Study On The Mechanism Of METTL3/YTHDF1 Modified NOD2 M6A Modification Regulating The Neuroinflammation In The White Matter In The Sepsis Mice

Background: Sepsis 3.0 defines sepsis as an imbalance in the host’s response to infection,resulting in life-threatening organ dysfunction.The central nervous system has always been considered as one of the earliest systems affected by sepsis.Sepsis associated encephalopathy is one of the serious complications of sepsis.Its obvious pathological characteristics are the activation of microglia and astrocyte,the release of a large number of inflammatory factors,leading to axonal damage,hypomyelination,and the destruction of the blood brain barrier.Sepsis associated encephalopathy including the acute brain injury and the long-term neurological dysfunction.Focusing on the long-term neurological dysfunction caused by sepsis is of great significance in improving the quality of life of sepstic patients after discharge and reducing readmission rates.It was pointed out that the phenotypic transformation of astrocyte into type A1 involved in the late inflammatory response depends on the activation of early microglia.Therefore,exploring the activation mechanism of microglia in the brain of sepsis is the key to reveal the mechanism of brain injury mediated by neuroinflammatory response.A Me RIP sequencing result of M0/M1/M2 microglia confirmed that there were a large number of m6 A modified genes with significant differences between different polarization states of microglia,indicating that the epigenetic modification of m6 A played an important role in microglia polarization.However,this study did not conduct corresponding experimental research or explore the corresponding mechanisms.Therefore,our study mainly discussed how the epigenetic modification of m6 A regulates the neuroinflammatory response in the periventricular white matter of sepsis brain mediated by the activation of microglia,and explored its mechanism.Objective: 1.To obtain reliable evidence that m6 A methylation is involved in the activation of microglia and causes neuroinflammation;2.In vivo and in vitro experiments confirmed that METTL3/YTHDF1 can increase the expression of NOD2 gene,promote the activation of M1 phenotype microglia,increase the release of inflammatory mediators,aggravate the neuroinflammatory response,cause white matter damage,and cause long-term neurological dysfunction by mediating the methylation of NOD2 m RNA m6 A modification;3.To clarify that NOD2 m RNA m6 A methylation of microglia in periventricular white matter promotes the activation of microglia to release inflammatory mediators through non classical NF-κB pathway.Methods: Sepsis mice model was established by cecal ligation and puncture(CLP).The expression of inflammatory factors in the corpus callosum was detected by immunofluorescence staining and western blot,and the neuroinflammatory response in sepsis mice was observed.Dot blot assay was used to detect the changes of m6 A methylation in the corpus callosum of septic mice,and real-time quantitative PCR assay was used to detect the changes of m6 A methylation related Writers,Erasers,Readers and NOD2 expression in the corpus callosum of septic mice.The newborn 1-3d C57 mice were used to culture primary microglia.After MDP treatment,the expression of inflammatory factors in primary microglia and BV2 microglia was detected by immunofluorescence staining and western blot,and the change of m6 A methylation level in primary microglia and BV2 microglia was detected by dot blot.Real-time quantitative PCR assay was used to detect the changes of Writers,Erasers,Readers and NOD2 expression related to m6 A methylation in primary microglia and BV2 microglia.The si-RNA transfection technique was used to knock down METTL3 and YTHDF1 on BV2 microglia,and the plasmid transfection technique was used to overexpress METTL3,YTHDF1 and NIK on BV2 microglia.After MDP treatment,the expression of inflammatory factors,M1/M2 microglia markers and NOD2 in BV2 microglia were detected by immunofluorescence staining and western blot.Me RIP-q PCR assay was used to detect the changes in NOD2 m RNA m6 A methylation after MDP,MDP+si-METTL3 treatment.Verify the correlation relationship between METTL3 protein and NOD2 m RNA through RIP experiments.The mice models of METTL3 and YTHDF1 knockdown were established by stereotactic injection of si-RNA into the brain.The expression of inflammatory factors,polar transformation of microglia and the expression of NOD2 in the corpus callosum of sepsis mice(CLP model)and MDP mice directly intervened by intracerebral injection were observed.Injecting IL-1β through stereotactic brain localization to establish the neuroinflammatory model,and the water maze test was used to evaluate the changes of cognitive functions.Results: 1.The sepsis mice model was established by cecal ligation and puncture(CLP).The obvious neuroinflammatory response occurred in the corpus callosum of sepsis mice,and microglia activated and secreted inflammatory factor TNF-α,i NOS and IL-1β increased.The activation of microglia was tended to M1 pro-inflammatory phenotype,while M2anti-inflammatory microglia decreased.The level of m6 A methylation in the corpus callosum of septic mice increased,and the expression of methyltransferase METTL3,reading protein YTHDF1 and NOD2 increased.2.In vitro: under the stimulation of MDP,the inflammatory factor TNF-α,i NOS and IL-1β increased.Knock down METTL3 and YTHDF1 respectively can reduce the expression of NOD2,reduce the activation of microglia,and reduce the inflammatory factor TNF-α,i NOS and IL-1β.The activation of M1 pro-inflammatory microglia decreased,and M2 anti-inflammatory microglia increased.3.Me RIP-q PCR experiment confirmed that after MDP stimulation,NOD2 m6 A modification increased,while knocking down METTL3 resulted in a decrease in NOD2 m6 A modification,and its regulated methylation sites may be 4297 or/and 4302 sites.The RIP experiment confirmed the interaction between METTL3 protein and NOD2 m RNA.4.In vitro: under the stimulation of MDP,overexpression of METTL3,YTHDF1 and NIK increased the expression of NOD2 protein,increased the activation of microglia and releases the inflammatory factor TNF-α,i NOS and IL-1β increased.The transformation of microglia into M1 pro-inflammatory phenotype stimulates neuroinflammatory response,which may be mediated by the non classical NF-κB signal pathway.5.In vivo: Brain stereotactic injection of si-RNA to establish a brain knockdown METTL3 and YTHDF1 mice model.Knocking down METTL3 and YTHDF1 can reduce the expression of NOD2 in the corpus callosum of septic mice and MDP direct intracerebral injection mice,while reducing the activation of microglia,reducing the release of inflammatory factors,reducing the activation of M1 pro-inflammatory microglia,and increasing the activation of M2 anti-inflammatory microglia.6.Intracerebral injection of IL-1β to establish a neuroinflammation model.Neuroinflammatory response can lead to long-term neurological dysfunction in animals.Conclusion: METTL3/YTHDF1 modified NOD2 m RNA m6 A methylation increased NOD2 expression,thereby activating non classical NF-κB pathway,leading to the activation of NF-κB subunit--Rel B enters the nucleus to promote the expression of inflammatory factor genes,promote the activation of M1 pro-inflammatory microglia,release a large number of inflammatory factors,and regulate the neuroinflammatory response in the periventricular white matter of sepsis.