Saroglitazar Mitigated Cyclophosphamide-Induced Testicular Injury: Crosstalk Between Oxidative Stress, Inflammation and Apoptosis

TL;DR

Saroglitazar helps reduce testicular damage caused by Cyclophosphamide by reducing oxidative stress, inflammation, and cell death in rats.

Contribution

This study demonstrates Saroglitazar's protective effects against Cyclophosphamide-induced testicular toxicity in rats.

Findings

Saroglitazar increased testis weight, sperm count, and hormone levels in rats exposed to Cyclophosphamide.

Saroglitazar reduced oxidative stress markers and increased antioxidant levels in testicular tissue.

Saroglitazar showed anti-inflammatory and anti-apoptotic effects by modulating key proteins and cytokines.

Abstract

Background: Cyclophosphamide (CYC) is an effective chemotherapeutic agent and immunosuppressant drug. Former research showed that CYC induces testicular toxicity through oxidative stress, inflammation and apoptosis. Saroglitazar (SAR) is a dual PPARα/γ agonist, used for treatment of diabetic dyslipidemia. Purpose: This study aimed to elucidate the protective impact of SAR against CYC-linked testicular toxicity. Methods: Randomly, thirty adult male rats were alienated into control group, SAR (4 mg/kg) group, CYC (200 mg/kg) group, CYC+SAR (2 mg/kg) group and CYC+SAR (4 mg/kg) group. SAR was orally administered at two doses (2 and 4 mg/kg) for 7 days. CYC was injected intraperitoneally at dose (200 mg/kg) at day 7. Results: In comparison to the CYC group, SAR at the dose of 2 and 4 mg/kg significantly increased testis weight, testicular index, sperm count, serum testosterone and serum luteinizing hormone. Additionally, SAR at both doses induced a significant reduction in testicular MDA content in addition to increased testicular levels of GSH and TAC. Furthermore, SAR markedly upregulated testicular levels of PPARγ, Nrf2 and HO-1 in addition to decreased testicular expression of NF-κB, IL-6 and TNF-α, illustrating its antioxidant and anti-inflammatory effect. SAR also significantly decreased testicular expression of caspase-3 and Bax and increased Bcl2 expression, indicating its anti-apoptotic effect. Conclusions: SAR at doses (2 and 4 mg/kg) could ameliorate CYC-induced testicular injury in rats, possibly through antioxidant, anti-inflammatory and anti-apoptotic effect.

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1. Introduction

Cyclophosphamide (CYC), one of the most commonly used alkylating agents, is a highly effective chemotherapeutic agent used for the treatment of a variety of malignant processes. CYC is also used widely in the management of autoimmune diseases including rheumatoid arthritis where it acts as an immunosuppressant [1,2]. CYC is a prodrug, metabolically activated to phosphoramide mustard and acrolein [3,4]. Phosphoramide mustard is the active metabolite, exerting its pharmacological action via cross-linking of DNA and RNA and hence inhibiting protein synthesis. Nevertheless, acrolein is a toxic metabolite, limiting the therapeutic use of CYC [5,6]. Though it is commonly used in clinical practice, CYC can lead to side effects, including nausea, vomiting, diarrhea, bone marrow depression, alopecia and hemorrhagic cystitis [7]. Former research showed that CYC induces multi-organ toxicities including the liver [8], the kidneys [9], the immune system [10] as well as male [11] and female reproductive systems [12].

CYC has central and peripheral impact on male fertility through downregulating the hypothalamic–pituitary–gonadal (HPG) axis, affecting testosterone synthesis and spermatogenesis [13]. CYC has been revealed to increase the risk of male infertility [14]; this may be attributed to its toxic metabolite, acrolein [11]. Acrolein, a reactive aldehyde, is able to generate reactive oxygen species (ROS), that has multiple influences, including inhibition of a variety of enzymes for testosterone production, DNA damage and lipid peroxidation, contributing to infertility [15,16]. Excessive ROS results in oxidative stress which contributes to pathophysiology of male infertility [17]. Testicular oxidative stress may affect microvascular blood flow and endocrine signaling in testes, contributing to germ cell apoptosis and hence hypo-spermatogenesis [18].

Nuclear factor erythroid 2-related factor 2 (Nrf2), a cytoprotective transcription factor, is a vital defense mechanism against oxidative stress [19]. Upon activation, Nrf2 binds to the antioxidant response element (ARE), endorsing transcription of antioxidant enzymes including heme oxygenase-1 (HO-1), and hence protecting cells against oxidative stress [20]. Additionally, Nrf2 upregulation is essential for normal spermatogenesis and sperm motility [21]. Peroxisome proliferator-activated receptor-γ (PPAR-γ) is a member of the nuclear hormone receptor family that controls the expression of numerous target genes and several metabolic processes [22]. Previous studies showed that PPAR-γ augments antioxidant defenses and attenuates inflammation, hence protecting testicular tissue. Additionally, PPAR-γ is essential for hormone and sperm regulation [23,24]. PPAR-γ also prompts nuclear factor-kappa B (NF-κB) inactivation, decreasing the production of pro-inflammatory mediators such as tumor necrosis factor-alpha (TNF-α) as well as interleukin-1β (IL-1β) [25]. The Bcl-2 family, an essential cell death regulator, controls apoptosis in cells [26]. The Bcl-2-associated X protein (Bax)/B-cell lymphoma-2 (Bcl-2) signaling pathway has an essential role in mitochondria-mediated apoptosis and hence affects the progression of CYC-induced testicular injury. CYC upregulates Bax expression and downregulates Bcl-2 expression [27].

Saroglitazar (SAR), a dual PPARα/γ agonist, is a medication approved in India since 2013 to manage diabetic dyslipidemia. It decreases triglycerides, LDL cholesterol, and total cholesterol, while also improving blood sugar control [28]. Beyond its benefits in diabetes, recent research showed that SAR can attenuate non-alcoholic steatohepatitis (NASH) via reducing fat buildup and scarring in the liver [29,30]. According to Afarin et al., SAR has an antioxidant effect via activating the Nrf2 pathway, increasing antioxidant enzymes production including HO-1. The same study also showed that SAR decreases pro-inflammatory markers including TNF-α, suggesting its anti-inflammatory impact [31]. Based on the antioxidant and anti-inflammatory impact of SAR, the present study aimed to elucidate the potential protective impact of SAR against CYC-linked testicular toxicity, possibly via modulation of PPAR-γ/Nrf2/HO-1 and Bax/Bcl2/caspase3 pathways.

2. Results

2.1. Impact of SAR2 and SAR4 on CYC-Induced Changes in Testis Weight, Testicular Index, Sperm Count, Sperm Viability, Serum Testosterone and Serum Luteinizing Hormone

CYC injection resulted in a significant decrease in testis weight and testicular index by 57% and 56%, respectively, in contrast to the control group. Oral administration of SAR_2_ and SAR_4_ resulted in significant 2- and 2.1-fold increases in testis weight, respectively, relative to the CYC group. Similarly, oral administration of SAR_2_ and SAR_4_ resulted in significant 2.1- and 2.2-fold increases in testicular index, respectively, in contrast to the CYC group (p < 0.05, Table 1).

Rats injected with CYC exhibited a significant decrease in sperm viability after 1-, 2- and 3 h by 42%, 47% and 55%, respectively, in comparison to the control group. Oral administration of SAR_2_ resulted in significant 1.4-, 1.52-, 1.8-fold increases in sperm viability after 1-, 2- and 3 h, respectively, relative to the CYC group. Similarly, oral administration of SAR_4_ resulted in significant 1.4-, 1.6-, 1.84-fold increases in sperm viability after 1-, 2- and 3 h, respectively, compared to the CYC group (p < 0.05, Table 2).

CYC injection exhibited a significant decrease in sperm count by 53% relative to the control group. Oral administration of SAR_2_ and SAR_4_ resulted in a significant increase in sperm count by 1.23- and 1.5-fold, respectively, compared to the CYC group (p < 0.05, Figure 1A). CYC injection induced a significant decrease in serum testosterone by 78.2% relative to the control group. Yet, oral administration of SAR_2_ and SAR_4_ resulted in significant 1.96- and 3.16-fold increases in serum testosterone, respectively, relative to the CYC group (p < 0.05, Figure 1B). Injection of CYC significantly decreased serum luteinizing hormone by 40.3% relative to the control group. Oral administration of SAR_2_ and SAR_4_ resulted in significant 1.21- and 1.39-fold increases in serum luteinizing hormone, respectively, relative to the CYC group (p < 0.05, Figure 1C). The impact of SAR_4_ was more profound on sperm count, serum testosterone and luteinizing hormone than SAR_2_ (Figure 1A–C).

2.2. Impact of SAR2 and SAR4 on CYC-Induced Changes in Oxidant/Antioxidant Balance

Rats injected with CYC resulted in oxidative stress as indicated by a significant 3.45-fold increase in MDA level along with a significant decrease in reduced GSH level and TAC by 78.6% and 77.3%, respectively, relative to the control group (p < 0.05, Figure 2). Oral administration of SAR_2_ and SAR_4_ induced a significant decrease in MDA level by 33% and 53.5%, respectively, relative to the CYC group (p < 0.05, Figure 2A). Also, administration of SAR_2_ and SAR_4_ resulted in significant 2.14- and 3.8-fold increases in GSH, respectively, relative to the CYC group (p < 0.05, Figure 2B).

Additionally, administration of SAR_2_ and SAR_4_ resulted in significant 1.41- and 1.69-fold increases in TAC, respectively, relative to the CYC group (p < 0.05, Figure 2C). The impact of SAR_4_ on MDA, GSH and TAC was more profound than SAR_2_ (p < 0.05, Figure 2).

2.3. Impact of SAR2 and SAR4 on CYC-Induced Changes in Peroxisome Proliferator-Activated Receptor Gamma (PPAR-γ), Nuclear Factor Erythroid 2-Related Factor 2 (Nrf2) and Heme Oxygenase 1 (HO-1)

CYC injection resulted in a significant decrease in PPAR-γ, Nrf2 and HO-1 by 80.7%, 76.4% and 75.7% relative to the control group (p < 0.05, Figure 3).

Oral administration of SAR_2_ and SAR_4_ resulted in significant 2.44- and 4.71-fold increases in PPAR-γ, respectively, compared to the CYC group (p < 0.05, Figure 3A). Additionally, oral administration of SAR_2_ and SAR_4_ significantly increased Nrf2 level by 2.65- and 4.20-fold, respectively, compared to the CYC group (p < 0.05, Figure 3B). Also, oral administration of SAR_2_ and SAR_4_ resulted in significant 2.38- and 4-fold increases in HO-1, respectively, relative to the CYC group (p < 0.05, Figure 3C). The impact of SAR_4_ was more potent on PPAR-γ, Nrf2 and HO-1 than SAR_2_ (p < 0.05, Figure 3A–C).

2.4. Impact of SAR2 and SAR4 on CYC-Induced Changes in Interleukin 6 (IL-6), Tumor Necrosis Factor-Alpha (TNF-α), Bcl2-Associated X Protein (BAX), B-Cell Lymphoma 2 (Bcl2)

CYC injection resulted in a significant increase in IL-6, TNF-α and BAX by 4.37-, 3.5- and 5.07-fold while eliciting a significant reduction in Bcl2 by 82% relative to the control group (p < 0.05, Figure 4).

SAR_2_ and SAR_4_ administration significantly decreased testicular level of IL-6 by 34% and 70%, respectively, relative to the CYC group (p < 0.05, Figure 4A). Similarly, oral administration of SAR_2_ and SAR_4_ elicited a significant decrease in TNF-α by 41% and 65%, respectively, relative to the CYC group (p < 0.05, Figure 4B). Also, administration of SAR_2_ and SAR_4_ caused a significant decrease in BAX by 40% and 70%, respectively, relative to the CYC group (p < 0.05, Figure 4C). Conversely, administration of SAR_2_ and SAR_4_ elicited significant 3.05- and 5.1-fold increases in testicular Bcl2 level, respectively, in comparison to the CYC group (p < 0.05, Figure 4D). The SAR_4_-mediated effect on IL-6, TNF-α, BAX and Bcl2 was more profound than SAR_2_ (p < 0.05, Figure 4A–D).

2.5. Impact of SAR 2 and SAR 4 on CYC-Induced Histopathological Changes in Testicular Tissues

Figure 5 shows photomicrographs of testicular sections from different treatment groups. The control group as well as SAR group displayed normal histological appearance of seminiferous tubular germ epithelial cells. The CYC group showed diffuse germ epithelial degeneration characterized by tubular vacuolation with scattered necrotic spermatocytes in addition to seminiferous tubular atrophy with intraluminal necrotic debris in other tubules and interstitial fibrosis, admixed with numerous leukocytic infiltrations and edema. CYC+SAR_2_ showed few necrotic spermatocytes with mild interstitial edema, admixed with low numbers of cellular infiltrates. The CYC+SAR_4_ group showed approximately normal arrangement of seminiferous tubules with numerous spermatozoa.

2.6. Impact of SAR2 and SAR4 on CYC-Induced Changes in Seminiferous Tubules Morphometry

The seminiferous epithelium height was 0.42 mm in the control group. CYC administration induced a significant decrease in height of the seminiferous epithelium by 29% relative to the control group. Conversely, oral administration of SAR_2_ and SAR_4_ elicited significant 1.4- and 1.6-fold increases in height of the seminiferous epithelium, respectively, relative to the CYC group (p < 0.05, Figure 6).

2.7. Impact of SAR2 and SAR4 on CYC-Induced Changes in Testicular Caspase-3 Expression

Figure 7A shows testicular section immunostaining against caspase-3. The control group displayed few faintly stained germ epithelial cells. The CYC group showed high expression of caspase in germ epithelial cells. CYC+SAR_2_ showed mild faintly stained epithelial cells. The CYC+SAR_4_ group showed few mild faintly stained germ epithelial cells.

Administration of CYC induced a significant seven-fold increase in caspase-3 expression relative to the control group. Administration of SAR_2_ and SAR_4_ significantly decreased caspase-3 expression by 83% and 84%, respectively, relative to the CYC group (p < 0.05, Figure 7B).

2.8. Impact of SAR2 and SAR4 on CYC-Induced Changes in Testicular NF-κB Expression

Figure 8A shows testicular section immunostaining against NF-κB. The control group displayed mild cytoplasmic and nuclear stained germ epithelial cells. The CYC group showed marked nuclear with cytoplasmic expression of NF-κB in germ epithelial cells. CYC+SAR_2_ showed moderate to high stained germ epithelial cells. The CYC+SAR_4_ group showed moderate faintly stained epithelial cells.

Administration of CYC induced a significant seven-fold increase in NF-κB expression relative to the control group. Oral administration of SAR_2_ and SAR_4_ significantly reduced NF-κB expression by 70% and 80%, respectively, relative to the CYC group. The impact of SAR_4_ on NF-κB expression was more potent than SAR_2_ (p < 0.05, Figure 8B).

3. Discussion

Cyclophosphamide (CYC) is a non-specific cell cycle cytotoxic drug as well as antiproliferative drug. It is widely used as a part of the treatment regimen of solid tumors [32]. Unfortunately, CYC is non selective for cancer cells; thus, its administration is associated with marked side effects including hemorrhagic cystitis, gonadotoxicity and nephrotoxicity, limiting its therapeutic use [33]. CYC can induce male infertility via central and peripheral mechanisms. Centrally, CYC has been reported to downregulate the hypothalamic–pituitary–gonadal (HPG) axis which controls male gonadal function, affecting spermatogenesis. Hypothalamus secretes gonadotropin releasing hormone (GnRH), stimulating secretion of luteinizing hormone from the pituitary gland, and hence enhancing testosterone production [34].

Our study revealed that CYC administration caused impairment of reproductive function as shown via a significant decrease in multiple parameters including sperm count, sperm viability, serum luteinizing hormone and serum testosterone. The marked decrease in serum testosterone levels indicates a major problem in the HPG axis. Histopathologically, CYC showed diffuse germ epithelial degeneration characterized by tubular vacuolation with scattered necrotic spermatocytes in addition to seminiferous tubular atrophy with intraluminal necrotic debris in other tubules and interstitial fibrosis, admixed with numerous leukocytic infiltrations and edema. These findings are in accordance with previous studies that show the gonadotoxic effects of CYC [35,36]. Yet, saroglitazar (SAR) administration could attenuate CYC-induced impairment of reproductive function, manifested by a marked increase in sperm count, sperm viability, serum luteinizing hormone and serum testosterone. These findings were further confirmed by histopathological results which showed marked improvement in testis architecture in SAR-treated groups. This could be attributed to the protective effect of SAR in terms of anti-inflammatory and antioxidant ability.

The testis is mostly prone to oxidative damage due to high levels of polyunsaturated fatty acids (PUFAs) besides the presence of various reactive oxygen species (ROS)-generating mechanisms. Yet, antioxidant enzymes protect the testis from the negative effects of oxidative damage [37]. CYC disrupts the balance of the redox system by producing excessive ROS and a marked reduction in antioxidant enzymes. CYC is metabolically converted by cytochrome P450 enzymes into active cytotoxic compounds including phosphoramide mustard and acrolein. Additionally, acrolein is considered a significant contributor to reproductive toxicity as it generates a high level of ROS and interferes with antioxidant enzymes, promoting oxidative damage and hence testicular failure [36]. Oxidative damage has a negative impact on male sex hormones, sperm concentration, sperm nuclear DNA integrity and sperm motility [38].

Our study data revealed a significant increase in malondialdehyde (MDA) content, an end product of lipid peroxidation, and a significant decrease in antioxidant enzymes including total antioxidant capacity (TAC) and glutathione (GSH) content in rats injected with CYC. The disturbance in oxidant/antioxidant balance was illustrated in former research [38,39].

Oral administration of SAR elicited a significant decline in MDA content along with a significant increase in TAC and GSH content, restoring the balance between oxidant and antioxidant and hence exerting antioxidant impact. These findings were in accordance with the study of Kushawaha et al., which showed that SAR could exert a neuroprotective effect against maximal electroshock seizure (MES)-induced epilepsy through its antioxidant effect [40]. Additionally, the study of Joharapurkar et al. revealed the ability of SAR to decrease oxidative stress in the rat retina, attenuating streptozotocin (STZ)-induced diabetic retinopathy [41]. The antioxidant effect of SAR could explain its protective role against CYC-induced testicular toxicity.

Peroxisome proliferator-activated receptor gamma (PPAR-γ) is a ligand-regulated nuclear receptor (PPAR) which is vital in regulating energy homeostasis in testis tissue. Also, it controls the HPG axis. PPAR-γ upregulates fatty acid metabolic target genes in sertoli cells, providing sertoli cells with adequate energy and hence enhancing spermatogenesis [42]. In our study, CYC injection showed a significant decrease in PPAR-γ expression compared to the control group. The study of Abu-Risha et al. displayed similar results [43]. Also, Abd El Tawab et al. indicated that CYC decreased testicular PPAR-γ [44]. However, our study revealed that oral administration of SAR upregulated CYC-lowered testicular PPAR-γ. SAR is a dual PPAR-α/γ agonist [45]. Owing to its PPAR-γ agonistic action, SAR could attenuate CYC-induced testicular damage and enhance spermatogenesis.

Nuclear factor erythroid 2 related factor 2 (Nrf2) has an important role in regulating cellular defense mechanisms against oxidative stress. Under physiological conditions, Nrf2 is located in the cytoplasm; yet, upon exposure to ROS, Nrf2 translocates to the nucleus, interacting with antioxidant response element (ARE) and hence increasing the expression of antioxidant enzymes including heme oxygenase-1 (HO-1). Consequently, Nrf2 activation helps to alleviate oxidative stress [46,47]. Former research revealed that Nrf2 expression is necessary for normal spermatogenesis as well as sperm motility [48]. The expression of Nrf2 and HO-1 was reduced in CYC-treated rats. Such results were in agreement with former studies [11,49]. Conversely, SAR-treated rats exhibited marked upregulation in testicular expression of Nrf2 and HO-1. These results are in accordance with a former study which reported that SAR attenuated diet-induced nonalcoholic steatohepatitis via activating the Nrf2 pathway [31]. Notably, according to Solano-Urrusquieta (2020) [50], there is a positive feedback loop between PPARγ and Nrf2. PPARγ is a target gene of Nrf2, consequently promoting its activation [50]. PPAR-γ and Nrf2/HO-1 signals are key cytoprotective pathways in oxidative stress [51].

Nuclear factor kappa B (NF-κB), a transcription factor, regulates pro-inflammatory gene expression. ROS activates NF-κB and subsequently promoting NF-κB-driven inflammatory genes including tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6) [52,53]. In our study, CYC injection caused a marked increase in NF-κB, TNF-α and IL-6 expression, indicating a state of inflammation.

In contrast, SAR showed a marked decrease in NF-κB, TNF-α and IL-6 levels, illustrating its anti-inflammatory effect. This finding agreed with a previous study which indicated that SAR exerted an anti-inflammatory effect in 5-Fluorouracil-induced hepatorenal damage in rats, mediated through a significant reduction in levels of NF-κB and TNF-α [54]. Additionally, the study of Makled and El-Kashef reported an anti-inflammatory effect of SAR in unilateral ureteral obstruction-induced renal fibrosis in rats through a marked decrease in levels of NF-κB and IL-6 [55]. The anti-inflammatory impact of SAR may also be attributed to its antioxidant effect and its ability to modulate PPAR-γ and Nrf2/HO-1 signals.

Besides oxidative stress and inflammation, apoptosis has an important role in CYC-induced testicular injury [56]. Oxidative stress and inflammation augment the activation of apoptotic pathways, enhancing testicular damage. Apoptosis is controlled through the expression of numerous proteins including a class of cysteine proteases known as caspases and the Bcl-2 family. The Bcl-2 family is classified into anti-apoptotic proteins as Bcl-2, and pro-apoptotic proteins as Bax [57]. The Bax/Bcl-2 signaling pathway has a vital role in mitochondria-mediated apoptosis, exacerbating the development of testicular injury [27]. Bax counteracts the cytoprotective effect of Bcl-2 [58]. Caspase, particularly caspase-3, has a crucial role in the activation of apoptosis. Caspase-3 is the most critical effector caspase and its activation triggers the hallmark of apoptosis [59]. Bax/Bcl-2 imbalance triggers the activation of caspase-3, hence exacerbating apoptosis [60].

CYC injection resulted in a marked increase in testicular expression of pro-apoptotic proteins, including caspase-3 and Bax, along with a marked decrease in the testicular level of anti-apoptotic protein, Bcl-2. These findings agreed with previous research [61,62].

SAR administration mitigated apoptosis, indicated through a significant decrease in expression of caspase-3 and Bax, along with a marked increase in the testicular level of Bcl-2. The former study of Amer et al. revealed a significant decrease in caspase-3 expression and a significant increase in Bcl-2 level in rats with dexamethasone-induced hepatic injury treated with SAR [63]. According to Kushawaha (2025), SAR could decrease the expression of Bax and increase Bcl-2 level, exerting a neuroprotective effect [40]. Furthermore, it has been revealed that Nrf2 activation regulates the Bax/Bcl2 ratio and thus attenuates apoptosis [64]. Accordingly, SAR attenuation of apoptosis may be related to activation of the Nrf2 antioxidant pathway.

Regarding our study, SAR ameliorated CYC-linked testicular injury, possibly through its antioxidant, anti-inflammatory and anti-apoptotic impact, mediated via modulation of PPARγ/Nrf2/HO-1, NF-κB/TNF-α/IL-6 and caspase-3/Bax/Bcl2 pathways. Further investigations are required to clinically prove its therapeutic effect.

4. Materials and Methods

4.1. Chemicals and Drugs

Cyclophosphamide (CYC): obtained as Endoxan^®^ powder from local pharmaceutical company, Astellas, Cairo, Egypt.

Saroglitazar (SAR): obtained as Bilypas^®^ tablet from Zydus, Ahmedabad, India.

4.2. Animals

Thirty adult male Sprague-Dawley (SD) rats (200–220 g) were acquired from “Medical Experimental Research Center”, Faculty of Medicine, Mansoura University, Egypt.

Animal care and procedures were in accordance with National Institutes of Health (NIH) guidelines and approval of “Animal Care and Use Committee”, Mansoura University, Egypt, with code number MU- ACUC (PHARM.MS.24.09.104).

4.3. Induction of Testicular Injury

Induction of testicular injury was via single intraperitoneal (i.p.) injection of CYC (200 mg/kg). CYC was prepared by dissolving in saline and injected intraperitoneally to rats at day 7 [65].

SAR was orally administered at 2 and 4 mg/kg. It was dissolved in carboxymethyl cellulose (CMC) for oral administration from day 1 to day 7 [66].

4.4. Experimental Design

Rats were randomly separated into 5 groups, (6 rats/group). The experimental protocol is illustrated in Table 3 and the attached related Figure 9.

Twenty-four hours post CYC injection, a 2-step euthanasia process followed; first, rats were anesthetized with secobarbital (50 mg/kg, i.p.) for blood collection followed by exsanguination via cardiac puncture. The blood samples were gathered via retro-orbital venous plexus and centrifuged for 10 min at 4000 g to obtain serum that was used for determination of testosterone as well as luteinizing hormone. In addition, the testes and the epididymis were gathered, rinsed in cold isotonic saline then weighed. The epididymis was isolated for sperm analyses. For further biochemical and molecular assessment, the right testis was homogenized in phosphate-buffered saline (10% w/v). For histopathological and immunohistochemical analysis, the left testis was preserved in Bouin’s fixative solution.

4.5. Semen Analysis

Cauda epididymis of the two testicles was separated, suspended in 2 mL saline (0.9%) and then incubated at 37 °C for 30 min to release the spermatozoa from the epididymal tubules for semen analysis [67]. The sperm concentration and viability were assessed for each sample. Each 0.5 mL of sperm sample was diluted with 9.5 mL of fixative (50 g of sodium bicarbonate and 10 mL of 35% (v/v) formalin in 1000 mL of purified water); (1:20) for sperm counting, then the hemocytometer was loaded with diluted sample allowing spermatozoa to settle in a humid chamber then counted in the middle square using 200× lens. Sperm count was expressed as the concentration of million/mL. One step eosin-nigrosine procedure was used to calculate sperm viability as percent [68].

4.6. Biochemical Assessment

Levels of testosterone and luteinizing hormones were determined in serum using commercial kits from (MyBioSource, San Diego, CA, USA, Cat. no. MBS282195) and (MyBioSource, CA, USA Cat. no. MBS590031), respectively, consistent with manufacturer’s instructions.

4.7. Determination of Oxidant/Antioxidant Balance

Testicular homogenate was used for determination of malondialdehyde (MDA) content, reduced glutathione (GSH) level in addition to total antioxidant capacity (TAC) using Biodiagnostic assay kits (Giza, Egypt) with cat.no. (# MD 25 29, # GR 25 11, and # TA 25 13), respectively, consistent with manufacturer’s instructions.

4.8. Determination of Tumor Necrosis Factor-Alpha (TNF-α), Interleukin 6 (IL-6), B-Cell Lymphoma 2 (Bcl2), Bcl2-Associated X Protein (Bax), Nuclear Factor Erythroid 2-Related Factor 2 (Nrf2), Peroxisome Proliferator-Activated Receptor Gamma (PPAR-γ) and Heme Oxygenase 1 (HO-1) via Enzyme-Linked Immunosorbent Assay (ELISA)

Testicular homogenate was used for determination of TNF-α (Elabscience, Wuhan, China, Cat. no. E0764Ra), IL-6 (Cloud-Clone Corp, Wuhan, China, Cat. no. SEA079Ra), Bcl2 (Elabscience, China, Cat. no. ER0762), Bax (Elabscience, China, Cat. no. ERO512), PPAR-γ (Elabscience, China, Cat. no. E-EL-R0724), Nrf2 (RayBiotech, Peachtree Corners, GA, USA, Cat. no. RD-NFE2L2-Ra) and HO-1 (CUSABIO, Wuhan, China, CSB-E08267r) via ELISA as instructed by the manufacturer.

4.9. Histopathological, Morphometrical and Immunohistochemical Analyses

For histopathological and immunohistochemical analyses, the left testicle was fixed in Bouin’s solution. All samples were dehydrated in ascending concentration of alcohol (70–100%) after 24 h. All tissues were then cleared in xylene, embedded in paraffin, and cut by the microtome at a thickness of 3 μm, and stained with hematoxylin and eosin (H&E) for evaluation of tissue injury.

Histopathological analyses were carried out by the pathologist in a blinded manner. As previously described, morphometric analysis of the testes was performed [68]. Software imageJ version 1.5r (NIH, Bethesda, MD, USA) was used for measurement of the diameter and the height of the seminiferous tubule epithelium at 100× magnification.

Caspase-3 and NF-κB expression was detected in testicular sections via immunohistochemical analyses. After deparaffinization and rehydration, antigen retrieval was performed. Tissue sections were incubated with rat polyclonal antibodies against caspase-3 (Servicebio, Wuhan, China, # GB 11532) and NF-κB (Abclonal, Wuhan, China, # A2547) overnight at 4 °C; then, the sections were washed and treated for 2 h at room temperature with a goat anti-rat secondary antibody (Genemed Biotechnologies, Torrance, CA, USA). For visualization, diaminobenzidine (DAB) staining was used and for examination of stained sections, a Leica light microscope was used. To minimize background staining and confirm specificity, appropriate control experiments were conducted [69]. In a blinded assessment, ImageJ software was used for quantification of positively stained regions. For each rat, mean of six readings from the left testicular sections was calculated.

4.10. Statistical Analysis

Data were displayed as mean ± standard error (mean ± S.E). To measure differences among groups, One-way analysis of variance (ANOVA) followed by Tukey–Kramer’s multiple comparison post hoc test was used. Statistical analyses were carried out using Graph Pad Prism software (Graph Pad Software Inc. V 8.4.2, La Jolla, CA, USA). Statistical significance was set at (p < 0.05).

5. Conclusions

Our study showed that SAR may have potential as a prophylactic agent to protect against CYC-induced testicular toxicity. Its dual PPARα/γ activity and antioxidant properties provide a mechanistic rationale for mitigating oxidative stress, inflammation and apoptosis in testicular tissue. SAR could be possibly co-administered with chemotherapy; yet, further studies are required to evaluate possible drug–drug interactions and potential impacts of SAR on chemotherapy efficacy before clinical application.

The limitations of study:

• The use of specific PPARγ or Nrf2 inhibitors would have provided stronger mechanistic evidence and greater depth to the proposed signaling pathways. Future studies incorporating pharmacological inhibition would be valuable in providing mechanistic depth.
• Our study evaluated the prophylactic effect of SAR against CYC-induced acute testicular toxicity. Although this model does not mimic the chronic or fractionated dosing regimens commonly used in clinical chemotherapy, it represents a relevant experimental model for evaluating acute testicular toxicity that may occur during intensive chemotherapy protocols. Chronic or fractionated CYC dosing regimens more closely simulate clinical exposure, and this will be considered in future investigations.
• The study did not investigate whether SAR affects the therapeutic efficacy or pharmacological actions of CYC. Further studies are needed to evaluate the possible impact of SAR on the pharmacological actions of CYC.
• The study demonstrated short-term protective effects of SAR; potential endocrine modulation and long-term reproductive outcomes were not assessed. Long-term studies assessing hormonal balance and reproductive safety are necessary to fully establish the clinical relevance and safety profile of SAR.