Silencing SLC31A1 attenuates high glucose plus copper-induced cuproptosis-like signaling, oxidative stress, and barrier dysfunction in human retinal microvascular endothelial cells

To examine whether SLC31A1 knockdown protects human retinal microvascular endothelial cells (HRMECs) exposed to high glucose and copper.

HRMECs were exposed to normal glucose (5 mmol/L) or high glucose (30 mmol/L), with or without 50 μmol/L CuSO₄ during the final 6h, and transfected with control or SLC31A1 small interfering RNA. Cell viability (cell counting kit-8) and adenosine triphosphate (ATP) content were measured. Oxidative stress was assessed by glutathione/oxidized glutathione ratio, malondialdehyde, superoxide dismutase, catalase, and reactive oxygen species. Intracellular copper and mRNA levels of SLC31A1, ferredoxin 1, lipoic acid synthetase, dihydrolipoamide S-acetyltransferase (DLAT), and tight junction genes were quantified. SLC31A1 protein and lipoylated DLAT were detected by Western blotting. Endothelial barrier function was evaluated by fluorescein isothiocyanate-dextran permeability and transendothelial electrical resistance.

High glucose alone caused modest loss of viability, ATP depletion, increased oxidative stress, downregulation of tight junction genes, and mild barrier impairment, with minimal additional effect of SLC31A1 knockdown. Copper supplementation under high glucose induced marked intracellular copper overload, enhanced DLAT lipoylation, severe ATP loss, oxidative injury, and pronounced barrier dysfunction. SLC31A1 knockdown significantly reduced copper accumulation, lipoylation of DLAT, and oxidative stress, preserved ATP and viability, and partially restored tight junction gene expression and barrier function, although none of these parameters returned to normal glucose levels.

SLC31A1-dependent copper influx appears to contribute to cuproptosis-associated mitochondrial energy failure, oxidative stress, and barrier breakdown in high glucose-exposed retinal endothelial cells, indicating that SLC31A1 may represent a potential therapeutic target for diabetic retinal microvascular protection.