Screening of natural compounds library for protection against endoplasmic reticulum stress in corneal endothelial cells.

Abstract

Purpose: Endoplasmic reticulum (ER) stress-mediated cell death contributes to many ocular diseases, including Fuchs corneal endothelial dystrophy (FECD). Natural compounds possess significant pharmacological activities necessary for vital cell signaling pathways and are considered protective in many ocular diseases such as diabetes, retinopathy, and glaucoma. However, there are limited studies examining the role of natural compounds in reducing sustained ER stress for improving corneal endothelial cell (CEnCs) survival. The purpose of this study is to identify readily available natural compounds to improve the viability of CEnCs under ER stress.

Methods: Human corneal endothelial cells (21T-HCEnC) were cultured and seeded in 384-well plates, pre-treated with 502 compounds from a natural product library (Enzo Screen-Well Natural Product library), and then subjected to ER stressor tunicamycin (1µg/mL) for 24 hours. Cell viability was measured using the Cell-Titer-Glo assay, and 74 compounds improved viability past the set threshold (≥ 30% increase in viability) and were selected for further dose-response testing. Further dose-response testing was performed using untreated F35T corneal endothelial cells, plated onto 384-well plates, and treated with a range of 6 log doses of each compound, from 10 pg/mL up to 1 μg/mL. After 24 hours of treatment, cell viability was measured again using the Cell-Titer Glo assay. Of the 4 natural compounds that improved viability past the set threshold at the lowest dose tested (0.00001 µg/mL), veratrine was analyzed further via western blotting, ranging from 10 pg/mL to 0.01 μg/mL. Apoptotic and unfolded protein response markers were also measured via western blotting.

Results: Of 502 compounds each administered at 0.01 µg/mL, we identified 74 compounds exhibiting 30% or more protection of 21T HCEnC cell viability in response to tunicamycin-induced stress. Of the 74 compounds that underwent further dose-response testing, 4 compounds improved cell viability by 30% or more at the lowest dose of compound tested compared to viability of F35T cells at baseline. Cevadine, or veratrine, was chosen for further analysis due to its function as a sodium channel agonist and its interaction with Na⁺/K⁺ ATPase channels. Pretreatment with veratrine 0.01 µg/mL, along with tunicamycin, downregulated expression levels of cleaved caspase 9, PARP, cytochrome C, Bax, and cleaved caspase 3. Western blots of FECD cell line under tunicamycin-induced ER stress, pretreated with veratrine 0.01 µg/mL, also showed reduced markers from all three ER unfolded protein response pathways.

Conclusions: This study provided insight into the role of natural compounds in regulating ER stress-mediated CEnC death associated with many corneal endothelial-associated diseases. Veratrine was found to have a protective effect on ER-stressed CEnCs, as indicated by reduced apoptotic signals and ER stress markers following veratrine treatment, suggesting a possible mechanism of action via mediating ER stress signals. Future studies are needed to clarify the mechanism and utility of veratrine in FECD.