Abstract
Purpose :
Oxidative stress may contribute to RPE cell damage in progressive degenerative diseases such as age-related macular degeneration (AMD). Nuclear factor erythroid 2-related factor 2 (NRF2) is a key modulator of the antioxidant response and represents a potential modifying factor to limit the extent of oxidative stress in the eye. We examined how modulation of NRF2 activation influenced the expression of tight junctional genes, trans-epithelial electrical resistance (TEER) and barrier permeability in an ARPE-19 cell model.
Methods :
ARPE-19 cells were treated with varying concentrations sulforaphane (SFN) or brusatol (BRL), or a combination, for 24 hours. Cell viability was assessed via resazurin reduction. Gene expression was determined by quantitative RT-PCR, using the comparative threshold cycle method and PPIA as the endogenous control. TEER and barrier permeability experiments were carried out on confluent monolayers cultured for up to four weeks in TransWell plates. Comparative studies with histone deacetylase inhibitors (HDACi) were used to assess the contribution of the known HDACi effect of SFN on the experimental readouts. The student’s t-test was used for statistical comparisons, with P<0.05 considered significant.
Results :
SFN and BRL appear well tolerated by ARPE-19 cells and modify the expression of classic antioxidant response genes (superoxide dismutase (SOD), thirredoxin-1 (TRX1)). The effects of BRL were only apparent at 100-200 nM, concentrations while those of SFN were concentration dependent (peak induction at 10 mM). SFN modulated the expression of several tight junction genes including Claudin-1 (CLDN1), occludin (OCCLDN) and zona occludens-1 (ZO1), again in a dose dependent manner. TEER was modestly, but significantly, reduced under these conditions. Analogous experiments with BRL resulted in a suppression of gene expression and an increase in TEER. Computational analysis identified potential antioxidant response elements (ARE) in the CLDN1 gene that may explain the observed effects on mRNA expression.
Conclusions :
We show that the natural compounds SFN and BRL can modulate tight junction proteins in ARPE-19 cells at low doses. The functional effects of SFN on TEER appear to be driven by the HDACi activity of SFN rather than the NRF2 effect, as the effects of other HDACi and SFN on TEER are consistent despite variable changes in gene expression.
This is a 2021 ARVO Annual Meeting abstract.