七氟醚对HT22小鼠海马神经元细胞DNA的损伤及其机制

目的：观察吸入麻醉药七氟醚对HT22小鼠海马神经元细胞DNA的损伤，阐明七氟醚诱导脑神经毒性的可能机制。方法：HT22小鼠海马神经元细胞随机分为空白对照组、2%七氟醚组（2%Sevo 6 h组、2%Sevo 12 h组和2%Sevo 24 h组）、4%七氟醚组（4%Sevo 6 h组、4%Sevo 12 h组和4%Sevo 24 h组）和8%七氟醚组（8%Sevo 6 h组、8%Sevo 12 h组和8%Sevo 24 h组），应用四甲基偶氮唑盐比色（MTT）法和乳酸脱氢酶（LDH）法分别检测各组HT22小鼠海马神经元细胞存活率和死亡率。根据实验结果将细胞分为生理盐水组、生理盐水+4%Sevo 12 h组、生理盐水+8%Sevo 12 h组、N-乙酰半胱氨酸组（NAC组）、NAC+4%Sevo 12 h组和NAC+8%Sevo 12 h组。采用MTT法和LDH法分别检测NAC预处理后各组HT22小鼠海马神经元细胞存活率和死亡率；应用单细胞凝胶电泳检测各组HT22小鼠海马神经元细胞DNA双链断裂情况；Western blotting法检测各组HT22小鼠海马神经元细胞中DNA损伤相关蛋白8-OHdG、ATM、p-ATM和γ-H2AX表达量；DCFH-DA法检测各组HT22小鼠海马神经元细胞中活性氧（ROS）水平。结果：与空白对照组比较，2%Sevo组HT22小鼠海马神经元细胞存活率和死亡率差异无统计学意义（P>0.05）；4%Sevo组和8%Sevo组HT22小鼠海马神经元细胞存活率降低（P<0.01），死亡率升高（P<0.01）。与4%Sevo组比较，同一时间8%Sevo组HT22小鼠海马神经元细胞存活率降低（P<0.05），死亡率升高（P<0.05）。与4%Sevo 6 h组比较，4%Sevo 12 h组和4%Sevo 24 h组HT22小鼠海马神经元细胞存活率降低（P<0.05），死亡率升高（P<0.05）；与4%Sevo 12 h组比较，4%Sevo 24 h组HT22小鼠海马神经元细胞存活率降低（P<0.05），死亡率升高（P<0.05）。与8%Sevo 6 h组比较，8%Sevo 12 h组和8%Sevo 24 h组HT22小鼠海马神经元细胞存活率降低（P<0.05），死亡率升高（P<0.05）；与8%Sevo 12 h组比较，8%Sevo 24 h组HT22小鼠海马神经元细胞存活率降低（P<0.05），死亡率升高（P<0.05）。与生理盐水组比较，生理盐水+4%Sevo 12 h组和生理盐水+8%Sevo 12 h组HT22小鼠海马神经元细胞内DNA双链断裂增多，DNA损伤相关蛋白8-OHdG、ATM、p-ATM和γ-H2AX表达量增加、ROS水平升高（P<0.05）。与生理盐水+4%Sevo 12 h组比较，NAC+4%Sevo 12 h组HT22小鼠海马神经元细胞存活率升高（P<0.01），死亡率降低（P<0.01），细胞中DNA双链断裂减少，DNA损伤相关蛋白8-OHdG、ATM、p-ATM和γ-H2AX表达量减少，海马神经元细胞中ROS水平明显降低（P<0.05）；与生理盐水+8%Sevo 12 h组比较，NAC+8%Sevo 12 h组HT22小鼠海马神经元细胞存活率升高（P<0.01），死亡率降低（P<0.01），细胞中DNA双链断裂减少，DNA损伤相关蛋白8-OHdG、ATM、p-ATM和γ-H2AX表达量减少，海马神经元细胞中ROS水平明显降低（P<0.05）。结论：七氟醚能够通过诱导DNA损伤导致HT22小鼠海马神经元细胞死亡，其机制可能与诱导海马神经元细胞中ROS积聚有关。

DNA damage of HT22 mouse hippocampal neuronal cells induced by sevoflurane and its mechanism

Objective: To observe the DNA damage of HT22 mouse hippocampal neuronal cells induced by inhaled anesthetic sevoflurane, and to clarify the possible mechanism of neurotoxicity induced by sevoflurane. Methods: The HT22 mouse hippocampal neuronal cells were randomly divided into blank control group, 2% sevoflurane groups (2%Sevo 6 h group, 2%Sevo 12 h group, 2%Sevo 24 h group), 4% sevoflurane groups (4%Sevo 6 h group, 4%Sevo 12 h group, 4%Sevo 24 h group) and 8% sevoflurane groups (8%Sevo 6 h group,8%Sevo 12 h group, 8%Sevo 24 h group).The survival rates and the mortalities of HT22 mouse hippocampal neuronal cells in various groups were measured by MTT method and LDH methods,respectively.According to the experiment results, the cells were divided into normal saline group, normal saline +4%Sevo 12 h group, normal saline+8%Sevo 12 h group, NAC group, NAC+4%Sevo 12 h group,and NAC+8%Sevo 12 h group.MTT method and LDH method were used to detect the survival rates and the mortalities of HT22 mouse hippocampal neuronal cells in various groups after NAC pretreatment; single cell gel electrophoresis was used to detect the DNA double-strand breaks in the HT22 mouse hippocampal neuronal cells in various groups; Western blotting method was used to detect the expression amounts of 8-OHdG, ATM, p-ATM, and γ-H2AX in the HT22 mouse hippocampal neuronal cells in various groups. DCFH-DA method was used to detect the levels of reactive oxygen species (ROS) in the HT22 mouse hippocampal neuronal cells in various groups. Results: Compared with blank control group, there were no significant differences in the survival rates and the mortalities of HT22 mouse hippocampal neuronal cells in 2% Sevo groups (P>0.05); the survival rates of HT22 mouse hippocampal neuronal cells in 4%Sevo groups and 8%Sevo groups were significantly decreased (P<0.01), and the mortalities were significantly increased (P<0.01). Compared with 4% Sevo groups, the survival rates of HT22 mouse hippocampal neuronal cells in 8% Sevo groups at the same time were decreased (P<0.05), and the mortalities were increased (P<0.05).Compared with 4% Sevo 6 h group, the survival rates of HT22 mouse hippocampal neuronal cells in 4% Sevo 12 h group and 4% Sevo 24 h group were decreased (P<0.05), and the mortalities were increased (P<0.05). The survival rate of HT22 mouse hippocampal neuronal cells in 4% Sevo 24 h group was lower than that in 4% Sevo 12 h group (P<0.05), and the mortality was higher (P<0.05).Compared with 8%Sevo 6 h group,the survival rates of HT22 mouse hippocampal neuronal cells in 8% Sevo 12 h group and 8% Sevo 24 h group were decreased (P<0.05),and the mortalities were increased (P<0.05); the survival rate of HT22 mouse hippocampal neuronal cells in 8%Sevo 24 h group was lower than that in 8%Sevo 12 h group (P<0.05), and the mortality was higher (P<0.05). Compared with normal saline group, the amounts of DNA double-strand breaks in the HT22 mouse hippocampal neuronal cells in normal saline + 4% Sevo 12 h group and normal saline + 8% Sevo 12 h group were increassed, the expression amounts of DNA damage-related proteins 8-OHdG, ATM, p-ATM, and γ-H2AX were increased, and the ROS levels were increased (P<0.05). Compared with normal saline + 4% Sevo 12 h group, the survival rate of hippocampal neurons of the HT22 mice in NAC + 4% Sevo 12 h group was increased (P<0.01), the mortality was reduced (P<0.01), the amount of DNA double-strand breaks in the hippocampal neuronal cells was reduced, the expression amount of DNA damage-related proteins 8-OHdG, ATM, p-ATM, γ-H2AX were decreased, and the ROS level in the hippocampal neuronal cells was significantly reduced (P<0.05). Compared with normal saline + 8% Sevo 12 h group, the survival rate of HT22 mouse hippocampal neuronal cells in NAC + 8% Sevo 12 h group was increased (P<0.01), the mortality was decreased (P<0.01), and the amount of DNA double-strand breaks in the hippocampal neuronal cells was reduced, the expression amounts of DNA damage-related proteins 8-OHdG, ATM, p-ATM,and γ-H2AX were decreased, and the ROS level in hippocampal neuronal cells was significantly reduced (P<0.05). Conclusion: Sevoflurane could cause the death of HT22 mouse hippocampal neuronal cells by inducing DNA damage,and its mechanism may be related to the intracellular ROS accumulation induced by sevoflurane.