Zuogui Wan preserves Leydig cell steroidogenesis via G6PD/NADPH/GSH-dependent ferroptosis inhibition in D-galactose-induced hypogonadism.

ETHNOPHARMACOLOGICAL RELEVANCE Zuogui Wan (ZGW), a classical formula for aging-related disorders, may delay reproductive decline, but its effects on late-onset hypogonadism (LOH) remain unclear.

Aim

OF THE STUDY To investigate the effects and underlying mechanisms of ZGW in LOH using rat and cellular models by integrating serum pharmacochemistry, network pharmacology, and transcriptomic analyses.

Materials And Methods

A D-galactose-induced accelerated aging rat model was established to evaluate the ameliorative effects of ZGW. Blood-absorbed constituents were identified by UHPLC-MS/MS, and candidate pathways and targets were screened by integrating network pharmacology, testicular transcriptomics, molecular docking, and molecular dynamics simulations. Mechanistic validation was performed in rat testicular tissues and TM3 Leydig cells, and the functional role of G6PD was further evaluated using G6PDi-1-mediated pharmacological inhibition and G6pdx knockdown/overexpression rescue experiments.

Results

ZGW ameliorated D-galactose-induced reproductive endocrine dysfunction and testicular aging-related injury in rats and attenuated injury in TM3 Leydig cells. Based on the identification of 36 blood-absorbed constituents, integrated analyses prioritized G6PD and suggested that it may be involved in redox regulation and ferroptosis-related mechanisms. In vivo and in vitro experiments showed that ZGW restored G6PD-related NADPH/GSH redox homeostasis, strengthened GPX4-dependent defense against lipid peroxidation, attenuated ferroptosis-related injury, and preserved mitochondrial function and StAR/CYP11A1-associated steroidogenesis. Both G6PDi-1 and G6pdx knockdown partially attenuated the effects of ZGW, whereas G6pdx rescue partially restored these changes.

Conclusions

ZGW may alleviate D-gal-induced LOH-like dysfunction by restoring G6PD-related NADPH/GSH homeostasis and GPX4-dependent lipid-peroxide defense. These effects may limit ferroptosis-related injury and preserve the mitochondrial conditions required for StAR/CYP11A1-associated steroidogenesis.