Pharmacological Evaluation of Bergapten in High Fat Diet and Letrozole Induced Polycystic Ovarian Syndrome in Wistar Rats

Pharmacological Evaluation of Bergapten in High Fat Diet and Letrozole Induced Polycystic Ovarian Syndrome in Wistar Rats | 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 Pharmacological Evaluation of Bergapten in High Fat Diet and Letrozole Induced Polycystic Ovarian Syndrome in Wistar Rats Abida Hussain, Ammara Saleem, Muhammad Furqan Akhtar This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7807066/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Polycystic ovarian syndrome (PCOS) is a complex multifactorial endocrine and metabolic disorder associated with hormonal imbalance, insulin resistance and ovarian dysfunction. Moreover, obesity and dietary imbalance act as major contributing factors. The present study was designed to evaluate pharmacological effects of bergapten in a high-fat diet (HFD) and letrozole-induced PCOS models using Wistar rats. Animals were divided into different experimental groups and treated with bergapten at three doses (5, 10 and 20 mg/kg), with control groups maintained on HFD and letrozole. Body weight deviation, blood glucose levels, histopathological examination, serum biochemical analysis and gene expression profiling were used to determine therapeutic efficacy of bergapten. Results exhibited significant decrease in blood glucose and body weight in all bergapten treatment groups than diseased control. Serum analysis demonstrated normalization of hormonal (FSH, LH, estrogen, progesterone, testosterone,) and metabolic biomarkers (adiponectin, leptin, CYP19A1, CYP11A1, ALT, AST, ALP, cholesterol, total lipids, TGs, HDL). While gene expression studies through qRT-PCR showed significant downregulation of pro-inflammatory mediators such as TNF-α, IL-6, IL-8, Keap1 and upregulation of Nrf-2 pathway. In addition, partial recovery of PPAR-γ expression in bergapten treatment groups. The marked improvement in ovarian and hepatic tissues morphology further supported medicinal value of bergapten in attenuating metabolic stress. Overall, findings exhibited that bergapten not only alleviates PCOS but also counteracts obesity-related disturbances caused by HFD, suggesting its potential as a therapeutic candidate. Further investigations are needed to validate its clinical relevance. PCOS obesity Keap1 Nrf-2 adiponectin bergapten 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 1. Introduction Polycystic ovarian syndrome (PCOS) is also recognized as “hyperandrogenic anovulation (HA) or Stein-Leventhal syndrome”, particularly common in reproductive-age women. PCOS is chronic and heterogeneous condition, clinically characterized by obesity, menstrual irregularities, infertility, hirsutism (Singh et al., 2023 ). Obesity is a chronic metabolic disorder characterized by excessive accumulation of adipose tissue that disrupts endocrine and metabolic homeostasis worldwide. In women, obesity not only predisposes to metabolic disorders (e.g., type 2 diabetes mellitus) but contributes significantly to reproductive abnormalities (e.g., infertility and polycystic ovarian syndrome (PCOS), menstrual dysfunction) and cardiovascular disease (El Hayek et al., 2016 ). Epidemiological studies consistently reported that majority of women with PCOS are overweight and having a high risk of infertility (Moazzam et al., 2024 ). Morphologically, hyperandrogenism is regarded as a major clinical manifestation of PCOS, disrupts follicular progression, develops anovulation and ovarian microcysts with multiple small follicles (≥10 of 2–9 mm) (Abinaya et al., 2019 ). The World Health Organization (WHO) reported that approximately 116 million women (3.4%) worldwide affected with PCOS (Deswal et al., 2020 ). The high prevalence of PCOS and its association with ovulatory dysfunction, metabolic disturbances and dermatological complications, highlights its substantial health and economic burden. Although PCOS can appear at any stage from “menarche onwards”, most diagnoses occur between 20–30 years of age (Salari et al., 2024 ). In 2017, it was estimated that PCOS affected 1.55 million reproductive-age women globally, accounting for 0.43 million of disease burden. Several studies have shown that both genetic predisposition and environmental determinants are central to PCOS development. Likewise, PCOS may also emerge early in adolescents with familial predisposition, irregular dietary habits, toxins and exposure to pollutants (Omar et al., 2025 ). Few researches have evidenced that various influential mechanisms in PCOS are endocrine disruption, prolonged inflammation, insulin insensitivity and androgen excess (Gupta & Khan, 2023 ). PCOS is an “oligogenic disorder” with multifactorial pathogenesis majorly involves impairment of hypothalamic-pituitary axis (HPA), pancreatic insulin regulation and ovarian activity. Although precise etiology remains elusive, strong associations with insulin resistance and obesity have been consistently explored yet (Ding et al., 2021 ; Shoaib et al., 2023 ). Obesity amplifies prevalence of glucose intolerance, dyslipidemia and ovulatory failure in PCOS, highlighting metabolic-reproductive overlap in its pathophysiology. The relationship between obesity and PCOS is multifaceted, as obesity acts both as a risk factor and as a provoking condition for syndrome. High-fat diet (HFD)-induced obesity enhances insulin resistance and hyperinsulinemia, all of which contribute to excessive androgen production by ovarian theca cells (Malik et al., 2024 ). Elevated androgens perpetuate folliculogenesis, leading to anovulation. Moreover, obesity-related reductions in sex hormone-binding globulin (SHBG) increase circulating free testosterone, further aggravating hyperandrogenic symptoms. In parallel, reduced adiponectin levels and elevated leptin concentrations in obese women may also worsen symptoms of PCOS (Ding et al., 2021 ; Kim, 2024 ). Bergapten (BP), is a naturally occurring furocoumarin found in various medicinal plants of Umbelliferae family such as Cnidii Fructus, Angelica dahurica, and Peucedanum ostruthium . This compound is also present in grapefruit juice, essential oil of bergamot and several other essential oils from citrus fruits (Liang et al., 2021 ). The chemical name of bergapten is 5-methoxypsoralen, which has a basic benzo-[alpha]-pyrone structure. Bergapten is recognized as a potent “photosensitizing compound” and exhibits diverse pharmacological actions including anticancer, antibacterial, hypolipidemic and anti-inflammatory activities (Aslam et al., 2022 ; Phucharoenrak & Trachootham, 2024 ). Despite, adipose tissue expansion in obesity activates irregular Keap1 signaling, which suppresses Nrf2-mediated antioxidant defenses. Consequently, a pro-oxidative environment generates leading towards granulosa cells apoptosis, mitochondrial dysfunction and reduced estrogen biosynthesis (Li et al., 2020 ; Zhou et al., 2021 ). In parallel, obesity triggers nuclear factors kappa-β (NF-κβ) activation, enhancing inflammatory responses of cytokines (e.g., IL-6, IL-8 and TNF-α). Importantly, clinical evidence suggests that addressing obesity should be a primary therapeutic target in PCOS management, as weight reduction improves insulin sensitivity, restores ovulation and endocrine balance (Wawrzkiewicz-Jałowiecka et al., 2021 ). Clinical data suggest that obesity not only increases risk of infertility but also prolongs time required to achieve pregnancy compared to non-obese women (Shoaib et al., 2023 ). Therefore, present study was designed to investigate therapeutic potential of bergapten in experimental models of polycystic ovary syndrome (PCOS) induced by letrozole administration in combination with a high-fat diet (HFD). Furthermore, research aimed to explore underlying mechanisms through which bergapten exerts its pharmacological effects, with particular attention to hormonal regulation, insulin sensitivity and inflammatory pathways in obese-PCOS rats. 2. Materials and Methods 2.1. Chemicals Analytical grade chemicals and reagents were used in whole study. Standard drug metformin (Glucophage 500mg) was purchased from Martin-Dow pharma, Pakistan. Letrozole (5g) was obtained from Saffron Pharmaceutical Faisalabad, Pakistan. Bergapten was acquired from Biosynth Slovakia. Preparation of solutions was done by using dimethyl sulfoxide (DMSO) and carboxyl-methyl cellulose (Sigma Aldrich, UK) ,10% formalin, chloroform from Merk, Germany. 2.2. Experimental Animals Healthy nulliparous female Wistar rats of n = 60 (100-150g) were acquired and housed in animal house of Department of Pharmacology, Government College University Faisalabad (GCUF). All animals were provided with standard laboratory conditions by following temperature (23 ± 2 o C), humidity (55 ± 5) and maintained 12 hours’ light/dark cycles, with unlimited supply of water and pallet diet. An acclimatization period of seven days was provided to adapt rats with laboratory environment. The experiment was conducted by following protocols of ethical committee of Government College University Faisalabad (GCUF). The study protocol was approved by Research Ethical Committee of Government College University Faisalabad Approval No. GCUF/ERC/633. All procedures involving animals followed guidelines of National Research Council and efforts were made to minimize any potential harm. 2.3. Obesity and PCOS induction The animals were randomly divided into two groups. Before studying initiation, body weight of animals was measured in grams. The normal control group animals (n = 12) were maintained on a standard pallet diet. Remaining animals (48) were administered on a high fat diet (HFD) including 60% fats, 20% carbohydrates and 20% protein for 12 weeks to induce obesity. REF Following HFD regimen, all animals were weighed weekly, and obesity status was determined using Lee obesity index with following formula. Lee obesity index = 3 cube roots (weight)/ length (cm)×1000 Animals with a Lee index > 320 were classified as obese and selected for further study (Shoaib et al., 2023 ). Letrozole treatment started after 2months and 9 days to induce PCOS in HFD taking animals. 6 animals out of 12 normal controls received a daily oral dose of 5 ml of 0.5% carboxymethyl cellulose (CMC) as vehicle, while other 6 normal animals were administered letrozole and served as letrozole control. While obese rats received letrozole (LET) 1mg/kg/day per oral (suspended in 0.5% CMC) once daily for 28 days to induce PCOS ((Hussain et al., 2022 ) . For PCOS confirmation, to perform vaginal cytology, Vaginal smear was taken after 21 days and studied estrous cycle, but no estrous cycle was observed under light microscope. Then letrozole was given for another 7days. Vaginal cytology performed and the estrous cycle was confirmed after 28 days. The weight deviation record was also being taken on a weekly basis. 2.4. Experimental Design After successful induction of PCOS and obesity, animals were divided into 7 groups(n = 6) normal control treated with normal diet and distilled water. Disease control obese were treated with high fat diet (HFD) and letrozole (LT), disease control PCOS were treated with letrozole (LT), obese + PCOS were treated with metformin 300 mg/kg (Standard control) and obese + PCOS were treated with different doses of bergapten 5mg/kg, 10mg/kg, 20mg/kg treatment. Treatment as mentioned below was started orally for a period of 28 days and their fasting blood glucose levels were measured after 28th day. The animals were allocated into groups as outlined below. Group I (normal control): received a normal chow diet. Group II (disease control obese): High fat diet (HFD) + Letrozole (LT) Group III (disease control PCOS): Letrozole (LT) Group IV (obese + PCOS): Metformin 300 mg/kg (Standard control) Group V (obese + PCOS): Bergapten 5mg/kg (Treatment) Group VI (obese + PCOS): Bergapten 10mg/kg (Treatment) Group VII (obese + PCOS): Bergapten 20mg/kg (Treatment) 2.5. Vaginal smear cytology Vaginal smears were collected by gently inserting a sterile micropipette into vaginal canal and flushing with 10–20 µL of sterile saline, after which fluid was aspirated and spread thinly onto pre-labeled glass slides, air-dried and stained with 0.1% crystal violet (methylene blue). Slides were examined under a light microscope at 40X, and difference was noted under microscope. 2.6. Oral glucose tolerance test (OGTT) After 28 days of drug treatment, rats were fasted overnight. Thirty minutes after administration of ongoing treatment,2g/kg glucose solution was administered orally. Blood samples were collected from tail vein using glucometer and readings were recorded at 0, 60, 120, and 180 minutes respectively. 2.7. Biochemical testing After completing treatment period, animals were anesthetized by pentobarbital sodium (40mg/kg, IP) blood sample was collected by cardiac puncture, and serum was separated and process for biochemical testing of lipids parameters such as total cholesterol (TC), triglycerides (TG), and levels of hormones including follicle-stimulating hormone (FSH), serum insulin, testosterone, luteinizing hormone (LH), estrogen and progesterone. 2.8. Estimation of ovary weight index (OWI) The anesthetized animals were euthanized through cervical dislocation, ovaries and liver were carefully removed. Ovaries were cleaned and weighed properly by using weighing balance. Afterwards, OMI was calculated by following formula: OWI = Ovary weight (mg)​/ Body weight (g)×100 2.9. Histopathological analysis Ovaries and liver were removed, rinsed with ice-cold normal saline and preserved in 10% formaldehyde solution. Using a rotary microtome, tissues (5µm thick) were embedded in paraffin wax, mounted on glass slides and stained with hematoxylin-eosin dye. While cytology of vaginal smear was analyzed with hematoxylin-eosin dye. Histological changes of ovaries (e.g., number /size of follicles and corpus luteum) and liver (e.g., hepatic inflammation, necrosis, fibrosis, scar tissues) were observed under a light microscope at magnifications of 40X and 100X (Moazzam et al., 2024 ; Younas et al., 2022 ). 2.10. Estimation of genetic biomarkers Genetic biomarkers such as Nrf-2/keap1, PPRA-γ, TNF, IL8, IL6, Cyp11A1 and Cyp19A1 were quantified to assess genetic alterations. Total RNA was isolated from serum samples using TRIzol reagent (Invitrogen, CA, USA) following manufacturer’s protocol. Complementary DNA (cDNA) was synthesized with a commercial reverse transcription kit and amplification was then carried out on quantitative real-time PCR System (Hussain et al., 2023 ) . 3. Results 3.1. Effect of bergapten on body weight The HFD control exhibited a marked (p < 0.001) increase in body weight compared to normal control, indicating persistent obesity. The weight of letrozole control group also elevated. All BA treated groups and standard control (300 mg/kg) showed a noticeable reduction in body weight, suggesting therapeutic efficacy Fig. 1 . BA treatment restored body weight dose dependently. 3.2. Effect of bergapten on oral glucose tolerance test (OGTT) In oral glucose tolerance test, glucose concentrations were measured at 0, 60, 120 and 180 minutes using a glucometer (Fig. 2 ). After 1h, 2h and 3h, all bergapten and metformin 300mg/kg treatment groups showed significantly (p < 0.001) decrease in glucose levels as compared to HFD control. Moreover, HFD control group exhibited noticeable (p < 0.001) increase in glucose levels relative to normal control. Blood glucose in letrozole control was also consistently increased. 3.3. Effect of bergapten on LDL, VLDL, total lipids and HDL The effect of bergapten on low-density lipoproteins (LDL), very low-density lipoproteins (VLDL), high density lipoproteins (HDL) and total lipids showed significant variations across experimental groups in Fig. 3 . HFD control (114.1 ± 4.15 mg/dl) showed marked elevation in LDL as compared to normal control (43.67 ± 2.43 mg/dl), confirming development of dyslipidemia. Standard control produced notable (p < 0.001) reduction in LDL when compared with normal control. Similarly, BA (5mg/kg,10mg/kg,20 mg/kg ) showed a highly significant decrease in LDL concentrations dose dependently relative to HFD control group. LDL concentration was also higher in letrozole control group (113.6 ± 3.51 mg/dl). The concentration of VLDL were escalated in HFD control (19.77 ± 2.03 mg/dl) than normal control (18.73 ± 1.88 mg/dl). In contrast, a significant (p < 0.001) decrease in VLDL levels was recorded in standard (metformin 300mg/kg) control group compared to normal control. Moreover, BA at 5, 10 and 20mg/kg (13.30 ± 1.72, 10.73 ± 1.15 and 9.73 ± 1.09 mg/dL) produced a marked (p < 0.001) decrease in VLDL values when compared with HFD control group. An elevation in VLDL levels was also noted in letrozole group. The concentrations of total lipids in HFD control had significantly elevated in PCOS-induced rats than normal control group (p < 0.001). Additionally, standard group also produced significant lipid-lowering effect (p < 0.0001) relative to normal control. Conversely, groups received different BA treatment showed a sharp (p < 0.001) decline in total lipids in comparison with HFD control. While LDL concentrations of total lipids was higher in letrozole control group (362.3 ± 6.02 mg/dL). Hence, all bergapten treatment groups restored LDL, VLDL and total lipids levels close normal control. Regarding HDL, HFD control group showed sharp reduction as compared to normal control. The levels of HDL were shown to be increase in standard control (30.90 ± 1.64 mg/dl) just similar to normal control. While, BA treatment at dose of 5 mg/kg (39.97 ± 1.69 mg/dl), 10 mg/kg (42.98 ± 2.21 mg/dl) and 20 mg/kg (64.48 ± 2.26 mg/dl) also produced a distinct rise in HDL concentrations as compared to HFD control. Thus, all BA treatment groups significantly exceeded HDL concentrations in a dose-dependent manner, indicating a strong HDL-elevating potential. The letrozole control group demonstrated obvious decrease in HDL levels. 3.4. Effect of bergapten on cholesterol and triglycerides HFD-disease control (91.07 ± 4.67 mg/dl) group exhibited significant elevation in cholesterol levels compared to normal control (77.92 ± 2.05 mg/dl). Standard control (58.18 ± 2.33 mg/dl) significantly reduced cholesterol levels similar to normal control. A dose-dependent hypocholesterolemia effect was observed with BA-5 mg/kg, BA-10 mg/kg and BA-20 mg/kg, led to notable decrease (p < 0.0001) than HFD control. Furthermore, letrozole control (101.9 ± 4.30 mg/dl) resulted in a marked (p < 0.0001) increase in cholesterol levels. According to effect on serum triglycerides (TGs), HFD control showed highest concentration (105.6 ± 5.69 mg/dl) as compared to normal control (40.00 ± 2.65 mg/dl). Metformin 300 mg/kg (43.22 ± 2.41 mg/dl) resulted in a substantial reduction in TGs levels just like normal control. Subsequently, three doses of bergapten (BA) treatment groups demonstrated significantly lower TGs levels in comparison to HFD control group. Letrozole showed more pronounced increase (p < 0.001) in TGs overall. 3.5. Effect of bergapten on cholesterol/HDL ratio HDL-cholesterol ratio displayed a strong therapeutic effect of bergapten in PCOS-induced rat model. The normal control group maintained this ratio with value of 1.83 ± 0.07, whereas HFD-disease control showed a marked decline in value as 1.51 ± 0.07 respectively. Standard control significantly improved (p < 0.001) HDL-cholesterol ratio to 1.86 ± 0.06, restoring values close to normal control. Bergapten 5 mg/kg (1.38 ± 0.05) and 10 mg/kg (1.54 ± 0.06) and 20 mg/kg (2.05 ± 0.11) produced modest increase (p < 0.01) in HDL-cholesterol ratio, when compared with HFD control. Values of letrozole control group were lessened to 1.31 ± 0.04 respectively (Fig. 4 ). 3.6. Effect of bergapten on FSH and LH Effect of bergapten on serum FSH and LH levels across experimental groups is presented in Fig. 5 . The normal control group recorded FSH levels as 1.21 ± 0.08 IU/mL whereas disease control shown a marked decline to 1.01 ± 0.09 IU/milk Metformin 300mg/kg treatment group (standard) restored FSH to near-normal control levels. All doses of BA treatment produced a dose-dependent rise (p < 0.001) in FSH, compared to HFD control. On the other hand, HFD-diseased control (1.09 ± 0.04) exhibited a sharp increase in LH than normal control (0.25 ± 0.02 mIU/mL). Standard control significantly reduced LH to 0.39 ± 0.04 mIU/mL approaching normal control levels. At 5 mg/kg BA, LH was lowered to 0.68 ± 0.03 mIU/m while BA at 10 mg/kg (0.95 ± 0.07 mIU/mL) and 20 mg/kg (0.87 ± 0.03 mIU/mL), maintained LH values markedly below HFD control. The significant (p < 0.0001) increase in LH and reduction in FSH was observed in letrozole control group. 3.7. Effect of bergapten on testosterone, progesterone and estrogen Bergapten showed modulatory effect on progesterone, testosterone and estrogen levels. Standard control group exhibited elevated concentrations of progesterone (2.88 ± 0.04 ng/mL) than disease control (1.92 ± 0.05 ng/mL). However, group received metformin 300mg/kg produced a marked elevation in progesterone just like normal control. BA administration further enhanced progesterone levels in a dose-dependent manner, with 5 mg/kg (3.20 ± 0.11 ng/mL), 10 mg/kg (3.04 ± 0.06 ng/mL) and 20 mg/kg (4.76 ± 0.10 ng/mL) respectively. All BA-treated groups produced superior results relative to HFD-diseased control, highlighting therapeutic efficacy of bergapten. Letrozole treatment group showed significant drop in progesterone levels as 1.80 ± 0.07 ng/mL. On the other side, standard control displayed strongest recovery of testosterone concentrations, similar to normal group. The levels of testosterone in each BA group showed significant improvement compared with HFD control diseased (p < 0.0001), though levels remained below normal control (3.51 ± 0.04). Resultantly, bergapten treatment approached same effect as of standard (3.04 ± 0.07 ng/mL) control overall. Levels of testosterone were suppressed in letrozole control group significantly (p < 0.001). Furthermore, treatment with metformin 300mg/kg, 5, 10 and 20mg/kg BA also lowered estrogen concentrations in a dose-responsive pattern with respect to HFD control, thus demonstrated significant therapeutic response (p < 0.001). While, metformin 300mg/kg treatment group showed sharp reduction than normal control, but close to reference range (15–350 pg/mL). Levels of estrogen was raised in letrozole control group (p < 0.001). 3.8. Effect of bergapten on adiponectin and leptin The change in serum concentration of adiponectin (ng/mL) and leptin (µg/mL) after bergapten administration are shown in Fig. 7 . Mean leptin concentration in normal control (4.12 ± 0.13) was below as compared to diseased control (12.57 ± 0.15), confirming leptin dysregulation via PCOS induction (p < 0.0001). Standard control and BA treatment significantly lowered leptin concentrations than disease control (p < 0.0001). Alternatively, normal control and HFD group displayed serum adiponectin levels as 17.23 ± 0.47, and 8.52 ± 0.27 respectively. Standard control effectively restored adiponectin (18.24 ± 0.46 µg/mL) levels thus outperforming normal control and closely match reference values. Moreover, BA 5, 10 and 20mg/kg produced strongest reduction in adiponectin, thus showed significant (p < 0.0001) improvement compared to HFD control. Overall, letrozole control (10.98 ± 0.14) groups exhibited considerable (p < 0.001) effects on leptin and adiponectin levels. 3.9. Effect of bergapten on Nfr-2, PPARγ and Keap-1 The levels of Nrf2, PPAR-γ, and Keap1 across various experimental groups including normal control, diseased control, letrozole control, standard control (300 mg/kg) and BA treatments (5, 10, and 20 mg/kg) is shown in Fig. 8 . Nrf2 expression was significantly elevated in normal control, while disease control showed obvious reduction (p < 0.01). Standard treatment restored Nrf2 expression near to values of normal control. While, BA (10 and 20 mg/kg) treatment groups caused a significant upregulation (p < 0.001) as compared to HFD control. PPAR-γ levels were elevated in HFD-disease control but remained low in normal control. Metformin 300mg/kg treatment group showed appreciable (p < 0.001) decrease in PPAR-γ expression. Furthermore, bergapten treatment groups significantly decreased PPAR-γ compared to HFD control (p < 0.001). Genetic expression of Keap1 was significantly (p < 0.001) downregulated in standard control group, comparable to normal control. While, upregulated in HFD control than normal control. Treatment with three different doses (5,10 and 20mg/kg) of BA also significantly and dose-dependently reduced Keap1 expression (p < 0.0001) compared to HFD-disease control, indicating suppression of Nrf2. In letrozole control group, trend of Nfr-2, PPAR-γ and Keap-1 levels was consistent with HFD control group. 3.10. Effect of bergapten on TNF-α, IL-6 and IL-8 The expression levels of pro-inflammatory cytokines TNF-α, IL-6 and IL-8 were assessed across different experimental groups in PCOS induced rats ( Fig. 9 ). For TNF-α, diseased control group exhibited highest expression than normal control. Conversely, metformin (300mg/kg) treated groups showed moderate reduction, equivalent to normal control group. However, BA-treatment groups (5, 10, and 20 mg/kg) showed dramatic decrease in TNF-α expression, that was statistically significant compared to HFD control (p < 0.01). IL-6 followed a similar pattern of TNF-α. For, IL-6 and IL-8, HFD-disease control group showed highest fold change than normal control. Meanwhile metformin (300mg/kg) treatment showed less fold change, resemble with normal control. BA treatments at all doses significantly (p < 0.001) suppressed expression of both genes (IL-6 and IL-8), thus produced prominent anti-inflammatory effect in PCOS-induced rats. Letrozole control group showed increase in all inflammatory markers (e.g., IL-8, IL6 and TNF-α) concentrations. 3.11. Effect on CYP19A1 and CYP11A1 The comparative effects of bergapten on serum CYP19A1 and CYP11A1 enzymes across all experimental groups are demonstrated in Fig. 10 . HFD control group exhibited a marked decrease in CYP19A1 expression than normal control. Metformin 300mg/kg exhibited sudden rise in CYP19A1 levels, consistent with normal control. While BA treatment groups (5, 10, 20mg/kg) showed significant (p < 0.001) upregulate CYP19A1 expression than HFD control. Data of CYP11A1 expression indicated that disease control (HFD) manifested elevation as compared to normal control. While standard control (metformin 300mg/kg) showed slight decline, close to normal control group. Treatment with bergapten (5, 10 and 20mg/kg) demonstrated downregulation of CYP11A1 compared to HFD control (p < 0.01). The net effect of letrozole control was induction of CYP19A1 expression (~ 3.0 fold), while suppression of CYP11A1 expression confirms earlier reports. 3.12. Vaginal Smear Cytology Vaginal Smear cytology, a type of exfoliative histology used to assess estrous cycle stage in PCOS-induced rats. In normal healthy control group (Fig. 11 ), smear shows predominantly nucleated epithelial cells with some presence of cornified cells and minimal leukocytes. cells appear polygonal, with some clusters and nuclei clearly visible. While in HFD diseased control group, four different stages of estrous cycle were analyzed. The proestrus phase corresponds to “follicular stage”, while estrus phase denotes “ovulation”. During estrus, smears typically show large, irregular, non-nucleated cornified epithelial cells. The metestrus stage is recognized by mixture of leukocytes along with both nucleated and non-nucleated cornified epithelial cells. In diestrus, a predominance of leukocytes with nucleated epithelial cells is observed (A). Following PCOS induction, this regular pattern becomes disrupted and estrous cycle shows irregularity, with a significant prolongation of metestrus stage. Smears in this condition display an abundance of leukocytes together with non-nucleated cornified epithelial cells, confirming disturbance caused by PCOS (B). NEC: normal epithelial cells; CFR: collapsed follicular remnants; LC: Leukocytes; FST: fibrotic stromal tissues; DEC: degenerated epithelial cells; SH: stromal hyperplasia; NR: necrotic remnants 3.13. Effect on OWI (%) The change in OWI after bergapten administration is shown in Fig. 12 . Mean value of OMI in normal control (0.038 ± 0.004) was below as compared to diseased control (0.049 ± 0.003), confirming ovarian changes via PCOS induction (p < 0.05). Standard control and BA treatment significantly reduce these alterations. 3.14. Histopathological Evaluation Histopathological analysis of liver and ovarian tissues of PCOS-induced rats treated with bergapten revealed largely preserved hepatic architecture with distinct morphological alterations linked to treatment modalities and disease induction (Fig. 13 ). Normal control group (a) exhibited healthy ovarian cortex with multiple healthy follicles, defined corpora luteum, compact vascularized stroma with no cystic formations. Disease control group (b) subjected to a high-fat diet exhibited multiple large, thin-walled cystic follicles with irregular granulosa cell layers and mild stromal edema. Letrozole treated animals (c) produced enlarged fluid-filled follicles with thin granulosa layers and absent oocytes, thus surrounding stroma was loose and edematous. Metformin (300 mg/kg) treatment group (d) displayed partial reversal of cystic changes, dense and hypercellular ovarian stroma with increased fibroblast-like cells. Treatment with bergapten demonstrated dose-dependent histological restoration. The group treated with 5 mg/kg (e) showed moderate preservation of follicular architecture with a reduced number of cystic follicles. While high doses of BA 10 mg/kg (f) and 20 mg/kg (g) showed near-normal ovarian histology, characterized by regression of cystic follicles, reappearance of corpora luteum and restoration of granulosa as well as thecal cells organization with undamaged architecture. 3.15. Hepatic tissues Histopathological analysis of liver sections across experimental groups revealed largely preserved hepatic architecture with specific alterations linked to treatment modalities and disease induction (Fig. 14 ). In normal control group (a), liver tissue exhibited well-organized hepatic cords, intact central veins and polygonal hepatocytes with centrally located nuclei with no fatty or inflammatory infiltration and necrosis. While HFD-disease control animal (b), surprisingly exhibited slight ballooning of hepatocytes with mildly dilated sinusoids. Occasional mononuclear cells near portal areas but no fatty liver deposits. In letrozole control animal (c), clear vacuoles within hepatocytes were seen under microscope with minimal disorganization of hepatic cords. Standard control group (d), treated with 300 mg/kg metformin showed no fatty infiltration but notable areas of hepatocellular necrosis, dense clusters of lymphocytes and macrophages around portal tracts and central veins were observed. Hepatocytes show signs of degeneration. Sinusoids are markedly dilated and kupffer cells appear enlarged in 5 mg/kg BA treatment group. While BA 10 mg/kg showed irregular hepatocytes, with minimal hepatic cord arrangement and fibrous strands may be faintly visible. Furthermore, BA 20 mg/kg portrayed widespread inflammatory infiltration with portal areas shown fibrotic expansion. 4. Discussion Obesity is globally organized metabolic disorder which aggravates pathogenesis of PCOS by promoting insulin resistance, hyperinsulinemia, oxidative stress and systemic inflammation, all of which contribute to ovarian dysfunction and hyperandrogenism (Li et al., 2019 ). The present study provides new insight into the complex interplay between obesity and polycystic ovary syndrome (PCOS), while also highlighting therapeutic potential of bergapten (a plant-derived furocoumarin) on letrozole and high-fat diet-induced PCOS in Wistar rats. In addition, study also pointed out that high-fat-diet induced obesity exacerbated reproductive and metabolic disturbances caused by letrozole, thereby mimicking multifactorial pathogenesis of PCOS. The high-fat-diet (HFD) and letrozole were administered among all animals except normal control group, to establish a reliable experimental model of obesity-associated PCOS. Review of literature has shown that letrozole (an aromatase inhibitor) causes estrogen biosynthesis disruption, follicular arrest, cystic changes, irregular estrous cycles and increases androgen production, thereby mimicking endocrine abnormalities observed in PCOS. While, HFD contributes to excessive weight gain, adipocytes accumulation and defective glycemic control (Kafali et al., 2004 ; Reddy et al., 2016 ). Consequently, combination of letrozole and HFD exacerbates persistent anovulation, hyperandrogenism, and dyslipidemia. Weight reduction is a potential approach for restoring ovulatory function and enhancing fertility in women with PCOS. Even modest weight loss, in range of 5–10% has been reported to improve gonadotropin balance, reduce hyperandrogenism and normalize menstrual cycle (Moran et al., 2010 ). In present study, rats treated with metformin and bergapten showed a significant decline in body weight compared to disease control group receiving letrozole and HFD, may reflect an improvement in metabolic efficiency and attenuation of adipose accumulation. In contrast, disease control group exhibited continuous weight gain, consistent with obesogenic and endocrine-disrupting effects of letrozole and HFD. Various studies reported that disturbances in glucose homeostasis are central to pathophysiology of obesity-related PCOS (Bednarz et al., 2022 ). In current study, administration of metformin and bergapten resulted in a marked reduction in fasting blood glucose levels, attributed to enhanced insulin sensitivity and improved glucose uptake, possibly mediated through activation of intracellular signaling pathways such as AMPK, which promotes lipid oxidation and reduces gluconeogenesis (Xin et al., 2016 ). In contrast, animals of letrozole and HFD group exhibited persistently elevated glucose levels suggested impaired glucose tolerance induced by excessive fat intake (Xin et al., 2016 ). Statistical analysis of biochemical markers further highlighting PCOS alteration and therapeutic impact of bergapten. In present study, dyslipidemia in diseased groups received high-fat diet altered metabolic features of PCOS more effectively than letrozole control alone. These findings align with evidence from studies of other natural and dietary interventions. For instance, short-chain fatty acids (SCFAs) such as sodium acetate, propionate, butyrate in letrozole-induced PCOS models significantly lower LDL, total cholesterol, elevate HDL and ameliorate VLDL levels (Acharya et al., 2024 ), mirroring lipid improvements seen with bergapten treatment in current study. Thus, lipid-normalizing dose-dependent effects observed with bergapten strongly suggest its effectiveness in counteracting metabolic dysfunction in PCOS. The vaginal smear cytology of healthy controls demonstrates a normal estrous cycle pattern with distinct phases. In contrast, PCOS-induced rats exhibited irregular cycle marked by prolonged metestrus (Thakor & Patel, 2014 ). The results of present study confirmed that letrozole induced PCOS significantly alters cyclic pattern in diseased control rats. Hormones act as key regulators of physiological processes, particularly in maintaining normal menstrual cycle. Follicular development and ovulation depend on proper function of ovary and balanced secretion of LH and FSH (Murray & Orr, 2020 ). In PCOS, follicles often fail to mature and release an egg that leads towards abnormalities in both follicular and luteal phases. As a result, not only infertility and irregular menstruation but metabolic disorders such as diabetes and cardiovascular complications also occur (Yang & Chen, 2024 ). Conventional therapies, including ovulation-inducing agents like clomiphene citrate, letrozole and metformin are widely used along with oral contraceptives for cycle regulation. However, these treatments often carry unwanted side effects (Sun et al., 2020 ). Therefore, attention is being directed toward herbal and natural alternatives which are generally considered safer. The bergapten treatment notably modulated endocrine and metabolic parameters with a clear dose-dependent improvement in serum leptin, FSH and adiponectin (ADP) levels in current study. Elevated leptin levels observed in disease control group were significantly reversed by bergapten, particularly at 20 mg/kg dose, suggesting restoration of leptin sensitivity and amelioration of hypothalamic-pituitary-adipose axis dysregulation in PCOS. Similarly, FSH levels which are often elevated in experimental PCOS model were significantly reduced by bergapten especially at 10 and 20 mg/kg doses, indicating a potential regulatory effect on gonadotropin secretion and folliculogenesis, aligning with improved ovarian function. Interestingly, serum adiponectin levels were paradoxically elevated in HFD and letrozole disease group possibly represent compensatory stress response. However, normalization of ADP levels with bergapten treatment could reflect progress in metabolic homeostasis and adipocyte functionality. In addition, “hyperandrogenism” is a hallmark of PCOS in which reduced levels of estrogen and progesterone are pointing to impaired steroidogenesis and disrupted ovulatory function. As luteinizing hormone (LH) levels rise, hypothalamic-pituitary-ovarian axis become aberrant leading towards endocrine complications e.g., amenorrhea and infertility (Sharma et al., 2023 ). In present study, HFD diseased and letrozole control groups showed a decline in progesterone and estrogen with a parallel rise in testosterone, indicating hormonal imbalance characteristic of PCOS. Metformin partially ameliorated these changes, while bergapten treatment significantly upregulate progesterone and estrogen levels and reduced testosterone in a dose-dependent fashion. The highest dose of BA (20 mg/kg) restored LH, progesterone, testosterone and estrogen concentrations similar to normal control, suggesting enhanced ovarian steroidogenesis. These findings highlight ability of bergapten to counteract letrozole-induced endocrine disruptions and optimize reproductive hormonal balance. Histopathological analysis of ovarian and hepatic tissues in present study revealed that bergapten-treatment groups ameliorate morphological alterations in PCOS-induced rats. Ovarian tissue from disease control group exhibited cystic follicles, attenuated granulosa layers, and absent corpora lutea validating successful induction of disease through high-fat diet and letrozole administration. In contrast, bergapten treatment, particularly at higher doses (20 mg/kg), substantially restored ovarian architecture with complete regression of cystic follicles and normalization of granulosa and thecal layers. These changes may suggest reactivation of ovulatory processes and reversal of follicular arrest. Such outcomes are in matching with protective effects of carvacol, which were shown to normalize ovarian structure in similar PCOS models via antioxidant and anti-inflammatory mechanisms (Gao & Li, 2023 ). Regarding liver tissues, in bergapten-treatment groups inflammation was markedly reduced, indicating suppression of hepatic injury and immune cell infiltration. The absence of necrosis demonstrated preservation of hepatocyte integrity with healthy hepatic cords, adequate vascularization ultimately displayed functional recovery of hepatic tissue. Notably, 20mg/kg exhibited indistinguishable repair of hepatocytes, highlighting safety of bergapten even under metabolic stress conditions. Thus, bergapten may confer hepatoprotective benefits possibly due to free radical scavenging properties, as supported by earlier literature (Phucharoenrak & Trachootham, 2024 ). Gene expression in PCOS is significant to assess alterations and underlying mechanisms in genes regulating hormones, metabolism and ovarian function. In present study, treatment with bergapten showed downregulation of pro-inflammatory mediators such as TNF-α, IL-8, IL-6 and Keap1 and enhanced modulation of steroidogenic enzymes including CYP19A1 and CYP11A1. The transcription factor Nrf2, known for orchestrating antioxidant defense, was significantly downregulated in disease control group, consistent with heightened oxidative stress in PCOS. Bergapten treatment, especially at lower doses (5 mg/kg) led to partial restoration of Nrf2 expression, suggests that bergapten exerts moderate antioxidant effects. However, expression of Keap-1, a negative regulator of Nrf2, was also declined in bergapten-treatment groups. Findings suggest persistent activation of Nrf2 antioxidant pathway, thereby potentially enhancing cellular defense against oxidative stress. The decline in TNF-α and IL-8 with bergapten treatment expressed limited immune cell infiltration and protection against ovarian tissue damage. While, lowering of IL-6 demonstrates improved metabolic balance and mitigation of insulin resistance (Hoene & Weigert, 2008 ). These effects positioned bergapten as a potent modulator of inflammatory milieu in PCOS. Bergapten particularly at 20 mg/kg dose, reversed dysregulation of CYP19A1 and CYP11A1 enzymes, reflects its strong potential to mediate estradiol synthesis and regulate androgen production, implies a beneficial effect on ovulatory function (Xiao et al., 2023 ). Moreover, bergapten administration at 10 mg/kg and 20 mg/kg doses resulted in a partial restoration of PPAR-γ, a key transcription factor regulating insulin sensitivity and lipid metabolism (Liu et al., 2018 ). This finding indicates its potential role in alleviating PCOS-associated metabolic complications. The superior performance of 20 mg/kg bergapten across all serum biomarkers and gene expression mainly revealed a threshold-dependent pharmacodynamic effect, with higher dose supports in reinstating normal physiological state of organs. However, a nonlinear trend was observed, where 10 mg/kg dose did not produce as strong effect as lower 5 mg/kg dose in certain outcomes, possibly due to receptor saturation, feedback inhibition or dose-limited bioavailability. Bergapten pharmacological effects are likely mediated through its antioxidant, anti-inflammatory, and possibly PPAR-γ modulating properties, which have been reported in earlier studies on metabolic syndrome and reproductive health (Rudrapal et al., 2024 ). These findings underscore bergapten as a dual-action agent for managing both reproductive and metabolic abnormalities associated with PCOS. Conclusion The present study demonstrated that bergapten exerts beneficial effects in a high-fat diet and letrozole-induced model of polycystic ovarian syndrome in Wistar rats. Biochemical analysis showed marked improvement in serum hormonal and metabolic parameters, while histopathological assessment revealed preservation of ovarian and hepatic tissue architecture. Gene expression studies indicated favorable modulation of inflammatory and metabolic biomarkers. Collectively, these findings suggest that bergapten possesses promising therapeutic properties against PCOS, supporting its potential role in restoring endocrine balance and metabolic function. Further studies require additional examination of its molecular targets, tissue-level effects and comparative efficacy against PCOS like metformin to validate its clinical relevance. Declarations Competing Interests The authors have no relevant financial or non-financial interests to disclose. Ethics approval The animal study design was reviewed and approved by the Ethical Review committee of GCU Faisalabad [(Ref. No. GCUF/ERC/633]. Laboratory animals were used in this study as per approved protocol. Funding The authors declare that no funds, grants, or other support were received during the preparation of this manuscript. Author Contribution All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by **Abida Hussain** , **Ammara Saleem** and **Muhammad Furqan Akhtar** . The first draft of the manuscript was written by **Abida Hussain** and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript. Acknowledgment: The authors sincerely thank the Department of Pharmacology, GCUF, for providing laboratory facilities and technical assistance during the experimental work. Special thanks to the animal house staff for their cooperation during the animal studies. The authors also appreciate the support and guidance of Ammara Saleem throughout the research. Data Availability The original contributions presented in the study are included in the article/Supplementary Material; further inquiries can be directed to the corresponding author. 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14:03:24","extension":"html","order_by":34,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":141875,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7807066/v1/471ede8d5eca04389081b630.html"},{"id":94408148,"identity":"9b26da03-e8b1-4088-a1b9-d473b651afd0","added_by":"auto","created_at":"2025-10-27 14:03:25","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":150983,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of bergapten on body weight in obese-PCOS rats. Results are shown as mean ± SD (n=6). One-way ANOVA followed by Tukey’s multiple comparison test, where ∗p\u0026lt;0.05, ∗∗p\u0026lt;0.01, ∗∗∗p\u0026lt;0.001 and ns p \u0026gt; 0.05 respectively. Significance of normal control with treatment groups is shown as \u003cstrong\u003e“a”\u003c/strong\u003ewhile significance of HFD control with treatment groups is shown as \u003cstrong\u003e“b”\u003c/strong\u003e and\u003cstrong\u003e \u003c/strong\u003esignificance of standard control (metformin 300 mg/kg) with treatment groups is shown as \u003cstrong\u003e“c”\u003c/strong\u003e.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7807066/v1/f05c08060d21610d74f11cfd.png"},{"id":94407993,"identity":"9abd76b8-196e-4b27-9cce-e1051ff43c12","added_by":"auto","created_at":"2025-10-27 14:03:20","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":153724,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of bergapten treatment on blood glucose levels in obese-PCOS rats. Results are shown as mean ± SD (n=6). One-way ANOVA followed by Tukey’s multiple comparison test, where ∗p\u0026lt;0.05, ∗∗p\u0026lt;0.01, ∗∗∗p\u0026lt;0.001 and ns p \u0026gt; 0.05 respectively. Significance of normal control with treatment groups is shown as \u003cstrong\u003e“a”\u003c/strong\u003e while significance of HFD control with treatment groups is shown as \u003cstrong\u003e“b” \u003c/strong\u003esignificance of standard control (metformin 300 mg/kg) with treatment groups is shown as \u003cstrong\u003e“c”\u003c/strong\u003e.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7807066/v1/b2c8429944a5cbc557f5966b.png"},{"id":94407623,"identity":"65a95fbd-4463-4bc5-9fa2-3865e86d9613","added_by":"auto","created_at":"2025-10-27 14:03:05","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1030198,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of bergapten treatment on (a) LDL levels, (b) VLDL levels, (c) HDL levels and (d) total lipids in obese-PCOS rats. Results are shown as mean ± SD (n=6). One-way ANOVA followed by Tukey’s multiple comparison test, where ∗p\u0026lt;0.05, ∗∗p\u0026lt;0.01, ∗∗∗p\u0026lt;0.001 and ns p \u0026gt; 0.05 respectively. Significance of normal control with treatment groups is shown as \u003cstrong\u003e“a”\u003c/strong\u003ewhile significance of HFD control with treatment groups is shown as \u003cstrong\u003e“b” \u003c/strong\u003eand\u003cstrong\u003e \u003c/strong\u003esignificance of standard control (metformin 300 mg/kg) with treatment groups is shown as \u003cstrong\u003e“c”\u003c/strong\u003e.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7807066/v1/3303072398964a1a83446167.png"},{"id":94408153,"identity":"19e1ea9f-ee39-4935-b97b-bb524b4a9951","added_by":"auto","created_at":"2025-10-27 14:03:26","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":451449,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of bergapten treatment on (a) cholesterol level, (b) cholesterol/HDL ratio and (c) triglyceride level in obese-PCOS rats. Results are shown as mean ± SD (n=6). One-way ANOVA followed by Tukey’s multiple comparison test, where ∗p\u0026lt;0.05, ∗∗p\u0026lt;0.01, ∗∗∗p\u0026lt;0.001 and ns p \u0026gt; 0.05 respectively. Significance of normal control with treatment groups is shown as \u003cstrong\u003e“a”\u003c/strong\u003e while significance of HFD control with treatment groups is shown as \u003cstrong\u003e“b” \u003c/strong\u003eand\u003cstrong\u003e \u003c/strong\u003esignificance of standard control (metformin 300 mg/kg) with treatment groups is shown as \u003cstrong\u003e“c”\u003c/strong\u003e.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-7807066/v1/83318ce1131132803440e04a.png"},{"id":94408166,"identity":"5752034f-513d-4e64-8605-fbf84335145e","added_by":"auto","created_at":"2025-10-27 14:03:27","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":584994,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of bergapten treatment on (a) FSH level and (b) LH level in obese-PCOS rats. Results are shown as mean ± SD (n=6). One-way ANOVA followed by Tukey’s multiple comparison test, where ∗p\u0026lt;0.05, ∗∗p\u0026lt;0.01, ∗∗∗p\u0026lt;0.001 and ns p \u0026gt; 0.05 respectively. Significance of normal control with treatment groups is shown as \u003cstrong\u003e“a”\u003c/strong\u003e while significance of HFD control with treatment groups is shown as \u003cstrong\u003e“b” \u003c/strong\u003eand significance of standard control (metformin 300 mg/kg) with treatment groups is shown as \u003cstrong\u003e“c”\u003c/strong\u003e.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-7807066/v1/cb1f6216faf986e94130a387.png"},{"id":94407990,"identity":"48b09ea2-3346-4d2e-979e-98b81ae5ec1c","added_by":"auto","created_at":"2025-10-27 14:03:20","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":470181,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of bergapten treatment on (a) progesterone level, (b) testosterone level and (c) estrogen level in obese-PCOS rats. Results are shown as mean ± SD (n=6). One-way ANOVA followed by Tukey’s multiple comparison test, where ∗p\u0026lt;0.05, ∗∗p\u0026lt;0.01, ∗∗∗p\u0026lt;0.001 and ns p \u0026gt; 0.05 respectively. Significance of normal control with treatment groups is shown as \u003cstrong\u003e“a”\u003c/strong\u003e while significance of HFD control with treatment groups is shown as \u003cstrong\u003e“b” \u003c/strong\u003eand significance of standard control (metformin 300 mg/kg) with treatment groups is shown as \u003cstrong\u003e“c”\u003c/strong\u003e.\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-7807066/v1/0f92d1b07d3856554c31c8b9.png"},{"id":94405653,"identity":"1197f8e9-7a87-4c29-9a48-cd15b057b0fc","added_by":"auto","created_at":"2025-10-27 14:01:59","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":573181,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of bergapten treatment on (a) adiponectin level and (b) leptin level in obese-PCOS rats. Results are shown as mean ± SD (n=6). One-way ANOVA followed by Tukey’s multiple comparison test, where ∗p\u0026lt;0.05, ∗∗p\u0026lt;0.01, ∗∗∗p\u0026lt;0.001 and ns p \u0026gt; 0.05 respectively. Significance of normal control with treatment groups is shown as \u003cstrong\u003e“a”\u003c/strong\u003e while significance of HFD control with treatment groups is shown as \u003cstrong\u003e“b” \u003c/strong\u003eand\u003cstrong\u003e \u003c/strong\u003esignificance of standard control (metformin 300 mg/kg) with treatment groups is shown as \u003cstrong\u003e“c”\u003c/strong\u003e.\u003c/p\u003e","description":"","filename":"floatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-7807066/v1/365d6863cd4c696297d887c0.png"},{"id":94407163,"identity":"2fb9c498-5159-4847-aaeb-51a918225627","added_by":"auto","created_at":"2025-10-27 14:02:44","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":408190,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of bergapten treatment on (a) Nrf2, (b) PPRA-g and (c) keap-1 in obese-PCOS rats. Results are shown as mean ± SD (n=6). One-way ANOVA followed by Tukey’s multiple comparison test, where ∗p\u0026lt;0.05, ∗∗p\u0026lt;0.01, ∗∗∗p\u0026lt;0.001 and ns p \u0026gt; 0.05 respectively. Significance of normal control with treatment groups is shown as \u003cstrong\u003e“a”\u003c/strong\u003e while significance of HFD control with treatment groups is shown as \u003cstrong\u003e“b” \u003c/strong\u003eand\u003cstrong\u003e \u003c/strong\u003esignificance of standard control (metformin 300 mg/kg) with treatment groups is shown as \u003cstrong\u003e“c”\u003c/strong\u003e.\u003c/p\u003e","description":"","filename":"floatimage9.png","url":"https://assets-eu.researchsquare.com/files/rs-7807066/v1/0e252945dea64bd30fe768ac.png"},{"id":94408110,"identity":"be8e6854-7e83-4c94-8cf8-5c49797fa0cf","added_by":"auto","created_at":"2025-10-27 14:03:25","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":795245,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of bergapten treatment on (a) TNF-α,\u003cstrong\u003e(b) IL-6\u003c/strong\u003e and (c) \u003cstrong\u003eIL-8 \u003c/strong\u003ein obese-PCOS rats. Results are shown as mean ± SD (n=6). One-way ANOVA followed by Tukey’s multiple comparison test, where ∗p\u0026lt;0.05, ∗∗p\u0026lt;0.01, ∗∗∗p\u0026lt;0.001 and ns p \u0026gt; 0.05 respectively. Significance of normal control with treatment groups is shown as \u003cstrong\u003e“a”\u003c/strong\u003ewhile significance of HFD control with treatment groups is shown as \u003cstrong\u003e“b” \u003c/strong\u003eand\u003cstrong\u003e \u003c/strong\u003esignificance of standard control (metformin 300 mg/kg) with treatment groups is shown as \u003cstrong\u003e“c”\u003c/strong\u003e.\u003c/p\u003e","description":"","filename":"floatimage10.png","url":"https://assets-eu.researchsquare.com/files/rs-7807066/v1/65d9669c77ef01cba9fcdb94.png"},{"id":94407372,"identity":"0c52e4dd-4c80-45ef-aaba-c23d4fbcd9a1","added_by":"auto","created_at":"2025-10-27 14:02:55","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":502835,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of bergapten treatment on (a) CYP19A1 and (b) CYP11A1 enzymes in obese-PCOS rats. Results are shown as mean ± SD (n=6). One-way ANOVA followed by Tukey’s multiple comparison test, where ∗p\u0026lt;0.05, ∗∗p\u0026lt;0.01, ∗∗∗p\u0026lt;0.001 and ns p \u0026gt; 0.05 respectively. Significance of normal control with treatment groups is shown as \u003cstrong\u003e“a”\u003c/strong\u003e while significance of HFD control with treatment groups is shown as \u003cstrong\u003e“b” \u003c/strong\u003eand\u003cstrong\u003e \u003c/strong\u003esignificance of standard control (metformin 300 mg/kg) with treatment groups is shown as \u003cstrong\u003e“c”\u003c/strong\u003e.\u003c/p\u003e","description":"","filename":"floatimage11.png","url":"https://assets-eu.researchsquare.com/files/rs-7807066/v1/3bcc6004772aa83a11ed9858.png"},{"id":94408151,"identity":"c7515eb9-7b3e-4d43-9fd3-7cc7565ea5b5","added_by":"auto","created_at":"2025-10-27 14:03:26","extension":"png","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":1031657,"visible":true,"origin":"","legend":"\u003cp\u003eVaginal smear cytology of normal control and obesity-induced PCOS rats at 40X (a)\u003cstrong\u003e \u003c/strong\u003enormal control group; (b) HFD-disease control.\u003c/p\u003e\n\u003cp\u003eNEC: normal epithelial cells; CFR: collapsed follicular remnants; LC: Leukocytes; FST: fibrotic stromal tissues; DEC: degenerated epithelial cells; SH: stromal hyperplasia; NR: necrotic remnants\u003c/p\u003e","description":"","filename":"floatimage12.png","url":"https://assets-eu.researchsquare.com/files/rs-7807066/v1/efee7af0bbc8e75dc352a9ef.png"},{"id":94405651,"identity":"f4dbca11-8728-468c-94bc-0c01cd038b76","added_by":"auto","created_at":"2025-10-27 14:01:59","extension":"png","order_by":12,"title":"Figure 12","display":"","copyAsset":false,"role":"figure","size":346206,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of bergapten treatment on OWI in obese-PCOS rats. Results are shown as mean ± SD (n=6). One-way ANOVA followed by Tukey’s multiple comparison test, where ∗p\u0026lt;0.05, ∗∗p\u0026lt;0.01, ∗∗∗p\u0026lt;0.001 and ns p \u0026gt; 0.05 respectively. Significance of normal control with treatment groups is shown as \u003cstrong\u003e“a”\u003c/strong\u003ewhile significance of HFD control with treatment groups is shown as \u003cstrong\u003e“b”\u003c/strong\u003e.\u003c/p\u003e","description":"","filename":"floatimage13.png","url":"https://assets-eu.researchsquare.com/files/rs-7807066/v1/f6043937a2d5c7d8617f2919.png"},{"id":94408008,"identity":"4388c893-c161-4c56-8164-f67c48ce1c4c","added_by":"auto","created_at":"2025-10-27 14:03:21","extension":"png","order_by":13,"title":"Figure 13","display":"","copyAsset":false,"role":"figure","size":777407,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of bergapten on histopathology of ovary in obesity-induced infertile rats at 40X \u003cstrong\u003e(\u003c/strong\u003ea\u003cstrong\u003e) \u003c/strong\u003enormal control group; \u003cstrong\u003e(\u003c/strong\u003eb\u003cstrong\u003e)\u003c/strong\u003e HFD-disease control; \u003cstrong\u003e(\u003c/strong\u003ec\u003cstrong\u003e)\u003c/strong\u003e Letrozole control; \u003cstrong\u003e(\u003c/strong\u003ed\u003cstrong\u003e)\u003c/strong\u003eStandard control (metformin 300 mg/kg); \u003cstrong\u003e(\u003c/strong\u003ee\u003cstrong\u003e) \u003c/strong\u003e5mg/kg BA treatments;\u003cstrong\u003e (\u003c/strong\u003ef)\u003cstrong\u003e \u003c/strong\u003e10mg/kg BA treatment; \u003cstrong\u003e(\u003c/strong\u003eg\u003cstrong\u003e)\u003c/strong\u003e 20mg/kg BA treatment\u003c/p\u003e","description":"","filename":"floatimage14.png","url":"https://assets-eu.researchsquare.com/files/rs-7807066/v1/208f548e0575cabf4325a1d7.png"},{"id":94406986,"identity":"e6796d17-1c6f-446c-8813-5d01960ad819","added_by":"auto","created_at":"2025-10-27 14:02:39","extension":"png","order_by":14,"title":"Figure 14","display":"","copyAsset":false,"role":"figure","size":749162,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of bergapten in on histopathology of Liver in obesity-induced infertile rats at 40X (a\u003cstrong\u003e)\u003c/strong\u003e; normal control group (b\u003cstrong\u003e)\u003c/strong\u003e; Disease Control HFD group \u003cstrong\u003e(\u003c/strong\u003ec\u003cstrong\u003e)\u003c/strong\u003e; Letrozole control (NHFS = non-high fat diet) \u003cstrong\u003e(\u003c/strong\u003ed\u003cstrong\u003e);\u003c/strong\u003e Standard control group (metformin 300 mg/kg) (e\u003cstrong\u003e)\u003c/strong\u003e; Treatment group 5 mg/kg doses (f\u003cstrong\u003e)\u003c/strong\u003e; Treatment group 10 mg/kg (g\u003cstrong\u003e)\u003c/strong\u003e; Treatment group 20 mg/kg.\u003c/p\u003e","description":"","filename":"floatimage15.png","url":"https://assets-eu.researchsquare.com/files/rs-7807066/v1/60c2532649252712d67cf8f9.png"},{"id":94505573,"identity":"e726a6af-6542-4e3b-a1c1-5062a65d118d","added_by":"auto","created_at":"2025-10-28 16:12:44","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":9149428,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7807066/v1/46dad2c3-3877-4c66-a7eb-2cd6525cca5c.pdf"},{"id":94489595,"identity":"bbe59b94-a90a-49d0-b6a2-deabb8598421","added_by":"auto","created_at":"2025-10-27 17:05:14","extension":"jpeg","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":113445,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7807066/v1/1f3f249ae24e9b7c8f51912d.jpeg"}],"financialInterests":"No competing interests reported.","formattedTitle":"Pharmacological Evaluation of Bergapten in High Fat Diet and Letrozole Induced Polycystic Ovarian Syndrome in Wistar Rats","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003ePolycystic ovarian syndrome (PCOS) is also recognized as \u0026ldquo;hyperandrogenic anovulation (HA) or Stein-Leventhal syndrome\u0026rdquo;, particularly common in reproductive-age women. PCOS is chronic and heterogeneous condition, clinically characterized by obesity, menstrual irregularities, infertility, hirsutism (Singh et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Obesity is a chronic metabolic disorder characterized by excessive accumulation of adipose tissue that disrupts endocrine and metabolic homeostasis worldwide. In women, obesity not only predisposes to metabolic disorders (e.g., type 2 diabetes mellitus) but contributes significantly to reproductive abnormalities (e.g., infertility and polycystic ovarian syndrome (PCOS), menstrual dysfunction) and cardiovascular disease (El Hayek et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Epidemiological studies consistently reported that majority of women with PCOS are overweight and having a high risk of infertility (Moazzam et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eMorphologically, hyperandrogenism is regarded as a major clinical manifestation of PCOS, disrupts follicular progression, develops anovulation and ovarian microcysts with multiple small follicles (\u0026ge;10 of 2\u0026ndash;9 mm) (Abinaya et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). The World Health Organization (WHO) reported that approximately 116\u0026nbsp;million women (3.4%) worldwide affected with PCOS (Deswal et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The high prevalence of PCOS and its association with ovulatory dysfunction, metabolic disturbances and dermatological complications, highlights its substantial health and economic burden. Although PCOS can appear at any stage from \u0026ldquo;menarche onwards\u0026rdquo;, most diagnoses occur between 20\u0026ndash;30 years of age (Salari et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). In 2017, it was estimated that PCOS affected 1.55\u0026nbsp;million reproductive-age women globally, accounting for 0.43\u0026nbsp;million of disease burden. Several studies have shown that both genetic predisposition and environmental determinants are central to PCOS development. Likewise, PCOS may also emerge early in adolescents with familial predisposition, irregular dietary habits, toxins and exposure to pollutants (Omar et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2025\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eFew researches have evidenced that various influential mechanisms in PCOS are endocrine disruption, prolonged inflammation, insulin insensitivity and androgen excess (Gupta \u0026amp; Khan, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). PCOS is an \u0026ldquo;oligogenic disorder\u0026rdquo; with multifactorial pathogenesis majorly involves impairment of hypothalamic-pituitary axis (HPA), pancreatic insulin regulation and ovarian activity. Although precise etiology remains elusive, strong associations with insulin resistance and obesity have been consistently explored yet (Ding et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Shoaib et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Obesity amplifies prevalence of glucose intolerance, dyslipidemia and ovulatory failure in PCOS, highlighting metabolic-reproductive overlap in its pathophysiology. The relationship between obesity and PCOS is multifaceted, as obesity acts both as a risk factor and as a provoking condition for syndrome. High-fat diet (HFD)-induced obesity enhances insulin resistance and hyperinsulinemia, all of which contribute to excessive androgen production by ovarian theca cells (Malik et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Elevated androgens perpetuate folliculogenesis, leading to anovulation. Moreover, obesity-related reductions in sex hormone-binding globulin (SHBG) increase circulating free testosterone, further aggravating hyperandrogenic symptoms. In parallel, reduced adiponectin levels and elevated leptin concentrations in obese women may also worsen symptoms of PCOS (Ding et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Kim, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eBergapten (BP), is a naturally occurring furocoumarin found in various medicinal plants of \u003cem\u003eUmbelliferae\u003c/em\u003e family such as \u003cem\u003eCnidii Fructus, Angelica dahurica, and Peucedanum ostruthium\u003c/em\u003e. This compound is also present in grapefruit juice, essential oil of bergamot and several other essential oils from citrus fruits (Liang et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The chemical name of bergapten is 5-methoxypsoralen, which has a basic benzo-[alpha]-pyrone structure. Bergapten is recognized as a potent \u0026ldquo;photosensitizing compound\u0026rdquo; and exhibits diverse pharmacological actions including anticancer, antibacterial, hypolipidemic and anti-inflammatory activities (Aslam et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Phucharoenrak \u0026amp; Trachootham, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eDespite, adipose tissue expansion in obesity activates irregular Keap1 signaling, which suppresses Nrf2-mediated antioxidant defenses. Consequently, a pro-oxidative environment generates leading towards granulosa cells apoptosis, mitochondrial dysfunction and reduced estrogen biosynthesis (Li et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Zhou et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). In parallel, obesity triggers nuclear factors kappa-β (NF-κβ) activation, enhancing inflammatory responses of cytokines (e.g., IL-6, IL-8 and TNF-α). Importantly, clinical evidence suggests that addressing obesity should be a primary therapeutic target in PCOS management, as weight reduction improves insulin sensitivity, restores ovulation and endocrine balance (Wawrzkiewicz-Jałowiecka et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Clinical data suggest that obesity not only increases risk of infertility but also prolongs time required to achieve pregnancy compared to non-obese women (Shoaib et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eTherefore, present study was designed to investigate therapeutic potential of bergapten in experimental models of polycystic ovary syndrome (PCOS) induced by letrozole administration in combination with a high-fat diet (HFD). Furthermore, research aimed to explore underlying mechanisms through which bergapten exerts its pharmacological effects, with particular attention to hormonal regulation, insulin sensitivity and inflammatory pathways in obese-PCOS rats.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1. Chemicals\u003c/h2\u003e\u003cp\u003eAnalytical grade chemicals and reagents were used in whole study. Standard drug metformin (Glucophage 500mg) was purchased from Martin-Dow pharma, Pakistan. Letrozole (5g) was obtained from Saffron Pharmaceutical Faisalabad, Pakistan. Bergapten was acquired from Biosynth Slovakia. Preparation of solutions was done by using dimethyl sulfoxide (DMSO) and carboxyl-methyl cellulose (Sigma Aldrich, UK) ,10% formalin, chloroform from Merk, Germany.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2. Experimental Animals\u003c/h2\u003e\u003cp\u003eHealthy nulliparous female \u003cem\u003eWistar\u003c/em\u003e rats of n\u0026thinsp;=\u0026thinsp;60 (100-150g) were acquired and housed in animal house of Department of Pharmacology, Government College University Faisalabad (GCUF). All animals were provided with standard laboratory conditions by following temperature (23\u0026thinsp;\u0026plusmn;\u0026thinsp;2 \u003csup\u003eo\u003c/sup\u003eC), humidity (55\u0026thinsp;\u0026plusmn;\u0026thinsp;5) and maintained 12 hours\u0026rsquo; light/dark cycles, with unlimited supply of water and pallet diet. An acclimatization period of seven days was provided to adapt rats with laboratory environment. The experiment was conducted by following protocols of ethical committee of Government College University Faisalabad (GCUF). The study protocol was approved by Research Ethical Committee of Government College University Faisalabad Approval No. GCUF/ERC/633.\u003c/p\u003e\u003cp\u003e All procedures involving animals followed guidelines of National Research Council and efforts were made to minimize any potential harm.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3. Obesity and PCOS induction\u003c/h2\u003e\u003cp\u003eThe animals were randomly divided into two groups. Before studying initiation, body weight of animals was measured in grams. The normal control group animals (n\u0026thinsp;=\u0026thinsp;12) were maintained on a standard pallet diet. Remaining animals (48) were administered on a high fat diet (HFD) including 60% fats, 20% carbohydrates and 20% protein for 12 weeks to induce obesity. REF\u003c/p\u003e\u003cp\u003eFollowing HFD regimen, all animals were weighed weekly, and obesity status was determined using Lee obesity index with following formula.\u003c/p\u003e\u003cp\u003e\u003cb\u003eLee obesity index\u003c/b\u003e\u0026thinsp;=\u0026thinsp;3 cube roots (weight)/ length (cm)\u0026times;1000\u003c/p\u003e\u003cp\u003eAnimals with a Lee index\u0026thinsp;\u0026gt;\u0026thinsp;320 were classified as obese and selected for further study (Shoaib et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eLetrozole treatment started after 2months and 9 days to induce PCOS in HFD taking animals.\u003c/p\u003e\u003cp\u003e6 animals out of 12 normal controls received a daily oral dose of 5 ml of 0.5% carboxymethyl cellulose (CMC) as vehicle, while other 6 normal animals were administered letrozole and served as letrozole control.\u003c/p\u003e\u003cp\u003eWhile obese rats received letrozole (LET) 1mg/kg/day per oral (suspended in 0.5% CMC) once daily for 28 days to induce PCOS ((Hussain et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) .\u003c/p\u003e\u003cp\u003eFor PCOS confirmation, to perform vaginal cytology, Vaginal smear was taken after 21 days and studied estrous cycle, but no estrous cycle was observed under light microscope. Then letrozole was given for another 7days. Vaginal cytology performed and the estrous cycle was confirmed after 28 days. The weight deviation record was also being taken on a weekly basis.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4. Experimental Design\u003c/h2\u003e\u003cp\u003eAfter successful induction of PCOS and obesity, animals were divided into 7 groups(n\u0026thinsp;=\u0026thinsp;6) normal control treated with normal diet and distilled water. Disease control obese were treated with high fat diet (HFD) and letrozole (LT), disease control PCOS were treated with letrozole (LT), obese\u0026thinsp;+\u0026thinsp;PCOS were treated with metformin 300 mg/kg (Standard control) and obese\u0026thinsp;+\u0026thinsp;PCOS were treated with different doses of bergapten 5mg/kg, 10mg/kg, 20mg/kg treatment.\u003c/p\u003e\u003cp\u003eTreatment as mentioned below was started orally for a period of 28 days and their fasting blood glucose levels were measured after 28th day. The animals were allocated into groups as outlined below.\u003c/p\u003e\u003cp\u003e\u003cul\u003e\u003cli\u003e\u003cp\u003eGroup I (normal control): received a normal chow diet.\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003eGroup II (disease control obese): High fat diet (HFD)\u0026thinsp;+\u0026thinsp;Letrozole (LT)\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003eGroup III (disease control PCOS): Letrozole (LT)\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003eGroup IV (obese\u0026thinsp;+\u0026thinsp;PCOS): Metformin 300 mg/kg (Standard control)\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003eGroup V (obese\u0026thinsp;+\u0026thinsp;PCOS): Bergapten 5mg/kg (Treatment)\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003eGroup VI (obese\u0026thinsp;+\u0026thinsp;PCOS): Bergapten 10mg/kg (Treatment)\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003eGroup VII (obese\u0026thinsp;+\u0026thinsp;PCOS): Bergapten 20mg/kg (Treatment)\u003c/p\u003e\u003c/li\u003e\u003c/ul\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.5. Vaginal smear cytology\u003c/h2\u003e\u003cp\u003eVaginal smears were collected by gently inserting a sterile micropipette into vaginal canal and flushing with 10\u0026ndash;20 \u0026micro;L of sterile saline, after which fluid was aspirated and spread thinly onto pre-labeled glass slides, air-dried and stained with 0.1% crystal violet (methylene blue). Slides were examined under a light microscope at 40X, and difference was noted under microscope.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e2.6. Oral glucose tolerance test (OGTT)\u003c/h2\u003e\u003cp\u003eAfter 28 days of drug treatment, rats were fasted overnight. Thirty minutes after administration of ongoing treatment,2g/kg glucose solution was administered orally. Blood samples were collected from tail vein using glucometer and readings were recorded at 0, 60, 120, and 180 minutes respectively.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e2.7. Biochemical testing\u003c/h2\u003e\u003cp\u003eAfter completing treatment period, animals were anesthetized by pentobarbital sodium (40mg/kg, IP) blood sample was collected by cardiac puncture, and serum was separated and process for biochemical testing of lipids parameters such as total cholesterol (TC), triglycerides (TG), and levels of hormones including follicle-stimulating hormone (FSH), serum insulin, testosterone, luteinizing hormone (LH), estrogen and progesterone.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e2.8. Estimation of ovary weight index (OWI)\u003c/h2\u003e\u003cp\u003eThe anesthetized animals were euthanized through cervical dislocation, ovaries and liver were carefully removed. Ovaries were cleaned and weighed properly by using weighing balance. Afterwards, OMI was calculated by following formula:\u003c/p\u003e\u003cp\u003eOWI\u0026thinsp;=\u0026thinsp;Ovary weight (mg)​/ Body weight (g)\u0026times;100\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e2.9. Histopathological analysis\u003c/h2\u003e\u003cp\u003eOvaries and liver were removed, rinsed with ice-cold normal saline and preserved in 10% formaldehyde solution. Using a rotary microtome, tissues (5\u0026micro;m thick) were embedded in paraffin wax, mounted on glass slides and stained with hematoxylin-eosin dye. While cytology of vaginal smear was analyzed with hematoxylin-eosin dye. Histological changes of ovaries (e.g., number /size of follicles and corpus luteum) and liver (e.g., hepatic inflammation, necrosis, fibrosis, scar tissues) were observed under a light microscope at magnifications of 40X and 100X (Moazzam et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Younas et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e2.10. Estimation of genetic biomarkers\u003c/h2\u003e\u003cp\u003eGenetic biomarkers such as Nrf-2/keap1, PPRA-γ, TNF, IL8, IL6, Cyp11A1 and Cyp19A1 were quantified to assess genetic alterations. Total RNA was isolated from serum samples using TRIzol reagent (Invitrogen, CA, USA) following manufacturer\u0026rsquo;s protocol. Complementary DNA (cDNA) was synthesized with a commercial reverse transcription kit and amplification was then carried out on quantitative real-time PCR System (Hussain et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) .\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003e3.1. Effect of bergapten on body weight\u003c/h2\u003e\u003cp\u003eThe HFD control exhibited a marked (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) increase in body weight compared to normal control, indicating persistent obesity. The weight of letrozole control group also elevated. All BA treated groups and standard control (300 mg/kg) showed a noticeable reduction in body weight, suggesting therapeutic efficacy Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. BA treatment restored body weight dose dependently.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003e\u003cb\u003e3.2. Effect of bergapten on oral glucose tolerance test (OGTT)\u003c/b\u003e\u003c/h2\u003e\u003cp\u003eIn oral glucose tolerance test, glucose concentrations were measured at 0, 60, 120 and 180 minutes using a glucometer (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). After 1h, 2h and 3h, all bergapten and metformin 300mg/kg treatment groups showed significantly (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) decrease in glucose levels as compared to HFD control. Moreover, HFD control group exhibited noticeable (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) increase in glucose levels relative to normal control. Blood glucose in letrozole control was also consistently increased.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003e3.3. Effect of bergapten on LDL, VLDL, total lipids and HDL\u003c/h2\u003e\u003cp\u003eThe effect of bergapten on low-density lipoproteins (LDL), very low-density lipoproteins (VLDL), high density lipoproteins (HDL) and total lipids showed significant variations across experimental groups in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e.\u003c/p\u003e\u003cp\u003eHFD control (114.1\u0026thinsp;\u0026plusmn;\u0026thinsp;4.15 mg/dl) showed marked elevation in LDL as compared to normal control (43.67\u0026thinsp;\u0026plusmn;\u0026thinsp;2.43 mg/dl), confirming development of dyslipidemia. Standard control produced notable (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) reduction in LDL when compared with normal control. Similarly, BA (5mg/kg,10mg/kg,20 mg/kg\u003cb\u003e)\u003c/b\u003e showed a highly significant decrease in LDL concentrations dose dependently relative to HFD control group. LDL concentration was also higher in letrozole control group (113.6\u0026thinsp;\u0026plusmn;\u0026thinsp;3.51 mg/dl).\u003c/p\u003e\u003cp\u003eThe concentration of VLDL were escalated in HFD control (19.77\u0026thinsp;\u0026plusmn;\u0026thinsp;2.03 mg/dl) than normal control (18.73\u0026thinsp;\u0026plusmn;\u0026thinsp;1.88 mg/dl). In contrast, a significant (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) decrease in VLDL levels was recorded in standard (metformin 300mg/kg) control group compared to normal control. Moreover, BA at 5, 10 and 20mg/kg (13.30\u0026thinsp;\u0026plusmn;\u0026thinsp;1.72, 10.73\u0026thinsp;\u0026plusmn;\u0026thinsp;1.15 and 9.73\u0026thinsp;\u0026plusmn;\u0026thinsp;1.09 mg/dL) produced a marked (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) decrease in VLDL values when compared with HFD control group. An elevation in VLDL levels was also noted in letrozole group.\u003c/p\u003e\u003cp\u003eThe concentrations of total lipids in HFD control had significantly elevated in PCOS-induced rats than normal control group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Additionally, standard group also produced significant lipid-lowering effect (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) relative to normal control. Conversely, groups received different BA treatment showed a sharp (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) decline in total lipids in comparison with HFD control. While LDL concentrations of total lipids was higher in letrozole control group (362.3\u0026thinsp;\u0026plusmn;\u0026thinsp;6.02 mg/dL). Hence, all bergapten treatment groups restored LDL, VLDL and total lipids levels close normal control.\u003c/p\u003e\u003cp\u003eRegarding HDL, HFD control group showed sharp reduction as compared to normal control. The levels of HDL were shown to be increase in standard control (30.90\u0026thinsp;\u0026plusmn;\u0026thinsp;1.64 mg/dl) just similar to normal control. While, BA treatment at dose of 5 mg/kg (39.97\u0026thinsp;\u0026plusmn;\u0026thinsp;1.69 mg/dl), 10 mg/kg (42.98\u0026thinsp;\u0026plusmn;\u0026thinsp;2.21 mg/dl) and 20 mg/kg (64.48\u0026thinsp;\u0026plusmn;\u0026thinsp;2.26 mg/dl) also produced a distinct rise in HDL concentrations as compared to HFD control. Thus, all BA treatment groups significantly exceeded HDL concentrations in a dose-dependent manner, indicating a strong HDL-elevating potential. The letrozole control group demonstrated obvious decrease in HDL levels.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003e3.4. Effect of bergapten on cholesterol and triglycerides\u003c/h2\u003e\u003cp\u003eHFD-disease control (91.07\u0026thinsp;\u0026plusmn;\u0026thinsp;4.67 mg/dl) group exhibited significant elevation in cholesterol levels compared to normal control (77.92\u0026thinsp;\u0026plusmn;\u0026thinsp;2.05 mg/dl). Standard control (58.18\u0026thinsp;\u0026plusmn;\u0026thinsp;2.33 mg/dl) significantly reduced cholesterol levels similar to normal control. A dose-dependent hypocholesterolemia effect was observed with BA-5 mg/kg, BA-10 mg/kg and BA-20 mg/kg, led to notable decrease (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) than HFD control. Furthermore, letrozole control (101.9\u0026thinsp;\u0026plusmn;\u0026thinsp;4.30 mg/dl) resulted in a marked (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) increase in cholesterol levels.\u003c/p\u003e\u003cp\u003eAccording to effect on serum triglycerides (TGs), HFD control showed highest concentration (105.6\u0026thinsp;\u0026plusmn;\u0026thinsp;5.69 mg/dl) as compared to normal control (40.00\u0026thinsp;\u0026plusmn;\u0026thinsp;2.65 mg/dl). Metformin 300 mg/kg (43.22\u0026thinsp;\u0026plusmn;\u0026thinsp;2.41 mg/dl) resulted in a substantial reduction in TGs levels just like normal control. Subsequently, three doses of bergapten (BA) treatment groups demonstrated significantly lower TGs levels in comparison to HFD control group. Letrozole showed more pronounced increase (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) in TGs overall.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003e3.5. Effect of bergapten on cholesterol/HDL ratio\u003c/h2\u003e\u003cp\u003eHDL-cholesterol ratio displayed a strong therapeutic effect of bergapten in PCOS-induced rat model. The normal control group maintained this ratio with value of 1.83\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07, whereas HFD-disease control showed a marked decline in value as 1.51\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07 respectively. Standard control significantly improved (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) HDL-cholesterol ratio to 1.86\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06, restoring values close to normal control. Bergapten 5 mg/kg (1.38\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05) and 10 mg/kg (1.54\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06) and 20 mg/kg (2.05\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11) produced modest increase (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) in HDL-cholesterol ratio, when compared with HFD control. Values of letrozole control group were lessened to 1.31\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04 respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003e3.6. Effect of bergapten on FSH and LH\u003c/h2\u003e\u003cp\u003eEffect of bergapten on serum FSH and LH levels across experimental groups is presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e. The normal control group recorded FSH levels as 1.21\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08 IU/mL whereas disease control shown a marked decline to 1.01\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09 IU/milk Metformin 300mg/kg treatment group (standard) restored FSH to near-normal control levels. All doses of BA treatment produced a dose-dependent rise (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) in FSH, compared to HFD control. On the other hand, HFD-diseased control (1.09\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04) exhibited a sharp increase in LH than normal control (0.25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02 mIU/mL). Standard control significantly reduced LH to 0.39\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04 mIU/mL approaching normal control levels. At 5 mg/kg BA, LH was lowered to 0.68\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03 mIU/m while BA at 10 mg/kg (0.95\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07 mIU/mL) and 20 mg/kg (0.87\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03 mIU/mL), maintained LH values markedly below HFD control. The significant (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) increase in LH and reduction in FSH was observed in letrozole control group.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003e3.7. Effect of bergapten on testosterone, progesterone and estrogen\u003c/h2\u003e\u003cp\u003eBergapten showed modulatory effect on progesterone, testosterone and estrogen levels. Standard control group exhibited elevated concentrations of progesterone (2.88\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04 ng/mL) than disease control (1.92\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05 ng/mL). However, group received metformin 300mg/kg produced a marked elevation in progesterone just like normal control. BA administration further enhanced progesterone levels in a dose-dependent manner, with 5 mg/kg (3.20\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11 ng/mL), 10 mg/kg (3.04\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06 ng/mL) and 20 mg/kg (4.76\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10 ng/mL) respectively. All BA-treated groups produced superior results relative to HFD-diseased control, highlighting therapeutic efficacy of bergapten. Letrozole treatment group showed significant drop in progesterone levels as 1.80\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07 ng/mL.\u003c/p\u003e\u003cp\u003eOn the other side, standard control displayed strongest recovery of testosterone concentrations, similar to normal group. The levels of testosterone in each BA group showed significant improvement compared with HFD control diseased (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), though levels remained below normal control (3.51\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04). Resultantly, bergapten treatment approached same effect as of standard (3.04\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07 ng/mL) control overall. Levels of testosterone were suppressed in letrozole control group significantly (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001).\u003c/p\u003e\u003cp\u003eFurthermore, treatment with metformin 300mg/kg, 5, 10 and 20mg/kg BA also lowered estrogen concentrations in a dose-responsive pattern with respect to HFD control, thus demonstrated significant therapeutic response (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). While, metformin 300mg/kg treatment group showed sharp reduction than normal control, but close to reference range (15\u0026ndash;350 pg/mL). Levels of estrogen was raised in letrozole control group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\u003ch2\u003e3.8. Effect of bergapten on adiponectin and leptin\u003c/h2\u003e\u003cp\u003eThe change in serum concentration of adiponectin (ng/mL) and leptin (\u0026micro;g/mL) after bergapten administration are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e. Mean leptin concentration in normal control (4.12\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13) was below as compared to diseased control (12.57\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15), confirming leptin dysregulation \u003cem\u003evia\u003c/em\u003e PCOS induction (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). Standard control and BA treatment significantly lowered leptin concentrations than disease control (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). Alternatively, normal control and HFD group displayed serum adiponectin levels as 17.23\u0026thinsp;\u0026plusmn;\u0026thinsp;0.47, and 8.52\u0026thinsp;\u0026plusmn;\u0026thinsp;0.27 respectively. Standard control effectively restored adiponectin (18.24\u0026thinsp;\u0026plusmn;\u0026thinsp;0.46 \u0026micro;g/mL) levels thus outperforming normal control and closely match reference values. Moreover, BA 5, 10 and 20mg/kg produced strongest reduction in adiponectin, thus showed significant (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) improvement compared to HFD control. Overall, letrozole control (10.98\u0026thinsp;\u0026plusmn;\u0026thinsp;0.14) groups exhibited considerable (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) effects on leptin and adiponectin levels.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\u003ch2\u003e3.9. Effect of bergapten on Nfr-2, PPARγ and Keap-1\u003c/h2\u003e\u003cp\u003eThe levels of Nrf2, PPAR-γ, and Keap1 across various experimental groups including normal control, diseased control, letrozole control, standard control (300 mg/kg) and BA treatments (5, 10, and 20 mg/kg) is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e. Nrf2 expression was significantly elevated in normal control, while disease control showed obvious reduction (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Standard treatment restored Nrf2 expression near to values of normal control. While, BA (10 and 20 mg/kg) treatment groups caused a significant upregulation (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) as compared to HFD control. PPAR-γ levels were elevated in HFD-disease control but remained low in normal control. Metformin 300mg/kg treatment group showed appreciable (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) decrease in PPAR-γ expression. Furthermore, bergapten treatment groups significantly decreased PPAR-γ compared to HFD control (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001).\u003c/p\u003e\u003cp\u003eGenetic expression of Keap1 was significantly (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) downregulated in standard control group, comparable to normal control. While, upregulated in HFD control than normal control. Treatment with three different doses (5,10 and 20mg/kg) of BA also significantly and dose-dependently reduced Keap1 expression (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) compared to HFD-disease control, indicating suppression of Nrf2. In letrozole control group, trend of Nfr-2, PPAR-γ and Keap-1 levels was consistent with HFD control group.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec23\" class=\"Section2\"\u003e\u003ch2\u003e\u003cb\u003e3.10. Effect of bergapten on TNF-α, IL-6 and IL-8\u003c/b\u003e\u003c/h2\u003e\u003cp\u003eThe expression levels of pro-inflammatory cytokines TNF-α, IL-6 and IL-8 were assessed across different experimental groups in PCOS induced rats (\u003cb\u003eFig.\u0026nbsp;9\u003c/b\u003e). For TNF-α, diseased control group exhibited highest expression than normal control. Conversely, metformin (300mg/kg) treated groups showed moderate reduction, equivalent to normal control group. However, BA-treatment groups (5, 10, and 20 mg/kg) showed dramatic decrease in TNF-α expression, that was statistically significant compared to HFD control (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01). IL-6 followed a similar pattern of TNF-α. For, IL-6 and IL-8, HFD-disease control group showed highest fold change than normal control. Meanwhile metformin (300mg/kg) treatment showed less fold change, resemble with normal control. BA treatments at all doses significantly (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) suppressed expression of both genes (IL-6 and IL-8), thus produced prominent anti-inflammatory effect in PCOS-induced rats. Letrozole control group showed increase in all inflammatory markers (e.g., IL-8, IL6 and TNF-α) concentrations.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec24\" class=\"Section2\"\u003e\u003ch2\u003e3.11. Effect on CYP19A1 and CYP11A1\u003c/h2\u003e\u003cp\u003eThe comparative effects of bergapten on serum CYP19A1 and CYP11A1 enzymes across all experimental groups are demonstrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e10\u003c/span\u003e. HFD control group exhibited a marked decrease in CYP19A1 expression than normal control. Metformin 300mg/kg exhibited sudden rise in CYP19A1 levels, consistent with normal control. While BA treatment groups (5, 10, 20mg/kg) showed significant (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) upregulate CYP19A1 expression than HFD control. Data of CYP11A1 expression indicated that disease control (HFD) manifested elevation as compared to normal control. While standard control (metformin 300mg/kg) showed slight decline, close to normal control group. Treatment with bergapten (5, 10 and 20mg/kg) demonstrated downregulation of CYP11A1 compared to HFD control (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01). The net effect of letrozole control was induction of CYP19A1 expression (~\u0026thinsp;3.0 fold), while suppression of CYP11A1 expression confirms earlier reports.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec25\" class=\"Section2\"\u003e\u003ch2\u003e3.12. Vaginal Smear Cytology\u003c/h2\u003e\u003cp\u003eVaginal Smear cytology, a type of exfoliative histology used to assess estrous cycle stage in PCOS-induced rats. In normal healthy control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e11\u003c/span\u003e), smear shows predominantly nucleated epithelial cells with some presence of cornified cells and minimal leukocytes. cells appear polygonal, with some clusters and nuclei clearly visible. While in HFD diseased control group, four different stages of estrous cycle were analyzed. The proestrus phase corresponds to \u0026ldquo;follicular stage\u0026rdquo;, while estrus phase denotes \u0026ldquo;ovulation\u0026rdquo;. During estrus, smears typically show large, irregular, non-nucleated cornified epithelial cells. The metestrus stage is recognized by mixture of leukocytes along with both nucleated and non-nucleated cornified epithelial cells. In diestrus, a predominance of leukocytes with nucleated epithelial cells is observed (A). Following PCOS induction, this regular pattern becomes disrupted and estrous cycle shows irregularity, with a significant prolongation of metestrus stage. Smears in this condition display an abundance of leukocytes together with non-nucleated cornified epithelial cells, confirming disturbance caused by PCOS (B).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eNEC: normal epithelial cells; CFR: collapsed follicular remnants; LC: Leukocytes; FST: fibrotic stromal tissues; DEC: degenerated epithelial cells; SH: stromal hyperplasia; NR: necrotic remnants\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec26\" class=\"Section2\"\u003e\u003ch2\u003e3.13. Effect on OWI (%)\u003c/h2\u003e\u003cp\u003eThe change in OWI after bergapten administration is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e12\u003c/span\u003e. Mean value of OMI in normal control (0.038\u0026thinsp;\u0026plusmn;\u0026thinsp;0.004) was below as compared to diseased control (0.049\u0026thinsp;\u0026plusmn;\u0026thinsp;0.003), confirming ovarian changes \u003cem\u003evia\u003c/em\u003e PCOS induction (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Standard control and BA treatment significantly reduce these alterations.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec27\" class=\"Section2\"\u003e\u003ch2\u003e3.14. Histopathological Evaluation\u003c/h2\u003e\u003cp\u003eHistopathological analysis of liver and ovarian tissues of PCOS-induced rats treated with bergapten revealed largely preserved hepatic architecture with distinct morphological alterations linked to treatment modalities and disease induction (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e13\u003c/span\u003e). Normal control group (a) exhibited healthy ovarian cortex with multiple healthy follicles, defined corpora luteum, compact vascularized stroma with no cystic formations. Disease control group (b) subjected to a high-fat diet exhibited multiple large, thin-walled cystic follicles with irregular granulosa cell layers and mild stromal edema. Letrozole treated animals (c) produced enlarged fluid-filled follicles with thin granulosa layers and absent oocytes, thus surrounding stroma was loose and edematous. Metformin (300 mg/kg) treatment group (d) displayed partial reversal of cystic changes, dense and hypercellular ovarian stroma with increased fibroblast-like cells. Treatment with bergapten demonstrated dose-dependent histological restoration. The group treated with 5 mg/kg (e) showed moderate preservation of follicular architecture with a reduced number of cystic follicles. While high doses of BA 10 mg/kg (f) and 20 mg/kg (g) showed near-normal ovarian histology, characterized by regression of cystic follicles, reappearance of corpora luteum and restoration of granulosa as well as thecal cells organization with undamaged architecture.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec28\" class=\"Section2\"\u003e\u003ch2\u003e3.15. Hepatic tissues\u003c/h2\u003e\u003cp\u003eHistopathological analysis of liver sections across experimental groups revealed largely preserved hepatic architecture with specific alterations linked to treatment modalities and disease induction (Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e14\u003c/span\u003e). In normal control group (a), liver tissue exhibited well-organized hepatic cords, intact central veins and polygonal hepatocytes with centrally located nuclei with no fatty or inflammatory infiltration and necrosis. While HFD-disease control animal (b), surprisingly exhibited slight ballooning of hepatocytes with mildly dilated sinusoids. Occasional mononuclear cells near portal areas but no fatty liver deposits. In letrozole control animal (c), clear vacuoles within hepatocytes were seen under microscope with minimal disorganization of hepatic cords. Standard control group (d), treated with 300 mg/kg metformin showed no fatty infiltration but notable areas of hepatocellular necrosis, dense clusters of lymphocytes and macrophages around portal tracts and central veins were observed. Hepatocytes show signs of degeneration. Sinusoids are markedly dilated and kupffer cells appear enlarged in 5 mg/kg BA treatment group. While BA 10 mg/kg showed irregular hepatocytes, with minimal hepatic cord arrangement and fibrous strands may be faintly visible. Furthermore, BA 20 mg/kg portrayed widespread inflammatory infiltration with portal areas shown fibrotic expansion.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eObesity is globally organized metabolic disorder which aggravates pathogenesis of PCOS by promoting insulin resistance, hyperinsulinemia, oxidative stress and systemic inflammation, all of which contribute to ovarian dysfunction and hyperandrogenism (Li et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). The present study provides new insight into the complex interplay between obesity and polycystic ovary syndrome (PCOS), while also highlighting therapeutic potential of bergapten (a plant-derived furocoumarin) on letrozole and high-fat diet-induced PCOS in \u003cem\u003eWistar\u003c/em\u003e rats. In addition, study also pointed out that high-fat-diet induced obesity exacerbated reproductive and metabolic disturbances caused by letrozole, thereby mimicking multifactorial pathogenesis of PCOS. The high-fat-diet (HFD) and letrozole were administered among all animals except normal control group, to establish a reliable experimental model of obesity-associated PCOS. Review of literature has shown that letrozole (an aromatase inhibitor) causes estrogen biosynthesis disruption, follicular arrest, cystic changes, irregular estrous cycles and increases androgen production, thereby mimicking endocrine abnormalities observed in PCOS. While, HFD contributes to excessive weight gain, adipocytes accumulation and defective glycemic control (Kafali et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Reddy et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Consequently, combination of letrozole and HFD exacerbates persistent anovulation, hyperandrogenism, and dyslipidemia.\u003c/p\u003e\u003cp\u003eWeight reduction is a potential approach for restoring ovulatory function and enhancing fertility in women with PCOS. Even modest weight loss, in range of 5\u0026ndash;10% has been reported to improve gonadotropin balance, reduce hyperandrogenism and normalize menstrual cycle (Moran et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). In present study, rats treated with metformin and bergapten showed a significant decline in body weight compared to disease control group receiving letrozole and HFD, may reflect an improvement in metabolic efficiency and attenuation of adipose accumulation. In contrast, disease control group exhibited continuous weight gain, consistent with obesogenic and endocrine-disrupting effects of letrozole and HFD.\u003c/p\u003e\u003cp\u003eVarious studies reported that disturbances in glucose homeostasis are central to pathophysiology of obesity-related PCOS (Bednarz et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). In current study, administration of metformin and bergapten resulted in a marked reduction in fasting blood glucose levels, attributed to enhanced insulin sensitivity and improved glucose uptake, possibly mediated through activation of intracellular signaling pathways such as AMPK, which promotes lipid oxidation and reduces gluconeogenesis (Xin et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). In contrast, animals of letrozole and HFD group exhibited persistently elevated glucose levels suggested impaired glucose tolerance induced by excessive fat intake (Xin et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eStatistical analysis of biochemical markers further highlighting PCOS alteration and therapeutic impact of bergapten. In present study, dyslipidemia in diseased groups received high-fat diet altered metabolic features of PCOS more effectively than letrozole control alone. These findings align with evidence from studies of other natural and dietary interventions. For instance, short-chain fatty acids (SCFAs) such as sodium acetate, propionate, butyrate in letrozole-induced PCOS models significantly lower LDL, total cholesterol, elevate HDL and ameliorate VLDL levels (Acharya et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), mirroring lipid improvements seen with bergapten treatment in current study. Thus, lipid-normalizing dose-dependent effects observed with bergapten strongly suggest its effectiveness in counteracting metabolic dysfunction in PCOS. The vaginal smear cytology of healthy controls demonstrates a normal estrous cycle pattern with distinct phases. In contrast, PCOS-induced rats exhibited irregular cycle marked by prolonged metestrus (Thakor \u0026amp; Patel, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). The results of present study confirmed that letrozole induced PCOS significantly alters cyclic pattern in diseased control rats.\u003c/p\u003e\u003cp\u003eHormones act as key regulators of physiological processes, particularly in maintaining normal menstrual cycle. Follicular development and ovulation depend on proper function of ovary and balanced secretion of LH and FSH (Murray \u0026amp; Orr, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). In PCOS, follicles often fail to mature and release an egg that leads towards abnormalities in both follicular and luteal phases. As a result, not only infertility and irregular menstruation but metabolic disorders such as diabetes and cardiovascular complications also occur (Yang \u0026amp; Chen, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Conventional therapies, including ovulation-inducing agents like clomiphene citrate, letrozole and metformin are widely used along with oral contraceptives for cycle regulation. However, these treatments often carry unwanted side effects (Sun et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Therefore, attention is being directed toward herbal and natural alternatives which are generally considered safer. The bergapten treatment notably modulated endocrine and metabolic parameters with a clear dose-dependent improvement in serum leptin, FSH and adiponectin (ADP) levels in current study. Elevated leptin levels observed in disease control group were significantly reversed by bergapten, particularly at 20 mg/kg dose, suggesting restoration of leptin sensitivity and amelioration of hypothalamic-pituitary-adipose axis dysregulation in PCOS. Similarly, FSH levels which are often elevated in experimental PCOS model were significantly reduced by bergapten especially at 10 and 20 mg/kg doses, indicating a potential regulatory effect on gonadotropin secretion and folliculogenesis, aligning with improved ovarian function. Interestingly, serum adiponectin levels were paradoxically elevated in HFD and letrozole disease group possibly represent compensatory stress response. However, normalization of ADP levels with bergapten treatment could reflect progress in metabolic homeostasis and adipocyte functionality.\u003c/p\u003e\u003cp\u003eIn addition, \u0026ldquo;hyperandrogenism\u0026rdquo; is a hallmark of PCOS in which reduced levels of estrogen and progesterone are pointing to impaired steroidogenesis and disrupted ovulatory function. As luteinizing hormone (LH) levels rise, hypothalamic-pituitary-ovarian axis become aberrant leading towards endocrine complications e.g., amenorrhea and infertility (Sharma et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). In present study, HFD diseased and letrozole control groups showed a decline in progesterone and estrogen with a parallel rise in testosterone, indicating hormonal imbalance characteristic of PCOS. Metformin partially ameliorated these changes, while bergapten treatment significantly upregulate progesterone and estrogen levels and reduced testosterone in a dose-dependent fashion. The highest dose of BA (20 mg/kg) restored LH, progesterone, testosterone and estrogen concentrations similar to normal control, suggesting enhanced ovarian steroidogenesis. These findings highlight ability of bergapten to counteract letrozole-induced endocrine disruptions and optimize reproductive hormonal balance.\u003c/p\u003e\u003cp\u003eHistopathological analysis of ovarian and hepatic tissues in present study revealed that bergapten-treatment groups ameliorate morphological alterations in PCOS-induced rats. Ovarian tissue from disease control group exhibited cystic follicles, attenuated granulosa layers, and absent corpora lutea validating successful induction of disease through high-fat diet and letrozole administration. In contrast, bergapten treatment, particularly at higher doses (20 mg/kg), substantially restored ovarian architecture with complete regression of cystic follicles and normalization of granulosa and thecal layers. These changes may suggest reactivation of ovulatory processes and reversal of follicular arrest. Such outcomes are in matching with protective effects of carvacol, which were shown to normalize ovarian structure in similar PCOS models \u003cem\u003evia\u003c/em\u003e antioxidant and anti-inflammatory mechanisms (Gao \u0026amp; Li, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Regarding liver tissues, in bergapten-treatment groups inflammation was markedly reduced, indicating suppression of hepatic injury and immune cell infiltration. The absence of necrosis demonstrated preservation of hepatocyte integrity with healthy hepatic cords, adequate vascularization ultimately displayed functional recovery of hepatic tissue. Notably, 20mg/kg exhibited indistinguishable repair of hepatocytes, highlighting safety of bergapten even under metabolic stress conditions. Thus, bergapten may confer hepatoprotective benefits possibly due to free radical scavenging properties, as supported by earlier literature (Phucharoenrak \u0026amp; Trachootham, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eGene expression in PCOS is significant to assess alterations and underlying mechanisms in genes regulating hormones, metabolism and ovarian function. In present study, treatment with bergapten showed downregulation of pro-inflammatory mediators such as TNF-α, IL-8, IL-6 and Keap1 and enhanced modulation of steroidogenic enzymes including CYP19A1 and CYP11A1. The transcription factor Nrf2, known for orchestrating antioxidant defense, was significantly downregulated in disease control group, consistent with heightened oxidative stress in PCOS. Bergapten treatment, especially at lower doses (5 mg/kg) led to partial restoration of Nrf2 expression, suggests that bergapten exerts moderate antioxidant effects. However, expression of Keap-1, a negative regulator of Nrf2, was also declined in bergapten-treatment groups. Findings suggest persistent activation of Nrf2 antioxidant pathway, thereby potentially enhancing cellular defense against oxidative stress. The decline in TNF-α and IL-8 with bergapten treatment expressed limited immune cell infiltration and protection against ovarian tissue damage. While, lowering of IL-6 demonstrates improved metabolic balance and mitigation of insulin resistance (Hoene \u0026amp; Weigert, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). These effects positioned bergapten as a potent modulator of inflammatory milieu in PCOS.\u003c/p\u003e\u003cp\u003eBergapten particularly at 20 mg/kg dose, reversed dysregulation of CYP19A1 and CYP11A1 enzymes, reflects its strong potential to mediate estradiol synthesis and regulate androgen production, implies a beneficial effect on ovulatory function (Xiao et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Moreover, bergapten administration at 10 mg/kg and 20 mg/kg doses resulted in a partial restoration of PPAR-γ, a key transcription factor regulating insulin sensitivity and lipid metabolism (Liu et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). This finding indicates its potential role in alleviating PCOS-associated metabolic complications.\u003c/p\u003e\u003cp\u003eThe superior performance of 20 mg/kg bergapten across all serum biomarkers and gene expression mainly revealed a threshold-dependent pharmacodynamic effect, with higher dose supports in reinstating normal physiological state of organs. However, a nonlinear trend was observed, where 10 mg/kg dose did not produce as strong effect as lower 5 mg/kg dose in certain outcomes, possibly due to receptor saturation, feedback inhibition or dose-limited bioavailability. Bergapten pharmacological effects are likely mediated through its antioxidant, anti-inflammatory, and possibly PPAR-γ modulating properties, which have been reported in earlier studies on metabolic syndrome and reproductive health (Rudrapal et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). These findings underscore bergapten as a dual-action agent for managing both reproductive and metabolic abnormalities associated with PCOS.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe present study demonstrated that bergapten exerts beneficial effects in a high-fat diet and letrozole-induced model of polycystic ovarian syndrome in \u003cem\u003eWistar\u003c/em\u003e rats. Biochemical analysis showed marked improvement in serum hormonal and metabolic parameters, while histopathological assessment revealed preservation of ovarian and hepatic tissue architecture. Gene expression studies indicated favorable modulation of inflammatory and metabolic biomarkers. Collectively, these findings suggest that bergapten possesses promising therapeutic properties against PCOS, supporting its potential role in restoring endocrine balance and metabolic function. Further studies require additional examination of its molecular targets, tissue-level effects and comparative efficacy against PCOS like metformin to validate its clinical relevance.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003ch2\u003eCompeting Interests\u003c/h2\u003e\u003cp\u003eThe authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003ch2\u003eEthics approval\u003c/h2\u003e\u003cp\u003e The animal study design was reviewed and approved by the Ethical Review committee of GCU Faisalabad [(Ref. No. GCUF/ERC/633]. Laboratory animals were used in this study as per approved protocol.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e\u003cp\u003eThe authors declare that no funds, grants, or other support were received during the preparation of this manuscript.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eAll authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by **Abida Hussain** , **Ammara Saleem** and **Muhammad Furqan Akhtar** . The first draft of the manuscript was written by **Abida Hussain** and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgment:\u003c/h2\u003e\u003cp\u003eThe authors sincerely thank the Department of Pharmacology, GCUF, for providing laboratory facilities and technical assistance during the experimental work. Special thanks to the animal house staff for their cooperation during the animal studies. The authors also appreciate the support and guidance of Ammara Saleem throughout the research.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe original contributions presented in the study are included in the article/Supplementary Material; further inquiries can be directed to the corresponding author.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAbinaya, S., Siva, D., Sabitha, R. and Achiraman, S. 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(2021) \u0026lsquo;The interplay between adipose tissue and vasculature: role of oxidative stress in obesity\u0026rsquo;, \u003cem\u003eFrontiers in Cardiovascular Medicine\u003c/em\u003e, 8, 650214.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"PCOS, obesity, Keap1, Nrf-2, adiponectin, bergapten","lastPublishedDoi":"10.21203/rs.3.rs-7807066/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7807066/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003ePolycystic ovarian syndrome (PCOS) is a complex multifactorial endocrine and metabolic disorder associated with hormonal imbalance, insulin resistance and ovarian dysfunction. Moreover, obesity and dietary imbalance act as major contributing factors. The present study was designed to evaluate pharmacological effects of bergapten in a high-fat diet (HFD) and letrozole-induced PCOS models using Wistar rats. Animals were divided into different experimental groups and treated with bergapten at three doses (5, 10 and 20 mg/kg), with control groups maintained on HFD and letrozole. Body weight deviation, blood glucose levels, histopathological examination, serum biochemical analysis and gene expression profiling were used to determine therapeutic efficacy of bergapten. Results exhibited significant decrease in blood glucose and body weight in all bergapten treatment groups than diseased control. Serum analysis demonstrated normalization of hormonal (FSH, LH, estrogen, progesterone, testosterone,) and metabolic biomarkers (adiponectin, leptin, CYP19A1, CYP11A1, ALT, AST, ALP, cholesterol, total lipids, TGs, HDL). While gene expression studies through qRT-PCR showed significant downregulation of pro-inflammatory mediators such as TNF-α, IL-6, IL-8, Keap1 and upregulation of Nrf-2 pathway. In addition, partial recovery of PPAR-γ expression in bergapten treatment groups. The marked improvement in ovarian and hepatic tissues morphology further supported medicinal value of bergapten in attenuating metabolic stress. Overall, findings exhibited that bergapten not only alleviates PCOS but also counteracts obesity-related disturbances caused by HFD, suggesting its potential as a therapeutic candidate. Further investigations are needed to validate its clinical relevance.\u003c/p\u003e","manuscriptTitle":"Pharmacological Evaluation of Bergapten in High Fat Diet and Letrozole Induced Polycystic Ovarian Syndrome in Wistar Rats","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-26 10:45:30","doi":"10.21203/rs.3.rs-7807066/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"4bb688b6-dedb-49f8-921b-2af53d77c917","owner":[],"postedDate":"October 26th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-10-27T14:22:06+00:00","versionOfRecord":[],"versionCreatedAt":"2025-10-26 10:45:30","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7807066","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7807066","identity":"rs-7807066","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}