Objectives: Systemic lupus erythematosus (SLE) is a multi-systemic disease with the unknown pathogenic mechanism. DNA demethylation is involved in SLE pathogenesis. Growth arrest and DNA damage inducible 45 alpha (Gadd45a) takes part in the process of DNA demethylation. Gadd45a is a DNA repair-related protein. This study aims to investigate the expressions of some proteins [including activation-induced cytidine deaminase (AID), thymine DNA glycosylase (TDG), and methyl-CpG-binding domain protein 4 (MBD4)] involving in base excision repair (BER) process in CD4+ T cells in patients with SLE, and to analyze the correlations between the above BER proteins and lupus disease.
Methods: From January 2019 to September 2020, 12 SLE patients and 12 healthy controls were recruited from Second Xiangya Hospital of Central South University. Peripheral blood mononuclear cells (PBMCs) were separated by Ficoll-Hypaque density gradient centrifugation and then CD4+ T cells were isolated via positive selection using Miltenyi beads. We measured the messenger RNA (mRNA) and protein expressions of AID, TDG, and MBD4 by real-time reverse transcription polymerase chain reaction (RT-PCR) and Western blotting, respectively.
Results: In contrast to controls, in SLE CD4+ T cells, the mRNA and protein expressions of AID were elevated (P=0.003, P=0.022, respectively); TDG protein expression was increased (P=0.017); and MBD4 protein level was reduced (P<0.001). No visible distinctions was found in the mRNA expressions of either TDG or MBD4 between the 2 groups (both P>0.05). The mRNA and protein expressions of AID and the protein levels of TDG were positively correlated with SLE disease activity index (SLEDAI). And the mRNA and protein expressions of MBD4 were negatively correlated with SLEDAI.
Conclusions: In SLE CD4+ T cells, the increased expressions of AID and TDG and the decreased MBD4 expression may participate in SLE pathogenic mechanism.
目的: 系统性红斑狼疮(systemic lupus erythematosus，SLE)是一种多系统疾病，其发病机制尚不清楚。DNA去甲基化参与SLE的发病机制，生长停滞和DNA损伤诱导型45α(Gadd45a)参与DNA的去甲基化过程。Gadd45a是一种DNA修复相关蛋白，因此，本研究旨在分析参与碱基切除修复(base excision repair，BER)过程的一些蛋白质，包括活化诱导胞嘧啶脱氨基化酶(activation-induced cytidine deaminase，AID)、胸腺嘧啶DNA糖基化酶(thymine DNA glycosylase，TDG)和甲基CpG结合蛋白4(methyl-CpG-binding domain protein 4，MBD4)在SLE患者CD4+ T细胞中的表达，并分析这些BER蛋白与SLE活动之间的相关性。方法: 2019年1月至2020年9月中南大学湘雅二医院收集12例SLE患者和12例健康体检者(对照组)。采用Ficoll-Hypaque密度梯度离心法分离外周血单个核细胞(peripheral blood mononuclear cells，PBMCs)，CD4免疫磁珠分离外周血CD4+ T细胞。通过实时反转录聚合酶链反应(real-time RT-PCR)和蛋白质印迹法检测AID、TDG和MBD4信使RNA(mRNA)和蛋白质的表达水平。结果: 与对照组相比，SLE患者CD4+ T细胞中AID mRNA和蛋白质表达升高(P=0.003，P=0.022)。SLE患者的CD4+ T细胞中TDG蛋白表达升高(P=0.017)，MBD4蛋白质表达降低(P<0.001)。两组之间TDG和MBD4 mRNA表达水平没有明显差异(均P>0.05)。AID mRNA和蛋白水平及TDG蛋白水平与系统性红斑狼疮疾病活动度(SLE disease activity index，SLEDAI)呈正相关，MBD4 mRNA和蛋白水平与SLEDAI呈负相关。结论: SLE患者CD4+ T细胞中AID和TDG表达增加，MBD4表达降低，它们可能参与了SLE的发病机制。.
目的: 系统性红斑狼疮(systemic lupus erythematosus，SLE)是一种多系统疾病，其发病机制尚不清楚。DNA去甲基化参与SLE的发病机制，生长停滞和DNA损伤诱导型45α(Gadd45a)参与DNA的去甲基化过程。Gadd45a是一种DNA修复相关蛋白，因此，本研究旨在分析参与碱基切除修复(base excision repair，BER)过程的一些蛋白质，包括活化诱导胞嘧啶脱氨基化酶(activation-induced cytidine deaminase，AID)、胸腺嘧啶DNA糖基化酶(thymine DNA glycosylase，TDG)和甲基CpG结合蛋白4(methyl-CpG-binding domain protein 4，MBD4)在SLE患者CD4 + T细胞中的表达，并分析这些BER蛋白与SLE活动之间的相关性。
方法: 2019年1月至2020年9月中南大学湘雅二医院收集12例SLE患者和12例健康体检者(对照组)。采用Ficoll-Hypaque密度梯度离心法分离外周血单个核细胞(peripheral blood mononuclear cells，PBMCs)，CD4免疫磁珠分离外周血CD4 + T细胞。通过实时反转录聚合酶链反应(real-time RT-PCR)和蛋白质印迹法检测AID、TDG和MBD4信使RNA(mRNA)和蛋白质的表达水平。
结果: 与对照组相比，SLE患者CD4 + T细胞中 AID mRNA和蛋白质表达升高( P=0.003， P=0.022)。SLE患者的CD4 + T细胞中TDG蛋白表达升高( P=0.017)，MBD4蛋白质表达降低( P<0.001)。两组之间 TDG和 MBD4 mRNA表达水平没有明显差异(均 P>0.05)。 AID mRNA和蛋白水平及TDG蛋白水平与系统性红斑狼疮疾病活动度(SLE disease activity index，SLEDAI)呈正相关， MBD4 mRNA和蛋白水平与SLEDAI呈负相关。
结论: SLE患者CD4 + T细胞中AID和TDG表达增加，MBD4表达降低，它们可能参与了SLE的发病机制。
Keywords: DNA demethylation; base excision repair; systemic lupus erythematosus.