Effects Of Glycyrrhiza Inflata Polysaccharides On Phenotype And Function Of Dendritic Cells And Their Preliminary Mechanism Study

Objective:Dendritic cells(DC)derived from bone marrow of mice were induced and cultured by adherent method in vitro.The effects of Glycyrrhiza inflata polysaccharides on phenotype maturation and antigen presenting function of DC and their preliminary mechanism of promoting DC immune function based on TLRs/NF-κB signaling pathway were studied.Methods:1)Primary cells were isolated from bone marrow of C57BL/6male mice and induced by 10 ng/mL IL-4 and ng/mL GM-CSF into immature DC.The phenotype of DC was identified by inverted microscopy,scanning electron microscopy and flow cytometry.2)Suspension cells were collected and cultured till the seventh day,and then treated with GiP and its purified component GiP-B1 at different concentrations for 24 and 48 hours respecti,vely.CCK-8 method was used to detect the toxicity of GiP-B1 and GiP to DC.FCM was used to detect the expression of CD11 c,MHC-II,CD80 and CD86.ELISA assay was used to detect the secretion of IL-12,IL-6,IL-1β and TNF-a in DC.WB was used to detect the effect of GiP-B1 and GiP on the migration ability of DC.3)The effects of GiP-B1 and GiP on DCs phenotype and secretion of IL-12,IL-6,IL-1βand TNF-α before and after blocking TLR2/4 monoclonal antibody were detected by FCM and ELISA methods,respectively.The methods of RT-PCR and WB were applied to detect the related genes expression(including MyD88,p65,TLR2 and TLR4)and the related proteins expression(including MyD88,TLR2,TLR4,p65,p-p65,IκB-α and p-IκB-α)in DC,respectively.Results:1)ImDC cultured in vitro formed colonies with semi-adherent growth and smooth surface.The expressions of surface markers CD11 c,MHC-II,CD80 and CD86 were relatively low.The volume of mature DC increased,thereupon their folds increased and the antennaes became longer,but the quantities of colonies decreased and a large number of cells suspended.The expressions of surface markers CD11 c,MHC-II,CD80 and CD86 were increased.2)GiP-B1 and GiP had no effects on the survival rate of DC treated by 24 and 48 hours.Within the experimental concentration range(100~400μg/mL),GiP-B1 and GiP could increase the expressions of DC surface markers,and had significant difference on MHC-II,CD80 and CD86(P<0.05)compared with blank control group.GiP-B1 and GiP could significantly promote the secretion of cytokines such as IL-12p40/p70(P<0.05),and could significantly increase the expression of CCR7 protein(P<0.05)in each group compared with blank control group.3)Compared with blank control group,anti-TLR2/4 could inhibit the expression of MHC-II and CD80,decrease the secretion of IL-6,IL-1β and TNF-α in DC induced by GiP-B1 and GiP(P<0.05).GiP-B1 and GiP with the contents of 100 and 200 μg/mL could up-regulate the mRAN levels of TLR4,TLR2,MyD88 and p65 and the phosphorylation protein level of p65 and IκB-a related to TLRs/NF-κB signaling pathway(P<0.05).Conclusion: GiP and its component GiP-B1 can significantly promote the phenotype and function maturation of DC,improve the ability of antigen presentation,and promote the immune function of DC.Their mechanism may be related to the activation of TLRs/NF-κB signaling pathway.