Hyperglycemia Promotes Polarization Of Macrophages To The M1 Phenotype And Aggravates Acute Pancreatitis Via Notch Signaling Pathway

Part 1:Hyperglycemia promotes polarization of macrophages to the M1 phenotype and aggravates acute pancreatitis.Objective:To observe the effects of hyperglycemia on pancreatic injury,pancreatic macrophage polarization and Notch activity levels in AP mice.Methods:FVB/N mice were randomly divided into three groups,control group(CON group),AP group,and AP with hyperglycemia treatment group;each group was further divided into three subgroups of 1d,3d,and 7d.The AP model was established by the cerulein,and 50%glucose(4 g/kg)was injected intraperitoneally to established the AP with hyperglycemia treatment model after the successful AP model.Blood glucose values were measured in each group of mice at 1d,3d,and 7d.H&E staining was used to assess the injury of the pancreatic tissue;immunohistochemistry(IHC)and immunofluorescence(IF)staining were used to label IL-1β,IL-6 and NF-κB to observe the degree of inflammatory infiltration;TUNEL staining and the protein level of cleaved-caspase 3 were used to assess the level of apoptosis in pancreatic tissue;immunofluorescence staining was used to detect the polarization ratio of pancreatic macrophages;Immunohistochemical staining for Rbpj expression and Western blotting for Rbpj and Hesl expression to verify the expression level of Notch activity;immunofluorescence staining for Hesl,CD68 to detect the Notch pathway expression in pancreatic macrophages.Results:H&E staining showed that compared with the control group,the APld mice had obvious edema,massive necrosis of glandular follicle cells,and significantly increased inflammatory cell infiltration in the mesenchyme;the tissue injury gradually recovered at AP7d;compared with the AP group,the injury was more severe in the AP with hyperglycemia treatment group at all indicate points(p<0.05).Compared with the control group,inflammatory cell infiltration and apoptosis increased significantly at APld and gradually recovered at AP7d,and at the corresponding time points,inflammatory infiltration was significantly more severe in the AP with hyperglycemia treatment group than in the AP group(p<0.05).Compared with the control group,pancreatic macrophage infiltration was significantly increased in the AP as well as AP with hyperglycemia treatment groups,and M1/M2 levels were significantly higher in the AP with hyperglycemia treatment group than in the AP group at the corresponding time points(p<0.05).The pancreatic Notch pathway was activated in the mouse AP model and was significantly higher in the AP with hyperglycemia treatment model(p<0.05).The Notch pathway was activated in pancreatic macrophages during AP,and Notch activity was positively correlated with the proportion of M1-type macrophages.Conclusion:Hyperglycemia exacerbated pancreatic injury and increased inflammatory infiltration in AP mice,prompting pancreatic macrophages to polarize toward M1 phenotype,which was associated with hyperglycemic overactivation of Notch signaling pathway.Part 2:Inhibition of Notch activity reduces M1-type polarization of macrophages after AP and attenuates the level of inflammationObjective:In vitro experiments confirmed that inhibition of Notch activity reduced the polarization of macrophages toward M1 type and attenuated inflammatory injury after AP and high glucose treatment.Methods:Mouse primary acinar cells were isolated and co-cultured with mouse bone marrow-derived macrophages in different glucose concentrations after cerulein stimulation,and the Notch signaling inhibitor,DAPT,was used to intervene in the high glucose group and the control group.The macrophage morphology was observed under microscope and verified by immunofluorescence staining.qPCR and Western blotting were performed to detect the levels of Notch pathway and the level of inflammatory factors;immunofluorescence staining was performed to verify the polarization ratio of macrophages and the expression level of Notch pathway in macrophages.Results:The morphology of macrophages changed into fusiformis after cerulein treatment compared with the control group,and high glucose treatment increased the proportion of fusiformis macrophages,and the proportion of fusiformis macrophages decreased after adding DAPT to inhibit Notch pathway activity;Western blotting and qPCR results showed that cerulein treatment increased Notch activity as well as the expression level of inflammatory factors.High glucose treatment significantly increased Notch activity as well as inflammatory factor expression levels(p<0.05),and DAPT treatment significantly decreased Notch activity as well as inflammatory factor expression levels(p<0.05).Immunofluorescence results showed that cerulein treatment polarized macrophages toward M1 type,and the proportion of M1 type macrophages was significantly increased by high glucose treatment(p<0.05),and DAPT decreased the proportion of M1 type macrophages(p<0.05).The proportion of M1 type macrophages was positively correlated with the expression level of Hesl in macrophages.Conclusion:In in-vitro AP model,high glucose significantly increased Notch activity,prompting macrophages to polarize toward M1 phenotype and increasing inflammatory factor levels;inhibition of Notch activity decreased the proportion of M1 type macrophages and reduced inflammatory factor levels.Part 3 Knockdown of Notchl in the RAW264.7 cell line further validation of the role of Notch pathway in macrophage polarizationObjective:Knockdown of Notchl levels using lentiviral transfection in the mouse macrophage cell line RAW264.7 to further validate the role of the Notch pathway in macrophage polarization.Methods:The mouse macrophage line RAW264.7 was transfected with lentivirus to knock down Notchl activity.Western Blotting as well as qPCR were used to verify the knockdown efficiency.qPCR methods were used to detect inflammatory factor levels of RAW264.7 cell line by stimulating with different concentrations of LPS.The cells were divided into CON group and Notchl knockdown group(shNotchl group),and then each group was divided into three subgroups:LPS absence,LPS group,and LPS with high glucose group.qPCR and Western blotting were used to detect Notch activity and inflammatory factor levels;Western blotting was used to detect iNOS expression levels.Results:Western blotting and qPCR results showed that LPS treatment increased Notch activity as well as inflammatory factor expression levels,and high glucose treatment significantly increased Notch activity as well as inflammatory factor expression levels(p<0.05),while Notchl knockdown significantly decreased Notch activity as well as inflammatory factor expression levels(p<0.05).Western blotting results showed that LPS treatment increased the level of macrophage polarization toward M1 phenotype,and high glucose treatment significantly increased the percentage of M1 phenotype macrophages(p<0.05),while Notchl knockdown significantly inhibited macrophage polarization toward M1 phenotype.Conclusion:In mouse macrophage lines,LPS treatment induced the release of inflammatory factors as well as macrophage polarization toward M1;high glucose treatment exacerbated this phenomenon;knockdown of Notchl signaling significantly inhibited inflammatory injury exacerbated by high glucose and suppressed macrophage polarization toward M1 phenotype.