Studies On Ferroptosis Of Lung Epithelial Cells By Seawater Drowning-induced Acute Lung Injury Via TNFAIP3-ACSL4 Pathway By SOX9 And Its Mechanism

BackgroundWith the significant strengthening of our maritime military force,drowning accidents have gradually received attention.Seawater drowning-induced acute lung injury may cause respiratory system failure,circulatory system failure and even death.How to reduce the lung injury caused by seawater drowning is of great significance to improve the clinical prognosis of seawater-drowning patients and reduce the military loss of forces.Recent studies have shown that the pathological process of acute lung injury is inseparable from oxidative stress.It has been pointed out that oxidative stress is closely related to ferroptosis.When a large amount of ferric ion accumulated in cells and tissues,redox homeostasis will be destroyed,which will lead to oxidative stress and then iron-dependent cell death.Ferroptosis is characterized by the accumulation of lipid ROS and by iron-dependent.In recent years,it has been discovered that ferroptosis is a key mechanism of cell death related to ischemia organ injury,neurodegeneration and cancer,which is fully involved in cell metabolism,oxidation,and various diseases such as lung ischemia reperfusion injury,Parkinson's disease,lung tumors and lung injury.Ferroptosis plays an important regulatory role in lung injury.However,whether ferroptosis is involved in the pathological changes of seawater-drowning acute lung injury and the specific mechanism of ferroptosis are still needed to explored.It has confirmed that the expression of TNFAIP3 is positively correlated with ferroptosis.It is found that SOX9 is the upstream transcription factor of TNFAIP3 by bio-information analysis and it can up-regulate the expression of TNFAIP3.Therefore,we hypothesized that knocking down SOX9 can inhibit the expression of TNFAIP3 and reduce ferroptosis which will alleviate seawater-induced acute lung injury.Objectives1.To construct a seawater drowning-induced lung injury cell model.To filter differential genes through RNA-sequencing technology and find out the differential genes those are highly expressed in the injured lung.2.To clarify the effect of TNFAIP3 on the ferroptosis of BEAS-2B cells induced by seawater stimulation.3.To verify the correlation of transcription factor and TNFAIP3 with ferroptosis by filtering the transcription factor SOX9 which regulates TNFAIP3 by the methods of bioinformatics analysis.4.To explore the effect of SOX9 and TNFAIP3-ACSL4 pathway on ferroptosis in mice with acute lung injury in vivo.Materials and methods1.Establishment of a cell model of seawater drowning-induced acute lung injury and verification of differentially expressed genesFirst of all,the conditions for establishing the BEAS-2B cell model of Seawater drowning-induced acute lung injury are needed to be explored:Seawater concentration and seawater stimulation time.The establishment conditions of the model is confirmed based on the results.Transcriptome sequencing is performed to search for differentially expressed genes.2.The effect of TNFAIP3 on ferroptosis in BEAS-2B cells induced by seawater drowningFlow cytometry is used to evaluate the death of seawater drowning cells.Flow cytometric of ROS is used to clarify the changes of ROS in seawater drowning cells to reflect the lipid peroxidation of cells;Ferrostatin-1,DFO,Z-V AD-FMK and GSK'872 are used to treat cells,detect cell death and determine the main factors of cell death;MDA and GSH kits are used to detect changes in intracellular content and reflect the level of ferroptosis.The JC-1 probe is used to detect the MMP.At the same time,Western blot was used to determine the TNFAIP3 and ferroptosis-related proteins TRF,GPX4,ACSL4 to explore the interaction between TNFAIP3 and ferroptosis.The expression of TNFAIP3 is up-regulated by plasmid-transfected BEAS-2B cells.The experiment methods are the same as the previous steps.The experiment is performed in BEAS-2B cells transfected with siRNA-TNFAIP3.The experiment methods are the same as the previous steps.3.The transcription factor SOX9 promotes ferroptosis in BEAS-2B cells through the TNFAIP3-ACSL4 pathwayUCSC website and JASPAR website are used to find transcription factor SOX9.Then luciferase reporter gene assay is used to verify the targeting relationship.The cells transfected with SOX9 plasmid or with SOX9 siRNA to identify TNFAIP3 of ferroptosis-related protein by western blot to verify the regulation of SOX9-TNFAIP3-ACSL4 pathway on ferroptosis.4.A Study on ferroptosis in mice with seawater drowning-induced acute lung injuryFirstly a mouse model of seawater drowning-induced acute lung injury is to be established.Blood gas analysis is performed and oxygenation index is calculated.After the mouse lung tissue is taken out,the pathological changes of the lung tissue is obsevered and the lung tissue damage is scored.The lung tissue of the mouse are taken out to weigh wet weight and dry weight so as to calculate the wet-to-dry ratio of the lung tissues.Immunofluorescence staining of ferroptosis and apoptosis related proteins in lung tissue sections is performed.Finally,Western blot is used to detect SOX9,TNFAIP3 and ferroptosis-related proteins to verify the conformity the results in vivo and vitro.Results1.Establishment of a cell model of seawater drowning-induced acute lung injury and verification of differentially expressed genesSeawater drowning lung injury BEAS-2B cell model,using DMEM medium is used to culture cells with 30%seawater concentration for 6h in the seawater drowning-induced acute lung injury BEAS-2B cell model.In transcriptome sequencing,the TNFAIP3 gene with a high multiple of gene expression difference and obvious significance is selected.2.The effect of TNFAIP3 on ferroptosis in BEAS-2B cells induced by seawater drowning2.1Correlation of TNFAIP3 with ferroptosis in seawater drowning BEAS-2B cells2.1.1 Seawater drowning resulted in significant cell death.The death of BEAS-2B cells under the condition of seawater drowning is closely related to ferroptosis which has a lower correlation with cell apoptosis and cell necrosis.BEAS-2B cells stimulated by seawater drowning will produce too much ROS.2.1.2 Seawater drowning can increase GSH consumption and decrease MDA.2.1.3 Seawater stimulation of BEAS-2B cells severely affects mitochondrial function and promotes ferroptosis.2.1.4 In the SW group,the expression level of TNFAIP,TRF,ACSL4 is significantly increased,the expression level of GPX4 is significantly decreased.2.1.5 All of the above results can be suppressed by ferroptosis inhibitors.2.2 Correlation of TNFAIP3 with ferroptosis in BEAS-2B cells transfected with plasmid2.2.1 Cells transfected with TNFAIP3 plasmid can induce cell death and produce too much ROS,and ROS is inhibited by Fer-1.2.2.2 Transfection of TNFAIP3 plasmid into BEAS-2B cells can cause the consumption of GSH and the increase of MDA.2.2.3 The BEAS-2B cells transfected with TNFAIP3 plasmid up-regulate the expression of TNFAIP3 and severely affected the mitochondrial function2.2.4 In BEAS-2B cells transfected with plasmids,the expression level of TNFAIP,TRF,ACSL4 is significantly increased,the expression level of GPX4 is significantly decreased.2.2.5 All of the above results can be suppressed by ferroptosis inhibitors.2.3 Correlation of TNFAIP3 with ferroptosis in BEAS-2B cells transfected with siRNA2.3.1 BEAS-2B cells transfected with siRNA can effectively inhibit cell death.Intracellular ROS in cells transfected with siRNA is Significantly decreased.2.3.2 MDA accumulation and GSH consumption can be reduced in cells transfected with siRNA.2.3.3 The BEAS-2B cells transfected with siRNA down-regulated the expression of TNFAIP3 and reduce the impact on mitochondrial function and ferroptosis.2.3.4 The expression level of TNFAIP,TRF,ACSL4 is significantly decreased,the expression level of GPX4 is significantly increased.The expression of TNFAIP3 is down-regulated,which restricts ferroptosis.3.The transcription factor SOX9 promotes ferroptosis in BEAS-2B cells through the TNFAIP3-ACSL4 pathway3.1 UCSC website and JASPAR website are used to find transcription factor SOX9.3.2 The expression of SOX9 is significantly up-regulated in seawater drowning BEAS-2B cells.3.3 In BEAS-2B cells transfected with SOX9 plasmid,the high expression of SOX9 can induce an increase in the expression of TNFAIP3.3.4 The high expression of SOX9 in seawater drowning BEAS-2B cells is accompanied by the high expression of TNFAIP3,which also induce ferroptosis;The decreased expression of SOX9 in cells transfected with SOX9 si-RNA is accompanied by a decrease in TNFAIP3 expression,which induce ferroptosis effectively inhibited.SOX9 regulates ferroptosis through the TNFAIP3-ACSL4 pathway.4.A Study on ferroptosis in mice with seawater drowning-induced acute lung injury4.1 Changes of Arterial blood gas analysis and Oxygenation Index in MiceIn the SW group,pH,pO2,SaO2 and OI decrease significantly,while pCO2,Lac,HCO3-increase significantly.The results suggest that respiratory oxygenation and reduced gas exchange have been significantly reduced in seawater drowning mice.The mice show obvious respiratory acidosis combined with metabolic acidosis,which has met the diagnostic criteria for acute lung injury.4.2 Lung tissue pathological changes and lung tissue injury scorePathological sections of lung tissues are stained with HE and observed under a microscope.Some alveolar walls are thickened,edema and hemorrhage,alveolar spaces has shrunk,alveoli is not expanded,lungs is solidified.Part of the alveoli fuse to form bullae.Obvious inflammatory cells infiltrated in the interstitium and alveolar space.The lung injury score of the SW group is significantly higher than that of the Ctl group.4.3 Wet to dry ratio of lung tissueThe W/D ratio of the lung tissue in the SW group is higher,which suggests that the lung edema of the mouse lung tissue from seawater drowning mouse is significantly increased.4.4 Immunofluorescence staining of ferroptosis-related proteins GPX4,TUNEL and PTGS2 in lung tissueThe expression of GPX4 is decreased.The expression of PTGS2 is increased in the lung tissue of seawater drowning mouse.4.5 Western blot of SOX9,TNFAIP3,GPX4 and ACSL4 in lung tissueCompared with the Ctl group,the protein expression of SOX9,TNFAIP3,ACSL4 increased in the lung tissue of seawater drowning mice,while the protein expression of GPX4 decreased in the SW group.The result is consistent with the cell experiment.Conclusion1.TNFAIP3 participates in the ferroptosis process of in seawater drowning BEAS-2B cells.The high expression of TNFAIP3 can induce ferroptosis;2.The transcription factor SOX9 targets and regulates ferroptosis through the TNFAIP3-ACSL4 pathway.The down-expression of SOX9 can reduce the occurrence of ferroptosis;3.The expression of SOX9,TNFAIP3 and ferroptosis-related protein in vivo is consistent with that in vitro.