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

Changes of hippocampal injuries in epileptic rats after intraperitoneal injection of topiramate and its mechanism

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。

Objective To observe the changes of hippocampal injuries in epileptic rats after intraperitoneal injection of topiramate （TPM）,and to explore its potential mechanisms.Methods Forty rats were randomly divided into the normal group,model group,low-dose TPM group,and high-dose TPM group.The rats in the model group,low-dose TPM group,and high-dose TPM group were treated with 10 mL/（kg·d） saline,40 mg/（kg·d） TPM and 80 mg/（kg·d） TPM,respectively,for 4 weeks to make the epileptic models.At 5 h after epileptic seizure,rats in the model group,low-dose TPM group,and high-dose TPM group were intraperitoneally injected with 10 mL/（kg＆#183;d） normal saline,40 mg/（kg＆#183;d） TPM,and 80 mg/（kg＆#183;d） TPM.After 4 weeks of continuous administration,rats were sacrificed to obtain the hippocampus tissues.HE staining was performed to observe the morphological changes of hippocampus.The differentially expressed miRNAs were obtained by miRNA microarray and were identified by real-time fluorescent quantitative PCR.TUNEL assay was used to detect apoptosis of hippocampal neurons and the number of apoptotic cells were counted.The expression levels of Caspase-8 were detected by Western blotting,immunohistochemistry （IHC） and immunofluorescence （ICC）.Results The hippocampus was obviously damaged in model group as compared with that of the normal group,whereas the injuries were alleviated in both low-dose TPM group and high-dose TPM group,especially the former one.A total of 12 miRNAs were up-regulated and 14 miRNAs were down-regulated in the model group as compared with those of the normal group.Interestingly,the miR-146a expression was significantly down-regulated both in low-and high-dose TPM groups as compared with that of the model group （all P〈0.05）.Compared with the normal group,the number of apoptotic cells in the CA1,CA3 and DG regions was significantly higher in the model group （all P〈0.05）.Meanwhile,the number of apoptotic cells was smaller in the low-dose TPM group and high-dose TPM group than that of the model group （all P〈0.05）.Western blotting showed that the Caspase-8 protein expression of the model group was higher than that of the normal group,and the Caspase-8 protein expression was successively decreased in the model group,high-dose,and low-dose TPM groups （all P〈0.01）.Similarly,ICC showed that the number of Caspase-8 positive cells in the CA1,CA3 and DG regions significantly increased in the model group as compared with that of the normal group （all P〈0.05）.Compared with the model group,the number of Caspase-8 positive cells in the low-dose TPM group and high-dose TPM group were dramatically reduced （all P〈0.05）.What＆#39;＆#39;s more,the results of IHC identified that the integrated option density （IOD） value was significantly higher in the model group as compared with that of the normal group.The value of IOD was successively decreased in the model group,low-dose TPM group,and high-dose TPM group （all P〈0.05）.Conclusions The intraperitoneal injection of TPM effectively alleviates hippocampal injuries in epileptic rats,inhibits neuronal apoptosis and down-regulates the expression of miR-146a and Caspase-8.Meanwhile,40 mg/（kg＆#183;d） TMP shows greater effect than 80 mg/（kg＆#183;d）.The anti-epileptic mechanism of TPM may be associated with the regulation of miRNA expression,down-regulation of Caspase-8,and reduction of neuronal apoptosis.