Astragalus polysaccharide regulates LPS-induced aerobic glycolysis and ferroptosis in MHS through Nrf2/Keap1 signaling pathway

Xue Xiang; Sun Zhaorui; Nie Shinan

doi:10.3969/j.issn.1002-1949.2025.08.009

Abstract：Objective To investigate the therapeutic potential of astragalus polysaccharides(APS)in modulating lipopolysaccharide(LPS)-induced aerobic glycolysis and ferroptosis in alveolar macrophages through the Nrf2/Keap1 signaling pathway.Methods An in vitro acute lung injury model was established by using LPS-stimulated alveolar macrophages.Cells were divided into five experimental groups:Control group,LPS group,Bardoxolone+LPS group,APS+LPS group,and ML385+APS+LPS group.Inflammatory cytokines[interleukin-6(IL-6),interleukin-1β(IL-1β),tumor necrosis factor-α(TNF-α)],oxidative stress markers[malonaldehyde(MDA),superoxide dismutase(SOD),reactive oxygen species(ROS)],glycolytic markers[lactate dehydrogenase(LDH),fructose 2,6-diphosphatase 3(PFKFB3),pyruvate kinase 2(PKM2),hexokinase 2(HK2)]and ferroptosis-related proteins[long chain acyl-CoA synthetase 4(ACSL4),cystine glutamate antiporter subunit(xCT),glutathione peroxidase 4(GPX4)and Nrf2/Keap1 signaling pathway components(nuclear/cytoplasmic Nrf2,Keap1)were analyzed by using enzyme-linked immunosorbent assay(ELISA),quantitative real-time polymerase chain reaction(RT-qPCR)and Western blot.Results In comparison to the Control group,the LPS group exhibited elevated levels of IL-6,IL-1 β and TNF-α,alongside increased concentrations of MDA and ROS,and a reduction in SOD levels.Additionally,the mRNA and protein expression of LDH,PFKFB3,PKM2,HK2,ACSCL4 were upregulated,whereas the expression of xCT,GPX4 were downregulated.Nuclear expression of Nrf2 and Keap1 also decreased.Conversely,compared to the LPS group,there was a reduction in the expression levels of IL-6,IL-1β and TNF-α,decreased levels of MDA and ROS,as well as an increase in SOD levels in the APS+LPS group.Furthermore,the expression levels of LDH,PFKFB3,PKM2,HK2,ACSL4 were downregulated,while the expression levels of xCT and GPX4 were upregulated.The nuclear expression levels of Nrf2 and Keap1 were increased.Bardoxolone,a Nrf2 activator,partially mitigated the metabolic reprogramming and ferroptosis in LPS group,whereas the therapeutic efficacy in the APS+LPS group was diminished by the Nrf2 inhibitor ML385.Conclusions APS can modulate the Nrf2/Keap1 signaling pathway,suppress LPS-induced aerobic glycolysis and ferroptosis in alveolar macrophages,and mitigate alveolar macrophage injury.