NSUN2-mediated m⁵C methylation of HSPB1 mRNA suppresses inflammation and ferroptosis in ischemic stroke via activating the NRF2/HO-1/NQO-1 pathway

Abstract

Ischemic stroke (IS), a leading cause of disability and mortality, is characterized by cerebral ischemia-reperfusion injury, inflammation, and ferroptosis. RNA 5-methylcytosine (m5C) modification is a dynamic epigenetic mark involved in various pathological processes, yet its role in IS remains unclear. This study aimed to investigate the role of m5C modification in IS and its underlying mechanisms. In vitro, human brain microvascular endothelial cells (HBMECs) were subjected to oxygen-glucose deprivation/reoxygenation (OGD/R), while in vivo, a transient middle cerebral artery occlusion (tMCAO) mouse model was established. Reverse transcription-quantitative polymerase chain reaction was used to analyze the mRNA levels of NSUN2 and heat shock protein family B member 1 (HSPB1). The contents of pro-inflammatory cytokines and ferroptosis-related indicators were measured using enzyme-linked immunosorbent assay and commercial kits. The expression of nuclear factor erythroid 2-related factor 2 (NRF2)/heme oxygenase-1 (HO-1)/NAD(P)H quinone dehydrogenase 1 (NQO-1) pathway proteins was detected by Western blot. RNA immunoprecipitation and dual-luciferase reporter assays were performed to assess the interaction between NSUN2 and HSPB1. Results showed that NSUN2 was downregulated in OGD/R-treated HBMECs and tMCAO mice. Furthermore, NSUN2 overexpression mitigated OGD/R-induced inflammation and ferroptosis. Mechanistically, NSUN2 mediated m5C methylation at site 621 in HSPB1 mRNA, enhancing its stability. Knockdown of HSPB1 abolished the protective effects of NSUN2, exacerbating inflammation and ferroptosis in OGD/R-treated HBMECs. Further investigations revealed that the NSUN2/HSPB1 axis exerted its protective role by activating the NRF2/HO-1/NQO-1 pathway. Inhibition of this pathway reversed the beneficial effects of HSPB1 overexpression. In tMCAO mice, NSUN2 overexpression reduced cerebral infarct volume, improved antioxidant capacity, and activated the NRF2/HO-1/NQO-1 pathway. In conclusion, NSUN2-mediated m⁵C methylation stabilized HSPB1 and activated the NRF2/HO-1/NQO-1 pathway, thereby mitigating inflammation and ferroptosis in IS. Targeting the NSUN2/HSPB1 axis may represent a novel therapeutic strategy for IS.