Study On The Mechanism Of Galectin-3 Affecting Macrophage Phagocytosis In Sepsis Mice

Objective:Galectin-3 gene knockout homozygous mice were screened by breeding and genotype identification of galectin-3 gene knockout heterozygous mice.To explore the mechanism of galectin-3 affecting macrophage phagocytosis in sepsis mice,we established sepsis mice models and compared the bacterial clearance ability,phagocyte acidification,cytokine expression and actin F/G ratio of macrophages in sepsis wild-type mice and sepsis galectin-3 gene knockout homozygotes.Method:1.Homozygous mice with galectin-3 gene knockout were screened by breeding and genotype identification of galectin-3 gene knockout heterozygotes.2.The model of bacterial sepsis was established by intraperitoneal injection of heat inactivation of Gram-negative Salmonella typhimurium.Peritoneal macrophages of mice were isolated and cultured.3.The levels of macrophage cytokines(IL-6,IL-10,TNF-α)in abdominal cavity of mice were determined by ELISA.4.Immunofluorescence labeling method was used to compare the pH of peritoneal macrophage phage and the fusion degree of peritoneal macrophage phage/lysosome between the two groups.5.Tissue homogenates prepared from peripheral blood and liver of mice were inoculated on a plate.The bacterial clearance rate of the two groups was evaluated by counting the cell colony units.6.F-actin and G-actin were separated by G-Actin/F-actin In Vivo Assay Biochem kit,and then precipitated(F-actin)and supernatant(G-actin)were detected by immunoblotting.The F/G ratio of macrophages in two groups was compared and analyzed to evaluate the aggregation of actin in two groups.Result:1.The expression of IL-6 and IL-10 in peritoneal macrophages of galectin-3 knockout mice was significantly lower than that of wild type mice(P<0.05).2.Phagocyte PH of galectin-3 knockout mice was significantly higher than that of wild type mice(P<0.05).3.Macrophage phages of wild-type mice were easily transported to lysosomes,while macrophage phages of galectin-3 knockout mice were not easily transported to lysosomes,the difference was statistically significant(P<0.05).4.With the prolongation of infection time,the number of bacterial colonies in peripheral blood of galectin-3 knockout mice was significantly higher than that of wild mice(P<0.05);the number of bacteria in liver of wild mice infected for 48 hours was lower than that infected for 12 hours(P<0.05),while the number of bacterial colonies in liver of galectin-3 knockout mice infected for 48 hours was significantly higher than that of wild mice infected for 12 hours(P<0.05).5.The F/G ratio of actin in wild type mice with the same infection time was higher than that in galectin-3 knockout mice(P<0.05).In addition,with the prolongation of infection time,the F/G ratio of macrophages in septic wild-type mice showed an increasing trend,while the F/G ratio of galectin-3 gene knockout mice showed no significant change.Conclusion:1.Galectin-3 participates in promoting the release of cytokines IL-6 and IL-10 from mouse peritoneal macrophages.2.Galectin-3 is involved in promoting the maturation of peritoneal macrophages in mice.3.Galectin-3 participates in promoting phagosome/lysosome fusion of mouse peritoneal macrophages.4.Galectin-3 participates in promoting the removal of bacteria by peritoneal macrophages in mice.5.Galectin-3 may promote macrophage phagocytosis by increasing the F/G ratio of actin.