FTO Alleviates CIRI By Inhibiting GADD45B M6A Methylation And Participating In The MAPK Pathway

Objective:Cerebral ischemia-reperfusion injury(CIRI)is a common and severe complication following acute ischemic stroke,leading to further damage and death of neurons due to its complex pathophysiological mechanisms.As a critical epigenetic modification,N6-methyladenosine(m6A)methylation has been identified to play roles in various neurodegenerative diseases,and its dysregulation may be closely linked with the progression of CIRI.This study aims to deepen our understanding of the molecular mechanisms underlying CIRI,with a particular focus on the role of m6 A methylation and its regulatory factor,Fat mass and obesity-associated protein(FTO).GADD45B is widely expressed in the nervous system and plays an important role in neuroprotection at the nervous system function,immunity,and neuroplasticity levels.GADD45B can reduce cell damage caused by neurological diseases including CIRI.In CIRI,the abnormal activation and inhibition of some pathways are closely related to the degree of cell damage,including the MAPK signaling pathway related to cell stress.By exploring the expression regulation of GADD45B by the m6 A methylation modification enzyme FTO in CIRI and its impact on the MAPK signaling pathway,the role of FTO in CIRI was revealed.We hope to explore the new mechanism of gene expression regulation in this pathological state and provide a theoretical basis and potential targets for the development of effective treatments.Methods:1.Rats were used as experimental animals to simulate CIRI,we adopted the focal cerebral ischemia-reperfusion(Middle cerebral artery occlusion/reperfusion,MCAO/R)model.TTC staining was used to evaluate cerebral infarction,and an m6 A RNA methylation quantification kit was employed to determine the m6 A levels in total RNA.High-throughput sequencing analysis(including MeRIP-Seq and RNA-Seq)was used to identify differentially methylated sites and their functions.RT-qPCR,Western blotting,and immunofluorescence methods were used to verify the expression and localization of key genes and proteins.In addition,differential gene expression analysis and GO and KEGG pathway enrichment analyses were conducted using information from public datasets.2.To clarify the role and mechanism of FTO in CIRI.Primary cerebral cortical neurons were extracted from rats and cultured under specific conditions.Immunofluorescence was used to identify the purity of neurons,ensuring the accuracy of the experiment.Subsequently,an oxygen deprivation/reoxygenation(OGD/R)cell model was constructed to simulate the condition of cerebral ischemia-reperfusion,and FTO overexpression vectors and inhibitor FB23-2 were used to explore the function of FTO under OGD/R conditions.After cell transfection,CCK-8 and flow cytometry techniques were used to assess cell viability and apoptosis.To further understand the molecular mechanism of FTO,MeRIP-qPCR and Western blotting techniques were employed to detect the methylation level of GADD45B and the expression of related proteins.3.To further validate the role and mechanism of FTO in CIRI model rats.We first constructed an FTO overexpression vector and delivered it to the cerebral hemisphere region of the MCAO/R model rats.TTC staining was used to evaluate the cerebral infarction in rats,and RT-qPCR was employed to detect the gene expression of FTO,GADD45B,METTL3,and ALKBH5.Hematoxylin-eosin(HE)staining was used to observe pathological changes in rat hippocampal tissue.Immunofluorescence double staining was utilized to assess the m6 A methylation level in the cerebral cortex and the expression of Neu N.Western blotting techniques were used to detect the expression of related proteins,while TUNEL staining and immunohistochemistry were used to assess cell apoptosis and the expression of key proteins.Results:1.CIRI rat model was successfully constructed by MCAO/R surgery and validated the model’s effectiveness with TTC staining,indicating the formation of cerebral infarction.Subsequently,MeRIP-seq and RNA-seq technologies demonstrated that compared to the control group,the m6 A level in CIRI rats significantly increased,especially in the coding sequences and stop codon regions.Further analysis revealed that under CIRI conditions,there are numerous differentially methylated m6 A peaks,mainly enriched in genes with "hyper-up" or "hypo-down" differential expression,indicating a positive correlation between m6 A modification and mRNA expression in CIRI.Notably,we found that FTO,an important m6 A demethylase,significantly decreased in expression in the cerebral cortex of CIRI rats,which might be one of the key factors leading to the increase in m6 A levels.Immunofluorescence staining further revealed that the expression of FTO in neurons and microglial cells post-CIRI was significantly reduced,especially in neurons.2.We delved deeper into the mechanism of FTO’s impact on CIRI.The expression of FTO was significantly downregulated in neurons of CIRI rats,and oe-FTO successfully transfected neurons and upregulated FTO expression.Overexpression of FTO significantly enhanced neuronal cell viability and inhibited apoptosis,revealing FTO’s crucial role in maintaining neuronal survival.Further studies found that FTO regulates GADD45B gene expression through m6 A methylation modification and affects key signaling molecules such as p38 and p90 RSK,suggesting that FTO may play a protective role in CIRI by affecting the methylation status of GADD45B and regulating the MAPK signaling pathway.3.These findings in the cellular model were further validated in the CIRI rat model.Overexpression of FTO significantly reduced the area of cerebral infarction,improved the damage to hippocampal neurons post-MCAO/R,and significantly weakened apoptosis.Additionally,FTO overexpression significantly reduced the m6 A methylation level and increased the number of neurons while enhancing the expression of the GADD45B gene.This indicates that FTO participates in the neuroprotective mechanism of CIRI by regulating the m6 A methylation status and GADD45B expression.Further Western blotting results support FTO’s regulatory effects on GADD45B methylation and the MAPK signaling pathway.Notably,the overexpression of FTO was able to reverse the expression changes of p38 and p90 RSK in MCAO/R rats,suggesting that FTO might influence the process of CIRI by regulating the MAPK signaling pathway.Conclusion:This study reveals the key role of FTO in the CIRI rat model,which can reduce cerebral infarct size and cell damage in CIRI.FTO is a key factor in regulating changes in m6 A levels in CIRI and can affect its expression by regulating the m6 A methylation modification of GADD45B mRNA.The expression of FTO is reduced in CIRI,and overexpression of FTO will reverse the expression changes of GADD45B and the activation degree of MAPK signaling pathway in CIRI,thereby achieving cell protection.These findings not only provide a new perspective for understanding the molecular mechanisms of CIRI but also offer a scientific basis for developing potential therapeutic targets for the disease.