解剖学报

解剖学报 ›› 2014, Vol. 45 ›› Issue (3): 321-327.doi: 10.3969/j.issn.0529-1356.2014.03.005

• 神经生物学 • 上一篇    下一篇

白藜芦醇对惊厥持续状态大鼠海马Toll样受体4、核因子-κB、Caspase-3表达的影响

周琴1*  张勤1 李光乾2   

  1. 1.浙江省人民医院儿科，杭州 310014; 2.杭州市儿童医院神经科， 杭州 310014
  • 收稿日期:2013-06-05 修回日期:2013-06-24 出版日期:2014-06-06 发布日期:2014-06-06
  • 通讯作者: 周琴 E-mail:zhouqin5803@126.com
  • 基金资助:

    白藜芦醇对惊厥持续状态大鼠海马TLR4/NF-кB信号通路的调控

Effects of resveratrol on the expression of Toll-like receptor 4, NF-кB and Caspase-3 in the hippocampus of the status convulsion rat

ZHOU Qin 1*  ZHANG Qin1  LI Guang-qian2   

  1. 1.Department of Pediatrics, Zhejiang Provincial People’s Hospital, Hangzhou 310014, China; 2.Department of Neurology，Hangzhou Children’s Hospital, Hangzhou 310014, China
  • Received:2013-06-05 Revised:2013-06-24 Online:2014-06-06 Published:2014-06-06

摘要：

目的 观察大鼠惊厥持续状态（SC）后脑组织海马中Toll样受体4（TLR4）、核因子-κB（NF-κB）和半胱氨酸门冬氨酸特异性蛋白酶3（Caspase-3）的表达及神经细胞凋亡的变化，并探讨白藜芦醇（Res）对三者的影响。 方法 106只SD大鼠随机分为对照组（A）、SE组（B）和白藜芦醇干预组（C），其中B组再随机分为B1～B4组（分别于惊厥后4h、24h、48h和72h处死），B组和C组采用氯化锂-匹罗卡品法制作大鼠SC模型，C组在大鼠惊厥终止后30min，给予30 mg/kg白藜芦醇腹腔注射，每天1次，连用3d，于惊厥后72 h处死。免疫组织化学法检测海马TLR4、NF-κB/p65蛋白的表达变化；RT-PCR技术检测海马TLR4、Caspase-3 mRNA表达的动态改变；TUNEL法检测神经细胞凋亡数的变化。 结果 免疫组织化学显示，B组各时间点TLR4蛋白的表达均明显高于A组（P＜0.05），并随时间延长明显增高；C组TLR4蛋白表达较B4组显著降低（P＜0.01）。B组可见NF-кB/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）。RT-PCR显示，B组各时间点TLR4、Caspase-3 mRNA的表达均较A组高（P＜0.05），且随时间延长逐步升高，惊厥后72h达高峰；C组海马TLR4、Caspase-3mRNA的表达明显低于B4组（P＜0.05）。B组在惊厥24h后海马CA1区TUNEL阳性细胞数已显著高于A组（P＜0.01），72h达高峰，而C组TUNEL阳性细胞数较B4组显著下降（P＜0.01）。 结论 SC后大鼠海马TLR4、NF-κB/p65和Caspase-3的表达增强。白藜芦醇可下调匹罗卡品致惊厥持续状态大鼠海马TLR4、NF-κB/p65和Caspase-3的表达，并使神经细胞凋亡减少；提示白藜芦醇对SC引起的脑损伤可能有保护作用。

关键词: 惊厥, 凋亡, Toll样受体4, Caspase-3, 白藜芦醇, 免疫组织化学, 大鼠

Abstract:

Objective To study the expression of toll-like receptor 4 (TLR4), nuclear factor κB (NF-кB) and Caspase-3 and the change of neuron apoptosis of the hippocampus in the status convulsion rat, and to explore the effect of resveratrol on them.Methods A total of 106 male Sprague-Dawley（SD）rats were randomly divided into the control (A), convulsion (B) and resveratrol treatment (C) groups. Group B was further randomly divided into four subset groups (B1-B4) which were executed at 4h, 24h, 48h, 72h after convulsion discontinued. Continuous epilepticus was induced by injecting lithium chloride and pilocarpine, and group C was daily injected with 30mg/kg resveratrol 30minutes after convulsion stopped for 3 days. TLR4 and NF-κB/p65 proteins were detected by immunohistochemistry(IHC); the expressions of TLR4 and Caspase-3 mRNA were detected by RT-PCR. The neuron apoptosis was observed by TUNEL. Results The IHC staining of TLR4 protein in group B was significantly higher than in group A (P<0.05), and that in group C was much lower than in B4 group (P<0.01). The expression of NF-кB/p65 showed that hippocampal neurons had positive expression in cell nuclei in group B compared with group A (P<0.05), and the expression of NF-кB/p65 protein in group C was much lower than that in group B4 (P<0.01). The mRNA expressions of TLR4 and Caspase-3 in the rat hippocampus of group B were significantly elevated than that in group A (P<0.05), and the tendency was increased gradually, reaching the peak at 72 hours after seizure, and that in group C was much lower than that in B4 group (P<0.01). The TUNEL positive cells in hippocampus CAl of B group were more than that of group A after the SC 24hours(P<0.01)，reached the highest level at the 72nd hour, and that in group C the TUNEL positive cells were lower than that in B4 group (P<0.01). Conclusion The expression of TLR4, NF-кB and Caspase-3 increased after SC. Resveratrol can down regulate the expression of TLR4, NF-кB and Caspase-3 in the hippocampus with pilocarpine-induced seizures, reduced the number of neuronal apoptosis.These results suggest that resveratrol may have a protective effect against the hippocampus damage caused by status convulsion.

Key words: Convulsion, Apoptosis, Toll like receptor 4, Caspase-3, Resveratrol, Immunohistochemistry, Rat