Rhein attenuates oxidative stress-induced injury in human ovarian granulosa cells by inhibiting the NF-κB-TNF-α/IL-6/PTGS2 inflammatory axis.

Polycystic ovary syndrome with insulin resistance (PCOS-IR) features oxidative stress, chronic inflammation, and lipid dysregulation that impair granulosa cells. This study evaluated whether Rhein mitigates oxidative/inflammatory injury in granulosa cells under PCOS-like conditions and explored its putative molecular basis. A PCOS-like metabolic injury model was established in human granulosa-like KGN cells using dihydrotestosterone, oleic acid, and palmitic acid. Reactive oxygen species (ROS), malondialdehyde (MDA), mitochondrial membrane potential (ΔΨm), and lipid accumulation were measured by DCFH-DA fluorescence, MDA assay, JC-1 staining, and Oil Red O, respectively. Western blotting assessed NF-κB, TNF-α, IL-6, and PTGS2. Network pharmacology, molecular docking, 100-ns molecular dynamics simulations were used to prioritize targets and evaluate binding stability. KEGG enrichment was used to infer related pathways. Rhein (10-20 μM) reduced ROS and MDA, partially restored ΔΨm, and lessened lipid accumulation versus model controls. Docking and MD results indicated stable binding of Rhein to NF-κB, TNF-α, IL-6, and PTGS2. Enrichment analysis suggested that the NF-κB-TNF-α/IL-6/PTGS2 inflammatory axis may constitute the core regulatory axis. Western blotting further confirmed downregulation of PTGS2, IL-6, TNF-α, and NF-κB protein expression. Rhein attenuates oxidative and inflammatory stress in KGN cells under PCOS-like metabolic injury, likely via modulation of the NF-κB-TNF-α/IL-6/PTGS2 inflammatory axis. These in vitro findings support Rhein as a pharmacological candidate for further investigation in reproductive endocrine disorders associated with insulin resistance.