Epilepsy represents a hazardous neurological disorder, underpinned by a pathophysiological process that is yet to be fully understood. Here, we aimed to elucidate the effect of methyl-CpG-binding domain protein 3 (MBD3) on hippocampal neuronal damage in epileptic mice by targeting the phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) pathway. The expression of MBD3 was determined by Western blot in a hippocampal neuronal culture (HNC) epileptic model established using the low Mg₂+ECF culture method. The interaction between MBD3 and DNA methyltransferase 1 (DNMT1) was determined via co-immunoprecipitation and mass spectrometry analysis. Bisulfite modification and sequencing was performed to evaluate the degree of methylation of triggering receptor expressed on myeloid cells 2 (TREM2). The viability and apoptosis of hippocampal neurons were detected by CCK-8 and TUNEL assays, respectively. Finally, the effect of MBD3 was verified in vivo. MBD3 was highly expressed in the HNC model of epilepsy, with its interaction with DNMT1 found to promote the hypermethylation of TREM2 at site cg25748868. Additionally, decreased TREM2 and inhibited PI3K/Akt pathway was observed in the HNC epileptic model. Simultaneous inhibition of MBD3 and DNMT1 decreased the methylation level at cg25748868, up-regulated TREM2 expression, and activated the PI3K/Akt pathway, thereby arresting neuronal damage. Inhibition of MBD3 reduced the level of epileptic seizures, down-regulated cg25748868 methylation, activated TREM2-mediated signaling pathways, and alleviated hippocampal neuronal damage in the acute seizure mouse models. The present study unveiled that MBD3 and DNMT1 synergistically enhanced hypermethylation of cg25748868 in TREM2, and promoted the onset of epilepsy via inhibition of the PI3K/Akt pathway.

癫痫是一种危险的神经系统疾病，其背后的病理生理过程尚未完全了解。在这里，我们旨在通过靶向磷脂酰肌醇 3-激酶/蛋白激酶 B (PI3K/Akt) 通路来阐明甲基 CpG 结合域蛋白 3 (MBD3) 对癫痫小鼠海马神经元损伤的影响。在使用低镁₂+ECF培养方法。MBD3 和 DNA 甲基转移酶 1 (DNMT1) 之间的相互作用是通过免疫共沉淀和质谱分析确定的。进行亚硫酸氢盐修饰和测序以评估在骨髓细胞 2 (TREM2) 上表达的触发受体的甲基化程度。分别通过CCK-8和TUNEL法检测海马神经元的活力和凋亡。最后，在体内验证了MBD3的作用。MBD3 在癫痫的 HNC 模型中高度表达，发现其与 DNMT1 的相互作用促进了 TREM2 在 cg25748868 位点的高甲基化。此外，在 HNC 癫痫模型中观察到 TREM2 减少和 PI3K/Akt 通路抑制。同时抑制 MBD3 和 DNMT1 降低了 cg25748868 的甲基化水平，上调了 TREM2 表达，并激活 PI3K/Akt 通路，从而阻止神经元损伤。MBD3 的抑制降低了癫痫发作的水平，下调了 cg25748868 甲基化，激活了 TREM2 介导的信号通路，并减轻了急性癫痫小鼠模型中的海马神经元损伤。本研究揭示 MBD3 和 DNMT1 协同增强 TREM2 中 cg25748868 的高甲基化，并通过抑制 PI3K/Akt 通路促进癫痫发作。