| Escherichia coli Nissle 1917(EcN)is a Gram-negative probiotic that has been gradually used in clinical treatment because of its significant remission effect on intestinal diseases.EcN can regulate the immune response of host intestinal epithelial cells by secreting extracellular vesicles.Macrophages are important immune cells in the innate immune response,but little is known about the way of the interaction between EcN and macrophages.In view of this,this experiment aims to explore the effects of outer membrane vesicles(OMVs)derived from EcN on the regulation of macrophage biological function and use proteomics technology to further explore the potential immune signal molecules which affect macrophage function,and provide a theoretical basis for the regulation mechanism of EcN in animal intestinal immune modulation.The experiment consists of two parts:(1)The EcN_OMVs were separated and purified by differential centrifugation and gradient density centrifugation.The particle size was analyzed by Laser nanoparticle analyzer and morphological structure was observed by transmission electron microscopy.In addition,the internalization of EcN_OMVs by macrophages was observed by fluorescence confocal microscopy.The number of pathogenic bacteria phagocyted by macrophages was determined by glass bead plate method.Then PBS(control Group),0.1 ?g/mL,1 ?g/mL,10 ?g/mL,and 20 ?g/mL EcN_OMVs stimulated RAW 264.7 cells for 16 h.Detection of TNF-?,IL-1 ?,IL-6,IL-12,IL-10 and NO contents and lactate dehydrogenase(LDH)activity in cell supernatants,and inducible nitric oxide synthase(iNOS)in cell lysates;(2)Analysis of EcN_OMVs protein components by highly sensitive LC–MS/MS.The research results are as follows:(1)EcN_OMVs with lipid bilayer structure were successfully isolated and purified from the culture supernatant of EcN,with a particle size ranging from 50 to 200 nm and a peak of 101.5 nm.(2)EcN_OMVs could be internalized by RAW 264.7 cells.And EcN_OMVs increased the bactericidal ability of RAW 264.7 cells after 5 h;Compared with the control group,the LDH activity of macrophages treated with different concentrations of EcN_OMVs was not significantly different(P>0.05);the secretion of NO by macrophages treated with EcN_OMVs was significantly increased(P<0.05),wherein the 0.1 ?g/mL EcN_OMVs group was significantly lower than that of the other three groups;the iNOS activity of 1 ?g/mL EcN_OMVs group was significantly higher than the control group(P<0.05);Inflammatory cytokines(IL-6,IL-12)were significantly increased in cells treated with EcN_OMVs at different concentrations(P < 0.05);the 1 ?g/mL and 10 ?g/mL EcN_OMVs groups significantly increased the level of IL-1 ?(P<0.05);1 ?g/mL EcN_OMVs group significantly improved the expression of TNF-?(P<0.05);EcN_OMVs group significantly increased the expression of IL-10(P<0.05).(3)The proteomics of EcN_OMVs were analyzed by LC–MS/MS.A total of 408 proteins were identified.Most of these proteins were found in the periplasmic space(40.69%)and cytoplasm(31.37%),followed by the outer membrane(10.78%),inner membrane(9.31%)and extracellular(7.84%).We used GO secondary classification and KEGG pathway analysis and found that these proteins are involved in normal survival of probiotics(envC,FepA,OmpA,carbohydrate process related enzyme proteins,etc.),adhesion(flgA,flgI and fliC,etc.)and immunity response(OmpA,OmpF and flgH)in the intestine;Conclusion: EcN can secrete extracellular vesicles as a signal molecule carrier to directly contact with RAW 264.7 cells,and also trigger pro-inflammatory and anti-inflammatory responses of RAW 264.7 cells,speculating that flagella-related proteins and outer membrane proteins loaded in the extracellular vesicles are related to the immune response. |
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