富马酸二甲酯通过激活NRF2/SLC7A11轴抑制肺上皮细胞铁死亡并调控巨噬细胞极化缓解高原低氧急性肺损伤OA

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<0.05),increased SOD activity and GSH level(P<0.05),elevated expression levels of SLC7A11 and glutathione peroxidase 4(GPX4)(P<0.05),along with markedly reduced lung injury score and inflammatory cytokine levels(P<0.05).② Proteomic analysis identified 5 377 proteins and 613 DEPs(302 up-regulated,311 down-regulated)in BEAS-2B cells,and among 9 ferroptosis-related DEPs,SLC7A11 exhibited the most significant alteration.In the cellular hypoxic ALI model,compared to the hypoxic injury group,DMF prophylaxis up-regulated SLC7A11 and GPX4 expression(P<0.05),increased GSH level and SOD activity,decreased the M1 macrophage ratio and inflammatory factor levels(P<0.05).③ SLC7A11 knockdown reversed DMF's effects by increasing MDA level(P<0.05),decreasing SOD activity and GSH level(P<0.05),suppressing GPX4 expression(P<0.05),and elevating M1 macrophage proportion and enhancing inflammatory cytokine secretion(P<0.05).Conversely,overexpression of SLC7A11 enhanced the protective effects of DMF.Compared with the hypoxia-DMF group,the hypoxia-DMF-overexpression group showed significantly increased GSH level,SOD activity,and GPX4 expression(P<0.05),along with a marked decrease in MDA content(P<0.05)in BEAS-2B cells.Meanwhile,SLC7A11 overexpression further alleviated the inflammatory response,as indicated by significantly reduced expression of IL-1β,IL-6 and TNF-α(P<0.05).Conclusion DMF attenuates high-altitude hypoxic ALI by suppressing ferroptosis in pulmonary epithelial cells via up-regulating the NRF2/SLC7A11 axis,modulating macrophage polarization,and mitigating inflammatory response,and thus,exerts a protective effect against hypoxic pulmonary injury.