基于JNK信号通路探讨左乙拉西坦对癫痫仔鼠海马神经元保护作用

Protective effect of levetiracetam on hippocampal neurons of epileptic offspring mice based on JNK signal pathway

目的 研究左乙拉西坦对癫痫仔鼠海马神经元的保护作用,并探讨可能机制。方法 50只仔鼠腹腔注射氯化锂联合皮下注射毛果芸香碱建立癫痫模型,随机分为模型组、低、中、高剂量实验组、联合组,各10只。另取10只仔鼠设为对照组。联合组灌胃左乙拉西坦40 mg·kg^(-1),尾静脉注射茴香霉素(anisomycin)2 mg·kg^(-1);低、中、高剂量实验组分别灌胃左乙拉西坦10、20、40 mg·kg^(-1),尾静脉注射等体积二甲基亚砜(DMSO)溶液;对照组、模型组灌胃等体积0.9%NaCl,尾静脉注射等体积DMSO溶液,均每天1次,干预7 d。用全自动生化分析仪检测脑组织氧化应激指标,用Tunel染色法检测海马区神经元凋亡率,用蛋白质印迹法检测脑组织半胱氨酸天冬氨酸酶3(caspase-3)蛋白表达水平。结果 对照组、模型组和低、中、高剂量实验组及联合组的脑组织丙二醛(MDA)水平分别为(6.24±0.75)、(11.52±1.32)、(10.26±1.19)、(8.95±1.20)、(7.54±1.16)和(9.62±1.14)nmol·mg^(-1),超氧化物歧化酶分别为(235.47±32.69)、(152.31±19.67)、(186.57±25.30)、(204.87±31.05)、(219.45±32.29)和(203.86±23.65)U·mg^(-1),海马区神经元凋亡率分别为(2.96±0.63)%、(18.56±2.71)%、(14.21±2.68)%、(9.21±1.53)%、(5.05±0.75)%和(8.29±2.41)%,脑组织caspase-3蛋白相对表达水平分别为0.12±0.02、0.78±0.08、0.62±0.07、0.50±0.05、0.32±0.04和0.49±0.05。模型组与对照组比较,低、中、高剂量实验组与模型组比较,联合组与高剂量实验组比较,上述指标的差异均有统计学意义(均P<0.05)。结论 左乙拉西坦可减轻癫痫仔鼠氧化应激,保护海马神经元,其作用机制可能与抑制c-Jun氨基末端激酶/c-Jun通路有关。

Objective To study the protective effect of levetiracetam on hippocampal neurons of epileptic offspring mice and explore the possible mechanism.Methods Fifty pups were injected intraperitoneally with lithium chloride combined with subcutaneous injection of pilocarpine to establish epilepsy models,and they were randomly divided into model group,experimental-L,-M,-H groups,and combined group,with 10 rats in each group.Another 10 pups were taken as the control group.The combination group was gavaged with levetiracetam 40 mg·kg^(-1)and injected of anisomycin 2 mg·kg^(-1) through the tail vein;the experimental-L,-M,-H groups were gavaged with levetiracetam 10,20,40 mg·kg^(-1)and injected of an equal volume of dimethyl sulfoxide(DMSO)solution through the tail vein;the control group and the model group were gavaged with equal volume of 0.9%NaCl,and injected of an equal volume of DMSO solution through the tail vein.The intervention lasted for 7 days once a day.The oxidative stress indicators in brain tissue were detected by automatic biochemical analyzer;the apoptosis rate of neurons in the hippocampus were detected by Tunel staining;the expression of caspase-3 protein in brain tissue were detected by Western blotting.Results The levels of malondialdehyde(MDA)in the brain tissue of the control group,model group,experimental-L,-M,-H groups and combination group were(6.24±0.75),(11.52±1.32),(10.26±1.19),(8.95±1.20),(7.54±1.16)and(9.62±1.14)nmol·mg^(-1);superoxide dismutase were(235.47±32.69),(152.31±19.67),(186.57±25.30),(204.87±31.05),(219.45±32.29)and(203.86±23.65)U·mg^(-1);the apoptosis rates of neurons in the hippocampus were(2.96±0.63)%,(18.56±2.71)%,(14.21±2.68)%,(9.21±1.53)%,(5.05±0.75)%and(8.29±2.41)%;the expression levels of caspase-3 protein in the brain tissues were 0.12±0.02,0.78±0.08,0.62±0.07,0.50±0.05,0.32±0.04 and 0.49±0.05.Compared between the model group and the normal group,between the experimental-L,-M,-H groups and the model group,between the combination group and the experimental-H group,the differences were statistically significant(all P<0.05).ConclusionL Ievetiracetam can reduce oxidative stress in epileptic offspring and protect hippocampal neurons.Its mechanism may be related to the inhibition of c-Jun N-terminal kinase/c-Jun pathway.