Cytoprotective effects of euxanthone against ox-LDL-induced endothelial cell injury is mediated via Nrf2.

Atherosclerosis (AS) is a chronic condition of the arterial vessels and a risk factor for myocardial infarction and stroke. Euxanthone is a xanthone compound extracted from Polygala caudata, and shows vasodilatory action. The aim of this study was to determine the potential pharmacological effects of euxanthone against oxidized low-density lipoprotein (ox-LDL)-induced endothelial cell injury.Human umbilical vein endothelial cells (HUVECs) were exposed to ox-LDL, following pre-treatment with different concentrations of euxanthone. Viability, apoptosis and DNA fragmentation were respectively assessed by CCK-8 assay, Annexin-V/PI staining and TdT-mediated dUTP Nick-End Labeling (TUNEL) assay. The cellular levels of malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px) were analyzed by enzyme linked immune-sorbent assays (ELISA), and reactive oxygen species (ROS) levels using dichlorodihydrofluorescin diacetate (DCFH) staining. Quantitative RT-PCR and Western blotting were respectively used to analyze the expression levels of specific mRNAs and proteins. HUVECs were transfected with Nrf2 siRNA to induce knockdown of the latter.Euxanthone pre-treatment rescued the HUVECs from ox-LDL-induced cytotoxicity, apoptosis and DNA fragmentation in a dose-dependent manner. In addition, euxanthone also significantly reversed ox-LDL-triggered loss of mitochondrial membrane potential (MMP), cytochrome C release from mitochondria to cytosol, cleavage of caspase-3 and PARP, and increase in Bax/Bcl-2 ratio. Pre-treatment with euxanthone markedly suppressed ox-LDL-induced ROS generation and inhibition of antioxidant enzymes, as well as the up-regulation of pro-inflammatory factors like MCP-1, IL-1β and TNF-α in the HUVECs. Euxanthone up-regulated and activated Nrf2 by repressing Keap1, and increased the expression of its downstream genes HO-1 and NQO-1. Nrf2 knockdown abrogated the cyto-protective, anti-apoptotic, anti-oxidant and anti-inflammatory effects of euxanthone in ox-LDL-treated HUVECs. Finally, euxanthone activated Nrf2 via the MAPK pathway and blocking the latter likewise negated the protective effects of euxanthone against cell ox-LDL.Euxanthone protected HUVECs against the oxidative and inflammatory damage induced by ox-LDL, indicating its potential as a novel therapeutic agent for AS.