Protective Effect of Propolis and Vitamin E on Testicular Dysfunction in Male Rats Induced by Cadmium Chloride

Introduction
Testicular dysfunction in male rats can manifest in various ways and may be caused by genetic, environmental, hormonal, and nutritional factors. Testicular dysfunction often refers to issues that affect the proper functioning of the testes, leading to impaired fertility or disruptions in reproductive health. Some potential causes and aspects of testicular dysfunction were observed in male rats (hormonal imbalances, genetic factors, nutritional deficiencies, and toxic exposures) (Agarwal et al., 2020; Usende et al., 2022; Sartorius & Handelsman, 2023).
Cadmium is divided into organic and inorganic compounds; it also belongs to the toxic substance group, along with lead, mercury, arsenic, and selenium. These substances are usually studied due to their use in a wide spectrum of sectors, especially agriculture, gas, and steelmaking. Vital applications include the discharge of cadmium waste into the environment and the consumption of food containing this element. With an increase in the amount of cadmium accumulated in the human body, the number of cases of poisoning due to this element also increases. Cadmium enters the body via the digestive or respiratory system (Bhattacharyya et al., 2023).
Cadmium usually accumulates in tissues, including the testes. Due to the long half-life of cadmium and its low excretion rate, it can cause a toxic effect that manifests in various ways, such as apoptosis, oxidative stress, and autographic cell death. Cadmium can alter Sertoli cells, which are crucial in the spermatogenesis process, and provide a barrier to the testis by damaging the cytoskeleton, leading to deformities in the structure of Sertoli cells, allowing the immune system to enter the testis and cause various problems, such as germ cell apoptosis, as well as decreased testosterone production by affecting Leydig cells by downregulating the genes that are responsible for testosterone production (Ali et al., 2022).
Propolis is a natural brownish-green resinous material that is produced by honeybees. Bees use Propolis to create a protective layer, fill cracks, and polish the cells of honeycomb walls (Zapata et al., 2022). Pro is a valuable natural compound capable of treating and preventing numerous illnesses (Handelsman, 2019). Many pharmacological actions of Propolis have been reported, such as hepatoprotective, anti-inflammatory, and antioxidant activities (Stojanović et al., 2020). Exhibiting immunomodulatory and anti-inflammatory properties is crucial to prevent testicular dysfunction since conditions, such as orchitis or testicular torsion are often accompanied by inflammation, and chronic inflammation can cause testicular dysfunction, due to its ingredients, phenols, flavonoids, and cinnamic acid derivatives (Magnavacca et al., 2022). Propolis is used to manufacture cosmetics and drugs to treat rhinitis, wounds, burns, and acne. It appears in various forms, including capsules, creams, mouthwash, toothpaste, and throat lozenges (Coppock, 2021). This study investigated the combined effects of Propolis and Vitamin E on cadmium chloride CdCl2-induced testicular dysfunction in male rats.
Several studies have shown that propolis can enhance wound healing through numerous mechanisms, including stimulating granulation tissue, fibroblasts, and angiogenesis. It also depresses inflammation (Salrian et al., 2022). Propolis is also known to have antibacterial effects, especially against Aenibacillus larvae and Melissococcus plutonius. The synergistic effect of Propolis and different extracts may enhance its therapeutic potential (Toutiaee et al., 2023). Other studies have shown that Propolis may play a role in immunization since combining it with silver nanoparticles can increase interleukin-4 and immunoglobulin concentrations in the body (Sekhi & Al-Samarraae, 2023).
Vitamin E was chosen to combine with propolis due to its well-documented antioxidant properties, which are the main mechanisms of testicular dysfunction induced by CdCl2 (Shi et al., 2024). While vitamin E provides antioxidant protection, and Propolis provides broader mechanisms to protect against testicular dysfunction, including anti-inflammatory and immunomodulatory pathways.
The present study was conducted to assess the potential therapeutic impact of Propolis and vitamin E in rats exposed to cadmium toxicity.

Materials and Methods

Experimental animals 
The animal model of this study was Albino Wistar rats, with a weight range of 250±25 g, The animals were kept in plastic cages, where there were five animals in each cage, with free access to drinking water and food; the conditions were under control, the humidity levels were 45±15%, temperature 25±3 and light cycle (14/10, light/dark), each group also was consisted of two cages, since each group contains 10 animals and each cage contains only five animals.

Drugs used in this study
Xylazine hydrochloride (100 mg/mL) and ketamine hydrochloride (100 mg/mL) were purchased from the Dechra Pharmaceuticals Company (UK). Propolis was purchased from YS Eco Bee Farms Company, USA. Cadmium Chloride was purchased from Sigma-Aldrich (USA).
Group 1 (G1 or negative control): rats received distilled water. 
Group 2 (G2 or positive control): Rats were administered 1 mg/kg of CdCl2 every 72 h for four weeks via the intraperitoneal (IP) route (Areba, 2020).
Group 3 (G3 or CdCl2 + propolis): Rats were treated with Propolis at 250 mg/kg body weight (BW) orally daily by a gavage needle (Singla et al., 2014), followed by CdCl2 at 1 mg/kg by IP injection every 72 h for four weeks
Group 4 (G4 or CdCl2+ Vit E): rats were administered a daily oral dose of 100 mg/kg BW of vitamin E using a gavage needle (Abouelghar et al., 2020), followed by CdCl2 at 1 mg/kg via IP injection every 72 h for four weeks

Parameters for investigation
Tissue samples were collected at the end of the study; the experimental rats were ethically sacrificed using ketamine at 10 mg/kg BW and xylazine at 40 mg/kg BW. Testes were collected from the rats and stored at -80 °C for seventy-two hours. One test was used for histopathological analysis, and the other was homogenized using cold polyphenylene sulfide (PPS) (PH 7.5) mixed with protease inhibitor to obtain a homogenizing solution cocktail. The homogenizing solution was centrifuged at 3000 rpm for 15 minutes in a cold centrifuge. The supernatant was collected in a sterile Eppendorf tube, discarded, and stored at -20 °C for 24 h. Elisa used the supernatant to evaluate this caspase- 3, tumor necrosis factor α (TNF-alpha), and testosterone levels.

Results

Statistical analysis
All results were analyzed using a one-way analysis of variance (ANOVA) test for statistical analysis, and post-hoc comparisons were performed using Tukey’s test to detect differences between groups.

The results of Caspase and TNF-α presented in Figures 1 and 2 demonstrated a significant (P≤0.05) increase in G2 compared to the control group, which indicates the toxic effect of CdCl2 on testicular dysfunction.

In contrast, testosterone in Figure 3 demonstrated a significant (P≤0.05) decrease in G2 compared to the control group. In contrast, caspase and TNF-α, presented in Figures 1 and 2, demonstrated a significant (P≤0.05) decrease in G3 and G4 compared to G2. Finally, testosterone in Figure 3 demonstrated a significant (P≤0.05) decrease in G3 and G4 compared to G2, which rules the therapeutic effect of Propolis.

Histopathological analysis
Figure 4 shows the histological sections of the testes in rats in the negative control group (Figure 4). The section shows the normal texture of testes, including seminiferous tubules (black arrow), interstitial cells of Leydig (red arrow), interstitial connective tissue (green arrow), spermatids (blue arrow), Sertoli cells (orange arrow), and primary spermatocytes (yellow arrow). The tissue is stained with hematoxylin and eosin (H&E), and the sections are captured using a digital camera and a light microscope at a 10X magnifier scale.

Figure 5 shows histopathological sections of the testes of rats treated with Cdcdl2 at 1 mg/kg BW. The section shows clear damage in the interstitial Leydig cells (black arrows) and severe damage in the interstitial connective tissue (red arrows). Sertoli cells and primary spermatocytes show clear necrotic changes (green arrows). The tissue is stained with H&E, and the sections are captured using a digital camera and a light microscope at a 10X magnifier scale.

Figure 6 shows the histological sections of the testes in rats in the propolis-treated group. The section shows normal texture without any significant occupied lesions (SOL) of the testes, including seminiferous tubules (black arrow), interstitial cells of Leydig (red arrow), interstitial connective tissue (green arrow), spermatids (blue arrow), Sertoli cells (orange arrow) and primary spermatocytes (yellow arrow). The tissue is stained with H&E, and the section is captured using a digital camera and a light microscope at a 10X magnifier scale.

Figure 7 shows the histological sections of the testes in rats in the vitamin E-treated group. The section shows normal texture without any SOL of testes, including seminiferous tubules (black arrow), interstitial cells of Leydig (red arrow), interstitial connective tissue (green arrow), spermatids (blue arrow), Sertoli cells (orange arrow) and primary spermatocytes (yellow arrow). The tissue is stained with H&E, and the sections are captured using a digital camera and a light microscope at a 10X magnifier scale.

Discussion
This study was conducted to evaluate the effectiveness of propolis and vitamin E in treating testicular dysfunction induced by CdCl2 by evaluating concentrations of TNF-α, caspase 3, and testosterone. G2, which also received CdCl2, showed a highly significant elevation in caspase and TNF-α levels compared to G1. Cadmium can exhibit toxic properties and the ability to disrupt cellular homeostasis to induce programmed cell death, as evidenced by an increase in caspase activity. (Đukić-Ćosić et al., 2020; Souza-Arroyo et al., 2022). Further evidence shows that the induction of a cellular immune response within the organism to cadmium exposure includes an increase in TNF-α, an inflammatory cytokine (Yang et al., 2021).
A significantly lower testosterone level observed in G2 than in G1 can thus provide an alert regarding the cadmium effect on reproductive performance. Testosterone is thus vital in many physiological processes of spermatogenesis and may reflect basic information on male fertility status. Its levels decline, probably due to the harmful cadmium influence on Leydig cells responsible for testosterone production. (Grande et al., 2022). This is supported by the findings of other authors, who showed that heavy metals can disrupt the endocrine system (Zhou et al., 2022).
Moreover, regarding caspase and TNF--α levels, treatments with propolis and vitamin E, along with CdCl2, have significantly lowered the levels in G3 and G4, respectively, compared to G2. This suggests that such treatments have protective effects against cadmium-induced toxicity. Propolis’s antioxidative properties could reduce oxidative stress and inflammation, leading to a lesser degree of apoptosis as expressed in reduced caspase levels. Correspondingly, vitamin E, a well-accepted antioxidant, can stabilize the cell membrane and inhibit oxidative damage, thereby reducing inflammation and apoptosis. (Mohamed et al., 2021; Mohamed et al., 2022)
However, testosterone levels were significantly reduced in groups 3 and 4 compared to group 2. This may be attributed to the fact that although propolis and vitamin E can play a role in reducing inflammation and apoptotic responses to a certain degree, they did not bring testosterone to the normal value. This would mean that such agents afford partial protection but are inadequate in surmounting the severe endocrine disturbances caused by cadmium exposure. This intimate relationship among the pathophysiological mechanisms of oxidative stress, inflammation, and hormonal regulation also points to this fact.
The results underlined the noxious effect of cadmium on both inflammatory markers and testosterone levels, underlining the health hazards possibly linked to cadmium exposure. The possible therapeutic benefit of Propolis and vitamin E underlines further possibilities of intervention that would require further investigation into the underlying mechanism and other protective measures.

Histopathologically, interstitial Leydig cells and connective tissue deterioration is accompanied by necrotic changes in Sertoli cells and primary spermatocytes. Pathological changes in the testes may be caused by several factors, including toxins and testicular torsion (Adamczewska et al., 2020; Abarikwu et al., 2021). These results also showed serious disturbances in the normal pattern and function of the testes, which are harmful to sperm production and fertility. Propolis is a resinous product collected by honeybees that have been reported to possess many biological activities, including antioxidant and anti-inflammatory properties (Seven et al., 2020). Considering these aspects, propolis appears to be one of the agents that can protect testicular tissues from damage at both the morphological and functional levels (Martins et al., 2021; Mega et al., 2022).
The absence of notable pathological alterations in this segment indicated that propolis therapy may have averted or remedied the type of harm detected in the positive control group sample. Vitamin E is one of the established antioxidants studied for efficiency in protecting the testicles. (El-Kotb et al., 2020; Abdel-Wahab et al., 2021). This coherence between the established ability of vitamin E to reduce oxidative stress and preserve testicular structure across different studies closely aligns with structurally intact presentations of testicular components. (Nowicka-Bauer & Nixon, 2020) (Mondal & Bandyopadhyay, 2024). This result supports using vitamin E as a treatment to maintain testicle health. 

Conclusion
This study demonstrated that propolis and vitamin E protected against CdCl2 by improving the optimal results according to physiological parameters and pathohistological sections.

Ethical Considerations

Compliance with ethical guidelines
All procedures used in this study were reviewed and approved by the Scientific Committee of the Faculty of Veterinary Medicine, University of Kufa, Kufa, Iraq, in compliance with the ethical principles of animal welfare (Code: UK.VET.2023.27152).

Funding
This research did not receive any grant from funding agencies in the public, commercial, or non-profit sectors.

Authors' contributions
All authors equally contributed to preparing this article

Conflict of interest
The authors declared no conflict of interest.

Acknowledgments
The authors thank the staff at Sigma Laboratory.

References

Abarikwu, S. O., Costa, G. M. J., de Lima E Martins Lara, N., Lacerda, S. M. S. N., & de França, L. R. (2021). Atrazine impairs testicular function in BalB/c mice by affecting Leydig cel Toxicology, 455, 152761. [DOI:10.1016/j.tox.2021.152761] [PMID]

Abdel-Wahab, A., Hassanin, K. M. A., Mahmoud, A. A., Abdel-Badeea, W. I. E., Abdel-Razik, A. H., & Attia, E. Z., et al. (2021). Physiological roles of red carrot methanolic extract and vitamin E to abrogate cadmium-induced oxidative challenge and apoptosis in rat testes: Involvement of the Bax/Bcl-2 ratio. Antioxidants (Basel, Switzerland), 10(11), 1653. [DOI:10.3390/antiox10111653] [PMID]

Abouelghar, G. E., El-Bermawy, Z. A., & Salman, H. M. S. (2020). Oxidative stress, hematological and biochemical alterations induced by sub-acute exposure to fipronil (COACH®) in albino mice and ameliorative effect of selenium plus vitamin E. Environmental Science and Pollution Research, 27(8), 7886–7900.[DOI:10.1007/s11356-019-06579-9] [PMID]

Adamczewska, D., Slowikowska-Hilczer, J., Marchlewska, K., & Walczak-Jedrzejowska, R. (2020). Features of gonadal dysgenesis and Leydig cell impairment in testes with Sertoli cell-only syndrome. Folia Histochemica et Cytobiologica, 58(2), 73–82. [PMID]

Agarwal, A., Leisegang, K., & Sengupta, P. (2020). Oxidative stress in pathologies of male reproductive disorders. In V. R. Preedy (Ed.), Pathology (pp. 15-27). Massachusetts: Academic Press. [DOI:10.1016/B978-0-12-815972-9.00002-0]

Ali, W., Ma, Y., Zhu, J., Zou, H., & Liu, Z. (2022). Mechanisms of Cadmium-Induced Testicular Injury: A risk to male fertility. Cells, 11(22), 3601. [DOI:10.3390/cells11223601][PMID]

Areba, (2020). Determination of antioxidant and metal chelating properties of tea (Camelia sinensis) in ameliorating cadmium induced toxicity in male Wistar rats [MA thesis]. Kenya: Egerton University. [Link]

Bhattacharyya, K., Sen, D., Laskar, P., Saha, T., Kundu, G., & Ghosh Chaudhuri, A., et al. (2021). Pathophysiological effects of cadmium (II) on human health-a critical review. Journal of Basic and Clinical Physiology and Pharmacology, 34(3), 249-261. [DOI:10.1515/jbcpp-2021-0173] [PMID]

Coppock, R. W. (2021). Bee products as nutraceuticals to nutraceuticals for bees. In R. C. Gupta, R. Lall & A. Srivastava (Eds.), Nutraceuticals (pp. 813-833). Massachusetts: Academic Press. [DOI:10.1016/B978-0-12-821038-3.00047-1]

Đukić-Ćosić, D., Baralić, K., Javorac, D., Djordjevic, A. B., & Bulat, Z. (2020). An overview of molecular mechanisms in cadmium toxicity. Current Opinion in Toxicology, 19, 56-62. [DOI:10.1016/j.cotox.2019.12.002]

Grande, G., Barrachina, F., Soler-Ventura, A., Jodar, M., Mancini, F., & Marana, R., et al. (2022). The role of testosterone in spermatogenesis: lessons from proteome profiling of human spermatozoa in testosterone deficiency. Frontiers in Endocrinology, 13, [DOI:10.3389/fendo.2022.852661][PMID]

Handelsman, Y. (2019). Rationale for the early use of sodium-glucose cotransporter-2 inhibitors in patients with type 2 diabetes. Advances in Therapy, 36(10), 2567-2586. [DOI:10.1007/s12325-019-01054-w][PMID]

Jabbar Sekhi, R., & Abbas Aboud Al-Samarraae, I. (2023). Propolis silver nanoparticles as an adjuvant in immunization of rats with citrobactor freundii antigens. Archives of Razi Institute, 78(3), 973–979. [PMID]

Kotb, S. M., El-Ghazouly, D. E., & Ameen, O. (2020). The potential cytoprotective effect of Vitamin C and Vitamin E on monosodium glutamate-induced testicular toxicity in rats. Alexandria Journal of Medicine, 56(1), 134-147. [DOI:10.1080/20905068.2020.1804311]

Magnavacca, A., Sangiovanni, E., Racagni, G., & Dell’Agli, M. (2022). The antiviral and immunomodulatory activities of propolis: An update and future perspectives for respiratory diseases. Medicinal Research Reviews, 42(2), 897-945. [DOI:10.1002/med.21866][PMID]

Martins, R. V., Silva, A. M., Duarte, A. P., Socorro, S., Correia, S., & Maia, C. J. (2021). Natural products as protective agents for male fertility. BioChem, 1(3), 122-147. [DOI:10.3390/biochem1030011]

Mega, O. O., Benneth, B. A., Edesiri, T. P., Rume, R. A., Victor, E., & Rotu, R. A., et al. (2022). Possible mechanisms involved in the testicular-protective property of quercetin in rats exposed to endosulfan toxicity. Pesticide Biochemistry and Physiology, 188, [DOI:10.1016/j.pestbp.2022.105224] [PMID]

Mohamed, E. K., Osman, A. A., Moghazy, A. M., Rahman, A. A. S. A., & AS, A. (2021). Propolis protective effects against doxorubicin-induced multi-organ toxicity via suppression of oxidative stress, inflammation, apoptosis, and histopathological alterations in female albino rats. Biointerface Research in Applied Chemistry, 12, 1762-1777. [Link]

Mohamed, H. K., Mobasher, M. A., Ebiya, R. A., Hassen, M. T., Hagag, H. M., & El-Sayed, R., et al. (2022). Anti-inflammatory, anti-apoptotic, and antioxidant roles of honey, royal jelly, and propolis in suppressing nephrotoxicity induced by doxorubicin in male albino rats. Antioxidants, 11(5), 1029. [DOI:10.3390/antiox11051029][PMID]

Mondal, S., & Bandyopadhyay, A. (2024). Antioxidants in mitigating phthalate-induced male reproductive toxicity: A comprehensive review. Chemosphere, 364, [DOI:10.1016/j.chemosphere.2024.143297] [PMID]

Nowicka-Bauer, K. & Nixon, B. (2020). Molecular changes induced by oxidative stress that impair human sperm motility. Antioxidants (Basel, Switzerland), 9(2), 134. [PMID]

Salrian, A. A., Behzadi, A., Oloumi, M. M., Abbasi, M. F., Delshad, S., & Moghadaszadeh, M. (2022). Amplification of wound healing by propolis and honey ointment in healthy and diabetic rat models; histopathological and morphometric findings. Archives of Razi Institute, 77(5), 1673-1681 [DOI:10.22092/ari.2022.357191.1991]

Sartorius, G. A., & Handelsman, D. J. (2023). Testicular dysfunction in systemic diseases. In E. Nieschlag, H. M. Behre, S. Kliesch& S. Nieschlag, (Eds), Andrology: Male reproductive health and dysfunction (pp. 503-542). Cham: Springer International Publishing. [DOI:10.1007/978-3-031-31574-9_34]

Seven, I., Seven, P. T., Baykalir, B. G., Ak, T. P., Kaya, S. O., & Yaman, M. (2020). Bee glue (propolis) improves reproductive organs, sperm quality and histological changes and antioxidant parameters of testis tissues in rats exposed to excess copper. Andrologia, 52(4). [DOI: 10.1111/and.13540]

Shi, Z., Wan, Y., Peng, M., Zhang, J., Gao, Z., Wang, X., & Zhu, F. (2024). Vitamin E: An assistant for black soldier fly to reduce cadmium accumulation and toxicity. Environment International, 185, [DOI:10.1016/j.envint.2024.108547] [PMID]

Singla, S., Kumar, N. R., & Kaur, J. (2014). In vivo studies on the protective effect of propolis on doxorubicin-induced dysfunction in liver of male rats. Toxicology International, 21(2), 191-195. [DOI:10.4103/0971-6580.139808][PMID]

Souza-Arroyo, V., Fabián, J. J., Bucio-Ortiz, L., Miranda-Labra, R. U., Gomez-Quiroz, L. E., & Gutiérrez-Ruiz, M. C. (2022).The mechanism of the cadmium-induced toxicity and cellular response in the liver. Toxicology, 480, [DOI:10.1016/j.tox.2021.153339] [PMID]

Stojanović, S. T., Najman, S. J., Popov, B. B., & Najman, S. S. (2020). Propolis: Chemical composition, biological and pharmacological activity-a review. Acta Medica Medianae, 59(2), 108-113. [Link]

Toutiaee, S., Mojgani, N., Harzandi, N., Moharrami, M., & Mokhberalsafa, L. (2023). Anti-Bacterial Activity of Four Distinct Propolis Extracts against P. larvae and M. plutonius; Etiological Agent of American and European Foulbrood Disease of Honeybees. Archives of Razi Institute, 78(3), 899-905.[DOI:10.22092/ari.2022.3602531]

Usende, I. L., Oyelowo, F. O., Adikpe, A. O., Emikpe, B. O., Nafady, A. A. H. M., & Olopade, J. O. (2022). Reproductive hormones imbalance, germ cell apoptosis, abnormal sperm morphophenotypes and ultrastructural changes in testis of African giant rats (Cricetomys gambianus) exposed to sodium metavanadate intoxication. Environmental Science and Pollution Research, 29(28), 42849-42861. [DOI:10.1007/s11356-021-18246-z][PMID]

Yang, Z., He, Y., Wang, H., & Zhang, Q. (2021). Protective effect of melatonin against chronic cadmium-induced hepatotoxicity by suppressing oxidative stress, inflammation, and apoptosis in mice. Ecotoxicology and Environmental Safety, 228, Advance online publication. [PMID]

Zapata, Z. T. C., Acosta-Leal, D. A., & Sanchez, A. M. Z. (2022). Physicochemical characterization of propolis collected by Apis mellifera L (Hymenoptera: Apidae) in Pacho and Bogotá Cundinamarca. Centrosur Agraria, 1(15), 123-138. [Link]

Zhou, Q., Chen, J., Zhang, J., Zhou, F., Zhao, J., & Wei, X., et al. (2022). Toxicity and endocrine-disrupting potential of PM2. 5: Association with particulate polycyclic aromatic hydrocarbons, phthalate esters, and heavy metals. Environmental Pollution, 292(Pt A), 118349. [DOI:10.1016/j.envpol.2021.118349] [PMID]