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Reactive oxygen species (ROS) which lead to oxidative stress affect ovarian function. Grape seed extract (GSE) could be proposed as an effective antioxidant, particularly due to its proanthocyanidin content. In this study, we investigated a dose effect (0, 0.01, 0.1, 1, 10, 50, and 100 μg/mL) of GSE and proanthocyanidin B2 (GSPB2) on the ROS content, cell proliferation, cell viability, and steroidogenesis in both primary luteinized granulosa cells (hGC) and the tumor granulosa cell line (KGN). The levels of ROS were measured using ROS-Glo assay. Cell proliferation and viability were evaluated by [3H]-thymidine incorporation and Cell Counting Kit-8 (CCK8) assay, respectively. Steroid secretion was evaluated by radioimmunoassay. We also analyzed the cell cycle component protein level and signaling pathways by immunoblot and the NOX4 mRNA expression by RTqPCR. From 0.1 to 1 μg/mL, GSE and GSBP2 reduced the ROS cell content and the NOX4 mRNA levels, whereas, GSE and GSBP2 increased the ROS cell content from 50 to 100 μM in both hGC and KGN. GSE and GSPB2 treatments at 50 and 100 μg/mL induced a delay in G 1 to S phase cell cycle progression as determined by fluorescence-activated cell sorting. Consequently, they reduced cell growth, cyclin D2 amount, and Akt phosphorylation, and they increased protein levels of p21 and p27 cyclin-dependent kinase inhibitors. These data were also associated with an increase in cell death that could be due to a reduction in Bcl-2-associated death promoter (BAD) phosphorylation and an increase in the cleaved-caspase-3 level. All these negative effects were not observed at lower concentrations of GSE and GSPB2 (0.01 to 10 μg/mL). Interestingly, we found that GSE and GSPB2 treatments (0.1 to 100 μg/mL) improved progesterone and estradiol secretion and this was associated with a higher level of the cholesterol carriers, StAR (steroidogenic acute regulatory protein), CREB (Cyclic adenosine monophosphate Response Element-binding protein), and MAPK ERK1/2 (Mitogen-Activated Protein Kinases Extracellular signal-Regulated Kinases 1/2) phosphorylation in both hGC and KGN cells. Taken together, GSE and GSPB2 (0.1-10 μg/mL) in vitro treatments decrease oxidative stress and increase steroidogenesis without affecting cell proliferation and viability in human granulosa cells.

international journal of molecular sciences

Effects of Grape Seed Extract and Proanthocyanidin B2 on In Vitro Proliferation, Viability, Steroidogenesis, Oxidative Stress, and Cell Signaling in Human Granulosa Cells