Fen-fang Hong, Fa-xian Guo, Ying Zhou, Qin-hua Min, Da-lei Zhang, Bei Yang, Wei-ying Zhou, Lei Wu, Zhi-ping Wei, Hui Liu, Shu-long Yang
Department of Physiology, College of Medicine, Nanchang University, Nanchang 310006, China; Department of Experimental Teaching, College of Medicine, Nanchang University, Nanchang 310006, China.
Department of Physiology, College of Medicine, Nanchang University, Nanchang 310006, China.
J Ethnopharmacol. 2015 Apr 2;163:203-9. doi: 10.1016/j.jep.2015.01.032. Epub 2015 Feb 4.
The pathogenesis of thromboangiitis obliterans (TAO) has not been fully elucidated until now. Shenfu injection (SFI), a traditional Chinese formula has been widely used clinically for the treatment of cardiovascular diseases for more than two decade. Our previous results first suggested that SFI can cause a significant therapeutic effect on experimental TAO model rats. This experiment was designed to further investigate the protective effect of SFI on VEC damaged by hydrogen peroxide (H2O2) oxidative stress in vitro.
The cell viability was evaluated by the MTT assay, the activities of SOD and GSH-PX and the content of MDA in the supernatants of the cultured ECV304 cells were evaluated by a colorimetry method, cell apoptosis was detected by flow cytometry and an AO/EB double staining method. The protein expressions of Bcl2, Bax and caspase-3 were examined by Western blotting.
When compared with control group, lower survival rate of ECV304 cells was observed in H2O2 group (p<0.01) ; 20μl/ml, 30μl/ml and 40μl/ml SFI increased the survival rate of ECV304 cells under H2O2 oxidative stress (p<0.05 and p<0.01). The activities of SOD and GSH-PX were higher and MDA level was lower in H2O2 group than those in control group. These effects of H2O2 on SOD, GSH-PX activities and MDA content were reversed by SFI in concentration-dependent way (p<0.05 and p<0.01). Flow cytometry and AO-EB double staining discovered that SFI pretreatment inhibited the ECV304 cells apoptosis. The protein expression of caspase3 in 30μl/ml and 40μl/ml SFI groups significantly decreased whereas Bcl2 protein expressions in 20μl/ml, 30μl/ml and 40μl/ml SFI groups were higher than H2O2 group, with Bax protein expression much lower than H2O2 group (p<0.05 and p<0.01).
Our findings suggest that SFI could prevent the ECV304 cells against H2O2 oxidative-stress by enhancing antioxidant enzyme activities, reducing the membrane lipid peroxidation, as well as upregulating antiapoptotic and downregulating apoptosis protein expressions.
直到现在，血栓闭塞性脉管炎（TAO）的发病机制仍未完全阐明。参附注射液（SFI），一种传统的中药配方，二十多年来一直在临床上广泛用于治疗心血管疾病。我们之前的结果首次表明，SFI对实验性TAO模型大鼠有显著治疗作用。本实验旨在进一步研究SFI对体外过氧化氢（H2O2）氧化应激损伤的血管内皮细胞（VEC）的保护作用。
采用MTT法评估细胞活力，比色法评估培养的ECV304细胞上清液中超氧化物歧化酶（SOD）和谷胱甘肽过氧化物酶（GSH-PX）的活性以及丙二醛（MDA）的含量，通过流式细胞术和AO/EB双重染色法检测细胞凋亡。采用蛋白质印迹法检测Bcl2、Bax和caspase-3的蛋白表达。
与对照组相比，H2O2组ECV304细胞存活率较低（p<0.01）；20μl/ml、30μl/ml和40μl/ml的SFI提高了H2O2氧化应激下ECV304细胞的存活率（p<0.05和p<0.01）。H2O2组SOD和GSH-PX的活性较高，MDA水平低于对照组。SFI以浓度依赖的方式逆转了H2O2对SOD、GSH-PX活性和MDA含量的这些影响（p<约0.05和p<0.01）。流式细胞术和AO-EB双重染色发现，SFI预处理可抑制ECV304细胞凋亡。30μl/ml和40μl/ml SFI组中caspase3的蛋白表达显著降低，而20μl/ml、30μl/ml和40μl/ml SFI组中Bcl2蛋白表达高于H2O2组，Bax蛋白表达远低于H2O2组（p<0.05和p<0.01）。
我们的研究结果表明，SFI可通过增强抗氧化酶活性、减少膜脂质过氧化以及上调抗凋亡蛋白表达和下调凋亡蛋白表达来保护ECV304细胞免受H2O2氧化应激的影响。
