Potential Effect of Etoricoxib in Reducing Inflammation in Methotrexate-Induced Pulmonary Injury in Rats: Role of Oxidative Stress and the TLR4/p38-MAPK/NF-κB Signaling Pathway

Abstract

Numerous chemotherapeutic medications can have hazardous effects on the lungs, which can result in severe lung diseases. Methotrexate (MTX) is prescribed for cancer and inflammation-related disorders; nevertheless, it is exceptionally highly toxic and has multiple kinds of adverse reactions, including pulmonary injury. Our work was designed to demonstrate the ability of etoricoxib (ETO) to mitigate MTX-induced lung injury in experimental animals. Adult male Wistar rats were separated into four groups. The first group consisted of healthy controls that received carboxymethyl cellulose (1 ml/day, p.o.), the second group received a single dose of MTX (20 mg/kg/day, i.p.), the third group received ETO (10 mg/kg/day, p.o.) for three weeks, and the fourth group first received a single MTX (20 mg/kg, i.p.) and then was treated with ETO for three weeks. Concomitant treatment with ETO and MTX improved the histological structure of the lung tissue. It significantly altered the levels of oxidant/antioxidant markers, such as malondialdehyde (MDA), heme oxygenase-1 (HO-1), reduced glutathione (GSH), and nuclear factor erythroid 2-related factor 2 (Nrf-2), in favor of antioxidants. Moreover, ETO can normalize the proinflammatory cascade, which includes tumor necrosis factor-alpha (TNF-α) and interleukin-1 beta (IL-1β). At the molecular level, ETO downregulated the protein expression of toll-like receptor 4 (TLR4), nuclear factor kappa-B (NF-κB), and p38 mitogen-activated protein kinase (p38 MAPK) in inflamed rat lungs. In conclusion, our findings indicate that oral administration of ETO ameliorates MTX-induced lung injury by inhibiting oxidative stress and suppressing the TLR4/NF-κB and TLR4/p38-MAPK inflammatory signaling pathways.

Keywords: ETO; Inflammation; MTX; NF-κβ; Oxidative stress; Pulmonary toxicity; TLR-4.

Fig. 1

Effects of ETO on the pulmonary oxidative stress markers A) MDA content, B) GSH level, C) NF-2 activity, and D) HO-1 activity in MTX-treated rats. The data are presented as the means ± S.Ds. (n = 12 mice). a, b Significantly different from the normal control and MTX groups, respectively, at P < 0.05. MTX: methotrexate; ETO: etoricoxib; MDA: malondialdehyde; GSH: reduced glutathione; NF-2: nuclear factor erythroid 2-related factor 2; HO-1: heme oxygenase-1

Fig. 2

Effects of ETO on the levels of the pulmonary proinflammatory markers A) IL-1B and B) TNF- in MTX-treated rats. The data are presented as the means ± S.Ds. (n = 12 mice). a, b Significantly different from the normal control and MTX groups, respectively, at P < 0.05. MTX: methotrexate; ETO: etoricoxib; TNF-α: tumor necrosis factor-alpha; IL-1β: interleukin-1 beta

Fig. 3

Effects of ETO on pulmonary protein expression levels of A) TLR4, B) p38MAPKN, and C) NF-kB in MTX-treated rats. The data are presented as the means ± S.Ds. (n = 12 mice). a, b Significantly different from the normal control and MTX groups, respectively, at P < 0.05. MTX: methotrexate; ETO: etoricoxib; TLR4: toll-like receptor 4; p38 MAPK: p38 mitogen-activated protein kinase; NF-κB: nuclear factor kappa-B

Fig. 4

Effects of ETO on pulmonary histopathological alterations in MTX-treated rats (n = 12 rats) examined via H&E staining. (A) A photomicrograph (× 400) of lung sections from the control group showing preserved alveolar structures. The alveoli are rather uniform in size and shape. The alveolar walls have average thickness with no thickening, edema, or inflammation (black arrows). (B-D) A photomicrograph (× 400) of the lung tissue of MTX-treated rats showing widespread interstitial and intra-alveolar hemorrhage (yellow arrow). Numerous congested capillaries of the alveolar wall were observed. Focal dense infiltration by mixed inflammatory cells, including neutrophils and lymphocytes (red arrows), was detected. Numerous hemosiderin-laden macrophages and hemosiderin particles were observed. Focal emphysema with disruption of the alveolar wall was detected (black arrow). (E) A photomicrograph (× 400) of lung sections from the ETO-treated group shows a lung structure like that of the negative control group (black arrows). (F) A photomicrograph (× 400) of lung sections from MTX-treated rats subjected to ETO showing mild residual inflammation of the lung tissue with relative thickening of the alveolar wall (black arrow). Mild residual congestion (red arrow). Mild residual hemorrhage and a few hemosiderin particles were also detected (yellow arrow)