Nesfatin-1 mitigates calcific aortic valve disease via suppressing ferroptosis mediated by GSH/GPX4 and ZIP8/SOD2 axes

Abstract Objective: Calcific aortic valve disease (CAVD) is a prevalent condition in the elderly population, lacking effective medical treatments. Nesfatin-1, a hypothalamic peptide generated from Nucleobindin 2 processing, has been implicated in both physiological and pathological calcification. In this study, we aimed to investigate the role of Nesfatin-1 in facilitating the transformation of aortic valve interstitial cells (AVICs) in relation to CAVD. Methods and Results: In vitro experiments demonstrated that Nesfatin-1 treatment attenuated osteogenic differentiation of AVICs, while in vivo studies showed that it slowed down CAVD progression. RNA-sequencing analysis of AVICs treated with or without Nesfatin-1 revealed enrichment of the Ferroptosis pathway among the top 15 Kyoto Encyclopedia of Genes and Genomes pathways. Further investigations confirmed increased ferroptosis in calcified valves and osteoblast-like AVICs, with Nesfatin-1 treatment reducing ferroptosis in AVICs. Among the genes involved in the Ferroptosis pathway, ZIP8 exhibited the most significant change upon Nesfatin-1 treatment. Silencing ZIP8 enhanced ferroptosis and osteogenic differentiation in AVICs. Additionally, silencing ZIP8 reduced intracellular Mn2+ concentration, as well as the expression and activity of superoxide dismutase (SOD2). Concordantly, silencing SOD2 further enhanced ferroptosis and osteogenic differentiation in AVICs. Moreover, Nesfatin-1 treatment increased glutathione peroxidase 4 (GPX4) expression and glutathione (GSH) levels in AVICs, as confirmed by Western blotting and GSH concentration estimation. Conclusion: In summary, Nesfatin-1 inhibits osteogenic differentiation of AVICs by attenuating ferroptosis. This reduction in ferroptosis levels is mediated by the GSH/GPX4 and ZIP8/SOD2 axes.