YOD1通过去泛素化作用稳定RIPK1，进而激活NF-κB并加剧心肌细胞缺氧/复氧损伤

Abstract

Background: Myocardial ischemia-reperfusion (I/R) injury represents a severe pathological process in cardiovascular diseases. This study aims to elucidate the mechanism of the yeast ovarian tumor (OTU) domain-containing protein 1 (YOD1) in cardiomyocyte injury. Methods: A hypoxia/reoxygenation (H/R) model was established using human AC16 cells to simulate I/R injury in vitro . Reverse transcription quantitative PCR (RT-qPCR) and western blotting were used to detect gene and protein expression. Cellular functions were evaluated using Cell Counting Kit-8 (CCK-8), lactate dehydrogenase (LDH), enzyme-linked immunosorbent assay (ELISA), flow cytometry, and biochemical kits. Protein interaction was validated through co-immunoprecipitation (Co-IP), ubiquitination assays, and cycloheximide (CHX) chase experiments. Results: YOD1 was up-regulated in H/R-induced AC16 cells ( p < 0.01). H/R decreased cell viability ( p < 0.001), superoxide dismutase (SOD) ( p < 0.001), and glutathione peroxidase 4 (GPX4) ( p < 0.001) and increased LDH ( p < 0.001), interleukin-6 (IL-6) ( p < 0.001), tumor necrosis factor-alpha (TNF-α) ( p < 0.001), reactive oxygen species (ROS) ( p < 0.001), malondialdehyde (MDA) ( p < 0.001), and Fe 2+ ( p < 0.001); these effects were alleviated by YOD1 knockdown ( p < 0.01). YOD1 knockdown ( p < 0.001) also reversed H/R-increased receptor-interacting serine/threonine kinase 1 (RIPK1) protein levels ( p < 0.001) and p-p65/p65 ( p < 0.001) and p-nuclear factor-kappa-B inhibitor alpha (p-IκBα)/IκBα ( p < 0.001) ratios. RIPK1 overexpression reversed the protective effects of YOD1 knockdown via activating the nuclear factor-kappa-B (NF-κB) pathway ( p < 0.01). Mechanistically, YOD1 interacted with RIPK1 and inhibited its K63-linked ubiquitination degradation ( p < 0.01). These statistical significances support the clinical potential of targeting YOD1 to mitigate inflammation, oxidative stress, and ferroptosis-related changes in myocardial