丹参多酚酸盐对过氧化氢诱导人内皮细胞EA.hy926氧化应激损伤的保护作用及机制
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摘要
目的:探讨丹参多酚酸盐(salvianolate)对过氧化氢诱导人脐静脉内皮细胞EA.hy926氧化损伤的保护作用及机制。方法:体外培养EA.hy926细胞,随机分为:正常对照组、损伤组和抗损伤(丹参多酚酸盐+过氧化氢)组。采用CCK-8法检测细胞活性,Transwell实验检测内皮细胞迁移能力;一氧化氮(nitric oxide,NO)试剂盒和ELISA法分别检测细胞培养液中NO和血管内皮生长因子(vascular endothelial growth factor,VEGF)的水平;流式细胞术检测细胞内超氧化物阴离子水平、线粒体跨膜电位和细胞凋亡情况;采用Western blot法检测caspase-3、cleaved-caspase3、Bcl-2、Bax、NF-κB和p53蛋白表达水平。结果:与损伤组比较,不同剂量的丹参多酚酸盐预处理可提高EA.hy926细胞的活性、降低内皮细胞的凋亡,并促进细胞的迁移(P<0.05);细胞上清液中VEGF和NO含量及细胞内线粒体膜电位水平显著升高(P<0.05);细胞内ROS水平显著下降;NF-κB、p53、Bax和cleaved caspase-3蛋白水平显著降低(P<0.05);Bcl-2蛋白水平显著升高(P<0.05)。结论:丹参多酚酸盐能减轻过氧化氢对EA.hy926细胞氧化应激的损伤作用,其机制可能与NF-κB通路的阻滞相关。
AIM: To investigate the effect of salvianolate on oxidative damage induced by hydrogen peroxide in human endothelial EA.hy926 cells.METHODS: EA.hy926 cells were cultured in vitro and divided into the following groups: control group, damage group, and anti-damage groups(salvianolate+damage groups). The cell viability was measured by CCK-8 assay. The migration ability of the EA.hy926 cells was detected by Transwell assay. The content of nitric oxide(NO) in the culture supernatant of the EA.hy926 cells was examined. The levels of vascular endothelial growth factor(VEGF) were detected by ELISA. The apoptosis,mitochondrial membrane potential and intracellular superoxide anion content of the EA.hy926 cells were analyzed by flow cytometry. The protein levels of caspase-3, cleaved caspase-3, Bcl-2, Bax, NF-κB and p53 were determined by Western blot. RESULTS: Compared with damage group, the viability of EA.hy926 cells pretreated with salvianolate at different concentrations was significantly increased(P<0.05). The apoptotic rate was significantly decreased(P<0.05). Savianolate enhanced the migration ability of the cells. The levels of VEGF, NO and mitochondrial transmembrane potential were increased(P<0.05), and the intracellular ROS level was significantly decreased(P<0.05). The protein levels of NF-κB, p53, Bax and cleaved caspase-3 were significantly decreased, and the protein level of Bcl-2 was markedly increased(P<0.05). CONCLUSION: Savianolate reduces the damage of EA.hy926 cells by hydrogen peroxide exposure, and its mechanism may be related to the blocking of NF-κB signaling pathway.
AIM: To investigate the effect of salvianolate on oxidative damage induced by hydrogen peroxide in human endothelial EA.hy926 cells.METHODS: EA.hy926 cells were cultured in vitro and divided into the following groups: control group, damage group, and anti-damage groups(salvianolate+damage groups). The cell viability was measured by CCK-8 assay. The migration ability of the EA.hy926 cells was detected by Transwell assay. The content of nitric oxide(NO) in the culture supernatant of the EA.hy926 cells was examined. The levels of vascular endothelial growth factor(VEGF) were detected by ELISA. The apoptosis,mitochondrial membrane potential and intracellular superoxide anion content of the EA.hy926 cells were analyzed by flow cytometry. The protein levels of caspase-3, cleaved caspase-3, Bcl-2, Bax, NF-κB and p53 were determined by Western blot. RESULTS: Compared with damage group, the viability of EA.hy926 cells pretreated with salvianolate at different concentrations was significantly increased(P<0.05). The apoptotic rate was significantly decreased(P<0.05). Savianolate enhanced the migration ability of the cells. The levels of VEGF, NO and mitochondrial transmembrane potential were increased(P<0.05), and the intracellular ROS level was significantly decreased(P<0.05). The protein levels of NF-κB, p53, Bax and cleaved caspase-3 were significantly decreased, and the protein level of Bcl-2 was markedly increased(P<0.05). CONCLUSION: Savianolate reduces the damage of EA.hy926 cells by hydrogen peroxide exposure, and its mechanism may be related to the blocking of NF-κB signaling pathway.
引文
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[14] 李兵.丹参多酚酸盐对过氧化氢所致乳鼠心肌细胞损伤保护作用的研究[J].中医药信息,2016,33(5):7-11.
[15] Beyfuss K,Hood DA.A systematic review of p53 regulation of oxidative stress in skeletal muscle[J].Redox Rep,2018,23(1):100-117.
[16] Jiao L,Jiang M,Liu J,et al.Nuclear factor-kappa B activation inhibits proliferation and promotes apoptosis of vascular smooth muscle cells[J].Vascular,2018,26(6):634-640.
[17] Pei X,Xiao J,Wei G,et al.Oenothein B induced human non-small cell lung cancer A549 cell death by ROS-mediated PI3K/Akt/NF-κB signaling pathway[J].Chem Biol Interact,2019,298:112-120.
[18] Xu J,Shen W,Pei B,et al.Xiao tan he wei decoction reverses MNNG-induced precancerous lesions of gastric carcinoma in vivo and vitro:regulation of apoptosis through NF-kappaB pathway[J].Biomed Pharmacother,2018,108:95-102.
[2] 樊春华,田宽,何菲,等.氰酸盐诱导人脐静脉内皮细胞氧化应激损伤[J].中国细胞生物学学报,2017,39(12):1550-1555.
[3] 戴青里,孙贵虎,闫斌,等.过氧化氢诱导HUVEs氧化应激模型的构建[J].昆明医科大学学报,2018,39(4):34-39.
[4] 李兵.丹参多酚酸盐对过氧化氢所致乳鼠心肌细胞氧化应激损伤影响及其机制[J].辽宁中医药大学学报,2016,18(12):28-31.
[5] 王玉荣,杨康,崔伟业,等.过氧化氢损伤A549细胞的作用[J].中国中药杂志,2018,43(5):1014-1020.
[6] 王春宇,姜民,侯冠宇,等.小柴胡汤对过氧化氢诱导的人皮肤成纤维细胞氧化应激损伤的保护作用[J].中国实验诊断学,2018,22(6):1074-1077.
[7] 金海南,刘莉园,金芳多,等.祁州漏芦水提取物对叔丁基过氧化氢诱导血管内皮细胞损伤的保护作用[J].中国药学杂志,2018,53(16):1366-1372.
[8] Li L,Liu H,Xu C,et al.VEGF promotes endothelial progenitor cell differentiation and vascular repair through connexin 43[J].Stem Cell Res Ther,2017,8:237.
[9] Bus P,Gerrits T,Heemskerk AC,et al.Endoglin mediates vascular endothelial growth factor-A-induced endothelial cell activation by regulating akt signaling[J].Am J Pathol,2018,188(12):2924-2935.
[10] Zhu M,Liu X,Wang Y,et al.YAP via interacting with STAT3 regulates VEGF-induced angiogenesis in human retinal microvascular endothelial cells[J].Exp Cell Res,2018,373(1-2):155-163.
[11] 何胜虎,燕建锋,袁彬,等.丹参多酚酸盐对高糖诱导的人脐静脉内皮细胞损伤的保护作用[J].中国动脉硬化杂志,2008,16(12):948-952.
[12] 王琳,李正飞,舒小军,等.丹参多酚酸盐对高同型半胱氨酸诱导的人脐静脉内皮细胞组织因子表达的干预作用[J].甘肃医药,2012,31(9):643-646.
[13] 李兵.丹参多酚酸盐对过氧化氢所致乳鼠心肌细胞凋亡的影响[J].天津中医药,2016,33(8):491-495.
[14] 李兵.丹参多酚酸盐对过氧化氢所致乳鼠心肌细胞损伤保护作用的研究[J].中医药信息,2016,33(5):7-11.
[15] Beyfuss K,Hood DA.A systematic review of p53 regulation of oxidative stress in skeletal muscle[J].Redox Rep,2018,23(1):100-117.
[16] Jiao L,Jiang M,Liu J,et al.Nuclear factor-kappa B activation inhibits proliferation and promotes apoptosis of vascular smooth muscle cells[J].Vascular,2018,26(6):634-640.
[17] Pei X,Xiao J,Wei G,et al.Oenothein B induced human non-small cell lung cancer A549 cell death by ROS-mediated PI3K/Akt/NF-κB signaling pathway[J].Chem Biol Interact,2019,298:112-120.
[18] Xu J,Shen W,Pei B,et al.Xiao tan he wei decoction reverses MNNG-induced precancerous lesions of gastric carcinoma in vivo and vitro:regulation of apoptosis through NF-kappaB pathway[J].Biomed Pharmacother,2018,108:95-102.