Protective Effect of BMSCs-derived Exosomes on Testicular Ischemia-reperfusion Injury in Rats

Background: Testicular Ischemia reperfusion injury(IRI) is a major pathophysiological process of surgical reduction after testicular torsion, and oxidative stress is the main injury factor. However, the role of BMSCs-derived exosomes in testicular IRI and its mechanism have not been reported. In this study, we investigated the protective effect of bone marrow mesenchymal stem cell-derived exosomes against testicular ischemia-reperfusion injury. Methods: BMSCs were isolated, cultured and identied by primary culture method. Exosomes derived from BMSCs were extracted by ultra-high speed centrifugation method. A testicular IRI model was established in male SD rats. Thirty healthy male SD rats were randomly divided into three groups. Group A: Sham group, group B: normal saline treatment group (I/R+NS), group C: BMSCs-derived exosomes (100 ug/mL) treatment group (I/R+ BMSCs-EXO). Finally take each side (left) of rat torsion by using optical microscope to detect testicular tissue pathology grade and ne structure of organization structure, adopt the method of biochemical determination of groups of testicular tissue MDA and NOS, SOD and CAT activity and T - AOC level. Results: BMSCs were successfully isolated and cultured from rat bone marrow, and exosomes secreted by BMSCs were successfully extracted. In animal model, Compared with the normal spermatogenic structure of testis in group A (Sham), the spermatogenic structure of testis in group B (I/R+NS) was obviously damaged to varying degrees, while the spermatogenic structure of testis in group C (I/R+ BMSCs-EXO) was improved to A certain extent (P<0.05). In the biochemical indexes of testis tissue, the contents of MDA and NOS in group B (I/R+NS) were signicantly increased compared with group A (Sham), while the activities of SOD and CAT and T-AOC were decreased compared with group A (Sham) (P<0.05). In the exosome-treated group C (I/R+ BMSCs-EXO), compared with the normal saline treatment group B, The contents of MDA and NOS were decreased to a certain extent, while the activities of SOD and CAT and the level of T-AOC were increased (P<0.05). Conclusion: BMSCs-derived exosomes can be absorbed by rat spermatogonia and have antioxidant and anti-inammatory protective effects against testicular ischemia-reperfusion injury (cid:0)


Introduction
Testicular torsion (TT), is a testicular torsion along the longitudinal axis of the spermatic cord [1], resulting in acute reduction or interruption of testicular blood supply, resulting in testicular tissue ischemia and necrosis. The incidence of testicular torsion is up to 158, which mostly occurs in teenagers under 24 years old [2][3][4][5][6]. Clinically, because it is di cult to distinguish the disease from scrotal in ammation in the early stage [1,2,7,8], domestic data show that the misdiagnosis rate of testicular torsion can be as high as 67.6%. TT is very easy to cause misdiagnosis and torsion testicular ischemia time is too long, tissue necrosis [9], and nally can only carry out surgery to remove necrotic testicles, resulting in testicular loss in patients; this disease is also one of the most common causes of testicular loss in adults, so it should be diagnosed and treated as soon as possible. At present, timely surgical reduction to restore the blood supply of torsional testis is the standard treatment for the disease. however, testicular torsion reduction is a typical (IRI) process of ischemia-reperfusion injury, which can increase testicular germ cell apoptosis [10], lead to varying degrees of testicular atrophy and spermatogenic dysfunction, and even lead to infertility. Therefore, even if there is timely surgical reduction, testicular ischemia-reperfusion injury is still inevitable. Although most researchers at home and abroad use some drugs to reduce testicular IRI, there is no ideal treatment in clinic [1,2,11].
The latest progress in stem cell research brings hope for the prevention and treatment of oxidative stress injury [1,2,11]. Because of its multi-directional differentiation potential, immunomodulatory activity, ability to migrate to the injured site, simple expansion in vitro, and a wide range of sources, it has been concerned by more and more researchers. Among them, bone marrow mesenchymal stem cells are easy to separate and culture, which has become a research hotspot in the eld of tissue injury and repair. at present, many studies have found that bone marrow mesenchymal stem cells can reduce oxidative stress injury in heart, brain, lung and other parts [14][15][16][17][18], but the mechanism of its effect is unknown. At present, more and more studies have shown that bone marrow mesenchymal stem cells alleviate oxidative stress injury mainly through exosome secreted by bone marrow mesenchymal stem cells [19][20][21][22][23].
Exosomes are extracellular vesicles with lipid bilayer membranes [24]. Their diameters range from 30 to 150nm and can be released by a variety of cells such as immune cells, vascular cells [25], nerve cells, stem cells and tumor cells. Exosomes have rich cellular speci city such as protein, lipid and nucleic acid, and can participate in a variety of physiological and pathological processes, such as antigen presentation [26,27], RNA transport, tissue repair, tumor metabolism, metabolism and so on. At present, some studies have shown that exosome can alleviate in ammation and oxidative stress injury, but whether exosome derived from bone marrow mesenchymal stem cells can improve testicular ischemiareperfusion injury has not been reported.
In this study, we explored a new function that exosome derived from bone marrow mesenchymal stem cells can alleviate oxidative stress caused by testicular ischemia-reperfusion injury. We proved that the exosome derived from bone marrow mesenchymal stem cells can pass through the blood-testis barrier to around the seminiferous tubule and reduce the oxidative stress injury caused by testicular ischemiareperfusion in testicular torsion model rats. These ndings reveal the reasons for the resistance of mesenchymal stem cells to oxidative stress injury and provide a new treatment for patients with testicular torsion to prevent and alleviate testicular ischemia-reperfusion after testicular reduction.

Ethics statement and Animals
This study was approved by the bioethics committees of Southern Medical University and the Third A liated Hospital of Southern Medical University, Guangzhou, China. All the rats we need[Male SD: 3 to 4weeks old] were purchased from Laboratory animal center of southern medical university and kept under speci c pathogen-free conditions. All animal experiments were approved by the Ethics Committee of Southern Medical University. Guidelines of the Institutional Animal Ethics Committee were followed when carrying out in vivo experiments. All efforts were made to minimize animal suffering.

Isolation of MSCs from mouse bone marrow
Healthy Male SD rats weighted about 80-120g were used to isolate bone marrow-derived MSCs which were killed by cervical dislocation. Under sterilized conditions The fur was disinfected with 75% alcohol and then The rats were anaesthetized using pentobarbital (50 mg/kg, intraperitoneal injection) to minimize suffering and sacri ced by cervical dislocation without recovery from anaesthesia. Tibias and femurs then were removed under sterile conditions. In the laboratory and under sterilized conditions, the femurs and tibias were cleaned off from the remaining muscle tissues with sterile surgical tools and washed few times with normal saline solution. The tip of the bone was inserted into the spiral tip bottom centrifuge tube(15ml)after ends of femurs and tibiasthe were cut. Bone marrow cell suspension was obtained by ushing marrow cavity using dispensable 1-ml syringe with low-glucose dulbecco's modi ed eagle medium (DMEM).Then bone marrow cell suspension was ltrated through 200-mesh sieve in order to remove bone debris. the sediment which Contains bone marrow cell was acquired after centrifugation of the ltered suspension at 1000 rpm/min for 5 min. Remove the supernatant, the sediment was suspended with A moderate amount of DMEM .bone marrow cells suspension were shifted into petri dishes On average and incubated at 37°C in a humidi ed atmosphere containing 5% CO2,adn 95% air with low-glucose DMEM plus 10% heat-inactivated fetal bovine serum (FBS, Gibco, Grand Island, NY, USA) and 1% penicillin-streptomycin (Beyotime Biotechnology, Shanghai, China).

Exosome isolation
In short, the exosomes were isolated using gradient centrifugation.in order to exclude exogenous exosome contamination, BMSCs at 4th to 8th passage was cultured with exosome-free serum and the cell culture medium was collected. the culture supernatants were cleared of cell debris and large vesicles by sequential centrifugation at 300g for 10 min, 1000g for 20 min, and 10,000g for 30 min, followed by ltration using 0.45um and 0.22um sterile lters Then, the cleared sample was centrifugal at 100,000g for two hour to pellet the exosomes. To get a more pure exosomes, Re-speeding in the same condition after removing the suspension the and the precipitating are suspended with phosphate buffer solution (PBS).The recovery of exosomes was estimated by measuring the protein concentration using the BCA Protein Assay Kit( Beyotime Biotech, Jiangsu, China).The obtained exosomal fraction was resuspended in PBS (500 ul, 1 mg/mL total protein).

Exosome characterization Western Blot
The concentrations of all the protein we extracted from cells, exosomes and tissues were determined using a BCA protein assay kit ( Beyotime Biotech, Jiangsu, China) according to manufacturer instructions. Subsequently, a certain amount of total protein[20ug-80ug] was heated to 95 °C for 10 min in 1 × DTT-containing sodium dodecyl sulfate (SDS) sample buffer and separated by 10% SDSpolyacrylamide gel electrophoresis, followed by transfer onto polyvinylidene uoride membranes Brie y, exosomes samples were vortexed and diluted to a nal dilution of 1 : 2000 in ltered moleculargrade H2O. Blank-ltered H2O was run as a negative control. Each sample analysis was conducted for 60 s and measured three times using Nanosight automatic analysis settings.
Transmission electron microscopy (TEM) TEM analysis was performed to con rm BMSCs-derived exosomes morphology. Brie y, exosomes samples were diluted with PBS to the appropriate concentration, and ~20-40 μL of PBS solution containing exosomes was transferred to a copper grid for incubation at room temperature for 5 min. Filter paper was used for absorbing unevaporated solution. exosomes samples were negatively stained with 4% phosphotungstic acid solution at room temperature for 5 min and dried at 65 °C for 10 min. Images of exosomes samples were obtained using a Hitachi H-7650 transmission electron microscope (Hitachi, Tokyo, Japan).
BMSCs-derived exosomes uptake by GC-1 cells Puri ed rBMSC-Exo were labeled with 1 μM Dil (Invitrogen) as previously described. Brie y, rBMSC-Exo were mixed with 1 μM Dil, and the exosome-dye suspension was incubated for 5 min with regular mixing.
Excess dye from the labeled exosomes was removed by ultracentrifugation at 100,000 g for 1 h at 4°C using a 70Ti rotor (Beckman Coulter), and the exosome pellets were washed three times by resuspending them in PBS. The nal pellets were resuspended in PBS. Dil-labeled exosomes were co-cultured with Spermatogonium cells(GC-1) for 6 h, then GC-1 were washed with PBS, and xed in 4% paraformaldehyde (PFA).The nucleus of cells were stained using medium containing 4,969-diamidino-2-phenylindole (DAPI; Vector Laboratories, USA). The uptake was observed by uorescence microscopy.

Animal experiments
Under the condition of Speci c pathogen Free(SPF) animal laboratory, Altogether 30 male Sprague -Dawley rats were randomly divided into 3 groups of 10 each:sham-operated control group ,experimental group(I/R) and Treated group(I/R+EXO).In order to induce the ischemia-reperfusion model of testis, rats were anesthetized with xylazine (20 mg/kg) and ketamine (50 mg/kg) after Overnight fasting In the sham-operated control group, rats underwent left scrotal exploration only with similar surgical procedures without the torsion and detorsion. And the experimental and Treated group were through a sub-inguinal incision the left testis was brought out and was rotated 720°clockwise and then inserted and xed into the scrotum with a 4/0 silk suture placed through the tunica albuginea and subcutaneous tissue. The incision was primarily closed with a 4/0 silk suture. After 4 hours, by using the same incision line, testis was counter rotated to the natural position and reinserted into the scrotum to induce reperfusion for 2 hours [28].After that ,the two groups ,experimental and Treated group, were respectively injected from the tail vein with the same amount of saline and 100ug/ml exosomes. At the end of the reperfusion period rats were decapitated and testis tissues were taken for biochemical analyses and histological evaluations. Biochemical and histological samples were blindly examined.

Distribution of exosome bodies in vivo (tracking)
According to the manufacturer's instructions, exosomes derived from the BMSCs were isolated as described above were labeled using PKH67 Fluorescent Cell Linker kits (Sigma-Aldrich, St. Louis, MO).The washed exosomal pellets from the 100 ml culture media were resuspended in 700 µl of Diluent C(exosomal solution). PKH67 dye (1 µl) was diluted in 250 µl of Diluent C (PKH67 solution). Then, 250 µl exosomal solution and 250 µl PKH67 solution were mixed in a 4.7 ml centrifugation tube. Samples were mixed gently for 4 min, and 4.2 ml of 1% BSA was added to bind the excess PKH67 dye. PKH67-labeled exosomes were ultracentrifuged at 120,000 x g for 3 h at 4˚C using the Optima Ultracentrifuge (Beckman Coulter).Exosomal pellets were washed three times in PBS by ultracentrifugation. Finally, PKH67-labeled exosomes were resuspended in D-MEM or RPMI-1640 medium. As the negative controls, no PKH67 control and no exosome control were prepared. Exosomes were collected by ultracentrifugation without PKH67 dye, and then D-MEM or RPMI-1640 medium were added to the centrifuged tubes (no PKH67 control). After PKH67 dye was washed by ultracentrifugation without exosomes, the supernatant was discarded and media described above were added to the centrifuged tubes (no exosome control).then the equal amount of exosome was injected into different mice through the tail vein.

Histological analysis
Testes were removed from the rats and xed with 10% formaldehyde. Thereafter, the testes were dehydrated with subsequent 70, 90, 96 and 100% ethanol and cleared with toluene. After overnight incubation of para n in a 60 °C incubator, testes were embedded and blocked in para n at room temperature. Aproximately 5 μm thick para n sections in midline area of the testis were stained with hematoxylin and eosin (H&E). In each section at least 30 seminiferous tubules were evaluated microscopically at × 200 magni cation. The rst seminiferous tubules were selected randomly and the others were taken by sliding the section towards the clockwise.

Biochemical indicators Analysis
An appropriate amount of testicular tissue and aseptic saline were prepared with a ratio of 1:9 to 10% tissue homogenate and then determination of the supernatant after centrifugation for various indicators. The testicular homogenates were assayed for total antioxidant capacity (T-AOC), superoxide dismutase (SOD),nitric oxide synthesis(NOS: tNOS and iNOS) Catalase (CAT) and malondialdehyde (MDA) by using colorimetry for all the oxidation indicators. The speci c determination principle of each index and the preparation method of reagent and the determination method of each indicator protein content were strictly carried out according to the instructions of the kit(Institute of Bioengineering Nanjing Jiancheng China).

Statistical analysis
For the all experiments, data are presented as the mean ± S.E.M. Tests for signi cant differences between the groups were performed using a t-test or one-way ANOVA with multiple comparisons (Fisher's pairwise comparisons) using GraphPad Prism 5.0 (GraphPad software, San Diego, CA, USA).A minimum p value of 0.05 was chosen as the signi cance level.

Characterization of exosomes
To con rm the presence of BMSCs-derived exosomes, exosome-marker proteins, including CD63,TSG101, ALIX and Calnexin were validated by western blot (Fig. 1C). In contrast, these exosomal-speci c proteins were absent from collected exosome-depleted fractions, whereas the endoplasmic reticulum marker Calnexin was detected in spermcell lysates. TEM results showed that exosomes contained lipid bilayerbound membranes, with size distributions peaking at 105 nm diameter according to NTA (Fig. 1A and  1C).
Exosomes derived from bone marrow mesenchymal stem cells can reduce testicular ischemiareperfusion injury in rats.
According to the literature, the rat testicular IRI model ( Fig. 2A) was successfully constructed. It can be found that compared with the sham operation group, the testicular injury of testicular IRI rats is more serious, showing a large number of exudation and vacuole formation in the lumen. The arrangement of spermatogenic cells is disordered, the cell level is unclear, and the seminiferous epithelium may be absent or exfoliated to some extent (Fig. 2B). On the basis of testicular IRI animal model, we injected BMSCsderived exosomes and PBS into the tail vein and found that compared with sham-operated group (Fig. 2C), both BMSCs-derived exosome treatment group (Fig. 2D) and PBS treatment group (Fig. 2E) had testicular ischemia-reperfusion injury, while BMSCs-derived exosome treatment group had less testicular tissue injury and less exudation compared with PBS treatment group. There are few vacuoles, the biblical cells are well arranged and the cell layers are lighter. The results showed that BMSCs-derived exosome could reduce testicular ischemia-reperfusion injury in testicular IRI rats.

Exosomes derived from bone marrow mesenchymal stem cells can improve the oxidative stress injury induced by testicular ischemia-reperfusion in rats
Then we detected the indexes of oxidative stress in testicular tissue of rats in three groups. Compared with sham operation group, the contents of T-AOC, SOD and CAT in testicular tissue of rats in BMSCs exosome treatment group and PBS treatment group decreased signi cantly (Fig. 3D, 3e, 3F), and the contents of T-AOC, SOD and CAT in PBS treatment group decreased signi cantly. Compared with PBS treatment group, the contents of T-AOC, SOD and CAT in testicular tissue decreased less in BMSCsderived exosome treatment group, indicating that BMSCs-derived exosome can resist oxidative stress injury caused by testicular ischemia-reperfusion injury in rats. In addition, compared with the sham operation group, the contents of MDA and NOS (including iNOS and TNOS) in testicular tissue in BMSCsderived exosome treatment group and PBS treatment group increased (Fig. 4A, 4B, 4C), and the contents of MDA and NOS (including iNOS and TNOS) in PBS treatment group increased signi cantly. Compared with the PBS treatment group, the contents of MDA and NOS (including iNOS and TNOS) in the testicular tissue of the BMSCs-derived exosome group increased less, indicating that the BMSCs-derived exosome can alleviate the oxidative stress injury caused by testicular ischemia-reperfusion injury in rats. In summary, from the perspective of biochemical level of oxidative stress related to testicular tissue, BMSCs-derived exosome can reduce the oxidative stress injury caused by testicular ischemia-reperfusion in testicular IRI rats. < / br > exosome bodies derived from bone marrow mesenchymal stem cells can pass through the blood-testis barrier of rats.
Exosome derived from bone marrow mesenchymal stem cells can pass through the blood-testis barrier of rats In order to explore how the exosome bodies derived from bone marrow mesenchymal stem cells play the role of antioxidation, we injected the exosome bodies labeled with green uorescent dye PKH67 through the tail vein. After a period of time, the testis were removed under aseptic operation and made into frozen sections. PKH67-labeled exosomes can be detected in testicular tissue under confocal microscope. The results show that exosome bodies derived from bone marrow mesenchymal stem cells can indeed reach testicular tissue (Fig. 4A, 4B), which is a key step for bone marrow mesenchymal stem cells to play a protective role.
Exosome derived from bone marrow mesenchymal stem cells can be absorbed by mouse spermatogonia.
Then we explored whether the exosome derived from bone marrow mesenchymal stem cells can be absorbed by rat spermatogonia, so as to protect the fertility of rats with testicular ischemia-reperfusion injury. We co-cultured the exosomes labeled with green uorescent dye PKH67 with mouse spermatogonia GC1 and xed them after a period of time. Under microscope, it was observed that some exosome bodies derived from bone marrow mesenchymal stem cells were absorbed by GC1 cells (Fig. 4C,   4D, 4e). The results showed that the exosome bodies derived from bone marrow mesenchymal stem cells could be absorbed by mouse spermatogonia so as to reduce the damage of spermatogonia under oxidative stress and protect the development of spermatozoa.

Discussion
The results of this study showed that the exosome derived from bone marrow mesenchymal stem cells could pass through the blood-testis barrier to the testicular tissue, and it was con rmed by cell experiments that they could be absorbed by spermatogonia. In rat testicular IRI model[28], compared with PBS treatment group and sham operation group after testicular reduction, although exosome derived from bone marrow mesenchymal stem cells could not prevent testicular ischemia-reperfusion injury, it could signi cantly improve the degree of testicular injury and protect the integrity and spermatogenic function of testicular tissue to a great extent through the detection of testicular histomorphology and related oxidative stress indexes. It provides a new idea for preventing ischemia-reperfusion injury after testicular torsion reduction. Testicular torsion, diagnosed mainly in adolescents, is an emergency that requires immediate treatment to save the testicles to prevent loss of testicular function, and about 30% of men with a history of torsion experience adult infertility [29][30][31][32]. Although surgical reduction of the affected testis is the main method, the subsequent ischemia-reperfusion injury is still inevitable, and the testicular tissue can still be worsened by ischemia-reperfusion injury. Current clinical experiments and research data show that the degree of ischemia-reperfusion injury after testicular torsion reduction is highly related to the prognosis after testicular torsion reduction. After reduction, too much oxygen is introduced into the tissue, resulting in excessive production of reactive oxygen species. The increase in the number of neutrophils and the excessive production of ROS in the testicular cycle lead to tissue damage by destroying the cell membrane. This process is called "Ihammer R damage", which is characterized by oxidative stress, and oxidative stress is the main cause of organ damage [33].
Many previous studies have shown that it is possible to improve the prognosis and protect testicular tissue and spermatogenic function by improving the oxidative stress injury caused by ischemia-reperfusion after testicular torsion reduction [34]. In this study, we con rmed that the exosome derived from bone marrow mesenchymal stem cells can reach around the seminiferous tubule through the bloodtestis barrier of rats, and further con rmed that exosome derived from bone marrow mesenchymal stem cells can reduce the degree of oxidative stress injury in testicular tissue and improve testicular ischemiareperfusion injury by HE staining and the content of related oxidative stress indexes.
At present, the mainstream research is to treat or alleviate testicular ischemia-reperfusion injury through antioxidant stress. some studies have shown that bone marrow mesenchymal stem cells can reduce the degree of oxidative stress injury caused by testicular ischemia-reperfusion, but the mechanism is unknown [1,2,7,8,10,11]. On the basis of the study of bone marrow mesenchymal stem cells, this study further clari ed that the exosome secreted by bone marrow mesenchymal stem cells play a major role, and the methods and corresponding indexes used are the same as those of previous studies. It can be fully explained that bone marrow mesenchymal stem cells rely on their secreted exosome bodies to improve testicular ischemia-reperfusion injury.
In this study, a preliminary study was made on the mechanism of exosome. It can reach around the seminiferous tubule through the blood-testis barrier, and the cell uptake experiment con rmed that the exosome body can be absorbed by spermatogonia, suggesting that the exosome body may be absorbed by testicular tissue and play a role. However, the deep mechanism of exosome body in this study is still shallow, and its mechanism can be further explored.
This study focuses on the kinetic energy study of exosome derived from bone marrow mesenchymal stem cells [24,35], but there is no control group for the effect of bone marrow mesenchymal stem cells in the experimental design, and the theoretical basis is still lacking, but combined with the results of previous studies and this experiment, to a certain extent, it can be concluded that the exosome derived from bone marrow mesenchymal stem cells has therapeutic effect.
In a word, the exosome derived from bone marrow mesenchymal stem cells can reduce testicular ischemia-reperfusion injury and reduce the level of oxidative stress in testicular tissue through the bloodtestis barrier possibly. At the same time, compared with bone marrow mesenchymal stem cells, exosome is more biosafety and has the value of clinical application.

Conclusions
This study shows that exocrine bodies derived from bone marrow mesenchyma may play the role of testicular ischemia-reperfusion through the blood-testis barrier, so as to reduce the tissue injury caused by testicular ischemia-reperfusion and protect testicular spermatogenesis. These ndings suggest that the exocrine derived from bone marrow mesenchymal stem cells can exert the function of antioxidant stress and have more biosafety, which can provide a therapeutic idea for clinical prevention or treatment of ischemia-reperfusion injury after testicular torsion reduction.   Exosome derived from bone marrow mesenchymal stem cells can pass through the blood-testis barrier of rats A-F: The contents of SOD, MDA, TNOS, T-AOC, iNOS and CAT in testes of rats in each group (mean ± standard deviation, n=4 or 5). **P<0.01, *P<0.05 compared with sham operation group, +). +**P<0.01, *P<0.05 compared with I/R+NS group.