基于Wnt/β-catenin信号探讨左乙拉西坦对神经元损伤的治疗作用

Effects of levetiracetam on the neuronal damage based on Wnt/β-catenin signal

目的:探究左乙拉西坦对神经元损伤的影响与机制。方法:体外研究以3%七氟醚构建原代海马神经元损伤模型,通过CCK8与台盼蓝染色检测细胞增殖与存活率,采用Western blotting检测Wnt/β-catenin信号蛋白表达,免疫荧光检测MAP2/Synaptophysin表达。体内研究以3%七氟醚构建幼鼠认知功能损伤模型,利用Morris水迷宫检测幼鼠学习、记忆能力,通过长时程电位反映突触可塑性,采用免疫荧光检测BrdU阳性表达,Western blotting检测蛋白表达。结果:体外研究中,七氟醚造成神经元细胞活力与细胞存活率降低,导致Wnt3a、β-catenin与p-GSK3β(Ser9)蛋白表达减少,MAP2/Synaptophysin表达下调。体内研究中,七氟醚造成幼鼠逃避潜伏期明显增加,且fEPSP斜率降低,BrdU表达减少,Wnt3a、β-catenin与p-GSK3β(Ser9)蛋白表达减少。然而,左乙拉西坦升高了神经元活力与细胞存活率,增加了Wnt3a、β-catenin与p-GSK3β(Ser9)蛋白表达,上调了MAP2/Synaptophysin表达。与此同时,左乙拉西坦减少了幼鼠的逃避潜伏期,上调了fEPSP斜率,增加了Wnt3a、β-catenin与p-GSK3β(Ser9)蛋白表达,且增加了海马DG区BrdU表达。结论:左乙拉西坦明显改善了七氟醚诱导的幼鼠认知功能损伤,其机制可能是通过激活Wnt/β-catenin信号提高海马神经元增殖,增加海马DG区神经发生。

OBJECTIVE To explore the effect and mechanism of levetiracetam on the damage of hippocampal neurons.METHODS 3%sevoflurane was arranged for primary hippocampal neuron injury model construction.Cell proliferation and viability were detected by CCK8 and trypan blue staining.The expression of Wnt/β-catenin signaling pathway proteins were detected by Western blot and the expression of MAP2/Synaptophysin were detected by fluorescence.Then,3%sevoflurane was used to construct cognitive impairment model,Morris water maze was used to detect learning and memory ability,long-term potential which reflecting synaptic plasticity was detected,immunofluorescence was used to detect the expression of BrdU,and Western blotting was used to detect the expression of Wnt/β-catenin signaling pathway.RESULTS In vitro,sevoflurane caused a decrease in the cell proliferation and viability of primary hippocampal neurons,resulted in a decrease in the expression of Wnt3 a,β-catenin and p-GSK3β(Ser9)proteins,and down-regulated MAP2/Synaptophysin expression.In vivo,sevoflurane caused an increase in the escape latency of infant rat and down-regulated of fEPSP slope.Sevoflurane also mediated a decrease in BrdU expression at DG area and resulted in a decrease of Wnt3 a,β-catenin and p-GSK3β(Ser9)expression in hippocampal.However,the treatment of levetiracetam increased the proliferation and viability of primary hippocampal neurons,increased the expression of Wnt3 a,β-catenin and p-GSK3β(Ser9)protein,and up-regulated the expression of MAP2/Synaptophysin.At the same time,levetiracetam treatment significantly reduced the escape latency of infant rats,up-regulated fEPSP slope,increased Wnt3 a,β-catenin and p-GSK3β(Ser9)protein expression.As well,levetiracetam increased the expression of BrdU in the hippocampal DG region.CONCLUSION Levetiracetam significantly improved the cognitive impairment of infant mice induced by sevoflurane whose mechanism may relate to neurogenesis and proliferation in hippocampal DG area through the Wnt/β-catenin signaling pathway activation.