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目的探讨新生期大鼠反复惊厥后海马丛生蛋白(Clusterin)的表达及溶酶体蛋白酶抑制剂Cathepsin B(CBI)对其表达的干预作用。方法 6日龄SD大鼠随机分为3组:对照组、惊厥组(RS组)、CBI组,每组6只。RS组新生大鼠每日吸入0.04～0.05 mL三氟乙醚诱导其惊厥发作5次,每次间隔30 min,连续9 d;对照组同样操作但不吸入三氟乙醚;CBI组惊厥前腹腔注射CBI(2μL,0.5 g.L-1),采用同样方法吸入三氟乙醚诱导其惊厥发作。于出生35 d(P35)处死3组大鼠,并取其海马组织。采用免疫印迹技术检测其Clusterin的表达。3组蛋白水平比较采用SPSS 17.0软件进行方差分析。结果 RS组海马中Clusterin表达(1.19±0.10)明显高于对照组(0.64±0.24),两两比较差异有统计学意义(P<0.01)。CBI组海马Clusterin表达(0.80±0.16)明显低于RS组,差异有统计学意义(P<0.01),CBI组海马Clusterin表达与对照组比较差异无统计学意义。结论惊厥后Clusterin蛋白表达水平明显上调,表明Clusterin参与了新生大鼠反复惊厥后所致脑损伤的分子机制,CBI对其表达有明显干预作用。
Objective To investigate the expression of clusterin and the effect of lysosomal protease inhibitor Cathepsin B (CBI) on the expression of clusterin in neonatal rats after recurrent seizures. Methods Six-day-old SD rats were randomly divided into 3 groups: control group, convulsion group (RS group), CBI group, 6 rats in each group. Rats in RS group were exposed to trifluoroethane (0.04-0.05 mL) for 5 times a day for seizures at intervals of 30 min for 9 consecutive days. The control group received the same operation but did not inhaled trifluoroacetic acid. The CBI group was injected intraperitoneally with CBI (2μL, 0.5gL-1), using the same method of inhalation of trifluoroethyl ether induced seizures. Three groups of rats were sacrificed 35 days after birth (P35), and their hippocampal tissues were obtained. The expression of Clusterin was detected by Western blotting. 3 protein levels were compared using SPSS 17.0 software ANOVA. Results The expression of Clusterin in RS group was significantly higher than that in control group (1.19 ± 0.10) (0.64 ± 0.24) (P <0.01). The expression of Clusterin in hippocampus of CBI group (0.80 ± 0.16) was significantly lower than that of RS group (P <0.01). There was no significant difference in Clusterin expression in CBI group as compared with control group. Conclusions The expression of Clusterin protein in convulsions is significantly increased, indicating that Clusterin is involved in the molecular mechanism of brain injury induced by recurrent seizures in neonatal rats. CBI has a significant intervention on its expression.