Targeting Oxidative Stress and Glycolytic Dysfunction: Nicotinamide Mononucleotide as a Promising Strategy to Prevent Ionizing Radiation Induced Ovarian Failure

Objective With advances in cancer diagnosis and therapy, survival rates of female patients have markedly improved. However, radiotherapy, a cornerstone in the treatment of colorectal and cervical cancers, poses a high risk of ovarian damage since the ovary is extremely sensitive to ionizing radiation(IR). Even low doses of IR may lead to infertility and endocrine dysfunction, even induce premature ovarian insufficiency (POI). Current protective strategies, such as ovarian transposition, gamete/ovarian tissue cryopreservation, hormone replacement therapy, or GnRH-a injections, are limited by technical, ethical, or clinical feasibility issues. Nicotinamide mononucleotide(NMN), a direct precursor of NAD⁺, has been validated to alleviate oxidative stress, restore energy metabolism, and protect ovarian function with high safety. In this study we aimed to systematically evaluate the protective role and mechanisms of NMN against IR induced ovarian dysfunction. Methods and Results This research integrated in vivo mouse experiments, ovarian transcriptomics, and in vitro granulosa cell validation to dissect the protective mechanisms of NMN. Animal studies: C57BL/6J female mice were subjected to 5Gy Cs-137 γ-irradiation to induce ovarian insufficiency. IR led to reduced ovarian size, follicular atresia, AMH↓/FSH↑, disrupted estrous cycles, NAD⁺ depletion, increased oxidative stress (SOD↓, MDA↑), impaired glycolysis (HK2, PKM2, LDHA↓), and apoptosis activation (BAX, Cleaved-Casp3↑). Oral NMN (500mg/kg/day) significantly reversed these phenotypes, restoring ovarian morphology, hormone levels, and metabolic balance, with no adverse effects in healthy mice. Transcriptomic analysis: RNA-seq revealed extensive DEGs in IR-exposed ovaries: upregulated genes enriched in DNA damage response, oxidative stress, and apoptosis pathways; downregulated genes involved in glycolysis, energy metabolism, and ovarian maintenance. NMN supplementation exerted a bidirectional correction effect, suppressing stress/apoptosis-related genes while restoring glycolysis and mitochondrial energy genes. KEGG enrichment highlighted overlapping pathways(oxidative stress, glycolysis, apoptosis, PI3K/AKT/HIF-1α), confirming the role of NMN in metabolic reprogramming and ovarian protection. Cellular validation: An H₂O₂ induced oxidative stress model in KGN cells reproduced IR-associated phenotypes(NAD⁺ depletion, ROS↑, Δψm loss, glycolysis inhibition, AMH/E2↓). NMN pretreatment improved antioxidant enzyme activity(SOD, CAT, T-GSH/GSSG), restored glycolytic flux(HK2, PKM2, LDHA, lactate/pyruvate), stabilized mitochondrial function, and enhanced hormone secretion. Inhibitors LY294002 and MK2206 abrogated NMN’s effects, verifying that PI3K/AKT/HIF-1α signaling is essential for NMN mediated ovarian protection. Conclusion The study indicated that NMN effectively mitigates IR induced ovarian insufficiency by restoring NAD⁺, reprogramming glycolysis, reducing oxidative stress, stabilizing mitochondrial function, and modulating the PI3K/AKT/HIF-1α axis. These findings highlight NMN is a safe and promising strategy for protecting female cancer patients’ ovarian function undergoing radiotherapy. Keywords: Ionizing radiation; Premature ovarian insufficiency; NMN; NAD⁺; Oxidative stress; Glycolysis; PI3K/AKT/HIF-1α