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目的探讨反复惊厥新生大鼠大脑皮层中自噬相关蛋白Beclin1的表达及3-甲基腺嘌呤(3-MA)对其表达的干预作用。方法出生6dSD大鼠随机分成惊厥组、3-MA组和对照组3组,每组36只。惊厥组新生大鼠每天吸入三氟乙醚诱导惊厥发作5次,每次间隔30min,连续9d;对照组新生大鼠同样操作,但不吸入三氟乙醚;3-MA组新生大鼠惊厥前腹腔注射3-MA(总量2μL),同样方法吸入三氟乙醚诱导惊厥。3组新生大鼠分别于最后1次惊厥后1.5h、3.0h、6.0h、12.0h、24.0h、48.0h处死并取其大脑皮层,采用蛋白质免疫印迹技术(Western blot)分别检测3组新生大鼠大脑皮层Beclin1的表达。结果1.5h、3.0h、6.0h、12.0h、24.0h惊厥组新生大鼠大脑皮层Beclin1的表达明显高于对照组同一时间点Beclin1的表达(t=2.249,2.426,3.002,2.385,1.542Pa<0.05),1.5h、3.0h、6.0h、12.0h3-MA组新生大鼠大脑皮层Beclin1的表达明显低于惊厥组同一时间点的表达(t=2.251,2.426,2.252,2.460Pa<0.05)。48.0h惊厥组Beclin1的表达与对照组比较无显著差异(t=1.502P>0.05),24.0h和48.0h3-MA组Beclin1的表达与惊厥组比较亦无明显差异(t=1.542,1.285Pa>0.05)。结论惊厥后Beclin1蛋白水平明显上调,表明自噬途径被激活,3-MA通过抑制Beclin1表达,参与自噬途径的调控。
Objective To investigate the expression of autophagy-related protein Beclin1 in the cerebral cortex of neonatal rats with recurrent seizures and the effect of 3-methyladenine (3-MA) on its expression. Methods Six-day-old SD rats were randomly divided into 3 groups: convulsion group, 3-MA group and control group, with 36 rats in each group. The convulsive group of neonatal rats inhaled trifluoroethyl ether daily induced convulsion seizures 5 times, each interval of 30min, continuous 9d; control group of new rats the same operation, but not inhaled trifluoroacetate; 3-MA group of neonatal rats pre-convulsive intraperitoneal injection 3-MA (total 2 μL), the same way inhalation of trifluoro-ether induced seizures. Three groups of neonatal rats were sacrificed at 1.5h, 3.0h, 6.0h, 12.0h, 24.0h and 48.0h after the last convulsion, respectively. The cerebral cortex was taken and the three newborn rats were detected by Western blot Expression of Beclin1 in rat cerebral cortex. Results The expression of Beclin1 in neonate rat cerebral cortex at 1.5h, 3.0h, 6.0h, 12.0h and 24.0h was significantly higher than that in the control group at the same time points (t = 2.249,2.426,3.002,2.385,1.542Pa < 0.05). The expression of Beclin1 in neonate rat cerebral cortex at 1.5h, 3.0h, 6.0h, 12.0h3-MA group was significantly lower than that at the same time point in convulsion group (t = 2.251,2.426,2.252,2.460Pa <0.05). The expression of Beclin1 in 48.0h convulsion group was not significantly different from that in control group (t = 1.502P> 0.05). The expression of Beclin1 in 24.0h and 48.0h3-MA group was also not significantly different from that in seizure group (t = 1.542,1.285Pa> 0.05). Conclusions Beclin1 protein levels were significantly upregulated after convulsions, indicating that autophagy pathway is activated. 3-MA is involved in the regulation of autophagy by inhibiting Beclin1 expression.