A mechanistic study on the repair of cadmium-induced male infertility using Yishen Tongluo formula based on network toxicology and experimental validation.

OBJECTIVE: This study employs network toxicology to screen for toxicological targets of cadmium chloride-induced male infertility, and validates the mechanism of Yishen Tongluo Formula in treating cadmium-induced male infertility through animal experiments.

METHOD: Employing network toxicology to search for potential target molecules in the ChEMBL database, and STITCH databases using 'cadmium chloride' as the keyword to identify potential targets. Using "male infertility" as the keyword, we searched for male infertility-related targets in the OMIM, PharmGKB, and GeneCards databases. The primary components of the Yishen Tongluo Formula were then input into the SwissTargetPrediction and PharmMapper databases to identify potential targets. Cadmium chloride, Yishen Tongluo Formula component targets, and male infertility-related disease targets were imported into Venny 2.1.0 to construct a protein-protein interaction (PPI) network by identifying intersections. Cadmium chloride-induced male infertility-related targets were imported into Metascape for Gene Ontology (GO) and KEGG pathway enrichment analysis. Molecular docking validation was performed, and a 'compound-target-pathway' network was constructed for result visualisation. In animal experiments, 33 mice were randomly divided into three groups: a control group, a cadmium chloride-exposed group (Cd group), and a Yishen Tongluo Formula group, with 11 mice per group. Cadmium chloride was diluted in purified water to a working solution of 0.3 mg/ml. Except for the control group, the Cd group and Yishen Tongluo Formula group received cadmium chloride via gavage at 10 ml/kg for 70 days. After successful modeling, from day 31 onward, the cadmium-exposed group and the herbal intervention group received daily gavage of 10 ml/kg cadmium chloride working solution at 8:00 a.m. At 6:00 pm daily, the herbal intervention group received 10 ml/kg of the Yishen Tongluo Formula decoction solution via protective gastric lavage, The Yishen Tongluo Formula group received 1.2 g/ml of the decoction solution via gastric lavage, while the toxic exposure group received the same volume of deionised water. The control group received the same volume of deionised water at the same time points. All three groups underwent continuous gastric lavage for 40 days. Following organ collection, the following parameters were assessed: sperm DNA fragmentation index (DFI), testicular index, testicular superoxide dismutase (SOD), malondialdehyde (MDA), adenosine triphosphate (ATP), nitric oxide (NO), and serum follicle-stimulating hormone (FSH), luteinizing hormone (LH), and testosterone (T) were assessed.

CONCLUSION: This study demonstrates that cadmium chloride induces male reproductive toxicity by targeting CFTR, SLC26A3, and SLC12A1 via the pancreatic secretion pathway, causing oxidative stress, endocrine disorders, sperm DNA damage, and testicular tissue injury. Yishen Tongluo Formula exerts therapeutic effects against cadmium-induced male infertility mainly by targeting TP53, which significantly improves testicular coefficient, reduces sperm DFI, enhances sperm quality, alleviates testicular oxidative stress, and restores the balance of reproductive hormones. These findings reveal the multi-target mechanism of Yishen Tongluo Formula in repairing cadmium-induced reproductive damage, provide a novel experimental basis for the treatment of heavy metal-related male infertility with traditional Chinese medicine, and suggest that further exploration of molecular mechanisms and clinical transformation will help develop safer and more effective intervention strategies for environmental toxin-induced reproductive dysfunction.