th Edition, 2011). In total, 32 female albino Wistar rats (age, 8–10 weeks) were obtained from the Experimental Research Centre of Suleyman Demirel University and housed in an environmentally controlled room at 21±1°C and 75±5% humidity, under a 12-h light/dark cycle. The animals were acclimatized for 1 week prior to the study, and had free access to standard laboratory feed and water.

Rats were divided into four groups. Since silibinin was solubilized in dimethyl sulfoxide (DMSO), an equal amount of DMSO (10%v/v) was included in the injections administered to the control group rats. The silibinin group rats received 100 mg/kg/day silibinin (Sigma-Aldrich, St. Louis, MO, USA), which was administered via intraperitoneal (i.p.) injection for 10 days (16). The MTX group rats were administered 10 mg/kg/day MTX (i.p.) on days 7–9, the commercially available form of the drug was used, it was taken from pharmacy with our own means (17). Finally, the MTX + silibinin group rats were cotreated with 100 mg/kg/day silibinin for 10 days and 10 mg/kg/day MTX for 3 days. In all the groups on day 14, anesthesia was induced by a single i.p. injection of 50 mg/kg ketamine (Ketalar®; Pfizer, Inc., Istanbul, Turkey) and 5 mg/kg xylasine (Rompun®; Bayer, Istanbul, Turkey). Blood samples were collected via cardiac puncture for serum analyses and the lungs were harvested for histological and immunohistochemical analysis.

All the biochemical analyses were performed in the Department of Biochemistry at Mugla Sıtkı Kocman University (Mugla, Turkey).

Serum activity levels of AST and ALT were calculated spectrophotometrically using Beckman Coulter kits and a UniCel DxC 800 Synchron autoanalyzer (Beckman Coulter, Inc., Brea, CA, USA).

Tissue samples were homogenized at 4,200 × g on ice in 5–10 ml cold buffer [20 mM HEPES buffer (pH 7.2) containing 1 mM EGTA, 210 mM mannitol and 70 mM sucrose per gram tissue]. Subsequently, the samples were centrifuged at 1,500 × g for 5 min at 4°C, and the supernatant was removed. In addition, the blood samples were centrifuged at 2,000 × g for 15 min at 4°C, after which the top yellow serum layer was pipetted off, without disturbing the white buffy coat. The serum was diluted 1:5 with sample buffer. The SOD activity was measured in the supernatant and serum using a SOD assay kit (Cayman Chemical Company, Inc., Ann Arbor, MI, USA) with an ELx-800 absorbance reader (Bio-Tek Instruments, Inc., Winooski, VT, USA). The assay was based on the detection of superoxide radicals generated by xanthine oxidase and hypoxanthine. One unit of SOD was defined as the quantity of enzyme required to induce 50% dismutation of the superoxide radical. The results are expressed in U/mg protein tissue for the liver tissue and U/ml for the serum.

Tissue samples were homogenized in 5–10 ml cold buffer [50 mM Tris-HCl (pH 7.5), 5 mM EDTA and 1 mM DTT], and centrifuged at 10,000 × g for 15 min at 4°C, after which the supernatant was removed. In addition, the blood samples were centrifuged at 700–1,000 × g for 10 min at 4°C, and the plasma was removed. GPx activity was measured in the liver tissue and plasma samples using a GPx assay kit (Cayman Chemical Company, Inc.) with an ELx-50 microplate strip washer. GPx activity was measured indirectly by a coupled reaction with glutathione reductase, where the oxidized glutathione was produced upon the reduction of hydroperoxide by GPx.

Tissue samples were homogenized in phosphate-buffered saline (pH 7.4) and centrifuged at 10,000 × g for 20 min to isolate the supernatant. A total NO assay was performed via spectrophotometry at 540 nm using a nitrate/nitrite colorimetric assay kit (Cayman Chemical Company, Inc.) with an ELx-50 microplate strip washer. The assay was based on nitrate and nitrite determinations, in which nitrate and nitrite were the stable end products of the reaction of NO with molecular oxygen. The total accumulation of nitrate and nitrite in the serum and liver tissue samples was evaluated, and expressed in µM/protein.

Quantitative detection of MPO activity was conducted using an enzyme-linked immunosorbent assay kit (MPO Instant ELISA; eBioscience, Inc., Vienna, Austria) in an ELx-50 microplate strip washer. The results are expressed in pg/ml/protein.

Histopathological analyses were performed in the Department of Pathology at Mugla Sıtkı Kocman University. Rat lung samples from the different groups were fixed in 10% neutral buffered formalin for 24 h. After washing with tap water, serial dilutions of alcohol (methyl, ethyl and absolute ethyl) were applied for dehydration. The specimens were cleared in xylene, and embedded in paraffin at 56°C in an oven for 24 h. Paraffin bees wax tissue blocks were prepared for sectioning at 5 µm thickness using a microtome. Lung tissues were stained with hematoxylin and eosin and observed under a BX46 light microscope (Olympus Corporation, Tokyo, Japan) for histopathological evaluation. Pulmonary damage was evaluated using six parameters, which included interstitial lymphocytic inflammation, interstitial fibrosis, type 2 pneumocyte infiltration, intraalveolar/interstitial macrophage existence, eosinophil existence and granuloma existence (18). Each parameter was scored semiquantitatively with regard to the severity using the following system: 0 (absent), no interstitial lymphocytic inflammation, fibrosis, type 2 pneumocyte infiltration, macrophages, eosinophils or granulomas; 1 (low), limited number of lymphocytes, type 2 pneumocytes, macrophages, eosinophils and granulomas, with minimal fibrosis; 2 (moderate), a variety of features in between a score of 1 and 3; and 3 (severe), diffuse infiltration of an excessive number of lymphocytes, type 2 pneumocytes, macrophages, eosinophils, granulomas and excessive, diffuse fibrosis (6–12).

SPSS software, version 21.0 (IBM SPSS, Armonk, NY, USA) and PAST software (http://folk.uio.no/ohammer/past/) were used for data analysis. For comparisons among multiple independent groups one-way analysis of variance (ANOVA) was used. If significance was identified in the test we used the Least Significant Difference (LSD) test for post-hoc analysis. Parametric methods were used for the analysis of the data with a normal distribution, while non-parametric methods were used for the analysis of the variations without a normal distribution. For comparisons among multiple independent groups, ANOVA (Robust test: Brown-Forsythe), one of the parametric methods was used, while for post-hoc analysis, Fisher's LSD test was used. For the comparison of categorical data, Pearson's χ² test was conducted using the Monte Carlo simulation technique. Quantitative data are expressed as the mean ± standard deviation, and categorical data are expressed as a number and percentage. Data were analyzed in the 95% confidence level, where P<0.05 was considered to indicate a statistically significant difference.

Serum levels of ALT and AST were significantly increased in the MTX group when compared with the control and silibinin groups (P<0.05; Table I). In the MTX + silibinin group, the ALT and AST levels in serum decreased significantly compared with the MTX group (P<0.05). Furthermore, in the MTX group, the SOD levels decreased significantly when compared with the control group (P<0.05). The levels of GPx and SOD increased in the MTX + silibinin group when compared with the MTX group, while the MPO values decreased significantly (P<0.05).

When compared with the control (Fig. 1) and silibinin groups (Fig. 2), the pulmonary damage appeared to be significantly increased in the MTX group (Fig. 3). Similarly, when the MTX + silibinin group was compared with the MTX group, the damage was observed to significantly decrease (Fig. 4 and Table II). MTX administration was shown to induce interstitial lymphocytic inflammation, interstitial fibrosis, type 2 pneumocyte hyperplasia and eosinophil infiltration in the pulmonary tissue. However, the MTX + silibinin group exhibited significantly decreased scores for interstitial lymphocytic inflammation, interstitial fibrosis and type 2 pneumocyte hyperplasia when compared with the MTX group, indicating that silibinin exerted beneficial effects (Table II).