4-octyl itaconate alleviates LPS-induced inflammation in Sertoli cells by inhibiting excessive autophagy

Bacterial orchitis is a major cause of male infertility, yet effective therapies remain limited. Although the itaconate derivative 4-octyl itaconate (4-OI) possesses potent anti-inflammatory properties, its role in testicular inflammation is unclear. Here, we investigated the protective effects and mechanisms of 4-OI in lipopolysaccharide (LPS)-induced inflammatory injury using Sertoli cells and a mouse model of acute orchitis. LPS activated NF-κB/NLRP3 signaling and induced excessive autophagy in Sertoli cells, resulting in oxidative stress, apoptosis, disruption of tight junctions, and functional impairment. 4-OI markedly suppressed NF-κB phosphorylation and NLRP3 activation, reduced mitochondrial oxidative stress, and improved cell viability. Mechanistically, 4-OI inhibited excessive autophagy by downregulating UNC-51-like kinase 1 (ULK1) and autophagy-related proteins, thereby limiting autophagic flux. Consequently, Sertoli cell functional markers, tight junction integrity, and mitochondrial homeostasis were restored. In vivo, 4-OI alleviated testicular histopathological damage, reduced germ cell apoptosis, improved sperm quality, preserved blood-testis barrier integrity, and enhanced spermatogenic activity. Collectively, these findings identify ULK1-associated excessive autophagy as a key mechanism of inflammatory testicular injury and demonstrate that 4-OI protects against orchitis-induced reproductive dysfunction, highlighting its therapeutic potential for inflammation-associated male infertility.