The aim of the present study was to assess the protective effect of silibinin against methotrexate (MTX)-induced pulmonary toxicity. Rats were divided into four groups (MTX, MTX + silibinin, silibinin and control. MTX was injected intraperitoneally (i.p) into female Wistar rats (10 mg/kg/day for 3 days), which resulted in significant increases in the serum levels of alanine aminotransferase, aspartate aminotransferase and oxidant enzymes, including nitric oxide and myeloperoxidase. Furthermore, significant reductions were detected in the serum activity levels of the antioxidative enzymes, glutathione peroxidase and superoxide dismutase, when compared with the control group. However, administration of silibinin (100 mg/kg/day for 10 days, i.p.) was shown to ameliorate the MTX-induced pulmonary toxicity, as indicated by the normalization of the oxidative stress parameters. Furthermore, silibinin treatment was demonstrated to reduce the histopathological changes associated with MTX. In conclusion, silibinin exhibited protective effects against MTX-induced pulmonary toxicity, which may be attributed to its antioxidant activity.
Methotrexate (MTX) is an antineoplastic agent that is associated with folic acid metabolism. MTX inhibits the synthesis of DNA, RNA, thymidylate and proteins. As a result of this activity, MTX is commonly used in cancer treatment, in addition to the treatment of non-neoplastic diseases, including rheumatoid arthritis and psoriasis (
Silibinin (C25H22O10; molecular weight, 482.44 g/mol) is isolated from the seeds of
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 (
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 (
SPSS software, version 21.0 (IBM SPSS, Armonk, NY, USA) and PAST software (
Serum levels of ALT and AST were significantly increased in the MTX group when compared with the control and silibinin groups (P<0.05;
When compared with the control (
The present study evaluated the interactions between silibinin and MTX, and their effects on lung tissue. The results indicate that treatment with silibinin ameliorated MTX-induced alterations in the serum ALT and AST levels. In addition, silibinin significantly mitigated the oxidation in the serum induced by MTX, as manifested by the decreased MPO and NO levels, accompanied by enhanced SOD and GPx activity. Furthermore, histopathological analysis demonstrated that administration of silibinin mitigated a number of the histopathological changes induced by MTX.
A previous study indicated that MTX exposure activates components of the mitogen-activated protein kinase (MAPK) signaling pathway (
Silibinin possesses marked antioxidative, anticancer, anti-inflammatory and cancer chemopreventive properties (
The potential for hepatic toxicity remains a concern for physicians who are increasingly using MTX (
In conclusion, silibinin was demonstrated to protect the lung tissue against MTX-induced pulmonary toxicity in rats. The antioxidant activity of silibinin may be the primary factor responsible for such pulmonary protective effects. Therefore, silibinin represents a potential candidate agent for the prevention of lung injury, which is a major and dose-limiting side effect of MTX therapy.
The authors thank Mugla Sıtkı Kocman University Hospital and School of Medicine.
Histological analysis of the control group pulmonary tissue (hematoxylin and eosin; magnification, x40).
Histological analysis of the silibinin group pulmonary tissue (hematoxylin and eosin; magnification, x100).
Histological analysis of the methotrexate group pulmonary tissue. (A) Interstitial lymphocytic inflammation and interstitial fibrosis were observed in the pulmonary tissue (hematoxylin and eosin; magnification, x100). (B) Type 2 pneumocyte hyperplasia was also observed in the tissue (magnification, x200).
Histological analysis of the methotrexate + silibinin group pulmonary tissue. Slight interstitial lymphocytic inflammation was observed in the pulmonary tissue, in addition to focal type 2 pneumocyte hyperplasia. Interstitial fibrosis was not observed (hematoxylin and eosin; magnification, x200).
Effect of silibinin on the biochemical parameters associated with MTX-induced pulmonary toxicity.
Group | ALT (U/L) | AST (U/L) | SOD (U/ml) | GPx (U/ml) | NO (µm/g) | MPO (ng/ml) |
---|---|---|---|---|---|---|
Control | 20.86±3.34 | 62.75±6.24 | 0.89±0.14 | 0.88±0.14 | 2.25±0.36 | 2.63±0.76 |
Silibinin | 16.25±2.60 | 49.38±7.90 | 3.54±0.57 | 0.24±0.04 | 3.05±0.49 | 2.70±0.53 |
MTX | 55.38±8.86 |
86.88±13.90 |
0.48±0.08 |
0.19±0.03 |
4.49±0.40 |
4.3±0.56 |
MTX + silibinin | 31.25±5.01 |
60.25±9.64 |
5.40±0.86 |
0.63±0.10 |
1.06±0.17 |
2.33±0.37 |
Results are presented as the mean ± standard deviation.
P<0.05, vs. control group
P<0.05, vs. silibinin group
P<0.05, vs. MTX group. MTX, methotrexate; ALT, alanine aminotransferase; AST, aspartate aminotransferase; SOD, superoxide dismutase; GPx, glutathione peroxidase; NO, nitric oxide; MPO, myeloperoxidase.
Histopathological damage parameters detected in the rat pulmonary tissue samples.
Histopathological parameter | Control | Silibinin | MTX | MTX + silibinin |
---|---|---|---|---|
Interstitial lymphocytic inflammation | 0.125 | 0.375 | 1.25 |
0.25 |
Interstitial fibrosis | 0 | 0 | 0.875 |
0 |
Type 2 pneumocyte infiltration | 0 | 0 | 0.5 |
0 |
Intraalveolar/interstitial macrophage existence | 0 | 0.375 | 0.5 | 0.125 |
Eosinophil existence | 0 | 0.5 | 0.875 |
0.125 |
Granuloma existence | 0 | 0 | 0.25 | 0 |
Results are presented as the mean ± standard deviation.
P<0.05, vs. control group
P<0.05, vs. silibinin group
P<0.05, vs. MTX group. MTX, methotrexate.