Wei Na, Lu Tan, Gu JianBang, Cai Huan
Department of Neurology, Shanghai Tenth People's Hospital Chongming Branch, 2866 Chongming Road, Shanghai 202157, China.
Department of Orthopaedics, The First Affiliated Hospital of Xinxiang Medical University, 88 Jiankang Road, Weihui, Henan 453100, China.
Brain Res Bull. 2025 Jan;220:111178. doi: 10.1016/j.brainresbull.2024.111178. Epub 2024 Dec 18.
This study aimed to clarify whether the neuroprotective effect of LXA4 is associated with the targeting of neutrophil extracellular traps (NETs) in ischemic stroke (IS).
The MCAO rat model was established to assess cerebral infarction, brain water content and neurological deficits. ELISA was employed to examine the activities of MPO, NE, MMP-9. RT-qPCR and western blot was performed to analyze molecular expressions. A luciferase reporter assay was performed to measure the effect of EGR1 on the METTL3 promoter. The formation of NETs and cell viability were evaluated using immunofluorescence staining and CCK8 assay, respectively.
LXA4 decreased cerebral infarction and brain water content, improved neurological deficits, and reduced the release of NETs-associated indicators (MPO, NE) in MCAO rats. LXA4 reduced NETs formation, MPO and NE levels in vitro. In addition, LXA4 reduced Fe levels while increasing GPX4, SLC7A11 protein expressions, as well as enhancing cell viability in vitro, suggesting the inhibitory effect of LXA4 on ferroptosis. Notably, METTL3 overexpression produced the opposite effects. Furthermore, the effects of METTL3 overexpression on NETs formation and ferroptosis were partially reversed by LXA4 treatment. The inhibition of METTL3 by LXA4 was found to be dependent on FPR2. In vivo experiments verified that LXA4 inhibited NETs formation through inhibition of METTL3 to alleviate brain injury.
This study demonstrates that LXA4 suppresses NETs formation through the FPR2-dependent regulation of METTL3, thereby alleviating brain injury in IS.
本研究旨在阐明脂氧素A4（LXA4）的神经保护作用是否与缺血性中风（IS）中中性粒细胞胞外陷阱（NETs）的靶向作用相关。
建立大脑中动脉闭塞（MCAO）大鼠模型以评估脑梗死、脑含水量和神经功能缺损。采用酶联免疫吸附测定（ELISA）检测髓过氧化物酶（MPO）、中性粒细胞弹性蛋白酶（NE）、基质金属蛋白酶-9（MMP-9）的活性。进行逆转录-定量聚合酶链反应（RT-qPCR）和蛋白质免疫印迹法分析分子表达。进行荧光素酶报告基因检测以测定早期生长反应蛋白1（EGR1）对甲基转移酶样3（METTL3）启动子的影响。分别使用免疫荧光染色和细胞计数试剂盒-8（CCK8）检测评估NETs的形成和细胞活力。
LXA4减少了MCAO大鼠的脑梗死面积和脑含水量，改善了神经功能缺损，并降低了NETs相关指标（MPO、NE）的释放。LXA4在体外减少了NETs的形成、MPO和NE水平。此外，LXA4降低了铁水平，同时增加了谷胱甘肽过氧化物酶4（GPX4）、溶质载体家族7成员11（SLC7A11）蛋白表达，并增强了体外细胞活力，提示LXA4对铁死亡有抑制作用。值得注意的是，METTL3过表达产生了相反的效果。此外，LXA4处理部分逆转了METTL3过表达对NETs形成和铁死亡的影响。发现LXA4对METTL3的抑制作用依赖于甲酰肽受体2（FPR2）。体内实验证实，LXA4通过抑制METTL3来抑制NETs形成，从而减轻脑损伤。
本研究表明，LXA4通过FPR2依赖的METTL3调节抑制NETs形成，从而减轻IS中的脑损伤。
