蒺藜皂苷对同型半胱氨酸诱导的人脐静脉内皮细胞氧化应激及炎症损伤的影响

Effects of the Saponins from Tribulus Terrestris L. on Oxidative Stress and Inflammation Injury Induced by Homocysteine in Human Umbilical Vein Endothelial Cells

目的:探讨蒺藜皂苷对同型半胱氨酸(Hcy)诱导的内皮细胞损伤的保护作用及其分子机制。方法:采用Hcy诱导人脐静脉内皮细胞(HUVECs)建立体外损伤模型,采用MTT法检测细胞增殖活力,生化方法检测超氧化物歧化酶(SOD)活性及丙二醛(MDA)含量,化学荧光法检测活性氧簇(ROS)水平,应用ELISA检测细胞培养上清中肿瘤坏死因子α(TNF-α)、细胞间黏附分子1(ICAM-1)、白细胞介素6(IL-6)、白细胞介素1β(IL-1β)水平,Real-time PCR检测TNF-α、核因子κB(NF-κB)p65mRNA表达,Western Blot检测NF-κB p65、p-IκBα、基质金属蛋白酶2(MMP-2)、NADPH氧化酶4(NOX4)蛋白表达。结果:Hcy刺激内皮细胞后可诱导氧化应激反应和炎症因子的大量释放。与模型组比较,蒺藜皂苷干预可显著增强细胞增殖活力和SOD活性,降低细胞内MDA含量和ROS、TNF-α、ICAM-1、IL-6、IL-1β水平,抑制TNF-α、NF-κB p65 mRNA和NF-κB p65、pIκBα、MMP-2、NOX4蛋白表达(P<0.05或P<0.01)。与蒺藜皂苷20μg·ml-1干预组比较,蒺藜皂苷20μg·ml^-1+BAY 11-7082组可显著降低TNF-α、ICAM-1、IL-6、IL-1β含量、细胞TNF-α、NF-κB p65 mRNA表达水平及NF-κB p65、p-IκBα、MMP-2、NOX4蛋白表达水平(P<0.05)。结论:蒺藜皂苷对Hcy诱导的内皮细胞氧化应激及炎症损伤具有保护作用,其作用机制可能与抑制NOX4表达,进而降低氧化应激诱导的NF-κB活化有关。

Objective:To investigate the protective effect of the saponins from Tribulus terrestris L.on human umbilical vein endothelial cells(HUVECs)injury induced by homocysteine(Hcy)and its molecular mechanism.Methods:An injury model of HUVECs was established by homocysteine.MTT assay was used to detect the cell proliferative activity.The biochemical method was applied to measure superoxide dismutase(SOD)activity and malondialdehyde(MDA)content,and the chemical fluorescence method was used to detect reactive oxygen species(ROS)level.The contents of tumor necrosis factorα(TNF-α),intercellular adhesion molecule 1(ICAM-1),interleukin-6(IL-6)and interleukin-1β(IL-1β)in cell culture supernatant were measured with enzyme-linked immunosorbent assay(ELISA).The expressions of TNF-αand nuclear factor-kappa B(NF-κB)p65 mRNA were detected with real-time PCR.Western blot was utilized to detect the expressions of NF-κB p65,p-IκBα,matrix metalloproteinase-2(MMP-2)and NADPH oxidase 4(NOX4)proteins.Results:Hcy stimulated the endothelial cells to induce oxidative stress and release inflammatory factors.Compared with the model group,the saponins from Tribulus terrestris L.could significantly enhance the cell proliferation activity and SOD activity,decrease MDA content and ROS,TNF-α,ICAM-1,IL-6 and IL-1βlevels,and inhibit TNF-αand NF-κB p65 mRNA and the expressions of NF-κB p65,p-IκBα,MMP-2 and NOX4 proteins(P<0.05 or P<0.01).Compared with the saponins from Tribulus terrestris L.20μg·ml^-1 intervention group,the combination of 20μg·ml-1 saponins and BAY 11-7082 could significantly reduce TNF-α,ICAM-1,IL-6 and IL-1βcontents,TNF-alpha and NF-κB p65 mRNA expression levels,and NF-κB p65,p-IκBα,MMP-2 and NOX4 protein expression levels(P<0.05).Conclusion:The saponins from Tribulus terrestris L.can protect HUVECs from oxidative stress and inflammation induced by Hcy.The effect may be related to inhibiting NOX4 expression and then reducing NF-κB activation induced by oxidative stress.