Effects of Buzhong Yiqi Decoction(补中益气汤)on VDR Methylation and PI3K-AKT-mTOR Pathway of Dendritic Cells
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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