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

Abstract

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.

Di-2-ethylhexyl phthalate (DEHP) induces apoptosis of mouse HT22 hippocampal neuronal cells via oxidative stress

“…Oxidative stress has been shown to induce apoptosis in cells such as mouse Leydig cells (Shen et al, 2019), germ cells (Yang et al, 2019; You et al, 2018b), human granulosa cells (Barbe et al, 2019), and human glioblastoma cells (Zappavigna et al, 2019). Oxidative stress has been reported to play an important role in DEHP-induced apoptosis of GC-2spd cells (Fu et al, 2017).…”

The Role of Oxidative Stress and Natural Antioxidants in Ovarian Aging

“…Similarly, in 3-nitropropionic acid (3-NPA)-induced oxidative ovarian damaged mice, PA significantly reduced the percentage of GC apoptosis and atretic follicles in ovarian tissues, increased the expression of antioxidant genes, and inhibited the expression of pro-apoptotic genes ( Zhang JQ et al, 2015 ). In addition, in multiple in vitro studies using animal and human GCs, PA and procyanidin B2 (B type of PA) exert a potent effect in terms of diminishing GC apoptosis and intracellular ROS production ( Zhang et al, 2016 ; Barbe et al, 2019 ). Taken together, the administration of PA has the potential to alleviate ovarian oxidative injury and delay ovarian aging mainly through antioxidant and anti-apoptotic activities.…”

A grape seed extract maternal dietary supplementation improves egg quality and reduces ovarian steroidogenesis without affecting fertility parameters in reproductive hens

“…In basal state (no IGF1 and LH stimulation), progesterone secretion is lower in granulosa cells from hens fed with a diet supplemented with GSE. In a recent paper, we showed a positive effect of GSE treatment on progesterone and oestradiol secretion by cultured human granulosa cells [49]. This discrepancy of the results between the two studies could be explained by the fact that the hen granulosa cells were indirectly exposed to GSE treatment (through maternal diet).…”