Saikosaponin A ameliorates acute lung injury by targeting eIF3f to regulate p62 ubiquitination and mitochondrial autophagy flux

SSA alleviates poly(I:C)-induced viral-like acute lung injury. • SSA restores mitochondrial homeostasis by recovering mitophagy flux. • eIF3f is identified as a direct molecular target of SSA. • SSA inhibits p62 K48-linked ubiquitination via eIF3f to relieve mitophagy blockade. Background Acute lung injury (ALI) is marked by an exaggerated inflammatory response, elevated oxidative stress, and impaired mitochondrial function, while effective targeted therapeutic strategies remain scarce. Saikosaponin A (SSA), a major active component of Bupleurum , exhibits anti-inflammatory activity, yet its direct targets and mechanisms in viral ALI remain unclear. Purpose This study aims to determine the mechanism of action and molecular target of SSA in viral ALI. Methods Poly(I:C)-induced viral-mimetic ALI models were established in both mice and BEAS-2B cells to explore the protective effects of SSA. Mitochondrial function was evaluated by detecting mitochondrial membrane potential, mitochondrial reactive oxygen species (MitoROS) levels, mitochondrial permeability transition pore (mPTP) opening, and oxygen consumption rate (OCR). Autophagy and mitophagy flux were further assessed via Western blotting, immunofluorescence staining, transmission electron microscopy (TEM), and the mCherry-GFP-LC3B reporter assay. Potential molecular targets of SSA were identified through drug affinity responsive target stability combined mass spectrometry (DARTS-MS), cellular thermal shift assay (CETSA), molecular docking, molecular dynamics simulation, and surface plasmon resonance (SPR), followed by validation using small interfering RNA (siRNA), single guide RNA (sgRNA), and adeno-associated virus (AAV)-mediated eif3f knockdown. Results SSA significantly mitigated poly(I:C)-triggered pulmonary inflammatory responses, oxidative stress, and lung tissue injury. Mechanistically, poly(I:C) induction caused mitochondrial dysfunction and mitophagy flux blockade, accompanied by the accumulation of LC3B and p62. SSA restored mitochondrial homeostasis by restoring mitophagy flux, rather than merely activating general autophagy. Further experiments identified eIF3f as a direct binding target of SSA. SSA suppressed abnormal K48-linked ubiquitination of p62 through targeting eIF3f, thereby maintaining p62 stability and alleviating mitophagy flux blockade. Deficiency of eIF3f markedly attenuated the protective effects of SSA. Conclusion SSA alleviates viral-like ALI by targeting eIF3f to regulate p62 ubiquitination and restore mitophagy flux, thereby identifying a novel SSA-eIF3f-p62 regulatory axis. During viral acute lung injury, excessive ROS production and inflammation severely impair mitochondrial function, leading to aberrant activation of mitophagy accompanied by marked blockage of autophagic flux. SSA targets eIF3f and regulates the ubiquitination-driven accumulation of p62, thereby modulating the p62-mediated selective autophagy pathway. This regulation restores mitophagic flux, improves mitochondrial function, and ultimately alleviates acute lung injury. Download: Download high-res image (179KB) Download: Download full-size image