Methyltransferase-like 3 silenced inhibited the ferroptosis development via regulating the glutathione peroxidase 4 levels in the intracerebral hemorrhage progression

This study examined the effects of methyltransferase-like 3 (METTL3) on ferroptosis during intracerebral hemorrhage (ICH) progression. The brain microvascular endothelial cells (BMVECs) were stimulated with oxygen and glucose deprivation (OGD) and hemin to establish an ICH model. Cell viability was tested using a CCK8 assay. The levels of Fe²⁺, glutathione, reactive oxygen species, LPO, and MDA were determined using the corresponding commercial kits. Cell death was analyzed using TUNEL and propidium iodide staining. The correlation between METTL3 and glutathione peroxidase 4 (GPX4) was analyzed using Spearman’s correlation test and further confirmed using the CHIP assay. Western blotting and RT-qPCR were performed to measure the relative expression levels. Mice were injected with 0.2 units collagenase IV to establish an ICH model <i>in vivo</i>. We found that the Fe²⁺, reactive oxygen species, LPO, and MDA levels were enhanced, and glutathione was depleted in OGD/H-treated BMVECs as well as in ICH mice. Additionally, cell viability and SLC7A11 protein levels decreased, and cell death and TFR1 protein levels increased in OGD/H-treated BMVECs. METTL3 silencing relieves OGD/H-induced injury in BMVECs. In addition, METTL3 was significantly negatively related to GPX4, which was further confirmed by the CHIP assay. Silencing of METTL3 decreased the N6-methyladenosine levels of GPX4 and increased its mRNA levels of GPX4. GPX4 knockdown neutralized the role of METTL3 in OGD/H-treated BMVECs. These results implied that ferroptosis occurred in the ODG/H-treated BMVECs and ICH mouse models. METTL3 silencing effectively suppressed ferroptosis by regulating N6-methyladenosine and mRNA levels of GPX4.

本研究探讨了甲基转移酶样3（methyltransferase-like 3, METTL3）在脑出血（intracerebral hemorrhage, ICH）进程中对铁死亡（ferroptosis）的调控作用。采用氧糖剥夺（oxygen and glucose deprivation, OGD）与氯化血红素（hemin）刺激脑微血管内皮细胞（brain microvascular endothelial cells, BMVECs），以构建ICH体外模型。通过CCK-8法检测细胞活力；使用商品化试剂盒检测Fe²+、谷胱甘肽、活性氧、脂质过氧化物（LPO）及丙二醛（MDA）的水平；采用TUNEL染色与碘化丙啶（propidium iodide, PI）染色分析细胞死亡情况。通过Spearman相关分析探究METTL3与谷胱甘肽过氧化物酶4（glutathione peroxidase 4, GPX4）的相关性，并通过染色质免疫沉淀实验（CHIP assay）进一步验证。采用蛋白质印迹（Western blotting）与实时定量聚合酶链反应（RT-qPCR）检测相关分子的相对表达水平。向小鼠颅内注射0.2单位IV型胶原酶（collagenase IV）以构建ICH体内模型。

研究结果显示：经OGD/H处理的BMVECs及ICH模型小鼠体内，Fe²+、活性氧、LPO及MDA水平均显著升高，谷胱甘肽含量耗竭；同时，经OGD/H处理的BMVECs中细胞活力与SLC7A11蛋白水平下调，细胞死亡及转铁蛋白受体1（TFR1）蛋白水平上调。沉默METTL3可缓解OGD/H诱导的BMVECs损伤。此外，METTL3与GPX4呈显著负相关，该结果经CHIP实验进一步验证。沉默METTL3可降低GPX4的N6-甲基腺苷（N6-methyladenosine, m6A）修饰水平，并上调其mRNA表达。敲低GPX4可抵消METTL3对OGD/H处理的BMVECs的保护作用。上述结果表明，OGD/H处理的BMVECs及ICH小鼠模型中均发生了铁死亡；沉默METTL3可通过调控GPX4的N6-甲基腺苷修饰及mRNA表达水平，有效抑制铁死亡。