Keap1-Nrf2 signaling activation by Bardoxolone-methyl ameliorates high glucose-induced oxidative injury in human umbilical vein endothelial cells.

Jing-Lei Yang,Yuan-Yuan Liu,Mei-Juan Dong,Li Mao,Qi Yuan,Shan Tang,Ri-Dong Zhang,Meng-Yue Sun

doi:10.18632/aging.103263

Abstract

In cultured human umbilical vein endothelial cells (HUVECs) high glucose (HG) stimulation will lead to significant cell death. Bardoxolone-methyl (BARD) is a NF-E2 p45-related factor 2 (Nrf2) agonist. In this study we show that BARD, at only nM concentrations, activated Nrf2 signaling in HUVECs. BARD induced Keap1-Nrf2 disassociation, Nrf2 protein stabilization and nuclear translocation, increasing expression of antioxidant response element (ARE) genes. BARD pretreatment in HUVECs inhibited HG-induced reactive oxygen species production, oxidative injury and cell apoptosis. Nrf2 shRNA or knockout (using a CRISPR/Cas9 construct) reversed BARD-induced cytoprotection in HG-stimulated HUVECs. Conversely, forced activation of Nrf2 cascade by Keap1 shRNA mimicked BARD’s activity and protected HUVECs from HG. Importantly, BARD failed to offer further cytoprotection against HG in the Keap1-silened HUVECs. Taken together, Keap1-Nrf2 cascade activation by BARD protects HUVECs from HG-induced oxidative injury.

Highlights

Vascular endothelial cell injury is a primary medical issue in the pathogenesis and progression of cardiovascular complications in diabetes mellitus patients [1, 2]
The input control results demonstrated that NF-E2 p45-related factor 2 (Nrf2) protein levels were elevated in BARD-treated Human umbilical vein endothelial cells (HUVECs) (Figure 1B), where Keap1 levels were unchanged (Figure 1B)
By testing the nuclear fraction proteins, we found that the Nrf2 protein was enriched in the nuclei of BARD (10100 nM)-treated HUVECs, with significant increase of antioxidant response element (ARE) activity (Figure 1D)

Summary

Introduction

Vascular endothelial cell injury is a primary medical issue in the pathogenesis and progression of cardiovascular complications in diabetes mellitus patients [1, 2]. Sustained high glucose (HG) exposure to vascular endothelial cells will induce robust reactive oxygen species (ROS) production and oxidative injury. It will induce calcium overload, lipid peroxidation, as well as profound protein and DNA damage. At last it will bind to antioxidant response element (ARE), essential for the basal and inducible expression of many different genes. These genes encode detoxification enzymes, antioxidant proteins and many other cytoprotective proteins [14,15,16,17,18]

Methods

Results

Conclusion