双氢青蒿素对MRL/lpr SLE小鼠CD4+T细胞基因组DNA甲基化水平的影响研究

摘要

目的探讨双氢青蒿素对MRL/lpr系统性红斑狼疮(SLE)小鼠CD4+T细胞基因组DNA甲基化水平的影响.方法从MRL/lpr SLE小鼠脾脏获取CD4+ T细胞,进行体外细胞双氢青蒿素干预培养.通过CCK-8实验检测双氢青蒿素的细胞毒性, 高效液相色谱技术检测CD4+ T细胞基因组DNA甲基化水平,RT-PCR和Western blot检测CD4+ T细胞DNA甲基化转移酶1(DNMT1)、生长阻滞和DNA损伤基因a(Gadd45a)mRNA和蛋白表达水平.结果双氢青蒿素对SLE小鼠CD4+ T的半抑制浓度(IC50)为62μmol/L.双氢青蒿素可抑制CD4+ T细胞增殖,且随着浓度的增加,对CD4+ T细胞增殖的抑制作用增强.双氢青蒿素干预后,CD4+T细胞基因组DNA甲基化水平显著升高;且双氢青蒿素浓度越高,基因组DNA甲基化水平越高.双氢青蒿素干预培养后,SLE小鼠CD4+ T细胞DNMT1 mRNA和蛋白水平均明显上调,而Gadd45a mRNA和蛋白水平均明显下调,其作用与浓度有关.结论双氢青蒿素可能通过DNA甲基化调控机制发挥治疗SLE的作用.%Objective To investigate the effect of dihydroatermisinin on genome DNA hypermethylation in CD4+ T cells of MRL/lpr SLE mice in vitro. Methods CD4+ T cells were isolated from spleen of MRL/lpr SLE mice and were cultured with dihydroartemisinin. Cell toxicity of dihydroartemisinin was tested by CCK-8 kits. Global DNA methylation in CD4+ T cells was examined with high-performance liquid chromatography (HPLC) analysis. The expression of DNMT1 and Gadd45a mRNA and protein were detected with RT-PCR and Western-blot, respectively. Results The IC50 of dihydroartemisin on CD4+ T cells of MRL/lpr mice was 62μmol/L. The proliferation of CD4+ T cells was inhibited by dihydroartemisinin in a concentration-dependent manner. Global DNA methylation in CD4+ T cells was increased after dihydroartemisinin treatment in a concentration-dependent manner. The expression of DNMT1 mRNA and protein was increased, while the expression of Gadd45a was decreased after dihydroartemisinin treatment, the effects were related to concentration of dihydroartemisinin. Conclusion Dihydroartemisinin can increase the genome DNA methylation in CD4+ T cells and inhibit CD4+ T cell proliferation in MRL/lpr SLE mice, indicating that it may be used to treat systemic lupus erythematosus.