细胞周期素依赖性激酶抑制剂olomoucine对大鼠癫痫持续状态后神经元凋亡的影响

Effects of cyclin dependent protein kinase inhibitor olomoucine on the neuronal apoptosis after status epilepticus:experiment with rats

目的探讨选择性细胞周期素依赖性激酶抑制剂 olomoucine 对癫痫持续状态(SE)后神经元凋亡的影响。方法建立氯化锂-匹罗卡品 SE 模型,随机分为生理盐水组、SE 组和 olomoucine干预组。应用免疫荧光化学法检测神经元核心抗原(NeuN)和周期素蛋白 B1表达,通过 TUNEL 方法检测神经元凋亡情况;应用逆转录-聚合酶链反应(RT-PCR)检测皮质及海马白介素-1β(IL-1β)和肿瘤坏死因子-α(TNF-α)mRNA 表达情况。统计方法采用方差分析与 t 检验。结果 (1)生理盐水组TUNEL 示阳性神经元稀少,SE 后显著增加(P<0.05),olomoucine 干预组明显少于 SE 组(P<0.05),尤其齿状回门区与生理盐水组相比差异没有统计学意义(P>0.05);(2)与 SE 组比较,olomoucine 干预组细胞周期素 B1阳性神经元数目减少,差异具有统计学意义(P<0.05);(3)生理盐水组 IL-1β、TNF-αmRNA 表达量稀少,SE 后显著增加(P<0.05),除皮质 TNF-αmRNA 外,olomoucine 干预组较 SE组均明显降低(P<0.05)。结论 olomoucine 可通过调控细胞周期,抑制癫痫持续状态后神经元重新进入细胞周期,以及抑制活化的胶质细胞分泌炎性细胞因子,抑制癫痫持续状态后神经元凋亡而减轻脑损伤。

Objective To investigate the effects of olomoucine, a cyclin dependent protein kinase （CDK） inhibitor, on the neuronal apoptosis after status epilepticus （SE）. Methods Lithium chloride was injected intraperitoneally, and pilecarpine was injected intraperitoneally after 18 h to 24 SD rats so as to cause SE. Twenty-two of the 24 rats developed SE and 2 of them died. The surviving 20 rats were then randomly divided into 2 equal groups： olomoucine group, injected intracerebroventricularly after the SE was terminated by diazepam and chloral hydrate once a day for 3 days, and SE group, infused intracerebroventricularly with DMSO solution Another 10 rats were injected intraperitoneally with normal saline and then infused intracerebroventricularly with DMSO solution to be used as control group. Six hours after SE attack 5 rats from each group were killed respectively with their brains taken out. Semiquantitative RT-PCR was used to detect the mRNA expression of anti-inflammatory cytokines, such as interleukin （IL）- 1β and tumor necrosis factor （TNF） -α. Three days later the other 5 rats in each group were killed with their entorhinal cortex and hippocampus taken out. TUNEL was used to observe the apoptosis. （IF） staining was used to detect the expression of neuronal nuclear nucleoprotein （NeuN） and cyclin B1. Results TUNEL showed that apoptotic neurons were rare in the control group and were numerous in the SE group, especially in the entorhinal cortex and the hylus of dentate gyrus, and the number of apoptotic neurons in the hylus of dentate gyrus of the olomoucine group was not significantly different from that of the control group （ P 〈 0. 05 ）, however, the number of apoptotic cells in the entorhinal cortex of the olomoucine group was still significantly higher than that of the control group （ P 〈 0.05 ）. IF staining demonstrated that in the control group the co-expression of NeuN and TUNEL-labeled cells was weak ; and in the SE group the co-expression of NeuN and TUNEL was significantly increased compared with that in the control group （P〈0. 05）. The number of cyclin B1 positive cells in the olomoucine group was 18.22 ±3.99, significantly lower than that of the SE group （24.57 ± 6.78, P 〈 0.05）. Semiquantitative RT-PCR showed that the IL-1β and TNF-α mRNA expression levels of the SE group were both significantly higher than those of the control group （ both P 〈 0.05 ）, and the IL-1β and TNF-α mRNA expression levels of the olomoucine group, except the TNF-α mRNA expression in the cortex,were all significantly lower than those of the SE group （ all P 〈 0.05 ）, and not significantly different from those of the control group （ all P 〉 0.05）. Conclusion Olomoucine treatment can inhibits cell cycle protein B1 expression, anti-inflammatory cytokines such as IL-1β and TNF-α secretion, thus decreasing neuronal death and providing neuroprotection after SE, which suggests a potential promising therapeutic way for epilepsy treatment.