Abstract

Acute lung injury (ALI) is a pulmonary disorder characterized by refractory hypoxemia, with acute respiratory distress syndrome (ARDS) representing the most severe form. In the post-pandemic era, ARDS remains associated with significant morbidity and mortality. Uncontrolled inflammatory responses and oxidative stress are recognized as key pathogenic mechanisms driving ALI. In this study, indoloquinolinone 3a , an indole-fused pirfenidone derivative, was investigated for its protective effect on lipopolysaccharide (LPS)-induced ALI, along with the underlying molecular mechanisms. In vivo , 3a notably attenuated pulmonary histological damage, decreased the lung wet/dry weight ratio and total protein concentration in bronchoalveolar lavage fluid (BALF), upregulated ZO-1 expression, suppressed neutrophil infiltration and myeloperoxidase (MPO) activity, decreased TNF-α, IL-6, IL-1β levels, downregulated cyclooxygenase-2 (COX-2) expression, and reduced PGE 2 content in ALI mice. In vitro , 3a markedly inhibited the LPS-stimulated inflammatory response by suppressing the NF-κB signaling pathway in both bone marrow-derived macrophages (BMDMs) and RAW 264.7 cells. Additionally, 3a enhanced anti-oxidant defense capacity in ALI mice by increasing glutathione (GSH), superoxide dismutase (SOD), and catalase (CAT) levels, while reducing reactive oxygen species (ROS) and malondialdehyde (MDA) levels. In LPS-stimulated macrophages, 3a also prevented ROS production via AMPK activation and upregulation of Nrf2 and key anti-oxidant enzymes. Moreover, AMPK inhibition abolished 3a 's suppressive effects on LPS-stimulated NF-κB p65 phosphorylation as well as iNOS and COX-2 expression in macrophages. Taken together, indoloquinolinone 3a ameliorated LPS-induced ALI, and the AMPK-mediated NF-κB inhibition and Nrf2 activation in macrophages may be responsible for the anti-inflammatory and anti-oxidant effects of indoloquinolinone 3a on LPS-induced ALI.