平肝止痫复方联合卡马西平对大鼠体外癫痫模型的保护作用

The protective effect of Pinggan Zhixian compound combined medicine with carbamazepine on rat epilepsy model in vitro

目的探讨平肝止痫复方联合卡马西平(CBZ)对大鼠癫痫(EP)细胞模型氧化应激反应及核因子E2相关因子2(Nrf2)-抗氧化反应元件(ARE)信号通路的影响。方法原代分离及培养大鼠海马神经元,将海马神经元分为5组,分别为对照组(control)、癫痫模型组(epilepsy)、大鼠空白血清组(BS)、CBZ含药血清组(CBZ)、平肝止痫复方联合CBZ含药血清组(PGZX+CBZ)。用无Mg^(2+)细胞外液诱导建立癫痫细胞模型后,通过不同浓度的血清继续处理24 h,利用CCK8试剂盒筛选后续实验最适血清浓度;利用商品化的试剂盒检测大鼠海马神经元超氧化物歧化酶(SOD)活性、丙二醛(MDA)及谷胱甘肽(GSH)含量;流式细胞术(FCM)的方法检测大鼠海马神经元中活性氧(ROS)含量;Western Blot检测大鼠海马神经元中B淋巴细胞瘤-2相关X蛋白(Bax)、B淋巴细胞瘤-2(Bcl-2)、半胱氨酸蛋白酶-3(Caspase-3)、醌氧化还原酶1(NQO1)、Nrf2蛋白含量。结果和epilepsy组相比,CBZ组及PGZX+CBZ组大鼠海马神经元中MDA、ROS、Bax和Caspase-3的含量均下降(P<0.05),SOD活性增加,GSH、Bcl-2、NQO1和Nrf2的含量则明显增加(P<0.05);和CBZ组相比,PGZX+CBZ组大鼠海马神经元中中MDA、ROS、Bax和Caspase-3的含量下降(P<0.05),SOD活性增加,GSH、Bcl-2、NQO1和Nrf2的含量则明显增加(P<0.05)。结论平肝止痫复方联合CBZ可能通过调节Nrf2/ARE信号通路增加机体抗氧化对大鼠癫痫细胞模型起保护作用。

Objective To explore the effects of Pinggan Zhixian compound combined with carbamazepine on oxidative stress and Nrf2-ARE signaling pathway in epileptic cell model.Methods Rat hippocampal neurons cells wereisolated and cultured in primary culture.They were divided into 5 groups:control group,epilepsy group(epilepsy),blank serum group(BS),carbamazepine serum group(CBZ),Pinggan Zhixian compound medicine combined with carbamazepine serum group(PGZX+CBZ).After the establishment of cell epilepsy model induced by magnesium-free extracellular fluid,the cells were treated with different concentrations of serum for 24 h.CCK8 kit assay was used to screen the optimal serum concentration;SOD activity,MDA and CAT content in rat hippocampal neurons cells were detected by commercial biochemical kit;ROS was detected by flow cytometry;Western blot was used to detect the relative protein expression of Bax,Bcl-2,caspase-3,NQO1,and Nrf2.Results Compared with the epilepsy group,the contents of MDA,ROS,Bax and caspase-3 in the CBZ and PGZX+CBZ group were all decreased(P<0.05),the contents of GSH,Bcl-2,Nrf2,NQO1,and SOD activity were significantly increased(P<0.05).Compared with the CBZ group,the contents of MDA,ROS,Bax and caspase-3 in the PGZX+CBZ group were all decreased(P<0.05),the contents of GSH,Bcl-2,Nrf2,NQO1,and SOD activity were significantly increased(P<0.05).Conclusion Pinggan Zhixian compound medicine combined with carbamazepine may play a protective role on hippocampal neurons cell model of epileptic rats by regulating Nrf2/ARE signal pathway and increasing antioxidation.