E3 Ubiquitin Ligase NEDD4L Regulates the TGF-β1/Smad Signaling Pathway to Mediate High-Glucose and High-Fat-Induced Ferroptosis of Hepatocytes.

Introduction: The aim of the study was to explore the molecular mechanism of E3 ubiquitin ligase neural precursor cell-expressed developmentally downregulated 4-like (NEDD4L) regulating high-glucose and high-fat-induced ferroptosis in hepatocytes via modulation of transforming growth factor (TGF)-β1/Smad signaling pathway.

Methods: Hepatocytes THLE-2 were cultured in high-glucose and high-fat medium to establish an in vitro nonalcoholic fatty liver disease model. This study detected cellular lipid deposition, cell viability, cellular superoxide dismutase (SOD) activity, glutathione (GSH), malondialdehyde (MDA), ferrous iron (Fe2+), reactive oxygen species (ROS) levels, and cellular mitochondrial membrane potential (MMP). Meanwhile, cellular NEDD4L, GPX4, ACSL4, SLC7A11, TGF-β1, TβRII, and p-Smad2/3 levels were detected by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot. In addition, TGF-β1-TβRII and NEDD4L-TβRII interactions were evaluated by co-immunoprecipitation.

Results: High-glucose and high-fat treatment led to ferroptosis in hepatocytes, manifested by decreased cell viability, SOD activity, and GSH level, increased MDA, Fe2+, and ROS levels, and reduced MMP. High-glucose and high-fat treatment downregulated NEDD4L expression in hepatocytes; by contrast, overexpression of NEDD4L alleviated ferroptosis in hepatocytes. NEDD4L inhibited TGF-β1 signaling by mediating TβRII ubiquitination and degradation. Besides, suppressed TGF-β1/Smad signaling pathway alleviated ferroptosis in hepatocytes, and NEDD4L could regulate hepatocyte ferroptosis by mediating TGF-β1/Smad signaling pathway.

Conclusion: NEDD4L can inhibit high-glucose and high-fat-induced ferroptosis in hepatocytes through suppressing the TGF-β1/Smad signaling pathway via mediating TβRII ubiquitination and degradation.