Open Veterinary Journal, (2024), Vol. 14(1): 304–315

Original Research

10.5455/OVJ.2024.v14.i1.27

Ameliorative effect of Odontonema cuspidatum extract against testicular damage induced by sodium nitrite in rats

Nesma H. Elsawy^1*, Samir A. Elshazly¹, Azza M. Elkattawy¹, Nasr E. Nasr¹, Essam A. Almadaly², Mohamed S. Refaey³, Khaled A. Kahilo¹, Mona Assas⁴, Walied Abdo⁵, Aml S. hashem⁶, Tarek K. Abouzed¹ and Doaa Abdullah Dorghamm¹

¹Department of Biochemistry, Faculty of Veterinary Medicine, Kafrelsheikh University, Kafrelsheikh, Egypt

²Department of Theriogenology, Faculty of Veterinary Medicine, Kafrelsheikh University, Kafrelsheikh, Egypt

³Department of Chemistry, Faculty of Science, Menoufia University, Shebin El-Koom, Egypt

⁴Fish Processing and Biotechnology Department, Faculty of Aquatic and Fisheries Sciences, Kafrelsheikh University, Kafrelsheikh, Egypt

⁵Department of Veterinary Pathology, Faculty of Veterinary Medicine, Kafrelsheikh University, Kafrelsheikh, Egypt

⁶Department of Biochemistry, Faculty of Veterinary Medicine, Alexandria University, Alexandria, Egypt

*Corresponding Author: Nesma Hussein Elsawy. Department of Biochemistry, Faculty of Veterinary Medicine, Kafrelsheikh University, Kafrelsheikh, Egypt. Email: Nesma.Alsawy_a009 [at] vet.kfs.edu.eg

Submitted: 01/10/2023 Accepted: 15/12/2023 Published: 31/01/2024

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ABSTRACT

Background: Sodium nitrite (NaNO₂) is a chemical substance used to enhance taste, add color, and keep food products fit for consumption for a longer time. NaNO₂ gives rise to a negative adverse effect on male reproductive function. Odontonema cuspidatum (OC) is a natural plant that possesses antioxidant capacity.

Aim: Our research evaluates the potential beneficial effect of OC extract on the harmful effects caused by NaNO₂ on the testicular tissue and sperm characteristics of male rats.

Methods: Four groups with a total of forty rats: the control, the NaNO₂-received group, the OC-administered group, and the fourth group received both NaNO₂ and OC. All groups were administered daily for two months. Sperm characteristics, testicular antioxidant status, qRT-PCR, and histopathological changes were evaluated.

Results: Coadministration of NaNO₂ and OC, in comparison with NaNO₂ alone, contributed to a notable enhancement in acrosomal integrity, decreasing sperm abnormalities and restoring serum testosterone levels. Moreover, such coadministration reduced the oxidative stress marker, malondialdehyde (MDA), and increased superoxide dismutase (SOD) in testicular tissue, lowering TNF-α gene expression, and increasing the expression of P450scc and StAR genes. In addition, the NaNO₂ and OC combination decreased the testicular histopathological changes and the Caspase-3 and Proliferating cell nuclear antigen (PCNA) immunoexpression in seminiferous tubules compared with the NaNO₂ group.

Conclusion: The extract of OC exhibited the ability to decrease oxidative stress and ameliorate the detrimental effects caused by NaNO₂.

Keywords: NaNO₂, Odontonema cuspidatum, Food additives, Testicular damage.

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Introduction

Food additives include coloring agents, flavoring agents, emulsifiers, stabilizers, and preservatives that can be either natural or synthetic (Khodjaeva et al., 2013). Currently, manufactured food additives have progressively overtaken natural food additives, and there are numerous harmful effects of food additives, such as the abuse of chemicals, their overuse, or even their toxicity.

Food additives undoubtedly provide consumers with a lot of flavor satisfaction; however, they could additionally pose health hazards to humans. Therefore, it is extremely important to limit the consumption of food additives (Wu et al., 2022).

NaNO₂ is a widely used food additive found in various processed meat as well as fish products (JørgenandMarianne, 2011). It is generally known that NaNO₂ is frequently employed in additives to enhance taste and add color (Wang et al., 2022).

In addition, various medical processes can be controlled through NaNO₂ (Deng, 2006). It has therapeutic uses for medical problems such as heart attack and bleeding stroke (Pluta et al., 2005). Earlier studies have shown that sodium nitrite (NaNO₂) prevents the presence of dangerous bacteria, particularly spores of Clostridium botulinum (Difonzo et al., 2022). Conversely, receiving too much NaNO₂ has a harmful effect by generating free radicals, which upset the equilibrium between oxidants and antioxidants and have deleterious biological effects (Jensen, 2007; Naik et al., 2006).

The uncontrolled use of NaNO₂ has been implicated in several adverse health effects (Ansari et al., 2017). Notably, excess NaNO₂ disrupts reproductive hormones (Pavlova et al., 2017), and raises inflammatory cytokines in the testis (Alyoussef and Al-Gayyar, 2016).

This prompted our investigation into the impact of NaNO₂ on rat spermatogenesis. The incidence of cancer in adults has been linked to NaNO₂ exposure, and the occurrence of childhood nasopharyngeal cancers, blood cancer, and neurological tumors has also been linked to it (Kozisek, 2007).

Recent years have seen a rise in popularity and widespread acceptance of the use of herbal medicines in the treatment of numerous disorders and dysfunctions (Oyagbemi and Odetola, 2010). The use of herbal remedies to treat illnesses has persisted for many generations (Chaughule and Barve, 2023). They are popular due to their low cost, cultural acceptance, and lack of drawbacks (Chaughule and Barve, 2023).

One of these herbal plants is Odontonema cuspidatum (OC), which belongs to the Acanthaceae family. Its leaf extract contains flavonoids, saponins, glycosides, tannins, steroids, and terpenoids (Pierre and Moses, 2015), so it has antioxidant activity against lipid peroxidation due to their high flavonoid content (Refaey et al., 2015).

Our research evaluates the potential beneficial impact of OC extract against testicular damage induced by NaNO₂.

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Materials and Methods

Chemicals

NaNO₂ was obtained from Merck KGaA (Darmstadt, Germany), with a purity of 96% (CAT NO. 7632-00-0). Absolute ethanol was purchased from Merck KGaA (Darmstadt, Germany). The formaldehyde solution was provided by PIOCHEM Co (Giza, Egypt), and chloroform was purchased from Fisher Scientific UK Ltd. (Leicestershire, UK). Glutaraldehyde (25% aqueous solution) was obtained from Oxford Lab Chem Maharashtra, India. The real-time SYBR Green Master Mix acquired by Applied Biotechnology Co. Macrogen Company (Korea) produced the PCR primers; the Primers 3 Program was used to build these sequences. Phosphate buffer (PBS) was purchased from Schwabach, Germany.

Kits

Serum sex hormones were assayed by the enzyme-linked immunosorbent assay (ELISA). Testosterone kits (Catalogue No. 201-11-5126), follicle-stimulating hormone (FSH) kits (Catalogue No. 201-11-0183), and luteinizing hormone (LH) kits were acquired from Sun Red Biotechnology Company, (Shanghai, China) (Catalogue No. 201-11-2119). Kits of malondialdehyde (MDA) (MD 25 29) and SOD (SD 25 21) were obtained from Biodiagnostics Company, (Cairo, Egypt). Enzynomics (Daejeon, Republic of Korea) provided the cDNA synthesis kit. Macrogen (Seoul, Republic of Korea) performed the synthesis of primers for β-actin, TNF-α, StAR, and p450scc.QIAGEN Company., (Maryland, USA) supplied the Qiazol lysis reagent. LobaChemie (Mumbai, India) provided the PBS.

Plant material

In October 2021, fresh aerial parts of OC were gathered from a private garden in Menoufia, Egypt. Therese Labib, the El Orman head (Botanical Garden), verified the plant material of O. cuspidatum.

Extraction procedure

To obtain the extract, methanol was macerated with 250 g of freshly powdered dried O. cuspidatum’s aerial portions at room temperature until complete exhaustion (1 l × 3). Afterward, low pressure was used to concentrate the methanolic extract at 40°C, the resulting yellowish–green viscous residues weighed 302 g. The methanolic extract was then dried and preserved at 4°C for use in in vivo studies.

Animals

The Animal House at the Veterinary Medicine Faculty, University of Kafr Elsheikh (Egypt) provided 40 adult male Wistar albino rats weighing between 120 and 140 g. These rats were housed in adequately ventilated metal cages with wood shavings bedding under regular hygienic conditions. They were provided with water ad libitum and a basal diet from Dakahlia Company, Egypt. Animals were exposed to a cycle of light and dark with a duration of 12 hours each and maintained at 20°C–25°C. Before starting the experiment, the animals were given two weeks to adapt to their new environment.

Design of the experiment

Four groups with a total of forty rats, every group consisted of 10 rats. All groups were given their respective doses orally via gastric tubes daily for 2 months. Control: This group received only half a milliliter of distilled H₂O (DW). NaNO₂: They were orally given 80 mg/kg of NaNO₂ soluble in DW (Akhzari et al., 2019).

OC: They were given 100 mg of OC extract dissolved in a 1% carboxymethylcellulose solution (Kasali et al., 2022).

NaNO₂ + OC: They simultaneously received 80 mg/kg NaNO₂ and 100 mg/kg OC extract.

Blood sampling

The rats were administered intramuscular anesthesia using xylazine (10 mg/kg) with ketamine (125 mg/kg) in a combination (Veilleux-Lemieux et al., 2013). Heparinized capillary tubes were used to collect blood from the eye’s medial canthus. We allowed the blood to coagulate, then centrifuged for 10 minutes at 3,500 rpm, collected serum, transferred it into Eppendorf tubes, then remained at −20°C up till measurement of the sex hormones.

Tissue sampling and homogenization

For euthanasia, the rats were decapitated, their testes and epididymis were taken, and the right testis was preserved for histopathological investigation in a 10% neutral buffered formalin solution. The left testis was divided into three parts. One part, together with the adjacent epididymis, was used to assess sperm parameters. The second part was rapidly frozen at −80°C to extract RNA for gene expression examination related to testicular function, whereas the third portion was preserved at −20°C to determine oxidative stress markers, 10% homogenate was prepared by adding 0.01 M PBS with pH 7.4, to this portion. Then, the mixture was subjected to homogenization at 12,000 rpm on ice. After homogenization, the tissue’s homogenate was centrifuged at 3,000 rpm for ten minutes at 4°C.

Assessment of body weight and testicular weight

Each rat’s final body weight in each group was measured. In addition, the testes of the rats were removed and weighed after euthanasia by decapitation.

Assessment of gonadosomatic index (GSI)

GSI=(Mean weight of the combined testes/Final body weight) x 100 (Mostafa-Hedeab et al., 2023).

Sperm characteristics

For the examination of sperm characteristics, the rats were euthanized by decapitation. A sterile scalpel blade was used to make a mid-caudoventral abdominal incision for post-mortem examination. Immediately after euthanasia, the testes were dissected and weighed along with the epididymis using a sensitive electronic weighing machine. The testes and epididymis were then sliced to collect sperm cells in freshly prepared warm 2.9% sodium citrate dihydrate solution.

Sperm motility

Sperm motility was evaluated using the method described (Aly and Azhar, 2013). A small amount of sperm mixture was put on a warm glass slide, and a cover slip measuring 18 × 18 mm was placed over it. The slide was examined using phase-contrast (400×) microscopy. The motility included two types: progressive (movement in straight lines or large circles) and total motility (all forms of sperm movement, including straight, circular, and oscillatory).

Plasma membrane integrity

The HOS-test assay was performed. This involved mixing the sperm mixture (100 μl) and hypoosmotic solution (1,000 μl) warmed to the appropriate temperature. To make the hypoosmotic solution, sodium citrate dihydrate (7.35 g) and fructose (1.351 g) were dissolved in 100 ml of DW, resulting in an osmolarity of 150 mOsm/L. Then, the sperm mixture was placed in an incubator at 37°C for 45 min. Subsequently, the sperm suspension (2 μl) was added to a glass slide with a cover slip. To view these slides, we utilized phase-contrast microscopy with a magnification power of 400×. The sperms showing swollen or curled tails were considered intact, while those lacking this feature were considered damaged. To ensure accurate results, any sperm cells exhibiting tail abnormalities were carefully identified to obtain precise HOS test-positive spermatozoa (Almadaly et al., 2016).

Acrosomal integrity

The retrieved sperm cells were fixed using a solution containing 2% Glutaraldehyde in Sodium Cacodylate buffer with a pH of 7.3 (Almadaly et al., 2012). Fifty microliters of Glutaraldehyde were mixed with 50 μl of the sperm suspension, followed by an incubation period of 30–60 minutes. After incubation, phase-contrast microscopy was utilized to examine a drop of 2 μl of the sperm suspension at a magnification power of 100×. Sperm cells showing a consistent and uniform apical edge were considered to have intact acrosomes, while those with a muffled or uneven apical edge were classified as having damaged acrosomes.

Sperm viability and morphology

The eosin–nigrosin stain evaluated the spermatozoa’s viability and morphology. After mixing an equal amount of sperm mixture and the dye, spread it onto a clean glass slide, and then allow it to dry on a warming plate. During the examination, sperm cells with unstained sperm heads were regarded as viable, while nonviable sperm showed the opposite (Talebi et al., 2007). Abnormal sperm cells were assayed using the stained smear under an oil immersion lens (Mortimer and Mortimer, 1992).

Assessment of testicular MDA level and SOD activity

Kits of MDA (MD 25 29) and SOD (SD 25 21) were obtained from Biodiagnostics Company (Cairo, Egypt). The colorimetric method is used to measure testicular MDA level and SOD activity according to Ohkawa et al. (1979) and Nishikimi (1975), respectively.

Hormonal analysis

Rat ELISA kits were used to measure Serum sex hormones, following their instructions. All hormonal kits were acquired from Sun Red Biotechnology Company (Shanghai, China).

Real-time polymerase chain reaction (RT-PCR)

RNA extraction

The testicular samples’ total RNA was extracted in all experimental groups using the QIAzol reagent. After dissolving the extracted RNA pellet in RNase-free water, the extracted RNA’s purity and concentration were estimated at absorbance at 260/280 nanometers by a NanoDrop 2000 spectrophotometer (Biochrom Ltd, Cambridge CB23 6DW, UK). The RNA’s integrity and purity have been examined by running the samples on a 2% agarose gel and analyzing the resulting bands. Only RNA samples that met high-quality criteria, including an absorbance ratio of 260/280 in the range of 1.8 and 2.0.

cDNA synthesis

The RNA was reverse transcribed using the TOPScriptTM RT cDNA synthesis kit. According to their guidelines, mix total RNA (1 μg) with 10 μl of 2X RT reaction Solution and enzymes (1 μl), then add RNase-free water to the final volume of 20 μl. Incubation of these combinations for 30 minutes at 50°C, followed by 10 minutes at 85°C was preceded.

Quantitative real-time polymerase chain reaction (qRT-PCR)

The protocol of qRT-PCR included heating at 92°C for 10 min to denature the samples followed by amplification for 40 cycles, where the samples underwent annealing at 55°C and extension at 72°C. To detect relative gene expression, we utilized the 2−ΔΔCT technique, which involved normalizing the results to the expression levels of β-actin. Primers for the targeted genes are described in Table 1.

Histopathology

Ten percent neutral buffered formalin was used to fix the testicular tissue and underwent the following standard paraffin embedding procedures for tissue preparation. After fixation, the tissues were subjected to a process of dehydration using a series of ethyl alcohol concentrations that gradually increased. Then, tissues were cleared with xylene, subsequently embedded in paraffin wax, after that was sliced into sections of 5 μm thickness, and subsequently subjected to staining using the hematoxylin and eosin (H&E) stain (Bancroft et al., 2013). The light microscope (Leica DFC295) was used to examine the tissue sections to evaluate any histopathological alterations in the testes among all experimental groups. We used the well-reproducible criteria formed by Johnsen (1970). For quantifying spermatogenesis, the Johnsen criteria scored using a ten-point system, Johnsen scores vary from 1 to 10, with 1 referring to no germ cells and 10 referring to the highest spermatogenesis activity.

Immunohistochemistry

The immunohistochemical staining procedures are detailed in the study conducted (Khalil et al., 2020) which consisted of several sequential steps. The first step involved dewaxing the sections before their exposure to 0.05 M citrate buffer (pH 6.8), which was necessary for antigen retrieval. The next step involved treating the sections with protein block and 0.3% H₂O₂ to mitigate nonspecific binding. Afterward, the sections underwent incubation with anti-NF-ĸB P65 (Santa Cruz, Cat# (F-6): sc-8008), diluted 1/100, caspase 3 antibodies (Invitrogen, Cat# PA5-77887), diluted 1/100, in addition to PCNA Polyclonal rabbit Dako, USA, PA5-32541 at a dilution of 1/100. After rinsing the tissue sections with phosphate-buffered saline, they were exposed to a goat anti-rabbit secondary antibody (Cat# K4003, Envision+™ System Horseradish Peroxidase Labeled Polymer; Dako) for 30 minutes at ambient temperature to detect polyclonal antibodies. Next, we used Mayer’s hematoxylin and the DAB kit to observe the sections. To represent the proportion of cells that exhibited positive results for the antibodies, staining labeling indices were determined out of 1,000 testicular spermatogenic cells.

Statistical analysis

Statistical analysis was done using GraphPad Prism version 9.5 (GraphPad Software Inc., San Diego, USA. The variance groups were identified by performing a one-way analysis of variance (ANOVA) and applying Tukey’s test. p < 0.05 was employed to determine the level of statistical significance. To facilitate the interpretation and evaluation of the results, quantitative variables, such as the mean ± standard error of the mean (SEM), are employed for representation.

Ethical approval

The Institutional Animal Care and Use Committee’s (IACUC) guidelines at the Veterinary Medicine Faculty, Kafr Elsheikh, Egypt, were followed with license number KFS-IACUC/134/2023.

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Results

The weight of the body and testicles in addition to the gonad somatic index

The final body weight was significantly reduced (p < 0.05) in the NaNO₂ group compared with the control group. NaNO₂ significantly increased the testis’s weight as well as the gonad somatic index (p < 0.05). The OC+ NaNO₂ group significantly reduced testicular weights and gonad somatic index and increased body weight returning them near control values (p < 0.05) (Table 2).

Table 1. Primers and reaction parameters.

Abbreviations: β-actin: Beta-actin; TNF-α: Tumor necrosis factor alpha; StAR: Steroidogenic acute regulatory protein; P450scc: Cytochrome P450 Family 11 Subfamily A, Member 1.

Sperm characteristics

Our findings listed in Table 3, showed that compared to the control group, the total and progressive motility of sperm were significantly reduced (p < 0.05) when exposed to NaNO₂ . It also had a significant negative impact on the sperm’s plasma membrane integrity and acrosomal integrity, as well as its viability, in addition, to significantly increasing abnormal sperm morphology. The OC showed a noticeable rise in sperm’s plasma membrane integrity as well as a significant decline in sperm abnormal morphology in contrast to the NaNO₂ at (p < 0.05). NaNO₂ + OC revealed notable amelioration in total motility, progressive motility, plasma membrane integrity, acrosomal integrity, and viability, in addition to a substantial decrease in abnormal morphology as contrasted with the NaNO₂ group (p < 0.05).

Table 2. The weight of the body and testicles, in addition to the gonad somatic index.

Values are presented as the Mean ± SEM. Different superscripts denote significant differences between groups at p < 0.05. OC: Odontonema cuspidatum extract.

Oxidative stress markers

In Table 4, NaNO₂ resulted in a significant elevation in MDA levels and a notable decrease in SOD activity compared to both the control and OC group, our findings of NaNO₂ were reversed in the NaNO₂ + OC group at (p < 0.05).

Hormonal analysis

In Table 5, serum testosterone was significantly decreased in response to NaNO₂ toxicity in contrast to the control; this impact was significantly neutralized in the NaNO₂ + OC group. NaNO₂-intoxicated rats’ group exhibited a notable increase in serum FSH and LH levels, these increases were restored with concurrent administration of NaNO₂ + OC. All results of hormonal analysis were significantly different at p < 0.05.

TNF-α, StAR, and cytochrome P450scc levels in the testicular tissue

As shown in Figure 1, all results of the gene expression analysis were significantly different at (p < 0.05). There was a significant increase in TNF-α levels and a significant decrease in P450scc (cholesterol side-chain cleavage) and StAR (Steroidogenic Acute Regulatory protein) levels in the NaNO₂ group compared to all other groups.

Table 3. The proportions of sperm characteristics of the six groups.

Values are expressed as Mean ± SEM. Different superscripts denote significant differences between groups at p < 0.05. OC: Odontonema cuspidatum extract.

Table 4. Oxidative stress markers in the experimental groups.

Values are expressed as Mean ± SEM. Different superscripts denote significant differences between groups at p < 0.05. Abbreviations: SOD, Superoxide dismutase; MDA, malondialdehyde; OC, Odontonema cuspidatum extract.

Table 5. The hormonal Analysis in the experimental groups.

Values are expressed as Mean ± SEM (standard error of the mean). different superscripts denote significant differences between groups at p < 0.05. Abbreviations: FSH, follicle-stimulating hormone; LH, luteinizing hormone; OC, Odontonema cuspidatum extract.

Fig. 1. Ameliorative impact OC extracts on the gene expression levels of TNF-±, StAR, and Cytochrome P450) in the testicular tissue on disturbance induced by NaNO₂ toxicity. The graphs represent the mean fold change ± SEM. *Significant difference as compared with the control rat groups at p < 0.001, # significant difference as compared with NaNO₂ rat group at p < 0.001. (A) (TNF-±), Tumor necrosis factor-alpha. (B) (StAR), Steroidogenic acute regulatory protein. (C) (P450scc), Cytochrome P450 Family 11 Subfamily A. (OC), Odontonema cuspidatum extract.

The group treated with NaNO₂ + OC exhibited significantly lower TNF-α levels and a significantly greater elevation in P450scc and StAR levels in contrast to NaNO₂.

Histopathological findings

The lesions of the testicular tissues are illustrated in Figure 2. The control group showed normal spermatogenic cell layers, the first layer is the primary spermatogenic cells followed by the secondary, tertiary, and spermatid layers. There was an obvious spermatogenesis associated with normal sperms within the seminiferous tubule lumen. NaNO₂ caused marked testicular degeneration extended along the spermatogenic layers. The spermatogenic cells within the different layers showed nuclear pyknosis, atrophy, and loss of adhesion between each other. In addition to severe edema associated with Leydig’s cell hyperplasia in the interstitial tissue, in the OC group, the testicular section had normal seminiferous tubules and a noticeable increase in spermatogenesis, accompanied by an increased number of sperm within the lumen of the seminiferous tubule. The testicular section of the NaNO₂ + OC group showed a marked decrease in degenerative changes compared to NaNO_2, mild nuclear pyknosis, mild edema in the interstitial tissue, and slight Leydig’s cell hyperplasia compared to the NaNO₂ group_. According to Johnsen’s score, the control and OC showed higher scores within the examined groups (p < 0.001), while the NaNO₂ showed a notable decrease in Johnsen’s score. At (p < 0.001), the NaNO₂ + OC revealed a significantly higher Johnsen’s score as contrasted with NaNO₂ (p < 0.001).

Fig. 2. Histopathological findings of the experimental groups. In the control group, the arrowhead and arrow indicate sperm within the lumen. In the NaNO₂ group, the white arrowhead reveals atrophic degenerated spermatogenic cells, the arrow indicates a spermatid layer, black arrowhead indicates interstitial edema. In the OC group, the white arrowhead reveals normal spermatogenic cells, the arrow indicates a spermatid layer. In the NaNO₂ + OC group, the white arrowhead indicates mild nuclear pyknosis of spermatogenic cells, the arrow indicates the presence of sperms within the lumen of the seminiferous tubule, and the black arrowhead shows mild to moderate interstitial edema. Scale bar=50 μm. The graph of Johnsen’s score represents the mean ± SEM. * Significant difference as compared with the control rat groups at p < 0.001, # Significant difference as compared with NaNO₂ rat group at p < 0.001. OC, Odontonema cuspidatum extract.

Fig. 3. The testicular sections immunostained for NF-κB p65 in the experimental groups. The arrow indicates a positive expression that markedly increases in the NaNO₂ group and decreases in the NaNO2 + OC group, the scale bar is set at 50 μm. The graph demonstrates the mean NF-κB p65 immunopositive cells (%) in each group represented in bars and the SEM represented in the T-shaped lines over the bars. * Significant difference as compared with the control rat groups at p < 0.001, # Significant difference as compared with NaNO₂ rat group at p < 0.001. Abbreviations: NF-κB p65, the nuclear factor kappa B; OC, Odontonema cuspidatum extract.

Immunohistochemical findings The testicular sections immunostained for NF-κB p65 were illustrated in Figure 3.

The control and OC groups showed slight NF-κB p65 immunostaining within the spermatogenic cells (p < 0.001). The NaNO₂ group showed marked NF-κB p65 immunostaining within the cytoplasm and nucleus of the spermatogenic cells (p < 0.001). The NaNO₂ + OC group showed a notable reduction in NF-κB p65 expression within the spermatogenic cells (p < 0.001).

In Figure 4, caspase-3 immunoexpression within the testicular tissue of the control and OC exhibited a slight expression in the spermatogenic cells’ cytoplasm, while NaNO₂ revealed an obvious nuclear and cytoplasmic expression of the seminiferous tubule’s germinal epithelium (p < 0.001). There was a remarkable decrease in caspase-3 immunoexpression within seminiferous tubules in the NaNO₂ + OC group (p < 0.001).

The testicular sections that were immunostained for PCNA are illustrated in Figure 5. There was a marked nuclear immunostaining within the spermatogenic lining epithelium of the control and OC groups (p < 0.001). NaNO₂ showed a marked decrease in PCNA immunostaining, which was only noticed within the primary spermatogonia (p < 0.001). The NaNO₂+ OC group showed a remarkable increase in PCNA antibodies within the germinal epithelium (p < 0.001).

Fig. 4. The testicular sections are immunostained for caspase-3 in the experimental groups. The arrow indicates mild caspase 3 expression in the Control and OC groups. The arrow in the NaNO₂ group indicates marked caspase 3 immunostaining within spermatogenic cells. NaNO₂ + OC group is showing a decrease in caspase 3 expression indicated by arrows. Scale bar=50 μm. The graph demonstrates the mean caspase-3immunopositive cells (%) in each group represented in bars and the SEM represented in the T-shaped lines over the bars. * Significant difference as compared with the control rat groups at p < 0.001, # Significant difference as compared with NaNO₂ rat group at p < 0.001. OC: Odontonema cuspidatum extract.

Fig. 5. The testicular sections are immunostained for PCNA in the experimental groups. The arrow indicates nuclear positive expression of PCNA antibody, scale bar=50 μm. The graph demonstrates the mean PCNA immunopositive cells (%) in each group represented in bars and the SEM represented in the T-shaped lines over the bars. * Significant difference as compared with the control rat groups at p < 0.001, # Significant difference as compared with NaNO₂ rat group at p < 0.001. Abbreviations: PCNA, Proliferating cell nuclear antigen; OC, Odontonema cuspidatum extract.

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Discussion

NaNO₂ is a primary preservative found in many foods, responsible for the red color of meat (Sindelar and Milkowski, 2012), and hinders the growth of Clostridium botulinum (Difonzo et al., 2022). However, it reacts with amines in the stomach resulting in the formation of nitrosamines that have harmful effects on body organs and have been linked to several types of human tumors (Hassan and Yousef, 2010).

The flavonoids of OC have an ameliorative impact against NaNO₂ toxicity due to their antioxidant activity; these flavonoids exert their activity as antioxidants through two mechanisms: First, the “direct mechanism” is scavenging and reduction to ROS, where the phenolic components of the flavonoid molecule transfer an electron or a hydrogen atom (Alov et al., 2015).

Second, the “indirect method” involves their phenolic moieties’ oxidation, these oxidative compounds interact with specific proteins rather than ROS directly to increase the endogenous antioxidant capacity of cells (Erlank et al., 2011; Dinkova-Kostova and Talalay, 2008); therefore, the flavonoid molecule itself does not initiate the antioxidant effect; rather, a metabolite that comes from the oxidation of the flavonoid molecule does (Bolton and Dunlap, 2017).

Verbascoside was also isolated from the same plant parts (Refaey et al., 2017), it inhibits lipid peroxidation (Liu, 2003) due to the binding with membrane phospholipids despite being hydrophilic (Funes et al., 2010). In addition, it also prevents oxidation of low-density lipoproteins (Cardinali et al., 2012), and demonstrated activity in reducing inflammation (Paola et al., 2011).

Our findings indicated that administering NaNO₂ led to a notable reduction in final body weights which aligns with the prior research (Akintunde et al., 2014) that reported an age-related rise in nitrite production. According to our results, rats’ testicular weight and gonadosomatic index were both increased by NaNO₂, and this was also consistent with Alyoussef and Al-Gayyar (2016), who reported that it could be caused by testicular anoxia or enhanced gonadotrophin due to a decrease in testosterone’s inhibiting effects.

The findings of the NaNO₂ group revealed a noticeable decline in sperm motility, integrity of both the plasma membrane and acrosome, and sperm viability, as well as a notable rise in abnormal sperm morphology, this might be explained by greater oxidative stress in this group since sperm’s plasma membrane is composed of a large quantity of polyunsaturated fatty acids (Bansal and Bilaspuri, 2011) and scavenger enzymes are present in their cytoplasm in little amounts (Saleh and HCLD, 2002; Sharma and Agarwal, 1996), the link between ROS and reduced motility might result from the depletion of the intracellular ATP and damaged axonemal tissue and immobilizes sperm (Bansal and Bilaspuri, 2011).

Our research also revealed that administering NaNO₂ reduced testosterone levels, indicating a disruption in steroidogenesis, this may be attributed to lower testicular StAR gene expression, proven in our gene expression analysis, which could have a role in inhibiting the synthesis of testosterone (Mosbah et al., 2018), cholesterol is transmitted to the inner mitochondrial membrane by the StAR, where it is converted by P450scc into pregnenolone, which is then converted into testosterone (Miller, 2017).

We showed a marked increase in FSH and LH concentrations in the NaNO₂ group in contrast to the control, inadequate feedback inhibition by testosterone can be used to explain this rise in gonadotropins (Alyoussef and Al-Gayyar, 2016). On the other hand, prior studies were inconsistent with our work using different doses (Darbandi et al., 2018; Adelakun et al., 2019), body steroid levels are disturbed dose dependently by NaNO₂ (Kuzhiumparambil and Fu, 2013). Treatment with OC in the NaNO₂ + OC group significantly improved these bad impacts by getting the rats’ serum back to its regular hormonal balance.

Administration of OC in the co-treated group significantly alleviated the impact of NaNO₂ on sperm characteristics due to the antioxidant activity of OC, concurrent with a marked decline in MDA and an increase in SOD activity.

The resulting oxidative stress is confirmed by the rise in MDA concentration, a marker for lipid peroxidation, and a distinct decrease in SOD activity in our biochemical findings (Table 5), which concurs with the previously discussed findings (Adelakun et al., 2019), which could be because of a concurrent rise in the production of free radicals such as H₂O₂ and OH in the NaNO₂ group’s testes.

We expanded our research to identify what’s behind the rise in oxidative stress; our results revealed that the NaNO₂ group significantly upregulated the expressions of the TNFα gene (pro-inflammatory cytokine), and reduced StAR and P450scc genetic expressions, the increase in pro-inflammatory cytokine levels can be due to cell peroxidation by NaNO₂.

This elevated TNFα level in the NaNO₂ group damages spermatogenesis. NaNO₂ + OC demonstrated a substantial drop in TNFα, confirming OC’s anti-inflammatory properties. The flavonoid part of OC also inhibits pro-inflammatory enzymes such as cyclooxygenase-2 and lipoxygenase and activates mitogen-activated protein kinase and protein kinase C (Santangelo et al., 2007). These effects were reversed when OC was administered in combination with NaNO₂.

The histopathological results of the NaNO₂ group revealed interstitial edema in the testes, leading to increased testicular weight, which is induced by tissue anoxia and hemic ischemia, accompanied by atrophy to germinal epithelium and arrest of spermatogenesis. The co-treatment in the OC+ NaNO₂ group decreased testicular edema and so significantly decreased testicular weights in addition to lowering the gonadosomatic index, returning them to near-average weights similar to the control group. In addition, our histological findings indicate the presence of Leydig’s cell hyperplasia, this hyperplasia is only to compensate for the decreased testosterone (Mylchreest et al., 2002), the decline in testosterone concentration is not reversed, it is the main androgenic hormone which is essential for secondary sexual characteristics, spermatogenesis, and sperm cell maturation, producing substantial fertility problems can be caused by a testosterone synthesis (Mohamed et al., 2017).

NF-ĸB P65 controls the gene transcription of inflammatory mediators and is essential for controlling inflammation (Ren et al., 2019), NF-ĸB P65 presents in the cytoplasm of healthy cells in the form of dimer comprising the protein subunits p50/p65 and its inhibitor (IkB), nevertheless, if the cell is exposed to oxidative damage, such as NaNO₂, this dimerization is disrupted, and IkB degraded in the proteasome (Doshi et al., 2012).

IkB degradation enables NF-ĸB P65 to enter quickly the nucleus and then attach to DNA to control the inflammation (Zhang and Sun, 2015), including TNFα (Zhao et al., 2017). We observed marked immunostaining of NF-ĸB P65 within the cytoplasm and nucleus of spermatogenic cells that indicated testicular inflammation which was reversed in the NaNO₂ + OC treated group.

Caspase-3 immunostaining in the testicular tissues that received NaNO₂ is notably increased and is regarded as the apoptosis’ last executor (Kumar et al., 2020). In considering this, it is crucial to remember that the increased oxidative stress induced by NaNO₂ causes DNA damage, ultimately resulting in apoptosis (Sherif and Al-Gayyar, 2013).

The equilibrium between germ cell proliferation, survival, and apoptosis is known to be necessary for successful spermatogenesis, and disruptions in the control of any of these may cause spermatogenesis to fail (Tan et al., 2007; Richburg, 2000).

PCNA is expressed in proliferating spermatogonia and spermatocytes and is an indicator of cellular proliferation (Tousson et al., 2011). As so, immunocytochemical analysis of PCNA is a useful indicator for assessing testicular germ proliferation. We observed a significant reduction in PCNA immunostaining expression in the NaNO2 group’s testes, while the NaNO₂ + OC group revealed an increase in PCNA in addition to lowering caspase immunoreactive cells.

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Conclusion

Our study shows evidence that OC extract ameliorates testicular damage induced by NaNO₂ by preventing oxidative stress, improving sperm parameters, and returning the proper balance of inflammatory cytokines.

Author contributions

Nesma H. Elsawy: writing, interpretation. Samir A. Elshazly and Azza M. Elkattawy: experiment design. Khaled A. Kahilo and Nasr E. Nasr: revision, editing. Mohamed S. Refaey: extract preparation. Mona Assas and Aml S. hashem: writing the manuscript. Essam A Almadaly: methodology, editing. Walied Abdo: histopathology, immunohistochemistry. Doaa Abdullah Dorghamm and Tarek K. Abouzed: data analysis. The completed version of this work was approved by all contributors.

Conflict of interest

All authors declare that there is no conflict of interest.

Funding

This work did not get any funding.

Data availability

All data are provided in the manuscript.

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