# Gui Shen Wan ameliorates PCOS-like cellular phenotypes by suppressing TNF-α-mediated inflammation and restoring the PI3K/Akt signaling pathway

## TL;DR

Gui Shen Wan, a traditional Chinese medicine, reduces inflammation and restores signaling in cells modeled after PCOS, offering a potential treatment mechanism.

## Contribution

The study identifies TNF-α suppression and PI3K/Akt pathway restoration as novel mechanisms for Gui Shen Wan's effects in PCOS-like cellular models.

## Key findings

- GSW-medicated serum improved cell viability and reduced apoptosis in a PCOS-like granulosa cell model.
- GSW suppressed inflammatory cytokines TNF-α and IL-6 while enhancing PI3K and Akt phosphorylation.
- Embelin and nobiletin, constituents of GSW, reproduced anti-inflammatory and signaling effects with a synergistic combined effect.

## Abstract

Polycystic ovary syndrome (PCOS) is associated with chronic low-grade inflammation and insulin signaling dysregulation. Gui Shen Wan (GSW), a traditional Chinese medicine formula, has been used empirically for ovarian dysfunction, yet its molecular basis remains incompletely defined. This study aimed to delineate the mechanism by which GSW modulates inflammation-linked insulin signaling in a PCOS-relevant granulosa cell model under metabolic stress, with a focus on the TNF-α/PI3K/Akt axis.

Network pharmacology based on serum-absorbed constituents identified by UPLC–MS/MS was integrated with in vitro validation using dexamethasone- and insulin–challenged human KGN cells to model selected PCOS-relevant cellular phenotypes. The effects of GSW-medicated serum on cell viability, apoptosis, hormone-associated readouts, and inflammatory cytokine production were assessed. PI3K/Akt signaling was examined by Western blotting, and a recombinant TNF-α rescue experiment was performed to probe mechanistic dependence. The effects of candidate constituents (embelin and nobiletin) were further evaluated.

Network pharmacology highlighted TNF and PI3K/Akt signaling as key pathways. In the KGN metabolic stress model, GSW-medicated serum dose-dependently improved cell viability, reduced apoptosis, and attenuated inflammatory cytokine output (TNF-α and IL-6), accompanied by increased phosphorylation of PI3K and Akt. Recombinant TNF-α markedly diminished the protective and signaling-activating effects of GSW, supporting a TNF-α–linked mechanism. Embelin and nobiletin reproduced key anti-inflammatory and signaling effects, and their co-application produced an enhanced combined effect at the tested concentrations.

These findings suggest that GSW mitigates PCOS-like granulosa cell dysfunction under metabolic stress by suppressing TNF-α–associated inflammatory signaling, thereby relieving inhibition of the PI3K/Akt pathway. Given the in vitro scope and the medicated-serum approach, the results should be interpreted as mechanistic insight rather than direct evidence of clinical efficacy, and they provide a rationale for subsequent in vivo validation.