低锌饮食对大鼠二次打击惊厥模型远期惊厥阈和锌转运体3的影响

Effects of low-zinc diet on long-term seizure threshold and zinc transporter 3 in rats with two-hit convulsion model

目的:探讨低锌饮食对大鼠二次打击惊厥模型远期惊厥阈、死亡率和锌转运体3(Zn T3)的影响。方法:60只27日龄(P27)健康雄性SD大鼠随机分为4组,惊厥组和惊厥低锌饮食组在P27腹腔注射氯化锂、在P28腹腔注射匹罗卡品致惊厥持续状态模型,观察大鼠惊厥发作情况,将达到Ⅳ级及以上痫性发作的视为造模成功。对照组和低锌饮食组在相同时间腹腔注射生理盐水。致惊厥前2天(即P27、P28) 4组均予常锌饮食(锌44 mg/kg)喂养,致惊厥后(即P28后)对照组和惊厥组继续予常锌饮食喂养,而低锌饮食组和惊厥低锌饮食组予低锌饮食(锌2. 7 mg/kg)喂养。2周后予青霉素二次打击,记录大鼠首次出现惊厥发作的时间(惊厥阈)、死亡时间及死亡率,观察时间为1 h。观察结束后取各组大鼠皮层组织,蛋白质印迹法检测Zn T3的表达。结果:大鼠的持续惊厥模型诱导成功率为72. 5%(29/40),死亡率为24. 1%(7/29)。惊厥第3天、5天、7天惊厥组、惊厥低锌饮食组体重明显低于对照组(P <0. 05);惊厥第9天惊厥低锌饮食组体重明显低于对照组(P <0. 05)。惊厥组和惊厥低锌饮食组血清锌浓度均明显低于对照组(P <0. 05)。低锌饮食组、惊厥组、惊厥低锌饮食组惊厥阈明显低于对照组(P <0. 05),惊厥低锌饮食组明显低于低锌饮食组和惊厥组(P <0. 05)。惊厥低锌饮食组生存时间明显低于对照组和低锌饮食组(χ~2分别为9. 994、6. 707,均P <0. 05)。对照组、低锌饮食组、惊厥组和惊厥低锌饮食组存活率分别为80%、60%、50%、20%,无统计学差异(P=0. 073)。低锌饮食组、惊厥组及惊厥低锌饮食组Zn T3蛋白表达明显低于对照组(P <0. 05),惊厥低锌饮食组明显低于惊厥组和低锌饮食组(P <0. 05)。结论:惊厥可致大鼠血清锌浓度降低,缺锌膳食能增加惊厥后再次打击时惊厥发作的易感性,二者互为增强效应,这种现象可能是通过下调Zn T3的表达,破坏大脑锌稳态而导致。

Objective: To investigate the effects of low-zinc diet on long-term seizure threshold,mortality and zinc transporter 3( ZnT3) in rats with two-hit convulsion model. Methods: A total of 60 postnatal day 27( P27) healthy male Sprague-Dawley rats were randomly divided into 4 groups. In the convulsion group and the convulsion with low-zinc diet group,the seizure persistence model were induced by intraperitoneal injection of lithium chloride in P27 and intraperitoneal injection of pilocarpine in P28. By observing the seizures in rats,the seizures of grade Ⅳ and above are considered successful. The control group and the low-zinc diet group were intraperitoneally injected with physiological saline at the same time. Two days before the convulsions( P27,P28) all rats were fed with the zinc diet( zinc 44 mg/kg).After the convulsions( after P28),the control group and the convulsion group continued to be fed with the zinc diet,while the low-zinc diet group and the convulsive low-zinc diet group were fed a low-zinc diet( zinc 2. 7 mg/kg). After 2 weeks,penicillin was given as a second strike,and the time of first seizures( seizure threshold),time of death and mortality were recorded. The observation time was 1 h. At the end of the observation,the cortical tissues of each group were isolated,and the expression of ZnT3 was detected by Western blotting. Results: The success rate of continuous convulsion model in rats was 72. 5%( 29/40),and the mortality rate was 24. 1%( 7/29). On the 3 rd,5 th,and 7 th day of convulsion,the body weight of the convulsion group and the convulsion with low-zinc diet group was significantly lower than that of the control group( P < 0. 05). The convulsion on the 9 th day was significantly lower than that of the control group( P < 0. 05). The serum zinc concentration in the convulsion group and the convulsion with low-zinc diet group were significantly less than those in the control group( P < 0. 05). The seizure threshold of the low-zinc diet group,the convulsion group and the convulsion low-zinc diet group was significantly lower than that of the control group( P < 0. 05),the convulsion low-zinc diet group was significantly lower than the low-zinc diet group and the convulsion group( P < 0. 05). The survival time of the convulsive low-zinc diet group was significantly lower than the control group and the low-zinc diet group( χ~2= 9. 994,6. 707,all P < 0. 05). The survival rates of the groups were 80%,60%,50%,and20%,there was no statistical difference( P = 0. 073). The expression of Zn T3 protein in the low-zinc diet group,the convulsion group and the convulsion low-zinc diet group were significantly lower than that of the control group( P < 0. 05),the convulsion low-zinc diet group was significantly lower than that of the convulsion group and the low-zinc diet group( P < 0. 05). Conclusion: Convulsion can reduce the serum zinc concentration in rats. The zinc deficiency diet can increase the susceptibility of two-hit seizure threshold in rats. This synergistic effect is related to the down-regulating the expression of ZnT3 and the zinc dys homeostasis in the brain.