Neuroprotection Effect Of α-Asarone In The Cell Model Of Intractble Epilepsy

Objective:To observe apoptosis effection in the cell model of intractble epilepsy which was exposed to magnesium-free extracellular solution media and discuss influences of -asarone in the cell model of intractble epilepsy on optical density (OD),apoptosis rate, apoptosis rate with mitochondrial damage and the influence of the ultrastructure of neurons. And investigate neuroprotection effect of -asarone in the cell model of intractble epilepsy.Methods:The hippocampal cell was isolated from the newborn rats in the 24 hours and cultured. The growth characteristics of hippocampal neurons in vitro was observed througt phase-contrast microscope.The neurons were identified by neurofilament proteins(NF) in the ninth day and cultured with 7.5,15,30,60,120mg/L -asarone. After 4 hours the hippocampal neurons were exposed to magnesium-free extracellular solution media for 3 hours and created the cell model of intractble epilepsy and then returned to maintainable media consisted of -asarone. The OD were detected by thiazolyl blue tetrazolium bromide MTT . Detected apoptosis rate at each time point and apoptosis rate of -asarone (15,60,120 g/ml) groups, apoptosis rate with mitochondrial damage by flow cytometry (FCM). The ultrastructural changes of neurons were observed by transmission electron microscopy .Results:1.The neurons of model group have a enrichment nuclear and the chromatin condense on the edge. There are a large number of vacuoles in the cytoplasm, endoplasmic reticulum, swelling mitochondrial and mitochondria cristae partial rupture in the electron microscope. The neurons of 120 g/mlasarone only have a little chromatin assembly in chromatin, a few of cytoplasm in vacuoles. The mitochondria were slightly swollen and there is no significant difference compared with the control group. 2. Compared with model group, the OD of -asarone group was significantly higher (P <0.01), and the survival rate of hippocampal neurons increased in a dose manner. 3. Compared with the control group,the apoptosis of model group at each time point (6h, 12h, 24h, 48h, 72h) increased notably (P <0.01). The apoptosis of model group increased significantly with time prolonging (P <0.01). Compared with model group, the apoptosis rate of -asarone groups (15 60 120 g/ml) were significantly lower (P <0.01) and have a dose manner. The apoptosis rate of 120 g/ml -asarone no significant difference compared with the control group (P> 0.05). 4. Compared with the control group, the apoptosis rate of model group with mitochondrial damage increased significantly (P <0.01). Compared with model group, apoptosis rate of - asarone(15,60,120 g/ml) groups with mitochondrial damage decreased (P <0.01), and apoptosis rate of 120 g/ml -asarone group have no difference in statistically significant ( P> 0.05)compared with the control group.The neurons of normal control group emit bright green and red fluorescence in the fluorescence microscopy. While the model group emits low red and high green fluorescence. The neurons in each group of -asarone emit bright red fluorescence gradually and the number of red cells gradually increased with higher concentrations, especially fluorescence of 120 g/ml - asarone group have no significant difference compared with the normal control group.Conclusions:1.The cell model of intractable epilepsy exist neuron apoptosis, with time prolonging, neuron apoptosis were more serious.2 The -asarone can lighten the neurons apoptosis in the cell model of intractble epilepsy in a dose-dependent manner.3.The -asarone can lighten the apoptosis of the cell model of intractble epilepsy by stabling inhibiting mitochondrial damage .