ABSTRACT

Background and Aim: BAJISAN is a traditional Chinese herbal formula used for male reproductive disorders; however, its bioactive constituents and mechanisms underlying improvements in sperm function remain insufficiently characterized. This study aimed to investigate the dose-dependent effects of BAJISAN extract (BJSE) on semen quality, sperm motility, reproductive hormones, and testicular molecular signaling in rabbits through integrated chemical profiling, network pharmacology, and in vivo validation.

Materials and Methods: Twenty-four male New Zealand rabbits were randomly allocated to four groups (n = 6/group): control and BJSE treatment at 150, 300, or 600 mg/kg body weight daily for 16 weeks. Chemical constituents were characterized using ultra-high-performance liquid chromatography coupled with quadrupole-Orbitrap mass spectrometry. Network pharmacology, pathway enrichment, and molecular docking were performed to identify potential bioactive targets. Semen quality and sperm kinematics were evaluated longitudinally using computer-assisted sperm analysis. Serum reproductive hormones, testicular histology, and gene and protein expression were assessed to investigate treatment-associated reproductive and molecular changes.

Results: Chemical profiling identified 29 major constituents, including five compounds confirmed using reference standards. Network analysis highlighted steroid hormone-associated targets and phosphatidylinositol 3-kinase/protein kinase B signaling. BJSE significantly improved sperm survival, acrosome integrity, motility, and kinematic parameters while reducing sperm abnormalities, with the strongest responses generally observed at 600 mg/kg (p < 0.05). Significant dose-response trends were identified for major semen quality outcomes. Treatment increased serum luteinizing hormone, follicle-stimulating hormone, and testosterone concentrations during earlier reproductive development, although the direction and magnitude of these hormonal differences changed by week 16; significant between-group differences persisted for FSH, LH, and testosterone. Histological examination suggested improved seminiferous epithelial organization. Testicular analyses demonstrated reduced phosphatase and tensin homolog (PTEN) expression and increased mechanistic target of rapamycin (mTOR), AKT serine/threonine kinase 1 (Akt1), and heat shock protein family A member 2 (HSPA2) expression at both transcript and protein levels (p < 0.05).

Conclusion: BJSE improved semen quality and sperm motility in rabbits in association with coordinated changes in PTEN, mTOR, Akt1, and HSPA2 expression. These findings provide preclinical evidence supporting further investigation of BJSE for male reproductive dysfunction. However, pathway activation and causal mechanisms require confirmation through phosphorylation analyses and targeted functional experiments.

Keywords: BAJISAN extract, male reproductive health, network pharmacology, PTEN/mTOR/Akt1/HSPA2 pathway, rabbit semen quality, sperm motility, testicular function, traditional Chinese medicine.