The Role Of ATP Synthase Inhibitor 1 In LPS-induced Acute Lung Injury By Regulating Mitophage

Background:Acute lung injury（ALI）is a type of lung injury caused by various diseases.Currently,there are many challenges in the treatment of ALI,and its molecular therapeutic targets are worth exploring.ATP synthase inhibitory factor 1（IF1）,as a physiological inhibitory factor of ATP synthase,can regulate mitochondrial function and cellular inflammation.Recently,IF1 has been found to improve ischemia reperfusion injury and reduce cellular inflammation in heart,tumor and nervous system diseases,but its protective effect on ALI remains unclear.Objective:To explore the role of IF1 in the acute lung injury induced by Lipopolysaccharide（LPS）.Methods:（1）Mouse alveolar macrophage cell line MH-S cells were cultured in vitro and stimulated with LPS（10ug/m L）for 12 h to establish an inflammation model.IF1 overexpression MH-S cell line was constructed by IF1 CRISPR Activation plasmid transfection.Control group,LPS treatment group,LPS+NC plasmid transfection（ACTNC）group and LPS+IF activating plasmid transfection（ACT-IF1）group were used to verify the effect of IF1 on the secretion of pro-inflammatory factors,mitochondrial function and mitophagy of MH-S cells.RT-qPCR and Western blot were used to verify the expression of IF1.RT-qPCR,Western blot and ELISA were used to detect the secretion of IL-1β,TNF-α and IL-6.JC-1 was used to detect mitochondrial membrane potential.Western blot was used to detect the levels of mitophagy-related proteins.Fluorescence confocal microscopy was used to detect the co-localization of mitochondrial tracer Mito-Tracker Red and autophagy protein LC3.Intracellular ROS levels were measured by Mito SOX^TM Red.（2）A mouse model of ALI was established by intratracheal injection of LPS（3mg/kg）,and IF1 overexpression mice were constructed by nasal drops of lentivirus.Sixteen C57BL/6 mice were randomly divided into four groups: Sham group,LPS treatment group,LPS+NC lentivirus infection（LV-NC）group and LPS+IF1 lentivirus infection（LV-IF1）group to observe the effect of IF1 on acute lung injury in mice.RT-qPCR and Western blot were used to verify the expression of IF1.HE staining was used to observe the morphological changes of lung tissue.The pathological injury score of lung tissue and wet/dry ratio（W/D）of lung tissue were compared.The levels of inflammatory factors in bronchoalveolar lavage fluid and lung tissue and the changes of mitophagyrelated proteins in lung tissue were detected.Results:（1）LPS promoted the secretion of pro-inflammatory cytokines IL-1β,TNF-α and IL-6by MH-S cells;The injection of LPS into the tracheal of mice resulted in the pathological damage of HE staining and the increase of wet/dry lung tissue in the sham operation group.（2）Mitochondrial autophagy was enhanced by LPS.Mitochondrial and autophagy protein LC3 colocalization increased,Westernblot detection of related protein changes,indicating the enhancement of mitochondrial autophagy.The increase of intracellular ROS and the decrease of mitochondrial membrane potential indicated that mitochondrial function was impaired.（3）LPS stimulation down-regulated the expression of IF1 in MH-S cells and lung tissues of mice.（4）Overexpression of IF1 can significantly reduce the secretion of pro-inflammatory cytokines IL-1β,TNF-α and IL-6 by MH-S cells.After overexpression of IF1,HE staining pathological damage and wet/dry lung tissue in mice were reduced compared with LPS group,and the secretion of pro-inflammatory factors IL-1β,TNF-α and IL-6in mice lung tissue was inhibited.（5）Overexpression of IF1 can inhibit mitochondrial autophagy and improve mitochondrial function.After overexpression of IF1,mitochondrial co-localization with LC3 decreased compared with LPS group,LC3II/LC3 I,Parkin and MFN2 protein levels decreased,while TOM20 and DRP1 protein levels increased,suggesting that mitochondrial autophagy activity was inhibited.The decrease of intracellular ROS and the recovery of mitochondrial membrane potential suggested that IF1 improved mitochondrial function.Conclusions:IF1 plays a protective role in LPS-induced acute lung injury by inhibiting mitochondrial autophagy in alveolar macrophages,improving mitochondrial function,and reducing the release of cellular inflammatory cytokines.