4-octyl itaconate protects goat Sertoli cells against LPS-induced mitochondrial dysfunction by modulating mitophagy-related processes

Sertoli cells are essential for maintaining spermatogenesis and testicular immune privilege; however, they are highly susceptible to inflammatory injury. Lipopolysaccharide (LPS)-induced inflammation disrupts mitochondrial homeostasis and impairs Sertoli cell function, yet the underlying mechanisms remain incompletely understood. In this study, we investigated the protective effects of 4-octyl itaconate (4-OI), a cell-permeable derivative of itaconate, on LPS-induced injury in goat Sertoli cells. Our results demonstrated that LPS exposure triggered significant apoptosis, oxidative stress, and mitochondrial dysfunction, accompanied by excessive activation of mitophagy. Mechanistically, LPS activated the nuclear factor kappa-B (NF-κB) signaling pathway while suppressing nuclear factor erythroid 2-related factor 2 (NRF2)-mediated antioxidant responses, leading to increased reactive oxygen species (ROS) production, mitochondrial fission, and loss of mitochondrial membrane potential. Notably, 4-OI pretreatment effectively inhibited NF-κB activation and enhanced NRF2-associated antioxidant responses, thereby reducing oxidative stress. Furthermore, 4-OI suppressed excessive mitophagy, as evidenced by decreased LC3B-II/I ratio, increased p62 accumulation, and reduced expression of mitophagy-related proteins including PINK1, Parkin, BNIP3, and FUNDC1. Inhibition of mitophagy partially rescued mitochondrial dysfunction, whereas pharmacological (rapamycin) activation of autophagy attenuated the protective effects of 4-OI. Importantly, 4-OI treatment preserved mitochondrial dynamics and maintained the expression of Sertoli cell phenotype- and barrier-associated proteins. Collectively, these findings demonstrate that excessive mitophagy is a critical mediator of LPS-induced mitochondrial dysfunction in Sertoli cells, and that 4-OI exerts protective effects by restoring mitochondrial homeostasis through suppression of mitophagy. This study provides new insights into the pathogenesis of testicular inflammation and highlights the potential cytoprotective effects of 4-OI against inflammatory injury in Sertoli cells.