Febuxostat alleviates testicular dysfunction induced by cyclophosphamide in rats via modulation of pyroptosis and mTOR autophagy axis

Abstract

Cyclophosphamide (CP) has long been employed in cancer treatment as well as in therapeutic regimens for autoimmune diseases. Despite its clinical value, one of its major drawbacks is testicular damage which may be due to disruption of antioxidant defenses, amplification of inflammatory responses, and suppression of autophagy. Febuxostat is a xanthine oxidase inhibitor with promising antioxidant, anti-inflammatory, and autophagy-inducing effects. The present study investigated whether febuxostat could mitigate CP-induced testicular toxicity in a rat model. Fifty male Sprague-Dawley rats were aligned into five groups: a control group, a CP-only group, and three CP-treated groups receiving febuxostat at doses of 5, 10, or 15 mg/kg/day. Seminal fluid, blood samples, and testicular tissues were collected and analyzed through biochemical assays and pathological examinations. Febuxostat dose-dependently restored the hormonal balance, enhanced antioxidant defenses, increased sirtuin-1 levels, and modulated both NOD-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome-driven pyroptosis and mammalian target of rapamycin (mTOR)-autophagy axis in the testicular tissues of CP-treated rats compared with CP-only rats. These improvements were consistently observed in the histopathological and immunohistochemical evaluations. Owing to its antioxidant, anti-inflammatory, autophagy-promoting, and pyroptosis-modulating effects, febuxostat may show promise as a potential therapeutic option for reducing CP-related gonadal dysfunction in males.

Introduction

Cyclophosphamide (CP) is an alkylating compound extensively applied in oncology as a chemotherapeutic agent and in the management of various autoimmune conditions¹. Its toxic effect arises from the ability to form DNA cross-links, which interfere with essential processes of replication and transcription, ultimately triggering cell death². Following administration, CP is metabolized in the liver, generating active compounds such as phosphoramide mustard and acrolein. These metabolites are responsible for its therapeutic benefits but are also linked to adverse toxic effects³.

Since CP primarily affects cells with rapid division, the germinal epithelium of the testes is highly susceptible, making testicular damage a frequent adverse effect that can limit its therapeutic application⁴. This adverse effect is commonly reflected in lowered sperm numbers, higher rates of abnormal morphology, and reduced viability and motility, which in severe cases can progress to permanent infertility⁵. The severity of these side effects is influenced by dosage and tends to be more evident in individuals undergoing long-term treatment, as is often required in cancer therapy⁶. Therefore, developing approaches to counteract CP-induced reproductive toxicity continues to be a major priority in the current scientific investigations⁷.

Silent information regulator 1 (SIRT1), also known as sirtuin-1, functions as a nicotinamide adenine dinucleotide (NAD)-dependent deacetylase. Through its histone-modifying activity, it shapes gene expression and governs a wide range of cellular processes⁸. Recent studies indicate that SIRT1 exerts a protective influence against CP-induced testicular damage, likely through its capacity to reduce oxidative stress, regulate inflammatory pathways, and maintain the balance between apoptosis and autophagy in testicular tissue⁹. Recent findings demonstrate that SIRT1 helps to limit chemotherapy-induced excess production of reactive oxygen species (ROS), maintains the integrity of testicular tissue, and supports both hormone synthesis and sperm development¹⁰. In addition, SIRT1 dampens pro-inflammatory signaling, likely through the deacetylation of high mobility group box 1 (HMGB1), a key regulator of inflammation within the testes¹¹. By restricting HMGB1 movement from the nucleus to the extracellular environment, SIRT1 prevents the activation of toll-like receptor 4 (TLR4) and receptors for advanced glycation end products (RAGE), thereby reducing downstream inflammatory signaling and limiting cytokine secretion¹². Moreover, SIRT1 disrupts nuclear factor kappa B (NF-κB) signaling and related pathways, thereby strengthening its anti-inflammatory and anti-apoptotic actions within the testes¹³.

Pyroptosis is a programmed cell death pathway closely linked to inflammatory responses¹⁴. It is marked by extensive cell swelling and rupture of the plasma membrane, followed by the release of numerous pro-inflammatory mediators¹⁵. In CP-induced testicular toxicity, pyroptosis is believed to significantly amplify tissue injury¹⁶. CP metabolites have been shown to drive excessive ROS generation and activate inflammasome pathways, which in turn trigger caspase-dependent mechanisms leading to pyroptotic cell death¹⁷. This cascade not only compromises the structural integrity of testicular tissue but also promotes the release of pro-inflammatory cytokines, thereby intensifying local inflammatory responses¹⁸. As a result, both spermatogenesis and steroidogenesis become markedly impaired, reducing overall fertility potential¹⁹. Recognizing the role of pyroptosis in this cellular toxicity underscores its value as a therapeutic target for safeguarding testicular function against CP exposure¹⁶.

Febuxostat, a xanthine oxidase inhibitor, is clinically prescribed for the management of hyperuricemia in gout patients²⁰. Beyond this established role, recent studies have emphasized its potent antioxidant, anti-apoptotic, and anti-inflammatory properties²¹. Acting as an antioxidant, febuxostat efficiently scavenges free radicals and quenches singlet oxygen, while also boosting endogenous antioxidant defenses across multiple tissues, thereby alleviating oxidative stress²². In terms of inflammation, it has been shown to modulate key signaling pathways, including NF-κB and the NOD-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome, resulting in reduced pro-inflammatory cytokine production and attenuation of chronic tissue inflammation²³. Additionally, febuxostat aids in restoring mitochondrial activity and shielding cells from lipid peroxidation, both of which are critical in the disease states marked by mitochondrial dysfunction²⁴. Combined with its established anti-apoptotic properties²⁵, these mechanisms point to febuxostat as a promising candidate for mitigating CP-induced testicular toxicity. Accordingly, this preclinical study aimed to evaluate the impact of febuxostat, administered at varying doses, on CP-induced gonadal dysfunction in male rats, with assessments conducted at both the biochemical and histopathological levels.

Materials and methods

Ethics and randomization

All animal procedures in this study complied with institutional regulations and adhered to the ARRIVE guidelines, the U.K. Animals (Scientific Procedures) Act of 1986, and the EU Directive 2010/63/EU on animal experimentation. Ethical approval was obtained from the Research Ethics Committee of the Faculty of Medicine, Tanta University, Egypt (Approval ID: 36264PR131013/10/25). To minimize selection bias and evenly distribute confounders, fifty male Sprague-Dawley rats were randomly assigned to five groups (ten rats each) using a computer-generated randomization method. The RAND function in Microsoft Excel was employed to generate random numbers, which determined group allocation. Throughout the study, the personnel responsible for animal care, biomarker analysis, and histopathological assessment were blinded to group assignments to ensure objectivity and reduce observer bias.

Chemicals, reagents, and drugs

Cyclophosphamide was purchased in a powder form (dissolved in distilled water) from TCI America, Portland, Oregon, United States (CAS RN: 6055-19-2, Product No. C2236). Febuxostat powder was supplied by Shanghai Nianxing Industrial Co., Ltd, Shanghai, China (CAS No.144060-53-7, Purity 98.0%) and was dissolved in 10% dimethyl sulfoxide (DMSO) solution, which was obtained from Santa Cruz Biotechnology, Inc., Dallas, Texas, United States (CAS No. 67-68-5, Catalog No. sc-202581, Purity ≥ 99%). The other reagents and chemicals used in this study were supplied by Henan Tianfu Chemical Co., Ltd., Zhengzhou, Henan 450,000, China.

The experimental animals

The current study was carried out on fifty adult male Sprague-Dawley rats (160–250 g), obtained from the animal facility of the Faculty of Pharmacy, Tanta University, Egypt. The animals were maintained in mesh wire cages under controlled conditions (temperature: 23 ± 2 °C, relative humidity: 56 ± 5%, and a 12-hour light/dark cycle). They were allowed free access to food and water and acclimatized to these conditions for ten days prior to experimentation. Following acclimatization, the rats were randomly allocated into five groups of ten animals each as depicted in Fig. 1. Except for the control group, all rats received CP (15 mg/kg/week) via gastric tube for eight weeks⁷. The control group was administered 0.25 mL of 10% DMSO solution daily by gastric tube throughout the same period. The remaining groups were treated with febuxostat at doses of 5, 10, and 15 mg/kg daily by gastric tube for eight weeks^26,27. Body weight and food consumption were recorded at the baseline and subsequently monitored on a weekly basis for the entire duration of the study.

Fig. 1.

The experimental procedures and the measured parameters of the study. Abbreviations: Akt, protein kinase B; β-HSD, beta hydroxysteroid dehydrogenase; CP, cyclophosphamide; FSH, follicle-stimulating hormone; HMGB1, high mobility group box 1; LH, luteinizing hormone; mTOR, mammalian target of rapamycin; NLRP3, NOD-like receptor family pyrin domain-containing 3; PI3K, phosphatidylinositol 3 kinase; RAGE, receptors for advanced glycation end products; SIRT1, silent information regulator 1; TLR4, toll-like receptor 4.

Collection and processing of blood and tissue specimens

At the 57th day of the study, rats were fasted overnight. In the next morning, they were anaesthetized with a single dose of intraperitoneal injection of thiopental sodium (50 mg/kg)²⁸. Blood samples were collected by cardiac puncture, after which they were transferred to centrifuge tubes. Centrifugation for ten minutes at 4 °C was carried out using a cooling centrifuge (5702/ 5702 R/5702 RH, Eppendorf India, Chennai, India). The resulting serum was used for quantification of the hormonal levels including follicle-stimulating hormone (FSH), luteinizing hormone (LH), and testosterone using sandwich ELISA kits (Invitrogen Bioservices India Private Limited (Thermofisher Scientific), Karnataka, India, catalog numbers EEL125, EEL122, and EELR017, respectively) according to the vendor’s manual.

Euthanization of animals was performed using cervical dislocation. Then, animals were opened by midline laparotomy for excision of the testis, epididymis, seminal vesicles, and prostate. The harvested tissues were washed with ice-cold saline and weighed. The testes were carefully divided into two segments. One segment was homogenized in 10 mL of 0.25 M sucrose solution using a Benchtop Tissue Homogenizer LMBTH-A100 (Labmate Scientific LLC 444 W Lake Street, Chicago, IL 60606, United States). The homogenized material was then transferred into a 100 mM phosphate buffer (pH 7.4) and subjected to centrifugation at 1006 ×g for fifteen minutes. The resulting supernatant was utilized for biochemical analysis of the testicular tissues, while the other segment was preserved and processed for histological examination under a light microscope. In the current work, all biochemical assays were performed with three biological replicates per group, and each measurement was repeated in triplicate technical replicates to ensure the reliability and reproducibility of data.

Assessment of the activity of 3 beta hydroxysteroid dehydrogenase type 1 (3β-HSD1) and 17 beta hydroxysteroid dehydrogenase type 3 (17β-HSD3) in the harvested testicular tissues

Sandwich ELISA kits obtained from Creative Diagnostics (Shirley, NY 11967, USA; catalog number DEIA-FN650) were employed to determine the activity of 3β-HSD1 in testicular tissue samples. For the evaluation of 17β-HSD3, double-antibody sandwich ELISA kits from Wuhan Fine Biotech Co. (Wuhan, 430074, Hubei, China; catalog number ER1743) were utilized. The activities of both enzymes were measured using the sandwich ELISA method following the manufacturers’ protocols. Optical density was recorded at 450 nm with a microplate reader, and the values were analyzed against the corresponding standard curve to quantify the activity of these enzymes.

Assessment of SIRT1 levels and the redox status of the testicular tissues

Sandwich ELISA kits from Biomatik (Ontario, N2C 1N6, Canada; Catalog number EKN48461) were employed to quantify SIRT1 levels in testicular tissue samples, following the manufacturer’s protocol. Malondialdehyde (MDA) content and superoxide dismutase (SOD) activity in the testicular tissues were quantified by colorimetric kits supplied by Biodiagnostic, Giza, Egypt (catalog numbers MD 25–29 and SD 25 21, respectively). The reduced glutathione (GSH) content of the testicular tissues was assayed by means of colorimetric kits supplied by Beijing Solarbio Science & Technology Co., Ltd. (Beijing, China; catalog number BC1170). The methodology of assessment of these parameters followed the vendors’ instructions.

Determination of the levels of NLRP3 inflammasome and gasdermin D in the harvested testicular tissue specimens

Both NLRP3 inflammasome and gasdermin D levels were assessed in the supernatant of the centrifuged homogenate of the testicular tissues using sandwich ELISA kits provided by Bio-Techne (Minneapolis, MN 55413, United States; catalog numbers NBP3-42294 and NBP3-43460, respectively).

Assessment of the levels of inflammatory mediators in the harvested testicular tissues

The levels of interleukin 1-beta (IL-1β) and IL-18 were assayed in the supernatant of the centrifuged homogenate of the testicular tissues using sandwich ELISA kits supplied by Novatein Biosciences Inc. (Woburn, MA 01801, United States; catalog numbers FM-E100054 and NB-E30650, respectively). Also, both tumor necrosis factor alpha (TNF-α) and monocyte chemoattractant protein-1 (MCP-1) levels in the testicular tissues were determined following the instructions enclosed with ELISA kits supplied by Proteintech Group, Inc., Rosemont, IL 60,018, United States (Catalog numbers KE20018 and KE20009, respectively).

Assessment of the HMGB1/RAGE/TLR4 axis in the harvested testicular tissue specimens

The levels of HMGB1 in the testicular tissues were assessed using kits supplied by AAA Biotech, LLC (La Jolla, CA 92038, United States; catalog number AAA252756) according to the vendor’s guide. RAGE levels were determined in the supernatant of the testicular tissue homogenate using kits purchased from LSBio (Newark, CA 94560, United States, catalog number LS-F37664) following the provider’s instructions. Reed Biotech was the supplier of sandwich ELISA kits used for biochemical estimation of TLR4 levels in the harvested testicular tissue specimens according to the vendor’s guide (Reed Biotech, East Lake High-tech Zone, Wuhan, China; catalog number RE2838R).

Assessment of the role of phosphatidylinositol 3 kinase (PI3K)/Protein Kinase B (Akt)/mammalian target of rapamycin (mTOR) signaling in the pathogenesis of CP-elicited testicular toxicity

Kits purchased from ELK Biotechnology CO., Ltd. (STE12 Sugar Land, TX 77478, United States; catalog number ELK8431) were the analytical tool used for assessment of PI3K levels in the supernatant of the homogenized testicular tissues. Both phospho-Akt (Phospho-Ser473) and phospho-mTOR (Ser2448) levels in the testicular tissue specimens were assayed using sandwich ELISA kits supplied by Cell Signaling Technology, Inc. (Boston, MA 02241–3843, United States, product numbers 80895 and 7976, respectively).

Determination of the testicular tissue content of beclin-1 and Light Chain 3-II (LC3-II) as autophagy mediators

The testicular tissue levels of the autophagy-related proteins, including beclin-1 and Light Chain 3-II (LC3-II), were quantified according to the instructions enclosed with sandwich ELISA kits purchased from AAA Biotech, LLC (La Jolla, CA 92038, United States; catalog numbers AAA87708 and AAA18933, respectively).

The afore-mentioned biochemical parameters in the testicular tissue specimens were assayed using a quantitative sandwich ELISA technique in which microplates were coated with antibodies specific to the target analyte. Standards or samples were added to the wells, followed by a biotin-labeled antibody against the same analyte. Subsequently, horseradish peroxidase (HRP)–conjugated avidin was added and incubated. The reaction was then developed with a TMB substrate, producing a color change in the wells containing the analyte–antibody complexes. The resulting color intensity was measured spectrophotometrically at the designated wavelength.

Determination of the characteristics of spermatozoa

The evaluation of the spermatozoa characteristics—including count, motility, viability, and morphological integrity—was conducted following established protocols²⁹. In brief, the cauda epididymis was dissected, and the sperm counts were determined using Nikon inverted routine microscope ECLIPSE Ts2 (Tokyo 140–8601, Japan) at 40× magnification. Motility was assessed within four minutes of sperm isolation by mixing one drop of the seminal fluid with one drop of 2.9% sodium citrate solution on a preheated glass slide maintained at 37 °C. Sperm viability and morphological abnormalities were analyzed using a staining mixture of 1% eosin-Y and 5% nigrosine. The stained preparations were then examined under Nikon inverted routine microscope ECLIPSE Ts2 (Tokyo 140–8601, Japan) at 200× magnification.

Histopathological evaluation of the harvested tissues

The testicular tissue samples were preserved in 10% formalin for 48 h, followed by ethanol dehydration and paraffin embedding. Using the BHTP-203 rotary microtome (Biolab Scientific Ltd., Markham, Ontario L3R 0B8, Canada), paraffin blocks were sectioned at a thickness of 5 μm. The sections were mounted on glass slides, stained with hematoxylin and eosin (H&E), and examined under Nikon inverted routine microscope ECLIPSE Ts2 (Tokyo 140–8601, Japan). For morphometric analysis, ten randomly selected high-power fields (×400) from each section were assessed, and the six most circular seminiferous tubules were measured to determine seminiferous tubule diameter (STD) and germinal epithelium thickness (GET). Spermatogenesis was further evaluated using Johnsen’s tubular biopsy score (JTBS) which is a histological grading system used to evaluate spermatogenesis within the seminiferous tubules of the testis³⁰. It is based on a scale from 1 to 10, where each score reflects the degree of germ cell development and the presence of mature spermatozoa. A score of 10 indicates complete and normal spermatogenesis with abundant spermatozoa, while lower scores represent progressive reduction in germ cell maturation, ranging from the presence of only spermatogonia or Sertoli cells to complete absence of the germ cells. Quantification of the histopathological alterations in the testicular tissues was performed with ImageJ software version 1.5.4, National Institutes of Health, Bethesda, MD, United States.

Identification of apoptosis and cellular proliferation in the testicular tissue using immunohistochemical analysis

Formalin-fixed, paraffin-embedded testicular tissue sections were processed to evaluate the expression of cleaved caspase-3 and proliferating cell nuclear antigen (PCNA) using immunohistochemistry. Tissue blocks were sectioned at 5 μm thickness with a BHTP-203 rotary microtome (Biolab Scientific Ltd., Markham, Ontario, Canada), mounted on glass slides, and incubated with primary antibodies: caspase-3 polyclonal antibody (Thermo Fisher Scientific, Waltham, MA, USA; catalog number PA5-114687) and PCNA polyclonal antibody (ProteinTech Group, Rosemont, IL, USA; catalog number 10205-2-AP). The staining procedures followed the manufacturer’s instructions. The immunolabeled sections were examined under a Nikon ECLIPSE Ts2 inverted microscope (Tokyo, Japan) at ×400 magnification across ten randomly selected representative fields per section, and the immune expression was expressed as (+) denoting mild, (++) denoting moderate, and (+++) indicating strongly positive expression. Quantitative analysis of immunohistochemical signals was performed using ImageJ software (version 1.5.4, National Institutes of Health, Bethesda, MD, USA).

Statistical evaluation

Statistical evaluation of the output results was performed using GraphPad Prism software, version 8 (GraphPad Software, LLC, San Diego, CA, USA). Results are expressed as mean ± standard deviation (SD). Normality of data distribution was verified with the Shapiro–Wilk test, and variance homogeneity was assessed using Bartlett’s test. When variances were equal, one-way analysis of variance (ANOVA) was applied, followed by Tukey’s post hoc comparison. For datasets with non-parametric values, the Kruskal-Wallis test was utilized in conjunction with Dunn’s test, and was expressed as median (Interquartile range, IQR). Differences were considered statistically significant at a threshold of p-value less than 0.05.

Results

Effect of varying doses of febuxostat on food intake and body weight in animals treated with CP

As illustrated in Fig. 2, administration of CP alone resulted in a marked reduction in both body weight and daily food consumption compared with the control group. Notably, treatment with febuxostat produced a dose-dependent improvement, leading to significant recovery in body weight gain and food intake among the CP-exposed rats when compared to those receiving CP alone.

Fig. 2.

Febuxostat dose-dependently abrogated CP-induced changes in (A) the daily food intake and (B) the body weight. Results are presented as mean ± standard deviation (SD) (number of animals = ten per group) and statistically analyzed using one-way ANOVA, confirmed by Tukey’s multiple comparison test. ^asignificant relative to the control, ^bsignificant relative to the CP-group, ^csignificant relative to the CP + FBX5 group, ^dsignificant relative to the CP + FBX10 group. Abbreviations: CP: cyclophosphamide; FBX5: febuxostat 5 mg/kg/day; FBX10: febuxostat 10 mg/kg/day; FBX15: febuxostat 15 mg/kg/day.

The impact of varying doses of febuxostat on the hormonal profile in CP-treated animals

As depicted in Fig. 3, the group treated with CP alone exhibited a significant decline in serum levels of FSH, LH, and testosterone when compared to the control group. Interestingly, treatment with febuxostat dose dependently reversed these changes, as demonstrated by a significant increase in the serum levels of FSH, LH, and testosterone when compared to the group treated with CP alone.

Fig. 3.

Febuxostat dose-dependently mitigated the serum hormonal changes induced by CP (A: FSH; B: LH; C: Testosterone). Results are presented as mean ± standard deviation (SD) (number of animals = ten per group) and statistically analyzed using one-way ANOVA, confirmed by Tukey’s multiple comparison test. p-value < 0.05 was considered significant. Abbreviations: CP, cyclophosphamide; FBX5: febuxostat 5 mg/kg/day; FBX10: febuxostat 10 mg/kg/day; FBX15: febuxostat 15 mg/kg/day; FSH, follicle-stimulating hormone; LH, luteinizing hormone.

The impact of varying doses of febuxostat on the activity of 3β-HSD1 and 17β-HSD3 in the testicular tissues of CP-treated animals

Figure 4 demonstrated that the group treated with CP alone exhibited a significant decline in 3β-HSD1 and 17β-HSD3 activity in the supernatant of the testicular tissue homogenate when compared to the control group. Meanwhile, febuxostat administration to the CP-treated animals led to a dose-related significant elevation in the activity of these enzymes in the testicular tissues when compared to the group treated with CP alone.

Fig. 4.

Febuxostat dose-dependently augmented (A) 3β-HSD1 and (B) 17β-HSD3 activities in CP-treated animals. Results are presented as mean ± standard deviation (SD) (number of animals = ten per group) and statistically analyzed using one-way ANOVA, confirmed by Tukey’s multiple comparison test. p-value < 0.05 was considered significant. Abbreviations: CP, cyclophosphamide; FBX5: febuxostat 5 mg/kg/day; FBX10: febuxostat 10 mg/kg/day; FBX15: febuxostat 15 mg/kg/day; 3β-HSD1, 3 beta hydroxysteroid dehydrogenase type 1; 17β-HSD3, 17 beta hydroxysteroid dehydrogenase 3.

The impact of varying doses of febuxostat on SIRT1 levels and the redox status of the testicular tissues in CP-treated animals

Treatment of animals with CP alone resulted in a significant decrease in the testicular tissue SIRT1 levels associated with a significant disruption of the redox balance – manifested by a significant elevation of the testicular tissue MDA and a considerable decline in tissue GSH levels and SOD activity- relative to the control rats. These changes were reversed with febuxostat treatment in a dose-dependent fashion, as evidenced by restoration of SIRT1 levels and mitigation of the oxidative insult of the testicular tissues when compared to CP-treated rats (Fig. 5).

Fig. 5.

Febuxostat dose-dependently restored the testicular tissue SIRT1 levels and the redox balance in CP-treated animals (A: SIRT1; B: MDA; C: GSH; D: SOD). Results are presented as mean ± standard deviation (SD) (number of animals = ten per group) and statistically analyzed using one-way ANOVA, confirmed by Tukey’s multiple comparison test. p-value < 0.05 was considered significant. Abbreviations: CP, cyclophosphamide; FBX5: febuxostat 5 mg/kg/day; FBX10: febuxostat 10 mg/kg/day; FBX15: febuxostat 15 mg/kg/day; GSH, reduced glutathione; MDA, malondialdehyde; SIRT1, sirtuin-1; SOD, superoxide dismutase.

The impact of varying doses of febuxostat on the testicular tissue levels of NLRP3 inflammasome and gasdermin D in CP-treated rats

Animals treated with CP alone exhibited a pronounced increase in NLRP3 inflammasome and gasdermin D in the supernatant of the testicular tissue homogenate compared to the control animals. Nevertheless, these changes were dose-dependently alleviated in the CP-groups treated with febuxostat when compared to the corresponding parameters in the group treated with CP alone (Fig. 6).

Fig. 6.

Febuxostat dose-dependently alleviated the CP-induced changes in the testicular tissue (A) NLRP3 inflammasome and (B) gasdermin D. Data are presented as mean ± standard deviation (SD) (number of animals = ten per group) and statistically analyzed using one-way ANOVA, confirmed by Tukey’s multiple comparison test. p-value < 0.05 was considered significant. Abbreviations: CP, cyclophosphamide; FBX5: febuxostat 5 mg/kg/day; FBX10: febuxostat 10 mg/kg/day; FBX15: febuxostat 15 mg/kg/day; NLRP3, nucleotide-binding domain, leucine-rich–containing family, pyrin domain–containing-3.

The impact of varying doses of febuxostat on the testicular tissue levels of inflammatory mediators

Regarding the inflammatory response, CP elicited a significant elevation of the testicular tissue levels of the proinflammatory cytokines, including IL-1β, IL-18, TNF-α, and MCP-1 relative to the control levels. Amazingly, treatment of CP-groups with febuxostat exhibited a unique ability to reduce the testicular tissue levels of these proinflammatory cytokines. This ability was proven to be dose-dependent (Fig. 7).

Fig. 7.

Febuxostat dose-dependently alleviated the CP-induced changes in the testicular tissue inflammatory mediators (A: IL-1β; B: IL-18; C: TNF-α; D: MCP-1). Data are presented as mean ± standard deviation (SD) (number of animals = ten per group) and statistically analyzed using one-way ANOVA, confirmed by Tukey’s multiple comparison test. p-value < 0.05 was considered significant. Abbreviations: CP, cyclophosphamide; FBX5: febuxostat 5 mg/kg/day; FBX10: febuxostat 10 mg/kg/day; FBX15: febuxostat 15 mg/kg/day; IL-1β, interleukin-1 beta; MCP-1, monocyte chemoattractant protein 1; TNF-α, tumor necrosis factor alpha.

The impact of varying doses of febuxostat on HMGB1/RAGE/TLR4 signaling in the testicular tissues of CP-treated animals

In the testicular tissues, CP exposure significantly augmented the HMGB1/RAGE/TLR4 signaling pathway, evidenced by significantly increased expression of HMGB1, RAGE, and TLR4 compared to the controls. Febuxostat administration at different doses mitigated these changes, with the strongest protective effect seen in rats treated with 15 mg/kg/day, surpassing the outcomes observed in lower-dose groups (Fig. 8).

Fig. 8.

Febuxostat dose-dependently abrogated the effects of CP administration on HMGB1/RAGE/TLR4 signaling in the testicular tissues (A: HMGB1; B: RAGE; C: TLR4). Data are presented as mean ± standard deviation (SD) (number of animals = ten per group) and statistically analyzed using one-way ANOVA, confirmed by Tukey’s multiple comparison test. p-value < 0.05 was considered significant. Abbreviations: CP, cyclophosphamide; FBX5: febuxostat 5 mg/kg/day; FBX10: febuxostat 10 mg/kg/day; FBX15: febuxostat 15 mg/kg/day; HMGB1, high mobility group box 1; RAGE, receptors of advanced glycation end products; TLR4, toll-like receptor 4.

The impact of varying doses of febuxostat on the testicular tissue levels of PI3K, phospho-Akt, and phospho-mTOR in CP-treated animals

As demonstrated in Fig. 9, administration of CP significantly stimulated the PI3K/Akt/mTOR signaling cascade within the testicular tissues, as reflected by elevated levels of PI3K, phosphorylated Akt, and mTOR relative to the control group. Notably, febuxostat therapy mitigated this stimulation in a dose-dependent fashion, effectively restoring these molecular markers closer to their baseline values.

Fig. 9.

Febuxostat dose-dependently interfered with PI₃K/Akt/mTOR signaling in the testicular tissues of CP-treated groups (A: PI3K; B: Phospho-Akt; C: Phospho-mTOR). Data are presented as mean ± standard deviation (SD) (number of animals = ten per group) and statistically analyzed using one-way ANOVA, confirmed by Tukey’s multiple comparison test. p-value < 0.05 was considered significant. CP: cyclophosphamide; FBX5: febuxostat 5 mg/kg/day; FBX10: febuxostat 10 mg/kg/day; FBX15: febuxostat 15 mg/kg/day; PI3K: phosphatidylinositol 3-kinase; Akt: Ak strain transforming; mTOR: mammalian target of Rapamycin.

The impact of varying doses of febuxostat on autophagy within the testicular tissues of CP-treated animals

In the present work, exposure to CP markedly lowered the levels of beclin-1 and LC3-II in testicular tissue relative to the control group. Administration of febuxostat at different doses successfully reversed these changes, with the greatest restoration seen in animals treated with 15 mg/kg/day, surpassing the effects observed at lower doses (Fig. 10).

Fig. 10.

Febuxostat dose-dependently augmented autophagy in the testicular tissues of CP-treated groups (A: Beclin-1; B: LC3-II). Data are presented as mean ± standard deviation (SD) (number of animals = ten per group) and statistically analyzed using one-way ANOVA, confirmed by Tukey’s multiple comparison test. p-value < 0.05 was considered significant. Abbreviations: CP, cyclophosphamide; FBX5: febuxostat 5 mg/kg/day; FBX10: febuxostat 10 mg/kg/day; FBX15: febuxostat 15 mg/kg/day; LC3-II, Light Chain 3 Type II.

The impact of varying doses of febuxostat on the spermatozoa characteristics in CP-treated animals

As depicted in Fig. 11, administration of CP alone led to a marked deterioration in the spermatozoa parameters, reflected by a significant rise in the percentage of abnormal and non-viable sperm, coupled with a pronounced decline in both sperm count and motility compared to the controls. Conversely, treatment with febuxostat produced a dose-dependent improvement in sperm quality, evidenced by a substantial reduction in the percentage of abnormal and dead sperm and a notable enhancement in the sperm count and motility relative to the CP-treated animals.

Fig. 11.

Febuxostat dose-dependently reversed the changes in the spermatozoa characteristics elicited by CP (A: Sperm count; B: Abnormal forms of sperms; C: The percentage of dead sperms; D: The percentage of sperm motility). Data are presented as mean ± standard deviation (SD) (number of animals = ten per group) and statistically analyzed using one-way ANOVA, confirmed by Tukey’s multiple comparison test. p-value < 0.05 was considered significant. Abbreviations: CP, cyclophosphamide; FBX5: febuxostat 5 mg/kg/day; FBX10: febuxostat 10 mg/kg/day; FBX15: febuxostat 15 mg/kg/day.

The impact of varying doses of febuxostat on the changes in the morphometric measurements and the histopathological picture of the reproductive organs elicited by CP administration

Administration of CP alone led to a significant decline in the testicular, epididymal, prostate, and seminal vesicles weight, STD, and GET relative to the control animals. Treatment with febuxostat at different doses significantly mitigated these negative effects, with the greatest recovery observed in the group receiving febuxostat (15 mg/kg/day), outperforming the improvements noted in groups given lower doses of febuxostat (Fig. 12).

Fig. 12.

Febuxostat dose-dependently mitigated the changes in the reproductive organs’ morphometric measurements elicited by CP (A: Testicular weight; B: Epididymal weight; C: Prostate weight; D: Seminal vesicles weight; E: Seminiferous tubules diameter; F: Germinal epithelium thickness). Data are presented as mean ± standard deviation (SD) (number of animals = ten per group) and statistically analyzed using one-way ANOVA, confirmed by Tukey’s multiple comparison test. p-value < 0.05 was considered significant. Abbreviations: CP, cyclophosphamide; FBX5: febuxostat 5 mg/kg/day; FBX10: febuxostat 10 mg/kg/day; FBX15: febuxostat 15 mg/kg/day; GET, germinal epithelium thickness; STD, seminiferous tubule diameter.

Regarding hematoxylin and eosin-stained sections, animals exposed solely to CP displayed significant alterations in the seminiferous tubules, characterized by disruption of the basal membrane, epithelial cell injury, extensive vacuolization, a sharp decline in the number of germ cell populations, and the presence of immature germinal epithelial cells scattered within the tubular lumens. These pathological changes were further intensified by pronounced vascular congestion and a significant reduction in spermatogenesis relative to the controls (Fig. 13A, B, and F). In contrast, treatment with febuxostat at different doses markedly mitigated these CP-induced abnormalities, promoting restoration of seminiferous tubule structure, alleviation of vascular congestion, and improvement in the spermatogenic activity (Fig. 13C–F). The most notable recovery was observed in the animals administered 15 mg/kg/day of febuxostat, which even closely resembled the histological profile of the control group.

Fig. 13.

Hematoxylin and eosin-stained testicular sections (×200, scale bar = 50 μm) revealed that (A) In the control group, seminiferous tubules maintained their typical histological organization, with intact spermatogenic cells and spermatozoa (thin arrows), as well as normal interstitial tissue (arrowhead); (B) In the CP group, there was marked degeneration of the interstitial tissue (arrowheads), pronounced vascular congestion (V), and severe epithelial damage within the seminiferous tubules (thin arrows) with accumulation of degenerated epithelial cells within the lumen (thick arrows); (C) CP group treated with FBX at 5 mg/kg/day showed partial improvement, with reduced interstitial tissue damage (arrowhead) and some seminiferous tubules retaining organized epithelial layers (thin arrows), though vascular congestion (V) and sloughed epithelial cells (thick arrows) remained evident; (D) Treatment with FBX at 10 mg/kg/day resulted in a greater number of well-preserved seminiferous tubules (thin arrows), recovery of interstitial tissue structure (arrowheads), and only mild vascular congestion (V); (E) Administration of FBX at 15 mg/kg/day restored the normal structure of the seminiferous tubules (thin arrows) and connective tissue septa (arrowhead); (F) The impact of CP with or without FBX on the Johnsen’s tubular biopsy score. The non-parametric values were expressed as median (Interquartile range, IQR) and compared using Kruskal-Wallis followed by Dunn’s test. p-value < 0.05 was considered significant. Abbreviations: CP, cyclophosphamide; FBX5: febuxostat 5 mg/kg/day; FBX10: febuxostat 10 mg/kg/day; FBX15: febuxostat 15 mg/kg/day; NS, non-significant.

The impact of the varying doses of febuxostat on the CP-induced changes in the immune expression of cleaved caspase-3 and PCNA in the testicular tissues

Animals treated with CP alone exhibited a pronounced increase in cleaved caspase-3 immune expression (Fig. 14A, B, and F) in association with a significant decline in PCNA immunoreactivity (Fig. 15A, B, and F) when compared to the controls. Nevertheless, treatment with febuxostat showed a dose-dependent restoration of both cleaved caspase-3 and PCNA expression levels to approximate the control values (Fig. 14C–F and 15C–F, respectively), highlighting the strong antiapoptotic potential of febuxostat alongside its ability to enhance cellular proliferation in the testicular tissues.

Fig. 14.

Immunohistochemical analysis of cleaved caspase-3 in testicular sections (×100, scale bar = 100 μm) demonstrated that (A) the control group exhibited only faint positive staining (red arrow, +), whereas the CP-treated group (B) showed intense caspase-3 expression (red arrows, +++). Animals receiving FBX at 5 or 10 mg/kg/day displayed a moderate level of immunoreactivity (red arrows, ++) (C and D, respectively), while (E) those treated with 15 mg/kg/day showed a mild response (red arrows, +). (F) A quantitative assessment of cleaved caspase-3 expression across the experimental groups, expressed as a percentage relative to the control, was performed on samples from ten animals per group. Results are presented as mean ± standard deviation (SD) and were statistically evaluated using one-way ANOVA followed by Tukey’s post hoc test, with significance set at p < 0.05. Abbreviations: CP, cyclophosphamide; FBX5: febuxostat 5 mg/kg/day; FBX10: febuxostat 10 mg/kg/day; FBX15: febuxostat 15 mg/kg/day.

Fig. 15.

PCNA immune expression in testicular tissue sections (×100, scale bar = 100 μm) in (A) the control rats exhibiting strong positive immune expression (red arrows, +++); (B) mild positive expression in the group treated with only CP (red arrow, +); (C) and (D) moderate positive PCNA immune expression in CP-treated animals receiving FBX at 5 and 10 mg/kg/day, respectively (red arrows, ++); (E) strong positive PCNA immune expression in CP-treated animals receiving 15 mg/kg/day FBX (red arrows, +++); and (F) A quantitative assessment of PCNA expression across the experimental groups, expressed as a percentage relative to the control, was performed on samples from ten animals per group. Results are presented as mean ± standard deviation (SD) and were statistically evaluated using one-way ANOVA followed by Tukey’s post hoc test, with significance set at p < 0.05. Abbreviations: CP, cyclophosphamide; FBX5: febuxostat 5 mg/kg/day; FBX10: febuxostat 10 mg/kg/day; FBX15: febuxostat 15 mg/kg/day; PCNA, proliferating cell nuclear antigen.

Discussion

In this study, CP-treated animals showed a marked decline in both final body weight and daily food consumption compared with the control group, aligning with the observations reported by Tohei et al.³¹. This reduction may be linked to metabolic disturbances that suppress appetite, decrease food intake, and elevate inflammatory cytokines—particularly interleukin-1 beta (IL-1β), which was markedly increased after CP administration³². IL-1β is recognized as a central mediator of chemotherapy-related weight loss, independent of tumor burden^33,34. Treatment with febuxostat successfully restored body weight and enhanced food intake, consistent with previous findings. These effects are attributed to its impact on insulin resistance, regulation of hepatic lipid metabolism, and modulation of inflammatory signaling—particularly through the suppression of IL-1β^35,36.

The reproductive function in males is governed by the hypothalamic–pituitary–gonadal (HPG) axis, where pulsatile release of gonadotropin-releasing hormone (GnRH) stimulates the pituitary to secrete FSH and LH, ultimately driving testosterone synthesis in Leydig cells³⁷. CP-induced testicular toxicity is strongly associated with suppression of the HPG axis, disrupting the hormonal regulation required for normal spermatogenesis³⁸. As a consequence, secretion of FSH and LH declines, impairing Sertoli cell support for sperm development and reducing Leydig cell stimulation for testosterone production². hormonal imbalance leads to reduced serum testosterone levels and further aggravates testicular dysfunction, ultimately contributing to infertility³⁹. Thus, suppression of the HPG axis plays a central role in intensifying CP-induced gonadal toxicity by creating a state of endocrine disruption³⁸. Supporting this, the activities of the key steroidogenic enzymes, 3β-HSD1 and 17β-HSD3, were diminished, indicating impaired testicular steroidogenesis⁴⁰. The processes driving these alterations are diverse and interconnected. CP metabolism produces harmful byproducts such as phosphoramide mustard and acrolein, which trigger DNA cross-linking and apoptosis in steroidogenic cells, thereby disrupting gonadal hormone synthesis⁴¹. CP also enhances reactive oxygen species (ROS) generation, lipid peroxidation, and mitochondrial impairment—events that interfere with the initial stages of steroidogenesis⁴². In addition, CP induces systemic inflammation, marked by increased IL-1β, IL-18, and TNF-α, which downregulate steroidogenic enzymes via NF-κB signaling¹⁸. In the present study, co-administration of febuxostat successfully mitigated these detrimental effects, normalizing FSH, LH, and testosterone levels along with 3β-HSD1 and 17β-HSD3 activity. This protective role is linked to febuxostat’s strong antioxidant and anti-inflammatory actions, which safeguard Leydig and Sertoli cells against ROS and cytokine-mediated damage, preserve enzyme function, and sustain the integrity of the HPG axis⁴³.

Beyond its systemic impact on body weight and endocrine regulation, CP (CP) also inflicted significant structural and functional damage on the male reproductive system in this study. This was reflected in decreased sperm count and motility, together with a higher proportion of abnormal spermatozoa—classic indicators of gonadal dysfunction. Such disturbances in spermatogenesis are well-documented following CP exposure and align with the spermotoxic effects of many chemotherapeutic drugs⁴⁴. Co-treatment with febuxostat markedly improved sperm quality, restoring parameters close to control levels. CP administration further disrupted testicular architecture, resulting in reduced STD, GET, spermatogenesis, and overall testicular weight. These changes signify germ cell loss, tubular degeneration, and impaired spermatogenesis, with androgen deficiency contributing to organ weight reduction⁴⁵. Febuxostat alleviated these abnormalities, likely through its antioxidant and cytoprotective mechanisms, which counteract lipid peroxidation and membrane injury caused by CP-induced free radicals⁴⁶.

Recent evidence indicates that CP compromises the antioxidant defense system by producing excessive ROS along with its toxic metabolite, acrolein, which overwhelms the intrinsic protective capacity of testicular cells⁴⁷. In the current study, this was demonstrated by a sharp decline in antioxidant enzymes and GSH levels in testicular tissue, coupled with a significant rise in MDA, a key marker of lipid peroxidation. CP-induced oxidative stress is a major contributor to gonadal dysfunction, partly through impaired microvascular blood flow that accelerates germ cell apoptosis⁴⁸. Febuxostat co-administration effectively lowered MDA concentrations, enhanced antioxidant enzyme activity, and restored GSH levels, highlighting its dual antioxidant action—direct ROS scavenging and activation of endogenous defense pathways. These properties position febuxostat as a promising agent to counter CP-driven redox imbalance⁴⁹.

Recent investigations have highlighted the pivotal role of SIRT1 in safeguarding testicular tissue against CP toxicity. CP induces oxidative stress and activates inflammatory pathways that impair spermatogenesis and damage testicular cells⁵⁰. A central mechanism of this toxicity involves the HMGB1/RAGE/TLR4 signaling axis. HMGB1 released from injured cells binds to RAGE, subsequently activating TLR4, which amplifies inflammation and cellular injury¹⁸. By strengthening antioxidant defenses and suppressing pro-inflammatory transcription factors, SIRT1 reduces HMGB1 release and limits RAGE/TLR4 activation, thereby preserving testicular integrity and curbing the progression of damage⁵⁰. In the present study, febuxostat treatment significantly upregulated testicular SIRT1 expression, which was associated with attenuation of CP-induced activation of the HMGB1/RAGE/TLR4 pathway and protection against testicular injury. These findings align with Abdel-Wahab et al.⁵¹ who reported that febuxostat’s modulation of SIRT1 and its downstream signaling contributes fundamentally to its cytoprotective effects against oxidative stress and inflammation.

Pyroptosis, an inflammatory form of programmed cell death, has emerged as a major contributor to CP-induced testicular injury¹⁶. CP provokes oxidative stress and cellular damage, which in turn activate the NLRP3 inflammasome. Once engaged, NLRP3 facilitates the conversion of pro-caspase-1 into its active form, driving the maturation of pro-inflammatory cytokines such as IL-1β and IL-18¹⁸. Concurrently, caspase-1 cleaves gasdermin D, releasing its N-terminal fragment that inserts into the plasma membrane to form pores. These pores compromise membrane integrity, leading to cell swelling, rupture, and the release of inflammatory mediators that exacerbate tissue damage⁵². In the testes, this signaling cascade results in germ cell depletion, impaired spermatogenesis, and structural deterioration, as observed in the current study following CP exposure. Thus, the NLRP3/gasdermin D pathway represents a central mechanism linking CP treatment to pyroptotic cell death and reproductive toxicity¹⁶. Notably, febuxostat administration in this study suppressed CP-induced activation of NLRP3/gasdermin D and its downstream mediators, consistent with previous reports suggesting that its antipyroptotic activity underlies its potent anti-inflammatory and apoptosis-attenuating effects^53,54.

Apoptosis and autophagy—often referred to as programmed cell death type II—are interconnected processes that jointly influence the survival and fate of testicular cells⁵⁵. A central regulator of these mechanisms is the PI3K/Akt/mTOR signaling pathway, which governs essential cellular functions in male reproduction, including growth, proliferation, survival, angiogenesis, transcription, translation, and metabolism⁵⁶. Disturbances in this pathway are among the most frequently observed alterations associated with male infertility, underscoring its importance as a therapeutic target in reproductive medicine⁵⁷. Within this context, autophagy was evaluated through beclin-1 and LC3-II, two essential markers that preserve testicular homeostasis by facilitating the clearance of damaged mitochondria, misfolded proteins, and defective organelles⁵⁸. Reduced expression of beclin-1 and LC3-II signifies impaired autophagy, which contributes to germ cell apoptosis, disrupted spermatogenesis, and overall testicular dysfunction⁵⁹. In the present study, CP administration activated the PI3K/Akt/mTOR pathway while simultaneously lowering beclin-1 and LC3-II levels, indicating suppression of autophagic activity. These findings are consistent with earlier reports linking CP-induced testicular injury to aberrant mTOR activation, a negative regulator of autophagy⁶⁰. Interestingly, contrasting evidence has been documented in other tissues, where CP exposure inhibited PI3K/Akt/mTOR signaling, suggesting tissue-specific differences in its regulatory effects⁶¹.

In this study, febuxostat treatment suppressed mTOR signaling, thereby lifting its inhibitory influence on autophagy. This was demonstrated by the increased expression of autophagy-related markers, including beclin-1 and LC3-II, which reflect enhanced autophagosome formation and improved autophagic flux. The observed upregulation indicates a direct cause-and-effect relationship, where inhibition of mTOR facilitated autophagy activation⁶². This protective effect is likely linked to the antioxidant capacity of febuxostat, which alleviates oxidative stress—the primary driver of mitochondrial injury and autophagy dysfunction⁶³. This mechanistic link provides a coherent explanation for how febuxostat restores cellular homeostasis and protects against pyroptotic cell death in the testicular tissue.

Cleaved caspase-3 serves as a pivotal executioner enzyme in apoptosis, responsible for degrading structural and regulatory proteins and driving the characteristic morphological changes of programmed cell death⁶⁴. In this study, CP administration markedly increased caspase-3 activation compared with controls, confirming its pro-apoptotic impact on testicular tissue. Co-treatment with febuxostat significantly reduced caspase-3 expression, consistent with its reported anti-apoptotic properties, which involve suppression of reactive oxygen species (ROS) generation and inhibition of mitochondrial-dependent apoptotic pathways⁶⁵.

Proliferating cell nuclear antigen (PCNA) serves as a vital marker of DNA replication and repair, and its expression mirrors the proliferative activity of germ cells within the testes⁶⁶. Under physiological conditions, PCNA ensures proper DNA synthesis during cell division, thereby supporting normal spermatogenesis⁶⁷. CP, however, disrupts this equilibrium by inducing oxidative stress and DNA damage, which suppress PCNA expression and compromise the regenerative potential of testicular tissue, as observed in the present study⁴. This suppression results in defective germ cell proliferation, impaired DNA repair processes, and progressive degeneration of the seminiferous tubules. Ultimately, CP-mediated interference with PCNA-dependent cellular proliferation contributes to fertility loss through disruption of testicular homeostasis¹⁶. Coinciding with our results, Hassan et al.⁶⁸ reported that febuxostat augments PCNA expression in the different body tissues, thereby playing a key role in cellular proliferation and regeneration.

While all doses of febuxostat (5, 10, and 15 mg/kg) demonstrated protective effects against CP-induced testicular dysfunction, the 15 mg/kg dose consistently produced the most pronounced improvements in the histological architecture, biochemical markers (SIRT1 activation, suppression of NLRP3 inflammasome-driven pyroptosis), and restoration of autophagy balance via the mTOR axis. Consequently, febuxostat at 15 mg/kg appears to be the optimal dose under the current experimental conditions, offering maximal therapeutic benefit. When calculating the human equivalent dose for the highest febuxostat dose used in the current study (15 mg/kg), it was found to fall within the clinically relevant therapeutic range of febuxostat, which is typically prescribed at 40–120 mg/day for treatment of gout⁶⁹. Therefore, this dose is translationally meaningful and supports the potential clinical applicability of febuxostat in mitigating chemotherapy-induced testicular toxicity.

Despite the promising findings of the present study, several limitations should be acknowledged. The relatively small sample size, species-specific differences between rats and humans in metabolism, immunity, and reproductive physiology, and the absence of evaluation of febuxostat’s potential adverse effects restrict the generalizability of the results. Furthermore, the current study didn’t address the extent to which febuxostat can influence the anticancer efficacy of CP, an essential consideration for the possible clinical implications of the results. The lack of a positive control group also limits comparative interpretation. Including a febuxostat-alone group would provide additional clarity in distinguishing the intrinsic pharmacological effects of febuxostat from its protective role against CP-induced toxicity. While our primary objective was to evaluate febuxostat’s modulatory impact within the disease model, previous studies have consistently reported minimal adverse or confounding effects of febuxostat when administered alone in rodents, supporting our interpretation⁴³. Finally, mechanistic insights were constrained by reliance on ELISA and immunohistochemistry without complementary validation using Western blotting, mRNA expression analysis, or studies in knock-out animals. Without phosphorylation-based validation, our findings should be interpreted as indicative rather than definitive mechanistic proof.

Conclusion

The results of the current experiments suggest that febuxostat may help to preserve fertility in male rats exposed to CP. As illustrated in Fig. 16, its protective role seems to stem from promoting autophagy and providing antioxidant, anti-inflammatory, and anti-apoptotic benefits, alongside modulating SIRT-1 expression and suppressing pyroptotic signals within the testicular tissues. Among the doses used in the present study, febuxostat at 15 mg/kg appears to be the optimal dose under the current experimental conditions, which achieved the maximal therapeutic benefit. These findings highlight febuxostat as a potential therapeutic option to counteract CP-induced testicular toxicity, thereby enhancing the CP’s safety in cancer therapy. Nonetheless, more extensive research using larger animal groups is needed to clarify the molecular mechanisms that may stand behind febuxostat’s dose-dependent effects. Future work should incorporate methods such as Western blotting, mRNA profiling of target proteins, and knock-out animal models. Also, future investigations incorporating a febuxostat-only group are warranted to delineate its standalone effects fully. Additionally, assessing febuxostat’s influence on CP-related testicular injury in cancer-bearing models is vital to determine its impact on the signaling pathways related to cancer biology.

Fig. 16.

The mechanisms by which febuxostat can prevent testicular dysfunction in cyclophosphamide-induced testicular toxicity in rats (This artwork was created using Reactome icon library and Smart Art Servier items). Abbreviations: HMGB1, high mobility group box 1; NLRP3, NOD-like receptor family pyrin domain-containing 3; RAGE, receptors for advanced glycation end products; ROS, reactive oxygen species; SIRT1, silent information regulator 1; TLR4, toll-like receptor 4. The red lines denote inhibition and the green lines denote activation.

Author contributions

Conceptualization and Data curation: Ahmed M. Kabel; Methodology, Investigation, Formal analysis, Visualization, Validation: Hanan Abdelmawgoud Atia, Hemat A. Elariny, Marwa H. Abdallah, Amany M. Khalifa, Doaa Hellal, Ali Alghubayshi, Ahmed M. Kabel; Funding acquisition: Hanan Abdelmawgoud Atia; Writing- Original draft preparation: Ahmed M. Kabel; Reviewing and Editing: Ahmed M. Kabel. All authors contributed to and approved the final manuscript.

Funding

This research was funded by Scientific Research Deanship at University of Ha’il, Saudi Arabia, through project number RG-25 106.

Data availability

Data will be made available from the corresponding author upon reasonable request.

Declarations

Competing interests

The authors declare no competing interests.

Ethical approval

The study protocol received ethical clearance from the Research Ethics Committee of the Faculty of Medicine, Tanta University, Egypt, under approval code 36264PR131013/10/25.