自噬抑制剂 3-甲基腺嘌呤调节复发性新生儿癫痫后大鼠大脑皮质中 LC3、Beclin-1 和 ZnTs 的表达。
Autophagy inhibitor 3-methyladenine regulates the expression of LC3, Beclin-1 and ZnTs in rat cerebral cortex following recurrent neonatal seizures.
机构信息
Neurology Laboratory, Soochow University Affiliated Children's Hospital; Laboratory of Aging and Nervous Diseases, Soochow University, Suzhou 215003, China.
出版信息
World J Emerg Med. 2010;1(3):216-23.
BACKGROUND
Autophagy is a homeostatic process for intracellular recycling of bulk proteins and aging organelles. Increased autophagy has now been reported in experimental models of traumatic brain injury, stroke and excitotoxicity, and in patients with Alzheimer's disease and critical illness. The role of autophagy in developmental epilepsy, however, is unknown. The present study was to investigate the effects of recurrent neonatal seizure, in the presence and absence of autophagy inhibitor 3-methyladenine (3-MA), on the acute phase gene expression of ZnTs, LC3 and Beclin-1 in rat cerebral cortex and the interaction among them.
METHODS
Thirty-six Sprague-Dawley neonatal rats at postnatal day 6(P6) were randomly divided into three groups: a recurrent-seizures group (RS, n=12), a 3-MA treated-seizure group (3-MA group, each rat pretreated with 3-methyladenine before seizures, 100nmol/μl/day, i.p., n=12) and a control group (n=12). At 1.5 and 6 hours after the last seizures, the mRNA levels of ZnT1-ZnT3, microtubule-associated protein 1A/1B light chain 3 (LC3) and beclin-1 were detected using the real-time RT-PCR method. The LC3 protein level was examined by Western blotting.
RESULTS
The levels of LC3, beclin-1 and ZnT-2 transcripts in the RS group elevated significantly at 1.5 and 6 hours after the last seizures compared with those in the control and 3-MA groups. At the interval of 1.5 hours, the mRNA level of ZnT-1 increased significantly after the last seizure compared with that in the control group. There was no significant difference in the transcript levels of ZnT-3 among the three groups. Linear correlation analysis showed that the expression of the five genes in the control group exhibited a significant inter-relationship. In the 3-MA group, however, the inter-relationship was only found between beclin-1 and ZnT-1. In the RS group, the inter-relationship was not observed.
CONCLUSIONS
The autophagy/lysosomal pathway is immediately activated along with the elevated expression of ZnT1 and ZnT2 in the cerebral cortex after recurrent seizures. 3-MA is involved in the regulation of the autophagy/lysosomal pathway and ZnTs by down-regulating the expression of LC3 and beclin-1.
背景
自噬是细胞内回收大量蛋白质和衰老细胞器的一种内稳态过程。现在已经在创伤性脑损伤、中风和兴奋毒性的实验模型以及阿尔茨海默病和危重病患者中报告了自噬增加。然而，自噬在发育性癫痫中的作用尚不清楚。本研究旨在研究复发性新生儿癫痫（存在和不存在自噬抑制剂 3-甲基腺嘌呤（3-MA））对大鼠大脑皮层急性相基因表达 ZnTs、LC3 和 Beclin-1 的影响，并研究它们之间的相互作用。
方法
36 只出生后 6 天（P6）的 Sprague-Dawley 新生大鼠随机分为三组：复发性癫痫组（RS 组，n=12）、3-MA 处理癫痫组（3-MA 组，每组大鼠在癫痫发作前 100nmol/μl/天，腹腔注射 3-甲基腺嘌呤，n=12）和对照组（n=12）。在最后一次癫痫发作后 1.5 和 6 小时，采用实时 RT-PCR 法检测 ZnT1-ZnT3、微管相关蛋白 1A/1B 轻链 3（LC3）和 Beclin-1 的 mRNA 水平。通过 Western blot 检测 LC3 蛋白水平。
结果
RS 组在最后一次癫痫发作后 1.5 和 6 小时 LC3、Beclin-1 和 ZnT-2 转录物水平明显升高，与对照组和 3-MA 组相比。在 1.5 小时的时间间隔内，与对照组相比，最后一次癫痫发作后 ZnT-1 的 mRNA 水平显著增加。三组之间 ZnT-3 的转录物水平无显著差异。线性相关分析显示，对照组中五个基因的表达存在显著的相互关系。然而，在 3-MA 组中，仅发现 Beclin-1 和 ZnT-1 之间存在相互关系。在 RS 组中，没有观察到相互关系。
结论
复发性癫痫后，大脑皮层中 ZnT1 和 ZnT2 的表达升高，自噬/溶酶体途径立即被激活。3-MA 通过下调 LC3 和 Beclin-1 的表达参与自噬/溶酶体途径和 ZnTs 的调节。
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