{"paper_id":"41758137-deb8-49ce-b574-3ff1975d088a","body_text":"Endometriosis is a chronic inflammatory disease and\none of the most common gynaecological disorders in\nwomen of childbearing age. It is characterised by the\ngrowth of endometrial-like tissue outside the uterus ( 1 ).\nAn estimated 6 to 10% of reproductive-age women are affected\nby endometriosis, and most suffer from pelvic pain\nand infertility ( 2 ). The pathogenesis of endometriosis has\nbeen widely studied, and various hypotheses have been\nproposed. The most commonly accepted theory is Sampson’s\ntransplantation theory, which describes retrograde\nmenstruation. This phenomenon can cause the transfer of\nendometrial cells to the peritoneal cavity through the fallopian\ntubes and result in their Placement in the peritoneal\ncavity ( 1 ). However, endometriosis does not occur in\nall women with recurrent retrograde menstrual bleeding,\nand this indicates the involvement of genetic, epigenetic,\nand environmental factors in this disease ( 3 ). According\nto Sampson’s theory, adhesion and proliferation of endometrial\ntissue, cellular invasion, and neoangiogenes are\nkey factors in the pathogenesis of endometriosis. Therefore,\ngrowth and angiogenesis factors such as insulin-like\ngrowth factors  (IGF1, IGF2)  and vascular endothelial\ngrowth factor  (VEGF)  play a critical role in ectopic endometrial\ncell proliferation ( 4 ).\nLong non-coding RNA (lncRNA)  H19  is one of the\nfirst identified imprinted lncRNAs expressed from the\nmaternal allele.  H19  is involved in the regulation of cell\nproliferation and differentiation and serves a critical role\nin various biological processes of different diseases ( 5 ).\nRecent research shows that decreased expression of  H19 \nalters stromal cell growth through IGF signalling in the\nendometrium of patients with endometriosis ( 6 ).\nNew agents can effectively improve endometriosis in\npatients. Considering that diet is a potential risk factor for\nthis disease, food compounds have recently been considered\nas therapeutic and preventive agents ( 7 ,  8 ). One of\nthese nutrients is curcumin, which is produced by the Curcuma\nlonga plant. Curcumin is a potent anti-inflammatory\nagent. Several studies have shown that curcumin has antiinflammatory,\nantioxidant, anti-cancer, and anti-angiogenic\neffects ( 9 ,  10 ).Curcumin can inhibit angiogenesis,\nproliferation, invasion, and metastasis of different cancers\nthrough targeting signalling pathways ( 11 ). In addition,\nthe anti-inflammatory, anti-angiogenic, anti-proliferative,\nand anti-invasive effects of curcumin on endometriosis\nhave been reported ( 12 - 15 ). In our previous study, we\nanalyzed the expression levels of  H19  lncRNA along with\ngenes involved in angiogenesis  (VEGF)  and proliferation\n (IGF1, IGF2) , in endometrial tissues from patients with\nendometriosis in comparison with healthy women. Increased\n VEGF  levels along with decreased  H19  lncRNA,\n IGF1 , and  IGF2  expressions were observed in the eutopic\nendometria of women with endometriosis ( 16 ). For the\ncurrent study, we took into consideration the role of an\ngiogenesis, proliferation factors, and migration in endometriosis\nand the anti-angiogenesis and anti-proliferation\neffects of curcumin. We intend to evaluate the impact of\ncurcumin on  VEGF, IGF1, IGF2,  and  H19  expressions,\nin addition to cell migration and proliferation in eutopic\nendometrial stromal cells (EU-ESCs) from women with\nendometriosis compared to normal endometrial tissues.\n\nDNase I, Dispase II, collagenase types І and IV,\nβ-mercaptoethanol, and curcumin were purchased from\nSigma Corporation, USA. DMED/F-12 (1:1), foetal bovine\nserum (FBS), GlutaMAX, non-essential amino acids\n(NEAA), penicillin-streptomycin, and phosphate-buffered\nsaline were purchased from Gibco, USA.\nThis experimental study was approved by the Ethics\nCommittee of Royan Institute (IR.ACECR.ROYAN.\nREC.1398.95) and written informed consent was obtained\nfrom participants before the endometrial biopsies. Based\non our previous study ( 17 ), we enrolled three women with\nendometriosis (case group) and three women without endometriosis\n(control group) in the current study. The age\nrange of the participants was 30 to 40 years. All women\nhad regular menstrual cycles and had not received any\nhormonal therapy for at least three months before endometrial\nsampling. Endometriosis was diagnosed by the\nexistence of endometriotic lesions during laparoscopy\nand after pathological examination. These endometriosis\npatients had stage ІІІ or ІV disease according to the\nrevised classification of the American Fertility Society.\nEndometrial specimens were collected from both groups\nduring the proliferative phase of their menstrual cycles.\nThe samples were obtained under sterile conditions using\na pipelle by an expert gynaecologist.\nThe endometrial specimens were placed in sterile medium\nand transferred to the laboratory. Tissues were\nwashed using washing medium with gentle stirring to\nremove blood cells and mucous. Then, the tissues were\ndissected into small pieces and incubated in DMEM/F-12\nthat included 10% FBS, collagenase type I (1 mg/ml), collagenase\ntype IV (1 mg/ml), DNase I (1 mg/ml), and Dis\npase II (4 mg/ml) for 30 minutes at 37°C. The epithelial\ncells were removed by serial filtration of the stromal cells\nthrough 70 and 40 µm sieves. The ESCs were obtained\nby centrifuging the cell suspension at 500 ×g for 5 minutes.\nThe resultant pellet was resuspended and cultured\nin a 25 cm 2  flask in DMEM/F‐12 medium that contained\n10% FBS, 1% GlutaMAX, 1% penicillin/streptomycin,\nand 1% NEAA, then incubated at 37°C in a humidified 5% CO2\nincubator. The culture medium was replaced\nevery  2 -3 days. All cultures were passaged three times,\nand when they reached 70-80% confluency, the cells were\nused for treatment with or without curcumin. Purity of\nthe stromal cells was assessed by flow cytometry and an\nantibody panel against CD29, CD31, CD45, CD73, and\nCD90 (all from Becton Dickinson Biosciences, USA),\nand immunofluorescent staining for vimentin ( 17 ).\nESC proliferation was assessed by the methylthiazole\ntetrazolium (MTT) test. The cells were seeded in 96-well\nculture plates at 5×10 3  cells/well. After the cells attached,\nthey were treated with various concentrations of curcumin\n(0-100 mM) for 72 hours. This time was selected based on\nthe doubling time of ESCs. MTT was performed as previously\nreported ( 18 ). Curcumin was dissolved in dimethyl\nsulphoxide (DMSO) and diluted in culture media to the\ndesired concentrations. The final concentration of DMSO\nwas less than 0.1%.\nThe wound-healing migration assay was used to measure\ncell migration. For this purpose, a line was scratched using\na pipette tip after the cells reached confluency. Then,\nthe detached cells were removed by washing them. Fresh\nmedia without curcumin or with different concentrations\nof curcumin were added to the plates. Representative photographs\nwere taken under an inverted light microscope\n(Olympus, Japan) at various times (0, 24, 48, and 72 hours).\nRNA was extracted from the cells using an RNeasy\nMicro kit (Qiagen, Germany) according to the manufacturer’s\ninstructions. DNase I (Takara, USA) was used to\nremove any DNA contamination. Both the concentration\nand purity of the RNA samples were evaluated using a\nNanodrop 2000 spectrophotometer (Thermo Scientific,\nUSA). Complementary DNA synthesis was performed using\na TaqMan reverse transcription kit (Takara, USA), according\nto the manufacturer’s instructions. Subsequently,\nreverse transcription quantitative polymerase chain reaction\n(RT-qPCR) was performed using a Step-One RT-PCR\n(AB Applied Biosystems, USA) with primers designed\nfor  VEGF, IGF1, IGF2,  and  H19  ( Table 1 ). The mean\nfold changes of these genes were calculated using the\n2 −ΔΔCT  algorithm, and their expressions were normalised\nto glyceraldehyde-3-phosphate dehydrogenase (GAPDH)\nas the internal standard.\nPrimer pairs used in this study\nAll experiments were performed in triplicate. The data\nwere analysed using GraphPad Prism software, version 8\n(GraphPad, San Diego, CA, USA) and the non-parametric\nWilcoxon test. Data are presented as medians and interquartile\nranges. P<0.05 indicated statistical significance.\nThe distance between the edges of the lines was measured\nwith ImageJ software, and Prism software was used to determine\nthe half maximal inhibitory concentration (IC 50 ).\n\nESCs incubated with various concentrations of curcumin\n(0-100 mM) for 72 hours showed that curcumin\ninhibited cell proliferation and migration in a time- and\ndose-dependent manner. The IC50\nvalue for curcumin at\n72 hours was 42.20 mM for the E 50 U-ESCs from endometriosis\npatients ( Fig .1 ). The wound healing assay was performed\nto evaluate the effects of curcumin on proliferation\nand migration of EU-ESCs. Curcumin significantly\ninhibited cell migration in cultured EU-ESCs compared\nto untreated EU-ESCs ( Fig .2A ). The inhibitory effect of\ncurcumin was time- and dose-dependent ( Fig .2B ). Based\non the cell viability results and IC 50 \nat 72 hours, we selected the\n30 mM dose of curcumin and an incubation time of\n72 hours for further assessment.\nCurcumin effect on cell proliferation. Effect of different doses of\ncurcumin (0-100 mM) on human EU-ESCs cultured  in vitro  after 72 hours\nof incubation. Curcumin inhibited the growth of EU-ESCs after 72 hours\nof treatment at different concentrations. The IC 50 \nvalue for curcumin at\n72 hours was 42.20 mM for EU-ESCs. IC 50 \n; Half maximal inhibitory concentration,\nEU-ESCs; Eutopic endometrial stromal cells, and OD; Optical\ndensity.\nGene expression analysis showed a decrease in VEGFA\nexpression in EU-ESCs following treatment with curcumin\n(P=0.250,  Fig .3A ). IGF1 expression increased in\nN-ESCs and EU-ESCs treated with curcumin compared\nto non-treated cells, but these increases were not significant\n(P=0.250,  Fig .3B ). IGF2 expression increased in\ncurcumin-treated EU-ESCs compared to those cultured\nwithout curcumin; however, it decreased in N-ESCs treated\nwith curcumin compared to non-treated cells. These\ndifferences were not significant (P=0.250,  Fig .3C ). Although\nN-ESCs and EU-ESCs had higher H19 expression\nafter curcumin treatment compared to the untreated conditions,\nthis increase in the curcumin-treated EU-ESCs\nand EU-ESCs was not significant (P=0.250,  Fig .3D ).  Table\n2  shows the descriptive statistics obtained from the\nnon-parametric test (Wilcoxon).\nEvaluation of migration of EU-ESCs by the scratch test.  A, B.  Cell migration of EU-ESCs was decreased by curcumin treatment in a time-and concen\ntration-dependent manner (scale bar: 500 μm). EU-ESCs; Eutopic endometrial stromal cells.\nGene expression patterns following treatment with curcumin. Relative mRNA expression levels of:  A.  VEGF ,\n  B. \n IGF1 ,  C. \n IGF2 , and  D.  H19  treated with\n30 mM curcumin for 72 hours were evaluated in cultured N-ESCs and EU-ESCs. Gene expression was analysed by qRT-PCR.  GAPDH  was used as a reference\ngene. The results are expressed as median and interquartile range. ns; P>0.05, N-ESCs; Normal endometrial stromal cells, EU-ESCs; Eutopic endometrial\nstromal cells, and qRT-PCR; Reverse transcription quantitative polymerase chain reaction.\nDescriptive statistics of the data\nThe descriptive statistics of data obtained from non-parametric test (Wilcoxon). N-ESCs;\nNormal endometrial stromal cells, EU-ESCs; Eutopic endometrial stromal cells, and IQR;\nInterquartile range.\n\nEndometriosis is a gynaecological disorder characterised\nby abnormal cell adhesion, invasion, growth, and\nproliferation of endometrial cells, along with neoangiogenesis,\nwhich leads to implantation in ectopic sites ( 19 ).\nIn our previous study, overexpression of  VEGF  in the\neutopic endometrium of women with endometriosis was\ndetected compared to the control endometrium. Expression\nof  H19  was lower in eutopic endometrial samples\ncompared with the control endometrium. The expression\nlevels of  IGF1  and  IGF2  were also decreased in the eutopic\nsamples compared to the control group ( 16 ). These\naltered patterns of expression suggest an impaired regulation\nof cellular growth and differentiation in endometriotic\ntissues. The aim of the present experimental study\nwas to determine if curcumin, as an anti-angiogenic and\nanti-proliferative agent, could affect the expression of\nthese genes.\nCurcumin (diferuloylmethane) is the main active poly\nphenol in turmeric, and has a low molecular weight. The\nchemical formula of curcumin is C 21 \nH 20 \nO 6 \n; it contains\n2-8% turmeric and was first identified in 1910 for its\nchemical properties ( 20 ,  21 ). Several studies have shown\nthe effects of curcumin on inflammation, invasion, angiogenesis,\ncell proliferation, and apoptosis ( 22 ,  23 ). In the\npresent study, we found that curcumin decreased cellular\nmigration and proliferation of EU-ESCs in a time- and\ndose-dependent manner. Consistent with the present results,\nZhang et al. ( 24 ) observed a dose-dependent, antiproliferative\neffect of curcumin in ESCs from patients with\nendometriosis. Curcumin inhibited cell proliferation in\novarian and endometrial cancer cells ( 25 ). It blocked the increase\nin size and weight of endometriosis lesions in endometriotic\nrats in a time- and dose-dependent manner ( 26 ).\nThese studies suggest that treatment with curcumin is associated\nwith decreased cell proliferation in endometriosis.\nIn the present study, curcumin decreased VEGF expression,\nwhich is consistent with previous studies. Zhang\net al. ( 27 ) showed that curcumin reduced VEGF protein\nexpression in ectopic tissues of a rat model with endometriosis.\nIn another study, curcumin reduced the survival\nof endometriotic stroma cells in vitro by reducing VEGF\nprotein expression ( 28 ). Also, the anti-angiogenic effect\nof curcumin has been reported in ovarian cancer ( 29 ).\nDownregulation of VEGF was attributed to curcumin\ntreatment, and might lead to a decrease in the ability of\nendometrial cells to implant at the ectopic sites.\nThe IGFs play a main role in regulating endometrial cell\ngrowth and differentiation ( 30 ). lncRNA H19 is involved\nin the regulation of cell proliferation and differentiation\n( 31 ). The current study results showed that curcumin increased\nthe expression levels of IGF1, IGF2, and H19.\nHowever, the effect of curcumin on the expressions of\nthese genes in endometriosis has not been studied. Other\nstudies have shown that curcumin reduces IGF1 and\nIGF2 expression in various diseases and cancers under in\nvivo and in vitro conditions ( 32 - 34 ). Similar to our results,\nonly one study showed an increase in IGF1 gene expression\nin curcumin-treated diabetic rats ( 35 ). Although\nthere are limited studies on the effect of curcumin on H19\ngene expression, these studies have shown that curcumin\nreduces its expression in tumour cells, and this was inconsistent\nwith our results ( 36 ,  37 ). This discrepancy may\nbe related to the diverse biological functions of H19 in\ncancer biology, which is known both as an oncogene and\na tumour suppressor gene ( 38 ). In addition, we previously\nobserved decreased expression of H19 in endometriosis\n( 16 ), and this finding suggests that H19 may have a suppressive\nrole in endometriosis. In the present study, the\nincreased H19 gene expression after curcumin treatment\nmight indicate the therapeutic effect of curcumin in endometriosis.\nHowever, in vivo investigations are needed,\nespecially in animal models.\nThe role of H19 in regulating cell proliferation and differentiation\nmay be due to its association with IGF1 and\nIGF2 ( 6 ,  38 ). We previously reported that the reduction in\nH19 in endometriosis lesions possibly caused decreased\nIGF1 and IGF2 expressions. This pattern implies that the\nendometriotic tissue may undergo a disturbance in cellular\ngrowth regulation and differentiation ( 16 ). In support of\nthis hypothesis, the present findings showed that although\ncurcumin treatment increases H19, IGF1, and IGF2 expressions\nin endometriotic tissue (EU-ESC), this pattern\nwas not completely detected in normal endometrium (NESC).\nIt implied that the EU-ESC of endometriosis has\ndifferent behaviour than N-ESC of normal endometrium\nin response to curcumin treatment.\nThus, the increased expressions of IGF1, IGF2, and\nH19 after curcumin treatment could be considered a new\nfinding of the present study. However, due to the limited\nstudies in this field and the inconsistent results of the effect\nof curcumin on other diseases, further investigations,\nespecially  in vivo  studies, are recommended to determine\nthe role of curcumin in endometriosis.\n\nOur data demonstrated that curcumin decreases cell migration\nand proliferation of endometriotic stromal cells in\na dose - and time-dependent manner. The present study\nalso showed that curcumin reduces the expression of\ngenes involved in angiogenesis. Another finding of this\nstudy was the increase in IGFs and H19 expressions in the\npresence of curcumin, which suggests that curcumin can\nbe an effective treatment for endometriosis.","source_license":"public-domain-us","license_restricted":false}