Abstract
Sulforaphane (SFN) has been considered as an indirect antioxidant and potential inducer of the Nrf2-ARE pathway. This study was conducted to investigate the protective role of SFN against oxidative stress in bovine granulosa cells (GCs). GCs were collected from antral follicles (4–8 mm) and cultured according to the experimental design where group 1 = control, group 2 = treated with SFN, group 3 = treated with hydrogen peroxide (H2O2), group 4 = pretreated with SFN and then with H2O2 (protective) and group 5 = treated with H2O2 followed by SFN treatment (rescuing). Results showed that SFN pretreatment significantly increases cell viability and reduces cytotoxicity in GCs under oxidative stress. Following H2O2 exposure, expression of NRF2 was found to be significantly increased (p < 0.05) in SFN-pretreated cells, while no significant differences were observed between group 3 and group 5, although the expression was significantly increased compared to the control group. Moreover, the relative abundance of the NRF2 downstream target antioxidant genes (CAT, PRDX1, SOD1 and TXN1) were higher (fold change ranged from 7 to 14, p < 0.05) in sulforaphane pretreated GCs. Low level of ROS and lipid accumulation and higher mitochondrial activity were observed in GCs pretreated with SFN, whereas no such changes were observed in GCs treated with SFN after exposure to oxidative stress (group 5). Thus, we suggest that SFN pretreatment effectively protects GCs against oxidative damage through the activation of the NRF2-ARE pathway, whereas addition of SFN during oxidative insult failed to rescue GCs.
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06 November 2018
There is an error in the original publication of this paper. Figures 1-6 were shown in the wrong version, thus corrected figures provided were in this article.
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The authors would like to thank Mr. Heinz Biörnsen and Mr. Mahmut Kaliber for their assistance during sample collection.
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The original version of this article was revised: There is an error in the Original Publication of this paper. Figures 1-6 were unupdated, thus corrected figures provided below:
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Fig. S1
The purity of GCs. GCs were collected according to the protocol mentioned in the “Materials and methods” section. Specific primers targeting FSHR, CYP17A1 and GAPDH were designed using primer3web version 4.0.4 (http://bioinfo.ut.ee/primer3/). Primer sequences are available in Supplementary Table 1. The polymerase chain reaction (PCR) was set in a thermocycler with thermal cycle conditions of pre-incubation at 95°C for 5 min, 35 cycles of denaturation at 95°C for 30 s, annealing at 55°C FSHR, 56°C GAPDH and 57°C CYP17A1 for 30 s, extension at 72°C for 1 min and a final extension at 72°C for 10 min. The corresponding PCR products were mixed with loading buffer and loaded into 2% agarose gel stained with Ethidium bromide (EtBr) and visualized using Chemidoc XRS (Bio-Rad) instrument (BIO-RAD, München, Germany) to detect the presence or absence of gene-specific bands. The results showed that the GC specific marker gene FSHR was detected at a higher level as indicated by a strong band, while no expression was detected for the CYP17A1 gene in GCs. These results confirmed that the isolation procedure of GCs was completely free of contamination from thecal cells. (PDF 117 kb)
Fig. S2
Expression of NRF2, KEAP1 and CASP3 in response to 10 μM SFN at different time points. Approximately 6 × 105 viable pure GCs were seeded in each well of a 6-well plate and grown until 40-50% confluence. Cells were then treated with 10 μM SFN for different time points (6 h, 12 h, 24 and 48 h) to identify the appropriate exposure time of SFN. We checked three different genes; NRF2 (inducer of the NRF2 antioxidant pathway), KEAP1 (negative controller of NRF2) and CASP3 (an indicator of apoptosis induction). The results showed that the expression of NRF2 was significantly higher in all exposure time points compared to the 6 h time point, however, the highest expression was observed in the 12 h and 24 h time points. On the other hand, the expression of KEAP1 was more or less similar across the time points. In addition, the expression of CASP3 was similar to control at 12 h and 24 h, however, it significantly increased at the 48 h time point. The expression analysis of these genes revealed that both the 12 h and 24 h exposure times are suitable for activating the antioxidant defense system without creating any damage to GCs. As most of the mammalian cell cycle is completed within 20-24 h, we decided to expose the GCs against 10 μM SFN for 24 h. (PDF 154 kb)
Fig. S3
Normality test of qPCR data. Normality of the qPCR data (Ct value) was tested using Microsoft Excel 2016. For this, all Ct values were organized in ascending order (smallest to largest). Afterwards, the cumulative distribution factor (CDF) was calculated using the 1 / (2*total count) formula for the first number, CDF1+2 / (2*total count) for the second number, CDF2+2 / (2*total count) for the third number and so on. Expected value was calculated using the NORM.INV(CDF, mean, standard deviation) formula and Z-score was calculated using the NORM.S.INV(CDF) formula. Normal probability plot for the Ct values shows that the data appear to be very close to being normally distributed. The actual Ct values (red) match very closely with the normally distributed (blue) Ct values that clearly indicate the data are normally distributed. (PDF 161 kb)
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Sohel, M.M.H., Amin, A., Prastowo, S. et al. Sulforaphane protects granulosa cells against oxidative stress via activation of NRF2-ARE pathway. Cell Tissue Res 374, 629–641 (2018). https://doi.org/10.1007/s00441-018-2877-z
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DOI: https://doi.org/10.1007/s00441-018-2877-z