Shock (Augusta, Ga.) · 2026
FTO Relieves Oxygen-glucose Deprivation/reoxygenation-induced Cerebral Ischemia-reperfusion Injury through the m6A Demethylation of Gpr84 mRNA.
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Li Qing, Huang Miao, Qu Changhua, Meng Xiangwu
Аннотация
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 model was also established for the study. Western blotting and quantitative polymerase chain reaction 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 tumor necrosis factor-α and interleukin-1β levels were quantified using quantitative polymerase chain reaction. 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 upregulated 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 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.
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 model was also established for the study. Western blotting and quantitative polymerase chain reaction 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 tumor necrosis factor-α and interleukin-1β levels were quantified using quantitative polymerase chain reaction. 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 upregulated 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 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.