Promotion of Treg/Th17 Balance in MRL/lpr Mice by Jianpi-Zishen Formula Via Modulation of DNMT1-Mediated Foxp3 Methylation
A total of 48 female MRL/lpr mice were included in this study and divided into six groups: JPZS group (Low, medium, and high doses), 5-aza-CdR group, and DC_517 group, with eight mice in each group. Eight female C57BL/6 mice were used as 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.The results indicate that JPZS significantly improved the disease development of MRL/lpr mice in a dose-dependent manner. Immunofluorescence staining and flow cytometry suggest that JPZS can increase the ratio of Treg/Th17. Research has found that Foxp3 is at a high methylation level in CD4+ T cells of the model group, and the transcription level of Foxp3 mRNA is downregulated; JPZS can downregulate Foxp3 methylation levels of CD4+ T cells in the model group. Further research has found that the level of Foxp3 methylation is closely related to Dnmt1 enzyme activity, and JPZS can downregulate Dnmt1 enzyme activity, thereby upregulating the transcription level of Foxp3 mRNA. Overall design: This study included a total of 48 female MRL/lpr mice, divided into six groups: JPZS group (low, medium, and high doses), 5-aza-CdR group, and DC_517 group, with 8 mice in each group. Use 8 female C57BL/6 mice as controls. The mice received corresponding intervention measures for eight weeks. CD4+T cells were isolated from the spleen and the DNA methylation level of Foxp3 gene was detected using MethylTarget sequencing technology (sulfite treatment combined with high-throughput sequencing).