# Oviductus Ranae alleviates D-galactose-induced ovarian aging by inhibiting ferroptosis and regulating the GPX4/ACSL4 pathway

## TL;DR

Oviductus Ranae, a traditional Chinese medicine, helps prevent ovarian aging by reducing cell death called ferroptosis and regulating key proteins like GPX4 and ACSL4.

## Contribution

This study reveals that Oviductus Ranae alleviates ovarian aging by inhibiting ferroptosis via the GPX4/ACSL4 pathway in rat models.

## Key findings

- Oviductus Ranae improved ovarian follicular development and hormone levels in aging rats.
- OR inhibited ferroptosis by upregulating GPX4 and downregulating ACSL4, reducing iron and lipid peroxidation.
- RNA sequencing showed OR affected genes involved in ovarian steroidogenesis and ferroptosis pathways.

## Abstract

Ovarian dysfunction caused by aging restricts female reproductive capacity and causes age-related health problems. Ferroptosis is an important mode of cell death during accelerated aging. Oviductus Ranae (OR), a traditional Chinese medicine, has been used to treat ovarian age-related diseases in women. However, the mechanisms through which OR mitigates ovarian aging, especially ferroptosis regulation, remain unclear. This study investigated the pharmacological effects and mechanisms of OR for ovarian aging in rats.

Sprague–Dawley female rats were divided into six groups: aging group induced with D-galactose; experiment groups treated with OR at low, middle, and high concentrations; positive control treated with estradiol valerate; and control group with no treatment. After 42 d, ovarian tissue and serum were collected for biochemical determination, hematoxylin and eosin staining, immunohistochemistry, enzyme-linked immunosorbent assay, reverse transcription-quantitative polymerase chain reaction, western blotting, and RNA sequencing analysis. To elucidate the mechanism underlying the effects of OR on ferroptosis in ovarian granulosa cells (OGCs), in vitro experiments were conducted.

Treatment with OR improved ovarian follicular development and serum hormone levels, reduced iron deposition, enhanced cell proliferation, and inhibited apoptosis in D-galactose-induced ovarian aging rats. Ferroptosis was alleviated in the ovaries and serum by elevated glutathione and decreased lipid peroxidation production. RNA-seq showed that OR induced changes in 2,768 genes that were involved in ovarian steroidogenesis, glutathione metabolism, lipid metabolism, and ferroptosis pathways. Notably, OR downregulated ACSL4, LPCAT3, and ALOX12 mRNA and protein expressions, while upregulating FTH1, FTL, SLC7A11, GSS, and GPX4, which serve as critical regulators in ferroptosis. OGCs pretreated with OR-containing serum before erastin exposure exhibited enhanced viability, reduced ferrous iron, total iron, and lipid peroxidation levels, improved antioxidant activity, and stabilized mitochondrial function, indicating effective ferroptosis inhibition. Furthermore, OR-containing serum pretreatment inhibited ferroptosis in OGCs by regulating the GPX4/ACSL4 axis.

OR resists ovarian damage and prevents aging through antiferroptosis mechanisms by regulating the GPX4/ACSL4 axis in OGCs and aging rats. These results highlight the potential of OR as a therapeutic agent for the prevention and treatment of ovarian aging.

The online version contains supplementary material available at 10.1186/s13048-025-01857-2.