基于DJ-1/Nrf2/HO-1信号通路探讨北柴胡地上部分多糖组分对癫痫小鼠脑内氧化应激的影响

Effect of polysaccharides from aerial parts of Bupleurum chinense on oxidative stress in brains of epileptic mice based on DJ-1/Nrf2/HO-1 signaling pathway

目的 探讨北柴胡Bupleurum chinense地上部分多糖组分(polysaccharides from aerial parts of B. chinense,ABP)对戊四唑致痫小鼠的预防作用及作用机制。方法 C57BL/6小鼠随机分为对照组、模型组、丙戊酸钠(125 mg/kg)组、癫痫宁片(1200 mg/kg)组和ABP高、低剂量(100、25 mg/kg)组,每组10只。各给药组ig相应药物,对照组和模型组ig等体积0.5%羧甲基纤维素钠溶液,1次/d,连续7 d。末次给药后60 min,除对照组外,其余各组ip戊四唑(60 mg/kg)。通过行为学评价ABP预防癫痫的作用;采用苏木素-伊红(HE)染色、免疫组化法检测海马神经元病理形态学变化;采用ELISA法测定海马组织中半胱氨酸天冬氨酸蛋白酶-3(cystein-asparate protease-3,Caspase-3)、胶质纤维酸性蛋白(glial fibrillary acidic protein,GFAP)、谷氨酸脱羧酶65(glutamic acid decarboxylase 65,GAD65)、GAD67、G蛋白偶联的内向整流钾通道1(G protein gated inwardly rectifying K channels 1,GIRK1)、N-甲基-D-天冬氨酸受体1(N-methyl-D-aspartic acid receptor,NMDAR1)、γ-氨基丁酸A型受体(γ-aminobutyric acid type A,GABA AR)水平;采用Western blotting检测海马组织中DJ-1、特异性蛋白1(specificity protein 1,SP1)、p-SP1、核因子E2相关因子2(nuclear factor erythroid 2-related factor 2,Nrf2)、血红素加氧酶-1(heme oxygenase-1,HO-1)蛋白表达。采用过氧化氢(hydrogen peroxide,H2O2)诱导的PC12细胞氧化应激模型进行验证。结果 与模型组比较,ABP明显改善小鼠癫痫症状,表现为躯体僵直率和死亡率降低等(P<0.01);海马锥体细胞排列规则,结构清晰,GFAP阳性表达面积明显降低;显著降低海马组织中Caspase-3、GFAP、GAD65、GAD67、GIRK1及NMDAR1水平(P<0.01),升高GABA AR水平(P<0.01);显著下调癫痫小鼠海马组织和H2O2诱导的PC12细胞中DJ-1、p-SP1、Nrf2及HO-1蛋白表达(P<0.01)。结论 ABP可以减轻癫痫发作期间DJ-1/Nrf2/HO-1介导的癫痫氧化应激,从而发挥预防癫痫的作用。

Objective To investigate the preventive effect and mechanism of polysaccharide from aerial parts of Bupleurum chinense(ABP) on pentylenetetrazole-induced epilepsy in mice. Methods C57BL/6 mice were randomly divided into control group, model group, sodium valproate(125 mg/kg) group, epileptic tablets(1200 mg/kg) group and ABP high-, low-dose(100, 25 mg/kg) groups,with 10 mice in each group. Each administration group was ig corresponding drug, control group and model group were ig equal volume 0.5% sodium carboxymethyl cellulose solution, once a day for 7 d. 60 min after the last administration, except for control group, other groups were ip pentylenetetrazole(60 mg/kg). The effect of ABP on preventing epilepsy was evaluated by behavior;The pathomorphological changes of hippocampal neurons were detected by hematoxylin eosin(HE) staining and immunohistochemistry;The levels of cystein-asparate protease-3(Caspase-3), glial fibrillary acidic protein(GFAP), glutamic acid decarboxylase 65(GAD65),GAD67, G protein gated inwardly rectifying K channels 1(GIRK1), N-methyl-D-aspartate acid receptor 1(NMDAR1) and γ-aminobutyric acid type A receptor(GABA AR) in hippocampus were detected by ELISA;Western blotting was used to detect DJ-1,specific protein 1(SP1), p-SP1, nuclear factor E2 related factor 2(Nrf2) and heme oxygenase-1(HO-1) protein expression in hippocampus. The oxidative stress model of PC12 cells induced by hydrogen peroxide(H2O2) was used to verified. Results Compared with model group, ABP significantly improved the epileptic symptoms of mice, which showed a decrease in rate of body stiffness and mortality(P < 0.01);Hippocampal pyramidal cells were regularly arranged with clear structure, and GFAP positive expression area was reduced;The levels of Caspase-3, GFAP, GAD65, GAD67, GIRK1 and NMDAR1 in hippocampus were significantly decreased(P < 0.01);DJ-1, p-SP1, Nrf2 and HO-1 protein expressions in hippocampus of epileptic mice and H2O2-induced PC12 cells were significantly decreased(P < 0.01). Conclusion ABP can reduce DJ-1/Nrf2/HO-1 mediated oxidative stress during epileptic seizures, thus playing a role in preventing epilepsy.