اثرات تجویز عصاره تانن کبراکو بر آسیب متعاقب ایسکمی/رپرفیوژن بیضه در موش صحرایی

Introduction

Testicular torsion has been known as one of the most serious disorders in the male reproduction system. The main pathophysiology of testicular torsion is ischemia/reperfusion (I/R) or cut-off of the blood supply that leads to testis injury (Moshkelani et al., 2020; Tanriverdi et al., 2021). In the I/R, interruption in blood supply leads to damage to tissues (Chouchani et al., 2016). In the testicular I/R, the generation of reactive oxygen species (ROS) leads to tissue damage during the reperfusion. Excessive production of ROS leads to decreased antioxidant defenses in the seminal plasma (Arena et al., 2017). Cellular antioxidants such as superoxide dismutase (SOD) and glutathione peroxidase (GPx) have essential effects in defense mechanisms against ROS-mediated cellular damage (Alabedi et al., 2021). Several anti-inflammatory agents, antioxidants, and free radical scavengers were applied for the treatment of testicular I/R-induced male infertility (Lee et al., 2021). However, because of side effects of the medicines, there are growing interest towards the application of medical plants in the treatment of testicular I/R (Hmood Jassim et al., 2021).

Several polyphenolic compounds are produced during the growth and maturation of plants, which have an important role in plant fate. These substances are known as plant secondary metabolites. Tannins are polyphenolic compounds extensively found in the plants. The term “tannin” refers to “tanning” of skin in leather industry. Tannins are usually classified, based on their molecular structure, into hydrolysable tannins (HT) and condensed tannins (CT) (Chen et al., 2019). Tannins have several biological properties including antimicrobial, anti-parasitic, antioxidant, anti-inflammatory, and antiviral effects (Buccioni et al., 2017). Red quebracho species (Schinopsis lorentzii and Schinopsis balansae) are the most common and plentiful sources of CT (14–26% of heartwood). Extracts are regularly made from the bark or heartwood and for leather tanning (Fraga-Corral et al., 2021). Quebracho tannin extract (QTE) is generally composed of 95% CT and 5% polysaccharides. QTE is a commercial source of CT and has been widely used for ruminant feeding to improve digestibility, nitrogen balance, energy partitioning, and milk production (Henke et al., 2016). Antioxidant activity of the tannin-rich plants are responsible for their direct inhibition of ROS production. ROS pathway plays a major role in the inflammatory response (Pinheiro et al., 2013).

Although antioxidant activity of the QTE is well known, there is no report on its possible effectiveness in treatment of testicular I/R. Thus this study aimed to determine the therapeutic effect of QTE on experimental testicular I/R injury in rats.

Materials and Methods

Animals and Experimental Groups

Thirty healthy adult male Wistar rats (weight 250-300 g) were purchased from Pasteur Institute. Animals were kept under constant room temperature (20±1°C) and relative humidity (42±1%) on a 12-hour light/dark cycle. All animals had free access to commercial food and water. Rats were randomly divided into 5 experimental groups (n=6). Groups were as follows: control group: animals intraperitoneally (ip) injected with saline, I/R group: rats subjected to 4-hour I/20-hour R periods, ip injected with saline 1 hour before R, group 3: rats subjected to 4-hour I/20-hour R periods, i.p. injected with 100 mg/kg of QTE 1 hour before R, group 4: animals subjected to 4-hour I/20-hour R periods, then 200 mg/kg of QTE ip injected 3 hours after I, and group 5: rats subjected to 4-hour I/20-hour R periods, then 400 mg/kg of QTE, ip injected 1 hour before R (Turkili et al., 2012; Koksal et al., 2012). This study was approved according to the Guidelines of the Animal Care provided by Research Committee of Islamic Azad University, Science and Research Branch, Tehran, Iran.

Extract Procedure

Commercial Quebracho bark powder was provided, then heated with 80% ethanol (v/v) to 80°C for 2 hours. The extraction continued for further 10 hours at 20°C. The extract was then filtered through cheese cloth and evaporated with a rotary evaporator. The aqueous extract was freeze-dried for 48 hours, and the dried extract was homogenized with a mortar and pestle (Ribnicky et al., 2009).

Experimental Protocol

All surgical procedures were performed under anesthesia by ip injection of ketamine hydrochloride (60 mg/kg) and xylazine hydrochloride (10 mg/kg) followed by experimental testicular I/R (Sahin et al., 2005). The upper left abdominal quadrant was approached through a midline laparotomy incision. During the surgical procedures, the body temperature was maintained with a heating pad. The testicular artery and vein of the left testis were occluded with a vascular clamp for 4 hours. Afterward, the clamp was removed, and the organ was allowed to reperfuse for 20 hours (Koksal et al., 2012). In the end, the rats were euthanized with an overdose injection of pentobarbital (300 mg/kg, ip), peritoneum was then opened, and left testis was removed for further investigations. The testicle was divided into two sagittal sections and one half was fixed in Bouin solution. The second half of the testicle tissue was stored at -80°C for biochemical analysis (Minutoli et al., 2005).

Tissue Processing

The tissue was fixed in Bouin solution (7.5 mL saturated picric acid, 2.65 mL glacial acetic acid, and 2.5 mL 7% formaldehyde), post-fixed in 70% alcohol, and embedded in paraffin blocks. A tissue section (5 µm) was obtained, deparaffinized, and stained with hematoxylin and eosin (H&E). The testicular tissue was evaluated in a random order with standard light microscopy by an observer who was unaware to which group the rat belonged (Dal Lago and Lucke, 1973). Then, testis tissue samples from the experimental rats were fixed in Bouin solution for complete fixation and processed for paraffin sectioning. A tissue section at 5 µm thickness was taken and stained with H&E. The testis sections were graded numerically to assess the degree of histological changes associated with seminiferous tubule injury as previously described by Johnsen (1970) as:

• 10: Complete spermatogenesis and perfect tubules
• 9: Many spermatozoa present but disorganized spermatogenesis
• 8: Only a few spermatozoa present
• 7: No spermatozoa but many spermatids present
• 6: Only a few spermatids present
• 5: No spermatozoa or spermatids present but many spermatocytes present
• 4: Only a few spermatocytes present
• 3: Only spermatogonia present
• 2: No germ cells present
• 1: Neither germ cells nor Sertoli cells present

Antioxidant Activity

At the end of the tests, testis samples were obtained and tissue MDA, SOD, GPx, and total antioxidant status (TAS) were determined using Zell Bio GmbH (Germany) assay kits.

Statistical analysis

Data were prepared in Excel, and the parametric data were analyzed with one-way analysis of variance (ANOVA) using the SPSS software version 16.0 for Windows (SPSS, Inc., Chicago, IL., USA). Data were expressed as mean ± standard error (SE). Where heterogenecity occurred, the groups were separated using Tukey’s multiple comparison test. The Kruskal-Wallis test was used to compare group medians for histopathological scores. P-value<0.05 was considered to denote significant differences between the groups.

Results

Histopathological Activity of the QTE

The effect of QTE on testicular histopathology is shown in Figures 1-6. As seen, the lowest effect was seen at QTE (100 mg/kg) while QTE (200 and 400 mg/kg) had better results on histological indexes compared to the control group (P<0.05). According to Figure 2, rats in the control group showed normal seminiferous tubules and spermatogenesis with spermatocytes, sertoli, and spermatozoa. Based on Figure 3, in I/R rats, seminiferous tubules were degenerated and loss of spermatogenesis with a few spermatocytes was observed. Based on the results shown in Figure 4, administration of the QTE (100 mg/kg) after I/R induction led to degenerated seminiferous tubules and loss of spermatogenesis with a few spermatocytes. While administration of the QTE (200 mg/kg) improved testis characteristics with a few normal seminiferous tubules and spermatocytes in seminiferous tubules in experimental I/R-induced rats (Figure 5). Administration of the QTE (400 mg/kg) to I/R rats improved testis characteristics with a few normal seminiferous tubules and spermatocytes in seminiferous tubules (Figure 6).

Effect of QTE on Antioxidant Activity

According to Table 1, tissue MDA levels significantly increased in I/R rats (P<0.05) while QTE (200 and 400 mg/kg) decreased the I/R-induced MDA level (P<0.05). Experimental I/R significantly decreased the tissue SOD and GPx concentrations compared to the control group (P<0.05). Administration of the QTE (200 and 400 mg/kg) significantly increased the tissue SOD activity in I/R rats (P<0.05). Furthermore, QTE (200 and 400 mg/kg) increased the tissue GPx levels in I/R rats (P<0.05). No significant difference was observed between the studied groups in terms of tissue TAS level (P>0.05).

Table 1. Effect of different levels Quebracho tannin extract on testis values of malondialdehyde, superoxide dismutase, glutathione peroxidase and total antioxidant status in I/R rat

I/R: Ischemia/reperfusion, Quebracho tannin extract: QTE, MDA: malondialdehyde, SOD: superoxide dismutase, GPx: glutathione peroxidase, TAS: total antioxidant status. Different letters (a-c) indicate significant differences between treatments (P<0.05).

Discussion

To the best of our knowledge, there are limited studies describing the role of QTE in oxidative damage and testicular pathology in testicular IR injury in rat. As observed in this study, I/R led to degenerated seminiferous tubules and loss of spermatogenesis. Administration of the QTE (200 and 400 mg/kg) to I/R rats showed many normal seminiferous tubules. Tissue MDA levels significantly increased in I/R rats while QTE decreased I/R-induced MDA level. Experimental I/R significantly decreased SOD and GPx activities. While administration of the QTE significantly increased tissue SOD and GPx activities in I/R rats.

Testicular torsion leads to ischemia and reperfusion with detorsion of the twisted testicle leading to morphological damage to testicular tissue. Additionally, post-ischemic reperfusion amplifies further tissue damage and apoptosis (Tuglu et al., 2015). Despite vast progresses during the past decade in this area, I/R injury remains a clinically challenging problem (Abdallah, 2021). In initial stage of I/R injury, ROS is released after reperfusion, then endothelial dysfunction or neutrophil infiltration triggers the oxidative damage (Zadeh et al., 2019). In this regard, Wei et al. (2011) reported 2 hours of unilateral testicular torsion followed by detorsion changes in MDA, SOD, and CAT activities. ROS include hydrogen peroxide and unstable free radicals with unpaired electrons in their outer orbits. Excessive ROS or decreased antioxidant defenses in the seminal plasma damages spermatozoa via oxidative stress (Shamsi-Gamchi et al., 2018). In this regard, Ahmadvand et al. (2017) reported that pretreatment with gallic acid as an antioxidant agent has protective effects on oxidative stress markers in renal I/R injury in rats. However, there is no report on the application of Quebracho on I/R injury and we were not able to compare our findings with previous reports. Recently, Reggi et al. (2020) demonstrated Quebracho, and chestnut tannin extracts exert growth inhibitory activity against E. coli as well as increasing cell viability modulation activities. Tannins can inhibit the growth of some pathogenic bacterial species without adverse effect on the physiological growth pattern of gastrointestinal tract. Moreover, dietary QT increased the antioxidant capacity of liver and plasma in sheep (López-Andrés et al., 2013). Supplementing the sheep with polyphenols and condensed tannins elevated SOD levels in the plasma (Sgorlon et al. 2006). Remarkably, Larrosa et al. (2010) showed that hydrolysable tannins in an indirect manner decrease the activity of antioxidant system in monogastric animals.

Oxidative stress, resulting in an imbalance between the production of oxygen free radicals and antioxidant capacity, damages the biological macromolecules and causes the disruption in normal metabolism and physiology (Zhang et al., 2012). Sperm membranes contain high levels of polyunsaturated fatty acids (PUFAs) in spermatozoa which are susceptible to be attacked by ROS (Hadwan et al., 2014). MDA is the end product of lipid peroxidation, and the elevation in testicle MDA level is a marker of extent of oxidative stress that leads to infertility (Hsieh et al., 2006). Our findings suggested treatment with QTE had a beneficial effect against I/R.

Conclusion

It can be concluded that QTE improves the antioxidant capacity of tissues via an indirect effect, possibly by enhancing the tissue endogenous antioxidant activity or by participating in the regeneration of other antioxidant compounds. This study was an animal model to investigate the antioxidant activity of QTE in testicular I/R injury. This information can be used as basic data for further investigations on antioxidant agents involved in human testicular torsion

Acknowledgments

Authors impress fully thanks to all co-workers.

Conflict of Interest

The authors declared no conflict of interest.