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目的探索西兰花多肽组分对过氧化氢(H_2O_2)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、8μg·mL~(-1))处理细胞24 h后,加入100μmol·L~(-1) H_2O_2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,H_2O_2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、8μg·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H_2O_2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 explore the protective effect of broccoli peptide on the injury of vascular endothelial cells induced by hydrogen peroxide (H 2 O 2) and its mechanism. Methods Separation and purification of broccoli polypeptide were obtained by using alcohol precipitation and Sephadex G-25 gel filtration chromatography. Cultured human vascular endothelial cells (EVC-304) in vitro, the cells were treated with different concentrations of broccoli polypeptide Ⅱ (1, 2, 4, 8 μg · mL -1) for 24 h, then treated with 100 μmol·L -1 ) H_2O_2 for 12 h. Cell viability was detected by MTT assay. Apoptosis and mitochondrial membrane potential of EVC-304 cells were detected by cell analyzer. The activity of superoxide dismutase (SOD) and malondialdehyde (MDA) were detected by Western blot. The expressions of activated caspase-3, Cyto C, Bax and Bcl-2 were detected by Western blot. Results Sephadex G-25 gel filtration chromatography obtained a total of four elution peaks, selected broccoli peptide fraction II for subsequent experiments. The experimental results showed that compared with the normal group, the cell survival rate was significantly decreased (P <0.01), the apoptosis rate was significantly increased (P <0.01), the mitochondrial membrane potential depolarized cells increased (P < 0.01). The content of MDA increased and the activity of SOD decreased. The expression of Cyto C and the ratio of Bax / Bcl-2 in activated caspase-3 and cytoplasm increased (P <0.01). Compared with H2O2-injured group, the viability of cells treated with 2,4,8μg · mL -1 broccoli peptide Ⅱ was significantly increased (P <0.01), and the apoptotic cells were significantly decreased (P <0.01) , The proportion of depolarized cells in mitochondrial membrane potential decreased (P <0.01), the content of MDA decreased, the activity of SOD increased, the expression of activated Caspase-3, Cyto C and the ratio of Bax / Bcl-2 decreased (P <0.01). Conclusion Broccoli polypeptide Ⅱ has a protective effect on the apoptosis of vascular endothelial cells induced by H 2 O 2, and its mechanism may be related to the protection of damaged mitochondrial function of vascular endothelial cells.