水蛭提取物对体外培养大鼠大脑皮层神经细胞缺氧性凋亡的影响

Effects of the antagonism of leech extract on the apoptosis of primary cultured neonate rat cerebral cortical neurons caused by hypoxia

目的研究水蛭提取物对体外缺氧性新生大鼠大脑皮层神经细胞凋亡的作用。方法采用NSE染色法,观察体外培养大脑皮层神经细胞的含量,TUNEL法检测神经细胞的凋亡率,Hochest 33342荧光染色法观察其对缺氧性神经细胞凋亡的保护作用,Bcl-2及Bax凋亡蛋白染色法对抗凋亡的可能机理进行研究。结果新生大鼠大脑皮层神经细胞原代培养NSE染色的结果显示其神经细胞含量为93.28%±1.18%;水蛭提取物给药组神经细胞凋亡率明显低于凋亡模型组;荧光染色除水蛭提取物组250μg.ml-1组外,各组神经细胞凋亡率明显降低;药物干预组表达Bcl-2的神经细胞较凋亡诱导模型组显著(P<0.01);水蛭提取物组中表达Bax的神经细胞较凋亡模型组略少,其1、2 mg.ml-1组与模型组有显著性差异。结论水蛭提取物具有明显的抗脑神经细胞凋亡的作用,抗凋亡机理与其对Bcl-2及Bax蛋白的调控有关。

OBJECTIVE To study the effects of leech extract on primary cultured neonate rat cerebral cortical neurons from the apop- tosis caused by hypoxia in vitro. METHODS NSE staining was used to determine the specific expression of neurons. TUNEL apoptotic staining was used to determine the changes of the apoptosis rate of neurons. Hochest 33342 fluorescent staining were used to observe the protections of leech extract on neurons from the necrosis caused by hypoxia. The expression changes of Bcl - 2 and Bax were directly observed for the investigation of the mechanism of anti - apoptosis of Leech Extract. RESULTS The enolase staining results showed that the average positive rate of primary cultured neonate rat cerebral cortical neurons was 93.28%± 1.18%. The TUNEL results displayed that the neurons apoptosis rate of leech extract controlled groups was decreased obviously. There was significant differences to compared with model group （P 〈 0. 05 or 0. 01 ）. Hoechst 33342 staining found the apoptosis rate of neurons in leech extract interfered groups decreased obviously except for 250250μg·ml-1 group and there was significant differences compared with model group（ P 〈 0.01 ）. Bcl - 2 staining results presented that the Bcl - 2 expressions of controlled groups were increased which was significantly different from model group（ P 〈 0.01 ）. Bax staining results showed that there were less Bax expressions of leech extract groups than model group, and only 1 mg. ml - l and 2 mg · ml - 1 groups of leech extract had significant differences compared to model group （ P 〈 0.05 or P 〈 0.01 ）. CONCLUSION There are obvious antagonism of apoptosis to the cerebral cortical neurons of Leech extract, and it＇ s mecha- nism of anti - apoptosis related to the regulations of Bcl - 2 and Bax protein.