The Immunomodulatory Effects And Possible Mechanisms Of ASPA80 And ASPA80-1 On Murine Macrophages And Dendritic Cells

Objective: To investigate the immunomodulatory activities and possible mechanisms of ASPA80 and ASPA80-1 in murine RAW264.7 macrophages and dendritic cells in vitro.Methods: 1.Morphological changes were observed by inverted microscope in RAW264.7 macrophages and dendritic cells which were treated with ASPA80 and ASPA80-1.2.The cell viability of dendritic cells was assessed by MTT assay after treated with ASPA80 and ASPA80-1.3.FCM was used to evaluate the expression levels of CD11 c,CD86 and MHC II in RAW264.7 macrophages which were treated with ASPA80 and ASPA80-1.4.FCM was used to evaluate the expression levels of CD86 and MHC II in dendritic cells which were treated with ASPA80 and ASPA80-1.5.Phagocytic assay was used to examine the phagocytosis ability of RAW264.7 macrophages which were treated with ASPA80 and ASPA80-1.6.FCM was used to examine the phagocytosis ability of dendritic cells which were treated with ASPA80 and ASPA80-1.7.Griess assay was used to test the NO production of RAW 264.7 macrophages and dendritic cells which were treated with ASPA80 and ASPA80-1.8.Western blotting assay was applied to analyze the expression levels of TLR2 and TLR4 in dendritic cells which were treated with ASPA80 and ASPA80-1.9.Western blotting assay was applied to analyze the expression levels of proteins relevant to MAPKs signaling pathways in RAW 264.7 macrophages and dendritic cells which were treated with ASPA80 and ASPA80-1.10.Western blotting assay was applied to analyze the expression levels of proteins relevant to NF-κB signaling pathways in RAW 264.7 macrophages and dendritic cells which were treated with ASPA80 and ASPA80-1.Results: 1.ASPA80 and ASPA80-1 induced morphological changes of RAW 264.7 macrophages and dendritic cells with more and longer protrusions.2.ASPA80 and ASPA80-1 had no cytotoxity in dendritic cells with concentration of 31.25-500 μg/mL.3.ASPA80 and ASPA80-1 upregulated the expression levels of CD11 c,CD86 and MHC II in RAW264.7 macrophages.4.ASPA80 and ASPA80-1 upregulated the expression levels of CD86 and MHC II in dendritic cells.5.ASPA80 and ASPA80-1 enhanced the phagocytosis ability of RAW 264.7 macrophages to neutral red and FITC-dextran,but decreased the phagocytosis ability of dendritic cells to FITC-dextran.6.ASPA80 and ASPA80-1 promoted the secretion of nitric oxide in a dose-dependent manner on RAW 264.7 macrophages and dendritic cells.7.ASPA80 and ASPA80-1 upregulated the expression levels of p-JNK,p-ERK,p-P38 and p-NF-κB,and promoted the translocation of the NF-κB to the nuclei of RAW264.7 macrophages.8.ASPA80 and ASPA80-1 upregulated the expression levels of TLR2,p-JNK,p-P38 and p-NF-κB,and promoted the translocation of the NF-κB to the nuclei of endritic cells,but had no effect on TLR4 and p-ERK.Conclusions: ASPA80 and ASPA80-1 enhanced immunomodulatory activity of RAW264.7 macrophages and dendritic cells in vitro.ASPA80 and ASPA80-1 activated RAW264.7 macrophages probably via activation of MAPKs and NF-κB signaling pathways,and ASPA80 and ASPA80-1 boosted the maturation of dendritic cells probably via the activation of TLR2,MAPKs and NF-κB signaling pathways.