油菜蜂花粉总黄酮对体外TBHP诱导细胞氧化损伤的保护作用  

Study on Protective Effect of Rape Bee Pollen Flavonoids on TBHP Induced EA.hy926 Cell Oxidative Damage in Vitro

作　　者：杨悦 刘鸣畅[1] 王凯[2] 吴黎明[2] 杨艳歌[1] 吴亚君[1] YANG Yue;LIU Ming-chang;WANG Kai;WU Li-ming;YANG Yan-ge;WU Ya-jun(Chinese Academy of Inspection and Quarantine,Beijing 100176,China;Institute of Apicultural Research,Chinese Academy of Agricultural Sciences,Beijing 100093,China)

机构地区：[1]中国检验检疫科学研究院,北京100176 [2]中国农业科学院蜜蜂研究所,北京100093

出　　处：《沈阳农业大学学报》2018年第1期108-113,共6页Journal of Shenyang Agricultural University

基　　金：中国检验检疫科学研究院基本科研项目(2017JK001)

摘　　要：为研究油菜蜂花粉总黄酮(rape bee pollen flavonoids,RBPF)抗心血管疾病的作用机制,以RBPF为研究对象,利用人工建立的血管内皮细胞氧化损伤模型,对RBPF的抗氧化活性进行初步研究,在体外水平探索RBPF对血管内皮细胞氧化损伤的保护作用。采用超声醇提及大孔树脂纯化的方法提取富集RBPF,同时采用过氧化氢叔丁醇(tert-Butyl hydroperoxide,TBHP)诱导细胞氧化损伤,建立人脐静脉内皮融合细胞系EA.hy926的氧化损伤模型,并利用此模型研究RBPF在细胞水平的抗氧化作用,以公认抗氧化性质良好的黄酮类化合物槲皮素作为阳性对照,CCK8法检测细胞活力。首先,用梯度稀释的RBPF及槲皮素作用于正常培养细胞,分析其细胞毒性,确定毒性阈值;然后,在毒性阈值范围内设置梯度浓度的槲皮素溶液作用于细胞损伤模型,确定槲皮素最佳抗氧化浓度剂量,并以此剂量组作为阳性对照组,与毒性阈值范围内梯度浓度的RBPF同时作用于细胞损伤模型,研究RBPF对血管内皮细胞氧化损伤的保护情况。结果表明:其一,体外血管内皮细胞EA.hy926氧化损伤模型的最佳建模条件确定为500μmol·L^(-1)TBHP作用3h;其二,细胞毒性试验结果表明,对于血管内皮细胞EA.hy926,RBPF的毒性阈值为24mg·L^(-1),阳性对照品槲皮素的毒性阈值为50mg·L^(-1);其三,CCK8法测定RBPF对细胞氧化损伤模型的保护作用,结果显示在6~18mg·L^(-1)剂量范围内,RBPF预处理对EA.hy926细胞氧化损伤模型具有明显的保护作用(p≤0.05),其抗氧化作用在低浓度(≤10mg·L^(-1))范围内呈上升趋势,在高浓度(≥14mg·L^(-1))范围内呈下降趋势,在10,12,14mg·L^(-1)3个剂量组其抗氧化性优于槲皮素(25mg·L^(-1)),RBPF浓度为10mg·L^(-1)时效果最佳。综上所述得出结论,对于血管内皮细胞氧化损伤模型,RBPF表现出了极好的体外抗氧化效果,可作为优质的预防心血管疾病的天然产物资源进行深入开发This paper preliminarily studied the antioxidant activity of Rape Bee Pollen Flavonoids(RBPF)by using an artificial vascular endothelial cell oxidative damage model and explored the protective effect of RBPF on this model in vitro.This paper used ultrasound-assisted ethanol extraction and macroporous resin purification methods to extract and enrich RBPF.Meanwhile,an oxidative damage model of human umbilical vein endothelial fusion cell line EA.hy926 was established by using tert-butyl hydroperoxide(TBHP)to induce oxidative damage.The model was used to study the antioxidant activity of RBPF at the cellular level with quercetin as a positive control,and the cell viability was detected by CCK8 method.First,gradient diluted RBPF and quercetin were used on normal cultured cells to analyze their cytotoxicity and determine their toxicity threshold.Then,the cell damage model was treated with a gradient of quercetin within the toxicity threshold range to determine the best antioxidant dose of quercetin.Finally,a gradient of RBPF under the toxicity threshold range was applied to the cell damage model,with the best antioxidant dose of quercetin as a positive group at the same time,to study the protective effect of RBPF on vascular endothelial cells oxidative damage.The results were as follows:First,the optimal modeling conditions for the oxidative damage model of vascular endothelial cells EA.hy926 in vitro were 500滋mol窑L-1 TBHP for 3 h.Second,the results of cytotoxicity test showed that,for EA hy926,the toxicity thresholds were 24 mg窑L-1 for RBPF and 50 mg窑L-1 for quercetin.Third,the protective effect of RBPF on the cell oxidation damage model was detected with CCK8 method.The results showed that,in the dose range of 6-18 mg窑L-1,RBPF pretreatment had a significant protective effect on EA.hy926 cell oxidative damage model(p臆0.05).The antioxidant activity of RBPF showed an rising trend in low concentration(臆10mg窑L-1)and decreased in high concentration(逸14 mg窑L-1).In three dose groups of 10,12 and 14 mg窑

关 键 词：油菜蜂花粉总黄酮 TBHP 人脐静脉内皮融合细胞EA.hy926 抗氧化 

分 类 号：S8[农业科学—畜牧兽医]