摘要目的 探讨Beclin-1、P62/SQSTM1、微管相关蛋白1轻链3(LC3)和unc-51样自噬活化激酶1 (ULK-1)等自噬相关分子mRNA水平在发育期大鼠癫痫脑损伤前后的变化.方法 21日龄雄性Sprague Daw-ley(SD)大鼠72只,按随机数字表法随机分为对照组和癫痫组.每组按时间点不同(3 h、6 h、12 h和48 h)随机分4个亚组,每个亚组9只大鼠.癫痫组采用腹腔注射卡因酸(12 mg/kg)诱导癫痫发作建立癫痫动物模型,对照组予等量9 g/L盐水腹腔注射.2组大鼠分别于注射卡因酸3 h、6 h、12 h和48 h后,予水合氯醛麻醉,处死并取出脑组织.Nissl染色法检测大鼠大脑皮层神经元损伤情况;脱氧核糖核苷酸末端转移酶介导的缺口末端标记法(TUNEL)检测癫痫大鼠大脑神经元凋亡情况;实时荧光定量聚合酶链反应(qPCR)检测Beclin-1、P62/SQSTM1、LC3、ULK-1 mRNA水平.结果 Nissl染色显示:随时间推移,癫痫组大鼠大脑皮层神经元尼氏体颜色变浅,结构紊乱;癫痫48 h组Nissl染色阳性细胞数[(82 ± 8)个]明显少于对照组[(122 ± 8)个],差异有统计学意义(F＝3. 768,P＝0. 01).TUNEL方法显示癫痫48 h组大鼠大脑皮层凋亡神经元[(13 ± 7)个]明显高于对照组[(2 ± 1)个],差异有统计学意义(t ＝ -3. 821,P ＝0. 003).qPCR显示 Beclin-1、P62/SQSTM1、LC3、ULK-1 mRNA水平在癫痫组12 h组(1. 70 ± 0. 75、1. 75 ± 0. 77、1. 52 ± 0. 43、7. 48 ± 6. 12)和癫痫48 h组(1. 63 ± 0. 43、1. 48 ± 0. 74、1. 74 ± 0. 55、7. 69 ± 5. 65)均明显高于对照组(1. 00、1. 00、1. 00、1. 00),差异均有统计学意义(F＝2. 820、3. 452、5. 811、5. 002,均P＜0. 05).结论 自噬的活化先于凋亡发生,自噬相关分子可能参与发育期大鼠癫痫所致脑损伤的发病过程.

abstractsObjective To explore the changes of Beclin-1,P62/SQSTM1,microtubule-associated protein 1 light chain 3 (LC3)and unc-51 like autophagy activating kinase 1 (ULK-1)in the brains of the rats in the deve-lopmental stage with epilepsy. Methods Seventy-two male Sprague Dawley (SD)rats aged 21 days were randomly divided into the control group and the epilepsy group. The rats in 2 groups were randomly subdivided into 4 groups according to the time intervals (3 h,6 h,12 h and 48 h),respectively,with 9 rats in each group. The rats in the epilep-sy group were injected with kainic acid (12 mg/kg)to induce epilepsy,and the rats in the control group were injected with equal volume of saline. The rats in 2 groups were anaesthetized and sacrificed. Then,the brain tissues of the rats were quickly removed according to the time intervals. The brain damages were determined by adopting Nissl staining method. The apoptotic cells were detected by Terminal - deoxynucleoitidyl transferase mediated nick end labeling (TUNEL)assays. The expressions of Beclin-1,P62/SQSTM1,LC3 and ULK-1 mRNA levels in cortex were mea-sured by using real-time quantitative polymerase chain reaction (qPCR)analysis. Results Nissl staining indicated that many neurons were damaged performing vague outline,irregularly aligned,pyknotic nuclei and shrunken somata in the epilepsy 48 h group. In addition,there was a huge loss of neurons in cortex in the epilepsy 48 h group [(82 ± 8)num-bers],compared with the control group [(122 ± 8)numbers],and the difference was statistically significant (F＝3. 768, P＝0. 01). The apoptotic cells tremendously increased in the epilepsy 48 h group [(13 ± 7)numbers],compared with the control group [(2 ± 1)numbers]by TUNEL analysis,and the diffe-rence was statistically significant (t＝ -3. 821, P＝0. 003). qPCR showed the mRNA levels of Beclin-1,P62/SQSTM1,LC3 and ULK-1 were upregulated in the epi-lepsy 12 h group (1. 70 ± 0. 75,1. 75 ± 0. 77,1. 52 ± 0. 43,7. 48 ± 6. 12)and the epilepsy 48 h group (1. 63 ± 0. 43, 1. 48 ± 0. 74,1. 74 ± 0. 55,7. 69 ± 5. 65),compared with the control group (1. 00,1. 00,1. 00,1. 00),and the differences were statistically significant (F＝2. 820,3. 452,5. 811,5. 002,all P＜0. 05). Conclusion The autophagy activates be-fore apoptosis occurs,and autophagy-related genes probably are involved in epilepsy-induced brain damage.