补中益气汤含药血清对树突状细胞VDR甲基化及PI3K-AKT-mTOR通路的影响

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

目的补中益气汤对树突状细胞(dendritic cells,DCs)免疫耐受机制的体外研究。方法小鼠树突状细胞株于体外进行培养,分为空白对照组(A组)、中药组(B组)、维生素D组(C组)、中药+维生素D组(D组)、中药+LY组(E组)、维生素D+LY组(F组)、中药+VitD+LY组(G组)。以15%含药血清,10μmol·L-1维生素D、1.5μmol·L-1LY294002(PI3K阻滞剂)分别干预7组细胞,于培养箱中分别培养24、48 h后,BSP法检测树突状细胞维生素D受体(vitamin D receptor,VDR)基因DNA甲基化情况;ELISA法检测细胞上清液中VDR、白细胞介素-10(interleukin-10,IL-10)的含量;流式细胞术检测细胞共刺激分子CD40、CD80、CD86、主要组织相容性复合物-Ⅱ(major histocom-patibility complex,MHC-Ⅱ)的表达水平;Western Blot、qPCR检测各组细胞PI3K-AKT-mTOR通路mRNA及蛋白情况。结果(1)中药及维生素D干预后,DCs上VDR的表达增多(P<0.001或P<0.05),VDR基因DNA甲基化情况显著降低(P<0.05)。(2)中药及维生素D能够提高DCs上清液中IL-10含量(P<0.05或P<0.001),阻滞剂作用后,DCs分泌IL-10的功能均降低(P<0.05或P<0.001)。(3)中药及维生素D能够降低DCs表面因子CD40、CD80、CD86及MHCD-Ⅱ的表达(P<0.05或P<0.001),阻滞剂作用后,CD40、CD80、CD86及MHCD-Ⅱ表达均提高(P<0.001)。(4)Western Blot、qPCR结果显示,中药及维生素D能够升高DCs PI3K-AKT-mTOR通路mRNA和蛋白的表达(P<0.05或P<0.001),抑制剂作用后,PI3K-AKT-mTOR通路mRNA和蛋白水平均下降,其中磷酸化蛋白降低显著(P<0.001)。(5)24 h与48 h两个时间点并未见明显差异(P>0.05)。结论补中益气汤通过抑制DCs VDR基因DNA甲基化,激活PI3K-AKT-mTOR通路,使其维持免疫耐受状态。

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+vitamin D group(group D),traditional Chinese medicine+LY group(group E),Vitamin D+LY group(F group)and traditional Chinese medicine+VitD+LY group(G group).The cells were treated with 15%traditional Chinese medicine,10μmol·L^(-1) vitamin D and 1.5μmol·L^(-1) LY294002(PI3K blocker).After being cultured in incubator for 24 h and 48 h,the DNA methylation 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 protein expression of PI3K-AKT-mTOR pathway in DCs(P<0.05 or P<0.001).After the inhibitor,the mRNA and protein level 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 maintain immune tolerance by inhibiting DNA methylation of VDR gene and activating PI3K-AKT-mTOR pathway.