Potential gonadal-beneficial effect of sitagliptin against paclitaxel-induced testicular dysfunction via mediating PERK/CHOP/NLRP3/Sestrin2 signaling pathway
Kareman M. El-Beheiry, Nagla A. El-Shitany, Magda El-Sayed El-Sayad, Alaa E. Elsisi
TL;DR
Sitagliptin may protect testicles from paclitaxel damage by reducing inflammation and stress through a specific signaling pathway.
Contribution
The study reveals a novel protective mechanism of sitagliptin against testicular dysfunction caused by paclitaxel.
Findings
Sitagliptin improves sperm count, motility, and viability while reducing abnormalities.
Sitagliptin reduces inflammation and oxidative stress by modulating the PERK/CHOP/NLRP3/Sestrin2 pathway.
Sitagliptin shows anti-apoptotic effects by decreasing cleaved caspase-3 and cytochrome c levels.
Abstract
Paclitaxel (PTX) is broadly prescribed to treat various malignancies. However, it induces negative impacts on many organs, including testes. This study explored the beneficial role of sitagliptin (SIT) in PTX-provoked testicular damage and the underlying mechanisms. Rats were allocated into four groups: (I) control, (II) PTX, (III) PTX + SIT5, and (IV) PTX + SIT10. Histopathological and ultrastructural analyses were conducted along with sperm analysis. Immunohistochemical examinations of NOD-like receptor protein 3 (NLRP3), cleaved caspase-3, caspase-3, cytochrome c (Cyt.c), and interleukin-1 beta (IL-1β) were assessed. Serum testosterone and testicular 17β-hydroxy steroid dehydrogenase (17β-HSD), sestrin2, phosphorylated protein kinase R-like ER kinase (pPERK), and C/EBP homologous protein (CHOP) were determined. SIT induced a remarkable increase in sperm count, motility, and…
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Figure 9- —Tanta University
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Taxonomy
TopicsTesticular diseases and treatments · Protein Degradation and Inhibitors · Cancer, Stress, Anesthesia, and Immune Response
Introduction
The worldwide burden of cancer occurrence and mortality is growing rapidly^1^. Chemotherapeutic medications exert anti-tumor actions by repressing the proliferation of dividing cells. Nevertheless, a correlation has been found between their use and the alteration in testicle function of cancer patients^2,3^.
Paclitaxel (PTX) is a chemotherapeutic drug that is often used as the first-line treatment for solid tumors such as pancreatic, lung, breast, and ovarian malignancies. It is one of the cytoskeletal agents that affects tubulin, hastening microtubule polymerization and inhibiting their dissociation, thereby hindering the progression of the cell cycle, mitosis, and the proliferation of cancer cells^4,5^. In addition to its beneficial and significant effects, several investigations have shown that PTX is harmful to various organs, including the liver, bone marrow, ovaries, immune system, kidneys, heart, and both the peripheral and central nervous systems^6–10^.
It is also clear that PTX has negative impacts on male reproduction and fertility. It caused pathological changes in testicular tissue, resulting in abnormalities in spermatogenesis, including reduced sperm count and motility with higher proportions of sperm malformation^11^. One of the mechanisms underlying the harmful effects of PTX is the stimulation of oxidative stress, which occurs by increasing the production of reactive oxygen species (ROS). ROS increase the DNA damage in the plasma membrane of sperm^12,13^.
Current studies have shown that endoplasmic reticulum stress (ER stress) controls the cellular homeostasis and apoptosis in male reproductive organs^14,15^. Some pathogenic or environmental elements, like variations in temperature and pH, DNA damage, and ROS, can all lead to ER stress. There are three mechanisms involved in ER stress: protein kinase RNA-like endoplasmic reticulum kinase (PERK), activating transcription factor 6 (ATF6), and inositol-requiring 1α (IRE1α) ^16^. When ER stress is exaggerated or chronic, it stimulates the unfolded protein response (UPR). UPR stimulation may hasten cell death. It has been stated that the ER stress-regulated apoptosis signaling pathway is critical for testicular apoptosis^17,18^, which can provoke testicular and spermatogenic dysfunctions. Also, a recent study has stated that PTX disrupts testicular tissue in rats by exacerbating ER stress^19^.
Sitagliptin (SIT) is a clinical anti-diabetic drug. It is a dipeptidyl peptidase-4 inhibitor that enhances the antihyperglycemic effect of incretin hormones by inhibiting the breakdown of glucagon-like peptide-1 (GLP-1), which in turn promotes insulin release in response to nutrient ingestion without causing hypoglycemia^20,21^. Some studies are uncovering the therapeutic benefits of SIT as a reno-protective agent against cyclosporine^22^ and 5-fluorouracil^23^. Additionally, SIT is considered hepatoprotective against thioacetamide^24^, methotrexate^21^, and hepatic ischemia^20^. Additionally, SIT exhibits a neuroprotective effect against the oxidative damage caused by cisplatin and cyclophosphamide in rats^25,26^. Interestingly, the aforementioned impacts have been linked to the antioxidant, anti-inflammatory, and anti-apoptotic effects of SIT.
PTX is known to cause gonadotoxicity primarily through oxidative and inflammatory mechanisms. Similarly, hyperglycemia in diabetes can induce oxidative stress and gonadal dysfunction^27,28^. Therefore, diabetic patients receiving PTX may experience aggravated reproductive toxicity. In this context, using SIT, an antidiabetic agent with proven antioxidant and anti-inflammatory properties, could provide dual protection against both diabetes- and chemotherapy-related gonadal injury.
To our knowledge, the impact of SIT on testicular damage induced by PTX has never been investigated. The current study aims to elucidate the potential benefits of SIT in mitigating PTX-provoked testicular damage in rats by mediating oxidative stress, ER stress, inflammation, and apoptosis pathways.
Materials and methods
Drugs and chemicals
Paclitaxel (Unitaxel, vial, 100 mg/16.67mL) was bought from Hikma Specialized Pharmaceuticals (Badr City, Egypt), and Sitagliptin was a gift from Adwia Pharmaceutical Company (Egypt). Every chemical utilized was of acceptable analytical quality.
Animals
The experiment used 20 adult male albino rats, weighing between 150 and 180 g. Rats were obtained from the animal house at Cairo University’s College of Veterinary Medicine (Cairo, Egypt). The rats were housed in standard rat cages with controlled lighting (a 12-hour light-dark cycle), temperature (22 ± 3 °C), and relative humidity (30–70%). Throughout the experiment, they had free access to food and drink. Before their handling in the study, all rats were given a week to acclimate. The experimental protocol was approved by the Research Ethics Committee of the Faculty of Pharmacy, Tanta University (REC-TP), adhering to the guidelines of the Council for International Organization of Medical Sciences (CIOMS) and local regulations (approval code: TP / RE/7/24 Ph-1). Euthanasia was performed in accordance with the American Veterinary Medical Association (AVMA) Guidelines for the Euthanasia of Animals (2020). The study is reported in accordance with the ARRIVE guidelines (PLoS Biol 8(6), e1000412, 2010).
Experimental design
The rats were randomly allocated to four groups by a simple randomization method (five per group), as shown in Table 1; Fig. 1(a and b).
Table 1. Experimental scheme.Experiment groupsApplicationsI (Control)Rats received 0.9% normal saline on the first day I.P., then P.O. daily for 14 consecutive days.II (PTX)Rats received PTX (8 mg/kg diluted in normal saline, I.P., as a single dose on day 1, followed by 0.9% normal saline for 14 consecutive days^57^.Ⅲ (PTX + SIT 5)Rats received PTX (8 mg/kg diluted in normal saline, I.P., as a single dose on day 1, followed by SIT (5 mg/kg, dissolved in normal saline, P.O., daily) for 14 consecutive days^58^.Ⅳ (PTX + SIT 10)Rats received PTX (8 mg/kg diluted in normal saline), I.P., as a single dose on day 1, followed by SIT (10 mg/kg, dissolved in normal saline, P.O., daily) for 14 consecutive days ^22^.PTX: paclitaxel and SIT: sitagliptin.
Fig. 1(a, b) Schematics of the experimental design and animal grouping. (a): Adult male albino rats were divided into four groups, and the number of animals in each group was five. (b): Blood and tissue sampling and techniques used. PTX; paclitaxel, SIT; sitagliptin, 17βHSD: 17 beta hydroxy steroid dehydrogenase, NLRP3: NOD-like receptor protein 3, MDA; malondialdehyde, GSH; reduced glutathione.
Tissue collection
On the 15th day, all rats were deeply anesthetized using pentobarbital (50 mg/kg, I.P.). Blood samples were obtained via a heart puncture and centrifuged for 15 min at 3000 rpm. The serum was appropriately separated and stored at -20 °C until it was needed for measuring serum testosterone. Subsequently, while rats reflex responses were completely absent, " fully unconscious”, they were humanely euthanized by cervical dislocation, in accordance with the American Veterinary Medical Association (AVMA) Guidelines for the Euthanasia of Animals (2020). The testes and epididymides were carefully isolated from connective tissue adhesions. One testis was divided into two halves; one half was fixed in 10% neutral buffered formalin for histopathological and immunohistochemical examination, and the second half was preserved with 2.5% glutaraldehyde for ultra-structure analysis. The other testis was gathered in liquid nitrogen and kept at − 80℃ for further biochemical analysis. The epididymis has been used to determine sperm counts, motility, abnormality, and viability. All investigators performing outcomes were blinded to the group allocations.
Preparation of epididymal sperm suspension (for sperm analysis)
The epididymides were carefully excised and cleared of surrounding fat and connective tissues. The cauda epididymis was then separated, gently rinsed with sterile normal saline to eliminate blood residues, and placed in a sterile Petri dish containing pre-warmed 0.9% NaCl at 37 °C. To release spermatozoa, the caudal tissue was finely minced and several shallow incisions were made using sterile scissors or a scalpel. The tissue fragments were subsequently incubated at 37 °C for 30 min, allowing spermatozoa to disperse naturally into the surrounding medium. Following incubation, the remaining tissue fragments were removed, and the sperm suspension was gently mixed to ensure homogeneity while avoiding bubble formation. The freshly prepared suspension was used immediately for sperm motility assessment and appropriately diluted for sperm count determination as well as viability and morphological analyses.^29^.
Sperm count
The improved Neubauer hemocytometer was used to count the sperm under a microscope. Five Thoma chambers were used for the counting.^30^.
Sperm motility
Semen samples were obtained from the cauda epididymis for spermatozoa analysis; the sperm motility was evaluated by diluting the semen sample with 0.9% NaCl. Three microscopic fields were analyzed for each semen sample utilizing a phase-contrast microscope (Leica DM500, Leica, Mikrosysteme, Vertrieb GmbH) set to 37 °C. The percent of sperm motility was recorded based on the number of progressively motile spermatozoa among the examined number of spermatozoa*100^30^.
Sperm viability and morphology
The percent of viability, unstained live sperm or stained dead sperm, and sperm abnormality could be calculated in the same semen field (300 sperm cells/sample) via staining it with a mixture of 10% nigrosine and 5% eosin stains and observing it with contrast microscopy at x 400 magnification The percentage viability was estimated by calculating the average value of the stained and unstained sperm cells^30^.
Determination of serum testosterone and testicular 17-βHSD content
Serum testosterone levels were measured using an ELISA kit (MyBioSource Southern California, San Diego, USA). 17β-Hydroxy steroid dehydrogenase type 3 (17-βHSD3) was evaluated in testicular tissue homogenate using ELISA kits purchased from Cloud Clone Corp., USA.
Histopathological investigation of testis tissue and scoring system
Following the experiment, the testes were separated, cleaned with saline, and set up for histological examination. Testis slices were preserved in a 10% formalin solution (pH 7.4) for 24 h before being treated in increasing degrees of alcohol and xylene. The tissues were lastly embedded in paraffin wax at 65 °C. A light microscope was used to examine tissue blocks that had been segmented at a thickness of 5 μm and stained with hematoxylin and eosin (H&E). The scoring of the grade of testicular injury and spermatogenesis quality was evaluated using the Johnsen testicular score, thickness of epithelial lining of seminiferous tubules, and number of Leydig cells^31^. The results were evaluated in 5 randomly selected microscopic fields using ImageJ 1.53t, Wayne Rasband and contributors, National Institutes of Health, USA.
Ultrastructural examination by transmission electron microscope (TEM)
Testicular sections from various groups were preserved in 2.5% cold-buffered glutaraldehyde. In accordance with a previously reported study, the samples were subsequently processed to obtain ultrathin sections for electron microscopy investigation^30^.
Assessment of testicular oxidative stress markers in the testis
The testis tissue’s malondialdehyde (MDA) content was measured using a colorimetric kit (Bio Diagnostic, Egypt) according to Ohkawa et al.^52^. In addition, testicular catalase was detected in testis homogenate according to Aebi^33^, using a colorimetric kit (Biodiagnostic, Egypt). These assays were evaluated by using a UV-Vis spectrophotometer T80, pg. Instruments. Testicular reduced glutathione (GSH) content was assessed in testicular tissue homogenate utilizing an ELISA kit purchased from MyBioSource, Southern California, San Diego (USA). Sestrin2 was assessed in testicular tissue homogenate utilizing ELISA kits bought from MyBioSource, Southern California, San Diego (USA), and all were used as stated by the manufacturer’s guidelines.
Assessment of testicular ER stress markers
C/EBP homologous protein (CHOP) was quantified in testicular tissue homogenate utilizing ELISA kits bought from MyBioSource, Southern California, San Diego (USA). Phosphorylated protein kinase R-like ER kinase (pPERK) was measured in testicular tissue homogenates using ELISA kits obtained from MESO SCALE DISCOVERY^®^, a division of Meso Scale Diagnostics, USA, and all were used according to the manufacturer’s instructions.
Quantitative real-time polymerase chain reaction (qRT-PCR) for NLRP3 and Sestrin2 genes
Testicular tissue samples were preserved at -80 °C and ground in liquid nitrogen. Tissue lysate was treated with Direct-zol RNA Miniprep Plus (Cat# R2072, ZYMO RESEARCH CORP. USA) to extract total RNA. Then, quantity and quality were determined by a Beckman dual spectrophotometer (USA). The extracted RNA was reverse-transcribed utilizing the SuperScript IV One-Step RT-PCR kit (Cat# 12594100, Thermo Fisher Scientific, Waltham, MA, USA). Then, PCR was performed in one step utilizing previously stated primers listed in Table 2. The prepared reaction mix samples were analyzed using real-time PCR (Step One Applied Biosystems, Foster City, USA). After the RT-PCR run, the data were expressed in cycle threshold (Ct). The PCR data sheet includes Ct values of the assessed gene (NLRP3 and Sestrin2) compared to the corresponding housekeeping gene (GAPDH). A control sample should be utilized to determine the gene expression of selected genes. The relative quantification (RQ) of each target gene is quantified and normalized to the housekeeping gene regarding the calculation of delta-delta Ct (2 ^-^^ΔΔCt^).^34^.
Table 2. Primers’ sequence of studied genes utilized in quantitative RT-PCR.GeneForward primer(5′–3′)Reverse primer(5′–3′)Accession numberNLRP3GTGGAGATCCTAGGTTTCTCTGCAGGATCTCATTCTCTTGGATCNC-086022Sestrin2TTGTGTTTGGCTGTGGGATACCGAGTTGTTCAATGGGTCTNC-086021.1GAPDHTGGATTTGGACGCATTGGTCTTTGCACTGGTACGTGTTGATNC-086022.1NLRP3: NOD-like receptor protein 3.
Immunohistochemical assessment for inflammatory and apoptotic parameters (NLRP3, IL-1β, cleaved caspase-3, caspase-3, and Cyt.c)
For immunohistochemical analysis, anti-caspase-3 monoclonal antibody (AB clonal, cat# A19664, USA), anti-cleaved caspase-3 polyclonal antibody (MyBioSource, Cat# MBS628492, USA), anti-NLRP3 polyclonal antibody (MyBioSource, cat# MBS9608075, USA), anti-cytochrome c monoclonal antibody (Novus Biologicals, cat# NB100-56503, USA), and anti-IL-1β monoclonal antibody (Santa Cruz Biotechnology, Cat# sc-52012, USA) were used to identify their targeted proteins via a standard immunohistochemical technique.
Scoring of immunohistochemistry results
Scoring of immunohistochemistry data by assessing the immunoreactive area percent in 5 microscopic fields utilizing Image J 1.53t, Wayne Rasband and contributors, National Institutes of Health, USA.
Statistical analysis
GraphPad Prism 9.5 Demo (GraphPad Software, San Diego, CA) was utilized for statistical analysis of various groups. Tukey multiple comparison tests were used after one-way analysis of variance (ANOVA) to compare data groups. Data were displayed in tables and figures as Mean ± SEM. In the results section, absolute values were expressed as mean (95% CI). A difference was considered significant when P < 0.05.
Results
Impact of SIT on epididymal sperm count, motility, abnormality, and viability
PTX impaired spermatogenesis, as evident in the decrease in sperm count with a mean of 74.7 (95% CI: 54–95.4), viability: 54% (95% CI: 43–64.8), motility: 11.7% (95% CI: 4.5–18.8) compared to the control group; 172 (95% CI: 152–192), 83.7% (95% CI: 76.5–90.8), and 58.3% (95% CI: 44–72.2), respectively (P < 0.05). PTX also increases sperm abnormal morphology with a mean of 28.7% (95% CI: 21–36.3), compared to the control group, 13.7% (95% CI: 9.8–17.5) (P < 0.05). SIT5 elevated sperm count;113.3(95% CI: 78.4–148.2), viability; 71.7% (95% CI: 67.9–75.5), motility; 28.3% (95% CI: 21.2–35.5) compared to the PTX group (P < 0.05). Additionally, SIT5 decreased the percentage of spermatozoa with abnormal morphology to 23.3% (95% CI: 19.5–27), compared to the PTX group (P < 0.05). While the SIT10-treated group induced a remarkable elevation in sperm count; 146.7 (95% CI: 135.2–158), viability; 80.3% (95% CI: 77.5–83.2), and motility; 50% (95% CI: 37.6–62.4) relative to the PTX group (P < 0.05). Furthermore, SIT10 decreased the percentage of spermatozoa with abnormal morphology to 16.3% (95% CI: 12.5–20), compared to the PTX group (P < 0.05). The findings of SIT10 were comparable to those of the control group, as demonstrated in Table 3.
Table 3. Impact of SIT on sperm parameters of PTX-treated rats.Experimental groupSperm count (millions/ml)Sperm viability (%)Sperm abnormality (%)Sperm motility (%)Control172.00 (95% CI: 152–192)83.67 (95% CI: 76.5–90.8)13.67 (95% CI: 9.8–17.5)58.33 (95% CI: 44–72.2)PTX55.00^a^ (95% CI: 54–95.4)54.00^a^ (95% CI: 43–64.8)28.67^a^ (95% CI: 21–36.3)11.67^a^ (95% CI: 4.5–18.8)PTX+SIT5113.3^b^ (95% CI: 78.4–148.2)71.67^b^ (95% CI: 67.9–75.5)23.33^b^ (95% CI: 19.5–27)28.33^b^ (95% CI: 21.2–35.5)PTX+SIT10146.7^bc^ (95% CI: 135.2–158)79.00^bc^ (95% CI: 77.5–83.2)15.00^bc^ (95% CI: 12.5–20)50.00^bc^ (95% CI: 37.6–62.4)PTX: paclitaxel, SIT: sitagliptin. Data shown are mean (95%CI). (n = 3/group).^a^Significant difference from control, P < 0.05.^b^Significant difference from PTX, P < 0.05.^c^Significant difference from SIT5, P < 0.05.
Impact of SIT on the level of serum testosterone and testicular 17β-HSD
The PTX group demonstrated a pronounced decline in serum testosterone, with a mean of 5.8 (95% CI: 4.9–6.7), compared to the control group, which had a mean of 25.2 (95% CI: 23.3–27.2) (P < 0.05). On the other hand, the SIT 5 and SIT 10 groups showed significant increases in serum testosterone, with means of 12.6 (95% CI: 10.4–14.9) and 18.8 (95% CI: 18.0–19.5), respectively, compared to the PTX group (p < 0.05). Interestingly, there is a significant difference between SIT5 and SIT10 (Fig. 2a).
Fig. 2. Effects of SIT on: (a) serum testosterone and (b) testicular 17βHSD content in PTX-intoxicated rats. The data presented are the mean ± SEM (n = 5/group). PTX: paclitaxel, SIT: sitagliptin, 17βHSD: 17β-hydroxy steroid dehydrogenase. ^a^ significant difference from control, P < 0.05, ^b^ significant difference from PTX, P < 0.05,^c^ significant difference from SIT5, P < 0.05.
Testicular 17β-HSD content was diminished in PTX-induced rats, with a mean of 3.7 (95% CI: 3.0–4.4) compared to the control group; 15.7 (95% CI: 14.6–16.8) (P < 0.05). Treatment with SIT 5 significantly enhanced testicular 17β-HSD content, with a mean of 7.5 (95% CI: 6.6–8.3), compared to the PTX group (P < 0.05). While SIT 10 significantly increased testicular 17β-HSD content with a mean of 11.17(95% CI: 10.2–12.2) compared to the PTX group, P ˂ 0.05, as illustrated in Fig. 2b. There is a significant difference between SIT5 and SIT10, P ˂ 0.05.
Histopathologic and microscopic score Findings
Histopathological analysis and lesion scoring were performed to elucidate the influence of the treatment on the cellular structures of the testicular tissue. The control group displayed normal seminiferous tubules, spermatocytes, and normally arranged Leydig cells (Fig. 3a). However, the PTX group revealed deterioration and nuclear pyknosis of spermatocytes, necrobiotic changes with a marked decline in the number of spermatogenic cells in some seminiferous tubules, in addition to vacuolar degeneration in Leydig cells (Fig. 3b). Furthermore, the SIT5 group revealed moderate necrobiotic changes in Leydig cells, a decrease in the number of spermatogenic cells (Fig. 3c). Notably, SIT10 showed mild vacuolar degeneration in Leydig cells (Fig. 3d).
Fig. 3. Representative photomicrographs of H&E-stained testicular tissue sections from different treatment groups. (a) The testicular section of the control group shows a normal histological structure of seminiferous tubules, spermatogenic cells, and Leydig cells. (b) The testicular section of the PTX group shows necrobiotic changes with a marked decrease in the number of spermatogenic cells (arrow) in some seminiferous tubules and vacuolar degeneration in Leydig cells (blue arrow). (c) The testicular section of the SIT5 group shows necrobiotic changes in Leydig cells (black arrow) and a decrease in the number of spermatogenic cells (blue arrow). (d) The testicular section of the SIT10 group shows mild vacuolar degeneration in Leydig cells (arrow). (e) Johnsen score. (f) Number of Leydig cells. (g) Thickness of the epithelial lining of the seminiferous tubules. The data displayed is mean ± SEM (n = 5/group). PTX: paclitaxel, SIT: sitagliptin. ^a^ significant difference from control, P < 0.05, ^b^ significant difference from PTX, P < 0.05, ^c^ significant difference from SIT5, P < 0.05.
PTX induced a marked decline in the Johnsen score (3.4/10) (95% CI: 2.7–4.1). While SIT5 and SIT10 revealed a remarkable improvement in the score (6.2/10) (95% CI: 5.2–7.2) and (8/10) (95% CI: 7.1–8.8), respectively, compared to the PTX group, P < 0.05. (Fig. 3e). There is a significant difference between SIT5 and SIT10, P < 0.05.
Regarding the number of Leydig cells, the PTX group showed a significant decrease, with a mean of 13.2 (95% CI: 10.8–15.6), compared to the control group, which had a mean of 30.4 (95% CI: 26.3–34.5). On the contrary, SIT5 and SIT10 revealed a notable increase in Leydig cell number 19.8 (95% CI: 18–21.6) and 25.6 (95% CI: 21.5–30), respectively, compared to the PTX group, P < 0.05. There is a significant difference between SIT5 and SIT10, P < 0.05. (Fig. 3f).
In addition, PTX induced a remarkable reduction in the thickness of the epithelial lining of seminiferous tubules by a mean of 22.6 (95% CI: 17.7–27.5), compared to the control group; 69.5 (95% CI: 65.6–73.2). In contrast, treatment with SIT5 and SIT10 resulted in a significant restoration of epithelial lining thickness, 34.4 (95% CI: 27.7-41.12) and 55.8 (95% CI: 49.4–62.2), respectively, compared to the PTX group, P < 0.05. There is a significant difference between SIT5 and SIT10, P < 0.05. (Fig. 3g).
Effects of SIT on testicular ultrastructure alterations caused by PTX
As shown in Fig. 4 (b1, b2, b3, b4), the PTX group shows an irregular basement membrane, spermatogonia with irregular cell membrane partly detached from the basement membrane, damaged nuclear membrane, and compact electron-dense granule mitochondria. Sertoli cells show their abnormal nucleus and disrupted cristae of mitochondria. A primary spermatocyte is also seen with an irregular-shaped nucleus and disorganized mitochondria, in addition to a late spermatid with euchromatic nuclei and the formed irregularly shaped acrosomal vesicle. Mitochondria have an abnormal shape and are distributed throughout the cytoplasm and vacuoles.
Fig. 4. Transmission electron microscope investigation of ultrathin sections of testicular tissue from various experimental groups. (a1, a2, a3, a4) The control group shows a normal spermatogonium with a normal nucleus, peripherally arranged mitochondria, a chromatoid body, normal rough endoplasmic reticulum, and a normal junction in between. Sertoli cells show their characteristic irregular nucleus with a prominent nucleolus. The cytoplasm contains normal rough endoplasmic reticulum, mitochondria, and well-developed junctional complexes. Primary spermatocytes exhibit normal characteristics, such as a fine cytoplasm with numerous normal mitochondria and a prominent, well-defined euchromatic nucleus. Furthermore, the control group shows normal spermatids with their characteristic complete acrosomal cap; its nucleus is euchromatic with finely granular chromatin and peripherally arranged vesicular mitochondria. (b1, b2, b3, b4) The PTX group shows spermatogonia with an irregular cell membrane, partly separated from the basement membrane, and a damaged nuclear membrane. (c1, c2, c3) The SIT5-treated group shows spermatogonia and Sertoli cells resting on a barely irregular basement membrane. (d1, d2, d3) The SIT10-treated group shows normal seminiferous tubules, spermatogonia, and Sertoli cells, as well as activated and fully developed spermatids with identifiable cell outlines and mitochondria in a typical arrangement. Spermatogonia (Sg) nucleus (N), mitochondria (m), chromatoid body (CH), rough endoplasmic reticulum (blue arrowhead), the junction (j), Sertoli cells (SC), nucleolus (Nu), primary spermatocyte (PS), spermatids (SP), acrosomal cap (AC), basement membrane (BM), intercellular gap (star), vacuoles (V), lysosomes (yellow arrowhead), little spaces (star), and Golgi apparatus (red arrow). (n = 3/group).
On the other hand, treatment with SIT5 exhibited spermatogonia and Sertoli cells resting on a barely irregular basement membrane. The Sertoli cell has a large euchromatic nucleus and some mitochondria with disrupted cristae. Additionally, the primary spermatocyte contains a large, spherical nucleus with heterochromatin masses dispersed throughout it. Its cytoplasm contains mitochondria and lysosomes, and small spaces are also observed between cells, as shown in Fig. 4(c1, c2, c3).
The SIT10-treated group exhibits normal seminiferous tubules, spermatogonia, and Sertoli cells, which are resting on the basement membrane. It also appeared to have little separation from the basement membrane. In addition, normal primary spermatocytes appear with large, rounded nuclei and electron-dense clumps of heterochromatin. Their cytoplasm contains mitochondria. Moreover, cells have normal cell-to-cell connections that reduce intracellular gaps, and spermatids are well-developed and active with distinct cell outlines and mitochondria that are arranged normally, as shown in Fig. 4 (d1, d2, d3).
SIT reduced oxidative stress parameters
Testicular MDA content was raised considerably by a mean of 91.5 (95% CI: 73.5–109.6) in the PTX-intoxicated group compared to the control group; 50.5 (95% CI: 48.1–53.1), P < 0.05 (Fig. 5a). SIT 5- and SIT 10-treated groups revealed a remarkable reduction in MDA content 62.2(95% CI: 59.2–71.3) and 63.2(95% CI: 57–69.4), respectively, compared to the PTX group, P < 0.05 (Fig. 5a). Interestingly, there was no significant difference between the control and SIT5 and SIT10, reflecting that treatment with SIT diminished testicular MDA nearly to the control.
Fig. 5. Effects of SIT oxidative stress markers. (a) MDA, (b) GSH, (c) Catalase, (d) Sestrin2 gene expression, and (e) Sestrin2 content in PTX-intoxicated rats. The data are displayed as mean ± SEM (n = 5/group). PTX: paclitaxel, SIT: sitagliptin, MDA: malondialdehyde, GSH: reduced glutathione. ^a^ significant difference from control, P < 0.05, ^b^ significant difference from PTX, P < 0.05, ^c^ significant difference from SIT5, P < 0.05.
Figure 5b illustrates that the testicular GSH content was significantly reduced by a mean of 2.2 (95% CI: 1.4–2.9) in PTX-induced rats compared to the control group; 7.3 (95% CI: 6.5–8.1), P < 0.05. The SIT 5 and SIT 10 groups induced a notable elevation in GSH content, 3.95 (95% CI: 3.5 to 4.4) and 4.88 (95% CI: 4.1 to 5.7), respectively, compared to the PTX group (Fig. 5b, P < 0.05).
The current experiment revealed that PTX induced a significant decrease in catalase activity by a mean of 3.2 (95% CI: 2.6–3.8) compared to the control group; 6.2 (95% CI: 5.1–7.1), P < 0.05. SIT 5 and SIT 10 showed a more distinct rise in catalase activity, 4.8 (95% CI: 3.8–5.8) and 6.4 (95% CI: 5.7–7.1), respectively, compared to the PTX group (Fig. 5c), P < 0.05. There is a significant difference between SIT5 and SIT10, P < 0.05.
Figure 5d exhibited that the PTX group caused a noteworthy drop in testicular Sestrin2 gene expression by a mean of 0.31 (95% CI: 0.16–0.45), compared to the control group; 1 (95% CI: 0.88–1.12). While SIT 5 and SIT 10 revealed a marked increase in testicular Sestrin2 gene expression, 0.52 (95% CI: 0.49–0.55) and 0.74 (95% CI: 0.68–0.79), respectively, compared to the PTX group, P < 0.05. Interestingly, there is a significant difference between the two dose levels (P < 0.05).
PTX induced a significant decline in Sestrin2 levels by a mean of 0.86 (95% CI: 0.66–1.05) compared to the control group; 4.2 (95% CI: 3.96–4.47). Conversely, SIT 5 and SIT 10 significantly increased Sestrin2 content by a mean of 1.76 (95% CI: 1.4–2.1) and 2.96 (95% CI: 2.6–3.3), respectively, compared to the PTX group (Fig. 5e), P < 0.05. Interestingly, a significant difference was observed between the two dose levels (P < 0.05).
Impact of SIT on ER stress markers (pPERK and CHOP)
The current findings revealed that PTX provoked a marked elevation in pPERK content by a mean of 14.6 (95% CI: 13.4–15.8) compared to the control group; 1.53 (95% CI: 1.3–1.8), P < 0.05. While SIT 5 and SIT 10 significantly reduced testicular pPERK content 9.25 (95% CI: 8.64–9.86) and 6.8 (95% CI: 6.1–7.5), respectively, compared to the PTX group (Fig. 6a), P ˂ 0.05. Additionally, there is a significant difference between the two dose levels, P < 0.05.
Fig. 6. Impact of SIT on ER stress markers. (a) testicular pPERK and (b) testicular CHOP in PTX-induced testicular dysfunction. The data are displayed as mean ± SEM (n = 5/group). PTX; paclitaxel, SIT; sitagliptin, pPERK; Phosphorylated Protein Kinase-Like Endoplasmic Reticulum Kinase, and CHOP; C/EBP homologs protein. ^a^ significant difference from control, P < 0.05, ^b^ significant difference from PTX, P < 0.05, ^c^ significant difference from SIT5, P < 0.05.
Figure 6b demonstrated that the PTX group significantly increased testicular CHOP content by a mean of 14.5 (95% CI: 13.8–15) compared to the control group; 2.3 (95% CI: 2.0–2.6), P < 0.05. However, treatment with SIT 5 and SIT 10 powerfully decreased testicular CHOP content, to 9.9 (95% CI: 8.9–10.9) and 6.1 (95% CI: 5.6–6.6), respectively, compared to the PTX group, P < 0.05. The findings were more prominent in SIT 10 than in SIT 5, P ˂ 0.05.
Impact of SIT on inflammatory parameters
PTX boosted a distinct rise in NLRP3 gene expression by a mean of 5.5 (95% CI: 4.3–6.7), compared to the control group; 1.003 (95% CI: 0.8–1.2), P < 0.05. Conversely, SIT 5- and SIT 10-treated groups significantly reduced testicular NLRP3 expression levels; 3.65 (95% CI: 3.0–4.3) and 2.02 (95% CI: 1.16–2.87), respectively, compared to the PTX group (Fig. 7), p ˂ 0.05. There is a significant difference between the two dose levels, P < 0.05.
Fig. 7. Impacts of SIT on testicular gene expression of NLRP3 in PTX-induced testicular dysfunction. The data are displayed as mean ± SEM (n = 3/group). PTX: paclitaxel; SIT: sitagliptin; NLRP3: NOD-like receptor protein 3. ^a^ significant difference from control, P < 0.05, ^b^ significant difference from PTX, P < 0.05, ^c^ significant difference from SIT5, P < 0.05.
As demonstrated in Fig. 8, there is a diffuse expression of NLRP3 in the PTX-incited group by a mean of 17.04% (95% CI: 15.35–18.7) compared to the control group; 0.5% (95% CI: 0.07–0.94), P < 0.05. Contrary to this, the percentage was extensively downregulated 12.3% (95% CI: 11.4–13.2), 9.3% (95% CI: 7.8–10.8), and after SIT5 and SIT10 treatment, respectively, compared to the PTX group, P < 0.05. Interestingly, a significant difference was observed between the SIT5 and SIT10 groups, P < 0.05.
Fig. 8. Impact of SIT on immunohistochemical expression of NLRP3 in PTX-provoked testicular toxicity. (a) Testicular sections of the control group presented negative NLRP3 expression in spermatogenic cells. (b) Testicular slices of the PTX group presented strong positive NLRP3 expression in spermatogenic cells (arrowhead). (c) Testicular slices of the SIT5 group revealed a remarkable decrease in NLRP3 expression in spermatogenic cells (arrowhead). (d) Testicular sections of the SIT10 group presented a more pronounced abrogation in NLRP3 expression in spermatogenic cells (arrowhead). (e) % of NLRP3 immunoreactive area. The data displayed is mean ± SEM (n = 5/group). PTX: paclitaxel, SIT: sitagliptin, and NLRP3: NOD-like receptor protein 3. ^a^ significant difference from control, P < 0.05, ^b^ significant difference from PTX, P < 0.05, ^c^ significant difference from SIT5, P < 0.05.
The impact of SIT on PTX-provoked inflammation was also illustrated by immunohistochemical staining of IL-1β (Fig. 9). PTX-induced inflammation was verified by a remarkable elevation in the percent of IL-1β immunoreactive area by a mean of 37.6 (95% CI: 35.7–39.5), compared to the control group; 0.55% (95% CI: 0.25–0.85), p < 0.05. Conversely, the percentage was considerably mitigated to 17% (95% CI: 15.92–18) and 14% (95% CI: 12.3–15.6) through treatment with SIT5 and SIT10, respectively, compared to the PTX group, P < 0.05. There was a significant difference between the two dose levels, P < 0.05.
Fig. 9. Effects of SIT on immunohistochemical staining of IL-1β in PTX-induced testicular toxicity. (a) Testicular sections of the control group presented negative expression of IL-1β in spermatogenic cells. (b) Testicular sections of the PTX group demonstrated extensive positive expression of IL-1β in spermatogenic cells (arrowhead). (c) Testicular slices of the SIT5 group revealed a remarkable reduction in positive expression of IL-1β in spermatogenic cells (arrowhead). (d) Testicular slices of the SIT10 group presented a prominent decrease in immunopositive expression of IL-1β in spermatogenic cells (arrowhead). (e) % of IL-1β immunoreactive area. The data displayed is mean ± SEM (n = 5/group). PTX; paclitaxel, SIT; sitagliptin ^a^ significant difference from control, P < 0.05, ^b^ significant difference from PTX, P < 0.05, ^c^ significant difference from SIT5, P < 0.05.
Impact of SIT on apoptotic markers
As shown in Fig. 10, there was a pronounced elevation in protein expression of cyt.c in the PTX group by a mean of 39% (95% CI: 36.5–41.3), compared to the control group; 0.73% (95% CI: 0.57–0.89), p < 0.05. Conversely, treatment with SIT5 and SIT10 induced a remarkable decline in cyt.c immunoreactive area 13.2% (95% CI: 11.7–14.7) and 6.6% (95% CI: 5.48–7.66), respectively, compared to the PTX group, P < 0.05. There is a significant difference between the two dose levels, P < 0.05.
Fig. 10. Effect of SIT on immunohistochemical expression of cyt.c in PTX-provoked testicular toxicity. (a) Testicular slices from the control group revealed negative cyt.c c expression in spermatogenic cells. (b) Testicular slices of the PTX group displayed strong positive cyt.c expression in spermatogenic cells (arrowhead). (c) Testicular sections of the SIT5 group showed moderate positive cyt.c expression in spermatogenic cells (arrowhead). (d) Testicular sections of the SIT10 group presented mild cyt.c expression in spermatogenic cells (arrowhead). (e) % of Cyt.c immunoreactive area. The data are displayed as mean ± SEM (n = 5/group). PTX: paclitaxel, SIT: sitagliptin. ^a^ significant difference from control, P < 0.05, ^b^ significant difference from PTX, P < 0.05, ^c^ significant difference from SIT5, P < 0.05.
PTX induced a marked elevation in the percent of caspase-3 immunoreactive area by a mean of 8.4% (95% CI: 6.5–10.2), compared to the control group; 0.35% (95% CI: 0.08–0.61), p < 0.05. Inversely, the percentage was markedly reduced to 4.88% (95% CI: 4.1–5.7) and 2.4% (95% CI: 1.86-3.0) through treatment with SIT5 and SIT10, respectively, compared to the PTX group, P < 0.05. There is a significant difference between the SIT5 and SIT10, P < 0.05 (Fig. 11).
Fig. 11. Effects of SIT on immunohistochemical expression of caspase-3 in PTX-provoked testicular toxicity. (a) Testicular sections of the control group showed negative expression of caspase-3 in spermatogenic cells. (b) Testicular slices of the PTX group displayed extensive diffuse expression of caspase-3 in spermatogenic cells (arrowhead). (c) Testicular slices of the SIT5 group revealed remarkable inhibition in the expression of caspase-3 in spermatogenic cells (arrowhead). (d) Testicular slices of the SIT10 group presented a mild immunopositive expression of caspase-3 in spermatogenic cells (arrowhead). (e) % of caspase-3 immunoreactive area. The data displayed is mean ± SEM (n = 5/group). PTX: paclitaxel, SIT: sitagliptin. ^a^ significant difference from control, P < 0.05, ^b^ significant difference from PTX, P < 0.05, ^c^ significant difference from SIT5, P < 0.05.
As illustrated in Fig. 12, the PTX group showed a significant elevation in the percent of cleaved caspase-3 immunoreactive area by a mean of 10.32% (95% CI: 8.4–2.2) compared to the control group; 0.69% (95% CI: 0.45–0.93), p < 0.05. Even so, SIT5 and SIT10 caused a marked reduction in the percentage of the immunoreactive area, 7.2% (95% CI: 5.5–8.87) and 2.9% (95% CI: 2.12–3.68), respectively, compared to the PTX group (P < 0.05). Additionally, there is a significant difference between the two dose levels, P < 0.05.
Fig. 12. Effects of SIT on immunohistochemical expression of cleaved caspase-3 in PTX-provoked testicular damage. (a) Testicular sections of the control group showed negative expression of cleaved caspase-3 in spermatogenic cells. (b) Testicular sections of the PTX group displayed marked diffuse positive expression of cleaved caspase-3 in spermatogenic cells (arrowhead). (c) Testicular sections of the SIT5 group presented mild positive expression of cleaved caspase-3 in spermatogenic cells (arrowhead). (d) Testicular sections of the SIT10 group presented a mild immunopositive expression of cleaved caspase-3 in spermatogenic cells (arrowhead). (e) % of cleaved caspase3 immunoreactive area. The data displayed is mean ± SEM (n = 5/group). PTX: paclitaxel, SIT: sitagliptin. ^a^ significant difference from control, P < 0.05, ^b^ significant difference from PTX, P < 0.05, ^c^ significant difference from SIT5, P < 0.05.
Discussion
Male infertility and even malfunctioning of the male reproductive organs might result from long-term usage of anticancer medications.^35^ The current results showed that SIT reversed PTX-induced testicular damage by suppressing pathways linked to oxidative stress, ER stress, inflammatory mediator release, and apoptotic pathways.
This study highlights the distinctive potential of SIT over other protective agents such as melatonin, hesperidin, and propolis. Unlike these experimental compounds, SIT is an approved antidiabetic drug with a well-established safety and pharmacokinetic profile at clinically relevant doses (5 and 10 mg/kg). These translationally relevant doses support its potential repurposing to mitigate PTX-induced testicular toxicity, particularly in diabetic cancer patients, offering a practical and clinically feasible protective strategy during chemotherapy.
PTX administration caused a marked drop in serum testosterone levels, which aligns with Okkay et al. ^35^. SIT (5 and 10 mg/kg) significantly restored testosterone levels, with a better impact observed at the 10 mg/kg dose of SIT^36^.
PTX also caused a significant decrease in sperm count, motility, and viability, while significantly increasing the number of abnormal spermatozoa^37,38^ SIT treatment significantly increased sperm motility, count, and viability while significantly reducing sperm abnormalities compared to the PTX group. These results are supported by other studies that describe how SIT can mitigate the toxicity caused by busulfan and methotrexate on the reproductive system and semen quality^36,39^.
The histological and TEM results, as shown in PTX and SIT-treated testes, provided additional confirmation of these findings. ^35,40,41^
Sestrin2 is a potent antioxidant protein that can accelerate the clearance of ROS under oxidative stress. It has been documented that Sestrin2 overexpression alleviated ER stress and inhibited cell apoptosis^42–44^. Recent data have verified that Sestrin2-/- mice testes exhibit exaggerated damage, as indicated by the aggressive loss of germ cells and higher oxidative stress levels compared to their wild-type counterparts after heat stress. Notably, Sestrin2-/- mice exhibited a significant increase in heat-induced spermatocyte apoptosis compared to the control.^45^.
PTX induced marked oxidative stress, initially triggering a compensatory rise in Sestrin2 that was later depleted under sustained stress. This depletion coincided with increased testicular lipid peroxidation (elevated MDA) and reduced antioxidant defenses (GSH and catalase), suggesting that PTX-induced oxidative imbalance contributes to its toxicity in non-target organs such as the testis.^46,47^.
SIT administration led to a significant restoration in testicular Sestrin 2 levels, which in turn reduced PTX-induced alterations in testicular oxidant balance and caused a notable decline in MDA, while increasing GSH levels and catalase activity.
In this study, SIT enhanced Sestrin2 expression, implying its involvement in the protective mechanism. Whether this regulation is direct or secondary to SIT’s antioxidant and anti-inflammatory effects remains unclear. Further focused studies are needed to elucidate the precise link between SIT and Sestrin2 signaling.
There is growing evidence that ER stress is associated with male infertility and decreased testicular steroidogenesis^15,48^. The two main enzyme groups involved in steroidogenesis are hydroxysteroid dehydrogenases, such as 17βHSD, and cytochrome P450 (CYPs). These enzymes are located on the inner mitochondrial and endoplasmic reticulum membranes of Leydig cells. Numerous studies have demonstrated that the steroidogenic function of Leydig cells is compromised by various factors, including aging, hormonal disruptors, and chemotherapy.^49,50^ The current experimental study explores PTX-induced aggravation in ER stress through a sharp increase in pPERK (active form of PERK) and CHOP, which agrees with a different experimental model of Zakaria et al. ^51^. This ER stress-induced disruption in testicular steroidogenesis resulted in a sharp decline in testicular 17βHSD with a subsequent decrease in testosterone level.
The NLRP3 inflammasome, an intracellular multiprotein complex, can be activated by various endogenous hazard signals. Its excessive activation leads to a sustained inflammatory response. Recent evidence has highlighted the pivotal role of the NLRP3 pathway in testicular dysfunction. Uncontrolled oxidative and ER stress within testicular tissue has been shown to activate the NLRP3 inflammasome, resulting in the release of pro-inflammatory cytokines such as IL-1β and IL-18, which in turn impair testicular structure, sperm integrity, and function.^52–54^.
In the present study, PTX was found to establish a mechanistic link between ER stress and the activation of the NLRP3 inflammasome pathway, culminating in elevated IL-1β production. Sustained ER stress further amplified apoptotic signaling through the transcription factor CHOP, thereby promoting programmed cell death, as evidenced by the increased testicular expression of Cyt.c, caspase-3, and cleaved caspase-3. Collectively, these findings underscore the growing recognition of ER stress, NLRP3 activation, and apoptosis as interrelated contributors to PTX-induced testicular dysfunction.
Furthermore, it has been demonstrated that Sestrin2 has cytoprotective effects by abrogating endoplasmic reticulum stress^55,56^. Our study showed this through treatment with SIT, which resulted in a remarkable restoration of sestrin2, accompanied by pronounced abrogation of pPERK, CHOP, NLRP3, and apoptotic markers. In addition, SIT exhibited a notable increase in 17β-HSD protein, indicating its role in regulating the steroidogenesis and testosterone production process.
Conclusion
This is the first in vivo investigation exploring the beneficial effect of SIT on PTX-induced testicular dysfunction and spermatogenesis disruption in rats. These valuable acts were mostly achieved by abrogation of oxidative stress, ER stress, inflammation, and apoptosis by modulating the Sestrin2/PERK/CHOP/NLRP3 signaling pathway as elucidated in Fig. 13. These results collectively could open the opportunity for more research to look at SIT’s effectiveness in the clinical context, especially for patients who have both diabetes mellitus and testicular damage.
Fig. 13A diagram summarizing the therapeutic effects of SIT against PTX-induced testicular dysfunction. MDA: malondialdehyde; GSH: reduced glutathione; 17β-HSD: 17β-hydroxysteroid dehydrogenase; PERK: Protein kinase-like endoplasmic reticulum kinase; CHOP: C/EBP homologs protein. IL-1β: interleukin-1β. NLRP3; NOD-like receptor protein 3. ROS: reactive oxygen species.
Recommendation
Based on the current findings in normoglycemic rats, it is recommended that future studies be conducted in diabetic models to further explore the effects of PTX and SIT on testicular function. Moreover, the mechanistic pathways through which SIT regulates Sestrin2 expression should be further clarified. Finally, clinical studies are encouraged to confirm the safety, efficacy, and translational potential of SIT as a repurposed adjuvant therapy during chemotherapy.
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