Potential Effects of Soy Isoflavones and Broccoli Extract on Oxidative Stress, Autophagy, and Apoptosis Gene Markers in Endometriosis

Endometriosis is a gynecological disease that occurs when the tissue similar to the lining of the uterus grows outside of the uterus, while its true prevalence is uncertain ( 1 ). It is unclear how this disease develops, but endometrial lesions , metastasis, metaplasia, blood reflux, and disorders of the immune system are involved ( 2 ). Endometriosis is frequently associated with pain, reduced quality of life, and infertility; and current treatments do not provide a sufficient cure despite of its long term management ( 3 ). The endometriosis associated symptoms include inflammation, asthma and osteoarthritis as well as multisite chronic pain or migraine ( 4 ). During endometriosis, immune response reduction, apoptosis impairment, and inflammation increase, result in the repeated tissue injury, neurogenesis, and chronic inflammation ( 5 ). Endometriosis, chronic and progressive forms, has a significant impact on the life quality of patients. While, endometriosis surgery, lesion removal, is a definitive treatment, with a high rate of recurrence , up to 50% , within five years of the procedure ( 6 ). Also, Surgery is associated with vascular, intestinal, and neurological complications, and pain may reappear if the lesions are not completely removed and pathogenetic mechanisms may not be treated by surgery and a long-term medical care must be prepared for the patients ( 7 ). Diphereline, a gonadotropin releasing hormone agonist (GnRHa), is a current treatment in women with endometriosis to impair the progression of endometriosis implants by blocking the hypothalamus-pituitary-ovary axis ( 8 , 9 ). The complexity of using GnRHa includes amenorrhea, musculoskeletal stiffness, hot flush, bone loss, hair loss, vaginal dryness, depression and headache. Pregnancy should be prevented during GnRHa treatment ( 10 , 11 ). Plant-derived agents are a potential safe option with a high level of efficiency to avoid adverse effects and preserve the chances of a successful pregnancy ( 12 ). Recent studies have suggested that the main effective components in broccoli and soybeans such as sulforaphane and isoflavones might induce cell death in cancer cells through regulating the essential mechanisms, such as oxidative stress, autophagy, and apoptosis which play important roles in the growth and progression of endometriosis ( 13 ). An essential function of apoptosis is to eliminate excess or dysfunctional cells. Pieces of evidence suggested that in healthy women, as a result of programmed cell death, endometrial cells expelled during menstruation cannot survive in ectopic locations and a decreased apoptosis can lead to endometriosis by allowing these cells to survive and implant ectopically ( 14 ). Oxidative stress is a term used to define an imbalance between the production of reactive oxygen species (ROS) and the body's natural defenses against their harmful effects. ROS molecules have the potential to damage cells and tissues in the body ( 15 ). Under normal circumstances, the body has ROS detoxification mechanisms and prevent them from causing harm. However, overproduction of ROS in the peritoneal environment causes the chain of events leading to endometriosis ( 16 , 17 ). Autophagy is a cellular process that helps to remove damaged organelles and proteins, thus maintaining cellular integrity and homeostasis. Apoptosis is essential for the removal of damaged or unnecessary cells and helps to regulate the growth of endometrial tissue. Therefore, understanding the role of oxidative stress, autophagy, and apoptosis in the context of endometriosis is essential to develop effective management and treatment strategies ( 18 ). Even though there are already treatment options for endometriosis such as surgery and hormone therapy, oral consumption of natural substances is a safe way to treat endometriosis ( 19 ). The present study aimed to explore the protective and therapeutic effects of broccoli extract and soy isoflavones (SI) as well as Diphereline by measuring the histopathological scores of the endometrial implants and mRNA expression level of the gene markers of autophagy, apoptosis, and oxidative stress within the tissue of peritoneal in endometriosis rat model.

To evaluate the persistence of epithelial cells in the graft, the pathological scores were applied ( 24 ). The persistence of epithelial cells (pathologic score) in grafts of Diphereline and BE+SI groups was significantly reduced in comparison with the control group (P≤0.001), and there was no substantial change in the score of the BE+SI treated group compared to the Diphereline group (P=0.903, Table 2 ). As it is illustrated in Figure 1, the well-preserved epithelial layer of endometrial implants (score 2-3) was evident in the control group, and also, a moderately persistent epithelial cell of endometrial implants (score 1-2) was observed in both groups, BE and SI and the epithelial layer of endometrial implants was poorly preserved (score 0-1) in BE+SI and D groups. The pathological score of endometrial implants SI; Soy isoflavone, BE; Broccoli extract, a ; Statistical comparison with the control group, and b ; Statistical comparison with Diphereline group. Hematoxylin-Eosin-stained biopsy of epithelial layer specimen at 250x magnification. i. Control group: well preserved (score 2-3), ii. BE, iii. SI groups: moderate preserved (score 1-2), iv: BE+SI and v. Diphereline (D) groups: poor preserved (score 0-1). SI; Soy isoflavone and BE; Broccoli extract. To evaluate the autophagy status in the treated groups, Becn1 and Lc3 mRNA levels were assessed. The expression levels of Becn1 in BE+SI, SI, and D treatment groups were significantly increased compared to the C group (P=0.0002, 0.0034, and 0.0054 respectively) ( Fig .2 ). Lc3 mRNA levels of BE+SI and D groups were significantly higher than C group (P=0.0004, and 0.0017 respectively). Although, SI and BE treatments lead to an increase in the mRNA expression level of Lc3 , the differences were not significant ( Fig .2 ). Real-time PCR for the mRNA expression of autophagy-related genes. A. Lc3 and B. Becn1 genes. In comparison with the control group ( 1 ) and in comparison with the Diphereline (D) group ( 2 ). **; P<0.01, ***; P<0.001. The details of mean ± SD and P values are presented in Table S1 (See sup- plementary Online Information at www.ijfs.ir). ns; Not significant, PCR; Polymerase chain reaction, SI; Soy isoflavone, and BE; Broccoli extract. Casp3, Bax , and Bcl-2 mRNA expression levels were evaluated as common markers of apoptosis. BE+SI, SI, and D groups showed a significant increase in the Casp3 mRNA expression levels in comparison with the C group (P<0.0001 for all). The Bax mRNA expression level was increased only after BE+SI administration (P=0.001, Fig .3 ). Other treatments could not change the Bax mRNA expression level in comparison with the control group. The Bcl-2 mRNA expression level has a reverse correlation with apoptosis induction. Administration of BE+SI, SI, and D significantly decreased the Bcl-2 mRNA levels in comparison with the C group (P<0.0001=0.0028, and =0.0017 respectively) ( Fig .3 ). Real-time PCR for the mRNA expression of apoptosis-related genes. A. Casp3 , B. Bax , and C. Bcl-2 genes. In comparison with the control group ( 1 ) and in comparison with the Diphereline (D) group ( 2 ). **; P<0.01, ***, ****; P<0.0001. The details of mean ± SD and P values are presented in Table S1 (See supplementary Online Information at www.ijfs.ir). ns; Not significant, PCR; Polymerase chain reaction, SI; Soy isoflavone, and BE; Broccoli extract. The expression level of Sod , as the oxidative stress status assessment marker, was significantly increased under BE+SI and SI treatments than the C group (P<0.0001 for both, Fig .4 ). However, BE and D administration did not change the Sod mRNA expression level. Real-time PCR for the mRNA expression of the Sod gene. In compari- son with the control group ( 1 ) and in comparison, with the Diphereline (D) group ( 2 ). ****; P<0.0001. The details of mean ± SD and P values are presented in Table S1 (See supplementary Online Information at www.ijfs. ir). ns; Not significant, PCR; Polymerase chain reaction, SI; Soy isoflavone, and BE; Broccoli extract.

While there are already clinical treatment options for endometriosis such as surgery and hormone therapy, the oral consumption of natural substances provides a safe alternative for treating the condition ( 26 ). The objective of this study was to investigate the potential therapeutic effects of BE, SI and the combination of these two nutraceuticals on progression of endometriosis. The underlying cellular mechanisms such as autophagy, apoptosis, and oxidative stress mechanisms in the pathogenesis and treatment of endometriosis was also considered, using a rat model of endometriosis. The supplementation of immunomodulators, antioxidants, and selective progesterone receptor modulators with antioxidant properties have been assessed as possible treatments for endometriosis. The results demonstrated that Sod gene expression was remarkably increased by BE+SI and SI treatments. The product of this gene is responsible for the dismutation of the superoxide radical. Similarly, a recent study found that an herbal compound, genistein, is capable of recovering SOD activity in an endometriosis mouse model ( 27 ). In the physiological state, cells strongly preserve the oxidative balance between oxidants and antioxidants; however, under the impact of various stimuli such as pharmaceuticals, toxins, radiations, etc., the oxidative balance is disturbed, which leads to the mechanism of oxidative stress ( 28 ). Along with the induced promotion in the level and activity of antioxidant defensive responses, excess oxidants can damage biomolecules and cellular structures, change a variety of signaling pathways, and contribute to the pathogenesis of different disorders such as endometriosis ( 29 ). In other hand, natural antioxidants, such as quercetin and soy isoflavones, that exhibit no/few undesired consequences are recommended for the management of endometriosis ( 30 , 31 ). More importantly, oxidative radicals can alter a variety of upstream signaling pathways and affect the flux of cellular processes. It was suggested that, oxidative radicals can alter autophagy by affecting upstream pathways, such as phosphoinositide 3-kinases (PI3K), mammalian target of rapamycin (mTOR), 5' AMP-activated protein kinase (AMPK), and nuclear factor erythroid 2– related factor 2 (NRF2) and are involved in the pathogenesis of endometriosis ( 32 ). The current study revealed that the administration of BE+SI remarkably increased mRNA levels of Lc3 and Becn1 . Previous studies have suggested that Becn1 is involved in the initiation of autophagic flux in mammals, while Lc3 significantly contributes to the completion of the autophagosome membrane and specific selection of cargoes ( 33 , 34 ). Therefore, it could be assumed that autophagy was disrupted in animals with endometriosis, in which the administration of the studied compounds resulted in the resumption of autophagic flux. Nevertheless, it has been documented widely that autophagy exhibits a dual role in cell fate, as it can participate in cell survival as well as programmed cell death ( 12 , 35 ). Thereby, to elucidate what happened, the current study aimed to consider the expression of genes involved in the apoptosis pathway. The obtained data demonstrated that in animal models of endometriosis, the expression of Casp3 and Bax reduced significantly, while Bcl-2 increased remarkably. The results of the current study reveal that apoptosis is deactivated during endometriosis that are compatible with previous studies ( 36 ), while the administrated compounds of this study can activate it. Excessive cell proliferation and escape from apoptosis are the main mechanisms involved in the development of endometrial tissue outside the uterus, which is the classic description of endometriosis ( 18 ). Concurrent with the findings of the present study, several reports have shown that a variety of herbals such as curcumin, quercetin, resveratrol, and naringin can confront endometriosis by the promotion of apoptosis, hence are considered promising therapeutic strategies in endometriosis management ( 37 ). In fact, it has been reviewed that phytochemicals and polyphenolic compounds participate in the treatment of endometriosis through different mechanisms such as anti-oxidative, anti-inflammatory, anti-proliferative and apoptotic, anti-angiogenic, anti-invasive, and modulating estrogenic effects ( 38 ). The administration of resveratrol, for example, could the ratio of Bcl-2/Bax gene expression in eutopic endometrial stromal cells from patients with endometriosis representing its therapeutic potential ( 39 ). Moreover, the antiproliferative properties of other herbal compounds are believed to be through the ROS-mediated induction of apoptosis via alteration in the expression of Bcl-2, Bax , and Casp3 ( 40 ). It is speculated that phytochemicals of the compounds studied in the present study such as Sulforaphane and Genistein, can be a promising approach for treating endometriosis by decreasing the oxidative stress and inducing the signaling pathways involved in apoptosis and autophagy. However, considering that the present study was conducted on a rat model of endometriosis, it is necessary to conduct further studies on human patients and in equivalent doses.

Due 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 consumption of broccoli extract mixed with soy isoflavons significantly reduced the histopathologic scores of the induced endometrial implants and also modulated the expression of the gene markers of oxidative stress, apoptosis, and autophagy in a female rat model of endometriosis. Accordingly, these compounds can be considered potential therapeutic agents for endometriosis. However, subsequent studies are needed to verify the beneficial effects of the natural products on regression of endometriosis.

In this experimental study, the care and control of the rats were approved by the Medical Ethics Committee of Shiraz University of Medical Sciences (IR.SUMS. REC.1398.1151). Forty five mature female Sprague-Dawley rats weighing 220 ± 20 g, and at the age of almost 8 weeks were used for this study. The rats were mature sexually and displayed normal estrous cycle changes in uterine histology using the Papanicolaou technique. The animals were kept at a constant temperature of 22°C with a humidity of 40-60% in the Animal Department of Shiraz University of Medical Sciences under standard conditions of cyclic light, 12 hours light/12 hours dark, for 1 week to acclimatize them. Their cages were cleaned and disinfected every week, and the rats were placed on a standard diet ( 20 ). The alcoholic extract of broccoli was prepared as described previously by Somasundaram ( 21 ). Each Soy tablet (6260232300192, Soya Gol, Gol Daru Company, Iran) includes 50 mg SI ≈ and 27 mg Genistein was dissolved in 1 ml saline 0.9%. Ectopic endometrium was induced surgically in the peritoneum of the studied rats ( 12 , 22 ). Briefly after mid-abdominal incision by anesthesia with isofluorane (26675- 46-7, Cyman Chemicals, USA), the left uterine horn was resected from the rats and was put in phosphate-buffered saline at 37°C. Eventually, a 5-mm-square segment was sectioned longitudinally. In this procedure, uterine tissue without myometrium was transplanted onto the right abdominal wall inner surface with the epithelium opposed to the peritoneum. Polypropylene sutures were used at two edges of the explanted endometrium to secure it (Prolene 6-0, 8695G, Ethicon, USA). Chromic catgut suture (3-0; 636H, 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 used to close the incision in the skin. Four weeks after the surgery, a second look laparotomy was performed to confirm the endometrial implants viability. The animals were given time in two weeks to recover and progress the induced endometriosis. After scarifying the rats and incising their abdomens, the explants were investigated for viability and size ( 12 ). A caliper was used to measure the surface area of the engrafted tissue. To eliminate the selection bias and achieve balance in baseline characteristics, a simple randomized method (throwing a dice) was used to divide the surgically induced endometriosis rat models randomly into five groups (nine rats in each group) including the control (C): (saline treatment: 0.5 ml of saline 0.9%), broccoli extract (BE: 3000 mg/kg), SI (1 tablet ≈ 50 mg isoflavones/kg), BE+SI: BE 3000 mg/kg+SI (1 tablet ≈ 50 mg isoflavones/kg) and, Diphereline (D: IM injection) [S.R. 11.25 mg (3 mg/kg, once during studied treatment)]. Diphereline (57773-63-4, IPSEN, France) was used as a standard hormonal treatment with known effects. The rats were sacrificed after six weeks of treatments and the endometrial implants were considered. The endometrial implants were fixed in formalin (F8775, Sigma Aldrich, Germany) for 48 hours and then embedded in paraffin wax box to transfer to pathology laboratory. 5 μm sections were provided to prepare tissue slides (three sections per sample). The pieces were heat-dried and stained with hematoxylin (H3136, SigmaAdrich, Germany) and eosin (109278, Merck, Germany) ( 23 ). The epithelial cells persistence in the endometriosis implants was scored according to Keenan et al. ( 24 ) scoring system: 0; No epithelial layer, 1 and 2; Poorly and moderately persistent epithelial cells, respectively, and 3; Well-preserved epithelial layers. Briefly, a piece of the tissue from the implanted area of each endometriosis-induced rats (n=45) were embedded in 1 ml RNAlater (YT9085, Yekta Tajhiz, Iran) to protect RNA degradation. Tissues were lysed with TRIzol reagent (YT9064, Yekta Tajhiz, Iran), and the RNX-PLUS reagent (EX6101, Sinaclon, Iran) was used for total RNA extraction as per the manufacturer’s instruction. Utilizing an Addbio cDNA synthesis kit (22701, Addbio, South Korea), purified RNA was used to create complementary DNA (cDNA). The messenger ribonucleic acid (mRNA) expression levels of Beclin-1 ( Becn1 ), Microtubule-associated proteins 1A/1B light chain 3B ( Lc3 ), caspase-3 ( Casp3 ), associated X protein ( Bax ), B-cell lymphoma/leukemia 2 (Bcl-2), superoxide dismutase ( Sod ), and β-actin in endometrial tissues of endometriosis-induced rats were evaluated ( Table 1 ) ( 25 ). β‐actin gene was used to normalize the gene expression levels. The mRNA expression levels of Lc3, Becn1, Casp3 , and Bcl-2 , genes calculated by ΔΔCt method through three independent experiments. Primer sequences used for real-time polymerase chain reaction The data was presented as mean ± SD. The Shapiro Wilks test was used to assess the normality assumption of data. The statistical differences between groups were evaluated by Kruskal-wallis followed by Dunn’s post-hoc tests using SPSS version 22 (IBM, USA). The P<0.05 was considered as statistically significant.