Dimethyl fumarate alleviates high-altitude hypoxic acute lung injury by activating the NRF2/SLC7A11 axis to inhibit pulmonary epithelial ferroptosis and regulate macrophage polarization

Abstract Objective To investigate the protective effects of dimethyl fumarate (DMF), an agonist of nuclear factor erythroid 2-related factor 2 (NRF2), against acute lung injury (ALI) induced by high-altitude hypoxia, in order to found a basis for developing novel preventive strategies against hypoxic pulmonary injury. Methods ① Twenty SPF-grade male Wistar rats (5 to 6 weeks old, 210 to 230 g) were randomly divided into (n=5): normoxic control (intraperitoneal injection of normal saline for 10 consecutive days followed by being placed in a normobaric normoxia chamber), hypoxic lung injury (intraperitoneal injection of normal saline for 10 consecutive days followed by being exposed to a hypobaric hypoxic chamber simulating 5 000 m altitude for 48 h), DMF control (intraperitoneal injection of DMF for 10 consecutive days followed by being placed in a normobaric normoxic chamber), and DMF prophylaxis (DMF preconditioning for 10 d followed by hypobaric hypoxic modelling) groups. HE staining was used to observe the histopathological changes in lung tissues to pathologically score the lung injury. ELISA was employed to detect the inflammatory factors to assess inflammatory responses, immunofluorescence assay was utilized to measure the generation of reactive oxygen species (ROS), and spectrophotometry was performed to determine the glutathione (GSH) and malondialdehyde (MDA) levels, as well as superoxide dismutase (SOD) activity. Western blotting and real-time polymerase chain reaction (RT-qPCR) were applied to analyze ferroptosis-related pathway proteins at protein and mRNA levels. ② Hypoxic lung injury cellular model and normoxic control model were established by co-culturing human bronchial epithelial (BEAS-2B) cells and TNF-α-activated human peripheral blood monocyte (THP-1)-derived macrophages under hypoxic and normoxic conditions for 48 h. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to compare the protein profiles in BEAS-2B cells between normoxic control and hypoxic injury groups. Bioinformatics analysis was carried out to identify the key differentially expressed proteins (DEPs). A hypoxic injury prophylaxis group was established by BEAS-2B cells being pretreated with DMF for 24 h and then co-cultured with THP-1 cells. Then the ferroptosis markers, macrophage polarization phenotypes, and inflammatory cytokines were detected in BEAS-2B cells. ③ Additionally, solute carrier family 7 member 11 (SLC7A11) gene was knocked down under DMF intervention to assess ferroptosis markers and macrophage-related inflammatory responses. Results ① Compared to the hypoxic injury group, the DMF prophylaxis group exhibited significantly decreased pulmonary ROS level and MDA content (P