Open Veterinary Journal, (2026), Vol. 16(4): 2471-2487

Research Articlewww.eldaghayes.com

10.5455/OVJ.2026.v16.i4.48

Citrullus colocynthis extract ameliorates hypothalamic-pituitary-gonadal axis dysfunction in diabetic rats

Sina Khalil Ismael^1,2* and Salam Haji Ibrahim³

¹Medical Laboratory Science Department, College of Health Sciences, Charmoo University, Chamchamal, Iraq

²Medical Laboratory Analysis Department, College of Health Sciences, Cihan University, Sulaymaniyah, Iraq

³College of Veterinary Medicine, University of Sulaimani, Sulaymaniyah, Iraq

*Corresponding Author: Sina Khalil Ismael. Medical Laboratory Science Department, College of Health Sciences, Charmoo University, Chamchamal, Iraq. Email: sina.khalllil [at] sulicihan.edu.krd

Submitted: 27/12/2025 Revised: 15/03/2026 Accepted: 26/03/2026 Published: 30/04/2026

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ABSTRACT

Background: Diabetes mellitus (DM) can negatively influence the hypothalamic–pituitary–gonadal axis (HPGA) and male fertility.

Aim: To investigate the effects of Citrullus colocynthis extract on HPGA regulation and pancreatic glucose transporter 2 (GLUT2) expression in streptozotocin-induced DM in male rats, together with sperm morphology and motility

Methods: Thirty-two adult male rats were divided into 4 groups of 8 animals each: negative control, positive control, treatment 1 (250 mg/kg body weight), and treatment 2 (500 mg/kg body weight) of CC extract, respectively. All groups were induced with DM, except group one. Treatments were given orally for 30 consecutive days. After animal scarification using deep anesthesia, blood samples were collected for hematological and biochemical analyses, and tissue samples were obtained for histopathological and immunohistochemical studies (pancreas) and semen analysis (testicles).

Results: Treatment 2 significantly (p ≤ 0.05) decreased blood glucose and glycated hemoglobin levels with marked elevation in serum insulin levels. The significant reduction of gonadotropin-releasing hormone in diabetic rats was substantially recovered by treatment 2, whereas no changes in luteinizing hormone, follicular stimulating hormone, and testosterone levels were recorded. Sperm motility was significantly increased in diabetic rats receiving treatment 1 of CC extract. Histopathological results have shown various alterations in the testis and pancreatic tissue in diabetic rats, which were improved almost to normal levels by different doses of CC extract. Immunohistochemical analysis showed complete restoration and normal localization of GLUT2 in pancreatic beta cells after treatment with CC extract.

Conclusion: CC extract ameliorates diabetic complications through a potent natural effect against HPGA dysfunction, pancreatic and testicular alteration, and GLUT2 overexpression in pancreatic beta cells.

Keywords: Diabetes mellitus, GLUT2, Hypothalamic-pituitary-gonadal axis, Plant extract, Semen analysis.

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Introduction

Diabetes mellitus (DM) is a metabolic condition marked by chronic elevation of blood glucose levels due to insufficient insulin release from pancreatic beta cells (PBC) and/or diminished responsiveness of tissues to insulin (EL-Gawish, 2023). Current estimates from the International Diabetes Federation indicate that about 463 million adults worldwide are living with DM, which might increase to nearly 578 million by 2030 (Butalia et al., 2016). DM is widely recognized as a contributing factor to male infertility, erectile dysfunction, retrograde ejaculation, and hypogonadism (Ostovan and Gol, 2015).

Under hyperglycemic conditions, a decline in testosterone triggers a compensatory rise in luteinizing hormone (LH) and follicle-stimulating hormone (FSH) secretions. Whenever hyperglycemia impairs hypothalamic cells responsible for producing LH-releasing hormone, disrupting their ability to respond to declining testosterone levels, leading to reduced testosterone production and secretion (Shi et al., 2017). Therefore, men with DM may exhibit decreased gonadotropin-releasing hormone (GnRH) receptor expression, which diminishes GnRH and leads to reduced LH and FSH production (Baccetti et al., 2002).

In DM, key reproductive parameters include spermatogenesis, sperm count and motility, seminal fluid volume, and testosterone (Bisht et al., 2017; Long et al., 2018). Moreover, histological alterations have been reported in both pancreatic and testicular tissues during DM (Shi et al., 2017), in which glucose transporter 2 (GLUT2) expression is dysregulated (Berger and Zdzieblo, 2020), leading to impaired glucose-stimulated insulin secretion (GSIS), reduced cellular glucose uptake, and persistent hyperglycemia (Al-Shaqha et al., 2015).

Traditionally, phytotherapy has been widely used in the management of DM (EL-Gawish, 2023), and plant extracts are considered more affordable and effective, with fewer adverse effects than conventional pharmaceutical agents (Tran et al., 2020). Citrullus colocynthis (CC), commonly referred to as colocynth, bitter apple, or wild gourd, is a perennial herbaceous species belonging to the Cucurbitaceae family (Kouadri and Satha, 2018). CC contains protein, amino acids, tannins, saponins, phenolics, flavanoids, glucosides, terpenoids, alkaloids, anthranol, steroids, cucurbitacins, and glycolipids (Afzal et al., 2023).

Prolonged administration of CC extracts has been shown to alleviate hyperglycemia by preserving and restoring PBCs and enhancing insulin synthesis and secretion (Drissi et al., 2021). Consequently, the resulting increase in insulin levels improves testosterone and mitigates the reproductive damage associated with DM (Ostovan and Gol, 2015). Despite growing evidence supporting the antidiabetic potential of CC, its role in regulating the hypothalamic–pituitary–gonadal axis (HPGA) and PBC structure and functions in DM remains poorly understood. Therefore, this study aims to evaluate the impact of CC extract on HPGA regulation in male rats with streptozotocin (STZ)-induced DM.

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Materials and Methods

Plant collection and extraction

Fresh CC fruit was obtained from Kalar District, Sulaymaniyah, Iraq, in July 2024. It was air-dried in a shaded area and grinded into fine powder (Owoade et al., 2018). The dried material was then extracted by mixing 100 g of the powder with 80% methanol using Soxhlet (Lichen, China) and incubated at 25°C for 72 hours in a shaker incubator (Biostellar, China). Then, the mixture was filtered several times through filter paper (125 mm) to remove solid particles, and the filtrate was evaporated at 40°C using a rotary evaporator (IKA, Germany). The remained aqueous extract was freeze-dried for 48 hours to obtain the powdered extract and stored in an airtight container at 20°C (Ibrahim, 2021).

Experimental animals

A total of 32 healthy male Wistar Albino rats (aged 10–12 weeks and weighed 250–300 g) were obtained from the animal house of the University of Human Development, Sulaymaniyah, Iraq. The rats were kept under controlled setting for 2 weeks for acclimatization, while receiving standard food and water ad libitum with 12-hour of light/dark and adequate airflow throughout the study. Then, the rats were assigned into four groups as Negative control (NC), Positive control (PC), Treatment 1 (T1), and Treatment 2 (T2), with eight rats per group. DM was experimentally induced in all groups (except NC) through a single intraperitoneal injection of freshly prepared STZ at a dose of 55 mg/kg (Afshari et al., 2021). After 96 hours post-STZ injection, overnight fasted rats were checked for blood glucose using a glucometer (On Call Plus, USA). Rats with fasting blood glucose (FBG) levels >300 mg/dl were classified as diabetic and included in the study. The NC and diabetic rats (DR) in the PC group were administered distilled water, while T1 and T2 were treated orally with CC extract at 250 and 500 mg/kg, respectively (Gurudeeban and Ramanathan, 2010) for 30 days.

Assessment of body weight

Body weight was measured on day 0 and day 30 to monitor any changes caused by treatments.

Blood test analysis

On day 31, the animals were deeply anesthetized using intraperitoneal injection of 0.2 ml of ketamine (30 mg/kg) and xylazine (20 mg/kg) after the rats were fasted overnight. Blood samples (5 ml) were collected through heart puncture, transferred into Ethylenediaminetetraacetic Acid tubes (1 ml) for measuring glycated hemoglobin (HbA1C), and into gel tubes for serum separation (4 ml). Subsequently, FBG, insulin, LH, FSH, testosterone, and GnRH were tested using the obtained serum (Barsiah et al., 2019). Biochemical analysis were performed using Cobas Pure Analyzer (Roche, Germany), while hormonal assays were conducted using an ELISA system (BioTek, USA).

Semen analysis

The sperm morphology was evaluated by removing the vas deference, rinsed in normal saline, prepared a semen smear, stained with eosin stain (Atomscientific, UK), washed with normal saline, and read under the microscope (Scarano et al., 2006). Additionally, sperm motility was assessed by separating the caudal epididymis from the left testis, placed in 2 ml normal saline at 37°C, cut into pieces, left for 30–60 seconds to facilitate the release of sperm, then 1–2 drops of semen suspension were added to a clean slide with coverslip and examined under the microscope (Pourheydar et al., 2021).

Histopathological analysis

After animal sacrification, the pancreas and right testis were promptly excised and fixed in 10% neutral buffered formalin (CDH, India) for 48 hours. Subsequently, tissues were processed routinely, embedded in paraffin, sectioned at a thickness of 4 μm, stained with hematoxylin and eosin, and examined under the microscope (Ibrahim, 2021).

Immunohistochemistry study

The paraffin-embedded pancreatic block section was used with a polyclonal GLUT2 antibody (Biorbyt, USA). Sections were mounted onto positively charged slides and incubated at 70°C overnight. Then, deparaffinized in xylene and graded ethanol solutions. Antigen retrieval was performed by heating the sections in a retrieval solution for 30 minutes. Endogenous peroxidase activity was blocked using a peroxidase blocking solution for 10 minutes. The sections were then incubated with the primary GLUT2 antibody (50 µl) for 1 hour, followed by 100 µl of the secondary antibody for an additional hour. Chromogen development was allowed for 5 minutes, then sections were counterstained with hematoxylin for 1 minute, then washed with tap water, dehydrated through an ascending series of ethanol, cleared in xylene, mounted with DPX, coverslip added, and read under a microscope. The distribution and staining intensity of GLUT2-immunopositive cells were quantified using the H-score system, and the positively stained PBCs were categorized as score 0 (no staining, 0%–5%), score 1 (6%–20%), score 2 (21%–40%), score 3 (41%–65%), and score 4 (positive staining, >65%).

Fourier-transform infrared (FTIR) assay

FTIR was performed to characterize the chemical structure and functional group of the CC extract using a PerkinElmer spectrophotometer (Waltham, MA, USA) in the range of 4,000–400 cm^–¹, employing the KBr pellet technique with a resolution of 4 cm^–¹ and 8 scans per sample.

Up and down procedure (UDP)

The acute oral toxicity of the CC extract was evaluated using UDP (Kariyil and Usha, 2020). Nineteen healthy male Albino rats (aged 10–12 weeks and weighed 150–250 g) were used. The CC extract was freshly suspended in normal saline and administered orally (2 ml). A gradual dosing strategy was used, with each animal dosed one at a time. The initial dose for the first animal was 500 mg/kg. Subsequent doses were adjusted based on the preceding animal’s outcome after 24 hours observation. If an animal survived, the next animal was administered a higher dose at a constant interval of 500 mg/kg. If an animal died, the next animal was administrated a lower dose by the same interval as 500, 1,000, 1,500, 2,000, 2,500, 3,000, 3,500, 4,000, 4,500, 5,000, 4,500, 5,000, 5,500, 5,000, 5,500, 6,000, 6,500, 7,000, and 7,500 mg/kg. The study was concluded when complete mortality occurred. Sequential dosing and observed outcomes (O for survival and X for death) were reported for each animal. The starting dose of 500 mg/kg was selected based on preliminary studies and literature reports of the subacute toxicity profile of the CC extract.

Statistical analysis

The Shapiro-Wilk test was used to determine the normality of the data. Then, statistical analyses were performed using one-way or two-way analysis of variance, followed by Tukey’s post hoc multiple comparison test, with one-sample t-tests applied when appropriate. The results are presented as the mean ± standard error of the mean (SEM). All statistical procedures were carried out using GraphPad Prism version 10.4.2, and a significance level of p < 0.05 was considered significant.

Ethical approval

The study protocol was approved by the ethics committee of Charmo University, Sulaimaniyah, Iraq (No. 21 on November 09, 2025). The methodology provided does not contain any potential ethical issues for animals or the environment. Measures were taken to minimize pain and discomfort, and the ARRIVE Guidelines for animal ethics were followed in all experiments.

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Results

Assessment of body weight

The NC group exhibited a significant increase in body weight (p < 0.05) over the course of the study. In contrast, the PC group showed a slight, nonsignificant decrease in body weight (p > 0.05) by day 30 compared with day 0. DR receiving T1 displayed no significant changes (p > 0.05) after 30 days of treatment. Conversely, DR receiving T2 demonstrated a significant reduction in body weight (p < 0.05) by the end of the experiment (Fig. 1).

Fig. 1. Body weight alteration in each group during the experimental period. A. Shows body weight of negative control group (NC) in day 0 and day 30. * p < 0.05 compared to Day 0 B. Shows body weight of positive control group (PC) and slight decrease in day 30 compared to day 0. C. Shows the effect of receiving treatment one (T1, 250 mg/kg of plant extract) on body weight with no significant (p > 0.05) change in day 0 and day 30. D. Shows the effect of receiving treatment 2 (T2, 500 mg/kg) of Cc extract on body weight. *p < 0.05 compared to day 0. Data are shown as mean ± SEM, n=8 for each group.

Glycemic and insulin hormone assessment

STZ administration induced a significant elevation (p < 0.05) in FBG relative to the NC group at 4 days post-injection (Fig. 2A). Treatment of DR with T1 did not produce a significant reduction in FBG (p > 0.05). Conversely, T2 resulted in a significant decrease (p < 0.05) in FBG, bringing them close to those in the NC group when compared to the PC group (Fig. 2B). STZ-induced DR exhibited a significant increase (p < 0.05) in HbA1C levels compared to the NC group. This elevation was not substantially reduced (p > 0.05) following treatment with T1; however, T2 produced a significant reduction (p < 0.05) in HbA1C relative to the PC group (Fig. 3A). T1 elicited only a slight, nonsignificant increase (p > 0.05) in insulin levels compared with the PC group. In contrast, T2 demonstrated a significant elevation (p < 0.05) in serum insulin relative to the PC group (Fig. 3B).

Fig. 2. Evaluation of fasting blood glucose (FBG) level. A. Shows FBG level prior to and after streptozotocin (STZ) injection, where STZ produce a significant increase in FBG 4 days post injection opposite to day 0. B. Shows influence of plant extract on FBG level in diabetic rats which was markedly reduced by treatment 2 (T2, 500 mg/kg) of plant extract in contrast to positive control group (PC). Treatment one (T1, 250 mg/kg) of plant extract produced non-significant decrease in FBG level. *p < 0.05 elevated compared to negative control (NC). #p < 0.05 decreased compared to PC. Data are shown as mean ± SEM, n=8 for FBG.

Fig. 3. Evaluation the impact of diabetes mellitus (DM) and graded doses of plant extract on glycated hemoglobin (HbA1C) and insulin levels. A. Shows the impact of DM on HbA1C which was significantly increased in positive control (PC) group, while receiving treatment 2 (T2, 500 mg/kg) of plant extract markedly decrease the HbA1C level and receiving treatment one (T1, 250 mg/kg) of plant extract non-significantly reduce HbA1C level. *p < 0.05 relative to negative control (NC). #p < 0.05 contrasted to PC. B. Shows that receiving treatment 2 (T2, 500 mg/kg) of plant extract markedly increase insulin hormone comparing to PC group, whereas treatment 1 (T1, 250 mg/kg) of plant extract slightly increase insulin hormone. *p < 0.05 in comparison with PC. Data are shown as mean ± SEM, n=8.

Hormones from hypothalamic–pituitary–gonadal axis

A significant reduction (p < 0.05) in serum GnRH concentrations in the PC group was found compared to NC group. T1 did not produce a substantial improvement in GnRH levels; however, T2 resulted in a significant elevation (p < 0.05) in serum GnRH, restoring levels close to those of the NC group (Fig. 4A). In addition, the PC group exhibited a slight, nonsignificant decrease (p > 0.05) in serum FSH, LH, and testosterone levels relative to the NC group. Although T1 and T2 induced mild increases in serum LH, FSH, and testosterone levels (p > 0.05) compared to the PC group (Fig. 4B–D).

Fig. 4. Hormonal assessment of hypothalamus-pituitary-gonadal-axis (HPGA). A. Shows significant decrease of GnRH in positive control (PC) in a relative to negative control (NC), while T1 (250 mg/kg) of plant extract insignificantly increase GnRH and T2 (500 mg/kg) of plant extract markedly increase GnRH. *p < 0.05 contrasted to NC. #p < 0.05 compared to PC. B. Shows that leutinizing hormone (LH) level in PC almost near to the NC, as well as graded concentrations of plant extract have no marked effect on increasing LH level. C. Shows that follicular stimulating hormone (FSH) level in PC approximately equivalent to NC, while 500 mg/kg of plant extract slightly increase FSH level and 250 mg/kg of plant extract almost near to NC. D. Shows that diabetes causes slight decrease in PC contrasting to NC; however, both 250 and 500 mg/kg of plant extract led to slightly increase of testosterone level in comparison to PC which is near the NC. Data are presented as the mean ± standard error of the mean (SEM), n=8.

Semen parameters

A significant reduction (p < 0.05) in sperm motility was observed in the PC group. This decline was significantly increased (p < 0.05) by treating DR with T1 relative to the PC group. This reduction was increased insignificantly (p > 0.05), almost similar to the NC group, by treating DR with T2 compared to untreated DR. Moreover, DM did not produce obvious changes in sperm morphology in any of the rats (Fig. 5B).

Fig. 5. Evaluation of semen parameters; A. Shows that sperm motility significantly reduced comparing to negative control (NC) group due to DM, since administration of T1 (250 mg/kg) of CC extract resulted in a significant improvement in sperm motility compared to positive control (PC), while administration of T2 (500 mg/kg) of Cc extract insignificantly increase sperm motility. * p < 0.05 versus NC. # p < 0.05 versus PC. B. Shows that sperm morphology didn’t markedly decrease in PC in a compare to NC, while receiving T1 (250 mg/kg) of CC extract cause a slight increase in normal sperm morphology, in contrast administration of 500 mg/kg of CC extract produce insignificant curing in sperm morphology comparing to PC. Data are shown as mean ± SEM, n=5 for sperm motility, sperm counting and sperm morphology

Histopathological study

Testicular tissue

The NC group showed normal architecture of the seminiferous tubules (STs) and interstitial components (Fig. 6a–d). DR displayed pronounced pathological alterations, including dilation of the ST, abnormal exfoliation of germinal epithelial cells, extensive degeneration of primary and secondary spermatocytes, severe spermatogenic arrest, germ cell depletion, a marked reduction in spermatozoa and round spermatids, and clear Leydig cell degeneration (Fig. 7a–d). Interestingly, T1 produced an obvious repair of ST abnormalities in the DR group relative to the PC group. It promoted the mitotic activity of spermatogenic and Leydig cells against diabetic-induced ST damage, showing only mild enlargement of ST with mild degeneration in germ cells, primary and secondary spermatocytes, with Sertoli cells and intact interstitial cells within normal, organized interstitial space (Fig. 8a–d). Moreover, T2 restored near normal histoarchitecture of the ST, exhibiting properly associated and cellular germinal layers along with intact interstitial cells (Fig. 9a–d). Furthermore, the testicular lesion score was significantly elevated (p < 0.05) in the PC group compared with the NC group, while both treatment groups (T1 and T2) showed a significant reduction in pathological scores (Fig. 10).

Fig. 6. Microscopic sections of seminiferous tubules (a–d) in the negative control group (NC) rats showed a well-organized arrangement of germ cells at various stages of spermatogenesis (yellow dashed lines), intact spermatogonia and spermatids (black and red arrows) and lumina appropriately filled with spermatozoa. The interstitial spaces were structurally preserved and contained normal, healthy Leydig cells (yellow arrows), (H & E stain).

Fig. 7. Microscopic sections of seminiferous tubules (ST) (a–d) in the positive control group (PC) revealed dilated ST with a disorganized germinal epithelium, hypospermatogenesis (yellow dashed line), exfoliated spermatogonia and spermatid (black and red arrows). The germinal epithelial cells exhibited pronounced degeneration, and the interstitial tissue contained degenerated Leydig cells, indicated by the yellow arrows, (H & E stain).

Fig. 8. Microscopic sections of seminiferous tubules (ST) (a–d) in diabetic rats received 250 mg/kg of plant extract presented normal arrangement of ST across various spermatogenic stages (yellow dashed lines), spermatozoa with mild hypospermatogenesis, mild degeneration of spermatogonia and spermatid (black and red arrows), and mild degeneration of Leydig cells (yellow arrows), (H & E stain).

Fig. 9. Microscopic sections of seminiferous tubules (ST) (a–d) in diabetic rats received 500 mg/kg of plant extract exhibited normal arrangement of ST across various spermatogenic stages (yellow dashed lines) and spermatozoa that filled the lumen with intact spermatogonia and spermatid (black and red arrows). Leydig cells in the interstitial space indicated by yellow arrows, (H & E stain).

Fig. 10. Shows pathological lesion scores of tests among the different groups. NC: Negative control; PC: Positive con-trol; T1: Treatment one (250 mg/kg of plant extract); T2: Treatment two (500 mg/kg of plant extract). Score 0: Absence of change, score 1: Change in <25%, score 2: Change in 26%–50%, score 3: Change in 51%–100%. Hypospermatogenesis, exfoliation of the germinal epithelium and hydropic degeneration are significantly altered in PC group in a comparison with NC group. However, hypospermatogenecis, exfoliation of the germinal epithelium and hydropic degeneration are significantly reduced in T1 and T2 in a comparison to PC group. *p < 0.05 increased compared to NC. #p < 0.05 decreased in compared to PC. Data are shown as mean ± SEM, n=8.

Pancreatic tissue

Microscopic examination of pancreatic tissue in the NC group demonstrated a normal lobular architecture comprising both exocrine and endocrine components. The endocrine region exhibited a centrally located cluster of beta cells surrounded by a peripheral mantle of alpha and gamma cells, with well-preserved parenchymal structure and orderly ductal organization (Fig. 11a–c). Conversely, the PC group exhibited pronounced pathological alterations, including degeneration of acinar cells accompanied by inflammatory infiltration, ballooning degeneration of endocrine cells, marked islet atrophy, and a marked reduction in the number of beta cells (Fig. 12a–d). Interestingly, these histological alterations in the pancreatic endocrine portion were improved by treating DR with T1 (Fig. 13a–c). This improvement was reinforced by treating DR with T2 that showed intact morphology and cellularity of the pancreatic exocrine and endocrine portions (Fig. 14a–d). Moreover, the scoring of pancreatic pathological lesions in the PC group was significantly elevated (p < 0.05) compared with the NC group, and T1 and T2 showed a significant reduction relative to the PC group (Fig. 15).

Fig. 11. Microscopic sections of pancreas in the negative control group (a–c) exhibited normal structural organization of tubuloacinar gland, intact exocrine (EX) and endocrine (En) portions, and ducts, (H & E stain).

Fig. 12. Microscopic sections of pancreas in the positive control group (a–d) displayed degeneration of exocrine acinar cells (EX), hyalinized arteriosclerosis (Bv), inflammatory reaction (black arrows), atrophy of islets (En), vacuolation of endocrine cells, and severe reduction of beta cells, (H & E stain).

Fig. 13. Microscopic sections of pancreas in treated rats with 250 mg/kg of plant extract (a–c) displayed degeneration of acinar cells (EX) and mild vacuolation of endocrine cells (En), (H & E stain).

Fig. 14. Microscopic sections of pancreas in treated rats with 500 mg/kg of plant extract (a–d) displayed intact acinar cells (EX) and endocrine cells (En), (H & E stain).

Fig. 15. Shows pathological lesion score of pancreas among the different groups. NC: Negative control; PC: Positive control; T1; Treatment one (250 mg/kg of plant extract); T2: Treatment two (500 mg/kg of plant extract). Score 0: absence of change, score 1: change in <25%, score 2: change in 26%–50%, score 3: change in 51%–100%. Arteriosclerosis, beta cells hypocellularity, exocrine degeneration, endocrine degeneration, islet atrophy, inflammation is significantly altered in PC group in a compare to NC group. However, arteriosclerosis, beta cells hypocellularity, exocrine degeneration, endocrine degeneration, islet atrophy, inflammation is significantly decreased in rats received T1 and T2 compared to PC group. *p < 0.05 increased relative to NC. #p < 0.05 decreased relative to PC. Results are summarized as mean ± SEM, n=8.

Immunohistochemical study

Pancreatic sections, specifically the islets of Langerhans, were analyzed using image analysis software (AHSQ). Immunoreactive cells displayed brown cytoplasmic GLUT2 granules, whereas the nuclei remained unstained and bluish in color. The PC group expressed GLUT2 in a moderate-to-strong intensity in the cytoplasm of PBCs (score=3, 60%) (Fig. 16). Conversely, no GLUT2 expression was observed in the NC group (score=0, 0%) (Fig. 17). T1 reduced GLUT2 expression in PBCs by 20% (score=2) (Fig. 18), while T2 improved PBCs function and completely reduced GLUT2 expression in the cytoplasm of PBCs (Fig. 19).

Fig. 16. Microscopic sections of immunohistochemistry of the pancreas in the negative control group (a–b) exhibited no positive GULT2 expression (Score 0).

Fig. 17. Microscopic sections of immunohistochemistry of the pancreas in the positive control group (a–b) exhibited diffuse moderate-strong immunopositive cell expression (GULT2), as indicated by black arrows, Score 3, 60%.

Fig. 18. Microscopic sections of immunohistochemistry of the pancreas of diabetic rats treated with 250 mg/kg of plant extract (a–b) exhibited focal weak immunopositive cell expression (GLUT2) as indicated by the black arrow, (Score 2, %20).

Fig. 19. Microscopic sections of immunohistochemistry of the pancreas in the diabetic rats treated with 500 mg/kg of plant extract (a–b) exhibited no positive cell expression (GLUT2) (Score 0, 0%).

FTIR analysis

The CC extract exhibited several distinct absorption peaks. The sharp, strong, and weak peaks correspond to the key functional groups, including O–H, C–H, C=O, C=C, and C–O (Fig. 20). These features suggest that flavonoids constitute the principal components of the extract. The broad peak detected at 3,411 cm^–¹ is characteristic of O–H stretching and is associated with hydroxyl functionalities in alcohols or phenolic structures. The two pronounced peaks at 2,974 and 2,933 cm^–¹ correspond to the C–H stretching of aliphatic –CH₃ and –CH₂ groups, indicating saturated hydrocarbon chains. The intense peak at 1,690 cm^–¹ represents C=O stretching and is indicative of carbonyl-containing molecules, a structural hallmark of many flavonoids. The band at 1,603 cm^–¹ reflects C=C stretching; it likely arises from aromatic ring systems or unsaturated alkenes. The peak at 1,384 cm^–¹ is attributed to C–H bending, while the signals at 1,076 and 1,050 cm^–¹ correspond to C–O stretching, which is commonly associated with ether linkages.

Fig. 20. Fourier transform infrared spectroscopy spectrum of the 80% methanolic extract of Citrullus colocynthis.

Up and down procedure

The results were as follows: 500 mg/kg (O), 1,000 mg/kg (O), 1,500 mg/kg (O), 2,000 mg/kg (O), 2,500 mg/kg (O), 3,000 mg/kg (O), 3,500 mg/kg (O), 4,000 mg/kg (O), 4,500 mg/kg (O), 5,000 mg/kg (X), 4,500 mg/kg (O), 5,000 mg/kg (O), 5,500 mg/kg (O), 5,000 mg/kg (O), 5,500 mg/kg (O), 6,000 mg/kg (O), 6,500 mg/kg (X), 7,000 mg/kg (X), and 7,500 mg/kg (X). However, the first mortality was at a concentration of 5,000 mg/kg, and subsequent mortality was at 6,500, 7,000, and 7,500 mg/kg. The estimated median lethal dose (LD50) of the CC extract was 5,564 mg/kg (Based on maximum likelihood) with 95% confidence intervals between 4,961 and 6,650 mg/kg.

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Discussion

In this study, body weight was significantly reduced in DR, which was probably due to proteolysis of the muscles, lipolysis of the adipose tissue, and impaired peripheral tissue glucose utilization (Kitabchi et al., 2009; Castellanos et al., 2020). Administration of 250 mg/kg of CC extract enhanced body weight improvement that attributed to the numerous bioactive molecules in CC that counteract diabetic actions (Dhakad et al., 2017). However, 500 mg/kg of CC extract did not recover body weight reduction in DR, which could be due to mild diarrhea and slight anorexia, with a decrease in food consumption as a result of an inhibitory effect of gastric acid secretion (Soufane et al., 2017). As well as through diabetic complications, including protein breakdown, lack of growth hormones, and thyroxine, and their effects on abolishing the anabolic activity of the body (Ostovan and Gol, 2015). In addition, CC extract markedly reduced the FBG level and HbA1C in the PC group, confirming the potent effect of CC extract on reducing hyperglycemia in DR due to containing a variety of active ingredients that serve a key function in maintaining and lowering FBG (Hussain et al., 2014) through an alpha-glucosidase inhibitory-dependent mechanism, which serves as a crucial enzyme involved in the biosynthesis of glycoproteins and the cleavage of glycosidic bonds (Ghauri et al., 2020), as well as through an inhibiting intestinal glucose uptake, through flavonoid dependent mechanism, since there are a wide range of free amino acid and flavonoid that could directly implicated in the insulinotropic activity of CC extract.

Earlier studies have reported that hyperglycemia impaired GnRH secretion and testicular function (Fathy et al., 2023), leading to reduced function of the HPGA, and production of LH and FSH, which are essential for maintaining Leydig and Sertoli cell populations and ensuring spermatogenesis (Shoorei et al., 2020). In agreement with these findings, this study pronounced that DM led to a considerable decrease in serum GnRH, although no marked variations were observed in LH, FSH, and testosterone levels. This outcome may be attributed to hypogonadotropic hypogonadism, which is associated with low or low-normal FSH and LH levels (Dandona and Rosenberg, 2010). High-dose CC extract revealed a good recovery in the GnRH level, suggesting a modulatory role of CC on HPGA in DR.

The outcomes of semen analysis in this investigation have shown that sperm motility was declined in DR driven by endocrine disorders, which causes HPGA hormones alteration, and neuropathy that may lead to seminal vesicles atonia, and increased oxidative stress that might cause DNA damage of sperm and compromised reproductive function (Zhu et al., 2017), while sperm morphology did not change, indicating that DM was not spermatoxic (Scarano et al., 2006). Interestingly, a low dose of CC extract enhanced an elevation of sperm motility, with a slight elevation in sperm morphology in DR, potentially through a wide range of antioxidants found in CC extract that play essentials role in abolishing oxidative stress and sperm cell damage (Marbat et al., 2023). High concentration of CC extract did not fully recover sperm morphology in DR, which might be due to the wide range of pro-oxidants found in CC extract (Reddy et al., 2015), and the influence of some polyphenols in higher concentration on the process of spermatogenesis (Maha et al., 2021; Sleiman et al., 2021).

Furthermore, histopathological study and lesion scoring of the testicles in DR revealed hypospermatogenesis, exfoliation of the germinal epithelium, and hydropic degeneration abnormalities through hyperglycemic-dependent effect on testicular functions (Kotian et al., 2019). These factors cause damage to nucleic acids, proteins, and biomembranes, leading to testicular injury and reproductive dysfunction (Abd El-Baky and Amin, 2011). Interestingly, both doses of CC extract improved testicular dysfunction by improving ST abnormalities and enhancing mitotic activity of spermatogenic cells and Leydig cells, via improving oxidative stress, decreasing Malondialdehyde and Glutathione disulfide, and enhancing catalase and glutathione activities (Mohammadzadeh et al., 2024). Moreover, histological study and lesion scoring in pancreatic tissue revealed morphological changes in DR that might be produced by the effects of proinflammatory cytokines, insulin deficiency, and microangiopathy (Hart et al., 2021). Treatment of DR with CC extract improved pancreatic tissues, suggesting that the insulinotropic and antioxidant effects of flavonoids in the extract, which promote the proliferation and regeneration of beta cells in the PBC and intraislet capillaries (Amin et al., 2017).

Moreover, the Immunohistochemistry (IHC) results demonstrated that GLUT2 expression in PBCs in the NC group was completely absent (H score=0, 0%), suggesting normal pancreatic function in preventing high FBG level, since GLUT2 is the “primary transporter of glucose” characterized by its low affinity and high capacity for glucose and expressed in PBCs required for GSIS (Thorens, 2015); therefore, it was not expressed in the NC group. However, there was over expression of GLUT2 (H score=3, 60%) on PBCs in DR through the accumulation of a high amount of glucose in the blood, ROS production (Sun et al., 2023), and impaired PBCs to produce adequate amount of insulin (Eguchi et al., 2021) due to STZ-induced damage to the nuclei or DNA of beta cells, when GLUT2 signaled for insulin production, the beta cells were unable to synthesize insulin (Thorens, 2015). Then, GLUT2 expression was significantly reduced (H score=2, 20%) by 250 mg/kg of CC extract and completely recovered back to normal with no GLUT2 expression by 500 mg/kg of CC extract in DR through antioxidant and ROS scavenging activity of the extract via flavonoid, saponin, and alkaloid-dependent mechanism (Meybodi, 2020). In addition to the present findings, another IHC study of GLUT2 in DM demonstrated that GLUT2 was expressed in the diabetic group, absent in the normal group, and decreased in the treated group (Kompella et al., 2025). On the other hand, the FTIR spectra are consistent with that of another study (Afzal et al., 2023), reinforcing the existence of flavonoid-rich constituents in the CC extract.

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Conclusion

This study provides evidence supporting the therapeutic effects of CC extract as a possible treatment candidate for DM related complications through improving glycemic control, restoring neuroendocrine function, preserving reproductive health, and protecting against oxidative damage, in conjunction with histological recoveries in tests and the pancreas. Furthermore, CC exerts a crucial antioxidant activity through the regulatory-dependent mechanism of GLUT2 expression in PBCs. However, additional studies are necessary to elucidate in detail the molecular pathways by which CC influences GLUT2 regulation and activity in DR.

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Acknowledgments

The authors would like to thank the College of Health Sciences, Cihan University, Animal House of the Human Development University, and High Quality Hospitals at Anwar Sheikha Medical City, Sulaymaniyah, Iraq, for their technical help and encouragement.

Conflict of interest

The authors declare no conflict of interest.

Funding

This research received no funding.

Authors’ contributions

SKI: Data curation, formal analysis, funding acquisition, investigation, methodology, and writing the original draft. SHI: Conceptualization, methodology, project administration, supervision, final draft review, and editing.

Data availability

Data are available from the authors and can be provided upon request.

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