Toll样受体4／核因子-κB信号通路在癫痫持续状态大鼠海马损伤中的作用

Role of TLR4/NF-κB signal pathway in the hippocampus injury of status epileptic rats

目的通过观察癫痫持续状态（status epilepticus，SE）后大鼠海马Toll样受体4（Toll—like receptor4，TLR4）及核因子-κB（nuclear factorκB，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72h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30min，给予100mg／kgPDTC腹腔注射，每天1次，连用3d。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／v65蛋白的表达变化；逆转录-聚合酶链反应（RT—PCR）技术检测海马TLR4mRNA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72h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±0．0329，B4组0．1604±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TLR4mRNA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72h达到高峰（1．242±0．100）；C组海马TLR4mRNA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。

Objective 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-κB 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 piloearpine, 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 C than in group B4. The expression of TLR4 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 TLR4 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 IM group （ t = - 0. 2578, P 〈 0. 01 ）. There was a coincidence between the expression of TLR4 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.