Testicular torsion (TT) is a urological emergency to which adolescents and young men are highly susceptible. It is estimated that the incidence of TT is about 1 in 4,000 per year [1]. The pathogenesis involves the rotation of the spermatic cord along its longitudinal axis, thereby blocking the blood supply to the testis, resulting in ischemic necrosis of the testis. The main clinical manifestations include redness and swelling of the scrotum, significant tenderness, disappearance of the cremaster muscle reflex, and lack of relief from scrotal elevation. The severity of testicular injury is determined by the time interval and degree of torsion. Blood flow interruption durimg testicular torsion leads to edema, bleeding, blocked arteries, and oxygen deprivation. The testicles are particularly susceptible to ischemia due to limited blood flow. The ipsilateral testis becomes less fertile due to this loss of function and may atrophy. As the main pathophysiological consequence of testicular torsion [2], testicular ischemia-reperfusion (I/R) injury often results in excessive reactivation of oxygen, destruction of spermatogonial cells [3], and ultimately male infertility or subfertility. An effective medical intervention aimed at alleviating testicular I/R injury and improving the prognosis of testicular torsion has always been the focus of the medical community [4].
Autophagy, present in all eukaryotes, is an intracellular degradation system regulated by autophagy-associated proteins (ATG), through which cellular material is delivered to and degraded in lysosomes. However, autophagy not only removes material components, but also repairs cells and maintains homeostatic production in the body. Autophagy is a complex cytoplasmic component degradation process that is upregulated during starvation to counteract nutrient deprivation [5]. It is programmed cell death of type II [6], which can be activated by various physiological and pathological factors to promote cell survival or lead to cell death [7, 8]. Autophagy serves as a defensive mechanism against environmental stress and is essential for various physiological and pathological processes. It is an induced and regulated process that determines cell survival or death in various ways [9]. Experiments have shown that I/R induced damage effectively triggers autophagy factors, and high levels of authophagy under high atherosclerotic shear stress might inhibit endothelial cell death and inflammation, thereby preventing atherosclerosis [10]. Microtubule- associated proteins 1A and 1B (LC3) and Beclin-1 are involved in autophagy. Treatment of LC3I to LC3II is related to the degree of authophagic granule formation. LC3II serves as an autophagic marker [11, 12]. Autophagy effectively degrades the autophagy protein p62, and the level of p62 negatively correlates with autophagy activity13. Apoptosis increases with autophagy inactivation, suggesting that increased autophagy can inhibit apoptosis [14].
This study regulates autophagy using rapamycin and 3-MA to explore its role in rat models with testicular I/R injury. Rapamycin, a macrolide antibiotic, that induces autophagy through inhibiting mTOR [15, 16]. 3-MA is PI3K inhibitor that inhibits the formation of primary autophagic vesicles and autophagosomes [17].
We studied activated autophagy in testicular I/R injury. 3-MA may aggravate testicular tissue damage and increase apoptosis, while rapamycin increases autophagy and reduces apoptosis. These mechanisms and the regulation of LC3II, Beclin-1, p62 and Caspase-3 were investigated.

Animals and Models

Male SD rats (200-250g/animal, n=10 for each group) were kept under a 12-hour light cycle at 20°C-22°C with a dark cycle. Animals were Intraperitoneal injected with of pentobarbital anaesthetized (45 mg/kg). Then, the rats were placed on a constant temperature operating board (37°C). During surgery, the scrotal skin was opened to release the left testicle. The left testicle was rotated clockwise 720° 1 hour, and four hours after reperfusion, all the rats were euthanized, and left testicular tissue was collected. Forty rats were divided into 4 groups (n=10): sham operation group (scrotal opening without testicular torsion), I/R group (the left testis rotated clockwise 720 ° for 1 hour, followed by four hours reperfusion), I/R+Rap group (rats given a tail vein injection of rapamycin (1 mg/kg) 15 minutes before torsion), I/R+ 3-MA group (3-MA(30 mg/kg) injected into tail vein 15 minutes before torsion, as previously described. After the experiment ended, the left testis was removed under anaesthesia, fixed in 10% phosphate-buffered formalin, and stored at -80 °C for further study.

Hematoxylin and eosin (HE) staining

After fixation with 4% paraformaldehyde, the paraffin blocks of renal tissue were cut into 5 mm thickness, and the sections were deparaffinized in xylene and dehydrated in alcohol. Then, they were stained in HE method.

Immunohistochemistry

Immunohistochemical staining kits were used to detect LC3-II and Caspase-3. After dehydration, the tissues were fixed with paraffin wax and sliced (4μm). The sections were deparaffinized, hydrated, followed by thermal antigen retrieval and blocking of endogenous peroxidases with 3% hydrogen peroxide. 4% goat serum was used to minimize non-specific staining. Samples were incubated with Beclin-1 and Caspase 3 antibodies (1: 200) at 4 ℃ overnight, followed by a 37 ℃ second incubation for 30 minutes. Each section was then treated with DAB chromogenic fluid. Hematoxylin was then used for counterstaining. Each sample was randomly selected from 5 different fields (400×) and the integrated optical density ratio (IOD) values were analyzed using image analysis software IP win32 (Acromag, Inc., Wixom, MI, USA) .

TUNEL staining

Apoptosis of spermatogonial cells was detected by TUNEL-mediated dUTP Nick terminal marker (TUNEL). The apoptosis detection kit was purchased from Roche, and the apoptotic nuclei were stained brown (DAB color). The negative control group used end transferase instead of PBS. The apoptosis index (AI) was calculated: 500 cells were counted to obtain the rate of positive cells.AI = 100% of positive cells/total cells.

Transmission electron microscopy

Approximately 1 mm3 of testicular tissue was taken from each animal, fixed at 4 ° C, washed, fixated, dehydrated, soaked, embedded, and sliced (60-90 nm each). Autophagosome ultrastructures were observed under transmission electron microscopy (Hitachi, Ltd., Tokyo, Japan).

Western blot

Total protein was extracted from testicular tissue according to manufacturer’s protocol using a Thermo Fisher Scientific reagent for mammalian proteins and quantified using a protein analysis kit (Hercules, Bio Rad, ca, USA) The proteins were electrophoretically separated, transferred to a PVDF membrane, and blocked with 5% nonfat emulsion at room temperature in Tris-buffered saline and Tween 20 buffer for 2 hours. The membrane was then incubated with primary antibodies (Cleaved Caspase 3 (CST, 1: 1000, #9664); LC3-II (CST, 1: 1000, #3868); Beclin-1 (CST, 1: 2000, #3495); P62 (Abcam, 1: 3000, ab109012); GAPDH (Abcam, 1: 1000, ab37168)) at 4°C overnight. After washing with TBST buffer, a secondary antibody (1: 2000, Wuhan DR Biological Engineering Co., Ltd.) was added and incubated at 37°C for 2 hours. The membrane was then washed, and the secondary antibody was detected for imaging.

RT-PCR

Total RNA was extracted and reversed into cDNA. The PCR reaction conditions were as follows: 10 min (94℃), 40 cycles of 15 s ( 94℃), 30 s (58℃), and 40 s (72℃). ABI 7500  and SYBR Green chemistry were used to detect LC3, Beclin-1, P62, and β-actin mRNA levels. The sequences are provided in Table 1. The PCR products were normalized to GAPDH levels, and the associated gene expression levels were calculated.

Statistical analysis

All data were analyzed using GraphPad Prism-7 and described as mean and standard deviations (mean ± SEM). One-way ANOVA was used to compare between groups. P<0.05 indicated statistical significance.

Testicular damage increased after testicular I/R injury

Histological scores increased significantly after I/R injury (Figure 1 A(b)). To evaluate  the activation level of autophagy, autophagy-related proteins LC3, Beclin-1 and p62 were detected by qRT-PCR and Western blot. After I/R injury, Beclin-1 positive cells increased (Figure 2). I/R injury also resulted in significant increase in LC3II and Beclin-1, and decreased expression of p62, but the decrease was not significant (Figure 3 and Figure 4). The structure of autophagic vacuoles was observed with a high-power (×5000) electron microscope, which is the gold standard for the detection of autophagy. Autophagic vacuoles were observed in the I/R group. (Figures 5 (A) and (B), white arrows). Taken together, these results confirm the occurrence of autophagy in this testicular I/R model. Apoptosis in testicular tissue is activated during I/R injury. TUNEL-positive cells were rarely observed in testicular tissue in the sham group (Figure 1A (e)). TUNEL-positive cells in testicular tissue sections increased in the I/R group compared to the sham group (Figure 1A (b)). Similarly, caspase-3 expression increased in the I/R group compared to the sham group (Figure 2).

MA aggravates testicular I/R damage, and rapamycin relieves testicular I/R damage

Immunohistochemical analysis showed that Beclin-1 and caspase-3 scores significantly increased compared with sham group. After rapamycin treatment, caspase-3 decreased significantly, while Beclin-1 was increased significantly. After 3-MA treatment, caspase-3 increased significantly, while Beclin-1 decreased significantly. (Figure 2) Compared with the sham group, LC3 and Beclin-1 mRNA expressions increased significantly, while p62 mRNA expression decreased significantly. After rapamycin treatment, LC3 and Beclin-1 mRNA expressions increased significantly, while the p62 mRNA expression was significantly decreased. After 3-MA treatment, LC3 and Beclin-1 mRNA expressions decreased significantly, while p62 mRNA expression increased significantly. (Figure 3) Western blot results showed that caspase-3, LC3, Beclin-1 and BNIP3 levels were increased significantly compared with sham group, while p62 level significantly decreased. After rapamycin treatment, caspase-3, LC3, Beclin-1 and BNIP3 levels was decreased significantly, while p62 level was increased significantly. After 3-MA treatment, LC3 and Beclin-1 levels increased significantly, while p62 level decreased significantly. (Figure 4) Observing the structure of autophagy vacuoles with a high-power (×5000) electron microscope. Autophagic vacuoles were observed in I/R group (Figures 5). Electron microscopy results show a decrease in autophagy vesicles and an increase in apoptosis after 3-MA pretreatment (Figures 5 (a, b, d), white arrows). The autophagic vacuoles in I/R Rap group is higher (Figure 5).

Testicular torsion (TT) is one of the common emergencies in neonates and adolescents, which can cause testicular injury and even infertility [18]. Although the rate of testicular recovery after surgery is between 42-88%, it is not clear whether testicular function is completely preserved [19]. Current clinical research shows that ischemia due to spermatic cord torsion in a unilateral testis results in specific and irreversible changes in ipsilateral and contralateral testicular tissue, and injury is positively correlated with ischemic time. These irreversible changes may be related to subsequent declines in fertility [20]. Although the mechanism of testicular tissue injury during torsion is not fully understood, studies so far have shown that the main pathophysiological event is I/R injury [21, 22]. The role of autophagy in testicular I/R injury is supported by reports on improved autophagy in Kidney injury, such as I/R injury [23, 24].
Our study successfully established an I/R injury model in rats to investigate the relationship between I/R injury and autophagy. This study found that autophagy was activated in testicular tissue after I/R injury. Inhibition of autophagy by 3-MA led to the aggravation of testicular tissue injury, while activation of autophagy by rapamycin was important in the testicular group. Autophagy promotes cell survival or leads to cell death by stimulating various factors [6-8, 20]. The basic level of autophagy is a self-feeding cellular process that degrades cytoplasmic proteins and subcellular organelles in lysosomes, recovers cytoplasmic components, regenerates cell components and energy, and maintains cell and tissue homeostasis [25, 26]. Autophagy is an evolutionarily conserved catabolic process that degrades misfolded or aggregated proteins. The results showed that testicular I/R injury activated autophagy. In this process, the formation of bilayer structures (autophagy) engulfs and transports cellular contents to lysosomes for degradation. LC3 and Beclin-1 proteins are autophagic markers. Compared with the sham operation, rapamycin or 3-MA can enhance or inhibit this activation, respectively, as demonstrated by autophagy-related protein expression. Autophagy-associated proteins increase with renal I/R injury [24 ], LC3-I is converted to LC3-II under phosphatidylethanolamine and participates in autophagy cell formation and promotes their fusion with lysosomes [27]. Beclin-1 is important for autophagy formation and autophagy and lysosome fusion [28]. Similarly, p62 (SQSTM1 or A170) is one of the key substrates of autophagy used to evaluate autophagy flux in some cases. This protein selectively enters the autophagosome by binding to LC3 and is efficiently degraded by autophagy. In addition, autophagic protein p62 participates in autophagy and is degraded, which negatively correlates with autophagic activity [10]. Expression of LC3-II and Belin-1 increased, and expression of p62 decreased in the I/R+Rap group, indicating that rapamycin activated autophagy, whereas the expression of LC3-II and Beclin-1 decreased and p62 increased, indicating that 3-MA inhibited autophagy. LC3 and Beclin-1 had highest expression, p62 had the lowest expression, and bilayer or multi-membrane autophagic vacuoles increased, suggesting that rapamycin activated autophagy. The results of electron microscopy also confirm this relationship. Autophagy's role in I/R injury has attracted more and more attention, but induced autophagy may also have two sides [10].
Studies have reported that increased autophagy activity can help reduce the inflammatory response caused by intestinal I/R29. Tubular injury and cell damage apoptosis are often associated with renal I/R injury [30]. Spermatogenic cells apoptosis is one of the important factors leading to infertility after testicular I/R injury. Apoptosis is often referred to as programmed cell death. Studies indicated that caspase-3 is positively correlated with cell apoptosis. Caspase-3 is a caspase effector, which is important in the process of apoptosis [31]. Compared with the I/R group, the  pathological score of testicular injury in the I/R+Rap group was also lower. With decreased expression of TUNEL-positive cells and decreased caspase-3 in this group, rapamycin can effectively inhibit apoptosis of testicular cells. In conclusion, the results show that autophagy is important for cell survival in I/R injury. Of course, this study still has some shortcomings. In this study, only the indicators related to autophagy were detected in this study, but the mechanim of the protective effect of I/R damage was not studied in depth. In future studies, investigating the mechanism of the protective effect of I/R damage will help the development and application of related therapeutic drugs.

Activation of autophagy exerts a protective effect in ischemic reperfusion injury by reducing rat testicular sperm cell apoptosis. At the same time, it can also be used as an adjunct to surgery to reduce the risk of postoperative infertility.

Acknowledgments

None.

Ethics approval

This study has been approved by the Experimental Animal Ethics Committee of the Third Hospital of Wuhan City (SY2019-027).

Data availability

The Data will be available upon request.

Funding

This study was supported by Key Project of General Program of Wuhan Municipal Health Commission (WX20A16).

Authors’ contribution

Hu Z and Zhang W designed the study. Cheng Q and Xu L analyzed the data and drafted the manuscript. Chen YY, Ning JZ and Cheng F revised the original manuscript. All the authors approved the final manuscript.

Competing interests

All authors declare that there is no conflict of interest in the publication of this study.

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