Lycium Barbarum Polysaccharides Promote Maturity Of Murine Dendritic Cells Through TLR4-NF?B/MAPK-Blimp1 Pathway

Lycium barbarum polysaccharides?LBP?is the main active ingredient of Chinese wolfberry.Our previous research found that LBP can promote the maturity of dendritic cell?DC?,showing a good adjuvant effect.However,the specific mechanism for promoting DC maturity is not clear.In this study we use TLR4 as the entry point to explore its molecular mechanism for promoting DC maturity.Objective:By studying the molecular mechanism by which LBP promotes DC maturation through TLR4,this will lay the foundation for the development of LBP as a new adjuvant.Methods:1.Observing the molecular mechanism of LBP promoting DC maturation and the effect of DC subtype-related transcription factors in vitroAseptically isolate mouse bone marrow-derived dendritic cells,Granulocyte-mononuclear colony-stimulating factor?GM-CSF?and recombinant murine interleukin-4?IL-4?were cultured for 7 days at this time as immature DC,then research as follows:1.1 Immature DC was stimulated with different concentrations of LBP,and 100ng/mL LPS was used as a positive control:After 24 hours of intervention,the expressions of cell surface molecules CD11c,MHCII,CD80,and CD86 were detected by Flow Cytometry?FCM?;Enzyme-linked immunosorbent assay?ELISA?was used to detect the levels of IL-6 and IL-4 in the cell culture supernatant.After 30minutes of intervention,the expression of mRNA and protein of NF?B,p38,Erk,JNK,and Blimp1 in the TLR4 pathway in DC was detected by Taqman-PCR and Western Blot?WB?respectively.At the same time,Taqman-PCR detected the expression levels of transcription factors Irf4,Irf5,Irf8 and Batf3 and WB detected the expression levels of transcription factors IRF4,IRF5,IRF8.1.2 Immature DC was pretreated with blocker of,TLR4,NF?B,Erk,JNK,p38 siRNA-Prdm1.These cells were stimulated with LBP for 30 min,and Taqman-PCR and WB were used to detect the expression levels of mRNA and protein of related molecules in the pathway.1.3 Immature DC was pretreated with ovalbumin?OVA?for 2 h and then stimulated with LBP for 24 h.After co-culture with CD4⁺T lymphocytes for 72 h,FCM detected the initial CD4⁺T cells to differentiate into Th1,Th2,Treg,Tfh cell ratio.2.Studying the effects of LBP on TLR4 pathway related molecules in DC and its subtype-related transcription factors in vivoC57BL/6 mice were intraperitoneally injected with different concentrations of LBP,and 1 mg·kg^-1·d LPS was intraperitoneally injected as a positive control.Seven days later,the expression levels of TLR4,NF?B,p-NF?B,p38,p-p38,Erk1/2,p-Erk1/2,JNK,p-JNK,Blimp1,IRF4,IRF5,IRF8,of spleen CD11c⁺DC was detected by WB.Results1.Observing the molecular mechanism of LBP promoting DC maturation in vitro1.1 LBP can promote the expression of CD11c,MHCII and co-stimulatory molecules CD80 and CD86 on the surface of DC and promote the secretion of IL-6 and IL-4 by DC.It also enhance the mRNA and phosphate protein expression level of TLR4,NF?B,p38,Erk1,Erk2,JNK,Blimp1,IRF4,IRF5,IRF8,Batf3 in DC.1.2 By blocking the key molecules TLR4,NF?B,p38,Erk,JNK and Blimp-1 in the TLR4 pathway,it can effectively interfere DC maturity which promote by LBP.1.3 DC treated with LBP significantly induced the differentiation of the naive CD4⁺T cells into Tfh cells,and increased the contents of IL-6,IL-4,and TNF in the cell culture supernatant.2.Studying the effect of LBP on TLR4 pathway related molecules in DC in vivoIn vivo experiments in mice showed that LBP enhanced the protein expression of TLR4,p-NF?B,p-p38,p-Erk1/2,Blimp1,IRF4 and IRF8 in CD11c⁺DC of mouse spleen.Conclusion:LBP can promote DC maturation through the TLR4-NF?B/Erk1/2-Blimp1 signaling pathway and induce the differentiation of cDC1s and cDC2s.LBP can promote the differentiation of DC-induced Tfh cells..