Integrated network toxicology, transcriptomics, and metabolomics revealed the potential mechanisms of aluminum-induced testicular injury.

Aluminum (Al) is a widespread environmental contaminant with multi-organ toxicity. Although aluminum-induced testicular toxicity has been reported in male rats, its underlying mechanism remains unclear. Different from conventional single-model or single-omics research, this study integrated in vitro and in vivo experiments, network toxicology, and multi-omics approaches to investigate aluminum-induced testicular toxicity and its underlying mechanisms. In vitro, aluminum reduced the viability of TM3 and TM4 cells in a concentration-dependent manner. In vivo, rats administered AlCl3·6H2O for 15 days showed decreased body weight and testicular index, testicular histopathological damage, and increased sperm abnormalities. Network toxicology analysis suggested that inflammation, oxidative stress, apoptosis, and the PI3K-Akt signaling pathway are suggested to be key pathways. Transcriptomics and metabolomics identified 287 differentially expressed genes and 39 metabolites, which were mainly involved in glycerophospholipid and amino acid metabolism. Multi-omics integration revealed disrupted amino acid, lipid, and energy metabolism, with significant enrichment of the PI3K-Akt signaling pathway, which was validated using qRT-PCR. These findings provide novel insights into aluminum-induced testicular toxicity and prevention of male reproductive dysfunction.