Fluorescent Labeling Of Polysaccharide From Fruit Of Physalis Alkekengi L.and Its Binding To TLR2 And TLR4 On Immune Cells

In recent years,the immunomodulation activity of the plant polysaccharides has to come into focus.It is reported that the recognition receptors and activation the associated signaling pathway by polysaccharides can mediate the immune response.The receptors of plant polysaccharide mainly include the complement receptor 3,TLRs,scavenger receptors,Dectin-1 and the mannose receptors.Our research shows the polysaccharide from Fruit of Physalis alkekengi L(PPSB)has a significant immunoenhancement effect in Candida albicans nucleic acid vaccines.The preliminary mechanism shows PPSB can facilitate murine bone marrow derived dendritic cells(BMDC)maturation,activating two signal pathway(MAPKs and NF-?B).However,the effect of targeting is unclear.By the aforementioned work,we study with PPSB-FITC as a probe to verify whether TLR2 and TLR4 of the immune cells(RAW264.7 and DC2.4)are the targets of PPSB,then to lay the foundation for the mechanism of immunologic enhancement by PPSB.The main research results are as follows:1.The synthesis of PPSB-FITCThe PPSB-FITC was obtained by fluorescence labeling of PPSB with tyramine reduction.Cytotoxicity results showed PPSB-FITC had no toxic effect on cells.When DC2.4 cells were incubated with PPSB-FITC and PPSB,the binding rate of PPSB-FITC to cells decreased significantly(P < 0.05).The results showed that FITC did not change the binding sites between PPSB and immune cells.These results suggest that PPSB-FITC can be used as a probe to study the binding sites between PPSB and immune cells.2.The binding of PPSB to TLR2 and TLR4 on immune cellsWe cultured DC2.4 and RAW264.7 with PPSB-FITC,and the binding of PPSB to TLR2 and TLR4 on immune cells was investigated by the following two methods:(1)Competitive binding experiments.PGN is the ligand of TLR2 on immune cells and LPS is the ligand of TLR4 on immune cells.The immune cells(DC2.4 and RAW264.7)were co-incubated with PGN or LPS,and then adding to PPSB-FITC.Then the binding fraction of PPSB-FITC and cells were detected by FCM.(2)Antibody inhibition experiments.The immune cells(DC2.4 and RAW264.7)were incubated with Anti-TLR2 or Anti-TLR4,adding to PPSB-FITC.Then the binding fraction of PPSB-FITC and cells were detected by FCM.(1)The binding of PPSB to TLR2 on immune cellsThe results of competitive binding tests showed that the binding rate of PPSB-FITC to immune cells DC2.4 and RAW264.7 in PGN group was significantly lower than control(P < 0.05),which indicated that PGN and PPSB-FITC were competitived binding immune cells.Antibody inhibition tests showed that the binding rate of PPSB-FITC to DC2.4 and RAW264.7 in Anti-TLR2 group was significantly lower than control group(P < 0.05),indicating the same binding sites were existed in Anti-TLR2 and PPSB.This means TLR2 was the binding site between PPSB and immune cells.These results suggest that PPSB can bind on the surface of immune cells by TLR2.(2)The combination of PPSB and the immune cell surface receptors TLR4The results of competitive binding tests showed that the binding rate of PPSB-FITC to immune cells DC2.4 and RAW264.7 in LPS group was evidently lower than control(P < 0.05),which indicated LPS and PPSB-FITC were competitived binding immune cells.The results of antibody inhibition tests showed that the binding rate of PPSB-FITC to immune cells DC2.4 and RAW264.7 in Anti-TLR4 group was markedly lower than control(P < 0.05).It is proved that TLR4 was the binding site between PPSB and immune cells.These results suggest that PPSB can bind to TLR4 which is on the surface of immune cells.In conclusion,the interaction sites of PPSB are TLR2 and TLR4 from immune cells.