An indoloquinolinone 3a alleviates lipopolysaccharide-induced acute lung injury by modulating the NF-κB and AMPK/Nrf2 signaling pathways

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 PGE₂ 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.