The Role Of DNA Methylation In Promoter Region Of Brain-derived Neurotrophic Factor Gene In Hydrogen Therapy Of Sepsis-associated Encephalopathy

ObjectiveSepsis-associated encephalopathy(SAE)is a central nervous system complication of sepsis,which can cause acute and long-term cognitive impairment and significantly increase the mortality of patients with sepsis.DNA methylation is an epigenetic modification.In the central nervous system,DNA dynamic methylation participates in the formation of synaptic plasticity and cognitive memory through gene regulation and other mechanisms.Brain-derived neurotrophic factor(BDNF)is one of the neurotrophic factors and a key signal component that regulates synaptic plasticity.The level of BDNF in the hippocampus of patients with cognitive impairment or animals is significantly decreased.Hydrogen is a kind of reducing agent,which has obvious therapeutic effect on SAE.However,the mechanism by which hydrogen inhalation is used to treat SAE is not fully understood.The purpose of this study was to explore whether DNA methylation plays a role in the pathogenesis of SAE and the changes of global DNA methylation level and expression of related enzymes in hippocampal tissue of mice treated with hydrogen.And to investigate whether DNA methylation changes in the promoter region of BDNF gene are involved in the treatment of SAE by hydrogen inhalation,in order to provide a new basis for the treatment of sepsis and the application of hydrogen gas.MethodsHealthy male C57BL/6 mice were used to establish SAE mouse model by cecal ligation and puncture(CLP).Morris water maze,Y maze and conditioned fear test were used to determine the cognitive function of mice.The hippocampal tissue from mice was used as research material.The global DNA methylation level was determined by colorimetry,and the m RNA expression levels of DNMTs,TETs and TDG were detected by RT-q RCR method.The concentrations of inflammatory factors(TNF-α,IL-1 β,HMGB1,IL-10)and oxidative stress indexes(SOD,MDA and CAT)were measured by ELISA method.S-Adenosylmethionine(SAM),a methyl donor,was injected intraperitoneally into septic mice to increase the level of global DNA methylation and to observe whether it could improve cognitive function,oxidative stress and inflammatory factors.After inhaling 2% hydrogen gas,the level of global DNA methylation in the hippocampus of mice was detected.The expression levels of m RNA and protein in DNMTs were detected by RT-q RCR,Western Blot and immunofluorescence to observe whether the therapeutic effect of hydrogen on SAE was related to the level of global DNA methylation and the change of DNMTs.The hippocampal tissues of SAE and sham-operated mice were sequenced by WGBS and RNA-seq,and combined analysis was carried out.The genes whose expression level was affected by DNA methylation in the hippocampus of mice during SAE were screened,and the BDNF gene closely related to cognitive function was selected from the screened genes for further detection.RT-q RCR and Western Blot methods were used to detect the m RNA expression level of BDNF exon IV and total BDNF in the hippocampus,as well as the changes of protein expression for BDNF in hippocampus during the treatment of SAE with hydrogen inhalation.The level of DNA methylation in the promoter region of exon IV of BDNF was detected by MASARRAY.To observe whether it was correlated with the changes of m RNA and protein levels.ResultsThe global DNA methylation levels in the hippocampus of sepsis mice decreased,and he m RNA expression levels of DNMTs,TETs and TDG in the hippocampus of septic mice changed significantly at 1,3 and 7 days after CLP.Administration of SAM decreased the levels of inflammatory factors(IL-6,TNF-αand HMGB1),increased the level of anti-inflammatory factors(IL-10),enhanced the activities of SOD and CAT,and decreased the expression level of MDA in the hippocampus of septic mice.It can significantly improve the cognitive impairment reflected by Morris water maze,Y maze and conditioned fear test in septic mice.After inhalation of 2% hydrogen gas,the global DNA methylation levels in the hippocampus of mice were increased,and the m RNA expression levels of DNMMTs,TETs and TDG,as well as the protein expression levels of DNMTs were changed to different degrees.By combined analysis of WGBS and RNA-SEQ sequencing data,a total of 70 genes whose expression levels were affected by DNA methylation were screened out in the hippocampus of SAE mice.After CLP,the protein expression levels of BDNF exon IV and total BDNF m RNA and BDNF decreased significantly,while the methylation levels of multiple Cp G sites in exon IV of BDNF increased.Hydrogen inhalation could significantly correct the methylation disorder in promoter region of BDNF exon IV and increase the expression levels of BDNF exon IV and total BDNF.ConclusionThe global DNA methylation levels in the hippocampus of septic mice changed significantly.Correcting the disorder of DNA methylation could improve the cognitive dysfunction and reduce the inflammatory reaction and oxidative stress in mice.Inhalation of 2% hydrogen gas could increase the global DNA methylation levels in the hippocampus of septic mice,and change the m RNA expression levels of DNMTs,TETs and TDG,as well as the protein expression level of DNMTs.The change of DNA methylation in the promoter region of exon IV of BDNF is closely related to the pathogenesis of SAE.Inhaling 2% hydrogen gas could reduce the level of DNA methylation in the promoter region of exon IV of BDNF and the expression of BDNF,indicating that DNA methylation in the promoter region of BDNF played an important role in hydrogen therapy of SAE.These results of this study provided new insights and support for hydrogen treatment of SAE.