A Study On Key Signaling Pathways And Genes In Transfusion-Related Acute Lung Injury Mediated By CD36 Antibody

Background and objectivesTransfusion-related acute lung injury(TRALI)is characterized by significant respiratory distress and hypoxia within 6 hours after transfusion,which can be lifethreatening in extreme cases.The pathophysiological mechanisms underlying TRALI are still controversial,and there is no specific targeted therapy in clinical practice.This study aims to investigate the pathophysiological mechanisms of TRALI and identify potential key genes using transcriptome sequencing and bioinformatics analysis.A TRALI mouse model will be established,and mouse lung tissues will be subjected to transcriptome sequencing.Differentially expressed genes will be analyzed and further validated,laying the foundation for a deeper understanding of TRALI mechanisms.MethodsIn this study,C57BL/6J mice were divided into three groups(Normal,LPS control,and TRALI)and a TRALI mouse model was established by intraperitoneal injection of lipopolysaccharide and anti-CD36 monoclonal antibody.An assessment of the modeling’s efficacy was conducted through indicators of acute lung injury,such as pathological alterations in lung injury,permeability of alveolar capillary walls,and inflammatory reactions linked to lung damage.After successful modelling,mRNA transcriptome sequencing was conducted on mouse lung tissue,followed by bioinformatics analysis to pinpoint key genes and pathways.Analysis of Gene Ontology,KEGG,and Reactome enrichment,protein-protein interaction network,hubgene,and hub-gene pathway enrichment are all included.The results were further validated using RT-qPCR,Western Blot,serum ELISA assay and fluorescence cross section.Results:1.Establishment of a CD36 antibody-mediated TRALI Mouse ModelThe CD36 antibody caused a significant reduction in body temperature and an increase in the dry/wet ratio of lung tissue in lung structure in CD36 antigen-positive mice compared to the controls.Microscopic examination revealed significant pathological changes in lung tissue structure.It was found that the total protein content,chemokines CXCL1,CXCL2,and pro-inflammatory cytokine TNF-α were significantly increased in bronchoalveolar lavage fluid from the lung tissues of mice.The variations among the groups were found to be statistically significant.The successful establishment of a mouse model with CD36 antibody-mediated transfusionassociated acute lung injury is suggested by this.2.Transcriptome Sequencing and Quality EvaluationTo investigate the pathogenesis of transfusion-associated acute lung injury mediated by the CD36 antibody,transcriptome sequencing was performed on lung tissue from three groups of mice(Normal,LPS,and TRALI groups).The quality of the sequencing was evaluated using NGS QC Toolkit,Bowtie2,and Tophat,and the results showed that the number of clean reads reached 5～8×109,the effective base ratio was as high as 99%,the Q30 ratio exceeded 92%,GC content ranged from 49.1%to 49.7%,and the genomic alignment rate was over 97.7%.The transcriptome sequencing results were reliable and provided a solid foundation for subsequent analysis.3.Analysis and validation of the sequencing results3.1 Significant pathway enrichment of DEGsTo explore the key pathways involved in CD36 antibody-mediated TRALI,879 differentially expressed genes(DEGs)between the LPS and TRALI groups were subjected to gene ontology(GO),KEGG,and Reactome enrichment analysis.The results shown that the cytokine-cytokine receptor interactions,IL-17 pathway and MAPK pathway play an essential role in TRALI.3.2 Analysis of key genes and experimental validation of MMP-9 expressionWe employed a PPI network diagram and Cytoscape 3.9.1 to investigate the essential genes implicated in this process and pinpoint possible targets with clinical significance.We obtained 10 potential hub-genes,including TNF,FOS,TLR2,CXCL1,CXCL2,IL-6,IL-10,CCL2,CCL4,and MMP-9,and then verified the expression of these hub-genes using RT-qPCR.After reviewing the literature and analyzing the key genes in related fields,we identified MMP-9 as the potential key gene for further investigation.Based on the results of RT-qPCR,all 10 hub-genes obtained from the analysis were upgraded after the occurrence of TRALI occurred.A Western Blot revealed a considerable rise in MMP-9 protein expression in the lung tissue of TRALI mice,while ELISA revealed a noteworthy rise in MMP-9 content in the serum of TRALI mice.Fluorescence results showed that MMP-9 was abundantly expressed in the neutrophils and mononuclear macrophages of lung tissue,with more expression on the surface of macrophage than on the surface of neutrophil surface.ConclusionIn this study,we successfully established an animal model of CD36 antibodymediated transfusion-related acute lung injury(TRALI)and identified ten key genes(TNF,IL-6,IL-10,CXCL1,CXCL2,FOS,CCL2,CCL4,TLR2,MMP-9)and core pathways(cytokine-receptor interaction pathway,IL-17 pathway,and MAPK pathway)that play crucial roles in the occurrence and development of TRALI.We conducted relevant validations as well.Based on the comprehensive results,MMP-9 emerged as a potential key gene involved in TRALI,associated with the IL-17 pathway and MAPK pathway.It has the potential to serve as a clinical biomarker or therapeutic target,but further research is still needed for validation.InnovationWe established the first mouse model of CD36 antibody-mediated TRALI.Currently,there is a lack of transcriptome sequencing analysis studies related to TRALI.This study revealed key genes and core pathways involved in the TRALI process and identified a potential target,MMP-9,which provides new insights for further mechanistic and clinical therapeutic research on TRALI.ProspectsBuilding upon existing research and the results of this study,further investigations can be carried out using techniques such as small interfering RNA transfection to locally knock down MMP-9 expression in mouse lung tissue,aiming to observe the occurrence of TRALI.MMP-9 knockout mice can be utilized to further explore the role of MMP-9 in TRALI.Additionally,additional in vitro and in vivo experiments can be conducted to investigate the relationship between MMP-9 and monocytes/macrophages and endothelial cells.