Antioxidant Effect Of Bromelain In Ischemia-Reperfusion Injury Of Polycystic Ovary Diseases: Experimental Study

Antioxidant Effect Of Bromelain In Ischemia-Reperfusion Injury Of Polycystic Ovary Diseases: Experimental Study | 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 correspondence Antioxidant Effect Of Bromelain In Ischemia-Reperfusion Injury Of Polycystic Ovary Diseases: Experimental Study Sevgi Ulusoy Tangül, Taylan ONAT, Demet AYDOĞAN KIRMIZI, Züleyha DOĞANYIĞIT, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4563351/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: Due to its increased volume, polycystic ovarian tissue(PCOT) is also more prone to torsion than normal tissue. In treating ovarian torsion, detorsion is applied, and oxygenation of hypoxic tissues is provided with detorsion. The oxygen radicals formed as a result cause tissue damage. Bromelain is a substance obtained from pineapple that can induce apoptosis and free radical formation in macrophages. Aim: This study aimed to evaluate the damage caused to the ovarian tissue by ischemia-reperfusion(I/R) in normal and polycystic ovaries and to investigate the role of bromelain in the damage. Study Design: Animal experimentation. Methods: First, PCOT was created by administering dihydroepiandrostenodione-sulfate to 24 rats. Then, the polycystic and normal tissue groups were divided into sham, I/R, and I/R-bromelain groups. After the procedure, all rats' ovarian and tubal tissues were taken and histopathological examination and MDA, TUNEL, NF-κB values were examined. Results : In this study, it was determined that there was a significant decrease in MDA, NF-κB values, apoptotic-cell rate evaluated by TUNEL in the groups in which bromelain was given in the ovaries of normal and PCOT rats with IR damage, compared to the group that was not provided. It was also shown that I/R damage in PCOT were more significant than in normal ovarian tissue. Conclusion : The ischemia perfusion damage in polycystic ovarian tissue may be higher than that of normal ovarian tissue. However, there needs to be a literature study investigating this issue. In these respects, this study is the first in the literature. Bromelain is a preferable agent in preventing I / R damage caused by ovarian torsion of PCOT. In addition, it is thought that Bromelain may function in treating ovary torsions, and further studies can be conducted on this subject. Bromelain ischemia reperfusion ovarian torsion polycystic ovary. Figures Figure 1 Figure 2 Figure 3 1. Introduction In ovary torsion, as in the exact mechanism of other torsions, continued arterial perfusion often leads to ovarian enlargement, and eventually, local bleeding, infarction, and necrosis develop [ 1 ]. Surgery has to be performed to restore blood flow to the adnexal area and limit the damage to the ovary and the tube [ 2 ]. By detorsion, hypoxic tissues are oxygenated, forming oxygen radicals and tissue damage [ 3 ]. Increased oxygen radical production and antioxidant consumption disrupt the oxidative antioxidative balance in favor of oxidative stress [ 4 ]. To support the antioxidative course, many agents were experimentally used for ischemia reperfusion injuries in ovary diseases [ 5 , 6 ]. For example, carotenoids, which should be taken by diet, are effective antioxidants. Lycopene is one of the 600 carotenoids [ 6 ]. Hydrogen-rich saline solutions are also one the most used antioxidants for ovary ischemia reperfusion. Selenium, vitamin C, protein, erythropoietin, vardenafil, and curcumin are other antioxidants used in ovary torsions [ 5 ]. Root bromelain derived from pineapple has recently gained significant clinical interest [ 7 ]. Studies in the literature show Bromelain's effect on ischemia reperfusion [ 8 ]. The ability of root bromelain to reduce apoptosis and free radical formation in macrophages, its anti-mycobacterial properties, and its effect on foamy macrophages have been demonstrated in the literature [ 9 ]. In a study conducted in a myocardial ischemia reperfusion model, the effect of Bromelain to limit myocardial damage was investigated, and it was found that it showed a bigger left ventricular functional improvement during reperfusion than with controls [ 10 ]. The literature has also shown that low doses of Bromelain reduce the I/R damage in the liver [ 8 ]. In the literature, Bromelain is also used to treat ovarian tumors [ 11 ]. However, no previous study has evaluated its effect on ovarian ischemia reperfusion injury. Due to its increased volume, polycystic ovarian tissue (PCOT) is more prone to torsion than healthy tissue, so the ischemia perfusion injury in polycystic ovarian tissue may be more severe than that in normal ovarian tissue. This study aimed to evaluate the damage caused by ischemia reperfusion in the polycystic ovaries and investigate the role of Bromelain in the damage caused. 2. Materials and Methods 2.1. Ethics The local animal studies ethics committee approved the experimental procedures used in this study (21.02.2019 and protocol number 344). All experiments were performed according to the Guidelines for the Care and Use of Laboratory Animals, which the National Institute of Health (USA) approved. 2.2. Animals In the study, 48 female Wistar albino rats, aged 2–3 months, weighing between 200–250 g, were used. The rats were placed in stainless steel cages until the experiment conveniently at the ambient temperature (24–25°C) and humidity environment (55–60%) controlled photoperiod (12:12 h light: dark) in a standard rodent chow and fed with water. 2.3. Surgical technique A total of 48 rats were randomly divided into six groups. Group 1 (P-S): Polycystic ovary sham, (n: 8) Group 2 (N-S): Normal ovary sham, (n: 8) Group 3 (P-IR): Polycystic ovary ischemia/reperfusion, (n: 8) Group 4 (N-IR): Normal ovary ischemia/reperfusion, (n: 8) Group 5 (P-IRB): Polycystic ovary ischemia/reperfusion + Bromelain (20 mg/kg intra-peritoneally), (n: 8) Group 6 (N-IRB): Normal ovary ischemia/reperfusion + Bromelaine (20 mg/kg intra-peritoneally). (n: 8) 2.3.1. Polycystic Ovary Formation : Dihydroepiandrostenodione sulfate (DHEA) was administered 60 mg/kg/day subcutaneously for 21 days to 24 rats, and polycystic ovarian tissue was formed. DHEA (Biosteron 25 mg; Lekam Pharmaceutical, Zakroczym, Poland) was dissolved in 0.2 ml/day of sesame oil and administered subcutaneously to rats at 60 mg/kg/day for 21 days. The PCOS pattern was proven by vaginal smear, similar to the model by EJ Kim et al. [ 12 ]. The rats were anesthetized using Ketamine (Ketalar; Parke Davis, Eczacibasi, Istanbul, Turkey) and Xylazine (Rompun, Bayer AG, Leverkusen, Germany) in combination. After anesthesia, the rats were placed in the supine position, and the lower abdomen was cleaned using 2% iodine alcohol for antisepsis. Then, a 2.5 cm longitudinal incision was made in the lower abdomen to visualize the right ovary. For the sham groups (P-S and N-S), only the abdomen was opened, and it was awaited for 3 hours. After 3 hours, the ovary and the tubal tissue were removed. To establish ischemia-reperfusion (I / R) groups (P-IR and N-IR), ischemia was created using a vascular clip approximately 1 cm below the adnexal structure containing the right tuba and ovarian vessels. The incision line was closed with 4/0 nylon, and after 3 hours, a re-laparotomy was performed. Reperfusion was created, and the ovaries were observed until they became pink. A reperfusion time of 3 hours was applied [ 6 ]. In the groups that received Bromelain (PIRB and NIRB), 20 mg/kg bromelain (Bromelain 25 g; J&K Scientific, China) was dissolved in distilled water and then administered intra-peritoneally before reperfusion [ 13 ]. During the surgical procedure, the vital signs of all rats remained stable. At the end of the study, the rats were sacrificed by decapitation according to the ethics committee principles. All tissues were divided into two; half were placed in formalin for histopathology examination, and the other half was stored at -80°C for biochemical examination. 2.4. Histological Analysis Ovarian samples obtained from the experimental groups were subjected to tissue tracking stages for histopathology analysis. Briefly, sections of 5 µm thickness obtained from tissue samples fixed in 10% formaldehyde solution and embedded in paraffin were stained with Harris Hematoxylin Eosin (H&E) and examined under a light microscope (Olympus BX53) [ 14 ]. Histopathology damage scoring was performed according to the Akdemir et al. scoring system for ovarian degeneration of follicles in the cortical area (cellular dispersion and degeneration of follicular cells), vascular occlusion, bleeding, edema, and infiltration of inflammatory cells [ 15 ]. Each criterion was scored as usual (0), mild (1), moderate (2), and severe (3). All results were statistically calculated as mean ± SD. 2.5. Immuno-histochemical Analysis The immuno-reactivity of NF-κβ (Cat: BT-MCA 1291, Bioassay Technology Laboratory, China) protein was determined in the ovary samples belonging to experimental groups using the Avidin-Biotin peroxidase method [ 14 , 16 ]. For this process, after deparaffinization of the sections obtained at 5µm thickness, citrate buffer was used to open the epitopes (pH: 6.0). The slides were then placed in 3% hydrogen peroxide solution in methanol to prevent endogenous peroxidase activity. Ultra V block solution was applied to avoid unspecific staining. Subsequently, the sections were incubated with NF-κβ p65 (phospho S536) primary antibody (Cat: MCA 1292, Bioassay Technology Laboratory, China) at four 0C overnight. Biotinylated secondary streptavidin-HRP (TP-125-HL Lot: PHL181115, Thermo SCIENTIFIC, USA) and DAB (TA-125-HD Lot: HD47396 Thermo SCIENTIFIC, USA) chromogens were applied, and the sections were counterstained with Gill Hematoxylin. It was dehydrated by passing through the increasing alcohol series and covered with a sealer called Stellan. The sections were then examined with an Olympus BX53 light microscope. The evaluation of the immune-reactivity levels was made with the Image J program. Ten different areas were evaluated for each slide. 2.6. Apoptosis (TUNEL) staining Apoptotic cells in the ovary sections taken from the subjects were determined using the Roche brand In Situ Cell Detection Apoptosis Fluorescein Kit (Roche-11684795910) [ 14 ]. The staining was performed according to the kit procedure. Ovary sections taken at a thickness of 5 µm were deparaffinized and then rehydrated and washed twice with PBS for 5 minutes. Then, 270° C in a microwave oven in 0.01 M 5% sodium citrate buffer for antigen recovery was left for 5 minutes, then allowed to cool at room temperature for 10 minutes. Tissues were washed with PBS for 3x5 minutes and then placed in the moisture chamber at 37 0 C, with the TUNEL reaction mixture coming out of the kit, and incubated in the oven for 75 minutes. Tissues were washed twice for 5 minutes with PBS and contrasted with DAPI (4 ', 6-diamidino-2-phenylindole). Tissues sealed with glycerol closure solution were visualized on the Olympus BX51 fluorescent microscope at 450–500 nm wavelength. For the apoptotic index, cells in 10 different areas were counted in 40X objective from each section. 2.7. Biochemical Analysis Subjects' ovary tissues were raised to -80 ° C. Tissues were homogenized before the study. Then, centrifugation was applied, and supernatants were transferred to Eppendorf tubes. The ELISA method used the Malondialdehyde (MDA) (Cat. No: Cat. No: 201-11-0157, Sun Red Biological Technology) kit, and quantities were determined in nmol/ml at 450 nm in the ELISA reader. 2.8. Statistical analysis The Kolmogorov-Smirnov test was used to identify the normal distribution of the data. One‐way analysis of variance and post hoc Tukey test were used to determine differences between groups. Results are presented as mean ± SEM. The SPSS/PC program (Version 20.0; SPSS, Chicago, IL) and GraphPad Prism 8.0 software were used for statistical analysis. P < 0.05 were considered as statistically significant. 3. Results 3.1. Histological Findings While normal growing follicles were observed in group 1, atretic follicles were found in group 3. In addition, degenerated oocytes and zona pellusida were observed in the cystic follicle [ 12 ] characterized by PCOS experimental model (Fig. 1 A). Using Akdemir et al.'s histopathological scoring system, statistical significance was observed in P-IRB and N-IRB, in which ovarian tissues were treated with Bromelain, either with PCOT or not (Table 1 ) [ 15 ]. Also, P-IRB had a significant result compared to N-S and N-IR. However, N-IRB was most important than P-IRB (Table 1 ). As shown in Fig. 1 A, hemorrhage was observed in and around atretic follicles in group 3. In group 4, it was observed that there was a lot of bleeding in the atretic follicles and surrounding tissues. In group 5, hemorrhage and the number of degenerated follicles have decreased. In group 6, antral follicles with normal histology were observed with significantly reduced bleeding. According to histopathologic scoring, statistically significant improvements were observed in the ovarian tissues of Bromelain treated groups. Table 1 Histopathological damage scores in ovarian samples belonging to the experimental groups Degeneration of follicles Vascular congestion Edema Infiltration of inflammatory cells P-S 0,54 0,54 0,54 0,41 N-S 0,54 a 0,51 a 0,54 0,52 P-IR 0,52 ab 0,55 ab 0,82 b 0,52 ab N-IR 0,55 ab 0,41 ab 0,75 b 0,52 ab P-IRB 0,63 b 0,52 bd 0,63 b 0,75 b N-IRB 0,63 cde 0,75 cd 0,52 acde 0,52 cde Data are expressed as mean ± SD. Two-way ANOVA analysis of variance and TUKEY post hoc comparison test were applied (a P < 0.05 was different from P-S; b P < 0.05was different from N-S; c P < 0.05 was different from P-IR; d P < 0.05 Different from N-IR; e P < 0.05 represents different from P-IRB). (P-S): Polycystic ovary sham, (N-S): Normal ovary sham, (P-IR): Polycystic ovary ischemia/reperfusion, (N-IR): Normal ovary ischemia/reperfusion, (P-IRB): Polycystic ovary ischemia/reperfusion + Bromelain, (N-IRB): Normal ovary ischemia/reperfusion + Bromelain) 3.2. Immuno-histochemical Findings NF-κβ expression was seen in specimens (Fig. 1 B). It is insignificant in P-IRB and N-IRB compared to P-S and N-S (Fig. 1 C). It was observed that the NF-κβ expression was significantly decreased in P-IRB and N-IRB compared to P-IR and N-IR ( P 0.05) had a significant increase compared to all other groups ( P < 0.05). 3.3. TUNEL (Apoptosis) Results Apoptosis decreased significantly in P-IRB and N-IRB, in which Bromelain had been administered, according to N-S and N-IR (Table 2 , Fig. 2 ). As expected, apoptosis increased significantly in the P-IR compared to P-S. Table 2 TUNEL positive cell numbers belonging to the groups. Groups P-S N-S P-IR N-IR P-IRB N-IRB p TUNEL Positive cell counts 5.36 ± 1.03 a 0.40 ± 0.50 b 8.80 ± 2.98 c 3.32 ± 4.11 d 1.04 ± 1.09 b 0.52 ± 0.58 b 0.0001 The data are expressed as mean + standard deviation. P < 0.05 was accepted as significant. There were no significant differences between the groups expressed with the same letter. (P-S): Polycystic ovary sham, (N-S): Normal ovary sham, (P-IR): Polycystic ovary ischemia/reperfusion, (N-IR): Normal ovary ischemia/reperfusion, (P-IRB): Polycystic ovary ischemia/reperfusion + Bromelain, (N-IRB): Normal ovary ischemia/reperfusion + Bromelain) 3.4. Biochemical Results MDA decreased significantly, and Bromelain was administered to PCOT ischemia P-IRB. Also, N-IRB reduced MDA significantly (Table 3 , Fig. 3 ). Table 3 Biochemical data results of the experimental groups Groups P-S N-S P-IR N-IR P-IRB N-IRB p MDA nmol/ml 3.03 ± 0.48 ab 2.56 ± 0.15 a 3.95 ± 0.97 b 3.28 ± 0.74 ab 2.65 ± 0.22 a 2.61 ± 0.21 a 0.0008 The data are expressed as mean + standard deviation. P < 0.05 was accepted as significant. There were no significant differences between the groups expressed with the same letter. (P-S): Polycystic ovary sham, (N-S): Normal ovary sham, (P-IR): Polycystic ovary ischemia/reperfusion, (N-IR): Normal ovary ischemia/reperfusion, (P-IRB): Polycystic ovary ischemia/reperfusion + Bromelain, (N-IRB): Normal ovary ischemia/reperfusion + Bromelain). 4. Discussion Emerging free oxygen radicals are the main factor causing ischemia reperfusion induced cell damage. Many different agents acting against these free oxygen radicals as antioxidant and anti-inflammatory substrates that support the protective mechanisms for all organs are tested experimentally, and their effectiveness has been evaluated [ 5 , 6 , 15 ]. In this study, we attempted to evaluate Bromelain for its antioxidant effects. Root bromelain, a widely used phytotherapeutic member of the sulfhydryl proteolytic enzyme family, is derived from Ananas comosus and has a significant clinical interest [ 7 ]. Bromelain comprises endo-peptidase, glycoprotein, and carbohydrates [ 17 ]. It has fibrinolytic, anti-thrombotic, and anti-inflammatory properties; these effects have been documented in animal and human studies [ 10 , 17 ]. Bromelain's anti-inflammatory effect is associated with its protease activity [ 18 ]. Furthermore, Bromelain has antioxidant activity [ 19 ]. Bromelain's therapeutic effects have been demonstrated in diseases such as angina pectoris, bronchitis, sinusitis, surgical trauma, thrombo-phlebitis, osteoarthritis, diarrhea, cancer, and cardiovascular disorders [ 20 ]. It has been reported in the literature that the use of recipes containing Bromelain together with N-acetyl cysteine and alpha lipoic acid in the treatment of endometriosis has an anti-inflammatory effect in vivo and in vitro environments [ 21 ]. Again, in a clinical study conducted on patients with endometriosis, it was stated that the use of preparations containing N-acetyl cysteine, alpha lipoic acid, and Bromelain reduced the level of pain and led to lower analgesic need [ 22 ]. Although the use and efficacy of Bromelain in many tissues have been demonstrated, to our knowledge, there are no studies investigating the effect of Bromelain on ischemia-reperfusion injury in ovarian torsion. In the literature, there are many studies on ovarian ischemia, edema, follicular cell damage, vascular occlusion, hemorrhage, neutrophil infiltration, histopathology evaluation, and apoptosis index [ 5 ]. The presence of cystic follicles, increased plasma testosterone concentration, and an increase in the number of atretic follicles have been shown in animal models with DHEA-induced PCOT compared to controls [ 23 ]. However, an ovarian torsion model created by PCOT has not been encountered in the literature; hence, the histopathology images that may occur after torsion are unknown. Nevertheless, in line with the data obtained from this study, we demonstrated that there might be less bleeding due to the scarcity of atretic follicles and the tissue around the follicles. In this study, vacuolization was observed in the cytoplasm of granulosa lutein cells in the corpus luteum in P-S. In P-IR, bleeding in atretic follicles and surrounding tissue was observed. When Bromelain was administered, the bleeding decreased in P-IR, and vacuolization in the cytoplasm of luteal cells in the corpus luteum and healthy primordial follicles were found. The accumulation of abnormal cellular free oxygen radicals and I / R damage damages cell proteins and membranes and causes damage by activating the pro-apoptotic pathways[ 6 ]. Nuclear factor kappa Beta (NF-κβ) is one of the pleiotropic transcription factors and markers that play an essential role in inflammatory processes. NF-κβ activation occurs through radical oxygen substrates (ROS) and cytokines. The NF-κβ signaling pathway may increase inflammation and apoptosis [ 24 ]. In this study, as expected, NF-κβ had similar intensity in both N-S and P-S ( P > 0.05), significantly increasing compared to all other groups. However, no difference was found between normal and PCOT ovarian tissues ( P < 0.05). Administration of Bromelain significantly reduced the NF-κβ ( P < 0.05). This indicates that Bromelain significantly reduced the apoptosis caused by ovarian torsion. Our results are consistent with the other results in the literature [ 24 – 26 ]. TUNEL tests showed that the number of apoptotic cells significantly increased in the direction of PCOT between normal and PCOT ovarian I / R tissues. It was determined that bromelain administration significantly reduced the number of apoptotic cells in both standard and PCOT ovarian tissue. Our results are consistent with the other results in the literature [ 27 , 28 ]. Interestingly, Bromelain was seen to reduce the number of apoptotic cells encountered in PCOT significantly. This suggests that using Bromelain in treating PCOT can open new horizons and that further studies can be conducted on this issue. Reperfusion of ischemic tissue leads to more severe tissue damage than ischemia [ 29 ]. Therefore, studies to prevent reperfusion injury gain importance [ 30 ]. Lipid peroxidation in the cell is the most harmful effect of free oxygen radicals, accompanied by a decrease in membrane potential and subsequent cell damage. Malondialdehyde (MDA), one of the end products of lipid peroxidation, causes severe cell damage [ 31 ]. In our study, MDA levels increased significantly in the P-IR. There was a significant difference in MDA levels between N-S and P-S ( P < 0.05) .The MDA levels decreased in the P-IRB, and no significant statistical difference was found in the NI-R compared to the P-IRB. Our results for Bromelain are similar to those in the literature [ 24 , 27 , 32 ]. The ischemia perfusion damage in polycystic ovarian tissue may be higher than that of normal ovarian tissue. However, there needs to be a literature study investigating this issue. In these respects, this study is the first in the literature. Bromelain can be used to prevent I/R injury due to ovarian torsion of PCOT. It is also thought that Bromelain may have a function in treating ovarian torsion, and further studies can be conducted on this subject. Declarations Funding information: Disclosure/Conflict of Interest Statement: There is no conflict of interest between the authors. Ethics approval: 21.02.2019–344 Author Contribution Conceptualization: Sevgi UT, Taylan O, Demet AK. Data curation: Sevgi UT, Züleyha D, Emin K, Aslı O. Formal analysis: Sevgi UT, Züleyha D, Emin K, Aslı O. Methodology: Sevgi UT, Taylan O, Demet AKSoftware: Sevgi UT, Taylan O, Demet AK, Atilla Ş. Validation: Sevgi UT, Taylan O, Demet AK, Atilla Ş, Salih S; Investigation: Sevgi UT, Taylan O, Demet AK; Writing - original draft: Sevgi UT, Züleyha D, Atilla Ş, Salih S. Writing - review & editing: Sevgi UT, Züleyha D, Atilla Ş, Salih S. References Asfour, V., R. Varma, and P. Menon, Clinical risk factors for ovarian torsion. Journal of obstetrics and gynaecology, 2015. 35 (7): p. 721-725. 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Behroozi-Lak, T., et al., Protective effects of intraperitoneal administration of nimodipine on ischemia–reperfusion injury in ovaries: Histological and biochemical assessments in a rat model. Journal of pediatric surgery, 2017. 52 (4): p. 602-608. Arikan, D.C., et al., Protective effect of tadalafil on ischemia/reperfusion injury of rat ovary. Journal of pediatric surgery, 2010. 45 (11): p. 2203-2209. 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. We do this by developing innovative software and high quality services for the global research community. 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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-4563351","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"correspondence","associatedPublications":[],"authors":[{"id":314889133,"identity":"1d3e037a-2412-40a1-b887-df2825a94c61","order_by":0,"name":"Sevgi Ulusoy Tangül","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAuUlEQVRIiWNgGAWjYPACGyBmbDxAipY0kJYGkrQcBpPEaZFv7zF+zdt23m5t+2GgLTU20QS1GJw5Y2bN23Y7eduZRKCWY2m5DQS1SKSlGYO0mB0AamFsOExYi/z8ZyAt55LNzj8kUgvDDebDj3nbDtiZ3SDWFoMzyccY55xLTjC7AbQlgRi/yLcfbP7wpszO3ux8+sMHH2psiHAYAwObBJBIBKtMIEI5CDB/ABL2RCoeBaNgFIyCkQgAH4dHWvWjVroAAAAASUVORK5CYII=","orcid":"","institution":"Yozgat Bozok University","correspondingAuthor":true,"prefix":"","firstName":"Sevgi","middleName":"Ulusoy","lastName":"Tangül","suffix":""},{"id":314889134,"identity":"59023a31-2442-48be-87b7-a8f2101f508d","order_by":1,"name":"Taylan ONAT","email":"","orcid":"","institution":"Inonu University","correspondingAuthor":false,"prefix":"","firstName":"Taylan","middleName":"","lastName":"ONAT","suffix":""},{"id":314889135,"identity":"080ebdf0-cd58-4a4b-b290-eb4dd60fa37b","order_by":2,"name":"Demet AYDOĞAN KIRMIZI","email":"","orcid":"","institution":"Ozel Saglık Hospital","correspondingAuthor":false,"prefix":"","firstName":"Demet","middleName":"AYDOĞAN","lastName":"KIRMIZI","suffix":""},{"id":314889136,"identity":"59ddab5c-1518-46dd-b9ae-1d54be4d60a4","order_by":3,"name":"Züleyha DOĞANYIĞIT","email":"","orcid":"","institution":"Yozgat Bozok University","correspondingAuthor":false,"prefix":"","firstName":"Züleyha","middleName":"","lastName":"DOĞANYIĞIT","suffix":""},{"id":314889137,"identity":"182968af-7a59-4e41-9ec1-2ec2098e73fe","order_by":4,"name":"Emin KAYMAK","email":"","orcid":"","institution":"Yozgat Bozok University","correspondingAuthor":false,"prefix":"","firstName":"Emin","middleName":"","lastName":"KAYMAK","suffix":""},{"id":314889138,"identity":"cf9eecb2-9f73-42a3-99e1-a8a3149eecac","order_by":5,"name":"Aslı OFLAMAZ","email":"","orcid":"","institution":"Yozgat Bozok University","correspondingAuthor":false,"prefix":"","firstName":"Aslı","middleName":"","lastName":"OFLAMAZ","suffix":""},{"id":314889139,"identity":"b29f23e9-9507-40a8-af1a-13a21de17187","order_by":6,"name":"Atilla Şenaylı","email":"","orcid":"","institution":"Yozgat Bozok University","correspondingAuthor":false,"prefix":"","firstName":"Atilla","middleName":"","lastName":"Şenaylı","suffix":""},{"id":314889140,"identity":"8e3857c9-ce7c-446e-aa40-d21ff2665545","order_by":7,"name":"Salih Somuncu","email":"","orcid":"","institution":"Medicana Ataşehir Hospital","correspondingAuthor":false,"prefix":"","firstName":"Salih","middleName":"","lastName":"Somuncu","suffix":""}],"badges":[],"createdAt":"2024-06-11 10:23:04","currentVersionCode":1,"declarations":{"humanSubjects":false,"vertebrateSubjects":false,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":false,"humanSubjectConsent":false,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":false},"doi":"10.21203/rs.3.rs-4563351/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4563351/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":58635752,"identity":"a4413924-892d-4f90-bdd7-a37e456782a0","added_by":"auto","created_at":"2024-06-19 06:43:03","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1021944,"visible":true,"origin":"","legend":"\u003cp\u003eA) H\u0026amp;E staining images of ovary tissues belonging to experimental groups. Group P-S (a), Group N-S(b), Group P-IR (c), Group N-IR (d), Group P-IRB (e), Group N-IRB (f), magnification 40X, bar = 20m (Yellow arrow: vacuolization seen in cytoplasm granulosa lutein cells; yellow star: healthy ovarian follicles; blue star: atretic follicles; yellow thin arrow: vascular congestion and bleeding). B) NF-κB immune staining images in ovarian tissue samples. C) The data shown in the histogram graph showing the intensity of NF-κB immune-reactivity are the mean. Expressed as ±SEM. The One-way ANOVA analysis of variance and the TUKEY post hoc multiple comparison test were applied (a P \u0026lt;0.05 different to Group P-S; b P \u0026lt;0.05 different to Group N-S; c P \u0026lt;0.05 different to Group P-IR; d P \u0026lt;0.05 different to Group N-IR and e P \u0026lt;0.05 displays difference to Group P-IRB).\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4563351/v1/aa670e82b82c8d3202f0357f.png"},{"id":58635749,"identity":"dc0d6f76-6f9d-4a36-80a6-3f075e5de7f0","added_by":"auto","created_at":"2024-06-19 06:42:52","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":202313,"visible":true,"origin":"","legend":"\u003cp\u003eTUNEL staining images were shown. A; P-S, B; N-S, C; P-IR, D; N-IR, E; P-IRB, F; N-IRB. Apoptotic cells were shown with yellow arrows. X400.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-4563351/v1/104058f670d618d2a8c13656.png"},{"id":58635748,"identity":"a9318ed4-5445-4db3-bde1-7b9b96229193","added_by":"auto","created_at":"2024-06-19 06:42:52","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":36146,"visible":true,"origin":"","legend":"\u003cp\u003eGraphics of biochemical result. \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 was accepted as significant. There were no significant differences between the groups expressed with the same letter.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-4563351/v1/463913990d1e7e4eed56595e.png"},{"id":58636316,"identity":"908b18b2-20db-4be7-8a37-17e0966c8f70","added_by":"auto","created_at":"2024-06-19 06:50:54","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1984582,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4563351/v1/1c67820a-1ace-4c4b-bb28-54cae57e5c73.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eAntioxidant Effect Of Bromelain In Ischemia-Reperfusion Injury Of Polycystic Ovary Diseases: Experimental Study\u003c/p\u003e","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eIn ovary torsion, as in the exact mechanism of other torsions, continued arterial perfusion often leads to ovarian enlargement, and eventually, local bleeding, infarction, and necrosis develop [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Surgery has to be performed to restore blood flow to the adnexal area and limit the damage to the ovary and the tube [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. By detorsion, hypoxic tissues are oxygenated, forming oxygen radicals and tissue damage [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Increased oxygen radical production and antioxidant consumption disrupt the oxidative antioxidative balance in favor of oxidative stress [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. To support the antioxidative course, many agents were experimentally used for ischemia reperfusion injuries in ovary diseases [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. For example, carotenoids, which should be taken by diet, are effective antioxidants. Lycopene is one of the 600 carotenoids [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Hydrogen-rich saline solutions are also one the most used antioxidants for ovary ischemia reperfusion. Selenium, vitamin C, protein, erythropoietin, vardenafil, and curcumin are other antioxidants used in ovary torsions [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eRoot bromelain derived from pineapple has recently gained significant clinical interest [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Studies in the literature show Bromelain's effect on ischemia reperfusion [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. The ability of root bromelain to reduce apoptosis and free radical formation in macrophages, its anti-mycobacterial properties, and its effect on foamy macrophages have been demonstrated in the literature [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. In a study conducted in a myocardial ischemia reperfusion model, the effect of Bromelain to limit myocardial damage was investigated, and it was found that it showed a bigger left ventricular functional improvement during reperfusion than with controls [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. The literature has also shown that low doses of Bromelain reduce the I/R damage in the liver [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. In the literature, Bromelain is also used to treat ovarian tumors [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. However, no previous study has evaluated its effect on ovarian ischemia reperfusion injury.\u003c/p\u003e \u003cp\u003eDue to its increased volume, polycystic ovarian tissue (PCOT) is more prone to torsion than healthy tissue, so the ischemia perfusion injury in polycystic ovarian tissue may be more severe than that in normal ovarian tissue. This study aimed to evaluate the damage caused by ischemia reperfusion in the polycystic ovaries and investigate the role of Bromelain in the damage caused.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Ethics\u003c/h2\u003e \u003cp\u003e The local animal studies ethics committee approved the experimental procedures used in this study (21.02.2019 and protocol number 344). All experiments were performed according to the Guidelines for the Care and Use of Laboratory Animals, which the National Institute of Health (USA) approved.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Animals\u003c/h2\u003e \u003cp\u003eIn the study, 48 female Wistar albino rats, aged 2\u0026ndash;3 months, weighing between 200\u0026ndash;250 g, were used. The rats were placed in stainless steel cages until the experiment conveniently at the ambient temperature (24\u0026ndash;25\u0026deg;C) and humidity environment (55\u0026ndash;60%) controlled photoperiod (12:12 h light: dark) in a standard rodent chow and fed with water.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Surgical technique\u003c/h2\u003e \u003cp\u003eA total of 48 rats were randomly divided into six groups.\u003c/p\u003e \u003cp\u003eGroup 1 (P-S): Polycystic ovary sham, (n: 8)\u003c/p\u003e \u003cp\u003eGroup 2 (N-S): Normal ovary sham, (n: 8)\u003c/p\u003e \u003cp\u003eGroup 3 (P-IR): Polycystic ovary ischemia/reperfusion, (n: 8)\u003c/p\u003e \u003cp\u003eGroup 4 (N-IR): Normal ovary ischemia/reperfusion, (n: 8)\u003c/p\u003e \u003cp\u003eGroup 5 (P-IRB): Polycystic ovary ischemia/reperfusion\u0026thinsp;+\u0026thinsp;Bromelain (20 mg/kg intra-peritoneally), (n: 8)\u003c/p\u003e \u003cp\u003eGroup 6 (N-IRB): Normal ovary ischemia/reperfusion\u0026thinsp;+\u0026thinsp;Bromelaine (20 mg/kg intra-peritoneally). (n: 8)\u003c/p\u003e \u003cp\u003e \u003cb\u003e2.3.1. Polycystic Ovary Formation\u003c/b\u003e: Dihydroepiandrostenodione sulfate (DHEA) was administered 60 mg/kg/day subcutaneously for 21 days to 24 rats, and polycystic ovarian tissue was formed. DHEA (Biosteron 25 mg; Lekam Pharmaceutical, Zakroczym, Poland) was dissolved in 0.2 ml/day of sesame oil and administered subcutaneously to rats at 60 mg/kg/day for 21 days. The PCOS pattern was proven by vaginal smear, similar to the model by EJ Kim et al. [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe rats were anesthetized using Ketamine (Ketalar; Parke Davis, Eczacibasi, Istanbul, Turkey) and Xylazine (Rompun, Bayer AG, Leverkusen, Germany) in combination. After anesthesia, the rats were placed in the supine position, and the lower abdomen was cleaned using 2% iodine alcohol for antisepsis. Then, a 2.5 cm longitudinal incision was made in the lower abdomen to visualize the right ovary.\u003c/p\u003e \u003cp\u003eFor the sham groups (P-S and N-S), only the abdomen was opened, and it was awaited for 3 hours. After 3 hours, the ovary and the tubal tissue were removed.\u003c/p\u003e \u003cp\u003eTo establish ischemia-reperfusion (I / R) groups (P-IR and N-IR), ischemia was created using a vascular clip approximately 1 cm below the adnexal structure containing the right tuba and ovarian vessels. The incision line was closed with 4/0 nylon, and after 3 hours, a re-laparotomy was performed. Reperfusion was created, and the ovaries were observed until they became pink. A reperfusion time of 3 hours was applied [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn the groups that received Bromelain (PIRB and NIRB), 20 mg/kg bromelain (Bromelain 25 g; J\u0026amp;K Scientific, China) was dissolved in distilled water and then administered intra-peritoneally before reperfusion [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eDuring the surgical procedure, the vital signs of all rats remained stable. At the end of the study, the rats were sacrificed by decapitation according to the ethics committee principles. All tissues were divided into two; half were placed in formalin for histopathology examination, and the other half was stored at -80\u0026deg;C for biochemical examination.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Histological Analysis\u003c/h2\u003e \u003cp\u003eOvarian samples obtained from the experimental groups were subjected to tissue tracking stages for histopathology analysis. Briefly, sections of 5 \u0026micro;m thickness obtained from tissue samples fixed in 10% formaldehyde solution and embedded in paraffin were stained with Harris Hematoxylin Eosin (H\u0026amp;E) and examined under a light microscope (Olympus BX53) [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Histopathology damage scoring was performed according to the Akdemir et al. scoring system for ovarian degeneration of follicles in the cortical area (cellular dispersion and degeneration of follicular cells), vascular occlusion, bleeding, edema, and infiltration of inflammatory cells [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Each criterion was scored as usual (0), mild (1), moderate (2), and severe (3). All results were statistically calculated as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Immuno-histochemical Analysis\u003c/h2\u003e \u003cp\u003eThe immuno-reactivity of NF-κβ (Cat: BT-MCA 1291, Bioassay Technology Laboratory, China) protein was determined in the ovary samples belonging to experimental groups using the Avidin-Biotin peroxidase method [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. For this process, after deparaffinization of the sections obtained at 5\u0026micro;m thickness, citrate buffer was used to open the epitopes (pH: 6.0). The slides were then placed in 3% hydrogen peroxide solution in methanol to prevent endogenous peroxidase activity. Ultra V block solution was applied to avoid unspecific staining. Subsequently, the sections were incubated with NF-κβ p65 (phospho S536) primary antibody (Cat: MCA 1292, Bioassay Technology Laboratory, China) at four 0C overnight. Biotinylated secondary streptavidin-HRP (TP-125-HL Lot: PHL181115, Thermo SCIENTIFIC, USA) and DAB (TA-125-HD Lot: HD47396 Thermo SCIENTIFIC, USA) chromogens were applied, and the sections were counterstained with Gill Hematoxylin. It was dehydrated by passing through the increasing alcohol series and covered with a sealer called Stellan. The sections were then examined with an Olympus BX53 light microscope. The evaluation of the immune-reactivity levels was made with the Image J program. Ten different areas were evaluated for each slide.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6. Apoptosis (TUNEL) staining\u003c/h2\u003e \u003cp\u003eApoptotic cells in the ovary sections taken from the subjects were determined using the Roche brand In Situ Cell Detection Apoptosis Fluorescein Kit (Roche-11684795910) [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. The staining was performed according to the kit procedure. Ovary sections taken at a thickness of 5 \u0026micro;m were deparaffinized and then rehydrated and washed twice with PBS for 5 minutes. Then, 270\u0026deg; C in a microwave oven in 0.01 M 5% sodium citrate buffer for antigen recovery was left for 5 minutes, then allowed to cool at room temperature for 10 minutes. Tissues were washed with PBS for 3x5 minutes and then placed in the moisture chamber at 37 \u003csup\u003e0\u003c/sup\u003eC, with the TUNEL reaction mixture coming out of the kit, and incubated in the oven for 75 minutes. Tissues were washed twice for 5 minutes with PBS and contrasted with DAPI (4 ', 6-diamidino-2-phenylindole). Tissues sealed with glycerol closure solution were visualized on the Olympus BX51 fluorescent microscope at 450\u0026ndash;500 nm wavelength. For the apoptotic index, cells in 10 different areas were counted in 40X objective from each section.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7. Biochemical Analysis\u003c/h2\u003e \u003cp\u003eSubjects' ovary tissues were raised to -80 \u0026deg; C. Tissues were homogenized before the study. Then, centrifugation was applied, and supernatants were transferred to Eppendorf tubes. The ELISA method used the Malondialdehyde (MDA) (Cat. No: Cat. No: 201-11-0157, Sun Red Biological Technology) kit, and quantities were determined in nmol/ml at 450 nm in the ELISA reader.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8. Statistical analysis\u003c/h2\u003e \u003cp\u003eThe Kolmogorov-Smirnov test was used to identify the normal distribution of the data. One‐way analysis of variance and post hoc Tukey test were used to determine differences between groups. Results are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM. The SPSS/PC program (Version 20.0; SPSS, Chicago, IL) and GraphPad Prism 8.0 software were used for statistical analysis. \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 were considered as statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Histological Findings\u003c/h2\u003e \u003cp\u003eWhile normal growing follicles were observed in group 1, atretic follicles were found in group 3. In addition, degenerated oocytes and zona pellusida were observed in the cystic follicle [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e] characterized by PCOS experimental model (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Using Akdemir et al.'s histopathological scoring system, statistical significance was observed in P-IRB and N-IRB, in which ovarian tissues were treated with Bromelain, either with PCOT or not (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Also, P-IRB had a significant result compared to N-S and N-IR. However, N-IRB was most important than P-IRB (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, hemorrhage was observed in and around atretic follicles in group 3. In group 4, it was observed that there was a lot of bleeding in the atretic follicles and surrounding tissues. In group 5, hemorrhage and the number of degenerated follicles have decreased. In group 6, antral follicles with normal histology were observed with significantly reduced bleeding. According to histopathologic scoring, statistically significant improvements were observed in the ovarian tissues of Bromelain treated groups.\u003c/p\u003e \u003cp\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\u003eHistopathological damage scores in ovarian samples belonging to the experimental groups\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\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 \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDegeneration of follicles\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eVascular congestion\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eEdema\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eInfiltration of inflammatory\u003c/p\u003e \u003cp\u003ecells\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP-S\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0,54\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e 0,54\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e 0,54\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0,41\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eN-S\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0,54\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e 0,51\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0,54\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0,52\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP-IR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0,52\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0,55\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0,82\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0,52\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eN-IR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0,55\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0,41\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0,75\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0,52\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP-IRB\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0,63\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0,52\u003csup\u003ebd\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0,63\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0,75\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eN-IRB\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0,63\u003csup\u003ecde\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0,75\u003csup\u003ecd\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0,52\u003csup\u003eacde\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0,52\u003csup\u003ecde\u003c/sup\u003e\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\u003eData are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD. Two-way ANOVA analysis of variance and TUKEY post hoc comparison test were applied (a P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was different from P-S; b P\u0026thinsp;\u0026lt;\u0026thinsp;0.05was different from N-S; c P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was different from P-IR; d P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 Different from N-IR; e P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 represents different from P-IRB). (P-S): Polycystic ovary sham, (N-S): Normal ovary sham, (P-IR): Polycystic ovary ischemia/reperfusion, (N-IR): Normal ovary ischemia/reperfusion, (P-IRB): Polycystic ovary ischemia/reperfusion\u0026thinsp;+\u0026thinsp;Bromelain, (N-IRB): Normal ovary ischemia/reperfusion\u0026thinsp;+\u0026thinsp;Bromelain)\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.2. Immuno-histochemical Findings\u003c/h2\u003e \u003cp\u003eNF-κβ expression was seen in specimens (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). It is insignificant in P-IRB and N-IRB compared to P-S and N-S (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). It was observed that the NF-κβ expression was significantly decreased in P-IRB and N-IRB compared to P-IR and N-IR (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). Only P-IR and N-IR (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05) had a significant increase compared to all other groups (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.3. TUNEL (Apoptosis) Results\u003c/h2\u003e \u003cp\u003eApoptosis decreased significantly in P-IRB and N-IRB, in which Bromelain had been administered, according to N-S and N-IR (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). As expected, apoptosis increased significantly in the P-IR compared to P-S.\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\u003eTUNEL positive cell numbers belonging to the groups.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"8\"\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 \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\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\" colname=\"c2\"\u003e \u003cp\u003eP-S\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eN-S\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eP-IR\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eN-IR\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eP-IRB\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eN-IRB\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003ep\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTUNEL Positive cell counts\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5.36\u0026thinsp;\u0026plusmn;\u0026thinsp;1.03\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.40\u0026thinsp;\u0026plusmn;\u0026thinsp;0.50\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e8.80\u0026thinsp;\u0026plusmn;\u0026thinsp;2.98\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3.32\u0026thinsp;\u0026plusmn;\u0026thinsp;4.11\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.04\u0026thinsp;\u0026plusmn;\u0026thinsp;1.09\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.52\u0026thinsp;\u0026plusmn;\u0026thinsp;0.58\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.0001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"8\"\u003eThe data are expressed as mean\u0026thinsp;+\u0026thinsp;standard deviation. \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was accepted as significant. There were no significant differences between the groups expressed with the same letter. (P-S): Polycystic ovary sham, (N-S): Normal ovary sham, (P-IR): Polycystic ovary ischemia/reperfusion, (N-IR): Normal ovary ischemia/reperfusion, (P-IRB): Polycystic ovary ischemia/reperfusion\u0026thinsp;+\u0026thinsp;Bromelain, (N-IRB): Normal ovary ischemia/reperfusion\u0026thinsp;+\u0026thinsp;Bromelain)\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.4. Biochemical Results\u003c/h2\u003e \u003cp\u003eMDA decreased significantly, and Bromelain was administered to PCOT ischemia P-IRB. Also, N-IRB reduced MDA significantly (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\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\u003eBiochemical data results of the experimental groups\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"8\"\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 \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\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\" colname=\"c2\"\u003e \u003cp\u003eP-S\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eN-S\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eP-IR\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eN-IR\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eP-IRB\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eN-IRB\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003ep\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMDA nmol/ml\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3.03\u0026thinsp;\u0026plusmn;\u0026thinsp;0.48\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.56\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3.95\u0026thinsp;\u0026plusmn;\u0026thinsp;0.97\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3.28\u0026thinsp;\u0026plusmn;\u0026thinsp;0.74\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2.65\u0026thinsp;\u0026plusmn;\u0026thinsp;0.22\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e2.61\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.0008\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 data are expressed as mean\u0026thinsp;+\u0026thinsp;standard deviation. \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was accepted as significant. There were no significant differences between the groups expressed with the same letter. (P-S): Polycystic ovary sham, (N-S): Normal ovary sham, (P-IR): Polycystic ovary ischemia/reperfusion, (N-IR): Normal ovary ischemia/reperfusion, (P-IRB): Polycystic ovary ischemia/reperfusion\u0026thinsp;+\u0026thinsp;Bromelain, (N-IRB): Normal ovary ischemia/reperfusion\u0026thinsp;+\u0026thinsp;Bromelain).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eEmerging free oxygen radicals are the main factor causing ischemia reperfusion induced cell damage. Many different agents acting against these free oxygen radicals as antioxidant and anti-inflammatory substrates that support the protective mechanisms for all organs are tested experimentally, and their effectiveness has been evaluated [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. In this study, we attempted to evaluate Bromelain for its antioxidant effects.\u003c/p\u003e \u003cp\u003eRoot bromelain, a widely used phytotherapeutic member of the sulfhydryl proteolytic enzyme family, is derived from Ananas comosus and has a significant clinical interest [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Bromelain comprises endo-peptidase, glycoprotein, and carbohydrates [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. It has fibrinolytic, anti-thrombotic, and anti-inflammatory properties; these effects have been documented in animal and human studies [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Bromelain's anti-inflammatory effect is associated with its protease activity [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Furthermore, Bromelain has antioxidant activity [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Bromelain's therapeutic effects have been demonstrated in diseases such as angina pectoris, bronchitis, sinusitis, surgical trauma, thrombo-phlebitis, osteoarthritis, diarrhea, cancer, and cardiovascular disorders [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. It has been reported in the literature that the use of recipes containing Bromelain together with N-acetyl cysteine and alpha lipoic acid in the treatment of endometriosis has an anti-inflammatory effect in vivo and in vitro environments [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Again, in a clinical study conducted on patients with endometriosis, it was stated that the use of preparations containing N-acetyl cysteine, alpha lipoic acid, and Bromelain reduced the level of pain and led to lower analgesic need [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Although the use and efficacy of Bromelain in many tissues have been demonstrated, to our knowledge, there are no studies investigating the effect of Bromelain on ischemia-reperfusion injury in ovarian torsion.\u003c/p\u003e \u003cp\u003eIn the literature, there are many studies on ovarian ischemia, edema, follicular cell damage, vascular occlusion, hemorrhage, neutrophil infiltration, histopathology evaluation, and apoptosis index [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. The presence of cystic follicles, increased plasma testosterone concentration, and an increase in the number of atretic follicles have been shown in animal models with DHEA-induced PCOT compared to controls [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. However, an ovarian torsion model created by PCOT has not been encountered in the literature; hence, the histopathology images that may occur after torsion are unknown. Nevertheless, in line with the data obtained from this study, we demonstrated that there might be less bleeding due to the scarcity of atretic follicles and the tissue around the follicles. In this study, vacuolization was observed in the cytoplasm of granulosa lutein cells in the corpus luteum in P-S. In P-IR, bleeding in atretic follicles and surrounding tissue was observed. When Bromelain was administered, the bleeding decreased in P-IR, and vacuolization in the cytoplasm of luteal cells in the corpus luteum and healthy primordial follicles were found.\u003c/p\u003e \u003cp\u003eThe accumulation of abnormal cellular free oxygen radicals and I / R damage damages cell proteins and membranes and causes damage by activating the pro-apoptotic pathways[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Nuclear factor kappa Beta (NF-κβ) is one of the pleiotropic transcription factors and markers that play an essential role in inflammatory processes. NF-κβ activation occurs through radical oxygen substrates (ROS) and cytokines. The NF-κβ signaling pathway may increase inflammation and apoptosis [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. In this study, as expected, NF-κβ had similar intensity in both N-S and P-S (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05), significantly increasing compared to all other groups. However, no difference was found between normal and PCOT ovarian tissues (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Administration of Bromelain significantly reduced the NF-κβ (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). This indicates that Bromelain significantly reduced the apoptosis caused by ovarian torsion. Our results are consistent with the other results in the literature [\u003cspan additionalcitationids=\"CR25\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTUNEL tests showed that the number of apoptotic cells significantly increased in the direction of PCOT between normal and PCOT ovarian I / R tissues. It was determined that bromelain administration significantly reduced the number of apoptotic cells in both standard and PCOT ovarian tissue. Our results are consistent with the other results in the literature [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Interestingly, Bromelain was seen to reduce the number of apoptotic cells encountered in PCOT significantly. This suggests that using Bromelain in treating PCOT can open new horizons and that further studies can be conducted on this issue.\u003c/p\u003e \u003cp\u003eReperfusion of ischemic tissue leads to more severe tissue damage than ischemia [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Therefore, studies to prevent reperfusion injury gain importance [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Lipid peroxidation in the cell is the most harmful effect of free oxygen radicals, accompanied by a decrease in membrane potential and subsequent cell damage. Malondialdehyde (MDA), one of the end products of lipid peroxidation, causes severe cell damage [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. In our study, MDA levels increased significantly in the P-IR. There was a significant difference in MDA levels between N-S and P-S (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) .The MDA levels decreased in the P-IRB, and no significant statistical difference was found in the NI-R compared to the P-IRB. Our results for Bromelain are similar to those in the literature [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe ischemia perfusion damage in polycystic ovarian tissue may be higher than that of normal ovarian tissue. However, there needs to be a literature study investigating this issue. In these respects, this study is the first in the literature.\u003c/p\u003e \u003cp\u003eBromelain can be used to prevent I/R injury due to ovarian torsion of PCOT. It is also thought that Bromelain may have a function in treating ovarian torsion, and further studies can be conducted on this subject.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eFunding information:\u003c/h2\u003e \u003cp\u003eDisclosure/Conflict of Interest Statement: There is no conflict of interest between the authors.\u003c/p\u003e \u003cp\u003e \u003cstrong\u003eEthics approval:\u003c/strong\u003e \u003cp\u003e21.02.2019\u0026ndash;344\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eConceptualization: Sevgi UT, Taylan O, Demet AK. Data curation: Sevgi UT, Z\u0026uuml;leyha D, Emin K, Aslı O. Formal analysis: Sevgi UT, Z\u0026uuml;leyha D, Emin K, Aslı O. Methodology: Sevgi UT, Taylan O, Demet AKSoftware: Sevgi UT, Taylan O, Demet AK, Atilla Ş. Validation: Sevgi UT, Taylan O, Demet AK, Atilla Ş, Salih S; Investigation: Sevgi UT, Taylan O, Demet AK; Writing - original draft: Sevgi UT, Z\u0026uuml;leyha D, Atilla Ş, Salih S. Writing - review \u0026amp; editing: Sevgi UT, Z\u0026uuml;leyha D, Atilla Ş, Salih S.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eAsfour, V., R. Varma, and P. Menon, \u003cem\u003eClinical risk factors for ovarian torsion.\u003c/em\u003e Journal of obstetrics and gynaecology, 2015. \u003cstrong\u003e35\u003c/strong\u003e(7): p. 721-725.\u003c/li\u003e\n \u003cli\u003eTasset, J., et al., \u003cem\u003eOvarian torsion in premenarchal girls.\u003c/em\u003e Journal of Pediatric and Adolescent Gynecology, 2019. \u003cstrong\u003e32\u003c/strong\u003e(3): p. 254-258.\u003c/li\u003e\n \u003cli\u003eSlater, T.F., \u003cem\u003eFree radical mechanisms in tissue injury.\u003c/em\u003e Cell function and disease, 1988: p. 209-218.\u003c/li\u003e\n \u003cli\u003eYildirim, A., et al., \u003cem\u003eCorrelation of ischemia-modified albumin levels and histopathologic findings in experimental ovarian torsion.\u003c/em\u003e Turkish journal of emergency medicine, 2016. \u003cstrong\u003e16\u003c/strong\u003e(1): p. 8-11.\u003c/li\u003e\n \u003cli\u003eGokalp, N., et al., \u003cem\u003eProtective effect of hydrogen rich saline solution on experimental ovarian ischemia reperfusion model in rats.\u003c/em\u003e Journal of pediatric surgery, 2017. \u003cstrong\u003e52\u003c/strong\u003e(3): p. 492-497.\u003c/li\u003e\n \u003cli\u003eKirmizi, D.A., et al., \u003cem\u003eThe effect of a natural molecule in ovary ischemia reperfusion damage: does lycopene protect ovary?\u003c/em\u003e Experimental animals, 2021. \u003cstrong\u003e70\u003c/strong\u003e(1): p. 37-44.\u003c/li\u003e\n \u003cli\u003eDave, S., \u003cem\u003eHexafluoroisopropanol Induced Helix Sheet Transition of Stem Bromelain Correlation to Function.\u003c/em\u003e Biophysical Journal, 2012. \u003cstrong\u003e102\u003c/strong\u003e(3): p. 453a-454a.\u003c/li\u003e\n \u003cli\u003eBahde, R., et al., \u003cem\u003eBromelain ameliorates hepatic microcirculation after warm ischemia.\u003c/em\u003e Journal of Surgical Research, 2007. \u003cstrong\u003e139\u003c/strong\u003e(1): p. 88-96.\u003c/li\u003e\n \u003cli\u003eMahajan, S., et al., \u003cem\u003eStem Bromelain\u0026ndash;Induced Macrophage Apoptosis and Activation Curtail Mycobacterium tuberculosis Persistence.\u003c/em\u003e The Journal of infectious diseases, 2012. \u003cstrong\u003e206\u003c/strong\u003e(3): p. 366-376.\u003c/li\u003e\n \u003cli\u003eJuhasz, B., et al., \u003cem\u003eBromelain induces cardioprotection against ischemia-reperfusion injury through Akt/FOXO pathway in rat myocardium.\u003c/em\u003e American journal of physiology-Heart and circulatory physiology, 2008. \u003cstrong\u003e294\u003c/strong\u003e(3): p. H1365-H1370.\u003c/li\u003e\n \u003cli\u003eGani, M.B.A., et al., \u003cem\u003eIn vitro antiproliferative activity of fresh pineapple juices on ovarian and colon cancer cell lines.\u003c/em\u003e International Journal of Peptide Research and Therapeutics, 2015. \u003cstrong\u003e21\u003c/strong\u003e: p. 353-364.\u003c/li\u003e\n \u003cli\u003eKim, E.-J., et al., \u003cem\u003eAn improved dehydroepiandrosterone-induced rat model of polycystic ovary syndrome (PCOS): Post-pubertal improve PCOS\u0026apos;s features.\u003c/em\u003e Frontiers in endocrinology, 2018. \u003cstrong\u003e9\u003c/strong\u003e: p. 735.\u003c/li\u003e\n \u003cli\u003eLotz-Winter, H., \u003cem\u003eOn the pharmacology of bromelain: an update with special regard to animal studies on dose-dependent effects.\u003c/em\u003e Planta medica, 1990. \u003cstrong\u003e56\u003c/strong\u003e(03): p. 249-253.\u003c/li\u003e\n \u003cli\u003eDoğanyiğit, Z., et al., \u003cem\u003eInvestigation of protective effects of apilarnil against lipopolysaccharide induced liver injury in rats via TLR 4/HMGB-1/NF-\u0026kappa;B pathway.\u003c/em\u003e Biomedicine \u0026amp; Pharmacotherapy, 2020. \u003cstrong\u003e125\u003c/strong\u003e: p. 109967.\u003c/li\u003e\n \u003cli\u003eAkdemir, A., et al., \u003cem\u003eProtective effect of oxytocin on ovarian ischemia-reperfusion injury in rats.\u003c/em\u003e Peptides, 2014. \u003cstrong\u003e55\u003c/strong\u003e: p. 126-130.\u003c/li\u003e\n \u003cli\u003eOkan, A., et al., \u003cem\u003eEvaluation of the protective role of resveratrol against sepsis caused by LPS via TLR4/NF‐\u0026kappa;B/TNF‐\u0026alpha; signaling pathways: Experimental study.\u003c/em\u003e Cell Biochemistry and Function, 2023. \u003cstrong\u003e41\u003c/strong\u003e(4): p. 423-433.\u003c/li\u003e\n \u003cli\u003ePavan, R., S. Jain, and A. Kumar, \u003cem\u003eProperties and therapeutic application of bromelain: a review.\u003c/em\u003e Biotechnology research international, 2012. \u003cstrong\u003e2012\u003c/strong\u003e.\u003c/li\u003e\n \u003cli\u003eZhou, Z., et al., \u003cem\u003eInhibition of epithelial TNF-\u0026alpha; receptors by purified fruit bromelain ameliorates intestinal inflammation and barrier dysfunction in colitis.\u003c/em\u003e Frontiers in immunology, 2017. \u003cstrong\u003e8\u003c/strong\u003e: p. 1468.\u003c/li\u003e\n \u003cli\u003eSaptarini, N.M., D. Rahayu, and I.E. Herawati, \u003cem\u003eAntioxidant activity of crude bromelain of pineapple (Ananas comosus (L.) Merr) crown from Subang District, Indonesia.\u003c/em\u003e Journal of Pharmacy and Bioallied Sciences, 2019. \u003cstrong\u003e11\u003c/strong\u003e(Suppl 4): p. S551-S555.\u003c/li\u003e\n \u003cli\u003eBayat, S., et al., \u003cem\u003eBromelain-loaded chitosan nanofibers prepared by electrospinning method for burn wound healing in animal models.\u003c/em\u003e Life sciences, 2019. \u003cstrong\u003e229\u003c/strong\u003e: p. 57-66.\u003c/li\u003e\n \u003cli\u003eAgostinis, C., et al., \u003cem\u003eThe combination of N-acetyl cysteine, alpha-lipoic acid, and bromelain shows high anti-inflammatory properties in novel in vivo and in vitro models of endometriosis.\u003c/em\u003e Mediators of inflammation, 2015. \u003cstrong\u003e2015\u003c/strong\u003e.\u003c/li\u003e\n \u003cli\u003eLete, I., et al., \u003cem\u003eEffectiveness of an antioxidant preparation with N-acetyl cysteine, alpha lipoic acid and bromelain in the treatment of endometriosis-associated pelvic pain: LEAP study.\u003c/em\u003e European Journal of Obstetrics \u0026amp; Gynecology and Reproductive Biology, 2018. \u003cstrong\u003e228\u003c/strong\u003e: p. 221-224.\u003c/li\u003e\n \u003cli\u003eMisugi, T., et al., \u003cem\u003eInsulin-lowering agents inhibit synthesis of testosterone in ovaries of DHEA-induced PCOS rats.\u003c/em\u003e Gynecologic and obstetric investigation, 2006. \u003cstrong\u003e61\u003c/strong\u003e(4): p. 208-215.\u003c/li\u003e\n \u003cli\u003eAli, F.F., A.F. Ahmed, and D.M.E. Ali, \u003cem\u003eUnderlying mechanisms behind the protective effect of angiotensin (1\u0026ndash;7) in experimental rat model of ovarian ischemia reperfusion injury.\u003c/em\u003e Life sciences, 2019. \u003cstrong\u003e235\u003c/strong\u003e: p. 116840.\u003c/li\u003e\n \u003cli\u003eM\u0026uuml;ller, A., et al., \u003cem\u003eComparative study of antitumor effects of bromelain and papain in human cholangiocarcinoma cell lines.\u003c/em\u003e International journal of oncology, 2016. \u003cstrong\u003e48\u003c/strong\u003e(5): p. 2025-2034.\u003c/li\u003e\n \u003cli\u003eHabashi, S.A., et al., \u003cem\u003eModulation of lipopolysaccharide stimulated nuclear factor kappa B mediated iNOS/NO production by bromelain in rat primary microglial cells.\u003c/em\u003e Iranian biomedical journal, 2016. \u003cstrong\u003e20\u003c/strong\u003e(1): p. 33.\u003c/li\u003e\n \u003cli\u003eUgurel, V., et al., \u003cem\u003eAntioxidant and antiapoptotic effects of erdosteine in a rat model of ovarian ischemia-reperfusion injury.\u003c/em\u003e Iranian journal of basic medical sciences, 2017. \u003cstrong\u003e20\u003c/strong\u003e(1): p. 53.\u003c/li\u003e\n \u003cli\u003eOzlem, K., et al., \u003cem\u003eProtective effect of Vaccinium myrtillus on ischemia-reperfusion injury in rat ovary.\u003c/em\u003e Taiwanese Journal of Obstetrics and Gynecology, 2018. \u003cstrong\u003e57\u003c/strong\u003e(6): p. 836-841.\u003c/li\u003e\n \u003cli\u003eZimmerman, B.J. and D.N. Granger, \u003cem\u003eReperfusion injury.\u003c/em\u003e Surgical Clinics of North America, 1992. \u003cstrong\u003e72\u003c/strong\u003e(1): p. 65-83.\u003c/li\u003e\n \u003cli\u003eIngec, M., et al., \u003cem\u003ePrevention of ischemia-reperfusion injury in rat ovarian tissue with the on-off method.\u003c/em\u003e Journal of physiology and pharmacology, 2011. \u003cstrong\u003e62\u003c/strong\u003e(5): p. 575.\u003c/li\u003e\n \u003cli\u003eBehroozi-Lak, T., et al., \u003cem\u003eProtective effects of intraperitoneal administration of nimodipine on ischemia\u0026ndash;reperfusion injury in ovaries: Histological and biochemical assessments in a rat model.\u003c/em\u003e Journal of pediatric surgery, 2017. \u003cstrong\u003e52\u003c/strong\u003e(4): p. 602-608.\u003c/li\u003e\n \u003cli\u003eArikan, D.C., et al., \u003cem\u003eProtective effect of tadalafil on ischemia/reperfusion injury of rat ovary.\u003c/em\u003e Journal of pediatric surgery, 2010. \u003cstrong\u003e45\u003c/strong\u003e(11): p. 2203-2209.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"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":"Bromelain, ischemia reperfusion, ovarian torsion, polycystic ovary.","lastPublishedDoi":"10.21203/rs.3.rs-4563351/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4563351/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground: \u003c/strong\u003eDue to its increased volume, polycystic ovarian tissue(PCOT) is also more prone to torsion than normal tissue. In treating ovarian torsion, detorsion is applied, and oxygenation of hypoxic tissues is provided with detorsion. The oxygen radicals formed as a result cause tissue damage. Bromelain is a substance obtained from pineapple that can induce apoptosis and free radical formation in macrophages. \u003cstrong\u003eAim: \u003c/strong\u003eThis study aimed to evaluate the damage caused to the ovarian tissue by ischemia-reperfusion(I/R) in normal and polycystic ovaries and to investigate the role of bromelain in the damage.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStudy Design: \u003c/strong\u003eAnimal experimentation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods: \u003c/strong\u003eFirst, PCOT was created by administering dihydroepiandrostenodione-sulfate to 24 rats. Then, the polycystic and normal tissue groups were divided into sham, I/R, and I/R-bromelain groups. After the procedure, all rats' ovarian and tubal tissues were taken and histopathological examination and MDA, TUNEL, NF-κB values were examined.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e: In this study, it was determined that there was a significant decrease in MDA, NF-κB values, apoptotic-cell rate evaluated by TUNEL in the groups in which bromelain was given in the ovaries of normal and PCOT rats with IR damage, compared to the group that was not provided. It was also shown that I/R damage in PCOT were more significant than in normal ovarian tissue.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion\u003c/strong\u003e: The ischemia perfusion damage in polycystic ovarian tissue may be higher than that of normal ovarian tissue. However, there needs to be a literature study investigating this issue. In these respects, this study is the first in the literature. Bromelain is a preferable agent in preventing I / R damage caused by ovarian torsion of PCOT. In addition, it is thought that Bromelain may function in treating ovary torsions, and further studies can be conducted on this subject.\u003c/p\u003e","manuscriptTitle":"Antioxidant Effect Of Bromelain In Ischemia-Reperfusion Injury Of Polycystic Ovary Diseases: Experimental Study","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-06-19 06:42:40","doi":"10.21203/rs.3.rs-4563351/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":"074656f5-c96e-4415-bf2a-971a3715de59","owner":[],"postedDate":"June 19th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-06-19T06:42:40+00:00","versionOfRecord":[],"versionCreatedAt":"2024-06-19 06:42:40","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4563351","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4563351","identity":"rs-4563351","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}