Puerarin ameliorates obesity-induced spermatogenic dysfunction by targeting GRP78/StAR to increase testosterone synthesis.

BACKGROUND

Testosterone synthesis dysfunction caused by obesity is a major contributor to male infertility, but effective therapeutic options remain limited. Puerarin (Pue), a bioactive compound from Pueraria lobata, has lipid-lowering and reproductive-protective properties. This study aimed to investigate the mechanisms by which Pue ameliorates obesity-associated spermatogenic impairment.

METHODS

A high-fat diet (HFD) mouse model was used to assess the effects of Pue supplementation on lipid metabolism, testosterone levels and spermatogenesis. Network pharmacology, transcriptomics, and activity-based protein profiling (ABPP) analyses were performed to explore potential pathways and protein targets. In addition, pull-down assays, immunofluorescence, and cellular thermal shift assays (CETSA) were used to validate target engagement, and the role of the identified target was further tested in palmitic acid (PA)-induced TM3 cells by using protein target inhibitors and protein target overexpression to verify the ability of Pue to ameliorate obesity-related testosterone synthesis dysfunction.

RESULTS

Pue supplementation markedly alleviated obesity-induced metabolic disorders by reducing body weight gain, adiposity, and hepatic lipid deposition while improving serum triglyceride (TG), total cholesterol (TC), high-density lipoprotein (HDL), and low-density lipoprotein (LDL) levels. Pue supplementation preserved testicular and epididymal morphology and significantly increased the serum testosterone level, sperm count, and sperm motility. Transcriptomic analysis revealed that Pue restored the expression of steroidogenic acute regulatory protein (StAR), cytochrome P450 family 11 subfamily A member 1 (CYP11A1), 3β-hydroxysteroid dehydrogenase (3β-HSD), and cytochrome P450 family 17 subfamily A member 1 (CYP17A1). Pull-down assays, molecular docking, and CETSAs revealed that glucose-regulated protein 78 (GRP78) is a Pue-binding target, with Pue forming hydrogen bonds at the TYR-65 residue. Coimmunoprecipitation (Co-IP) and colocalization assays verified the specific GRP78-StAR interaction. Pue binding enhanced the chaperone activity of GRP78, stabilized StAR and upregulated its expression. The inhibition of GRP78 reduced StAR expression and testosterone secretion in TM3 cells, whereas Pue supplementation reversed these effects, and GRP78 overexpression further enhanced the therapeutic effect of Pue.

CONCLUSION

Pueraria root alleviates obesity-induced spermatogenic dysfunction by improving lipid metabolism, upregulating the expression of key testosterone enzymes, and promoting testosterone synthesis through the targeting of GRP78 by StAR expression. These findings reveal a novel mechanism through which Pue restores androgen biosynthesis and provide a scientific basis for the development of Pueraria-derived interventions for obesity-associated male infertility.