| Objective:Sepsis is a common severe consequence of trauma,and sepsis induced by Staphylococcus aureus(S.aureus)has a high mortality rate;however,the mechanism of action is still unknown.The purpose of this study is to investigate the relationship between traumatic patients infected with Staphylococcus aureus and the incidence rate of sepsis and multiple organ dysfunction syndrome(MODS),as well as the mechanism of action,in order to provide a new strategy and foundation for the early diagnosis of sepsis and targeted immunotherapy in clinical practice.Methods:1.1,574 cases of trauma patients were obtained from the "Trauma Patient Biological Sample Resource Database and Clinical Research Database" to study the general distribution features of patients with sepsis and the incidence and mortality of sepsis in patients infected with various kinds of pathogenic bacteria.2.To construct a sepsis mouse model infected with Staphylococcus aureus,observe the damage of essential organs in the mice,conduct high throughput detection of inflammatory factors in the peripheral blood of the sepsis model mice,and observe the changes of inflammatory factors.To explore the targeted therapeutic effect of IL-6monoclonal antibody in septic mice and to observe its impact on the survival curve and body weight of septic mice.The expression of costimulatory molecule CD28 on T lymphocytes in peripheral blood of superantigen-induced sepsis was analyzed by flow cytometry.Proteomic analysis was performed on CD3+ and CD28+ T cells to observe the changes in the expression level of differential proteins.3.To validate the proteomic results,we explored the role of ferroptosis in multiple organ injury in sepsis based on the GEO dataset analysis and in vivo experiments.Four sepsis data sets were downloaded from the GEO database: two training sets(GSE26378 and GSE26440)and two validation sets(GSE11755 and GSE11281).A bioinformatics study revealed 63 genes associated with iron death.For functional annotation,the GO and KEGG pathway enrichment analyses were performed.The protein-protein interaction(PPI)network and cluster analysis were then built in order to get hub-FRG(a ferrotropis-related gene)and investigate its association with immune regulation.Finally,using a sepsis animal model,the biochemical enzymology of peripheral blood and pathological staining of important organs of model mice were detected,the expression of hub-FRG in major organs was confirmed using a Western blot and immunofluorescence test,and the function of mitochondria in major organs was determined using an ATP test.Results:1.Patients infected with Staphylococcus aureus are more likely to develop sepsis than other G+ bacteria infected individuals.The death rate in staphylococcus aureus superantigen caused sepsis was considerably greater than in other types of sepsis.In addition,the main manifestations of multiple organ function syndrome in patients with sepsis induced by staphylococcus aureus superantigen were lungs(81.25%),liver(68.75%)and kidney(37.5%).2.By establishing different types of sepsis mouse models,it was observed that the lung,liver,and kidney damage in sepsis mice induced by Staphylococcus aureus was significantly aggravated compared to those in Escherichia coli and LPS groups;The detection of inflammatory factors showed that compared with the control group(Escherichia coli group and LPS group sepsis mice),the level of IL-6 expression in the peripheral blood of staphylococcus aureus induced sepsis mice was significantly increased Targeted therapy with IL-6 monoclonal antibodies in different sepsis mice has shown that IL-6 monoclonal antibodies can effectively improve the survival time of sepsis mice induced by Staphylococcus aureus,Escherichia coli,and LPS,respectively,and can improve their body weight.Furthermore,flow cytometry analysis showed that the expression of costimulatory molecule CD28 on CD3+ T lymphocytes in the peripheral blood of staphylococcus aureus induced septic mice increased significantly;In addition,The proteomic study of CD3+ CD28+ T cells revealed 127 key differentially expressed proteins(Key-DEPs).The top ten Key-DEPs were discovered using Cytohubba software.These are defined as hub differentially expressed proteins(hub-DEPs),and they include Gpx1,Hsp90b1,Gpx4,Hspa5,Ywhab,Atp2a2,P4 hb,Glud1,Ywhag,and Got2.Moreover,Cytoscape Clue GO observed the biological processes of DEPs and KEGG analysis,which primarily centered on the biological regulation of ferroptosis,lysosomes,glutathione metabolism,and protein processing in the endoplasmic reticulum.3.Based on the GEO dataset,the validation proteomics results above show: In the GSE26378 and GSE26440 data sets,DEGs and FRGs intersected to produce 63DE-FRGs.DE-FRGs were highly enriched in ferroptosis,mitochondria,autophagy,and oxidoreductase activity,according to GO and KEGG analyses.PPI had three essential MCODE modules,and four core-FRGs(FTH1,GPX4,ACSL1,and ACSL6)were created by crossing with distinct mitochondrial function relation genes(DE-MFRGs).The validation set is then used to filter out Hub-FRG(GPX4).These results are consistent with the proteomic analysis.Then,the ROC analysis revealed that GPX 4 is a important value biomarker in the diagnosis of sepsis.In addition,four subtypes with different expressions of ferroptosis were obtained by consensus clustering analysis.The study of immune cell infiltration and immune checkpoints in Cluster1(sepsis)and Cluster2(control)showed that compared with Cluster2,the expression level of immune cells in Cluster1 was significantly up-regulated in seven species and significantly down-regulated in ten species.In addition,the gene expression level of the immune checkpoint was significantly up-regulated in 2(CD274 and HAVCR2)and significantly down-regulated in 1(TIGIT).Pearson correlation analysis revealed that GPX4 was highly correlated with neutrophils,T cells CD8,and immune checkpoints.Lastly,animal experiments confirmed that the biochemical enzyme detection in the liver and kidney of mice with staphylococcus aureus sepsis was significantly higher than in the control group,and HE staining revealed that the liver,kidney,and lung of mice with staphylococcus aureus sepsis were significantly damaged compared to the control group.Besides,ATP test showed that ATP expression was substantially reduced in the liver and kidney of septic mice compared with the control group.Western blot analysis confirmed that the expression level of GPX4 in the liver and kidney tissues of sepsis model mice induced by S.aureus was significantly lower than that of the control group.Immunofluorescence is consistent with Western blot.Conclusions:This study analyzed the relationship between staphylococcus aureus and mortality in patients with sepsis through a clinical database and found that compared with other G+ bacteria,patients with staphylococcus aureus infection are more likely to develop sepsis and have a higher mortality rate.Then,the study confirms that IL-6 plays a key role in S.aureus-induced sepsis cytokine storms and is a novel potential therapeutic target for sepsis.Finally,the study shows that the ferroptosis-related gene GPX4 is critical for sepsis-induced multiple organ injury and is significantly associated with immune cell infiltration and immune checkpoint gene expression.GPX4 is a potential immunotherapy target-point and a biomarker with significant diagnostic value in S.aureus-induced sepsis. |
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