Sirtuin 1-dependent oleanolic acid protection attenuates oxidative stress-induced granulosa cell dysfunction and ovarian damage

Premature ovarian insufficiency (POI) is characterized by impaired ovarian function before the age of 40 and is closely associated with granulosa cell dysfunction, excessive oxidative stress, inflammation, and accelerated depletion of the ovarian follicular reserve. Oleanolic acid (OA), a naturally occurring pentacyclic triterpenoid found in several medicinal plants, has been reported to exert antioxidant and anti-inflammatory effects in various disease models. Although previous studies have suggested potential protective effects of OA in ovarian granulosa cells under oxidative stress conditions, the molecular mechanisms underlying these effects remain largely unclear. Therefore, this study investigated whether OA protects against oxidative stress-induced granulosa cell dysfunction and ovarian injury through modulation of the SIRT1/NF-κB signaling pathway. Human KGN cells and mouse primary granulosa cells were exposed to hydrogen peroxide (H 2 O 2 ) to establish an in vitro oxidative stress model. Cells were treated with OA, and cell viability, intracellular ROS accumulation, antioxidant enzyme activity, antioxidant protein expression, NF-κB signaling activation, inflammatory cytokine expression, and follicular development-related gene expression were analyzed. To determine whether SIRT1 mediates the effects of OA, SIRT1 knockdown was performed using shRNA. In vivo, female mice were subjected to 3-nitropropionic acid (3-NP)-induced oxidative ovarian injury and treated with OA. Ovarian morphology, follicle counts, serum anti-Müllerian hormone (AMH) levels, and ovarian SIRT1/NF-κB expression were evaluated. OA significantly improved granulosa cell viability under H 2 O 2 -induced oxidative stress by reducing intracellular ROS accumulation and restoring antioxidant defense through increased SOD and catalase activity and upregulation of SOD2, catalase, and GPx-1 expression. OA also inhibited NF-κB p65 nuclear translocation, reduced phosphorylation of NF-κB p65 and IκBα, and decreased the expression of inflammatory mediators including NLRP3, TNF-α, IL-1β, and IL-6. In addition, OA restored the expression of genes associated with granulosa cell function and follicular development. Mechanistically, OA increased SIRT1 expression, whereas SIRT1 knockdown attenuated the inhibitory effect of OA on NF-κB signaling. In the 3-NP-induced ovarian injury model, OA preserved follicular development, reduced follicular atresia, restored serum AMH levels, increased ovarian SIRT1 expression, and suppressed NF-κB activation. OA protects granulosa cells and ovarian tissue from oxidative stress-induced inflammatory injury by restoring SIRT1 expression and suppressing NF-κB signaling in a SIRT1-dependent manner. These findings suggest that OA may help preserve granulosa cell function, support follicular development, and maintain ovarian reserve under oxidative stress-associated ovarian dysfunction.