The Study Of The Extracellular Polysaccharide From Antarctic Marine Bacteria Enhancing Immune Activity And Its Molecular Mechanisms In Macrophages

Objective: To study the extracellular polysaccharide from Antarctic marine bacteriaPseudoaltermonas sp.S-5enhancing immune function of RAW264.7cells and its associatedmolecular mechanisms.Method:1. Using MTT assay to detect the cellular viability of RAW264.7cells treated byPEP, RAW264.7cells were treated with different concentrations of PEP for24hours andthen using the MTT assay to detect the cellular viability.2. The study of the PEP enhancing immune activity of RAW264.7cells,RAW264.7cells inthe resting state were treated with the certain concentration of PEP, the change in cellmorphology at different time points (1,3,6,9,12h) was observed by using phase contrastmicroscope. Using neutral red phagocytosis assay to detect PEP influencing phagocyticactivity of RAW264.7cells, RAW264.7cells were treated with different concentrations ofPEP for24hours and then using the neutral red phagocytosis assay to evaluate phagocyticactivity. Using Griess reagent assay to detect PEP influencing NO production in RAW264.7cells, RAW264.7cells were treated with different concentrations of PEP for24hours andthen using the Griess reagent assay to detect the production of NO. Using ELISA assay todetect PEP influencing the secretion of TNF-α in RAW264.7cells, RAW264.7cells weretreated with different concentrations of PEP for24hours and then using the enzyme-linked immunosorbent assay to detect the production of TNF-α.3. Exploring the molecular mechanisms of PEP enhancing immune activity in RAW264.7cells.Immunofluorescence staining was taken to find PEP induce the subunit P65of NF-κB nucleartranslocation occurring. The Western blotting technique was taken to detect IκBa degradationand phosphorylation of P38MAPK.After pretreatment cells with P38and NF-κB inhibitor(SB203580, BAY11-7082) respectively. NO and TNF-α production changes were detected.Result:1.MTT assay shows that2.5-500μg/ml PEP shows no cytotoxicity to RAW264.7cells.2. In negative control group, most of Raw264.7cells are round, a few of them show fusiform,nine hours after PEP treatment, compared with control group, cells in PEP treated groupshows morphological change significantly. They increase in volume and intracellular vesiclesparticles, cells begin to show irregμlar polygons, some of cells extend pseudopods showingtypical active state.Neutral red phagocytosis assay shows that2.5-200μg/mlPEP cansignificantly enhance the phagocytic activity of RAW264.7cells, compared with the controlgroup,200μg/ml PEP can improve phagocytic activity up to140.2%.Griess reagent assayshows that PEP can significantly induce RAW264.7cells to produce NO.2.5-200μg/ml doserange of PEP can stimulate RAW264.7cells to increase production of NO significantly, cellstreated with200μg/ml PEP can induce NO production as well as2μg/ml LPS.compared withthe control group,200μg/ml PEP cells can increase the amount of NO up to270.8%. ELISAassay shows that10-200μg/ml PEP can significantly improve the amount of TNF-α secretedby RAW264.7cells, compared with the control group,200μg/ml PEP cells can increase theamount of TNF-α up to551.3%.3. Immunofluorescence staining technique shows that in the control group, the fluorescence ofP65subunit are distributed loose, while the fluorescence of P65subunit in PEP treated groupare focus on nucleus, this result indicates that PEP can induce nuclear translocation of P65subunit to activate nuclear transcription factor NF-κB. Western blotting shows that PEP cansignificantly promote the degradation of IκBa and phosphorylation of P38MAPK.These results indicate that PEP can activate nuclear transcription factor NF-κB and P38MAPK inRAW264.7cells. When the cells were pretreated with NF-κB and P38inhibitors (BAY11-7082, SB203580), NO and TNF-α production significantly decrease when compared withtreated by PEP alone.These results indicate that PEP can activate RAW264.7cells throughP38MAPK and NF-κB signal transduction pathways.Conclusion: Antarctic marine bacteria Pseudoaltermonas sp.S-5extracellular polysaccharidePEP can activate RAW264.7cells in the resting state to induce cell morphological change,increasing the production of NO and TNF-α, enhancing phagocytic activity and PEP promoteimmune activity of RAW264.7cells through P38MAPK and NF-κB signal transductionpathways.