Objective To study the immune tolerance mechanism of dendritic cells(DCs)by Buzhong Yiqi Decoction(补中益气汤)in vitro.Methods The dendritic cell lines from mice were cultured in vitro and divided into seven groups:blank control group(A group),traditional Chinese medicine group(B group),vitamin D group(C group),traditional Chinese medicine+vita-min D group(group D),traditional Chinese medicine+LY group(group E),Vitamin D+LY group(F group)and traditional Chi-nese medicine+VitD+LY group(G group).The cells were treated with 15％traditional Chinese medicine,10 μmol·L-1 vi-tamin D and 1.5 μmol·L-1 LY294002(PI3K blocker).After being cultured in incubator for 24 h and 48 h,the DNA methyla-tion of vitamin D receptor(VDR)gene of dendritic cells was detected by BSP.The contents of VDR and interleukin-10(IL-10)in cell supernatant were detected by ELISA.The expression levels of cell costimulatory molecules CD40,CD80,CD86 and major histocompatibility complex-Ⅱ(MHC-Ⅱ)were detected by flow cytometry.The mRNA and protein of PI3K-AKT-mTOR pathway were detected by Western Blot and qPCR.Results(1)After the intervention of traditional Chinese medicine and vitamin D,the expression of VDR in DCs increased(P＜0.001 or P＜0.05),and the DNA methylation of VDR gene decreased significantly(P＜0.05).(2)Traditional Chinese medicine and vitamin D can increase the content of IL-10 in the supernatant of DCs(P＜0.05 or P＜0.001),but the secretion of IL-10 by DCs decreases after blocking agent(P＜0.05 or P＜0.001).(3)Traditional Chinese medicine and vitamin D can reduce the expressions of DCs CD40,CD80,CD86 and MHCD-Ⅱ(P＜0.05 or P＜0.001),but the expressions of CD40,CD80,CD86 and MHCD-Ⅱ were increased after blocking agent(P＜0.001).(4)Western Blot and qPCR showed that traditional Chinese medicine and vitamin D can increase the mRNA and pro-tein expression of PI3K-AKT-mTOR pathway in DCs(P＜0.05 or P＜0.001).After the inhibitor,the mRNA and protein lev-el of PI3K-Akt-mTOR pathway decreased,especially the phosphorylated protein decreased significantly(P＜0.001).(5)There was no significant difference between 24 h and 48 h(P＞0.05).Conclusion Buzhong Yiqi Decoction can make DCs main-tain immune tolerance by inhibiting DNA methylation of VDR gene and activating PI3K-AKT-mTOR pathway.
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