甘氨酸转运体1抑制剂对癫痫模型小鼠痫性发作和认知障碍的作用

Effect of glycine transporter 1 inhibitor on epileptic seizures and cognitive dysfunction in epilepsy mice

目的探讨不同剂量甘氨酸转运体1抑制剂M22对戊四氮致癫痫模型小鼠痫性发作及认知障碍的作用。方法110只C57BL/6小鼠按照随机数字表法分为6组:正常对照组(CON组,n=10)、模型组(Mod组,n=20)、M22-1组(n=20)、M22-2组(n=20)、M22-3组(n=20),M22-4组(n=20)。采用腹腔注射戊四氮PTZ (30 mg/kg)制备癫痫小鼠模型,连续注射2周。CON组小鼠腹腔注射生理盐水(10 ml/kg),于注射PTZ前30 min,模型组给予腹腔注射生理盐水(10 ml/kg)。M22-1组,M22-2组,M22-3组,M22-4组干预组分别腹腔注射M22 10 mg/kg、20 mg/kg、40 mg/kg、80 mg/kg,连续2周。采用癫痫发作Racine分级标准观测癫痫发作等级;采用Morris水迷宫实验评估小鼠的学习记忆能力;Western blot检测小鼠大脑皮层凋亡相关蛋白Bcl-2、Bax和Cyt-c的相对表达量。结果(1)M22-2组和M22-3组小鼠模型死亡率、点燃率、痫性发作等级及强直阵挛发作率均明显低于Mod组、M22-1组及M22-4(均P<0.05),且M22-3组抑制痫性发作的作用显著优于其他M22组(P<0.05)。(2)定向航行实验中各组小鼠的逃避潜伏期随着时间均呈缩短趋势,于第4天,与Mod组小鼠的逃避潜伏期[(30.24±9.46)s]比较,M22-3组小鼠的逃避潜伏期[(16.05±5.72)s]明显缩短,差异有统计学意义(t=20.36,P<0.05)。空间探索实验中,与Mod组比较,M22-3组小鼠的穿越站台次数[(6.45±3.62)次,(3.23±2.47)次;t=38.63,P=0.004]增多,目标象限活动时间[(21.53±6.38)s,(11.52±3.15)s;t=37.53,P<0.05)]变长,均差异有统计学意义。(3)Western blot结果显示,M22-3组小鼠的Bcl-2显著高于Mod组(P<0.05),Bax及Cyt-c显著低于Mod组(均P<0.05)。而M22-1及M22-4组小鼠Bcl-2、Bax和Cyt-c与模型组比较差异无统计学意义(均P>0.05)。结论M22(40 mg/kg)有显著的抗癫痫作用,且能有效改善癫痫小鼠的认知功能障碍,可能与抑制小鼠神经元凋亡有关。

Objective To study the effect of glycine transporter 1 inhibitor M22 on epileptic seizures and cognitive dysfunction in epilepsy mice. Methods A total of 110 C57BL/6 mice were randomly divided into Normal control group (CON group, n=10), Model group (Mod group n=20), M22-1 group (n=20), M22-2 group (n=20), M22-3 group (n=20), M22-4 group (n=20) according to weight.The chronic epileptic model was established by intraperitoneal injection of PTZ(30 mg/kg). The mice in CON group was injected with normal saline(10 mg/kg). The mice in Mod group were intraperitoneally injected with normal saline (10 ml/kg) and were injected with PT2 30 min later.The mice in M22-1 group, M22-2 group, M22-3 group, M22-4 group were intraperitoneally injected with M22 of corresponding dose(10 mg/kg, 20 mg/kg, 40 mg/kg, 80 mg/kg)respectively, lasting for 2 weeks.Epilepsy seizures of mice in each group were recorded.The learning and memory function of epilepsy mice were evaluated by Morris water maze test .Then the mice were sacrificed and the apoptosis related proteins Bcl-2, Bax, Cyt-c in the cerebral cortex of mice were measured by Western blot. Results (1)The mortality kindling rate, epileptic seizure grade and rate of tonic clonus in M22-2 and M22-3 group were significantly lower than those in Mod, M22-1 and M22-4 group(all P<0.05).(2) In the directional navigation experiment, the escape latency of mice in each group decreased with time. On the 4th day, the escape latency of mice in M22-3 group was significantly shorter than that in Mod group, and the difference was statistically significant ((30.24±9.46),(16.05±5.72), t=20.36, P<0.05). In space exploration experiment, compared with Mod group, M22-3 group had more times of crossing platform ((6.45±3.62),(3.23±2.47), t=38.63, P=0.004) and longer time of target quadrant activity((21.53±6.38) s,(11.52±3.15) s, t=37.53, P<0.05).(3)It was showed by Western blotting that the relative expression levels of Bcl-2 in M22-3 group were significantly higher than those in Mod group(P<0.05), while the Bax and Cyt-c in M22-3 group were significantly lower than those in Mod group(P<0.05). There was no significant difference in Bcl-2, Bax and Cyt-c between M22-1 group, M22-4 group and model group(P>0.05). Conclusion M22 (40 mg/kg) has significant anti-epileptic effect and can effectively improve the cognitive dysfunction of epileptic mice, which may be related to the inhibition of neuronal apoptosis in mice.