{"paper_id":"d0f519a9-26b0-4e3d-962e-b68146211c0a","body_text":"1\nVol.:(0123456789)Scientific Reports |         (2021) 11:6054  | https://doi.org/10.1038/s41598-021-85512-y\nwww.nature.com/scientificreports\nThe effects of resveratrol \non the expression of VEGF, TGF‑β, \nand MMP‑9 in endometrial stromal \ncells of women with endometriosis\nTahereh Arablou1, Naheed Aryaeian1, Sepideh Khodaverdi2, Roya Kolahdouz‑Mohammadi1, \nZahra Moradi3, Nesa Rashidi3 & Ali‑Akbar Delbandi3,4*\nResveratrol is a phytochemical with anti‑angiogenic, anti‑inflammatory, and antioxidant properties. \nThe present study has evaluated the effect of resveratrol on the expression of vascular endothelial \ngrowth factor (VEGF), transforming growth factor‑β (TGF‑β) and matrix metalloproteinase‑9 (MMP‑\n9) as factors related to endometriosis progression. Thirteen eutopic (EuESCs) and 8 ectopic (EESCs) \nendometrial stromal cells from women with endometriosis and 11 control endometrial stromal cells \n(CESCs) were treated with resveratrol (100 µM) for 6, 24 and 48 h. The gene and protein expression \nlevels of VEGF, TGF‑β, and MMP‑9 were measured using real‑time PCR and ELISA methods, \nrespectively. Results showed that the basal gene and protein expression of VEGF and MMP‑9 \nwere higher in EESCs compared to EuESCs and CESCs (P < 0.01 to  < 0.001 and P < 0.05 to  < 0.01 \nrespectively). Also, resveratrol treatment decreased the gene and protein expression of VEGF and \nMMP‑9 in EuESCs, EESCs and CESCs (P < 0.05 to  < 0.01 and P < 0.05 to  < 0.01 respectively) and gene \nand protein expression of TGF‑β in EESCs and EuESCs (P < 0.05 to  < 0.01). The effect of resveratrol in \nreduction of VEGF gene expression was statistically more noticeable in EESCs compared to EuESCs \nand CESCs (P < 0.05). According to the findings, resveratrol may ameliorate endometriosis progression \nthrough reducing the expression of VEGF, TGF‑β, and MMP‑9 in endometrial stromal cells (ESCs).\nAbbreviations\nVEGF  Vascular endothelial growth factor\nTGF-β  Transforming growth factor-β\nMMP-9  Matrix metalloproteinase-9\nESCs  Endometrial stromal cells\nEuESCs  Eutopic endometrial stromal cells\nEESCs  Ectopic endometrial stromal cells\nCESCs  Control endometrial stromal cells\nEM  Endometriosis\nPF  Peritoneal fluid\nGnRH  Gonadotropin-releasing hormone\nLMP  Last menstrual period\nDMEM  Dulbecco’s modified Eagle’s medium\nLPS  Lipopolysaccharide\nRT-PCR  Reverse transcription polymerase chain reaction\nELISA  Enzyme-linked immunoassay\nPCR  Polymerase chain reaction\nGAPDH  Glyceraldehyde 3-phosphate dehydrogenase\nNF-κB  Nuclear factor-κB\nMAP-kinase  Mitogen-activated protein kinase\nACE-I  Angiotensin-converting-enzyme inhibitors\nOPEN\n1Department of Nutrition, School of Public Health, Iran University of Medical Sciences, Tehran, Iran. 2Endometriosis \nResearch Center, Iran University of Medical Science, Tehran, Iran. 3Department of Immunology, School of \nMedicine, Iran University of Medical Sciences, Tehran, Iran. 4Immunology Research Center, Immunology and \nInfectious Disease Institute, Iran University of Medical Sciences, Tehran, Iran. *email: delbandi.ak@iums.ac.ir\n\n2\nVol:.(1234567890)Scientific Reports |         (2021) 11:6054  | https://doi.org/10.1038/s41598-021-85512-y\nwww.nature.com/scientificreports/\nROS  Reactive oxygen species\nRNS  Reactive nitrogen species\nAP-1  Activator protein 1\nPhytochemicals are a large group of biologically active compounds found in  plants1. Intake of dietary phyto-\nchemicals has been associated with health benefits and disease  prevention2.\nResveratrol is a polyphenolic phytochemical belongs to the stilbenoid  class3. Its main sources are grapes, \nberries, peanuts, and some other  plants4. In recent decades, resveratrol is considered because of its beneficiary \neffects on oxidative  stress5,  inflammation3, tumor  progression6, aging and  angiogenesis7.\nEndometriosis (EM), one of the most prevalent gynecological disorders, is characterized by the growth \nof endometrial stroma and glands outside the uterine  cavity8. Its main symptoms are chronic pelvic pain and \n infertility9. To date, several theories have tried to explain the pathogenesis of the disease; among them, Sampson’s \nretrograde menstruation theory is more  reputable10. This theory stated that translocation of the endometrial \ncells into the peritoneal cavity through fallopian tubes leads to their adhesion, angiogenesis, and growth in the \nperitoneum and formation of the ectopic  lesions10,11. So, according to this theory, growth and angiogenesis fac-\ntors play vital roles in disease progression.\nVascular endothelial growth factor (VEGF) is shown to play a crucial role in angiogenesis in peritoneal \nendometriosis. It is secreted by the eutopic endometrium, ectopic endometriotic tissue, and peritoneal fluid \n(PF)  macrophages12,13. Previous studies reported the higher concentration of VEGF in PF from patients with \nendometriosis compared to non-endometriotic controls and its correlation with disease  stages14,15.\nTransforming growth factor-β (TGF-β) is one of the most potent growth factors and monocytes chemoattract-\nants. It can induce fibrosis and angiogenesis in ectopic implants and causes endometriosis  progression16,17. The \nPF of women with stage III and IV of endometriosis has higher levels of TGF-β compared to women with milder \nendometriosis, and a significant decrease in concentrations was achieved after treatment with a gonadotropin-\nreleasing hormone (GnRH)  agonist18.\nMatrix metalloproteinase-9 (MMP-9) is a member of proteinases that plays an essential role in the remod -\neling of the extracellular  matrix19. The overexpression of MMP-9 in ectopic endometrial lesions primary seems \nto exacerbate the angiogenesis and invasion of ectopic  implants20,21.\nIt was claimed that resveratrol could ameliorate endometriosis  progression22. Its effect is due to suppression \nthe expression of growth  factors23, decrease cell  proliferation24, reduction of the size of the ectopic  implant25, \ninduction of  apoptosis26, reduction of the  inflammation25 and oxidative  stress27, and inhibition the invasion, \nadhesion, and angiogenesis of endometriotic ectopic  lesions22. Also, Resveratrol has shown to suppress the \nexpression of VEGF and MMP-9 in rat-induced  endometriosis25 and decrease the expression of VEGF , TGF-β, \nand MMP-9 in other  diseases28–30.\nConsidering the importance of VEGF , TGF-β and MMP-9 in the development of EM and the inhibitory \neffect of resveratrol on the expression of these factors in other cell types in different diseases, and given that to \ndate no study has been performed on the effect of resveratrol on gene and protein expression of these factors in \nESCs, the aim of the present study was to investigate the effect of resveratrol treatment on the gene and protein \nexpression of VEGF , TGF-β and MMP-9 in ectopic (EESCs) and eutopic (EuESCs) endometrial stromal cells in \nwomen with endometriosis in comparison with non-endometriotic controls (CESCs).\nMaterials and methods\nStudy population. The present study was performed on 40 patients with peritoneal endometriosis and \n15 non-endometriotic controls. The inclusion criteria were: being at reproductive age (19–45 years old), at the \nproliferative phase of the menstrual cycle, the III-IV stages of peritoneal endometriosis according to the revised \nAmerican Fertility Society system (rAFS)31 for patients and non-endometriotic lesions for control group based \non the laparoscopy. The control group was undergone laparoscopy for diagnostic reasons and because of benign \ngynecological problems.\nThe exclusion criteria were: any history of malignancy, autoimmune or metabolic disorders, taking any hor-\nmonal medications or dietary supplements within the last three months before the surgery, pregnancy, lactation, \nand cigarette smoking.\nAll individuals signed written informed consent before participating in the study, and all participants’ privacy \nwas respected. The study protocols was approved by the Ethics Committee for Medical Research of Iran Uni-\nversity of Medical Sciences (Code: IR.IUMS.rec.1395.9221324203). All methods were performed in accordance \nwith the relevant guidelines and regulations.\nSample collection. All sample collection and tissue extraction details were explained  earlier23. Ectopic \nand eutopic endometrial samples were collected using laparoscopic sampling and biopsy curette, respectively. \nAll endometriotic cysts (endometrioma) size were ≥ 5 cm in diameter. Tissue samples were put in sterile tubes \ncontaining Dulbecco’s modified Eagle’s medium (DMEM)-F12 (Sigma-Aldrich, St. Louis, MO, USA) culture \nmedium with 1% Penicillin–Streptomycin antibiotics (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) \nand quickly transferred to the laboratory on ice. A part of all samples was taken to the pathology laboratory to \nconfirm endometriosis. The phase of the menstrual cycle was confirmed by the histological dating of ectopic \nendometrial implants. In the case of virgins and other patients with only the ectopic tissue, the confirmation of \nthe cycle phase done by the last menstrual period (LMP).\nIsolation, culture and purification of endometrial stromal cells (ESCs). The digestion of endome-\ntrial tissue samples and the purification and culture of stromal cells performed as we described  earlier23. Briefly, \n\n3\nVol.:(0123456789)Scientific Reports |         (2021) 11:6054  | https://doi.org/10.1038/s41598-021-85512-y\nwww.nature.com/scientificreports/\nin the sterile condition, ectopic and eutopic endometrial tissues from endometriotic women and normal endo-\nmetrial tissues from the control women were minced into smaller pieces and digested in the presence of 2 mg/\nml Collagenase A (Roche, Pleasanton, CA, USA) and 300 mg/ml DNase (Roche, Pleasanton, CA, USA). Then \nthe obtained cells were cultured in T25 culture flasks (SPL Life Sciences, Korea), ans stored in an atmosphere \nof 5% CO2 at 37 °C in DMEM-F12 (Sigma-Aldrich, St. Louis, MO, USA) containing 1% Penicillin–Strepto-\nmycin antibiotics (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) and 10% fetal bovine serum (FBS) \n(Gibco, Thermo Fisher Scientific, Waltham, MA, USA) and adherent stromal cells were allowed to multiply. The \ncultured cells were passaged three times and when they reached to about 80% confluency they were used for \nthe treatment. Some tissue samples especially ectopic tissues were excluded due to the cultural contamination, \ninproper pathology results, or not obtained the desired cells. At the end, from 40 endometriotic and 15 non-\nendometriotic control tissues, 8 ectopic, 13 eutopic, and 11 control tissues were treated. The purification of the \nESCs was approved by immunofluorescent staining and flow cytometry. These cells were characterized as a panel \nof  vimentin+,  nestin+,  cytokeratin− ,  CD10+,  CD44+,  CD73+,  CD105+,  CD34− , and  CD45−  cells. as we described \n earlier23.\nTreatment of endometrial stromal cells with resveratrol. Based on the results of MTT test and the \npilot  study23, all ESCs from the three study groups were seeded 30 ×  104 in 24-well plates (SPL Life Sciences, \nKorea) to have confluency about 80% for resveratrol treatment. After 3 h treated with the pre-determined opti-\nmized concentration of 100 µM resveratrol (Sigma-Aldrich, St. Louis, MO, USA) and stimulated with 100 ng/\nml Lipopolysaccharide (LPS) (Sigma-Aldrich, St. Louis, MO, USA)32, and incubated for three-time points 6, 24 \nand 48 h.\nExtraction of RNA and quantitative real‑time PCR. For RNA isolation all ESCs were stored in Tri-\nzol (Qiagen, Hilden, Germany) at −80  °C. Total RNA was isolated according to the manufacturer’s instruc-\ntions. Extracted RNA was reverse transcribed to complementary DNA (cDNA) using reverse transcription-\npolymerase chain reaction (RT-PCR) kit (Fermentas, Thermo Fisher Scientific, Waltham, MA, USA). The gene \nexpressions of VEGF, TGF-β, and MMP-9 were quantified by real-time PCR with Syber premix Extaq (Biofact, \nDaejeon, Korea) according to the protocol by Rotor-Gene Q (Qiagen, Hilden, Germany). The gene expressions \nwere normalized using Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA as an internal control. \nThe primer pairs and the size of the amplicons are shown in Table  1. The PCR conditions were mentioned in \ndetails  earlier23. It included a holding step on 95° for 15 min (for enzyme activation), which followed by 40 cycles \nof 95 °C for the 20 s, extension at 60 °C for 40 s (GAPDH at 58 °C for 40 s) and the melting step at 60° to 99°. All \nreactions were run in duplicate.\nMeasurement of VEGF, TGF‑β, and MMP‑9 protein. The concentration of VEGF , TGF-β, and MMP-9 \nprotein in the cell supernatant was examined by a standard enzyme-linked immunoassay (ELISA) kit (Duoset; \nR&D Systems, Minneapolis, MN, USA) according to the manufacturer’s protocol.\nStatistical analysis. Statistical analyses were carried out using GraphPad Prism software 6.01 (GraphPad \nSoftware. Inc). Based on the results of the Kolmogorov–Smirnov test, all data was analyzed using the non-par -\nametric tests, including the Wilcoxon sign-ranked test, Mann–Whitney, and Kruskal–Wallis tests. For the gene \nexpression analysis, the fold change and relative expression were compared by calculating the  2−∆Δct  and  2−∆ct , \nrespectively. P-value < 0.05 was considered as the statistically significant level.\nResults\nThe Basal expression of VEGF gene and protein in ESCs. Based on the results of real-time PCR, \nthe VEGF gene was expressed significantly more in EESCs compared to EuESCs and CESCs (Both P < 0.01) in \nthe basic state (Fig. 1a). Moreover, according to the results of ELISA, the VEGF protein had significantly higher \nexpression in EESCs compared to EuESCs and CESCs (P < 0.01 and P < 0.001 respectively) (Fig. 1b).\nTable 1.  The VEGF , TGF-β, MMP-9 and GAPDH primers sequences. IGF-1: Insulin-like growth factor-1; \nHGF: Hepatocyte growth factor; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; bp: Base pair.\nSequence Name Accession No Sequence 5′ to 3’ Amplicon Size (bp)\nVEGF-Sense NM_001204384.1\nNM_001171622.1\nTTG CCT TGC TGC TCT ACC TCCA \n126\nVEGF-Anti-sense GAT GGC AGT AGC TGC GCT GATA \nTGF-β-Sense NM_000660.6\nXM_011527242.2\nTGG TGG AAA CCC ACA ACG AA\n113\nTGF-β-Anti-sense GAG CAA CAC GGG TTC AGG TA\nMMP-9-Sense\nNM_004994.2\nGCA CGA CGT CTT CCA GTA CC\n124\nMMP-9- Anti-sense CAG GAT GTC ATA GGT CAC GTAGC \nGAPDH- Sense NM_001289745.2\nNM_002046.6\nGCA CCG TCA AGG CTG AGA AC\n138\nGAPDH—Anti-sense TGG TGA AGA CGC CAG TGG A\n\n4\nVol:.(1234567890)Scientific Reports |         (2021) 11:6054  | https://doi.org/10.1038/s41598-021-85512-y\nwww.nature.com/scientificreports/\nThe Basal expression of TGF‑β gene and protein in ESCs. According to the results of real-time PCR \nand ELISA the basal gene and protein expression of TGF-β had no statistically significant differences among \nEESCs, EuESCs and CESCs (Fig. 1c,d).\nFigure 1.  The basal expression levels of VEGF , TGF-β and MMP-9 genes and proteins in ESCs. The basal \nexpression of VEGF , TGF-β and MMP-9 genes and proteins were measured in EESCs (n = 8) and EuESCs \n(n = 13) from endometriotic women and CESCs from non-endometriotic controls (n = 11) by real-time PCR \nand ELISA. (a) The basal expression of VEGF gene, (b) The basal expression of VEGF protein, (c) The basal \nexpression of TGF-β gene, (d) The basal expression of TGF-β protein, (e) The basal expression of MMP-9 gene, \n(f) The basal expression of MMP-9 protein. *P-value < 0.05, ** P-value < 0.01 and *** P-value < 0.001 by non-\nparametric tests. VEGF: Vascular endothelial growth factor, TGF-β: Transforming growth factor-β, MMP-9: \nMatrix metalloproteinase-9, ESCs: Endometrial stromal cells, EuESCs: Eutopic endometrial stromal cells, \nEESCs: Ectopic endometrial stromal cells, CESCs: Control endometrial stromal cells.\n\n5\nVol.:(0123456789)Scientific Reports |         (2021) 11:6054  | https://doi.org/10.1038/s41598-021-85512-y\nwww.nature.com/scientificreports/\nThe Basal expression of MMP‑9 gene and protein in ESCs. Analysis of real-time PCR and ELISA \nmethods revealed that the basal expression of MMP-9 gene and protein was significantly more in EESCs in com-\nparison with EuESCs (Both gene and protein P < 0.05) and CESCs (Both gene and protein P < 0.01) (Fig. 1e,f).\nResveratrol decreased the expression of VEGF gene in all ESCs. The real-time PCR method dem-\nonstrated that treatment with resveratrol (100 µM) reduced the expression of VEGF gene significantly in EESCs \nat 24 (P < 0.05) and 48 h (P < 0.01) and in EuESCs and CESCs only at 48 h (Both P < 0.05) (Table 2). Also, the \neffect of 100 µM resveratrol treatment was more noticeable in EESCs in comparison with EuESCs and CESCs at \n48 h (Both P < 0.05) (Supplementary file).\nResveratrol decreased the expression of TGF‑β gene in EuESCs and EESCs. The TGF-β gene \nexpression had significant reduction by treatment with resveratrol (100  µM) in EuESCs and EESCs at 48  h \n(P < 0.05 and P = 0.01 respectively). The TGF-β gene expression had no significant changes in EuESCs and EESCs \nat 6 and 24 h, and in CESCs at all three time intervals (Table 2). There was no significant difference in the effect \nof resveratrol treatment between EESCs and EuESCs at 48 h (Supplementary file).\nResveratrol decreased the expression of MMP‑9 gene in all ESCs. The MMP-9 gene expression was \nsignificantly reduced by resveratrol (100 µM) in EuESCs at 24 (P < 0.01) and 48 h (P < 0.05) and in EESCs and \nCESCs at 48 h (Both P < 0.05). The gene expression of MMP-9 did not show significant changes in EuESCs at 6 h, \nand in EESCs and CESCs at 6 and 24 h (Table 2). In addition, resveratrol had a greater effect on EESCs compared \nwith EuESCs and CESCs at 48 h, but this was not statistically significant (Supplementary file).\nResveratrol decreased the expression of VEGF protein in EuESCs and EESCs. The use of ELISA \nmethod revealed that the protein expression of VEGF was significantly reduced in EuESCs and EESCs at 48 h by \nTable 2.  The effect of resveratrol on VEGF, TGF-β and MMP-9 gene expression in ESCs. ESCs from \nendometriotic women (8 EESCs and 13 EuESCs) and non-endometriotic controls (11 CESCs) were cultured \nwith or without 100 µM resveratrol. After 6, 24, and 48 h, the gene expression of VEGF, TGF-β and MMP-9 \nwere examined using real-time PCR. Data were analyzed by non-parametric tests. *P-value < 0.05 is statistically \nsignificant. VEGF: Vascular endothelial growth factor, TGF-β: Transforming growth factor-β, MMP-9: Matrix \nmetalloproteinase-9, ESCs: Endometrial stromal cells, EuESCs: Eutopic endometrial stromal cells, EESCs: \nEctopic endometrial stromal cells, CESCs: Control endometrial stromal cells.\nGene ESC type Treatment time Fold change P-value\nVEGF\nEuESCs\n6 h 0.79 vs 1 0.67\n24 h 0.93 vs 1 0.78\n48 h 0.44 vs 1 0.04*\nEESCs\n6 h 0.50 vs 1 0.07\n24 h 0.45 vs 1 0.04*\n48 h 0.19 vs 1 0.007*\nCESCs\n6 h 0.99 vs 1 0.64\n24 h 0.72 vs 1 0.99\n48 h 0.43 vs 1 0.04*\nTGF-β\nEuESCs\n6 h 1.20 vs 1 0.12\n24 h 1.36 vs 1 0.10\n48 h 0.63 vs 1 0.02*\nEESCs\n6 h 0.87 vs 1 0.46\n24 h 0.55 vs 1 0.15\n48 h 0.63 vs 1 0.01*\nCESCs\n6 h 1.03 vs 1 0.25\n24 h 1.20 vs 1 0.25\n48 h 1.15 vs 1 0.32\nMMP-9\nEuESCs\n6 h 0.53 vs 1 0.49\n24 h 0.20 vs 1 0.002*\n48 h 0.58 vs 1 0.04*\nEESCs\n6 h 0.93 vs 1 0.84\n24 h 1.81 vs 1 0.43\n48 h 0.08 vs 1 0.03*\nCESCs\n6 h 0.49 vs 1 0.15\n24 h 1.42 vs 1 0.25\n48 h 0.27 vs 1 0.03*\n\n6\nVol:.(1234567890)Scientific Reports |         (2021) 11:6054  | https://doi.org/10.1038/s41598-021-85512-y\nwww.nature.com/scientificreports/\n100 µM resveratrol (P < 0.05 and P < 0.01 respectively). Resveratrol treatment had no significant effect at 6 and \n24 h in these cells. The VEGF protein expression did not change significantly in CESCs at any of the treatment \ntimes (Fig. 2). Although, the effect of resveratrol treatment on reducing VEGF protein expression in EESCs was \ngreater than that of EuESCs, this difference was not statistically significant. (Supplementary file).\nResveratrol decreased the expression of TGF‑β protein in EuESCs and EESCs. The effect of \ntreatment with 100 µM resveratrol on the expression of TGF-β protein was the same as its gene expression. Res-\nveratrol could reduce the expression of this factor in EuESCs and EESCs at 48 (Both P < 0.05). The expression of \nTGF-β protein showed no significant changes at 6 and 24 h in EuESCs and EESCs, and at any treatment times \nin CESCs (Fig. 3). The effect of resveratrol treatment between EESCs and EuESCs had no significant difference \nat 48 h (Supplementary file).\nResveratrol decreased the expression of MMP‑9 in all ESCs. Resveratrol (100 µM) decreased sig-\nnificantly the MMP-9 protein in EuESCs, at 24 and 48 h and in EESCs and CESCs at 48 h (All P < 0.05). The \nMMP-9 protein production had no significant changes at 6 in EuESCs at 6 and 24 h in EESCs and CESCs (Fig. 4). \nIn addition, the effect of treatment with 100 µM resveratrol at 48 h on the reduction of MMP-9 protein produc-\ntion was not statistically significant among three groups (Supplementary file).\nFigure 2.  Resveratrol Decreased the Expression of VEGF Protein in EuESCs and EESCs. ESCs from \nendometriotic women (8 EESCs and 13 EuESCs) and non-endometriotic controls (11 CESCs) were cultured \nwith or without of 100 µM resveratrol. After 6, 24, and 48 h, the protein expression of VEGF was examined \nusing ELISA. (a) 6 hr (Res + vs Res-), (b) 24 hr (Res + vs Res-), (c) 48 hr (Res + vs Res-). *P-value < 0.05, ** \nP-value < 0.01 by non-parametric tests. VEGF: Vascular endothelial growth factor, EuESCs: Eutopic endometrial \nstromal cells, EESCs: Ectopic endometrial stromal cells, CESCs: Control endometrial stromal cells.\n\n7\nVol.:(0123456789)Scientific Reports |         (2021) 11:6054  | https://doi.org/10.1038/s41598-021-85512-y\nwww.nature.com/scientificreports/\nDiscussion\nThe results revealed that the basal expression of VEGF and MMP-9, but not TGF-β in EESCs were significantly \nhigher compared to EuESCs and CESCs. To date, some studies assessed the concentration of these factors in PF \nor endometrial implants of the endometriotic  patients21,33–35 and according to our knowledge, the present study \nis the first to compare the expression of VEGF , TGF-β and MMP-9 in EESCs, EuESCs, and CESCs.\nThe findings of the previous studies, consistent with the present study, have shown that VEGF expression in \nendometrial tissue and PF of patients with endometriosis is increased compared to controls, although, it does not \ndiffer significantly between the different stages of the  disease33,36. In the only discordant study, the VEGF concen-\ntration in PF of patients with genital endometriosis and healthy control women was not significantly  different34. \nVEGF receptors gene expression was also higher in ectopic endometrial lesions than in eutopic  tissue21,37.\nVEGF is one of the most important angiogenic factors in endometriosis. It can increase cell proliferation, \ncell migration, and vascular  permeability13,38. The most important cells secreting this factor in endometriosis are \neutopic and ectopic stromal cells, peritoneal macrophages, and neutrophils that increase the expression of this \nfactor in response to elevated inflammatory  conditions13. Increased levels of reactive oxygen species (ROS) due to \noxidative stress in endometriosis can also increase VEGF expression and its angiogenesis in in-vivo and in-vitro39.\nThe few studies that have examined the expression of TGF-β in endometriosis have shown contradictory \nfindings. For example, in a study of Sokolov et al. the concentration of TGF-β in PF did not differ significantly \nbetween the women with genital endometriosis and healthy  controls34, but two other studies, showed that levels \nof TGF-β in serum and PF were higher in patients than in controls, and this level, especially in PF , increased \nwith increasing severity of the  disease35,40.\nFigure 3.  Resveratrol Decreased the Expression of TGF-β Protein in EuESCs and EESCs. ESCs from \nendometriotic women (8 EESCs and 13 EuESCs) and non-endometriotic controls (11 CESCs) were cultured \nwith or without of 100 µM resveratrol. After 6, 24, and 48 h, the protein expression of TGF-β was examined \nusing ELISA. (a) 6 hr (Res + vs Res-), (b) 24 hr (Res + vs Res-), (c) 48 hr (Res + vs Res-). *P-value < 0.05 by non-\nparametric tests. TGF-β: Transforming growth factor-β, EuESCs: Eutopic endometrial stromal cells, EESCs: \nEctopic endometrial stromal cells, CESCs: Control endometrial stromal cells.\n\n8\nVol:.(1234567890)Scientific Reports |         (2021) 11:6054  | https://doi.org/10.1038/s41598-021-85512-y\nwww.nature.com/scientificreports/\nSeveral studies have shown the role of TGF-β in regulating the immune system and  inflammation41. TGF-β \nenhances the growth and angiogenesis of ESCs, especially ectopic cells, and plays an essential role in the develop-\nment of endometriotic  lesions13. Increased expression of this factor in endometriosis seems to occur in response \nto increased inflammatory conditions and oxidative stress in the peritoneal  cavity42. As is evident in our study, \nthere was no significant difference in TGF-β expression in the stromal cells of the study groups. Previous studies \nreported that peritoneal mesothelial cells are the most important source of this factor in peritoneum-related dis-\neases such as peritoneal endometriosis, followed by peritoneal macrophages, ectopic endometrial tissue including \n ESCs17, it appears that the increased expression of this factor in the serum and PF of patients with endometriosis \nthan in controls has been reported in some previous  studies35,40, may be due to the increased production of this \ncytokine by peritoneal mesothelial cells and then other sources and in the meantime, the ESCs evaluated in the \npresent study, have less role in the production of this factor. Previous studies have also shown that the concentra-\ntion of TGF-β in the peritoneum of individuals with endometriosis changes during the menstrual cycle and its \nhighest concentration is seen in the secretory phase and in the premenstrual  phase18,41. However, in our study, \nwe measured the expression of TGF-β in the proliferative phase.\nIn the case of MMP-9, the only study comparing the expression of this factor in ectopic endometrial lesions \nwith eutopic endometrium is Machado’s study on an induced model of endometriosis in rats and reported find-\nings consistent with the present  study21.\nStudies have shown that chronic inflammation increases MMP-9 expression. Expression of MMP-9 by EESCs \nand EuESCs increases in endometriosis in response to inflammatory conditions in the peritoneal cavity, which \nis higher in ectopic than eutopic lesions and activation of the NF-κB and MAP-kinase signaling and other \nFigure 4.  Resveratrol Decreased the Expression of MMP-9 Protein in all ESCs. ESCs from endometriotic \nwomen (8 EESCs and 13 EuESCs) and non-endometriotic controls (11 CESCs) were cultured with or without of \n100 µM resveratrol. After 6, 24, and 48 h, the protein expression of MMP-9 was examined using ELISA. (a) 6 hr \n(Res + vs Res-), (b) 24 hr (Res + vs Res-), (c) 48 hr (Res + vs Res-). *P-value < 0.05 by non-parametric tests. MMP-\n9: Matrix metalloproteinase-9, EuESCs: Eutopic endometrial stromal cells, EESCs: Ectopic endometrial stromal \ncells, CESCs: Control endometrial stromal cells.\n\n9\nVol.:(0123456789)Scientific Reports |         (2021) 11:6054  | https://doi.org/10.1038/s41598-021-85512-y\nwww.nature.com/scientificreports/\ninflammatory pathways, as well as to increased oxidative  stress43,44; This assists the replacement, growth, and \ninvasion of endometriotic  implants20,21. Increased production and activity of MMP-9 increase the degradation \nand regeneration of extracellular matrix, angiogenesis, and VEGF  secretion19,45.\nThe present study revealed that the gene and protein expression of VEGF , TGF-β, and MMP-9 in EESCs and \nEuESCs were reduced by resveratrol treatment. According to our knowledge, this is the first study to investigate \nthe effect of resveratrol on VEGF expression in ESCs of patients with endometriosis. In the only animal study, \nresveratrol significantly reduced VEGF expression in endometriosis-induced  rats25. Other previous in-vivo and \nin-vitro studies on the effect of resveratrol on VEGF expression in other diseases have also reported findings \nconsistent with the present  study29,46–49.\nThe mechanisms of the effect of resveratrol on VEGF expression seems to be through activation of the sir -\ntuin-1 molecule and inhibition of the NF-κB  pathway50. Resveratrol can also inhibit VEGF through ACE-I-like \nactivity. Thus, resveratrol inhibits positive feedback between angiotensin-II and VEGF . The in-vitro studies have \nshown that ACE-I-like factors can inhibit VEGF-induced endothelial cell migration and invasion and inhibit \nVEGF mRNA  expression49,51. Resveratrol may also block the VEGF receptor response pathway by reducing \nMAP-kinase phosphorylation and inhibiting VEGF-induced angiogenesis by blocking tyrosine phosphorylation \nin the cadherin  molecule46. Besides, resveratrol reduces VEGF expression and its invasion and angiogenesis by \npreventing the production and eliminating the ROS and reactive nitrogen species (RNS)39,52.\nIt seems that the difference in the effect of resveratrol on the reduction of VEGF  gene expression in EESCs \ncompared to EuESCs and CESCs is due to differences in inflammatory and micro-environmental conditions \nof these cells. Previous studies have shown that EESCs, EuESCs, and CESCs differ in cytokine expression, cell \nproliferation, invasion, metastasis, and response to nutritional  interventions53,54.\nThe present study is the first to investigate the effect of resveratrol on TGF-β expression in ESCs of patients \nwith endometriosis, and it is not possible to compare the results with similar studies. Therefore, the findings \nof this study were compared with those of animal studies on the effect of resveratrol on TGF-β levels in other \ndiseases. Most of these studies consistent with the present study have shown that resveratrol can decrease TGF-β \ngene and protein  expression28,55,56. In the only inconsistent study, a single-dose intraperitoneal injection of res-\nveratrol had no significant effect on TGF-β levels in rats with acute liver injury, possibly due to the amount and \ntiming of the  intervention57.\nResveratrol has been reported to inhibit TGF-β transcription by blocking the NF-κB  pathway55. Resveratrol \ncan also reduce TGF-β expression by blocking the activator protein 1 (AP-1) molecule and removing ROS and \nreducing oxidative  stress58. Resveratrol also down-regulates TGF-β expression and activity by down-regulating \nTGF-β signaling pathway molecules, including, Smad-2, 3,4 59. TGF-β is a pro-fibrotic factor that can increase \nthe production of type IV collagen and  fibrin28. Resveratrol treatment can prevent TGF-β-induced fibrotic tissue \ngrowth in ectopic  lesions55.\nThe present study is the first to assess the effect of resveratrol treatment on MMP-9 expression in ESCs. The \nonly animal study that investigated the effect of resveratrol on MMP-9 expression in endometriosis also reported \nthe same  results25. Other in-vivo and in-vitro studies also shown that resveratrol decreases MMP-9 mRNA and \nprotein expression and suppresses the activity of this  enzyme30,60,61. In the only inconsistent study, Gweon and \nKim reported that resveratrol at different concentrations increased the activity and expression of MMP-9 in \nhuman fibrosarcoma cells. The cause of this contradictory finding may be the different inflammatory  condition62.\nMMP-9 is one of the proteins whose expression is enhanced by activation of the NF-κB pathway. It appears \nthat resveratrol decreases the expression of this factor by suppressing the expression and activity of the NF-κB \n pathway60. Resveratrol inhibits NF-κB transcriptional activity by blocking phosphorylation and degradation of \nthe IκB inhibitor molecule, thereby inhibiting NF-κB translocation and DNA binding and preventing expression \nof inflammatory cytokines and growth factors and angiogenesis including MMP-963. Resveratrol can also prevent \nMMP-9 expression by decreasing TGF-β expression, inhibiting MAP-kinase signaling pathway, reabsorption of \nROS, and reducing oxidative  stress64,65.\nThe present study had some advantages and limitations: As we mentioned earlier, it was the first study \ninvestigated the basal gene and protein expression and also the effect of resverstrol treatment on the gene and \nprotein expression of VEGF , TGF-β and MMP-9 in ectopic (EESCs), and eutopic (EuESCs) endometrial stro-\nmal cells of women with endometriosis in comparison with non-endometriotic controls (CESCs). One of the \nlimitations was that the present study was carried out only in the severe (III and IV) stages of the EM and at \nthe proliferative phase. Also, it would have been better if we could assess the MMP-9 activity. It is also better to \ninvestigate the effect of resveratrol treatment on the expression of VEGF , TGF-β and MMP-9 in the peritoneal \nfluid mononuclear cells (PFMCs) and mesothelial cells as the important sources of these factors. Moreover, in \norder to better determine the effect of resveratrol on EM, further studies are needed on the effect of resveratrol \ntreatment on cell proliferation, angiogenesis, invasion, adhesion, apoptosis, and other processes involved in the \npathogenesis of EM.\nConclusion\nThe present study showed that the basal gene and protein expression of VEGF and MMP-9 were higher in EESCs \ncompared to EuESCs and CESCs. The treatment of EESCs and EuESCs with resveratrol could reduce the gene and \nprotein expression of VEGF , TGF-β, and MMP-9. Further in-vitro and in-vivo studies are needed to determine \nthe possible beneficial effects of resveratrol on EM progression.\nData availability\nAll data generated or analysed during this study are included in this published article (and its Supplementary \nInformation files).\n\n10\nVol:.(1234567890)Scientific Reports |         (2021) 11:6054  | https://doi.org/10.1038/s41598-021-85512-y\nwww.nature.com/scientificreports/\nReceived: 9 September 2020; Accepted: 2 March 2021\nReferences\n 1. Tyagi, S. et al. Pytochemicals as candidate therapeutics: an overview. Int. J. Pharm. Sci. Rev. Res. 3(1), 53–55 (2010).\n 2. Melanie-Jayne, H. & Monique, S. The role of phytochemicals as micronutrients in health and disease. Curr. Opin. Clin. Nutr. Metab. \nCare 17(6), 558–566 (2014).\n 3. Poulsen, M. et al. (2015) Resveratrol and inflammation: Challenges in translating pre-clinical findings to improved patient out-\ncomes. Biochimica et Biophysica Acta Mol Basis Dis 6, 1124–1136 (1852).\n 4. Harikumar, K. B. & Aggarwal, B. B. Resveratrol: a multitargeted agent for ageassociated chronic diseases. Cell Cycle 7(8), 1020–1035 \n(2008).\n 5. Fukui, M. et al. Mechanism for the protective effect of resveratrol against oxidative stress-induced neuronal death. Free Radical \nBiol. Med. 49(5), 800–813 (2010).\n 6. Noh, K. et al. Resveratrol suppresses tumor progression via the regulation of indoleamine 2,3-dioxygenase. Biochem. Biophys. Res. \nCommun. 431(2), 348–353 (2013).\n 7. Kasiotis, K. et al. Resveratrol and related stilbenes: Their anti-aging and anti-angiogenic properties. Food Chem. Toxicol. 61, 112–120 \n(2013).\n 8. Ashrafi, M. et al. Evaluation of Risk Factors Associated with Endometriosis in Infertile Women. Int J Fertil Steril 10(1), 11–21 \n(2016).\n 9. Laganà, A. et al. Unus pro omnibus, omnes pro uno: A novel, evidence-based, unifying theory for the pathogenesis of endome -\ntriosis. Med Hypotheses. 103, 10–20 (2017).\n 10. Sourial, S., Tempest, N. & Hapangama, D. K. Theories on the Pathogenesis of Endometriosis. Int. J. Reprod. Med. 2014, 179515 \n(2014).\n 11. Gupta, S. et al. Role of oxidative stress in endometriosis. Reprod. Biomed. Online 13(1), 126–134 (2006).\n 12. Burney, R. O. & Giudice, L. C. Pathogenesis and pathophysiology of endometriosis. Fertil. Steril. 98(3), 511–519 (2012).\n 13. Gazvani, R. & Templeton, A. Peritoneal environment, cytokines and angiogenesis in the pathophysiology of endometriosis. Repro-\nduction 123, 217–226 (2002).\n 14. McLaren, J. et al. Vascular endothelial growth factor (VEGF) concentrations are elevated in peritoneal fluid of women with endo-\nmetriosis. Hum Reprod. 11(1), 220–223 (1996).\n 15. Mahnke, J., Dawood, M. & Huang, J. Vascular endothelial growth factor and interleukin-6 in peritoneal fluid of women with \nendometriosis. Fertil Steril. 73(1), 166–170 (2000).\n 16. Hull, M. et al. Host-derived TGFB1 deficiency suppresses lesion development in a mouse model of endometriosis. Am J Pathol  \n180, 880–887 (2012).\n 17. Y oung, V . J. et al. The role of TGF-β in the pathophysiology of peritoneal endometriosis. Hum. Reprod. Update  23(5), 548–559 \n(2017).\n 18. Kupker, W ., Schultze-Mosgau, A. & Diedrich, K. Paracrine changes in the peritoneal environment of women with endometriosis. \nHum. Reprod. 4, 719–723 (1998).\n 19. London, C. A. et al. A novel antisense inhibitor of MMP-9 attenuates angiogenesis, human prostate cancer cell invasion and \ntumorigenicity. Cancer Gene Ther. 10, 823–832 (2003).\n 20. Weigel, M. T. et al. Differential expression of MMP-2, MMP-9 and PCNA in endometriosis and endometrial carcinoma. Eur. J. \nObst. Gynecol. Reprod. Biol. 160, 74–78 (2012).\n 21. Machado, D. E. et al. Higher expression of vascular endothelial growth factor (VEGF) and its receptor VEGFR-2 (Flk-1) and \nmetalloproteinase-9 (MMP-9) in a rat model of peritoneal endometriosis is similar to cancer diseases. J. Experim. Clin. Cancer \nRes. 29(4), 1 (2010).\n 22. Kolahdouz-Mohammadi, R. & Arablou, T. Resveratrol and endometriosis: In vitro and animal studies and underlying mechanisms \n(Review). Biomed. Pharmacother. 91, 220–228 (2017).\n 23. Arablou, T. et al. Resveratrol reduces the expression of insulin-like growth factor-1 and hepatocyte growth factor in stromal cells \nof women with endometriosis compared with nonendometriotic women. Phytother. Res. 33(4), 1044–1054 (2019).\n 24. Rudzitis-Auth, J., Menger, M. D. & Laschke, M. W . Resveratrol is a potent inhibitor of vascularization and cell proliferation in \nexperimental endometriosis. Hum. Reprod. 28(5), 1339–1347 (2013).\n 25. Tekin, Y . B. et al. Is resveratrol a potential substitute for leuprolide acetate in experimental endometriosis?. Eur. J. Obst. Gynecol. \nReprod. Biol. 184, 1–6 (2015).\n 26. Taguchi, A. et al. Resveratrol Enhances Apoptosis in Endometriotic Stromal Cells. Am J Reprod Immunol. 75(4), 486–492 (2016).\n 27. Y avuz, S. et al. Resveratrol successfully treats experimental endometriosis through modulation of oxidative stress and lipid per -\noxidation. J. Cancer Res. Ther. 10(2), 324–329 (2014).\n 28. Rahal, K. et al. Resveratrol has antiinflammatory and antifibrotic effects in the peptidoglycan-polysaccharide rat model of Crohn’s \ndisease. Inflamm. Bowel Dis. 18(4), 613–623 (2012).\n 29. Nagineni, C. N. et al. Resveratrol suppresses expression of VEGF by human retinal pigment epithelial cells: potential nutraceutical \nfor age-related macular degeneration. Aging Dis. 5(2), 88–100 (2014).\n 30. Cheng, G. et al. Resveratrol inhibits MMP-9 expression by up-regulating PPAR α expression in an oxygen glucose deprivation-\nexposed neuron model. Neurosci. Lett. 451(2), 105–108 (2009).\n 31. AmericanFertilitySociety, Revised American. Fertility Society classification of endometriosis. Fertil Steril  43, 351–352 (1985).\n 32. Rashidi, N. et al. Lipopolysaccharide- and lipoteichoic acid-mediated pro-inflammatory cytokine production and modulation of \nTLR2, TLR4 and MyD88 expression in human endometrial cells. J. Reprod. Infertil. 16, 72–81 (2015).\n 33. Ying-ying, W . & Xiao-ling, F . The expression of hepatocyte growth factor (HGF) and vascular epithelial growth factor (VEGF) in \nperitoneal fluid of patients with endometriosis. J. Clin. Res. 1, 1 (2007).\n 34. Sokolov, D. I. et al. Study of cytokine profile and angiogenic potential of peritoneal fluid in patients with external genital endome-\ntriosis. Bull. Exp. Biol. Med. 140(5), 541–544 (2005).\n 35. Pizzo, A. et al. Behaviour of cytokine levels in serum and peritoneal fluid of women with endometriosis. Gynecol. Obstet. Invest.  \n54, 82–87 (2002).\n 36. Sel’kov, S. A. et al. Local production of interleukins and growth factors in external genital endometriosis. Bull. Experim. Biol. Med. \n139(4), 444–447 (2005).\n 37. Y erlikaya, G. et al. Comprehensive study of angiogenic factors in women with endometriosis compared to women without endo-\nmetriosis. Eur. J. Obst. Gynecol. Reprod. Biol. 204, 88–98 (2016).\n 38. Rocha, A. L. L., Reis, F . M. & Taylor, R. N. Angiogenesis and Endometriosis. . Obst. Gynecol. Int. 2013, 1 (2013).\n 39. Kuroki, M. et al. Reactive oxygen intermediates increase vascular endothelial growth factor expression in vitro and in vivo. J. Clin. \nInvest. 98(7), 1667–1675 (1996).\n 40. Y oung, V . J. et al. Transforming growth factor-b induced Warburg-likemetabolic reprogramming may underpin the development \nof peritoneal endometriosis. Clin. Endocrinol. Metab. 99(9), 3450–3459 (2014).\n\n11\nVol.:(0123456789)Scientific Reports |         (2021) 11:6054  | https://doi.org/10.1038/s41598-021-85512-y\nwww.nature.com/scientificreports/\n 41. Y oung, V . J. et al. The peritoneum is both a source and target of TGF-b in women with endometriosis. PLoS ONE 9(9), e106773 \n(2014).\n 42. Zhao, W . et al. Oxidative stress mediates cardiac fibrosis by enhancing transforming growth factor-beta1 in hypertensive rats. Mol. \nCell. Biochem. 317(1–2), 43–50 (2008).\n 43. Reddy, K. B. et al. Mitogen-activated protein kinase (MAPK) regulates the expression of progelatinase B (MMP-9) in breast epi -\nthelial cells. Int. J. Cancer 82(2), 268–273 (1999).\n 44. Yu, F. et al. Induction of MMP-9 expression and endothelial injury by oxidative stress after spinal cord injury. J. Neurotrauma \n25(3), 1 (2008).\n 45. Zheng, H. et al. Expressions of MMP-2, MMP-9 and VEGF are closely linked to growth, invasion, metastasis and angiogenesis of \ngastric carcinoma. Anticancer Res. 26, 3579–3584 (2006).\n 46. Garvin, S., Llinger, K. O. & Dabrosin, C. Resveratrol induces apoptosis and inhibits angiogenesis in human breast cancer xenografts \nin vivo. Cancer Lett. 231, 113–122 (2006).\n 47. Zhang, H. & Y ang, R. Resveratrol inhibits VEGF gene expression and proliferation of hepatocarcinoma cells. Hepatogastroenterol-\nogy 61(130), 410–412 (2014).\n 48. Yu, H. et al. Resveratrol inhibits VEGF expression of human hepatocellular carcinoma cells through a NF-kappa B-mediated \nmechanism. Hepatogastroenterology 57(102–103), 1241–1246 (2010).\n 49. Zehai, T., Xin-yue, L. & Ping, Z. Resveratrol Inhibits the Secretion of Vascular Endothelial Growth Factor and Subsequent Prolif-\neration in Human Leukemia U937 Cells. J. Huazhong Univ. Sci. Technol. 27(5), 508–512 (2007).\n 50. Kundu, O. K. & Surh, Y .-J. Cancer chemopreventive and therapeutic potential of resveratrol: mechanistic perspectives. Cancer \nLett. 269, 243–261 (2008).\n 51. Saijonmaa, O. et al. Upregulation of angiotensin-converting enzyme by vascular endothelial growth factor. Am. J. Physiol. Heart \nCirc. Physiol. 280, H885–H891 (2001).\n 52. Lastra, C. A. N. D. L. & Villegas, I. Resveratrol as an antioxidant and pro-oxidant agent: mechanisms and clinical implications. \nBiochem. Soc. Trans. 35(5), 1156–1160 (2007).\n 53. Delbandi, A.-A. et al. Eutopic and ectopic stromal cells from patients with endometriosis exhibit differential invasive, adhesive, \nand proliferative behavior. Fertil. Steril. 100(3), 761–769 (2013).\n 54. Delbandi, A.-A. et al. 1,25-dihydroxy vitamin D3 modulates endometriosis-related features of human endometriotic stromal cells. \nAm. J. Reprod. Immunol. 75, 461–473 (2016).\n 55. Chávez, E. et al. Resveratrol prevents fibrosis, NF-κB activation and TGF-β increases induced by chronic CCl4 treatment in rats. \nJ. Appl. Toxicol. 28, 35–43 (2008).\n 56. Chen, K.-H. et al. Resveratrol ameliorates early diabetic nephropathy associated with suppression of augmented TGF-β/smad and \nERK1/2 signaling in streptozotocin-induced diabetic rats. Chem. Biol. Interact. 190(1), 45–53 (2011).\n 57. Chan, C.-C. et al. Regulation by resveratrol of the cellular factors mediating liver damage and regeneration after acute toxic liver \ninjury. J. Gastroenterol. Hepatol. 29, 603–613 (2014).\n 58. Kim, S. et al. Autoinduction of transforming growth factor 13 is mediated by the AP-1 complex. Mol. Cell. Biol. 10(4), 1492–1497 \n(1990).\n 59. Whyte, L. et al. Molecular mechanisms of resveratrol action in lung cancer cells using dual protein and microarray analyses. Cancer \nRes. 67(24), 12007–12017 (2007).\n 60. Banerjee, S., Bueso-Ramos, C. & Aggarwal, B. B. Suppression of 7,12-dimethylbenz(a)anthracene-induced mammary carcinogen-\nesis in rats by resveratrol: role of nuclear factor-kappaB, cyclooxygenase 2, and matrix metalloprotease 9. Can. Res. 62, 4945–4954 \n(2002).\n 61. Sun, C.-Y . et al. Resveratrol as a novel agent for treatment of multiple myeloma with matrix metalloproteinase inhibitory activity. \nActa Pharmacol. Sin. 27(11), 1447–1452 (2006).\n 62. Gweon, E. J. & Kim, S. J. Resveratrol induces MMP-9 and cell migration via the p38 kinase and PI-3K pathways in HT1080 human \nfibrosarcoma cells. Oncol. Rep. 29, 826–834 (2013).\n 63. Kundu, J. K. & Surh, Y .-J. Molecular basis of chemoprevention by resveratrol:NF-B and AP-1 as potential targets. Mutat. Res. 555, \n65–80 (2004).\n 64. Yu, R. et al. Resveratrol inhibits phorbol ester and UV-induced activator protein 1 activation by interfering with mitogen-activated \nprotein kinase pathways. Mol. Pharmacol. 60(1), 217–224 (2001).\n 65. Kim, E.-S., Kim, M.-S. & Moon, A. TGF-β-induced upregulation of MMP-2 and MMP-9 depends on p38 MAPK, but not ERK \nsignaling in MCF10A human breast epithelial cells. Int. J. Oncol. 25(5), 1375–1382 (2004).\nAcknowledgements\nWe thank all the participants in the present study. The study was funded by Iran University of Medical Sciences \nwith the grant number 28107.\nAuthor contributions\nT.A., R.K.M., N.A. and A.A.D. designed the project and planned the experiments. S.K.H. contributed to sample \npreparation. T.A., R.K.M., Z.M. and N.R. carried out the experiments. T.A., A.A.D., R.K.M., and Z.M. contrib-\nuted to the data analysis and interpretation of the results. T.A. wrote the first draft of the manuscript. N.A. and \nA.A.D. critically reviewed the paper. A.A.D. supervised the project. All authors read and approved the final \nversion of the manuscript.\nCompeting interests \nThe authors declare no competing interests.\nAdditional information\nSupplementary Information The online version contains supplementary material available at https ://doi.\norg/10.1038/s4159 8-021-85512 -y.\nCorrespondence and requests for materials should be addressed to A.-A.D.\nReprints and permissions information is available at www.nature.com/reprints.\nPublisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and \ninstitutional affiliations.\n\n12\nVol:.(1234567890)Scientific Reports |         (2021) 11:6054  | https://doi.org/10.1038/s41598-021-85512-y\nwww.nature.com/scientificreports/\nOpen Access  This article is licensed under a Creative Commons Attribution 4.0 International \nLicense, which permits use, sharing, adaptation, distribution and reproduction in any medium or \nformat, as long as you give appropriate credit to the original author(s) and the source, provide a link to the \nCreative Commons licence, and indicate if changes were made. The images or other third party material in this \narticle are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the \nmaterial. If material is not included in the article’s Creative Commons licence and your intended use is not \npermitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from \nthe copyright holder. To view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/4.0/.\n© The Author(s) 2021","source_license":"CC0","license_restricted":false}