| Flammulina velutipes is a common edible fungus which possess numerous biological activities,such as anti-oxidation,anti-tumor and immunoregulation.Polysaccharide is one of the most important substances of Flammulina velutipes.The immunoregulatory activity of Flammulina velutipes polysaccharide(FVP)has been reported,however,there are relatively few studies on the receptors of Flammulina velutipes polysaccharide on macrophages.Therefore,the purpose of this study is to treat mouse mononuclear macrophage RAW264.7 with FVP to explore the receptors of FVP on RAW264.7 cells and the mechanisms of FVP as immunomodulator,hoping to provide a certain experimental basis and theoretical basis for the study of immunoregulatory activity of FVP.Methods: The effect of FVP on the activity of RAW264.7 cells were detected by MTT assay.The effect of FVP on the phagocytic activity of RAW264.7 cells was detected by neutral red method.The change of cell morphology after FVP treatment was observed under the inverted microscope.Griess method was used to detect the secretion of NO.The levels of cytokines(TNF-? and IL-1?)in RAW264.7 cells supernatant was detected by ELISA.The FVP receptors on RAW264.7 cells were detected by fluorescence microscope and flow cytometry.The immune-related factors iNOS,TNF-? and IL-1? mRNA and TLR4 and MR mRNA expression levels were detected by qPCR.Western blot was used to detect the expression levels of ?-Actin,TLR4,MR,ERK1/2,p-ERK1/2,p38,p-p38,JNK,p-JNK,IkB?,p-IkB? and p65.Immunofluorescence assay was used for detection of p65 nuclear translocation.Results are as follows:1.12.5,25,50 and 100 ?g/mL FVP had no cytotoxicity on RAW264.7 cells,and 12.5 ?g/mL FVP could significantly promote RAW264.7 cell proliferation.Thus we chose the doses of 12.5-100 ?g/mL of FVP for subsequent experiments.2.Neutral red experiment results showed that 12.5 ?g/mL FVP could enhance the phagocytic activity of RAW264.7 cells;the morphology of RAW264.7 cells changed from resting to activated mode after FVP treatment;FVP promoted the secretion of NO and IL-1? in RAW264.7 cells and the expression of iNOS and IL-1? mRNA in a concentration-dependent manner.The secretion of TNF-? and TNF-? mRNA expression increased first and then decreased,reaching the highest value at 50 ?g/mL.3.FVP could bind to RAW264.7 cells.TLR4 and MR receptor inhibitors could not only inhibit the binding of the FVP and cells,but also inhibit FVP-induced NO,TNF-? and IL-1? secretion in RAW264.7 cells.In addition,high concentrations of FVP could significantly up-regulate the expression of TLR4 mRNA and protein,and all doses of FVP could significantly up-regulate MR protein expression,while the expression of MR mRNA also increased.4.FVP could activate MAPK signaling pathway in RAW264.7 cells,and the expression of p-ERK1/2 and p-p38 proteins reached the highest at 15 min after FVP treatment,and the increase of p-JNK protein expression lasted up to 30 min;Phosphorylation of ERK1/2 and JNK proteins increased dose-dependently after FVP treament,while the phosphorylation level of p38 increased first and then decreased,reaching the highest value at 50 ?g/mL;MAPK inhibitor could significantly inhibit the secretion of NO,TNF-? and IL-1? in RAW264.7 cells induced by FVP,among which JNK inhibitor(SP600125)had the best inhibitory effect.5.FVP stimulated phosphorylation of IkB? protein in RAW264.7 cells,and NF-?B signaling pathway was activated.Western blot and immunofluorescence experiments showed that FVP promoted p65 nuclear translocation;NF-kB inhibitor had a significant inhibitory effect on FVP-induced immune response in RAW264.7 cells.Conclusion: FVP activates RAW264.7 cells to generate an immune response through TLR4,MR/MAPK/NF-kB signaling pathways.This study not only reveals the FVP receptors on macrophages and the mechanisms FVP exerting immunoregulatory effect,but also provides experimental basis and theoretical basis for the study of immunoregulatory activity of Flammulina velutipes polysaccharides. |
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