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reperfusion injury in rats
Journal of Pediatric Surgery xxx (2016) xxx–xxx
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Protective effect of pterostilbene on testicular ischemia/reperfusion injury in rats Hyung Joon Kim a, Jae-Won Lee a, Bo Ram Hwang a, Young-Ah Lee b, Jong-In Kim a, Yoon Ju Cho c, Hyun jhung Jhun d, Jin Soo Han a,⁎ a
The Institute for the 3Rs & Department of Laboratory Animal Medicine, College of Veterinary Medicine, Konkuk University, 1 Hwayang-dong, Gwangjin-gu, Seoul 143-701, Republic of Korea Department of Animal Science, Shingu College University, 2 Geumgwang-dong, Jungwon-gu, Seongnam-si, Gyeonggi-do 462-743, Republic of Korea Department of Pet Science, Seojeong College University, Yongam-ri, Eunhyeon-m yeon, Yangju-si, Gyeonggi-do 482-777, Republic of Korea d Laboratory Animal Research Center, Konkuk University, 1 Hwayang-dong, Gwangjin-gu, Seoul 143-701, Republic of Korea b c
a r t i c l e
i n f o
Article history: Received 27 July 2015 Received in revised form 28 December 2015 Accepted 21 January 2016 Available online xxxx Key words: Antioxidant Apoptosis Caspases Testicular torsion
a b s t r a c t Purpose: To evaluate protective effect of pterostilbene against testicular ischemia/reperfusion (I/R) injury, which results in increased formation of oxidative stress, leading to testicular apoptosis and impaired spermatogenesis. Methods: Thirty two pubertal male Sprague–Dawley rats weighing 180–220 g were selected and randomly divided into the following four groups: group A (normal control group), group B (sham-operated group), group C (induced I/R injury group), group D (induced I/R injury group receiving pterostilbene treatment). Johnsen's scores and mean seminiferous tubule diameters were evaluated for histopathologic assessment; germinal cell apoptosis was evaluated by the transferase dUTP nick end labeling (TUNEL) assay and immunohistochemistry for caspases. Malondialdehyde (MDA) levels were assessed as an indicator of oxidative stress and total antioxidant capacity (TAC) was measured. Results: Germ cell apoptosis and MDA level significantly increased whereas TAC significantly decreased in group C; moreover, abnormal morphology and impaired spermatogenesis were observed in group C. In contrast, treatment with pterostilbene inhibited lipid peroxidation and apoptosis induced by ROS and restored the antioxidant capacity in group D. Conclusions: These results show that treatment with pterostilbene may be a promising therapy for testicular I/R injury. © 2016 Elsevier Inc. All rights reserved.
Testicular torsion is a human urologic emergency induced by torsion of the spermatic cord. It causes ischemia, immediate circulatory changes, testicular dysfunction, and fertility loss. Therefore, it should be reperfused within an appropriate critical time interval for maintaining testicular functions such as production of hormones and sperm. Although reperfusion is necessary for the survival of testicular cells and tissues, it is associated with reactive oxygen species (ROS) and reactive nitrogen species (RNS) production that induce a cascade of events causing testicular ischemia/reperfusion (I/R) injury [1]. It is assumed that reperfusion itself leads to more severe cell and tissue damage than ischemia-induced reduction in oxygen [2]. Pterostilbene is a natural compound found primarily in blueberries and Pterocarpus marsupium (PM) heartwood [3]. It belongs to the group of phytoalexins, which are produced by plants in response to pathogens such as bacteria or fungi [4]. Pterostilbene has been reported to reduce oxidative stress and ROS generation and upregulate the expression of various antioxidant enzymes such as glutathione (GSH), glutathione-S-transferase (GST), superoxide dismutase (SOD),
glutathione peroxidase (GPx), and catalase [3]. Furthermore, it ameliorates I/R injury in the heart [5]. Therefore, pterostilbene is also expected to ameliorate I/R injury in the testis, however, there have been no reports on the protective effect of pterostilbene on testicular I/R injury thus far. In the present study, the authors evaluated the protective effects of pterostilbene against testicular I/R injury. Histopathological assessment was conducted after hematoxylin–eosin staining. Additionally, the location and extent of apoptosis were examined by using the transferase dUTP nick end labeling (TUNEL) assay. Immunohistochemistry (IHC) revealed that the protective mechanism of pterostilbene involved caspase-3, 8, and 9 expression. Finally, malondialdehyde (MDA) levels were measured as an indicator of lipid peroxidation and testicular total antioxidant capacities (TACs) were evaluated.
1. Materials and methods 1.1. Experimental rat model
⁎ Corresponding author: The Institute for the 3Rs & Department of Laboratory Animal Medicine, College of Veterinary Medicine, Konkuk University, Seoul 143-701, Republic of Korea. Tel.: +82 2 2049 6114; fax: +82 2 454 3932. E-mail address: [email protected] (J.S. Han).
All experimental procedures and protocols were approved by the Institutional Animal Care and Use Committee (IACUC) of Konkuk University and conformed to the National Institutes of Health Guide for the
http://dx.doi.org/10.1016/j.jpedsurg.2016.01.009 0022-3468/© 2016 Elsevier Inc. All rights reserved.
Please cite this article as: Kim HJ, et al, Protective effect of pterostilbene on testicular ischemia/reperfusion injury in rats, J Pediatr Surg (2016), http://dx.doi.org/10.1016/j.jpedsurg.2016.01.009
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H.J. Kim et al. / Journal of Pediatric Surgery xxx (2016) xxx–xxx
Care and Use of Laboratory Animals. Thirty-two pubertal male Sprague– Dawley rats (180–220 g) were purchased from KOATECH (Gyeonggi do, Korea) and randomly divided into the following four groups: group A, a normal control group with no surgical stress and I/R; group B, a shamoperated control group with surgical stress and no I/R; group C, an I/R group without treatment; and group D, an I/R group treated with pterostilbene (BP1175, Chengdu, Sichuan, China). All surgical procedures were performed under deep anesthesia induced by intramuscular injection of xylazine (5 mg/kg) and Zoletil (40 mg/kg). After midline incision of the scrotum, the left testis was exteriorized. In group A, the testis was promptly extracted. In group B, the exteriorized testis was promptly returned to the scrotum, and a 4-0 polyamide monofilament nonabsorbable surgical suture was used to close the tunica albuginea and the midline incision in the scrotum. In groups C and D, the left testis with the spermatic cord was rotated 720° in the clockwise direction and attached to the scrotum with a 4-0 surgical suture in order to maintain the ischemic state. After 5 h of ischemia, the rotated testis was released by rotating it counterclockwise to induce reperfusion [6]. Pterostilbene (50 mg/kg) was simultaneously injected intraperitoneally in group D. The concentration of pterostilbene that showed therapeutic effect during the pilot test was used for the experiments. After the surgery, ketoprofen (5 mg/kg) was injected subcutaneously for reducing pain. In groups B, C, and D, the testis was extracted by using the same anesthetic protocol 24 h after reperfusion. 1.2. Histopathological evaluation of the testis The testicular tissue was fixed in modified Davidson's fluid (mDF) [7]. Paraffin-embedded tissue was cut into 4-μm-thick slices and attached to slides at 60 °C. Tissue sections were stained with hematoxylin and eosin. Johnsen's scores and mean seminiferous tubular diameters (MSTDs) were used for histopathological evaluation. Ten continuous seminiferous tubules were evaluated, and the mean diameter was calculated. Each seminiferous tubule was assigned a score ranging from 10 to 1 according to the Johnsen's scoring system; the scoring is performed based on the state of germinal epithelium and the presence of germinal cells in the seminiferous tubules [8]. MSTD was calculated from 20 seminiferous tubular diameters [9]. 1.3. Evaluation of germinal cell apoptosis Apoptosis of germinal cells was evaluated with the TUNEL assay using the In Situ Cell Death Detection Kit (Roche Applied Sciences, Mannheim, Germany) following the manufacturer's instructions. After the tissue sections were attached to poly-L-lysine-coated slides at 60 °C, they were deparaffinized and rehydrated. Antigen retrieval was performed using a microwave oven. The slides were boiled in 10 mM citrate buffer, pH 6, in the microwave oven for 10 min. After rinsing with phosphate-buffered saline (PBS) twice, the sections were incubated in the blocking solution for 10 min. After three rinses with PBS, the sections were incubated in the TUNEL reaction mixture at 37 °C for 1 h. Following three more rinses with PBS, 50 μL of converter-POD was added, and the sections were incubated in a humidified chamber for 30 min at 37 °C. After rinsing with PBS three times, the sections were treated with 3,3′-diaminobenzidine (DAB) for 10 s, followed by counterstaining with hematoxylin. The degree of apoptosis was evaluated based on the apoptotic indices (AI) 1 and 2. AI-1 was defined as the mean number of apoptotic TUNEL-positive cells per positive tubule section in 100 tubules, and AI-2 was defined as the ratio of the number of positive tubules to 100 tubules [6]. The testicular tissues were examined by two qualified pathologists blinded to their origin. 1.4. Evaluation of testicular lipid peroxidation MDA levels were evaluated with the thiobarbituric acid assay using a commercially available kit (Cell Biolabs, San Diego, CA, USA) according
to the manufacturer's instructions. Each tissue was resuspended at 100 mg/mL in PBS, and 10 μL of 100× BHT (butylated hydroxytoluene) was added to 1 mL of sample to prevent further oxidation. All samples were homogenized on ice and centrifuged at 10,000g for 5 min to collect the supernatant. A dilution series of MDA standards was prepared in the concentration range of 125 to 0 μM by diluting in distilled water. Each MDA-containing sample and standard was assayed in duplicates. Onehundred microliters of samples and standards was added to microcentrifuge tubes. One-hundred microliters of sodium dodecyl sulfate lysis solution was added to both the samples and the MDA standards, and the samples were incubated for 5 min at room temperature. One-hundred microliters of TBA (thiobarbituric acid) reagent was added to both the samples and standards. After incubating for an hour at 95 °C, the tubes were cooled to room temperature in an ice bath for 5 min. All samples were centrifuged at 3000 rpm for 15 min and the supernatants were separated. Supernatants of the samples and standards were transferred to a 96-well microplate. The spectrophotometric scans were performed at 532 nm (Microplate Reader, Tecan Austria GmbH 5082 Grödig, Austria). 1.5. Evaluation of testicular total antioxidant capacity TAC was evaluated by using a commercially available kit (Cell Biolabs, San Diego, CA, USA) following the manufacturer's instructions. Each tissue was homogenized in cold PBS and centrifuged at 10,000g for 10 min at 4 °C to collect the supernatant. A dilution series of uric acid was prepared in the concentration range of 1 to 0 mM by diluting 2 mM uric acid antioxidant standard in deionized water. Uric acid standards and samples were assayed in duplicates. Twenty microliters of the diluted uric acid standards and samples was added to the 96-well microtiter plate. After adding 180 μL of 1× reaction buffer to each well, initial absorbance was read. The reading was repeated after adding 50 μL of 1× copper ion reagent to each well and incubating for 5 min to initiate the reaction; 50 μL of the 1× stop solution was used to terminate the reaction in each well. The spectrophotometric scans were performed at 490 nm. 1.6. Evaluation of the pattern of immunohistochemical staining for active caspases Anti-active caspase-3 antibody (ab2302), anti-caspase-8 antibody (ab4052), and anti-caspase-9 antibody (ab32539) from Abcam, Cambridge, UK, were used to evaluate the expression level of caspases. After attaching to a poly-L-lysine-coated slide at 60 °C, tissue sections were deparaffinized and rehydrated. Antigen retrieval was performed using a microwave oven. The slides were boiled in 10 mM citrate buffer, pH 6, in the microwave oven for 10 min. The sections were then incubated in 3% H2O2 for 30 min at room temperature. After rinsing in PBS for 5 min, the sections were incubated with the blocking solution for 30 min. After the blocking solution was removed, the primary antibody to active caspase diluted 50:1 was added, and the sections were incubated in a humidified chamber at 4 °C overnight. The sections were then rinsed in PBS for 5 min and incubated with diluted biotinylated “universal” secondary antibody (Vector Laboratories, Burlingame, CA, USA) for 30 min. After rinsing in PBS for 5 min, the sections were incubated with the VECTASTAINN R.T.U Elite ABC reagent (Vector Laboratories, Burlingame, CA, USA) for 30 min. After the DAB substrate (DAKO North America, Carpinteria, CA, USA) was applied to the sections for 10 min, they were counterstained with hematoxylin. The level of caspase expression was evaluated based on the mean number of positive cells per positive tubule in 100 tubules (PC/PT), the ratio of the number of positive tubules to 100 tubules (PT/CT), and the mean number of positive cells per 100 tubules (PC/CT). The testicular tissues were examined by two qualified pathologists blinded to their origin.
Please cite this article as: Kim HJ, et al, Protective effect of pterostilbene on testicular ischemia/reperfusion injury in rats, J Pediatr Surg (2016), http://dx.doi.org/10.1016/j.jpedsurg.2016.01.009
H.J. Kim et al. / Journal of Pediatric Surgery xxx (2016) xxx–xxx Table 1 Mean seminiferous tubule diameter (MSTD) values and Johnsen's scores in the left testis. Group
MSTD
A B C D
277.44 238.55 203.76 247.59
3
(P b 0.05; Table 2). In group D, the MDA level significantly decreased compared to that in group C (P b 0.05).
Johnsen's score ± ± ± ±
6.84* 13.05*,† 8.11† 17.03*,†
9.18 8.88 5.38 9.14
± ± ± ±
0.26* 0.32* 0.19† 0.29*
MSTD: mean seminiferous tubule diameter. Groups: A, normal control; B, sham control (surgical stress); C, ischemia/reperfusion; D, ischemia/reperfusion with pterostilbene treatment. ⁎ P b 0.05 vs. group C. † P b 0.05 vs. group A.
2.4. Testicular total antioxidant capacity values The TAC in the left testis significantly reduced in group C compared to that in the normal control and sham control groups (P b 0.05; Table 2). In group D, the TAC significantly increased compared to that in group C (P b 0.05).
2.5. Comparison of the expression levels of active caspase-3 1.7. Statistical analysis One-way analysis of variance with the Tukey's test was used for multiple comparisons. All data were expressed as means ± SD, and the significance level was set at 0.05. The results were compared between groups by using statistical software. IBM® SPSS® Statistics 22.0 was used for data analysis. 2. Results 2.1. Assessment of spermatogenesis Table 1 shows the MSTD values and Johnsen's scores for all the groups. The MSTD values and Johnsen's scores in group C were significantly lower than those in groups A and B (P b 0.05). The MSTD values and Johnsen's scores in group D were significantly greater than those in group C (P b 0.05). 2.2. Evaluation of germinal cell apoptosis The extent of germinal cell apoptosis expressed as AI-1 and AI-2 was evaluated by using the TUNEL assay and microscopy (Fig. 1). AI-1 and AI-2 in group C increased significantly compared to those in group A (Table 2), while those in group B were comparable to those in group A. In group D, wherein rats received pterostilbene after reperfusion, AI-1 and AI-2 decreased significantly compared to the values in group C (P b 0.05). 2.3. Testis malondialdehyde levels The MDA level in the left testis significantly increased in group C compared to that in the normal control and sham control groups
Immunohistochemistry for caspase-3 revealed a significant increase (P b 0.05) of caspase-3 expression in group C compared to that in groups A and group B. The calculated ratio in group D reduced significantly compared to that in group C (P b 0.05).
2.6. Comparison of the expression levels of active caspase-8 Immunohistochemistry for caspase-8 revealed a significant increase (P b 0.05) of caspase-8 expression in group C compared to that in groups A and group B. The calculated ratio in group D reduced significantly compared to that in group C (P b 0.05).
2.7. Comparison of the expression levels of active caspase-9 Immunohistochemistry for caspase-9 revealed a significant increase (P b 0.05) of caspase-9 expression in group C compared to that in groups A and group B. The calculated ratio in group D reduced significantly compared to that in group C (P b 0.05).
2.8. Comparison of the expression levels of caspases Immunohistochemistry for caspase-3, 8, and 9 (Fig. 2) revealed a significant increase (P b 0.05) of PC/PT, PT/CT, and PC/CT in group C compared to those in groups A and B; a significant decrease (P b 0.05) in these parameters was detected in group D compared to those in group C. A comparison of the values of PC/CT of each caspase in group C showed that caspase-3 expression was significantly higher than that of caspase-8, and caspase-9 expression was significantly higher than that of caspase-3.
Fig. 1. Hematoxylin and eosin (H&E) staining and detection of apoptotic cells in the left testis in the 4 groups H&E staining. Groups A and B: Normal testicular structure, well-defined seminiferous tubule. Group C: Hypospermatogenesis, severely impaired seminiferous tubules. Group D: Normal testicular structure and well-defined seminiferous tubule comparable to that in groups A and B. TUNEL assay: There were few apoptotic cells in groups A and B. The number of apoptotic cells increased significantly in group C. The number of apoptotic cells decreased significantly in group D. Groups: A, normal control; B, sham control; C, ischemia/reperfusion; D, ischemia/reperfusion with pterostilbene treatment.
Please cite this article as: Kim HJ, et al, Protective effect of pterostilbene on testicular ischemia/reperfusion injury in rats, J Pediatr Surg (2016), http://dx.doi.org/10.1016/j.jpedsurg.2016.01.009
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H.J. Kim et al. / Journal of Pediatric Surgery xxx (2016) xxx–xxx
Table 2 Apoptotic indices 1 and 2 (AI-1 and AI-2), malondialdehyde (MDA) levels, and total antioxidant capacity (TAC) values in the left testis. Group
AI-1
A B C D
1.20 1.52 43.99 1.92
AI-2 ± ± ± ±
0.22* 0.48* 6.00† 0.34*
0.07 0.06 0.96 0.13
MDA ± ± ± ±
0.16* 0.02* 0.04† 0.06*
1.02 1.33 6.78 1.15
± ± ± ±
TAC 0.08* 0.32* 1.19† 0.10*
1042.61 1082.48 444.20 1150.76
± ± ± ±
141.31* 85.12* 22.80† 100.38*
AI-1 (apoptotic index 1): the mean number of apoptotic TUNEL-positive cells per positive tubule in 100 tubules. AI-2 (apoptotic index 2): the ratio of the number of positive tubules to 100 tubules. MDA levels are expressed in μM. TAC values are expressed in μM of copper reducing equivalents. Groups: A, normal control; B, sham control (surgical stress); C, ischemia/reperfusion; D, ischemia/reperfusion with pterostilbene treatment. ⁎ P b 0.05 vs. group C. † P b 0.05 vs. group A.
3. Discussion Testicular torsion is a urologic emergency. Late presentation or failure to diagnose and correctly manage this condition leads to testicular injury and subfertility [10]. Testicular torsion should be treated within a proper critical time interval to maintain testicular function because the time interval between torsion and detorsion is an important factor affecting the extent of germinal cell damage [11]. Although it is known that testicular function can be restored through rapid diagnosis and treatment, a study has reported that 29%–50% of patients with immediate restoration of blood flow to the testis develop infertility [12,13]. This infertility is caused by the imbalance between restoration of oxygen supply and mitochondrial respiratory function, resulting in the excessive generation of superoxide in mitochondria. Under these circumstances, free oxygen radical scavenging enzymes, such as SOD, CAT, and GPx are needed to cope with oxidative stress and oxidative damage in the testicular tissue [14]. Even if the levels of these enzymes increase following testicular I/R, this response is insufficient to adequately counteract the excessive ROS and RNS generation occurring during reperfusion. The increased levels of ROS and RNS cause DNA fragmentation, lipid peroxidation, and protein inactivation [15,16]. Therefore, scavenging these substances during testicular I/R injury has beneficial effects that may lead to better outcomes. It has been recently reported that treatment with resveratrol, an analogue of pterostilbene, reduced germinal cell apoptosis in a rat model of testicular I/R injury induced by left testis torsion and detorsion surgery [17]. Similar to resveratrol, pterostilbene has anti-inflammatory, antioxidant, and anticancer activity [3].
To the best of our knowledge, this is the first study to investigate the effect of pterostilbene against testicular I/R injury. It has been demonstrated that pterostilbene administration results in the recovery of testicular function to a near normal level in the testicular I/R rat model. Consistent with the results of earlier studies, testicular I/R induced deterioration of the structure of seminiferous tubules, reduced their diameter, and impaired spermatogenesis, in addition to increasing testicular MDA levels and apoptotic cell numbers, upregulating the expression levels of caspases, the key factors of the apoptotic pathway, and decreasing testicular TAC [18]. At least 1 h of 720° testicular torsion is required to induce ischemia in the rat testis [19]. In contrast, it has been reported that 4 h of testicular torsion is required to induce ischemia in the human testis [11]. In this study, 720° testicular torsion for maintained for 5 h to mimic the human condition. In addition, the analyses were conducted 24 h after reperfusion because germ cell apoptosis was shown to be highest at this time point [20]. In agreement with other studies using rat testicular I/R injury models, the MSTD significantly decreased in group C compared to that in groups A and B [4]. Spermatogenesis also significantly decreased in group C compared to that in groups A and B, as evaluated using Johnsen's scores. Finally, the MSTD and Johnsen's score in group D significantly increased compared to the corresponding values in group C; these parameters (in group D) were similar to those observed for groups A and B. The processes observed in the testis were related to apoptosis induced by oxidative stress. In this study, the mean number of apoptotic cells in a seminiferous tubule (AI-1) and the ratio of the number of positive tubules to 100 tubules (AI-2), as well as the degree of lipid peroxidation (MDA) significantly increased in group C compared to the corresponding values in groups A and B. In addition, the TAC level decreased in group C compared to that in groups A and B. Finally, in group D, AI-1, AI-2, and MDA levels significantly decreased, and the TAC level increased compared to the corresponding values in group C; however, these values were similar to those observed for groups A and B. The above results are similar to those of other studies and permit us to conclude that testicular I/R induces excessive oxidative stress and exhausts the antioxidant system, causing germinal cell apoptosis, and administration of pterostilbene reverses these changes [21]. Caspase-3 is part of the common pathway of apoptosis stimulation. A dose-dependent activation and inhibition of caspase-3 activity by pterostilbene have been demonstrated in vitro [18]. Accordingly, IHC for caspase-3 was performed in each group to determine whether the apoptosis induced by testicular torsion/detorsion is mediated via the common pathway, and whether pterostilbene affects this pathway.
Fig. 2. Comparison of the levels of caspase expression. A: Caspase-3-, 8-, and 9-positive cells per positive tubule in 100 tubules were calculated. B: Caspase-3, 8-, and 9-positive tubules per 100 tubules were calculated. C: Caspase-3, 8-, and 9-positive tubules per 100 tubules were calculated. Groups: A, normal control; B, sham control; C, ischemia/reperfusion; D, ischemia/ reperfusion with pterostilbene treatment *P b 0.5 vs. group C, †P b 0.5 vs. group A.
Please cite this article as: Kim HJ, et al, Protective effect of pterostilbene on testicular ischemia/reperfusion injury in rats, J Pediatr Surg (2016), http://dx.doi.org/10.1016/j.jpedsurg.2016.01.009
H.J. Kim et al. / Journal of Pediatric Surgery xxx (2016) xxx–xxx
This study used a dose that was determined to reduce apoptosis in a pilot test and other studies. The results of IHC in the study groups showed that the expression level of caspase-3 significantly increased in group C compared to the expression levels in groups A and B, although the expression level of caspase-3 in the testis is generally low. Importantly, the expression level of caspase-3 significantly increased in group D compared to that in group C. Caspase-8 participates in the extrinsic pathway and caspase-9 in the intrinsic pathway of apoptosis [22]. In this study, IHC for caspase-8 and 9 was performed in each group to determine whether the apoptosis induced by testicular torsion/detorsion is mediated via the intrinsic or the extrinsic pathway, and whether pterostilbene affects either of these pathways. The expression level of caspase-9 and 8 was found to be significantly upregulated in group C compared to their corresponding values in groups A and B. Furthermore, this level significantly increased in group D compared to that in group C. However, comparison of the values of PC/CT of each caspase in group C revealed that the caspase-9 expression was significantly higher than that of caspase-8, and caspase-8 expression was even significantly lower than caspase-3 expression. These results and other studies may permit us to conclude that germinal cell apoptosis induced by testicular I/R occurred through the intrinsic pathway via caspase-9 rather than the extrinsic pathway via caspase-8, and pterostilbene inhibited apoptosis by suppressing the expression of caspase-3 and caspase-9 [23]. However, this study has some limitations. As mentioned earlier, testicular torsion can occur in newborns, children, and adolescents. Since testicular torsion can also occur in prepubertal children or neonates, studies on neonatal or prepubertal rats are needed. However, this study uses pubertal rats to mimic pubertal children or adolescents and to evaluate the protective effect of pterostilbene on testicular I/R injury. Therefore, it is unclear whether pterostilbene will show the same protective effect on testicular I/R injury in neonatal or prepubertal rats. In addition, this study evaluates only the short-term effect of pterostilbene. Therefore, additional study is required for evaluating the protective effect on testicular I/R injury in neonatal or prepubertal rats, and long-term effect of pterostilbene on I/R injury in rats. 4. Conclusions These findings demonstrate that pterostilbene protects against testicular I/R injury, which results in increased formation of oxidative stress, leading to testicular apoptosis and impaired spermatogenesis. In conclusion, treatment with pterostilbene may be a promising therapy for testicular I/R injury.
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Acknowledgments This study was supported by Konkuk University in 2014. References [1] Mogilner JG, Elenberg Y, Lurie M, et al. Effect of dexamethasone on germ cell apoptosis in the contralateral testis after testicular ischemia–reperfusion injury in the rat. Fertil Steril 2006;85:1111–7. [2] Hanci V, Erol B, Bektaş S, et al. Effect of dexmedetomidine on testicular torsion/ detorsion damage in rats. Urol Int 2009;84:105–11. [3] McFadden D. A review of pterostilbene antioxidant activity and disease modification. Oxid Med Cell Longev 2013;2013. [4] Langcake P, Cornford C, Pryce R. Identification of pterostilbene as a phytoalexin from Vitis vinifera leaves. Phytochemistry 1979;18:1025–7. [5] Lv M, Liu K, Fu S, et al. Pterostilbene attenuates the inflammatory reaction induced by ischemia/reperfusion in rat heart. Mol Med Rep 2015;11:724–8. [6] Lee J-W, Kim J-I, Lee Y-A, et al. Inhaled hydrogen gas therapy for prevention of testicular ischemia/reperfusion injury in rats. J Pediatr Surg 2012;47:736–42. [7] Latendresse JR, Warbrittion AR, Jonassen H, et al. Fixation of testes and eyes using a modified Davidson's fluid: comparison with Bouin's fluid and conventional Davidson's fluid. Toxicol Pathol 2002;30:524–33. [8] Sukhotnik I, Voskoboinik K, Lurie M, et al. Effect of testicular ischemia–reperfusion on recruitment of neutrophils, E-selectin expression and germ cell apoptosis in the contralateral testis in a rat. Pediatr Surg Int 2007;23:479–85. [9] Tunçkıran A, Çayan S, Bozlu M, et al. Protective effect of vascular endothelial growth factor on histologic changes in testicular ischemia–reperfusion injury. Fertil Steril 2005;84:468–73. [10] Visser A, Heyns C. Testicular function after torsion of the spermatic cord. BJU Int 2003;92:200–3. [11] Kapoor S. Testicular torsion: a race against time. Int J Clin Pract 2008;62:821–7. [12] Ringdahl E, Teague L. Testicular torsion. Am Fam Physician 2006;74:1739–43. [13] Bartsch G, Frank S, Marberger H, et al. Testicular torsion: late results with special regard to fertility and endocrine function. J Urol 1980;124:375–8. [14] Uz E, Söğüt S, Şahin Ş, et al. The protective role of caffeic acid phenethyl ester (CAPE) on testicular tissue after testicular torsion and detorsion. World J Urol 2002;20: 264–70. [15] Hayashida K, Sano M, Ohsawa I, et al. Inhalation of hydrogen gas reduces infarct size in the rat model of myocardial ischemia–reperfusion injury. Biochem Biophys Res Commun 2008;373:30–5. [16] Kuroda S, Siesjö B. Reperfusion damage following focal ischemia: pathophysiology and therapeutic windows. Clin Neurosci 1996;4:199–212. [17] Yuluğ E, Türedi S, Karagüzel E, et al. The short term effects of resveratrol on ischemia–reperfusion injury in rat testis. J Pediatr Surg 2014;49:484–9. [18] Porter AG, Jänicke RU. Emerging roles of caspase-3 in apoptosis. Cell Death Differ 1999;6:99–104. [19] Turner T, Brown K. Spermatic cord torsion: loss of spermatogenesis despite return of blood flow. Biol Reprod 1993;49:401–7. [20] Lysiak JJ, Turner SD, Turner TT. Molecular pathway of germ cell apoptosis following ischemia/reperfusion of the rat testis. Biol Reprod 2000;63:1465–72. [21] Ozbek O, Altintas R, Polat A, et al. The protective effect of apocynin on testicular ischemia–reperfusion injury. J Urol 2015;193:1417–22. [22] Elmore S. Apoptosis: a review of programmed cell death. Toxicol Pathol 2007;35: 495–516. [23] Lysiak JJ, Zheng S, Woodson R, et al. Caspase-9-dependent pathway to murine germ cell apoptosis: mediation by oxidative stress, BAX, and caspase 2. Cell Tissue Res 2007;328:411–9.
Please cite this article as: Kim HJ, et al, Protective effect of pterostilbene on testicular ischemia/reperfusion injury in rats, J Pediatr Surg (2016), http://dx.doi.org/10.1016/j.jpedsurg.2016.01.009
Contents lists available at ScienceDirect
Journal of Pediatric Surgery journal homepage: www.elsevier.com/locate/jpedsurg
Protective effect of pterostilbene on testicular ischemia/reperfusion injury in rats Hyung Joon Kim a, Jae-Won Lee a, Bo Ram Hwang a, Young-Ah Lee b, Jong-In Kim a, Yoon Ju Cho c, Hyun jhung Jhun d, Jin Soo Han a,⁎ a
The Institute for the 3Rs & Department of Laboratory Animal Medicine, College of Veterinary Medicine, Konkuk University, 1 Hwayang-dong, Gwangjin-gu, Seoul 143-701, Republic of Korea Department of Animal Science, Shingu College University, 2 Geumgwang-dong, Jungwon-gu, Seongnam-si, Gyeonggi-do 462-743, Republic of Korea Department of Pet Science, Seojeong College University, Yongam-ri, Eunhyeon-m yeon, Yangju-si, Gyeonggi-do 482-777, Republic of Korea d Laboratory Animal Research Center, Konkuk University, 1 Hwayang-dong, Gwangjin-gu, Seoul 143-701, Republic of Korea b c
a r t i c l e
i n f o
Article history: Received 27 July 2015 Received in revised form 28 December 2015 Accepted 21 January 2016 Available online xxxx Key words: Antioxidant Apoptosis Caspases Testicular torsion
a b s t r a c t Purpose: To evaluate protective effect of pterostilbene against testicular ischemia/reperfusion (I/R) injury, which results in increased formation of oxidative stress, leading to testicular apoptosis and impaired spermatogenesis. Methods: Thirty two pubertal male Sprague–Dawley rats weighing 180–220 g were selected and randomly divided into the following four groups: group A (normal control group), group B (sham-operated group), group C (induced I/R injury group), group D (induced I/R injury group receiving pterostilbene treatment). Johnsen's scores and mean seminiferous tubule diameters were evaluated for histopathologic assessment; germinal cell apoptosis was evaluated by the transferase dUTP nick end labeling (TUNEL) assay and immunohistochemistry for caspases. Malondialdehyde (MDA) levels were assessed as an indicator of oxidative stress and total antioxidant capacity (TAC) was measured. Results: Germ cell apoptosis and MDA level significantly increased whereas TAC significantly decreased in group C; moreover, abnormal morphology and impaired spermatogenesis were observed in group C. In contrast, treatment with pterostilbene inhibited lipid peroxidation and apoptosis induced by ROS and restored the antioxidant capacity in group D. Conclusions: These results show that treatment with pterostilbene may be a promising therapy for testicular I/R injury. © 2016 Elsevier Inc. All rights reserved.
Testicular torsion is a human urologic emergency induced by torsion of the spermatic cord. It causes ischemia, immediate circulatory changes, testicular dysfunction, and fertility loss. Therefore, it should be reperfused within an appropriate critical time interval for maintaining testicular functions such as production of hormones and sperm. Although reperfusion is necessary for the survival of testicular cells and tissues, it is associated with reactive oxygen species (ROS) and reactive nitrogen species (RNS) production that induce a cascade of events causing testicular ischemia/reperfusion (I/R) injury [1]. It is assumed that reperfusion itself leads to more severe cell and tissue damage than ischemia-induced reduction in oxygen [2]. Pterostilbene is a natural compound found primarily in blueberries and Pterocarpus marsupium (PM) heartwood [3]. It belongs to the group of phytoalexins, which are produced by plants in response to pathogens such as bacteria or fungi [4]. Pterostilbene has been reported to reduce oxidative stress and ROS generation and upregulate the expression of various antioxidant enzymes such as glutathione (GSH), glutathione-S-transferase (GST), superoxide dismutase (SOD),
glutathione peroxidase (GPx), and catalase [3]. Furthermore, it ameliorates I/R injury in the heart [5]. Therefore, pterostilbene is also expected to ameliorate I/R injury in the testis, however, there have been no reports on the protective effect of pterostilbene on testicular I/R injury thus far. In the present study, the authors evaluated the protective effects of pterostilbene against testicular I/R injury. Histopathological assessment was conducted after hematoxylin–eosin staining. Additionally, the location and extent of apoptosis were examined by using the transferase dUTP nick end labeling (TUNEL) assay. Immunohistochemistry (IHC) revealed that the protective mechanism of pterostilbene involved caspase-3, 8, and 9 expression. Finally, malondialdehyde (MDA) levels were measured as an indicator of lipid peroxidation and testicular total antioxidant capacities (TACs) were evaluated.
1. Materials and methods 1.1. Experimental rat model
⁎ Corresponding author: The Institute for the 3Rs & Department of Laboratory Animal Medicine, College of Veterinary Medicine, Konkuk University, Seoul 143-701, Republic of Korea. Tel.: +82 2 2049 6114; fax: +82 2 454 3932. E-mail address: [email protected] (J.S. Han).
All experimental procedures and protocols were approved by the Institutional Animal Care and Use Committee (IACUC) of Konkuk University and conformed to the National Institutes of Health Guide for the
http://dx.doi.org/10.1016/j.jpedsurg.2016.01.009 0022-3468/© 2016 Elsevier Inc. All rights reserved.
Please cite this article as: Kim HJ, et al, Protective effect of pterostilbene on testicular ischemia/reperfusion injury in rats, J Pediatr Surg (2016), http://dx.doi.org/10.1016/j.jpedsurg.2016.01.009
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H.J. Kim et al. / Journal of Pediatric Surgery xxx (2016) xxx–xxx
Care and Use of Laboratory Animals. Thirty-two pubertal male Sprague– Dawley rats (180–220 g) were purchased from KOATECH (Gyeonggi do, Korea) and randomly divided into the following four groups: group A, a normal control group with no surgical stress and I/R; group B, a shamoperated control group with surgical stress and no I/R; group C, an I/R group without treatment; and group D, an I/R group treated with pterostilbene (BP1175, Chengdu, Sichuan, China). All surgical procedures were performed under deep anesthesia induced by intramuscular injection of xylazine (5 mg/kg) and Zoletil (40 mg/kg). After midline incision of the scrotum, the left testis was exteriorized. In group A, the testis was promptly extracted. In group B, the exteriorized testis was promptly returned to the scrotum, and a 4-0 polyamide monofilament nonabsorbable surgical suture was used to close the tunica albuginea and the midline incision in the scrotum. In groups C and D, the left testis with the spermatic cord was rotated 720° in the clockwise direction and attached to the scrotum with a 4-0 surgical suture in order to maintain the ischemic state. After 5 h of ischemia, the rotated testis was released by rotating it counterclockwise to induce reperfusion [6]. Pterostilbene (50 mg/kg) was simultaneously injected intraperitoneally in group D. The concentration of pterostilbene that showed therapeutic effect during the pilot test was used for the experiments. After the surgery, ketoprofen (5 mg/kg) was injected subcutaneously for reducing pain. In groups B, C, and D, the testis was extracted by using the same anesthetic protocol 24 h after reperfusion. 1.2. Histopathological evaluation of the testis The testicular tissue was fixed in modified Davidson's fluid (mDF) [7]. Paraffin-embedded tissue was cut into 4-μm-thick slices and attached to slides at 60 °C. Tissue sections were stained with hematoxylin and eosin. Johnsen's scores and mean seminiferous tubular diameters (MSTDs) were used for histopathological evaluation. Ten continuous seminiferous tubules were evaluated, and the mean diameter was calculated. Each seminiferous tubule was assigned a score ranging from 10 to 1 according to the Johnsen's scoring system; the scoring is performed based on the state of germinal epithelium and the presence of germinal cells in the seminiferous tubules [8]. MSTD was calculated from 20 seminiferous tubular diameters [9]. 1.3. Evaluation of germinal cell apoptosis Apoptosis of germinal cells was evaluated with the TUNEL assay using the In Situ Cell Death Detection Kit (Roche Applied Sciences, Mannheim, Germany) following the manufacturer's instructions. After the tissue sections were attached to poly-L-lysine-coated slides at 60 °C, they were deparaffinized and rehydrated. Antigen retrieval was performed using a microwave oven. The slides were boiled in 10 mM citrate buffer, pH 6, in the microwave oven for 10 min. After rinsing with phosphate-buffered saline (PBS) twice, the sections were incubated in the blocking solution for 10 min. After three rinses with PBS, the sections were incubated in the TUNEL reaction mixture at 37 °C for 1 h. Following three more rinses with PBS, 50 μL of converter-POD was added, and the sections were incubated in a humidified chamber for 30 min at 37 °C. After rinsing with PBS three times, the sections were treated with 3,3′-diaminobenzidine (DAB) for 10 s, followed by counterstaining with hematoxylin. The degree of apoptosis was evaluated based on the apoptotic indices (AI) 1 and 2. AI-1 was defined as the mean number of apoptotic TUNEL-positive cells per positive tubule section in 100 tubules, and AI-2 was defined as the ratio of the number of positive tubules to 100 tubules [6]. The testicular tissues were examined by two qualified pathologists blinded to their origin. 1.4. Evaluation of testicular lipid peroxidation MDA levels were evaluated with the thiobarbituric acid assay using a commercially available kit (Cell Biolabs, San Diego, CA, USA) according
to the manufacturer's instructions. Each tissue was resuspended at 100 mg/mL in PBS, and 10 μL of 100× BHT (butylated hydroxytoluene) was added to 1 mL of sample to prevent further oxidation. All samples were homogenized on ice and centrifuged at 10,000g for 5 min to collect the supernatant. A dilution series of MDA standards was prepared in the concentration range of 125 to 0 μM by diluting in distilled water. Each MDA-containing sample and standard was assayed in duplicates. Onehundred microliters of samples and standards was added to microcentrifuge tubes. One-hundred microliters of sodium dodecyl sulfate lysis solution was added to both the samples and the MDA standards, and the samples were incubated for 5 min at room temperature. One-hundred microliters of TBA (thiobarbituric acid) reagent was added to both the samples and standards. After incubating for an hour at 95 °C, the tubes were cooled to room temperature in an ice bath for 5 min. All samples were centrifuged at 3000 rpm for 15 min and the supernatants were separated. Supernatants of the samples and standards were transferred to a 96-well microplate. The spectrophotometric scans were performed at 532 nm (Microplate Reader, Tecan Austria GmbH 5082 Grödig, Austria). 1.5. Evaluation of testicular total antioxidant capacity TAC was evaluated by using a commercially available kit (Cell Biolabs, San Diego, CA, USA) following the manufacturer's instructions. Each tissue was homogenized in cold PBS and centrifuged at 10,000g for 10 min at 4 °C to collect the supernatant. A dilution series of uric acid was prepared in the concentration range of 1 to 0 mM by diluting 2 mM uric acid antioxidant standard in deionized water. Uric acid standards and samples were assayed in duplicates. Twenty microliters of the diluted uric acid standards and samples was added to the 96-well microtiter plate. After adding 180 μL of 1× reaction buffer to each well, initial absorbance was read. The reading was repeated after adding 50 μL of 1× copper ion reagent to each well and incubating for 5 min to initiate the reaction; 50 μL of the 1× stop solution was used to terminate the reaction in each well. The spectrophotometric scans were performed at 490 nm. 1.6. Evaluation of the pattern of immunohistochemical staining for active caspases Anti-active caspase-3 antibody (ab2302), anti-caspase-8 antibody (ab4052), and anti-caspase-9 antibody (ab32539) from Abcam, Cambridge, UK, were used to evaluate the expression level of caspases. After attaching to a poly-L-lysine-coated slide at 60 °C, tissue sections were deparaffinized and rehydrated. Antigen retrieval was performed using a microwave oven. The slides were boiled in 10 mM citrate buffer, pH 6, in the microwave oven for 10 min. The sections were then incubated in 3% H2O2 for 30 min at room temperature. After rinsing in PBS for 5 min, the sections were incubated with the blocking solution for 30 min. After the blocking solution was removed, the primary antibody to active caspase diluted 50:1 was added, and the sections were incubated in a humidified chamber at 4 °C overnight. The sections were then rinsed in PBS for 5 min and incubated with diluted biotinylated “universal” secondary antibody (Vector Laboratories, Burlingame, CA, USA) for 30 min. After rinsing in PBS for 5 min, the sections were incubated with the VECTASTAINN R.T.U Elite ABC reagent (Vector Laboratories, Burlingame, CA, USA) for 30 min. After the DAB substrate (DAKO North America, Carpinteria, CA, USA) was applied to the sections for 10 min, they were counterstained with hematoxylin. The level of caspase expression was evaluated based on the mean number of positive cells per positive tubule in 100 tubules (PC/PT), the ratio of the number of positive tubules to 100 tubules (PT/CT), and the mean number of positive cells per 100 tubules (PC/CT). The testicular tissues were examined by two qualified pathologists blinded to their origin.
Please cite this article as: Kim HJ, et al, Protective effect of pterostilbene on testicular ischemia/reperfusion injury in rats, J Pediatr Surg (2016), http://dx.doi.org/10.1016/j.jpedsurg.2016.01.009
H.J. Kim et al. / Journal of Pediatric Surgery xxx (2016) xxx–xxx Table 1 Mean seminiferous tubule diameter (MSTD) values and Johnsen's scores in the left testis. Group
MSTD
A B C D
277.44 238.55 203.76 247.59
3
(P b 0.05; Table 2). In group D, the MDA level significantly decreased compared to that in group C (P b 0.05).
Johnsen's score ± ± ± ±
6.84* 13.05*,† 8.11† 17.03*,†
9.18 8.88 5.38 9.14
± ± ± ±
0.26* 0.32* 0.19† 0.29*
MSTD: mean seminiferous tubule diameter. Groups: A, normal control; B, sham control (surgical stress); C, ischemia/reperfusion; D, ischemia/reperfusion with pterostilbene treatment. ⁎ P b 0.05 vs. group C. † P b 0.05 vs. group A.
2.4. Testicular total antioxidant capacity values The TAC in the left testis significantly reduced in group C compared to that in the normal control and sham control groups (P b 0.05; Table 2). In group D, the TAC significantly increased compared to that in group C (P b 0.05).
2.5. Comparison of the expression levels of active caspase-3 1.7. Statistical analysis One-way analysis of variance with the Tukey's test was used for multiple comparisons. All data were expressed as means ± SD, and the significance level was set at 0.05. The results were compared between groups by using statistical software. IBM® SPSS® Statistics 22.0 was used for data analysis. 2. Results 2.1. Assessment of spermatogenesis Table 1 shows the MSTD values and Johnsen's scores for all the groups. The MSTD values and Johnsen's scores in group C were significantly lower than those in groups A and B (P b 0.05). The MSTD values and Johnsen's scores in group D were significantly greater than those in group C (P b 0.05). 2.2. Evaluation of germinal cell apoptosis The extent of germinal cell apoptosis expressed as AI-1 and AI-2 was evaluated by using the TUNEL assay and microscopy (Fig. 1). AI-1 and AI-2 in group C increased significantly compared to those in group A (Table 2), while those in group B were comparable to those in group A. In group D, wherein rats received pterostilbene after reperfusion, AI-1 and AI-2 decreased significantly compared to the values in group C (P b 0.05). 2.3. Testis malondialdehyde levels The MDA level in the left testis significantly increased in group C compared to that in the normal control and sham control groups
Immunohistochemistry for caspase-3 revealed a significant increase (P b 0.05) of caspase-3 expression in group C compared to that in groups A and group B. The calculated ratio in group D reduced significantly compared to that in group C (P b 0.05).
2.6. Comparison of the expression levels of active caspase-8 Immunohistochemistry for caspase-8 revealed a significant increase (P b 0.05) of caspase-8 expression in group C compared to that in groups A and group B. The calculated ratio in group D reduced significantly compared to that in group C (P b 0.05).
2.7. Comparison of the expression levels of active caspase-9 Immunohistochemistry for caspase-9 revealed a significant increase (P b 0.05) of caspase-9 expression in group C compared to that in groups A and group B. The calculated ratio in group D reduced significantly compared to that in group C (P b 0.05).
2.8. Comparison of the expression levels of caspases Immunohistochemistry for caspase-3, 8, and 9 (Fig. 2) revealed a significant increase (P b 0.05) of PC/PT, PT/CT, and PC/CT in group C compared to those in groups A and B; a significant decrease (P b 0.05) in these parameters was detected in group D compared to those in group C. A comparison of the values of PC/CT of each caspase in group C showed that caspase-3 expression was significantly higher than that of caspase-8, and caspase-9 expression was significantly higher than that of caspase-3.
Fig. 1. Hematoxylin and eosin (H&E) staining and detection of apoptotic cells in the left testis in the 4 groups H&E staining. Groups A and B: Normal testicular structure, well-defined seminiferous tubule. Group C: Hypospermatogenesis, severely impaired seminiferous tubules. Group D: Normal testicular structure and well-defined seminiferous tubule comparable to that in groups A and B. TUNEL assay: There were few apoptotic cells in groups A and B. The number of apoptotic cells increased significantly in group C. The number of apoptotic cells decreased significantly in group D. Groups: A, normal control; B, sham control; C, ischemia/reperfusion; D, ischemia/reperfusion with pterostilbene treatment.
Please cite this article as: Kim HJ, et al, Protective effect of pterostilbene on testicular ischemia/reperfusion injury in rats, J Pediatr Surg (2016), http://dx.doi.org/10.1016/j.jpedsurg.2016.01.009
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H.J. Kim et al. / Journal of Pediatric Surgery xxx (2016) xxx–xxx
Table 2 Apoptotic indices 1 and 2 (AI-1 and AI-2), malondialdehyde (MDA) levels, and total antioxidant capacity (TAC) values in the left testis. Group
AI-1
A B C D
1.20 1.52 43.99 1.92
AI-2 ± ± ± ±
0.22* 0.48* 6.00† 0.34*
0.07 0.06 0.96 0.13
MDA ± ± ± ±
0.16* 0.02* 0.04† 0.06*
1.02 1.33 6.78 1.15
± ± ± ±
TAC 0.08* 0.32* 1.19† 0.10*
1042.61 1082.48 444.20 1150.76
± ± ± ±
141.31* 85.12* 22.80† 100.38*
AI-1 (apoptotic index 1): the mean number of apoptotic TUNEL-positive cells per positive tubule in 100 tubules. AI-2 (apoptotic index 2): the ratio of the number of positive tubules to 100 tubules. MDA levels are expressed in μM. TAC values are expressed in μM of copper reducing equivalents. Groups: A, normal control; B, sham control (surgical stress); C, ischemia/reperfusion; D, ischemia/reperfusion with pterostilbene treatment. ⁎ P b 0.05 vs. group C. † P b 0.05 vs. group A.
3. Discussion Testicular torsion is a urologic emergency. Late presentation or failure to diagnose and correctly manage this condition leads to testicular injury and subfertility [10]. Testicular torsion should be treated within a proper critical time interval to maintain testicular function because the time interval between torsion and detorsion is an important factor affecting the extent of germinal cell damage [11]. Although it is known that testicular function can be restored through rapid diagnosis and treatment, a study has reported that 29%–50% of patients with immediate restoration of blood flow to the testis develop infertility [12,13]. This infertility is caused by the imbalance between restoration of oxygen supply and mitochondrial respiratory function, resulting in the excessive generation of superoxide in mitochondria. Under these circumstances, free oxygen radical scavenging enzymes, such as SOD, CAT, and GPx are needed to cope with oxidative stress and oxidative damage in the testicular tissue [14]. Even if the levels of these enzymes increase following testicular I/R, this response is insufficient to adequately counteract the excessive ROS and RNS generation occurring during reperfusion. The increased levels of ROS and RNS cause DNA fragmentation, lipid peroxidation, and protein inactivation [15,16]. Therefore, scavenging these substances during testicular I/R injury has beneficial effects that may lead to better outcomes. It has been recently reported that treatment with resveratrol, an analogue of pterostilbene, reduced germinal cell apoptosis in a rat model of testicular I/R injury induced by left testis torsion and detorsion surgery [17]. Similar to resveratrol, pterostilbene has anti-inflammatory, antioxidant, and anticancer activity [3].
To the best of our knowledge, this is the first study to investigate the effect of pterostilbene against testicular I/R injury. It has been demonstrated that pterostilbene administration results in the recovery of testicular function to a near normal level in the testicular I/R rat model. Consistent with the results of earlier studies, testicular I/R induced deterioration of the structure of seminiferous tubules, reduced their diameter, and impaired spermatogenesis, in addition to increasing testicular MDA levels and apoptotic cell numbers, upregulating the expression levels of caspases, the key factors of the apoptotic pathway, and decreasing testicular TAC [18]. At least 1 h of 720° testicular torsion is required to induce ischemia in the rat testis [19]. In contrast, it has been reported that 4 h of testicular torsion is required to induce ischemia in the human testis [11]. In this study, 720° testicular torsion for maintained for 5 h to mimic the human condition. In addition, the analyses were conducted 24 h after reperfusion because germ cell apoptosis was shown to be highest at this time point [20]. In agreement with other studies using rat testicular I/R injury models, the MSTD significantly decreased in group C compared to that in groups A and B [4]. Spermatogenesis also significantly decreased in group C compared to that in groups A and B, as evaluated using Johnsen's scores. Finally, the MSTD and Johnsen's score in group D significantly increased compared to the corresponding values in group C; these parameters (in group D) were similar to those observed for groups A and B. The processes observed in the testis were related to apoptosis induced by oxidative stress. In this study, the mean number of apoptotic cells in a seminiferous tubule (AI-1) and the ratio of the number of positive tubules to 100 tubules (AI-2), as well as the degree of lipid peroxidation (MDA) significantly increased in group C compared to the corresponding values in groups A and B. In addition, the TAC level decreased in group C compared to that in groups A and B. Finally, in group D, AI-1, AI-2, and MDA levels significantly decreased, and the TAC level increased compared to the corresponding values in group C; however, these values were similar to those observed for groups A and B. The above results are similar to those of other studies and permit us to conclude that testicular I/R induces excessive oxidative stress and exhausts the antioxidant system, causing germinal cell apoptosis, and administration of pterostilbene reverses these changes [21]. Caspase-3 is part of the common pathway of apoptosis stimulation. A dose-dependent activation and inhibition of caspase-3 activity by pterostilbene have been demonstrated in vitro [18]. Accordingly, IHC for caspase-3 was performed in each group to determine whether the apoptosis induced by testicular torsion/detorsion is mediated via the common pathway, and whether pterostilbene affects this pathway.
Fig. 2. Comparison of the levels of caspase expression. A: Caspase-3-, 8-, and 9-positive cells per positive tubule in 100 tubules were calculated. B: Caspase-3, 8-, and 9-positive tubules per 100 tubules were calculated. C: Caspase-3, 8-, and 9-positive tubules per 100 tubules were calculated. Groups: A, normal control; B, sham control; C, ischemia/reperfusion; D, ischemia/ reperfusion with pterostilbene treatment *P b 0.5 vs. group C, †P b 0.5 vs. group A.
Please cite this article as: Kim HJ, et al, Protective effect of pterostilbene on testicular ischemia/reperfusion injury in rats, J Pediatr Surg (2016), http://dx.doi.org/10.1016/j.jpedsurg.2016.01.009
H.J. Kim et al. / Journal of Pediatric Surgery xxx (2016) xxx–xxx
This study used a dose that was determined to reduce apoptosis in a pilot test and other studies. The results of IHC in the study groups showed that the expression level of caspase-3 significantly increased in group C compared to the expression levels in groups A and B, although the expression level of caspase-3 in the testis is generally low. Importantly, the expression level of caspase-3 significantly increased in group D compared to that in group C. Caspase-8 participates in the extrinsic pathway and caspase-9 in the intrinsic pathway of apoptosis [22]. In this study, IHC for caspase-8 and 9 was performed in each group to determine whether the apoptosis induced by testicular torsion/detorsion is mediated via the intrinsic or the extrinsic pathway, and whether pterostilbene affects either of these pathways. The expression level of caspase-9 and 8 was found to be significantly upregulated in group C compared to their corresponding values in groups A and B. Furthermore, this level significantly increased in group D compared to that in group C. However, comparison of the values of PC/CT of each caspase in group C revealed that the caspase-9 expression was significantly higher than that of caspase-8, and caspase-8 expression was even significantly lower than caspase-3 expression. These results and other studies may permit us to conclude that germinal cell apoptosis induced by testicular I/R occurred through the intrinsic pathway via caspase-9 rather than the extrinsic pathway via caspase-8, and pterostilbene inhibited apoptosis by suppressing the expression of caspase-3 and caspase-9 [23]. However, this study has some limitations. As mentioned earlier, testicular torsion can occur in newborns, children, and adolescents. Since testicular torsion can also occur in prepubertal children or neonates, studies on neonatal or prepubertal rats are needed. However, this study uses pubertal rats to mimic pubertal children or adolescents and to evaluate the protective effect of pterostilbene on testicular I/R injury. Therefore, it is unclear whether pterostilbene will show the same protective effect on testicular I/R injury in neonatal or prepubertal rats. In addition, this study evaluates only the short-term effect of pterostilbene. Therefore, additional study is required for evaluating the protective effect on testicular I/R injury in neonatal or prepubertal rats, and long-term effect of pterostilbene on I/R injury in rats. 4. Conclusions These findings demonstrate that pterostilbene protects against testicular I/R injury, which results in increased formation of oxidative stress, leading to testicular apoptosis and impaired spermatogenesis. In conclusion, treatment with pterostilbene may be a promising therapy for testicular I/R injury.
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Please cite this article as: Kim HJ, et al, Protective effect of pterostilbene on testicular ischemia/reperfusion injury in rats, J Pediatr Surg (2016), http://dx.doi.org/10.1016/j.jpedsurg.2016.01.009