The Influence Of Exopolysaccharide From Trichoderma Pseudokoningii On The Immune Activity Of DC2.4 Cells And Its Molecular Mechanisms

Objective: To study effect of exopolysaccharide from Trichoderma pseudokoningii on DC2.4 maturity and function from marrow of mice and role in the NF-κB signaling pathway.Methods: 1. Using MTT assay to detect the cellular viability of DC2.4 cells treated by EPS, DC2.4 cells were treated with different concentrations of EPS for 24 hours and then use the MTT assay to detect the cellular viability. 2. DC2.4 cells in the resting state were treated with the certain concentration of EPS, the change in cell morphology at different time points was observed by using phase contrast microscope. 3. Using FACS assay to detect EPS influencing surface marker CD11 c, CD86, CD80, MHC-II expression and phagocytic activity of DC2.4 cells. 4. Using ELISA assay to detect EPS influencing the secretion of IL-12p70. 5. Immunofluorescence staining was taken to find EPS induce the subunit p65 of NF-κB nuclear translocation occurring. 6. The Western blotting technique was taken to detect IκBa degradation and phosphorylation of p38 MAPK. 7. After pretreatment cells with p38 and NF-κB inhibitor(SB 203580, BAY 11-7082) respectively IL-12p70 production change was detected.Results: 1. MTT assay shows that DC2.4 cells treated with 2.5-400 μg/m L EPS has no cytotoxicity. 2. In negative control group, most of DC2.4 cells are semi-adherent,astro-shaped、tadpole shaped or fusiform shaped, ten hours after EPS treatment, compared with control group, cells in EPS treated group shows branches increased、thickening、round、gradually enlarged, polygon-shaped and distinctively astro-shaped. 3. FACS analysis shows, DC2.4 in CD11 c, CD86, CD80, MHC-II expression were 35.31%、43.49% 、 64.21% 、 53.66%, compared with control group was increased significantly.FACS analysis mannose receptor-mediated DC2.4 for FITC-dextran phagocytosis,EPS group was 27.38%, compared with control group, cells in EPS treated group shows phagocytosis decreased significantly. 4. ELISA assay shows that EPS group content of 151 pg /m L and compared with control group, can significantly improve the amount of IL-12 secreted by DC2.4 cells 5. Immunofluorescence staining technique shows that in the control group, the fluorescence of p65 subunit are distributed loose, while the fluorescence of p65 subunit in EPS treated group are focus on nucleus, this result indicates that EPS can induce nuclear translocation of p65 subunit to activate nuclear transcription factor NF-κB. 6. Western blotting shows that EPS can significantly promote the degradation of IκBa and phosphorylation of p38 MAPK.These results indicate that EPS can activate nuclear transcription factor NF-κB and p38 MAPK in DC2.4 cells. 7. When the cells were pretreated with NF-κB and p38 inhibitors( BAY 11-7082, SB203580), IL-12p70 production significantly decrease when compared with treated by EPS alone, the decline were 46.34% and 42.68%,These results indicate that EPS can activate DC2.4 cells through p38 MAPK and NF-κB signal transduction pathways.Conclusion:1. EPS can promote the maturation of DC2.4 in morphology and increase the co-expression of CD-11c、DC80、DC86 and MHC class II molecules on DC2.4 surface. 2. EPS can inhibit the endocytic capacity of DC2.4. 3. EPS can increase secretion of IL-12p70 of DC2.4. 4. EPS promote immune activity of DC2.4cells through p38 MAPK and NF-κB signal transduction pathways.