Muramyl Dipeptide Induces Reactive Oxygen Species Generation Through the NOD2/COX-2/NOX4 Signaling Pathway in Human Umbilical Vein Endothelial Cells

Abstract

Vascular endothelium dysfunction caused by oxidative stress accelerates the pathologic process of cardiovascular diseases. NOD2, an essential receptor of innate immune system, has been demonstrated to play a critical role in atherosclerosis. Here, the aim of our study was to investigate the effect and underlying molecular mechanism of muramyl dipeptide (MDP) on NOX4-mediated reactive oxygen species (ROS) generation in human umbilical vein endothelial cells (HUVECs). The 2,7-dichlorofluorescein diacetate staining was to measure the intracellular ROS level and showed MDP-promoted ROS production in a time- and dose-dependent manner. The mRNA and protein levels of NOX4 and COX-2 were detected by real-time polymerase chain reaction and western blot. Small interfering RNA (siRNA) was used to silence NOD2 or COX-2 gene expression and investigate the mechanism of NOD2-mediated signaling pathway in HUVECs. Data showed that MDP induced NOX4 and COX-2 expression in a time- and dose-dependent manner. NOD2 knock-down suppressed upregulation of COX-2 and NOX4 in HUVECs treated with MDP. Furthermore, silence of COX-2 in HUVECs downregulated the NOX4 expression after MDP stimulation. Collectively, we indicated that NOD2 played a leading role in MDP-induced COX-2/NOX4/ROS signaling pathway in HUVECs, which was a novel regulatory mechanism in the progress of ROS generation.