Fluoride-induced male reproductive toxicity: Dual-tissue pyroptosis in the gut-testis axis mediated by inflammatory cytokines.

Excessive fluoride impairs male reproductive function, and the gut-testis axis is potentially an important pathway. However, the molecular mechanism remains unclear. This study aimed to investigate whether fluoride damaged the male reproductive system through the gut-testis axis and to identify the molecular pathways involved. We first established in vivo rat models that were treated with sodium fluoride (NaF) and underwent fecal microbiota transplantation (FMT), and then constructed in vitro indirect co-culture models. In NaF-treated rats, our results showed disrupted testicular tissue structure, significantly decreased sperm quality and serum testosterone levels, elevated estradiol, and downregulated expression of tight junction proteins (ZO-1 and Occludin). NaF damaged intestinal physical, chemical, and microbial barriers, characterized by decreased ZO-1, Occludin, and MUC2, increased relative abundances of Firmicutes and Proteobacteria, and elevated intestinal permeability. Higher levels of IL-1β and TNF-α were found in colon, testis, and serum of NaF-treated rats. Transcriptomic analysis revealed significant enrichment of the NF-κB signaling pathway in both the colon and testis. Quantitative analysis and immunofluorescence exhibited the alteration of NF-κB-mediated pyroptosis pathway, with upregulation of Cle-Casp-1, NLRP3, ASC, GSDMD, GSDMD-N in both the colon and testis. The elevation of LDH levels was observed in serum and cell supernatant. Critically, FMT alleviated these damages. Concurrently, in vitro experiments confirmed that NaF induced similar inflammatory responses and pyroptosis, and these effects were mitigated by shikonin. In conclusion, fluoride may impair male reproduction by activating the NF-κB-mediated pyroptosis pathway with the gut-testis axis.