Melatonin protect against pregabalin-induced gonadotoxicity via anti-oxidative, anti- inflammatory, anti-apoptotic, enzymatic and hormonal regulatory mechanisms

Melatonin protect against pregabalin-induced gonadotoxicity via anti-oxidative, anti- inflammatory, anti-apoptotic, enzymatic and hormonal regulatory mechanisms | 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 Melatonin protect against pregabalin-induced gonadotoxicity via anti-oxidative, anti- inflammatory, anti-apoptotic, enzymatic and hormonal regulatory mechanisms Ayodeji Folorunsho Ajayi, Motolani Susan Borisade, Precious Oyedokun, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5034037/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 12 Feb, 2025 Read the published version in BMC Pharmacology and Toxicology → Version 1 posted 12 You are reading this latest preprint version Abstract Background: The therapeutic value of pregabalin in the management of different pathological states like sleep, anxiety and bipolar disorders, fibromyalgia, epilepsy, among others, cannot be overemphasized. Nevertheless, the gonadotoxicity of this drug remains a point of concern. Contrarily, melatonin, an endogenous hormone is known for its favourable effects on the reproductive tissues following different insults. Thus, this study aimed to examine the impact of melatonin on male Wistar rats exposed to pregabalin. Methods: A total of sixty male Wistar rats weighing between 120-140g were assigned randomly to six groups, with each group consisting of ten rats. The control group was given 0.5ml of normal saline orally, whereas melatonin alone and increasing dosages of pregabalin were delivered at 10, 150, and 300 mg/kg/BW orally, respectively. At the specified dosages, two groups were simultaneously treated with melatonin and low and high doses of pregabalin. All treatments lasted for 56 days. With the excepton of the hormones, biomarkers were assayed in the testicular and epididymal tissues. Results: Pregabalin resulted in notable decreases in the percentage body weight, testicular weight, relative testicular weight, FSH, LH, testosterone, 3β-HSD, 17β-HSD, SOD, catalase, and GSH, as compared to the control group. However, these effects were mitigated in the groups who received melatonin in conjunction with pregabalin. Overall, the administration of melatonin had no negative impact on the levels and activities of the biomarkers. Pregabalin caused significant elevations in lactate, pyruvate, LDH, GGT, MDA, caspase, IL-1β, NFk, TNF-a, and distorted testicular histoarchitecture, but this effects was blunted in the group that were co-administered with melatonin. The impact of the two doses of pregabalin on all the biomarkers exhibited an irregular combination. The histological findings were parallel to the biochemical assays. Conclusion: Conclusively, melatonin has a protective effect against pregabalin-induced gonadotoxicity via anti-oxidative, anti-inflammatory, anti-apoptotic, and enzymatic and hormonal regulatory mechanisms. Clinical trial number : not applicable Melatonin pregabalin oxidative stress inflammation hormones enzymes testes Epipidymis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Introduction Infertility is defined as the inability to achieve pregnancy after engaging in regular sexual intercourse at least twice a week for duration of one year [ 1 ]. Male infertility is usually characterized by dysfunctional sperm parameters and may account for 50% of all infertility cases [ 1 ]. Akang and co explained an infertility belt stretching beyond Sub-saharan Africa and a high prevalence of 20 to 40 percent [ 2 ]. The frequency of secondary male infertility was estimated to be 49 percent. In Nigeria, research indicates that male infertility is responsible for 40 to 50 percent of all infertility cases [ 3 ]. The testicular function is tightly regulated by the hypothalamo-pituitary-gonadal axis (HPG). This regulatory processes can be distorted by exposure to drugs [ 4 ] such as ketoconazole, chemotherapy, gabapentinoids, and opioids [ 1 ] resulting in hypogonadism and ultimately impaired semen quality. Testicular dysfunction could also be as a result of injury, trauma, and diseases such as mumps, varicocele, and orchitis [ 1 ] and could occur following prolonged use of pregabalin with associated hypogonadism, and spermato-toxicity leading to infertility [ 5 ]. Pregabalin (PG) is a novel anticonvulsant medication that falls under the category of gabapentinoids. It has been found to be beneficial in treating sleep disorders, generalised anxiety disorder, fibromyalgia, and epilepsy [ 6 , 7 ]. Pregabalin received FDA approval in 2004 as an efficacious treatment for neuropathic pain associated with diabetic peripheral neuropathy, spinal cord injury, and postherpetic neuralgia [ 8 ]. Studies also show that pregabalin may be effective in the management of bipolar disorder, chronic pruritus, chronic cough, and restless leg syndrome [ 9 ]. According to Kamel and, the administration of pregabalin produces side effects, including nausea, vomiting, dry mouth, stomach cramps, constipation and diarrhea, flatulence, and abdominal distention [ 10 ]. Nevertheless, there is a scarcity of evidence in literature regarding the potential impacts of this medication on male reproductive function. Unlike pregabalin, melatonin is a neurohormone that is naturally produced by the pineal gland located behind the third ventricle in the brain [ 11 ]. The hormone is associated with the regulation of physiological functions such as sleep cycle, immune function, homeostasis, glucose regulation [ 12 , 13 ] and is beneficial to the cardiovascular system [ 14 ]. Melatonin controls the release of gonadotropin releasing hormone (GnRH), luteinizing hormone (LH), testosterone, and the development of the testes [ 15 ]. According to Yu and co, it has been documented as a powerful antioxidant that possesses both lipophilic and hydrophilic characteristics [15. Melatonin was demonstrated to exert ameliorative effects in tramadol-induced reproductive toxicity by preventing oxidative damage, mitochondrial injury, and apoptosis [ 16 ]. Although there are meagre research reports demonstrating spermatotoxicty following chronic use of pregabalin, no study has reported its effects on testicular steroidogenesis and the possible ameliorative potential of melatonin when co-administered with the drug. The present study focuses on hormonal, oxidative, apoptotic, steroidogenic and inflammatory markers, and also testicular and epididymal histoarchitecture in male rats exposed to melatonin and pregabalin. Methods and methodology Chemicals Pregabalin (CAS no: 148553-50-8) and melatonin (CAS no: 73-31-4) were obtained from Pfizer pharmaceutical industry, USA. All the other compounds utilised in this investigation were of conventional analytical grades. Animal care and experimental design A total of sixty (60) adult male Wistar rats weighing between 120-140g were obtained from reputable commercial breeders and housed in the Animal House Facility of the Department of Physiology, Ladoke Akintola University of Technology, Ogbomoso, Nigeria. The subjects were maintained under controlled conditions, specifically a temperature range of 28–31°C, a light/dark cycle of 12 hours, and a relative humidity range of 50–70%. The rats were kept in plastic cages, provided with unlimited access to ordinary pellets, and given unrestricted access to water. The animals are provided with compassionate care in line with the laboratory animal care principles of the National Medical Research Council and the guidelines outlined in the National Academy of Sciences' "Guide for the Care and Use of Laboratory Animals" (National Institute of Health Publication no. 80–23, updated 1978). The rats would be randomly assigned to six groups, with ten rats in each group. The control group was administered a placebo of 0.1 ml of 0.9% normal saline. Pregabalin was delivered orally at two different doses: a low dose of 150 mg/kg/BW and a high dose of 300 mg/kg/BW. Melatonin was also administered orally at a dose of 10 mg/kg/BW, either alone or in combination with pregabalin. Body weights were measured on a weekly basis using a precise digital electronic weighing scale (Bioevopeak, Shandong, China) that was calibrated for accuracy. Therapeutic dose equivalents to account for the weight difference in animals, in order to get the appropriate therapeutic doses for the rat model. The treatment regimen consisted of a daily administration for duration of eight (8) weeks. The ethical reference number for the present study is AERCFBMSLAUTECH: 011/09/20231. Clinical trial number not applicable Sample collection After 24 hours of administering the last dose of the The doses of pregabalin and melatonin administered were determined by scaling the human experiment; the animals were anaesthetised with pentobarbital sodium (40 mg/kg BW, i.p.) [ 17 ] and then euthanised before collecting blood through heart puncture and removing the testes. The mass of each testis was documented. The gonadosomatic index was calculated by dividing the paired testicular weight by the body weight and multiplying the result by 100 [ 2 ]. The blood samples were collected in bottles containing heparin and then centrifuged at a speed of 3500rpm for a duration of 10 minutes. The centrifugation was carried out at a temperature of -4o℃using a refrigerated centrifuge manufactured by Bio-Gene Technology Ltd., located in Grandtech Centre, Shatin, Hong Kong. The plasma samples that were divided into discrete portions were utilised to evaluate several biochemical parameters. The tissue from the left testicle was broken down into a uniform mixture using a phosphate buffer solution, while the right testicle was kept in bouin's fluid before being examined histologically. Biochemical assay Estimation of serum levels of reproductive hormones The measurement of serum and testicular testosterone was conducted using the ELISA kit from Monobind Inc. USA, with the product number 4806-300A. The serum FSH was analysed using an ELISA kit (Monobind Inc. USA; product number: 506-300A), whereas the serum LH was analysed using another ELISA kit (Monobind Inc. USA; product number: 625-300A). Estimation of testicular enzymes, lactate and pyruvate The testicular enzymatic activity of 17β-hydroxysteroid dehydrogenase and 3β-hydroxysteroid dehydrogenase were assessed using a standard kit (Teco Diagnostics, N Lakeview Ave, Anaheim, CA 92807, United States) according to the manufacturer's instructions. The levels of lactate dehydrogenase (LDH) and gamma-glutamyl transferase (GGT) activities, as well as lactate and pyruvate concentrations, in the testicular homogenate were measured using a standard kit provided by Agappe Diagnostics Ltd., located at Agappe Hills, Pattimattom PO, Kochi, Kerala 683562, India. Estimation of testicular oxidative markers The concentrations of malondialdehyde (MDA), as well as the enzymatic activity of superoxide dismutase (SOD) and catalase, were measured in the testicular and epididymis tissues, following the methods described [ 2 ]. The level of reduced glutathione (GSH), as well as the activities of glutathione peroxidase (GPx) and glutathione-S-transferase (GST) in the testicular and epididymis tissues, were measured using the procedures described [ 18 – 20 ]. Assessment of markers of inflammation The concentrations of nuclear factor-kappa B ( NF-kB), tumour necrosis factor-α (TNF-α) and interleukin-1β (IL-1 β) in the testicular and epididymis tissues were determined by ELISA kits according per manufacturer’s guideline (Elabscience Biotechnology Company Ltd., Wuhan, Hubei, China). Histology The testicular tissues were preserved in Bouin's solution, dried using a series of alcohol solutions, clarified using xylene, embedded in paraffin wax, cut into thin sections (2–3µm thick) using a microtome, attached to glass slides, stained with haematoxylin-eosin, and examined using a digital light microscope (Olympus CH; Olympus, Tokyo, Japan) at various levels of magnification. Photomicrographs were captured using a Sony digital camera (Model: DSC-W710). Statistical analysis The data was analysed utilising the Statistical Package for the Social Sciences (SPSS), and the results were reported as the mean value plus or minus the standard deviation. The data was analysed using one-way analysis of variance (ANOVA) followed by the Tukey test for post-hoc comparisons among several groups. Statistical significance was determined for intergroup differences at a significance level of P < 0.05. Results Effect of Melatonin on Body Weight Change, Testicular Weight, and Relative Testicular Weight Figure 1a-c illustrates the alterations in body weight, testicular weight, and relative testicular weight among the Control and test groups (Melatonin, Low dose Pregabalin, High dose Pregabalin, Low dose Pregabalin + Melatonin, High dose Pregabalin + Melatonin). The administration of melatonin had no significant effect on the body weight of the treated rats compared to the control group (Fig. 1a). Exposure to pregabalin, whether at low or high dosages, resulted in a substantial reduction in body weight gain, testicular weight, and relative testicular weight in the treated rats compared to the control and melatonin-treated rats. Nevertheless, administering melatonin to rats treated with pregabalin at both doses leads to a notable rise in body weight growth, testicular weight, and relative testicular weight in comparison to their corresponding doses of pregabalin therapy, as seen in Fig. 1a-c. Effect of Melatonin on the Testicular Concentration of Reproductive Hormones in Pregabalin-Exposed Male Rats Figure 2a-c displays the variations in the levels of reproductive hormones in the testicles of both the Control and test groups. The groups include Melatonin, Low dose Pregabalin, High dose Pregabalin, Low dose Pregabalin + Melatonin, and High dose Pregabalin + Melatonin. Administration of melatonin does not have a significant effect on the testicular concentration of hormones in the treated rats compared to the control group. Exposure to pregabalin, whether at low or high dosages, led to a significant reduction in the concentration of testosterone, luteinizing hormone, and follicle stimulating hormone in the testicles of rats, as compared to rats in the control group and those treated with melatonin. Nevertheless, administering melatonin to rats exposed to pregabalin at both doses leads to a notable elevation in testicular hormone levels compared to the corresponding doses of pregabalin treatment (Fig. 2a-c). Effect of Melatonin on the Testicular activities of Steroidogenic Enzymes and tissue injury markers in Pregabalin-Exposed Male Rats Figure 3a-f displays the alterations in testicular activity of steroidogenic enzymes for the Control group and other test groups, including Melatonin, Low dose Pregabalin, High dose Pregabalin, Low dose Pregabalin + Melatonin, and High dose Pregabalin + Melatonin. Administration of melatonin does not have a significant effect on the testicular activity of steroidogenic enzymes 3 and 17 βeta-Hydrosteroid Dehydrogenase, as compared to the control group. Exposure to pregabalin, at both low and high doses, resulted in a significant decrease in testicular enzyme activity compared to rats in the control group and rats treated with melatonin. Nevertheless, administering melatonin to rats exposed to pregabalin at both levels leads to a noteworthy augmentation in testicular activity of the markers, in comparison to the corresponding doses of pregabalin therapy. Administration of melatonin at both dosages considerably reduces the testicular activity of these enzymes in comparison to both the control group and the group of rats treated with melatonin alone (see Fig. 3a and 3b). Figures 3c-f depict the alterations in the concentration of lactate, pyruvate, lactate dehydrogenase, and Gamma-glutamyl transferase in the testicles of both the Control and test groups. The test groups include those treated with Melatonin, Low dose Pregabalin, High dose Pregabalin, Low dose Pregabalin + Melatonin, and High dose Pregabalin + Melatonin. The testicular activity of lactate, pyruvate, lactate dehydrogenase, and Gamma-glutamyl transferase was considerably elevated in rats exposed to both low and high dosages of pregabalin, compared to rats in the control group and those treated with melatonin. Nevertheless, administering melatonin to rats exposed to pregabalin at both doses results in a noteworthy reduction in testicular levels of lactate, pyruvate, lactate dehydrogenase, and Gamma-glutamyl transferase, compared to the corresponding doses of pregabalin. Effect of Melatonin on the Epididymal Tissues Injury Markers in Pregabalin-Exposed Male Rats Figures 4a-d depicts the alterations in the concentration of lactate, pyruvate, lactate dehydrogenase, and Gamma-glutamyl transferase in the epididymis of both the Control and test groups. The test groups include those treated with Melatonin, Low dose Pregabalin, High dose Pregabalin, Low dose Pregabalin + Melatonin, and High dose Pregabalin + Melatonin. Administration of melatonin does not cause a significant change in the concentration of the parameters in the epididymis of the treated rats compared to the control group. Exposure to pregabalin, whether at low or high doses, led to a substantial rise in the levels of lactate, pyruvate, lactate dehydrogenase, and Gamma-glutamyl transferase in the epididymis, as compared to rats that were not exposed to pregabalin or were treated with melatonin. Nevertheless, the administration of melatonin to rats exposed to pregabalin at both doses results in a notable reduction in the testicular levels of lactate, pyruvate, lactate dehydrogenase, and Gamma-glutamyl transferase, in comparison to the corresponding doses of pregabalin alone. malondialdehyde (MDA) level, superoxide dismutase (SOD) and catalase activities, level of reduced glutathione, and glutathione peroxidase (Gpx). Glutathione-S-transferase (GST) and caspase 3 activitie Effect of Melatonin on the Concentration of Oxidative Stress Markers and Caspase 3 Activities in Testicular and Epididymal Tissues of Pregabalin-Exposed Male Rats Figure 5a-f displays the variations in the testicular levels of oxidative stress parameters for the Control group and the test groups (Melatonin, Low dose Pregabalin, High dose Pregabalin, Low dose Pregabalin + Melatonin, High dose Pregabalin + Melatonin).The injection of pregabalin resulted in a rise in malondialdehyde concentration and glutathione-s-transferase activity, while causing a decrease in the activities of antioxidant enzymes, including superoxide dismutase, catalase, reduced glutathione, and glutathione-peroxidase, as compared to the control and melatonin only groups. Nevertheless, administering melatonin to rats exposed to pregabalin resulted in a notable reduction in testicular malondialdehyde levels and glutathione-s-transferase activity. Additionally, there was a significant increase in the activities of superoxide dismutase, catalase, reduced glutathione, and glutathione-peroxidase compared to the low and high dose pregabalin groups. Figure 5g displays the alterations in the testicular activity of Caspase 3 for the Control group and other test groups, including Melatonin, Low dose Pregabalin, High dose Pregabalin, Low dose Pregabalin + Melatonin, and High dose Pregabalin + Melatonin. Exposure to pregabalin, at both low and high dosages, resulted in a significant increase in testicular Caspase 3 activity compared to rats in the control group and rats treated with melatonin. Nevertheless, administration of melatonin to rats exposed to pregabalin at both levels leads to a noteworthy decrease in testicular activity of Caspase 3, compared to the corresponding doses of pregabalin treatment. Figure 6a-f displays the variations in the epididymal concentration of oxidative stress parameters for the Control group and the test groups (Melatonin, Low dose Pregabalin, High dose Pregabalin, Low dose Pregabalin + Melatonin, High dose Pregabalin + Melatonin).The injection of pregabalin resulted in an increase in malondialdehyde content, while causing a decrease in the activity of antioxidant enzymes such as superoxide dismutase, catalase, reduced glutathione, glutathione-peroxidase, and glutathione-s-transferase, as compared to the control and melatonin only groups. Nevertheless, administering melatonin to rats exposed to pregabalin at both doses resulted in a notable reduction in epididymal malondialdehyde concentration. Additionally, there was a significant increase in the activities of superoxide dismutase, catalase, reduced glutathione, glutathione-peroxidase, and glutathione-s-transferase compared to the corresponding groups that received only pregabalin. Figure 6g illustrates the variations in the epididymal activity of Caspase 3 for the Control group and the test groups (Melatonin, Low dose Pregabalin, High dose Pregabalin, Low dose Pregabalin + Melatonin, High dose Pregabalin + Melatonin). Exposure to pregabalin, whether at low or high dosages, resulted in a significant increase in epididymal Caspase 3 activity compared to rats that were not exposed to pregabalin or were given with melatonin. Nevertheless, administering melatonin to rats exposed to pregabalin at both levels leads to a noteworthy decrease in epididymal activity of Caspase 3, in comparison to the corresponding doses of pregabalin administration. Effect of Melatonin on the Testicular Concentration of Inflammatory Parameters in Pregabalin-Exposed Male Rats Figure 7a-c shows the changes in the testicular concentration of inflammatory markers for Control and test groups The options available are melatonin, low-dose pregabalin, high-dose pregabalin, low-dose pregabalin combined with melatonin, and high-dose pregabalin combined with melatonin.The results indicate that the injection of Pregabalin led to an increase in inflammatory markers in the testes of rats, as evidenced by an elevation in the nuclear respiratory kappa B (NF-kB), tumour necrosis factor (TNF-α), and interleukin 1B (IL-1β) cytokine. Nevertheless, administering melatonin to rats exposed to pregabalin at both levels results in a noteworthy decrease in testicular inflammatory markers compared to the corresponding doses of pregabalin alone. In Table 1 , the treatment of Pregabalin resulted in an increase in inflammatory markers in the rat epididymis, as indicated by the elevation of nuclear respiratory kappa B (NF-kB), tumour necrosis factor (TNF-α), and cytokine interleukin 1B (IL-1β). However, the treatment of pregabalin-exposed rats at both doses with melatonin causes a significant reduction in epididymal inflammatory markers when compared to their respective pregabalin only treated groups. Table 1 Effect of melatonin on inflammation indices in the epididymis of the pregabalin treated male Wistar rats Group/Parameters NF-kB (pg/mg protein) TNF-α (pg/mg protein) IL-1β (pg/mg protein) Ctrl 14.40 ± 2.41 39.20 ± 2.49 17.80 ± 2.17 Mel 13.20 ± 2.39 38.80 ± 1.79 16.80 ± 2.49 LD PG 27.80 ± 2.28 a,b 69.20 ± 2.17 a,b 41.40 ± 2.30 a,b HD PG 41.20 ± 1.92 a,b,c 89.00 ± 1.58 a,b,c 58.00 ± 2.34 a,b,c LD PG + Mel 21.40 ± 1.82 a,b,c,d 45.60 ± 2.41 a,b,c,d 24.80 ± 2.49 a,b,c,d HD PG + Mel 32.00 ± 2.24 a,b,d,e 69.40 ± 2.61 a,b,d,e 35.40 ± 1.95 a,b,c,d,e Values (n = 5) are expressed as mean ± SEM. Bars carrying - a represent significance ( p < 0.05) when compared to ctrl (control), - b represent significance ( p < 0.05) when compared to Mel. (melatonin), - c represents significance ( p < 0.05) when compared to LD PG (Low dose of pregabalin), and - d represent significance ( p < 0.05) when compared to HD PG (High dose of pregabalin). Histopathological examination of the effect of melatonin on the testicular tissue and epididymis of Wistar rats following chronic exposure to pregabalin Photomicrographs display cross-sectional views of testicular tissue. The structure of the testicles is well-maintained in both the control group and the group of rats treated with melatonin. The morphology and dimensions of the seminiferous tubules (shown by the black arrow) appear to be within the expected range. Nevertheless, pregabalin caused alterations in the structure of the testicles. The seminiferous tubules exhibit a distorted and diminished appearance. The germ cells (shown by the red arrow) are shed. The lumens of the seminiferous tubules contain a small amount of fully developed sperm cells (shown by a red circle). The interstitial space exhibits distortion, characterised by regions of congestion and diminished size (shown by the green arrow). Melatonin improved these degenerative alterations. The germ cells (shown by the red arrow) are in different stages of development, with some discarded germ cells observed. The seminiferous tubules contain a small number of fully developed sperm cells (shown by red circles) within their lumens. The interstitial area exhibits Leydig cells within the expected range (shown by the green arrow) Fig. 8. The mice treated with control and melatonin had well-preserved epididymal structure, with the epithelium and lumen remaining intact, and a high number of fully developed sperm cells present. The pregabalin treated showed thickened epithelial layer with multiple areas of degeneration seen with the lumen showing little mature sperm (Fig. 9). The luminal diameter of the rats administered just with melatonin exhibited a substantial increase in comparison to the control animals (Table 2 ). The luminal diameter of the rats treated with pregabalin at both doses showed a substantial reduction compared to the group treated with melatonin alone (Table 2 ). Table 2 Table showing the Histo-morphometry properties of the epididymis in the treated rats Group/Parameters Tubular Diameter (µm) Luminal Diameter (µm) Epithelial Height (µm) Ctrl 279.0 ± 4.27 254.7 ± 6.51 19.74 ± 1.70 Mel 292.4 ± 24.42 294.0 ± 11.31 a 19.46 ± 0.52 LD PG 290.6 ± 14.77 218.5 ± 6.36 b 22.67 ± 0.00 a,b HD PG 300.7 ± 26.04 249.9 ± 17.25 b 25.93 ± 0.91 a,b LD PG + Mel 269.5 ± 8.59 246.5 ± 0.64 b 15.81 ± 1.03 a,c,d HD PG + Mel 290.2 ± 4.43 270.7 ± 6.60 c 18.25 ± 0.65 c,d Data are expressed as mean ± standard deviation (n = 5). Data marked with superscript a , are statistically significant (p < 0.05) when compared to control; Data marked with superscript b , are statistically significant (p < 0.05) when compared to melatonin treated group. Data marked with superscript c , are statistically significant (p < 0.05) when compared to Low-dose Pregabalin treated group. Data marked with superscript d , are statistically significant (p < 0.05) when compared to High-dose Pregabalin treated group. The height of the epithelium layer in the rats treated with pregabalin at both doses was considerably greater than that of the rats in the control group and the rats treated with melatonin alone (Table 2 ). Melatonin treatment on the pregabalin exposed rats significantly reduced the epithelial height when compared to the corresponding pregabalin treatments (Table 2 ). Discussion The current investigation observed notable decreases in the body weight of the rats who received pregabalin treatment. Curiously, this discovery was similarly observed when evaluating both the testicular weight and the relative testicular weight of the rats. The reduced body weights could be secondary to decrease food intake as a result of the inhibition of the feeding center in the hypothalamus. This could possibly be linked to the reported effects of pregabalin on the brain [ 21 ]. Moreover, the weight loss [ 22 ] could also be due to altered energy metabolism at the cellular levels and hence inefficient processing of the intake food substances. Failure to measure the daily food intake is considered as one of the limitations of this study. The drug has the potential for addiction [ 23 ]; hence, there could be accompanying reduction in appetite. The observed substantial decrease in testicular weight could potentially be accounted for by the mechanism discussed earlier. The occurrence of tissue death and the resulting decrease in the total tissue size of the gonads has undoubtedly led to the observed considerable decline in testosterone levels in the groups treated with pregabalin [ 24 ]. The observed atrophy of the testicular and epididymal tissues demonstrated in the micrograph and the infiltration of these tissues with inflammatory cells further affirms the peripheral effects of pregabalin. The Leydig cells were subjected to oxidative assault, and the hypothalamic-pituitary-gonadal (HPG) axis was suppressed. This was confirmed by the notable decrease in the plasma levels of FSH and LH following the administration of pregabalin, indicating a significant reduction in testosterone levels. The weak signalling action of the low plasma levels of gonadotrophins on the testicular tissue has no potent effect to drive the process involved in the peripheral synthesis of the testosterone to completion. FSH is known to stimulate testicular growth and health and hence it is indirectly important for testosterone synthesis, unlike LH which has a direct effect on Leydig cells in the testes. In the groups simultaneously treated with melatonin, the observed effects of pregabalin on the body and testicular weights and testicular and epididymal histoarchitecture were significantly prevented. Melatonin has been reported to increase the plasma level of FSH and LH, following the stimulation of the release of GNRH at the level of hypothalamus, which subsequently activates the gonadotrophs in the anterior pituitary [ 25 ]. 3β-HSD and 17β-HSD are crucial enzymes involved in the synthesis, breakdown, and conversion of steroid hormones [ 26 ]. The observed changes in enzyme activity in the testicular tissue after the injection of pregabalin support the large decrease in testosterone levels in the plasma. Additionally, these changes indicate a disruption in the HPG axis, which can result in many endocrine disorders. The co-administration of pregabalin with melatonin may avoid enzymatic imbalances, possibly due to the antioxidant properties of melatonin, which can scavenge free radicals. Previous studies have suggested that reactive species might disrupt the structure and activities of enzymes. The treatment of pregabalin resulted in enzymatic dysfunction, which was accompanied by increased levels of lactate and pyruvate, as well as elevated activity of LDH and GGT in the testicular tissue. The observed changes are known to be present in different metabolic abnormalities, including lactic acidosis, tissue necrosis, and hepatic dysfunction. These abnormalities lead to a reduction in the removal of lactate from the blood and the inhibition of pyruvate dehydrogenase (PDH), an enzyme that converts pyruvate to acetyl-CoA. An impairment in the functional role of PDH leads to the buildup of pyruvate in the bloodstream. The delivery of pregabalin resulted in considerable changes in lactate and pyruvate levels, as well as LDH and GGT activities. However, these changes were mostly averted in the groups who received simultaneous treatment with melatonin. The effects of the graded dosages of pregabalin on the biomarkers indicated above were inconsistent. The efficacy of the medicine is not consistently superior with a higher dosage compared to a lower dosage. Literature has documented the hepatoprotective and antioxidant properties of melatonin in response to various environmental challenges [ 27 – 29 ]. The correlation between the levels of lactate and pyruvate and the activities of LDH and GGT in the epididymal tissue and the results of the biomarker assays in the testicular tissue is expected. It is important to mention that the group treated with simply melatonin did not show any significant changes in the biomarkers listed above, compared to the control group, at the given dose of melatonin. This phenomenon was also noted during the evaluation of the antioxidant and apoptotic indicators. However, at higher doses and for longer duration of administration, there might be some mild accompanying adverse effects [ 30 ]. The current research found that the use of pregabalin caused lipid peroxidation, which could potentially damage the cell membrane, lead to cell death, and disrupt the balance of the antioxidant system. This was observed through significant reductions in the activity of different antioxidant enzymes in the testicular and epididymal tissues. The pro-oxidative effects of pregabalin are consistent with the findings[ 31 ]. Taha and colleagues specifically observed that the medication induces genotoxicity through the reversal of the BAX/BCL2 ratio, increased levels of p38 MAPK, and impairment of the antioxidant system [ 31 ]. The increase in caspase 3 activity after the injection of pregabalin is consistent with its ability to generate free radicals and operate as a pro-oxidant. Caspase-3 is a crucial enzyme involved in the final stage of programmed cell death, known as apoptosis. It has a crucial function in the regulated elimination of cells during apoptosis. Therefore, dysregulation in the activity of this enzyme can instigate different pathological cascade. Shokry equally observed an elevation in the activities of caspase 3 following the administration of pregabalin [ 22 ]. It is noteworthy to assert that the effect of pregabalin on the activities of caspase 3 was significantly prevented in the groups simultaneously treated with melatonin, possible due to the antioxidant action of the hormone [ 32 ]. While the specific mechanism through which melatonin exerts its anti-oxidative effects remains unclear, it is clear that the hormone can inhibit lipid peroxidation, as measured by the level of MDA in testicular and epididymal tissues, and enhance the oxidative enzyme system. This was confirmed by the notable increases in the levels of CAT, SOD, Gpx, GSH, and GST enzymes, as well as the considerable decrease in the activity of the apoptotic marker caspase 3 [ 33 ]. By donating electrons, melatonin can deactivate harmful free radicals, therefore preventing oxidative damage to the body tissues. The disturbance of the antioxidant system following the administration of pregabalin was accompanied by the increase in the level of inflammatory markers in the testicular and epididymal tissues. Oxidative stress promotes inflammation through the activation of pro-inflammatory transcription factors, activation of inflammasomes, inactivation of anti-inflammatory molecules, direct damage to cellular components, and the recruitment of immune cells. The inflammatory effect of pregabalin was evidenced by the notable elevations in the levels of NF-kB, TNF-α, and IL-1β in the tissue. While literature does contain evidence on the anti-inflammatory properties of pregabalin [ 34 , 35 ], the possible pro-inflammatory action of this drug is no doubt secondary to its pro-oxidative effects. Conclusion Melatonin has a protective effect against pregabalin-induced gonadotoxicity via anti-oxidative, anti-inflammatory, anti-apoptotic, and enzymatic and hormonal regulatory mechanisms. Abbreviations ELISA Enzyme linked immunosorbent assay FSH Follicle stimulating hormone GGT Gamma glutamyl transferase GnRH Gonadotropin-releasing hormone Gpx Glutathione peroxidase IL-1β Interleukin-1β LDH Lactate dehydrogenase LH Luteinizing Hormone MDA Malondialdehyde PG Pregabalin SOD Superoxide dismutase TNF-α Tumor necrosis factor-alpha Declarations Ethics approval and consent to participate : The ethical reference number for the present study is AERCFBMSLAUTECH: 011/09/20231. Approval was given by the Ethics Review Committee of the Faculty of Basic Medical Sciences, LAUTECH, Ogbomoso, Oyo state, Nigeria. Consent for publication : not applicable. Consent to Participate : not applicable. Competing interests : the authors have no competing interests as defined by BMC, or other interests that might be perceived to influence the results and/or discussion reported in this paper. Funding: The authors confirm that they have not received any financial assistance or support for the research, preparation, and publication of this article. Author Contribution A.F.A. conceptualize the work. A.F.A., M.S.B., W.J.A. and L.O.A wrote the main manuscript text. P.O. and O.P.A. prepared figures 1-9. All authors reviewed the manuscript Acknowledgements: not applicable. Availability of data and materials: No datasets were generated or analysed during the current study. References Sharma A, Minhas S, Dhillo WS, Jayasena CN. Male infertility due to testicular disorders. J Clin Endocrinol Metab. 2021;106(2). 10.1210/clinem/dgaa781 . PMID: 33295608; PMCID: PMC7823320. Akhigbe R, Ajayi A. Testicular toxicity following chronic codeine administration is via oxidative DNA damage and up-regulation of NO/TNF-α and caspase 3 activities. PLoS ONE. 2020;15(3). 10.1371/journal.pone.0224052 . Agarwal A, Mulgund A, Hamada A, Chyatte MR. A unique view on male infertility around the globe. 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Gonadal steroid hormones and the hypothalamo-pituitary-adrenal axis. Front Neuroendocrinol. 2014;35(2):197–220. 10.1016/j.yfrne.2013.11.001 . Epub 2013 Nov 16. PMID: 24246855; PMCID: PMC5802971. Olayaki LA, Alagbonsi IA, Abdulrahim AH et al. Melatonin prevents and ameliorates lead-induced gonadotoxicity through antioxidative and hormonal mechanisms. Toxicol Ind Health. 2018;34(9):596–608. 10.1177/0748233718773508 . PMID: 29759042. Odetayo AF, Adeyemi WJ, Olayaki LA. In vivo exposure to bisphenol F induces oxidative testicular toxicity: role of Erβ and p53/Bcl-2 signaling pathway. Front Reprod Health. 2023;5:1204728. 10.3389/frph.2023.1204728 . PMID: 37601897; PMCID: PMC10433915. Mathes AM. Hepatoprotective actions of melatonin: possible mediation by melatonin receptors. World J Gastroenterol. 2010;16(48):6087–97. 10.3748/wjg.v16.i48.6087 . PMID: 21182223; PMCID: PMC3012585. Oleshchuk O, Ivankiv Y, Falfushynska H, Mudra A, Lisnychuk N. Hepatoprotective effect of melatonin in toxic liver injury in rats. Med (Kaunas). 2019;55(6):304. 10.3390/medicina55060304 . PMID: 31238587; PMCID: PMC6631928. Barbarossa A, Carrieri A, Carocci A. Melatonin and related compounds as antioxidants. Mini Rev Med Chem. 2024;24(5):546–565. 10.2174/1389557523666230627140816 . PMID: 37366352. Andersen LP, Gögenur I, Rosenberg J, Reiter RJ. The safety of melatonin in humans. Clin Drug Investig. 2016;36(3):169 – 75. 10.1007/s40261-015-0368-5 . PMID: 26692007. Taha SHN, Zaghloul HS, Ali AAER, Gaballah IF, Rashed LA, Aboulhoda BE. The neurotoxic effect of long-term use of high-dose pregabalin and the role of alpha tocopherol in amelioration: implication of MAPK signaling with oxidative stress and apoptosis. Naunyn Schmiedebergs Arch Pharmacol. 2020;393(9):1635–48. Epub 2020 May 6. PMID: 32377769. Chrustek A, Olszewska-Słonina D. Melatonin as a powerful antioxidant. Acta Pharm. 2020;71(3):335–354. 10.2478/acph-2021-0027 . PMID: 36654092. Estaras M, Ortiz-Placin C, Castillejo-Rufo A, Fernandez-Bermejo M, Blanco G, Mateos JM, Vara D, Gonzalez-Cordero PL, Chamizo S, Lopez D, Rojas A, Jaen I, de Armas N, Salido GM, Iovanna JL, Santofimia-Castaño P, Gonzalez A. Melatonin controls cell proliferation and modulates mitochondrial physiology in pancreatic stellate cells. J Physiol Biochem. 2023;79(1):235–49. 10.1007/s13105-022-00930-4 . Epub 2022 Nov 5. PMID: 36334253; PMCID: PMC9905253. Hummig W, Kopruszinski CM, Chichorro JG. Pregabalin reduces acute inflammatory and persistent pain associated with nerve injury and cancer in rat models of orofacial pain. J Oral Facial Pain Headache. 2014 Fall;28(4):350-9. 10.11607/ofph.1317 . PMID: 25347171. Kilic FS, Kaygisiz B, Aydin S, Yildirim C, Karimkhani H, Oner S. Pregabalin attenuates carrageenan-induced acute inflammation in rats by inhibiting proinflammatory cytokine levels. Eurasian J Med. 2018;50(3):156–9. 10.5152/eurasianjmed.2018.17261 . PMID: 30515034; PMCID: PMC6263227. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 12 Feb, 2025 Read the published version in BMC Pharmacology and Toxicology → Version 1 posted Editorial decision: Revision requested 29 Nov, 2024 Reviews received at journal 05 Nov, 2024 Reviews received at journal 30 Oct, 2024 Reviewers agreed at journal 30 Oct, 2024 Reviews received at journal 25 Oct, 2024 Reviewers agreed at journal 23 Oct, 2024 Reviewers agreed at journal 15 Oct, 2024 Reviewers invited by journal 15 Oct, 2024 Editor invited by journal 13 Sep, 2024 Editor assigned by journal 12 Sep, 2024 Submission checks completed at journal 12 Sep, 2024 First submitted to journal 04 Sep, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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mechanisms","fulltext":[{"header":"Introduction","content":"\u003cp\u003eInfertility is defined as the inability to achieve pregnancy after engaging in regular sexual intercourse at least twice a week for duration of one year [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Male infertility is usually characterized by dysfunctional sperm parameters and may account for 50% of all infertility cases [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Akang and co explained an infertility belt stretching beyond Sub-saharan Africa and a high prevalence of 20 to 40 percent [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. The frequency of secondary male infertility was estimated to be 49 percent. In Nigeria, research indicates that male infertility is responsible for 40 to 50 percent of all infertility cases [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe testicular function is tightly regulated by the hypothalamo-pituitary-gonadal axis (HPG). This regulatory processes can be distorted by exposure to drugs [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e] such as ketoconazole, chemotherapy, gabapentinoids, and opioids [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e] resulting in hypogonadism and ultimately impaired semen quality. Testicular dysfunction could also be as a result of injury, trauma, and diseases such as mumps, varicocele, and orchitis [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e] and could occur following prolonged use of pregabalin with associated hypogonadism, and spermato-toxicity leading to infertility [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e \u003cp\u003ePregabalin (PG) is a novel anticonvulsant medication that falls under the category of gabapentinoids. It has been found to be beneficial in treating sleep disorders, generalised anxiety disorder, fibromyalgia, and epilepsy [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Pregabalin received FDA approval in 2004 as an efficacious treatment for neuropathic pain associated with diabetic peripheral neuropathy, spinal cord injury, and postherpetic neuralgia [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Studies also show that pregabalin may be effective in the management of bipolar disorder, chronic pruritus, chronic cough, and restless leg syndrome [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. According to Kamel and, the administration of pregabalin produces side effects, including nausea, vomiting, dry mouth, stomach cramps, constipation and diarrhea, flatulence, and abdominal distention [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Nevertheless, there is a scarcity of evidence in literature regarding the potential impacts of this medication on male reproductive function.\u003c/p\u003e \u003cp\u003eUnlike pregabalin, melatonin is a neurohormone that is naturally produced by the pineal gland located behind the third ventricle in the brain [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. The hormone is associated with the regulation of physiological functions such as sleep cycle, immune function, homeostasis, glucose regulation [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e] and is beneficial to the cardiovascular system [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Melatonin controls the release of gonadotropin releasing hormone (GnRH), luteinizing hormone (LH), testosterone, and the development of the testes [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. According to Yu and co, it has been documented as a powerful antioxidant that possesses both lipophilic and hydrophilic characteristics [15. Melatonin was demonstrated to exert ameliorative effects in tramadol-induced reproductive toxicity by preventing oxidative damage, mitochondrial injury, and apoptosis [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAlthough there are meagre research reports demonstrating spermatotoxicty following chronic use of pregabalin, no study has reported its effects on testicular steroidogenesis and the possible ameliorative potential of melatonin when co-administered with the drug. The present study focuses on hormonal, oxidative, apoptotic, steroidogenic and inflammatory markers, and also testicular and epididymal histoarchitecture in male rats exposed to melatonin and pregabalin.\u003c/p\u003e"},{"header":"Methods and methodology","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eChemicals\u003c/h2\u003e \u003cp\u003ePregabalin (CAS no: 148553-50-8) and melatonin (CAS no: 73-31-4) were obtained from Pfizer pharmaceutical industry, USA. All the other compounds utilised in this investigation were of conventional analytical grades.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eAnimal care and experimental design\u003c/h2\u003e \u003cp\u003eA total of sixty (60) adult male Wistar rats weighing between 120-140g were obtained from reputable commercial breeders and housed in the Animal House Facility of the Department of Physiology, Ladoke Akintola University of Technology, Ogbomoso, Nigeria. The subjects were maintained under controlled conditions, specifically a temperature range of 28\u0026ndash;31\u0026deg;C, a light/dark cycle of 12 hours, and a relative humidity range of 50\u0026ndash;70%.\u003c/p\u003e \u003cp\u003eThe rats were kept in plastic cages, provided with unlimited access to ordinary pellets, and given unrestricted access to water. The animals are provided with compassionate care in line with the laboratory animal care principles of the National Medical Research Council and the guidelines outlined in the National Academy of Sciences' \"Guide for the Care and Use of Laboratory Animals\" (National Institute of Health Publication no. 80\u0026ndash;23, updated 1978).\u003c/p\u003e \u003cp\u003eThe rats would be randomly assigned to six groups, with ten rats in each group. The control group was administered a placebo of 0.1 ml of 0.9% normal saline. Pregabalin was delivered orally at two different doses: a low dose of 150 mg/kg/BW and a high dose of 300 mg/kg/BW. Melatonin was also administered orally at a dose of 10 mg/kg/BW, either alone or in combination with pregabalin. Body weights were measured on a weekly basis using a precise digital electronic weighing scale (Bioevopeak, Shandong, China) that was calibrated for accuracy. Therapeutic dose equivalents to account for the weight difference in animals, in order to get the appropriate therapeutic doses for the rat model. The treatment regimen consisted of a daily administration for duration of eight (8) weeks. The ethical reference number for the present study is AERCFBMSLAUTECH: 011/09/20231.\u003c/p\u003e \u003cp\u003e \u003cstrong\u003eClinical trial number\u003c/strong\u003e \u003cp\u003enot applicable\u003c/p\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eSample collection\u003c/h2\u003e \u003cp\u003eAfter 24 hours of administering the last dose of the\u003c/p\u003e \u003cp\u003eThe doses of pregabalin and melatonin administered were determined by scaling the human experiment; the animals were anaesthetised with pentobarbital sodium (40 mg/kg BW, i.p.) [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e] and then euthanised before collecting blood through heart puncture and removing the testes. The mass of each testis was documented. The gonadosomatic index was calculated by dividing the paired testicular weight by the body weight and multiplying the result by 100 [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe blood samples were collected in bottles containing heparin and then centrifuged at a speed of 3500rpm for a duration of 10 minutes. The centrifugation was carried out at a temperature of -4o℃using a refrigerated centrifuge manufactured by Bio-Gene Technology Ltd., located in Grandtech Centre, Shatin, Hong Kong. The plasma samples that were divided into discrete portions were utilised to evaluate several biochemical parameters. The tissue from the left testicle was broken down into a uniform mixture using a phosphate buffer solution, while the right testicle was kept in bouin's fluid before being examined histologically.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eBiochemical assay\u003c/h2\u003e \u003cdiv id=\"Sec7\" class=\"Section3\"\u003e \u003ch2\u003eEstimation of serum levels of reproductive hormones\u003c/h2\u003e \u003cp\u003eThe measurement of serum and testicular testosterone was conducted using the ELISA kit from Monobind Inc. USA, with the product number 4806-300A. The serum FSH was analysed using an ELISA kit (Monobind Inc. USA; product number: 506-300A), whereas the serum LH was analysed using another ELISA kit (Monobind Inc. USA; product number: 625-300A).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eEstimation of testicular enzymes, lactate and pyruvate\u003c/h2\u003e \u003cp\u003eThe testicular enzymatic activity of 17β-hydroxysteroid dehydrogenase and 3β-hydroxysteroid dehydrogenase were assessed using a standard kit (Teco Diagnostics, N Lakeview Ave, Anaheim, CA 92807, United States) according to the manufacturer's instructions.\u003c/p\u003e \u003cp\u003eThe levels of lactate dehydrogenase (LDH) and gamma-glutamyl transferase (GGT) activities, as well as lactate and pyruvate concentrations, in the testicular homogenate were measured using a standard kit provided by Agappe Diagnostics Ltd., located at Agappe Hills, Pattimattom PO, Kochi, Kerala 683562, India.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eEstimation of testicular oxidative markers\u003c/h2\u003e \u003cp\u003eThe concentrations of malondialdehyde (MDA), as well as the enzymatic activity of superoxide dismutase (SOD) and catalase, were measured in the testicular and epididymis tissues, following the methods described [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. The level of reduced glutathione (GSH), as well as the activities of glutathione peroxidase (GPx) and glutathione-S-transferase (GST) in the testicular and epididymis tissues, were measured using the procedures described [\u003cspan additionalcitationids=\"CR19\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eAssessment of markers of inflammation\u003c/h2\u003e \u003cp\u003eThe concentrations of nuclear factor-kappa B \u003cb\u003e(\u003c/b\u003eNF-kB), tumour necrosis factor-α (TNF-α) and interleukin-1β (IL-1 β) in the testicular and epididymis tissues were determined by ELISA kits according per manufacturer\u0026rsquo;s guideline (Elabscience Biotechnology Company Ltd., Wuhan, Hubei, China).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eHistology\u003c/h2\u003e \u003cp\u003eThe testicular tissues were preserved in Bouin's solution, dried using a series of alcohol solutions, clarified using xylene, embedded in paraffin wax, cut into thin sections (2\u0026ndash;3\u0026micro;m thick) using a microtome, attached to glass slides, stained with haematoxylin-eosin, and examined using a digital light microscope (Olympus CH; Olympus, Tokyo, Japan) at various levels of magnification. Photomicrographs were captured using a Sony digital camera (Model: DSC-W710).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eThe data was analysed utilising the Statistical Package for the Social Sciences (SPSS), and the results were reported as the mean value plus or minus the standard deviation. The data was analysed using one-way analysis of variance (ANOVA) followed by the Tukey test for post-hoc comparisons among several groups. Statistical significance was determined for intergroup differences at a significance level of P\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eEffect of Melatonin on Body Weight Change, Testicular Weight, and Relative Testicular Weight\u003c/h2\u003e \u003cp\u003eFigure 1a-c illustrates the alterations in body weight, testicular weight, and relative testicular weight among the Control and test groups (Melatonin, Low dose Pregabalin, High dose Pregabalin, Low dose Pregabalin\u0026thinsp;+\u0026thinsp;Melatonin, High dose Pregabalin\u0026thinsp;+\u0026thinsp;Melatonin).\u003c/p\u003e \u003cp\u003eThe administration of melatonin had no significant effect on the body weight of the treated rats compared to the control group (Fig.\u0026nbsp;1a). Exposure to pregabalin, whether at low or high dosages, resulted in a substantial reduction in body weight gain, testicular weight, and relative testicular weight in the treated rats compared to the control and melatonin-treated rats. Nevertheless, administering melatonin to rats treated with pregabalin at both doses leads to a notable rise in body weight growth, testicular weight, and relative testicular weight in comparison to their corresponding doses of pregabalin therapy, as seen in Fig.\u0026nbsp;1a-c.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eEffect of Melatonin on the Testicular Concentration of Reproductive Hormones in Pregabalin-Exposed Male Rats\u003c/h2\u003e \u003cp\u003eFigure 2a-c displays the variations in the levels of reproductive hormones in the testicles of both the Control and test groups. The groups include Melatonin, Low dose Pregabalin, High dose Pregabalin, Low dose Pregabalin\u0026thinsp;+\u0026thinsp;Melatonin, and High dose Pregabalin\u0026thinsp;+\u0026thinsp;Melatonin. Administration of melatonin does not have a significant effect on the testicular concentration of hormones in the treated rats compared to the control group. Exposure to pregabalin, whether at low or high dosages, led to a significant reduction in the concentration of testosterone, luteinizing hormone, and follicle stimulating hormone in the testicles of rats, as compared to rats in the control group and those treated with melatonin. Nevertheless, administering melatonin to rats exposed to pregabalin at both doses leads to a notable elevation in testicular hormone levels compared to the corresponding doses of pregabalin treatment (Fig.\u0026nbsp;2a-c).\u003c/p\u003e \u003cp\u003e \u003cb\u003eEffect of Melatonin on the Testicular activities of Steroidogenic Enzymes and tissue injury markers in Pregabalin-Exposed Male Rats\u003c/b\u003e \u003c/p\u003e \u003cp\u003eFigure 3a-f displays the alterations in testicular activity of steroidogenic enzymes for the Control group and other test groups, including Melatonin, Low dose Pregabalin, High dose Pregabalin, Low dose Pregabalin\u0026thinsp;+\u0026thinsp;Melatonin, and High dose Pregabalin\u0026thinsp;+\u0026thinsp;Melatonin. Administration of melatonin does not have a significant effect on the testicular activity of steroidogenic enzymes 3 and 17 βeta-Hydrosteroid Dehydrogenase, as compared to the control group. Exposure to pregabalin, at both low and high doses, resulted in a significant decrease in testicular enzyme activity compared to rats in the control group and rats treated with melatonin. Nevertheless, administering melatonin to rats exposed to pregabalin at both levels leads to a noteworthy augmentation in testicular activity of the markers, in comparison to the corresponding doses of pregabalin therapy. Administration of melatonin at both dosages considerably reduces the testicular activity of these enzymes in comparison to both the control group and the group of rats treated with melatonin alone (see Fig.\u0026nbsp;3a and 3b).\u003c/p\u003e \u003cp\u003eFigures 3c-f depict the alterations in the concentration of lactate, pyruvate, lactate dehydrogenase, and Gamma-glutamyl transferase in the testicles of both the Control and test groups. The test groups include those treated with Melatonin, Low dose Pregabalin, High dose Pregabalin, Low dose Pregabalin\u0026thinsp;+\u0026thinsp;Melatonin, and High dose Pregabalin\u0026thinsp;+\u0026thinsp;Melatonin. The testicular activity of lactate, pyruvate, lactate dehydrogenase, and Gamma-glutamyl transferase was considerably elevated in rats exposed to both low and high dosages of pregabalin, compared to rats in the control group and those treated with melatonin. Nevertheless, administering melatonin to rats exposed to pregabalin at both doses results in a noteworthy reduction in testicular levels of lactate, pyruvate, lactate dehydrogenase, and Gamma-glutamyl transferase, compared to the corresponding doses of pregabalin.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eEffect of Melatonin on the Epididymal Tissues Injury Markers in Pregabalin-Exposed Male Rats\u003c/h2\u003e \u003cp\u003e \u003cb\u003eFigures 4a-d\u003c/b\u003e depicts the alterations in the concentration of lactate, pyruvate, lactate dehydrogenase, and Gamma-glutamyl transferase in the epididymis of both the Control and test groups. The test groups include those treated with Melatonin, Low dose Pregabalin, High dose Pregabalin, Low dose Pregabalin\u0026thinsp;+\u0026thinsp;Melatonin, and High dose Pregabalin\u0026thinsp;+\u0026thinsp;Melatonin. Administration of melatonin does not cause a significant change in the concentration of the parameters in the epididymis of the treated rats compared to the control group. Exposure to pregabalin, whether at low or high doses, led to a substantial rise in the levels of lactate, pyruvate, lactate dehydrogenase, and Gamma-glutamyl transferase in the epididymis, as compared to rats that were not exposed to pregabalin or were treated with melatonin. Nevertheless, the administration of melatonin to rats exposed to pregabalin at both doses results in a notable reduction in the testicular levels of lactate, pyruvate, lactate dehydrogenase, and Gamma-glutamyl transferase, in comparison to the corresponding doses of pregabalin alone.\u003c/p\u003e \u003cp\u003e \u003cb\u003emalondialdehyde (MDA) level, superoxide dismutase (SOD) and catalase activities, level of reduced glutathione, and glutathione peroxidase (Gpx). Glutathione-S-transferase (GST) and caspase 3 activitie\u003c/b\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eEffect of Melatonin on the Concentration of Oxidative Stress Markers and Caspase 3 Activities in Testicular and Epididymal Tissues of Pregabalin-Exposed Male Rats\u003c/b\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eFigure 5a-f\u003c/b\u003e displays the variations in the testicular levels of oxidative stress parameters for the Control group and the test groups (Melatonin, Low dose Pregabalin, High dose Pregabalin, Low dose Pregabalin\u0026thinsp;+\u0026thinsp;Melatonin, High dose Pregabalin\u0026thinsp;+\u0026thinsp;Melatonin).The injection of pregabalin resulted in a rise in malondialdehyde concentration and glutathione-s-transferase activity, while causing a decrease in the activities of antioxidant enzymes, including superoxide dismutase, catalase, reduced glutathione, and glutathione-peroxidase, as compared to the control and melatonin only groups. Nevertheless, administering melatonin to rats exposed to pregabalin resulted in a notable reduction in testicular malondialdehyde levels and glutathione-s-transferase activity. Additionally, there was a significant increase in the activities of superoxide dismutase, catalase, reduced glutathione, and glutathione-peroxidase compared to the low and high dose pregabalin groups.\u003c/p\u003e \u003cp\u003eFigure 5g displays the alterations in the testicular activity of Caspase 3 for the Control group and other test groups, including Melatonin, Low dose Pregabalin, High dose Pregabalin, Low dose Pregabalin\u0026thinsp;+\u0026thinsp;Melatonin, and High dose Pregabalin\u0026thinsp;+\u0026thinsp;Melatonin. Exposure to pregabalin, at both low and high dosages, resulted in a significant increase in testicular Caspase 3 activity compared to rats in the control group and rats treated with melatonin. Nevertheless, administration of melatonin to rats exposed to pregabalin at both levels leads to a noteworthy decrease in testicular activity of Caspase 3, compared to the corresponding doses of pregabalin treatment.\u003c/p\u003e \u003cp\u003eFigure 6a-f displays the variations in the epididymal concentration of oxidative stress parameters for the Control group and the test groups (Melatonin, Low dose Pregabalin, High dose Pregabalin, Low dose Pregabalin\u0026thinsp;+\u0026thinsp;Melatonin, High dose Pregabalin\u0026thinsp;+\u0026thinsp;Melatonin).The injection of pregabalin resulted in an increase in malondialdehyde content, while causing a decrease in the activity of antioxidant enzymes such as superoxide dismutase, catalase, reduced glutathione, glutathione-peroxidase, and glutathione-s-transferase, as compared to the control and melatonin only groups. Nevertheless, administering melatonin to rats exposed to pregabalin at both doses resulted in a notable reduction in epididymal malondialdehyde concentration. Additionally, there was a significant increase in the activities of superoxide dismutase, catalase, reduced glutathione, glutathione-peroxidase, and glutathione-s-transferase compared to the corresponding groups that received only pregabalin.\u003c/p\u003e \u003cp\u003eFigure 6g illustrates the variations in the epididymal activity of Caspase 3 for the Control group and the test groups (Melatonin, Low dose Pregabalin, High dose Pregabalin, Low dose Pregabalin\u0026thinsp;+\u0026thinsp;Melatonin, High dose Pregabalin\u0026thinsp;+\u0026thinsp;Melatonin). Exposure to pregabalin, whether at low or high dosages, resulted in a significant increase in epididymal Caspase 3 activity compared to rats that were not exposed to pregabalin or were given with melatonin. Nevertheless, administering melatonin to rats exposed to pregabalin at both levels leads to a noteworthy decrease in epididymal activity of Caspase 3, in comparison to the corresponding doses of pregabalin administration.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eEffect of Melatonin on the Testicular Concentration of Inflammatory Parameters in Pregabalin-Exposed Male Rats\u003c/h2\u003e \u003cp\u003e \u003cb\u003eFigure 7a-c\u003c/b\u003e shows the changes in the testicular concentration of inflammatory markers for Control and test groups The options available are melatonin, low-dose pregabalin, high-dose pregabalin, low-dose pregabalin combined with melatonin, and high-dose pregabalin combined with melatonin.The results indicate that the injection of Pregabalin led to an increase in inflammatory markers in the testes of rats, as evidenced by an elevation in the nuclear respiratory kappa B (NF-kB), tumour necrosis factor (TNF-α), and interleukin 1B (IL-1β) cytokine. Nevertheless, administering melatonin to rats exposed to pregabalin at both levels results in a noteworthy decrease in testicular inflammatory markers compared to the corresponding doses of pregabalin alone. In Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, the treatment of Pregabalin resulted in an increase in inflammatory markers in the rat epididymis, as indicated by the elevation of nuclear respiratory kappa B (NF-kB), tumour necrosis factor (TNF-α), and cytokine interleukin 1B (IL-1β). However, the treatment of pregabalin-exposed rats at both doses with melatonin causes a significant reduction in epididymal inflammatory markers when compared to their respective pregabalin only treated groups.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eEffect of melatonin on inflammation indices in the epididymis of the pregabalin treated male \u003cem\u003eWistar\u003c/em\u003e rats\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGroup/Parameters\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNF-kB\u003c/p\u003e \u003cp\u003e(pg/mg protein)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTNF-α\u003c/p\u003e \u003cp\u003e(pg/mg protein)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eIL-1β\u003c/p\u003e \u003cp\u003e(pg/mg protein)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCtrl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e14.40\u0026thinsp;\u0026plusmn;\u0026thinsp;2.41\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e39.20\u0026thinsp;\u0026plusmn;\u0026thinsp;2.49\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e17.80\u0026thinsp;\u0026plusmn;\u0026thinsp;2.17\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMel\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e13.20\u0026thinsp;\u0026plusmn;\u0026thinsp;2.39\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e38.80\u0026thinsp;\u0026plusmn;\u0026thinsp;1.79\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e16.80\u0026thinsp;\u0026plusmn;\u0026thinsp;2.49\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLD PG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e27.80\u0026thinsp;\u0026plusmn;\u0026thinsp;2.28\u003csup\u003ea,b\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e69.20\u0026thinsp;\u0026plusmn;\u0026thinsp;2.17\u003csup\u003ea,b\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e41.40\u0026thinsp;\u0026plusmn;\u0026thinsp;2.30 \u003csup\u003ea,b\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHD PG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e41.20\u0026thinsp;\u0026plusmn;\u0026thinsp;1.92\u003csup\u003ea,b,c\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e89.00\u0026thinsp;\u0026plusmn;\u0026thinsp;1.58 \u003csup\u003ea,b,c\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e58.00\u0026thinsp;\u0026plusmn;\u0026thinsp;2.34 \u003csup\u003ea,b,c\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLD PG\u0026thinsp;+\u0026thinsp;Mel\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e21.40\u0026thinsp;\u0026plusmn;\u0026thinsp;1.82\u003csup\u003ea,b,c,d\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e45.60\u0026thinsp;\u0026plusmn;\u0026thinsp;2.41 \u003csup\u003ea,b,c,d\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e24.80\u0026thinsp;\u0026plusmn;\u0026thinsp;2.49 \u003csup\u003ea,b,c,d\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHD PG\u0026thinsp;+\u0026thinsp;Mel\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e32.00\u0026thinsp;\u0026plusmn;\u0026thinsp;2.24\u003csup\u003ea,b,d,e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e69.40\u0026thinsp;\u0026plusmn;\u0026thinsp;2.61\u003csup\u003ea,b,d,e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e35.40\u0026thinsp;\u0026plusmn;\u0026thinsp;1.95 \u003csup\u003ea,b,c,d,e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"4\"\u003eValues (n\u0026thinsp;=\u0026thinsp;5) are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM. Bars carrying - a represent significance (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) when compared to ctrl (control), - b represent significance (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) when compared to Mel. (melatonin), - c represents significance (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) when compared to LD PG (Low dose of pregabalin), and - d represent significance (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) when compared to HD PG (High dose of pregabalin).\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eHistopathological examination of the effect of melatonin on the testicular tissue and epididymis of Wistar rats following chronic exposure to pregabalin\u003c/b\u003e \u003c/p\u003e \u003cp\u003ePhotomicrographs display cross-sectional views of testicular tissue. The structure of the testicles is well-maintained in both the control group and the group of rats treated with melatonin. The morphology and dimensions of the seminiferous tubules (shown by the black arrow) appear to be within the expected range. Nevertheless, pregabalin caused alterations in the structure of the testicles. The seminiferous tubules exhibit a distorted and diminished appearance. The germ cells (shown by the red arrow) are shed. The lumens of the seminiferous tubules contain a small amount of fully developed sperm cells (shown by a red circle). The interstitial space exhibits distortion, characterised by regions of congestion and diminished size (shown by the green arrow). Melatonin improved these degenerative alterations. The germ cells (shown by the red arrow) are in different stages of development, with some discarded germ cells observed. The seminiferous tubules contain a small number of fully developed sperm cells (shown by red circles) within their lumens. The interstitial area exhibits Leydig cells within the expected range (shown by the green arrow) Fig.\u0026nbsp;8.\u003c/p\u003e \u003cp\u003eThe mice treated with control and melatonin had well-preserved epididymal structure, with the epithelium and lumen remaining intact, and a high number of fully developed sperm cells present. The pregabalin treated showed thickened epithelial layer with multiple areas of degeneration seen with the lumen showing little mature sperm (Fig.\u0026nbsp;9).\u003c/p\u003e \u003cp\u003eThe luminal diameter of the rats administered just with melatonin exhibited a substantial increase in comparison to the control animals (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The luminal diameter of the rats treated with pregabalin at both doses showed a substantial reduction compared to the group treated with melatonin alone (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eTable showing the Histo-morphometry properties of the epididymis in the treated rats\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGroup/Parameters\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTubular Diameter (\u0026micro;m)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eLuminal Diameter (\u0026micro;m)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eEpithelial Height (\u0026micro;m)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCtrl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e279.0\u0026thinsp;\u0026plusmn;\u0026thinsp;4.27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e254.7\u0026thinsp;\u0026plusmn;\u0026thinsp;6.51\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e19.74\u0026thinsp;\u0026plusmn;\u0026thinsp;1.70\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMel\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e292.4\u0026thinsp;\u0026plusmn;\u0026thinsp;24.42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e294.0\u0026thinsp;\u0026plusmn;\u0026thinsp;11.31\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e19.46\u0026thinsp;\u0026plusmn;\u0026thinsp;0.52\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLD PG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e290.6\u0026thinsp;\u0026plusmn;\u0026thinsp;14.77\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e218.5\u0026thinsp;\u0026plusmn;\u0026thinsp;6.36\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e22.67\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003csup\u003ea,b\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHD PG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e300.7\u0026thinsp;\u0026plusmn;\u0026thinsp;26.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e249.9\u0026thinsp;\u0026plusmn;\u0026thinsp;17.25\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e25.93\u0026thinsp;\u0026plusmn;\u0026thinsp;0.91\u003csup\u003ea,b\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLD PG\u0026thinsp;+\u0026thinsp;Mel\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e269.5\u0026thinsp;\u0026plusmn;\u0026thinsp;8.59\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e246.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.64\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e15.81\u0026thinsp;\u0026plusmn;\u0026thinsp;1.03\u003csup\u003ea,c,d\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHD PG\u0026thinsp;+\u0026thinsp;Mel\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e290.2\u0026thinsp;\u0026plusmn;\u0026thinsp;4.43\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e270.7\u0026thinsp;\u0026plusmn;\u0026thinsp;6.60\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e18.25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.65\u003csup\u003ec,d\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"4\"\u003e\u003cem\u003eData are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (n\u0026thinsp;=\u0026thinsp;5). Data marked with superscript\u003c/em\u003e \u003csup\u003e\u003cem\u003ea\u003c/em\u003e\u003c/sup\u003e, \u003cem\u003eare statistically significant (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) when compared to control; Data marked with superscript\u003c/em\u003e \u003csup\u003e\u003cem\u003eb\u003c/em\u003e\u003c/sup\u003e, \u003cem\u003eare statistically significant (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) when compared to melatonin treated group. Data marked with superscript\u003c/em\u003e \u003csup\u003e\u003cem\u003ec\u003c/em\u003e\u003c/sup\u003e, \u003cem\u003eare statistically significant (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) when compared to Low-dose Pregabalin treated group. Data marked with superscript\u003c/em\u003e \u003csup\u003e\u003cem\u003ed\u003c/em\u003e\u003c/sup\u003e, \u003cem\u003eare statistically significant (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) when compared to High-dose Pregabalin treated group.\u003c/em\u003e\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThe height of the epithelium layer in the rats treated with pregabalin at both doses was considerably greater than that of the rats in the control group and the rats treated with melatonin alone (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Melatonin treatment on the pregabalin exposed rats significantly reduced the epithelial height when compared to the corresponding pregabalin treatments (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe current investigation observed notable decreases in the body weight of the rats who received pregabalin treatment. Curiously, this discovery was similarly observed when evaluating both the testicular weight and the relative testicular weight of the rats. The reduced body weights could be secondary to decrease food intake as a result of the inhibition of the feeding center in the hypothalamus. This could possibly be linked to the reported effects of pregabalin on the brain [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Moreover, the weight loss [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e] could also be due to altered energy metabolism at the cellular levels and hence inefficient processing of the intake food substances. Failure to measure the daily food intake is considered as one of the limitations of this study. The drug has the potential for addiction [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]; hence, there could be accompanying reduction in appetite. The observed substantial decrease in testicular weight could potentially be accounted for by the mechanism discussed earlier. The occurrence of tissue death and the resulting decrease in the total tissue size of the gonads has undoubtedly led to the observed considerable decline in testosterone levels in the groups treated with pregabalin [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. The observed atrophy of the testicular and epididymal tissues demonstrated in the micrograph and the infiltration of these tissues with inflammatory cells further affirms the peripheral effects of pregabalin. The Leydig cells were subjected to oxidative assault, and the hypothalamic-pituitary-gonadal (HPG) axis was suppressed. This was confirmed by the notable decrease in the plasma levels of FSH and LH following the administration of pregabalin, indicating a significant reduction in testosterone levels. The weak signalling action of the low plasma levels of gonadotrophins on the testicular tissue has no potent effect to drive the process involved in the peripheral synthesis of the testosterone to completion. FSH is known to stimulate testicular growth and health and hence it is indirectly important for testosterone synthesis, unlike LH which has a direct effect on Leydig cells in the testes. In the groups simultaneously treated with melatonin, the observed effects of pregabalin on the body and testicular weights and testicular and epididymal histoarchitecture were significantly prevented. Melatonin has been reported to increase the plasma level of FSH and LH, following the stimulation of the release of GNRH at the level of hypothalamus, which subsequently activates the gonadotrophs in the anterior pituitary [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e3β-HSD and 17β-HSD are crucial enzymes involved in the synthesis, breakdown, and conversion of steroid hormones [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. The observed changes in enzyme activity in the testicular tissue after the injection of pregabalin support the large decrease in testosterone levels in the plasma. Additionally, these changes indicate a disruption in the HPG axis, which can result in many endocrine disorders. The co-administration of pregabalin with melatonin may avoid enzymatic imbalances, possibly due to the antioxidant properties of melatonin, which can scavenge free radicals. Previous studies have suggested that reactive species might disrupt the structure and activities of enzymes. The treatment of pregabalin resulted in enzymatic dysfunction, which was accompanied by increased levels of lactate and pyruvate, as well as elevated activity of LDH and GGT in the testicular tissue. The observed changes are known to be present in different metabolic abnormalities, including lactic acidosis, tissue necrosis, and hepatic dysfunction. These abnormalities lead to a reduction in the removal of lactate from the blood and the inhibition of pyruvate dehydrogenase (PDH), an enzyme that converts pyruvate to acetyl-CoA. An impairment in the functional role of PDH leads to the buildup of pyruvate in the bloodstream. The delivery of pregabalin resulted in considerable changes in lactate and pyruvate levels, as well as LDH and GGT activities. However, these changes were mostly averted in the groups who received simultaneous treatment with melatonin. The effects of the graded dosages of pregabalin on the biomarkers indicated above were inconsistent. The efficacy of the medicine is not consistently superior with a higher dosage compared to a lower dosage. Literature has documented the hepatoprotective and antioxidant properties of melatonin in response to various environmental challenges [\u003cspan additionalcitationids=\"CR28\" citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. The correlation between the levels of lactate and pyruvate and the activities of LDH and GGT in the epididymal tissue and the results of the biomarker assays in the testicular tissue is expected. It is important to mention that the group treated with simply melatonin did not show any significant changes in the biomarkers listed above, compared to the control group, at the given dose of melatonin. This phenomenon was also noted during the evaluation of the antioxidant and apoptotic indicators. However, at higher doses and for longer duration of administration, there might be some mild accompanying adverse effects [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe current research found that the use of pregabalin caused lipid peroxidation, which could potentially damage the cell membrane, lead to cell death, and disrupt the balance of the antioxidant system. This was observed through significant reductions in the activity of different antioxidant enzymes in the testicular and epididymal tissues. The pro-oxidative effects of pregabalin are consistent with the findings[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Taha and colleagues specifically observed that the medication induces genotoxicity through the reversal of the BAX/BCL2 ratio, increased levels of p38 MAPK, and impairment of the antioxidant system [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. The increase in caspase 3 activity after the injection of pregabalin is consistent with its ability to generate free radicals and operate as a pro-oxidant. Caspase-3 is a crucial enzyme involved in the final stage of programmed cell death, known as apoptosis. It has a crucial function in the regulated elimination of cells during apoptosis. Therefore, dysregulation in the activity of this enzyme can instigate different pathological cascade. Shokry equally observed an elevation in the activities of caspase 3 following the administration of pregabalin [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. It is noteworthy to assert that the effect of pregabalin on the activities of caspase 3 was significantly prevented in the groups simultaneously treated with melatonin, possible due to the antioxidant action of the hormone [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. While the specific mechanism through which melatonin exerts its anti-oxidative effects remains unclear, it is clear that the hormone can inhibit lipid peroxidation, as measured by the level of MDA in testicular and epididymal tissues, and enhance the oxidative enzyme system. This was confirmed by the notable increases in the levels of CAT, SOD, Gpx, GSH, and GST enzymes, as well as the considerable decrease in the activity of the apoptotic marker caspase 3 [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. By donating electrons, melatonin can deactivate harmful free radicals, therefore preventing oxidative damage to the body tissues.\u003c/p\u003e \u003cp\u003eThe disturbance of the antioxidant system following the administration of pregabalin was accompanied by the increase in the level of inflammatory markers in the testicular and epididymal tissues. Oxidative stress promotes inflammation through the activation of pro-inflammatory transcription factors, activation of inflammasomes, inactivation of anti-inflammatory molecules, direct damage to cellular components, and the recruitment of immune cells. The inflammatory effect of pregabalin was evidenced by the notable elevations in the levels of NF-kB, TNF-α, and IL-1β in the tissue. While literature does contain evidence on the anti-inflammatory properties of pregabalin [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e], the possible pro-inflammatory action of this drug is no doubt secondary to its pro-oxidative effects.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eMelatonin has a protective effect against pregabalin-induced gonadotoxicity via anti-oxidative, anti-inflammatory, anti-apoptotic, and enzymatic and hormonal regulatory mechanisms.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003e\u003cp\u003eELISA Enzyme linked immunosorbent assay\u003c/p\u003e \u003cp\u003eFSH Follicle stimulating hormone\u003c/p\u003e \u003cp\u003eGGT Gamma glutamyl transferase\u003c/p\u003e \u003cp\u003eGnRH Gonadotropin-releasing hormone\u003c/p\u003e \u003cp\u003eGpx Glutathione peroxidase\u003c/p\u003e \u003cp\u003eIL-1β Interleukin-1β\u003c/p\u003e \u003cp\u003eLDH Lactate dehydrogenase\u003c/p\u003e \u003cp\u003eLH Luteinizing Hormone\u003c/p\u003e \u003cp\u003eMDA Malondialdehyde\u003c/p\u003e \u003cp\u003ePG Pregabalin\u003c/p\u003e \u003cp\u003eSOD Superoxide dismutase\u003c/p\u003e \u003cp\u003eTNF-α Tumor necrosis factor-alpha\u003c/p\u003e \u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003cstrong\u003e \u003cb\u003eEthics approval and consent to participate\u003c/b\u003e:\u003c/strong\u003e \u003cp\u003eThe ethical reference number for the present study is AERCFBMSLAUTECH: 011/09/20231. Approval was given by the Ethics Review Committee of the Faculty of Basic Medical Sciences, LAUTECH, Ogbomoso, Oyo state, Nigeria.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003e \u003cb\u003eConsent for publication\u003c/b\u003e:\u003c/strong\u003e \u003cp\u003enot applicable.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eConsent to Participate\u003c/b\u003e:\u003c/strong\u003e \u003cp\u003enot applicable.\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003e Competing interests\u003c/b\u003e:\u003c/h2\u003e \u003cp\u003ethe authors have no competing interests as defined by BMC, or other interests that might be perceived to influence the results and/or discussion reported in this paper.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding:\u003c/h2\u003e \u003cp\u003eThe authors confirm that they have not received any financial assistance or support for the research, preparation, and publication of this article.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eA.F.A. conceptualize the work. A.F.A., M.S.B., W.J.A. and L.O.A wrote the main manuscript text. P.O. and O.P.A. prepared figures 1-9. All authors reviewed the manuscript\u003c/p\u003e\u003ch2\u003eAcknowledgements:\u003c/h2\u003e \u003cp\u003enot applicable.\u003c/p\u003e\u003ch2\u003eAvailability of data and materials:\u003c/h2\u003e \u003cp\u003eNo datasets were generated or analysed during the current study.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSharma A, Minhas S, Dhillo WS, Jayasena CN. Male infertility due to testicular disorders. J Clin Endocrinol Metab. 2021;106(2). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1210/clinem/dgaa781\u003c/span\u003e\u003cspan address=\"10.1210/clinem/dgaa781\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. PMID: 33295608; PMCID: PMC7823320.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAkhigbe R, Ajayi A. 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PMID: 30515034; PMCID: PMC6263227.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-pharmacology-and-toxicology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"phat","sideBox":"Learn more about [BMC Pharmacology and Toxicology](http://bmcpharmacoltoxicol.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/phat/Default.aspx","title":"BMC Pharmacology and Toxicology","twitterHandle":"@BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Melatonin, pregabalin, oxidative stress, inflammation, hormones, enzymes, testes, Epipidymis","lastPublishedDoi":"10.21203/rs.3.rs-5034037/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5034037/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe therapeutic value of pregabalin in the management of different pathological states like sleep, anxiety and bipolar disorders, fibromyalgia, epilepsy, among others, cannot be overemphasized. Nevertheless, the gonadotoxicity of this drug remains a point of concern. Contrarily, melatonin, an endogenous hormone is known for its favourable effects on the reproductive tissues following different insults. Thus, this study aimed to examine the impact of melatonin on male Wistar rats exposed to pregabalin.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA total of sixty male Wistar rats weighing between 120-140g were assigned randomly to six groups, with each group consisting of ten rats. The control group was given 0.5ml of normal saline orally, whereas melatonin alone and increasing dosages of pregabalin were delivered at 10, 150, and 300 mg/kg/BW orally, respectively. At the specified dosages, two groups were simultaneously treated with melatonin and low and high doses of pregabalin. All treatments lasted for 56 days. With the excepton of the hormones, biomarkers were assayed in the testicular and epididymal tissues.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePregabalin resulted in notable decreases in the percentage body weight, testicular weight, relative testicular weight, FSH, LH, testosterone, 3β-HSD, 17β-HSD, SOD, catalase, and GSH, as compared to the control group. However, these effects were mitigated in the groups who received melatonin in conjunction with pregabalin. Overall, the administration of melatonin had no negative impact on the levels and activities of the biomarkers. Pregabalin caused significant elevations in lactate, pyruvate, LDH, GGT, MDA, caspase, IL-1β, NFk, TNF-a, and distorted testicular histoarchitecture, but this effects was blunted in the group that were co-administered with melatonin. The impact of the two doses of pregabalin on all the biomarkers exhibited an irregular combination. The histological findings were parallel to the biochemical assays.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConclusively, melatonin has a protective effect against pregabalin-induced gonadotoxicity via anti-oxidative, anti-inflammatory, anti-apoptotic, and enzymatic and hormonal regulatory mechanisms.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eClinical trial number\u003c/strong\u003e: not applicable\u003c/p\u003e","manuscriptTitle":"Melatonin protect against pregabalin-induced gonadotoxicity via anti-oxidative, anti- inflammatory, anti-apoptotic, enzymatic and hormonal regulatory mechanisms","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-12-23 12:32:52","doi":"10.21203/rs.3.rs-5034037/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-11-29T11:33:07+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-11-05T14:58:49+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-10-30T11:10:20+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"113262106383205945673250807456312164289","date":"2024-10-30T10:00:30+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-10-25T22:22:20+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"236486371877125148087391283348019712890","date":"2024-10-23T16:16:14+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"166669536470739827799872123034867310369","date":"2024-10-15T17:44:08+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-10-15T13:01:44+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2024-09-13T16:08:02+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-09-13T03:08:06+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-09-13T03:07:24+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Pharmacology and Toxicology","date":"2024-09-04T21:55:38+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-pharmacology-and-toxicology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"phat","sideBox":"Learn more about [BMC Pharmacology and Toxicology](http://bmcpharmacoltoxicol.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/phat/Default.aspx","title":"BMC Pharmacology and Toxicology","twitterHandle":"@BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"5ac327bd-cc70-408b-9c60-5ae9c923cb5a","owner":[],"postedDate":"December 23rd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-02-17T16:00:34+00:00","versionOfRecord":{"articleIdentity":"rs-5034037","link":"https://doi.org/10.1186/s40360-025-00863-w","journal":{"identity":"bmc-pharmacology-and-toxicology","isVorOnly":false,"title":"BMC Pharmacology and Toxicology"},"publishedOn":"2025-02-12 15:57:20","publishedOnDateReadable":"February 12th, 2025"},"versionCreatedAt":"2024-12-23 12:32:52","video":"","vorDoi":"10.1186/s40360-025-00863-w","vorDoiUrl":"https://doi.org/10.1186/s40360-025-00863-w","workflowStages":[]},"version":"v1","identity":"rs-5034037","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5034037","identity":"rs-5034037","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}