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Open AccessArticle

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

by Alix Barbe 1,2,3,4, Christelle Ramé 1,2,3,4, Namya Mellouk 1,2,3,4, Anthony Estienne 1,2,3,4, Alice Bongrani 1,2,3,4, Adeline Brossaud 1,2,3,4, Antonella Riva 5, Fabrice Guérif 6, Pascal Froment 1,2,3,4 and Joëlle Dupont 1,2,3,4,*
1
INRA, UMR 85 Physiologie de la Reproduction et des Comportements, F-37380 Nouzilly, France
2
CNRS, UMR 7247 Physiologie de la Reproduction et des Comportements, F-37380 Nouzilly, France
3
Department of Animal Physiology, University of François Rabelais, F-37041 Tours, France
4
Institut Français du Cheval et de l'Equitation, F-37380 Nouzilly, France
5
INDENA S.p.A, 12-20139 Milan, Italy
6
Service de Médecine et Biologie de la Reproduction, Hospital of Tours, F-37044 Tours, France
*
Author to whom correspondence should be addressed.
Int. J. Mol. Sci. 2019, 20(17), 4215; https://doi.org/10.3390/ijms20174215
Received: 19 June 2019 / Revised: 20 August 2019 / Accepted: 24 August 2019 / Published: 28 August 2019
(This article belongs to the Section Bioactives and Nutraceuticals)
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 G1 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. View Full-Text
Keywords: human granulosa cells; grape seed extract; oxidative stress; steroidogenesis; cell signaling; apoptosis human granulosa cells; grape seed extract; oxidative stress; steroidogenesis; cell signaling; apoptosis
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Barbe, A.; Ramé, C.; Mellouk, N.; Estienne, A.; Bongrani, A.; Brossaud, A.; Riva, A.; Guérif, F.; Froment, P.; Dupont, J. Effects of Grape Seed Extract and Proanthocyanidin B2 on In Vitro Proliferation, Viability, Steroidogenesis, Oxidative Stress, and Cell Signaling in Human Granulosa Cells. Int. J. Mol. Sci. 2019, 20, 4215.

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