Effects Of Sevoflurane On Learning And Cognition Of Juvenile Rats And Its Molecular Mechanism

Part ?:Effects of sevoflurane on cognitive function in juvenile ratsObjective:To investigate the potential effects of sevoflurane anesthesia on learning and cognitive function in juvenile rats,and the effect of sevoflurane on BDNF expression in hippocampus.Methods:Four-week-old rats were exposed to 3.5%sevoflurane for 1 hour per day for 2 weeks.The rats were exposed to a mixture of oxygen and air in the control group.Morris water maze behavior test was used to evaluate the learning and memory ability of each group of rats,and the quantitative expression of BDNF in hippocampus tissue of model rats was quantified by RT-qPCR experiment.Results:The Morris water maze experiment found that the escape time gradually decreased with the extension of the experimental days in all groups.Compared with the NC group,the escape time in Sev group rats was statistically significant,starting from the second day of the experiment.The escape time in the Sev group was significantly longer than that in the NC group(P<0.05).In the space exploration test,the rats in the Sev group(4.83±0.75)traversed the platform less than the rats in the NC group within 90 seconds(7.50±1.05)(P<0.05).PCR results showed that the expression level of BDNF in hippocampus of Sev group was significantly lower than that of NC group(P<0.01).Conclusion:Sevoflurane can cause down-regulation of BDNF gene expression in hippocampus of juvenile rats,further affect the development of the nervous system,and impair the learning and memory abilities of juvenile rats in the short term.Part ?:RNA-Seq analysis of multi-organ transcriptome in rat model of sevofluraneObjective:Sevoflurane is an inhalation anesthetic used to induce and maintain general anesthesia in pediatric surgery,but few studies have focused on multi-organ toxicity and the mechanisms.This paper uses transcriptome sequencing to explore the molecular characteristics of sevoflurane anesthesia on multiple organ damage in rats,to find potential signal pathways of sevoflurane anesthesia leading to cognitive impairment in rats,and to construct a gene regulatory network.Methods:Anesthesia model of inhaled sevoflurane was established by exposing juvenile rats to 3.5%sevoflurane.The control group rats were exposed to a mixture of oxygen and air,for 7 days and 1 hour per day.Rat lung,cerebral cortex,hippocampus and heart tissue were transcriptome sequenced(RNA-seq),and IP A(Ingenuity Pathway Analysis)tool was used to analyze and investigate the effect of sevoflurane inhalation anesthesia on gene expression in lung,cerebral cortex,hippocampus and heart.The R-project related software package was used to analyze the differentially expressed genes(DEGs),gene function(GO)and pathway(KEGG),and CytoScape was used to draw the gene regulatory network.Results:The results of RNA sequencing analysis showed that the transcriptome of lung,hippocampus,and cerebral cortex changed more than that of heart,and the number of DEGs between the normal group and sevoflurane group in the heart was the least.IPA analysis results show that a variety of stress signaling pathways,such as inflammatory pathways,are activated in the lung and cerebral cortex,and axon-guided signaling pathways in the hippocampus are highly abundant.The biological function enrichment and apoptosis-related function enrichment of DEGs provide strong evidence for the harmful effects of sevoflurane on multi-organ development and induction of cell death;BCKDHB and SCN1B and other genes play a role in the gene regulatory network of tissue damage caused by sevoflurane.Conclusion:This is a study of toxicological evaluation of sevoflurane using a juvenile rat model.Sevoflurane inhalation anesthesia mainly causes tissue damage by inducing activation of inflammatory pathways,induces neuronal apoptosis,further affects organ development,and causes learning and cognitive dysfunction.Genes such as BCKDHB and SCN1B play a key regulatory role.This study provides some new experimental evidence for possible brain and lung injury in children caused by sevoflurane from the perspective of molecular mechanism.Part ?:The effect of BCKDHB gene interference on nerve cellsObjective:To explore the effect of BCKDHB gene on neuron apoptosis and migration using gene interference technology.Methods:Fetal rats were used to culture neuronal primary cells,and MAP2 antibodies were used to identify the cells.The BCKDHB overexpression and interference plasmids were constructed,and BCKDHB overexpression and interference cells were obtained by plasmid transformation and expansion culture.And tested the expression of BCKDHB gene and protein in each experimental group by PCR and WB experiments,and verified that the interference of BCKDHB expression was successful;finally,the apoptosis and neuronal cell apoptosis after BCKDHB gene interference were detected by AV/PI apoptosis experiment and Transwell test.Change in migration capacity.Results:The MAP2 immunofluorescence staining of isolated and cultured cells showed the characteristics of primary neurons.The results of fluorescent quantitative PCR and WB experiments showed that the overexpression of BCKDHB gene and the construction of interfering plasmids were successful,which had significant regulatory effects on BCKDHB gene and protein expression The results of apoptosis experiments show that overexpression of BCKDHB gene can significantly promote the apoptosis ability of neurons,and inhibition of BCKDHB expression can reduce the ability of neurons to apoptosis;Transwell test results show that BCKDHB gene has an effect on cell migration No significant impact.Conclusion:BCKDHB gene is closely related to neuronal apoptosis,indicating that mitochondrial-dependent apoptosis may be an important manifestation of sevoflurane injury.