托吡酯腹腔注射后癫痫大鼠海马组织损伤变化及其机制探讨

发布时间：2018-03-17 10:46

本文选题：癫痫　切入点：托吡酯　出处：《山东医药》2017年27期 　论文类型：期刊论文

【摘要】：目的观察托吡酯(TPM)腹腔注射后癫痫大鼠海马组织损伤改变,并探讨其可能机制。方法将40只大鼠依据随机数字表法分为正常组、模型组、TPM低剂量组、TPM高剂量组。模型组、TPM低剂量组、TPM高剂量组采用腹腔注射氯化锂180 mg/kg、匹罗卡品30 mg/kg制作癫痫模型。模型组、TPM低剂量组、TPM高剂量组大鼠分别于癫痫发作后5 h经腹腔注射生理盐水10 m L/(kg·d)、TPM 40 mg/(kg·d)、TPM 80 mg/(kg·d)。各组连续用药4周后处死大鼠取海马组织,HE染色观察大鼠海马组织病理变化;应用基因芯片技术筛选各组差异表达的miRNA,并用实时荧光定量PCR法进行验证;采用TUNEL法检测海马组织凋亡细胞,并计算凋亡细胞数;分别采用Western blotting法、免疫荧光法和免疫组化法检测各组大鼠海马组织中的Caspase-8。结果与正常组相比,模型组海马组织损伤明显,TPM低剂量组和TPM高剂量组海马组织损伤较模型组减轻,且TPM低剂量组损伤最轻。基因芯片检测发现模型组出现12个表达上调的miRNA和14个表达下调的miRNA,经实时荧光定量PCR验证,只有miR-146a在各组表达的差异有统计学意义。模型组、TPM高剂量组、TPM低剂量组、正常组海马组织中miR-146a相对表达量依次降低(P均0.05)。细胞凋亡检测发现,与正常组相比,模型组大鼠CA1区、CA3区和DG区凋亡细胞细胞数高于正常组(P均0.05),而TPM高剂量组和TPM低剂量组凋亡细胞数低于模型组(P均0.05)。Western blotting结果显示,模型组Caspase-8蛋白相对表达量高于正常组,模型组、TPM高剂量组、TPM低剂量组Caspase-8蛋白相对表达量依次降低(P均0.01)。免疫荧光结果显示,与正常组相比,模型组大鼠CA1区、CA3区和DG区Caspase-8阳性细胞数高于正常组(P均0.05),TPM高剂量组和TPM低剂量组Caspase-8阳性细胞数低于模型组(P均0.05)。免疫组化结果显示,模型组IOD值高于正常组,模型组、TPM高剂量组、TPM低剂量组IOD值依次降低(P均0.05)。结论 TPM腹腔注射可减轻癫痫大鼠海马组织病理改变,减少海马组织细胞凋亡,下调海马组织中miR-146a和Caspase-8的表达,其中40 mg/(kg·d)的TPM较80 mg/(kg·d)效果更加明显。TPM的抗癫痫作用机制可能与调节miRNA表达、减少细胞凋亡、下调Caspase-8表达有关。
[Abstract]:Objective to observe the changes of hippocampal injury in epileptic rats after intraperitoneal injection of topiramate (TPM) and to explore its possible mechanism. Model group TPM low dose group TPM high dose group, model group TPM low dose group TPM high dose group by intraperitoneal injection of lithium chloride 180 mg / kg, pilocarpine 30 mg/kg to make epilepsy model. Five hours after seizure, the rats were injected intraperitoneally with normal saline (10 mL / kg 路d ~ (-1)) for 40 mg/(kg 路d ~ (-1) TPM 80 mg/(kg 路d ~ (-1). After 4 weeks of continuous administration, the rats were sacrificed to observe the pathological changes of hippocampus tissue by HE staining. The differentially expressed miRNAs were screened by gene chip technique and verified by real-time fluorescence quantitative PCR. The apoptotic cells in hippocampus were detected by TUNEL method and the number of apoptotic cells was calculated. Western blotting method was used respectively. Caspase-8 in hippocampal tissue of rats in each group was detected by immunofluorescence and immunohistochemistry. Results compared with the normal group, the hippocampal tissue damage in the model group was significantly reduced compared with the model group in the low dose group and the high dose group of TPM. Gene chip analysis showed that 12 up-regulated miRNA and 14 down-regulated miRNAs were found in the model group, which were verified by real-time fluorescence quantitative PCR. Only the difference of miR-146a expression in each group was statistically significant. The relative expression of miR-146a in hippocampal tissues of the model group and the normal group decreased in turn, and the relative expression of miR-146a in the hippocampus of the normal group was decreased in turn. The results of apoptosis detection showed that compared with the normal group, the relative expression of miR-146a in the model group was significantly lower than that in the control group. The number of apoptotic cells in the CA1 CA3 and DG regions of the model group was higher than that in the normal group (P 0.05), while the number of apoptotic cells in the high dose TPM group and the low dose TPM group was lower than that in the model group. The results showed that the relative expression of Caspase-8 protein in the model group was higher than that in the normal group. The relative expression of Caspase-8 protein decreased in turn in the model group with high dose of TPM and low dose group (P < 0.01). The immunofluorescence results showed that, compared with the normal group, the relative expression of Caspase-8 protein in the model group was significantly lower than that in the normal group. The number of Caspase-8 positive cells in the CA1 CA3 and DG regions in the model group was higher than that in the normal group (P 0.05) and the number of Caspase-8 positive cells in the low-dose TPM group was lower than that in the model group (P 0.05). The immunohistochemical results showed that the IOD value in the model group was higher than that in the normal group. Conclusion Intraperitoneal injection of TPM can attenuate pathological changes of hippocampus, decrease apoptosis of hippocampal cells and down-regulate the expression of miR-146a and Caspase-8 in hippocampus of epileptic rats. The antiepileptic mechanism of 40 mg/(kg 路d TPM is more obvious than that of 80 mg/(kg 路d). The mechanism may be related to the regulation of miRNA expression, the reduction of apoptosis and the down-regulation of Caspase-8 expression.
【作者单位】：延边大学附属医院;延边大学;
【基金】：吉林省医药卫生科研计划项目(C2017103)
【分类号】：R742.1

【相似文献】