Inulin Mitigates Reproductive Toxicity from Early Life GenX Exposure in Mice by Inhibiting LPS-TLR4-Mediated Necroptosis: Exploring the Gut–Testis Axis

Hexafluoropropylene oxide dimer acid (HFPO-DA or GenX), a common short-chain substitute for perfluorooctanoic acid, affects mammalian development. However, the effects of GenX on male reproductive development remain incompletely understood. In this study, pregnant mice were administered GenX (2 mg/kg bw/d) via gavage and/or inulin (1% w/v) in the drinking water until lactation concluded. At the 16th week after birth, male littermates were necropsied. Early-life exposure to GenX in mice led to gut microbiota imbalance, colonic tissue alterations, reduced expression of goblet cells and tight junction proteins (ZO-1, occludin, and claudin 5), and triggered an inflammatory response. Moreover, increased serum LPS levels, histopathological alterations in testicular tissue, reduced testosterone levels, and decreased expression of blood–testis barrier proteins (ZO-1, occludin, N-cadherin, and β-catenin) were resulted from early-life exposure to GenX. Additionally, there was an upregulation of inflammatory factors TNF-a and IL-1β, along with a higher incidence of sperm abnormalities. Transcriptomic analysis revealed an enrichment of necroptosis in the testes of the GenX group. Consistently, overexpression of RIPK1, RIPK3, TLR4, and p-ERK, and decreased expression of caspase-8 confirmed the participation of necroptosis in GenX-induced testicular injury. As a potent prebiotic, inulin balanced intestinal homeostasis and attenuated GenX-induced testicular injury by modulating LPS-TLR4-mediated necroptosis. This study offers new insights into the impact of early-life GenX exposure on male reproductive risk and evaluates the potential of inulin as a dietary modification strategy.