黄芪甲苷在N-甲基-D-天冬氨酸受体介导的神经元兴奋性损伤中的保护作用

AstragalosideⅣprotects neurons in N-methyl-D-aspartate receptor-mediated neuronal excitatory injury

目的探讨黄芪甲苷(astragalosideⅣ)对N-甲基-D-天冬氨酸受体(N-methyl-D-aspartic acid receptor,NMDAR)介导的神经元兴奋性损伤的保护作用。方法选择新生0~1 d的C57/BL6J小鼠,分离海马区神经元进行原代细胞培养,将培养的神经元随机分为对照组、N-甲基-D-天冬氨酸(N-methyl-D-aspartic acid,NMDA)损伤模型组、氧-糖剥夺(oxygen and glucose deprivation,OGD)模型组、NMDAR抑制剂氯胺酮组及黄芪甲苷组。采用Hoechst-33342染色观察各组神经元死亡情况,采用ELISA法检测各组神经元乳酸脱氢酶(lactate dehydrogenase,LDH)释放情况;采用Ca^(2+)成像观察不同条件下〔加入NMDA;NMDAR抑制剂APV(100μmol/L)预处理+NMDA;黄芪甲苷(100μmol/L)预处理+NMDA;无Ca^(2+)+NMDA〕神经元内钙离子浓度;采用Western blot测定各组神经元活化型半胱氨酸天冬氨酸蛋白水解酶-3(cysteinyl aspartate specific proteinase-3,Caspase-3)的表达。另选择出生后4~6周的C57BL/6雄性小鼠,制备包含海马的冠状脑切片,使用电压钳观察黄芪甲苷应用前后突触后膜电流的变化。结果与应用黄芪甲苷(100μmol/L)前比较,应用黄芪甲苷(100μmol/L)后海马CA1区锥体细胞微小兴奋性突触后电流的平均峰值降低〔(9.96±0.43)pA比(12.63±0.45)pA;t=3.741,P<0.05〕,平均频率〔(0.52±0.03)Hz比(0.68±0.05)Hz;t=2.933,P<0.05〕下降;与NMDA损伤模型组相比,黄芪甲苷组神经元凋亡率降低,LDH释放减少,活化型Caspase-3表达减低,细胞内钙离子内流减轻(均P<0.05);在体外OGD模型实验中,黄芪甲苷亦表现出同样的神经元保护作用。结论黄芪甲苷在NMDAR介导的神经元兴奋性损伤中具有保护作用,其作用机制与抑制NMDAR有关。

Objective To investigate the protective effect of astragalosideⅣon N-methyl-D-aspartic acid receptor(NMDAR)-mediated neuronal excitotoxicity injury.Methods Hippocampal neurons isolated from newborn C57/BL6J mice aged 0-1 d were used for primary cell culture.The cultured cells were randomly divided into control group,N-methyl-D-aspartic acid(NMDA)injury model group,oxygen-glucose deprivation(OGD)model group,NMDAR inhibitor ketamin intervention group,and astragalosideⅣintervention group.Hoechst-33342 staining was used to observe the apoptosis of neurons in each group,and ELISA was used to detect the release of lactate dehydrogenase(LDH);Ca^(2+)imaging was used to observe the intracellular calcium concentration of neurons under different conditions[addition of NMDA;pretreatment with NMDAR inhibitor APV(100μmol/L)+NMDA;astragalosideⅣ(100μmol/L)pretreatment+NMDA;extracellular fluid without Ca^(2+)+NMDA];Western blot was used to determine the expression of activated cysteinyl aspartate specific proteinase-3(cleaved caspase-3)in neurons of each group.Another group of C57BL/6 male mice aged 4-6 weeks was used to prepare coronal brain slices containing the hippocampus,and the changes in postsynaptic membrane currents were observed using a voltage clamp.Results Compared with the application of astragalosideⅣ(100μmol/L),the average peak of miniature excitatory postsynaptic currents in hippocampal CA1 pyramidal cells decreased[(9.96±0.43)pA vs.(12.63±0.45)pA;t=3.741,P<0.05],and the average frequency[(0.52±0.03)Hz vs.(0.68±0.05)Hz;t=2.933,P<0.05]decreased.Compared with the NMDA injury model group,the astragalosideⅣgroup had a lower rate of neuronal apoptosis,less LDH release,lower expression of cleaved caspase-3,and a decrease in intracellular calcium influx(all P<0.05).In the in vitro ischemia model OGD,astragalosideⅣshowed a similar neuroprotective effect.Conclusions AstragalosideⅣhas a protective effect on NMDAR-mediated neuronal excitotoxicity injury,and the mechanism is related to inhibition of NMDAR.