Resveratrol Protects HUVECs from Oxidized-LDL Induced Oxidative Damage by Autophagy Upregulation via the AMPK/SIRT1 Pathway
Resveratrol Protects HUVECs from Oxidized-LDL Induced Oxidative Damage by Autophagy Upregulation via the AMPK/SIRT1 Pathway
复制标题
白藜芦醇通过 AMPK/SIRT1 途径上调自噬，保护 HUVEC 免受氧化 LDL 诱导的氧化损伤
DOI：
10.1007/s10557-013-6442-4
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发表时间：
2013-06-01
影响因子：
3.4
通讯作者：
Wu, Weikang
中科院分区：
文献类型：
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作者：
Guo, Hualei;Chen, Yanling;Wu, Weikang
白藜芦醇可通过激活sirtuin诱导基础自噬。本研究采用氧化低密度脂蛋白（ox-LDL）诱导人脐静脉内皮细胞（HUVECs）氧化损伤24 h，观察白藜芦醇对氧化低密度脂蛋白（ox-LDL）诱导的HUVECs氧化损伤的影响，并探讨自噬在其中的作用。采用MTT比色法和超氧化物歧化酶（SOD）活性测定法观察不同浓度白藜芦醇对ox-LDL诱导的HUVECs氧化损伤的影响。通过检测微管相关蛋白1轻链3（LC 3）和隔离体1（SQSTM 1/P62）的蛋白表达来检测各组自噬水平。电镜和荧光显微镜下观察自噬体的形态。Western blot检测沉默信息调节因子1（Sirt 1）和AMP激活的蛋白激酶α 1（AMPK）的表达。自噬抑制剂3-甲基腺嘌呤（3-MA）和Sirt 1抑制剂6-氯-2，3，4，用9-tetrahydro-1H-Carbazole-1-carbamamide（EX 527）证实自噬在白藜芦醇的这种作用中的作用及其途径（对照组的72.9 ± 1.7%）和SOD活性（14.37 ± 0.21 U/ml）分别为对照组的83.4 ± 1.4%和16.41 ± 0.27 U/ml。这种效应伴随着自噬的上调和Sirt 1蛋白表达的增加以及苏氨酸172（p-AMPK）上AMPK磷酸化。3-MA和EX 527都消除了白藜芦醇对细胞活力的保护作用，分别为对照组的80.4 +/-2.7%和73.9 +/-1.1%。3-MA抑制自噬激活，而Sirt 1在mRNA和蛋白水平的表达没有任何变化。EX 527抑制Sirt 1的表达并减少自噬的上调。加入3-MA或EX 527对p-AMPK蛋白水平无影响，白藜芦醇对ox-LDL引起的HUVECs氧化损伤有保护作用。这种作用是通过AMPK/Sirt 1途径由Sirt 1依赖性自噬介导的。
Resveratrol could induce basal autophagy through the activation of sirtuin. In this study, we investigated the effect of resveratrol on oxidative injury of human umbilical endothelial vein cells (HUVECs) induced by oxidized low-density lipoprotein (ox-LDL) and the role of autophagy in this effect.HUVECs were exposed to 100 mg/L ox-LDL for 24 h to cause oxidative injury. The effect of different concentrations of resveratrol on oxidative damage in HUVECs treated with ox-LDL was evaluated by MTT assay and superoxide dismutase (SOD) activity test. The autophagic level in different groups was measured by the protein expression of microtubule-associated protein 1 light chain 3 (LC3) and sequestosome 1 (SQSTM1/P62). Autophagosomes were observed under electron microscope and fluorescence microscope (by MDC staining). The expression of silencing information regulator1 (Sirt1) and AMP activated protein kinase alpha 1 (AMPK) was investigated by Western blot. Autophagy inhibitor 3-methyladenine (3-MA) and Sirt1 inhibitor 6-Chloro-2,3,4,9-tetrahydro-1H-Carbazole-1-carboxamide (EX527) were used to confirm the role of autophagy in this effect of resveratrol and the pathway involved.Resveratrol reversed the decreases in cell viability (72.9 +/- 1.7 % of the control group) and SOD activity (14.37 +/- 0.21 U/ml) caused by ox-LDL at 83.4 +/- 1.4 % of the control group and 16.41 +/- 0.27 U/ml respectively. This effect accompanied by upregulation of autophagy and increased protein expression of Sirt1 and AMPK phosphorylation on threonine 172 (p-AMPK). Both 3-MA and EX527 abolished the protective effect of resveratrol in cell viability, at 80.4 +/- 2.7 % and 73.9 +/- 1.1 % of the control group respectively. 3-MA inhibited autophagy activation without any change of Sirt1 expression at both the mRNA and protein level. EX527 suppressed the expression of Sirt1 and diminished the upregulation of autophagy. Addition of 3-MA or EX527 could not affect the protein level of p-AMPK.Resveratrol protected HUVECs from oxidative damage caused by ox-LDL. This effect was mediated by Sirt1-dependent autophagy via the AMPK/ Sirt1 pathway.