Li Ming, Pang Lijun, Li Yunfei, Chen Junjie, Shang Shuangshuang, Huang Chuanbing
Department of Rheumatology, The First Affiliated Hospital of Anhui University of Chinese Medicine, Hefei, Anhui, China.
Center for Xin'an Medicine and Modernization of Traditional Chinese Medicine of Institute of Herbal Medicine (IHM), Hefei, Anhui, China.
Front Immunol. 2025 Aug 21;16:1631631. doi: 10.3389/fimmu.2025.1631631. eCollection 2025.
This study aimed to investigate whether Jianpi-Zishen Formula (JPZS) modulates the Treg/Th17 balance in MRL/lpr mice through regulation of DNA methyltransferase 1 (DNMT1)-mediated forkhead box P3 (Foxp3) methylation, and to elucidate its potential mechanism for improving immune homeostasis in systemic lupus erythematosus (SLE).
Forty-eight female MRL/lpr mice were randomized into six groups (n=8/group): JPZS (low/medium/high doses), 5-aza-CdR (DNMT inhibitor), DC_517 (DNMT1 inhibitor), and model control. Eight C57BL/6 mice served as healthy controls. The mice were subjected to the corresponding intervention measures for eight weeks. The impact of JPZS on the disease progression of MRL/lpr mice was evaluated using enzyme-linked immunosorbent assay (ELISA) and serum biochemical parameters. Moreover, immunofluorescence staining and flow cytometry were employed to investigate alterations in the proportions of Tregs and Th17 cells. CD4 T cells were isolated from the spleen for subsequent investigation, including quantitative real-time PCR, western blotting, and determination of DNA methylation levels. Furthermore, the enzymatic activity of CD4 T cell-specific DNA methyltransferases was quantified using an EpiQuik DNMT detection kit.
JPZS significantly improved the disease development of MRL/lpr mice in a dose-dependent manner. Flow cytometry and immunofluorescence indicated JPZS promoted Treg/Th17 rebalancing. Research has found that is at a high methylation level in CD4 T cells of the model group, and the transcription level of mRNA is downregulated; JPZS can downregulate methylation levels of CD4 T cells in the model group. Further research has found that the level of methylation is closely related to Dnmt1 enzyme activity, and JPZS can downregulate Dnmt1 enzyme activity, thereby upregulating the transcription level of mRNA.
JPZS may restore Treg/Th17 balance in SLE via DNMT1-regulated demethylation, suggesting an epigenetic mechanism for its immunomodulatory effects.
本研究旨在探讨健脾滋肾方（JPZS）是否通过调节DNA甲基转移酶1（DNMT1）介导的叉头框蛋白P3（Foxp3）甲基化来调节MRL/lpr小鼠的Treg/Th17平衡，并阐明其改善系统性红斑狼疮（SLE）免疫稳态的潜在机制。
将48只雌性MRL/lpr小鼠随机分为6组（每组n = 8）：JPZS（低/中/高剂量）组、5-氮杂胞苷（DNMT抑制剂）组、DC_517（DNMT1抑制剂）组和模型对照组。8只C57BL/6小鼠作为健康对照组。对小鼠进行相应干预措施8周时间。采用酶联免疫吸附测定（ELISA）和血清生化参数评估JPZS对MRL/lpr小鼠疾病进展的影响。此外，采用免疫荧光染色和流式细胞术研究Tregs和Th17细胞比例的变化。从脾脏中分离CD4 T细胞用于后续研究，包括定量实时聚合酶链反应、蛋白质印迹法以及DNA甲基化水平测定。此外，使用EpiQuik DNMT检测试剂盒定量CD4 T细胞特异性DNA甲基转移酶的酶活性。
JPZS以剂量依赖方式显著改善MRL/lpr小鼠的疾病发展。流式细胞术和免疫荧光表明JPZS促进Treg/Th17重新平衡。研究发现模型组CD4 T细胞中[此处原文缺失相关基因名称]处于高甲基化水平，且[此处原文缺失相关基因名称]mRNA的转录水平下调；JPZS可下调模型组CD4 T细胞的[此处原文缺失相关基因名称]甲基化水平。进一步研究发现[此处原文缺失相关基因名称]甲基化水平与Dnmt1酶活性密切相关，且JPZS可下调Dnmt1酶活性，从而上调[此处原文缺失相关基因名称]mRNA的转录水平。
JPZS可能通过DNMT1调节的[此处原文缺失相关基因名称]去甲基化作用恢复SLE中的Treg/Th17平衡，提示其免疫调节作用的一种表观遗传机制。
