{"paper_id":"9bf3e862-6e9c-4318-830e-2a98d7e2350d","body_text":"Oxidative stress (OS) occurs when there is a disruption in redox homeostasis, a state which\nrefers to the natural capacity of a cell to handle the challenges that produce electrophile\nmolecules such as reactive oxygen species (ROS) ( 1 ). Indeed, excessive ROS production takes\nplace during OS and results in damage to cellular components including DNA, proteins, and\nlipids. Hydrogen peroxide (H 2  O 2 ), superoxide (•O 2 ), and\nhydroxyl radical (•OH), the most important types of ROS, are produced as byproducts during\ncellular metabolism activities ( 2 ). These factors participate in the modulation of molecular\nand biological mechanisms in reproduction ( 3 ). However, excessive production of ROS leads to\nthe disruption of endogenous redox homeostasis and consequently activation of cell death\nmechanisms such as apoptosis ( 4 ). Therefore, ROS are considered as a double-edged sword ( 5 ).\nIn this manner, an adapted antioxidant system conducts the elimination of excessive ROS and\nmaintains the balance between ROS production and cellular antioxidant capacity, which\nfinally protects the cell from the damages of OS ( 1 ).\nIt has been revealed that ROS plays a regulatory role\nin the female reproductive system, especially during\nfolliculogenesis, steroidogenesis, oocyte maturation, and\nluteolysis ( 6 ). However, when the level of ROS exceeds\nthe normal value, as describes above as an OS condition,\nseveral reproductive disorders may occur including\npolycystic ovary syndrome (PCOS) ( 7 ). Hence, there is a link between OS and pathophysiology of disorders\nof the female reproductive system ( 6 ). Granulosa cells\n(GCs), somatic steroidogenic cells surrounding the\noocyte, play a vital role in oocyte maturation ( 8 ). These\ncells produce nutrients and growth factors needed for the\noocyte maturation and have a complicated antioxidant\nsystem preserving oocytes from the damages of OS ( 9 ).\nNevertheless, a high level of ROS production caused by\ndisruption of the intrinsic antioxidant system in GCs,\nleads to apoptosis in GCs and contributes to poor oocyte\nmaturation especially in the cases of PCOS ( 10 ).\nKelch‑like ECH‑associated protein 1 (KEAP1)‑nuclear\nfactor erythroid 2‑related factor 2 (NRF2)‑antioxidant\nresponse element (ARE) signaling pathway is one of\nthe most important mechanisms in OS regulation ( 11 ).\nNRF2, is an essential transcription factor for the\nexpression of different antioxidant genes and thus the\nprimary cellular mean against OS ( 12 ). Under normal\nconditions, KEAP1 molecule deactivates NRF2 by\nbinding to it and sequestering it from nuclei, where\nits target genes reside ( 11 ). During OS, oxidation\nof cysteine residue inactivates KEAP1, leading\nto activation and translocation of NRF2 into the\nnucleus where it binds to ARE promoter region ( 13 ).\nConsequently, NRF2 enhances cellular antioxidant\ncapacity by upregulating the gene expression of\ndownstream antioxidant enzymes such as superoxide\ndismutase (SOD) and catalase (CAT), the first-line\ndefense enzymes in the elimination process of ROS\n( 14 ). It has been reported that NRF2-ARE pathway\nplays an essential role against OS in murine, bovine,\nand human GCs ( 15 - 18 ). Therefore, induction of this\nsignaling pathway using exogenous activators may\npotentially be a beneficial route for the management of\nROS generation in GCs.\nSulforaphane (SFN) is an organic isothiocyanate with\nantioxidant activity found in the Brassicaceae family, e.g.\nbroccoli ( 19 ). SFN has a lipophilic nature and translocates\ninto cells by passive diffusion due to its low molecular\nweight, and has various impacts such as antioxidant, anti-apoptotic, and anti-inflammatory effects ( 20 ). Several\n in vivo  and in vitro studies have indicated that SFN can\ninduce NRF2 pathway and promotes the downstream\nantioxidant genes ( 16 ,  21 ). It has also been reported that\nSFN improves cell viability and decreases the cytotoxicity\nin bovine OS-induces GCs ( 16 ). Nevertheless, there is no\nevidence of its protective effects on human OS-induced\nGCs. This study aimed to explore the effects of SFN in\nactivation of NRF2-ARE pathway and its downstream\nantioxidant enzymes, SOD and CAT, in OS-induced\ncultured human GCs.\n\nThis experimental study was conducted on GCs of\nhealthy women aged from 20-38 years old referred to\nthe Infertility Department of Shariati Hospital, Tehran,\nIran. All the participants had a normal ovulatory function\nand menstrual cycle (25-35 days), without a history of\nPCOS, endometriosis, hirsutism, menstrual disorders,\nhyperprolactinemia, or hormonal therapy. The GCs\nwere isolated from follicular fluid during ovum pickup.\nThe Research Ethics Committee of Tehran University\nof Medical Sciences approved the study (IR.TUMS.\nMEDICINE.REC.1397.230) and informed consent was\nobtained from all participants. A total of 12 individuals\nparticipated in the study.\nFor ovarian hyperstimulation, gonadotropin-releasing hormone (GnRH) antagonist protocol\nwas performed in all participants. Briefly, recombinant follicle stimulating hormone\n(rFSH,150-225 IU, Gonal-F ® , Merck Serono SA, Switzerland) was administered on\nday 3 from the beginning of the menstrual cycle and sustained until at least 2 follicles\nachieve the size of 14 to 15 mm. To assess the follicular growth, transvaginal\nultrasonography was performed. Next, GnRH antagonist (0.25 mg, Cetrotide ® ,\nMerck Serono SA, Switzerland) was administered and continued until the dominant follicles\nachieve the size of 18 mm. Human chorionic gonadotropin (hCG,  10 ,000 IU,\nChoriomon ® , IBSA, Lugano, Switzerland) was utilized for the final maturation\nof the oocytes. After 36 hours of hCG administration, transvaginal ultrasound-guided\nfollicular aspiration was performed for oocyte retrieval. The follicles with a size above\n18 mm were used for the isolation of GCs.\nHuman GCs were isolated and purified as previously\ndescribed ( 22 ), which provides the highest percentage\nyield of live purified GCs using density gradient\ncentrifugation. In brief, the isolated follicular fluids\nwere pooled from differentpatients to reduce the\nvariability of individual samples. Then, 3000 rpm\ncentrifugation was performed for 10 minutes to remove\nthe supernatant and the pellet was resuspended in 2.5\nml of Dulbecco’s Modified Eagle Medium: Nutrient\nMixture F-12 (DMEM/F-12, Gibco, USA). GCs\nwere isolated using Ficoll-Paque Plus solution (GE\nHealthcare, United Kingdom), followed by another\ncentrifugation at 3000 rpm for 10 min. Next, GCs were\nwashed and cultured in a complete medium containing\nDMEM/F-12 supplemented with 10% heat-inactivated\nfetal bovine serum (FBS, Gibco, USA), 100 U/ml of\npenicillin (Gibco, USA), 100 mg/ml of (Gibco, USA),\n2 mmol/l of glutmax (Sigma-Aldrich, USA), and 2 mg/\nml of amphotericin B (PAN Biotech, Germany) at 37°C\nand 5% CO 2 \n. The medium was changed after 48 hours.\nAfter 48 hours of culture, the cells were divided into 4\nexperimental groups:\nGroup 1: GCs were cultured for 24 hours in the presence of dimethyl sulfoxide (DMSO) as the vehicle of SFN\nGroup 2: GCs were cultured for 22 hours and then\nexposed to 200 µM of H 2  \nO 2 \nfor another 2 hours to induce\nOS\nGroup 3: GCs were cultured for 24 hours in the presence\nof SFN\nGroup 4: GCs pretreated with SFN for 22 hours and\nthen exposed to 200 µM of H 2  \nO 2 \nfor another 2 hours\nThe dose and duration of H 2  \nO 2 \nexposure were selected\naccording to our recently published model for OS\ninduction in human GCs ( 23 ).\n3 - ( 4 ,  5 - d i m e t h y l t h i a z o l-  2 - y l) - 2 , 5-diphenyltetrazolium\nbromide (MTT) (Alfa Aesar by Thermo Fisher Scientific, England) assay was conducted to\nevaluate the cytotoxicity of SFN (S4441, Sigma-Aldrich, USA) and to determine its optimal\ndose for treating cells. First, SFN (stock concentration: 5 mg/mL) was diluted in DMSO\n(Sigma-Aldrich, USA) and different concentrations of it were prepared by diluting in the\nculture medium including 5, 10, 15, 20, 25, and 30 µM. Then, GCs were seeded on a 96-well\nplate at a density of 1×10 4  cells per well and exposed to the mentioned doses\nof SFN for 24 hours. Furthermore, we also pretreated GCs with the mentioned concentrations\nof SFN for 22 hours and then exposed them to 200 µM of H 2   O 2  for 2\nhours to determine an optimal dose of SFN for protecting cells against H 2  \nO 2  induced OS. Next, 0.5 mg/ml of MTT solution was added to the wells and the\ncells were incubated at 37°C for 4 hours in dark. Then the produced colorful crystals were\ndissolved in DMSO and, the optical density (OD) was measured at 570 nm wavelength using a\nmicroplate reader (BioTek, USA).\nThe intracellular ROS levels of experimental groups were measured by flow cytometric\nanalysis using 2ˊ-7ˊ-Dichlorodihydrofluorescein diacetate (DCFH-DA, Sigma-Aldrich, USA)\nfluorescent probe according to the manufacturer’s protocol. In detail, GCs were seeded at\na density of 2×10 5  cells per well in a 6 well plate and treated as described\nabove. Next, the cells were incubated with DCFH-DA at a concentration of 1 µM for 30\nminutes at 37°C and then washed and resuspended in phosphate-buffered saline (PBS). The\nfluorescence intensity was measured in the FL-1 channel at a wavelength between 500 and\n530 nm by BD FACScan flow cytometry (Becton Dickinson, USA). About 10,000 cells were\nanalyzed for each group. Data were analyzed using Flowjo software (Flowjo 7.6.1).\nThe Annexin V-fluorescein isothiocyanate (FITC)/ propidium iodide (PI) apoptosis\ndetection kit (Thermo Fisher Scientific, USA) was used as per the manufacturers’ protocol\nto define the total apoptotic cells and distinguish apoptosis from necrosis. In brief, GCs\nwere seeded at a density of 2×10 5  cells per well in a 6-well plate in the\ndefined groups. Then, cells were suspended in 1X annexin-binding buffer and, incubated\nwith Annexin V-FITC and PI at room temperature for 15 minutes in dark. The fluorescence\nemission was evaluated using BD FACScan flow cytometry (Becton Dickinson, USA) and the\nresults were analyzed using Flowjo software (Flowjo 7.6.1). Briefly, the following\ncriteria were used for interpretation of the data obtained from flow cytometry:\nQ1 area: Annexin V-negative, PI-positive GCs are\nnecrotic GCs,\nQ2 area: Annexin V-positive, PI-positive GCs are late\napoptotic GCs,\nQ3 area: Annexin V-positive, PI-negative GCs are early\napoptotic GCs,\nQ4 area: Annexin V-negative, PI-negative GCs are\nviable GCs.\nThe total RNA was extracted by TRIzol reagent (Life Technologies, USA) based on the\nmanufacturer’s instructions. Then, cDNA was synthesized using 1 μg of total RNA by a\nFirst-Strand cDNA Synthesis Kit (Thermo Fisher Scientific, USA). Quantitative real-time\npolymerase chain reaction (PCR) was conducted to investigate the levels of target mRNAs\nusing a RealQ Plus Master Mix Green (Ampliqon, Denmark) by Applied Biosystems StepOne\nreal-time PCR (Applied Biosystems, USA). The 2 -ΔΔCt  method was applied for data\nanalysis.  GAPDH  was considered as a housekeeping gene. The specific\nprimers for target genes are provided in Table 1.\nSpecific primers used for quantitative real-time polymerase\nchain reaction\nTotal cellular proteins were extracted using a\nReadyPrep™ Protein Extraction Kit (Bio-Rad, USA)\nusing the manufacturer’s protocol. Bradford reagent\n(Bio-Rad, USA) was applied to estimate the protein\nconcentration. Next, lysates (20 μg of protein) were\nloaded and resolved on 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and\ntransferred to a polyvinylidene difluoride (PVDF)\nmembranes (Bio-Rad, USA). After blocking in a\nsolution of 3% skim milk, the membranes were\nincubated with each primary antibody at 4°C overnight.\nThe used antibodies were as follow: i. Antibody against\nSOD (ab16831, Abcam, UK), ii. Antibody against CAT\n(ab16731, Abcam, UK), iii. Antibody against NRF2\n(ab137550, Abcam, UK), and iv. Antibody against\nβ-actin (ab8226, Abcam, UK). After washing the blots,\nincubation with corresponding horseradish peroxidase-conjugated secondary antibodies (Abcam, UK) was\nperformed at room temperature for 1 hour. Signals were\ndetected using an enhanced chemiluminescent (ECL)\ndetection system (Amersham Pharmacia Biotech,\nUK). β-actin was used to normalize the relative band\ndensities of SOD, CAT, and NRF2. Data were analyzed\nusing ImageJ software (V1.48, NIH, USA).\nAll data were presented as mean values ± standard\ndeviation (SD). The normality of the data was checked\nby the Kolmogorov-Smirnov test. The comparisons\nof the groups’ means were conducted by One-way\nANOVA and the suitable post-hoc test. The SPSS\nv.19 software (Chicago, IL, USA) was used for the\nstatistical analysis of the data. P<0.05 was considered\nstatistically significant.\n\nTo discover an optimal concentration of SFN for\nprotecting cells against OS-induced cytotoxicity,\nwe pretreated GCs with different concentrations of\nSFN, and cell viability was measured by MTT assay.\nThe results revealed a significant reduction in the\nviability of GCs at concentrations of ≥ 25 µM of SFN\nin comparison to the control group after 24 hours\n(P≤0.01,  Fig .1A ). Hence, GC cells were treated with\n5, 10, 15, and 20 µM of SFN for 22 hours and then\nexposed to 200 µM of H 2  \nO 2 \nfor 2 hours.\nResults showed that SFN pretreatment can protect GCs\nfrom H 2  \nO 2 \ncytotoxicity (P<0.001,  Fig .1B ). We chose\n10 µM as the optimal protective concentration of SFN\nagainst H 2  \nO 2 \n-induced OS in GCs to be used in the next\nexperiments.\nSFN cytotoxicity evaluation and its protective effect against H 2   O 2 \n-induced OS in GCs.  A.  A significant reduction in the viability of GCs\nwas detected in SFN-treated cells at concentrations of ≥ 25 µM in comparison to the\ncontrol group.  B.  SFN pretreatment at concentrations of 5, 10, and 15 µM\nprotected GCs from H 2   O 2  cytotoxicity. Results are presented as\nthe mean ± SD of 3 independent experiments. **; P<0.01, ***; P<0.001,\nSFN; Sulforaphane, H 2   O 2  ; Hydrogen peroxide, OS; Oxidative\nstress, GCs; Granulosa cells, and OD; Optical density.\nTo determine the intracellular ROS levels in the GCs\nexposed to H 2  \nO 2 \n, a DCFH-DA fluorescent probe was\nused. The results confirmed that H 2  \nO 2 \ntreatment induced\na significant elevation in the level of intracellular ROS\nwhen compared to the control group (mean fluorescence\nintensity: 290.33 vs. 183.67). However, this value\nsignificantly decreased in the group pretreated with\nSFN and then exposed to the H 2  \nO 2 \n(mean fluorescence\nintensity: 214.67,  Fig .2 ).\nProtective effects of SFN on H 2   O 2  -induced intracellular ROS production\nin GCs.  A.  Flow cytometry using a DCFH-DA fluorescent probe showed\nalteration in the level of intracellular ROS in the experimental groups.\n B.  H 2   O 2  treatment induced a significant\nelevation in the level of intracellular ROS compared to the control group, whereas\npretreatment with SFN before H 2   O 2  exposure significantly\ndecreased the level of intracellular ROS. Results are presented as the mean ± SD of 3\nindependent experiments. ***; P<0.001, SFN; Sulforaphane, H 2  \nO 2  ; Hydrogen peroxide, and GCs; Granulosa cells.\nThe annexin V/PI staining method was utilized to investigate cell death pathways\n(necrosis or apoptosis) of GCs in the experimental groups by flow cytometry. Our focus was\non the detection of late apoptotic cells (annexin V +  /PI + ) that\ntypically locate in the Q2 area as described in the method section. Results demonstrated\nthat the percentage of the cells in the late apoptotic state was significantly higher in\nthe group exposed to H 2   O 2  (50.37%) in comparison to the control\ngroup (26.40%). The percentage of annexin V +  /PI +  cells\nsignificantly decreased in the group pretreated with SFN and then exposed to H 2\n  O 2  (27.83%,  Fig .3 ).\nProtective effects of SFN on H 2   O 2  -induced GCs death.  A. \nAnnexin V/PI assay showed a difference in cell death pathways (necrosis and apoptosis)\nof GCs in the experimental groups.  B.  The percentage of cells in the late\napoptotic state was significantly higher in the group exposed to H 2  \nO 2  in comparison to the control group, whereas the percentage of late\napoptotic cells significantly decreased in the group pretreated with SFN and then\nexposed to the H 2   O 2  . The results are presented as the mean ±\nSD of 3 independent experiments. ***; P<0.001, SFN; Sulforaphane, H 2\n  O 2  ; Hydrogen peroxide, PI; Propidium iodide, and GCs; Granulosa\ncells.\nThe results so far showed that H 2   O 2  treatment elevated ROS\nproduction and apoptosis in GCs; however, SFN pretreatment protected GCs against these\ndetrimental effects of H 2   O 2  . Therefore, it was assumed that OS\nregulation pathways were activated. To investigate the alterations in gene and protein\nexpression level of key regulator genes in the pathway, including  NRF2,\nSOD , and  CAT , quantitative real-time PCR ( Fig .4 ) and western\nblot analysis ( Fig .5 ) were performed, respectively.\nResults showed that H 2   O 2  treatment caused an increase in the\nexpression of NRF2 and SOD at both mRNA and protein levels (P<0.05). However, while\nthe level of  CAT  gene expression was significantly higher in the H 2\n  O 2  -exposed group (P<0.001); it was not reflected at the protein\nlevel.\nEvaluation of the mRNA expression level of NRF2, SOD, and  CAT  in the\nexperimental groups by quantitative real-time PCR.  GAPDH  was utilized\nas the internal standard for the normalization of the data. H 2  \nO 2  treatment caused a significant increase in the expression of\n NRF2, SOD , and  CAT  at mRNA level compared to the\ncontrol group. Likewise, SFN pretreatment increased the mRNA expression of\n NRF2, SOD , and  CAT  compared to both control and\nH 2   O 2  -treated groups. Results are presented as mean ± SD. *;\nP<0.05, **; P<0.01, ***; P<0.001,  NRF2 ; Nuclear\nfactor erythroid 2-related factor 2,  SOD ; Superoxide dismutase,\n CAT ; Catalase, SFN: Sulforaphane, H 2   O 2  ;\nHydrogen peroxide, and PCR; Polymerase chain reaction.\nEvaluation of the protein expression level of NRF2, SOD, and CAT in the experimental.\n A.  NRF2, SOD, and CAT protein levels were measured by western blot.\n B.  The bands’ densities of NRF2, SOD, and CAT proteins were normalized\nin comparison to β-actin and analyzed by the semiquantitative method. H 2  \nO 2  treatment caused a significant increase in the expression of NRF2 and\nSOD proteins but had no significant effect on CAT protein expression. A significantly\nhigher expression of NRF2, SOD, and CAT proteins was observed in SFN+H 2  \nO 2  treated group compared to both control and H 2  \nO 2  -exposed groups. Values are presented as the mean ± SD. *;\nP<0.05, **; P<0.01, ***; P<0.001. NRF2; Nuclear factor-E2-related\nfactor 2, SOD; Superoxide dismutase, CAT; Catalase, SFN; Sulforaphane, and H 2\n  O 2  ; Hydrogen peroxide.\nSFN treatment in both SFN alone and SFN+H 2   O 2  groups\nsignificantly increased the mRNA expression of  NRF2, SOD , and \nCAT  compared to the control group (P<0.001). Moreover, SFN significantly\naugmented the mRNA expression of  NRF2, SOD , and  CAT  in\nthe SFN+H 2   O 2  treated group compared to the H 2  \nO 2  -exposed group (P<0.001). At the protein levels, we also observed\nsignificantly higher expression of NRF2 and SOD in both SFN and SFN+H 2  \nO 2  treated groups compared to the control group.\nWhen the protein levels of these genes were assessed,\nNRF2 and SOD showed a significant increase in the\nSFN+H 2  \nO 2 \ntreated group compared to the group exposed to\nH 2  \nO 2 \n(P<0.001). CAT did not show a significant difference at\nthe protein level in the group treated with SFN compared to\nthe control group. However, its protein level was significantly\nelevated in the SFN+H 2  \nO 2 \ntreated group compared to the\ncontrol (P<0.01) and H 2  \nO 2 \n-exposed groups (P<0.05).\n\nThe present study investigated the effect of SFN on\nNRF2-ARE pathway and the downstream antioxidant\nenzymes, SOD and CAT, in human GCs under H 2  \nO 2 \n-\ninduced OS condition. Following the determination of\nnontoxic doses of SFN, we first studied the protective\neffects of these concentrations on GCs viability under\nH 2  \nO 2 \n-induced OS to choose the optimal dose. The\nresults revealed that SFN has a protective effect at\nconcentrations of 5, 10, and 15 μM, and 10 μM was\nchosen as the optimal dose to be used for the rest of the\nstudy. Then, we investigated the effect of SFN on the\nintracellular ROS production, apoptosis, mRNA, and\nprotein expression levels of NRF2, SOD, and CAT in\nGCs treated by H 2  \nO 2 \n. The main finding of the present\nstudy was that the protective effect of SFN against H 2  \nO 2 \n-\ninduced intracellular ROS production and cell death may\nbe conducted through mediating the NRF2-ARE pathway\nand its downstream antioxidant enzymes including SOD\nand CAT at both mRNA and protein levels.\nAs mentioned above, GCs are important cells during\nfollicular development and oocyte maturation. In the\nprocess of follicular rupture and ovulation, a great level\nof ROS is produced by neutrophils and macrophages at\nthe site of follicular rupture, where GCs come to direct\ncontact with ( 7 ). GCs are sensitive to the damage caused\nby this OS, so lack of a protective system results in a\nwide spectrum of disorders in GCs which have inevitable\neffects on the fertility of the oocyte ( 10 ). Therefore, there\nis a need for the presence of an antioxidant system in\nGCs against oxidative damage, apoptosis, and follicular\natresia during the ovulatory process. GCs have a complex\nantioxidant system that defends oocytes from the damage\nof homeostasis imbalances ( 9 ).\nGCs are equipped with both enzymatic and non-enzymatic antioxidant systems that are pivotal\nfor their survival under OS conditions ( 24 ). Among several endogenous antioxidants involved\nin this manner, the NRF2-ARE pathway has drawn attention in GCs recently ( 16 ,  18 ). During\nthe follicular growth and ovulation, GCs establish an inherent defense system including the\nNRF2-ARE pathway against various stressors ( 25 ). The first-line defense of the antioxidant\nsystem is identified as SOD and CAT enzymes regulated by a promoter sequence recognized as\nARE in the NRF2-ARE pathway. These antioxidant enzymes are induced when NRF2, as the main\ntranscription factor, is stimulated and translocated into the nucleus for attaching to the\nARE region and thereby inducing their expression in OS condition ( 26 ). Compounds like SFN,\nas a natural product targeting this enzymatic system, have achieved growing attention in\nthis context and seem to have potential effects against OS in various cell types ( 16 ,  21 ).\nIndeed, numerous studies have shown that SFN is able to increase the expression of phase II\nantioxidant system enzymes and protects against oxidative damage in different types of cells\n( 27 ,  28 ). For this purpose, we intended to explore the protective effects of SFN in human\nGCs under the condition of OS. It should be noted that one of the most common models for OS\ninduction  in vitro  is H 2   O 2  exposure ( 29 ). Our\nrecent study developed a similar model for OS induction in human GCs using H 2  \nO 2  at the concentration of 200 µM by 2 hours incubation.\nIn line with our findings regarding the noncytotoxic\neffect of SFN at the concentration of 10 μM, other studies\nhave also reported that 10 μM of SFN is not toxic and\ncan be considered as the optimal dose to investigate its\nprotective effects ( 30 ). Indeed, we showed that 5-20 µM\nof SFN had no adverse effect on GCs viability, whereas\nhigher concentrations (≥25 µM) displayed cytotoxic\neffects identified by the lower number of viable cells. This\nfinding was almost supported by a study that introduced\nhigher concentrations of SFN (>15 μM) as a cytotoxic\ndose leading to cell loss in bovine GCs ( 31 ).\nHerein, we showed that 10 μM of SFN protects GCs\nagainst H 2  \nO 2 \n-induced OS and the following apoptosis\nas supported by the previous studies ( 16 ). For instance,\nCarrasco-Pozo et al. ( 32 ) reported that 10 μM of SFN\ncan protect pancreatic beta cell line MIN6 against OS\ninduced by high levels of cholesterol. According to the\npresent study, the observed effect of SFN on attenuating\nH 2  \nO 2 \n-induced ROS production and apoptosis seems to be\nmediated by the NRF2-ARE pathway as the results showed\na remarkable higher expression of NRF2 at both gene and\nprotein levels in GCs treated with a medium concentration\nof SFN. This effect may be modulated by both direct\nand indirect effects of SFN on the expression of Nrf2\nas described in previous studies ( 33 ). Indeed, the direct\neffect of SFN on the NRF2 promoter hypomethylation\nwas reported before ( 34 ). Moreover, in the indirect effect\nof SFN, the overexpression of NRF2 may result from\nthe process of its positive autoregulation ( 16 ). In addition,\nit is reported that this powerful antioxidant modifies\ncysteine residues of Keap1 chemically and enhances the\ndissociation of the NRF2-KEAP1 complex ( 35 ).\nRemarkably, our selected downstream antioxidant\nenzymes, SOD and CAT, were also upregulated at both\ngene and protein levels after treatment with SFN. In line with our data, a study reported that SFN treatment at a\nsimilar concentration (10 μM) induces the NRF2-ARE\npathway and the expression of SOD and CAT almost 2\nto 5 folds in bovine GCs compared to the control group.\nThey also indicated that a higher concentration of SFN\n(20 μM) is cytotoxic and induces the accumulation of\nROS and cell death in GCs. Hence, they propose a dose-dependent antioxidative effect of SFN in these cells ( 31 ).\nAnother study also supported these findings regarding the\ninducible effect of SFN on the expression of Nrf2 and its\ndownstream target antioxidant genes (SOD and CAT) in\nbovine GCs ( 16 ).\nOur findings are also consistent with the data described\nin other model systems. For instance, it was reported\nthat SFN was able to induce the expression of NRF2 and\nreduce the production of intracellular ROS in rat lung\nepithelial cells ( 36 ). SFN treatment also induced the\nNRF2-ARE pathway and SOD and CAT, and reduced\ncell death in rats with stress urinary incontinence ( 37 ).\nMoreover, SFN induced the NRF2-ARE pathway and\nCAT expression in a dose-dependent manner in human\nand rat lens epithelial cells (LECs) and aging human\nlenses which were halted after ARE area mutation,\nconfirming the SFN-induced NRF2-ARE pathway ( 38 ).\nWe also observed that the protective effect of SFN\nagainst intracellular ROS production and cell death was\nremarkable when cells were treated with H 2  \nO 2 \nand not\nSFN alone. One interesting finding of the study is that\nH 2  \nO 2 \ninduced the expression of NRF2 and antioxidant\nsystem enzymes which was remarkably amplified when\ncooperated with SFN treatment. This points toward this\nhypothesis that a low or moderate level of ROS acts as\nan activator of stress-responsive mechanisms, but an\nextra level of ROS, with damaging effects, needs the\npresence of powerful antioxidants to scavenge them\nthrough inducing the gene expression of antioxidant\nenzymes such as SOD and CAT as downstream factors\nof NRF2-ARE pathway.\nHence, these explanations along with our data support the protective role of SFN in\npreserving GCs against H 2   O 2  -induced OS. Then, we may reach this\npoint that administration of foods rich in SFN, such as broccoli sprout with approximately\n7.5 g of SFN, may have beneficial effects in improving disorders linked to the process of\novulation. Moreover, the current study may bring to mind a potential role for SFN in\nproviding a noble strategy for treating PCOS, infertility, and other ovarian diseases linked\nto oxidative damage and improving the quality of ovarian follicles. It should be noted that\nthe use of SFN as a therapeutic agent was demonstrated in cancer therapy before ( 39 ).\nTherefore, different  in vivo  and  in vitro  studies have\nprovided convincing evidence for using SFN to induce the antioxidant enzymes in several\ntypes of cells in the OS condition. One important notion is the appropriate dose of SFN for\nclinical usage as its different concentrations display different consequences ( 40 ). Here, we\nfound 10 μM of SFN as an optimal dose in our  in vitro  study in human GCs.\nConversely, a high concentration of SFN is cytotoxic to GCs as indicated by a previous study\nshowing higher levels of intracellular ROS and lower viability of bovine GCs ( 31 ). Hence,\nthe exact dose for use in clinical practice must be well-recognized in pre-clinical or\n in vivo  studies.\nThe present study is one of the first studies, to the best\nof our knowledge, to draw attention to the possible role\nof SFN in protecting human GCs against H 2  \nO 2 \n-induced\nOS; however, the limitations deserve to be declared. An\nimportant point which warrants consideration is the need\nfor using a specific inhibitor of Nrf2, such as Trigonelline\n( 23 ), to specify the study pathway as a target of SFN\nand to improve the validation of the results. Therefore,\nwe propose the use of specific inhibitors in future\ninvestigations associated with this subject.\n\nThe present study indicated that SFN induces the\nexpression of NRF2 and its downstream antioxidant\nenzymes, SOD and CAT, at both gene and protein\nexpression levels in human GCs under OS conditions.\nMoreover, SFN reduces the levels of intracellular ROS\nand the apoptosis rate of the GCs. It is tempting to\nspeculate that the stimulation of the NRF2-ARE pathway\nby SFN attenuates the damage by OS in human GCs via\nthe activation of SOD and CAT. Hence, this study may\nhave applicable information for improving the outcomes\nof assisted reproduction cycles, especially in PCOS\npatients.","source_license":"public-domain-us","license_restricted":false}