Activation of Nrf2 signaling by 4-octyl itaconate attenuates the cartilaginous endplate degeneration by inhibiting E3 ubiquitin ligase ZNF598

Abstract

Objective: Cartilaginous endplate (CEP) degeneration is the main early manifestations of intervertebral disc degeneration (IVDD), and is closely related to the oxidative stress. Nrf2 (nuclear factor E2-related factor 2, NFE2L2) is a vital transcriptional factor of cellular antioxidant and anti-inflammatory responses. We aimed to illustrate whether the Nrf2 which was increased in expression by 4-octyl itaconate (4OI) could attenuate intervertebral disc degeneration through suppressing macrophage associated inflammation and catabolism of cartilaginous endplate.

Methods: Firstly, we detected the expression of Nrf2 in human degenerative CEPs. Then, we performed in vitro, ex vivo and in vivo (a rat-tail puncture model) experiments to explore the role of 4OI in IVDD. Also, by cell co-culture experiments, we demonstrated 4OI restrained the macrophage-associated inflammatory responses. Finally, through western blotting and immunoprecipitation (IP) assay, we clarified the ZNF598-mediated ubiquitination of Nrf2.

Results: We found decreased expression of Nrf2 in human degenerative CEPs. Using a rat IVDD model(n = 6), 4OI significantly ameliorated the progression of IVDD by MR images and histological analysis. Immunofluorescence results reveal that catabolism of CEPs and macrophage-associated inflammation are suppressed by 4OI treatment. Mechanistically, the 4OI increases Nrf2 expression and inhibits the secretion of inflammatory factors (IL-1β) by Lipopolysaccharide (LPS)-induced macrophages, thus preventing the inflammatory-related CEP degeneration. Meanwhile, 4OI suppresses the reactive oxygen species (ROS) production and catabolism of LPS-induced rat CEP cells. In addition, 4OI inhibits the ZNF598-dependent ubiquitination of Nrf2 in LPS-induced rat CEP cells.

Conclusions: 4OI may alleviate IVDD by suppressing CEP degeneration and macrophage-associated inflammation. 4OI may be an alternative therapy for degenerative CEPs/IVDs.

Keywords: 4-Octyl itaconate; Cartilaginous endplate; Inflammation; Intervertebral disc degeneration; Nrf2.