Expression Of CD177~+ PMNs In Peripheral Blood Of AP Patients And CD 177 Inhibits Neutrophil Extracellular Trap Formation And Protects Against Acute Pancreatitis In Mice

Background:Acute pancreatitis(AP)is a clinical non-inflammatory infectious disease characterized by local pancreatic injury,which is mainly treated conservatively in the early stage.Severe acute pancreatitis.The clinical mortality rate remains high,and it is crucial to seek new therapeutic targets from basic research for clinical application to protect and slow down the disease progression of AP.Mild acute pancreatitis(MAP)accounts for the majority of AP and usually recovers within 1-2 weeks after hospitalization with a low morbidity and mortality rate.In addition,about 15-20%of patients with AP have an inflammatory response that is not limited to the pancreas itself,but may involve the peripancreatic tissues or distal organs(lungs,kidneys,etc.)and progress to severe acute pancreatitis(SAP),which has a high clinical mortality rate in the early stages of SAP.For systemic inflammatory response syndrome,the mortality rate is as high as 40%-70%.Neutrophils are the body’s first line of defense,and neutrophils are rapidly recruited to inflammation,releasing neutrophil extracellular traps(NETs)to efficiently bind,phagocytose,and kill microorganisms.However,in addition to their central role in antimicrobial innate immunity,NETs have been shown to play a deleterious role in the pathophysiology of many infectious and non-infectious diseases.NETs have been shown to have a facilitative role in the progression of AP to SAP.D177 encodes the neutrophil membrane glycoprotein(gp)NB1,which belongs to the leukocyte antigen 6(Ly-6)supergene family.CD 177 binds to β2 integrin and recognizes platelet endothelial cell adhesion molecule 1(PECAM-1).CD177 has been reported to play a key role in the development of several inflammatory diseases,such as arthritis,systemic lupus erythematosus(SLE),pathogen-induced colitis and inflammatory bowel disease(IBD);however,the function of CD 177 in regulating the formation and development of NETs in AP remains unclear.In addition,no studies have so far investigated the role of recombinant human CD 177 protein(rhCD177)in attenuating the inflammatory response and AP pathogenesis.The aim of this study was to investigate the expression of CD 177 in the peripheral blood of AP patients and its role in the pathogenesis of AP,which will provide a theoretical reference for future prediction and clinical targeting of SAP.Objective:1.To detect the frequency of CD45+,CD11b+,CD16+,CD177+cells in peripheral blood PBMC of the healthy people and AP patients.2.To study the effect of rhCD177 on phorbol12-myristate13-acetate(PMA)-induced NETs model in vitro.3.To study the effect of rhCD177 on the Cae-induced AP mouse model.Methods:We collected peripheral blood samples from AP patients and Healthy controls.The levels of CD45+,CD11b+,CD16+,CD177+cells in peripheral blood were detected by flow cytometry(FCM).2.We extracted bone marrow cells from mice,and constructed NETs using PMA in vitro.With the addition of rhCD177,the formation of NET was observed by flow cytometry and immunofluorescence.3.Detect the level of inflammatory factors in the cell supernatant by ELISA to observe the effect of rhCD177 on NETs.4.Extract the proteins from the above cells and perform Western Blot(WB)to analyze the expression of NETs marker PAD4 after adding rhCD177.5.Construct AP mouse model using Cae:divide the experimental animals into normal control group,model group and treatment group.model group and treatment group.The serum of the mice was kept for serum enzymatic level detection.6.Pancreatic tissues were extracted for fixed dehydration,and HE-stained paraffin sections were prepared to observe the effect of rhCD 177 on AP pathology.7.Immunohistochemical staining(IHC)was performed on pancreatic and lung tissues simultaneously to observe the changes of macrophage and neutrophil infiltration in the tissues.8.Peripheral blood and pancreatic tissues of mice were extracted for Immune cells were extracted from peripheral blood and pancreatic tissues of mice,and the infiltration of immune cells in pancreatic tissues was detected by flow cytometry.9.Pancreatic tissue proteins were extracted and WB was performed to observe the changes in the expression of NETs marker PAD4.Results:1.Flow cytometry showed that the frequency of CD45+,CD11b+,CD16+,CD177+ cells decreased(P<0.001)and was positively correlated with disease severity.2.and MPO production were significantly increased in the PMA-induced NETs model group compared with the control group,and the application of rhCD177 significantly inhibited the above related phenomena,thus protecting against acute pancreatitis.3.The expression levels of TNF-α and IL-6 in cell supernatants were significantly decreased by ELISA assay after the addition of rhCD177(P<0.01).4.It was found by WB results that the expression of PAD4 was significantly decreased after the application of rhCD177 compared to the model group(P<0.01).5.It was found by HE staining and serum enzymatic levels that rhCD177 improved the disease severity of acute pancreatitis in mice induced by rainfarin.rhCD177 significantly protected against acute pancreatitis pancreatic histopathology results and improved serum enzymology and serum amylase levels(P<0.001).6.Immunofluorescence by IHC,revealed that:immune cell infiltration was increased in pancreatic and lung tissues of Cae-induced AP model mice;exogenous supplementation of rhCD177 effectively improved inflammatory manifestations and significantly reduced neutrophil and macrophage infiltration(P<0.01).7.After extracting immune cells from peripheral blood and pancreatic tissues,it was found by flow cytometry Immune cell infiltration in peripheral blood and pancreatic tissue was significantly decreased after the use of rhCD177(P<0.01).8.Extraction of pancreatic tissue proteins revealed that the expression of protein PAD4 was significantly decreased in the treated group(P<0.01).Conclusion:The expression of CD177+neutrophil levels was elevated in peripheral blood of AP patients and increased with disease severity;rhCD177 could exert a protective effect against AP and alleviate AP-induced lung injury by inhibiting NETs.This predicts that CD 177 may be a potential therapeutic strategy to prevent or inhibit AP exacerbations.