Li Rongqun, Zhuang Aiwen, Ma Jiawei, Ji Lina, Hou Xiaoli, Chen Hongbo, Pan Xiaoping, Liu Wenhong
School of Basic Medical Sciences, Zhejiang Chinese Medical University, Hangzhou, China.
Institute of TCM Literature and Information, Zhejiang Academy of Traditional Chinese Medicine, Hangzhou, China.
In Vitro Cell Dev Biol Anim. 2018 Dec;54(10):692-704. doi: 10.1007/s11626-018-0295-x. Epub 2018 Oct 26.
How genomic DNA methylation and methyl CpG-binding protein 2 (MeCP2) gene expression affect the pathogenesis of systemic lupus erythematosus (SLE) remains poorly understood. Traditional Chinese medicine has a unique effect in the treatment of SLE patients. This study aimed to investigate the effect of Jieduquyuziyin prescription (JP)-treated rat serum on the gene expression of MeCP2 in Jurkat T cells and its role in the pathogenesis of SLE. Jurkat T cells were harvested, and drug-containing serum was prepared. The ferulic acid and paeoniflorin content in the drug-containing serum were determined by liquid chromatography-mass spectrometry (LC-MS/MS). 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assays were used to screen the optimal concentration of drug-containing serum. The DNA methylation level in Jurkat T cells was detected with a Methylamp™ Total DNA Methylation Kit. The methylation status of the MeCP2 promoter region was detected using bisulfite modification and methylation-specific PCR (MSP). Real-time PCR was used to measure MeCP2 mRNA expression. Western blotting and flow cytometry were done to detect MeCP2 protein expression in Jurkat cell nuclei. Paeoniflorin and ferulic acid were detected in the drug-containing serum of JP-treated rats. The results showed that cell growth was affected in the high serum-containing drug group. The experimental results showed that JP and prednisone acetate increased the level of genomic DNA methylation and MeCP2 gene promoter region methylation in Jurkat cells. MeCP2 mRNA and protein levels were also increased in the JP and prednisone acetate groups. Furthermore, flow cytometry revealed that the expression of MeCP2 protein in Jurkat T cell nuclei was higher in the drug group than the blank control group, and these results were consistent with the western blot analysis results. Our study found that there is a negative correlation between drug-containing serum and cell survival rate. JP upregulated the levels of DNA methylation, MeCP2 mRNA and protein as effectively as prednisone acetate and thus may activate the MeCP2 gene by increasing the methylation level, thereby inhibiting the pathogenesis of SLE. Therefore, JP may potentially be used to treat SLE patients. The Jurkat T lymphocyte in vitro experiments provided a foundation to study the effects of JP on the lupus mouse CD4 T cell methylation mechanism and to further explore the pathogenesis of SLE.
基因组DNA甲基化和甲基化CpG结合蛋白2（MeCP2）基因表达如何影响系统性红斑狼疮（SLE）的发病机制仍知之甚少。中医在治疗SLE患者方面具有独特疗效。本研究旨在探讨解毒祛瘀滋阴方（JP）含药血清对Jurkat T细胞中MeCP2基因表达的影响及其在SLE发病机制中的作用。收集Jurkat T细胞并制备含药血清。采用液相色谱-质谱联用（LC-MS/MS）法测定含药血清中阿魏酸和芍药苷的含量。采用3-(4,5-二甲基噻唑-2-基)-5-(3-羧甲氧基苯基)-2-(4-磺基苯基)-2H-四唑（MTS）法筛选含药血清的最佳浓度。用Methylamp™全基因组DNA甲基化试剂盒检测Jurkat T细胞中的DNA甲基化水平。采用亚硫酸氢盐修饰和甲基化特异性PCR（MSP）检测MeCP2启动子区域的甲基化状态。采用实时PCR检测MeCP2 mRNA表达。通过蛋白质印迹法和流式细胞术检测Jurkat细胞核中MeCP2蛋白的表达。在JP治疗大鼠的含药血清中检测到芍药苷和阿魏酸。结果显示，高含药血清组细胞生长受到影响。实验结果表明，JP和醋酸泼尼松可提高Jurkat细胞中基因组DNA甲基化水平和MeCP2基因启动子区域甲基化水平。JP组和醋酸泼尼松组中MeCP2 mRNA和蛋白水平也升高。此外，流式细胞术显示，药物组Jurkat T细胞核中MeCP2蛋白的表达高于空白对照组，这些结果与蛋白质印迹分析结果一致。本研究发现含药血清与细胞存活率呈负相关。JP上调DNA甲基化、MeCP2 mRNA和蛋白水平的效果与醋酸泼尼松相当，因此可能通过提高甲基化水平激活MeCP2基因，从而抑制SLE的发病机制。因此，JP可能有潜力用于治疗SLE患者。Jurkat T淋巴细胞体外实验为研究JP对狼疮小鼠CD4 T细胞甲基化机制的影响及进一步探索SLE的发病机制奠定了基础。
