Differential Modulation of Oxidative Stress and VEGF-A Secretion by Saffron Repron® Pretreatment in ARPE-19 Cells Exposed to High Glucose

Background: Hyperglycemia contributes to diabetic retinopathy by promoting oxidative stress, inflammatory signaling, retinal pigment epithelial (RPE) dysfunction, and vascular endothelial growth factor A (VEGF-A) secretion. Saffron Repron® (sR), selected (and patented) according to its chemical composition, has been shown to activate neuroprotective responses. This study investigated whether sR pretreatment modulates high glucose-induced oxidative damage, antioxidant and inflammatory signaling, VEGF-A secretion, and paracrine endothelial activation in ARPE-19 cells. Methods: ARPE-19 cells were pretreated with sR (40 μg/mL for 24 h), washed, and exposed to high-glucose (HG) (25 mM) for 24 or 96 h. Cell viability and cytotoxicity were assessed using colorimetric assays, reactive oxygen species (ROS) using a luminescence assay, superoxide dismutase 2 (SOD2), nuclear factor erythroid 2–related factor 2 (NRF2), KEAP1, nuclear factor kappa B (NF-κB), and IκBα expression by western blotting, catalase activity by spectrophotometry, and VEGF-A secretion by bioluminescent immunoassay. Human microvascular endothelial cells (HMEC-1) were exposed to ARPE-19 conditioned media for 24 h, after which viability and ROS levels were evaluated. Data were analyzed using one-way analysis of variance followed by Tukey’s post-hoc test. Results: sR alone did not substantially affect basal ARPE-19 viability or VEGF-A secretion. HG increased cytotoxicity and ROS levels at 24 h and reduced viability, while increasing the cytotoxicity/viability ratio and ROS levels at 96 h. sR reduced HG-induced cytotoxicity, restored viability at 96 h, and normalized ROS levels. However, SOD2 expression and catalase activity were reduced by HG and/or sR, indicating that sR-mediated ROS reduction was not associated with induction of these antioxidant defenses. Total NRF2 expression and the NRF2/KEAP1 ratio remained unchanged, although sR prevented HG-induced KEAP1 upregulation at 24 h. HG altered the NF-κB/IκBα balance and increased VEGF-A secretion at both time points; sR did not prevent increase in VEGF-A at 24 h but significantly reduced the HG-induced increase at 96 h. Conditioned media from HG-treated ARPE-19 cells increased HMEC-1 viability and ROS levels, whereas media from sR-pretreated HG cells attenuated these effects. Conclusions: Pretreatment with sR protects ARPE-19 cells from HG-induced cytotoxicity and oxidative stress primarily by reducing ROS levels rather than by inducing NRF2/SOD2-associated antioxidant responses. sR pretreatment also attenuated the pro-oxidative paracrine effects of HG-exposed ARPE-19 RPE model on endothelial cells, supporting further investigation of its potential protective effects in models of hyperglycemic retinal stress.