Cathepsin B抑制剂对发育期惊厥大鼠海马自噬相关蛋白表达的调节

The expression of beclin-1, cathepsin B and bcl-2 following recurrent neonatal seizures in hippocam-pus antophagia and its regulation by cathepsin-B inhibitor

目的探讨新生期大鼠反复惊厥后海马自噬、凋亡相关蛋白Beclin-1，Cathepsin B和Bcl-2的表达及溶酶体蛋白酶抑制剂CBI对表达的干预作用。方法实验在苏州大学神经科学研究所进行。日龄6d的Sprague-Dawley大鼠90只随机（随机数字法）分为3组，每组30只：对照组、反复惊厥组及CBI组。反复惊厥组（RS）每日吸入三氟乙醚诱导惊厥发作5次，每次间隔30min，连续9d；对照组同样操作但不吸人三氟乙醚；CBI组于惊厥前腹腔注射cathepsin B（2μL，0．5μg／μL），同样方法吸人三氟乙醚诱导惊厥发作。各组分别于末次惊厥后1．5h，3h，6h，24h和生后35d5个时间点[（n=6／（每点，每组）]取海马组织，采用免疫印迹技术检测自噬相关蛋白Becfin-1，Cathepsin B和Bcl-2的表达，采用方差分析进行统计。结果对照组和CBI组同一时间点Beclin-1，Cathepsin B和Bcl-2的表达差异无统计学意义，惊厥组（1．5h，3h，6h，24h）海马组织中Beclin-1和Cathepsin B的表达明显高于对照组和CBI组同一时间点的Beclin-1表达（F值分别为Beclin-1：7．44，6．58，5．43，9．69，P〈0．05；CathepsinB：4．62，2．02，3．49，4．86）；而惊厥组（1．5h，3h，6h，24h）Bcl-2的表达则明显低于对照组和CBI组同一时间点Bcl-2的表达（F值分别为4．34，3．79，4．88，4．42，P〈0．05）。结论发育期大鼠反复惊厥后海马神经元自噬信号通路激活，同时抗凋亡基因Bcl-2蛋白水平显著下调，表明自噬信号途径与凋亡途径共同激活，协调参与发育期大鼠惊厥急性期兴奋毒性脑损伤的病理生理机制。

Objective To explore the dynamic expressions of autophagia and apoptosis associated protein Beclin-1, Cathepsin B and Bcl-2 in hippocampus and the intervention efficacy of cathepsin-B inhibitor （CBI） after recurrent neonatal seizure. Method Ninty 6-day-old SD rats were randomly （random number） divided into the recurrent neonatal seizure group （RS group, n =30） , CBI-treated seizure group （CBI group, n = 30） and the control group （n = 30）. Rats in RS group were subjected to 55 attacks of seizure induced by using flurothyl during the consecutive 9 days beginning on the 6 th postnatal day （P6）. In CBI group, CBI （2 μL, 0.5 μg/μL） was administered every day before seizures induced. Western blot was employed to determine the protein level at different intervals （1.5 h,3 h,6 h,24 h） after the last convulsion. Results There were higher expressions of Beclin-1 and Cathepsin B, and lower expressions of Bcl-2 expression in RS group（ 1.5 h,3 h,6 h and 24 h）than those at the same time in control group （P 〈 0.05）. Beelin- 1 and Cathepsin B expressions were lower while Bel-2 expressions were higher in CBI group at the intervals of 1.5 h,3 h,6 h and 24 h compared with those in RS group （P 〈 0.05）. Conciusions Autophagie and apoptotie pathways were activated immediately after recurrent neonatal seizures as indicated by expression changes of Beelin-1, Cathepsin B and Bel-2 in hippocampus, which suggests a synergistic effect of the two pathways in the pathophysiology of the long-term brain damage of neonates resulted from the adverse effects of recurrent neonatal seizures.