Department of Pathophysiology, Zhongshan Medical School, Sun Yat-sen University, 74 Zhongshan Er Road, Guangzhou, Guangdong 510080, China.
Cardiovasc Drugs Ther. 2013 Jun;27(3):189-98. doi: 10.1007/s10557-013-6442-4.
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 kinaseα1 (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.
白藜芦醇可以通过激活沉默信息调节因子 1（Sirtuin）诱导基础自噬。本研究旨在探讨白藜芦醇对氧化型低密度脂蛋白（ox-LDL）诱导的人脐静脉内皮细胞（HUVEC）氧化损伤的影响，并探讨自噬在此作用中的机制。
用 100mg/L ox-LDL 孵育 HUVEC 24h 造成氧化损伤模型。MTT 法和超氧化物歧化酶（SOD）活性检测不同浓度白藜芦醇对 ox-LDL 诱导的 HUVEC 氧化损伤的影响。通过微管相关蛋白 1 轻链 3（LC3）和自噬相关蛋白 P62（SQSTM1）的蛋白表达来测量不同组的自噬水平。用电子显微镜和荧光显微镜（通过 MDC 染色）观察自噬体。Western blot 检测 Sirt1 和 AMP 激活蛋白激酶α1（AMPK）的表达。使用自噬抑制剂 3-甲基腺嘌呤（3-MA）和 Sirt1 抑制剂 6-氯-2,3,4,9-四氢-1H-咔唑-1-甲酰胺（EX527）来确认自噬在白藜芦醇的这种作用中的作用及其涉及的途径。
白藜芦醇使 ox-LDL 导致的细胞活力（对照组的 72.9±1.7%）和 SOD 活性（对照组的 14.37±0.21U/ml）分别增加至对照组的 83.4±1.4%和 16.41±0.27U/ml。这种作用伴随着自噬的上调以及 Sirt1 和 AMPK 磷酸化在苏氨酸 172 位（p-AMPK）的蛋白表达增加。3-MA 和 EX527 分别使白藜芦醇对细胞活力的保护作用消失，分别为对照组的 80.4±2.7%和 73.9±1.1%。3-MA 抑制自噬激活，但不改变 Sirt1 的 mRNA 和蛋白表达。EX527 抑制 Sirt1 的表达，并减弱自噬的上调。添加 3-MA 或 EX527 均不影响 p-AMPK 的蛋白水平。
白藜芦醇可减轻 ox-LDL 引起的 HUVEC 氧化损伤。这种作用是通过 AMPK/Sirt1 通路介导的 Sirt1 依赖性自噬来实现的。
