丙酮酸乙酯通过Nrf2途径抑制谷氨酸对大鼠小脑颗粒神经元的毒性作用

Ethyl pyruvate protects against glutamate excitotoxicity through Nrf2 pathway in rat cerebellar granule neuron model

目的:探讨丙酮酸乙酯是否对谷氨酸诱导的新生大鼠小脑颗粒神经元具有保护作用及其作用机制。方法:体外原代培养新生7 d大鼠小脑颗粒神经元模型。将培养皿中成熟细胞模型随机分为空白对照组、谷氨酸组和谷氨酸+丙酮酸乙酯组。通过MTT法及FDA/PI荧光双染色法测定细胞存活率,用显微镜观察细胞形态并用核染色法分析核形态学变化;通过Fluo-3/AM检测细胞内游离钙浓度([Ca^2+]i);通过H2-DCF-DA染色检测细胞内ROS含量;通过DHE染色法检测细胞内超氧阴离子含量;通过免疫组化和Western blot法检测细胞核和细胞浆中核因子E2相关因子2(Nrf2)和血红素加氧酶1(HO-1)的表达水平;通过抗氧化反应元件(ARE)诱导转录并采用双萤光素酶报告基因实验检测转录活性。结果:(1)丙酮酸乙酯可剂量依赖性(0.1~5 mmol/L)拮抗谷氨酸的兴奋性毒性作用(P<0.05);(2)丙酮酸乙酯(2.5和5 mmol/L)+谷氨酸30 min可阻滞[Ca^2+]i升高(P<0.05);(3)H2-DCF-DA染色中,谷氨酸+丙酮酸乙酯组ROS含量低于谷氨酸组(P<0.05);DHE染色中,谷氨酸+丙酮酸乙酯组超氧阴离子含量低于谷氨酸组(P<0.05);(4)与谷氨酸组比较,丙酮酸乙酯+谷氨酸组6 h后细胞浆内Nrf2降低,细胞核内Nrf2浓度升高,12 h仍显著升高(P<0.05);丙酮酸乙酯+谷氨酸组HO-1升高的时间较单独谷氨酸组早,且升高的幅度显著(P<0.05);丙酮酸乙酯组ARE诱导的萤光素酶转录活性显著增强。结论:丙酮酸乙酯能够抑制谷氨酸诱导的体外培养新生大鼠小脑颗粒神经元死亡,其机制与通过抑制[Ca^2+]i升高和激活Nrf2/ARE/HO-1通路而拮抗谷氨酸的兴奋性毒性作用有关。

AIM:To investigate the protective effect of ethyl pyruvate on glutamate-induced rat cerebellar granule neurons and its mechanism.METHODS:The pure culture system of cerebellar granule neurons in vitro isolated from 7-day-old neonatal rats was established.The neurons were randomly divided into 3 groups:control group,glutamate group,and glutamate+ethyl pyruvate group.The cell viability was measured by MTT assay and FDA/PI double fluorescent staining.Cellular morphological changes were assessed by contrast microscopy.Nuclear morphological changes were assessed by Hoechst 33258 staining.The intracellular free calcium concentration([Ca^2+]i)was detected by Fluo-3/AM.H2-DCF-DA staining method was used to detect intracellular reactive oxygen species(ROS),and DHE staining method was used to detect intracellular superoxide anion content.The levels of nuclear factor E2-related factor 2(Nrf2)and heme oxygenase-1(HO-1)in the cytoplasm and nucleus were detected by immunohistochemical staining and Western blot.The dual-luciferase reporter assay was used to detect the transcriptional activity induced by antioxidant response element(ARE).RESULTS:(1)The protective effects of ethyl pyruvate against excitotoxicity induced by glutamate were dose-dependent(0.1~5 mmol/L,P<0.05).(2)Ethyl pyruvate(2.5 and 5.0 mmol/L)+glutamate blocked the increase in[Ca^2+]i in 30 min.(3)The ROS content in ethyl pyruvate+glutamate group was lower than that in glutamate group(P<0.05).The intracellular superoxide anion content in ethyl pyruvate+glutamate group was lower than that in glutamate group(P<0.05).(4)Compared with glutamate group,the expression of Nrf2 was decreased in the cytoplasm and increased in the nucleus in ethyl pyruvate+glutamate group(P<0.05),while the expression of HO-1 was increased more significantly and rapidly in ethyl pyruvate+glutamate group than that in glutamate group(P<0.05).The transcriptional activity induced by ARE was increased in ethyl pyruvate group.CONCLUSION:Ethyl pyruvate protects neonatal rat cerebellar granule neurons against the excitotoxicity of glutamate via inhibiting the elevation of[Ca^2+]i and activating the Nrf2/ARE/HO-1 pathway.