The Research About The Mechenism Of Monocytes/Macrophages In Acute Pancreatitis

OBJECTIVES:At present,there is no effective strategy for clinical targeted therapy in acute pancreatitis.There are still many limitations in the understanding of the mechanism of its injury and recovery.In recent years,immune cells,especially monocytes/macrophages,have been found to play an important role in the pathogenesis of acute pancreatitis.Our previous study found that M1 macrophages are involved in the acute injury of acute pancreatitis and carbon monoxide（CO）plays a protective role against AP by inhibiting M1.Next,my research will focus on the pathogenesis and therapy of acute pancreatitis including:1)further explore the molecular mechanism of the protective effect of carbon monoxide from the perspective of mononuclear cell infiltration;2)explore the phenotype and mechanism llof monocytes/macrophage in this process based on the point of injury recovery of acute pancreatitis.From the above two parts of study,we can further understand the immune pathology of acute pancreatitis,providing new ideas and theoretical basis for the development of immunotherapy.METHODS:1)In the 1^st part,firstly,the number of monocytes in the early stage of AP was observed by flow cytometry after constructing the AP model induced by caerulin under CO intervention.Secondly,the expression of genes and proteins of the chemokines and cytokines in the pancreas of CO intervention group/control group was analyzed by real-time fluorescence quantitative PCR and multi-cytokine kit,by which the differentially expressed cytokines/chemokines were determined.Finally,whether monocytes directly affected monocytes was determined by injection of CO pretreated monocytes into normal AP mice,and the molecular mechanism of CO’s impact on monocyte migration was futher determined by endocytosis assay,Ca²⁺release assay,a4β1 integrin-specific ligand（VLA-4）binding assay and co-localization immunofluorescence staining.2)In the 2^nd part:firstly,we established the model of AP recovery model and the key time points of AP recovery were established by H&E and Ki67 immunofluorescence staining analysis.Secondly,the numbers and phenotypes of macrophages during the recovery stage of AP were further determined by immunofluorescence staining and flow cytometry analysis.Furthermore,macrophage liposome depletion experiment was administrated to verify the direct effect of macrophages in the acute pancreatitis recovery.Finally,high-throughput RNA-seq analysis of pancreatic macrophages in the key points of AP recovery was adopted to explore the mechanism of macrophage M2 polarization and acinar cell proliferation during acute pancreatitis recovery process.RESULT:1)At the early stage of AP injury,CO affects the migration of(CD11b⁺Ly-6C^hi)inflammatory monocyte migration from blood to inflammatory pancreas by blocking the endocytosis of CCL2/CCR2 signaling axis and the expression of CCL2 in pancreas.2)At the recovery stage,acute pancreatitis was promoted by macrophages polarization to the M2 type through IL4RA.CONCLUSION:1)Carbon monoxide can impair the CD11b⁺Ly-6C^himonocyte migration from the blood to inflammatory pancreas in the early stage of acute pancreatitis.2)M2-type polarization of macrophages can promote the recovery of acute pancreatitis.