{"paper_id":"26ae7ad3-20da-41f5-b01a-0d8021c13d5f","body_text":"Endometriosis is a gynecological disease that occurs\nwhen the tissue similar to the lining of the uterus grows\noutside of the uterus, while its true prevalence is uncertain ( 1 ). It is unclear how this disease develops, but endometrial lesions , metastasis, metaplasia, blood reflux,\nand disorders of the immune system are involved ( 2 ).\nEndometriosis is frequently associated with pain, reduced\nquality of life, and infertility; and current treatments do\nnot provide a sufficient cure despite of its long term management ( 3 ).\nThe endometriosis associated symptoms include inflammation, asthma and osteoarthritis as well as multisite\nchronic pain or migraine ( 4 ).\nDuring endometriosis, immune response reduction, apoptosis impairment, and inflammation increase, result in\nthe repeated tissue injury, neurogenesis, and chronic inflammation ( 5 ).\nEndometriosis, chronic and progressive forms, has a\nsignificant impact on the life quality of patients. While,\nendometriosis surgery, lesion removal, is a definitive\ntreatment, with a high rate of recurrence , up to 50%\n, within five years of the procedure ( 6 ). Also, Surgery\nis associated with vascular, intestinal, and neurological\ncomplications, and pain may reappear if the lesions are\nnot completely removed and pathogenetic mechanisms\nmay not be treated by surgery and a long-term medical\ncare must be prepared for the patients ( 7 ). Diphereline,\na gonadotropin releasing hormone agonist (GnRHa), is a\ncurrent treatment in women with endometriosis to impair\nthe progression of endometriosis implants by blocking\nthe hypothalamus-pituitary-ovary axis ( 8 ,  9 ). The complexity of using GnRHa includes amenorrhea, musculoskeletal stiffness, hot flush, bone loss, hair loss, vaginal\ndryness, depression and headache. Pregnancy should be\nprevented during GnRHa treatment ( 10 ,  11 ). Plant-derived agents are a potential safe option with a high level\nof efficiency to avoid adverse effects and preserve the\nchances of a successful pregnancy ( 12 ). Recent studies have suggested that the main effective components\nin broccoli and soybeans such as sulforaphane and isoflavones might induce cell death in cancer cells through\nregulating the essential mechanisms, such as oxidative\nstress, autophagy, and apoptosis which play important\nroles in the growth and progression of endometriosis\n( 13 ). An essential function of apoptosis is to eliminate\nexcess or dysfunctional cells. Pieces of evidence suggested that in healthy women, as a result of programmed\ncell death, endometrial cells expelled during menstruation cannot survive in ectopic locations and a decreased\napoptosis can lead to endometriosis by allowing these\ncells to survive and implant ectopically ( 14 ).\nOxidative stress is a term used to define an imbalance\nbetween the production of reactive oxygen species (ROS)\nand the body's natural defenses against their harmful effects. ROS molecules have the potential to damage cells\nand tissues in the body ( 15 ). Under normal circumstances, the body has ROS detoxification mechanisms and prevent them from causing harm. However, overproduction\nof ROS in the peritoneal environment causes the chain\nof events leading to endometriosis ( 16 ,  17 ). Autophagy\nis a cellular process that helps to remove damaged organelles and proteins, thus maintaining cellular integrity\nand homeostasis. Apoptosis is essential for the removal\nof damaged or unnecessary cells and helps to regulate the\ngrowth of endometrial tissue. Therefore, understanding\nthe role of oxidative stress, autophagy, and apoptosis in\nthe context of endometriosis is essential to develop effective management and treatment strategies ( 18 ).\nEven though there are already treatment options for\nendometriosis such as surgery and hormone therapy, oral\nconsumption of natural substances is a safe way to treat\nendometriosis ( 19 ). The present study aimed to explore\nthe protective and therapeutic effects of broccoli extract\nand soy isoflavones (SI) as well as Diphereline by measuring the histopathological scores of the endometrial implants and mRNA expression level of the gene markers\nof autophagy, apoptosis, and oxidative stress within the\ntissue of peritoneal in endometriosis rat model.\n\nIn this experimental study, the care and control of the\nrats were approved by the Medical Ethics Committee\nof Shiraz University of Medical Sciences (IR.SUMS.\nREC.1398.1151).\nForty five mature female Sprague-Dawley rats weighing 220 ± 20 g, and at the age of almost 8 weeks were\nused for this study. The rats were mature sexually and displayed normal estrous cycle changes in uterine histology\nusing the Papanicolaou technique.\nThe animals were kept at a constant temperature of 22°C\nwith a humidity of 40-60% in the Animal Department of\nShiraz University of Medical Sciences under standard\nconditions of cyclic light, 12 hours light/12 hours dark,\nfor 1 week to acclimatize them. Their cages were cleaned\nand disinfected every week, and the rats were placed on a\nstandard diet ( 20 ).\nThe alcoholic extract of broccoli was prepared as described previously by Somasundaram ( 21 ). Each Soy tablet (6260232300192, Soya Gol, Gol Daru Company, Iran)\nincludes 50 mg SI ≈ and 27 mg Genistein was dissolved\nin 1 ml saline 0.9%.\nEctopic endometrium was induced surgically in the peritoneum of the studied rats ( 12 ,  22 ).\nBriefly after mid-abdominal incision by anesthesia with isofluorane (26675-\n46-7, Cyman Chemicals, USA), the left uterine horn was\nresected from the rats and was put in phosphate-buffered\nsaline at 37°C. Eventually, a 5-mm-square segment was\nsectioned longitudinally. In this procedure, uterine tissue\nwithout myometrium was transplanted onto the right abdominal wall inner surface with the epithelium opposed to\nthe peritoneum. Polypropylene sutures were used at two\nedges of the explanted endometrium to secure it (Prolene\n6-0, 8695G, Ethicon, USA). Chromic catgut suture (3-0;\n636H, Ethicon, USA) was used to close the midline abdominal incision. A horizontal mattress suture of polyglactin 910 (coated Vicryl 4-0; J507G, Ethicon, USA) was\nused to close the incision in the skin. Four weeks after\nthe surgery, a second look laparotomy was performed to confirm the endometrial implants viability. The animals\nwere given time in two weeks to recover and progress the\ninduced endometriosis.\nAfter scarifying the rats and incising their abdomens, the explants were investigated for viability and size ( 12 ). A caliper\nwas used to measure the surface area of the engrafted tissue.\nTo eliminate the selection bias and achieve balance in baseline characteristics, a simple randomized method (throwing\na dice) was used to divide the surgically induced endometriosis rat models randomly into five groups (nine rats in\neach group) including the control (C): (saline treatment: 0.5\nml of saline 0.9%), broccoli extract (BE: 3000 mg/kg), SI (1\ntablet ≈ 50 mg isoflavones/kg), BE+SI: BE 3000 mg/kg+SI\n(1 tablet ≈ 50 mg isoflavones/kg) and, Diphereline (D: IM\ninjection) [S.R. 11.25 mg (3 mg/kg, once during studied\ntreatment)]. Diphereline (57773-63-4, IPSEN, France) was\nused as a standard hormonal treatment with known effects.\nThe rats were sacrificed after six weeks of treatments and\nthe endometrial implants were considered.\nThe endometrial implants were fixed in formalin\n(F8775, Sigma Aldrich, Germany) for 48 hours and then\nembedded in paraffin wax box to transfer to pathology\nlaboratory. 5 μm sections were provided to prepare tissue slides (three sections per sample). The pieces were\nheat-dried and stained with hematoxylin (H3136, SigmaAdrich, Germany) and eosin (109278, Merck, Germany)\n( 23 ). The epithelial cells persistence in the endometriosis\nimplants was scored according to Keenan et al. ( 24 ) scoring system: 0; No epithelial layer, 1 and 2; Poorly and\nmoderately persistent epithelial cells, respectively, and 3;\nWell-preserved epithelial layers.\nBriefly, a piece of the tissue from the implanted area of\neach endometriosis-induced rats (n=45) were embedded\nin 1 ml RNAlater (YT9085, Yekta Tajhiz, Iran) to protect\nRNA degradation. Tissues were lysed with TRIzol reagent\n(YT9064, Yekta Tajhiz, Iran), and the RNX-PLUS reagent\n(EX6101, Sinaclon, Iran) was used for total RNA extraction\nas per the manufacturer’s instruction. Utilizing an Addbio\ncDNA synthesis kit (22701, Addbio, South Korea), purified\nRNA was used to create complementary DNA (cDNA).\nThe messenger ribonucleic acid (mRNA) expression levels of Beclin-1\n( Becn1 ), Microtubule-associated proteins 1A/1B light chain 3B\n( Lc3 ), caspase-3 ( Casp3 ), associated X protein\n( Bax ), B-cell lymphoma/leukemia 2 (<italic>Bcl-2</italic>), superoxide\ndismutase ( Sod ), and β-actin in endometrial tissues of\nendometriosis-induced rats were evaluated ( Table 1 ) ( 25 ). β‐actin gene was used to\nnormalize the gene expression levels. The mRNA expression levels of  Lc3, Becn1,\nCasp3 , and  Bcl-2 , genes calculated by ΔΔCt method through\nthree independent experiments.\nPrimer sequences used for real-time polymerase chain reaction\nThe data was presented as mean ± SD. The Shapiro\nWilks test was used to assess the normality assumption\nof data. The statistical differences between groups were\nevaluated by Kruskal-wallis followed by Dunn’s post-hoc\ntests using SPSS version 22 (IBM, USA). The P<0.05 was\nconsidered as statistically significant.\n\nTo evaluate the persistence of epithelial cells in the\ngraft, the pathological scores were applied ( 24 ).\nThe persistence of epithelial cells (pathologic score)\nin grafts of Diphereline and BE+SI groups was significantly reduced in comparison with the control group\n(P≤0.001), and there was no substantial change in\nthe score of the BE+SI treated group compared to the\nDiphereline group (P=0.903,  Table 2 ). As it is illustrated\nin Figure 1, the well-preserved epithelial layer of endometrial implants (score 2-3) was evident in the control\ngroup, and also, a moderately persistent epithelial cell of\nendometrial implants (score 1-2) was observed in both\ngroups, BE and SI and the epithelial layer of endometrial\nimplants was poorly preserved (score 0-1) in BE+SI and\nD groups.\nThe pathological score of endometrial implants\nSI; Soy isoflavone, BE; Broccoli extract,  a  ; Statistical comparison with the control\ngroup, and  b ; Statistical comparison with Diphereline group.\nHematoxylin-Eosin-stained biopsy of epithelial layer specimen at 250x magnification.  i.\n Control group: well preserved (score 2-3),  ii.  BE,\n iii.  SI groups: moderate preserved (score 1-2), iv: BE+SI and v.\nDiphereline (D) groups: poor preserved (score 0-1). SI; Soy isoflavone and BE;\nBroccoli extract.\nTo evaluate the autophagy status in the treated groups,  Becn1  and\n Lc3  mRNA levels were assessed. The expression levels of\n Becn1  in BE+SI, SI, and D treatment groups were significantly increased\ncompared to the C group (P=0.0002, 0.0034, and 0.0054 respectively) ( Fig .2 ).\n Lc3  mRNA levels of BE+SI and D groups were significantly higher than C\ngroup (P=0.0004, and 0.0017 respectively). Although, SI and BE treatments lead to an\nincrease in the mRNA expression level of  Lc3 , the differences were not\nsignificant ( Fig .2 ).\nReal-time PCR for the mRNA expression of autophagy-related genes.  A.\n Lc3  and  B.  Becn1  genes. In\ncomparison with the control group ( 1 ) and in comparison with the Diphereline (D) group\n( 2 ). **; P<0.01, ***; P<0.001. The details of mean ± SD and P values are\npresented in Table S1 (See sup- plementary Online Information at www.ijfs.ir). ns; Not\nsignificant, PCR; Polymerase chain reaction, SI; Soy isoflavone, and BE; Broccoli\nextract.\nCasp3, Bax , and  Bcl-2  mRNA expression levels were\nevaluated as common markers of apoptosis. BE+SI, SI, and D groups showed a significant\nincrease in the  Casp3  mRNA expression levels in comparison with the C\ngroup (P<0.0001 for all). The Bax mRNA expression level was increased only after\nBE+SI administration (P=0.001,  Fig .3 ). Other treatments could not change the\n Bax  mRNA expression level in comparison with the control group. The\n Bcl-2  mRNA expression level has a reverse correlation with apoptosis\ninduction. Administration of BE+SI, SI, and D significantly decreased the\n Bcl-2  mRNA levels in comparison with the C group\n(P<0.0001=0.0028, and =0.0017 respectively) ( Fig .3 ).\nReal-time PCR for the mRNA expression of apoptosis-related genes.  A.\n Casp3 ,  B.  Bax , and  C.\n Bcl-2  genes. In comparison with the control group ( 1 ) and in\ncomparison with the Diphereline (D) group ( 2 ). **; P<0.01, ***, ****;\nP<0.0001. The details of mean ± SD and P values are presented in Table S1 (See\nsupplementary Online Information at www.ijfs.ir). ns; Not significant, PCR; Polymerase\nchain reaction, SI; Soy isoflavone, and BE; Broccoli extract.\nThe expression level of  Sod , as the oxidative stress status assessment\nmarker, was significantly increased under BE+SI and SI treatments than the C group\n(P<0.0001 for both,  Fig .4 ). However, BE and D administration did not change the\n Sod  mRNA expression level.\nReal-time PCR for the mRNA expression of the Sod gene. In compari- son with the control group ( 1 ) and in comparison, with the Diphereline\n(D) group ( 2 ). ****; P<0.0001. The details of mean ± SD and P values are\npresented in Table S1 (See supplementary Online Information at www.ijfs.\nir). ns; Not significant, PCR; Polymerase chain reaction, SI; Soy isoflavone,\nand BE; Broccoli extract.\n\nWhile there are already clinical treatment options for\nendometriosis such as surgery and hormone therapy, the\noral consumption of natural substances provides a safe\nalternative for treating the condition ( 26 ). The objective\nof this study was to investigate the potential therapeutic\neffects of BE, SI and the combination of these two nutraceuticals on progression of endometriosis. The underlying cellular mechanisms such as autophagy, apoptosis,\nand oxidative stress mechanisms in the pathogenesis and\ntreatment of endometriosis was also considered, using a\nrat model of endometriosis.\nThe supplementation of immunomodulators, antioxidants, and selective progesterone receptor\nmodulators with antioxidant properties have been assessed as possible treatments for\nendometriosis. The results demonstrated that  Sod  gene expression was\nremarkably increased by BE+SI and SI treatments. The product of this gene is responsible for\nthe dismutation of the superoxide radical. Similarly, a recent study found that an herbal\ncompound, genistein, is capable of recovering SOD activity in an endometriosis mouse model\n( 27 ). In the physiological state, cells strongly preserve the oxidative balance between\noxidants and antioxidants; however, under the impact of various stimuli such as\npharmaceuticals, toxins, radiations, etc., the oxidative balance is disturbed, which leads\nto the mechanism of oxidative stress ( 28 ). Along with the induced promotion in the level and\nactivity of antioxidant defensive responses, excess oxidants can damage biomolecules and\ncellular structures, change a variety of signaling pathways, and contribute to the\npathogenesis of different disorders such as endometriosis ( 29 ). In other hand, natural\nantioxidants, such as quercetin and soy isoflavones, that exhibit no/few undesired\nconsequences are recommended for the management of endometriosis ( 30 ,  31 ). More importantly,\noxidative radicals can alter a variety of upstream signaling pathways and affect the flux of\ncellular processes. It was suggested that, oxidative radicals can alter autophagy by\naffecting upstream pathways, such as phosphoinositide 3-kinases (PI3K), mammalian target of\nrapamycin (mTOR), 5' AMP-activated protein kinase (AMPK), and nuclear factor erythroid 2–\nrelated factor 2 (NRF2) and are involved in the pathogenesis of endometriosis ( 32 ).\nThe current study revealed that the administration of  BE+SI  remarkably\nincreased mRNA levels of  Lc3  and  Becn1 . Previous studies\nhave suggested that  Becn1  is involved in the initiation of autophagic flux\nin mammals, while  Lc3  significantly contributes to the completion of the\nautophagosome membrane and specific selection of cargoes ( 33 ,  34 ). Therefore, it could be\nassumed that autophagy was disrupted in animals with endometriosis, in which the\nadministration of the studied compounds resulted in the resumption of autophagic flux.\nNevertheless, it has been documented widely that autophagy exhibits a dual role in cell\nfate, as it can participate in cell survival as well as programmed cell death ( 12 ,  35 ).\nThereby, to elucidate what happened, the current study aimed to consider the expression of\ngenes involved in the apoptosis pathway.\nThe obtained data demonstrated that in animal models of endometriosis, the expression of\n Casp3  and  Bax  reduced significantly, while\n Bcl-2  increased remarkably. The results of the current study reveal that\napoptosis is deactivated during endometriosis that are compatible with previous studies\n( 36 ), while the administrated compounds of this study can activate it. Excessive cell\nproliferation and escape from apoptosis are the main mechanisms involved in the development\nof endometrial tissue outside the uterus, which is the classic description of endometriosis\n( 18 ). Concurrent with the findings of the present study, several reports have shown that a\nvariety of herbals such as curcumin, quercetin, resveratrol, and naringin can confront\nendometriosis by the promotion of apoptosis, hence are considered promising therapeutic\nstrategies in endometriosis management ( 37 ). In fact, it has been reviewed that\nphytochemicals and polyphenolic compounds participate in the treatment of endometriosis\nthrough different mechanisms such as anti-oxidative, anti-inflammatory, anti-proliferative\nand apoptotic, anti-angiogenic, anti-invasive, and modulating estrogenic effects ( 38 ). The\nadministration of resveratrol, for example, could the ratio of  Bcl-2/Bax\n gene expression in eutopic endometrial stromal cells from patients with\nendometriosis representing its therapeutic potential ( 39 ). Moreover, the antiproliferative\nproperties of other herbal compounds are believed to be through the ROS-mediated induction\nof apoptosis via alteration in the expression of  Bcl-2, Bax , and\n Casp3  ( 40 ).\nIt is speculated that phytochemicals of the compounds\nstudied in the present study such as Sulforaphane and\nGenistein, can be a promising approach for treating endometriosis by decreasing the oxidative stress and inducing the signaling pathways involved in apoptosis and\nautophagy. However, considering that the present study\nwas conducted on a rat model of endometriosis, it is necessary to conduct further studies on human patients and in\nequivalent doses.\n\nDue to the limitation of routine treatment for endometriosis such as surgery and hormone therapy, natural substances can be consumed safely to treat this condition. Dietary\nconsumption of broccoli extract mixed with soy isoflavons significantly reduced the histopathologic scores of\nthe induced endometrial implants and also modulated the\nexpression of the gene markers of oxidative stress, apoptosis, and autophagy in a female rat model of endometriosis.\nAccordingly, these compounds can be considered potential\ntherapeutic agents for endometriosis. However, subsequent\nstudies are needed to verify the beneficial effects of the\nnatural products on regression of endometriosis.","source_license":"CC0","license_restricted":false}