{"paper_id":"008b7566-560f-4751-b580-074c9216fc71","body_text":"Endometriosis is a chronic disorder characterised by abnormal growth and adherence of functional endometrial tissue outside the uterine cavity. It is a common cause of chronic pelvic pain and infertility in reproductive-aged women,\nwith estimates suggesting that 20-50% of infertile women\nsuffer from endometriosis and 30-50% of women with endometriosis are sub-fertile or infertile ( 1 ). Chaichian et al.\n( 2 ) reported that the prevalence of endometriosis among 441\ninfertile Iranian women who underwent laparoscopy is 18.6.\nGenetic and epigenetic factors play important roles in the development of endometriosis\n( 3 ). DNA methyltransferases (DNMTs), namely  DNMT1, DNMT3A  and\n DNMT3B  that maintain CpG methylation, along with histone deacetylases\n(HDACs), play central roles in epigenetic regulation of cancers ( 4 ). Previous studies found\nthat aberrant expressions of the  DNMT1, DNMT3A, DNMT3B,  and\n HDAC1  genes were more prominent in women with endometriosis, as this\ndisease exhibits malignant-like features ( 5 ).\nOverexpression of cyclooxygenase-2 (<italic>COX-2</italic>) and the subsequent increase in\nprostaglandin E2 level are the main causes of dysmenorrhea and recurrence of endometriotic\nlesions ( 6 ). Matrix metalloproteinase-2 ( MMP2 ) is another well-known factor\nthat plays a role in endometriosis development ( 7 ).  MMP2  contributes to the\ndegradation of collagens and other components of the extracellular matrix and basement\nmembrane, which results in disseminated malignancies ( 8 ). A similar mechanism is likely\ninvolved in the pathogenesis of endometriosis ( 7 ).\nDespite various medical and surgical strategies used for\nthe management of endometriosis, current approaches are\nnot completely effective and often have major side effects\n( 9 ). Therefore, novel medical agents obtained from traditional medicine may prove effective in targeting the biological pathways involved in endometriosis and improve\ntreatment outcomes.\nCeratonia siliqua L ., commonly known as carob, is an evergreen tree that\nbelongs to the Le-guminosae family, and is widely grown in Mediterranean countries. It has\nbeen used in folk medicine to treat diabetes mellitus, dyslipidaemia, and various\ngastrointestinal ailments ( 10 ). Furthermore, indigenous people in Middle Eastern countries\nhave used this natural product since ancient times to improve fertility ( 11 ). The main\nconstituents of  Ceratonia siliqua  are gum, protein, and polyphenols, of\nwhich the latter is mainly responsible for anti-inflammatory, antioxidant, and anti-ulcer\nactivities ( 12 ). Elbouzidi et al. ( 13 ) evaluated the antioxidant and antimicrobial\nproperties of carob to inhibit the proliferation of breast cancer cells (MCF-7, MDA-MB-231,\nand MDA-MB-436) and studied its genotoxicity. It has been traditionally used for medicinal\npurposes ( 10 ,  12 ); moreover, there is limited research on the potential uses of carob, and\nthey are often considered a by product of carob production.\nThe aim of this study is to investigate the potential therapeutic effect of carob\n(<italic>Ceratonia si-liqua L</italic>.) pod extract (CPE) on gene and peptide expressions\nof  DNMT1, DNMT3A, DNMT3B, HDAC1, MMP2 , and  COX-2  in\nendometrial mesenchymal stromal/stem cells (EnMSCs) isolated from women with\nendometriosis-associated infertility (EAI).\n\nIn this experimental study, EnMSCs were obtained from\nthe endometrium, endometrioma, and deep infiltrative endometriosis (DIE) samples of 10 women diagnosed with\nEAI and compared to EnMSCs derived from the endometrium of one endometriosis-free normal woman as the control group (C-EnMSCs). DIE samples were obtained from\nthe rectovaginal septum and deep retroperitoneal portions\nof the women with EAI, and ectopic endometrial cells were\nderived from their ovarian endometrioma (OMA).\nThese 10 women were candidates for laparoscopic\nendometriosis surgery who referred to the Laparoscopy\nClinic of the Obstetrics and Gynaecology Department at\nShiraz University of Medical Sciences (Shiraz, Iran) from\n2018 to 2019. Women who received gonadotropin-releasing hormone (GnRH) agonist, progesterone, or other hormones three months before the study were excluded. All\nincluded cases had surgical and histological evidence of\nstage III or IV endometriosis according to the American\nSociety of Reproductive Medicine classification ( 14 ). Endometrial samples were obtained by dilatation and curettage from the endometrial cavity as ectopic endometrial\ncells. Samples of eutopic endometrial cells were taken\nfrom OMA and DIE lesions by laparoscopic intervention. Eutopic and ectopic endometrial samples were taken by\nthe laparoscopic surgical team of Shiraz University of\nMedical Sciences. Eutopic endometrial cells were also\nobtained from a normal, endometriosis-free woman who\nwas scheduled for benign gynaecologic surgery by dilatation and curettage to serve as the C-EnMSCs.\nThe cellular samples were classified into three groups\n( Table 1 ). Group 1 consisted of eutopic endometrial cells\nderived from the EAI women (E-EnMSCs); group 2 included ectopic endometrial cells derived from OMA and\nDIE of the EAI women (OMA-EnMSCs.); and group 3\nincluded eutopic endometrial cells taken from a normal,\nendometriosis-free woman (C-EnMSCs.).\nGrouping of different samples of endometrial mesenchymal stromal/stem cells (EnMSCs) for evaluation of different  Ceratonia siliqua L .\npod extract (CPE) concentrations\nCPE was purchased from an herbal market in Shiraz, Iran. The plant material was\nauthenticated by a botanist and assigned voucher specimen number (PM-1292), and deposited\nin the herbarium of the Phytopharmaceutical Department, Shiraz University of Medical\nSciences, Shiraz, Iran. The CPE was prepared as follows:  Ceratonia\nsiliqua  pods were ground into a powder and sieved through a 50 µm mesh. Then,\n100 g of the prepared CPE was added to 500 mL of 70% ethanol and the suspension was shaken\nat room temperature for 24 hours. After filtration, the supernatant, at a concentration of\n200 000 μg/mL, was stored at -20°C for the next steps of the study. Gregoriou et al. ( 15 )\nshowed that the total phenolic content (TPC) varies according to the maturity of the\nplant.\nThe total phenolic content (TPC) of the CPE was evaluated by the Folin-Ciocalteu method\n( 16 ). Briefly, a 5 μL aliquot of the CPE (at different concentrations in methanol) was\nmixed with 158 μL of dH 2  O followed by the addition of 10 μL of Folin-Ciocalteu\nreagent. Then, 30 μL of 15% sodium carbonate was added. The mixture was incubated at room\ntemperature for 45 minutes in the dark and the absorbance was measured at 765 nm using a\nmicroplate reader (POLARstar Omega, BMG Labtech, Germany). A standard curve was generated\nbased on a prepared standard gallic acid solution (0, 10, 50, 100, 150, 250, and 500\nmg/mL), which revealed a linear regression of R2 >0.99. The phenolic level was expressed\nas milligrams of gallic acid equivalents (GAE) per gram of dry plant extract (mg GAE/g dry\nextract). Each sample was assessed in triplicate ( 17 ).\nThe CPE was lyophilised and analysed with an Agilent\n7890B Gas chromatography-mass spectroscopy (GC-MS)\nsystem. Electron ionisation mass spectra were obtained\nwith a scan range of m/z 50-500 using electrons with\nan energy of 70 eV and a filament emission of 0.5 mA.\nSeparation was carried out using an HP-5MS UI column\nwith dimensions of 30 m×0.25 mm i.d. and a film thickness of 0.5 μm. Helium was used as a carrier gas with a\nflow rate of 0.8 mL/minutes. The GC oven temperature\nwas increased at a rate of 5°C/minutes from 80°C after\n3 minutes of sample injection and held at 250°C for 10\nminutes. The transfer line, ion source, and injection port\nof the gas chromatograph of 5977MSD were maintained\nat 250°C, 220°C and 240°C, respectively. The separated\ncompounds were identified by comparing them with the\ncompound data in the National Institute of Standards and\nTechnology (NIST MS database) library ( 18 ).\nThe  in vitro  antioxidant activity of the CPE was measured based on the\ndecolourisation of the 2,2-Diphenyl1-picrylhydrazyl (DPPH) as described previously ( 19 ).\nIn brief, 20 μL of different concentrations of the crude CPE or quercetin, as the positive\ncontrol, were added to 180 μL of 110 µM DPPH radical dissolved in methanol. The reagent\nwas then incubated in the dark for 30 minutes. Methanol was used as a blank solution. The\npale-yellow colour of the reduced DPPH radical was measured at an optical density of 517\nnm at room temperature using a microplate reader (POLARstar Omega, BMG Labtech, Germany).\nThe volume of an-tioxidant required to produce a 50% reduction of the DPPH was recorded as\nthe half maximal inhibitory concentration (IC 50 ) value. A lower IC 50 \nvalue indicated a stronger DPPH scavenging capacity ( 20 ).\nProliferation capacity and cell viability of the E-EnMSCs,\nOMA-EnMSCs, and C-EnMSCs were determined using\nthe MTT assay (Sigma-Aldrich, USA). Briefly, cells were\nseeded in 96-well plates at a density of 5000 cells/well. After\n24 hours of incubation, different concentrations of CPE (0-\n20 000 μg/mL) were added to the wells and incubated for\none, three, and seven days. At the end of each incubation\nperiod, MTT solution (5 mg/mL) was added to the medium\nand the cells were further incubated for another four hours\nat 37°C. The MTT solution was then replaced with 200 μL of dimethyl sulfoxide for 30 minutes. The absorbance was\nmeasured at 570 nm using an ELISA plate reader (Bio‐Rad,\nHercules, CA, USA). The cell viability percentage was calculated using the following formula: (%)=[100×(sample\nabsorbance)/(control absorbance)]. Each test was repeated\nthree times. The means of the data were calculated, and CPE\nconcentrations of 0.8 and 2 μg/mL were selected based on\nthe viability results and compared to the C-EnMSCs ( 21 ).\nThe EnMSCs were isolated according to modifications to previously described methods ( 22 ).\nBriefly, tissues were minced into small pieces of about 1-2 mm 3  and digested\nwith 1 mg/mL of collagenase type 1 (Cat. no. 17100-017, Gibco, USA) in Dulbecco’s\nmodified Eagle medium (DMEM, Biovet, Bulgaria) that contained 10% foetal bovine serum\n(FBS, Biovet, Bulgaria) for one hour with constant stirring. The resultant cell suspension\nwas passed through a 40 µm nylon sieve to remove undigested aggregates. Red blood cells\nwere removed using Ficoll (Ref. 002041600, Gibco, USA). The cell suspension was then\nseeded in T25 culture flasks and incubated in DMEM/F12 medium (Sigma‐Aldrich, UK) that\ncontained 10% FBS, 100 μg/mL penicillin, and 100 U/mL streptomycin (Sigma, USA) until the\ncells reached 80% confluency. These cells were used for the subsequent experiments.\nInterestingly, the DIE cells did not show any growth and proliferation in the culture\nmedium, and no stem cells could be obtained from those samples. However, the ESC isolation\nprocedure was successfully performed on eutopic endometrial cells from the EAI, normal\ncontrol women, and ectopic OMA.\nNext, we assessed cell purity in the three groups (E-EnMSCs, OMA-EnMSCs, and C-EnMSCs). Flow cytometry was\nused to categorise the passage-3 cells based on their expressions of cell surface markers. The resultant cell suspension\nwas washed in blocking solution, cold phosphate-buffered\nsaline (PBS) that contained 10% FBS, for 20 minutes. Then,\nthe cells were labelled with FITC-conjugated anti-CD45,\nanti-CD90, anti-CD73, and an-ti-CD31 antibodies (all from\nAbcam, Cambridge, UK). Subsequently, the cells were\nwashed twice and resuspended in cold PBS. The percentage\nof positive or negative cells was evaluated using a calibrated\nFACS device (FACSCalibur™, BD Biosciences) and analysed by FlowJo software (BD Biosciences).\nNext, we evaluated the multipotency of the isolated cells by differentiating them into\ntwo mesenchymal lineagesosteocytes and adipocytes. Briefly, 1×10 4  passage-3\ncells/ cm 2  were cultured in 24-well plates. Once the cells reached\napproximately 80% confluency, the growth medium was changed to osteogenic medium (DMEM-LG\nsupplemented with 10% FBS, 2 mM L-glutamine, 100 U/mL penicillin, 100 μg/mL streptomycin,\n100 nM dexamethasone, 0.2 mM L-ascorbate, and 10 mM β-glycerophosphate) and adipogenic\nmedium (DMEM-LG supplemented with 10% FBS, 2 mM L-glutamine, 100 U/mL penicillin, 100\nμg/mL streptomycin, 60 μM indomethacin, 1 μM dexamethasone, 0.5 mM IBMX, and 5 μg/mL\ninsulin solution). The osteogenic and adipogenic cells were incubated for 28 and 21 days,\nrespectively, with half of the medium replaced every three days. The differentiated cell\nlines were fixed in 4% paraformaldehyde and stained with Alizarin Red S and Oil Red O to\nobserve differentiated osteoblasts and adipocytes, respectively ( 23 ).\nTotal RNA was extracted from the E-EnMSCs, OMAEnMSCs, and C-EnMSCs after seven days of\nexposure to CPE using a FavorPrep™ Blood/Cultured Cell Total RNA kit (Favorgen, Ping-Tung,\nTaiwan) according to the manufacturer’s instructions. The quantity and quality of the\nobtained RNA were measured using a Nanodrop™ spectrophotometer (Thermo Fisher Scientific,\nWilmington, DE, USA) based on a 260/280 nm optical density ratio, and then stored at -80˚C\nuntil cDNA synthesis. cDNA synthesis was performed using a RevertAid™ First Strand cDNA\nSynthesis kit (Thermo Fisher Scientific, Inc., Waltham, MA, USA) according to the\nmanufacturer’s protocols. The synthesized cDNA was used as a template for PCR\namplification. The expression levels of  MMP2, COX-2, HDAC1, DNMT1,\nDNMT3A , and  DNMT3B  were quantified by realtime PCR, which was\nperformed using an ABI Biosystems StepOne and RealQ Plus 2x Master Mix Green (Ampliqon\nA/S, Odense, Denmark). Primers were designed based on human DNA sequences obtained from\nthe gene bank Primer-Blast online program ( Table S1 , See Supplementary Online Information\nat  www.ijfs.ir ). The housekeeping gene TATA-binding protein (TBP) was used as a reference\nto normalise the expression values. The following quantitativePCR conditions were used: 10\nminutes at 94˚C, followed by 40 cycles of 15 seconds at 94˚C, 60 seconds at 60˚C, and\nfinally a melting curve stage was performed to determine the specificity of the product.\nAdditionally, to confirm the results, real-time PCR products were analysed on 2% ultrapure\nagarose gel electrophoresis (Sigma, USA) according to the manufacturer’s instructions\n( 24 ).\nBased on the results of the MTT cell viability and proliferation assay, the most effective concentrations of the CPE\n(0.8 and 2 μg/mL) were selected and used for Western blot\nanalysis. Specifically, the E-EnMSCs, OMA-EnMSCs, and\nC-EnMSCs were treated with CPE; after 72 hours, the cells\nwere lysed with RIPA buffer that consisted of 50 mM trisHCl (pH=8.0), 0.4% Nonidet P-40, 120 mM NaCl, 1.5 mM\nMgCl2, 2 mM phenylmethylsulfonyl fluoride, 80 μg/mL leupeptin, 3 mM NaF, and 1 mM DTT, at 4°C for 20 minutes.\nThe lysed components were then centrifuged at 12 000 ×g for 20 minutes at 4°C. The protein content of the resultant solution was measured by the Bradford protein assay. The proteins were then transferred to microporous polyvinylidene\ndifluoride membranes (Millipore, France), which were incubated with 5% BSA (Sigma, USA) as a blocking buffer for\none hour at room temperature. Subsequently, the membranes\nwere incubated overnight at 4°C with primary antibodies.\nSamples were immunoblotted (1:200) with anti-beta-actin,\nanti-MMP2, anti-COX-2, anti-HDAC1, anti-DNMT1, antiDNMT3A, and anti-DNMT3B antibodies (Cell Signaling\nTechnology, Danvers, MA, USA). The membranes were\nwashed three times (10 minutes each) with Tween buffer,\nand then incubated with horseradish peroxidase (HRP)-\nconjugated goat anti-mouse or rabbit secondary antibodies.\nAfter four washes, the excess antibodies were removed from\nthe membranes, and HRP activities were detected using ECL\nPlus Chemiluminescence Reagent (Amersham, Chalfont,\nUK) according to the manufacturer’s protocol.\nWe selected 62 compounds detected in the CPE and six proteins for the docking process.\nThe 3D structure of the ligands and receptors were obtained from the PubChem databases\nof the Protein Data Bank (PDB). The downloaded receptors included  COX-2, MMP2,\nDNMT3B, DNMT1, DNMT3A , and  HDAC1  with PDB codes 3nt1, 3ayu,\n3flg, 3os5, 4qbs, and 4bkx. The geometry of the ligands was optimised using HyperChem\nsoftware version 8.0.10. The receptors were prepared using Chimera 1.15 by removing all\nnon-standard residues, water, and original hydrogens, and by adding polar hydrogen,\ncharges, and bond orders. Lastly, the outputs were suitably formatted for the docking\nprocess.\nThe generation of a grid box is a crucial step in the\ndocking process. Grid boxes with spacing of 0.375 Å\nwere generated using AutoGrid at the active site of each\nreceptor selected based on CASTp analysis.\nAutoDock Vina 1.1.2 was used to investigate the interactions between the target receptors and selected ligands.\nAfter completing the docking process, the conformation\nwith the lowest binding affinity and RMSD ≤2 Å was selected as the best.\nThe following critical parameters were considered in\nthe sample size calculation. Standard deviation (σ): The\nstandard deviation reflects the variability within each\ntreatment group. We obtained estimates of the standard\ndeviation for each specific gene expression of interest\nfrom preliminary experiments.\nDesired effect size (δ): The effect size represents the\nminimum difference in gene expression that we considered to have biological and clinical significance. It was\ndetermined based on prior research findings and expected\nbiological significance.\nLevel of confidence (α): We selected a 95% confidence level,\nwhich corresponded to a sig-nificance level (α) of 0.05, and indicated our willingness to accept a 5% chance of a type I error.\nPower (1-β): We aimed for a statistical power (1-β) of\n80% or higher, which represented our ability to correctly\ndetect true effects with a type II error rate of 20% or less.\nThe sample size for each treatment group was calculated using the following formula:\nn = (2(Z α/2 +Z β ) 2  ×(σ 2 ))/(δ 2 )\nWhere:\nn=Required sample size per treatment group.\nZ α/2 =Z-score for the chosen level of confidence (e.g., 1.96 for a 95%\nconfidence level).\nZβ\n=Z-score for the desired statistical power (e.g., 0.84\nfor an 80% power).\nσ=Estimated standard deviation.\nδ=Desired effect size.\nFinal sample size: We calculated the required sample size\nfor each treatment group after plug-ging in the specific values for each gene of interest and using the above formula.\nAll statistical analyses were performed using IBM SPSS\nStatistics 26 software (SPSS Inc., Chicago, IL, USA). Results are presented as mean ± standard deviation. Differences\nbetween groups were evaluated by one-way ANOVA followed by the Duncan test. In order to ensure the validity of\nour one-way ANOVA analyses, we rigorously assessed the\nkey assumptions, including independence of observations,\nnormality of residuals, homogeneity of variances, and independence of groups, as detailed in the Assumption Checks\nsubsection. P<0.05 indicated statistical significance. Graphs\nwere prepared using GraphPad Prism software (v7.0a,\nGraphPad Software, Inc., San Diego, CA, USA).\nThe study was approved by the Institutional Review Board\nand the Ethics Committee of Shiraz University of Medical\nSciences, Shiraz, Iran (IR.SUMS.REC.1397.555). Informed\nconsent was obtained from all donors after a thorough explanation of the study design prior to sample collection.\n\nThe results showed that the human EnMSCs in all three groups were positive for the standard mesenchymal markers CD73 and CD90, and negative for the hematopoietic\nmarker CD45 and endothelial marker CD31. Surface antigen marker analysis of the endometrial cell cultures revealed relatively homogenous cell populations that were\nfree from contaminant cells ( Fig .S1A , See Supplementary\nOnline Information at www.ijfs.ir).\nThe EnMSCs in the C-EnMSCs were plastic adherent\ncells that exhibited a spindle-shaped, fibroblast-like morphology with radial or helical growth patterns ( Fig .S1B ,\nSee Supplementary Online Information at www.ijfs.ir). EEnMSCs and OMA-EnMSCs isolated from different patients also showed typical spindle-shaped and fibroblastlike morphological features.\nThe C-EnMSCs, E-EnMSCs, and OMA-EnMSCs cultured in osteogenic and adipogenic media for one month\nand three weeks, respectively, were positive for Alizarin\nRed S and Oil Red O staining. The red brilliant stain in the\nculture plate indicated the calcium deposits by EnMSCs\nthat had differentiated into osteoblasts. The lipid droplets\nappeared in the cytoplasms of the EnMSCs, which confirmed their multipotent capabilities ( Fig .S1B , See Supplementary Online Information at www.ijfs.ir).\nPolyphenols are one of the important constituents of hydrochloric CPE. Therefore, the TPC was evaluated using\nthe Folin-Ciocalteu assay. The results revealed the presence of 18.13 ± 2.47 mg of pure phenol in 1 g of dry CPE.\nSeveral compounds with different biological activities\nwere observed in the GC-MS profile of the CPE ( Fig .S2 ,\nSee Supplementary Online Information at www.ijfs.ir). In\ndetail, 60 compounds were identified in the CPE, four of\nwhich had anticancer biological activities based on previous studies. Moreover, four compounds had cytotoxic\neffects, and another four showed antioxidant properties\nin accordance with previous studies. In addition, six of\nthe CPE compounds had anti-inflammatory biological effects. Other biological activities were also observed in the\nCPE compounds and included antimicrobial, antiseptic,\nand muscle relaxant effects based on previous studies ( Table S2 , See Supplementary Online Information at  www.\nijfs.ir ).\nThe antioxidant potency and reduction capability of the DPPH radicals were determined by\na reduction in absorbance at 517 nm that was induced by the antioxidants. A high\nantioxidant capacity was reported for the CPE, with an IC50 value of 0.63 ± 0.11 mg/mL,\ncompared to quercetin (positive control), which showed an IC 50  value of 1.40 ±\n2.89 mg/mL.\nThe cell viability percentages of EnMSCs exposed to\nvarious concentrations of CPE after one, three, and seven days were repeated three times, and the mean values\nwere calculated. CPE concentrations higher than 2 μg/\nmL did not result in acceptable EnMSC viability after\nseven days and were excluded. EnMSC viability in CPE\nconcentrations of less than 2 μg/mL were not significantly different. Thus, we chose the 2 and 0.8 μg/mL concentrations based on the proliferation and viability results\nfor the following gene studies. One-way ANOVA and the\nDuncan test showed a statistically significant decrease in\nproliferation and viability between the 2 and 0.8 μg/mL\nconcentrations in C-EnMSCs compared to the untreated\n(0 μg/mL) group ( Fig .1 ).\nEndometrial mesenchymal stromal/stem cell (EnMSCs) viability ac- cording to the MTT assay results with different  Ceratonia siliqua L . pod\nextract (CPE) concentrations. Lines above the columns indicate statisti- cally significant differences. Data are analysed by one-way ANOVA and the\nDuncan test (mean ± SD). *; P<0.05, C-EnMSCs; Control-EnMSCs, E-EnM- SCs; Endometriosis-EnMSCs, and OMA-EnMSCs; Endometrioma-EnMSCs.\nTotal RNA was extracted from E-EnMSCs, OMAEnMSCs, and C-EnMSCs after seven days of exposure\nto CPE. cDNA synthesis was performed, and quantitative\nPCR was used to determine gene expression levels. Gene\nexpression levels at CPE concentrations of 0.8 μg/mL and\n2 μg/mL were compared to the C-EnMSCs ( Fig .2 ).\nTreatment with 0.8 μg/mL of CPE significantly reduced  MMP-2  gene\nexpression in OMA-EnMSCs (P=0.022,  Fig .2A ). However, the differences were not\nstatistically significant (P>0.05) for the E-EnMSCs and C-EnMSCs ( Fig .2A ).\n COX-2  gene expression significantly downregulated in the E-EnMSCs\n(P=0.022) and upregulated in the C-EnMSCs (P=0.022) after treatment with 0.8 and 2 μg/ mL\nof CPE, respectively ( Fig .2B ).\nHDAC1  had significantly lower expression in the EEnMSCs after treatment\nwith 2 μg/mL of CPE (P=0.022).  HDAC1  gene expression showed a\nnonsignificant decrease after CPE treat-ment with both concentrations in the OMA-EnMSCs\ncompared to the C-EnMSCs (P>0.05,  Fig .2C ). When compared to the C-EnMSCs,\n DNMT1  gene expression decreased after treat-ment with 0.8 and 2 μg/mL\nCPE in the E-EnMSCs and OMA-EnMSCs. However, the dif-ferences did not show any statistical\nsignificance (P>0.05,  Fig .2D ).\nDNMT3A  gene expression significantly increased with the 2 μg/mL\nconcentration of CPE in the C-EnMSCs (P=0.034). However, its expression after treatment\nwith E-EnMSCs and OMA-EnMSCs was comparable to the C-EnMSCs ( Fig .2E ). Treatment with 0.8\nμg/mL of CPE reduced  DNMT3B  gene expression in OMA-EnMSCs (P=0.034).\nTreatment with 2 μg/mL of CPE had no significant effect on  DNMT3B  gene\nexpression ( Fig .2F ).\nDifferent  Ceratonia siliqua L . pod extract (CPE) concentrations were used to\ndetermine the relative mRNA expression levels (mean ± SD) of gene expressions.\n A.  MMP2 ,  B. \n COX-2 ,  C.  HDAC1 ,  D.\n DNMT1 ,  E.  DNMT3A  and  F.\n DNMT3B , and in the three study groups: i. Endometrial\nmesenchymal stromal/ stem cells (EnMSCs) derived from the endometrium of endometrioma\npatients (OMA-EnMSCs), ii. Deep infiltrative en-dometriosis (DIE) samples of women\nwith endometriosis associated infertility (E-EnMSCs), and iii. EnMSCs derived from the\nendometrium of an endometriosis free, normal woman (C-EnMSCs). The lines above the\ncolumns show significant differences (P<0.05). Data are analysed by one-way\nANOVA and the Duncan test.\nWestern blot analysis demonstrated that the related antibodies decreased in the E-EnMSCs, OMA-EnMSCs, and\nC-EnMSCs after treatment with CPE. COX-2, HDAC1,\nDNMT1, DNMT3A, and DNMT3B expressions decreased in the E-EnMSCs, OMA-EnMSCs, and C-EnMSCs after treatment with 0.8 and 2 μg/mL of CPE (Fig.\nS3, See Supplementary Online Information at www.ijfs.ir,\n Fig .3 ). Although there was a decrease in MMP2 expression in the OMA-EnMSCs, this result was not observed\nin E-EnMSCs and C-EnMSCs. Rather, MMP2 expression\nincreased in the E-EnMSCs and C-EnMSCs ( Fig .3 ).\nGC-MS analysis of CPE identified 14 bioactive molecules that were investigated for their interactions with\nsix target proteins through the docking process. The docking process produced multiple possible conformations for\neach ligand-protein complex, and we selected the most\nstable complex with the lowest binding affinity, ΔG [U\ntotal in kcal/mol]. The results of the docking process are\npresented in  Table 2 . The binding affinity of the selected\nconfor-mation ranged from -3.6 to -7.8 Kcal/mol.\nDiphenyl sulfone, 3,4-dihydro-2(1H)-isoquinoline carboximidamide, methanone\n(1-hydroxycyclohexyl)phenyl, and 4-imidazolidinone,1-benzoyl-2-(1-methylethyl)- 3-methyl\nexhibited the highest affinity to COX-2 with ΔG values of -7.4, -7.3, -7.2, and -7.1\nKcal/mol, respectively. 4-Imidazolidinone,1-benzoyl-2-(1-methylethyl)-3-methyl; ben-zene,\n1, 3, 5-tris(1-methylpropyl)-; 3.4-dihydro2(1H)-isoquinoline carboximidamide; naphthalene,\n1, 2, 3, 4-tetrahydro-1-isopropyl-1, 2, 4, 4, 7-pentamethyl-; naphtha-lene, decahydro-2,\n3-dimethyl-; benzoic acid, 4-heptyl-, 4-cyanophenyl ester; and methanone,\n(1-hydroxycyclohexyl)phenyl showed the highest affinity to DNMT3A with ΔG values of -5.8,\n-5.6, -5.5, -5.4, -5.4, -5.4, and -5.4 Kcal/mol. Diphenyl sulfone; methanone,\n(1-hydroxycyclohexyl)phenyl-; 3.4-dihydro-2(1H)-isoquinoline carboximidamide; and\n4-imidazolidinone,1- benzoyl-2-(1-methylethyl)-3-methyl exhibited the highest affinity to\n MMP2  with ΔG values of -6.8, -6.7, -6.6, and -6.6 Kcal/mol. The best\naffinity to  HDAC1  was observed for benzoic acid, 4-heptyl-,4-cyanophenyl\nester with ΔG of -6 Kcal/mol. The highest affinity to  DNMT1  was related\nto naphtha-lene, 1, 2, 3, 4-tetrahydro-1-isopropyl-1, 2, 4, 4, 7-pentamethyl-with ΔG of\n-7.8 Kcal/mol. Penta-noic acid, octyl ester; hexadecane; 2-methyl-5-(1-\nmethylethenyl)-2-cyclohexen-1-ol; diethy-leneglycol dimethacrylate; naphthalene;\n9-octadecenoic acid (Z)-, methyl ester; and naphthalene, decahydro-2,3-dimethyl-; showed\nthe highest affinity to  DNMT3B  with a ΔG of -7.5, -7.4, -7.4, -7.2, -7.2,\n-7.1, and -7.1 Kcal/mol, respectively. The intermolecular interactions of these compounds\nare depicted in Figure 4A-X.\nRelative peptide expression levels (mean ± SD).  A.  MMP-2,  B.  COX-2,\n C.  HDAC1,  D.  DNMT1,  E.  DNMT3A, and  F.\n DNMT3B expressions and  G.  Sodium dodecyl sulfate–polyacrylamide\ngel electrophoresis (SDS-PAGE) of analyzed peptides in endometrial mesenchymal\nstromal/stem cells (EnMSCs) derived from endometrium of endometrioma patients\n(OMA-EnMSCs) and deep infiltrative endometriosis (DIE) samples of women with\nendometriosis associated infertility (E-EnMSCs), and EnMSCs derived from endometrium\nof an endometriosis-free, normal woman (C-EnMSCs) at different  Ceratonia\nsiliqua L . pod extract (CPE) concentrations.\nBinding affinity of ligands in  Ceratonia siliqua L . pod extract (CPE) to receptors\nDNMT; DNA methyltransferase, HDAC1; Histone deacetylase 1, COX-2; Cyclooxygenase-2, and MMP2; Matrix metalloproteinase-2.\nThe 3D plot of the binding sites and 2D plot of interactions of DNA methyltransferase 3A\n( DNMT3A ), cyclooxygenase-2 (<italic>COX-2</italic>), histone\ndeacetylase 1 ( HDAC1 ), matrix metalloproteinase-2\n( MMP2 ),  DMNT1 , and  DNMT3B  with\ndifferent molecules of the  Ceratonia siliqua L . pod extract (CPE).\nInteraction of  DNMT3A  with:  A.  4-imidazolidinone,\n1-benzoyl-2-(1-methylethyl)-3-methyl,  B.  Benzene,\n1,3,5-tris(1-methylpropyl)-,  C.  3, 4-dihydro2(1H)-isoquinoline\ncarboximidamide,  D.  Naphthalene,1,2,3,4-tetrahydro-1-isopropyl-1, 2, 4,\n4, 7-pentamethyl-,  E.  naphthalene, decahydro-2, 3-dimethyl-,  F.\n Benzoic acid, 4-heptyl-, 4-cyanophenyl ester, and  G.\n Methanone,(1-hydroxycyclohexyl)phenyl. Interaction of  COX-2 \nwith:  H.  Diphenyl sulfone; I) 3,4-dihydro-2(1H)-isoquinoline\ncarboximidamide,  J.  Methanone,(1-hydroxycyclohexyl)phenyl, and\n K.  4-imidazolidinone, 1-benzoyl-2-(1-methylethyl)- 3-methyl.\nInteraction of  HDAC1  with:  L.  Benzoic acid,\n4-heptyl-,4-cyanophenyl ester. Interaction of  MMP2  with:\n M.  Diphenyl sulfone,  N.  Methanone, (1-hydroxycyclohexyl)\nphenyl-,  O.  3,4-dihydro-2(1H)-isoquinoline carboximidamide, and\n P.  4-imidazolidinone,1-benzoyl-2-(1-methylethyl)-3-methyl. Interaction\nof  DMNT1  with:  Q.  Naphthalene,\n1,2,3,4-tetrahydro-1-isopropyl-1, 2, 4, 4, 7-pentamethyl. Interaction of\n DNMT3B  with:  R.  Pentanoic acid, octyl ester,  S.\n Hexadecane,  T.  2-methyl-5-(1-methylethenyl)-2-cyclohexen-1-ol,\n U.  Diethylene glycol dimethacrylate,  V.  Naphthalene, and\n W.  9-octadecenoic acid (Z)-, methyl ester, and  X. \nNaphthalene, decahydro-2, 3-dimethyl.\nDiphenyl sulfone, 3,4-dihydro-2(1H)-isoquinoline carboximidamide, methanone\n(1-hydroxycyclohexyl)phenyl, and 4-imidazolidinone,1-benzoyl-2-(1- methylethyl)-3-methyl\nexhibited the highest affinity to  COX-2  with ΔG values of -7.4, -7.3,\n-7.2, and -7.1 Kcal/mol, respectively. 4-Imidazolidinone,1-benzoyl-2-\n(1-methylethyl)-3-methyl; benzene,1,3,5-tris (1-methylpropyl)-; 3.4-dihydro-2\n(1H)-isoquinoline carboximidamide; naphtha-lene,1, 2, 3, 4-tetrahydro-1-isopropyl-1, 2, 4,\n4, 7-pentamethyl-; naphthalene,decahydro-2, 3-dimethyl-; benzoic acid, 4-heptyl-,\n4-cyanophenyl ester; and methanone, (1-hydroxycyclohexyl) phenyl showed the highest\naffinity to DNMT3A with ΔG values of -5.8, -5.6, -5.5, -5.4, -5.4, -5.4, and -5.4 Kcal/\nmol. Diphenyl sulfone; methanone, (1-hydroxycyclohexyl)phenyl-;\n3.4-dihydro-2(1H)-isoquinoline carboximidamide; and 4-imidazolidinone,1-benzoyl-2-(1-\nmethylethyl)-3-methyl exhibited the highest affinity to  MMP2  with ΔG\nvalues of -6.8, -6.7, -6.6, and -6.6 Kcal/ mol. The best affinity to\n HDAC1  was observed for benzoic acid, 4-heptyl-,4-cyanophenyl ester with\nΔG of -6 Kcal/mol. The highest affinity to  DNMT1  was related to\nnaphthalene,1,2,3,4-tetrahydro-1-isopropyl-1,2,4,4,7- pentamethyl-with ΔG of -7.8\nKcal/mol. Penta-noic acid, octyl ester; hexadecane; 2-methyl-5-(1-methylethenyl)-\n2-cyclohexen-1-ol; diethy-leneglycol dimethacrylate; naphthalene; 9-octadecenoic acid\n(Z)-, methyl ester; and naphtha-lene, decahydro-2,3-dimethyl-; showed the highest affinity\nto  DNMT3B  with a ΔG of -7.5, -7.4, -7.4, -7.2, -7.2, -7.1, and -7.1\nKcal/mol, respectively. The intermolecular interactions of these compounds are depicted in\nFigure 4A-X.\n\nGC-MS analysis of the CPE identified 60 bioactive\ncompounds with various properties, some of which had\nanticancer (4 compounds), cytotoxic (4 compounds), and\nanti-inflammatory (6 compounds) effects. The focus of this\nstudy was to evaluate the compounds that had anti-cancer\nand anti-inflammatory properties. Previous studies have\ndemonstrated the potent biological anticancer effects of\noctacosane, 9-octadecenoic acid (Z)-, methyl ester, phenol,\n2,4-bis( 1 , 1 -dimethylethyl)-, and naphthalene ( 25 - 27 ). Additionally, the phytochemical profile of the CPE showed\nhigh levels of phenolic content. Overall, both GC-MS and\nphytochemical analyses suggest that CPE has potential anticancer effects. This finding is consistent with previous\nstudies that reported high amounts of phenolic content and\nanticancer properties in CPEs ( 28 ,  29 ).\nFurthermore, the CPE also exhibited anti-inflammatory\neffects in six of the identified compounds, which is consistent with previous studies on CPEs that reported antiinflammatory properties. The antioxidant, cell cytotoxic,\nand antimicrobial properties of CPE have also been reported in previous studies ( 12 ,  19 ).\nThe ideal medical treatment for endometriosis has yet to be identified, and current\nmedications often have relatively short-term effects. Additionally, hormonal drugs have many\nundesirable side effects and can induce an amenorrhoeic state by creating a hypo-oestrogenic\nenvironment ( 30 ). Therefore, finding novel treatment agents is necessary to improve\nfertility and other complications of endometriosis. The present study evaluated the impact\nof CPE on genes related to invasion and attachment ( MMP2 ), inflammation\n(<italic>COX-2</italic>), and epigenetic mechanisms (<italic>DNMTs</italic> and\n<italic>HDAC1</italic>) involved in endometriosis.\nUncontrolled regeneration of free radicals is a critical contributor to aging because it\nattacks various biomolecules, membranes, lipids, proteins, enzymes, and DNA, and leads to\noxidative stress and cell death. In cases of infertility, overproduction of free radicals\nand oxidative damage can lead to compromised follicles in endometriosis-associated fecundity\nimpairment ( 31 ). Antioxidants are stable components that donate an electron or hydrogen to\ncounteract the effects of free radicals and terminate the chain reaction before molecules\nare damaged. Also, the radical scavenging property of antioxidants delays or inhibits\ncellular damage. Phenolic compounds are regarded as powerful molecules with increased\npotential to neutralise free radicals ( 32 ). In this study, TPC analysis confirmed the\npresence of phenolic antioxidants in  Ceratonia siliqua , which may be\nresponsible for its therapeutic effects. Also, the hydroalcoholic CPE was screened for its\n in vitro  antioxidant activity and the results showed promising\nantioxidant potency. Phytochemical analysis suggests natural antioxidants are a promising\nnovel therapy to reduce the EAI rate ( 31 ). The biological activities and pharmacological\nproperties of  Ceratonia siliqua  may be linked to its high phenolic content,\nespecially gallic acid, catechin, epicatechin gallate, epigallocatechin gallate, and\nquercetin glycosides ( 12 ). Biometal cofactors such as Cu, Fe, Mn and Zn, which are essential\nfor antioxidant enzymes, are found in  Ceratonia siliqua  ( 33 ).\nIn the present study, total RNA was extracted from EEnMSCs, OMA-EnMSCs, and C-EnMSCs after\nexposure to CPE. cDNA synthesis and quantitative-PCR, gene expression levels were\ncalculated. The gene expression levels at CPE concentrations of 0.8 μg/mL and 2 μg/mL were\ncompared to those of the C-EnMSCs. Our data demonstrated that treatment of E-EnMSCs with CPE\nsuppressed  COX-2  expression. Previous studies have suggested that\n COX-2  overexpression plays a crucial role in endometriosis-associated\npain and in the pathogenesis and development of this disease through marked enhancement of\ninflammatory mediators ( 6 ). Moreover, the protein expressions of EEnMSCs, OMA-EnMSCs, and\nC-EnMSCs were evaluated after treatment with 0.8 μg/mL and 2 μg/mL of CPE. There was a\ndecrease in COX-2 protein expression after treatment with both concentrations compared to\nthe C-EnMSCs.\nEndometriosis is an inflammatory disease that is associated with various alterations in\nimmune cells and inflammatory cytokines. This subclinical inflammation is mainly responsible\nfor the pain and infertility associated with this disease ( 34 ). Flavonoids and tannins,\nwhich are the main phenolic compounds found in  Ceratonia siliqua , have\nanti-inflammatory activities because they inhibit the expressions of inflammatory mediators\nsuch as cytokines, inducible nitric oxide synthase, and  COX-2  ( 35 ). The\nelevated expression of  COX-2  in both endometrial and endometriotic cells of\nwomen with endometriosis plays a critical role in the pathogenesis of pain and infertility\n( 36 ), and down-regulation of  COX-2  by CPE may reduce dis-ease-associated\npain and improve fertility outcomes. Further  in vivo  studies are required\nto investigate the clinical applications of CPE in endometriosis.\nA growing body of evidence has suggested that endometriosis is an epigenetic disorder ( 36 ).\nTwo predominant epigenetic mechanisms are DNA methylation, which occurs through\n DNMTs , and histone modification by  HDACs  ( 37 ).\nConsistent with previous studies ( 38 ), we observed an increased level of\n DNMT3A  expression in the E-EnMSCs from women with endometriosis compared\nto those in the C-EnMSCs. However, the role of other DNMTs in the pathogenesis of\nendometriosis remains controversial ( 38 ). The results of the current study reveal that\n DNMT3B  gene expression levels can be significantly reduced after\ntreatment with 0.8 μg/mL of CPE. However,  HDAC1  expression only reduced in\nE-EnMSCs exposed to 2 μg/mL of CPE; no significant efficacy of CPE was observed in terms of\nreducing HDAC1 expression in OMA-EnMSCs. Western blot analysis showed that the DNMT3A and\n-3B pro-tein expressions decreased in all three cell types after treatment with 0.8 and 2\nμg/mL of CPE. Additionally, HDAC1 protein expression also showed a decline after treatment\nat both concentrations. In contrast with gene expression, a reduction in protein expression\nof DNMT3B and HDAC1 was higher after exposure to 2 μg/mL of CPE than 0.8 μg/mL.\nIn this regard, HDAC inhibitors have been suggested as attractive treatment targets for\nendometriosis.  In vitro  and  in vivo  studies of human\nendometrial stromal and epithelial cells and animal models of endometriosis, respectively,\nthat were treated with different HDAC inhibitors have shown anti-proliferative activities\nand reduced impacts on lesion size ( 39 ).\nMMP2  acts as a key enzyme associated with tumour metastasis through the\ndegradation of extracellular matrix components ( 7 ).  MMPs  are essential\nenzymes in the ectopic implantation of endometrial tissue, and our study revealed that the\n MMP2  gene expression level in OMAEnMSCs could be modulated by 0.8 μg/mL\nof CPE. Moreover, the protein expression of MMP2 was also decreased in OMA-EnMSCs. However,\nthis downregulation of MMP2 protein expression was not observed in CEnMSCs and E-EnMSCs. The\neffectiveness of  Ceratonia siliqua  in the current study was based on\n in vitro  treatment of endometriosis. Therefore, well-designed animal and\nhuman trials are recommended to achieve conclusive re-sults on the efficacy and safety of\nthis natural product for endometriosis.\nTwo noteworthy limitations of our study were the limited sample size and lack of budget to\ncarry out further  in vivo  evaluations. Furthermore, while the antioxidative\nand antiinflammatory effects of  Ceratonia siliqua  have been evaluated,\nfurther biochemical studies are necessary to assess the parameters that induced these\neffects in our study.\nA computational study showed that not only one specific compound had the highest affinity to different proteins, which made it impossible to identify the most effective specific compound. The combination of several\ncompounds can be involved in the inhibition of proteins.\nThis difference can be attributed to the role of conformation and intermolecular interactions in the formation of\nthe ligand-protein complex ( 40 ). According to the docking scores, 14 of the 60 studied compounds showed good\naffinity to different proteins. However, among them, three\ncompounds, 3,4-dihydro-2(1H)-isoquinoline carboximidamide, 4-imidazolidinone,1-benzoyl-2-(1-methylethyl)-\n3-methyl, and methanone (1-hydroxycyclohexyl)phenyl,\nshowed good affinity to three proteins. Hydrogen bonds,\nvan der Waals interactions, and hydrophobic interactions play a role in the binding of compounds to proteins.\nTherefore, it can be inferred that the inhibitory effect of\nCPE is mostly related to these compounds.\nWe briefly touched upon the limitations of this study; however, further elaboration could provide\na clearer picture of potential areas for improvement. More detailed discussion on the\nspecific pathways and potential interactions involved in the mechanism of action of\n Ceratonia siliqua  could provide more insight into its therapeutic\neffects. Further exploration of potential clinical implications that include dosage regimens\nand considerations for patient populations could be beneficial. Finally, a comprehensive\ndiscussion on the current state of endometriosis treatment and how  Ceratonia\nsiliqua  compares to other treatments may provide more context for its potential\nimpact.\n\nWe demonstrated that the CPE could reduce the viability and proliferation of OMA-EnMSCs and\nC-EnMSCs, as well as decrease the expressions of key enzymes and epigenetic factors involved\nin endometriosis pathogenesis, including  MMP2, DNMT3A, DNMT3B , and\n HDAC1 . Our study also identified several compounds in the CPE that showed\ngood affinity to different proteins involved in endometriosis pathogenesis, which suggests\nthat a combination of these compounds could be responsible for the inhibitory effects of\nCPE.","source_license":"CC0","license_restricted":false}