Proteomic Analysis Of Bronchoalveolar Lavage Fluid From ARDS Patients Based On ITRAQ Quantitative Technology

Background Acute respiratory distress syndrome(ARDS)is a poor prognosis and life-threatening disease,the most severe form of acute lung injury,resulting in high mortality.ARDS is a dynamic syndrome with multiple etiologies,and timely diagnosis and follow-up of the syndrome process is difficult.The underlying cellular-molecular mechanisms are currently unknown,and there are no definitive biomarkers available for the diagnosis and treatment of ARDS.Therefore,there is an urgent need for rapid diagnosis,clinical monitoring,and exploration of potential drug targets for ARDS to improve patient survival and quality of life.Objective To investigate the potential of bronchoalveolar lavage fluid(BALF)in assessing the severity of lung injury,predicting prognosis,monitoring response to treatment,and exploring potential drug targets in patients with ARDS caused by severe pneumonia by quantifying the proteomics of BALF before and after treatment using commonly used bioinformatics analysis methods.Methods From the perspective of clinical disease regression,we collected paired BALFs samples from three ARDS patients in the acute inflammatory and proliferative recovery phases by i TRAQ quantitative technique analysis.Proteins with the same expression pattern between two different pathophysiological processes in the three patients were identified as co-upregulated and co-downregulated proteins(CUDPs),and differentially expressed proteins(DEPs)were selected from CUDPs with fold change > 1.2 or fold change <0.83 and P value < 0.05.Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)analyses were applied to determine the enrichment functions and enrichment pathways of CUDPs.Protein-protein interaction(PPI)networks were generated in the STRING database and core proteins were identified by Cytoscape software.Treatment of A549 cells with lipopolysaccharide(LPS)to mimic the inflammatory environment of alveolar epithelial cells in ARDS.Results We identified 374 proteins(98 co-up-regulated and 276 co-down-regulated proteins)and 53 differentially expressed proteins.Gene ontology analysis showed that 374 proteins were significantly enriched in neutrophil-mediated immune response,neutrophil activation,neutrophil activation involved in immune response,and neutrophil degranulation.And KEGG analysis showed that 374 proteins were significantly enriched in coronavirus disease COVID-19 and ribosome interaction pathway,etc.Protein interaction combined with Cytoscape software analysis suggested6 key proteins: RPSA,RPS5,RPS25,PSMD8,EIF3 E,PSMB1,and that RPSA was the most important core differential protein,with protein levels downregulated in ARDS patients during the recovery improvement phase(and upregulated during the acute inflammatory phase).In addition,we further confirmed by cellular assays that both m RNA and protein levels of RPSA were significantly increased in A549 cells stimulated with lipopolysaccharide in vitro to mimic the inflammatory environment,and RPSA changes in LPS-stimulated cell culture supernatants were also significantly elevated as detected by ELISA.Conclusion 1.Proteomic analysis of bronchoalveolar lavage fluid may reveal potential biomarkers and therapeutic targets for ARDS. 2.RPSA is a candidate for ARDS biomarker and therapeutic target.