Effect Of Regulating FUNDC1 Autophagy-Mediated CGAS/STING Pathway In Oleic Acid-Induced Acute Lung Injury

Background The pathogenesis of acute lung injury is unclear,the diagnosis and treatment are not specific and the clinical prognosis is poor.Its pathogenesis involves the aggregation,activation and release of a variety of inflammatory cytokines and chemokines of various key effector cells,resulting in lung and even multiple organ injuries.cGAS/STING signaling pathway is a newly discovered immune pathway in recent years.The existence of dsDNA in cytoplasm can activate this pathway and trigger a cascade reaction,producing pro-inflammatory cytokines and type I interferon.This process can enhance the body’s resistance,but excessive activation will lead to serious inflammatory reaction,destroy autoimmune and even produce diseases.Some studies have confirmed that cGAS-STING signaling pathway is activated in acute lung injury,which is related to mtDNA.We suppose that mitophagy has correlation with mtDNAcGAS/STING signaling pathway in acute lung injury.This study will explore the mechanism of mitophagy and cGAS/STING pathway in oleic acid-induced acute lung injury.Methods A mouse model of acute lung injury was established by tail vein injection of oleic acid.Pulmonary microvascular endothelial cells were treated with different concentrations of oleic acid and select the optimal concentration for modeling.First,we verify the mechanism of cGAS-STING signaling pathway in oleic acid-induced acute lung injury.The experimental subjects were randomly divided into three groups,including the control group,the acute lung injury group and the inhibitor group.The cGAS inhibitor RU.521 was intraperitoneally injected into the inhibitor group one hour before modeling.The mice were sacrificed 24 hours after modeling and hematoxylin eosin staining was performed on the lung tissue to compare the pathological changes of lung injury.Western blot was used to quantitatively analyze the protein expression differences of pathways cGAS,STING,pTBK1,pIRF3 and pNF-κB.The expressions of STING and pNF-κB in the lung tissue were observed by immunohistochemistry.The serum IFN-β expression level was detected by ELISA.The cell was divided into the same groups as mice.The morphological changes of cells after oleic acid stimulation were observed under the microscope.The LDH level of cell supernatant was detected by ELISA method to assess the change of vascular endothelial permeability.The expressions of F-actin and tight junction protein ZO-1 as well as cGAS and STING in endothelial cells of each group were observed by confocal microscopy.In order to further explore the correlation between mitophagy and cGAS-STING pathway in oleic acid-induced acute lung injury.FUNDC1 knockout mouse model was constructed,the cells were targeted and silenced by siRNA.The subjects were divided into the control group,the acute lung injury group,the FUNDC1-control group and the FUNDC1-acute lung injury group.The autophagosomes were observed by electron microscope and the expression level of mtDNA was detected by qPCR.The other detection methods were the same as above,including the differences in pathology,cytomorphologic changes,permeability changes,pathway proteins and IFN-β were compared among different groups.Results Compared with the control group,the pathological changes of lung injury in the tail vein injection of oleic acid group were significantly aggravated.The expressions of cGAS,STING,pIRF3 and pNF-κB in lung tissues of mice with acute lung injury were significantly higher than those in the control group.serum IFN-β is also increased.After oleic acid stimulation,the morphology of lung microvascular endothelial cells changed from polygonal to circular and the expression of LDH in cell supernatant was increased.The confocal observation showed that the expressions of endothelial F-actin and tight junction protein ZO-1 were decreased,indicating that the endothelial permeability was worse.The injection of inhibitor could effectively improve the above conditions.After the FUNDC1 gene was knocked out to inhibit mitochondrial autophagy,it could be seen that the autophagosomes were significantly reduced and the mtDNA in cell supernatant was increased.The expression levels of pathway proteins in lung tissues and cells of FUNDC1-acute lung injury group were higher than those in the wild-type acute lung injury group and the expression of serum IFN-β was also increased.Conclusion cGAS/STING pathway is activated in oleic acid-induced acute lung injury and the inflammatory response is severe.Inhibition of this pathway may improve the inflammatory response.The silence of FUNDC1 inhibits mitophagy,subsequently the accumulated mtDNA activates cGAS/STING pathway to aggravate the pathological damage and inflammatory response in acute lung injury group,suggesting that mitophagy may have a protective effect in oleic acid-induced acute lung injury through cGAS/STING pathway.