银杏叶标准提取物对H_2O_2诱导PC12细胞凋亡的保护作用

Protective Effect of EGB761 on H202-induced Damage of PC12 Cells

目的:探讨银杏叶标准提取物EGB761对过氧化氢(H2O2)诱导大鼠肾上腺髓质嗜铬瘤细胞(PC12细胞)凋亡的保护作用及可能的机制。方法:PC12细胞常规培养生长至对数期,进行如下分组试验:正常对照组(NS),H2O2组(H2O2,200μmol/LH2O2),低剂量EGB761组(GL,10μg/mlEGB761+H2O2),中剂量EGB761组(GM,20μg/mlEGB761+H2O2),高剂量EGB761组(GH,50μg/mlEGB761+H2O2)。用四甲基偶氮唑盐(MTT)比色法测定PC12细胞活力;流式细胞仪测定PC12细胞凋亡率;分光光度计测定过氧化氢酶(CAT)、谷胱甘肽过氧化物酶(GSH-Px)、总超氧化物岐化酶(T-SOD)活力和抗氧化能力(T-AOC)以及丙二醛(MDA)含量;Western blot法测定胞浆内Bcl-2、Bax以及Caspase-3的表达。结果:与正常对照组比较,H2O2组的PC12细胞的活力明显降低,细胞凋亡率显著增加(P<0.05)。与H2O2组比较,EGB761低、中、高组的PC12细胞活力显著增强,且呈剂量依赖关系(P<0.05);GM组和GH组CAT,GSH-Px,T-SOD和活性和T-AOC均显著提高(P<0.05),MDA含量显著下降(P<0.05),GL组T-SOD活力和T-AOC水平升高以及MDA含量下降(P<0.05),而CAT和GSH-Px活力无显著变化(P<0.05)。与H2O2组比较,EGB761低、中、高组PC12细胞的Bcl-2的表达量增加,Bax的表达量下降,Bcl-2/Bax的比值显著增加(P<0.05),Caspase-3的表达显著降低(P<0.05)。结论:H2O2可诱导PC12细胞凋亡,EGB761能显著抑制H2O2诱导PC12细胞凋亡并对细胞有保护作用;EGB761有较强的抗氧化能力,通过其抗氧化作用改善H2O2诱导的氧化应激状态,并能显著提高Bcl-2/Bax的比值,抑制Caspase-3的活化,发挥保护作用。

Objective：To investigate the protective effect of Standard extract of Ginkgo biloba （EGB761） on hydrogen peroxide （H2O2） induced damage of catecholaminnergic cell line（PC12 cell） and the possible molecular mechanisms.Methods：The differentiated PC12 cells were cultured with H-DMEM medium and divided into 5 groups： normal H-DMEM medium group （NS）; 200μmol/L H2O2 group （H2O2）; low-dose EGB761 group （GL, 10μg/ml EGB761 ＋ H2O2）; moderate-dose EGB761 group （GM, 20μg/ml EGB761 ＋ H2O2）; high-dose EGB761 group （GH, 50μg/ml EGB761 ＋ H2O2）.After corresponding treatment, PC12 cell viability was assayed by MTT, PC12 cell apoptosis rate was determined by flow cytometry.the level of five antioxidative indexes including CAT, GSH-Px, T-AOC, T-SOD and MDA were measured by visible spectrophotometry, the expression of Bcl-2, Bax and Caspase-3 were determinated by Western blot.Results：Compared with the NS group, PC12 cell viability of H2O2 group was significantly reduced, the rate of PC12 cell apoptosis exposure to H2O2 was significantly increased （P 0.05）. while EGB761 of different dose significantly ameliorated above indexes（P0.05）. When compared with H2O2 group, GM and GH groups increased the four antioxidative indexes of （CAT, GSH-Px, T-AOC and T-SOD） and decreased the content of MDA （P 0.05）.Low dose of EGB761 only increased T-SOD activity and T-AOC levels and decreased MDA content （P 0.05）, but it had no evident effects on the activities of CAT and GSH-Px （P 0.05）. EGB761 of different dose significantly increased the expression of Bcl-2 and significantly decreased the expression of Bax, the ratio of Bcl-2/Bax was significantly increased （P 0.05）, The expression of Caspase-3 was strikingly lower than that the H2O2 group （P 0.05）.Conclusion：H2O2 induced the differentitated PC12 cells apoptosis. EGB761 had potent effect on cytoprotective action and significantly inhibited PC12 cells apoptosis induced by H2O2. EGB761 has a powerful antioxidative capacity and can significantly increase the ratio of Bcl-2/Bax and inhibit the activation of Caspase-3. Therefore, EGB761 can inhibit H2O2-induced oxidative stress and correct the abnormal expression of apoptotic genes and abnormal ativation of apoptotic effect molecules Caspase-3.The results from this study provide an experimental evidence for the potential use of EGB761 in clinical treatment of neurodegenerative diseases.