摘要目的 通过观察癫疴持续状态(status epilepticus,SE)后大鼠海马Toll样受体4(Toll-like receptor 4,TLR4)及核因子-κB(nuclear factor KB,NF-κB)的表达;并观察应用NF-κB抑制剂吡咯烷二硫代氨基甲酸盐(PDTC)后,对TLR4/NF-κB信号通路及海马损伤的影响,探讨TLR4/NF-κB信号通路在SE后大鼠海马损伤的发生发展过程中的作用.方法 106只SD大鼠随机分为对照组(A组)、SE组(B组)和PDTC干预组(C组),其中B组再随机分为B1～B4组(分别于惊厥后4、24、48和72 h处死),B组和C组采用氯化锂.匹罗卡品法制作大鼠SE模型,C组在大鼠惊厥终止后30 min,给予100 mg/kg PDTC腹腔注射,每天1次,连用3 d.光镜下观察大鼠海马病理学改变;免疫组织化学法检测海马TLR4和NF-κB/p65蛋白的表达变化;逆转录-聚合酶链反应(RT-PCR)技术检测海马TLR4 mRNA表达的动态改变.结果 长程惊厥发作后,脑内神经元损伤存在动态变化,在72 h内随着观察时间的延长,神经元损伤逐渐加重,C组改变较B4组明显减轻.B组各时间点TLR4蛋白的表达(B1组0.1287±0.0260,B2组0.1296±0.0285,B3组0.1330 4-0.0329,B4组0.1604 4-0.0457)均明显高于A组(0.0964±0.0324,t=0.0641～0.3236,P<0.05),并随时间延长明显增高;C组TLR4蛋白表达(0.1271±0.0330)较B4组显著降低(t=-0.0334,P<0.01).B组可见NF-κB/p65蛋白在胞核内有不同程度表达,与A组比较差异有统计学意义(P<0.05);C组与B4组比较,NF-κB/p65蛋白表达水平明显降低(P<0.01).B组各时间点TLR4 mRNA的表达均较A组(0.268±0.072)高(P<0.05),且随时间延长逐步升高,惊厥后72 h达到高峰(1.242±0.100);C组海马TLR4 mRNA的表达(0.984±0.263)明显低于B4组(t=-0.2578,P<0.05).各组TLR4的表达情况与NF-κB/p65一致.结论 ,TLR4及NF-κB/p65在SE后的大鼠海马中表达升高,NF-κB抑制剂PDTC可以下调TLR4的表达,并减轻惊厥后海马病理损伤程度,提示TLR4/NF-κB信号通路在惊厥后海马损伤的发生发展过程中起促进作用.

abstractsObjective To observe the expression of TLR4 and NF-κB in hippocampus injury of status epileptic rats and to study the regulating effect of PDTC on TLR4/NF-KB signal pathway and hippocampus injury, and to explore the role of TLR4/NF-κB signal pathway in the hippocampus injury of SE rats. Methods A hundred and six male Sprague-Dawley (SD) rats were randomly divided into control group (A), convulsion group (B), PDTC group (C), and group B were randomly divided into 4 subset groups (B1-B4), which would be executed at 4, 24, 48 and 72 hours after convulsion. Continuous epilepticus was induced by injecting lithium chloride and pilocarpine, and group C were daily injected with 100 mg/kg PDTC 30 minutes after convulsion stopped for 3 days. Then the histopathology changes in hippocampus were viewed by HE staining, TLR4 and NF-κB/p65 protein were detected by immunohistochemistry (IHC), the expression of TLR4 mRNA were detected by RT-PCR. Results Neuronal injury was observed after a long time of convulsion, and the change was increased gradually 72 hours after seizure, which was milder in group Cthan in group B4. The expression of TLB4 protein in group B (B1-B4 were 0.1287±0. 0260, 0. 1296± 0. 0285, 0. 1330±0. 0329 and 0. 1604±0. 0457, respectively) was significantly higher than in group A (0.0964±0.0324, t =0.0641-0.3236, all P<0.05), and that in group C (0.1271±0.0330) was much lower than in B4 group (t = -0. 0334, P <0. 01). The IHC staining of NF-κB/p65 showed that hippocampal neurons had positive expression in cell nucleus in group B compared with the group A (P < 0. 05), and the expression of NF-κB/p65 protein in group C was much lower than that in group B4 (P < 0. 01). The mRNA expression of TLB4 in rat hippocampus of group B were significantly elevated than that in group A (0. 268±0. 072, P < 0. 05), and the tendency was increased gradually, reaching the peak at 72 hours after seizure (1. 242±0. 100), and that in group C (0. 984±0. 263) was much lower than that in B4 group (t=-0.2578, P<0.01). There was a coincidence between the expression of TLB4 and NF-κB/ p65. Conclusions The increased expression of TLR4 and NF-κB/p65 in SE rat hippocampus may play an promotion role on the development of the hippocampus injury; PDTC can down regulate the expression of TLR4, and lessen the pathologic changes of hippocampus.