Screening Of Key Genes Involved In Sepsis Based On Bioinformatics Analysis And Its Mechanism

Background Sepsis is a multi-organ dysfunction caused by a dysregulated host response to severe infection,and can progress to septic shock in severe cases.The high mortality rate of sepsis has always been the focus of medical research.In the past 20 years,a large number of research data had improved understanding of the pathophysiology of sepsis,and gradually clarified the role of regulation of inflammatory pathway and immunosuppression in sepsis.However,the pathogenesis and molecular mechanism of vascular endothelial cell injury caused by sepsis are still unclear.The bioinformatics technology has recently emerged to help us obtain differentially expressed genes(Degs)between subjects with sepsis and normal subjects,and analyze them by protein-protein interaction(PPI)networks,along with the validation of mechanisms,which facilitate a further exploration of novel molecular mechanisms of sepsis and provide new ideas and methods for the prevention and treatment of sepsis.Objective The key genes involved in the pathogenesis of sepsis were screened by bioinformatics analysis of the Gene Expression Omnibus(GEO)dataset,and their expression and regulation in vascular endothelial cells in the subjects with sepsis were investigated to explore their potential mechanisms of action in the pathogenesis of sepsis.Methods 1.The method of bioinformatic analysis was implemented.Specifically,the gene microarray expression profiles of sepsis were obtained from the GEO database,the microarray dataset GSE64457 was downloaded from the neutrophil sample data of 15 infectious shock cases and 8 healthy volunteers,the differentially expressed genes(DEGs)of neutrophils from the infectious shock cases and healthy volunteers were statistically analyzed by R-studioru software,and the protein-protein interaction(PPI)network of differential genes was constructed by the Search Tool for the Retrieval of Interaction Gene(STRING)database as a search tool,and visualized in a software(Cytoscape)which graphically displayed the network and allowed for the analysis and editing functions,so as to identify key genes expected to be studied by analyzing the correlation between known pathways and mechanisms of action of the screened key genes and their pathophysiological changes in vascular injury caused by sepsis.2.The expression level of the screened key genes in human neutrophils was detected by fluorescence quantitative PCR to validate the results of bioinformatic analysis.3.Human umbilical vein endothelial cells(HUVECs)were stimulated by serum from patients with sepsis and normal volunteers,and intervened by key gene inhibitors.The change of VCAM-1 gene expression in HUVEC was detected by fluorescence quantitative PCR,the change of VCAM-1 protein in supernatant was detected by ELISA,the adhesion rate between peripheral blood mononuclear cells(PBMCs)and HUVEC was detected,and the change of p38 MAPK and ERK1/2 in HUVEC was detected by Western blot.Results 1.In this study,seven HUB genes(MMP8,HP,ARG1,FOLR3,QSOX1,PGLYRP1 and OSCAR)were identified.The analysis result of biological process revealed that these genes were mainly enriched in inflammatory reaction and immune reaction,and MMP8 was found to be associated with endothelial cell injury by analyzing the correlation between known pathways and mechanisms of action of the screened key genes and their pathophysiological changes in vascular injury caused by sepsis.2.Fluorescence quantitative PCR showed that the expression of MMP8 gene in neutrophils in the blood of patients with sepsis was significantly higher than that of the group of normal volunteers(22.9±16.77 vs 2.36±2.16,p<0.05).3.After stimulation of HUVEC with serum from patients with sepsis and normal volunteers for 6 h,the expression of VCAM-1 gene was found to be significantly higher than that of normal serum stimulation group(4.51±1.45 vs 1.00±0.11,p<0.05),and the protein expression was significantly increased(2360±34.15 vs 576.3±19.58,p<0.05).Through the adhesion test of peripheral blood mononuclear cells,it was found that the adhesion rate of peripheral blood mononuclear cells stimulated by serum from patients with sepsis was significantly higher than that of normal serum(479.3±47.96% vs.100%,p<0.05).The relative expression of p38 MAPK(0.69±0.04 vs 0.30±0.02,p<0.05)and ERK1/2(0.68±0.05 vs 0.35±0.02,p<0.05)proteins was found to be significantly higher after stimulation with sepsis serum by WB assay.Through pretreatment with MMP8 inhibitor for 1 h,followed by stimulation with serum from patients with sepsis,a significant decrease was observed in VCAM-1 gene expression in endothelial cells(1.01±0.05 vs 0.36±0.15,p<0.05),VCAM-1 protein content in the supernatant(2381±50.76 vs 1941±6.23,p<0.05),and adhesion rate of peripheral blood mononuclear cells(479.3±47.96% vs 210.7±13.65%,p<0.05).Also,the intracellular p38 MAPK(0.83±0.03 vs 0.32±0.04,p<0.05)and ERK1/2(0.55±0.02 vs 0.24±0.07,p<0.05)protein content was significantly decreased.Conclusion MMP8,a key gene screened by bioinformatic analysis,was involved in the production and release of vascular endothelial cell adhesion molecule 1(VCAM-1)in sepsis and caused an increase in the adhesion number of peripheral blood mononuclear cells,a role that may be related to the activation of p38 MAPK and ERK1/2 signaling pathways by MMP8.