Total flavonoids of Epimedium ameliorate testicular damage via keap1-Nrf2/ARE signalling and multi-target regulation.

This study investigates the protective effects of total flavonoids of Epimedium (TFE) against obesity-induced testicular damage in mice. The potential pharmacological mechanisms were predicted using network pharmacology, molecular docking and molecular dynamics simulation. In vivo experiments were conducted in mice randomly assigned to five groups: a normal diet group, a high-fat diet (HFD) group, two TFE groups, and a positive control group. Biomarkers including dihydrotestosterone and testosterone were measured to evaluate testicular function, while lipid profiles were assessed. Oxidative stress was determined through glutathione (GSH), superoxide dismutase (SOD), and malondialdehyde (MDA) assays, and protein expression of heme oxygenase-1 (HO-1), NAD(P)H quinone dehydrogenase 1 (NQO1), nuclear factor erythroid 2-related factor 2 (Nrf2), and Kelch-like ECH-associated protein 1 (Keap1) was analysed. Network pharmacology identified 29 flavonoids and 288 key targets. Molecular docking revealed that 22 active components exhibited strong binding activity, with PLCZ1-isoginkgetin/bilobetin showing the most stable conformation. TFE treatment significantly improved testicular function and mitigated obesity-induced male infertility. It enhanced sperm quality, reversed HFD-induced reductions in sperm count and motility, decreased malformation rates, normalized serum testosterone and dihydrotestosterone levels, ameliorated oxidative stress by enhancing antioxidant enzyme activity (GSH) and reducing lipid peroxidation (MDA), and improved lipid profiles by lowering triglycerides (TG) while increasing high-density lipoprotein cholesterol (HDL-C). Mechanistically, TFE upregulated Nrf2 pathway components (HO-1, NQO1) and modulated Keap1 expression. Collectively, these findings establish that the protective effects of TFE against obesity-induced testicular damage are mediated by the coordinated regulation of oxidative stress and key testicular targets.