Study On The Role Of Transcription Factor KLF4 In Protecting Against Seawater Drowning-induced Acute Lung Injury By Regulating Ferroptosis

BackgroundWith the vigorous development of China’s marine industry,naval construction continues to move toward the deep blue,seawater drowning accidents also continue to attract people’s attention.Seawater drowning-induced acute lung injury(Seawater drowninginduced acute lung injury,SWD-ALI)is caused by the damage of gas exchange barrier caused by seawater inhalation,resulting in a large amount of fluid leakage into the alveolar interstitium and alveolar cavity,and rapid accumulation of acute pulmonary edema,which may even develop into acute respiratory distress syndrome.The study of seawater drowninginduced acute lung injury from the molecular mechanism can provide a theoretical basis for related drug development and clinical treatment of injured patients.Ferroptosis is a new form of programmed cell death(Programmed cell death,PCD)characterized by excessive iron accumulation followed by an unbalanced redox state.Ferroptosis differs from the already reported PCD in having unique morphological features and biochemical processes.The process of ferroptosis is precisely regulated by numerous signaling pathways including iron metabolism,lipid metabolism,and amino acid metabolism,and is involved in the development of multiple diseases.Recent studies have found that ferroptosis is closely associated with the onset and development of acute lung injury,including infection-related acute lung injury,radiation-related acute lung injury,acute lung injury caused by ischemia-reperfusion,and seawater drowning-induced acute lung injury.Krüppel-like factor 4(Krüppel-like factor 4,KLF4)is a eukaryotic transcription factor that has been found to regulate a variety of cellular processes and participate in the development of multiple diseases;studies have shown that KLF4 can play a protective role in acute lung injury,and increased KLF4 expression ameliorates the pathological state and inflammatory response to LPS-induced acute lung injury in mice.Recent studies have found that KLF4 can upregulate SLC7A11 at the protein level,thereby regulating ferroptosis.Does increased KLF4 expression have a protective effect on SWD-ALI? Are the effects related to the regulation of ferroptosis by KLF4?Objectives1.To construct a seawater drowning-induced acute lung injury C57BL/6 mouse model and preliminarily explore its correlation with ferroptosis.2.To explore the effect of transcription factor KLF4 on seawater stimulation-induced ferroptosis in BEAS-2B cells.3.To investigate the effect of increased expression of KLF4 on ferroptosis in seawater drowning-induced acute lung injury in mice and the effect on lung injury in mice.Materials and methods1.Establishment of a model of acute lung injury by seawater drowning in mice and preliminary investigation of ferroptosisA mouse SWD-ALI model was constructed by tracheal intubation in the form of seawater drip,and the success of the model construction was evaluated by blood gas analysis,lung wet/dry mass ratio,lung injury score,alveolar lavage fluid inflammatory factors,total protein,and total cell count.The ROS content,MDA content,GSH content of lung tissue,and ferroptosis-related protein content were measured to determine whether ferroptosis occurred in this model of lung injury.2.Study on the effect of transcription factor KLF4 on seawater stimulationinduced ferroptosis in BEAS-2B cellsWe searched the UCSC website for 2000 base sequences upstream of SLC7A11 and predicted transcription factors that might target the promoter region of SLC7A11 by bioinformatic analysis on the JASPAR website,and finally screened KLF4 was screened among the differentially expressed genes that were highly expressed in the seawaterstimulated group by comparing the transcript sequencing results with those of previous SWD-ALI cell model.KLF4-silenced BEAS-2B cells after si RNA transfection exhibited weaker resistance in response to seawater stimulation: higher cell mortality,higher ROS content,more severe MDA accumulation,and more severe GSH depletion,as well as lower expression of SLC7A11 and GPX4 compared to the seawater-stimulated group alone.Plasmid DNA transfected BEAS-2B cells with high KLF4 expression showed strong resistance in response to seawater stimulation,with significantly lower cell mortality,lower ROS and MDA content,and higher GSH content,as well as increased expression of SLC7A11 and GPX4 compared to the seawater stimulation-only group.The above studies suggest that the transcription factor KLF4 can indeed regulate seawater stimulation-induced ferroptosis in BEAS-2B cells and play a protective role in the acute injury of BEAS-2B cells induced by seawater stimulation,which may be achieved by targeting and regulating SLC7A11.3.Study on the effect of KLF4 on ferroptosis in acute lung injury induced by seawater drowning in miceCompared with the control group,the expression of KLF4 increased and the expression of SLC7A11 and GPX4 decreased in the seawater drowning group;the expression of SLC7A11 and GPX4 in lung tissue increased,and the content of ROS,MDA and GSH in lung tissue decreased in the seawater drowning+APTO-253 treated group compared with the seawater drowning group;meanwhile,the pathological damage score decreased,the lung wet/dry mass ratio decreased,p O2 increased,p CO2 decreased,p H increased,and oxygenation index increased.Thus,in vivo upregulation of KLF4 was shown to inhibit iron death in SWD-ALI mice and exert a protective effect against lung injury.Results1.Establishment of a model of acute lung injury by seawater drowning in mice and preliminary investigation of ferroptosisThe transoral tracheal intubation of seawater was able to successfully construct seawater flooding lung injury,with significant damage to the gross lung appearance,significantly higher pathological damage scores,increased lung wet/dry mass ratio,acidosis and type II respiratory failure characterized by hypoxemia and hypercapnia,and a significant decrease in oxygenation index in the experimental group compared to the control group,consistent with the hallmarks of acute lung injury and even acute respiratory distress syndrome.Total BALF protein was elevated,total cell count was increased,and the levels of all inflammatory factors were elevated to varying degrees.The above pathophysiological changes were time-dependent,and the relevant indexes gradually deviated from normal values after seawater stimulation,reaching extreme values at roughly 6 hours and then returning to normal levels.In the seawater inhalation group,the GSH content of lung tissue was reduced and the ROS and MDA contents were increased,and the changes could be reversed by iron death inhibitors,and the characteristic expression of iron death-related genes suggested the correlation between iron death and seawater drowning lung injury.2.Study on the effect of transcription factor KLF4 on ferroptosis induced by seawater stimulation in BEAS-2B cellsWe searched the UCSC website for 2000 base sequences upstream of SLC7A11 and predicted transcription factors that might target the promoter region of SLC7A11 by raw letter analysis on the JASPAR website,and finally screened KLF4 among the differentially expressed genes in the seawater-stimulated group by comparing the transcript sequencing results with those of the previous seawater flooded lung injury cell model.KLF4 silencing after si RNA transfection BEAS-2B cells exhibited weaker resistance in response to seawater stimulation: higher cell mortality,higher ROS content,more severe MDA accumulation and GSH depletion,as well as lower protein expression levels of SLC7A11 and GPX4 compared to the seawater stimulation-only group.Plasmid-transfected KLF4 high expression BEAS-2B cells showed strong resistance in response to seawater stimulation,with significantly lower cell mortality,lower ROS and MDA content,and higher GSH content,as well as increased protein expression levels of SLC7A11 and GPX4 compared to the seawater stimulation-only group.The above study showed that the transcription factor KLF4 could indeed regulate seawater stimulation-induced iron death in BEAS-2B cells and play a protective role in the acute injury of BEAS-2B cells induced by seawater stimulation,which may be achieved by targeting and regulating SLC7A11.3.Study on the effect of KLF4 on ferroptosis in acute lung injury induced by seawater drowning in miceCompared with the control group,the expression of KLF4 was increased and the expression of SLC7A11 and GPX4 was decreased in the mice in the seawater drowning group.Compared with the seawater drowning group,in the seawater drowning + APTO-253 treated group,the expression of SLC7A11 and GPX4 in lung tissue was increased,and the ROS content,MDA content and GSH content in lung tissue were decreased,while the lung injury score,lung wet/dry mass ratio,Pa O2,Pa CO2,p H,and oxygenation index were increased.Thus,it was demonstrated that high expression of KLF4 at the in vivo level could inhibit ferroptosis in SWD-ALI mice and exert a protective effect against lung injury.Conclusion1.KLF4 has a regulatory effect on seawater stimulation-induced ferroptosis in BEAS-2B cells,and KLF4 expression is negatively correlated with ferroptosis;this may be due to the role of KLF4 in the regulation of SLC7A11.2.High expression of KLF4 inhibited the occurrence of ferroptosis in seawater drowning-induced acute lung injury and exerted a protective effect against lung injury in mice.