Pharmacological Activation of Nrf2 by Rosolic Acid Attenuates Endoplasmic Reticulum Stress in Endothelial Cells.

Karan Naresh Amin,Takayoshi Suzuki,Ramkumar Kunka Mohanram,M R Ganesh,Daoud Ali,Koustav Sarkar,Palanisamy Rajagru,Ayman Mahmoud

doi:10.1155/2021/2732435

Abstract

Endoplasmic reticulum (ER) plays a key role in the folding, modification, and trafficking of proteins. When the homeostasis of the ER is disturbed, un/misfolded proteins accumulate in the ER which leads to ER stress. Sustained ER stress results in apoptosis, which is associated with various diseases. Nuclear factor erythroid 2-related factor 2 (Nrf2) is a major transcription factor in redox homeostasis by regulating various genes associated with detoxification and cell-protective mechanisms. We found that Rosolic acid (RA) treatment dose-dependently activates Nrf2 in endothelial cells using the enzyme fragment complementation assay. The cytoprotective role of RA against ER stress-induced endothelial apoptosis and its molecular mechanism was explored in the present study. The Nrf2 and its target genes, as well as ER stress marker expressions, were measured by qPCR in ER stress-exposed endothelial cells. The contribution of Nrf2 in RA-mediated defense mechanism in endothelial cells was established by knockout studies using Nrf2-CRISPR/Cas9. The treatment with RA to ER stress-induced endothelial cells exhibited activation of Nrf2, as demonstrated by Nrf2 translocation and reduction of ER stress markers. We found that the Nrf2 knockout sensitized the endothelial cells against ER stress, and further, RA failed to mediate its cytoprotective effect. Proteomic studies using LC-MS/MS revealed that among the 1370 proteins detected, we found 296 differentially regulated proteins in ER stress-induced endothelial cells, and RA administration ameliorated 71 proteins towards the control levels. Of note, the ER stress in endothelial cells was attenuated by the treatment with the RA, suggesting the role of the Nrf2 activator in the pathological conditions of ER stress-associated diseases.

Highlights

The endothelium is the main component of the barricade for transporting many important biological compounds from the blood to the inner layer of cells
We have developed a high-throughput screening system based on luciferase complementation to screen Nuclear factor erythroid 2-related factor 2 (Nrf2) activators and demonstrated its role against oxidative and cytokine stress in pancreatic beta cells through Nrf2 activation [21,22,23,24,25]
To determine whether the protective efficacy of Rosolic acid (RA) was facilitated via activation of Nrf2, we have developed Nrf2-knockout endothelial cells using CRISPR/Cas9

Summary

Introduction

The endothelium is the main component of the barricade for transporting many important biological compounds from the blood to the inner layer of cells. ER stress has increased more attention as a crucial factor connecting and congregating molecular relationships among oxidative stress, endothelial cell dysfunction, and insulin resistance and, is considered an auspicious drug target for controlling diabetes and cardiovascular problems. Numerous therapeutic strategies have been developed to combat ER stress-induced endothelial cell dysfunction [10,11,12]. Activation of Nrf by small molecules have been demonstrated as a new promising therapeutic approach for counteracting endothelial apoptosis [16,17,18,19]. Few Nrf activators showed promising results against hyperglycemia-induced endothelial dysfunction (ED) [16]. We reported quercetin, a potent Nrf activator that showed a cytoprotective effect on endothelial cells against ER stress-induced ED [26]. To offer better understanding of the underlying molecular mechanism, we have carried out a proteomic study on the RAtreated ER stress-induced endothelial cells using electrospray ionization tandem mass spectrometry (LC-MS/MS)

Materials and Methods

Results

40 Thapsigargin

Discussion

Conclusions

Conflicts of Interest