Background: Cerebral ischemia-reperfusion injury (CIRI) is a critical determinant of therapeutic outcome in ischemic stroke and can lead to further neurological complications. Given the critical role of RNA modifications in human disease, this study aimed to investigate the molecular mechanisms of CIRI associated with m6A methylation. Methods: The study analyzed differentially expressed genes between brain tissues from the Sham operation group and the ischemic/reperfusion injury group of mice through the GSE23163 dataset. An in vitro model of CIRI was established by exposing HT22 mouse hippocampal neuronal cells to oxygen-glucose deprivation/reoxygenation (OGD/R). The middle cerebral artery occlusion/reperfusion (MCAO/R) model was also established for the study. Western blotting and qPCR were performed to examine protein and mRNA expression, respectively. Cell function was detected using cell viability assay and flow cytometry. Oxidative stress was assessed by detecting reactive oxygen species, malondialdehyde and superoxide dismutase. Inflammatory TNF-α and IL-1β levels were quantified using qPCR. Methylated RNA immunoprecipitation assay was performed to confirm m6A modification. Gene interaction was analyzed by RNA immunoprecipitation assay. Results: G protein-coupled receptor 84 (Gpr84) was up-regulated in brain tissues of CIRI mice and OGD/R-induced HT22 cells. Gpr84 knockdown mitigated OGD/R-induced oxidative stress, inflammatory response, and apoptosis. Mechanistically, fat mass and obesity-associated protein (FTO) governed m6A demethylation modification to inhibit Gpr84 mRNA stability. OGD/R-induced cell injury was also attenuated after FTO overexpression. Moreover, insulin-like growth factor 2 mRNA binding protein 1 (IGF2BP1) stabilized Gpr84 mRNA by acting as an m6A reader. FTO overexpression relieved OGD/R-induced oxidative stress, inflammatory response, and apoptosis and brain injury by suppressing Gpr84. Conclusion: FTO-mediated m6A demethylation modification reduced Gpr84 mRNA stability, thereby alleviating OGD/R-induced CIRI.
Li et al. (Mon,) studied this question.