Gene Regulation of Neutrophils Mediated Liver and Lung Injury through NETosis in Acute Pancreatitis.

Abstract

Acute pancreatitis (AP) is one of the most common gastrointestinal emergencies, often resulting in self-digestion, edema, hemorrhage, and even necrosis of pancreatic tissue. When AP progresses to severe acute pancreatitis (SAP), it often causes multi-organ damage, leading to a high mortality rate. However, the molecular mechanisms underlying SAP-mediated organ damage remain unclear. This study aims to systematically mine SAP data from public databases and combine experimental validation to identify key molecules involved in multi-organ damage caused by SAP. Retrieve transcriptomic data of mice pancreatic tissue for AP, lung and liver tissue for SAP, and corresponding normal tissue from the Gene Expression Omnibus (GEO) database. Conduct gene differential analysis using Limma and DEseq2 methods. Perform enrichment analysis using the clusterProfiler package in R software. Score immune cells and immune status in various organs using single-sample gene set enrichment analysis (ssGSEA). Evaluate mRNA expression levels of core genes using reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry. Validate serum amylase, TNF-α, IL-1β, and IL-6 levels in peripheral blood using enzyme-linked immunosorbent assay (ELISA), and detect the formation of neutrophil extracellular traps (NETs) in mice pancreatic, liver, and lung tissues using immunofluorescence. Differential analysis reveals that 46 genes exhibit expression dysregulation in mice pancreatic tissue for AP, liver and lung tissue for SAP, as well as peripheral blood in humans. Functional enrichment analysis indicates that these genes are primarily associated with neutrophil-related biological processes. ROC curve analysis indicates that 12 neutrophil-related genes have diagnostic potential for SAP. Immune infiltration analysis reveals high neutrophil infiltration in various organs affected by SAP. Single-cell sequencing analysis shows that these genes are predominantly expressed in neutrophils and macrophages. FPR1, ITGAM, and C5AR1 are identified as key genes involved in the formation of NETs and activation of neutrophils. qPCR and IHC results demonstrate upregulation of FPR1, ITGAM, and C5AR1 expression in pancreatic, liver, and lung tissues of mice with SAP. Immunofluorescence staining shows increased levels of neutrophils and NETs in SAP mice. Inhibition of NETs formation can alleviate the severity of SAP as well as the levels of inflammation in the liver and lung tissues. This study identified key genes involved in the formation of NETs, namely FPR1, ITGAM, and C5AR1, which are upregulated during multi-organ damage in SAP. Inhibition of NETs release effectively reduces the systemic inflammatory response and liver-lung damage in SAP. This research provides new therapeutic targets for the multi-organ damage associated with SAP.