Lipoxin A4 suppresses neutrophil extracellular traps formation through the FPR2-dependent regulation of METTL3 in ischemic stroke.

Abstract

Background: 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).

Methods: 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.

Results: 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 Fe²⁺ 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.

Conclusion: This study demonstrates that LXA4 suppresses NETs formation through the FPR2-dependent regulation of METTL3, thereby alleviating brain injury in IS.