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Updated: Jun 7, 2026
Induction and Analysis of Oxidative Stress in Sleeping Beauty Transposon-Transfected Human Retinal Pigment Epithelial Cells
Published on: December 11, 2020
H₂O₂-induced oxidative stress modulates methotrexate and dexamethasone-dependent regulation of NLRP3-associated
Zahra Souri1, Mohammad Malekahmadi2
1Department of Cell and Molecular Biology & Microbiology, Faculty of Biological Science and Technology, University of Isfahan, Isfahan, Iran.
Purpose:
Oxidative stress-driven inflammation is a central mechanism in retinal diseases. This study investigated how methotrexate (MTX) and dexamethasone (DEX), alone or in combination, modulate NLRP3-related gene expression and HLA class I expression in primary human retinal pigment epithelial (RPE) cells under basal and oxidative stress conditions.
Methods:
Primary human RPE cells were isolated from cadaveric donor eyes and treated with MTX (100 µg/mL), DEX (50 µg/mL), or their combination for 24 h under normal culture conditions or following H₂O₂-induced oxidative stress (100 µM). mRNA expression of NLRP3, PYCARD, CASPASE-1, HLA-A, and HLA-B was quantified by qPCR. ELISA measured IL-1β and IL-18 secretion.
Results:
Under basal conditions, MTX and DEX downregulated NLRP3 and HLA-A/B mRNA expression while increasing PYCARD and CASPASE-1 transcripts. Combination treatment reduced IL-1β secretion, whereas MTX and combination therapy decreased IL-18 levels. In oxidative stress conditions, MTX and combination therapy upregulated NLRP3 and CASPASE-1 mRNA, while HLA-A/B and PYCARD transcripts were primarily increased by combination treatment. IL-1β levels remained unchanged, and IL-18 was reduced only in the combination group.
Conclusions:
MTX and DEX modulate NLRP3-associated and HLA class I transcriptional responses in RPE cells in a redox-dependent manner. Although oxidative stress modulated drug-associated gene expression profiles, these transcriptional changes were not paralleled by corresponding alterations in secreted inflammatory cytokine levels, suggesting that the context-dependent nature of inflammatory signaling and cytokine release in RPE cells should be taken into consideration.
