| Objective: This study aims to explore the effect of FADS1 on macrophages phagocytosis in Escherichia coli bacterial infection,further explore the mechanism of its regulation of lipid metabolism and inflammatory signaling pathway,so as to provide new metabolic ideas for the pathophysiological process of macrophages response to bacterial infection,and provide potential therapeutic targets for the prevention,diagnosis and treatment of bacterial infection.Method:(1)Construct FADS1 knockdown cell models on cell lines and mouse bone marrow-derived macrophages by lentiviral transfection,transient transfection,and verify the effect by q RT-PCR,Western blot,and liquid chromatography-mass spectrometry.Immunofluorescence assay,agar plate culture,and q RT-PCR are used to detect the phagocytic killing effect and inflammatory factors expression of macrophages on Escherichia coli in vitro.(2)The levels of total cholesterol,free cholesterol,and cholesterol esters in macrophages are measured using a cholesterol kit.The levels of SREBP2 system protein in macrophages are detected by western blotting,including the precursor,mature,and nuclear-translocated parts of SREBP2(p SREBP2,m SREBP2,and n SREBP2),as well as SCAP and LDLR.The effect of FADS1 on macrophage uptake of LDL is validated by immunofluorescence assays.(3)To explore the relationship between FADS1-mediated cholesterol metabolism and phagocytosis,methyl-β-cyclodextrin and combination of methyl-β-cyclodextrin with free cholesterol are used to clear/overload cholesterol content in macrophage.The protein levels of m TOR pathway,including m TOR,phosphorylated m TOR,S6 K,phosphorylated S6 K,4EBP1,and phosphorylated 4EBP1,are detected by western blotting.Detect the changes of these protein levels after treatment of cholesterol clearance/overload to explore the relationship between FADS1-mediated changes in cholesterol metabolism and m TOR pathway.Rapamycin is used to inhibit m TOR activity to further verify its regulation of macrophage phagocytosis.(4)Conduct oral gavage of FADS1 inhibitor in mice and construct mice subcutaneous Escherichia coli infection model.Animal fluorescence imager,agar plate culture,and enzyme linked immunosorbent assay are used to detect the fluorescence signals of subcutaneous bacteria,residual bacterial quantity,chemokine secretion and peripheral monocyte in mice.Result:(1)Compared with the control group,FADS1 knockdown can promote the phagocytosis and killing ability of macrophages against Escherichia coli in vitro,and promote the up-regulation of IL-1β,IL-6and TNF-α expression levels of inflammatory factors in macrophages.(2)Compared with the control group,FADS1 knockdown can increase the contents of total cholesterol and free cholesterol in macrophages,up-regulate the expression levels of m SREBP2,n SREBP2,SCAP and LDLR proteins,and enhance the uptake of low density lipoprotein in macrophages.(3)Phagocytosis was significantly inhibited by the depletion of intracellular free cholesterol,while the effect was reversed by the overload of cholesterol.Compared with the control group,FADS1 knockdown activated the m TOR pathway and up-regulated the expression levels of phosphorylated m TOR,phosphorylated S6 K and phosphorylated4EBP1 proteins.Clearance of intracellular free cholesterol significantly reduced the expression of phosphorylated S6 K and phosphorylated4EBP1 proteins,while overload cholesterol reversed the effect.The intervention of rapamycin in macrophages inhibited the phagocytosis mediated by FADS1.(4)Inhibition of FADS1 function in mice promoted the clearance of subcutaneous Escherichia coli lesions,increased the secretion of chemokine MCP-1 and the number of peripheral blood monocytes.Conclusion:(1)Knockdown of FADS1 can promote macrophage phagocytic clearance of Escherichia coli and inflammatory responses.(2)Knockdown of FADS1 can increase trhe intracellular cholesterol levels by activating the SREBP2 system,thereby activating the m TOR pathway and mediating the phagocytosis.(3)Inhibition of FADS1 function in mice can promote the clearance of subcutaneous Escherichia coli lesions and increased the chemotaxis of monocytes.Figures:22;tables:16;reference:131... |
PDF Full Text Request