The protective effect of Ganoderma lucidum on testicular torsion/detorsion-induced ischemia-reperfusion (I/R) injury 1

Abstract Purpose To investigate the protective effect of Ganoderma lucidum on testicular torsion/detorsion (T/D)-induced ischemia-reperfusion (I/R) injury. Methods Thirty male Wistar albino rats were randomly categorized into 3 groups: Group 1: sham, Group 2 ( T/D): 2,5 hours of ischemia and 7 days of reperfusion, Group 3 (T/D+ G. lucidum ): 2,5 hours of ischemia and 7 days of reperfusion and 7 days of 20 mg/kg via gastric gavage G. lucidum polysaccharides per day. Biochemical assays of Malondialdehyde (MDA), superoxide dismutase (SOD), Catalase (CAT), Glutathione (GSH) levels , histopathology and expression levels of VEGF and Bcl-2 with immunohistochemical methods were examined in testicular tissue. Results G. lucidum treatment was found to have prevented the T/D-induced I/R injury by decreasing MDA levels of the testis. SOD, CAT and GSH activities were decreased in group 2, while they were increased in group 3 (p<0.001) and significant improvement in the tube diameter was observed in group 3. Bcl-2-positive germinal cells were lowered in group 3 compared to the group 2. VEGF expression showed an increase in group 2, whereas it decreased in group 3. Conclusion The antioxidant G. lucidum is thought to induce angiogenesis by reducing the apoptotic effect in testicular torsion-detorsion.


■ Introduction
Testicular torsion is among the most common urological emergencies observed in children. The annual incidence of spermatic cord torsion is 4.5 in 100,000 males aged 1-25 years 1 . This condition may cause ischemia and loss of the testis. Testicular damage varies depending on the grade and duration of the torsion 2 . Testicular torsion decreases blood flow to the testis, causing tissue ischemia. The accumulation of reactive oxygen species (ROS) is the underlying pathologic mechanism of testicular torsion followed by ischemia, and it has been observed to lead to infertility in several cases 3 . ROS is known to react with proteins, lipids, carbohydrates and nucleic acids, causing impaired cell function, DNA damage and apoptosis. The extent of testicular tissue damage is related to both the degree of twisting and the rapidity of surgical intervention to counter-rotate both the testis and spermatic cord to re-establish normal blood flow to the organ 4 . Previous studies using a rat model of testicular torsion have demonstrated that a 1-hour, 720° rotation of the testes followed by reperfusion results in permanent loss of spermatogenesis despite the return of blood flow. If treated within 6 hours of presenting with pain, there is a good chance of saving the affected testicle, as 90%-100% testicles will be saved. If treated within 6-12 h, 20%-50% testicles will be saved depending on the degree of the torsion, and if treated within 12-24 h, 0%-10% testicles will be saved 5 . Delayed surgery may result in orchiectomy or diminished fertility, and even if the testis is detorsed in time, infertility risk is the most worrisome complication 6 .
Ganoderma lucidum (G. lucidum) is a mushroom belonging to the Polyporaceae family of Basidiomycota and has been widely used as a traditional medicine for thousands of years, particularly in Asian countries 7 . The fruiting bodies, cultured mycelia and spores of G. lucidum contain a variety of bioactive chemical substances such as polysaccharides, triterpenoids and proteins 8 . Different experimental studies and modern clinical trials suggest that these active compounds isolated from G. lucidum have anti-inflammatory, antioxidant, antitumor and immunomodulatory activities 9 .
Though several compounds have been used to heal ischemia-reperfusion (I/R) injury in animal models of testicular torsion, few are available for use 10 . However, to date, there is no study on the effect of G. lucidum on a rat testicular torsion model. In our study, biochemical assays of malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT), glutathione (GSH) levels, histopathology and expression levels of vascular endothelial growth factor (VEGF) and B-cell GSH levels of biochemical parameters. Secondary outcome measure consisted of histopathologic and immunohistochemical changes.

Surgical procedure
The rats were anesthetized by an intramuscular injection of ketamine hydroxide (50 mg/ kg) and xylazine (10 mg/ kg) under aseptic conditions. The tunica vaginal was removed with the help of a forceps to allow the testicular tissue to be visible. A scrotal pocket was created to place the testicle back into the scrotum after torsion. To create torsion, the left testis was rotated at 720°degrees clockwise around the longitudinal axis of spermatic cord for 2.5 hours. To prevent detorsion, the testis was fixed into the scrotal pocket by passing 4/0 non-traumatic absorbable suture through the dartos and testicular tunica albuginea. After 2.5 hours of torsion, the suture that fixed testis to dartos was cut for detorsion and the reperfusion period of the following 7 days.

Biochemical analysis
MDA, SOD, CAT and GSH levels were examined in testicular tissue. Tissue samples were homogenized with ice-cold 150 mMKC. MDA levels were assayed for products of lipid peroxidation, and the results were expressed as nmol MDA/g tissue 11 . The SOD activity in the tissue was measured using the RANSOD kit (Randox Laboratories, Crumlin, UK). GSH was determined by the spectrophotometric method based on the use of Ellman's reagent, and the results were expressed as μmol glutathione/g tissue 12 . CAT activity was determined by the spectrophotometric method based on the ability of hydrogen peroxide to form a stable stained complex with molybdenum salts 13 .

Immunohistochemical technique
Formaldehyde-fixed testis tissue was embedded in paraffin wax for further immunohistochemical examination. Sections were deparaffinized in xylene. The antigen retrieval process was performed twice in citrate buffer solution (pH 6.0), first for 7 minutes, and then for 5 minutes, and was boiled in a microwave oven at 700 W. They were then allowed to cool to room temperature for 30 minutes and washed twice in distilled water for 5 minutes. Endogenous peroxidase activity was blocked in 0.1% hydrogen peroxide for 20 minutes. Ultra V block (Cat. No. 85-9043, Invitrogen, Carlsbad, California, USA) was applied for 10 minutes prior to the application of primary antibodies for Bcl-2 (cat: PA5-20068, Invitrogen, Carlsbad, California, USA) and VEGF (cat: PA5-16754, Invitrogen, Carlsbad, California, USA). Secondary antibody (Cat. No. 85-9043, Invitrogen, Carlsbad, California, USA) was applied for 20 minutes. Slides were then exposed to streptavidin-peroxidase for 20 minutes. Chromogen diaminobenzidine (DAB, Cat. No. 34002, Invitrogen, Carlsbad, California, USA) was used. Control slides were prepared as mentioned above, but by omitting the primary antibodies. After counter staining with hematoxylin and washing in tap water for 8 minutes and in distilled water for 10 minutes, sections were examined using a light microscope (Zeiss, Germany).

Statistical analysis
Statistical analyses were conducted using SPSS (Version 22.0, SPSS Inc., Chicago, IL, USA). Descriptive statistics are presented as median (min-max) and mean ± standard deviation (SD). The normal distribution of the data was evaluated by Shapiro-Wilks test. Since data normality distribution was met, the groups were compared by an analysis of variance (ANOVA) followed by Tukey's multiple comparison tests to determine which groups differed with pairwise comparison. The priori sample size and post-hoc power analyses were calculated using the G-power (Version 3.1) package. The sample size was calculated for ANOVA test, which was used to test the main hypothesis of the study. As a result of the sample size analysis performed using previous study knowledge, it was found that 30 rats, 10 in three different groups, needed to be involved in the study to reveal the significant differences in the groups using 80% power (1-β=0.80), α=0.05 error (95% confidence interval) with a two-sided hypothesis. Values of p<0.05 were considered statistically significant.

Biochemical results
MDA levels in the testicular tissues of rats in the T/D group were significantly increased compared to the sham group, while the MDA levels in the T/D+G. lucidum group were significantly decreased compared to the T/D group. G. lucidum treatment prevented the T/D-induced augmentation of MDA levels in the testis. The SOD, CAT and GSH activities of testicular tissue significantly decreased in the T/D group compared to the sham group. These values were significantly increased in the T/D+G. lucidum group compared to the T/D group. The MDA, SOD, CAT and GSH levels of the sham, T/D and T/D + G. lucidum groups are shown in Table 1 (Fig. 1).
The protective effect of Ganoderma lucidum on testicular torsion/ detorsion-induced ischemia-reperfusion (I/R) injury Doğan G et al.

Histopathologic results
Testicular tubule diameter measurements were evaluated among the groups. There was a significant difference between the sham, T/D and T/D+G.lucidum groups (p<0.001). According to the tube diameter measurement results, a significant improvement in the tube diameter was observed in the group treated with G.lucidum after T/D injury (Table 2, Fig. 2).  Testicular tissue was histologically examined under light microscope for all groups. In the sham group, seminiferous tubules had normal structure. Spermatic cells in seminiferous tubules were regular oval and mitotically active towards the lumen. The large luminal faces of Sertoli cells were regularly seen. The membrane thickness was normal. The intertubular space, connective tissue cells, blood vessels and Leydig cells were normal (Fig. 3A). In the T/D group, basal membrane thickness of tubules increased. Pyknosis and deteriorated nuclei of the spermatogenic cells, attenuated maturation in the sperm cells and degenerated Sertoli cells were observed. Hemorrhage and dilatation in the blood vessels of the interstitial area, increased connective tissue and degenerated Leydig cells were prominent (Fig. 3B). The T/D+G. lucidum group showed a decrease in the basal membrane thickness of tubules compared to the T/D group. Degenerated and deteriorated spermatic cells were evident in some tubules, along with mitotic increase. Little hemorrhage was observed in the blood vessels of the interstitial regular connective tissue cells and heterochromatin Leydig cells (Fig. 3C).

Immunohistochemical results
In the sham group, negative VEGF expression was observed in germ and Sertoli cells, vascular interstitial endothelial cells and Leydig cells (Fig. 3D). In the T/D group, the expression of VEGF in the apical faces of spermatogenic cells and Sertoli cells was positive. VEGF expression was positive in endothelial, some inflammatory, Leydig cells and interstitial blood vessels (Fig.3E). In the T/D+G. lucidum group, positive VEGF expression was observed in a small number of spermatogenic cells, particularly Sertoli cells. However, it was negative in interstitial endothelial cells and Leydig cells (Fig. 3F).
Bcl-2 expression was negative in spermatogenic cells and Sertoli cells in the sham group, while it was positive in several small spermatid cells near the lumen (Fig. 3G). In the T/D group, intense Bcl-2 expression was observed in spermatogenic cells with strong mitotic activity in the basal layers of tubules. Significant expression of Bcl-2 was observed in the luminal faces of some Sertoli cells (Fig. 3H). In the basal and apical regions of the tubules, Bcl-2 reaction was positive in a few spermatogenic cells, whereas it was negative in Sertoli and Leydig cells. Bcl-2-positive germinal cells were also fewer in the G. lucidumtreated group compared to the T/D group (Fig. 3I).

■ Discussion
In our study, MDA levels were found to be significantly higher in the T/D group compared to the Sham and T/D+G. lucidum groups. The SOD, CAT and GSH levels were found to be significantly lower in the T/D group compared to the Sham and T/D+ G. lucidum groups. There was a decrease in the basal membrane thickness of tubules in the T/D+ G. lucidum group compared to the T/D group. While the T/D group was observed to have erupted spermatogenic cells with impaired nuclei, weakened maturation in the sperm cells and degenerated Sertoli cells, degenerated and impaired spermatic cells and mitotic increase were evident in some tubules of the T/D+ G. lucidum group. Bleeding and dilatation of blood vessels in the interstitial region, increased connective tissue and impaired Leydig cells were evident in the T/D group, whereas a small amount of bleeding was observed in the blood vessels of the interstitial regular connective tissue cells and the heterochromatin Leydig cells in the T/D+ G. lucidum group.
In the present study, we did not study the effect of G. lucidum on testicular I/R injury at different doses or different administration times. Detailed randomized controlled prospective clinical studies are required to assess the efficacy of G. lucidum on testis torsion.
Ozbek et al. 14 suggested in an experimental study that testicular torsion significantly decreased SOD and CAT levels when compared to the control groups. In our study, the SOD and CAT activities in testicular tissue significantly increased in the T/D+ G. lucidum group compared to the T/D group. Mestrovic et al. suggested that GSH activities in the ipsilateral testes of the treatment group were significantly higher than those in the T/D group 15 . In our study, GSH activities in testicular tissue significantly increased in the T/ D+ G. lucidum group compared to the T/D group. Results from I/R studies of G. lucidum in brain, kidney and heart comply with the results of our study with decreased MDA and increased SOD and GSH 16 . In the literature, there are testis I/R studies using apocynin, nifedipine and urapidil. The results of these studies comply with ours with these medications reducing MDA levels and increasing SOD and GSH, showing antioxidant effect 14,15,17 . Histological alterations similar to the results in our study were also demonstrated in another testicular I/R study conducted by Ozbek et al. 14 19 suggested that administering VEGF before reperfusion might have the potential to decrease long-term histologic damage after testicular torsion. In our study, VEGF expression was positive in the T/D group in the endothelial cells in the interstitial blood vessels, in some inflammatory cells and Leydig cells, in the apical faces of the spermatogenic cells and in Sertoli cells. In the T/D+ G. lucidum group, positive VEGF expression was observed in a small number of spermatogenic cells, especially in Sertoli cells, and it was negative in interstitial endothelial cells and Leydig cells. This may indicate that G. lucidum has a protective effect on VEGF expression linked to the testis. Kanter et al. 12 stated that sensitivity to modulated apoptotic factors was higher in spermatogonia and spermatocytes, whereas Sertoli and Leydig cells were highly resistant. A study showed that the amount of VEGF and Bcl-2 both decreased in testicular tissue, showing positive correlation in diabetic rats, and that both proteins were involved in cell proliferation, apoptosis and angiogenesis in the pathophysiology of diabetes 13 . In our study, intense Bcl-2 expression was observed in spermatogenic cells with strong mitotic activity in the basal layers of tubules in the T/D group. Bcl-2 expression was also found to be higher in Sertoli cells. Bcl-2-positive germinal cells were fewer in the G. lucidum-treated group compared to the T/D group. G.lucidum administered after T/D injury was estimated to decrease the effect of Bcl-2 expression and inhibit apoptosis. A similar study by Sumii et al. 20 showed that urocortin reduced Bcl-2 expression and had an antiapoptotic effect.

■ Conclusions
G. lucidum administration may decrease oxidative stress and histopathological damage. G. lucidum has a significant protective effect against testicular T/D damage in rats. This protective effect is thought to be mainly due to its antioxidative property, and it is thought to induce angiogenic effect and decrease apoptotic development. The use of G. lucidum therapy after reperfusion may be an alternative to germ cell degeneration resulting from testicular torsion and associated infertility.