Abstract

Oxidative stress is a major driver of granulosa cell (GC) dysfunction and disrupted steroidogenesis. However, the mechanisms underlying oxidative stress–induced progesterone synthesis impairment and potential intervention strategies remain inadequately defined. In this study, RNA-seq re-analysis revealed that hydrogen peroxide (H₂O₂) markedly altered gene expression in mouse granulosa cells (mGCs), particularly affecting pathways related to progesterone biosynthesis. In vitro experiments confirmed that H₂O₂ suppresses progesterone production via downregulation of Sirtuin 1 (SIRT1). Conversely, β-nicotinamide mononucleotide (NMN) supplementation increased intracellular NAD⁺ levels, and upregulated SIRT1 and Nicotinamide phosphoribosyl transferase (NAMPT) expression. This regulation enhanced cell proliferation, stimulated the expression of progesterone biosynthetic enzymes, and promoted NAMPT transcriptional activity. Notably, the beneficial effects of NMN were diminished upon SIRT1 inhibition. Similarly, inhibition of NAMPT attenuated the positive regulatory effect of NMN, leading to a marked reduction in SIRT1 levels and a consequent suppression of progesterone secretion. Thus, SIRT1 activation induces NAMPT expression, which in turn feeds back to enhance SIRT1 activity and support progesterone biosynthesis. Together, these findings suggest that NMN alleviates H₂O₂-induced progesterone synthesis disruption in mGCs through activation of the NAD⁺/SIRT1/NAMPT axis, providing a potential therapeutic strategy for oxidative stress–related reproductive dysfunction.