Patterns of proliferation and fibrosis in a rat model of endometriosis following administration of Allium cepa

Endometriosis is a common gynecological disease among reproductive-age women. Numerous hypotheses exist regarding the pathogenesis of endometriosis. In Turkey, the consumption of Allium cepa (commonly known as the “onion cure”) is a popular treatment employed to alleviate a variety of gynecological disorders.

In this study, our objective was to assess the therapeutic mechanisms of the onion bulb A. cepa using an autologous endometrio- sis model in Sprague-Dawley rats. Previous research has shown that A. cepa possesses anti-inflammatory, antioxidant, and antiapoptotic properties. We evaluated the pathological condition of endometriotic implants by employing hematoxylin-eosin staining and Ki67 immuno- histochemistry analysis. Transforming growth factor-beta 1 (TGF-β1) and alpha-smooth muscle actin (α-SMA) have been identified as profi- brotic markers that are highly overexpressed in endometriotic tissues relative to eutopic endometrial tissue. Furthermore, TGF-β1 influences the differentiation and progression of endometriosis. To quantify profibrotic activity, we measured TGF-β1 and α-SMA using the immunosor- bent assay method.

Lower histologic evaluation scores for endometriotic implants were observed in the group receiving high-dose A. cepa relative to the other groups. Ki67 expression was reduced following the high-dose A. cepa regimen, which consisted of 30% A. cepa and 70% normal feed. However, no statistically significant differences in TGF-β1 or α-SMA levels were observed among the groups (p=0.7 and p=0.778, re- spectively).

The findings suggest that A. cepa could serve as a therapeutic agent in endometriosis treatment, as evidenced by the reduction in proliferative potential. Nevertheless, A. cepa was not associated with significantly lower levels of endometriosis-associated TGF-β1 or α-SMA.

Alpha-smooth muscle actin; Fibrosis; Ki67; Proliferation; Transforming growth factor beta1

Endometriosis is a common inflammatory gynecological patholo- gy characterized by the ectopic implantation of endometrial tissues [1]. Endometriosis can damage the tissue through inflammatory, proliferative, and fibrogenic processes. This condition is estimated to affect nearly 10% of the general female population and between 25% and 40% of women who are infertile [2]. Furthermore, endome- triosis is present in approximately 70% of women with chronic pelvic pain [3]. The primary clinical manifestations of endometriosis include chronic pelvic pain, dysmenorrhea, dyspareunia, and infertility. The pathogenesis of endometriosis is multifactorial and continues to provoke debate concerning its pathophysiology and treatment strategies. A variety of theories contribute to our understanding of endometriosis, including the roles of inflammatory mediators, infec- tious agents, endocrine factors, and proangiogenic elements. Recent studies have focused on the inflammatory and immune mechanisms underlying endometriosis. Increased oxidative damage, as evidenced by elevated levels of malondialdehyde, superoxide anions, and hy- drogen peroxide, has also been implicated in the disease process [4]. Recommended treatments encompass medical therapies, surgical interventions, herbal remedies, and specific anti-inflammatory and antioxidant agents. Fibrosis refers to a loss of tissue function due to repetitive damage. This condition is marked by increased activity of myofibroblasts and heightened collagen production, causing the primary tissue to lose its functionality [5]. Histologically, fibrosis in endometriosis is evi- denced by the envelopment of endometrial glands and stroma by dense fibrous tissue [6]. Furthermore, fibrosis contributes to the degra- dation of both the vasculature and tissue functions, potentially lead- ing to resistance against medical treatments and hormonal suppres- sive therapies [7]. Transforming growth factor-beta 1 (TGF-β1) and al- pha-smooth muscle actin (α-SMA) have been identified as key profi- brogenic biomarkers that can be used to assess myofibroblast activity and the extent of fibrosis in endometriotic tissue [8]. Ki67, a nuclear protein, is intimately associated with cell division, as it is present during all active phases of the cell cycle (G1, S, G2, and mitosis) but is conspicuously absent in the resting phase, G0 [9]. Ki67 is a crucial marker for determining a cell’s proliferative capacity. Therefore, due to its role in signaling proliferative activity in neoplastic diseases, an in- creased level of Ki67 is considered a key factor for predicting the prog- nosis and the risk of recurrence in endometriosis cases [10]. Allium cepa, commonly known as the onion bulb, is a member of the Amaryllidaceae family [11]. Prior research has explored its anti- cancer and antioxidant properties [12]. Studies have shown that A. cepa possesses anti-inflammatory effects, as evidenced by reduced levels of prostaglandin E2, nuclear factor kappa-light-chain-enhancer of activated B cells, and thromboxanes, as well as the inhibition of chemotaxis [13]. Furthermore, A. cepa has been reported to amelio- rate cellular degeneration and mitigate damage caused by ischemia and reperfusion in ovarian tissue [14]. Quercetin, flavonoids, and or- ganosulfur compounds have been identified as the primary active components in A. cepa that contribute to its anti-inflammatory ef- fects. The literature also describes the inhibition of endometriotic proliferation through the regulation of the cyclin pathway by querce- tin molecules [15]. The objective of the present study is to investigate whether A. cepa can improve the histological parameters of endo- metriotic lesion proliferation and the levels of profibrotic mediators.

This study received approval from the Dokuz Eylül University Lab- oratory Animals Local Ethics Committee (Approval No: 41-2020). Twenty-one adult Sprague-Dawley rats, each weighing between 220 and 250 g, were acquired from the Dokuz Eylül University Experi- mental Animal Laboratory. The trial was conducted in accordance with the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines. The rats were housed in standard cages with sawdust bedding, maintained at a room temperature of 22±2 °C, and kept under a 12/12-hour dark/light cycle throughout the trial. To deter- mine the estrus phase of the rats, vaginal smears were collected four times daily. Each procedure was documented using a digital camera. The 21 rats were randomly divided into three groups of seven ani- mals each. Surgical procedures were carried out under sterile condi- tions. Anesthesia was induced with an intraperitoneal injection of ketamine hydrochloride (50 mg/kg, Ketalar; Pfizer Inc.) and xylazine hydrochloride (7 mg/kg, Alfazyne; Alfasan International BV). All labo- ratory procedures are detailed in Figure 1. 1. Surgical procedure (step 1: implantation of endometriotic tissue) Before the incision was made, the abdominal wall was shaved and cleansed with a 10% povidone-iodine solution. With the rat under anesthesia, a sterile incision measuring 2 cm was made along the midline of the lower abdomen. The uterine horns, adnexa, and ova- • Implantation of right uterine horn into the peritoneum Implanting endometriotic tissues Validation of endometriotic implants A. cepa regimen Evaluation • Relaparotomy is performed at day 14 to establish the model • Low-dose Allium cepa regimen: 10% A. cepa+90% normal feed • High-dose A. cepa regimen: 30% A. cepa+70% normal feed • A. cepa is given by oral gavage per day for 21 days • Histological examination • TGF-β1 and α-SMA concentrations measured by ELISA • Immunohistochemistry of Ki67 expression Figure 1. Explanation of the endometriosis model. TGF-β1, transforming growth factor-beta 1; α-SMA, alpha-smooth muscle actin; ELISA, enzyme-linked immunosorbent assay. www.eCERM.org 113 H Kula et al. A. cepa administration in an endometriosis model ries were identified in each animal. Autologous uterine tissue (1 cm long) was harvested from the distal end of the right uterine horn. This tissue was then immersed in a phosphate-buffered saline solu- tion for 2 minutes. Subsequently, the endometrium was isolated, and endometrial fragments measuring 5×5 mm were implanted into the abdominal wall (Figure 2). The abdominal wall and skin were closed using 3.0 polyglactin su- tures. Following sterilization of the abdominal wall with 10% povi- done-iodine, heating pads were applied until the animal regained consciousness. To induce the formation of endometriotic implants in autologous tissues, 50 μg/kg of estrogen was administered subcuta- neously to the rats twice weekly [16]. 2. Validation of endometriotic implants Relaparotomy was scheduled for day 14 to validate the presence of endometriotic implants on the inner abdominal wall, coinciding with the estrus phase in rats. The anesthetic and surgical protocols from step 1 were repeated, from the initial incision to the closure of the abdominal cavity. To determine the estrus phase, daily vaginal smears were conducted. Three rats were excluded from the remain- der of the study due to the inadequate development of endometri- otic lesions. 3. Preparation of A. cepa Feed fortified with A. cepa was incorporated into the rats’ regular diet. The A. cepa onion bulbs were peeled, sliced, and air-dried for 1 week before being ground into a fine powder. Group A (n=6), desig- nated as the Sham group, was administered only a 0.9% sodium chlo- ride solution via oral gavage along with their standard diet. Group B (n=6), the low-dose group, received a mixture of 10% A. cepa and 90% standard feed, while group C (n=6), the high-dose group, was given a blend of 30% A. cepa and 70% standard feed through oral ga- vage daily for 21 days. The A. cepa-supplemented diet was initiated after the stabilization period following relaparotomy. 4. Effect of A. cepa on endometriotic implants Under anesthesia, the rats were euthanized, and their endometri- otic implants were harvested for both histopathological and bio - chemical assessments. Concurrently, blood samples were obtained for biochemical analysis. The collected endometriotic implants were subjected to histological and biochemical examinations. For histo- logical evaluation, the tissues were preserved in 10% formalin. 5. Histological examination Endometriotic implants were embedded in paraffin blocks follow- ing formalin fixation. Tissue sections, 5 mm thick, were prepared, then stained with hematoxylin-eosin and examined under light mi- croscopy (CX-41; Olympus). Blinded histological assessment was conducted by a histologist with experience in a prior study of rat en- dometriosis, and the findings were documented photographically [17,18]. The epithelial lining of the endometrial implants underwent semi-quantitative evaluation based on a method previously outlined in the literature [17,18]. The grading was as follows: grade 0 indicated an absence of epithelium, with a corresponding score of 0; grade 1 represented a poorly preserved epithelium, characterized by the presence of only occasional epithelial cells, and was assigned a score of 1; grade 2 denoted a moderately preserved epithelium accompa- nied by leukocyte infiltration, with a score of 2; and grade 3 de - scribed a well-preserved epithelial lining, which received a score of 3. 6. Biochemical examination Whole blood was drawn into blood collection tubes without anti- coagulant and then allowed to clot naturally at 25 °C for 30 minutes. Following coagulation, the samples were centrifuged at 2,000 ×g and 4 °C for 15 minutes. The upper layer of yellow serum was then carefully collected and stored at −80 °C. Upon thawing, the serum concentrations of TGF-β1 and α-SMA were measured using an en- zyme-linked immunosorbent assay kit (BTLAB, catalog numbers E1688Ra and E2330Ra). All procedures were performed in strict ac- cordance with the instructions provided with the kit. Figure 2. Macroscopic visualization of endometriotic implants. https://doi.org/10.5653/cerm.2023.06261114 Clin Exp Reprod Med 2024;51(2):112-119 7. Immunohistochemical analysis Sequential 5-μm-thick sections were cut from the paraffin blocks. For antigen retrieval, these sections were boiled for 9 minutes in 0.01 mol/L sodium citrate buffer at a pH of 6. To suppress endogenous peroxidase activity, the sections were then incubated in a 3% hydro- gen peroxide solution for 10 minutes. Immunohistochemical stain- ing was carried out using the avidin-biotin immunoperoxidase method, employing the Ki67 rabbit polyclonal antibody (catalog no. NB500-170SS; Novus Biologicals) as the primary antibody. 8. Statistical analysis Statistical analysis of the data gathered during the study was con- ducted using SPSS ver. 26.0 (IBM Corp.). Means and standard devia- tions were calculated for the evaluation, with biochemical and para- metric data presented as mean±standard deviation. Differences among groups were assessed using the Kruskal-Wallis test, and the source of any differences was further investigated with the Mann- Whitney U test. A p-value of less than 0.05 was considered indicative of statistical significance.

Throughout the trial, we observed no adverse effects—such as hair loss, fatigue, or loss of appetite—in rats fed A. cepa. All the ani- mals survived until the conclusion of the study, and none exhibited signs of wound infection. Figure 3 displays representative histopathological images of endo- metriotic implants. Table 1 presents the mean scores from the histo- pathological evaluation of the implants following the treatment pe- riod. A significant reduction in the mean histopathological evalua- tion score was observed for group C (1.14±1) relative to groups A and B (2.71±0.7 vs. 2.28±1.2, respectively; p<0.05). The levels of tissue biomarkers (TGF-β1 and α-SMA) were assessed across groups. The data for TGF-β1 and α-SMA levels are presented in Table 2. Notably, we observed no statistically significant difference in TGF-β1 levels among groups A, B, and C (633.21±89.49 vs. 697.66±76.53 vs. 636.08±156.11, p=0.7). Similarly, α-SMA levels exhibited no signifi- cant changes following treatment with A. cepa in groups A, B, and C (36.26±6.81 vs. 32.84±5.75 vs. 33.98±8.90; p=0.778). Immunohisto- chemical analysis revealed that Ki67, a marker of cell proliferation, was highly expressed in all areas of the endometriotic implantation tissues in group A and in the low-dose A. cepa group (group B). However, Ki67 expression was diminished in group C, which received a high-dose of A. cepa (30% A. cepa+70% normal feed), as depicted in Figure 4.

In this study, we demonstrated that high-dose A. cepa intake sig- nificantly reduced histological evaluation scores and decreased Ki67 expression in a surgically induced rat model. However, the levels of the profibrotic mediators TGF-β1 and α-SMA did not change signifi- cantly following A. cepa administration. Endometriosis is a common gynecological disorder characterized by chronic pelvic pain, infertility, pelvic organ dysfunction, and im- Figure 3. Histopathologic images of the ovaries under hematoxylin and eosin staining (×10 magnification). (A) Endometriosis only. (B) Endometriosis+low-dose Allium cepa. (C) Endometriosis+high-dose A. cepa. Table 1. Mean histopathological evaluation scores Group Histopathological evaluation score Endometriosis only 2.71 ± 0.7 Endometriosis+low-dose Allium cepa 2.28 ± 1.2 Endometriosis+high-dose A. cepa 1.14 ± 1.0 a) Values are presented as mean±standard deviation. a) p<0.05. AA BB CC www.eCERM.org 115 H Kula et al. A. cepa administration in an endometriosis model pairment in daily activities and overall quality of life. The clinical manifestations of endometriosis are driven by chronic inflammation, which is evidenced by elevated local and systemic levels of inflam- matory chemokines and cytokines [19]. These mediators play key roles in histopathological cell damage and the clinical symptoms ob- served. Despite this, the underlying mechanisms of endometriosis remain incompletely understood, and definitive treatment protocols are a subject of ongoing research. Inflammatory mediators such as tumor necrosis factor alpha, interleukin 1β (IL-1β), IL-6, IL-12, prosta- glandin E2, thromboxane, and others are also involved in the patho- physiology of endometriosis [20]. The progression of this condition is similarly impacted by inflam- matory mediators, cytokines, and angiogenic and fibrogenic factors. The regulation of these mediators may reduce the severity of endo- metriosis and alleviate gynecological symptoms. Strategies that tar- get antiproliferative, proapoptotic, autophagic, anti-cell migration and invasion, antifibrotic, and anti-angiogenic mechanisms have been explored to inhibit the progression of endometriosis [21]. Fur- thermore, modulators of the immune system and inhibitors of an- giogenesis have been studied as alternatives to hormonal therapy and nonsteroidal anti-inflammatory drugs (NSAIDs) for the improve- ment of endometriosis-related clinical outcomes [22]. Deep endometriosis is the primary predisposing factor for poor outcomes. It is characterized by a rigid and compromised pelvic structure resulting from extensive fibrosis and the activation of myo- fibroblasts [23]. The recurrent tissue injury and repair (ReTIAR) theory is among the most prevalent explanations for deep endometriosis and the associated fibrosis. The key mechanisms of ReTIAR include overexpression of TGF-β1, THY-1, and peroxisome proliferator-acti- vated receptor gamma; the transformation of fibroblasts into myofi- broblasts; and recurrent tissue injury [24]. In the present study, hematoxylin-eosin staining revealed a mor- phological enhancement in group C (which received a high-dose A. cepa regimen) relative to the other groups. Observations of epitheli- al integrity, inflammation, glandular structures, and vasculature in endometriosis tissue were less frequent in group C. These findings corroborate a significant decrease in histological features associated with endometriosis following the intake of A. cepa. A. cepa contains a variety of chemical constituents, including fla- vonoids (such as quercetin), lipophilic antioxidants, and isoliquiriti- genin (ISL) [25]. These components are associated with a range of therapeutic benefits, including analgesic, antitumor, antihyperlipid- emic, and antithrombotic effects [17]. Two studies have been con- ducted on the impact of A. cepa on endometriosis [15,26]. The find- ings from these studies indicate that quercetin and ISL not only im- prove endometriosis-associated clinical symptoms and lesions but also may influence antiproliferative and anti-inflammatory agents within endometriotic tissue. Additionally, ISL exhibits a proapoptotic Table 2. Levels of TGF-β1 and α-SMA by group Variable Endometriosis only Endometriosis+low-dose Allium cepa Endometriosis+high-dose A. cepa p-value TGF-β1 633.21 ± 89.49 697.66 ± 76.53 636.08 ± 156.11 0.705 α-SMA 36.26 ± 6.81 32.84 ± 5.75 33.98 ± 8.90 0.778 Values are presented as mean±standard deviation. TGF-β1, transforming growth factor-beta 1; α-SMA, alpha-smooth muscle actin. Figure 4. Immunohistochemical analysis of Ki67 (×10 magnification). (A) Endometriosis only. (B) Endometriosis+low-dose Allium cepa. (C) Endometriosis+high-dose A. cepa. AA BB CC https://doi.org/10.5653/cerm.2023.06261116 Clin Exp Reprod Med 2024;51(2):112-119 effect on endometriotic cells. These results align with prior research that has documented histological improvements in endometriotic tissue. The presence of fibrosis within endometriotic tissue can lead to anatomical distortion in the pelvis and chronic pelvic pain. Further- more, TGF-β not only plays a critical role in fibrosis but also influences apoptosis, disease progression, and cellular differentiation in endo- metriosis [27]. Myofibroblast-like cells that are positive for α-SMA have been observed within fibrotic areas in ovarian, deep, and su- perficial endometriotic implants. One study noted markedly elevat- ed levels of fibrotic mediators, such as α-SMA and fibronectin, in en- dometriotic lesions relative to the eutopic endometrium [28]. Conse- quently, TGF-β and α-SMA, along with associated profibrotic signal- ing pathways, may serve as potential therapeutic targets for the treatment of endometriosis. In the literature, two studies have investigated the levels of TGF-β1, α-SMA, and fibrosis in rats with endometriosis treated with various substances [23,29]. These studies suggest that targeting the antifi- brotic mechanism could be considered as an alternative treatment approach for endometriosis. However, we did not observe significant changes in the levels of the profibrotic mediators TGF-β and α-SMA in our surgically induced endometriosis rat model. Previous research has indicated that the activation of the fibrino- gen pathway by fibrotic mediators in endometriosis leads to reduced effectiveness of cyclooxygenase (COX) inhibitors. This reduction in drug efficacy is attributed to lower levels of prostaglandin E2 and di- minished expression of COX-2 [30]. Another study demonstrated that A. cepa exhibits anti- inflammatory and analgesic effects rela- tive to indomethacin [31]. That research indicated that A. cepa is as- sociated with a significantly shorter pain reaction time on pain stim- ulation tests compared to indomethacin. The findings suggest that the diminished analgesic efficacy of NSAIDs in the presence of A. cepa within endometriotic lesions could be a consequence of fi - brosis. Ki67, which can be detected through immunohistochemistry, serves as a marker of cellular proliferation. This enables the assess- ment of activity levels within endometriotic lesions, providing valu- able insights. An increase in Ki67 expression suggests that cells are becoming autonomous and may exert an influence on adjacent tis- sues [32]. Such an increase may be linked to the aggressiveness of endometriosis. Previous research has suggested that an increase in cell number may indicate uncontrolled cell division, and a correlation has been found between the severity of endometriosis and Ki67 ex- pression levels [33]. In our study, a high-dose A. cepa regimen (com- prising 30% A. cepa and 70% standard feed) resulted in a reduction of Ki67 expression compared to that observed in the other groups. Moreover, elevated Ki67 expression has been more commonly ob- served in infertile women, with a strong correlation between Ki67 levels and postoperative pregnancy. Notably, 66.7% of patients with high Ki67 expression have been found to achieve pregnancy within 1 year after endometriosis surgery, underscoring the importance of endometrial resection in the treatment of infertility [34]. Our findings suggest that treatment with A. cepa may reduce Ki67 expression in lesions, which could be advantageous for infertile patients and avoid the need for surgical intervention during the follow-up period. In conclusion, A. cepa may enhance histological outcomes as re- flected by evaluation scores and reduce Ki67 expression when ad- ministered in high doses. However, no statistically significant change was detected in the levels of profibrotic mediators. A. cepa appears to exert a more pronounced effect on the proliferation aspect of en- dometriosis rather than on antifibrotic characteristics. Conflict of interest No potential conflict of interest relevant to this article was report- ed. ORCID Hakan Kula https://orcid.org/0000-0003-1443-5796 Orkun Ilgen https://orcid.org/0000-0002-0296-8504 Sefa Kurt https://orcid.org/0000-0002-5144-0634 Filiz Yılmaz https://orcid.org/0000-0003-0505-3905 Author contributions Data curation: HK, OI. Formal analysis: OI, SK. Funding acquisition: HK, SK. Methodology: HK, OI. Project administration: SK. Visualiza- tion: FY. Writing-original draft: HK, FY. Writing-review & editing: SK.

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