The Effect Of Sevoflurane On The Expression Of ChAT、AChE And α7nAChR In Hippocampal Neurons

Objective:This experiment took HT-22 mouse hippocampal neuron cells as the research object.After HT-22 cells were treated with 2.6%sevoflurane for 2 h,4 h,and 6 h,the cell morphology,cell viability,and HT22 cells were detected Changes in the relative expression of Ch AT,ACh E,α7n ACh R protein and m RNA,discuss the effect of sevoflurane on the morphology and activity of hippocampal neurons from the cellular level,and verify the relationship between sevoflurane and the cholinergic system.Methods:HT-22 cells were cultured to the logarithmic growth phase for subsequent experiments.The HT-22 cells were divided into a control group（Con group）and a 2.6%sevoflurane treatment group（Sev group）.Sevoflurane treatment time imitated the clinical short-term,medium-term,and long-term use of sevoflurane anesthesia,respectively,2 h,4h,and 6 h.They were divided into Sev2 h,Sev4 h,Sev6 h,Con2 h,Con4 h,and Con6 h groups.The Con group was placed in an incubator with CO₂0.1 L/min+air 1.9 L/min（5%CO₂,95%air）.The Sev group was placed in a cell sevoflurane incubator with CO₂0.1L/min+air 1.9 L/min+2.6%sevoflurane,adjusted the sevoflurane volatilization tank scale to keep the concentration at 2.6%,and sevoflurane treatment 2 h,4 h and 6 h.After the treatment,the cells were taken out and the following experiments were carried out:（1）Cell morphology observation:Observe the cell morphology under an optical microscope at40X,100X,200X,400X and take photos.The observation content includes cell morphology,axis Cone shape,cell adhesion,whether cells are shedding,large cells in suspension.（2）Cell viability test:use CCK-8 method to determine cell viability.（3）Detection of the relative expression of Ch AT,ACh E,andα7n ACh R m RNA in hippocampal neuron HT-22 cells:RT-PCR was used to detect the relative expression of Ch AT,ACh E,andα7n ACh R m RNA in each group.（4）Detection of Ch AT,ACh E,andα7n ACh R proteins in hippocampal neuron HT-22 cells:ELISA was used to detect the changes in the expression of Ch AT,ACh E,andα7n ACh R proteins in each group.Results:（1）Observation of cell morphology:After HT-22 cells were treated with 2.6%sevoflurane,there was no significant change in cell morphology in the Con2 h group,Con4h group,Con6 h group and Sev2 h group.Compared with the Con4 h group and the Con6 h group,in the Sev4 h group and Sev6 h group,the cell bodies and dendrites of hippocampal neurons shrank,stumps appeared,the cells did not show a regular round shape,and there were gaps between the cells and the bottom of the culture flask.Cells adhere to the wall and do not stretch,and are easier to fall off from the bottom.Observed under a high-power microscope,granular material appeared between the cell bodies.（2）Cell viability test:（1）Comparison of cell viability between groups:Compared with the control group,after each group treated HT-22 cells with 2.6%sevoflurane,the cell viability was significantly reduced（P<0.05）.（2）Comparison results within the cell viability group:After 2.6%sevoflurane treatment of HT-22 cells,the cell viability of Sev4 h group and Sev6 h group was significantly lower than that of Sev2 h group,and cell viability of Sev6 h group was lower than that of Sev4 h group（P<0.05）),there was no statistically significant difference within the group at other time points（P>0.05）.（3）Detection of the relative expression levels of Ch AT,ACh E,andα7n ACh R m RNA in hippocampal neuron HT-22 cells:（1）Comparison of the relative expression levels of Ch AT,ACh E,andα7n ACh R m RNA between groups:Compared with the Con2 h group,Con4 h group,and Con6 h group,Sev2 h group,The relative expressions of Ch AT,ACh E,andα7n ACh R m RNA in the Sev4 h group and Sev6 h group were reduced,and the difference was statistically significant（P<0.05）.（2）The relative expression of Ch AT,ACh E,α7n ACh R m RNA within the group:a.Compared with the Con2 h group,the relative expression of Ch AT m RNA in the Con4 h group and the Con6 h group decreased,and the difference was statistically significant（P<0.05）.There was no statistically significant difference within the group at the time point（P>0.05）.b.Compared with the Sev2 h group,the relative expression of ACh E m RNA in the Sev6 h group decreased,and the differences were statistically significant（P<0.05）,and there were no statistically significant differences within the groups at other time points（P>0.05）.c.Compared with the Sev2 h group,the relative expression ofα7n ACh R m RNA in the Sev6 h group increased,and the relative expression ofα7n ACh R m RNA in the Sev4 h group decreased.Compared with the Sev4 h group,the relative expression ofα7n ACh R m RNA in the Sev6 h group increased.Compared with the Con2 h group,The relative expression ofα7n ACh R m RNA in the Con6 h group decreased.Compared with the Con4 h group,the relative expression ofα7n ACh R m RNA in the Con6 h group decreased,and the differences were statistically significant（P<0.05）.There was no statistical difference within the group at other time points.Significance（P>0.05）.（4）Detection of Ch AT,ACh E,α7n ACh R protein in hippocampal neuron HT-22 cells:（1）Comparison of Ch AT,ACh E,α7n ACh R protein expression between groups:Compared with Con2 h group,Con4 h group,Con6 h group,Sev2 h group,Sev4 h The expression of Ch AT and ACh E protein in the Sev6 h group and the Sev6 h group decreased.Compared with the Con4 h group and the Con6 h group,theα7n ACh R protein expression in the Sev4h group and Sev6 h group decreased,and the difference was statistically significant（P<0.05）.There was no statistically significant difference between the groups at the time point（P>0.05）.（2）Ch AT,ACh E,α7n ACh R protein expression group comparison results:a.Compared with Sev2 h group,Sev6 h group Ch AT protein expression decreased,compared with Con2 h group,Con6 h group Ch AT protein expression decreased,the difference was statistically significant（P<0.05）,there was no significant difference within the group at other time points（P>0.05）.b.Compared with the Sev2 h group,the ACh E protein expression in the Sev6 h group was reduced.Compared with the Sev4 h group,the ACh E protein expression in the Sev6 h group was reduced.Compared with the Con2 h group,the ACh E protein expression in the Con4 h group was reduced,and the difference was statistically significant.（P<0.05）,there was no significant difference within the group at other time points（P>0.05）.c.Compared with the Sev2 h group,theα7n ACh R protein expression in the Sev4 h group and the Sev6 h group decreased.Compared with the Con4h group,theα7n ACh R protein expression in the Con6 h group decreased,and the difference was statistically significant（P<0.05）.There was no statistically significant difference within the group（P>0.05）.Conclusion:After 2.6%sevoflurane treatment of hippocampal cell HT-22 cells,the morphology changed,the cell activity decreased,and the expression of Ch AT、ACh E、α7n ACh R protein and m RNA was inhibited.