Yu Manshu, Zhao Junyi, Shan Yun, Dai Huibo, Tang Lei, Sheng Li, Zhang Lu, Sheng Meixiao
Renal Division, Affiliated Hospital of Nanjing University of Chinese Medicine, Nanjing, 210029, China.
Institute of Literature in Chinese Medicine, Nanjing University of Chinese Medicine, Nanjing, 210023, China.
Sci Rep. 2025 May 6;15(1):15786. doi: 10.1038/s41598-025-96709-w.
Peritoneal dialysis (PD) is a successful renal replacement therapy for end-stage renal disease. Continuous infiltration of bioincompatible PD fluid causes mesothelial-mesenchymal transition (MMT) of peritoneal mesothelial cells (PMCs), leading to peritoneal fibrosis (PF). DNA methylation has been characterized as an important regulatory mechanism on multiple fibrosis. However, the mechanisms by which DNA methylation regulates PF are not fully understood resulting in a lack of disease-modifying drugs. Astragalus membranaceus (Astragalus) is naturally phytomedicine that has immunoregulation properties. The study aimed to elucidate the underlying mechanisms of Astragalus in regulating DNA methylation and anti-PF capabilities. In vivo PD rat models were established by inducing with high-glucose PD fluid and Astragalus was intraperitoneal injection. Global DNA methylation sequencing was used to compare the DNA methylation status between control and PF rat models. Methylation profiles and KEGG analysis were identified a possible methylated target gene and its correlation pathway. Through real-time PCR and western blotting, candidate markers and pathways were validated in vivo and in vitro. Chromatin immunoprecipitation and luciferase assays were used to identify the prediction of DNA methyltransferase (Dnmts) binding with methylated target gene. The functions of the validated pathways were further investigated using the knockdown or overexpression strategy. In vivo and in vitro, Astragalus treatment showed a protective effect against PF and Dnmts, characterized by improving pathological manifestation, ameliorating MMT markers, and reducing Dnmt1/3a proteins. Inhibitor of DNA-binding 2 (ID2) was investigated in target gene by integrating the mRNA and methylation profiles involved in PF and Astragalus treatment. PF induced the methylation of ID2 that resulted in recruitment of the Dnmt3a and decreased ID2 expression. The increased ID2 expression in response to Astragalus is a consequence of demethylation in promoter. In addition, phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) signaling pathway correlated with PF, knockdown or overexpression of ID2 regulated this pathway and MMT of PMCs. Astragalus ameliorated PF by targeting Dnmt3a mediated ID2 promoter via PI3K/Akt signaling pathway. The epigenetic regulation of DNA methylation existed the critical role in attenuating PF.
腹膜透析（PD）是一种成功的终末期肾病肾脏替代疗法。生物不相容的腹膜透析液持续浸润会导致腹膜间皮细胞（PMC）发生间皮 - 间充质转化（MMT），进而导致腹膜纤维化（PF）。DNA甲基化已被认为是多种纤维化过程中的重要调节机制。然而，DNA甲基化调节PF的机制尚未完全明确，这导致缺乏改善疾病的药物。黄芪是一种具有免疫调节特性的天然植物药。本研究旨在阐明黄芪调节DNA甲基化及抗PF能力的潜在机制。通过高糖腹膜透析液诱导建立体内腹膜透析大鼠模型，并腹腔注射黄芪。采用全基因组DNA甲基化测序比较对照和PF大鼠模型之间的DNA甲基化状态。通过甲基化谱和KEGG分析确定可能的甲基化靶基因及其相关途径。通过实时PCR和蛋白质印迹法在体内和体外验证候选标志物和途径。采用染色质免疫沉淀和荧光素酶测定法鉴定DNA甲基转移酶（Dnmts）与甲基化靶基因结合的预测情况。使用敲低或过表达策略进一步研究已验证途径的功能。在体内和体外，黄芪治疗对PF和Dnmts均显示出保护作用，其特征为改善病理表现、改善MMT标志物以及降低Dnmt1/3a蛋白水平。通过整合PF和黄芪治疗相关的mRNA和甲基化谱，对靶基因中的DNA结合抑制因子2（ID2）进行了研究。PF诱导ID2甲基化，导致Dnmt3a募集并降低ID2表达。黄芪作用后ID2表达增加是启动子去甲基化的结果。此外，磷脂酰肌醇3激酶（PI3K）/蛋白激酶B（Akt）信号通路与PF相关，ID2的敲低或过表达调节该通路以及PMC的MMT。黄芪通过PI3K/Akt信号通路靶向Dnmt3a介导的ID2启动子来改善PF。DNA甲基化的表观遗传调控在减轻PF中起关键作用。
