Effects Of Sevoflurane On DNA Damage Of HT22 Mouse Hippocampal Neuronal Cells

Objective:To observe the effect of inhaled anesthetic sevoflurane on DNA damage of the HT22 mouse hippocampal neuronal cells,and to clarify the possible mechanism of sevoflurane-induced developmental neurotoxicity in the brain.Grouping:Selecting HT22 mouse hippocampal neuronal cells as the research object.The cells were randomly divided into blank control groups,sevoflurane groups and N-acetylcysteine(NAC)groups.The blank control groups were not treated with drugs.Sevoflurane groups were treated with different concentrations of sevoflurane(2%,4%,8%)for 6 h,12 h and 24 h.NAC groups were pretreated with 5 mmol / L NAC for 1 h,and then treated with different concentrations of sevoflurane(4%,8%)for 12 h.Method:1?MTT assay was used to detect the survival rates of the HT22 mouse hippocampal neuronal cells.2?LDH assay was used to detect the mortalities of the HT22 mouse hippocampal neuronal cells.3?DNA double-strand damage of the HT22 mouse hippocampal neuronal cells were detected by single-cell gel electrophoresis.4?Western blot was used to detect the expression amounts of 8-OHd G,ATM,p-ATM,and ?-H2 AX in the the HT22 mouse hippocampal neuronal cells.5?DCFH-DA was used to detect the levels of reactive oxygen species(ROS)in the HT22 mouse hippocampal neuronal cells.Results:1?Sevoflurane decreased the activity of the HT22 mouse hippocampal neuronal cells.Compared with the control group,the activity of HT22 mouse hippocampal neuronal cells was significantly decreased after 4% and 8% sevoflurane treatment(P<0.01).Sevoflurane inhibited the survival of HT22 mouse hippocampal neuronal cells in a dose-dependent manner.Treated with sevoflurane for the same time,as the concentration of sevoflurane increased,the survival rates of HT22 cells showed a downward trend(P<0.05).Sevoflurane has a time-dependent effect on the survival of HT22 mouse hippocampal neuronal cells.HT22 cells were treated with 4%sevoflurane for 6,12 and 24 hours,and the survival rates of HT22 cells decreased gradually with the prolongation of time(P<0.05).The HT22 cells were treated with4% sevoflurane for 6,12,24 hours.With the extension of time,the survival rates of HT22 cells gradually decreased(P<0.05).NAC pretreatment could reduce the inhibition of sevoflurane on the the HT22 mouse hippocampal neuronal cells.Compared with 4% and 8% sevoflurane groups,the survival rates of HT22 mouse hippocampal neuronal cells pretreated with NAC were significantly higher(P<0.01).2?Sevoflurane resulted in the death of the HT22 mouse hippocampal neuronal cells.Compared with the control group,the cell death rates of HT22 mouse hippocampal neuronal cells treated with 4% and 8% sevoflurane were significantly higher(P<0.01).Sevoflurane induced the death of HT22 mouse hippocampal neuronal cells in a dose-dependent manner.The mortalities of HT22 cells increased with the increase of sevoflurane concentration(P<0.05).Sevoflurane also induced HT22 mouse hippocampal neuronal cells death in a time-dependent manner.HT22 cells were treated with 4% sevoflurane for 6,12 and 24 hours,and the mortalities of HT22 cells increased gradually with time(P<0.05).When HT22 cells were treated with 8% sevoflurane for 6,12 and 24 hours,the death rates of HT22 cells also increased with time(P<0.05).NAC pretreatment can inhibit the damage of sevoflurane to HT22 mouse hippocampal neuronal cells and reduce cells mortalities.Compared with 4% and 8% sevoflurane groups,the mortalities of HT22 mouse hippocampal neuronal cells in NAC pretreatment groups were significantly lower(P< 0.01).3?Sevoflurane induced DNA double-strand breaks in HT22 mouse hippocampal neuronal cells.Single cell gel electrophoresis showed that DNA migration of HT22 mouse hippocampal neuronal cells was prolonged by sevoflurane,indicating that DNA double-strand damage increased.Compared with the blank control group,DNA migration of HT22 cells in 4% and 8% sevoflurane groups were significantly prolonged.NAC can reduce the DNA double-strand damage of HT22 mouse hippocampal neuronal cells caused by sevoflurane,and NAC pretreatment can significantly shorten the length of DNA migration.Compared with 4% and 8%sevoflurane groups,the DNA double-strand migration length of HT22 mouse hippocampal neuronal cells in NAC pretreatment groups decreased significantly.4 ? Sevoflurane promoted the expressions of DNA damage related proteins8-OHd G,ATM,p-ATM and ?-H2 AX in HT22 mouse hippocampal neuronal cells in a dose-dependent manner.Compared with the 2% Sevoflurane group,as the concentration of sevoflurane increasing,the expression levels of 8-OHd G,ATM,p-ATM,and ?-H2 AX in HT22 mouse hippocampal neuronal cells increased gradually(P<0.05).NAC pretreatment significantly reduced the expressions of 8-OHd G,ATM,p-ATM and ?-H2 AX.Compared with 4% and 8% sevoflurane groups,the expressions of DNA damage related proteins 8-OHd G,ATM,p-ATM and ?-H2 AX in HT22 mouse hippocampal neuronal cells in NAC pretreatment groups decreased(P<0.05).5 ? Sevoflurane promoted ROS accumulation in HT22 mouse hippocampal neuronal cells.DCFH-DA staining showed that sevoflurane enhanced the green fluorescence expression of HT22 mouse hippocampal neuronal cells,indicating that ROS accumulation in the cells increased.Compared with the control group,ROS accumulation in HT22 mouse hippocampal neuronal cells were significantly increased in 4% and 8% sevoflurane groups(P<0.05).Sevoflurane promoted the accumulation of ROS in HT22 mouse hippocampal neuronal cells in a dose-dependent manner.Compared with 4% sevoflurane 12 h group,ROS accumulation in HT22 cells of 8%sevoflurane 12 h group increased significantly(P<0.05).NAC could inhibit the accumulation of ROS in HT22 mouse hippocampal neuronal cells caused by sevoflurane.Compared with 4% and 8% sevoflurane groups,the expression of green fluorescence in HT22 mouse hippocampal neuronal cells in NAC pretreatment groups reduced dramatically,indicating that ROS level decreased significantly(P<0.01).Conclusion:1?Sevoflurane induced the death and inhibited the survival of the HT22 mouse hippocampal neuronal cells in a time and dose-dependent manner.2?Sevoflurane induced DNA double-strand breaks and promoted the expressions of DNA damage related proteins 8-OHd G,ATM,p-ATM,and?-H2 AX in HT22 mouse hippocampal neuronal cells.3?Sevoflurane can induce DNA damage by increasing the accumulation of ROS in hippocampal neuronal cells,and eventually lead to the death of hippocampal neuronal cells.