Effects and mechanisms of NMB knockdown on cell proliferation, apoptosis and steroidogenesis in porcine ovarian granulosa cells.

Abstract

Neuromedin B (NMB), a neuropeptide, is increasingly recognized as a potential regulator of female reproductive processes. However, its role in porcine ovarian function and follicular development remains unclear. Given the indispensable roles of ovarian granulosa cells (GCs) in follicular growth, endocrine homeostasis, and oocyte maturation, this study investigated the expression pattern of NMB in porcine ovaries and explored the effects of NMB knockdown on GC function and the underlying mechanisms. The results indicated that NMB was expressed in porcine follicles at all development stages (<3 mm, 3-5 mm, >5 mm), with its expression level increasing with follicle diameter; furthermore, NMB was primarily expressed in GCs. Functional assays further revealed that NMB knockdown reduced cell viability and suppressed the expression of Cyclin B1 and PCNA; induced cell apoptosis by upregulating Bax and Caspase-3 expression while downregulating Bcl-2 expression; impaired E2 and P4 synthesis by reducing STAR, CYP11A1, HSD3B1, and CYP19A1 expression. Mechanistically, NMB knockdown compromised mitochondrial Ca2+ uptake and disrupted PKA/CREB signaling, as reflected by decreased expression levels of total and phosphorylated PKA and CREB proteins. Importantly, the functional impairments induced by NMB knockdown were alleviated by exogenous NMB supplementation. Collectively, these findings suggest that NMB may serve as an important regulator of cell viability, apoptosis and steroidogenesis through mitochondrial Ca2+ uptake and the PKA/CREB axis in GCs, thereby providing new mechanistic insights into follicular development and potential targets for improving porcine reproductive efficiency.