| Objectives:To investigate the role and mechanism of P-selectin glycoprotein ligand-1（PSGL-1）in the occurrence and development of severe acute pancreatitis（SAP）.Methods:We collected the peripheral blood samples from patients who is diagnosed as SAP from January 2018 to January 2019 in the people’s Hospital of Zhengzhou University.We collected the peripheral blood samples from healthy people during the same time.We used flow cytometry to detect the number of monocytes/macrophages and neutrophils in the two groups.The expression of PSGL-1 on the surface of these cells was detected by same method,too.The mouse model of severe acute pancreatitis was established in PSGL-1 knockout mice(PSGL-1-/-),and the amylase level and pancreatic pathological injury were detected.IL-6 and IL-1βin peripheral blood of mice were detected by ELISA Expression level of IL-6 and IL-1βin pancreatic tissue were detected by western blot（WB）.We used TUNEL assay to detect the apoptosis of pancreatic acinar cells in AP mouse model.The number of monocytes/macrophages and neutrophils from peripheral blood of AP mouse model was detceted by flow cytometry.The expression of PSGL-1 on the surface of monocytes/macrophages and neutrophils in AP mouse model was detected by the same method.The infiltration of monocytes/macrophages and neutrophils in pancreatic tissue was detected by immunohistochemical staining.We use Cae to stimulate AR42J cell line to construct AP model in vitro.For detecting the ability of adhesion with monocytes/macrophages and endothelial cell,we created a co-culture system.Si RNA was constructed and transfected into RAW264.7 cells,the expression of PSGL-1 m RNA was detected by q PCR,and the expression of PSGL-1,ARG1 and i NOS protein was detected by Western Blot.Using transcriptome sequencing technology to detect the changes of downstream genes after knockdown of PSGL-1,and further analyzes the changes of downstream signal pathways through GO analysis,KEGG analysis and GSEA analysis.Results:1.Compared with the healthy group,the SAP group has higher quantitative monocytes/macrophages and neutrophils number significantly.The SAP group has higher expressive PSGL-1 on the surface of monocytes/macrophages and neutrophils compared with the healthy group.2.The SAP mouse model group has higher expressive PSGL-1 on the surface of monocytes/macrophages and neutrophils than control group.3.Serum amylase level,IL-6 and IL-1βexpression,pathological injury of pancreatic tissue in PSGL-1-/-mice with severe pancreatitis were significantly lower than that of wild-type mice.4.The infiltration of monocytes/macrophages and neutrophils in the pancreas of PSGL-1-/-mice with severe pancreatitis was significantly lower than that of wild-type mice.5.The adhesion between peripheral blood monocytes/macrophages and endothelial cells in PSGL-1-/-mice was significantly lower than that in control wild-type mice.6.Knockdown of PSGL-1 expression can inhibit the differentiation of monocytes/macrophages into M1 type and promote the differentiation of monocytes/macrophages into M2 type7.Transcriptome sequencing,GO analysis,KEGG analysis and GSEA analysis showed that PSGL-1 may induce monocytes/macrophages to differentiate into M2type by regulating Wnt signaling pathway.Conclusion:PSGL-1 promotes the infiltration of monocytes/macrophages and neutrophils into pancreatic tissue by mediating the adhesion of monocytes/macrophages and neutrophils to endothelial cells,and may regulate the differentiation of monocyte macrophages into M2 type by activating Wnt signaling pathway. |
