Ambrisentan ameliorates methotrexate-induced pulmonary injury in rats: involvement of ET-1/ETA, NF-κB, and MMP-9/TIMP-1 pathways

Methotrexate (MTX) is an effective chemotherapeutic agent; however, its clinical application is limited by serious pulmonary toxicity. This study evaluated the protective effect of ambrisentan (AMB) against MTX-induced lung injury and elucidated the underlying mechanisms.

Male Wistar rats were randomly divided into control, AMB₁₀, MTX, AMB₅ + MTX, and AMB₁₀ + MTX groups. AMB was administered orally for 7 days, with MTX (20 mg/kg, i.p.) given during the last 3 days. On the 8th day, serum, lung tissues, and bronchoalveolar lavage fluid (BALF) were collected and analyzed.

AMB markedly suppressed MTX-induced acute lung injury, as evidenced by reduction in serum and BALF lactate dehydrogenase (LDH), BALF protein leakage, and inflammatory cell infiltration. AMB restored the oxidant/antioxidant balance by lowering malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE) levels, while enhancing total antioxidant capacity (TAC). Mechanistically, AMB inhibited the endothelin-1 (ET-1)/endothelin type-A receptor (ETA) axis and suppressed its downstream signaling kinases, phosphorylated extracellular signal-regulated kinase 1 and 2 (p-ERK1/2) and phosphorylated protein kinase B (p-Akt), thereby attenuating NF-κB activation and reducing pro-inflammatory cytokines, tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6). Moreover, AMB preserved extracellular matrix (ECM) homeostasis by suppressing matrix metalloproteinase-9 (MMP-9) and tissue inhibitor of metalloproteinases-1 (TIMP-1) upregulation. These biochemical and molecular findings were further confirmed by histopathological improvement in lung architecture.

AMB protects against MTX-induced acute lung injury by modulating ET-1/ETA-mediated ERK1/2 and Akt signaling that activates NF-κB, thereby mitigating oxidative stress, inflammation, and restoring ECM homeostasis. These findings support AMB as a promising adjunctive therapy for MTX-treated patients.