目的 探索西兰花多肽组分对过氧化氢(H₂O₂)所致血管内皮细胞损伤的保护作用及其机制。方法 采用醇沉,Sephadex G-25凝胶过滤层析获得分离西兰花多肽组分。体外培养人静脉血管内皮细胞EVC-304,经不同浓度西兰花多肽组分Ⅱ(1、2、4、8 μg·mL^-1)处理细胞24 h后,加入100 μmol·L^-1 H₂O₂再进行培养12 h。四甲基偶氮唑蓝(MTT)法检测细胞存活率,细胞分析仪检测EVC-304细胞凋亡和线粒体膜电位的变化,试剂盒法检测超氧化物歧化酶(SOD)活性和丙二醛(MDA)含量,Western blot法检测细胞内激活型Caspase-3、细胞色素C(Cyto C)、Bax和Bcl-2蛋白表达变化。结果 Sephadex G-25凝胶过滤层析共获得4个洗脱峰,选择西兰花多肽组分Ⅱ用于后续实验。实验结果表明,同正常组相比,H₂O₂损伤组细胞存活率显著降低(P<0.01),细胞凋亡率显著升高(P<0.01),线粒体膜电位去极化细胞比例升高(P<0.01),MDA含量增加,SOD活性减少,激活型Caspase-3、细胞质中Cyto C表达水平,Bax/Bcl-2比值升高(P<0.01)。与H₂O₂损伤组相比,2、4、8 μg·mL^-1西兰花多肽组分Ⅱ处理组细胞存活率明显升高(P<0.01),凋亡细胞率明显下降(P<0.01),线粒体膜电位去极化细胞比例下降(P<0.01),MDA含量下降,SOD活性升高,细胞内激活型Caspase-3,Cyto C表达,Bax/Bcl-2比值下降(P<0.01)。 结论 西兰花多肽组分Ⅱ对H₂O₂所致血管内皮细胞凋亡具有保护作用,其机制可能与保护血管内皮细胞受损线粒体功能相关。

OBJECTIVE To investigate the protective effects of broccoli polypeptide component on vascular endothelial cells injury induced by hydrogen peroxide (H₂O₂). METHODS The broccoli polypeptide was isolated by methods of alcohol precipitation and sephadex G-25 gel filtration chromatography. The EVC-304 cells were cultured in logarithmic growth phase and incubated with 100 μmol·L^-1 H₂O₂ for another 12 h after pretreatment with broccoli polypeptide component (1, 2, 4, 8 μg·mL^-1) for 24 h. Cell viability was determined by MTT assay, the change of cell apoptosis and mitochondrial membrane potential were measured with cytoanalyzer, the activity of SOD and the level of MDA were detected with the kit and the expression levels of Cleaved Caspase-3, Cyto C, Bax and Bcl-2 were detected by Western blotting. RESULTS Four main elution peaks were obtained after Sephadex G-25 gel filtration chromatography and the broccoli polypeptide components Ⅱ was selected subsequent experiments. The experimental results demonstrate that compared with the normal control group, the survival rate of H₂O₂-treated cells was decreased and the apoptosis ratio was increased significantly (P<0.01), mitochondrial membrane potential depolarization ratio was increased significantly (P<0.01), mitochondrial membrane potential depolarization ratio decreased (P<0.01), intracellular SOD activity decreased and MDA content increased (P<0.01), the proportion of cleaved caspase-3, Cyto C and Bax/Bcl-2 were increased (P<0.01). Compared with H₂O₂-treated group, the survival rate of cells in broccoli polypeptide component Ⅱ- pretreated groups was increased significantly (P<0.01), the apoptosis rate was decreased significantly (P<0.01). The SOD activity increased and MDA concentration decreased (P<0.01). The expression of intracellular cleaved caspase-3, Cyto C and Bax/Bcl-2 were decreased (P<0.01). CONCLUSION Broccoli polypeptide component Ⅱ has protective effect on hydrogen peroxide-induced vascular endothelial cells apoptosis and the possible mechanism may be associated with protection the function of mitochondrion.