枸杞多糖在siNrf2胸主动脉血管内皮细胞氧化应激 损伤中的作用

Effect of Lycium Barbarum Polysaccharide on Oxidative Stress Injury of siNrf2 Thoracic Aortic Vascular Endothelial Cells

目的探讨枸杞多糖(lycium barbarum polysaccharide,LBP)对血管紧张素Ⅱ(AngⅡ)诱导主动脉血管内皮细胞氧化应激损伤的保护机制。方法通过应用小干扰RNA[siRNA(50 nmol)]转染大鼠胸主动脉内皮细胞(rat thoracic aortic endothelial cell,RTAEC),对Nrf2进行基因沉默,检测转染效率、筛选最佳siRNA后,对沉默Nrf2基因分组:空白对照组、AngⅡ组、AngⅡ+LBP组、沉默空载组(siNC组)、siNC+AngⅡ+LBP组、沉默Nrf2组(siRNA组)、siRNA+AngⅡ+LBP组。Western blot检测Nrf2、P-Nrf2的蛋白表达水平在AngⅡ+LBP组与siRNA+AngⅡ+LBP组的变化。ELISA检测丙二醛(MDA)、谷胱甘肽(GSH)、过氧化氢酶(CAT)、总抗氧化能力(T-AOC)的含量变化。结果转染效率检测结果显示:荧光显微镜下观察到带有荧光标记的siRNA转染内皮细胞的效率在80%以上。与空白对照组比较,siRNA5组的Nrf2蛋白表达降低(P<0.05)。与空白对照组比较,siRNA5组的Nrf2的mRNA表达降低(P<0.05)。转染目的siRNA实验结果显示:与AngⅡ+LBP组比较,siRNA+AngⅡ+LBP组的Nrf2蛋白表达下降(P<0.05)。与AngⅡ+LBP组比较,siRNA+AngⅡ+LBP组的MDA的表达水平升高(P<0.05),GSH、CAT、T-AOC的表达水平降低(P均<0.01)。结论LBP可能通过增加内皮细胞中Nrf2的蛋白表达水平,提高其抗氧化应激能力,达到保护血管内皮细胞功能的作用。

Objective To explore the effect and protective mechanism of lycium barbarum polysaccharide(LBP)on oxidative stress(angiotensinⅡ,AngⅡ)induced aortic endothelial cell damage.Methods Rat thoracic aortic endothelial cell(RTAEC)was transfected with small interfering RNA(siRNA 50 nmol)to silence Nrf2 gene.After detecting the transfection efficiency and screening the best siRNA,the silenced Nrf2 gene was divided into blank control group,angiotensinⅡgroup(AngⅡ),angiotensinⅡ+lycium barbarum polysaccharide group(AngⅡ+LBP),silencing empty group(siNC),silencing empty group+angiotensinⅡ+lycium barbarum polysaccharide group(siNC+AngⅡ+LBP),silencing Nrf2 group(siRNA),silencing Nrf2+angiotensinⅡ+lycium barbarum polysaccharide group(siRNA+AngⅡ+LBP).The changes of protein expression levels of Nrf2 and p-nrf2 in AngⅡ+LBP group and siRNA+AngⅡ+LBP group were detected by WB.Malondialdehyde(MDA),glutathione(GSH),catalase(CAT)and total antioxidant capacity(T-AOC)were detected by ELISA.Results The transfection efficiency test showed that the transfection efficiency of fluorescent labeled siRNA into endothelial cells was more than 80%under fluorescence microscope.Compared with the blank control group,the expression of Nrf2 protein in siRNA5 group decreased(P<0.05).Compared with the blank control group,the mRNA expression of Nrf2 in siRNA5 group decreased(P<0.05).The results of transfection target siRNA showed that the protein expression of Nrf2 in siRNA+AngⅡ+LBP group decreased(P<0.05)compared with AngⅡ+LBP group.Compared with AngⅡ+LBP group,the expression level of MDA in siRNA+AngⅡ+LBP group increased(P<0.05),the expression levels of GSH,CAT and T-AOC decreased(P all<0.01).Conclusion LBP may protect the function of vascular endothelial cells by increasing the protein expression level of Nrf2 in endothelial cells and improving its anti-oxidative stress ability.