Exploring The Role And Mechanism Of Pyroptosis In COVID-19 Based On Bioinformatics And Immunofluorescence

Part One The mechanisms and roles of pyroptosis-related factors in patients with COVID-19 based on bioinformatics Objective To explore the expression differences of pyroptosis-related genes in COVID-19 patients,as well as the immune pathways and immune cell infiltration in the pathological process of COVID-19.Methods The transcriptome data GSE and GSE were downloaded from the GEO database,and the "limma" package was used to analyze the differentially expressed genes of the healthy control group and the COVID-19 group,as well as the differentially expressed pyroptosis-related genes in nasopharyngeal epithelial cells from mild,moderate and severe COVID-19 patients,Inflammatory pathways and immune cells involved in the pathological response to COVID-19 were analyzed using GO analysis and KEGG enrichment.Results NOD-like receptor signaling pathway,TNF signaling pathway,IL-17 signaling pathway,Toll-like receptor binding,viral receptor activity,natural killer cell-mediated cytotoxicity,positive regulation of cytokine production,NF-κB transcription factor activity The positive regulation of IL-1β production,the positive regulation of T cell activation and other pathways were significantly different between the control group and the COVID-19 group.Compared with healthy controls,COVID-19 patients had higher M0 levels of neutrophils,B memory cells,T memory cells and macrophages,but lower levels of B cells and monocytes.Pyroptosis-related genes GZMA,GZMB and Caspase3 were differentially expressed in nasopharyngeal epithelial cells of control group,mild,moderate and severe COVID-19 patients（P<0.05）;Caspase8 was expressed in nasopharyngeal epithelial cells of mild,moderate and severe COVID-19 patients There were differences in expression in cells（P<0.05）;IL-18 was differentially expressed in nasopharyngeal epithelial cells in control group,mild,moderate and severe COVID-19 patients（P<0.05）;HMGB-1 was expressed in blood monocytes in mild and moderately severe patients There were differences in expression（P<0.05）.Conclusion Upon SARS-CoV-2 infection of the host,the expression of pyroptosis-related factors in nasopharyngeal epithelial cells and monocytes is up-regulated,and the immune pathways related to pyroptosis are activated.Part Two A study on pyroptosis-related factors in lung and spleen of patients with COVID-19 Objective The purpose of this study is to investigate the role of pyroptosis-related inflammatory factors in the pathogenesis of COVID-19.Methods The expression of inflammatory factors in the lungs and spleens of patients with COVID-19 is studied by the tissue immunofluorescence method and analyzed by SPSS statistical software 20.Results The positive rates of NF-κB,NLRP-3,IL-18 and GSDMD in the lung tissues from the control group and COVID-19 group were 9.8% vs 73.4%（P=0.000）,5.5% vs 63.6%（P=0.000）,24.4% vs 76.2%（P=0.000）,and 17.5% vs 46.8%（P=0.000）,respectively.The positive rates of NF-κB,NLRP-3,IL-18,HMGB-1 and GSDMD in the spleen tissues from the control group and COVID-19 group were 20.6% vs 71.2%（P=0.000）,18.9% vs 72.0%（P=0.000）,15.2% vs 64.8%（P=0.000）,27.6% vs 69.2%（P=0.000）,and 23% vs 48.8%（P=0.000）,respectively.The rates of the lung and spleen CD68 positive cells in the control group and the COVID-19 group were 16.7% vs 63.3%（P=0.000）,22.1% vs 54.0%（P=0.000）,respectively.The positive rates of SARS-CoV-2 S protein in the CD68 positive cells of the lung and spleen in the control group and COVID-19 group were 2.5% vs 56.8%（P=0.000）;3.0% vs 64.9%（P=0.000）respectively.The rates of NF-κB positive nuclei in the control group and COVID-19 group were 13.4% vs 51.4%（P=0.000）in the lung,and 38.2% vs 59.3%（P=0.000）in the spleen respectively.The rates of HMGB-1 positive cytoplasm in the control and the COVID-19 group were 19.7% vs 50.3%（P=0.000）in the lung,and 12.3% vs 45.2%（P=0.000）in the spleen,respectively.Conclusions The targets of SARS-CoV-2 are both lung and spleen,where increased macrophages could be involved in the up-regulation of pyroptosis-related inflammatory factors such as NF-κB,HMGB-1,NLRP-3,IL-18,and GSDMD.