Abstract: Objective: To investigate the antagonistic effect and mechanisms of chlorogenic acid (CGA) on testicular dysfunction induced by the food contact chemical bisphenol F (BPF). Methods: Core targets were identified using Gene Ontology (GO) functional enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses based on network toxicology and network pharmacology approaches, followed by validation via molecular docking. A subacute exposure model in rats was established by continuous oral administration of BPF for three months. Hematoxylin-eosin (HE) staining was used to observe pathological changes in testicular and epididymal tissues, and Papanicolaou staining was used to examine sperm morphology in the rat epididymis. An enzyme-linked immunosorbent assay (ELISA) was used to measure the serum levels of C-reactive protein (CRP), interleukin-1β (IL-1β), malondialdehyde (MDA), and testosterone (T). Western blotting (WB) analysis was used to detect the expression levels of proteins in the phosphoinositide 3-kinase/protein kinase B (PI3K/AKT) signaling pathway, as well as the expression levels of estrogen receptor 1 (ESR1), B-cell lymphoma 2 (BCL-2), and BCL-2-associated X protein (BAX). Results: We identified 75 overlapping targets for testicular damage induced by CGA and BPF, and the core targets included ESR1, BCL-2, AKT1, and HSP90AA1. GO functional analysis showed that CGA was primarily involved in biological processes such as oxidative stress response, peptide hormone signaling, and apoptosis regulation; KEGG pathway enrichment indicated that the PI3K/AKT signaling pathway was a key pathway. Molecular docking revealed that the binding energies between the target proteins and the small molecules were all ≤ –5.0 kcal/mol, and CGA exhibited significantly stronger binding affinities for key targets such as ESR1, BCL-2, EGFR, and HSP90AA1 compared with BPF. Animal experiments demonstrated that compared with the BPF group, the CGA group showed significantly lower serum levels of MDA, IL-1β, and CRP in rats (P < 0.01), while T levels were significantly elevated (P < 0.01). Furthermore, CGA intervention significantly improved BPF-induced testicular histopathological damage and sperm abnormalities, with the most pronounced effects observed in the high-dose group. Protein analysis of the key PI3K/AKT signaling pathway in testicular tissue revealed significant upregulation of p-PI3K/PI3K, p-AKT/AKT, and BCL-2/BAX protein expression (P < 0.05), and the number of TUNEL-positive cells was significantly reduced (P < 0.01). Conclusion: CGA may mitigate BPF-induced testicular injury by multi-target regulation of the PI3K/AKT signaling axis, alleviating oxidative stress and inhibiting apoptosis.

Key words: chlorogenic acid; bisphenol F; testicular injury; apoptosis; phosphoinositide 3-kinase/protein kinase B signaling pathway