Molecular Mechanism Of Melatonin Alleviates Septic ARDS By Inhibiting Macrophage Ferritinophagy

BACKGROUND:Acute respiratory distress syndrome（ARDS）is a critical clinical syndrome posing a major challenge to modern medicine with high morbidity and mortality.As the main intrinsic immune cells in the lung,macrophages participate in the entire pathogenic trajectory of ARDS and play key roles in regulating the inflammatory response in ARDS.Studies have confirmed that macrophages are rich in iron stores,excessive iron accumulation can cause M1 polarization and ferroptosis in macrophages,thus having an impact on ARDS.Ferritinophagy is a process mediated by nuclear receptor coactivator 4（NCOA4）.In this process,ferritins are transported to lysosomes for degradation then ferric ions bound to ferritins released as free irons,thereby promoting ferroptosis.Melatonin（MT）primarily secreted by the pineal gland that exerts different regulatory effects.MT’s endogenous antioxidant effects and chelating properties can effectively alleviate cytotoxicity caused by metal accumulation.The the resent study aimed to discuss whether MT could alleviate septic ARDS by inhibiting macrophage ferritinophagy and to explore the specific mechanisms involved,providing new approaches and strategies for the treatment of septic ARDS.METHODS:In the present study,ARDS models in cells were constructed with RAW264.7 cells stimulated with Lipopolysaccharide（LPS）and ARDS animal models were constructed with intraperitoneal injection of LPS.The therapeutic effect of MT on ferritinophagy was also observed on cellular and animal models.The key proteins of ferritinophagy,NCOA4,Glutathione peroxidase 4（GPX4）,Ferritin heavy chain 1（FTH1）and Microtubule-associated protein 1 light chain 3（LC3）were detected by western blot.Relevant indicators including glutathione（GSH）,malondialdehyde（MDA）,intracellular Fe²⁺and mitochondrial membrane potential were measured by the assay kits.Subsequently,we used melatonin receptor antagonists,ferroptosis agonist,autophagy agonist on cells,and transfected cells with small interfering RNA and lentivirus to further investigate the specific pathways of the pulmonary protective effects of MT.RESULTS:After LPS stimulation of RAW264.7 cells,expression of LC3 II/I was elevated,expression of GPX4 and FTH1 were decreased,intracellular GSH content and cell proliferation capacity were decreased,MDA,intracellular Fe²⁺and lipid peroxidation level were elevated,with altered mitochondrial morphology and diminished mitochondrial membrane potential,which were attenuated markedly with MT treatment.The therapeutic effect of MT on LPS-stimulated RAW264.7 cells was significantly attenuated after knockdown of melatonin receptor 1（MT1）.Moreover,Erastin and rapamycin weakened the effect of MT on ferroptosis,as indicated by decreased cell proliferation level,GPX4 and FTH1 protein expressions,and intracellular GSH,increased cell lipid peroxidation level,cell mitochondrial membrane potential,intracellular Fe²⁺and MDA levels.Subsequently,we found that the therapeutic effect of MT was similarly inhibited by lentiviral overexpression of NCOA4.In addition,in animal models,we found that MT could alleviate the extent of septic lung injury in mice.The lung injury score and protein concentration of mouse broncho alveolar lavage fluid（BALF）were relieved after MT treatment.The expression of ferritinophagy-related proteins and associated Fe²⁺,GSH,and MDA of alveolar macrophages showed consistent results.In addition,the treatment of MT was attenuated with the use of Erastin in advance in vivo.CONCLUSION:MT can alleviate septic ARDS by inhibiting macrophage NCOA4-ferritinophagy,providing a new idea for future clinical treatment of septic ARDS.