Adipose-Derived Mesenchymal Stromal Cell-Derived Exosomes Alleviate Testicular Torsion Injury via Activation of PI3K/AKT and MAPK/ERK1/2 Pathways

Background: Exosomes have became a new therapeutic method in recent years. Adipose-derived mesenchymal stromal cell-derived exosomes (ADSC-Exos) have shown great potential in the treatment of ischemia-reperfusion injury. However, there is no research on whether ADSC-Exos can alleviate testicular torsion injury. Therefore, we investigated the protective effect of ADSC-Exos in alleviating testicular torsion-detorsion injury. Methods: ADSC-Exos were isolated by ultracentrifugation and injected into the torsion-detorsion-affected testes of rats. H&E staining and sperm quality were used to evaluate the therapeutic effects of ADSC-Exos, and the level of tissue oxidative stress was determined using malondialdehyde (MDA) and superoxide dismutase (SOD). In addition, TUNEL staining and immunohistological analyses (Ki67, cleaved Caspase-3, IL-6, IL-10, CCR7, and CD163) were used to clarify the effects of ADSC-Exos on spermatogenic cell proliferation, apoptosis, and the inammatory microenvironment in vivo. Possible signaling pathways were predicted using sequencing technology and bioinformatics analysis. The signaling pathways were validated by proliferation (EdU), migration (transwell and scratch test), and apoptosis (ow cytometry, TUNEL, and western blot) in vitro. Results: ADSC-Exos alleviated testicular torsion-detorsion injury by attenuating oxidative stress and inammatory responses. In addition, ADSC-Exos promoted the proliferation and migration of spermatogenic cells and inhibited their apoptosis by activating the PI3K/AKT and MAPK/ERK1/2 signaling pathways. Conclusions: Overall, we demonstrated that ADSC-Exos can alleviate testicular torsion injury and provided a new approach for treating testicular torsion injury. superoxide dismutase, ROS: reactive oxygen Eagle’s serum, Transmission transferase nick end Gene Ontology, KEGG: Kyoto of Genes and


Introduction
Testicular torsion is a urological emergency characterized by acute scrotal pain with nausea and vomiting, which often occurs in male children [1,2]. Early detection, early diagnosis, and early treatment are key to avoiding testicular necrosis [3,4]. The primary pathological mechanism of testicular torsion is ischemia-reperfusion (I/R) injury. Changes in microvascular blood ow cause the release of proin ammatory cytokines and production of reactive oxygen species (ROS) [5]. This leads to disorganization of the spermatogenic cell structure and function, or even apoptosis, leading to spermatogenic impairment [6,7]. Thus, early surgery combined with antioxidants, anti-in ammatory cytokines, or other drugs is an important means of improving the prognosis of testicular torsion.
Adipose-derived mesenchymal stromal cells (ADSCs) play a profound role in preclinical studies [8,9]. Abundant data have demonstrated that ADSCs have many roles in the treatment of organ I/R injury, such as anti-in ammatory, antioxidant, and anti-apoptotic roles [10][11][12][13]. Local injection of ADSCs has been shown to rescue testicular torsion-induced infertility [14,15]. However, recent studies have found that transplanted mesenchymal stromal cells (MSCs) do not survive effectively in the ischemic Rat epididymal tissues were cut into 1 mm3 cubes and immersed in 0.9% NaCl at 37 ℃ for 20 min to extract the spermatozoa. Sperm quality (quantity, morphology, and motility) was assessed using the WHO sperm analysis method [21]. The morphology and motility of 200 sperm in each group were evaluated.

Histopathological and immunohistological analyses
Testicular tissues were xed in Davidson's xative (Beyotime) and the tissue sections were stained with hematoxylin and eosin (H&E). Johnsen's score (Table 1) was used to evaluate the spermatogenic function [22]. Fifty seminiferous tubules were examined in each testis.

Biochemical analysis
The malondialdehyde (MDA) in testicular tissue was determined by colorimetry measurement using the Lipid Peroxidation MDA Assay Kit (Beyotime). Superoxide dismutase (SOD) in testicular tissue was detected using the Cu/Zn-SOD, and Mn-SOD assay kits with WST-8 (Beyotime).

Cell culture and treatment
The GC-1 spg cell line was purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). The cells were cultured in DMEM with high glucose (Sigma) supplemented with 10% FBS, and 1% penicillin and streptomycin at 37 ℃ with 5% CO2. To establish the I/R model in vitro, 1 × 106 cells were cultured in glucose-free DMEM (Sigma) in a 1% O2 environment for 18 h, followed by reoxygenation with normal O2 in complete medium with or without exosomes.

ADSC-Exos internalization analysis
ADSC-Exos were labeled using PKH26 dye (Sigma). After ultracentrifugation, PKH-26 labeled ADSC-Exos were added to GC-1 spg cells cultured in exosome-depleted medium. The nuclei were counterstained with DAPI after 24 h, and the internalization of ADSC-Exos was observed under a uorescence microscope.

Proliferation of GC-1 spg cells
For cell proliferation analysis, 10 μM 5-ethynyl-2-deoxyuridine (EdU) was added to the GC-1 spg cell culture medium for 30 min. Then, the cells were xed and stained using an EdU assay kit (UE, China). Next, cell proliferation was observed under a uorescence microscope after DAPI counterstaining of the nuclei.

Migration of GC-1 spg cells
For cell migration analysis, we chose the scratch test and transwell assays. Brie y, after hypoxic injury with a serum-free culture medium, a scratch was made through the cultured cells. The extent of cell migration was measured after 0 and 24 h.
Each group of cells after hypoxic injury was digested for transwell assays. A total of 1 × 105 GC-1 spg cells were cultured in the upper chambers. The reoxygenation medium was added to the lower chambers. The upper chamber was xed with paraformaldehyde and stained with crystal violet after 24 h.

Flow cytometry analysis
To analyze the apoptosis, cells were collected and stained with the FITC Annexin V Apoptosis Detection Kit (Becton-Dickinson, USA), and then analyzed by ow cytometry. Becton-Dickinson in-house FACSDiva software was used to analyze the data.
Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay In vivo, apoptosis of spermatogenic cells was detected using a TUNEL apoptosis assay kit (Wanleibio, Shenyang, China). Brie y, para n sections of testicular tissues were incubated with 50 μL TUNEL reaction mixture. The sections were dehydrated and xed after hematoxylin counterstaining of the nuclei. Apoptosis of spermatogenic cells was evaluated under a light microscope.
In addition, we performed a TUNEL assay in vitro. Brie y, GC-1 spg cells from each group were xed and stained with a TUNEL Assay Apoptosis Detection Kit (UE, China). Next, apoptosis was observed under a uorescence microscope after DAPI counterstaining of the nuclei.

Micro RNA (miRNA) sequencing and data analysis
The sequencing of miRNA in ADSC-Exos was performed by OE Biotech Company (Shanghai, China). Brie y, 20 ng of exosomal RNA was extracted and sequenced using an Illumina HiSeq 2500 instrument (Illumina, CA, USA) (n = 3). The top 50 highly expressed miRNAs in ADSC-Exos were predicted to target genes using miRanda software. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses of target genes were performed using DAVID (https://david.ncifcrf.gov/), and KOBAS 3.0 (http://kobas.cbi.pku.edu.cn/kobas3/), respectively. The results of the analyses were visualized using R software.

Statistical analysis
Data were expressed as the mean ± standard deviation (SD). Statistical analysis for multiple groups were conducted by the Tukey-Kramer t-test. P < 0.05 was considered statistically signi cant.

Alleviation of testicular torsion-detorsion injury by ADSC-Exos
H&E staining showed that normal testes showed normal testicular structure, normal morphology of seminiferous tubules, and many mature sperm. However, 3 d after torsion-detorsion injury, severe injuries were observed in the testes, which manifested as seminiferous tubule disorders, unclear boundaries, interstitial edema, and few sperm. In contrast, the histological appearance of the testes was signi cantly improved after treatment with ADSC-Exos. Seven days after torsion-detorsion injury, the ADSC-Exostreated group had signi cantly more spermatogenic cells compared with the IR group, and there was a more orderly arrangement ( Figure 2A). The spermatogenic function was quanti ed using Johnsen's score and was substantially improved in the ADSC-Exos group on days 3 and 7 ( Figure 2B).
To further determine whether ADSC-Exos could improve spermatogenesis after torsion-detorsion injury, we extracted sperm from the epididymis of each rat. Analysis of sperm parameters showed that testicular torsion-detorsion led to poor sperm quality. The quantity, mobility, and morphology of sperm were signi cantly decreased in the I/R group. However, treatment with ADSC-Exos signi cantly improved the sperm quality ( Figure 2C-F). Furthermore, the level of MDA in the I/R group was signi cantly increased, while the level of SOD was decreased. ADSC-Exos treatment reduced MDA levels and increased SOD levels ( Figure 2G, H).

Protection of spermatogenic cell activity by ADSC-Exos
We next investigated whether ADSC-Exos affected spermatogenic cell activity after testicular torsiondetorsion injury. Immunohistochemistry revealed that the expression of Ki67 in spermatogenic cells was decreased after testicular torsion-detorsion injury. However, treatment with ADSC-Exos signi cantly increased the number of Ki67+ spermatogenic cells ( Figure 3A). Apoptosis marker TUNEL staining revealed massive spermatogenic cell apoptosis after testicular torsion-detorsion injury. The number of apoptotic spermatogenic cells in the ADSC-Exos group on days 3 and 7 was lower than that in the I/R group ( Figure 3B). As expected, cleaved Caspase-3 (a marker of apoptosis) immuno uorescence staining showed the same results as TUNEL staining ( Figure 3C). ADSC-Exos therefore promote spermatogenic cell proliferation and reduce apoptosis after testicular torsion-detorsion injury ( Figure 3D).

The miRNA sequencing and bioinformatics analysis of ADSC-Exos
To further understand how ADSC-Exos alleviate testicular torsion-detorsion injury, we sequenced the miRNA in ADSC-Exos. The top 50 miRNAs detected in ADSC-Exos are shown in Figure 4A. Next, we performed functional enrichment analysis of their possible target genes using GO and KEGG pathway enrichment analyses ( Figure 4B). The biological process (BP) was mainly enriched in "regulation of cell adhesion", the cellular component (CC) was mainly enriched in "proteinaceous extracellular matrix", the molecular function (MF) was mainly enriched in "SH3 domain binding" ( Figure 5A). KEGG pathway enrichment analyses showed that "PI3K/AKT", and "MAPK" were the main signaling pathways ( Figure   5B). Therefore, we made a bold assertion that ADSC-Exos alleviate testicular torsion-detorsion injury via the PI3K/AKT, and MAPK/ERK1/2 signaling pathways and proceeded to test this hypothesis.
ADSC-Exos regulated GC-1 spg cell proliferation and migration by activating PI3K/AKT and MAPK/ERK1/2 signaling pathways We evaluated the effects of ADSC-Exos on the proliferation and migration of GC-1 spg cells using different concentrations of ADSC-Exos, as well as pathway inhibitors. The EdU assays showed that ADSC-Exos promoted GC-1 spg cell proliferation after I/R injury in a dose-dependent manner, and LY294002 and PD98059 signi cantly attenuated this effect ( Figure 7A, D). Similarly, the transwell assays ( Figure 7B, E), and scratch test ( Figure 7C, F) showed that ADSC-Exos promoted GC-1 spg cell migration after I/R injury, which could also be suppressed by LY294002 and PD98059.
ADSC-Exos protected GC-1 spg cells against apoptosis by activating PI3K/AKT and MAPK/ERK1/2 pathways signaling pathways Flow cytometry and TUNEL assays were used to detect apoptosis in the GC-1 spg cells. After I/R injury, the number of apoptotic cells in GC-1 spg cells signi cantly increased, whereas treatment with ADSC-Exos clearly decreased the apoptosis of cells. LY294002 and PD98059 inhibited the anti-apoptotic effects of ADSC-Exos ( Figure 8A, B, F, J). In addition, western blotting showed that ADSC-Exos could increase the low expression of Bcl-2, and decrease the high expression of Bax caused by I/R. Similarly, LY294002 and PD98059 attenuated the regulation of Bcl-2 and Bax expression by ADSC-Exos (Figure 8C-E, G-I).
ADSC-Exos regulate the in ammatory response induced by testicular torsion-detorsion injury We next investigated whether ADSC-Exos affected the early in ammatory response after testicular torsion injury. Immuno uorescence showed that a large amount of IL-6 (a pro-in ammatory factor) aggregated within the testicular tissue on day 3 after testicular torsion-detorsion injury and decreased on day 7 ( Figure 9A). However, compared with the I/R group, both ADSC-Exos groups were signi cantly decreased on days 3 and 7. In contrast, IL-10 (an anti-in ammatory factor) was increased in the ADSC-Exos group ( Figure 9B). In addition, we found that CCR7+ (M1 macrophage marker), and CD163+ (M2 macrophage marker) cells increased after testicular torsion-detorsion injury. However, compared with the I/R group, there were signi cantly less CCR7+ cells in the ADSC-Exos group, and conversely, CD163+ cells were signi cantly increased ( Figure 9C, D). The results of the quantitative analyses are shown in Figure 9E.

Discussion
Testicular torsion is a major cause of testicular loss in male adolescents [3]. Effective anti-oxidation and anti-in ammatory adjuvant therapy are the main means of reducing I/R injury after testicular torsion. Recent studies have shown that ADSC-Exos can effectively alleviate I/R injury of the cerebrum and myocardium [23]. In this study, we found that ADSC-Exos can reduce oxidative stress, inhibit in ammation, promote proliferation and migration, and prevent apoptosis in the testes.
The physiological properties of spermatogenic cells, which are on the borderline of hypoxia, make them sensitive to changes in blood ow [24]. In the present study, the rat testes were severely damaged after I/R injury, whereas local injection of ADSC-Exos signi cantly improved the pathological features of testes. The ROS induced by I/R exceed the scavenging ability of antioxidant enzymes, leading to spermatogenic cell apoptosis. MDA is the end product of ROS and is a reliable indicator of the level of ROS [25]. SOD protects cells against superoxide radical damage by dismutating superoxide radicals into H2O2 and O2 [26]. Although antioxidants can reduce the level of oxidative stress induced by testicular I/R injury [27][28][29], antioxidants alone cannot improve spermatogenesis after injury [15]. Fortunately, transplantation of the uncultured adipose-derived stromal vascular fraction can promote spermatogenesis while reducing oxidative stress levels after testicular torsion [30]. As expected, in the present study, ADSC-Exos signi cantly reduced MDA levels, increased SOD levels, and improved sperm quality (quantity, morphology, and motility) after testicular torsion injury.
After testicular torsion-detorsion injury, ROS production is accompanied by the activation of apoptotic pathways [31]. Apoptosis induced by testicular torsion-detorsion injury occurs in all spermatogenic cells, of which apoptosis of primary spermatocytes is the main reason for impaired fertility [32]. In the present study, TUNEL staining and cleaved Caspase-3 immuno uorescence analyses indicated that after testicular torsion injury, the caspase-dependent apoptosis pathways were activated and spermatogenic cells were largely apoptotic, whereas treatment with ADSC-Exos signi cantly attenuated the degree of apoptosis in spermatogenic cells. Seven days after injury, the number of apoptotic cells signi cantly decreased, indicating that apoptotic spermatogenic cells had died. However, H&E staining indicated that the number of spermatogenic cells increased after ADSC-Exos treatment. Therefore, we investigated the effects on the proliferation of spermatogenic cells. Ki67 is a cell proliferation marker. Immunohistochemistry showed that in normal testicular tissue, spermatogenic cell proliferation mainly occurred in primary spermatocytes. The proliferative capacity of primary spermatocytes was inhibited after testicular torsion-detorsion injury. Following treatment with ADSC-Exos, the expression of Ki67 in spermatogenic cells was obviously increased. Interestingly, we also found that the proliferative effect of ADSC-Exos on spermatogenic cells was not restricted to primary spermatocytes. These results suggest that ADSC-Exos inhibit spermatogenic cell apoptosis and promote their proliferation after testicular torsion-detorsion injury.
Since miRNAs are the main components through which exosomes function [33], we sequenced miRNAs in ADSC-Exos to understand how ADSC-Exos alleviate testicular torsion-detorsion injury. The target genes were predicted and subjected to bioinformatics analysis. The enrichment analyses showed that PI3K/AKT and MAPK pathways may play major roles. Therefore, we hypothesized that ADSC-Exos alleviate testicular torsion-detorsion injury by activating the PI3K/AKT and MAPK/ERK1/2 pathways. To con rm our hypothesis, we examined the association between ADSC-Exos and PI3K/AKT and MAPK/ERK1/2 signaling pathways. We found that ADSC-Exos upregulated the decreased expression of p-AKT and p-ERK1/2 induced by I/R injury. In addition, ADSC-Exos promoted proliferation and migration and inhibited the apoptosis of GC-1 spg cells after I/R injury. Pathway inhibitors (LY294002 and PD98059) were used to further investigate the roles of the PI3K/AKT and MAPK/ERK1/2 pathways in ADSC-Exos alleviated I/R injury. LY294002 and PD98059 effectively inhibited the expression of p-AKT and p-ERK1/2, respectively, and blocked the protective effects of ADSC-Exos against GC-1 spg cell I/R injury. This supports the assertion that ADSC-Exos alleviated testicular torsion-detorsion injury by promoting activation of the PI3K/AKT and MAPK/ERK1/2 signaling pathways.
The in ammatory response is an important pathological mechanism of I/R injury [34]. ROS-induced redox changes lead to the release of in ammatory cytokines [35]. Sevil et al. reported that the expression of TNF-α and IL-6 was increased, and that of IL-10 was decreased after testicular torsion [32]. Turner et al. found that TNF-α, IL-6, and IL-1β released by macrophages exacerbated in ammation in testicular torsion-detorsion injury [36]. Recent studies have con rmed that the polarization of macrophages determines their role in in ammation [37][38][39]. In addition, previous studies have con rmed that ADSC-Exos modulate macrophage polarization from the pro-in ammatory M1 to the anti-in ammatory M2 phenotype in vitro [40][41][42]. In the present study, treatment with ADSC-Exos reduced the number of CCR7 + M1 macrophages and increased the CD163 + M2 macrophages after testicular torsion injury. As we expected, the expression of the pro-in ammatory cytokine, IL-6, was decreased, whereas the antiin ammatory cytokine, IL-10, was increased after ADSC-Exos treatment. ADSC-Exos therefore alleviate in ammation induced after testicular torsion injury by modulating macrophages.
In general, exosomes are used to repair damaged tissues via intravenous injection. Because of the physiological blood-testis barrier, transplanted exosomes may not be able to enter the testis via the circulation. Thus, we chose local injections to transplant ADSC-Exos. In addition, exosomes have little immunogenicity compared to stromal cell transplantation. Especially in ethically relevant organs such as the testis, the transplantation of exosomes has more advantages than stromal cells.
There are some limitations to the current study. First, we only evaluated the early e cacy of ADSC-Exos in ameliorating testicular torsion-detorsion injury, and the long-term e cacy, such as fertility function, still requires further study. Second, the optimal dose for the treatment of testicular torsion-detorsion injury requires further study. Third, although we predicted and validated the pathway by which ADSC-Exos play a role by sequencing, the speci c miRNA still needs to be identi ed in subsequent studies.

Conclusions
This study showed that ADSC-Exos can alleviate testicular torsion-detorsion injury by reducing oxidative stress and promoting M2 polarization of macrophages to inhibit in ammation. ADSC-Exos activated the PI3K/AKT and MAPK/ERK1/2 pathways to promote proliferation and migration and inhibit apoptosis of spermatogenic cells. Our study raises the feasibility of ADSC-Exos in alleviating testicular torsiondetorsion injury and provides a new avenue for treating testicular torsion injury.

Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Ethics approval and consent to participate
The experimental protocol about animals was approved by the Harbin Medical University Ethics Committee.

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