Methylene blue mitigates lung injury in HCA rats by regulating macrophage pyroptosis via Nrf2/HO-1 and NLRP3 pathways

Methylene blue (MB) has antioxidant properties, yet its role in acute lung injury (ALI) induced by hypothermic circulatory arrest (HCA) remains unexplored. This study investigates MB's effects and underlying regulatory mechanisms in an HCA rat model. Rats received an intravenous bolus of MB (1 mg/kg) 15 min before HCA induction. Physiological parameters were monitored, and bronchoalveolar lavage fluid (BALF) was collected 2 h postoperatively to assess total protein levels, inflammatory cells, and cytokines. Histopathological lung damage was evaluated using hematoxylin-eosin (H&E) and TUNEL staining. Inflammatory markers and oxidative stress indicators were measured via ELISA and dihydroethidium (DHE) staining. Alveolar macrophages (AMs) were isolated to analyze polarization using flow cytometry and immunofluorescence double staining. Pyroptosis in AMs was detected with Yo-Pro-1 and Hoechst 33342 staining. Additionally, Western blotting was performed to examine the nuclear factor erythroid-2 related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) pathway, Nod-like receptor protein 3 (NLRP3) inflammasome, and pyroptosis-related proteins. Following HCA, rats exhibited significant blood gas abnormalities, structural lung damage, increased pathological scores, and higher apoptosis rates. However, MB mitigated these effects, improving physiological parameters and reducing lung histopathology scores. MB also lowered proinflammatory cytokine levels, increased SOD and GSH-Px activity, promoted AM polarization toward the M2 phenotype, and decreased pyroptosis. Mechanistically, MB activated the Nrf2/HO-1 pathway while inhibiting NLRP3 inflammasome activation. Notably, Nrf2 inhibitors and NLRP3 agonists weakened MB's protective effects by promoting inflammasome activation and pyroptosis, whereas Nrf2 agonists and NLRP3 inhibitors enhanced MB's beneficial impact. In conclusion, MB attenuates HCA-induced ALI by modulating AM polarization and pyroptosis via Nrf2/HO-1 pathway activation and NLRP3 inflammasome inhibition.