Effect of beta-glucan on oxidative stress, inflammation, hormonal and histopathological changes in dehydroepiandrosterone-induced polycystic ovary syndrome

Effect of beta-glucan on oxidative stress, inflammation, hormonal and histopathological changes in dehydroepiandrosterone-induced polycystic ovary syndrome | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Effect of beta-glucan on oxidative stress, inflammation, hormonal and histopathological changes in dehydroepiandrosterone-induced polycystic ovary syndrome Hande Yüce, Neşe Başak Türkmen, Muhterem Aydın, Aslı Taşlıdere, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4448893/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 11 You are reading this latest preprint version Abstract A class of dietary fibers and biologically active polysaccharides from natural sources, beta-glucans (βTGs) have bioactive capabilities. The anti-tumor, anti-inflammatory, prebiotic, anti-obesity, anti-allergic, anti-microbial, antiviral, anti-osteoporotic, and immunomodulating effects of βTGs are well documented. Although many biological activities of βTG have been proven, its mechanism in DHEA-induced PCOS has not been investigated. We aimed to investigate the protective effects of βTG treatment on PCOS and its capacity to reverse PCOS-induced changes. Female Sprague-Dawley (SD) rats were divided into four groups at random (n = 8): control, PCOS, PCOS + βTG, and βTG groups. Biochemical markers linked to oxidative stress, antioxidant state, inflammation, cytokines, and hormone levels were assessed. Analyses using immunohistochemistry and histopathology were also carried out. Membrane array analysis detected growth factors, cytokine, and chemokine protein profiles. βTG did not cause any change in body, uterus, and ovarian weights in rats. βTG normalized the deviations in the oestrus cycle caused by PCOS. It was observed that βTG had a positive effect on the reproductive system. βTG can reduce the inflammatory response in PCOS rats by decreasing inflammatory cytokines. Oxidative stress was significantly reduced, whereas antioxidant enzyme activities were significantly elevated in the βTG group. βTG also prevented histopathological alterations. βTG induced the expression of some essential proteins, including bNGF, TIMP-1, Agrin, CINC-1, BDNF, and FGF-2 (bFGF). The results of this study showed that treatment with βTG protects against oxidative stress, inflammation, hormone imbalance, and histopathological damage in ovarian tissue caused by PCOS. antioxidant beta-glucan dehydroepiandrosterone inflammation oxidative stress polycystic ovary syndrome. Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 Figure 13 Figure 14 Figure 15 Figure 16 1. INTRODUCTION Polycystic ovary syndrome (PCOS), whose incidence has increased in recent years, is a gynecological disease and also an endocrine disease that negatively affects fertility and paves the way for the formation of many different diseases. Polycystic ovary, which is common, especially among women of reproductive age, causes infertility by causing ovulation disorder. In addition, if untreated, it progresses and leads to many secondary diseases, such as hypertension and cardiovascular diseases. 1 Objective of PCOS treatment: reducing the effects of androgen-related symptoms, treatment of menstrual cycle and related irregularities, and infertility treatment. Symptoms and severity differ in each woman. Therefore, treatment methods are determined individually for each patient. It is now widely acknowledged that oxidative stress plays a crucial part in the pathogenesis of numerous illnesses, 2 – 4 including PCOS. Although the production and spread of intracellular reactive oxygen species (ROS) are tightly regulated by extremely sophisticated antioxidant enzymatic and non-enzymatic systems, 5 understanding oxidative stress mechanisms is crucial for developing PCOS prevention and treatment strategies. 6 PCOS presents diagnostic difficulties due to the incomplete explanation of its etiology and phenotypic diversity. The diagnosis of PCOS is usually based on subjective criteria such as clinical and ultrasonographic findings. First of all, the fact that AMH measurements used to determine ovarian reserve are higher than usual in hyperandrogenism cases has attracted attention, leading to the thought that AMH measurements may have a place in the diagnosis of PCOS. AMH is inhibitory in folliculogenesis, explaining the cause of PCOS anovulation. This is the key to high AMH in PCOS. It is natural for women with PCOS to have an increase in LH, and this is in parallel with the AMH level. However, unlike LH, AMH level indicates increased antral follicle number and has diagnostic value in PCOS. 7 , 8 Recent research has demonstrated a link between PCOS incidence and low-grade chronic inflammation. 9 Proinflammatory cytokines such as interleukin-6 (IL-6) and IL-1, as well as tumor necrosis factor-alpha (TNF-α), are crucial mediators indicating the existence of inflammation. 10 TNF-α, IL-6, and IL-1 are important in reproductive physiology. Events such as ovarian steroid production, fertilization, implantation, follicular maturation, and ovulation are all affected in women with PCOS. 11 The protective effects of natural compounds on diseases have been the subject of many studies. 12 – 15 (Beta-glucans (βTGs) are a class of polysaccharides derived from numerous species of fungi, mushrooms, and cereal grains. They have various pharmacological effects, including antioxidant, antibacterial, antitumor, free radical scavenging, anti-lipid, immunomodulation, antifungal, and radioprotective activities. 16 Previous research has shown that βTG has various pharmacological effects, but the impact of βTG on a rat PCOS model has not been examined. First, to the best of our knowledge, it is the first study to assess the regressions of oxidative stress, inflammation, sexual cycles, and histopathological damage due to βTG treatment in PCOS. In our previous study, we found that PCOS is not only related to reproductive pathology but also a systemic complication whose etiopathogenesis has not yet been fully elucidated and that nerolidol is effective in controlling the biochemical, apoptotic, histopathological, and metabolic changes in PCOS. 17 2. MATERIALS AND METHODS 2.1. Drugs and chemicals We purchased beta-glucan from Sigma (9012-72-0; St. Louis, MO) and dehydroepiandrosterone (DHEA) from Avanti, Polar Lipids, INC. in the United States (Cas no: 53-43-0). All chemicals that we used for the biochemical, histological, and immunohistochemical analyses were provided by Sigma. The Millipore Autopure WR600A system was used to purify the water used to make the solutions (Millipore, Ltd., Burlington, MA). 2.2. Animals and experimental protocol Thirty-two female Sprague-Dawley rats (aged 21 days) were used in the study, which the Experimental Animals Institute at İnönü University conducted. The study was approved by the Institutional Animal Ethics Committee of the same university (Approval no. 2020/2–8). A 12-hour light/12-hour dark cycle, 40–60% relative humidity, and an ambient temperature of 20–24°C were all maintained for all animals in a well-ventilated setting. Four experimental groups of eight SD pre-pubertal rats each were randomly assigned. Control group: sesame oil per day was injected subcutaneously at 0.2 ml for 21 days. PCOS group: 60 mg/kg/day DHEA diluted with 0.2 ml sesame oil was injected subcutaneously for 21 days. 18 P + βTG group: 60 mg/kg/day DHEA was injected subcutaneously for 21 days, and βTG was given orally at 50 mg/kg/day 22 days later for 21 days. βTG group: Rats were treated orally with βTG (50 mg/kg/day) dissolved in 0.2 ml sesame oil for 21 days. At the end of the treatment period, ketamine/xylazine anesthesia was administered to all rats. 2.3. Estrous cycles, ovarian, uterus, and body weights The body weights of the rats were weighed every day during the experiment. After sacrification, the ovarian and uterine tissues of the rats were weighed. Three different types of vaginal epithelial cells controlled the estrous cycle. 19 Every day between 9:00 and 10:00, vaginal smears were performed. The method of Giemsa staining was employed to analyze cell morphology. 2.4. Biochemical analysis Anti-mullerian hormone (AMH), follicle-stimulating hormone (FSH), testosterone (T), luteinizing hormone (LH), tumor necrosis factor-alpha (TNF-alpha), interleukin-1 beta (IL-1 beta), and interleukin-6 (IL-6) were quantified using commercially available ELISA kits. 2.5. Determination of oxidative stress Yagi's approach was used to determine the lipid peroxidation index. 20 After centrifuging for 10 minutes while cooling at 3000 rpm, the tissue homogenate was precipitated with 10% trichloroacetic acid (TCA) in a boiling water bath for 15 minutes. TBA was added to the supernatant and then incubated for 50 minutes at 95°C. At 532 nm, the pink color complex's absorbances were determined spectrophotometrically. Nmol/mg protein was used to express the results. 2.6. Determination of GSH levels and SOD, CAT, and GPx activities Superoxide dismutase activity was determined according to the method of Sun et al. 21 Catalase activity was assayed by the method developed by Aebi 22 . The glutathione content was determined using the Sedlak and Lindsay method. 23 Glutathione peroxidase activity was measured using the Paglia and Valentine method. 24 2.10. Histopathological assay The ovarian tissue samples were embedded in paraffin and treated in 10% formalin to be examined under a light microscope. Sections of tissues fixed in paraffin were cut into 5 µm thick pieces. They were captured on slides and hematoxylin-eosin stained (H-E). 25 2.11. Immunohistochemical analysis Ovarian tissue samples were fixed in neutral formalin for caspase 3 immunoreactivity studies. After routine tissue follow-ups, paraffin sections 5 µm thick were taken from paraffin-embedded tissue samples to polylysine-coated slides. The antigens were then exposed by placing the sections in a citrate solution (pH 7.6) and heating them for 20 minutes in a microwave. It was chilled for 20 minutes, and then the sections were incubated in 0.3% H2O2 for 7 minutes. Then, the sections were washed with PBS. After carefully wiping and drying around each section, the blocking solution (Ab4051, Abcam, USA.) was dripped onto it to prevent nonspecific binding and incubated for two hours. Following a PBS rinse, they were treated with biotinylated goat anti-polyvalent and streptavidin peroxidase. Slides were dehydrated and counterstained for 1 minute with Mayer's hematoxylin before being stained for 15 minutes with chromogen + substrate. The caspase-3 kit was used following the manufacturer's instructions. 2.12. Rat growth factor chemokine and cytokine analysis Growth factor, chemokine, and cytokine analysis of ovarian tissues were examined using protein-membrane arrays. Protein isolation was conducted using the Lysis Buffer of the array kit, and protein concentrations were measured by bicinchoninic acid assay. An equal amount of protein samples were administered to the membranes and incubated in the blocking buffer for 30 min. Membranes were washed t times after overnight incubation with protein samples. Horseradish peroxidase (HRP) was applied to membranes for 2 hours at room temperature, after which a chemiluminescent system was used to detect the presence of a protein (Bio-Rad Biotechnology Inc., USA). The Image Lab software measured protein intensities. 26 2.13. Statistical analysis For all statistical calculations, GraphPad Prism version 8.0.0 for Windows was used. ClustVis ( https://biit.cs.ut.ee/clustvis/ ) was used to create the principal component analysis (PCA) for all experiments. 27 Shapiro-Wilk test was used to assess fit for normal distribution. Mann-Whitney U, Kruskal-Wallis, Conover, and Friedman tests were used for statistical analysis. p < 0.05 was considered statistically significant. 3. RESULTS 3.1. Effects of beta-glucan on ovarian, uterus, and body weights The right ovary weight, uterine weight, and total genital weight of the PCOS group were significantly higher than those of the control group. The uterus and ovarian weights of the control, PCOS, P + βTG, and βTG groups did not differ statistically (Fig. 1 ). 3.2. Effects of beta-glucan on estrous cycle The fractional survival data between the groups shows that the sexual cycles obtained proceed in a particular order. There was no deviation in the control, βTG, and PCOS + βTG groups. A significant deviation was observed in the PCOS group (Fig. 2 ). The estrous cycle is examined in four consecutive periods. In the first period (proestrus), nucleated epithelial cells are dense. These cells can be seen together or separately. In some areas, cornified cells can be seen. The second period (estrus) is characterized by cornified squamous epithelial cells. Cells often appear in clumps. In the third period (metestrus), leukocytes are usually dominant. A collection of cornified cells with a few nucleated epithelia is seen. In the fourth period (diestrus), leukocytes increase (Fig. 3 ). 3.3. Effects of beta-glucan on hormone levels Reproductive hormone levels were determined to examine the relationship between βTG and PCOS further. AMH has the feature of inhibiting follicle formation and causes androgen production indirectly by inhibiting FSH activity or by blocking aromatase activity. Thus, the concentration of AMH in the serum is proportional to the number of antral follicles and menstrual disorders. AMH levels are high in women with PCOS. Serum levels of AMH, T, and LH were significantly higher in the PCOS group than in the control group. The increased levels of LH, testosterone, and androstenedione in the serum, yet low or normal FSH levels and abnormal estrogen secretion, have created an endocrine profile accepted by many researchers in PCOS. In this study, we found a significant decrease in serum levels of FSH in the PCOS group compared to the control group. The PCOS group significantly increased AMH, LH, and T serum levels. Serum levels of FSH were found to be substantially reduced in the PCOS group compared with the P + βTG and βTG groups. The prevalent PCOS symptoms of hyperandrogenism and excessive LH production are signs that the PCOS model was developed successfully in the current study. In PCOS rats, treatment with βTG dramatically decreased the LH, AMH, and T blood levels, demonstrating that βTG could raise the levels of PCOS' reproductive hormones (Fig. 4 ). These findings suggested that βTG might have a correcting impact on a female's reproductive endocrine. 3.4. Effects of beta-glucan on cytokines It was discovered how βTG affected the expression of inflammatory cytokines (Figure. 5). 3.5. Biochemical results In Table 2, the biochemical outcomes were displayed. SOD activity and TBARS levels between the control and βTG groups, as well as between the PCOS + βTG groups, were not significantly different. TBARS was considerably lower in the control group compared to the PCOS group. In comparison to the PCOS group, SOD activity was noticeably higher in the control group. The control, PCOS + βTG, and βTG groups all had considerably higher GSH and CAT levels and GPx activity than the PCOS group (Fig. 6 ). 3.6. Histopathological results Table 3 provides the histopathologic damage score. The treatment groups' ovarian morphologies varied significantly from one another. Table 3 The difference in ovarian tissue's histological grade between the groups. Groups Histopathological score (Mean ± SE) Control 0.30 ± 0.07 a PCOS 2.04 ± 0.12 b PCOS + βTG 1.20 ± 0.08 c βTG 0.77 ± 0.06 a The treatment groups' ovarian morphologies varied significantly. The histological appearance of the ovaries in the control (Fig. 7 A, 7 B) and βTG (Fig. 7 C, 7 D) groups was normal. In the control and βTG groups, various follicle types and corpus luteum were seen in the ovary with a normal histological appearance. Secondary follicles (Fig. 7 A, 7 C) and multilaminate primary follicles (Fig. 6 B, 6 D) were observed in the control and βTG groups. Atretic follicles were found (thick black arrows) (Fig. 8 A, 8 C, 8 D), vascular congestion (black asterisks) (8A, 8B, 8C, 8D), mononuclear cell infiltration (white asterisks) (Fig. 8 B, 8 D), vacuolization (Fig. 8 D, 8 E), edema (Fig. 8 D), hemorrhage in corpus luteum (Fig. 8 E) were observed. As opposed to the PCOS group, the PCOS + βTG group had significantly worse ovarian follicle shape, fewer mononuclear cell infiltration, atretic follicles, vacuolization, vascular congestion, edema, and bleeding. Different secondary follicles (white arrows) (Fig. 9 A, Fig. 9 B) were observed in the PCOS + βTG group. 3.7. Immunohistochemistry results Figure 10 shows the immunohistochemistry expression of caspase-3. The PCOS + βTG group had fewer stained (positive) cells than the PCOS group. 3.8. Protein array results Protein profiles of rat growth factor, cytokine, and chemokine arrays in control, PCOS, P + βTG, and βTG groups were analyzed (Fig. 11 ). Cluster analysis of all proteins revealed that the controls, PCOS, P + βTG and βTG clustered together (Fig. 11 A). Principal component analysis showed a separate distribution for PCOS and all the other experimental groups (PC1: 50.7% and PC2: 26.4%) (Fig. 11 B). Correlation coefficient analysis was used to demonstrate the beneficial activity of βTG. Compared to the control, the stand-alone βTG application exhibited correlation coefficient values of 0.72 and 0.75, respectively (Fig. 11 C). The chemokine profile demonstrated a separation of PCOS from other experimental groups (Fig. 12 A). Similar outcomes for principal component analysis were attained (PC1: 59% and PC2: 39.1%) (Fig. 12 B). Control and P + βTG had a 0.71 correlation coefficient value (Fig. 12 C), confirming the cluster and PCA analysis. P + βTG was separated from all experimental groups according to the cluster analysis in the growth factor array (Fig. 13 A). Similar results (PC1: 92.2% and PC2: 5.7%) were attained (Fig. 13 B). Correlation coefficient analysis demonstrated the beneficial effect of βTG. Stand-alone P + βTG and βTG had a 0.78 correlation coefficient value (Fig. 13 C). In the cytokine 1 array analysis, the groups’ control, PCOS, P + βTG, and βTG were all grouped (Fig. 14 A). Similar outcomes for principal component analysis were attained (PC1: 71.5% and PC2: 18.4%) (Fig. 14 B). Control and P + βTG had a 0.84 correlation coefficient value (Fig. 14 C). PCOS group was separated from all experimental groups in the cytokine 2 array (Fig. 15 A, 15 B). Control was clustered separately (PC1: 66.7% and PC2: 22.7%) (Fig. 15 B). According to correlation coefficient analysis, experimental groups did not correlate (Fig. 15 C). βTG adminisration increased bNGF, TIMP-1, TNF-α, CINC-1, BDNF and bFGF leves in ovary tissues. βTG treatment decreased CTACK, LIX, and PDGF-AA (Fig. 16 ). 4. DISCUSSION Oxidative stress and changes in antioxidant capacity cause toxicity in many organ systems. 28 Oxidative stress, which is involved in many pathologies, also plays a role in initiating and exacerbating the pathological process of PCOS. 29 Increased antioxidant enzyme activity, including catalase, glutathione peroxidase, superoxide dismutase, and glutathione, may help prevent oxidative stress in PCOS. The decrease in antioxidants such as SOD, CAT, GSH, and GPx may affect the physiological processes of the ovary, such as ovulation and egg development. 30 Antioxidant enzyme activities, such as CAT and GPx, are detected at increased levels in growing follicles to preserve ovarian function. 31 Increased ROS levels in women with PCOS are accompanied by low GSH levels. 32 In the ovaries of PCOS patients, GSH levels, CAT, SOD, and GPx are found in the ovaries. A decrease in endogenous antioxidant levels is observed due to oxidative stress triggered by PCOS. 33 However, some researchers have observed different results. In the study of Nawrocka-Rutkowska et al., while MDA levels, an indicator of oxidative stress, were found to be high in women with PCOS, CAT levels were also found to be high. 34 While Sabuncu et al. 35 found SOD levels higher in women with PCOS than in the control group. On the other hand, Zhang et al. found low SOD levels in PCOS. 36 Luteinizing hormone and FSH are secreted by the pituitary gland and promote ovulation. While LH levels increase in patients with PCOS, FSH levels decrease. 37 Contrary to previous studies 38 , 39 , in our study, we observed a significant decrease in serum FSH levels and an increase in LH levels in rats with the PCOS model. We observed elevated serum levels of AMH and T in PCOS rats. Specifically, we found that serum sex hormone levels, including T, AMH, LH, and FSH, were similar in the βTG-treated group to those in the control group. These results emphasized that βTG positively affects hormones by normalizing the changes in serum sex hormone levels in PCOS. Administration of βTG brought the protein profile of the target tissue closer to the control samples. Standalone βTG or βTG + PCOS groups exerted a similar protein profile compared to the baseline in cluster analysis. In addition, βTG induced the expression of some essential proteins, including bNGF, TIMP-1, Agrin, CINC-1, BDNF, and FGF-2 (bFGF). A previously published study observed decreased expression of TIMP-1 PCOS patients 40 TIMP-1 increase in response to βTG supplementation might be related to the potential therapeutic activity of βTG. In a previously published report, FGF-2 injection had protective effects in a mouse model of PCOS 41 , which is consistent with our results that βTG increased the FGF-2 expression. Although there was no significant relation between PCOS and CTACK (cutaneous T-cell attracting chemokine), according to the literature,42 βTG decreased the CTACK, an inflammatory regulator. Suppression of inflammation by βTG might contribute to the treatment process of PCOS. 5. CONCLUSION Taken together, all the results of this study showed that treatment with βTG stabilizes serum sex hormones and normalizes the abnormal oestrus cycles that are a hallmark of PCOS. These results suggest that βTG has a protective role in PCOS. In our literature review, we came across several studies that tested the effect of natural compounds in treating PCOS. However, we have not previously come across a study investigating the relationship between βTG and PCOS. In this respect, our study will be the first to investigate the effects of βTG on PCOS. Declarations CONFLICT OF INTEREST The authors declare no conflict of interest. FUNDING The İnönü University Department of Scientific Research Projects funded this study (Project number: TCD-2020-2090). Author Contribution N.B.T, M.A., O.Ç. conceptualized and designed. A.T., H.Y., S.Ş., D.A.Ö., S.A. carried out the experiments. A.D. examined the information. S.Ü. wrote the manuscript. References Leon LIR, Anastasopoulou C, Mayrin JV. In StatPearls. StatPearls Publishing; 2022. Polycystic Ovarian Disease. Aktay G, Gürsoy ŞÖ, Uyumlu U, Ünüvar S, İlhan N. Protective effect of atorvastatin on oxidative stress in streptozotocin-induced diabetic rats independently their lipid-lowering effects. J Biochem Mol Toxicol. 2019;33(5):e22295. 10.1002/jbt.22295 . GÜRSOY Ş, ceritli m, ÜNÜVAR, S., AKTAY G. Effects of Captopril on Cell Damage and Liver Damage. Volume 40. Türkiye Klinikleri Tip Bilimleri Dergisi; 2020. pp. 320–5. ÜNÜVAR S, Gursoy Ş, Berk A, Kaymaz B, Ilhan N, AKTAY G. Antioxidant Effect of a Dihydropyridine Calcium Antagonist Nitrendipine in Streptozotocin-Induced Diabetes. Volume 57. JOURNAL OF EVOLUTIONARY BIOCHEMISTRY AND PHYSIOLOGY; 2020. pp. 126–33. 1. Türkmen NB, Yüce H, Şahin Y, Taşlıdere AÇ, Özek DA, Ünüvar S, Çiftçi O. Protective effect of resveratrol against pembrolizumab-induced hepatotoxicity and neurotoxicity in male rats. J Biochem Mol Toxicol. 2023;37(3):e23263. Mohammadi M. Oxidative Stress and Polycystic Ovary Syndrome: A Brief Review. Int J Prev Med. 2019;10:86. Bonakdaran S, Khorasani ZM, Davachi B, Khorasani JM. The effects of calcitriol on improvement of insulin resistance, ovulation and comparison with metformin therapy in PCOS patients: a randomized placebo-controlled clinical trial. Iran J Reprod Med. 2012;10(5):465. Durlinger AL, Gruijters MJ, Kramer P, et al. Anti-Müllerian hormone attenuates the effects of FSH on follicle development in the mouse ovary. Endocrinology. 2001;142(11):4891–9. Deligeoroglou E, Vrachnis N, Athanasopoulos N, et al. Mediators of chronic inflammation in polycystic ovarian syndrome. Gynecol Endocrinol. 2012;28(12):974–8. Xia YH, Yao L, Zhang ZX. Correlation between IL-1β, IL-1Ra gene polymorphism and occurrence of polycystic ovary syndrome infertility. Asian Pac J Trop Med. 2013;6(3):232–6. Deshpande RR, Chang MY, Chapman JC, Michael SD. Alteration of cytokine production in follicular cystic ovaries induced in mice by neonatal estradiol injection. Am J Reprod Immunol. 2000;44(2):80–8. Eyol E, Tanriverdi Z, Karakus F, Yilmaz K, Unuvar S. Synergistic anti-proliferative effects of cucurbitacin i and irinotecan onhuman colorectal cancer cell lines. Clin Exp Pharmacol. 2016;6(5):1000219. Yıilmaz K, Karakuş F, Eyol E, Tosun E, Yıilmaz İ, Ünüvar S. Cytotoxic Effects of Cucurbitacin I and Ecballium elaterium on Breast Cancer Cells. Nat Prod Commun. 2018;13(11):1445–8. Karakuş F, Yilmaz K, Eyol E, ÜNÜVAR S. (2019). Combination of 2 Bioactive Compounds for Treatment of Breast Cancer: Triterpenoid Cucurbitacin I and Phenolic CAPE. NATURAL PRODUCT COMMUNICATIONS, vol.14, no.6. Yüce H, Şahin Y, Türkmen NB, et al. Apoptotic, Cytotoxic and Antimigratory Activities of Phenolic Compounds. J Evol Biochem Phys. 2022;58:1819–33. Mirończuk-Chodakowska I, Kujawowicz K, Witkowska AM. Beta-Glucans from Fungi: Biological and Health-Promoting Potential in the COVID-19 Pandemic Era. Nutrients. 2021;13(11):3960. Türkmen NB, Yüce H, Aydın M, Taşlıdere A, Doğan A, Özek DA, Hayal TB, Yaşar Ş, Çiftçi O, Ünüvar S. Nerolidol attenuates dehydroepiandrosterone-induced polycystic ovary syndrome in rats by regulating oxidative stress and decreasing apoptosis. Life Sci. 2023;315:121380. Roy S, Mahesh VB, Greenblatt RB. Effect of dehydroepiandrosterone and delta 4-androstenedione on the reproductive organs of female rats: production of cystic changes in the ovary. Nature. 1962;196:42–3. Marcondes FK, Bianchi FJ, Tanno AP. Determination of the estrous cycle phases of rats: some helpful considerations. Braz J Biol. 2002;62(4):609–14. Yagi K. Simple assay for the level of total lipid peroxides in serum or plasma. Methods Mol Biol. 1998;108:101–6. Sun Y, Oberley LW, Li Y. A simple method for clinical assay of superoxide dismutase. Clin Chem. 1988;34(3):497–500. Aebi H. Catalase in vitro. Methods Enzymol. 1984;105:121–6. Sedlak J, Lindsay RH. Estimation of total, protein-bound, and nonprotein sulfhydryl groups in tissue with Ellman's reagent. Anal Biochem 1968;25(1):192–205. Paglia DE, Valentine WN. Studies on the quantitative and qualitative characterization of erythrocyte glutathione peroxidase. J Lab Clin Med. 1967;70(1):158–69. Türkmen NB, Yüce H, Taşlıdere A, Şahin Y, Ayhan İ. 18β-glycyrrhetinic acid attenuates global cerebral ischemia/reperfusion-induced cardiac damage in C57BL/J6 mice. Braz J Pharm Sci [Internet]. 2022;58:e21219. Şenkal S, Hayal TB, Sağraç D, et al. Human ESC-derived Neuromesodermal Progenitors (NMPs) Successfully Differentiate into Mesenchymal Stem Cells (MSCs). Stem Cell Rev Rep. 2022;18(1):278–93. Metsalu T, Vilo J. ClustVis: a web tool for visualizing clustering of multivariate data using Principal Component Analysis and heatmap. Nucleic Acids Res. 2015;43(1):566–70. Türkmen NB, Yüce H, Şahin Y, Taşlıdere AÇ, Özek DA, Ünüvar S, Çiftçi O. Protective effect of resveratrol against pembrolizumab-induced hepatotoxicity and neurotoxicity in male rats. J Biochem Mol Toxicol. 2023;37(3):e23263. 10.1002/jbt.23263 . Murri M, Luque-Ramírez M, Insenser M, Ojeda-Ojeda M, Escobar-Morreale HF. Circulating markers of oxidative stress and polycystic ovary syndrome (PCOS): a systematic review and meta-analysis. Hum Reprod Update. 2013;19(3):268–88. Lu J, Wang Z, Cao J, Chen Y, Dong Y. A novel and compact review on the role of oxidative stress in female reproduction. Reprod Biol Endocrinol. 2018;16(1):80. Sun Y, Li S, Liu H, et al. Association of GPx1 P198L and CAT C-262T Genetic Variations With Polycystic Ovary Syndrome in Chinese Women. Front Endocrinol (Lausanne). 2019;10:771. Donà G, Sabbadin C, Fiore C, et al. Inositol administration reduces oxidative stress in erythrocytes of patients with polycystic ovary syndrome. Eur J Endocrinol. 2012;166(4):703–10. Hong Y, Yin Y, Tan Y, Hong K, Zhou H. The Flavanone, Naringenin, Modifies Antioxidant and Steroidogenic Enzyme Activity in a Rat Model of Letrozole-Induced Polycystic Ovary Syndrome. Med Sci Monit. 2019;25:395–401. Nawrocka-Rutkowska J, Szydłowska I, Jakubowska K, et al. Assessment of the Parameters of Oxidative Stress Depending on the Metabolic and Anthropometric Status Indicators in Women with PCOS. Life (Basel). 2022;12(2):225. Sabuncu T, Vural H, Harma M, Harma M. Oxidative stress in polycystic ovary syndrome and its contribution to the risk of cardiovascular disease. Clin Biochem. 2001;34(5):407–13. Zhang D, Luo WY, Liao H, Wang CF, Sun Y. Sichuan Da Xue Xue Bao Yi Xue Ban. 2008;39(3):421–3. Laganà AS, Garzon S, Casarin J, Franchi M, Ghezzi F. Inositol in Polycystic Ovary Syndrome: Restoring Fertility through a Pathophysiology-Based Approach. Trends Endocrinol Metab. 2018;29(11):768–80. Qiu Z, Dong J, Xue C, et al. Liuwei Dihuang Pills alleviate the polycystic ovary syndrome with improved insulin sensitivity through PI3K/Akt signaling pathway. J Ethnopharmacol. 2020;250:111965. Onalan G, Pabuçcu R, Goktolga U, Ceyhan T, Bagis T, Cincik M. Metformin treatment in patients with polycystic ovary syndrome undergoing in vitro fertilization: a prospective randomized trial. Fertil Steril. 2005;84(3):798–801. Lahav-Baratz S, Kraiem Z, Shiloh H, Koifman M, Ishai D, Dirnfeld M. Decreased expression of tissue inhibitor of matrix metalloproteinases in follicular fluid from women with polycystic ovaries compared with normally ovulating patients undergoing in vitro fertilization. Fertil Steril. 2003;79(3):567–71. Moayeri A, Rostamzadeh A, Raoofi A, Rezaie MJ, Abasian Z, Ahmadi R. Retinoic acid and fibroblast growth factor-2 play a crucial role on modulation of sex hormones and apoptosis in a mouse model of polycystic ovary syndrome induced by estradiol valerate. Taiwan J Obstet Gynecol. 2020;59(6):882–90. Chen H, Zhang Y, Li S, et al. The Association Between Genetically Predicted Systemic Inflammatory Regulators and Polycystic Ovary Syndrome: A Mendelian Randomization Study. Front Endocrinol (Lausanne). 2021;12:731569. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 02 Aug, 2024 Reviews received at journal 02 Aug, 2024 Reviews received at journal 30 Jul, 2024 Reviewers agreed at journal 26 Jul, 2024 Reviews received at journal 06 Jun, 2024 Reviewers agreed at journal 03 Jun, 2024 Reviewers agreed at journal 02 Jun, 2024 Reviewers invited by journal 02 Jun, 2024 Editor assigned by journal 21 May, 2024 Submission checks completed at journal 21 May, 2024 First submitted to journal 20 May, 2024 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. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-4448893","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":309714144,"identity":"31a0706e-f714-4a3a-851d-15d211c13a6b","order_by":0,"name":"Hande Yüce","email":"","orcid":"","institution":"İnönü University Malatya","correspondingAuthor":false,"prefix":"","firstName":"Hande","middleName":"","lastName":"Yüce","suffix":""},{"id":309714145,"identity":"478c6a10-c47e-4190-b122-531ff6dddecb","order_by":1,"name":"Neşe Başak Türkmen","email":"","orcid":"","institution":"İnönü University Malatya","correspondingAuthor":false,"prefix":"","firstName":"Neşe","middleName":"Başak","lastName":"Türkmen","suffix":""},{"id":309714146,"identity":"94b9d4b9-07ba-4ce4-9f75-4ae41317b32b","order_by":2,"name":"Muhterem Aydın","email":"","orcid":"","institution":"Fırat University","correspondingAuthor":false,"prefix":"","firstName":"Muhterem","middleName":"","lastName":"Aydın","suffix":""},{"id":309714147,"identity":"2fbce3a3-d70f-4e8a-9dd5-fb5061367fb4","order_by":3,"name":"Aslı Taşlıdere","email":"","orcid":"","institution":"İnönü University","correspondingAuthor":false,"prefix":"","firstName":"Aslı","middleName":"","lastName":"Taşlıdere","suffix":""},{"id":309714148,"identity":"e51c34ee-c2da-4bb1-8553-9dd83f76fae0","order_by":4,"name":"Dilan Aşkın Özek","email":"","orcid":"","institution":"İnönü University Malatya","correspondingAuthor":false,"prefix":"","firstName":"Dilan","middleName":"Aşkın","lastName":"Özek","suffix":""},{"id":309714149,"identity":"293e2dbe-2c9f-4002-9a7b-43aea7f8803b","order_by":5,"name":"Selinay Şenkal","email":"","orcid":"","institution":"Yeditepe University","correspondingAuthor":false,"prefix":"","firstName":"Selinay","middleName":"","lastName":"Şenkal","suffix":""},{"id":309714150,"identity":"48fc7b8c-73b5-4a07-b539-b813ee819d90","order_by":6,"name":"Sümeyye Aslan","email":"","orcid":"","institution":"İnönü University Malatya","correspondingAuthor":false,"prefix":"","firstName":"Sümeyye","middleName":"","lastName":"Aslan","suffix":""},{"id":309714151,"identity":"1d5ee2eb-abe8-44be-b5bb-0ef629c9a1f7","order_by":7,"name":"Ayşegül Doğan","email":"","orcid":"","institution":"Yeditepe University","correspondingAuthor":false,"prefix":"","firstName":"Ayşegül","middleName":"","lastName":"Doğan","suffix":""},{"id":309714152,"identity":"7735e663-f307-429d-a7c5-d2db02610581","order_by":8,"name":"Osman Çiftçi","email":"","orcid":"","institution":"Pamukkale University","correspondingAuthor":false,"prefix":"","firstName":"Osman","middleName":"","lastName":"Çiftçi","suffix":""},{"id":309714154,"identity":"1a21dfa2-fe66-4959-aaef-44f57b7072c0","order_by":9,"name":"Songül Ünüvar","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA/ElEQVRIiWNgGAWjYNCCAhijQgLKYCOkxQDGOIOkhYcoLYxtMBE8WuTbew+/5jFgSNwukWP4uXKeRR6/dI8Bw4eywwz20gewm3/mXJo1SMvOGTnGkme3SRRLzjljwDjj3GEGHr4E7FokcsyMQVo23EhLkGzcJgFk5Bgw87YBteBwmfwMhJbkn41zoFr+4tHCcCPH+DFES/IxycYGqBZGPFoMzpwxY5xjIGG84czjY5YNxyQSZ85IKzjYcy6dh+cMrhDrMf7wpsJGdsPxxOabDTV1if0SyRsf/CizlmPvweEwYBQAo08CVegAA/6YZP6AR3IUjIJRMApGAQMDADXPV5BbTxYpAAAAAElFTkSuQmCC","orcid":"","institution":"İnönü University Malatya","correspondingAuthor":true,"prefix":"","firstName":"Songül","middleName":"","lastName":"Ünüvar","suffix":""}],"badges":[],"createdAt":"2024-05-20 11:42:44","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4448893/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4448893/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":57632235,"identity":"2d0e3577-5cf4-46cc-9812-ae87b3bda120","added_by":"auto","created_at":"2024-06-03 15:10:07","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":316948,"visible":true,"origin":"","legend":"\u003cp\u003eBody (A), ovary (B), and uterus weights (C) in PCOS rats.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4448893/v1/c217d7c45b181c64dcd5c34e.png"},{"id":57633638,"identity":"14f7d4a2-fcaa-412b-9f2a-7e4c2f59875f","added_by":"auto","created_at":"2024-06-03 15:26:07","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":213690,"visible":true,"origin":"","legend":"\u003cp\u003eGiems stained estrous cycle (Scale bars = 10 μm).\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-4448893/v1/5f8a9fe2c4c5fd6bde081c49.png"},{"id":57632884,"identity":"eba4ebfc-35a1-44f5-8080-e230bb1e64fb","added_by":"auto","created_at":"2024-06-03 15:18:07","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":310782,"visible":true,"origin":"","legend":"\u003cp\u003eEstrous cycle in the control group (A), PCOS group (B), P+βTG group (C), and βTG (D) group.\u003c/p\u003e","description":"","filename":"3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4448893/v1/276a526048674cc50a0308bd.jpeg"},{"id":57632239,"identity":"f5154e45-d5bc-491f-b7cb-23e836cbbf7f","added_by":"auto","created_at":"2024-06-03 15:10:07","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":85797,"visible":true,"origin":"","legend":"\u003cp\u003ePCOS, polycystic ovarian syndrome; βTG, beta-glucan; AMH, antimullerian hormone; mUI/ml each; FSH, luteinizing hormone; mUI/ml each; T, testosterone; ng/cl each. Each value was presented as the mean SEM (n=8). Using one-way ANOVA and Tukey's test as a post-hoc analysis, the probability value is p0.05. The results were significant for the control group (*) and the PCOS group (#), respectively.\u003c/p\u003e","description":"","filename":"4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4448893/v1/5525d25ab07a89c02bb027e5.jpeg"},{"id":57632886,"identity":"f881acf1-4153-4536-a891-57f423c7c662","added_by":"auto","created_at":"2024-06-03 15:18:07","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":74362,"visible":true,"origin":"","legend":"\u003cp\u003ePCOS: polycystic ovarian syndrome; βTG: beta-glucan; IL-1: interleukin 1 beta; IL-6: interleukin 6; TNF-: tumor necrosis factor-alpha; pg/ml, g/dl, ng/L. Each value was presented as the mean SEM (n=8). Using one-way ANOVA and Tukey's test as a post-hoc analysis, the probability value is p0.05. Significant for the control group (*) and the PCOS group (#), respectively.\u003c/p\u003e","description":"","filename":"5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4448893/v1/147c9d90946a743c7e9d84b9.jpeg"},{"id":57632233,"identity":"0d21e0e8-ac09-4916-83df-b7630a6bc071","added_by":"auto","created_at":"2024-06-03 15:10:07","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":135722,"visible":true,"origin":"","legend":"\u003cp\u003eEach value was presented as the mean SEM (n=8). Using one-way ANOVA and Tukey's test as a post-hoc analysis, the probability value is 0.05. (βTG: Beta-glucan, CAT: Catalase GSH: Glutathione GPx: Glutathione peroxidase PCOS: Polycystic ovary syndrome, SOD: Superoxide dismutase, TBARS: Thiobarbituric acid reactive substances, SEM: Standard error of the mean).\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-4448893/v1/ebd442342e7998282d3e0a72.png"},{"id":57633639,"identity":"f8babd2a-182c-4013-a43c-a0ca41bfe0f6","added_by":"auto","created_at":"2024-06-03 15:26:07","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1110152,"visible":true,"origin":"","legend":"\u003cp\u003eThe ovaries of the Control and βTG groups displayed normal histological appearance. The ovary's corpus luteum and many follicle types were visible. In the control and βTG groups, secondary follicles (A, C) and multilaminar primary follicles (B, D) were seen. A, C: H-E; X20; B, D: H-E; X40.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-4448893/v1/84c0da68dee8fdcbecb6ed31.png"},{"id":57632243,"identity":"6e385e63-9732-48b8-a3a1-094ced57eff8","added_by":"auto","created_at":"2024-06-03 15:10:07","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":1476134,"visible":true,"origin":"","legend":"\u003cp\u003eAtretic follicles (thick black arrows) were found in the PCOS group, along with vascular obstruction (black asterisks) in the A, B, C, and D, mononuclear cell infiltration (white asterisks) in the B, D, vacuolization in the D, and hemorrhage in the D. A, D: H-E; X20, B, C: H-E; X10, E: H-E; X40.\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-4448893/v1/a57aa5ce4fa1fd2cb7128373.png"},{"id":57632249,"identity":"b0f34839-6219-4d21-bc2b-837698253bd7","added_by":"auto","created_at":"2024-06-03 15:10:08","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":567933,"visible":true,"origin":"","legend":"\u003cp\u003eDifferent secondary follicles (white arrows) (A, B) were observed in the PCOS+βTG group. A: H-E; X20, B: H-E; X40.\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-4448893/v1/7b72ed92c70831f8d275d6f9.png"},{"id":57633640,"identity":"15bbc55b-e505-4607-8e4f-21f48e2bfcfd","added_by":"auto","created_at":"2024-06-03 15:26:07","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":2273077,"visible":true,"origin":"","legend":"\u003cp\u003eCaspase-3 immunohistochemical expression in the control, PCOS, PCOS+βTG, and βTG groups (A, B, C, and D). A, B, C, D: Caspase-3; X20.\u003c/p\u003e","description":"","filename":"10.png","url":"https://assets-eu.researchsquare.com/files/rs-4448893/v1/ae3a4da2ff6482b1e15401ad.png"},{"id":57632247,"identity":"7e15a929-02e8-4770-a93e-c1df8f740212","added_by":"auto","created_at":"2024-06-03 15:10:08","extension":"png","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":77971,"visible":true,"origin":"","legend":"\u003cp\u003eOvarian tissue's overall protein composition profile. Experimental sample representation using heat maps and hierarchical clustering (A). Analyses of the principal components (B) (PCA). Calculations of correlation coefficients (C).\u003c/p\u003e","description":"","filename":"11.png","url":"https://assets-eu.researchsquare.com/files/rs-4448893/v1/dd4a894448fba62bfbcac579.png"},{"id":57632890,"identity":"72e8320c-ebf2-42b5-a14b-e167c75a5fee","added_by":"auto","created_at":"2024-06-03 15:18:08","extension":"png","order_by":12,"title":"Figure 12","display":"","copyAsset":false,"role":"figure","size":79295,"visible":true,"origin":"","legend":"\u003cp\u003eOvarian tissue chemokine array profile. Experimental sample representation using heat maps and hierarchical clustering (A). Analyses of the principal components (B) (PCA). Calculations of correlation coefficients (C).\u003c/p\u003e","description":"","filename":"12.png","url":"https://assets-eu.researchsquare.com/files/rs-4448893/v1/4576b6304a373857473b82eb.png"},{"id":57632246,"identity":"8dd826fd-fe41-4a1d-86ac-0dd71f20507a","added_by":"auto","created_at":"2024-06-03 15:10:08","extension":"png","order_by":13,"title":"Figure 13","display":"","copyAsset":false,"role":"figure","size":59205,"visible":true,"origin":"","legend":"\u003cp\u003eProfile of growth factors in ovarian tissue. Experimental sample representation using heat maps and hierarchical clustering (A). Analyses of the principal components (B) (PCA). Calculations of correlation coefficients (C).\u003c/p\u003e","description":"","filename":"13.png","url":"https://assets-eu.researchsquare.com/files/rs-4448893/v1/5491cff876e2d980d9a21f4a.png"},{"id":57632888,"identity":"1f547956-9e38-44b7-9e56-975184670e84","added_by":"auto","created_at":"2024-06-03 15:18:07","extension":"png","order_by":14,"title":"Figure 14","display":"","copyAsset":false,"role":"figure","size":75101,"visible":true,"origin":"","legend":"\u003cp\u003eOvarian tissue cytokine 1 array profile. Experimental sample representation using heat maps and hierarchical clustering (A). Analyses of the principal components (B) (PCA). Calculations of correlation coefficients (C).\u003c/p\u003e","description":"","filename":"14.png","url":"https://assets-eu.researchsquare.com/files/rs-4448893/v1/e6cb262dab2f45a43c9d7a84.png"},{"id":57632245,"identity":"42f8a12b-6c48-4b11-be4a-4fb1195a2cbb","added_by":"auto","created_at":"2024-06-03 15:10:08","extension":"png","order_by":15,"title":"Figure 15","display":"","copyAsset":false,"role":"figure","size":83083,"visible":true,"origin":"","legend":"\u003cp\u003eOvarian tissue cytokine 2 array profile. Experimental sample representation using heat maps and hierarchical clustering (A). Analyses of the principal components (B) (PCA). Calculations of correlation coefficients (C).\u003c/p\u003e","description":"","filename":"15.png","url":"https://assets-eu.researchsquare.com/files/rs-4448893/v1/f4f5c9bb857c990969255b06.png"},{"id":57632240,"identity":"4500fe85-f222-4919-a124-c8d862e63bc6","added_by":"auto","created_at":"2024-06-03 15:10:07","extension":"png","order_by":16,"title":"Figure 16","display":"","copyAsset":false,"role":"figure","size":532767,"visible":true,"origin":"","legend":"\u003cp\u003eCytokine Array 1, Cytokine Array 2, Chemokine Array, and Growth Factor Array analysis.\u003c/p\u003e","description":"","filename":"16.png","url":"https://assets-eu.researchsquare.com/files/rs-4448893/v1/199b97379f85ce8312a632f8.png"},{"id":57634086,"identity":"ba532d16-5ec5-4053-9142-76db87242b07","added_by":"auto","created_at":"2024-06-03 15:34:11","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":7886430,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4448893/v1/b2f90ac1-d1c2-4193-8a1d-dc9d1998dae6.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Effect of beta-glucan on oxidative stress, inflammation, hormonal and histopathological changes in dehydroepiandrosterone-induced polycystic ovary syndrome","fulltext":[{"header":"1. INTRODUCTION","content":"\u003cp\u003ePolycystic ovary syndrome (PCOS), whose incidence has increased in recent years, is a gynecological disease and also an endocrine disease that negatively affects fertility and paves the way for the formation of many different diseases. Polycystic ovary, which is common, especially among women of reproductive age, causes infertility by causing ovulation disorder. In addition, if untreated, it progresses and leads to many secondary diseases, such as hypertension and cardiovascular diseases.\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e Objective of PCOS treatment: reducing the effects of androgen-related symptoms, treatment of menstrual cycle and related irregularities, and infertility treatment. Symptoms and severity differ in each woman. Therefore, treatment methods are determined individually for each patient. It is now widely acknowledged that oxidative stress plays a crucial part in the pathogenesis of numerous illnesses,\u003csup\u003e\u003cspan additionalcitationids=\"CR3\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e including PCOS. Although the production and spread of intracellular reactive oxygen species (ROS) are tightly regulated by extremely sophisticated antioxidant enzymatic and non-enzymatic systems,\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e understanding oxidative stress mechanisms is crucial for developing PCOS prevention and treatment strategies.\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003ePCOS presents diagnostic difficulties due to the incomplete explanation of its etiology and phenotypic diversity. The diagnosis of PCOS is usually based on subjective criteria such as clinical and ultrasonographic findings. First of all, the fact that AMH measurements used to determine ovarian reserve are higher than usual in hyperandrogenism cases has attracted attention, leading to the thought that AMH measurements may have a place in the diagnosis of PCOS. AMH is inhibitory in folliculogenesis, explaining the cause of PCOS anovulation. This is the key to high AMH in PCOS. It is natural for women with PCOS to have an increase in LH, and this is in parallel with the AMH level. However, unlike LH, AMH level indicates increased antral follicle number and has diagnostic value in PCOS.\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e Recent research has demonstrated a link between PCOS incidence and low-grade chronic inflammation.\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e Proinflammatory cytokines such as interleukin-6 (IL-6) and IL-1, as well as tumor necrosis factor-alpha (TNF-α), are crucial mediators indicating the existence of inflammation.\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e TNF-α, IL-6, and IL-1 are important in reproductive physiology. Events such as ovarian steroid production, fertilization, implantation, follicular maturation, and ovulation are all affected in women with PCOS.\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eThe protective effects of natural compounds on diseases have been the subject of many studies.\u003csup\u003e\u003cspan additionalcitationids=\"CR13 CR14\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e (Beta-glucans (βTGs) are a class of polysaccharides derived from numerous species of fungi, mushrooms, and cereal grains. They have various pharmacological effects, including antioxidant, antibacterial, antitumor, free radical scavenging, anti-lipid, immunomodulation, antifungal, and radioprotective activities.\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e Previous research has shown that βTG has various pharmacological effects, but the impact of βTG on a rat PCOS model has not been examined. First, to the best of our knowledge, it is the first study to assess the regressions of oxidative stress, inflammation, sexual cycles, and histopathological damage due to βTG treatment in PCOS. In our previous study, we found that PCOS is not only related to reproductive pathology but also a systemic complication whose etiopathogenesis has not yet been fully elucidated and that nerolidol is effective in controlling the biochemical, apoptotic, histopathological, and metabolic changes in PCOS.\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e"},{"header":"2. MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Drugs and chemicals\u003c/h2\u003e \u003cp\u003eWe purchased beta-glucan from Sigma (9012-72-0; St. Louis, MO) and dehydroepiandrosterone (DHEA) from Avanti, Polar Lipids, INC. in the United States (Cas no: 53-43-0). All chemicals that we used for the biochemical, histological, and immunohistochemical analyses were provided by Sigma. The Millipore Autopure WR600A system was used to purify the water used to make the solutions (Millipore, Ltd., Burlington, MA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Animals and experimental protocol\u003c/h2\u003e \u003cp\u003eThirty-two female Sprague-Dawley rats (aged 21 days) were used in the study, which the Experimental Animals Institute at İn\u0026ouml;n\u0026uuml; University conducted. The study was approved by the Institutional Animal Ethics Committee of the same university (Approval no. 2020/2\u0026ndash;8). A 12-hour light/12-hour dark cycle, 40\u0026ndash;60% relative humidity, and an ambient temperature of 20\u0026ndash;24\u0026deg;C were all maintained for all animals in a well-ventilated setting.\u003c/p\u003e \u003cp\u003eFour experimental groups of eight SD pre-pubertal rats each were randomly assigned. Control group: sesame oil per day was injected subcutaneously at 0.2 ml for 21 days. PCOS group: 60 mg/kg/day DHEA diluted with 0.2 ml sesame oil was injected subcutaneously for 21 days.\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e P\u0026thinsp;+\u0026thinsp;βTG group: 60 mg/kg/day DHEA was injected subcutaneously for 21 days, and βTG was given orally at 50 mg/kg/day 22 days later for 21 days. βTG group: Rats were treated orally with βTG (50 mg/kg/day) dissolved in 0.2 ml sesame oil for 21 days. At the end of the treatment period, ketamine/xylazine anesthesia was administered to all rats.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Estrous cycles, ovarian, uterus, and body weights\u003c/h2\u003e \u003cp\u003eThe body weights of the rats were weighed every day during the experiment. After sacrification, the ovarian and uterine tissues of the rats were weighed. Three different types of vaginal epithelial cells controlled the estrous cycle.\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e Every day between 9:00 and 10:00, vaginal smears were performed. The method of Giemsa staining was employed to analyze cell morphology.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Biochemical analysis\u003c/h2\u003e \u003cp\u003eAnti-mullerian hormone (AMH), follicle-stimulating hormone (FSH), testosterone (T), luteinizing hormone (LH), tumor necrosis factor-alpha (TNF-alpha), interleukin-1 beta (IL-1 beta), and interleukin-6 (IL-6) were quantified using commercially available ELISA kits.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Determination of oxidative stress\u003c/h2\u003e \u003cp\u003eYagi's approach was used to determine the lipid peroxidation index.\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e After centrifuging for 10 minutes while cooling at 3000 rpm, the tissue homogenate was precipitated with 10% trichloroacetic acid (TCA) in a boiling water bath for 15 minutes. TBA was added to the supernatant and then incubated for 50 minutes at 95\u0026deg;C. At 532 nm, the pink color complex's absorbances were determined spectrophotometrically. Nmol/mg protein was used to express the results.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6. Determination of GSH levels and SOD, CAT, and GPx activities\u003c/h2\u003e \u003cp\u003eSuperoxide dismutase activity was determined according to the method of Sun et al.\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e Catalase activity was assayed by the method developed by Aebi\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. The glutathione content was determined using the Sedlak and Lindsay method.\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e Glutathione peroxidase activity was measured using the Paglia and Valentine method.\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.10. Histopathological assay\u003c/h2\u003e \u003cp\u003eThe ovarian tissue samples were embedded in paraffin and treated in 10% formalin to be examined under a light microscope. Sections of tissues fixed in paraffin were cut into 5 \u0026micro;m thick pieces. They were captured on slides and hematoxylin-eosin stained (H-E).\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.11. Immunohistochemical analysis\u003c/h2\u003e \u003cp\u003eOvarian tissue samples were fixed in neutral formalin for caspase 3 immunoreactivity studies. After routine tissue follow-ups, paraffin sections 5 \u0026micro;m thick were taken from paraffin-embedded tissue samples to polylysine-coated slides. The antigens were then exposed by placing the sections in a citrate solution (pH 7.6) and heating them for 20 minutes in a microwave. It was chilled for 20 minutes, and then the sections were incubated in 0.3% H2O2 for 7 minutes. Then, the sections were washed with PBS. After carefully wiping and drying around each section, the blocking solution (Ab4051, Abcam, USA.) was dripped onto it to prevent nonspecific binding and incubated for two hours. Following a PBS rinse, they were treated with biotinylated goat anti-polyvalent and streptavidin peroxidase. Slides were dehydrated and counterstained for 1 minute with Mayer's hematoxylin before being stained for 15 minutes with chromogen\u0026thinsp;+\u0026thinsp;substrate. The caspase-3 kit was used following the manufacturer's instructions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.12. Rat growth factor chemokine and cytokine analysis\u003c/h2\u003e \u003cp\u003eGrowth factor, chemokine, and cytokine analysis of ovarian tissues were examined using protein-membrane arrays. Protein isolation was conducted using the Lysis Buffer of the array kit, and protein concentrations were measured by bicinchoninic acid assay. An equal amount of protein samples were administered to the membranes and incubated in the blocking buffer for 30 min. Membranes were washed t times after overnight incubation with protein samples. Horseradish peroxidase (HRP) was applied to membranes for 2 hours at room temperature, after which a chemiluminescent system was used to detect the presence of a protein (Bio-Rad Biotechnology Inc., USA). The Image Lab software measured protein intensities.\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.13. Statistical analysis\u003c/h2\u003e \u003cp\u003eFor all statistical calculations, GraphPad Prism version 8.0.0 for Windows was used. ClustVis (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://biit.cs.ut.ee/clustvis/\u003c/span\u003e\u003cspan address=\"https://biit.cs.ut.ee/clustvis/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) was used to create the principal component analysis (PCA) for all experiments.\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e Shapiro-Wilk test was used to assess fit for normal distribution. Mann-Whitney U, Kruskal-Wallis, Conover, and Friedman tests were used for statistical analysis. p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. RESULTS","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Effects of beta-glucan on ovarian, uterus, and body weights\u003c/h2\u003e \u003cp\u003eThe right ovary weight, uterine weight, and total genital weight of the PCOS group were significantly higher than those of the control group. The uterus and ovarian weights of the control, PCOS, P\u0026thinsp;+\u0026thinsp;βTG, and βTG groups did not differ statistically (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.2. Effects of beta-glucan on estrous cycle\u003c/h2\u003e \u003cp\u003eThe fractional survival data between the groups shows that the sexual cycles obtained proceed in a particular order. There was no deviation in the control, βTG, and PCOS\u0026thinsp;+\u0026thinsp;βTG groups. A significant deviation was observed in the PCOS group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe estrous cycle is examined in four consecutive periods. In the first period (proestrus), nucleated epithelial cells are dense. These cells can be seen together or separately. In some areas, cornified cells can be seen. The second period (estrus) is characterized by cornified squamous epithelial cells. Cells often appear in clumps. In the third period (metestrus), leukocytes are usually dominant. A collection of cornified cells with a few nucleated epithelia is seen. In the fourth period (diestrus), leukocytes increase (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.3. Effects of beta-glucan on hormone levels\u003c/h2\u003e \u003cp\u003eReproductive hormone levels were determined to examine the relationship between βTG and PCOS further. AMH has the feature of inhibiting follicle formation and causes androgen production indirectly by inhibiting FSH activity or by blocking aromatase activity. Thus, the concentration of AMH in the serum is proportional to the number of antral follicles and menstrual disorders. AMH levels are high in women with PCOS. Serum levels of AMH, T, and LH were significantly higher in the PCOS group than in the control group. The increased levels of LH, testosterone, and androstenedione in the serum, yet low or normal FSH levels and abnormal estrogen secretion, have created an endocrine profile accepted by many researchers in PCOS. In this study, we found a significant decrease in serum levels of FSH in the PCOS group compared to the control group. The PCOS group significantly increased AMH, LH, and T serum levels. Serum levels of FSH were found to be substantially reduced in the PCOS group compared with the P\u0026thinsp;+\u0026thinsp;βTG and βTG groups. The prevalent PCOS symptoms of hyperandrogenism and excessive LH production are signs that the PCOS model was developed successfully in the current study. In PCOS rats, treatment with βTG dramatically decreased the LH, AMH, and T blood levels, demonstrating that βTG could raise the levels of PCOS' reproductive hormones (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). These findings suggested that βTG might have a correcting impact on a female's reproductive endocrine.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.4. Effects of beta-glucan on cytokines\u003c/h2\u003e \u003cp\u003eIt was discovered how βTG affected the expression of inflammatory cytokines (Figure. 5).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.5. Biochemical results\u003c/h2\u003e \u003cp\u003eIn Table\u0026nbsp;2, the biochemical outcomes were displayed. SOD activity and TBARS levels between the control and βTG groups, as well as between the PCOS\u0026thinsp;+\u0026thinsp;βTG groups, were not significantly different. TBARS was considerably lower in the control group compared to the PCOS group. In comparison to the PCOS group, SOD activity was noticeably higher in the control group. The control, PCOS\u0026thinsp;+\u0026thinsp;βTG, and βTG groups all had considerably higher GSH and CAT levels and GPx activity than the PCOS group (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e3.6. Histopathological results\u003c/h2\u003e \u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e3\u003c/span\u003e provides the histopathologic damage score. The treatment groups' ovarian morphologies varied significantly from one another.\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 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eThe difference in ovarian tissue's histological grade between the groups.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\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 \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\u003eHistopathological score (Mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SE)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eControl\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.30\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003ePCOS\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.04\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003ePCOS\u0026thinsp;+\u0026thinsp;βTG\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.20\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eβTG\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.77\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003csup\u003ea\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\u003eThe treatment groups' ovarian morphologies varied significantly. The histological appearance of the ovaries in the control (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA, \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB) and βTG (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC, \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD) groups was normal. In the control and βTG groups, various follicle types and corpus luteum were seen in the ovary with a normal histological appearance. Secondary follicles (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA, \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC) and multilaminate primary follicles (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB, \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD) were observed in the control and βTG groups.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAtretic follicles were found (thick black arrows) (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA, \u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eC, \u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eD), vascular congestion (black asterisks) (8A, 8B, 8C, 8D), mononuclear cell infiltration (white asterisks) (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eB, \u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eD), vacuolization (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eD, \u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eE), edema (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eD), hemorrhage in corpus luteum (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eE) were observed.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAs opposed to the PCOS group, the PCOS\u0026thinsp;+\u0026thinsp;βTG group had significantly worse ovarian follicle shape, fewer mononuclear cell infiltration, atretic follicles, vacuolization, vascular congestion, edema, and bleeding. Different secondary follicles (white arrows) (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eA, Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eB) were observed in the PCOS\u0026thinsp;+\u0026thinsp;βTG group.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e3.7. Immunohistochemistry results\u003c/h2\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003e shows the immunohistochemistry expression of caspase-3. The PCOS\u0026thinsp;+\u0026thinsp;βTG group had fewer stained (positive) cells than the PCOS group.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e3.8. Protein array results\u003c/h2\u003e \u003cp\u003eProtein profiles of rat growth factor, cytokine, and chemokine arrays in control, PCOS, P\u0026thinsp;+\u0026thinsp;βTG, and βTG groups were analyzed (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e11\u003c/span\u003e). Cluster analysis of all proteins revealed that the controls, PCOS, P\u0026thinsp;+\u0026thinsp;βTG and βTG clustered together (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e11\u003c/span\u003eA). Principal component analysis showed a separate distribution for PCOS and all the other experimental groups (PC1: 50.7% and PC2: 26.4%) (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e11\u003c/span\u003eB). Correlation coefficient analysis was used to demonstrate the beneficial activity of βTG. Compared to the control, the stand-alone βTG application exhibited correlation coefficient values of 0.72 and 0.75, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e11\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe chemokine profile demonstrated a separation of PCOS from other experimental groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e12\u003c/span\u003eA). Similar outcomes for principal component analysis were attained (PC1: 59% and PC2: 39.1%) (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e12\u003c/span\u003eB). Control and P\u0026thinsp;+\u0026thinsp;βTG had a 0.71 correlation coefficient value (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e12\u003c/span\u003eC), confirming the cluster and PCA analysis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eP\u0026thinsp;+\u0026thinsp;βTG was separated from all experimental groups according to the cluster analysis in the growth factor array (Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e13\u003c/span\u003eA). Similar results (PC1: 92.2% and PC2: 5.7%) were attained (Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e13\u003c/span\u003eB). Correlation coefficient analysis demonstrated the beneficial effect of βTG. Stand-alone P\u0026thinsp;+\u0026thinsp;βTG and βTG had a 0.78 correlation coefficient value (Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e13\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn the cytokine 1 array analysis, the groups\u0026rsquo; control, PCOS, P\u0026thinsp;+\u0026thinsp;βTG, and βTG were all grouped (Fig.\u0026nbsp;\u003cspan refid=\"Fig14\" class=\"InternalRef\"\u003e14\u003c/span\u003eA). Similar outcomes for principal component analysis were attained (PC1: 71.5% and PC2: 18.4%) (Fig.\u0026nbsp;\u003cspan refid=\"Fig14\" class=\"InternalRef\"\u003e14\u003c/span\u003eB). Control and P\u0026thinsp;+\u0026thinsp;βTG had a 0.84 correlation coefficient value (Fig.\u0026nbsp;\u003cspan refid=\"Fig14\" class=\"InternalRef\"\u003e14\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003ePCOS group was separated from all experimental groups in the cytokine 2 array (Fig.\u0026nbsp;\u003cspan refid=\"Fig15\" class=\"InternalRef\"\u003e15\u003c/span\u003eA, \u003cspan refid=\"Fig15\" class=\"InternalRef\"\u003e15\u003c/span\u003eB). Control was clustered separately (PC1: 66.7% and PC2: 22.7%) (Fig.\u0026nbsp;\u003cspan refid=\"Fig15\" class=\"InternalRef\"\u003e15\u003c/span\u003eB). According to correlation coefficient analysis, experimental groups did not correlate (Fig.\u0026nbsp;\u003cspan refid=\"Fig15\" class=\"InternalRef\"\u003e15\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eβTG adminisration increased bNGF, TIMP-1, TNF-α, CINC-1, BDNF and bFGF leves in ovary tissues. βTG treatment decreased CTACK, LIX, and PDGF-AA (Fig.\u0026nbsp;\u003cspan refid=\"Fig16\" class=\"InternalRef\"\u003e16\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. DISCUSSION","content":"\u003cp\u003eOxidative stress and changes in antioxidant capacity cause toxicity in many organ systems.\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e Oxidative stress, which is involved in many pathologies, also plays a role in initiating and exacerbating the pathological process of PCOS.\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e Increased antioxidant enzyme activity, including catalase, glutathione peroxidase, superoxide dismutase, and glutathione, may help prevent oxidative stress in PCOS. The decrease in antioxidants such as SOD, CAT, GSH, and GPx may affect the physiological processes of the ovary, such as ovulation and egg development.\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e Antioxidant enzyme activities, such as CAT and GPx, are detected at increased levels in growing follicles to preserve ovarian function.\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e Increased ROS levels in women with PCOS are accompanied by low GSH levels.\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e In the ovaries of PCOS patients, GSH levels, CAT, SOD, and GPx are found in the ovaries. A decrease in endogenous antioxidant levels is observed due to oxidative stress triggered by PCOS.\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e However, some researchers have observed different results. In the study of Nawrocka-Rutkowska et al., while MDA levels, an indicator of oxidative stress, were found to be high in women with PCOS, CAT levels were also found to be high.\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e While Sabuncu et al.\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e found SOD levels higher in women with PCOS than in the control group. On the other hand, Zhang et al. found low SOD levels in PCOS.\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eLuteinizing hormone and FSH are secreted by the pituitary gland and promote ovulation. While LH levels increase in patients with PCOS, FSH levels decrease.\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e Contrary to previous studies\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e,\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e, in our study, we observed a significant decrease in serum FSH levels and an increase in LH levels in rats with the PCOS model. We observed elevated serum levels of AMH and T in PCOS rats. Specifically, we found that serum sex hormone levels, including T, AMH, LH, and FSH, were similar in the βTG-treated group to those in the control group. These results emphasized that βTG positively affects hormones by normalizing the changes in serum sex hormone levels in PCOS.\u003c/p\u003e \u003cp\u003eAdministration of βTG brought the protein profile of the target tissue closer to the control samples. Standalone βTG or βTG\u0026thinsp;+\u0026thinsp;PCOS groups exerted a similar protein profile compared to the baseline in cluster analysis. In addition, βTG induced the expression of some essential proteins, including bNGF, TIMP-1, Agrin, CINC-1, BDNF, and FGF-2 (bFGF). A previously published study observed decreased expression of TIMP-1 PCOS patients \u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e TIMP-1 increase in response to βTG supplementation might be related to the potential therapeutic activity of βTG. In a previously published report, FGF-2 injection had protective effects in a mouse model of PCOS\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e, which is consistent with our results that βTG increased the FGF-2 expression. Although there was no significant relation between PCOS and CTACK (cutaneous T-cell attracting chemokine), according to the literature,42 βTG decreased the CTACK, an inflammatory regulator. Suppression of inflammation by βTG might contribute to the treatment process of PCOS.\u003c/p\u003e"},{"header":"5. CONCLUSION","content":"\u003cp\u003eTaken together, all the results of this study showed that treatment with βTG stabilizes serum sex hormones and normalizes the abnormal oestrus cycles that are a hallmark of PCOS. These results suggest that βTG has a protective role in PCOS. In our literature review, we came across several studies that tested the effect of natural compounds in treating PCOS. However, we have not previously come across a study investigating the relationship between βTG and PCOS. In this respect, our study will be the first to investigate the effects of βTG on PCOS.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eCONFLICT OF INTEREST\u003c/h2\u003e \u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFUNDING\u003c/h2\u003e \u003cp\u003eThe İn\u0026ouml;n\u0026uuml; University Department of Scientific Research Projects funded this study (Project number: TCD-2020-2090).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eN.B.T, M.A., O.\u0026Ccedil;. conceptualized and designed. A.T., H.Y., S.Ş., D.A.\u0026Ouml;., S.A. carried out the experiments. A.D. examined the information. S.\u0026Uuml;. wrote the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eLeon LIR, Anastasopoulou C, Mayrin JV. In StatPearls. StatPearls Publishing; 2022. Polycystic Ovarian Disease.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAktay G, G\u0026uuml;rsoy Ş\u0026Ouml;, Uyumlu U, \u0026Uuml;n\u0026uuml;var S, İlhan N. Protective effect of atorvastatin on oxidative stress in streptozotocin-induced diabetic rats independently their lipid-lowering effects. J Biochem Mol Toxicol. 2019;33(5):e22295. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1002/jbt.22295\u003c/span\u003e\u003cspan address=\"10.1002/jbt.22295\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eG\u0026Uuml;RSOY Ş, ceritli m, \u0026Uuml;N\u0026Uuml;VAR, S., AKTAY G. Effects of Captopril on Cell Damage and Liver Damage. Volume 40. T\u0026uuml;rkiye Klinikleri Tip Bilimleri Dergisi; 2020. pp. 320\u0026ndash;5.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e\u0026Uuml;N\u0026Uuml;VAR S, Gursoy Ş, Berk A, Kaymaz B, Ilhan N, AKTAY G. Antioxidant Effect of a Dihydropyridine Calcium Antagonist Nitrendipine in Streptozotocin-Induced Diabetes. Volume 57. JOURNAL OF EVOLUTIONARY BIOCHEMISTRY AND PHYSIOLOGY; 2020. pp. 126\u0026ndash;33. 1.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eT\u0026uuml;rkmen NB, Y\u0026uuml;ce H, Şahin Y, Taşlıdere A\u0026Ccedil;, \u0026Ouml;zek DA, \u0026Uuml;n\u0026uuml;var S, \u0026Ccedil;ift\u0026ccedil;i O. Protective effect of resveratrol against pembrolizumab-induced hepatotoxicity and neurotoxicity in male rats. J Biochem Mol Toxicol. 2023;37(3):e23263.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMohammadi M. Oxidative Stress and Polycystic Ovary Syndrome: A Brief Review. Int J Prev Med. 2019;10:86.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBonakdaran S, Khorasani ZM, Davachi B, Khorasani JM. The effects of calcitriol on improvement of insulin resistance, ovulation and comparison with metformin therapy in PCOS patients: a randomized placebo-controlled clinical trial. Iran J Reprod Med. 2012;10(5):465.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDurlinger AL, Gruijters MJ, Kramer P, et al. Anti-M\u0026uuml;llerian hormone attenuates the effects of FSH on follicle development in the mouse ovary. Endocrinology. 2001;142(11):4891\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDeligeoroglou E, Vrachnis N, Athanasopoulos N, et al. Mediators of chronic inflammation in polycystic ovarian syndrome. Gynecol Endocrinol. 2012;28(12):974\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXia YH, Yao L, Zhang ZX. Correlation between IL-1β, IL-1Ra gene polymorphism and occurrence of polycystic ovary syndrome infertility. Asian Pac J Trop Med. 2013;6(3):232\u0026ndash;6.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDeshpande RR, Chang MY, Chapman JC, Michael SD. Alteration of cytokine production in follicular cystic ovaries induced in mice by neonatal estradiol injection. Am J Reprod Immunol. 2000;44(2):80\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEyol E, Tanriverdi Z, Karakus F, Yilmaz K, Unuvar S. Synergistic anti-proliferative effects of cucurbitacin i and irinotecan onhuman colorectal cancer cell lines. Clin Exp Pharmacol. 2016;6(5):1000219.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYıilmaz K, Karakuş F, Eyol E, Tosun E, Yıilmaz İ, \u0026Uuml;n\u0026uuml;var S. Cytotoxic Effects of Cucurbitacin I and Ecballium elaterium on Breast Cancer Cells. Nat Prod Commun. 2018;13(11):1445\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKarakuş F, Yilmaz K, Eyol E, \u0026Uuml;N\u0026Uuml;VAR S. (2019). Combination of 2 Bioactive Compounds for Treatment of Breast Cancer: Triterpenoid Cucurbitacin I and Phenolic CAPE. NATURAL PRODUCT COMMUNICATIONS, vol.14, no.6.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eY\u0026uuml;ce H, Şahin Y, T\u0026uuml;rkmen NB, et al. Apoptotic, Cytotoxic and Antimigratory Activities of Phenolic Compounds. J Evol Biochem Phys. 2022;58:1819\u0026ndash;33.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMirończuk-Chodakowska I, Kujawowicz K, Witkowska AM. Beta-Glucans from Fungi: Biological and Health-Promoting Potential in the COVID-19 Pandemic Era. Nutrients. 2021;13(11):3960.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eT\u0026uuml;rkmen NB, Y\u0026uuml;ce H, Aydın M, Taşlıdere A, Doğan A, \u0026Ouml;zek DA, Hayal TB, Yaşar Ş, \u0026Ccedil;ift\u0026ccedil;i O, \u0026Uuml;n\u0026uuml;var S. Nerolidol attenuates dehydroepiandrosterone-induced polycystic ovary syndrome in rats by regulating oxidative stress and decreasing apoptosis. Life Sci. 2023;315:121380.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRoy S, Mahesh VB, Greenblatt RB. Effect of dehydroepiandrosterone and delta 4-androstenedione on the reproductive organs of female rats: production of cystic changes in the ovary. Nature. 1962;196:42\u0026ndash;3.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMarcondes FK, Bianchi FJ, Tanno AP. Determination of the estrous cycle phases of rats: some helpful considerations. Braz J Biol. 2002;62(4):609\u0026ndash;14.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYagi K. Simple assay for the level of total lipid peroxides in serum or plasma. Methods Mol Biol. 1998;108:101\u0026ndash;6.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSun Y, Oberley LW, Li Y. A simple method for clinical assay of superoxide dismutase. Clin Chem. 1988;34(3):497\u0026ndash;500.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAebi H. Catalase in vitro. Methods Enzymol. 1984;105:121\u0026ndash;6.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSedlak J, Lindsay RH. Estimation of total, protein-bound, and nonprotein sulfhydryl groups in tissue with Ellman's reagent. Anal Biochem 1968;25(1):192\u0026ndash;205.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePaglia DE, Valentine WN. Studies on the quantitative and qualitative characterization of erythrocyte glutathione peroxidase. J Lab Clin Med. 1967;70(1):158\u0026ndash;69.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eT\u0026uuml;rkmen NB, Y\u0026uuml;ce H, Taşlıdere A, Şahin Y, Ayhan İ. 18β-glycyrrhetinic acid attenuates global cerebral ischemia/reperfusion-induced cardiac damage in C57BL/J6 mice. Braz J Pharm Sci [Internet]. 2022;58:e21219.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eŞenkal S, Hayal TB, Sağra\u0026ccedil; D, et al. Human ESC-derived Neuromesodermal Progenitors (NMPs) Successfully Differentiate into Mesenchymal Stem Cells (MSCs). Stem Cell Rev Rep. 2022;18(1):278\u0026ndash;93.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMetsalu T, Vilo J. ClustVis: a web tool for visualizing clustering of multivariate data using Principal Component Analysis and heatmap. Nucleic Acids Res. 2015;43(1):566\u0026ndash;70.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eT\u0026uuml;rkmen NB, Y\u0026uuml;ce H, Şahin Y, Taşlıdere A\u0026Ccedil;, \u0026Ouml;zek DA, \u0026Uuml;n\u0026uuml;var S, \u0026Ccedil;ift\u0026ccedil;i O. Protective effect of resveratrol against pembrolizumab-induced hepatotoxicity and neurotoxicity in male rats. J Biochem Mol Toxicol. 2023;37(3):e23263. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1002/jbt.23263\u003c/span\u003e\u003cspan address=\"10.1002/jbt.23263\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMurri M, Luque-Ram\u0026iacute;rez M, Insenser M, Ojeda-Ojeda M, Escobar-Morreale HF. Circulating markers of oxidative stress and polycystic ovary syndrome (PCOS): a systematic review and meta-analysis. Hum Reprod Update. 2013;19(3):268\u0026ndash;88.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLu J, Wang Z, Cao J, Chen Y, Dong Y. A novel and compact review on the role of oxidative stress in female reproduction. Reprod Biol Endocrinol. 2018;16(1):80.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSun Y, Li S, Liu H, et al. Association of GPx1 P198L and CAT C-262T Genetic Variations With Polycystic Ovary Syndrome in Chinese Women. Front Endocrinol (Lausanne). 2019;10:771.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDon\u0026agrave; G, Sabbadin C, Fiore C, et al. Inositol administration reduces oxidative stress in erythrocytes of patients with polycystic ovary syndrome. Eur J Endocrinol. 2012;166(4):703\u0026ndash;10.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHong Y, Yin Y, Tan Y, Hong K, Zhou H. The Flavanone, Naringenin, Modifies Antioxidant and Steroidogenic Enzyme Activity in a Rat Model of Letrozole-Induced Polycystic Ovary Syndrome. Med Sci Monit. 2019;25:395\u0026ndash;401.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNawrocka-Rutkowska J, Szydłowska I, Jakubowska K, et al. Assessment of the Parameters of Oxidative Stress Depending on the Metabolic and Anthropometric Status Indicators in Women with PCOS. Life (Basel). 2022;12(2):225.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSabuncu T, Vural H, Harma M, Harma M. Oxidative stress in polycystic ovary syndrome and its contribution to the risk of cardiovascular disease. Clin Biochem. 2001;34(5):407\u0026ndash;13.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang D, Luo WY, Liao H, Wang CF, Sun Y. Sichuan Da Xue Xue Bao Yi Xue Ban. 2008;39(3):421\u0026ndash;3.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLagan\u0026agrave; AS, Garzon S, Casarin J, Franchi M, Ghezzi F. Inositol in Polycystic Ovary Syndrome: Restoring Fertility through a Pathophysiology-Based Approach. Trends Endocrinol Metab. 2018;29(11):768\u0026ndash;80.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eQiu Z, Dong J, Xue C, et al. Liuwei Dihuang Pills alleviate the polycystic ovary syndrome with improved insulin sensitivity through PI3K/Akt signaling pathway. J Ethnopharmacol. 2020;250:111965.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOnalan G, Pabu\u0026ccedil;cu R, Goktolga U, Ceyhan T, Bagis T, Cincik M. Metformin treatment in patients with polycystic ovary syndrome undergoing in vitro fertilization: a prospective randomized trial. Fertil Steril. 2005;84(3):798\u0026ndash;801.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLahav-Baratz S, Kraiem Z, Shiloh H, Koifman M, Ishai D, Dirnfeld M. Decreased expression of tissue inhibitor of matrix metalloproteinases in follicular fluid from women with polycystic ovaries compared with normally ovulating patients undergoing in vitro fertilization. Fertil Steril. 2003;79(3):567\u0026ndash;71.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMoayeri A, Rostamzadeh A, Raoofi A, Rezaie MJ, Abasian Z, Ahmadi R. Retinoic acid and fibroblast growth factor-2 play a crucial role on modulation of sex hormones and apoptosis in a mouse model of polycystic ovary syndrome induced by estradiol valerate. Taiwan J Obstet Gynecol. 2020;59(6):882\u0026ndash;90.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen H, Zhang Y, Li S, et al. The Association Between Genetically Predicted Systemic Inflammatory Regulators and Polycystic Ovary Syndrome: A Mendelian Randomization Study. Front Endocrinol (Lausanne). 2021;12:731569.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"journal-of-ovarian-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jovr","sideBox":"Learn more about [Journal of Ovarian Research](http://ovarianresearch.biomedcentral.com)","snPcode":"13048","submissionUrl":"https://submission.nature.com/new-submission/13048/3","title":"Journal of Ovarian Research","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"antioxidant, beta-glucan, dehydroepiandrosterone, inflammation, oxidative stress, polycystic ovary syndrome.","lastPublishedDoi":"10.21203/rs.3.rs-4448893/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4448893/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eA class of dietary fibers and biologically active polysaccharides from natural sources, beta-glucans (βTGs) have bioactive capabilities. The anti-tumor, anti-inflammatory, prebiotic, anti-obesity, anti-allergic, anti-microbial, antiviral, anti-osteoporotic, and immunomodulating effects of βTGs are well documented. Although many biological activities of βTG have been proven, its mechanism in DHEA-induced PCOS has not been investigated. We aimed to investigate the protective effects of βTG treatment on PCOS and its capacity to reverse PCOS-induced changes. Female Sprague-Dawley (SD) rats were divided into four groups at random (n\u0026thinsp;=\u0026thinsp;8): control, PCOS, PCOS\u0026thinsp;+\u0026thinsp;βTG, and βTG groups. Biochemical markers linked to oxidative stress, antioxidant state, inflammation, cytokines, and hormone levels were assessed. Analyses using immunohistochemistry and histopathology were also carried out. Membrane array analysis detected growth factors, cytokine, and chemokine protein profiles. βTG did not cause any change in body, uterus, and ovarian weights in rats. βTG normalized the deviations in the oestrus cycle caused by PCOS. It was observed that βTG had a positive effect on the reproductive system. βTG can reduce the inflammatory response in PCOS rats by decreasing inflammatory cytokines. Oxidative stress was significantly reduced, whereas antioxidant enzyme activities were significantly elevated in the βTG group. βTG also prevented histopathological alterations. βTG induced the expression of some essential proteins, including bNGF, TIMP-1, Agrin, CINC-1, BDNF, and FGF-2 (bFGF). The results of this study showed that treatment with βTG protects against oxidative stress, inflammation, hormone imbalance, and histopathological damage in ovarian tissue caused by PCOS.\u003c/p\u003e","manuscriptTitle":"Effect of beta-glucan on oxidative stress, inflammation, hormonal and histopathological changes in dehydroepiandrosterone-induced polycystic ovary syndrome","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-06-03 15:10:02","doi":"10.21203/rs.3.rs-4448893/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-08-02T12:37:49+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-08-02T04:43:06+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-07-30T10:42:27+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"277910592699748040854852502807679544936","date":"2024-07-26T10:08:58+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-06-06T18:20:31+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"166669536470739827799872123034867310369","date":"2024-06-03T05:04:19+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"199297805662156242027403580768858129590","date":"2024-06-03T01:47:49+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-06-03T00:32:35+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-05-22T01:01:04+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-05-21T05:48:54+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Ovarian Research","date":"2024-05-20T11:41:28+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-ovarian-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jovr","sideBox":"Learn more about [Journal of Ovarian Research](http://ovarianresearch.biomedcentral.com)","snPcode":"13048","submissionUrl":"https://submission.nature.com/new-submission/13048/3","title":"Journal of Ovarian Research","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"1b4e86e7-950d-4a59-ba84-17826006b14f","owner":[],"postedDate":"June 3rd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2024-08-18T01:08:26+00:00","versionOfRecord":[],"versionCreatedAt":"2024-06-03 15:10:02","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4448893","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4448893","identity":"rs-4448893","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}