The effect of broccoli extract and soy isoflavones on endometrial implants and oxidative parameters in female rat model of endometriosis

The effect of broccoli extract and soy isoflavones on endometrial implants and oxidative parameters in female rat model of endometriosis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article The effect of broccoli extract and soy isoflavones on endometrial implants and oxidative parameters in female rat model of endometriosis Soudabeh Sabetian, Parimah Archin Dialameh, Nader Tanideh, Behrooz Gharesifard, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2369250/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background: Endometriosis is a gynecological disorder characterized by the growth of endometrial tissue outside the uterine cavity that is associated with chronic pelvic pain, subfertility and 1-3% increased risk of ovarian cancer. The purpose of the study was to investigate the effect of broccoli extract (BE) alone and in combination with soy isoflavones (SI) on endometrial implants in female rat. Method: In this study, endometriosis was induced surgically in 40 mature female rats. The rats were divided into 5 groups that were treated by oral gavage for 6 weeks with 0.5 ml of saline 0.9%/day (control group), BE (3000 mg/kg/day), SI (50mg/kg/day), BE/soy isoflavones (BE 3000 mg/kg/day + soy isoflavones 50mg/kg/day) and diphereline as a standard medication (3 mg/kg) intramuscularly. At the end of treatments, the volume and histopathology of the endometrial implants were compared among the 5 groups. The serum levels of oxidative parameters including Superoxide dismutase (SOD), Malondialdehyde (MDA) and tumor necrosis factor alpha (TNF-α) were also compared between the groups. Results: The volume of the implants significantly decreased in diphereline group (p = 0.002). The histopathological grade of endometrial implants in BE/SI and diphereline group were significantly decreased compared to the control group (p = 0.001). The serum levels of SOD in BE group were enhanced significantly in comparison to the control group (p = 0.034). Conclusion: BE in combination with SI decreased the growth and histopathologic grades of transplanted endometrial implants. These herbal compounds may have the potential to be used as an alternative medication for the treatment of endometriosis Figures Figure 1 Figure 2 Figure 3 Introduction Endometriosis is a medical condition that occurs when the tissues of uterine lining grow in other pelvic organs, such as the ovaries or fallopian tubes [ 1 ]. Bleeding the endometrial tissue outside the uterus may cause pain, dysmenorrhea, and infertility [ 2 ]. Endometriosis occur in a unique environmental condition characterized by enhanced hormonal concentrations, inflammation, and oxidative stress [ 3 ]. Gonadotropin releasing hormone agonists (GnRHa) such as diphereline, as standard hormonal treatment, can regress endometriosis by suppression of hypothalamic-pituitary-gonadal (HPG) axis and ovarian estrogen production. The side effects of GnRha include amenorrhea, hot flush, depression, headache, hair loss, musculoskeletal stiffness, vaginal dryness and bone loss [ 4 ]. Although surgical removal of the lesions or induction of a hypoestrogenic state is used as the current treatment, recurrence of lesions is common and is less than ideal for women in the reproductive ages [ 5 ]. Therefore, additional strategies are required for the treatment of endometriosis [ 6 ]. Epidemiologic studies represent an anticancer effect of diet-derived bioactive sulforaphane against different type of cancers such as thyroid and ovarian cancer [ 7 ]. Cruciferous vegetables, particularly broccoli has a high content of sulforaphane, derived from the breakdown of glucoraphanin, that is capable of modulating the secretion of the proinflammatory cytokines such as interleukin-1 beta (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor alpha (TNF-α) [ 8 , 9 ]. Although the exact pathogenic mechanisms of endometriosis remain unknown [ 10 ], it shows a tumoral behavior with tendency to inappropriate growth and adhesion formation [ 11 ]. There are a few reports on the use of dietary components as chemopreventive agents to improve endometriosis [ 12 ]. Soy isoflavones (SI) are people's daily diet with antitumoral functions; there are reports on a correlation between high levels of SI in the urine with decreased risk of endometriosis [ 13 , 14 ]. SI triggered regression of endometriotic implants, marked by decreasing size of the lesions and also prohibited the increase in proinflammatory cytokines, NF-kB activation and estrogen receptor expression in a mice model of endometriosis [ 15 ]. Superoxide dismutase (SOD) is an important enzyme involved in antioxidant system [ 16 ]. SOD catalyzes the dismutation of superoxide into hydrogen peroxide and oxygen [ 17 ]. SOD shows a decreased activity in the plasma of women with endometriosis, suggesting a decreased antioxidant capacity in these women [ 18 ]. Malondialdehyde (MDA) is an organic compound that occurs naturally as the enol, and is a marker for oxidative stress [ 19 ]. A higher level of MDA in the serum of women with endometriosis was previously observed [ 20 ]. It has been demonstrated that SI and sulforaphane modulate oxidative stresses and enhance antioxidative status against oxidative damage to the cells [ 21 , 22 ]. In the current study, we aim to compare anti-progressive effects of broccoli extract, soy isoflavones and combination of these compounds on endometriosis lesions, inflammatory cytokines including TNF-α and antioxidant marker including MDA and SOD within peritoneal tissue in rat model of induced endometriosis. The achieved results were compared with diphereline treatment. Methods Experimental case-control study Animals In this study, 45 mature female Sprague- Dawley rats (almost 8 weeks old, weighting 220 ± 20 g) were collected and housed at 22 ° C with a 12-h light/dark cycle and had free access to standard rodent chow and tap water. Preparation of broccoli extract and soy isoflavones solution Broccoli sprouts was air-dried, powdered and an alcoholic extract was produced by using ethanol (80%) and percolation method. Then, solvent was completely removed by drying under reduced pressure in a rotary evaporator. The extract was stored at 4° C until use. Soy tablet (Soya Gol, Gol Daru Company, Iran) was dissolved in saline 0.9% (1 tablet includes 50 mg soy isoflavones (≈ 27 mg Genistein)) Induction of endometriosis Endometriosis was induced surgically using the method described by Vernon and Wilson with little modifications [ 25 ]. Briefly, all the female rats were anesthetized using ketamine hydrochloride 10% (100 mg/kg, Alfasan, Netherlands) and xylazine 2% (10 mg/kg, Alfasan, Netherlands). Then, abdomen was opened through 4 cm midline incision starting from 1 cm below the xiphoid. The left horn of uterus was ligated at both the uterotubal and cervical junction ends and removed. A longitudinal cut was made through the uterine horn. By a punch biopsy, 4 round pieces of the distal part of uterine tissue was excised (4× 4× 1 mm) and placed in warm sterile saline 0.9%. Two implants were sutured with proline 5 − 0, one on the left and the other on the right side of the peritoneal cavity on the areas of well-visible vasculature with endometrial surface facing the peritoneum. Finally, the abdominal muscles, fascia and skin were sutured. Then, chlortetracycline was sprayed on the incisions site and animals was allowed to recover from anesthesia. Six weeks after the first surgery, a second look laparotomy was performed and the viability of endometrial implants was confirmed by observation of good vascular supply and pinkish colored tissue in contrast to necrosis and fibrosis (Fig. 1 ). Treatments At this stage, 45 female rats were divided into 5 groups (9 rats in each group). The control group was treated by 0.5 ml of saline 0.9%/day, the second group by BE (3000 mg/kg/day), the third group by SI (1 tablet ≈ 50mg isoflavones/kg/day)) and the fourth group was taken BE enriched with soy isoflavones (BE 3000 mg/kg/day + soy isoflavones (1 tablet ≈ 50mg soflavones/kg/day)). All the treatments of four groups were administered by oral gavage. The fifth group was received a single IM injection of diphereline S.R. 11.25 mg (3 mg/kg, Ipsen, France). Six weeks after the treatments, the rats were sacrificed, and the endometrial implants were evaluated. Measurement of the volume of endometriosis lesions The length, width and height of each endometrial lesion was measured in five groups using collis rulers. The volume of the endometriotic nodules was calculated by ellipsoid volume formulation (π/6×length× width× height). Pathologic scoring of implants All of the endometrial lesions were fixed in formalin, placed in paraffin, cut into 5 µ m sections, stained with hematoxylin-eosin and evaluated by pathologist. The persistence of epithelial cells in the grafts was scored based on the scoring system applied by Keenan et al accordingly, score 0, 1, 2 and 3 show no epithelial layer, poorly, moderately and well-preserved epithelial layers, respectively [ 26 ]. Determination of inflammatory cytokines and antioxidant markers Tissue samples from the lower anterior abdominal wall were taken and homogenized in Trizol reagent (Life Technologies, Burlington, Ontario, Canada). Each homogenate was centrifuged for 30 min at 800 g. Total protein in this extract was assayed using a standard protein assay method (BioRad DC Protein Assay BioRad Laboratories, Mississauga, Canada). Equal quantities of protein were used to determine SOD activity by Activity Assay Kit (ZellBio GmbH, Germany), MDA levels by MDA Assay Kit (crystal day Biotech Co., Ltd, China), TNF-&alpha by Rat TNF alpha ELISA Kit (crystal day Biotech Co., Ltd, China). Analysis was done according to the detail procedures in the kit. Statistical analysis Data are presented as mean ± SD and the difference between groups was analyzed using proper tests (parametric or non-parametric) by SPSS according to distribution of the data. Only probability values of P < 0.05 was considered significant. The flow chart of the study is displayed in Fig. 2 . Results The mean of volume values of endometrial implants in Diphereline group was significantly decreased (P = 0.002) and the implants' volume was stepped down considerably in BE/SI group compared to control group even though the difference was not significant (Table 1 ). Table 1 The mean volume of endometrial implants Groups Implants' volume (mm 3 ) Mean ± SD p value 1* p value 2* Control 86.77 ± 73.13 ----- ----- Broccoli Extract 56.60 ± 52.12 0.36 0.01 Soy Isoflavone 78.46 ± 73.72 0.80 0.03 Broccoli Extract/Soy Isoflavone 42.02 ± 31.88 0.13 ≤ 0.01 Diphereline 2.22 ± 2.07 ≤ 0.01 ----- *p value 1: Comparing to Control; p value 2: Comparing to Diphereline The histopathological score of endometrial implants in BE/SI and diphereline group were significantly decreased compared to the control group (p = 0.001) and there was no significant change in the score in rats treated with BE/SI compared to diphereline group (p = 0.90) (Table 2 ) & Fig. 3 . Table 2 The histopathological grade of endometrial implants Groups Pathological score Mean ± SD p value 1* p value 2* Control 2.08 ± 0.44 ---- ------ Broccoli Extract 1.66 ± 0.44 0.08 0.02 Soy Isoflavone 1.80 ± 0.32 0.17 ≤ 0.01 Broccoli Extract/Soy Isoflavone 1.2 ± 0.41 ≤ 0.01 0.90 Diphereline 1.2 ± 0.27 ≤ 0.01 ----- *p value 1: Comparing to Control; p value 2: Comparing to Diphereline D & E) Broccoli Extract/Soy Isoflavone and Soy Isoflavone groups respectively: Poorly preserved epithelial layer of endometrial implants (grade 1). Table 3 The level of serum MDA and SOD Groups MDA (nmol/ml) SOD (U/ml) TNF-α (pg/ml) Mean ± SD p value Mean ± SD p value Mean ± SD p value Control 2.90 ± 1.024 ----- 92.00 ± 20.43 ----- 20.63 ± 10.50 ----- Broccoli Extract 2.86 ± 0.817 0.23 112.07 ± 14.87 0.03 21.74 ± 11.39 0.85 Soy Isoflavone 2.75 ± 1.05 0.63 92.97 ± 26.84 0.98 19.41 ± 11.98 0.83 Broccoli Extract/Soy Isoflavone 2.81 ± 0.711 0.06 101.72 ± 17.08 0.24 25.77 ± 10.61 0.38 Diphereline 2.27 ± 0.438 0.19 96.38 ± 29.35 0.81 15.18 ± 4.66 0.24 The serum level of MDA in the studied groups was not significantly different and the level of serum SOD in broccoli group was enhanced significantly in comparison to the control group (p = 0.034) (Table 3 ). Discussion Endometriosis is characterized by the existence of endometrial tissue outside the uterus and exact physiopathology is not well known [ 23 ]. Surgical and hormonal treatment of endometriosis have inappropriate side effects and high rates of recurrence. Recent studies have demonstrated the beneficial effect of herbal medicine against endometriosis [ 24 ]. In the current study, we compared the therapeutic effects of broccoli extract and soy isoflavones on endometriosis lesions, inflammatory cytokines including TNF-α and serum levels of antioxidant markers including MDA and SOD in a rat model of induced endometriosis. The achieved results were compared with hormone therapy (gonadotropin-releasing hormone: diphereline). Endometriosis is associated with inflammation along with oxidative stress, thereby weakening the oxidative and inflammatory potential is the main strategy of the effective therapeutic roles of phytochemicals on the control of endometriosis [ 25 , 26 ]. Sulforaphane, a compound derived from broccoli sprouts is an agent to mediate anti-oxidant and anti-inflammatory activity [ 27 ]. Soy isoflavones including genistein and daidzein modulate the estrogen receptor and inhibit cell growth and inflammation in endometriosis [ 28 ]. It was demonstrated the potential preventive effects of isoflavones on endometriosis progression through the control of apoptosis dysregulation, the inhibition of NF-κB and COX-2 pathways in the peritoneal tissues [ 29 ]. In this study, the mean implants' volume in the treatment with broccoli extract mixed with soy isoflavones (42.02 ± 31.88 mm 3 ) was decreased considerably compared with control (86.77 ± 73.13 mm 3 ) even though the difference was not significant statistically (p = 0.13). Interestingly, the histopathological score of Broccoli/Soy group showed poorly epithelial layers with significant regression compared to the control group. The results may imply the synergistic phytopreventive effects of isoflavones and sulforaphane on endometriosis development. SOD is an important enzyme in antioxidant system that catalyzes the dismutation of superoxide into hydrogen peroxide and oxygen [ 30 ]. Activity of SOD was shown to be decreased in the plasma of women with endometriosis, suggesting a declined antioxidant capacity in these women [ 31 ]. In the present study, the serum level of SOD was increased significantly after broccoli exctract treatment and was enhanced insignificantly in other studied groups. It was reported that broccoli exctract protects the cells from H2O2-induced oxidative stress through enhancing Nrf2/Keap1 signaling pathway consequently augmenting antioxidant enzyme such as SOD [ 32 ]. Isoflavone supplementation was illustrated to enhance antioxidant action via weakening of exercise-induced oxidative stress [ 33 ]. The previous studies have demonstrated that serum MDA was significantly reduced by dietary Supplement of Broccoli and soy isoflavones [ 34 ]. The current findings showed no significant effects of the studied treatments on MDA. Previous studies have represented that the volume of endometriosis lesions was significantly decreased simultaneously with a reduction in the level of IL-6, TNF-α, estradiol and vascular endothelial growth factor (VEGF) within serum and peritoneal fluid [ 35 ]. It has been corroborated that dietary supplements including sulforaphane and isoflavones decrease the levels of anti-inflammatory cytokines such as TNF-α and nuclear factor kappa B (NF-κB), that are involved in the mechanism of inflammation [ 36 , 37 ]. However, Serum levels of TNF-α showed no significant statistical difference among the treated groups in the current study. These incompatibilities might be due to the treatment duration and dosages of the compounds. Dipherelin showed the lowest and BE/ SI showed the highest levels of TNF-α in our study. Conclusion Dietary consumption of broccoli extract in combination with soy isoflavones significantly reduced the histopathologic grades of endometrial implants in female rat model of endometriosis. The volume of endometrial implants was decreased considerably in the treated rats by mixed broccoli extract and soy isoflavones. The serum level of SOD was significantly increased by broccoli extract treatment and insignificantly in other studied groups. The findings of the current study show the potential preventive effect of the combination of broccoli extract and soy isoflavones on endometriosis. Further studies are required to prove the beneficial properties of these compounds on the clinical symptoms of endometriosis. Declarations Acknowledgements None. Funding This article has been financially supported by the Vice Chancellor for Research of Shiraz University of Medical Sciences with the grant number of 97-01-50-19167. Author information Authors and Affiliations Infertility Research Center, Shiraz University of Medical Sciences, Shiraz, Iran Soudabeh Sabetian, Behrooz Gharesifard, Bahia Namavar Jahromi Department of Obstetrics and Gynecology, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran Parimah Archin Dialameh, Bahia Namavar Jahromi Department of Pharmacology, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran Nader Tanideh Department of Immunology, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran Behrooz Gharesifard, Moslem Ahmadi, Maryam Valibeigi Department of Pathology, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran Perikala Vijayananda Kumar, Sarah Siahbani Contributions SSa and BNJ contributed to the study design and conceptualization. SSa, PAD, NT, BGh, MA, MV, PVK and SSi provided technical support and critically reviewed the manuscript for its intellectual content. SSa and BNJ reviewed the literature and performed the analysis. All authors read and approved the final manuscript. Corresponding author Correspondence to Bahia Namavar Jahromi. Ethics declarations Ethics approval and consent to participate The Medical Ethics Committee of Shiraz University of Medical Sciences approved this study with the reference number IR.SUMS.REC.1398.176. Consent for publication Not applicable. Competing interests The authors declare that they have no competing interests. References Arafah M, Rashid S, MJ.A.i.AP Akhtar. Endometriosis: a comprehensive review. 2021;28(1):30–43. Español F, Endometriosis is a disease where tissue similar to the lining of the uterus grows outside the uterus, causing pain and/or infertility (1). Endometriosis affects roughly 10%(190 million) of reproductive age women and girls globally (2). It is a chronic disease associated with severe, life-impacting pain during periods, sexual intercourse, bowel movements and/or urination, chronic pelvic pain, abdominal bloating, nausea. 2021. Assaf L, Eid AA, Nassif JJLS, Role of ampk/mtor, mitochondria, and ros in the pathogenesis of endometriosis. 2022: p. 120805. Jahromi BN, et al., Comparing the effects of glycyrrhiza glabra root extract, a cyclooxygenase-2 inhibitor (celecoxib) and a gonadotropin-releasing hormone analog (diphereline) in a rat model of endometriosis . 2019. 13(1): p. 45. Clemenza S, et al. Endometriosis-Induced Pain: The Treatment Strategy , in Endometriosis Pathogenesis, Clinical Impact and Management . Springer; 2021. pp. 41–50. Guo Y, et al., Complementary and alternative medicine for dysmenorrhea caused by endometriosis: a review of utilization and mechanism. 2021. 2021 . Deldar Abad Paskeh M, et al. Target cancer stem cells Diet agents: important therapeutic strategy against Hum malignancies. 2021;22(21):11669. Mangla B, et al., Sulforaphane: A review of its therapeutic potentials, advances in its nanodelivery, recent patents, and clinical trials. 2021. 35 (10): p. 5440–5458. Moon S-J, et al., The anti-arthritis effect of sulforaphane, an activator of Nrf2, is associated with inhibition of both B cell differentiation and the production of inflammatory cytokines . 2021. 16(2): p. e0245986. Polak G, et al. Environ Factors Endometr. 2021;18(21):11025. Sun Y, Liu GJJoC. Endometriosis-associated Ovarian Clear Cell Carcinoma: A Special Entity? 2021;12(22):6773. George BP, Chandran R, Abrahamse HJA. Role of phytochemicals in cancer chemoprevention: insights. 2021;10(9):1455. Kim I-SJA. Current perspectives on the beneficial effects of soybean isoflavones and their metabolites for humans . 2021. 10(7): p. 1064. Fan Y, et al. Intake of Soy, Soy Isoflavones and Soy Protein and Risk of. Cancer Incidence and Mortality. 2022;9:847421. Cai X, et al., Phytoestrogens for the management of endometriosis: findings and issues. 2021. 14 (6): p. 569. Cecerska-Heryć E, et al., Are antioxidant enzymes essential markers in the diagnosis and monitoring of cancer patients–a review . 2021. 93: p. 1–8. Senthilkumar M, Amaresan N, Sankaranarayanan A. Estimation of superoxide dismutase (SOD) , in Plant-microbe interactions . Springer; 2021. pp. 117–8. Udayana IGNBS, et al. Association of Endometriosis and Oxidative Stress. 2022;4(5):109–13. Labib H, et al., Keap1/Nrf2 pathway in sodium fluoride-induced cardiac toxicity and the prophylactic role of vitamin C versus platelet-rich plasma . 2022. 81(3): p. 663–678. Da Broi MG, et al., The Impact of Controlled Ovarian Stimulation on Serum Oxidative Stress Markers in Infertile Women with Endometriosis Undergoing ICSI . 2022. 11(6): p. 1161. Yadav E, et al. Polyphenols as Modulators of Oxidative Stress in Cancer Disease , in Polyphenols-based Nanotherapeutics for Cancer Management . Springer; 2021. pp. 143–88. Jurcau AJN, The role of natural antioxidants in the prevention of dementia—where do we stand and future perspectives. 2021. 13 (2): p. 282. Marcolongo P, et al., Regulation of autophagy in the uterus: from physiological processes to endometriosis and uterine fibroids. 2021. Meresman GF, Götte M. and M.W.J.H.r.u. Laschke, Plants as source of new therapies for endometriosis: a review of preclinical and clinical studies . 2021. 27(2): p. 367–392. Iftikhar M, et al. Assessment of Lipid Profile in Females Suffering from Endometriosis . in Proceedings . 2022. Cacciottola L, Donnez J, M.-M.J.I.J.o.M S, Dolmans. Can endometriosis-related oxidative stress pave the way for new treatment targets? 2021. 22(13): p. 7138. Mahn A, Castillo AJM. Potential of sulforaphane as a natural immune system enhancer: A review . 2021. 26(3): p. 752. Maksymowicz M, et al., The role of soy in development of breast, ovarian, endometrial and prostate gland cancer . 2021. 11(9): p. 180–186. Gołąbek A, Kowalska K, Olejnik AJN. Polyphenols as a Diet Therapy Concept for Endometriosis—Current Opinion and Future Perspectives . 2021. 13(4): p. 1347. Fujii J, Homma T, Osaki TJA. Superoxide Radicals in the Execution of Cell Death. 2022;11(3):501. Lu J, et al., Emerging hallmarks of endometriosis metabolism: A promising target for the treatment of endometriosis. 2022: p. 119381. Lee D-Y, Song M-Y, Kim E-HJA. Role of oxidative stress and Nrf2/keap1 signaling in colorectal cancer: Mechanisms and therapeutic perspectives with phytochemicals . 2021. 10(5): p. 743. Zeng Z, et al., Effects of dietary strategies on exercise-induced oxidative stress: a narrative review of human studies . 2021. 10(4): p. 542. Nazari-Khanamiri F, MJ.J.o.FB Ghasemnejad‐Berenji. Cellular and molecular mechanisms of genistein in prevention and treatment of diseases: An overview . 2021. 45(11): p. e13972. Lu F, et al., Antibiotic Therapy and Vaginal Microbiota Transplantation Reduce Endometriosis Disease Progression in Female Mice via NF-κB Signaling Pathway . 2022. 9. Pérez-Torres I, et al., Oxidative stress, plant natural antioxidants, and obesity. 2021. 22 (4): p. 1786. Zamani-Garmsiri F, et al., Polyphenols: Potential anti‐inflammatory agents for treatment of metabolic disorders. 2022. 36 (1): p. 415–432. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-2369250","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":159888224,"identity":"5ca8b119-a42a-4119-8f05-71de161ecb7a","order_by":0,"name":"Soudabeh Sabetian","email":"","orcid":"","institution":"Infertility Research Center, Shiraz University of Medical Sciences, Shiraz, Iran","correspondingAuthor":false,"prefix":"","firstName":"Soudabeh","middleName":"","lastName":"Sabetian","suffix":""},{"id":159888225,"identity":"9b15a72c-4705-431d-aeba-07387ef455b2","order_by":1,"name":"Parimah Archin Dialameh","email":"","orcid":"","institution":"Department of Obstetrics and Gynecology, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran","correspondingAuthor":false,"prefix":"","firstName":"Parimah","middleName":"Archin","lastName":"Dialameh","suffix":""},{"id":159888226,"identity":"24c1bc3d-4e0d-4805-8d73-1c44062b9163","order_by":2,"name":"Nader Tanideh","email":"","orcid":"","institution":"Department of Pharmacology, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran","correspondingAuthor":false,"prefix":"","firstName":"Nader","middleName":"","lastName":"Tanideh","suffix":""},{"id":159888227,"identity":"8e37998e-6aa9-430d-a11b-5460af0a1f85","order_by":3,"name":"Behrooz Gharesifard","email":"","orcid":"","institution":"Department of Immunology, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran","correspondingAuthor":false,"prefix":"","firstName":"Behrooz","middleName":"","lastName":"Gharesifard","suffix":""},{"id":159888228,"identity":"11cd8aa1-e184-44a0-886a-ab5df25507a5","order_by":4,"name":"Moslem Ahmadi","email":"","orcid":"","institution":"Department of Immunology, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran","correspondingAuthor":false,"prefix":"","firstName":"Moslem","middleName":"","lastName":"Ahmadi","suffix":""},{"id":159888229,"identity":"a60c5bbe-23df-4994-b51f-43e09e1dd0d4","order_by":5,"name":"Maryam Valibeigi","email":"","orcid":"","institution":"Department of Immunology, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran","correspondingAuthor":false,"prefix":"","firstName":"Maryam","middleName":"","lastName":"Valibeigi","suffix":""},{"id":159888230,"identity":"7fa086c3-cdae-4a93-a69a-7f5c2938f608","order_by":6,"name":"Perikala Vijayananda Kumar","email":"","orcid":"","institution":"Department of Pathology, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran","correspondingAuthor":false,"prefix":"","firstName":"Perikala","middleName":"Vijayananda","lastName":"Kumar","suffix":""},{"id":159888231,"identity":"85cdadc5-96d4-41ce-b706-1a372191eab3","order_by":7,"name":"Sarah Siahbani","email":"","orcid":"","institution":"Department of Pathology, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran","correspondingAuthor":false,"prefix":"","firstName":"Sarah","middleName":"","lastName":"Siahbani","suffix":""},{"id":159888232,"identity":"820e8959-cc0f-4389-b37d-c8a1c0a5d8fd","order_by":8,"name":"Bahia Namavar Jahromi","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA1klEQVRIiWNgGAWjYHAD5gNAQkKGFC1sCSAtPKRo4TEAkwTVmbOfTvzMU3Mvz7z9zOdXN2oseBjYDx/dgE+LZU/uZmmeY8XFMmdyt1nnHAM6jCct7QY+LQYHcjdIzmBLSJwhwbvNOIcNqEWCxwy/lvNvN/+c8Q+kheeZcc4/YrTcyN0m8bENrIX5cW4bUVrebrP42JdQLMGTZsac2yfBw0bQL+dzN99I+JaQJ8F++PHnnG91cvzsh4/h1QIDCUDMJgFisRGjHKaF+QOxqkfBKBgFo2BkAQDYykcGPTOpnwAAAABJRU5ErkJggg==","orcid":"","institution":"Department of Obstetrics and Gynecology, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran","correspondingAuthor":true,"prefix":"","firstName":"Bahia","middleName":"Namavar","lastName":"Jahromi","suffix":""}],"badges":[],"createdAt":"2022-12-12 09:14:27","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-2369250/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-2369250/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":30384245,"identity":"2f8f74ce-bcd2-43c5-9b2c-c14c5e3fd2e8","added_by":"auto","created_at":"2022-12-15 16:16:53","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":54408,"visible":true,"origin":"","legend":"\u003cp\u003eInduction of endometriosis. A) The first laparotomy: transplant of endometrial implants on the peritoneum. B) The second look surgery: necropsy of a rat in the control group showing growth of implanted lesions of endometriosis.\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-2369250/v1/05097e89ac952f2e6b3a4c3e.jpeg"},{"id":30384244,"identity":"d0088588-a8b0-4d36-9bd7-d1df230a2816","added_by":"auto","created_at":"2022-12-15 16:16:53","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":100050,"visible":true,"origin":"","legend":"\u003cp\u003eThe flow chart of the study\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-2369250/v1/2b62d75bab963c20a557b8d0.jpeg"},{"id":30384246,"identity":"6566d4da-523e-440d-b21d-4a560e00d9fc","added_by":"auto","created_at":"2022-12-15 16:16:53","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":222690,"visible":true,"origin":"","legend":"\u003cp\u003eSpecimens of the treated groups stained by hematoxylin-eosin (scale bar: 100 μm). A) Control group: well preserved epithelial layer of endometrial implants (grade 3), B \u0026amp; C) Broccoli Extract and Soy Isoflavone groups respectively: Moderately preserved epithelial layer of endometrial implants (grade 2).\u003c/p\u003e\n\u003cp\u003eD \u0026amp; E) Broccoli Extract/Soy Isoflavone and Soy Isoflavone groups respectively: Poorly preserved epithelial layer of endometrial implants (grade 1).\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-2369250/v1/2976a16c99e7d4568ec7bf4e.jpeg"},{"id":30468441,"identity":"9042fce5-3259-4c11-8e15-6cfcd1387242","added_by":"auto","created_at":"2022-12-17 16:29:32","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":483491,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2369250/v1/2088ecc3-21b8-446a-8358-cffe0bc80e7b.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"The effect of broccoli extract and soy isoflavones on endometrial implants and oxidative parameters in female rat model of endometriosis","fulltext":[{"header":"Introduction","content":"\u003cp\u003eEndometriosis is a medical condition that occurs when the tissues of uterine lining grow in other pelvic organs, such as the ovaries or fallopian tubes [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Bleeding the endometrial tissue outside the uterus may cause pain, dysmenorrhea, and infertility [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Endometriosis occur in a unique environmental condition characterized by enhanced hormonal concentrations, inflammation, and oxidative stress [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Gonadotropin releasing hormone agonists (GnRHa) such as diphereline, as standard hormonal treatment, can regress endometriosis by suppression of hypothalamic-pituitary-gonadal (HPG) axis and ovarian estrogen production. The side effects of GnRha include amenorrhea, hot flush, depression, headache, hair loss, musculoskeletal stiffness, vaginal dryness and bone loss [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Although surgical removal of the lesions or induction of a hypoestrogenic state is used as the current treatment, recurrence of lesions is common and is less than ideal for women in the reproductive ages [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Therefore, additional strategies are required for the treatment of endometriosis [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Epidemiologic studies represent an anticancer effect of diet-derived bioactive sulforaphane against different type of cancers such as thyroid and ovarian cancer [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Cruciferous vegetables, particularly broccoli has a high content of sulforaphane, derived from the breakdown of glucoraphanin, that is capable of modulating the secretion of the proinflammatory cytokines such as interleukin-1 beta (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor alpha (TNF-α) [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Although the exact pathogenic mechanisms of endometriosis remain unknown [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], it shows a tumoral behavior with tendency to inappropriate growth and adhesion formation [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. There are a few reports on the use of dietary components as chemopreventive agents to improve endometriosis [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Soy isoflavones (SI) are people's daily diet with antitumoral functions; there are reports on a correlation between high levels of SI in the urine with decreased risk of endometriosis [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. SI triggered regression of endometriotic implants, marked by decreasing size of the lesions and also prohibited the increase in proinflammatory cytokines, NF-kB activation and estrogen receptor expression in a mice model of endometriosis [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Superoxide dismutase (SOD) is an important enzyme involved in antioxidant system [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. SOD catalyzes the dismutation of superoxide into hydrogen peroxide and oxygen [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. SOD shows a decreased activity in the plasma of women with endometriosis, suggesting a decreased antioxidant capacity in these women [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Malondialdehyde (MDA) is an organic compound that occurs naturally as the enol, and is a marker for oxidative stress [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. A higher level of MDA in the serum of women with endometriosis was previously observed [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. It has been demonstrated that SI and sulforaphane modulate oxidative stresses and enhance antioxidative status against oxidative damage to the cells [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. In the current study, we aim to compare anti-progressive effects of broccoli extract, soy isoflavones and combination of these compounds on endometriosis lesions, inflammatory cytokines including TNF-α and antioxidant marker including MDA and SOD within peritoneal tissue in rat model of induced endometriosis. The achieved results were compared with diphereline treatment.\u003c/p\u003e "},{"header":"Methods","content":" \u003cp\u003eExperimental case-control study Animals\u003c/p\u003e \u003cp\u003eIn this study, 45 mature female Sprague- Dawley rats (almost 8 weeks old, weighting 220\u0026thinsp;\u0026plusmn;\u0026thinsp;20 g) were collected and housed at 22 \u0026deg; C with a 12-h light/dark cycle and had free access to standard rodent chow and tap water.\u003c/p\u003e \u003cp\u003ePreparation of broccoli extract and soy isoflavones solution\u003c/p\u003e \u003cp\u003eBroccoli sprouts was air-dried, powdered and an alcoholic extract was produced by using ethanol (80%) and percolation method. Then, solvent was completely removed by drying under reduced pressure in a rotary evaporator. The extract was stored at 4\u0026deg; C until use. Soy tablet (Soya Gol, Gol Daru Company, Iran) was dissolved in saline 0.9% (1 tablet includes 50 mg soy isoflavones (\u0026asymp;\u0026thinsp;27 mg Genistein))\u003c/p\u003e \u003cp\u003eInduction of endometriosis\u003c/p\u003e \u003cp\u003eEndometriosis was induced surgically using the method described by Vernon and Wilson with little modifications [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Briefly, all the female rats were anesthetized using ketamine hydrochloride 10% (100 mg/kg, Alfasan, Netherlands) and xylazine 2% (10 mg/kg, Alfasan, Netherlands). Then, abdomen was opened through 4 cm midline incision starting from 1 cm below the xiphoid. The left horn of uterus was ligated at both the uterotubal and cervical junction ends and removed. A longitudinal cut was made through the uterine horn. By a punch biopsy, 4 round pieces of the distal part of uterine tissue was excised (4\u0026times; 4\u0026times; 1 mm) and placed in warm sterile saline 0.9%. Two implants were sutured with proline 5\u0026thinsp;\u0026minus;\u0026thinsp;0, one on the left and the other on the right side of the peritoneal cavity on the areas of well-visible vasculature with endometrial surface facing the peritoneum. Finally, the abdominal muscles, fascia and skin were sutured. Then, chlortetracycline was sprayed on the incisions site and animals was allowed to recover from anesthesia. Six weeks after the first surgery, a second look laparotomy was performed and the viability of endometrial implants was confirmed by observation of good vascular supply and pinkish colored tissue in contrast to necrosis and fibrosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTreatments\u003c/p\u003e \u003cp\u003eAt this stage, 45 female rats were divided into 5 groups (9 rats in each group). The control group was treated by 0.5 ml of saline 0.9%/day, the second group by BE (3000 mg/kg/day), the third group by SI (1 tablet\u0026thinsp;\u0026asymp;\u0026thinsp;50mg isoflavones/kg/day)) and the fourth group was taken BE enriched with soy isoflavones (BE 3000 mg/kg/day\u0026thinsp;+\u0026thinsp;soy isoflavones (1 tablet\u0026thinsp;\u0026asymp;\u0026thinsp;50mg soflavones/kg/day)). All the treatments of four groups were administered by oral gavage. The fifth group was received a single IM injection of diphereline S.R. 11.25 mg (3 mg/kg, Ipsen, France). Six weeks after the treatments, the rats were sacrificed, and the endometrial implants were evaluated.\u003c/p\u003e \u003cp\u003eMeasurement of the volume of endometriosis lesions\u003c/p\u003e \u003cp\u003eThe length, width and height of each endometrial lesion was measured in five groups using collis rulers. The volume of the endometriotic nodules was calculated by ellipsoid volume formulation (π/6\u0026times;length\u0026times; width\u0026times; height).\u003c/p\u003e \u003cp\u003ePathologic scoring of implants\u003c/p\u003e \u003cp\u003eAll of the endometrial lesions were fixed in formalin, placed in paraffin, cut into 5 \u0026micro; m sections, stained with hematoxylin-eosin and evaluated by pathologist. The persistence of epithelial cells in the grafts was scored based on the scoring system applied by Keenan et al accordingly, score 0, 1, 2 and 3 show no epithelial layer, poorly, moderately and well-preserved epithelial layers, respectively [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eDetermination of inflammatory cytokines and antioxidant markers\u003c/p\u003e \u003cp\u003eTissue samples from the lower anterior abdominal wall were taken and homogenized in Trizol reagent (Life Technologies, Burlington, Ontario, Canada). Each homogenate was centrifuged for 30 min at 800 g. Total protein in this extract was assayed using a standard protein assay method (BioRad DC Protein Assay BioRad Laboratories, Mississauga, Canada). Equal quantities of protein were used to determine SOD activity by Activity Assay Kit (ZellBio GmbH, Germany), MDA levels by MDA Assay Kit (crystal day Biotech Co., Ltd, China), TNF-\u0026amp;alpha by Rat TNF alpha ELISA Kit (crystal day Biotech Co., Ltd, China). Analysis was done according to the detail procedures in the kit.\u003c/p\u003e \u003cdiv id=\"Sec2\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eData are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD and the difference between groups was analyzed using proper tests (parametric or non-parametric) by SPSS according to distribution of the data. Only probability values of P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered significant. The flow chart of the study is displayed in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003eThe mean of volume values of endometrial implants in Diphereline group was significantly decreased (P\u0026thinsp;=\u0026thinsp;0.002) and the implants' volume was stepped down considerably in BE/SI group compared to control group even though the difference was not significant (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eThe mean volume of endometrial implants\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGroups\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e \u003cp\u003eImplants' volume (mm\u003csup\u003e3\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ep value 1*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ep value 2*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e86.77\u0026thinsp;\u0026plusmn;\u0026thinsp;73.13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-----\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-----\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBroccoli Extract\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e56.60\u0026thinsp;\u0026plusmn;\u0026thinsp;52.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSoy Isoflavone\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e78.46\u0026thinsp;\u0026plusmn;\u0026thinsp;73.72\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.80\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.03\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBroccoli Extract/Soy Isoflavone\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e42.02\u0026thinsp;\u0026plusmn;\u0026thinsp;31.88\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026le;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDiphereline\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.22\u0026thinsp;\u0026plusmn;\u0026thinsp;2.07\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026le;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-----\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"4\"\u003e*p value 1: Comparing to Control; p value 2: Comparing to Diphereline\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThe histopathological score of endometrial implants in BE/SI and diphereline group were significantly decreased compared to the control group (p\u0026thinsp;=\u0026thinsp;0.001) and there was no significant change in the score in rats treated with BE/SI compared to diphereline group (p\u0026thinsp;=\u0026thinsp;0.90) (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) \u0026amp; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eThe histopathological grade of endometrial implants\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eGroups\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e \u003cp\u003ePathological score\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003ep value 1*\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003ep value 2*\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e2.08\u0026thinsp;\u0026plusmn;\u0026thinsp;0.44\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e----\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e------\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBroccoli Extract\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e1.66\u0026thinsp;\u0026plusmn;\u0026thinsp;0.44\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.02\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSoy Isoflavone\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e1.80\u0026thinsp;\u0026plusmn;\u0026thinsp;0.32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026le;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBroccoli Extract/Soy Isoflavone\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e1.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.41\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026le;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.90\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDiphereline\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e1.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026le;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-----\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"4\"\u003e*p value 1: Comparing to Control; p value 2: Comparing to Diphereline\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eD \u0026amp; E) Broccoli Extract/Soy Isoflavone and Soy Isoflavone groups respectively: Poorly preserved epithelial layer of endometrial implants (grade 1).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eThe level of serum MDA and SOD\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eGroups\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eMDA (nmol/ml)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003eSOD (U/ml)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003eTNF-α (pg/ml)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003ep value\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003ep value\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eMean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003ep value\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e2.90\u0026thinsp;\u0026plusmn;\u0026thinsp;1.024\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-----\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e92.00\u0026thinsp;\u0026plusmn;\u0026thinsp;20.43\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-----\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e20.63\u0026thinsp;\u0026plusmn;\u0026thinsp;10.50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-----\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBroccoli Extract\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e2.86\u0026thinsp;\u0026plusmn;\u0026thinsp;0.817\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e112.07\u0026thinsp;\u0026plusmn;\u0026thinsp;14.87\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e21.74\u0026thinsp;\u0026plusmn;\u0026thinsp;11.39\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.85\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSoy Isoflavone\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e2.75\u0026thinsp;\u0026plusmn;\u0026thinsp;1.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.63\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e92.97\u0026thinsp;\u0026plusmn;\u0026thinsp;26.84\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.98\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e19.41\u0026thinsp;\u0026plusmn;\u0026thinsp;11.98\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.83\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBroccoli Extract/Soy Isoflavone\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e2.81\u0026thinsp;\u0026plusmn;\u0026thinsp;0.711\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e101.72\u0026thinsp;\u0026plusmn;\u0026thinsp;17.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e25.77\u0026thinsp;\u0026plusmn;\u0026thinsp;10.61\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.38\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDiphereline\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e2.27\u0026thinsp;\u0026plusmn;\u0026thinsp;0.438\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e96.38\u0026thinsp;\u0026plusmn;\u0026thinsp;29.35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.81\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e15.18\u0026thinsp;\u0026plusmn;\u0026thinsp;4.66\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.24\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThe serum level of MDA in the studied groups was not significantly different and the level of serum SOD in broccoli group was enhanced significantly in comparison to the control group (p\u0026thinsp;=\u0026thinsp;0.034) (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eEndometriosis is characterized by the existence of endometrial tissue outside the uterus and exact physiopathology is not well known [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Surgical and hormonal treatment of endometriosis have inappropriate side effects and high rates of recurrence. Recent studies have demonstrated the beneficial effect of herbal medicine against endometriosis [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. In the current study, we compared the therapeutic effects of broccoli extract and soy isoflavones on endometriosis lesions, inflammatory cytokines including TNF-α and serum levels of antioxidant markers including MDA and SOD in a rat model of induced endometriosis. The achieved results were compared with hormone therapy (gonadotropin-releasing hormone: diphereline). Endometriosis is associated with inflammation along with oxidative stress, thereby weakening the oxidative and inflammatory potential is the main strategy of the effective therapeutic roles of phytochemicals on the control of endometriosis [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Sulforaphane, a compound derived from broccoli sprouts is an agent to mediate anti-oxidant and anti-inflammatory activity [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Soy isoflavones including genistein and daidzein modulate the estrogen receptor and inhibit cell growth and inflammation in endometriosis [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. It was demonstrated the potential preventive effects of isoflavones on endometriosis progression through the control of apoptosis dysregulation, the inhibition of NF-κB and COX-2 pathways in the peritoneal tissues [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. In this study, the mean implants' volume in the treatment with broccoli extract mixed with soy isoflavones (42.02\u0026thinsp;\u0026plusmn;\u0026thinsp;31.88 mm\u003csup\u003e3\u003c/sup\u003e) was decreased considerably compared with control (86.77\u0026thinsp;\u0026plusmn;\u0026thinsp;73.13 mm\u003csup\u003e3\u003c/sup\u003e) even though the difference was not significant statistically (p\u0026thinsp;=\u0026thinsp;0.13). Interestingly, the histopathological score of Broccoli/Soy group showed poorly epithelial layers with significant regression compared to the control group. The results may imply the synergistic phytopreventive effects of isoflavones and sulforaphane on endometriosis development. SOD is an important enzyme in antioxidant system that catalyzes the dismutation of superoxide into hydrogen peroxide and oxygen [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Activity of SOD was shown to be decreased in the plasma of women with endometriosis, suggesting a declined antioxidant capacity in these women [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. In the present study, the serum level of SOD was increased significantly after broccoli exctract treatment and was enhanced insignificantly in other studied groups. It was reported that broccoli exctract protects the cells from H2O2-induced oxidative stress through enhancing Nrf2/Keap1 signaling pathway consequently augmenting antioxidant enzyme such as SOD [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Isoflavone supplementation was illustrated to enhance antioxidant action via weakening of exercise-induced oxidative stress [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. The previous studies have demonstrated that serum MDA was significantly reduced by dietary Supplement of Broccoli and soy isoflavones [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. The current findings showed no significant effects of the studied treatments on MDA. Previous studies have represented that the volume of endometriosis lesions was significantly decreased simultaneously with a reduction in the level of IL-6, TNF-α, estradiol and vascular endothelial growth factor (VEGF) within serum and peritoneal fluid [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. It has been corroborated that dietary supplements including sulforaphane and isoflavones decrease the levels of anti-inflammatory cytokines such as TNF-α and nuclear factor kappa B (NF-κB), that are involved in the mechanism of inflammation [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. However, Serum levels of TNF-α showed no significant statistical difference among the treated groups in the current study. These incompatibilities might be due to the treatment duration and dosages of the compounds. Dipherelin showed the lowest and BE/ SI showed the highest levels of TNF-α in our study.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eDietary consumption of broccoli extract in combination with soy isoflavones significantly reduced the histopathologic grades of endometrial implants in female rat model of endometriosis. The volume of endometrial implants was decreased considerably in the treated rats by mixed broccoli extract and soy isoflavones. The serum level of SOD was significantly increased by broccoli extract treatment and insignificantly in other studied groups. The findings of the current study show the potential preventive effect of the combination of broccoli extract and soy isoflavones on endometriosis. Further studies are required to prove the beneficial properties of these compounds on the clinical symptoms of endometriosis.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eAcknowledgements\u003c/p\u003e\n\u003cp\u003eNone.\u003c/p\u003e\n\u003cp\u003eFunding\u003c/p\u003e\n\u003cp\u003eThis article has been financially supported by the Vice Chancellor for Research of Shiraz University of Medical Sciences with the grant number of 97-01-50-19167.\u003c/p\u003e\n\u003cp\u003eAuthor information\u003c/p\u003e\n\u003cp\u003eAuthors and Affiliations\u003c/p\u003e\n\u003cp\u003eInfertility Research Center, Shiraz University of Medical Sciences, Shiraz, Iran\u003c/p\u003e\n\u003cp\u003eSoudabeh Sabetian, Behrooz Gharesifard, Bahia Namavar Jahromi\u003c/p\u003e\n\u003cp\u003eDepartment of Obstetrics and Gynecology, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran\u003c/p\u003e\n\u003cp\u003eParimah Archin Dialameh, Bahia Namavar Jahromi\u003c/p\u003e\n\u003cp\u003eDepartment of Pharmacology, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran\u003c/p\u003e\n\u003cp\u003eNader Tanideh\u003c/p\u003e\n\u003cp\u003eDepartment of Immunology, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran\u003c/p\u003e\n\u003cp\u003eBehrooz Gharesifard, Moslem Ahmadi, Maryam Valibeigi\u003c/p\u003e\n\u003cp\u003eDepartment of Pathology, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran\u003c/p\u003e\n\u003cp\u003ePerikala Vijayananda Kumar, Sarah Siahbani\u003c/p\u003e\n\u003cp\u003eContributions\u003c/p\u003e\n\u003cp\u003eSSa and BNJ contributed to the study design and conceptualization. SSa, PAD, NT, BGh, MA, MV, PVK and SSi provided technical support and critically reviewed the manuscript for its intellectual content. SSa and BNJ reviewed the literature and performed the analysis. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003eCorresponding author\u003c/p\u003e\n\u003cp\u003eCorrespondence to Bahia Namavar Jahromi.\u003c/p\u003e\n\u003cp\u003eEthics declarations\u003c/p\u003e\n\u003cp\u003eEthics approval and consent to participate\u003c/p\u003e\n\u003cp\u003eThe Medical Ethics Committee of Shiraz University of Medical Sciences approved this study with the reference number IR.SUMS.REC.1398.176.\u003c/p\u003e\n\u003cp\u003eConsent for publication\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003eCompeting interests\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eArafah M, Rashid S, MJ.A.i.AP Akhtar. Endometriosis: a comprehensive review. 2021;28(1):30\u0026ndash;43.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEspa\u0026ntilde;ol F, \u003cem\u003eEndometriosis is a disease where tissue similar to the lining of the uterus grows outside the uterus, causing pain and/or infertility (1). Endometriosis affects roughly 10%(190 million) of reproductive age women and girls globally (2). It is a chronic disease associated with severe, life-impacting pain during periods, sexual intercourse, bowel movements and/or urination, chronic pelvic pain, abdominal bloating, nausea.\u003c/em\u003e 2021.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAssaf L, Eid AA, Nassif JJLS, \u003cem\u003eRole of ampk/mtor, mitochondria, and ros in the pathogenesis of endometriosis.\u003c/em\u003e 2022: p.\u0026nbsp;120805.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJahromi BN, et al., \u003cem\u003eComparing the effects of glycyrrhiza glabra root extract, a cyclooxygenase-2 inhibitor (celecoxib) and a gonadotropin-releasing hormone analog (diphereline) in a rat model of endometriosis\u003c/em\u003e. 2019. 13(1): p.\u0026nbsp;45.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eClemenza S, et al. \u003cem\u003eEndometriosis-Induced Pain: The Treatment Strategy\u003c/em\u003e, in \u003cem\u003eEndometriosis Pathogenesis, Clinical Impact and Management\u003c/em\u003e. Springer; 2021. pp.\u0026nbsp;41\u0026ndash;50.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGuo Y, et al., \u003cem\u003eComplementary and alternative medicine for dysmenorrhea caused by endometriosis: a review of utilization and mechanism.\u003c/em\u003e 2021. \u003cb\u003e2021\u003c/b\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDeldar Abad Paskeh M, et al. Target cancer stem cells Diet agents: important therapeutic strategy against Hum malignancies. 2021;22(21):11669.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMangla B, et al., \u003cem\u003eSulforaphane: A review of its therapeutic potentials, advances in its nanodelivery, recent patents, and clinical trials.\u003c/em\u003e 2021. \u003cb\u003e35\u003c/b\u003e(10): p.\u0026nbsp;5440\u0026ndash;5458.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMoon S-J, et al., \u003cem\u003eThe anti-arthritis effect of sulforaphane, an activator of Nrf2, is associated with inhibition of both B cell differentiation and the production of inflammatory cytokines\u003c/em\u003e. 2021. 16(2): p.\u0026nbsp;e0245986.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePolak G, et al. Environ Factors Endometr. 2021;18(21):11025.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSun Y, Liu GJJoC. Endometriosis-associated Ovarian Clear Cell Carcinoma: A Special Entity? 2021;12(22):6773.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGeorge BP, Chandran R, Abrahamse HJA. Role of phytochemicals in cancer chemoprevention: insights. 2021;10(9):1455.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKim I-SJA. \u003cem\u003eCurrent perspectives on the beneficial effects of soybean isoflavones and their metabolites for humans\u003c/em\u003e. 2021. 10(7): p.\u0026nbsp;1064.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFan Y, et al. Intake of Soy, Soy Isoflavones and Soy Protein and Risk of. Cancer Incidence and Mortality. 2022;9:847421.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCai X, et al., \u003cem\u003ePhytoestrogens for the management of endometriosis: findings and issues.\u003c/em\u003e 2021. \u003cb\u003e14\u003c/b\u003e(6): p.\u0026nbsp;569.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCecerska-Heryć E, et al., \u003cem\u003eAre antioxidant enzymes essential markers in the diagnosis and monitoring of cancer patients\u0026ndash;a review\u003c/em\u003e. 2021. 93: p.\u0026nbsp;1\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSenthilkumar M, Amaresan N, Sankaranarayanan A. \u003cem\u003eEstimation of superoxide dismutase (SOD)\u003c/em\u003e, in \u003cem\u003ePlant-microbe interactions\u003c/em\u003e. Springer; 2021. pp.\u0026nbsp;117\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eUdayana IGNBS, et al. Association of Endometriosis and Oxidative Stress. 2022;4(5):109\u0026ndash;13.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLabib H, et al., \u003cem\u003eKeap1/Nrf2 pathway in sodium fluoride-induced cardiac toxicity and the prophylactic role of vitamin C versus platelet-rich plasma\u003c/em\u003e. 2022. 81(3): p.\u0026nbsp;663\u0026ndash;678.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDa Broi MG, et al., \u003cem\u003eThe Impact of Controlled Ovarian Stimulation on Serum Oxidative Stress Markers in Infertile Women with Endometriosis Undergoing ICSI\u003c/em\u003e. 2022. 11(6): p.\u0026nbsp;1161.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYadav E, et al. \u003cem\u003ePolyphenols as Modulators of Oxidative Stress in Cancer Disease\u003c/em\u003e, in \u003cem\u003ePolyphenols-based Nanotherapeutics for Cancer Management\u003c/em\u003e. Springer; 2021. pp.\u0026nbsp;143\u0026ndash;88.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJurcau AJN, \u003cem\u003eThe role of natural antioxidants in the prevention of dementia\u0026mdash;where do we stand and future perspectives.\u003c/em\u003e 2021. \u003cb\u003e13\u003c/b\u003e(2): p.\u0026nbsp;282.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMarcolongo P, et al., \u003cem\u003eRegulation of autophagy in the uterus: from physiological processes to endometriosis and uterine fibroids.\u003c/em\u003e 2021.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMeresman GF, G\u0026ouml;tte M. and M.W.J.H.r.u. Laschke, \u003cem\u003ePlants as source of new therapies for endometriosis: a review of preclinical and clinical studies\u003c/em\u003e. 2021. 27(2): p.\u0026nbsp;367\u0026ndash;392.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eIftikhar M, et al. \u003cem\u003eAssessment of Lipid Profile in Females Suffering from Endometriosis\u003c/em\u003e. in \u003cem\u003eProceedings\u003c/em\u003e. 2022.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCacciottola L, Donnez J, M.-M.J.I.J.o.M S, Dolmans. \u003cem\u003eCan endometriosis-related oxidative stress pave the way for new treatment targets?\u003c/em\u003e 2021. 22(13): p.\u0026nbsp;7138.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMahn A, Castillo AJM. \u003cem\u003ePotential of sulforaphane as a natural immune system enhancer: A review\u003c/em\u003e. 2021. 26(3): p.\u0026nbsp;752.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMaksymowicz M, et al., \u003cem\u003eThe role of soy in development of breast, ovarian, endometrial and prostate gland cancer\u003c/em\u003e. 2021. 11(9): p.\u0026nbsp;180\u0026ndash;186.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGołąbek A, Kowalska K, Olejnik AJN. \u003cem\u003ePolyphenols as a Diet Therapy Concept for Endometriosis\u0026mdash;Current Opinion and Future Perspectives\u003c/em\u003e. 2021. 13(4): p.\u0026nbsp;1347.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFujii J, Homma T, Osaki TJA. Superoxide Radicals in the Execution of Cell Death. 2022;11(3):501.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLu J, et al., \u003cem\u003eEmerging hallmarks of endometriosis metabolism: A promising target for the treatment of endometriosis.\u003c/em\u003e 2022: p.\u0026nbsp;119381.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLee D-Y, Song M-Y, Kim E-HJA. \u003cem\u003eRole of oxidative stress and Nrf2/keap1 signaling in colorectal cancer: Mechanisms and therapeutic perspectives with phytochemicals\u003c/em\u003e. 2021. 10(5): p.\u0026nbsp;743.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZeng Z, et al., \u003cem\u003eEffects of dietary strategies on exercise-induced oxidative stress: a narrative review of human studies\u003c/em\u003e. 2021. 10(4): p.\u0026nbsp;542.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNazari-Khanamiri F, MJ.J.o.FB Ghasemnejad‐Berenji. \u003cem\u003eCellular and molecular mechanisms of genistein in prevention and treatment of diseases: An overview\u003c/em\u003e. 2021. 45(11): p.\u0026nbsp;e13972.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLu F, et al., \u003cem\u003eAntibiotic Therapy and Vaginal Microbiota Transplantation Reduce Endometriosis Disease Progression in Female Mice via NF-κB Signaling Pathway\u003c/em\u003e. 2022. 9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eP\u0026eacute;rez-Torres I, et al., \u003cem\u003eOxidative stress, plant natural antioxidants, and obesity.\u003c/em\u003e 2021. \u003cb\u003e22\u003c/b\u003e(4): p.\u0026nbsp;1786.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZamani-Garmsiri F, et al., \u003cem\u003ePolyphenols: Potential anti‐inflammatory agents for treatment of metabolic disorders.\u003c/em\u003e 2022. \u003cb\u003e36\u003c/b\u003e(1): p.\u0026nbsp;415\u0026ndash;432.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-2369250/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-2369250/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eBackground: Endometriosis is a gynecological disorder characterized by the growth of endometrial tissue outside the uterine cavity that is associated with chronic pelvic pain, subfertility and 1-3% increased risk of ovarian cancer. The purpose of the study was to investigate the effect of broccoli extract (BE) alone and in combination with soy isoflavones (SI) on endometrial implants in female rat.\u003c/p\u003e\n\u003cp\u003eMethod: In this study, endometriosis was induced surgically in 40 mature female rats. The rats were divided into 5 groups that were treated by oral gavage for 6 weeks with 0.5 ml of saline 0.9%/day (control group), BE (3000 mg/kg/day), SI (50mg/kg/day), BE/soy isoflavones (BE 3000 mg/kg/day + soy isoflavones 50mg/kg/day) and diphereline as a standard medication (3 mg/kg) intramuscularly. At the end of treatments, the volume and histopathology of the endometrial implants were compared among the 5 groups. The serum levels of oxidative parameters including Superoxide dismutase (SOD), Malondialdehyde (MDA) and tumor necrosis factor alpha (TNF-α) were also compared between the groups.\u003c/p\u003e\n\u003cp\u003eResults: The volume of the implants significantly decreased in diphereline group (p = 0.002). The histopathological grade of endometrial implants in BE/SI and diphereline group were significantly decreased compared to the control group (p = 0.001). The serum levels of SOD in BE group were enhanced significantly in comparison to the control group (p = 0.034).\u003c/p\u003e\n\u003cp\u003eConclusion: BE in combination with SI decreased the growth and histopathologic grades of transplanted endometrial implants. These herbal compounds may have the potential to be used as an alternative medication for the treatment of endometriosis\u003c/p\u003e","manuscriptTitle":"The effect of broccoli extract and soy isoflavones on endometrial implants and oxidative parameters in female rat model of endometriosis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2022-12-15 16:16:49","doi":"10.21203/rs.3.rs-2369250/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"eaf37437-5fd8-427e-a7b3-b2a21624f4a6","owner":[],"postedDate":"December 15th, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2022-12-17T16:29:20+00:00","versionOfRecord":[],"versionCreatedAt":"2022-12-15 16:16:49","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-2369250","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2369250","identity":"rs-2369250","version":["v1"]},"buildId":"_2-kVJe1T_tPrBINL-cwx","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}