Study Of Umbilical Cord Mesenchymal Stem Cell Derived Exosomes Alleviating Seawater Induced Lung Injury By Inhibiting Necroptosis Through MiR-143-3p/NFATc2 Pathway

BackgroundDrowning is the third leading cause of accidental injury deaths.Over the past decade,it has resulted in over 2.5 million deaths.The proportion of seawater drowning varies in different countries and regions,and the number of seawater drownings may continue to increase with the increase in coastal tourism,offshore operations,and the needs of marine national defense.The lung is the target organ easily damaged during seawater drowning.One-third of non-fatal drowning patients will be diagnosed with acute lung injury(ALI)or acute respiratory distress syndrome(ARDS),also known as seawater aspiration-induced acute lung injury(SW-ALI).ALI/ARDS is a serious life-threatening clinical syndrome characterized by refractory hypoxic respiratory failure and bilateral diffuse infiltrates on chest imaging.In the process of ALI/ARDS,the destruction of the alveolar epithelial-vascular endothelial barrier function ultimately leads to the exudation of protein-rich fluid into the alveolar cavity and pulmonary interstitium.Currently,there is no effective drug treatment for ALI/ARDS,and mainly supportive treatments such as mechanical ventilation,prone positioning,extracorporeal membrane oxygenation(ECMO),and glucocorticoids are adopted.Mesenchymal stem cell(MSC)-based therapy is a promising treatment for ALI/ARDS.Preclinical studies have shown that MSCs can effectively alleviate lung injury in animal models.However,the application of MSCs is limited by issues such as tumorigenesis,immunogenicity,and low transplantation rates.MSC-derived exosomes(MSC-Exo)are cell vesicles secreted by MSCs which inherit the functions of MSCs and have even better pharmacokinetics.MSC-Exo may exert regulatory effects on inflammatory signaling pathways,promote tissue repair and regeneration,and have anti-microbial effects via the transfer of bioactive substances.Necroptosis is the result of a series of stimuli that activate RIPK1,RIPK3,and MLKL,causing cells to undergo regulated cell death.Necroptosis induction factors are similar to apoptosis and serve as alternative death pathways for cells when apoptosis is blocked.Morphologically,it is similar to necrosis,manifested by organelle swelling and plasma membrane rupture.Increasing evidence suggests that necroptosis plays an important role in the progression of ALI/ARDS.Furthermore,recent researches have shown that hyperosmotic stress and hyperglycemia can induce necroptosis in cells.The nuclear factor of activated T cells(NFAT)transcription factor family,consisting of five subtypes(NFAT1-NFAT5),regulates the activation and differentiation of T cells,as well as important genes related to the cell cycle and death.Through bioinformatics analysis,it was found that NFATc2 is a transcription factor upstream of the receptor interacting serine threonine kinase 1(RIPK1)signaling pathway.NFATc2 can upregulate the expression of RIPK1.Therefore,it is hypothesized that UMSC-Exo could alleviate seawater-induced lung injury by influencing the expression of NFATc2?Objectives1.Optimize the cell model conditions of seawater-induced lung injury,isolate and purify umbilical cord mesenchymal stem cell derived exosomes(UMSC-Exo),and verify whether UMSC-Exo can alleviate seawater-induced lung injury in the cell model;2.Explore the possible mechanism by which UMSC-Exo alleviates seawater-induced lung injury in the cell model;3.Validate whether UMSC-Exo can alleviate seawater-induced lung injury in mice.Materials and methods1.Construct a cell model of seawater induced lung injury,identify the extracted exosomes,and study the effect of UMSC-Exo on seawater induced lung injuryFirstly,the effects of seawater volume and stimulation durations on Bronchial Epithelium transformed with Ad12-SV40 2B(BEAS-2B)cell viability were examined using a CCK8(Cell Counting Kit-8)assay to determine the conditions for establishing a seawater-induced lung injury cell model.The supernatant of UMSC cultured with exocrine free serum was collected and purified using standard differential centrifugation.Western Blot was used to detect the exosomal marker proteins,transmission electron microscopy was used to observe the morphology of exosome,and nanoparticle tracking were to analyze the particle size of exosome.After induction with seawater-containing medium,the normal medium was replaced and UMSC-Exo was added.The ability of BEAS-2B cells to uptake UMSC-Exo was tested using PKH-67staining.The effect of UMSC-Exo on seawater-induced cell damage was studied by examining cell viability using the CCK8 assay,staining mitochondrial membrane potential with JC-1,testing cell membrane integrity with propidium iodide(PI)staining,and examining cell death using Annexin V assay.2.Mechanism study of UMSC-Exo alleviating seawater-induced lung injury cell modelFirstly,screen the major cell death type and key regulatory genes in seawater-induced BEAS-2B cell injury.Based on the cell model of seawater-induced lung injury and optimal treatment concentration of UMSC-Exo to alleviate seawater-induced lung injury,transcriptome sequencing was performed.Gene set enrichment analysis(GSEA)was used to identify the enriched cell death type after seawater induction,while inhibited by UMSC-Exo treatment was identified as necroptosis.The expression level of necroptosis executing protein p-MLKL,MLKL,p-RIPK3,RIPK1,RIPK3,and apoptotic marker protein Caspase-8 in seawater-induced lung injury were further verified by WB.Identifying differential genes that were upregulated after seawater induction but downregulated after UMSC-Exo treatment,and transcription factors that could target and regulate necroptosis were found through bioinformatics analysis.The key gene NFATc2 was determined by comparing the two sets.The changes in NFATc2in UMSC-Exo alleviating seawater-induced BEAS-2B cell injury were validated by WB and qPCR.Transcription factor NFATc2’s targeted regulatory effect on RIPK1 was verified by luciferase assay.After si-NFATc2 transfection in BEAS-2B,the role of NFATc2 in sea water-induced necroptosis was verified by CCK8,JC-1 staining,PI staining,flow cytometry,and WB.Secondly,we screened miRNAs in UMSC-Exo that can target NFATc2.Search for miRNA sequencing datasets of umbilical cord mesenchymal stem cell exosomes from the GEO database,select the top 100 expressed miRNAs and take the intersection.From the databases(Target Scan,miRDB,miRWalk,mir DIP,miRTar Base),screen for miRNAs that can regulate NFATc2 and compare them with miRNAs expressed in UMSC-Exo.Select candidate miRNAs that can target and regulate NFATc2,and compare their expression levels with those in normal BEAS-2B cells.Design and construct miRNA mimics,verify them as targeting NFATc2 using WB and qPCR methods.Further verify the targeting effect of the miRNA on NFATc2 through a dual luciferase assay.Use CCK8,JC-1 staining,PI staining,flow cytometry,and WB to verify the effect of the designed miRNA mimics on NFATc2 expression and necroptosis during seawater-induced cell damage.3.Validation study of UMSC-Exo inhibiting necroptosis and alleviating seawater induced acute lung injury in miceThe mouse SW-ALI model was established by slowly injecting seawater through an oral tracheal tube.The treatment group received UMSC-Exo treatment after 6 hours of seawater injection.Lung tissue pathological changes were observed using HE staining and the severity of lung injury was assessed by quantifying the lung tissue injury score.Blood gas analysis was performed by collecting blood samples from the abdominal aorta to evaluate gas exchange disorders.Lung edema was evaluated by measuring the wet-to-dry weight ratio of lung tissue.Chest X-ray was used to evaluate lung exudation.Inflammatory factors in bronchoalveolar lavage fluid were detected to evaluate inflammation.Immunohistochemical analysis and immunofluorescence staining were performed on lung tissue sections targeting the purpose gene of necroptosis.Finally,Western Blot was used to detect NFATc2 and necroptosis-related proteins to verify whether the in vivo experimental results were consistent with the cell experimental results.Results1.Establishing the conditions for seawater-induced lung injury cell model,extracting exosomes that meet the standards,and using UMSC-Exo to alleviate seawater-induced BEAS-2B cell injuryStimulation of BEAS-2B cells with a 20%seawater-containing medium for 6hours can induce a 50%decrease in cell viability,which was used as the condition for the seawater-induced lung injury cell model.Western Blot results showed that UMSC-Exo expressed CD9,CD63,and Alix but did not express Calnexin.Transmission electron microscopy showed that the exosomes had a single-layer membrane structure and a semi-spherical shape with a concave on one side.Particle size analysis showed that the extracted UMSC-Exo had a diameter mainly ranging from 60 to 120 nm.Tracing experiments showed that UMSC-Exo could be uptaken by BEAS-2B cells.Compared to the seawater-induced group,cell viability was restored after UMSC-Exo treatment,JC-1 staining showed weakened green fluorescence and enhanced red fluorescence,propidium iodide(PI)staining showed decreased red fluorescence,and the number of dead cells decreased.The therapeutic effect of UMSC-Exo was enhanced with increasing dosage,and the optimal therapeutic concentration was found to be 50μg/ml.2.miR143-3p derived from UMSC-Exo alleviates seawater-induced necroptosis by targeting the regulation of NFATc2.First,NFATc2 is the key gene in regulating seawater induced necroptosis(1)UMSC-Exo attenuates seawater induced cell damage by inhibiting necroptosisGSEA analysis showed that the cell death type induced by sea water stimulation while inhibited after treatment with UMSC-Exo in BEAS-2B cells was necroptosis(P<0.05).WB results showed that after seawater induction,the expression of necroptosis executive proteins p-MLKL,p-RIPK3,and RIPK1 increased,while RIPK3 expression was decreased,and the expression of Caspase-8,which inhibits necrotic apoptosis,was decreased,with no significant change in MLKL expression.After adding UMSC-Exo,the expression of p-MLKL,p-RIPK3,and RIPK1 decreased,while the expression of RIPK3 increased,while the expression of Caspase-8 increased.The change in MLKL expression was not significant.(2)Screening differentially expressed genes in seawater induced BEAS-2B cell damage by transcriptome sequencingAfter seawater induction,there were 26 genes that were upregulated,and downregulated after treatment with UMSC-Exo.Among them were 4 transcription factors:NFATc2,LTF,MAFF,and NR4A3.The heatmap showed the transcription factors included in the top 50 differentially significant genes:NFATc2,LTF,and MAFF.The volcano plot showed that the gene with the greatest differential significance was NFATc2.(3)Bioinformatics analysis was performed to identify transcription factors that may target necroptosis and its validationThe PROMO database was used to predict transcription factors for RIPK1,and four transcription factors that were differentially screened by transcription sequencing were compared.The predicted transcription factor NFATc2 was found to be a key gene in regulating necrotic apoptosis.The JASPAR website was used to search for the binding site sequence of NFATc2 in the RIPK1 promoter and design a binding site mutation sequence.The luciferase experiment showed that the NFATc2 plasmid could bind to the wild-type RIPK1 promoter sequence and enhance fluorescence.(4)Validation of NFATc2 expression in UMSC-Exo treated seawater-induced lung injury cell model.WB and qPCR analysis found that the expression levels of NFATc2 m RNA and protein increased after seawater induction,and the levels decreased after UMSC-Exo treatment.(5)Relationship between NFATc2 and necroptosis in BEAS-2B cells transfected with si RNA1)Transfection of si-NFATc2 resulted in a decrease in NFATc2 m RNA and protein expression levels;2)Transfection of si-NFATc2 resulted in a reduction of seawater-induced cell viability decrease;3)Transfection of si-NFATc2 resulted in a decrease in seawater-induced mitochondrial membrane potential decrease;4)Transfection of si-NFATc2 resulted in a decrease in seawater-induced cell membrane damage;5)Transfection of si-NFATc2 resulted in a decrease in seawater-induced cell death;6)Transfection of si-NFATc2 resulted in a decrease in the expression of p-MLKL,p-RIPK3,and RIPK1,an increase in RIKP3 expression,and an increase in Caspase-8expression in cells after seawater induction.The above results suggest that si-NFATc2 transfection results in downregulation of NFATc2 expression,negative regulation of expression of necroptosis related proteins,and inhibition of necroptosis occurrence.Secondly,UMSC-Exo targets NFATc2 through miR143-3p.(1)Screening results of miRNA targeting NFATc2 in UMSC-ExoScreening results of miRNA regulating NFATc2 in UMSC-Exo.Among the top100 expressed miRNAs in the GSE159814 and GSE69909 datasets,there were 51shared miRNAs.Six miRNAs predicted to potentially bind to the 3’UTR of the NFATc2gene were obtained from the Target Scan,miRDB,miRWalk,mir DIP,and miRTar Base databases,and four candidate miRNAs,including miR-7-5p,miR-143-3p,miR-221-3p,and miR-222-3p were obtained by taking the intersection of both sets of miRNAs.The expression levels of these four candidate miRNAs were low in normal BEAS-2B cells.(2)Validation of candidate miRNAs targeting and regulating NFATc2 in UMSC-ExoAfter transfection of four candidate miRNA mimics into cells,the mimic of miR-143-3p decreased the m RNA and protein expression levels of NFATc2.The binding site sequence of miR-143-3p on the 3’UTR of the NFATc2 gene m RNA was searched and wild-type and mutant binding site vectors were designed and constructed.Luciferase experiments showed that miR-143-3p mimics significantly reduced the fluorescence intensity of the wild-type 3’UTR,indicating that miR-143-3p can target and bind to the 3’UTR of NFATc2.(3)Effect of miR-143-3p on seawater induced necroptosis in BEAS-2B cells1)Similar to the effect of UMSC-Exo,miR-143-3p mimics can then alleviate the decrease in cell viability induced by seawater;2)Similar to the effects of UMSC-Exo,miR-143-3p mimics can increase the mitochondrial membrane potential induced by seawater.;3)Similar to the effects of UMSC-Exo,miR-143-3p mimics can reduce the membrane damage induced by seawater;4)Similar to the effects of UMSC-Exo,miR-143-3p mimics can reduce cell death induced by seawater;5)Similar to the effect of UMSC-Exo,seawater induces necroptosis.After transfection with miR-143-3p mimics,cell death induced by seawater can be inhibited.3.In vivo verification study of UMSC-Exo inhibiting necroptosis and alleviating acute lung injury induced by seawater in mice(1)Changes of arterial blood gas in miceSeawater induced lung injury in mice can lead to significant decreases in pH,pO2,and SaO2,while increases in pCO2,and lactic acid HCO3~-.After UMSC-Exo treatment,pH,pO2,and SaO2 increased,while pCO2,and lactic acid HCO3~-decreased.(2)Changes in chest X-rays of mice.Seawater induction can cause diffuse exudation in bilateral lung of mice,and after UMSC-Exo treatment,the exudation in bilateral lung decreased.(3)Pathological changes and lung tissue injury scores in miceAfter seawater induction,there were obvious neutrophil infiltrations,thickening of the alveolar septum,collapse of the alveoli,and increased alveolar and interstitial hemorrhage in mouse lung tissue.After UMSC-Exo treatment,neutrophil infiltration decreased,alveolar septum thickness decreased,and alveolar and interstitial hemorrhage reduced.The lung injury score of mouse lung tissue increased after seawater induction,and decreased after UMSC-Exo treatment.(4)Wet-to-dry weight ratio of mice lung tissue.After seawater induction,the wet-to-dry weight ratio of mice lung tissue increased.After UMSC-Exo treatment,the wet-to-dry weight ratio decreased.(5)Detection of inflammatory factors in mouse alveolar lavage fluidAfter seawater induction,the levels of inflammatory factors IL-1β,IL-6,IL-18,and TNF-αin mouse lung lavage fluid significantly increased,and decreased significantly after UMSC-Exo treatment.(6)Immunohistochemistry of NFATc2,p-MLKL,RIPK1 in lung tissueThe expression of NFATc2,p-MLKL,and RIPK1 in mouse lung tissue increased after seawater induction and decreased after UMSC-Exo treatment.(7)Immunofluorescence of NFATc2,p-MLKL,RIPK1 in lung tissueThe fluorescence and expression of NFATc2,p-MLKL,and RIPK1 increased in mouse lung tissue after seawater induction,and decreased after UMSC-Exo treatment(8)Western blot detection of NFATc2,p-MLKL,p-RIPK3,MLKL,RIPK3,RIPK1,and Caspase-8 in lung tissueThe expression of NFATc2,p-MLKL,p-RIPK3,and RIPK1 increased in mouse lung tissue after seawater induction,while RIPK3 and Caspase-8 decreased.After UMSC-Exo treatment,the expression of NFATc2,p-MLKL,p-RIPK3,and RIPK1decreased slightly,while RIPK3 and Caspase-8 increased slightly.Conclusion1.UMSC-Exo can alleviate seawater induced BEAS-2B cell injury.2.UMSC-Exo inhibits necroptosis and alleviates seawater-induced BEAS-2B cell damage by targeting regulating NFATc2 via miR-143-3p.3.In vivo experiments show that UMSC-Exo can alleviate acute lung injury induced by seawater in mice.The expression of NFATc2 and necroptosis-related proteins is consistent with the results of in vitro cell experiments.