3-甲基腺嘌呤对反复惊厥新生大鼠大脑皮层自噬相关蛋白Beclin1表达的影响

Effects of 3-Methlyadenine on Dynamic Expression of Cerebral Cortex Autophagy-Associated Protein Beclin1 in Newborn Rats with Recurrent Seizure

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 explore the dynamic expression of autophagy - associated protein Beclinl in cerebral cortex in newborn rats with recurrent seizure and the influence of 3 - methlyadeine （3 - MA）on Beclinl expression. Methods One hundred and eight rats were di- vided into the recurrent neonatal seizure group （RS group, n = 36）, the 3 -MA- treated seizure group （3 -MA group, n = 36） and control group （ n = 36 ）. Rats in RS group were subjected to 5 seizures with flurothyl during the consecutive 9 days beginning on postnatal day 6 （P6）. In 3 - MA group, 3 - MA （2μL） was injected each day before seizure was induced. Western blot analysis was used to determine Beclinl protein level in cerebral cortex at different time points （ 1.5 h,3.0 h ,6.0 h,24.0 h and 48.0 h after the last convulsion）. Results Beclinl protein level in RS group at the time point of （1.5 h,3.0 h,6.0 h,12.0 h,24.0 h）was significantly higher than those at the corresponding time point in control group（ t = 2. 249 ,2. 426 ,3. 002,2. 385 ,1. 542 P,〈0.05）. The protein level in 3 - MA group at the time point（1. 5 h, 3.0 h,6.0 h,12.0 h）was decreased significantly compared with those in RS group （t =2.251,2.426,2.252,2.460 P 〈0.05）. There was no significant difference of Beclinl expression at 48.0 h between RS group and control group （ t = 1. 502 P 〉 0.05 ）, and the protein level at 24.0 h and 48.0 h was also not significantly different between 3 - MA group and RS group （ t = 1. 542,1. 285 Pn 〉 0.05 ）. Conclusions Autop-hagy/lysosomal pathway was activated immediately after recurrent seizures as indicated by the elevated expression of Beclinl in cerebral cortex. 3 - MA was involved in the regulation of autophagy/lysosomal pathway by down - regulating the expression of Beclinl.