β-细辛醚对谷氨酸诱导损伤的脑皮层神经元凋亡线粒体膜电位和超微结构的影响

Effect of β-asarone on mitochondrial membrane potentials and utrastructural changes induced apoptosis by glutamate in cultured rat cortical neurons

目的探讨β-细辛醚对谷氨酸(Glu)诱导损伤的脑皮层神经元凋亡、线粒体膜电位(mitochon-drial membrane potential,MMP)和超微结构的影响。方法体外培养乳鼠脑皮层神经元,β-细辛醚(7.5、15、30μg/ml)预给药4h,荡洗后加入终浓度为40mmol/L的Glu继续培养16h,采用流式细胞仪检测细胞凋亡率、MMP,并用透射电镜观察超微结构的变化。结果40mmol/L Glu组凋亡率明显增加(P<0.01),7.5、15、30μg/mlβ-细辛醚均能不同程度地抑制皮层神经元凋亡,30μg/mlβ-细辛醚抑制神经元凋亡的作用较7.5、15μg/mlβ-细辛醚明显(P<0.01),10μmol/L尼莫地平与30μg/mlβ-细辛醚抑制凋亡的作用相当(P>0.05);40mmol/L Glu组MMP明显低于正常对照组(P<0.01),10μmol/L尼莫地平组和30μg/mlβ-细辛醚组MMP明显高于40mmol/LGlu组(P<0.05,P<0.01),而与正常对照组比较无明显差别(P>0.05);电镜下40mmol/LGlu组的脑皮层神经元见染色质边聚、线粒体肿胀、部分内质网扩张,而30μg/mlβ-细辛醚组神经元则仅见少量内质网扩张、线粒体轻度肿胀。结论β-细辛醚可抑制Glu诱导的脑皮层神经元凋元,其作用机制可能与稳定细胞线粒体膜电位有关。

Objective To study the effect of β-asarone on apoptosis, mitochondrial membrane potentials （MMP）and ultrastructural changes induced by glutamate in cultured rat cortical neurons. Methods Rat cortical neurons were cultured in vivo, cultured with β-asarone for 4 hours in 12^th to 14^th day, then cultured with 40 mmol/L glutamate for 16 hours after washing, the MMP and apoptosis ratio were detected by flow cytometry, and mitochondriai ultrastructure changes were observed under transmission electron microscope. Results Apoptosis rate was significantly increased in neurons after exposure to glutamate for 16 hours （compare with control, P〈0. 01）,7. 5, 15, 30μg/ml β-asarone can check cortical neuronal apoptosis in distinct level, the effect of check apoptosis of 30 μg/ml β-asarone was more significant than 7. 5 μg/mlβ-asarone or 15 μg/ml β-asarone （P〈0. 01）, the effect of check apoptosis of 30 μg/ml β-asarone and 10 μmol/L Nimodipine was in the same manner （P〈0. 05）. MMP decreased obviously in neurons cultured with glutamate（compare with control, P〈0. 01）, MMP in neurons cultured with 30 μg/mlβ-asarone or 10 μmol/L Nimodipine was higher than which in neurons cultured with glutamate（P〈0. 05, P〈0. 01）, compare with normal control, there was no significant difference in MMP between neurons cultured with 30 μg/ml β-asarone or 10 μmol/L Nimodipine （P〈0. 05）. Most of neuronal ultrastruture obviously changed, including agminated chromatin, swelling mitochondrium and distended endoplasmic reticulum （ER）in neurons exposure to 40 mmol/L glutamate, there were little changes of mitochondria ultrastructure in neurons cultured with 30 μg/mlβ-asarone. Conclusions β-asarone inhibits glutamate-induced apoptosis in rat cortical neurons. The mechanism is related to maintaining mitochondrial membrance potential.