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

LU Fen-ping; SU Ning; ZHU Ming-ming; ZHENG Zong-mei; SHEN Yu-jun; LIU Ting-yu; SHI Hong-fei

doi:10.13422/j.cnki.syfjx.2018050088

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

更新时间：2024-01-04

- Effect of Hydroxysafflor Yellow A Combined with Astragaloside-Ⅳ on Apoptosis of Endothelial Cells Induced by TNF-
- Chinese Journal of Experimental Traditional Medical Formulae Vol. 24, Issue 5, Pages: 88-94(2018)
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- DOI：10.13422/j.cnki.syfjx.2018050088
  CLC： R285
- Published：2018
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LU Fen-ping, SU Ning, ZHU Ming-ming, et al. Effect of Hydroxysafflor Yellow A Combined with Astragaloside-Ⅳ on Apoptosis of Endothelial Cells Induced by TNF-[J]. Chinese journal of experimental traditional medical formulae, 2018, 24(5): 88-94.
DOI：

LU Fen-ping, SU Ning, ZHU Ming-ming, et al. Effect of Hydroxysafflor Yellow A Combined with Astragaloside-Ⅳ on Apoptosis of Endothelial Cells Induced by TNF-[J]. Chinese journal of experimental traditional medical formulae, 2018, 24(5): 88-94. DOI： 10.13422/j.cnki.syfjx.2018050088.

摘要

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（0，10，20，50 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）对细胞活力的影响以及HSYA联合As-Ⅳ（1×10-5mol · L-1）对TNF-α（20 μg · 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20 μg · 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。

Abstract

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

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

1×10-4mol·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.

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