Integrated Network Pharmacology and Experimental Study on Shikonin‐Induced Male Reproductive Toxicity

Shikonin (SHK), a natural naphthoquinone compound with diverse pharmacological activities, exerts potent inhibitory effects on multiple tumor cell types. However, its impact on the male reproductive system lacks clarity. This investigation applied an integrated network pharmacology and experimental validation strategy to systematically evaluate the potential male reproductive toxicity of SHK. Computational analyses identified 59 overlapping targets between SHK and male infertility-associated genes, with functional enrichment implicating apoptosis regulation and oxidative stress as central mechanisms. To validate these predictions, in vitro experiments were performed to assess the toxicity of SHK on mouse testicular tissues and human sperm. Results demonstrated that SHK reduced testicular tissue viability and disrupted the histological structure of seminiferous tubules by modulating apoptosis-related protein expression and inducing cellular apoptosis. Furthermore, SHK decreased viability, motility, mitochondrial membrane potential (MMP), and B-cell lymphoma-2 (BCL-2)/BCL-2-associated X (BAX) ratios while elevating reactive oxygen species (ROS) levels in human sperm. Critically, co-treatment with ascorbic acid (AA), a potent antioxidant, attenuated SHK-induced ROS overproduction, partially restored sperm motility and viability, and mitigated testicular tissue damage. In summary, SHK triggered mitochondrial apoptosis in the testes and sperm via ROS-mediated oxidative stress, while AA counteracted this toxicity by suppressing oxidative stress.