藁本内酯对谷氨酸诱导的PC12细胞凋亡的保护作用  

Protective effect of ligustilide against glutamate-induced apoptosis in PC12 cells

作　　者：吴倩[1] 汪宁[1] 王艳[1] 汪光云 朴新新 

机构地区：[1]新安医学省部共建教育部重点实验室,安徽省中医药科学院中药药效与安全性评价研究所,安徽中医药大学药学院,安徽合肥230012

出　　处：《药学学报》2015年第2期162-168,共7页Acta Pharmaceutica Sinica

基　　金：国家自然科学基金资助项目(81374005);十二五科技支撑计划项目(2012BAI26B03);2013年度安徽省学术及技术带头人及后备人选科研活动经费资助项目

摘　　要：研究藁本内酯对谷氨酸诱导PC12细胞凋亡的保护作用。采用MTT比色法检测细胞存活率;Annexin V-FITC/propidium iodide(PI)双染法流式细胞术(flow cytometry,FCM)检测细胞凋亡率;Fluo-3/AM荧光染色法检测钙离子含量;Western blot法检测线粒体和胞浆中细胞色素C(cytochrome C,Cyt C)及凋亡相关蛋白Caspase-3、Bcl-2、Bax的表达。结果显示,谷氨酸具有细胞毒性,其半数抑制浓度(IC50)为15 mmol·L-1;不同浓度藁本内酯(1、5、15μmol·L-1)预处理可显著提高细胞存活率。与正常对照组相比,谷氨酸单独给药组的细胞凋亡率为13.39%;藁本内酯给药组(1、5、15μmol·L-1)使细胞的凋亡率分别减少到9.06%、6.48%和3.82%。并且藁本内酯可以显著减少谷氨酸所致的钙离子内流,Western blot结果显示,藁本内酯可能通过减少线粒体Cyt C的释放,抑制Caspase-3活性,同时升高Bcl-2/Bax比值来保护谷氨酸诱导的PC12细胞凋亡。结果表明,藁本内酯对谷氨酸诱导PC12细胞的凋亡具有保护作用。该保护作用可能与抑制细胞内钙离子内流及阻断Cyt C从线粒体释放入胞浆有关。To investigate the neuroprotective of ligustilide（LIG） against glutamate-induced apoptosis of PC12 cells, cell viability were examined by MTT assay. Flow cytometry was applied to assay cell apoptosis rate. Intracellular calcium concentration was measured by using fluorescent dye Fluo-3/AM. Cytochrome C（Cyt C）, Caspase-3, Bax and Bcl-2 protein expression were assayed by western blot. The results showed that glutamate is cytotoxic with an inhibitory concentration 50（ID50） of 15 mmol·L-1. Pretreatment with LIG（1, 5, 15 μmol·L-1） significantly improved cell viability. The apoptosis rate in glutamate-induced PC12 cells was 13.39%, and decreased in the presence of LIG（1, 5, 15 μmol·L-1） by 9.06%, 6.48%, 3.82%, separately. Extracellular accumulation of Ca2＋ induced by glutamate were significantly reduced by LIG. The results of western blot manifested that pretreatment LIG could decrease the release of Cyt C from mitochondria, down-regulate Caspase-3 protein expression and up-regulate Bcl-2/Bax ratio, thereby protects PC12 cells from apoptosis. In summary, LIG had protective effect on glutamate-induced apoptosis in PC12 cells through attenuating the increase in intracellular Ca2＋ concentration, and inhibiting the release of Cyt C from mitochondria to cytoplasm.

关 键 词：藁本内酯 PC12细胞 谷氨酸 钙离子 细胞色素C 细胞凋亡 

分 类 号：R285.5[医药卫生—中药学]