TNF-α/NF-κB activation and PI3K/AKT suppression contribute to radiation-induced dysfunction in KGN granulosa-like cells

Background Radiation-induced ovarian dysfunction has been extensively documented; however, the molecular pathways responsible for granulosa cell injury remain largely unresolved. We hypothesized that radiation-induced dysfunction in granulosa-like cells involves TNF-α/NF-κB inflammatory activation and PI3K/AKT signaling suppression and examined this hypothesis using pharmacological modulation of these pathways. Methods Human KGN granulosa-like cells were exposed to different radiation doses to establish a dose-dependent injury model. Cell viability, apoptosis, intracellular reactive oxygen species production, hormone secretion, and granulosa cell functional markers were assessed. TNF-α/NF-κB inflammatory signaling and PI3K/AKT signaling were examined using western blotting, RT-qPCR, ELISA, immunofluorescence, and functional assays. TNF-α blockade, recombinant TNF-α treatment, and PI3K inhibition were further used to evaluate the functional involvement of TNF-α signaling and AKT-dependent survival signaling. Results At 24 h, irradiation also dose-dependently increased apoptosis and intracellular reactive oxygen species accumulation while downregulating AMH, FSHR, and CYP19A1 expression and shifting apoptosis-related protein expression toward a pro-apoptotic pattern. In the 4 Gy mechanistic model, irradiation increased TNF-α expression, NF-κB p65 phosphorylation and nuclear translocation, and TNF-α and IL-6 production, while reducing PI3K and AKT phosphorylation. Inhibition of TNF-α markedly ameliorated irradiation-induced cellular dysfunction. Specifically, this intervention improved cell survival, preserved mitochondrial membrane potential, recovered the expression of granulosa cell–associated functional markers, enhanced E2 production, and promoted AKT activation. It also shifted the apoptotic protein profile toward cell survival by suppressing Bax and cleaved caspase-3 levels while upregulating Bcl-2 expression. In contrast, PI3K inhibition aggravated radiation-induced functional impairment and significantly weakened the protective effects of TNF-α blockade, supporting the partial involvement of PI3K/AKT-dependent anti-apoptotic signaling. Conclusion TNF-α/NF-κB inflammatory activation and PI3K/AKT signaling suppression contribute to radiation-induced dysfunction in KGN granulosa-like cells. TNF-α blockade may exert protective effects by partially restoring AKT-dependent anti-apoptotic signaling. These findings provide in vitro mechanistic evidence supporting TNF-α signaling as a potential target for radiation-induced granulosa cell injury, although validation in primary granulosa cells and in vivo models is required.