Department of Pharmacy, Nanfang Hospital, Southern Medical University, No. 1838, Guangzhou Boulevard (North), Guangzhou 510515, China; Department of Pharmacy, Shenzhen Hospital, Southern Medical University, Shenzhen, China.
Department of Pharmacy, Nanfang Hospital, Southern Medical University, No. 1838, Guangzhou Boulevard (North), Guangzhou 510515, China.
Phytomedicine. 2017 Oct 15;34:115-126. doi: 10.1016/j.phymed.2017.08.010. Epub 2017 Aug 17.
Paeoniflorin, a monoterpene glycoside, exerts protective vascular effects, showing good antioxidant properties. However, whether Paeoniflorin has protective effect against the oxidative damage induced by advanced oxidation protein products (AOPPs) in Human umbilical vein endothelial cells (HUVECs) is unknown, as is the underlying mechanism.
The present study was designed to investigate the effect of Paeoniflorin on oxidative damage of HUVECs and elucidate its underlying molecular mechanisms.
The fluorescence intensity of 2', 7'-dichlorofluorescein-diacetate (DCFH-DA) staining was detected for intracellular reactive oxygen species (ROS) production. The increases mitochondrial membrane potential (MMP) was measured via flow cytometry and confocal microscopy using MitoTracker® Deep Red/ MitoTracker® Green staining. The intracellular adenosine triphosphate (ATP) was measured by ATP Determination Kit according to the manufacturer's protocol. Nox2, Nox4, hypoxia inducible factor-1α (HIF-1α), vascular endothelial growth factor (VEGF) and nuclear factor-κB (NF-κB) p65 expressions were detected by western blot.
Our results showed that Paeoniflorin increases MMP and ATP levels of HUVECs induced by AOPPs, and attenuates NF-κB p65 expression on HUVECs might mainly result from its antioxidant capability by suppressing ROS production. Moreover, we also found that Paeoniflorin can suppress HIF-1α and VEGF protein expression through a decrease of ROS production via down-regulation of Nox2/Nox4 expression in HUVECs. AOPP-induced RAGE mRNA up-regulation was blocked by Paeoniflorin treatment in HUVECs.
Our results provided the first experimental that Paeoniflorin protects against AOPP-induced oxidative damage in HUVECs, mainly through a mechanism involving a decrease in ROS production by the inhibition of Nox2/Nox4 and RAGE expression; restored ATP depletion and mitochondria dysfunction via ROS suppression; and down-regulated HIF-1α/VEGF, possibly via the ROS-NF-κB axis.
芍药苷是一种单萜糖苷，具有保护血管的作用，表现出良好的抗氧化特性。然而，芍药苷是否对人脐静脉内皮细胞（HUVEC）中由晚期氧化蛋白产物（AOPPs）引起的氧化损伤具有保护作用尚不清楚，其潜在机制也不清楚。
本研究旨在探讨芍药苷对 HUVEC 氧化损伤的影响，并阐明其潜在的分子机制。
通过 2'，7'-二氯荧光素二乙酸酯（DCFH-DA）染色检测细胞内活性氧（ROS）的产生来检测荧光强度。通过流式细胞术和使用 MitoTracker® Deep Red/MitoTracker® Green 染色的共聚焦显微镜测量线粒体膜电位（MMP）的增加。根据制造商的方案，通过 ATP 测定试剂盒测量细胞内三磷酸腺苷（ATP）的含量。通过 Western blot 检测 Nox2、Nox4、缺氧诱导因子-1α（HIF-1α）、血管内皮生长因子（VEGF）和核因子-κB（NF-κB）p65 的表达。
我们的结果表明，芍药苷增加了 AOPPs 诱导的 HUVECs 的 MMP 和 ATP 水平，并通过抑制 ROS 产生来减轻 HUVECs 中 NF-κB p65 的表达，这主要归因于其抗氧化能力。此外，我们还发现，芍药苷通过下调 Nox2/Nox4 的表达抑制 ROS 产生，可抑制 HIF-1α 和 VEGF 蛋白的表达。芍药苷处理可阻断 AOPP 诱导的 HUVECs 中 RAGE mRNA 的上调。
我们的研究结果首次提供了芍药苷可防止 AOPP 诱导的 HUVECs 氧化损伤的实验证据，这主要是通过抑制 Nox2/Nox4 和 RAGE 表达来减少 ROS 产生的机制实现的；通过抑制 ROS 来恢复 ATP 耗竭和线粒体功能障碍；并通过 ROS-NF-κB 轴下调 HIF-1α/VEGF，可能通过 ROS-NF-κB 轴实现。
