GYY4137

Comparison of Intraperitoneal and Intratesticular GYY4137 Therapy for the Treatment of Testicular Ischemia Reperfusion Injury in Rats

Li-jia CHEN1†, Jin-zhuo NING1†, Fan CHENG1#, Ting RAO1, Wei-min YU1, Yuan RUAN1, Jun-feng WU1, Ren-gui LI1, Rong-xin GENG2
1Department of Urology, Renmin Hospital of Wuhan University, Wuhan 430060, China
2Department of Neurosurgery, Renmin Hospital of Wuhan University, Wuhan 430060, China

Huazhong University of Science and Technology 2020

Summary: The efficacy of intraperitoneal GYY4137 therapy and intratesticular GYY4137 therapy in an experimental rat model was investigated. Four groups were set up as the sham-operation group, torsion/detorsion (T/D) group, T/D plus intraperitoneal GYY4137 (G-IP) group, and T/D plus intratesticular GYY4137 (G-IT) group. In order to establish a testicular T/D model, the left testis was operated and the rotation reached 720° clockwise which lasted 1 h before reperfusion. The G-IP group accepted 100 µmol/kg of GYY4137 intraperitoneally 30 min after testicular rotation, while the G-IT group was treated with the same dose by intratesticular injection. Six h after detorsion, the testis was collected and subsequently assessed. The T/D group showed significant changes in histology and an enhancement in the level of oxidative stress and apoptosis compared to the sham-operation group. The expression of Caspase-3 and Bax turned out to be strengthened by T/ D and relatively decreased with GYY4137 treatment in both the G-IP and G-IT groups. Moreover, the Bcl-2 expression was inhibited in the T/D group, and promoted by GYY4137 in the G-IP and G-IT groups. GYY4137, moderating these observed changes, displayed a more protective effect with G-IT therapy than G-IP therapy.This study indicated that the efficacy of intratesticular therapy with GYY4137 is better than that of intraperitoneal therapy, which may provide a more valuable approach for testicular torsion therapy.

Key words: testicular torsion; GYY4137; apoptosis; oxidative stress; intratesticular therapy

Testicular torsion, which accounts for most primary cases of urological emergency, can lead to severe acute ischemic injury in the testis[1, 2]. Reperfusion therapy, such as testicular detorsion, is essential for repair of the ischemic tissue. However, the process may result in ischemia reperfusion injury (IRI) affiliated with the overproduction of reactive oxygen species (ROS), which further contributes to subfertility or infertility[3]. A range of agents have been proposed as valuable in the treatment of testicular IRI in animal models[4].
Hydrogen sulfide (H2S) has attracted impressive attention as a novel endogenous transmitter of gas[5]. In combination with carbon monoxide (CO) and nitric oxide (NO), H2S exerts protective effects on cell growth regulation[6]. However, the limitation of appropriate H2S donors has restricted the development of effective H2S clinical applications. In recent years, sodium hydrosulfide (NaHS) has been found significantly useful in clinical trials for its high rate of H2S discharge, but it is unable to replicate the natural production of H S suitably[7]. Morpholin-4-ium 4 methoxyphenyl (morpholino) phosphinodithioate (GYY4137), a newly introduced H2S donor, can release H2S slowly and steadily under physiological acid-base environment and temperature and works stably. Previous articles have seen that GYY4137 therapy shows a correspondingly protective effect against IRI in other viscera[8, 9], which may affirm the effective impact of GYY4137 on the treatment of IRI following testicular torsion/detorsion (T/D). This study verified that GYY4137 possessed the ability to alleviate IRI after reperfusion, and two different routes of administration were compared to explore a novel therapeutic approach to manage testicular torsion.

1 MATERIALS AND METHODS

1.1 Animals

Wistar rats (male, n=40, weighing 250–300 g) we used in our study were purchased from the Hubei Center for Disease Control (China). Before the research, same food and water was given to each animal. Four groups of rats were raised under normal room temperature (22°C). The Guide for the Care and Use of Laboratory Animals was used to standardize all operations.

1.2 Experimental Grouping and Surgical Procedures

We randomly divided 40 rats into 4 groups (n=10 for each group): the sham-operation group (A), T/D group (B), T/D+intraperitoneal GYY4137 (G-IP) group (C) and T/D+intratesticular GYY4137 (G-IT) group (D). 2% sodium phenobarbital (50 mg/kg) was injected intraperitoneally for anesthesia in all rats.

For group A, the testis was found and gained through a left-sided scrotal surgery. After collection, we used 5/0 silk to sew up the incision without additional intervention. For group B, the aim was to explore how testicular IRI influenced the testis of rats on the same side. A left-sided surgery was performed as group A. The testis was rotated 720° clockwise to bring out the torsion and then sutured to the skin for 1 h. Afterward, the testis was restored to normal position, and 5/0 silk was used to do the wound closure.

For group C, GYY4137 (100 µmol/kg) (Sigma- Aldrich, USA) was given intraperitoneally to the rats at the initial phase of testicular torsion with other surgical procedures identical to group B. For group D, the rats were given an intratesticular administration of GYY4137 (100 µmol/kg) in addition to the operations performed in group B.

After 4 h of reperfusion, the left testis of rats in 4 groups was collected and longitudinally cut into 2 parts. 4% paraformaldehyde was used to keep one section for tissue fixation. The other part was speedily frozen and placed at –80°C for subsequent experiment.

1.3 HE Staining

After the surgical procedure, 4% paraformaldehyde was used to fix half of the testis tissue and paraffin was prepared for embedment. Testis sections (5 μm) were then dealt with hematoxylin and eosin (H&E) for staining and examined under a microscope.

1.4 Immunohistochemistry

Antibodies for immunohistochemistry including rabbit anti-Caspase-3 (sc-7148), rabbit anti-Bax (sc- 493) and rabbit anti-Bcl-2 (sc-7382) were obtained from Santa Cruz Biotechnology Co. Ltd. (USA). The instructions of manufacturer were well followed in all experimental steps and the Olympus BX50 light microscope (Olympus Corporation, Japan) was used to assess and compare the staining intensity.

1.5 Biochemical Evaluation

Testicular epididymal superoxide dismutase (SOD) activity and malondialdehyde (MDA) content were tested spectrophotometrically using MDA and SOD assay kits following the manufacturer’s instructions. The MDA content was tested at an absorbance of 532 nm and showed an expression of nmol/g protein.Testicular SOD activity was detected at an absorbance of 550 nm and associated with U/mg protein expression.

1.6 TUNEL Assays

Apoptosis in spermatogenic cells was evaluated using the transferase-mediated dUTP nick-end labelling (TUNEL) method with a detection kit (Roche, Germany). Nuclei that were stained brown were observed and counted as TUNEL-positive cells. Every field consisted of approximately 200 cells, and 5 high-power fields of vision were stochastically chosen from each slide. The apoptosis index (AI) was defined as follows: AI=(Positive cells/Total cells counted)×100%.

1.7 Western Blotting

A bicinchoninic acid assay was used to extract and quantify all the testis tissue proteins. Concisely, 40 µg/ lane protein samples were set up for gel electrophoresis. 10% sodium dodecyl sulfate-polyacrylamide gel (SDS- PAGE) was used for separation by electrophoresis. Then a polyvinylidene difluoride membrane was prepared for transfer.

The membrane was blocked using 5% nonfat milk in TBST buffer at 37°C for 2 h and incubated at 4°C overnight with the following major antibodies: Bax (1:500 dilution; Santa Cruz, USA), Bcl-2 (1:1000 dilution; Santa Cruz, USA) and Caspase-3 (1:1000 dilution; Santa Cruz, USA). After 3 times of TBST buffer washes, the incubation of membranes was undertaken with secondary antibodies conjugated with horseradish peroxidase for 1 h. An ECL kit (Pierce Biotechnology, China) was used to visualize evert specific band. The absorbance (A) values were judged by ImageJ software (NIH, USA).

1.8 Quantitative Reverse Transcription-polymerase Chain Reaction (qRT-PCR)

TRIzol reagent (Invitrogen Life Technologies, USA) was used to gain total RNA in the testis samples, and the RNA concentration was examined by spectrophotometry. A cDNA synthesis kit (Promega Corporation, USA) was used for the synthesis of first-strand cDNA according to the instructions of manufacturer. Then, an Applied Biosystems SYBR Green mix kit (Applied Biosystems, USA) and the ABI 7900 Real-Time PCR system (Applied Biosystems Life Technologies, USA) were applied to fulfill qPCR. The primers were designed as follows: Bax (forward, 5′-TCACACCGATGTCCTGACGA-3′, and reverse, 5’- CTGTCATGCAGATGGTCATC-3′); and Caspase-3 (forward, 5′-TGGACTGCGGTATTGAGACA-3′, and reverse, 5′-GCGCAAAGTGACTGGATGAA-3′). GAPDH worked as a housekeeping gene. The data were detected as a ratio of the gene to GAPDH mRNA (forward, 5′-ACAGCAACAGGGTGGTGGAC-3′, and reverse, 5′-TTTGAGGGTGCAGCGAACTT-3′). PCR was un- dertaken with 40 cycles of 94°C for 30 s, followed by 56°C for 30 s and 72°C for 25 s, using the ABI 7900 Real-Time PCR system (Applied Biosystems Life Technologies, USA).

1.9 Statistical Analysis

All data are expressed as the mean±standard deviation. SPSS 20.0 (SPSS Inc., USA) was used for statistical analysis. The means were compared with the help of a one-way analysis of variance followed by the Student-Newman-Keuls test for different groups. The result was viewed as statistically significant when P<0.05. All studies were finished at least 3 times. 2 RESULTS 2.1 GYY4137 Ameliorates Histopathological Changes after Testicular T/D The HE-stained testis sections revealed no obvious morphological changes in group A. Testicular T/D led to a severe decline in spermatogenic function in group B, including epithelial cell abscission in the lumen, disorder of spermatogenic cells arrangement and wide damage to the seminiferous epithelium. Nevertheless, GYY4137 treatment appeared to alleviate significant testicular damage in group C and group D, with group D showing a more tangible result than that of group C (fig. 1). 2.2 GYY4137 Regulates a Decrease in MDA Content and Increase in SOD Activity after Testicular T/D For the sake of analyzing the oxidative stress level caused by testicular T/D, we detected the MDA content and SOD activity in testis samples. Compared to group A, a marked increase in the MDA content was observed in group B, while the SOD activity showed an obvious decrease. GYY4137 reduced the pathologic findings associated with oxidative stress of testicular T/D in group C and group D. However, this effect seemed to be augmented by intratesticular administration (fig. 2). 2.3 GYY4137 Inhibits Spermatogenic Cell Apoptosis after Testicular T/D To detect apoptosis and confirm the AI, the expression of Bax, Bcl-2 and Caspase-3 in spermatogenic cells of testis was detected by immunohistochemistry, Western blotting, RT-qPCR and TUNEL assays. In group B, Caspase-3 and Bax expression was up-regulated, and Bcl-2 expression was down-regulated compared to that in group A. In addition, TUNEL-positive spermatogenic cells increased markedly. GYY4137 inhibited the expression of the aforementioned genes in group C and group D. Additionally, a smaller number of TUNEL- positive spermatogenic cells were seen. However, the decrease in apoptosis in group D appeared to be more obvious than that in group C, and the protective effect of GYY4137 was relatively larger in group D (fig. 3). 3 DISCUSSION Testicular torsion is one of the usual urologic emergency, which is mainly provoked by twisting of the spermatic cord in infants and adolescents[3, 10]. The main pathophysiological mechanism of testicular T/D is ischemia followed by reperfusion that results in damage to the testis generated by the bending of the spermatic cord[11]. The histological damage resulting from IRI in testes has been reported in previous experiments with different time periods, degrees of torsion and durations of reperfusion[4, 12, 13]. In this study, we selected a rat model in which a testicular torsion procedure lasting 1 h with 720°clockwise rotation was performed followed by a detorsion procedure lasting 4 h[12]. The dosage we selected in this study was learned from former research[14]. After collecting samples, we measured the impact of testicular IRI on histological changes in the ipsilateral testes. The results showed serious damage of testicular spermatogenic function resulted from IR injury, which has been authenticated in other researches[15, 16]. GYY4137, however, has shown a magnificent effect in healing the aforementioned histological injury remarkably after testicular T/D. Fig. 1 Microphotographs of testis tissues in four groups (hematoxylin and eosin staining, ×200) A: the sham-operation group: no obvious morphological changes. B: the T/D group: significant damage of spermatogenic function, such as extensive seminiferous epithelium injury, the vacuoles formation, and the derangement of spermatogenic cells. C: the G-IP group: The changes of spermatogenic cells and spermatogenic epithelium were less than those in T/D group. D: the G-IT group: The protective effect of GYY4137 was enhanced. Fig. 2 MDA content and SOD activity in four groups Bars represent the mean±standard error of the mean (n=10). One-way ANOVA analysis of variance was used to evaluate statistical significance. As compared with group A, the MDA content was obviously increased in group B, while the SOD activity was significantly reduced. GYY4137 improved the level of oxidative stress in testicular T/D. This effect was augmented by intratesticular administration. A: group A; B: group B; C: group C; D: group D. *P<0.05 vs. group B, #P<0.05 vs. group C. Fig. 3 Apoptosis index (AI) in four groups and the expression of Bax, Bcl-2 and Caspase-3 detected by immunohistochemistry, Western blotting and RT-qPCR (×200) GAPDH was used to show equal amounts of protein loading in each lane. The means were compared using one-way ANOVA analysis of variance. In group B, TUNEL-positive cells were widely observed as compared with group A. GYY4137 decreased the number of TUNEL-positive spermatogenic cells in group C and group D, while fewer TUNEL-positive spermatogenic cells were observed in group D. Besides, the expression of Caspase-3 and Bax increased markedly in group B, while the expression of Bcl-2 decreased compared to that in group A. GYY4137 inhibited the expression of the aforementioned genes in group C and group D. However, the decrease in apoptosis in group D appeared to be more obvious than that in group C, and the protective effect of GYY4137 was relatively larger in group D. A: group A; B: group B; C: group C; D: group D. *P<0.05 vs. group B, P<0.05 vs. group C. IR injury is mainly caused by virtue of the over- production of ROS in addition to neutrophil recruitment, lipid peroxidation, alteration of the microvascular blood flow and other pathophysiological processes[17], and mammalian testes are extremely susceptible to ROS and lipid peroxidation because of the rich content of testicular polyunsaturated fatty acids[18, 19]. In normal cases, the ROS accumulation occurs at a low level with the help of the antioxidant defense system[20]. During ischemia-reperfusion, however, an abundance of reactive oxygen species is produced such as superoxide anions, hydrogen peroxide, nitric oxide, and hypochlorous acid, which can result in the loss of cell viability and DNA damage, even death[21, 22]. ROS is hard to calculate directly due to their high reactivity and short life span[23]. MDA is a stable end product of lipid peroxidative decomposition generated by ROS[24]. Therefore, it is typically used as a sensitive index of ROS in many pathological situations, including IR injury. SOD, an important active substrate in the process of cell growth and differentiation, provides cell protection from harmful injury[25]. It is also known that antioxidants can benefit the impaired testes after IR injury following testicular torsion[26]. Our study proved that treatment with GYY4137 can ease the oxidative stress in testicular T/D by decreasing the expression of MDA and promoting the expression of SOD, and the intratesticular GYY4137 group showed a more consistent alleviation. As such, our data are in accordance with those of other investigations[27, 28]. Apoptosis has a marked effect on the process of spermatozoa development[29]. However, a large number of spermatogenic cells may end up undergoing apoptosis during testicular IR injury, which consequently leads to defective testicular function and results in loss of the affected testicle[30]. Many other studies have shown that the incidence of apoptosis may rise ipsilaterally in the testicle, and the extent of apoptosis turns out to be related to the duration of testicular ischemia[31, 32]. In our study, the present results demonstrated that the number of apoptotic cells was greatly decreased in group C and group D, which were dealt with GYY4137 treatment, as compared with that in the T/D group, while apoptosis of spermatogenic cells seemed to be more relieved in group D than in group C, as explained by the TUNEL assay results. In addition, it has been generally acknowledged that the major pathway of cell apoptosis has a bearing on the intrinsic signaling pathway, for example, the mitochondrial signal pathway, which is accurately regulated and controlled by the expression of genes[33]. Bcl-2 proteins turns out to be a family of pro- and anti- apoptotic proteins which control an important step in the mitochondrial apoptotic pathway, among which Bcl-2 and Bax were demonstrated to be the two main anti-apoptosis factors[34]. In some recent studies, these two proteins have been found to play a significant role in spermatogenic cell apoptosis during IR injury. The results of previous studies have provided tremendous insights into our understanding, showing that the ratio of Bcl-2/Bax greatly impacts spermatogenesis in normal cells and may affect the presence of apoptosis in impaired cells[35, 36]. Furthermore, Caspase-3 is an inactive zymogen acting in the cytoplasm and becomes the intersecting point of numerous apoptotic pathways. The occurrence of cell apoptosis was found to be significantly affected by the activation of Caspase-3, which resulted in changes in cell shrinkage, chromatin condensation and degradation of DNA[37, 38]. To form a rich understanding of the apoptosis pathway during testicular T/D, the aforementioned apoptosis-related factors have been taken into account. In the results, we found an increase of Caspase-3 and Bax expression, while the expression of Bcl-2 was down-regulated in group B compared to that in group A, as measured by immunohistochemistry, Western blotting and RT-qPCR assays. The previous research has indicated that the decrease in the ratio of Bcl-2/Bax remaining after Bcl- 2 and Bax have performed their functions in cells may become a signal of cell apoptosis and subsequently activate the mitochondrial permeability transition pore (MPTP). Caspase-3 activation then starts, and apoptotic programming follows. In addition, the release of some caspase activators from mitochondria is regulated by the Bcl-2 protein. Finally, it may cause spermatogenic cell apoptosis after IR injury in the rat. All of the aforementioned evidence demonstrates the effect of the mitochondrial signaling pathway on apoptosis induced by GYY4137[39]. In conclusion, our study validated that GYY4137 contributes to the inhibition of oxidative stress and spermatogenic cell apoptosis. Discovering new IR injury treatment regimens to supplement surgery in clinical environments is a matter of cardinal significance. For this reason, intratesticular administration of GYY4137 for IR injury deserves sufficient attention compared with traditional intraperitoneal administration by injection, since it was revealed to be more efficient and may be a potential alternative solution for testicular torsion. Conflict of Interest Statement The authors declare that they have no competing interests. REFERENCES 1 Ning JZ, Li W, Cheng F, et al. The protective effects of GYY4137 on ipsilateral testicular injury in experimentally varicocele-induced rats. Exp Ther Med, 2018,15(1):433-439 2 Zhou L, Song K, Xu L, et al. 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