ShenRongGuBenHuanShao Pill ameliorates oligoasthenospermia by alleviating oxidative stress and DNA damage via activation of the PI3K/Akt/mTOR pathway

Ethnopharmacological relevance: ShenRongGuBenHuanShao Pill (SRGBHSP) is a classic kidney-tonifying traditional Chinese medicine formula with a clinical history of nearly a century for treating oligoasthenospermia (OA). However, systematic investigations into its therapeutic effects and underlying mechanisms remain lacking.

Aims of study: This study aims to investigate the therapeutic effects of SRGBHSP on OA and to elucidate its underlying mechanisms.

Methods: Network pharmacology was employed to predict the potential targets of SRGBHSP for treating OA. Subsequently, H₂O₂-induced GC-1 cells injury model and cyclophosphamide-induced OA rats' model were established. The therapeutic mechanism of SRGBHSP was then validated using a specific inhibitor. Finally, a multi-omics analysis was conducted to provide a systems-level understanding of the underlying mechanism.

Results: Network pharmacology analysis identified 1276 potential targets of SRGBHSP for OA. Mechanistically, SRGBHSP alleviated oxidative stress, mitochondrial dysfunction, and DNA damage in GC-1 cells by activating the PI3K/Akt/mTOR pathway. In OA rats, SRGBHSP administration significantly improved semen quality, restored testicular/epididymal histology, and normalized hepatorenal function and hormone levels. The reversal of these benefits by the PI3K Inhibitor LY294002 confirmed the functional necessity of this pathway. Integrative multi-omics analyses unveiled a novel systemic dimension to its action. SRGBHSP restructured gut microbiota function (enhancing carbohydrate/amino acid metabolism) and reprogrammed host metabolism, particularly the arachidonic acid pathway. Correlation networks linked these gut and metabolic changes to the activation of testicular signaling, supporting the operation of a "gut-testis" axis. This axis involved modulation of the AMPK/mTOR/PPAR transcriptional program and the cAMP/PKA/PI3K/cGMP/PKG protein network. In conclusion, SRGBHSP ameliorates OA not only by direct activation of the PI3K/Akt/mTOR pathway but also through a multi-organ mechanism involving the gut-testis axis, providing a comprehensive mechanistic framework for its therapeutic efficacy.

Conclusions: This study concludes that the therapeutic effect of SRGBHSP on OA is mediated through the activation of the PI3K/Akt/mTOR signaling pathway. Beyond this core pathway, SRGBHSP orchestrates a systemic response that involves maintaining gut-testis axis homeostasis and modulating gene expression at both the transcriptional and translational levels.

Metabolomics; Oligoasthenospermia; PI3K/Akt/mTOR signaling pathway; Proteomics; ShenRongGuBenHuanShao pill; Transcriptomics.