发育期大鼠青霉素点燃后大脑皮质自噬标记蛋白LC-3表达及E-64d干预效应的研究

The expression of LC-3 following penicillin-induced developmental seizures in cortex and the intervention by E-64d in young rat

目的研究发育期大鼠反复惊厥后大脑皮层中自噬标记蛋白LC-3的表达,以及E-64d对其表达的影响。方法生后21 d的SD大鼠随机分成惊厥组、对照组、干预组3组,每组24只。惊厥组大鼠隔日腹腔注射青霉素(4.8×106U/kg),连续6次,诱导惊厥发作;对照组大鼠予同等剂量的生理盐水腹腔注射;干预组大鼠于腹腔注射青霉素诱导惊厥发作前,腹腔注射E-64d(4μg)。惊厥组和干预组大鼠于末次惊厥后3 h、12 h、24 h和出生51 d处死并取其大脑皮质,对照组大鼠也在相应时间点取大脑皮质。采用蛋白质免疫印迹技术(Western-blot)分别检测大鼠大脑皮质中自噬标记蛋白LC-3的表达。结果惊厥组大鼠于末次惊厥后3 h、12 h、24 h大脑皮层LC3II/LC3I较对照组升高,差异有统计学意义(t=2.091～10.353,P均<0.05);干预组与惊厥组大鼠比较,LC3II/LC3I降低,差异有统计学意义(t=2.911～5.980,P均<0.05)。在出生51 d时,三组大鼠间差异无统计学意义(P均>0.05)。结论大鼠惊厥急性期LC-3蛋白明显上调,自噬途径被激活,而E-64d参与了自噬途径的调控。

Objective To explore the dynamic expression of autophagy-associated protein LC-3 in cerebral cortex in young rats with recurrent seizure and the effect of E-64d intervention on LC-3 expression. Methods SD rats, 21- day-old （P21）, were randomly divided into three groups of 24 rats each, recurrent seizure group, control group and intervention group. Penicillin （4.8 × 10^6 U/kg） were administrated in recurrent seizure and intervention groups by intraperitoneal injection six times, once every other day to induce seizure; E-64d （4 μg） was administrated in intervention group by intraperitoneal injection before inducing seizure. Normal saline were administrated in control group. The rats had been slaughtered to take the cerebral cortex at 3 h, 12 h, 24 h after the last induced seizure and at 51-day-old （P51）. The expressions of LC-3 protein in cerebral cortex at different time points were detected by Western-blot. Results At 3 h, 12 h and 24 h after the last induced seizure, the ratio of LC3II/LC3I in cerebral cortex was significantly increased in recurrent seizure group than that in control group （t = 2.091 - 10.353, P 〈 0.05）, and significantly decreased in intervention group than that in recurrent seizure group （t = 2.911 - 5.980, P 〈 0.05）. At P51, the ratio of LC3Ⅱ/ LC3I were not significhntly different among three groups （P 〉 0.05）. Conclusions Autophagy/lysosomal pathway were activated immediately after recurrent seizures as indicated by the elevated expression of LC-3 in cerebral cortex. E-64d was involved in the regulation of autophagy/lysosomal pathway.