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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。
Objective To observe the changes of TLR4, NF-κB and Caspase-3 in the hippocampus of rats after convulsion (SC) And the change of apoptosis of nerve cells, and to explore the effect of resveratrol on them. Methods One hundred and sixty SD rats were randomly divided into control group (A), SE group (B) and resveratrol intervention group (C). Group B was randomly divided into B1 ~ B4 group , 48h and 72h). Rats in group B and group C were induced by lithium chloride-pilocarpine. Rats in group C received intraperitoneal injection of 30 mg / kg resveratrol 30 min after termination of seizure. 1 day, once every 3d, 72 h after convulsion. The expressions of TLR4 and NF-κB / p65 in the hippocampus were detected by immunohistochemistry. The changes of the mRNA expression of TLR4 and Caspase-3 in the hippocampus were detected by RT-PCR. The changes of apoptosis were detected by TUNEL. Results Immunohistochemistry showed that the expression of TLR4 protein in group B was significantly higher than that in group A at each time point (P <0.05), and significantly increased with time. The expression of TLR4 in group C was significantly lower than that in group B4 (P <0.01). In group B, NF-кB / p65 protein was expressed in different degrees in the nucleus, which was significantly different from that in group A (P <0.05). The expression of NF-κB / p65 protein in group C was significantly lower than that in group B4 (P <0.01). The mRNA expression of TLR4 and Caspase-3 at each time point in group B was higher than that in group A at each time point (P <0.05), and gradually increased with time and peaked at 72 hours after seizure. The expressions of TLR4 and Caspase- 3 mRNA expression was significantly lower than the B4 group (P <0.05). In group B, the number of TUNEL positive cells in hippocampal CA1 area was significantly higher than that in group A (P <0.01) at 24h and reached the peak at 72h in group B, while the number of TUNEL positive cells in group C was significantly lower than that in group B4 (P <0.01). Conclusion The expression of TLR4, NF-κB / p65 and Caspase-3 in the hippocampus of rats after SC increase. Resveratrol down-regulated the expression of TLR4, NF-κB / p65 and Caspase-3 in the hippocampus of convulsant-induced convulsion induced by pilocarpine, and decreased the apoptosis of neurons. It suggested that resveratrol could induce brain injury induced by SC May have a protective effect.