Ergothioneine Alleviated the Apoptosis of HK Cells by Regulating Ferroptosis.

Abstract

Introduction/objective: Ergothioneine (EGT) is a naturally occurring antioxidant with protective effects on various human cell types. The impact of this substance on HK-2 cells, a human renal proximal tubular epithelial cell line, and the associated molecular mechanisms remain incompletely elucidated.

Methods: The present study aims to elucidate the effects of EGT on apoptosis induced by RAS-selective Lethal Molecule 3 (RSL3) and Erastin in HK-2 cells, as well as the potential mechanisms involved. The renal cortical proximal tubular epithelial HK-2 cells were cultured and exposed to RSL3 and Erastin, with or without EGT treatment. Cell viability and apoptosis were assessed using the Cell Counting Kit-8 (CCK-8) assay, while the detection of ferrous ion content and mitochondrial membrane potential changes was accomplished through the utilisation of flow cytometry and the JC-1 staining method, respectively. Furthermore, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis, proteomic analysis, and western blotting were employed to explore the molecular pathways involved.

Results: The data demonstrated that RSL3 and Erastin exhibited a substantial inhibitory effect on HK-2 cell proliferation, concomitant with the accumulation of intracellular ferrous ions and a shift in mitochondrial membrane potential. The EGT treatment effectively mitigated RSL3- and Erastin-induced apoptosis in HK-2 cells. GO and KEGG enrichment analysis demonstrated that EGT suppressed pathways and functions associated with oxidative stress during ferroptosis. Proteomics analysis further demonstrated that EGT modulated various biological processes, molecular functions, and cellular components in HK-2 cells. The protective mechanism of EGT against RSL3- and Erastin-induced HK-2 cells was potentially mediated through regulation of ferroptosis. The results of the western blot analysis demonstrated that EGT modulated the expression levels of Glutathione Peroxidase 4 (GPX4) and Solute Carrier Family 7 member 11 (SLC7A11) in HK-2 cells.

Conclusion: These data demonstrated that EGT exerts an alleviating effect on the apoptosis of HK-2 cells induced by RSL3 and Erastin by modulating ferroptosis. These findings suggest that EGT has the potential to serve as a therapeutic candidate for the treatment of kidney diseases in the future.