热性惊厥大鼠海马IL-6表达及丙戊酸对其甲基化水平的干预研究

Expression of interleukin-6 in hippocampus of rat with febrile seizures and the effect of valproic acid administration on its methylation level

目的检测热性惊厥大鼠海马白细胞介素-6（interleukin-6，IL-6）表达及启动子甲基化状态，探讨丙戊酸（valproic acid，VPA）对其DNA甲基化的影响。方法21日龄sD大鼠24只，随机分为对照组和热性惊厥组，每组12只，通过热水浴（43．5℃，4min）方法构建热性惊厥大鼠模型。Racine分级法进行行为学评分，脑电图检测惊厥发作情况。实时荧光定量PER检测大鼠海马IL-6mRNA水平，酶联免疫吸附实验检测IL-6蛋白浓度，重亚硫酸氢盐测序检测IL-6启动子甲基化状态。培养大鼠神经胶质瘤C6细胞系，分为对照组、VPA（0．2mol／L）组、热处理组（43．5cc，1h）、热处理＋VPA组（43．5℃，1h，VPA：0．2mol／L），重亚硫酸氢盐测序检测VPA对IL-6启动子区域甲基化状态的影响。结果Racine行为学评分及脑电图结果显示大鼠经过热水浴处理后神经元放电频率和幅度增加，惊厥发作潜伏期缩短。与对照组相比，热性惊厥大鼠海马组织中IL-6mRNA[对照组：（0．98±0．34）；热性惊厥组：（2．85±0．39）；P〈0．05）]及蛋白表达[对照组：（2824．33±169．20）pg／ml；热性惊厥组：（3514．58±86．4）pg／ml；P〈0．01）]均显著增加；热性惊厥组大鼠海马IL-6启动子区域甲基化水平显著低于对照组[IL-6基因甲基化百分率：对照组：100％（25／25）；热性惊厥组：84％（21／25）；（P〈0．05）]。VPA单独处理对C6细胞系上IL-6基因甲基化水平无显著影响，但能显著增加C6细胞热诱导后IL-6基因甲基化水平[IL-6基因甲基化百分率：对照组：100％（25／25）；VPA组：100％（25／25）；热处理组：84％（21／25）；热处理＋VPA组：96％（24／25）]。结论在热性惊厥大鼠海马中IL-6表达增加，IL-6启动子区甲基化水平降低，VPA处理可增加热处理后IL-6基因甲基化水平。

Objective To investigate the level of interleukin-6 （IL-6） and the effect of valproic acid（VPA） administration on IL-6 promoter methylation,further to explore the epigenetic mechanism in febrile seizures. Methods Sprague-Dawley （SD） rats （21 day） were randomly divided into control group （n = 12） and febrile seizure （FS） group （n= 12）,and seizures were generated using hot bath methods and evaluated by racine score and electroencephalogram （EEG）.The IL-6 protein and mRNA levels were detected using ELISA and quantitative reverse transcription polymerase chain reaction （qRT-PCR） , respectively. Rat C6 cell line was cultured and randomly divided into control, VPA（0.2 mol/L）, hyperthermia（43.5 ℃, 1 h）, byperthermia （43.5 ℃, 1 h）＋VPA （0.2 mol/L） groups.The methylation status of IL-6 promoter in FS rats and the effect of VPA on IL-6 methylation in C6 cell line were examined by bisulfite sequencing polymerase chain reaction （BSP） to determine the epigenetic regulation of IL-6 level in FS. Results Racine score and EEG results showed that the frequency and amplitude of neuronal discharge increased after hot water bath in rats, and the latency of convulsion was shorter.The expression levels of IL-6 mRNA and protein were significantly increased in Fs group compared with those in control group （ mRNA ： Control ： 0.98± 0.34 ; FS ： 2.85 ± 0.39 ,P〈0.05 ; Protein： Control： （ 2 824.33± 169.20） pg/ml; FS： （ 3 514.58± 86.4） pg/ml, P〈0.01 ） .The methylation of IL-6 promoter in FS group （ 84% （ 21/25 ） ） was lower than that in control group （ 100% （ 25/ 25） ） （P〈0.05）.A significant increase in methylation of IL-6 promoter was observed in C6 cell line from VPA combined with hyperthermia （ 96% （ 24/25 ） ） but no change was showed in that from VPA alone （ 100% （25/25） ） compared with that from control（ 100% （25/25） ）. Conclusion IL-6 level is up-regulated in FS rats, and the hypomethylation or demethylation of the IL-6 promoter region might increase the IL-6 level.VPA administration can elevate its methylation level,which provides a strong experimental basis for the future study of the epigenetic mechanism in FS.