{"paper_id":"36f3fd0f-0e13-4c79-acc2-0aa3f00cc7f8","body_text":"Oxidative stress as a result of disruption in reduction-oxidation (redox) homeostasis is an unavoidable threat\nfor different human cells. When a great amount of\nreactive oxygen species (ROS) is generated, cells become\nmore sensitive to outcomes of oxidative stress including\napoptosis and damages to major organic molecules. For\na better understanding it should be noted that oxidative\nstress is a caused by as an imbalance between the\nproduction of ROS and antioxidant scavengers levels\n( 1 ). In normal states, a complex antioxidant system with\nseveral important defense enzymes, protects cells against\noxidative stress through scavenging ROS and maintaining\nthe redox homeostasis. Among these endogenous\nscavengers, nuclear factor-E2-related factor 2 (NRF2)/\nKelch-like ECH-associated protein 1 (KEAP1)-antioxidant\nresponse element (ARE) pathway and its underlying\nmechanism involving phase II enzymes including\nglutamate-cysteine ligase (GCL), heme oxygenase 1\n(HO1), and NAD(P)H quinone dehydrogenase 1 (NQO1),\nregulate antioxidant responses. GCL holoenzyme is an\nimportant antioxidant in glutathione biosynthesis with\ntwo different subunits, GCLC as a catalytic subunit and\nGCLM as a modifier subunit. HO1 cleaves the heme ring\nand leads to the formation of biliverdin and subsequently,\nbilirubin as potent antioxidants. Moreover, an excess\namount of heme sensitizes cells to apoptosis. NQO1,\nas a flavoprotein can be produced under different stress\nconditions particularly oxidative stress in order to reduce\nquinones to hydroquinones and prevent the formation of\nsubsequent ROS ( 2 ). Therefore, when ROS production is\nincreased, NRF2 as a key transcription factor, translocates\ninto the nucleus and enhances the expression of phase II\nantioxidant enzymes by attaching to ARE region but under\nnormal conditions, KEAP1 binds to NRF2 and facilitates its degradation through ubiquitination. Any disturbance\nin the function of this pathway results in an inability to\nneutralize the oxidative stress and following damages to\nmultiple cells ( 3 ).\nIt is widely known that oxidative stress is highly correlated with chronic inflammation,\nage-related diseases, cancer, and infertility in both men and women. Over the past years,\nconsiderable effort has been made to increase the success rate of infertility treatments.\nOxidative stress is regarded as an imperative factor affecting the success rates of\n in vitro  fertilization (IVF) especially in granulosa cells (GCs) of women\nwith polycystic ovarian syndrome (PCOS) ( 4 ). GCs surround the oocyte within the developing\novarian follicles and are key cells for the production of steroids as well as growth factors\nrequired for ovarian follicles growth and function. Although maintenance of normal\nphysiological levels of ROS is important for successful fertilization and regulation of\nspermatozoa maturation, capacitation, hyperactivation, acrosomal reaction, chemotactic\nprocesses, and sperm-oocyte fusion, the overproduction of ROS has been linked to many\nfertility complications caused by damaging many organic molecules. Since oxidative stress\nreverses well maturation of GCs and embryo quality, a great attempt must be made for\nmanagement of ROS generation ( 5 ).\nAstaxanthin (AST,  3 ,3′-dihydroxy-β,β′-carotene-4,4′- dione) is a powerful carotenoid\npigment naturally found in orange and red fruits. AST with multiple health benefits has a\nwide range of biological activities including antioxidant, anti-apoptotic,\nanti-inflammatory, and neuroprotective effects. AST with a great antioxidant capacity can\ninhibit oxidative damage and then, protect different cells from most pathological\nconditions. Previous studies reported that AST shows significant antioxidant activities not\nonly through radical scavenging but also by inducing the expression of NRF2 and its\ndownstream target genes, to promote the antioxidant defense in human cells ( 6 ). However,\nthere is no study indicating the protective role of AST in human GCs against oxidative\nstress. Since oxidative damage is a major cause of GCs and oocytes apoptosis and subsequent\ninfertility in women, we intended to examine the possible role of AST in protecting cultured\nprimary human GCs against hydrogen peroxide (H 2  O 2 )-induced oxidative\nstress through up-regulation of NRF2 pathway and subsequent activation of phase II enzymes\nincluding GCL, HO1, and NQO1. Furthermore, trigonelline (Trig) was used as an inhibitor of\nNRF2 ( 7 ) to express the link between NRF2-ARE pathway and AST-induced phase II enzymes\nexpression.\n\nThis study was approved by the Ethics Committee\nof Tehran University of Medical Sciences (IR.TUMS.\nMEDICINE.REC.1395.730) and written informed consent\nwas obtained from all contributors before initiation of the\nresearch.\nOur study was an experimental study. GCs used in\nthe current study were provided from follicular fluid of\nhealthy women aged between 20-38 years old, who had\na regular menstrual cycle and healthy ovulatory function,\nwere not taking took no hormonal drugs and underwent\nIVF for tubal and male infertility in the Infertility\nDepartment of Shariati Hospital affiliated with Tehran\nUniversity of Medical Sciences (TUMS). A history of\nPCOS, autoimmune diseases, menstrual disturbance,\nendometriosis, hirsutism, and hyperprolactinemia was\nregarded as exclusion criteria.\nPurification of GCs was done according to previous studies ( 8 ). First, to eliminate\nthe individual’s effects, follicular fluids obtained from different participants were\npooled and centrifuged at 3000 rpm for 10 minutes. The cell pellet was resuspended in\nDulbecco’s Modified Eagle Medium F-12 (DMEM/F-12, Gibco, Finland), then layered over\nFicoll-Paque (GE Healthcare Biosciences, Uppsala, Sweden) and centrifuged at 3000 rpm for\n10 minutes. We collected GCs from the interphase, and washed, and cultured them in a\ncomplete medium that contained DMEM/F-12 supplemented with 10% (v/v) heat-inactivated\nfetal bovine serum (FBS, Gibco, South America), 100 mg/ml of streptomycin (Gibco by Life\nTechnologies, Auckland, New Zealand), 100 U/ml of penicillin (Gibco by Life Technologies,\nAuckland, New Zealand), 2 mmol/l of glutamax (Sigma, St Louis, MO, USA), and 2 mg/ml of\namphotericin B (PAN Biotech, Berlin, Germany) at 37˚C in a humidified atmosphere\ncontaining 5% CO 2  , for different experiments. The medium was freshly changed\nevery other day. In the present study, we defined the study groups as control, cells\ntreated with dimethyl sulfoxide (DMSO, Sigma, Germany), H 2  O 2  , AST,\nAST+H 2  O 2  , and Trig+AST+H 2  O 2 .\nTo evaluate the viability of GCs after treatment with H 2  O 2  and AST\n(Sigma, China) and to determine an optimum dose for GCs treatment,\n3-( 4 , 5 -dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Alfa Aesar by Thermo\nFisher Scientific, Germany) test was conducted according to previous studies ( 9 ,  10 ). It\nshould be noted that AST was prepared by dissolving in DMSO. The test depends on the\nability of viable cells to reduce tetrazolium bromide by mitochondrial dehydrogenase to\nproduce formazan crystals ( 11 ). Briefly, GCs were cultured in a 96- well plate at a\ndensity of 1×10 4  cells per well and treated with H 2  O 2 \n(Fluka, Germany) at concentrations of 100, 150, 200, 300, and 400 µM for 2 hours at 37˚C,\nto evaluate the cell viability after oxidative stress. Next, MTT solution at a\nconcentration of 0.5 mg/ml was added to each well and plates were dark incubated at 37˚C\nfor 4 hours. DMSO was used for dissolving the produced colorful crystals and the optical\ndensity (OD) of samples was estimated at 570 nm by a microplate reader (EONTM, BioTek,\nUSA) with a background control as the blank. Moreover, GCs were treated with various\nconcentrations of AST (0, 5, 10, and 20 µM) for 24 hours at 37˚C using MTT assay for\nevaluating the cell viability as described above. Finally, the MTT assay was conducted\nafter pretreating cells with different concentrations of AST (0, 5, and 10 µM) for 24\nhours followed by H 2  O 2  treatment at a concentration of 200 µM for\nanother 2 hours at 37˚C to determine the optimal dose of AST for next steps.\nThe intracellular ROS levels were calculated using 2’-7’-Dichlorodihydrofluorescein\ndiacetate (DCFH-DA, Sigma, Switzerland) fluorescent probe by flow cytometry ( 12 ). Briefly,\ncells were cultured at a density of 2×10 5  cells per well in the presence or\nabsence of 5 µM of AST for 24 hours and then, exposed to 200 µM H 2 \nO 2  for another 2 hours at 37˚C. In addition, Trig (Sigma, Switzerland) at a\nconcentration of 0.1 µM was used as an inhibitor of NRF2, 1 hour before treatment with\nAST. Then, GCs were incubated with 1 µM of DCFH-DA for 30 minutes at 37˚C and resuspended\nin phosphate buffered saline (PBS, Sigma, Germany). Flow cytometry was applied for\ndetecting the fluorescence intensity at the wavelength of 525nm (FL1-H) band-pass filter\nbased on mean fluorescence intensity of 10,000 cells. FlowJo 7.6.1 was applied for\nanalyzing the results.\nThe Annexin V FITC-Propidium Iodide (PI) Apoptosis Detection Kit (Invitrogen by Thermo\nFisher Scientific eBioscience) was applied to determine the total cell apoptosis according\nto the manufacturers’ protocol. In details, GCs were cultured in a six-well plate at a\ndensity of 1×10 5  cells per well in the presence or absence of 5 µM of AST for\n24 hours and then, treated with 200 µM H 2  O 2  for another 2 hours at\n37˚C. Moreover, the effect of Trig at a concentration of 0.1 µM on GCs viability was\nmeasured. Next, GCs were resuspended in 1X Annexin-Binding buffer and a dark incubation at\nroom temperature for 15 minutes was accomplished after adding Annexin V-FITC and PI. The\nfluorescence emission was measured by flow cytometry. Staining for apoptosis was performed\nas described by the manufacturer. Annexin V-negative, PI-negative stained cells: viable\ncells; Annexin V-positive, PI-negative stained cells: early apoptotic cells; Annexin\nV-positive, PI-positive stained cells: late apoptotic cells; and Annexin V-negative,\nPI-positive stained cells: necrotic cells ( 13 ). The stained cells were analyzed by FlowJo\nsoftware.\nTotal RNA was extracted from cells using TRIzol reagent (Life Technologies, Gaithersburg,\nMD, USA) as explained by the manufacturer. Next, 1 μg of total RNA was applicable for cDNA\nsynthesis using a First-Strand cDNA Synthesis Kit (Thermo scientific, Foster City, CA,\nUSA) based on the manufacturer’s protocol. Real-time polymerase chain reaction (real-time\nPCR) was conducted to quantitate mRNA levels by the RealQ plus 5x Master Mix Green\n(Bio-Rad Laboratories, Hercules, CA, USA) using an Applied Biosystem StepOne real-time\nPCR, according to the manufacturers’ protocol.  GAPDH  was used as an\ninternal standard for normalizing the expression levels of our studied genes using the\n2 -ΔΔCt  method in order to obtain the relative fold change results ( 14 ). All\nsamples were analyzed in triplicate. The primers used in the present study are shown in\nTable 1.\nForward and reverse primers used for real-time polymerase\nchain reaction\nGCs were lysed by a Protein Extraction Kit\n(Active Motif Inc., Carlsbad, CA, USA) based on\nthe manufacturer’s protocol. The insoluble material\nwas removed by centrifugation at 15000×g for\n10 minutes at 4˚C. After collecting supernatants,\nBradford reagent (Bio-Rad, Foster City, MI, USA)\nwas used to determine the protein concentrations.\nProtein lysates at a concentration of 20 µg/µl were\nsubjected to sodium dodecyl sulfate-polyacrylamide\ngel electrophoresis (SDS-PAGE) and then, transferred\nto polyvinylidene difluoride membranes (Bio-Rad).\nAfter blocking with 5% BSA in TBST buffer at 4˚C\novernight, the membranes were blotted with primary\nantibodies including antibody against NRF2 (1:750; GeneTex, USA), antibody against KEAP1 (1:750;\nAbcam, Cambridge, MA, USA), and antibody against\nβ-actin (1:500; Santa Cruz Biotechnology, CA, USA),\nat 4˚C overnight. Next, the blots were washed and\nincubated with corresponding horseradish peroxidase\n(HRP)-linked secondary antibodies (rabbit anti-mouse\nIgG, ab97046, 1:5000; Abcam, Cambridge, UK) for 2\nhours at room temperature. Protein bands were developed\nusing a chemiluminescence system (ECL-plus, Lumigen,\nInc., Southfield, MI, USA) ( 15 ). β-actin was used as an\ninternal protein to normalize the expression of target\nproteins NRF2 and KEAP1. Data were analyzed by the\nImageJ software.\nWe used a TransAM NRF2 Transcription Factor ELISA Kit (Active Motif Inc., Carlsbad, CA,\nUSA) to evaluate the binding activity of NRF2 to DNA according to the manufacturer’s\nprotocol. In detail, we incubated 2.5 μg of nuclear extracts in a 96-well plate after ARE\noligonucleotides immobilization. By washing and adding an NRF2 antibody, we incubated the\nplate again and finally used a HRP-linked secondary antibody to provide colorimetric data.\nA microplate reader was applied for detecting the absorbance at 450 nm. A 450 \nindicated the binding activity of NRF2-ARE.\nThe Statistical Package for Social Sciences 22 (SPSS 22,\nInc., Chicago, IL, USA) was used for the statistical analysis\nof all results. The Kolmogorov–Smirnov test was used for\ntesting the normalization of data. For multiple comparisons\nbetween groups, Mann-Whitney U-test was used for\nnonparametric data. Results are shown as mean ± standard\ndeviation. Values of P<0.05 were regarded as significant.\n\nTo determine the viability of GCs following treatment with various concentrations of\nAST (0, 5, 10, and 20 µM), we used MTT assay which showed a significant cell death at a\nconcentration of 20 µM compared to the control group. Moreover, we also used MTT assay for\ndetermining the most appropriate concentration of H 2  O 2  for\ninduction of oxidative stress. Thus, we treated GCs with different concentrations of\nH 2  O 2  (100, 150, 200, 300, and 400 µM) for 2 hours. We detected a\nreduction of cell viability up to 50% at 200 µM and higher concentrations after 2 hours\n(P<0.001). Finally, we pretreated our studied cells with various concentrations of\nAST (0, 5, and 10 µM) for 24 hours followed by an extra treatment with 200 µM\nH 2  O 2  for 2 hours, to find the optimal dose of AST for protecting\nGCs from oxidative damage as provided in Figure 1. Here, the optimal dose of AST with the\nbest protective effect was 5 µM for 24 hours.\nIntracellular ROS levels were evaluated by a DCFH-DA fluorescent probe. For this\npurpose, we pretreated cells with 5 µM of AST for 24 hours and then, treated them with 200\nµM of H 2  O 2  for another 2 hours. Here, we observed a significant\nincrease in ROS generation using a DCF fluorescence, in the H 2  O 2 \n-treated group (mean fluorescence: 215 vs. 93) as shown in Figure 2, which was remarkably\nreduced to 50% after pretreatment with AST in the AST+H 2  O2 and\nTrig+AST+H 2  O 2  groups (P<0.01 and P<0.001,\nrespectively). The fluorescence intensity of GCs was significantly decreased after AST\npretreatment in all AST-treated groups.\nH 2  O 2  and AST toxicity measurement and the protective effect of AST against\nH 2  O 2  in GCs.  A.  To evaluate oxidative stress\nconditions; GCs were treated with various concentrations of H 2 \nO 2  (100, 150, 200, 300, and 400 µM) for 2 hours.  B.  To\ndetermine AST toxicity on GCs, various concentrations of AST (0, 5, 10, and 20 µM)\nwere used for 24 hours.  C.  To evaluate the protective effects of AST on\noxidative stress conditions, GCs were treated with various concentrations of AST (0,\n5, and 10 µM) for 24 hours, next treated with 200 μM of H 2  O 2 \nfor another 2 hours. Results are demonstrated as the mean ± SD. *; P<0.05, **;\nP<0.01, and ***; P<0.001, GCs; Granulosa cells, AST; Astaxanthin, and\nOD; Optimal density.\nROS induction.  A, B.  AST protects GCs from H 2  O 2  -mediated ROS\ngeneration. GCs were pretreated with 5 µM of AST for 24 hours, and then treated with\n200 µM of H 2  O 2  for another 2 hours. GCs were also treated with\n0.1 µM of Trig 1 hour before the exposure to AST. Intracellular ROS levels were\nevaluated by flow cytometry using a DCFH-DA fluorescent probe. Values are presented as\nthe median fluorescence ± SD of 3 independent experiments. *; P<0.05, **;\nP<0.01, ***; P<0.001, ROS; Reactive oxygen species, AST; Astaxanthin,\nGCs; Granulosa cells, DMSO; Dimethyl sulfoxide, and Trig; Trigonelline.\nH 2  O 2  exposure as a common model used for the induction of\noxidative damage, increases cellular apoptosis. To determine the protective effect of AST\nagainst H 2  O 2  -induced apoptosis, GCs were pretreated with 5 μM of\nAST as the optimal dose for 24 hours and then, treated with 200 µM of H 2 \nO 2  for 2 hours. The annexin V/PI staining was performed to determine the\ntotal GCs apoptosis due to H 2  O 2  treatment with or without\npretreatment of AST by using flow cytometry. As provided in  Figure 3 , annexin V+/PI- as an\nindicator of early apoptotic cells percentage showed a higher apoptosis rate in the\nH 2  O 2  -treated GCs in comparison with the control group which was\nsignificantly reduced after pretreatment with AST (P<0.05). Moreover, GCs were\ntreated with 0.1 µM of Trig as a known inhibitor of NRF2 1 hour before treatment with AST.\nRemarkably, the apoptosis rate was still significantly lower in the Trig+AST+H 2 \nO 2  GCs compared to H 2  O 2  -treated GCs (P<0.01)\nbut Trig treatment slightly increased the percentage of early apoptotic cells compared to\nthe AST+H 2  O 2  group (P<0.05).\nFirstly, our results indicated that H 2  O 2  treatment resulted in\ninduced expression of NRF2. The protein levels of KEAP1 as an endogenous inhibitor of\nNRF2, was also increased after H 2  O 2  exposure, however, it was not\nsignificant. Moreover, pretreatment with AST significantly induced  NRF2 \nexpression at both mRNA and protein levels in cells with or without H 2 \nO 2  exposure (P<0.01). Moreover, AST increased NRF2 activity and its\nconnection to ARE region in DNA compared to the H 2  O 2  -treated GCs\nwithout AST exposure (P<0.05) which was induced by Trig treatment as an inhibitor\nof NRF2 (P<0.05). AST also reduced KEAP1 protein levels in cells with or without\nH 2  O 2  exposure (P<0.05). Furthermore, Trig resulted in a\nsignificant decrease in  NRF2  gene expression in GCs treated with\nH 2  O 2  (P<0.01,  Fig.4A-C ). It also significantly reduced\ngene and protein expression of  NRF2  compared to the H 2 \nO 2  -treated group after AST pretreatment (P<0.01). The protein levels\nof KEAP1 were significantly induced in the Trig+AST+H 2  O 2  group in\ncomparison to the H 2  O 2  - treated group with or without AST\npretreatment (P<0.01). However, as provided in Figure 4, the higher expression of\n NRF2  at both mRNA and protein levels and the lower protein levels of\nKEAP1 were still observed as significant after Trig exposure in the Trig+AST+H 2 \nO 2  GCs compared to the H 2  O 2  -treated GCs.\nTo determine whether the effect of AST on the activation of NRF2/ARE pathway is\nfollowed by a higher gene expression of phase II enzymes including  GCLC, GCLM,\nHO1,  and  NQO1 , we conducted real-time PCR. The gene expression\nof these antioxidant enzymes was increased after exposure to H 2  O 2 \nin comparison with the control group. Our results demonstrated that pretreatment with 5 µM\nof AST for 24 hours, significantly enhanced the gene expression of phase II enzymes in the\nH 2  O 2  -treated and untreated groups ( Fig.5 ). Moreover, Trig as an\ninhibitor of NRF2, significantly attenuated this effect on the mRNA levels of\n GCLC, GCLM , and  HO1  in the H 2  O 2 \n-treated GCs after pretreatment with AST (P<0.01). Therefore, these findings\nsupport the role of phase II antioxidant enzymes in the protective effects of AST through\n NRF2  up-regulation.\nAST protects GCs from H 2  O 2  -induced apoptosis.  A.  The\napoptosis rate was measured by an annexin V/PI double staining test and flow\ncytometry. The quadrants on annexin V/PI dot plots classed as: Annexin V-negative,\nPI-negative staining cells: viable cells; Annexin V-positive, PI-negative staining\ncells: early apoptotic cells; Annexin V-positive, PI-positive staining cells: late\napoptotic cells; and Annexin V-negative, PI-positive staining cells: necrotic cells.\n B.  The quantitative data are shown as median ± SD of three independent\nexperiments. *; P<0.05, **; P<0.01, AST; Astaxanthin, GCs; Granulosa\ncells, DMSO; Dimethyl sulfoxide, and Trig; Trigonelline.\nEvaluation of NRF2 mRNA, protein, and activity and KEAP1 protein levels after AST and\nH 2  O 2  treatment in GCs.  A.  NRF2 and KEAP1 protein\nlevels were evaluated by western blot after treatment with 5 µM of AST for 24 hours\nand then treatment with 200 µM of H 2  O 2  for another 2 hours. The\nband densities of NRF2 and KEAP1 were normalized against β-actin.  B, C. \nReal-time PCR was conducted to evaluate the expression of  NRF2  mRNA.\n GAPDH  was used as an internal standard for normalization.\n D.  Effects of AST on DNA binding activity of NRF2. The molecular\nweights of NRF2, KEAP1, and β-actin are reported to be 61, 70, and 42 kDa,\nrespectively. The data are indicated as the mean ± SD of 3 independent experiments. *;\nP<0.05, **; P<0.01, AST; Astaxanthin, GCs; Granulosa cells, PCR;\nPolymerase chain reaction, DMSO; Dimethyl sulfoxide, and Trig; Trigonelline.\nEvaluation of  GCLC, GCLM, Ho1,  and  NQO1  mRNAs after AST and\nH 2  O 2  treatment in GCs. After treatment with 5 µM of AST for\n24 hours and then treatment with 200 µM of H 2  O 2  for another 2\nhours,  A. \n GCLC,  B. \n GCLM,  C.  Ho1,  and  D.\n NQO1  mRNAs were measured using real-time PCR.\n GAPDH  was used as an internal standard for normalization. The data\nare indicated as the mean ± SD of 3 independent experiments. *; P<0.05, **;\nP<0.01, AST; Astaxanthin, GCs; Granulosa cells, PCR; Polymerase chain reaction,\nDMSO; Dimethyl sulfoxide, and Trig; Trigonelline.\n\nIn the present study, we intended to examine the effects of AST on H 2 \nO 2  -induced oxidative stress in primary human GCs through investigating the\nexpression of  NRF2, KEAP1  and downstream phase II enzymes including\n GCL, HO1,  and  NQO1 . The main finding of our study was\nthe stimulatory effect of AST on the gene and protein levels and the nuclear localization of\nNRF2 along with its inhibitory effect on KEAP1 protein levels. Importantly, we indicated\nthat AST pretreatment suppressed ROS generation and cell death in GCs under the conditions\nof oxidative stress. Moreover, we revealed that using Trig as an inhibitor of\n NRF2 , reduced the protective effects of AST by decreasing\n NRF2  expression and activity and the gene expression of phase II enzymes.\nHowever, its inhibitory role did not completely remove the protective effects of AST on GCs.\nTherefore, our study may support a key role of the NRF2/ARE pathway in stimulating\nantioxidant enzymes induced by AST pretreatment in GCs.\nAs mentioned before, oxidative stress plays an important role in GCs related disorders like\nPCOS and may have a notable influence on IVF outcome. Therefore, developing an accurate\nmodel of oxidative stress is essential in different studies. One of the most applicable\nmodels used for establishing oxidative stress is the treatment of cultured cells like\nprimary human GCs with H 2  O 2  ( 4 ). Here, we used the model of\nH 2  O 2  -induced oxidative stress in GCs for investigating the\nantioxidant efficiency against oxidative damage. 200 µM of H 2  O 2  for 2\nhours was determined to induce oxidative stress in GCs for the next experiments. In our\nrecent study, we showed that a concentration of 200 μM of H 2  O 2  for\n2 hours, promotes oxidative stress in GCs ( 16 ). A recent similar study also used 200, 400,\nand 600 μM of H 2  O 2  for 48 hours to induce oxidative stress in GCs and\nevaluate the expression of  NRF2  and associated antioxidant enzymes ( 17 ). In\naddition, another study used 200 µM of H 2  O 2  for 24 hours to create\nthe same condition in retinal pigment epithelial cells ( 18 ). Furthermore, 400 µM of\nH 2  O 2  for 48 hours was used to trigger the model of oxidative stress\nin GCs ( 17 ). Our chosen concentration of H 2  O 2  for creating the model\nof oxidative stress was almost supported by other studies too in different cultured cells\nlike human keratinocytes ( 19 ).\nAntioxidant enzymes are produced more under\nconditions of excess ROS production to neutralize\noxidative stress and return the homeostasis of cells like\nGCs and then, regulate ovarian follicles growth and\nfunction. Earlier studies emphasized the importance of\nNRF2 and KEAP1 in the regulation of GCs condition\nat several phases of follicles ( 17 ). Since the exact\nmechanisms underlying the interaction between oxidative\nstress and antioxidant defense in human GCs, are largely\nunknown and need more comprehensive investigations,\nhere, we intended to explore the role of NRF2/ARE\npathway in protecting GCs against ROS production and\napoptosis by using the natural carotenoid pigment, AST.\nRecently, AST has been under great attention for its\nvarious biological functions including ROS scavenging,\nanti-inflammatory, anti-apoptotic, and anti-oxidative\neffects ( 20 ). AST is a powerful carotenoid antioxidant\nhaving many potential applications in human health\nprotection. Although the exact mechanism of AST in\nreducing oxidative damage is largely unknown, the role\nof NRF2/ARE pathway activation for these anti-oxidative\neffects was illustrated ( 21 ). However, the protective effect\nof AST on human GCs against oxidative stress is still\nelusive. Here, we firstly determined the best protective\nconcentration of AST. The optimal dose of AST we used\nwas 5 µM for 24 hours which was also supported by\nother researches done under similar concentrations. For\ninstance, 5 µM of AST was able to protect keratinocytes\nand peritoneal mesothelial cells from oxidative damage\n( 22 ,  23 ). Likewise, AST at concentrations of 5 and 6.25\nμM increased the expression of phase II antioxidant\nenzymes in other types of cells ( 24 ).\nOne of the most important results of our study is that AST can inhibit ROS production and\nprotect cells from apoptosis. The intracellular ROS levels generated by H 2 \nO 2  were significantly lower in GCs after pretreatment with 5 µM AST. Several\nstudies demonstrated that AST acts as a potent free radical scavenger and reduces the amount\nof intracellular ROS in several types of human cells. For instance, AST at a concentration\nof 5 μM protected peritoneal mesothelial cells by scavenging glucose-induced ROS ( 23 ).\nInterestingly, another study showed that AST at concentrations of 10 and 20 μM decreased ROS\nproduction in retinal pigment epithelial cells ( 18 ). Moreover, 2 μM of AST decreased fatty\nacid-induced ROS production in human lymphocytes ( 25 ). Several studies also reported the\npreventive effects of AST on ROS production in human neuroblastoma cells ( 26 ). AST at\nconcentrations of 10 and 100 μM also scavenged intracellular ROS in retinal ganglion cells\n( 27 ). Putting these together, we may conclude that AST as a direct scavenger of free\nradicals like H 2  O 2  , has beneficial effects on improving viability of\nprimary human GCs.\nIn our study, flow cytometry analysis showed a possible role of AST in decreasing\nH 2  O 2  -induced GCs early and late apoptosis through its\nanti-oxidative and anti-apoptotic properties. The present study suggests that AST\nup-regulated NRF2/ARE pathway and as a result, suppressed the apoptosis rate of GCs induced\nby intracellular ROS. Along with our data, other studies also suggested the inhibitory role\nof AST on apoptosis in different human cell types including keratinocytes treated with 5 µM\nAST ( 22 ), alveolar epithelial cells treated with 8 µM AST ( 28 ), and human neuroblastoma\ncells treated with 20 μM AST ( 29 ). Therefore, AST can be used for protecting GCs from\napoptosis through its scavenging activity and then, to strengthen the ability to reproduce\nin women. Moreover, as high levels of NRF2 were observed in the presence of AST followed by\na decrease in ROS production and cell damage, we may suggest NRF2 as a survival protein in\nfollicular development.\nAmong many endogenous antioxidant components involved in maintaining cellular homeostasis,\nNRF2- ARE pathway and its underlying targets phase II enzymes GCL, HO1, and NQO1, are of\ngreat importance ( 2 ). They are induced under conditions of oxidative stress when NRF2 as a\nkey transcription factor, is translocated into the nucleus for binding to ARE region leading\nto enhanced expression of phase II antioxidant enzymes. But until the homeostasis of the\ncell remains normal, NRF2 is inactivated by its endogenous inhibitor, KEAP1 protein ( 30 ). As\ndescribed earlier, the phase II enzymes induced by this pathway consist of several\nantioxidants. Here, we intended to evaluate the effects of AST treatment on mRNA and protein\nexpression and the activity of NRF2 as well as the gene expression of  GCLC, GCLM,\nHO1,  and  NQO1 . Furthermore, we evaluated the protein levels of\nKEAP1 to investigate whether the effects of AST on the NRF2-ARE pathway is dependent on NRF2\ninhibitor or not. Our study demonstrated that AST pretreatment induced the gene and protein\nlevels of  NRF2  but reduced the protein levels of KEAP1 in GCs in the\npresence or absence of H 2  O 2  . Moreover, measurement of NRF2 activity\nshowed that AST was able to significantly increase NRF2 translocation to the nucleus and its\nconnection to the ARE consensus site (5′-GTCACAGTGACTCAGCAGAATCTG-3′) compared to\nH 2  O 2  -treated GCs without AST. Then, AST stimulates NRF2-ARE\npathway by both enhancing the gene expression and activity of NRF2 and decreasing KEAP1\nprotein levels. Interestingly, a higher level of NRF2 in the nucleus may result from its\nup-regulation and lower levels of KEAP1 protein as its intracellular inhibitor.\nWe also observed a significant increase in the gene expression of phase II enzymes after\nAST pretreatment in H 2  O 2  -treated and untreated groups which followed\nby subsequent protection of GCs against oxidative damage and cell death. Some studies\nsupport our findings including a recent study that indicated the potential role of AST in\nincreasing the nuclear localization of NRF2 and subsequent expression of\n NQO1  and  HO1  in glomerular mesangial cells ( 31 ).\nInterestingly, AST induced the gene expression of  HO1  followed by the\nactivation of NRF2 nuclear translocation in human umbilical vein endothelial cells which was\nreduced after using NRF2 specific small interfering RNA (siRNA) for its inhibition ( 32 ).\nFurthermore, AST reduced the levels of KEAP1 protein which triggered its dissociation from\nNRF2 and increased the nuclear localization of NRF2 in the kidney of diabetic rats ( 33 ).\nAnother investigation on the brain after experimental subarachnoid hemorrhage showed that\nAST can activate NRF2-ARE pathway and subsequent gene expression of  HO1  and\n NQO1  enzymes, then ameliorated oxidative stress ( 34 ). Moreover, AST at a\nconcentration of 6.25 μM produced the highest gene expression of  NRF2,\nNQO1 , and  HO1  compared to other concentrations of AST in HepG2\ncells ( 24 ). In another similar study on retinal pigment epithelial cells, researchers\nreported a higher NRF2 nuclear localization and GCLC, GCLM, HO1, and NQO1 expression after\ntreatment with 5, 10, and 20 μM of AST ( 18 ). In view of our findings and the others, we may\nremark the importance of NRF2-ARE pathway in the activation of its downstream phase II\nantioxidant enzymes for the protection of ovarian follicles, preventing women from oxidative\nstress-related disorders, and increasing the success rates of IVF. Here, we pointed towards\nthe activation of NRF2-ARE pathway by AST as well as other antioxidants such as phenolic\ncompounds and carotenoids ( 35 ). Finally, we hope to achieve a better pregnancy result by\napplying AST as an inducer of NRF2/ARE pathway to neutralize oxidative stress and apoptosis\nin human GCs.\nMoreover, Trig as an alkaloid derived from niacin (vitamin B3) was added at a concentration\nof 0.1 µM to express the importance of NRF2 in the protective effects of AST and describe\nthe link between NRF2-ARE pathway and AST-induced phase II enzymes expression ( 7 ). Here, we\nobserved a significant decrease in the expression of  NRF2  at both mRNA and\nprotein levels as well as the gene expression of phase II enzymes by adding Trig to\nH 2  O 2  -treated and untreated GCs after AST pretreatment. Likewise,\nTrig treatment induced the levels of endogenous inhibitor of NRF2, KEAP1 protein along with\na reduction in NRF2 activity in H 2  O 2  -treated GCs after AST\npretreatment. This underlines the remarkable role of AST-induced NRF2/ARE pathway in\nstimulating phase II enzymes. However, our results revealed that the protective effects of\nAST on our studied target expression remained significant after Trig treatment compared to\nH 2  O 2  -treated GCs which highlighted the effectiveness of AST and\nthe inability of Trig to completely erase the protective effects of AST. Furthermore, there\nare other studies regarding the inhibitory role of Trig on the nuclear accumulation of NRF2\nprotein in different types of cells ( 36 ). According to our data and those reported by\nprevious studies, it seems likely that Trig inhibits NRF2 pathway and its downstream\nantioxidant enzymes mostly by inhibition of NRF2 nuclear accumulation. Putting these\nfindings together, our study established the importance of NRF2/ARE pathway in the related\nantioxidant defense induced by AST regarding the possibility that Trig has an inverse\ninfluence on the stimulatory role of AST on GCLC, GCLM, HO1, and NQO1 expression.\nAltogether, in this study, we showed that AST as a protective natural factor promotes gene\nand protein levels of NRF2 and inhibits the protein levels of KEAP1 in primary human GCs. We\nmay consider this mechanism for the inhibition of H 2  O 2  -induced\napoptosis and intracellular ROS generation by AST treatment. Hence, here for the first time,\nwe showed that AST inhibits H 2  O 2  -induced apoptosis and intracellular\nROS generation through a mechanism by which NRF2 induces the expression of antioxidant\nenzymes such as GCL, HO1, and NQO1 in GCs. Therefore, the current study provides supporting\ndata considering the possible role of AST in presenting a noble therapeutic strategy for\ninfertility, PCOS and other ovarian diseases related to oxidative damage. These results show\nthat AST as a radical scavenger and an anti-apoptotic factor, probably protects primary\nhuman GCs against H 2  O 2  -induced oxidative stress and cell death via\nregulating NRF2 and related factors and thus, improves the development of the ovarian\nfollicles.\nThe limitations of the present study included using Trig as an inhibitor of\n NRF2 . Because this agent is not capable of completely suppressing\n NRF2  as provided in our results, a more applicable and specific material\nmust be applied for complete inhibition of  NRF2  to investigate its role in\nactivating downstream antioxidant defense. Therefore, we suggest the use of a specific siRNA\nfor this propose in future studies related to this topic.\n\nOur study demonstrated that AST promotes gene and\nprotein levels of NRF2 and inhibits the protein levels\nof KEAP1 in primary human GCs. It seems likely that\nactivation of NRF2 by AST may attenuate oxidative stress in\nhuman GCs through activation of downstream antioxidant\nenzymes including GCL, HO1, and NQO1 and may produce\nbetter outcomes of IVF and reproduction in women.","source_license":"CC-BY-4.0","license_restricted":false}