摘要目的:探讨黄芪甲苷是否通过锌离子(Zn 2+)调控线粒体相关内质网膜(MAMs)发挥神经保护作用，并阐明可能的机制。 方法:常规培养PC12细胞并分为7组。对照组：细胞常规培养；2-DG组：用50 μmol/L 2-DG处理30 min；黄芪甲苷+2-DG组：用50 μmol/L黄芪甲苷处理20 min后再用50 μmol/L 2-DG处理30 min；黄芪甲苷组：用50 μmol/L黄芪甲苷处理20 min；TPEN+黄芪甲苷+2-DG组：用10 μmol/L TPEN处理10 min后再用50 μmol/L黄芪甲苷处理20 min，接着用50 μmol/L 2-DG处理30 min；TPEN组：10 μmol/L TPEN处理10 min；TPEN+2-DG组：用10 μmol/L TPEN处理10 min后再用50 μmol/L 2-DG处理30 min。通过Western blotting实验检测葡萄糖调节蛋白(GRP)78、GRP94、含半胱氨酸的天冬氨酸蛋白水解酶(Caspase)-8、B细胞受体相关蛋白31(Bap31)、线粒体分裂蛋白1(Fis1)的表达，采用Annexin V-FITC/PI试剂盒检测细胞凋亡水平，采用免疫荧光法检测Fis1蛋白的表达，采用线粒体荧光探针TMRE检测线粒体通透性转移孔(mPTP)开放情况。结果:(1)与对照组相比，2-DG组GRP78、GRP94、Caspase-8、Bap31、Fis1蛋白表达以及Annexin V-FITC/PI染色的绿色荧光强度明显增加；与2-DG组相比，黄芪甲苷+2-DG组上述5种蛋白表达以及Annexin V-FITC/PI染色的绿色荧光强度明显降低；与黄芪甲苷+2-DG组相比，TPEN+黄芪甲苷+2-DG组上述5种蛋白表达以及Annexin V-FITC/PI染色的绿色荧光强度明显增强；差异均有统计学意义( P<0.05)。(2)与对照组相比，2-DG组Fis1蛋白荧光强度明显增强；与2-DG组相比，黄芪甲苷+2-DG组Fis1蛋白荧光强度明显减弱；与黄芪甲苷+2-DG组相比，TPEN+黄芪甲苷+2-DG组Fis1蛋白荧光强度明显增强；与对照组相比，TPEN+2-DG组Fis1蛋白荧光强度明显增强；差异均有统计学意义( P<0.05)。(3)与对照组相比，2-DG组TMRE荧光强度明显减弱；与2-DG组相比，黄芪甲苷+2-DG组TMRE荧光强度明显增强；与黄芪甲苷+2-DG组相比，TPEN+黄芪甲苷+2-DG组TMRE荧光强度明显减弱；与对照组相比，TPEN+2-DG组TMRE荧光强度明显减弱；差异均有统计学意义( P<0.05)。 结论:黄芪甲苷能够通过Zn 2+抑制内质网应激诱导的PC12细胞凋亡，阻止mPTP开放发挥神经保护作用，其作用机制可能与Fis1、Bap31表达有关。

abstractsObjective:To investigate whether astragaloside IV (AS-IV) exerts neuroprotective effects via zinc ion (Zn 2+) modulation of mitochondria-associated endoplasmic reticulum membranes (MAMs) and to elucidate the possible mechanisms. Methods:PC12 cells (rat adrenal pheochromocytoma cells) were routinely cultured; they were divided into 7 groups: control group (routinely cultured), 2-DG group (treated with 2-DG at 50 μmol/L for 30 min), astragaloside IV+2-DG group (treated with 50 μmol/L astragaloside IV for 20 min, and then treated with 50 μmol/L 2-DG for 30 min), astragaloside IV group (treated with 50 μmol/L astragaloside IV for 20 min), N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (a zinc chelator, TPEN)+astragaloside IV+2-DG group (treated with 10 μmol/L TPEN for 10 min, 50 μmol/L astragaloside IV for 20 min, and then 50 μmol/L 2-DG for 30 min); TPEN group (treated with 10 μmol/L TPEN for 10 min), TPEN+2-DG group (treated with 10 μmol/L TPEN for 10 min and then treated with 50 μmol/L 2-DG for 30 min). The expressions of glucose-regulated protein (GRP)78, GRP94, cysteine-containing aspartate proteolytic enzyme caspases-8, B cell receptor associated protein 31 (BAP31) and mitochondrial fission protein 1 (Fis1) were detected by Western blotting. Annexin V-FITC/PI kit was used to detect the apoptosis level. Immunofluorescence was used to detect the Fis1 protein expression, and mitochondrial fluorescence probe TMRE was used to detect the opening of mitochondrial permeability transition pore (mPTP).Results:(1) Compared with the control group, the 2-DG group had significantly increased expressions of GRP78, GRP94, Caspase-8, Bap31 and Fis1, and green fluorescent intensity of Annexin V-FITC ( P<0.05); compared with the 2-DG group, the astragaloside IV+2-DG group had significantly decreased expressions of GRP78, GRP94, Caspase-8, Bap31 and Fis1, and green fluorescent intensity of Annexin V-FITC ( P<0.05); compared with the astragaloside IV+2-DG group, the TPEN+astragaloside IV+2-DG group had significantly increased expressions of GRP78, GRP94, Caspase-8, Bap31 and Fis1, and green fluorescent intensity of Annexin V-FITC ( P<0.05). (2) Compared with the control group, the 2-DG group had significantly enhanced Fis1 protein fluorescent intensity ( P<0.05); the astragaloside IV+2-DG group had significantly decreased Fis1 protein fluorescent intensity compared with 2-DG group ( P<0.05); Compared with astragaloside IV+2-DG group, TPEN+astragaloside IV+2-DG group had significantly enhanced Fis1 protein fluorescence intensity ( P<0.05); compared with the control group, the TPEN+2-DG group had significantly enhanced Fis1 protein fluorescent intensity ( P<0.05). (3) TMRE fluorescence intensity in 2-DG group was significantly decreased compared with control group ( P<0.05); TMRE fluorescence intensity in astragaloside IV+2-DG group was significantly enhanced compared with 2-DG group ( P<0.05); TMRE fluorescence intensity in TPEN+astragaloside IV+2-DG group was significantly decreased compared with astragaloside IV+2-DG group (P<0.05); TMRE fluorescence intensity in TPEN+2-DG group was significantly decreased compared with the control group ( P<0.05). Conclusion:Astragaloside IV can exert neuroprotective effects through Zn 2+ inhibiting endoplasmic reticulum stress-induced apoptosis and preventing mPTP opening in PC12 cells, whose mechanism may be related to Fis1 and Bap31 expressions.