Targeting TLR4 and Regulating Keap1/Nrf2 Pathway with Andrographolide to suppress inflammation and ferroptosis in LPS-induced acute lung injury

Acute lung injury (ALI) is a severe inflammatory lung disease with a high mortality rate, often caused by sepsis. The pathophysiology of ALI involves inflammation, oxidative stress, and ferroptosis, a recently identified form of regulated cell death. This study aims to investigate the potential therapeutic effects of andrographolide (AG), a bioactive compound from Andrographis, on LPS-induced inflammation and ferroptosis. The research utilized in vitro experiments with RAW264.7 cells and in vivo studies using a mouse model of LPS-induced ALI. The results demonstrate that AG exhibited notable efficacy in suppressing the production of pro-inflammatory cytokines and inhibiting ferroptosis in RAW264.7 cells induced by LPS. Furthermore, AG administration conferred a protective effect in ALI mice induced by LPS by reducing lung edema, diminishing inflammatory cell infiltration, and mitigating ferroptosis in lung tissues. These effects were mediated through the modulation of the TLR4/Keap1/Nrf2 pathway. Molecular docking simulations demonstrated the binding sites of AG to the TLR4 protein (KD value: −33.5 kcal/mol), and the findings were subsequently validated using the CETSA and SPR assay. Collectively, these findings demonstrate that AG exerts anti-inflammatory and anti-ferroptosis effects in LPS-induced ALI by targeting TLR4 and modulating the Keap1/Nrf2 pathway. This study highlights AG as a potential therapeutic agent for managing ALI and offers new insights into its mechanisms of action.