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目的:观察羟基红花黄色素A(hydroxysafflower yellow A,HSYA)联合黄苗甲苷-Ⅳ(astragaloside-Ⅳ,As-Ⅳ)对人重组肿瘤坏死因子tumor necrosis factor-α,TNF-α)诱导人胳静脉内皮细胞EA.hy926调亡的影响。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~(-1),1×10~(-5),1×10~(-1)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~(-5)mol·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)蛋内表达的影响,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Bd-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1-ISYA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 hydroxysafflor yellow A (HSYA) combined with astragaloside-Ⅳ (As-Ⅳ) on the induction of human recombinant human tumor necrosis factor-α (TNF-α) Effects of apoptosis on endothelial cell EA. Hy926. Methods: MTT assay was used to detect the expression of TNF-α (0, 10, 20 and 50 μg · L -1), HSYA (0,1 × 10 -6, (-1, 1 × 10 -5, 1 × 10 -1 mol·L -1), As-Ⅳ (0,1 × 10 -6, 5 × 10 -1) The effect of HSYA combined with As-Ⅳ (1 × 10 ~ (-5), 1 × 10 ~ (-4) mol·L -1) 5) mol·L -1 on cell viability induced by TNF-α (20μg · L -1). Western blot was used to detect the effect of different concentrations of TNF-α on the expression of Bcl-2, Bax, Bax and activated aspartate protease-3 (cleaved Caspase-3) and activated cleaved Caspase-9, and whether HSYA combined with As-Ⅳ can improve the expression of these proteins. The effect of HSYA combined with As-Ⅳ on Bcl-2 gene expression induced by TNF-α was detected by Real-time PCR. The expression of Bcl-2 and Bax protein induced by TNF-α by HSYA combined with As-Ⅳ was analyzed by immunofluorescence Impact. RESULTS: Compared with the blank group, TNF-α induced EA.hy926 cell viability decreased at 20μg · L -1, up-regulated the expression of Bax, cleaved Caspase-3 and cleaved Caspase-9 protein and down-regulated Bcl-2 protein Compared with TNF-α group, HSYA combined with As-Ⅳ pretreatment could decrease the cell viability induced by TNF-α, inhibit the expression of Bax, cleaved Caspase-3 and cleaved Caspase-9 and increase the expression of Bcl- Expression, and 1-ISYA combined with As-Ⅳ pretreatment group of these indicators better than HSYA or As-IV pretreatment group. Conclusion: HSYA combined with As-Ⅳ can inhibit the apoptosis of EA.hy926 cells induced by TNF-α, and its mechanism is better than that of monotherapy. The mechanism may be related to the down-regulation of Bax, cleaved Caspase-3, cleaved Caspase- 2 expression related.