羟基红花黄色素A联合黄芪甲苷-Ⅳ对TNF-α诱导内皮细胞凋亡的影响

Effect of Hydroxysafflor Yellow A Combined with Astragaloside-Ⅳ on Apoptosis of Endothelial Cells Induced by TNF-α

目的：观察羟基红花黄色素A（hydroxysafflower yellow A,HSYA）联合黄芪甲苷-Ⅳ（astragaloside-Ⅳ,As-Ⅳ）对人重组肿瘤坏死因子-α（tumor necrosis factor-α,TNF-α）诱导人脐静脉内皮细胞EA.hy926凋亡的影响。方法：噻唑蓝（MTT）比色法检测不同浓度TNF-α（0,10,20,50μg·L^（-1））,HSYA（0,1×10^（-6）,5×10^（-6）,1×10^（-5）,1×10^（-4）mol·L^（-1））,As-Ⅳ（0,1×10-6,5×10^（-6）,1×10^（-5）,1×10^（-4）mol·L^（-1））对细胞活力的影响以及HSYA联合As-Ⅳ（1×10-5mol·L^（-1））对TNF-α（20μg·L^（-1））损伤细胞活力的影响。蛋白免疫印迹法（Western blot）检测不同浓度TNF-α对B细胞淋巴瘤/白血病-2（Bcl-2）,Bcl-2相关X蛋白（Bax）,活化的天冬氨酸蛋白水解酶-3（cleaved Caspase-3）,活化的天冬氨酸蛋白水解酶-9（cleaved Caspase-9）蛋白表达的影响,以及HSYA联合As-Ⅳ对上述蛋白表达是否具有改善作用。实时荧光定量PCR（Real-time PCR）检测HSYA联合As-Ⅳ对TNF-α诱导Bcl-2基因表达的影响,免疫荧光技术分析HSYA联合As-Ⅳ对TNF-α诱导Bcl-2,Bax蛋白表达的影响。结果：与空白组比较,TNF-α在20μg·L^（-1）时可诱导EA.hy926细胞活力下降,上调Bax,cleaved Caspase-3,cleaved Caspase-9蛋白的表达,下调Bcl-2蛋白的表达;与TNF-α组比较,HSYA联合As-Ⅳ预处理组可减少TNF-α诱导的细胞活力下降,抑制Bax,cleaved Caspase-3,cleaved Caspase-9蛋白的表达,增加Bcl-2蛋白的表达,且HSYA联合As-Ⅳ预处理组对上述指标的改善效果优于HSYA或As-Ⅳ单独预处理组。结论：HSYA联合As-Ⅳ对TNF-α诱导的EA.hy926细胞凋亡具有抑制作用,且效果优于单药,其机制可能与其下调Bax,cleaved Caspase-3,cleaved Caspase-9和上调Bcl-2的表达有关。

Objective： To observe the effect of hydroxysafflower yellow A（ HSYA） combined with astragaloside-Ⅳ（ As-Ⅳ） on the apoptosis of human umbilical vein endothelial cells EA. hy926 induced by human recombinant tumor necrosis factor-α（ TNF-α）. Method： The effects of different concentrations of TNF-α（ 0,10,20,50 μg·L^（-1））,HSYA（ 0,1 × 10^（-6）,5 × 10^（-6）,1 × 10^（-5）,1 × 10^（-4） mol·L^（-1））,As-Ⅳ（ 0,1 × 10^（-6）,5 × 10^（-6）,1 × 10^（-5）,1 × 10-4 mol·L^（-1）） on the cell viability of EA. hy926,as well as the effect of HSYA combined with As-Ⅳ（ 1 × 10-5 mol·L^（-1）） on the viability of EA. hy926 cells induced by TNF-α（ 20 μg·L^（-1）） were measured by thiazole blue method（ MTT） assay; Western blot assay was used to detect the effect of different concentrations of TNF-α on the expressions of B-cell lymphoma-2（ Bcl-2）,Bcl-2 associated X protein（ Bax）,cleaved cysteine asparate protease-3（ Caspase-3） and cleaved Caspase-9 proteins,and the effect of HSYA combined with As-Ⅳ in improving expressions of the above proteins. Real-time PCR assay was used to observe the effect of HSYA combined with As-Ⅳ on the expression of Bcl-2 gene induced by TNF-α. Immunofluorescence technique was used to analyze the effect of HSYA combined with As-Ⅳ on the expressions of Bcl-2 and Bax proteins induced by TNF-α. Result：Compared with the blank group,TNF-α could induce the activity of EA. hy926 cells at 20 μg·L^（-1）,up-regulate the expressions of Bax,cleaved Caspase-3 and cleaved Caspase-9,and down-regulate the expression of Bcl-2. Compared with TNF-α group,HSYA combined with As-Ⅳ pretreatment group could decrease the cell viability induced by TNF-α,inhibit the expressions of Bax,cleaved Caspase-3 and cleaved Caspase-9,and increase the expression of Bcl-2 induced by TNF-α. The effect of HSYA combined with As-Ⅳ pretreatment group was better than that of single use of HSYA or As-Ⅳ pretreatment group. Conclusion： HSYA combined with As-Ⅳ has an inhibitory effect on the apoptosis of EA. hy926 cells induced by TNF-α,with a better effect than that of single drug. The mechanism may be related to the down-regulation of expressions of Bax,cleaved Caspase-3 and cleaved Caspase-9 and the up-regulation of Bcl-2 expression.