| Because of its high mortality and disability rate,ischemic stroke has become one of the important diseases that threaten human health and has brought serious economic burden to the society and the country.At present,there are two kinds of treatment: intravenous thrombolysis and mechanical thrombectomy,but the mortality rate of patients is still high after receiving the above treatment.after a large number of studies,it is found that the existence of ischemia-reperfusion injury is one of the important reasons for this situation.Ischemia-reperfusion injury refers to the further aggravation of tissue injury after the restoration of blood flow and reperfusion in ischemic tissue.Epigenetic modifications,such as histone modification,DNA modification and RNA modification,have been reported to play an important role in ischemia-reperfusion injury.Methylation modification accounts for a large proportion of RNA modification types,while M6 A methylation modification is the most common in M6 A,M5C,M1 C,m6Am and other methylation modifications.M6 A methylation modification is mainly regulated by methyltransferases(such as METTL3,METTL4,etc.),demethylases(FTO,ALKBH5,etc.)and binding proteins(IGF2BP1-3).The genes involved in the regulation of M6 A methylation are closely related to the occurrence and development of inflammation.FTO is the first protein with demethylase activity,and FTO also plays an important role in ischemia-reperfusion injury.It has been found that after ischemia-reperfusion injury in liver,heart and other organs,the expression of FTO in the injured tissue is significantly decreased,and overexpression of FTO can significantly alleviate the tissue injury caused by ischemia-reperfusion.Inflammation plays an important role in ischemia-reperfusion injury.The results from clinical studies found that neutrophil/lymphocyte ratio is an effective index to evaluate the prognosis of ischemic stroke,which indicates the importance of inflammation.FTO regulates the process of inflammation.In previous studies,FTO gene silencing can promote the transformation of macrophages into pro-inflammatory M1 macrophages,and then increase the occurrence of inflammatory response,while overexpression of FTO can inhibit the activation of NF-KB/NLRP3 inflammatory complex and related inflammatory signal pathways,and reduce the production of related inflammatory factors.On the basis of the above studies,we speculate that demethylase FTO may play an important role in cerebral ischemia-reperfusion injury.Therefore,we intend to explore the following three aspects: 1)to determine the change of FTO expression level after cerebral ischemia-reperfusion injury;2)the effect of FTO overexpression on cerebral ischemia-reperfusion injury;3)the specific mechanism of FTO regulating cerebral ischemia-reperfusion injury.Part 1: Down-regulation of M6 A demethylase FTO expression in brain tissue after ischemia-reperfusion injury Objective: The animal model of cerebral ischemia-reperfusion injury was established to detect the changes of m6 A methylation level and the regulation of m6 A methylation related genes after cerebral ischemia-reperfusion injury.This would provide a theoretical basis for exploring the role of m6 A methylation in cerebral ischemia-reperfusion injury.Methods: The mouse model of ischemia-reperfusion injury was established by middle cerebral artery occlusion,and the infarct size was evaluated by TTC staining of brain tissue sections,and the change of M6 A methylation level in brain tissue after injury was detected.Furthermore,the changes of the expression of METTL3,METTL4,WTAP,ALKBH5 and FTO,which regulate M6 A methylation,in brain tissue after ischemia-reperfusion injury was also detected from the level of both RNA and protein aspect.Results: The TTC staining of brain tissue in the ischemia-reperfusion injury group showed a significant increase in the white part(that is,the infarcted part),but there was no obvious white infarcted area in the sham operation group.The level of m6 A methylation in the cerebral cortex after ischemia-reperfusion injury was significantly higher than that in the sham operation group.No matter at the RNA level or protein level,there was no difference in the expression of METTL3,METTL4,WTAP and ALKBH5 between the sham operation group and the ischemia-reperfusion injury group,but only the expression of FTO decreased significantly in the brain tissue after ischemia-reperfusion injury.Part 2: Overexpression of FTO attenuates inflammation after cerebral ischemia-reperfusion injury Objective: FTO was overexpressed in brain tissue,and the effects of overexpression of FTO on cerebral ischemia-reperfusion injury and inflammatory molecules and signal pathways were observed,so as to clarify the important role of FTO in cerebral ischemia-reperfusion injury.Methods: Adenovirus transfection was used to observe the effect of FTO overexpression on the methylation level of M6 A in brain tissue and the size of cerebral infarction after reperfusion injury.The animal model of cerebral ischemia-reperfusion injury was established to observe the effect of overexpression of FTO on the infarct size caused by injury.The effect of FTO overexpression on the expression of inflammatory factors after ischemia-reperfusion injury was detected from the levels of RNA and protein,and the effect on IKB/NF-KB signal pathway was observed.Results: Adenovirus transfection can significantly increase the expression of FTO in brain tissue.The overexpression of FTO can significantly reduce the level of M6 A methylation in brain tissue after ischemia-reperfusion injury,and significantly reduce the area of cerebral infarction after injury.Overexpression of FTO can significantly reduce the expression of inflammatory factors after cerebral ischemia-reperfusion injury.After ischemia-reperfusion injury,the expression of IKB in the cytoplasm decreased,while the expression of NF-KB in the nucleus increased.After overexpression of FTO,the expression of IKB in the cytoplasm increased significantly,while the expression of NF-KB in the nucleus decreased significantly.Part 3: Overexpression of FTO attenuates inflammation after cerebral ischemia-reperfusion injury by mediating pri-miR-155 m6 A modification and regulating miR-155.Objective: To clarify the regulatory relationship between FTO and miR-155,and to explore the mechanism of FTO reducing inflammation and improving cerebral ischemia-reperfusion injury.Methods: Firstly,the effect of overexpression of FTO on the expression of pri-miR-155 and miR-155 was observed.Secondly,FTO overexpression adenovirus and miR-155 mimic adenovirus were constructed,and FTO and miR-155 were overexpressed in brain tissue at the same time.The effect of miR-155 overexpression on pri-miR-155 m6 A methylation level was observed.At the same time,the effects of miR-155 overexpression on FTO overexpression reducing cerebral infarction area and inflammatory cytokines were observed.Results: The overexpression of FTO decreased the expression of miR-155 and increased the expression of pri-miR-155 in brain tissue after ischemia-reperfusion injury.The level of M6 A in pri-miR-155 increased significantly after ischemia-reperfusion injury,and overexpression of FTO could significantly decrease the level of M6 A in pri-miR-155.Transfection of FTO could significantly reduce the infarct size after ischemia-reperfusion injury,while co-transfection of miR-155 mimic and FTO could partially counteract the effect of FTO on reducing infarct size and inflammatory cytokines TNF-and IL-1 β.After ischemia-reperfusion injury,the expression of IKB in the cytoplasm decreased,while the expression of NF-KB in the nucleus increased significantly.After transfection of FTO,the expression of IKB in the cytoplasm increased significantly,while the expression of NF-KB in the nucleus decreased significantly.When co-transfected with miR-155 mimic and FTO,it could significantly counteract the effect of FTO overexpression on IKB activation and NF-KB inhibition.Conclusion: Overexpression of FTO reduces the level of pri-miR-155 m6 A methylation modification,inhibits the maturation of miR-155,and then reduces the inflammatory response after cerebral ischemia-reperfusion injury. |
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