Abstract
Purpose :
To determine the role of the Trx/TrxR redox signaling pathway in human retinal pigment epithelieum (RPE) dysfunction and cell death mechanisms.
Methods :
A human RPE cell line (APRE-19) was cultured in DMEM/F12 medium and treated with auranofin (4 μM, an inhibitor of TrxR) for 4 and 24 h. Mitochondrial and lysosomal functions, cellular oxidative stress and NLRP3 inflammasome activity were measured using cell assays, Western blotting and confocal microscopy. Antioxidants and anti-inflammatory compounds were tested for blocking auronofin effects. Cell death mechanisms (LDH release to culture media) were determined using necroptosis, ferroptosis and pyroptosis inhibitors. P<0.05 was considered significant in statistical analysis.
Results :
Auranofin causes mitochondrial dysfunction (Δψm↓ and ATP↓), oxidative stress (H2O2↑) and mitophagic flux to lysosomes. Lysosomal enzyme (cathepsin L) activity is reduced while caspase-1 (NLRP3 inflammasome) activity is enhanced in ARPE-19. These effects of auranofin on ARPE-19 are inhibited by N-acetylcysteine (5 mM, NAC) and partly by a combination of SS31 (mitochondrial anti-oxidant) and anti-inflammatory drugs (amlexanox and tranilast). Auranofin causes LDH release, which is not inhibited by either ferrostatin-1 or necrostatin-1 (ferroptosis and necroptosis inhibitors, respectively). Conversely, auranofin-induced LDH release is reduced by MCC950 and Ac-YVAD-cmk (NLRP3 and Caspase-1 inhibitors, respectively), suggesting a pro-inflammatory cell death by pyroptosis.
Conclusions :
The Trx/TrxR redox system is critical for RPE function and cell viability. We previously showed that thioredoxin-interacting protein (TXNIP) is strongly induced in diabetic retinopathy (DR) and inhibits the Trx/TrxR system. Therefore, the results suggest that the TXNIP-Trx-TrxR redox pathway may participate in RPE dysfunction in DR.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.