SIRT3-mediated mitophagy by deacetylating ATP5F1A involved in the protective effects of SIGMAR1/Sigma-1 receptor against ferroptosis and microvascular hyperpermeability in lipopolysaccharide-induced acute lung injury

Previous studies have shown that SIGMAR1/Sigma-1 receptor (sigma non-opioid intracellular receptor 1) provides protective effects against lipopolysaccharide (LPS)-induced acute lung injury (ALI), however the underlying mechanism remains unclear. A recent study highlighted SIGMAR1's protective role against ferroptosis but did not fully elucidate the mechanism involved. Endothelial ferroptosis, which significantly affects microvascular permeability, has garnered increasing attention in research. In this context, we aimed to investigate how SIGMAR1 mitigates endothelial ferroptosis in ALI induced by LPS. PRE-084 (SIGMAR1 activator) inhibited endothelial ferroptosis and microvascular hyperpermeability in ALI induced by LPS; however, this effect was blocked by mitophagy inhibition. Knockout of sigmar1 worsened microvascular hyperpermeability and endothelial ferroptosis, but these effects were mitigated by activating SIRT3 (sirtuin 3). Conversely, inhibiting SIRT3 blocked the upregulation of SIGMAR1-mediated mitophagy and limited endothelial ferroptosis in ALI induced by LPS. In addition, LPS exposure led to the acetylation of lysine 498 in ATP5F1A/ATP5A1 (ATP synthase F1 subunit alpha). Importantly, downregulating ATP5F1A acetylation prevented the SIRT3 inhibition from blocking the effects of SIGMAR1 in facilitating mitophagy and preventing ferroptosis. Interestingly, downregulating ATP5F1A acetylation or activation of SIRT3 did not alter the effects of PRE-084 on ALI when mitophagy was inhibited, suggesting that SIGMAR1's ALI protective effects involve ATP5F1A- or SIRT3-dependent mitophagy. In conclusion, our findings indicate that SIGMAR1 alleviates endothelial ferroptosis and microvascular hyperpermeability in LPS-induced ALI through SIRT3-mediated mitophagy. Furthermore, the deacetylation of ATP5F1A at lysine 498 by SIRT3 is essential for SIGMAR1-mediated PRKN/parkin-dependent mitophagy.Abbreviations: ALI, acute lung injury; ARDS, acute respiratory distress syndrome; ATP, adenosine triphosphate; ATP5F1A, ATP synthase F1 subunit alpha; BCA, bicinchoninic acid; EB, Evans blue dye; ECM, endothelial cell medium; FBS, fetal bovine serum; FITC, fluorescein isothiocyanate; Fer-1, ferrostatin-1; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GFP-LC3, green fluorescent protein-microtubule associated protein 1 light chain 3 alpha; GPX4, glutathione peroxidase 4; GSH, glutathione; GSSG, glutathione disulfide; KO, knockout; LPS, lipopolysaccharide; LRRK2, leucine rich repeat kinase 2; MDA, malondialdehyde; MPMVECs, mouse pulmonary microvascular endothelial cells; MTT, 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide; PBS, phosphate-buffered saline; PECAM1/CD31, platelet and endothelial cell adhesion molecule 1; PRKN, parkin RBR E3 uniquitin protein ligase; ROS, reactive oxygen species; RSL3, RAS-selective lethal 3; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; SIGMAR1, sigma non-opioid intracellular receptor 1; SIRT3, sirtuin 3; siRNA, small interfering RNA; TUNEL, terminal deoxyribonucleotidyl transferase-mediated deoxyuridine 5-triphosphate-digoxigenin nick end labeling; VHP, vascular hyperpermeability; W:D, wet:dry; WT, wild type.