Quantitative Proteomics Reveals The Mechanisms Of Hydrogen-Conferred Protection Against Hyperoxia-Induced Injury In Type Ⅱ Alveolar Epithelial Cells

Objective:The objective of this study was to characterize differentially regulated proteins and biological processes in hydrogen-treated hyperoxic primary type II alveolar epithelial cells(AECIIs)to elucidate the protective mechanism of hydrogen using quantitative proteomics.Methods:AECIIs were divided into three groups that were cultured for 24 h in three different conditions: control(21% oxygen),hyperoxia(95% oxygen),and hyperoxia + hydrogen treatment(95% oxygen with hydrogen dissolved in the medium).Morphologic examination,flow cytometric analysis,cell viability assessment and analysis of the expression of apoptosis-associated proteins Bax and Bcl-2 as well as AECI markers(AQP5,T1α)and an AECII marker(SP-C)were performed for each group.The TMT labeling quantitative proteome technique was used to detect changes in the protein expression profile,and bioinformatics analysis wasperformed.Results:(1)The cells in the normoxia group proliferated and prolonged significantly,and the cytoplasmic particulate matter was abundant.In the hyperoxia group,nucleus pyknosis and cytoplasmic particulate matter decreased significantly.Morphology was improved by hydrogen treatment.Compared with the normoxia group,the expression of BAX and SPC in the hyperoxia group increased significantly,and the expression of Bcl-2,AQP5 and T1α decreased significantly.Increased apoptosis,decreased viability and survival,and inhibited transdifferentiation of AECIIs into AECIs were observed in the hyperoxia group.Hydrogen plays a protective role in hyperoxia-induced damage in AECIIs,as evidenced by improved morphology,reduced apoptosis,increased viability and survival,and enhanced transdifferentiation of AECIIs into AECIs.(2)A total of 5782 proteins were identified in our study,of which 162 were significantly altered in abundance after hyperoxia exposure,and 97 were significantly altered in abundance in response to hydrogen treatment,29 proteins were commonly shared between both comparisons.The Gene Ontology and KEGG enrichment analyses identified a large number of proteins and biological processes that may responsible for the protective effect of hydrogen,including VEGFA,PDGFB,IGFBP3,EDN1,NADPH oxidase,the coagulation cascade,etc.Conclusions: Molecular hydrogen protects AECIIs from hyperoxicinjury by complex mechanisms involving a variety of proteins and biological processes,such as VEGFA,PDGFB,IGFBP3,EDN1,NADPH oxidase and the coagulation cascade.These findings suggest novel pathways that need to be investigated as possible therapeutic targets for hyperoxia-induced lung injury.