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

Calcific aortic valve disease (CAVD) predominantly affects the elderly and currently lacks effective medical treatments. Nesfatin-1, a peptide derived from the cleavage of Nucleobindin 2, has been implicated in various calcification processes, both physiological and pathological. This study explores the impact of Nesfatin-1 on the transformation of aortic valve interstitial cells (AVICs) in CAVD. In vitro experiments showed that Nesfatin-1 treatment mitigated the osteogenic differentiation of AVICs. Corresponding in vivo studies demonstrated a deceleration in the progression of CAVD. RNA-sequencing of AVICs treated with and without Nesfatin-1 highlighted an enrichment of the Ferroptosis pathway among the top pathways identified by the Kyoto Encyclopedia of Genes and Genomes analysis. Further examination confirmed increased ferroptosis in both calcified valves and osteoblast-like AVICs, with a reduction in ferroptosis following Nesfatin-1 treatment. Within the Ferroptosis pathway, ZIP8 showed the most notable modulation by Nesfatin-1. Silencing ZIP8 in AVICs increased ferroptosis and osteogenic differentiation, decreased intracellular Mn2+ concentration, and reduced the expression and activity of superoxide dismutase (SOD2). Furthermore, the silencing of SOD2 exacerbated ferroptosis and osteogenic differentiation. Nesfatin-1 treatment was found to elevate the expression of glutathione peroxidase 4 (GPX4) and levels of glutathione (GSH), as confirmed by Western blotting and GSH concentration assays. In summary, Nesfatin-1 effectively inhibits the osteogenic differentiation of AVICs by attenuating ferroptosis, primarily through the GSH/GPX4 and ZIP8/SOD2 pathways. A schematic model is presented to demonstrate the protective effects of Nesfatin-1 against aortic valve calcification in aortic valve interstitial cells (AVICs). This protection occurs through the regulation of ferroptosis, facilitated by the GSH/GPX4 and ZIP8/SOD2 axes. [Display omitted] •Nesfatin-1 could inhibit AVICs osteogenic differentiation in vitro.•Nesfatin-1 treatment reduces AVICs' osteogenic differentiation, a potential therapy for CAVD.•Ferroptosis is involved in Nesfatin-1 inhibiting AVICs osteogenic differentiation.•GSH/GPX4 and ZIP8/SOD2 axes mediate the reduced ferroptosis level.