Effect of Jieduquyuziyin prescription-treated rat serum on MeCP2 gene expression in Jurkat T cells
Effect of Jieduquyuziyin prescription-treated rat serum on MeCP2 gene expression in Jurkat T cells
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DOI：
10.1007/s11626-018-0295-x
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发表时间：
2018-12-01
影响因子：
2.1
通讯作者：
Liu, Wenhong
中科院分区：
文献类型：
--
作者：
Li, Rongqun;Zhuang, Aiwen;Liu, Wenhong
基因组DNA甲基化和甲基cpg结合蛋白2 （MeCP2）基因表达如何影响系统性红斑狼疮（SLE）的发病机制尚不清楚。中药对SLE患者的治疗有独特的效果。本研究旨在探讨解毒祛瘀子饮方（JP）处理大鼠血清对Jurkat T细胞MeCP2基因表达的影响及其在SLE发病机制中的作用。收获Jurkat T细胞，制备含药血清。采用液相色谱-质谱联用技术（LC-MS/MS）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-羧基甲氧基苯基）-2-（4-磺苯基）- 2h -四氮唑（MTS）法筛选含药血清的最佳浓度。使用Methylamp总DNA甲基化试剂盒检测Jurkat T细胞的DNA甲基化水平。利用亚硫酸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。Real-time PCR检测MeCP2 mRNA表达。Western blotting和流式细胞术检测MeCP2蛋白在Jurkat细胞核中的表达。在jp处理大鼠含药血清中检测到芍药苷和阿魏酸。结果表明，高血清含药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细胞基因组DNA甲基化和MeCP2基因启动子区甲基化水平。JP组和醋酸泼尼松组MeCP2 mRNA和蛋白水平均升高。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，这与western blot分析结果一致。我们的研究发现含药血清与细胞存活率呈负相关。JP与醋酸泼尼松一样上调DNA甲基化、MeCP2 mRNA和蛋白水平，可能通过增加甲基化水平激活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，进一步探讨SLE的发病机制提供了基础。
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.