Study On Epigenetic Regulation Mechanism Of Central Nerve Ion Channels In Rats Under General Anesthesia With Emulsified Isoflurane

The mechanism of general anesthesia is very complicated and has not yet been fully elucidated.With the rapid development of molecular biology technology,the research on the mechanisms of general anesthesia has made remarkable progress."Lipid theory","Synaptic theory","Protein theory",and"Ion channel theory"have been put forward.However,the mechanism of general anesthetics exerting anesthetic effect by affecting the structure and function of central and peripheral nerve channels,and thus affecting the transmission of neurotransmitters remains to be studied.General anesthetics can act on the ion channel family and its subunits.The ion channels are divided into voltage-gated channels and ligand-gated channels.In recent years,ion channel theory and related signal conduction have perfected the anesthesia theory.Epigenetic mechanism decribes the changes gene expression without changing the actual inheritance or DNA sequence.Its main regulation includes DNA methylation,histone modification,and non-coding RNA regulation.This study explored the effect of DNA methylation on the expression of ion channel proteins in rats anesthetized with emulsified isoflurane.This study also used emulsified isoflurane to treat the cultured rat cerebral cortical neurons in vitro,detected changes in neuronal gene expression and DNA methylation,and further explored the regulation of epigenetic isoflurane on genes related to anesthesia with emulsified isoflurane.1.Effect of emulsified isoflurane anesthesia on ion channels in cerebral cortex in ratsIn this work,8%emulsified isoflurane was used to anesthetize SD rats by constant flow injection through the tail vein.The cerebral cortex parietal lobe tissues in rats after anesthesia were collected,the expressions of ion channel-related genes and proteins were detected,and the influence of epigenetic regulation mechanism on the regulation of related genes was explored.32 SD rats were randomly divided into 4 groups with 8 rats in each group.In the experiment,constant flow of treated drugs were injected into the tail vein in rats,including normal saline,a high dose of 8%emulsified isoflurane,a low dose of 8%emulsified isoflurane and fat emulsion.After 25 minutes of treatment,the rats were sacrificed,and cerebral cortex tissues were collected for histological examination and gene expression detection.The results showed that emulsified isoflurane was an good injectable anesthetic,and has little effect on the respiratory and circulatory systems.Compared with the blank control group or the fat injection group,in rats anesthetized with emulsified isoflurane,theγ-aminobutyric acid A receptorα1 subunit(GABA_Aα1)andμ-Opioid Receptor 1（OPRM1）m RNA expressions increased significantly,while N-methyl-D-aspartate receptor subunit 1（NMDAR1）and N-methyl-D-aspartate Receptor Subunit 2B（NR2B）expression levels were significantly reduced.At the same time,the expression of GABA_Aα1 and NMDAR1 protein increased significantly,while the expression of OPRM1 decreased significantly.DNA methylation detection showed that the methylation rate of GABA_Aα1 and OPRM1 promoter regions decreased significantly after emulsified isoflurane anesthesia while NDMAR1increased.The results indicated that emulsified isoflurane affected the expression of related proteins and performed anesthesia by changing the methylation of the promoter region of the protein encoding protein in the cerebral cortex.2.The effect of emulsified isoflurane treatment on ion channelsIn this study,rat cerebral cortex neurons were cultured in vitro,and the obtained cells were identified by immunofluorescence staining.After these work,the cells were treated with8%emulsified isoflurane,and CCK-8 kit was used to detect cell viability after drug treatment.The secretion products of cells were detected by ELISA.Compared with the blank control or the fat emulsion treatment group,the expression of GABA increased significantly,the expression of NMDA decreased significantly after the emulsified isoflurane treatment,and the expression of Endomorphin-2（EM-2）did not significant changes.Real-time fluorescent quantitative PCR was used to detect the expression levels of genes related to the GABA_A/STEP61 signaling pathway,and it was found that the expression of GABA_Aα1 and OPRM1 increased significantly after emulsified isoflurane treatment,while NMDAR1 and NR2B were the opposite.At the same time,the signal molecules of P38-MAPK signal pathway increased significantly after emulsified isoflurane treatment.Western blotting was used to examine the level of changes in related proteins,and GABA_Aα1 and OPRM1 protein expression increased significantly after emulsified isoflurane treatment,while NMDAR1decreased.After treatment with emulsified isoflurane,the expression levels of major cytokines in the P38-MAPK signaling pathway increased significantly.The detection of the DNA methylation level in the promoter region of the target gene revealed that the methylation rate of the DNA promoter region encoding GABA_Aα1 and OPRM1 decreased significantly after emulsified isoflurane treatment,and NMDAR1 increased.The results showed that 8%emulsified isoflurane can change the DNA methylation level of rat cerebral cortex neurons,regulate the expression of ion channel proteins,and control the expressions of anesthesia-related proteins through GABA_A/STEP61 and P38-MAPK signaling pathways.