茶黄素激活Nrf2/HO-1通路保护血管内皮细胞氧化应激损伤

Theaflavin Activates Nrf2/HO-1 Pathway to Alleviate Oxidative Stress Injury in Vascular Endothelial Cells

为探讨茶黄素(Theaflavin,TF)对过氧化氢(H_2O_2)诱导的血管内皮细胞氧化应激损伤的保护效应及作用机制,将人脐静脉血管内皮细胞HUVEC分为对照组、损伤组(0.2 mmol·L^(-1) H_2O_2处理)和TF预处理组(2.0、5.0、10.0μmol·L^(-1)TF和0.2 mmol·L^(-1) H_2O_2处理)。损伤组和TF组均以H_2O_2处理24 h,其中TF组先以不同浓度TF预处理2 h,对照组均以定量溶剂处理。以MTT法测定细胞活力,DCFH-DA探针测定活性氧(ROS)水平,流式细胞术检测细胞凋亡,Western blot测定蛋白表达水平,相应试剂盒测定乳酸脱氢酶(LDH)、一氧化氮(NO)、丙二醛(MDA)含量及超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、谷胱甘肽过氧化氢酶(GSH-Px)活性。结果显示,与对照组相比,损伤组细胞活力显著降低,LDH释放量增加,NO水平降低,胞内ROS水平升高,MDA增加,抗氧化酶活力降低,细胞凋亡升高;TF预处理能够显著提高细胞活力,降低LDH水平,维持NO水平,降低胞内ROS水平及MDA的产生,提高抗氧化酶活力,并抑制细胞凋亡;进一步研究显示,TF能够激活Nrf2/HO-1通路,且Nrf2抑制剂会显著降低TF的内皮细胞保护效应。可见,TF能够有效抑制H_2O_2诱导的血管内皮细胞氧化应激损伤,其机制至少部分是通过激活Nrf2/HO-1通路实现的。

To investigate the protective effect of theaflavin(TF)on injury of human umbilical vein endothelial cells(HUVEC)induced by hydrogen peroxide(H2O2),HUVECs were divided into control group,injury group(0.2 mmol·L-1 H2O2 treatment)and TF pretreatment groups(2.0,5.0,10.0μmol·L-1+0.2 mmol·L-1 H2O2 treatment).The TF pretreatment groups were pretreated with TF for 2 h.Then,both the injury group and the TF groups were treated with H2O2 for 24 h,while the control group was treated with solvent.Cells activity was detected by the MTT method.The levels of lactic dehydrogenase(LDH),nitric oxide(NO),malondialdehyde(MDA)and the activities of superoxide dismutase(SOD),catalase(CAT),glutathione peroxidase(GSH-Px)were measured by corresponding detection kits.Reactive oxygen species(ROS),cells apoptosis and protein expression levels were detected using DCFH-DA dying,flow cytometer and Western blot.The results show that cells activity was dramatically decreased in the injury group,and the levels of LDH,cellular ROS,MDA and cells apoptosis increased,while the level of NO and the activities of antioxidant enzymes were declined.TF pretreatment could increase cells’viability,decrease the level of LDH,maintain the level of NO,and inhibit the increments of ROS and MDA,as well as cells apoptosis.Further study indicated that TF treatment could activate Nrf2/HO-1 signaling pathway,and the inhibitor of Nrf2 could reduce the protective effects of TF on HUVEC cells.In conclusion,TF could alleviate oxidative stress injury in vascular endothelial cells induced by H2O2.The mechanism is at least partly associated with the activation of Nrf2/HO-1 pathway.