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

BMC Pharmacology and Toxicology volume 26, Article number: 30 (2025) Cite this article

Abstract

Background

The therapeutic value of pregabalin in managing various pathological states, such as sleep, anxiety, and bipolar disorders, fibromyalgia, epilepsy, and others, cannot be overstated. Nevertheless, the gonadotoxicity of this drug remains a concern. In contrast, melatonin, an endogenous hormone, is known for its beneficial effects on reproductive tissues following various 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 and 140 g, were randomly assigned to six groups, with each group consisting of ten rats. The control group was given 0.5 ml of normal saline orally, whereas melatonin was administered alone at 10 mg/kg/BW, and pregabalin was delivered at low and high doses of 150 and 300 mg/kg/BW orally, respectively. At the specified dosages, rats were also treated simultaneously with low and high doses of pregabalin in combination with melatonin. All treatments lasted for 56 days. Biomarkers were assayed in the testicular and epididymal tissues, while hormones were assayed in the serum.

Results

Pregabalin treatment resulted in notable decreases in the percentage body weight change, 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 administered melatonin in conjunction with pregabalin. Pregabalin treatment also caused significant elevations in lactate, pyruvate, LDH, GGT, MDA, caspase, IL-1β, NF-κB, and TNF-α, and distorted testicular histoarchitecture, but these effects were blunted in the group co-administered with pregabalin and melatonin. The histological findings paralleled the biochemical assays.

Conclusion

Conclusively, melatonin has a protective effect against pregabalin-induced gonadotoxicity through anti-oxidative, anti-inflammatory, anti-apoptotic, enzymatic, and hormonal regulatory mechanisms.

Clinical trial number

Not applicable.

Peer Review reports

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 a dysfunctional reproductive process and accounts for approximately 50% of infertility cases [1].

The testicular function is tightly regulated by the hypothalamo-pituitary-gonadal axis (HPG). These regulatory processes can be distorted by exposure to drugs [3, 4], such as ketoconazole, chemotherapy, gabapentinoids, and opioids [1], resulting in hypogonadism and ultimately impaired semen quality. Testicular dysfunction can also result from injury, trauma, and diseases such as mumps, varicocele, and orchitis [1]. Additionally, it can occur following prolonged use of pregabalin, which is associated with 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, generalized 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 managing bipolar disorder, chronic pruritus, chronic cough, and restless leg syndrome [9]. According to Kamel et al. [10], the administration of pregabalin produces side effects, including nausea, vomiting, dry mouth, stomach cramps, constipation, diarrhea, flatulence, and abdominal distention [10]. Nevertheless, there is a scarcity of evidence in the 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 the sleep cycle, immune function, homeostasis, and 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 et al., 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 spermatotoxicity 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, as well as 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 used in this investigation were of conventional analytical grade.

Animal care and experimental design

Sixty (60) Wistar rats (male aged 9–11 weeks old weighing between 160 and 180 g were obtained from reputable commercial laboratory rats breeder and housed in the Animal House Facility of the Department of Physiology, Ladoke Akintola University of Technology, Ogbomoso, Nigeria. The rats kept in plastic cages, provided with unlimited access to ordinary pellets, and given unrestricted access to water and maintained and cared for under standard laboratory conditions 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). All experiments were carried out as approved by the Research Ethics and Review Committee of Faculty of Basic Medical Sciences, LAUTECH with the approval reference number AERCFBMSLAUTECH: 011/09/20231.

The rats were randomly assigned to six (6) groups of n = 5. The control group administered with a placebo of 0.5 ml of 0.9% normal saline. Pregabalin were delivered orally at two different doses: a low dose of 150 mg/kg/BW and a high dose of 300 mg/kg/BW [17]. Melatonin was also administered orally at a dose of 10 mg/kg/BW [18], 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.

Sample collection

Twenty-four (24) hours after administering the last dose, the animals were anesthetized with pentobarbital sodium (40 mg/kg BW, i.p.) and then euthanized for collection of blood through heart puncture. The testes and epididymis were removed, and their weights were 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 3500 rpm for a duration of 10 min. The centrifugation was carried out at a temperature of -4 ℃ using a refrigerated centrifuge manufactured by Bio-Gene Technology Ltd., located in Grandtech Centre, Shatin, Hong Kong. The plasma samples, divided into discrete portions, were utilized 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 for histological processing.

Biochemical assay

Estimation of serum levels of reproductive hormones and testicular enzymes activities

The measurement of serum and testicular testosterone was conducted following the detailed procedure of the ELISA kit from Monobind Inc., USA, with the product number 4806–300 A. The serum FSH and LH were estimated using ELISA kits (Monobind Inc., USA; product numbers: 506–300 A and 625–300 A, respectively). The testicular enzymatic activities 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.

Estimation of the testicular oxidative stress markers

The malondialdehyde (MDA) level in the testicular tissues was assayed as previously reported [20, 21]. Also, testicular antioxidant activities, including superoxide dismutase (SOD), catalase (CAT), glutathione (GSH), glutathione peroxidase (GPx), and glutathione-S-transferase (GST), were assayed based on established methods [19,20,21,22].

Briefly, the marker of oxidative stress, MDA, was determined based on the generated amount of thiobarbituric acid reactive substance (TBARS) during lipid peroxidation. This method involves the reaction between 2-thiobarbituric acid (TBA) and malondialdehyde, a by-product of lipid peroxidation, following the analysis of the pink chromogen complex [(TBA) 2-malondialdehyde adduct] formed upon heating at acidic pH. 200 µl of the sample was first treated with 500 µl of trichloroacetic acid (TCA) to remove proteins and centrifuged at 3,000 rpm for 10 min. 1 ml of 0.75% TBA was added to 0.1 ml of the supernatant and heated in a water bath at 100 °C for 20 min, and cooled with ice water. The absorbance of the sample/standard was then read at 532 nm using a spectrophotometer and compared to a blank. The concentration of TBARS was determined by extrapolating from a standard curve.

For SOD, a 1:10 dilution of the sample was made using 1 ml of sample with 9 ml of distilled water. 0.2 ml of the diluted sample was then added to 2.5 ml of 0.05 M carbonate buffer (pH 10.2) and the reaction was initiated by adding 0.3 ml of freshly prepared 0.3 mM adrenaline. The mixture was mixed and the increase in absorbance was monitored at 480 nm every 30 s for 150 s using a spectrophotometer. A reference cuvette containing 2.5 ml buffer, 0.3 ml substrate (adrenaline), and 0.2 ml water was also used.

In estimating catalase activities, a 1:19 dilution of the sample was made by mixing 1 ml of the supernatant of the tissue homogenate with 19 ml of diluted water. 4 ml of hydrogen peroxide (H2O2) solution (800 µmoles) and 5 ml of phosphate buffer were added to a 10 ml flat-bottom flask. 1 ml of the diluted enzyme preparation was mixed into the reaction followed by gentle swirling at 37 °C. The samples of the reacting mixture were withdrawn at 60 s intervals, and the H2O2 content was determined by blowing 1 ml of the sample into 2 ml dichromate/acetic acid reagent. Catalase levels in the sample were determined by comparing the absorbance at 653 nm to that of a catalase standard.

For determining GSH, an aliquot of the tissue sample homogenates was deproteinized by adding an equal volume of 4% sulfosalicylic acid, before centrifuging at 4,000 rpm for 5 min. 0.5 ml of the supernatant was then added to 4.5 ml of Ellman’s reagent. A blank was prepared prior by mixing 0.5 ml of the diluted precipitating agent with 4.5 ml of Ellman’s reagent.

The level of GSH was calculated by measuring the absorbance at 412 nm. For GPx, the tissue homogenate was incubated at 37 °C for 3 min, before adding 0.5 ml of 10% trichloroacetic acid (TCA) and the mixture was centrifuged at 3,000 rpm for 5 min. The supernatant was mixed with 2 ml of phosphate buffer and 1 ml of 5′-5′-dithiobis-2-dinitrobenzoic acid (DTNB) solution, and the absorbance was measured at 412 nm using a blank as reference.

The GPx activity was extrapolated from the plot against the standard curve to determine the concentration of remaining GSH from the curve. The activity of glutathione-S-transferase in testicles was also measured. This method utilizes the enzyme’s high activity with 1-chloro-2,4-dinitrobenzene as a substrate. The assay was performed at 37 °C for 60 s, and the absorbance was read at 340 nm after comparing it with a blank sample.

Assessment of markers of inflammation in the testis

The concentrations of nuclear factor-kappa B (NF-kB), tumour necrosis factor-α (TNF-α), and interleukin-1β (IL-1β) in the testicular tissues were determined using ELISA kits according to the manufacturer’s guidelines (Elabscience Biotechnology Company Ltd., Wuhan, Hubei, China).

Estimation of epididymal tissues lactate dehydrogenase (LDH), gamma-glutamyl transferase (GGT) lactate and pyruvate

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., Agappe Hills, Pattimattom PO, Kochi, Kerala 683562, India.

Histology

The testicular tissues were preserved in Bouin’s solution, dried using a series of alcohol solutions, clarified with xylene, embedded in paraffin wax, cut into thin Sects. (2–3 μm thick) using a microtome, attached to glass slides, stained with haematoxylin and 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 were analyzed using GraphPad Prism, Version 10.0. The results were reported as the mean value plus or minus the standard deviation using one-way analysis of variance (ANOVA) followed by the Tukey test for post-hoc comparisons among several groups for statistical analysis, with the significance level set at P < 0.05.

Results

Effect of melatonin on body weight change, reproductive organs weight

The exposure to pregabalin, at both doses, caused a significant (p < 0.05) reduction in body weight gain and relative testicular weight, but also a reduction in relative epididymal weight at the high dose (Fig. 1a-c) in the treated rats compared to the control and melatonin-treated rats. Nevertheless, administering melatonin to rats treated with pregabalin at both doses led to a notable rise in body weight growth, relative epididymal, and testicular weight in comparison to their corresponding doses of pregabalin treatment (Fig. 1a-c).

Fig. 1
figure 1

Effects of melatonin administration on the (a) body weight (b) relative epididymal weight (c) relative testicular weight of pregabalin-exposed male Wistar rats. Data are expressed as mean ± standard deviation (n = 5). Bars carrying superscript a are statistically significant (p < 0.05) when compared to Ctrl; b are statistically significant (p < 0.05) when compared to Mel; c are statistically significant (p < 0.05) when compared to LD PG; d are statistically significant (p < 0.05) when compared to HD PG; e are statistically significant (p < 0.05) when compared to LD PG + Mel

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Effect of melatonin on the serum concentration of reproductive hormones and testicular steroidogenic enzymes activities of pregabalin-exposed male rats

Administration of melatonin does not have a significant effect on the serum reproductive hormone concentration compared to the control group (Fig. 2a-c). However, exposure to pregabalin at both doses significantly (p < 0.05) reduced the serum hormone (LH, FSH, and testosterone) concentrations compared to the control group and melatonin-treated groups (Fig. 2a-c). 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).

Fig. 2
figure 2

Effects of melatonin administration on serum (a) LH (b) FSH (c) testosterone concentrations of pregabalin-exposed male Wistar rats. Data are expressed as mean ± standard deviation (n = 5). Bars carrying superscript a are statistically significant (p < 0.05) when compared to Ctrl; b are statistically significant (p < 0.05) when compared to Mel; c are statistically significant (p < 0.05) when compared to LD PG; d are statistically significant (p < 0.05) when compared to HD PG; e are statistically significant (p < 0.05) when compared to LD PG + Mel

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Also, supplementation with melatonin had no significant effect on the testicular steroidogenic enzymes’ activities (3βHSD and 17βHSD) when compared to the control group (Fig. 3a and b). The exposure to pregabalin at both doses significantly (p < 0.05) reduced the testicular steroidogenic enzymes’ activities (3βHSD and 17βHSD) when compared to the control group and melatonin-treated groups (Fig. 3a-b). However, administering melatonin to rats exposed to pregabalin at both doses led to a notable increase in the activities of the testicular steroidogenic enzymes (3βHSD and 17βHSD) when compared to their corresponding doses of pregabalin treatment (Fig. 3a and b).

Fig. 3
figure 3

Effects of melatonin administration on testicular activities of (a) 3βHSD (b) 17βHSD of pregabalin-exposed male Wistar rats. Data are expressed as mean ± standard deviation (n = 5). Bars carrying superscript a are statistically significant (p < 0.05) when compared to Ctrl; b are statistically significant (p < 0.05) when compared to Mel; c are statistically significant (p < 0.05) when compared to LD PG; d are statistically significant (p < 0.05) when compared to HD PG; e are statistically significant (p < 0.05) when compared to LD PG + Mel

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Effect of melatonin on the oxidative stress markers and caspase 3 activities in testicular tissues of pregabalin-exposed male rats

Pregabalin treatment significantly (p < 0.05) increased the testicular MDA concentration, while significantly (p < 0.05) reducing the antioxidant enzymes (SOD, CAT, GSH, GPx, and GST) when compared to the control and melatonin-only groups (Fig. 4a-f). Nevertheless, administering melatonin to rats exposed to pregabalin resulted in a notable reduction in testicular MDA and significantly (p < 0.05) increased the antioxidant activities (Fig. 4a-f).

Fig. 4
figure 4

Effects of melatonin administration on testicular redox status (a) MDA (b) SOD (c) Catalase (d) GSH (e) GPx (f) GST of pregabalin-exposed male Wistar rats. Data are expressed as mean ± standard deviation (n = 5). Bars carrying superscript a are statistically significant (p < 0.05) when compared to Ctrl; b are statistically significant (p < 0.05) when compared to Mel; c are statistically significant (p < 0.05) when compared to LD PG; d are statistically significant (p < 0.05) when compared to HD PG; e are statistically significant (p < 0.05) when compared to LD PG + Mel

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Effect of melatonin on the epididymal tissues energy metabolism

Exposure to pregabalin, at both doses, led to a significant (p < 0.05) rise in the levels of lactate, pyruvate, and lactate dehydrogenase in the epididymis, as compared to the control and melatonin-treated groups (Fig. 5a-c). However, melatonin supplementation to the rats exposed to pregabalin at both doses resulted in a notable reduction in the testicular levels of lactate, pyruvate, and lactate dehydrogenase in comparison to the corresponding doses of pregabalin alone (Fig. 5a-c).

Fig. 5
figure 5

Effects of melatonin administration on epididymal tissue (a) lactate (b) Pyruvate (c) LDH activities of pregabalin-exposed male Wistar rats. Data are expressed as mean ± standard deviation (n = 5). Bars carrying superscript a are statistically significant (p < 0.05) when compared to Ctrl; b are statistically significant (p < 0.05) when compared to Mel; c are statistically significant (p < 0.05) when compared to LD PG; d are statistically significant (p < 0.05) when compared to HD PG; e are statistically significant (p < 0.05) when compared to LD PG + Mel

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Effect of melatonin on the testicular inflammatory and apoptotic parameters in pregabalin-exposed male rats

The administration of pregabalin causes a significant (P < 0.05) increase in inflammatory markers (NF-κβ, TNF-α, IL-1β, and GGT) and caspase-3 activity (Fig. 6a-e). Administration of melatonin to rats exposed to pregabalin at both doses results in a noteworthy decrease in testicular inflammatory markers compared to their corresponding pregabalin treatment doses.

Fig. 6
figure 6

Effects of melatonin administration on the testicular tissue inflammatory markers (a) NFk-β (b) TNF-α (c) IL-1β and apoptotic (d) GGT (e) Caspase-3 activities of pregabalin-exposed male Wistar rats. Data are expressed as mean ± standard deviation (n = 5). Bars carrying superscript a are statistically significant (p < 0.05) when compared to Ctrl; b are statistically significant (p < 0.05) when compared to Mel; c are statistically significant (p < 0.05) when compared to LD PG; d are statistically significant (p < 0.05) when compared to HD PG; e are statistically significant (p < 0.05) when compared to LD PG + Mel

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Histopathological examination of the effect of melatonin on the testicular tissue and epididymis of Wistar rats following chronic exposure to pregabalin

Histopathological findings revealed features consistent with normal testicular and epididymal tissues in animals in the control and melatonin-treated groups, whereas their counterparts in the pregabalin exposure group exhibited histological features suggesting cellular reaction to injury and inflammatory response (Figs. 7 and 8). Additionally, the luminal diameter and epithelial height of the epididymal tissues of rats administered with melatonin exhibited a substantial increase compared to the control animals (Table 1). In contrast, the luminal diameter and epithelial height of rats treated with pregabalin at both doses showed a substantial reduction compared to the group treated with melatonin alone (Table 1).

Fig. 7
figure 7

Effect of Melatonin on the Histology of the Testes in Pregabalin-Exposed Male Wistar Rats. The Control and Melatonin Groups showed normal seminiferous tubules (black arrow) with germ cells (red arrow) at varying degrees of maturation. Mature sperm cells (red circle) were present in the lumens. In contrast, the Low-Dose Pregabalin Group exhibited distorted tubules (black arrow), sloughed germ cells (red arrow), and scanty mature sperm cells (red circle). However, the Pregabalin + Melatonin Groups showed improved histological features, with normal seminiferous tubules (black arrow) and Leydig cells

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Fig. 8
figure 8

Photomicrographs of the left epididymis of the rats (H&E stains) at x 100. The control and melatonin only treated shows a well-preserved epithelial layer. The tubules (black arrow) are well-outlined with thickened epithelial layer and numerous mature sperm cells within the lumens (red arrow). Also the interstitium appear normal. Both pregabalin treated doses also shows a well-preserved architecture. The tubules (black arrow) are well-outlined with thickened epithelial layer with multiple areas of degeneration. The lumens contain numerous mature sperm cells (red arrow)

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Table 1 Table showing the histo-morphometry properties of the epididymis in the treated rats
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Discussion

The current investigation observed significant decreases in the body weight of rats that 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 decreased 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 [23]. Moreover, the weight loss [24] could also be due to altered energy metabolism at the cellular level and hence inefficient processing of ingested food substances. Failure to measure daily food intake is considered one of the limitations of this study. The drug has the potential for addiction [25]; hence, there could be an accompanying reduction in appetite. The occurrence of tissue death and the resulting decrease in the total tissue size of the gonads undoubtedly led to the observed considerable decline in testosterone levels in the groups treated with pregabalin [26]. The observed atrophy of the testicular and epididymal tissues demonstrated in the micrograph and the infiltration of these tissues with inflammatory cells further affirm the peripheral effects of pregabalin. Also, the Leydig cells were subjected to oxidative assault, and the hypothalamic-pituitary-gonadal (HPG) axis activities were suppressed following pregabalin exposure, in line with previous studies [27, 28]. 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. 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 reversed, in line with previous suggestions of the role of melatonin in male reproductive functions [15]. However, there are also controversies regarding the role of melatonin in regulating the plasma level of gonadotropic hormones [26], following the stimulation of the release of GNRH at the level of the hypothalamus, which subsequently activates the gonadotrophs in the anterior pituitary [27, 28].

3β-HSD and 17β-HSD are crucial enzymes involved in the synthesis, breakdown, and conversion of steroid hormones [29]. 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 with 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 administration 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 that 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 [30,31,32]. 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 note that the group treated with melatonin alone 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 a longer duration of administration, there might be some mild accompanying adverse effects [33].

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 [34]. 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 [34]. 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 cascades. Shokry also observed an elevation in the activities of caspase 3 following the administration of pregabalin [24]. 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, possibly due to the antioxidant action of the hormone [35]. 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 [36]. By donating electrons, melatonin can deactivate harmful free radicals, thereby preventing oxidative damage to body tissues.

The disturbance of the antioxidant system following the administration of pregabalin was accompanied by an increase in the levels 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 notable elevations in the levels of NF-kB, TNF-α, and IL-1β in the tissue. Although literature does contain evidence on the anti-inflammatory properties of pregabalin [37, 38], the possible pro-inflammatory action of this drug undoubtedly results from its pro-oxidative effects.

Conclusion

Pregabalin treatment induced significant reproductive toxicity in rats, characterized by decreased body and testicular weights, increased oxidative stress, inflammation, and apoptosis, accompanied by disruption of the hypothalamic-pituitary-gonadal axis. Co-administration of melatonin mitigated these effects through antioxidant, anti-inflammatory, anti-apoptotic, enzymatic, and hormonal regulatory mechanisms in rats. These findings suggest that melatonin may serve as a useful adjunct in mitigating the gonadotoxic effects of pregabalin. Therefore, further studies on the use of melatonin as a protective agent against pregabalin-induced reproductive toxicity in humans are recommended.

Data availability

No datasets were generated or analysed during the current study.

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

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Authors and Affiliations

  1. Department of Physiology, Ladoke Akintola University of Technology, Ogbomoso, Oyo State, Nigeria

    Ayodeji Folorunsho Ajayi, Motolani Susan Borisade, Precious Oyedokun & David Tolulope Oluwole

  2. Department of Physiology, Adeleke University, Ede, Osun State, Nigeria

    Ayodeji Folorunsho Ajayi & Wale Johnson Adeyemi

  3. Anchor Biomed Research Institute, Ogbomoso, Oyo State, Nigeria

    Ayodeji Folorunsho Ajayi

  4. Department of Physiology, Babcock University, Ilishan Remo, Ogun State, Nigeria

    Oyedayo Phillips Akano

  5. Department of Biochemistry, Ladoke Akintola University of Technology, Ogbomoso, Oyo State, Nigeria

    Lydia Oluwatoyin Ajayi

  6. Department of Physiology, Crescent University, Abeokuta, Abeokuta, Nigeria

    David Tolulope Oluwole

  7. Department of Physiology, University of Ilesa, Ilesa, Osun State, Nigeria

    David Tolulope Oluwole

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Contributions

A.F.A. conceptualize the work. A.F.A., M.S.B., W.J.A. and L.O.A wrote the main manuscript text. D.T.O., P.O. and O.P.A. prepared Figs. 1, 2, 3, 4, 5, 6, 7 and 8. All authors reviewed the manuscript.

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Correspondence to Ayodeji Folorunsho Ajayi.

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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.

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Ajayi, A.F., Borisade, M.S., Oyedokun, P. et al. Melatonin protect against pregabalin-induced gonadotoxicity via anti-oxidative, anti-inflammatory, anti-apoptotic, enzymatic and hormonal regulatory mechanisms in rats. BMC Pharmacol Toxicol 26, 30 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s40360-025-00863-w

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BMC Pharmacology and Toxicology

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