Exosome Therapy on Cisplatin-Induced Testicular Tissue Toxicity 

Exosome Therapy on Cisplatin-Induced Testicular Tissue Toxicity | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Exosome Therapy on Cisplatin-Induced Testicular Tissue Toxicity Halime TOZAK YILDIZ, Kübra Tuğçe KALKAN, Numan BAYDİLLİ, Zeynep Burçin GÖNEN, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4011219/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Backround: Cisplatin is a significant chemotherapy drug that has been used for different cancer types. The infertility and gonadotoxicity caused by metabolites resulting from cisplatin metabolism are two of the most serious side effects of cisplatin. Testicular dysfunction is generally caused by oxidative stress and autophagy that are induced by cisplatin. This study focused on potential exosomes' regenerative, antioxidant, and autophagy-regulating properties might be used to minimize testicular damage resulting from cisplatin. Methods For cisplatin toxicity, one dose of 7.5mg/kg was injected on first day. Exosom (80x10 6 /100µl) was injected single dose on sixth day. To examine potential therapeutic effects of the exosome, tissue samples were taken on eighth day. For examination of testicular morphology, tissues were stained with Hematoxylin&Eosin. Sperm motility and number were examined. Autophagy markers and SF-1,StAR expressions, were evaluated immunohistochemistry. Oxidative stress, inhibin and testosterone levels were analysed. Results Cisplatin-induced damage caused to decrease in germinal epithelium, edema in interstitial areas, and degeneration in seminiferous tubules. Sperm motility and quality decreased. When compared to control groups, there was an increase in the expression of Beclin-1, p62, LC3-2, SF-1, and StAR. MDA activity increased,GSH-PX, SOD, and CAT activity decreased. Testosterone and inhibin levels decreased in serum. Tissue morphology was returned to normal, autophagy marker upregulation decreased, and SF-1 and StAR expressions increased in the group that received exosome, following cisplatin. There was a decrease in oxidative stress and increase in hormone levels Conclusion These findings suggest that the exosome may offer a promising approach to minimizing Cis-induced reproductive toxicity and tissue damage by reducing autophagy and improving oxidative stress. Exosome cisplatin testicular damage autophagy Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 INTRODUCTION Cisplatin (Cis) is an effective antineoplastic drug used to treat a variety of cancers. Antineoplastic drugs cause oxidative stress in the biological system( 1 – 3 ). It has been reported that tissue damage in the reproductive system is caused by reactive oxygen metabolites produced during Cis metabolism. Its gonadotoxic effects include the formation of primordial follicles and the degradation of granulosa cells in ovaries, as well as a reduction in spermatogenesis in the testicles. ( 3 – 12 ) Cis causes cell cycle arrest and apoptosis by creating intra/inter-chain cross-covalent connections in the DNA doublechain, which inhibits DNA synthesis ( 13 ). Testicular damage caused by Cis affects leydig cells and seminiferous tubules, spermatogenic cells, and Sertoli cells, resulting in decreased sperm count, morphology, chromatin quality, and motility. ( 4 , 14 ) Exosomes, a novel mode of cell-cell communication, are nanovesicles of endocytic origin that range in size 30-150nm. Extracellular and amniotic fluids with various cell types, including blood, urine, and those released by the hydrothorax, saliva, breast milk, sperm, and cerebral fluids. ( 15 , 16 ) Exosomes contain many bioactive molecules, such as proteins, lipids, mRNAs, miRNAs, tRNA, genomicDNA, cDNA, and mtDNA. Exosomes, which are extracellular organelles, play an important role in the overall information flow within cells.( 17 ) Exosomes initiate cell-to-cell contact by delivering the proteins, miRNAs and enzymes they carry to the target cell. Exosomes are extracellular organelles that function in both the paracrine and endocrine systems. ( 18 ) Exosomes can be phagocytosed by another cell type, endocytosed, or directly fusioned by other cells. ( 19 ) The autophagy mechanism is a physiological process that serves as a quality control system in cells by destroying pathogens, long-lived proteins, damaged macromolecules, and degraded organelles. ( 20 ) The major objective is to offer an alternative source for intracellular structures and a substrate for energy production in order to maintain cell survival. If the cell's environment is unable to provide it with the nutrition it requires, the cell must consume it internally in order to survive. Additionally, cellular stress caused by pathogen infection, hypoxia, nutritional deprivation, or reactive oxygen species (ROS) can cause autophagy to occur. The majority of research has been on how hunger-induced autophagy helps cells survive. The independent relationship between autophagy and other cell death pathways or processes, such as apoptosis, is tight. ( 21 ) Cellular autophagy can be induced by anti-tumor drugs that kill tumor cells. Although several oncoproteins also suppress autophagy, many tumor suppressor genes increase it. ( 22 ) Testicular damage, oxidative stress, and the activation of apoptotic signaling pathways are the main symptoms of cis-induced gonadotoxicity. ( 3 , 4 , 13 , 14 ) Spermatogenic series in testicles frequently leads to harm by inducing apoptosis in Sertoli and Leydig cells, which is one of the condition's adverse effects. Bone marrow-derived mesenchymal stem cell exosomes are active paracrine components that have a great potential for damaged tissue repair, suppress inflammatory responses, and modulate the immune system. ( 23 , 24 ) Due to the large range of components they carry, exosomes have promise in the field of regenerative medicine. ( 24 – 26 ) No studies examining the therapeutic effects of exosomes were found in the literature, despite the fact that there are studies examining the impact of several stem cells on testicular injury in experimental Cis studies in rats. In this study aimed the potential therapeutic effects of bone marrow-derived MSC exosomes on cis-induced testicular damage through the autophagy mechanism and oxidative stress. METHODS Animals and experimental groups The study was started with 12 eight-week-old male Wistar albino rats (weight: 150–210 g). The rats were kept in a 25°C room with a 12-hour light/dark cycle, free access to water, and a standard diet from the Experimental and Clinic Research Center at Erciyes University in Kayseri, Turkey. The adult rats were randomly divided into 4 groups, with 8 rats in each group: Control group: untreated control. Exosome (Exo) group: Exosomes (80x10 6 /100μl) were injected intravenous (i.v.) in a single dose to rats on the fifth day of the experiment. Cis group: Cisplatin (7,5 mg/kg in 0.9% saline solution)(Koçak Pharma Company, Turkey) was injected intraperitoneally (i.p.) in a single dose to rats on the first day of the experiment to induce cisplatin testicular toxicity (25 mg/50 ml) vials. (27,28) Cisplatin+Exosome (Cis+Exo) group: Cis (7,5 mg/kg in 0.9% saline solution) was injected i.p. in a single dose to rats on the first day to induce Cis. Exosomes (80x10 6 /100μl) were injected i.v. in a single dose into rats on the fifth day. On the 8th day of the study, rats in the groups were sacrificed by cervical dislocation after testicular and blood samples were taken under xylazine (10 mg/kg, IP) and ketamine (60 mg/kg, IP) general anaesthesia. Experiments were carried out following the Declaration of Helsinki and the National Institutes of Health's (USA) Guide for the Care and Use of Laboratory Animals. All experimental protocols were approved by the Local Ethic Committee of Animal Experiments of Erciyes University (approval no. 22/185). Isolation of Exosomes from Mesenchymal Stem Cells Rat bone marrow-derived mesenchymal stem cells were thawed in a 37°C water bath before being seeded into 75 cm² cell culture dishes (TPP, Switzerland) that were acquired from Erciyes University Genome and Stem Cell Centre. A serological pipette was used to remove the media from the MSCs to facilitate the generation of exosomes after their density exceeded 90%. After one day, serum-free media was added and the cell secretomes were extracted. Exosome production was performed from the obtained secretome using ExoQuick-TC (#EXOTC50A-1, System Bio sciences, USA) kit. The collected secretomes were centrifuged at 3000x g for 15 minutes, as per the kit instructions, and the cells and their wastes sank to the bottom. One-fifth of the volume of the supernatant was added to a fresh tube containing the supernatant and the exosome isolation solution. Vortex was used to assist homogenize it. After that, it was incubated for 24 hours at +4ºC. Following incubation, the mixture was centrifuged for 30 minutes at +4ºC at 1500x g. After centrifugation, the pellet was resuspended in PBS, and the supernatant was discarded. A microvesicle quantitation kit (EXOCET Exosome Quantitation Assay- EXOCET96A-1 SBI, USA) was used to measure the quantity of exosomes that were obtained before they were given to experimental animals. The Glomax Multi ELISA absorbance reader (Promega, USA) was used to read absorbance values at 450 nm following the quantification process that followed the kit's instructions. The amount of exosomes was calculated using standards. (29 ) Exosomes diluted 80x10 6 /100μl were injected to every animal in the study. Assessment of sperm parameters 10 µL of the supernatant containing epididymal sperm was diluted in a 990 µL solution of paraformaldehyde and sodium citrate for this purpose. Approximately 10 µL of the diluted material was transferred to a hemocytometer (Neubauer chamber), and the pelletized cells on the chamber's surface were counted using a 400 x light microscope. The sperm concentration was calculated using the number of counted cells and hemocytometer dimensions. Thus, the concentration was reported in millions of sperm per mL, and the hemocytometer dimensions were expressed in millions of sperm per mL, as described by Badkoobeh et al. in 2013. A drop of about 20 µL of sperm solution was placed on the microscope slides and swiped for sperm morphological analysis. After drying, the slides were stained with eosin-nigrosin (1% eosin Y and 5% nigrosine). They were viewed in optical microscopy (BX51, Olympus Corp. Tokyo Japan) at 400 x after drying. A differential count of 200 spermatozoa per slide was done, and alterations in the head, middle piece, and tail were detected. Badkoobeh et al. reported the data as a proportion of normal cells. (30) Histopathological analysis The testicles were fixed in 10% neutral formalin for 48 hours, treated with ethanol , and then embedded in paraffin blocks for histological examinations. Sections 5 μm thick were then cut from a paraffin block (Leica, RM 2000, Germany) and stained with hematoxylin and eosin (H&E) to examine the general histomorphological structure of testis tissues . Standard light microscopy was used to analyze and assess the testicular tissue in a blinded manner light microscope. The testicular tissue was scored according to Johnsen's criteria after being stained with H&E. (31) The Johnsen’s testicular biopsy score (JBTS) was determined by sampling five locations in each section's 20 seminiferous tubules. According to the number of cells and how mature they were, JBTS was calculated for each tubule (Table 1). Moreover, as a measure of damage, the diameter of the seminiferous tubule was measured at a scale of 200 x using the ImageJ software (Bethesda, MD). For the rat, the typical tubule diameter was estimated after measuring the seminiferous tubule diameters in 10 distinct places Immunohistochemistry The streptavidin-biotin-peroxidase method was used for the immunohistochemistry staining kit (Lab VisionTM UltraVisionTM Large Volume Detection System: anti-polyvalent, HRP, TA-125-HL). Beclin-1, p62, LC3-2, steroidogenic factor-1 (SF-1), and steroidogenic acute regulatory protein (StAR) expressions in testicular tissues were identified using this technique. Testes were cut into 5-μm sections, deparaffinized in xylene, and then rehydrated using a graduated ethanol series. After rehydration, the sections were washed in deionized water, phosphate-buffered saline (PBS) was used to rinse the sections, and then 3% hydrogen peroxide was applied for 5 minutes. All sections were then saturated in 5% sodium citrate buffer and heated at 600 W for 5 minutes to initiate antigen retrieval. Beclin-1 primary antibody diluted 1:500 (Novus, NB500-249), p62 primary antibody diluted 1:200 (Anti-SQSTM-1, H00008878), LC3-2 primary antibody diluted 1:750 (Cell Signaling, 12741 S), SF-1 primary antibody diluted 1:150 (NBP2-46247; Novus Biologicals), and StAR primary antibody diluted 1:250 (bs-3570R; Bioss Inc.) were all applied to the sections in an immunohistochemical chamber for an overnight duration at 4°C. Following that, biotinylated secondary antibodies were incubated with the sections. Then, after the washing process, they were treated with 3,3'-diaminobenzidine tetrahydrochloride substrate (TA-060-HDX, Thermo Fisher Scientific, Waltham, MA, USA) to make the immunoreactivity visible. Gill's hematoxylin was used as a counterstain on the sections to enhance nuclear staining. Under a light microscope, each section was evaluated, and microscopic images of five different testicular tissue regions were obtained. Beclin-1, LC3-2, p62, SF-1, and StAR immunoreactivity intensities were measured using the Image J software (NIH; Washington, USA). Elisa Testicular tissues stored at -80 ºC for biochemical evaluation were transferred to PBS (0.01M, pH:7.4) for homogenate preparation. Each homogenized sample was centrifuged at 4 ºC at 10000 rpm for 10 min. For the ELISA method, the obtained supernatants were aliquoted. Glutathione Peroxidase (GSH-PX) (Sunred Bio, Cat. No: 201-11-5104), Superoxide Dismutase (SOD), Catalase (CAT) (Sunred Bio, Cat. No: 201-11-5106), and tumor necrosis factor-alpha (TNF-α) (Sunred Bio, Cat. No: 201-11-0765) were measured to determine the antioxidant levels of testicular tissue. The levels of serum inhibine (INHB) and testosterone were assessed. Furthermore, the level of malondialdehyde (MDA) (Sunred Bio, Cat. No: 201-11-0157) was measured in the testis tissue. Statistical Analysis of the Data The GraphPad Prism 9 program was used to evaluate the outcomes. The Shapiro-Wilk test was performed to determine if the data were normally distributed. A one-way analysis of variance (ANOVA) was used to analyze data that was regularly distributed. The source of the difference was then confirmed using the Tukey posthoc test. The significance level was set at P < 0.05. RESULTS Exo reduced the pathological changes in Cis-damaged testicular tissues As the testicular sections stained with H&E were evaluated, the control group's seminiferous tubules were organized into the various stages of spermatogenesis and germinal epithelium. The components of this epithelium are the spermatogonia, spermatids, and spermatocytes. In the interstitial location, Leydig cells were observed. The Cis group demonstrated degenerative alterations in the seminiferous tubule, dilatation, a reduction in the number of germ cells, irregularities, and lumen shedding. The seminiferous tubules of the Exo groups are arranged regularly, smilar to control group. While edema was observed in the Cis+Exo group, tubule degeneration was reduced, and the germinal epithelium became more regular (Figure 1A). The experimental group’s testicular injuries were scored using JTBS (Table 1). The Cis group scored significantly lower than the other groups. There is a significant difference between Cis and Cis+Exo (p<0.001 for all comparisons) (Figure 1B). Seminiferous tubule diameters were also evaluated to assess testicular injury. The Cis group had significantly smaller seminiferous tubule diameters than the control group and exo group. Seminiferous tubule diameter increased statistically in the Cis+Exo group compared to the Cis group. (p<0.05) (Figure 1C). There is a significant difference between the control, Exo, Cis (p<0.001), and Cis+Exo groups (p<0.05). The Effect of Exo Sperm Quality Analysis After Cis therapy, there was a significant decrease in the number of sperm compared to the control group (p = 0.000) and Exo group (p = 0.007). Between the Cis and Cis+Exo groups, there was a significant increase (p<0.001) (Table 2). Sperm motility analysis showed a significant decrease after Cis therapy when compared to the control group and Exo group (p<0.05). The Cis+Exo group shows an increase in the Cis group, but the difference is not statistically significant. (Table 2). Cis-induced autophagy is supressed by Exo The contribution of MSC-derived exosomes on the expression levels of autophagy proteins Beclin-1, p62, and LC3-2 following Cis-giving was investigated in this research. In the control groups, immunohistochemical evaluation of autophagy markers indicated a weakly positive cytoplasmic reactivity (Figure 2). The Cis group immunoreactivity intensity for Beclin-1, p62, and LC3-2 was elevated substantially in comparison with the control group (p<0.001 for all comparisons). Following Cis, Exo-treated rats showed a significant decrease in the autophagy markers Beclin-1 (p<0.05), p62, and LC3-2 (p<0.001) (Figure 3). SF-1 and StAR İmmunoreactivity SF-1 has been shown to modulate the expression of the key protein StAR, which plays a role in the synthesis of testosterone. Leydig cells in the interstitial area, the control groups expressed SF-1 and StAR immunoreactivity (Figure 4A). The Cis group had considerably lower immunoreactive Leydig cell intensity than the control group (p<0.001). SF-1 immunoreactivity in Leydig cells showed a significant increase (p<0.05) in Cis+Exo group rats treated with Exo after Cis (Figure 4B). The StAR expression in the Cis+Exo group did not significantly increase. Effect of Exo on testicular oxidative stress and serum hormone levels Testicular oxidative stress injury brought on by cisplatin was discovered by measuring antioxidant and oxidant enzyme levels in this study. When compared to the control group, testicular GSH-PX concentration decreased significantly (p<0.05), and oxidative stress was dramatically initiated, as indicated by an elevated MDA level (p<0.01). However, compared to controls, it decreased activity, SOD, and CAT (p<0.001). MDA production was reduced, and MDA imbalance was improved in the Cis+Exo group treated with Exo following Cis (p<0.001). Exo-therapy increased GSH-PX concentration in the Cis+Exo group rats (p<0.05) and significantly decreased oxidative stress by decreasing testicular MDA (p<0.001). When compared to the control group, there was a significant rise in testicular TNF-α expression in the Cis-treated group (p<0.05). The Exo-treated Cis+Exo group blocked the increase in testicular TNF-α expression compared to the Cis group, but there was no statistically significant difference (Figure 5). Reduced SF-1 and StAR immunoreactivity in Leydig cells resulted in decreased testosterone levels. INHB regulates sperm production by inhibiting the pituitary gland from secreting too much follicle-stimulating hormone (FSH). The presence of androgens in the blood increases inhibin levels, promoting spermatogenesis. Reduced testosterone levels also have a deleterious impact on INHB levels. Cis therapy decreased serum testosterone (p<0.005) and INHB levels (p<0.001) significantly (p<0.001). When compared to the Cis group, Exo therapy increased serum testosterone (p<0.005) and INHB levels (p<0.001) (Figure 6). DISCUSSION Cis, a chemotherapy drug used to treat several cancers, causes damage to tissue in the testes despite its potent antitumoral activity. Because chemotherapy agents attack dividing cells, the spermatogenesis process is particularly affected. Previous studies reported that exposure to Cis reduced reproductive function. ( 2 , 5 , 11 , 12 , 14 , 32 ) This study's histological, immunohistochemical, and biochemical results demonstrated Cis's damaging impact on testicular tissue. Antitumoral drugs that destroy cancer cells, like Cis, damage tissue by causing cells to undergo autophagy and apoptosis. ( 1 – 3 , 6 , 10 – 14 , 22 , 27 , 28 ) Exosomes of BM-derived MSCs have been shown to have therapeutic potential in different articles studying tissue damage and degenerative disorders. ( 15 , 19 , 33 – 35 ) Exosomes function as extracellular organelles with a paracrine/endocrine role, transporting proteins, miRNAs, and enzymes to target cells. ( 18 ) Many studies have reported that MSC exosomes have antiapoptotic, anti-inflammatory, and regenerative effects. ( 17 , 18 , 33 – 35 ) Cis therapy changed the testicular morphology, induced autophagy, and decreased hormone levels, sperm count, and oxidative stress. A single dose of exo-therapy was shown to have considerable regenerative and antioxidant effects, as well as increased autophagic activity. Hormone levels increased significantly, although sperm motility and count were unaffected. The Cis group exhibited degeneration, atrophied tubular epithelium, cavities, and dilatation of seminiferous tubules. A decrease in tubule spermatogenic cells, germ cell infiltration into the lumen, and interstitial edema were reported. These findings were supported by a decrease in the JTBS score and tubule diameter. In studies, it was reported that seminiferous tubule diameters, germinal epithelial thickness, and testosterone synthesis decreased in Cis-treated rats; testicular atrophy and infertility may develop accordingly. ( 36 – 38 ) Exosomes are highly effective active paracrine components that play a vital role in cell communication and have a high potential for tissue repair ( 23 ). Exosomes establish cell-cell interactions by delivering proteins, miRNAs, and enzymes to the target cell. Exosomes, as extracellular organelles, thus serve a paracrine/endocrine role. ( 18 ) Exosomal proteins and microRNAs have a wide range of biochemical and physiological functions, including communication, structure and interactions, inflammation, exosome biogenesis, tissue repair and regeneration, and metabolism. ( 16 , 17 , 34 ) Exo treatment reduced seminiferous tubule degeneration and restored germinal epithelial cell structure. Edema continues in this group. Additionally, the JTBS socket and tubule diameters increased in this group. Cis, an alkalizing chemical, cross-links to the inner and outer chains of DNA, generating 'adduct' forms that can be seen as twisted DNA chains. Sperm are presumably impacted by this mechanism, as is the testicular structure. ( 36 ) In studies, Cis treatment caused a significant decrease in sperm count and sperm motility in rat testes. ( 27 , 37 , 38 ) In this study, sperm motility and count were significantly decreased by Cis therapy. A significant increase in sperm count was detected in the Cis + Exo group that received Exo therapy, but sperm motility did not significantly increase. Sperm count and motility increased when Exo was given as only one dose on the third day following Cis therapy, but motility did not significantly increase. Because exosome uptake is dependent on intracellular and microenvironmental acidity and tissue injury is frequently characterized by tissue acidosis, exosomes would be preferentially endocytosed by cells in an injured tissue, implying that exosomes could be home to injured tissues. ( 35 , 39 ) Furthermore, exosomes are small, nanometer-sized particles that can easily be carried via blood and other bodily fluids, as indicated by the quantity of exosomes seen in most biological fluids, and MSC exosomes can interact with cells in a paracrine and endocrine way. As a result, exosomes have the biophysical capacity to orchestrate MSC interactions with a variety of cell types in both local and distant environments. However, the ability to evoke appropriate cellular responses after tissue injury in order to restore and maintain tissue microenvironment homeostasis would be dependent on the biochemical capability of its protein and RNA cargo. ( 33 ) Autophagy, which is essential for cellular adaptability or survival, can be triggered by a variety of stressors, including metabolic stress, endoplasmic reticulum (ER) stress, and chemotherapeutic drugs. ( 40 ) An intense energy is expended in the process of sprematogenesis. In this process, starvation, chemical exposure and radiation negatively affect this process. The connection between apoptotic and autophagic molecular mechanisms in these pathophysiological conditions has been shown by the studies carried out. ( 41 , 42 ) Cis-induced oxidative stress and tissue acidosis in testicular tissue stimulated the apoptotic pathway and autophagy process. Autophagy induced by Cis in testis was detected by increased Beclin-1, LC3-2 and p62 proteins. Exo therapy at a single dose significantly improved in tissue repair via regulating Cis-induced autophagy. Previous studies have showed that exos protected against testicular ischemia/reperfusion injury in rats through antioxidant, anti-inflammatory, and antiapoptotic mechanisms ( 43 , 44 ). It has also been reported that antitumoural drugs trigger the autophagy process. ( 22 ) Exosomes have been shown to have immunoregulatory, anti-inflammatory, regenerative, and anti-apoptotic capacities in therapeutic applications, however it is unclear whether or if such characteristics regulate autophagy. ( 19 ) Oxidative stress, defined as an imbalance between free radicals and the antioxidant defense system, is crucial in disease development. ( 45 ) Chemotherapy drugs cause mitochondrial damage. This results in an increase in reactive oxygen species and, as a result, an increase in oxidative stress. ( 46 ) This accumulation of ROS would operate as a cell chemoattractant, attracting MSCs and exosomes to the damage site. ( 47 ) As a result of the accumulation of ROS at the injury site, MSCs and their exosomes are drawn to the area by this chemoattractant. ( 34 , 48 ) The levels of antioxidant enzymes such as GSH-PX, MDA, SOD, and CAT were evaluated in male rats to evaluate the therapeutic effects of Exo on testicular toxicity caused by Cis-induced oxidative stress. The Cis-induced group's GSH-PX tissue concentration, SOD, and CAT activities in testicular homogenate were significantly lower than the control group. Cis increased MDA levels, a marker of testicular lipid peroxidation. TNF-α, an inflammatory marker in the testis, was significantly increased. The results confirmed oxidative stress, which is similar to previous research on gonadal damage caused by cisplatin.( 2 , 3 , 12 , 27 , 28 , 36 ) The exo-treated Cis group's GSH-PX concentration and SOD and CAT activities improved significantly. MDA levels were significantly reduced. Single doz exo caused protection against the up-regulation of testicular TNF-α levels in Cis-inducted rats. However, no significant decrease in TNF-α level was obtained. TNF-α plays an active role as an inflammatory cytokine in Cis-induced testicular damage. ( 49 , 50 ) An increase in TNF-α levels results in impaired spermatogenesis and decreased testosterone levels. Increased ROS production following damage to mitochondria contributes as well to this process by initially apoptosis. ( 51 ) Studies have shown that exosomes are able to penetrate the body's barrier and reach the damaged area. ( 52 ) Exosomes with antiapoptotic, anticancer, and antioxidant qualities have been reported to lower oxidative stress in the area of damage. ( 19 , 44 , 53 , 54 ) The primary functions of the testicles are the production of testosterone and sperm. ( 55 ) Leydig cells in the interstitial tissue of the testes are responsible for the secretion of testosterone. ( 56 ) In this study, the group that received Cis testosterone had lower testosterone levels. The decrease in SF-1 and StAR protein expression supports the decrease in testosterone levels. NHB is a key hormone that regulates reproductive function. The INHB hormone is a key regulator of FSH, which is released by sertoli cells in the testis. ( 57 , 58 ) FSH stimulates the seminiferous tubules and the Sertoli cells. It is in charge of starting the spermatogenesis process. Testosterone is required for spermatogenesis to continue. ( 59 ) INHB and FSH secretion become negatively correlated during secondary Sertoli cell development and spermatogenesis. Decreased serum INHB indicates sperm damage and infertility. As a result, it is used to measure male spermatogenesis ( 60 , 61 ). Inhibins also serve as growth factors in Leydig cells via paracrine mechanisms. INHB, in conjunction with testosterone, regulates spermatogenesis indirectly. ( 62 ) Previous research has shown that CP intake has a direct effect on the Leydig cells, resulting in cellular death and a decrease in secretory activity. ( 28 , 63 ) Because of damaged seminiferous tubule epithelium and low testosterone levels, serum INHB levels were considerably lower in the Cis group compared to the control group. INHB levels significantly increased in the exo-treated group after Cis. This recovery process was supported by tissue regeneration in the seminiferous tubule and interstitial areas. CONCLUSION Exosomes have several advantages as a cell-free therapy. In recent years, it has shown potential in the treatment of several human diseases. The key advantages of exosomes are that they are natural, can be derived from a person's own cells, and can be targeted to specific cells or tissues. This study examined the therapeutic efficacy of bone marrow-derived mesenchymal stem cell exosomes on cisplatin-induced testicular damage in rats. Exosomes, a regeneratif product with anti-cancer efficacy, were found to reduce cisplatin-induced testicular damage in rats. Accordingly, exosomes might be an encouraging agent in treating Cis-induced testiculer damage and degeneration of the germ cell layer. This study shows that a single dose exosomes may treat Cis-induced testicular damage by reducing oxidative stress and regulating enhanced autophagy. In order to see more effective and long-term results, new studies may design to include repeated doses, different dose applications and more localised applications. Studies on the regulation of autophagy at a more molecular level may be more informative to understand its therapeutic effect. Declarations ACKNOWLEDGMENT We thank the staff of Erciyes University ERÜ Experimental Research and Application Centre for the care of the rats in this study. We would like to thank the Gevher Nesibe Genome and Stem Cell Institute of Erciyes University for their support in the generation of BM-MSCs and the exosomes derived from these cells. For this study, no funding were provided. CONFLİCT OF INTEREST STATEMENT The other authors have no conflict of interest to disclose. ETHICS STATEMENT Experiments were carried out following the Declaration of Helsinki and the National Institutes of Health's (USA) Guide for the Care and Use of Laboratory Animals. All experimental protocols were approved by the Local Ethic Committee of Animal Experiments of Erciyes University (approval no. 22/185). DATA AVAİLABİLİTY The data in this study were obtained from experiments, not from the internet, and therefore are not suitable for data sharing. AUTHORS’ CONTRİBUTİONS H.T.Y.: Conceptualization, data curation, resources, writing-original draft, and writing-review & editing. K.T.K: Data curation, formal analysis, methodology, and software. N.B.: Methodology, supervision, and validation. Z.B.G.: Conceptualization, methodology, and validation. O.C.M.: Formal analysis, methodology, supervision, and validation. E.K.: Formal analysis, methodology, and software. G.O.O.: Investigation, project administration, and validation. A.Y.: Conceptualization, supervision, visualization, writing and review & editing. FUNDİNG There was no funding source for this work. ORCID Halime Tozak Yıldız: https://orcid.org/0000-0003-4310-6238 References McSweeney KR, Gadanec LK, Qaradakhi T, Ali BA, Zulli A, Apostolopoulos V. Mechanisms of cisplatin-induced acute kidney injury: Pathological mechanisms, pharmacological ınterventions, and genetic mitigations. Cancers. Basel. 2021 Mar 29;13(7):1572. Moradi M, Goodarzi N, Faramarzi A, Cheraghi H, Hashemian AH, Jalili C. Melatonin protects rats testes against bleomycin, etoposide, and cisplatin-induced toxicity via mitigating nitro-oxidative stress and apoptosis. Biomed Pharmacother. 2021 Jun;138:111481. Sallam AO, Rizk HA, Emam MA, Fadl SE, Abdelhiee EY, Khater H. et al. The ameliorative effects of l-carnitine against cisplatin-induced gonadal toxicity in rats. Pak Vet J. 2021;41(1):147-151. Available from: http://dx.doi.org/10.29261/pakvetj/2020.082. Mackie EJ, Radford M, Shalet SM. Gonadal function following chemotherapy for childhood Hodgkin’sdisease. MedPeidatrOncol. 1996;27:74-8. Cherry SM, Hunt PA, Hassold TJ. Cisplatin disrupts mammalian spermatogenesis, but does not affect recombination or chromosome segregation. Mutat Res. 2004;564(2):1115–1128. Dasari S, Njiki S, Mbemi A, Yedjou CG, Tchounwou PB. pharmacological effects of cisplatin combination with natural products in cancer chemotherapy. Int J Mol Sci. 2022 Jan 28;23(3):1532. Kelland L. Theresurgence of platinum-based cancer chemotherapy. NatRevCancer. 2007;7(8):573-84. Li X, Kang X, Deng Q, Cai J, Wang Z. Combination of a GnRH agonist with an antagonist prevent sflare-upeffects and protects primordial ovarian follicles in therat ovary from cisplatin-induced toxicity: a controlled experimental animal study. Reprod Biol Endocrino. 2013;11-16. Boekelheide K. Mechanisms of toxic damage to spermatogenesis. J Natl Cancer Inst Monogr. 2005;34:6–8. Amin A, Hamza A. Effects of ginger and roselle on cisplatin induced reproductive toxicity in rats. Asian J Androl. 2006;8(5):607–12. Amin A, Hamza AA, Kmbal A, Daoud S. Herbal extracts counteract cisplatin-mediated cell death in rat testis. Asian J Androl. 2008;10(2):291–7. 39. Mesbahzadeh B, Hassanzadeh-Taheri M, Aliparast Ms, Baniasadi P, Hosseini M . The protective effect of crocin on cisplatin-induced testicular impairment in rats. BMC Urol. 2021;21:117. Available from: https://doi.org/10.1186/s12894-021-00889-2. Sueishi K, Mishima K, Makino K, Itoh Y, Tsuruya K, Hirakata H. Protection by a radical scavenger edaravone against cisplatin-induced nephrotoxicity in rats. Eur J Pharmacol. 2002;451:203–8. Whirledge SD, Garcia JM, Smith RG, Lamb DJ. Ghrelin partially protects against cisplatin-induced male murine gonadal toxicity in a GHSR-1a-dependent manner. Biol Reprod. 2015 Mar;92(3):76. Chen B, Li Q, Zhao B, Wang Y. Stem cell-derived extracellular vesicles as a novel potential therapeutic tool for tissue repair. Stem Cells Transl Med. 2017 Sep; 6(9);6(9):1753-1758. Maia J, Caja S, Strano Moraes MC, Couto N, Costa-Silva B. Exosome-based cell-cell communication in the tumor microenvironment. Front Cell Dev Biol. 2018 Feb;6:18. Zhang J, Guan J, Niu X, Hu G, Guo S, Li Q, et al. Exosomes released from human induced pluripotent stem cells-derived MSCs facilitate cutaneous wound healing by promoting collagen synthesis and angiogenesis. J Transl Med. 2015 Feb 1;13:49. Xie Y, Chen Y, Zhang L, Ge W. The roles of bone-derived exosomes and exosomal micro RNA s in regulating bone remodelling. Journal of Cellular and Molecular Medicine. 2017;21(5):1033-1041. Rani S, Ryan AE, Griffin MD, Ritter T. Mesenchymal stem cell derived extracellular vesicles: toward cell-free therapeutic applications. Molecular Therapy. 2015;23:812-823. Anding AL, Baehrecke EH. Autophagy in cell life and cell death. Apoptosis and Development. 2015;114:67–91. Galluzzi L, Vitale I, Abrams JM, Alnemri ES, Baehrecke EH, Blagosklonny MV. Molecular definitions of cell death subroutines: Recommendations of the nomenclature committee on cell death. Cell Death and Differentiation. 2012;19(1):107–120. Xiao G. Autophagy and Nf-Kb: Fight for fate. Cytokine Growth Factor Rev. 2007 Jun-Aug;18(3-4):233-43. Toh WS, Lai RC, Zhang B, Lim SK. MSC exosome works through a protein-based mechanism of action. Biochem Soc Trans. 2018 August 20;46(4):843–853. Toh WS, Zhang BIN, Lai RC, Lim SK. Immune regulatory targets of mesenchymal stromal cell exosomes/small extracellular vesicles in tissue regeneration. Cytotherapy. 2018;20:(12):1419-1426. Bang C and Thum T. Exosomes: new players in cell–cell communication. The International Journal of Biochemistry & Cell Biology. 2012;44:2060-2064. Raposo G and Stoorvogel W. Extracellular vesicles: exosomes, microvesicles, and friends. The Journal of Cell Biology. 2013;200:373-383. Abdel-Latif R, Fathy M, Anwar HA, Naseem M, Dandekar T, Othman EM. Cisplatin-induced reproductive toxicity and oxidative stress: Ameliorative effect of kinetin. Antioxidants. 2022;11(5):863. Available from: https://doi.org/10.3390/antiox11050863. Afsar T, Razak S, Khan MR, Almajwal A. Acacia hydaspica ethyl acetate extract protects against cisplatin-induced DNA damage, oxidative stress and testicular injuries in adult male rats. BMC Cancer. 2017;17(1):883. Bahar D, Gonen ZB, Gumusderelioglu M, Onger ME, Tokak EK, Ozturk-Kup F, et al. Repair of rat calvarial bone defect by using exosomes of umbilical cord-derived mesenchymal stromal cells embedded in chitosan/hydroxyapatite scaffolds. Int J Oral Maxillofac Implants. 2022 Sep-Oct;37(5):943-950. Badkoobeh P, Parivar K, Kalantar SM, Hosseini SD, Salabat A. Effect of nano-zinc oxide on doxorubicin- induced oxidative stress and sperm disorders in adult male Wistar rats. Iran J Reprod Med. 2013 May;11(5):355-64. Johnsen SG. Testicular biopsy score count–a method for registration of spermatogenesis in human testes: normal values and results in 335 hypogonadal males. Hormones. 1970;1:2–25. Shabani R, Ashtari K, Behnam B, Izadyar F, Asgari H, Asghari Jafarabadi M, et al. In vitro toxicity assay of cisplatin on mouse acute lymphoblastic leukaemia and spermatogonial stem cells, Andrologia. 2016;48:(5):584–94. Lai RC, Yeo RW, Lim SK. Mesenchymal stem cell exosomes. Semin Cell Dev Biol. 2015 Apr;40:82-8. Lai RC, Tan SS, Teh BJ, Sze SK, Arslan F, de Kleijn DP, Choo A, Lim SK. Proteolytic potential of the msc exosome proteome: ımplications for an exosome-mediated delivery of therapeutic proteasome. Int J Proteomics. 2012;2012:971907. Parolini I, Federici C, Raggi C, Lugini L, Palleschi S, De Milito A, et al. Microenvironmental pH is a key factor for exosome traffic in tumor cells. J Biol Chem. 2009 Dec 4; 284:(49):34211-22. Narayana K, Al-Bader M, Mousa A, Khan KM. Molecular effects of chemotherapeutic drugs and their modulation by antioxidants in the testis. Eur J Pharmacol 2012;674:207-216. Prihatno SA, Padeta I, Larasati AD, Sundari B, Hidayati A, Fibrianto, et al. Effects of secretome on cisplatin-induced testicular dysfunction in rats. Vet. World. 2018;11:1349. Gholami Jourabi F, Yari S, Amiri P, Heidarianpour A, Hashemi H. The ameliorative effects of methylene blue on testicular damage induced by cisplatin in rats. Andrologia. 2021 Feb;53(1):e13850. Grimshaw MJ, Balkwill FR. Inhibition of monocyte and macrophage chemotaxis by hypoxia and inflammation--a potential mechanism. Eur J Immunol. 2001 Feb;31(2):480-9. Yoshida GJ. Therapeutic strategies of drug repositioning targeting autophagy to induce cancer cell death: from pathophysiology to treatment. J. Hematol. Oncol. 2017;10:67. Zhang M, Jiang M, Bi Y, Zhu H, Zhou Z, Sha J. Autophagy and apoptosis act as partners to induce germ cell death after heat stress in mice. Plos One. 2012;7:7. Gallagher LE, Williamson LE, Chan EY. Advances in autophagy regulatory mechanisms. Cells 2016; 5: 1–30. Zhang W, Yang C, Guo W, Guo X, Bian J, Zhou Q, et al. [Protective effect of bone marrow mesenchymal stem cells-derived exosomes against testicular ischemia-reperfusion injury in rats]. Nan Fang Yi Ke Da Xue Xue Bao. 2018 Jul 30;38(8):910-916. Liu H, Shi M, Li X, Lu W, Zhang M, Zhang T, et al. Adipose mesenchymal stromal cell-derived exosomes Prevent testicular torsion injury via activating PI3K/AKT and MAPK/ERK1/2 pathways. Oxid Med Cell Longev. 2022 Jun 16;2022:8065771. Afsar T, Razak S, Khan MR, Almajwal A. Acacia hydaspica ethyl acetate extract protects against cisplatin-induced DNA damage, oxidative stress and testicular injuries in adult male rats. BMC Cancer. 2017 Dec 21;17(1):883. Dasari S, Tchounwou PB. Cisplatin in cancer therapy: molecular mechanisms of action. Eur J Pharmacol. 2014 Oct 5;740:364-78. Cocetta V, Ragazzi E, Montopoli M. Mitochondrial Involvement in Cisplatin Resistance. Int J Mol Sci. 2019 Jul 10;20(14):3384. Wan M, Li C, Zhen G, Jiao K, He W, Jia X, et al. Injury-activated transforming growth factor β controls mobilization of mesenchymal stem cells for tissue remodeling. Stem Cells. 2012 Nov;30(11):2498-2511. Aly HA, Eid BG. Cisplatin induced testicular damage through mitochondria mediated apoptosis, inflammation and oxidative stress in rats: Impact of resveratrol. Endocr. J. 2020;67:969–980. Wang L, He Y, Li Y, Pei C, Olatunji OJ, Tang J, et al. Protective effects of nucleosides-rich extract from cordyceps cicadae against cisplatin ınduced testicular damage. Chem Biodivers. 2020 Nov; 17(11):e2000671. Ferreiro ME, Amarilla MS, Glienke L, Méndez CS, González C, Jacobo PV, et al. The inflammatory mediators TNF-α and nitric oxide arrest spermatogonia GC-1 cell cycle. Reprod. Biol. 2019;19:329–339. Alvarez-Erviti L, Seow Y, Yin H, Betts C, Lakhal S, Wood MJ. Delivery of siRNA to the mouse brain by systemic injection of targeted exosomes. Nat Biotechnol. 2011 Apr;29(4):341-5. Arslan F, Lai RC, Smeets MB, Akeroyd L, Choo A, Aguor EN, et al. Mesenchymal stem cell-derived exosomes increase ATP levels, decrease oxidative stress and activate PI3K/Akt pathway to enhance myocardial viability and prevent adverse remodeling after myocardial ischemia/reperfusion injury. Stem Cell Res. 2013 May;10(3):301-12. Guo XB, Zhai JW, Xia H, Yang JK, Zhou JH, Guo WB, et al. Protective effect of bone marrow mesenchymal stem cell-derived exosomes against the reproductive toxicity of cyclophosphamide is associated with the p38MAPK/ERK and AKT signaling pathways. Asian J Androl. 2021 Jul-Aug;23(4):386-391. Hassanzadeh-Taheri M, Hosseini M. Comments on “The improvement efects of Gordonia bronchialis on male fertility of rats with diabetes mellitus induced by Streptozotocin". Pharm Sci. 2020;26:93–5. Hoivik EA, Lewis AE, Aumo L, Bakke M. Molecular aspects of steroidogenic factor 1 (SF-1). Mol Cell Endocrinol. 2010;315:27–39. Ying SY. Inhibins, activins, and follistatins: gonadal proteins modulating the secretion of follicle-stimulating hormone. Endocr Rev 1988:267-93. Available from: https://doi.org/10.1210/edrv-9-2-267. Han, Y., Jiang, T., Liu, A., & Liu, L. Role and regulatory mechanism of inhibin in animal reproductive system. Theriogenology. 2023;202:10-20. Yang X. To analyze the relationship between semen quality and male FSH, Inh B level. Modern Med Health Res. 2021:113-5. Andersson AM, Juul A, Petersen JH, Müller J, Groome NP, Skakkebaek NE. Serum inhibin B in healthy pubertal and adolescent boys: relation to age, stage of puberty, and follicle-stimulating hormone, luteinizing hormone, testosterone, and estradiol levels. J Clin Endocrinol Metabol. 1997;3976-81. Available from: https://doi.org/10.1210/jcem.82.12.4449. Pfaff T, Rhodes J, Bergmann M, Weinbauer GF. Inhibin B as a marker of sertoli cell damage and spermatogenic disturbance in the rat. Birth defects research. Part B, Developmental and reproductive toxicology. 2013; p. 91-103. Available from: https://doi.org/10.1002/bdrb.21046. de Kretser DM, Buzzard JJ, Okuma Y, O'Connor AE, Hayashi T, Lin SY, Morrison JR, Loveland KL, Hedger MP. The role of activin, follistatin and inhibin in testicular physiology. Mol Cell Endocrinol. 2004;225(1-2):57-64. Tian M, Liu F, Liu H, Zhang Q, Li L, Hou X, et al. Grape seed procyanidins extract attenuates Cisplatin-induced oxidative stress and testosterone synthase inhibition in rat testes. Syst Biol Reprod Med. 2018;64:246–59. Additional Declarations No competing interests reported. Supplementary Files Table1and2.docx TABLE 1. Johnsen testicular biopsy score TABLE 2. Sperm count and motility. Effect of exosomes on sperm count (million/mL),and sperm motility (%) in testis tissues. Values are expressed as mean ± SD; *p<0.05, **p<0.01, ***p<0.001 . 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4011219","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":276618143,"identity":"fa8439d6-1125-43de-b947-f2affd06933f","order_by":0,"name":"Halime TOZAK YILDIZ","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABA0lEQVRIiWNgGAWjYBACAyA+8IBNgoGfgYfhAExUAq8WNqCWBKAWyQZULQZ4tTAkAAmDAzwIUbxazOV7DA8klFnYGx8/e/DAx7Z78uYMzAdv8zD8ycelxbKNx+BAwjkJZrMzeQkHZ7YVG+5sYEu25mEwsGzA5bBjQC2JbRJsZgdyDA7znElg3HCAx0waqAWny2BaeIz73xgc/nMmwX7DAf5vRGmRMJAA2sJQkZAItIWNgJa0ApBfDCRuvDE42FORkLzhMJux5RwDY9xaDh/e/OFDWZ09f3+O8YcfBgm2G443P7zxpkIOdygzcKDLMYONwq2BgYH9AT7ZUTAKRsEoGAUMDACZb1W2KfmgKwAAAABJRU5ErkJggg==","orcid":"","institution":"Kirsehir Ahi Evran University","correspondingAuthor":true,"prefix":"","firstName":"Halime","middleName":"TOZAK","lastName":"YILDIZ","suffix":""},{"id":276618144,"identity":"b5b1fc60-0324-4013-b134-ab020fa9cade","order_by":1,"name":"Kübra Tuğçe KALKAN","email":"","orcid":"","institution":"Kirsehir Ahi Evran University","correspondingAuthor":false,"prefix":"","firstName":"Kübra","middleName":"Tuğçe","lastName":"KALKAN","suffix":""},{"id":276618145,"identity":"ddb8ac0d-1950-4676-82bb-eaabf2cb8af9","order_by":2,"name":"Numan BAYDİLLİ","email":"","orcid":"","institution":"Erciyes University","correspondingAuthor":false,"prefix":"","firstName":"Numan","middleName":"","lastName":"BAYDİLLİ","suffix":""},{"id":276618146,"identity":"91661e82-1ca2-4294-9ac3-bea0e08383e0","order_by":3,"name":"Zeynep Burçin GÖNEN","email":"","orcid":"","institution":"Erciyes University","correspondingAuthor":false,"prefix":"","firstName":"Zeynep","middleName":"Burçin","lastName":"GÖNEN","suffix":""},{"id":276618147,"identity":"637be334-5da4-4c35-b095-0c154997c83a","order_by":4,"name":"Özge CENGİZ MAT","email":"","orcid":"","institution":"Erciyes University","correspondingAuthor":false,"prefix":"","firstName":"Özge","middleName":"CENGİZ","lastName":"MAT","suffix":""},{"id":276618148,"identity":"6d21527c-effc-4ad7-8197-d9cd7ec584fb","order_by":5,"name":"Eda OKUR","email":"","orcid":"","institution":"Erciyes University","correspondingAuthor":false,"prefix":"","firstName":"Eda","middleName":"","lastName":"OKUR","suffix":""},{"id":276618149,"identity":"7017a8cd-836e-4a0d-936c-2ae537ac9464","order_by":6,"name":"Gözde Özge ÖNDER","email":"","orcid":"","institution":"Erciyes University","correspondingAuthor":false,"prefix":"","firstName":"Gözde","middleName":"Özge","lastName":"ÖNDER","suffix":""},{"id":276618150,"identity":"4950486e-bc42-4543-b5a3-f5a79990b379","order_by":7,"name":"Arzu YAY Prof","email":"","orcid":"","institution":"Erciyes University","correspondingAuthor":false,"prefix":"","firstName":"Arzu","middleName":"YAY","lastName":"Prof","suffix":""}],"badges":[],"createdAt":"2024-03-04 09:03:36","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4011219/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4011219/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":52232933,"identity":"536747e8-9f74-4d42-99cd-3dbf9a21d435","added_by":"auto","created_at":"2024-03-08 06:35:34","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":670768,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHistological of testis sections.\u003c/strong\u003e (Olympus BX51, Tokyo, Japan X20: Scale bar; 200 µm, X40: Scale bar; 100 µm). Effects of exosomes on seminiferous tubules (ST) and interstitial tissue with Leydig cells (black asterisks) in testis tissues. Representative Hematoxylin and Eosin stained sections from indicated groups. Normal histology was observed in the control group. The Cis group experienced degenerative seminiferous tubule (dST) features. Black arrow; decreased number of germ cells, Red asterisks; lumen shedding, Blue asterisks; edema.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-4011219/v1/f8963c7f21e71b0775898334.png"},{"id":52232937,"identity":"3755c926-f906-4199-9dfd-906955bc5d9c","added_by":"auto","created_at":"2024-03-08 06:35:34","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":198447,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eJTBS and Tubule Diameters\u003c/strong\u003e (a). The general Johnsen's testicular biopsy scoring for all groups. (b). Seminiferous tubule diameter measurement for all groups. *, p\u0026lt;0.05, **; p\u0026lt;0.01, **; p\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-4011219/v1/fdd1eedea42aacef248ac447.png"},{"id":52232940,"identity":"104a6871-0ad2-4533-b9d4-114c85db989a","added_by":"auto","created_at":"2024-03-08 06:35:34","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2543107,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eImmunohistochemical analysis of testis sections \u003c/strong\u003e(Olympus BX51, Tokyo, Japan. X40: Scale bar; 100 µm). Levels of autophagy markers in the testes of control and treated group. Exosome treatment ameliorated expression of Beclin-1, P62, and LC3-2 levels in the testes of cisplatin-induced rats. Avidin-biotin peroxidase technique.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-4011219/v1/5d11c1e3f7aa489119578128.png"},{"id":52233685,"identity":"2315f12f-5f3b-4845-be27-cbb18e09b2e0","added_by":"auto","created_at":"2024-03-08 06:43:34","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":234560,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eİmmunoreactivity intensity of autophagy proteins. \u003c/strong\u003eImpact of exosome Beclin-1, p62, and LC3/2 in the testes tissue of rats treated with cisplatin. *,p\u0026lt;0.05, **p\u0026lt;0.01, ***p\u0026lt;0.001 .\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-4011219/v1/9996f6feec088851ae2e07ec.png"},{"id":52232939,"identity":"65d2e5a6-69f7-4e6d-8f3d-082a31838453","added_by":"auto","created_at":"2024-03-08 06:35:34","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":504045,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eImmunohistochemical analysis of\u003c/strong\u003e \u003cstrong\u003eSF-1 and StAR expression in interstitial area of testes\u003c/strong\u003e(Olympus BX51, Tokyo, Japan. X40: Scale bar; 100 µm). (a). SF-1 and StAR expression in all experimental groups is shown in the illustrative figure. Avidin-biotin peroxidase technique. (b). Immunoreactivity intensity of SF-1 and StAR in interstitial area of testes tissue. *, p\u0026lt;0.05, **; p\u0026lt;0.01, ***; p\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-4011219/v1/063cd59bd1c0883a93c64386.png"},{"id":52232938,"identity":"ad793460-146a-4137-95b0-165f89a18235","added_by":"auto","created_at":"2024-03-08 06:35:34","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":403751,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eStatistical analysis of testicular oxidative stress. \u003c/strong\u003eThe effects exosome on oxidative stress parameters and apoptotic marker in cisplatin-induced testis damaged in rats. MDA, SOD, CAT, GSH-Px and TNF-α testes tissue level of the experimental groups. ; *, p\u0026lt;0.05, **; p\u0026lt;0.01, ***; p\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"floatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-4011219/v1/fd7f8b1edb728712ce67341d.png"},{"id":52232935,"identity":"92456a7c-7d97-47a1-8ba5-4274754fc02b","added_by":"auto","created_at":"2024-03-08 06:35:34","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":189327,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eStatistical analysis of\u003c/strong\u003e \u003cstrong\u003eserum hormone levels\u003c/strong\u003e Serum testesteron and serum INHB level of the experimental groups. ; *, p\u0026lt;0.05, **; p\u0026lt;0.01, ***; p\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"floatimage9.png","url":"https://assets-eu.researchsquare.com/files/rs-4011219/v1/acd3fc5f542f04cb72fbae70.png"},{"id":52634294,"identity":"28206d76-e11a-4fc4-937f-1e644bcc381c","added_by":"auto","created_at":"2024-03-13 21:37:36","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4355042,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4011219/v1/c2fa4681-d901-4707-8280-ddbcf846d377.pdf"},{"id":52233684,"identity":"c2a963fe-5061-4303-9a8d-f76dedd9dc84","added_by":"auto","created_at":"2024-03-08 06:43:34","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":115192,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTABLE 1.\u003c/strong\u003e \u003cstrong\u003eJohnsen testicular biopsy score\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTABLE 2. Sperm count and motility. \u003c/strong\u003eEffect of exosomes on\u003cstrong\u003e s\u003c/strong\u003eperm count (million/mL),and sperm motility\u003cstrong\u003e (%) \u003c/strong\u003ein testis tissues\u003cstrong\u003e. \u003c/strong\u003eValues are expressed as mean ± SD;\u003cstrong\u003e \u003c/strong\u003e*p\u0026lt;0.05, **p\u0026lt;0.01, ***p\u0026lt;0.001 .\u003c/p\u003e","description":"","filename":"Table1and2.docx","url":"https://assets-eu.researchsquare.com/files/rs-4011219/v1/a882570b2cdea64f6498aacd.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Exosome Therapy on Cisplatin-Induced Testicular Tissue Toxicity ","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eCisplatin (Cis) is an effective antineoplastic drug used to treat a variety of cancers. Antineoplastic drugs cause oxidative stress in the biological system(\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). It has been reported that tissue damage in the reproductive system is caused by reactive oxygen metabolites produced during Cis metabolism. Its gonadotoxic effects include the formation of primordial follicles and the degradation of granulosa cells in ovaries, as well as a reduction in spermatogenesis in the testicles. (\u003cspan additionalcitationids=\"CR4 CR5 CR6 CR7 CR8 CR9 CR10 CR11\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e) Cis causes cell cycle arrest and apoptosis by creating intra/inter-chain cross-covalent connections in the DNA doublechain, which inhibits DNA synthesis (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). Testicular damage caused by Cis affects leydig cells and seminiferous tubules, spermatogenic cells, and Sertoli cells, resulting in decreased sperm count, morphology, chromatin quality, and motility. (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e)\u003c/p\u003e \u003cp\u003eExosomes, a novel mode of cell-cell communication, are nanovesicles of endocytic origin that range in size 30-150nm. Extracellular and amniotic fluids with various cell types, including blood, urine, and those released by the hydrothorax, saliva, breast milk, sperm, and cerebral fluids. (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e) Exosomes contain many bioactive molecules, such as proteins, lipids, mRNAs, miRNAs, tRNA, genomicDNA, cDNA, and mtDNA. Exosomes, which are extracellular organelles, play an important role in the overall information flow within cells.(\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e) Exosomes initiate cell-to-cell contact by delivering the proteins, miRNAs and enzymes they carry to the target cell. Exosomes are extracellular organelles that function in both the paracrine and endocrine systems. (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e) Exosomes can be phagocytosed by another cell type, endocytosed, or directly fusioned by other cells. (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e)\u003c/p\u003e \u003cp\u003eThe autophagy mechanism is a physiological process that serves as a quality control system in cells by destroying pathogens, long-lived proteins, damaged macromolecules, and degraded organelles. (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e) The major objective is to offer an alternative source for intracellular structures and a substrate for energy production in order to maintain cell survival. If the cell's environment is unable to provide it with the nutrition it requires, the cell must consume it internally in order to survive. Additionally, cellular stress caused by pathogen infection, hypoxia, nutritional deprivation, or reactive oxygen species (ROS) can cause autophagy to occur. The majority of research has been on how hunger-induced autophagy helps cells survive. The independent relationship between autophagy and other cell death pathways or processes, such as apoptosis, is tight. (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e) Cellular autophagy can be induced by anti-tumor drugs that kill tumor cells. Although several oncoproteins also suppress autophagy, many tumor suppressor genes increase it. (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e)\u003c/p\u003e \u003cp\u003eTesticular damage, oxidative stress, and the activation of apoptotic signaling pathways are the main symptoms of cis-induced gonadotoxicity. (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e) Spermatogenic series in testicles frequently leads to harm by inducing apoptosis in Sertoli and Leydig cells, which is one of the condition's adverse effects. Bone marrow-derived mesenchymal stem cell exosomes are active paracrine components that have a great potential for damaged tissue repair, suppress inflammatory responses, and modulate the immune system. (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e) Due to the large range of components they carry, exosomes have promise in the field of regenerative medicine. (\u003cspan additionalcitationids=\"CR25\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e) No studies examining the therapeutic effects of exosomes were found in the literature, despite the fact that there are studies examining the impact of several stem cells on testicular injury in experimental Cis studies in rats. In this study aimed the potential therapeutic effects of bone marrow-derived MSC exosomes on cis-induced testicular damage through the autophagy mechanism and oxidative stress.\u003c/p\u003e"},{"header":"METHODS","content":"\u003cp\u003e\u003cstrong\u003eAnimals and experimental groups\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study was started with 12 eight-week-old male Wistar albino rats (weight: 150\u0026ndash;210 g). The rats were kept in a 25\u0026deg;C room with a 12-hour light/dark cycle, free access to water, and a standard diet from the Experimental and Clinic Research Center at Erciyes University in Kayseri, Turkey. The adult rats were randomly divided into 4 groups, with 8 rats in each group:\u003c/p\u003e\n\u003cp\u003eControl group: untreated control.\u003c/p\u003e\n\u003cp\u003eExosome (Exo) group: Exosomes (80x10\u003csup\u003e6\u003c/sup\u003e/100\u0026mu;l) were injected intravenous (i.v.) in a single dose to rats on the fifth day of the experiment.\u003c/p\u003e\n\u003cp\u003eCis group: Cisplatin (7,5 mg/kg in 0.9% saline solution)(Ko\u0026ccedil;ak Pharma Company, Turkey) was injected intraperitoneally (i.p.) in a single dose to rats on the first day of the experiment to induce cisplatin testicular toxicity (25 mg/50 ml) vials. (27,28)\u003c/p\u003e\n\u003cp\u003eCisplatin+Exosome (Cis+Exo) group: Cis (7,5 mg/kg in 0.9% saline solution) was injected i.p. in a single dose to rats on the first day to induce Cis. Exosomes (80x10\u003csup\u003e6\u003c/sup\u003e/100\u0026mu;l) were injected i.v. in a single dose into rats on the fifth day.\u003c/p\u003e\n\u003cp\u003eOn the 8th day of the study, rats in the groups were sacrificed by cervical dislocation after testicular and blood samples were taken under xylazine (10 mg/kg, IP) and ketamine (60 mg/kg, IP) general anaesthesia.\u003c/p\u003e\n\u003cp\u003eExperiments were carried out following the Declaration of Helsinki and the National Institutes of Health\u0026apos;s (USA) Guide for the Care and Use of Laboratory Animals. All experimental protocols were approved by the Local Ethic Committee of Animal Experiments of Erciyes University (approval no. 22/185).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;Isolation of Exosomes from Mesenchymal Stem Cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRat bone marrow-derived mesenchymal stem cells were thawed in a 37\u0026deg;C water bath before being seeded into 75 cm\u0026sup2; cell culture dishes (TPP, Switzerland) that were acquired from Erciyes University Genome and Stem Cell Centre. A serological pipette was used to remove the media from the MSCs to facilitate the generation of exosomes after their density exceeded 90%. After one day, serum-free media was added and the cell secretomes were extracted.\u003c/p\u003e\n\u003cp\u003eExosome production was performed from the obtained secretome using ExoQuick-TC (#EXOTC50A-1, System Bio sciences, USA) kit. The collected secretomes were centrifuged at 3000x g for 15 minutes, as per the kit instructions, and the cells and their wastes sank to the bottom. One-fifth of the volume of the supernatant was added to a fresh tube containing the supernatant and the exosome isolation solution. Vortex was used to assist homogenize it. After that, it was incubated for 24 hours at +4\u0026ordm;C. Following incubation, the mixture was centrifuged for 30 minutes at +4\u0026ordm;C at 1500x g. After centrifugation, the pellet was resuspended in PBS, and the supernatant was discarded.\u003c/p\u003e\n\u003cp\u003eA microvesicle quantitation kit (EXOCET Exosome Quantitation Assay- EXOCET96A-1 SBI, USA) was used to measure the quantity of exosomes that were obtained before they were given to experimental animals. The Glomax Multi ELISA absorbance reader (Promega, USA) was used to read absorbance values at 450 nm following the quantification process that followed the kit\u0026apos;s instructions. The amount of exosomes was calculated using standards. (29 )\u0026nbsp;Exosomes diluted 80x10\u003csup\u003e6\u003c/sup\u003e/100\u0026mu;l were injected to every animal in the study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;Assessment of sperm parameters\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e10 \u0026micro;L of the supernatant containing epididymal sperm was diluted in a 990 \u0026micro;L solution of paraformaldehyde and sodium citrate for this purpose. Approximately 10 \u0026micro;L of the diluted material was transferred to a hemocytometer (Neubauer chamber), and the pelletized cells on the chamber\u0026apos;s surface were counted using a 400 x light microscope. The sperm concentration was calculated using the number of counted cells and hemocytometer dimensions. Thus, the concentration was reported in millions of sperm per mL, and the hemocytometer dimensions were expressed in millions of sperm per mL, as described by Badkoobeh et al. in 2013. A drop of about 20 \u0026micro;L of sperm solution was placed on the microscope slides and swiped for sperm morphological analysis. After drying, the slides were stained with eosin-nigrosin (1% eosin Y and 5% nigrosine). They were viewed in optical microscopy (BX51, Olympus Corp. Tokyo Japan) at 400 x after drying. A differential count of 200 spermatozoa per slide was done, and alterations in the head, middle piece, and tail were detected. Badkoobeh et al. reported the data as a proportion of normal cells. (30)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;Histopathological analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe testicles were fixed in 10% neutral formalin for 48 hours, treated with ethanol , and then embedded in paraffin blocks for histological examinations. Sections 5 \u0026mu;m thick were then cut from a paraffin block (Leica, RM 2000, Germany) and stained with hematoxylin and eosin (H\u0026amp;E) to examine the general histomorphological structure of testis tissues . \u0026nbsp;Standard light microscopy was used to analyze and assess the testicular tissue in a blinded manner light microscope. The testicular tissue was scored according to Johnsen\u0026apos;s criteria after being stained with H\u0026amp;E. (31) The Johnsen\u0026rsquo;s testicular biopsy score (JBTS) was determined by sampling five locations in each section\u0026apos;s 20 seminiferous tubules. According to the number of cells and how mature they were, JBTS was calculated for each tubule (Table 1). Moreover, as a measure of damage, the diameter of the seminiferous tubule was measured at a scale of 200 x using the ImageJ software (Bethesda, MD). For the rat, the typical tubule diameter was estimated after measuring the seminiferous tubule diameters in 10 distinct places\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;Immunohistochemistry\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe streptavidin-biotin-peroxidase method was used for the immunohistochemistry staining kit (Lab VisionTM UltraVisionTM Large Volume Detection System: anti-polyvalent, HRP, TA-125-HL). Beclin-1, p62, LC3-2, steroidogenic factor-1 (SF-1), and steroidogenic acute regulatory protein (StAR) expressions in testicular tissues were identified using this technique. Testes were cut into 5-\u0026mu;m sections, deparaffinized in xylene, and then rehydrated using a graduated ethanol series. After rehydration, the sections were washed in deionized water, phosphate-buffered saline (PBS) was used to rinse the sections, and then 3% hydrogen peroxide was applied for 5 minutes. All sections were then saturated in 5% sodium citrate buffer and heated at 600 W for 5 minutes to initiate antigen retrieval. Beclin-1 primary antibody diluted 1:500 (Novus, NB500-249), p62 primary antibody diluted 1:200 (Anti-SQSTM-1, H00008878), LC3-2 primary antibody diluted 1:750 (Cell Signaling, 12741 S), SF-1 primary antibody diluted 1:150 (NBP2-46247; Novus Biologicals), and StAR primary antibody diluted 1:250 (bs-3570R; Bioss Inc.) were all applied to the sections in an immunohistochemical chamber for an overnight duration at 4\u0026deg;C. Following that, biotinylated secondary antibodies were incubated with the sections. Then, after the washing process, they were treated with 3,3\u0026apos;-diaminobenzidine tetrahydrochloride substrate (TA-060-HDX, Thermo Fisher Scientific, Waltham, MA, USA) to make the immunoreactivity visible. Gill\u0026apos;s hematoxylin was used as a counterstain on the sections to enhance nuclear staining. Under a light microscope, each section was evaluated, and microscopic images of five different testicular tissue regions were obtained. Beclin-1, LC3-2, p62, SF-1, and StAR immunoreactivity intensities were measured using the Image J software (NIH; Washington, USA).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;Elisa\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTesticular tissues stored at -80 \u0026ordm;C for biochemical evaluation were transferred to PBS (0.01M, pH:7.4) for homogenate preparation. Each homogenized sample was centrifuged at 4 \u0026ordm;C at 10000 rpm for 10 min. For the ELISA method, the obtained supernatants were aliquoted. Glutathione Peroxidase (GSH-PX) (Sunred Bio, Cat. No: 201-11-5104), Superoxide Dismutase (SOD), Catalase (CAT) (Sunred Bio, Cat. No: 201-11-5106), and tumor necrosis\u0026nbsp;factor-alpha (TNF-\u0026alpha;) (Sunred Bio, Cat. No: 201-11-0765) were measured to determine the antioxidant levels of testicular tissue. The levels of serum inhibine (INHB) and testosterone were assessed. Furthermore, the level of malondialdehyde (MDA) (Sunred Bio, Cat. No: 201-11-0157) was measured in the testis tissue.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;Statistical Analysis of the Data\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe GraphPad Prism 9 program was used to evaluate the outcomes. The Shapiro-Wilk test was performed to determine if the data were normally distributed. A one-way analysis of variance (ANOVA) was used to analyze data that was regularly distributed. The source of the difference was then confirmed using the Tukey posthoc test. The significance level was set at P \u0026lt; 0.05.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cp\u003e\u003cstrong\u003eExo reduced the pathological changes in Cis-damaged testicular tissues\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAs the testicular sections stained with H\u0026amp;E were evaluated, the control group\u0026apos;s seminiferous tubules were organized into the various stages of spermatogenesis and germinal epithelium. The components of this epithelium are the spermatogonia, spermatids, and spermatocytes. In the interstitial location, Leydig cells were observed. The Cis group demonstrated degenerative alterations in the seminiferous tubule, dilatation, a reduction in the number of germ cells, irregularities, and lumen shedding. The seminiferous tubules of the Exo groups are arranged regularly, smilar to control group. While edema was observed in the Cis+Exo group, tubule degeneration was reduced, and the germinal epithelium became more regular (Figure 1A).\u003c/p\u003e\n\u003cp\u003eThe experimental group\u0026rsquo;s testicular injuries were scored using JTBS (Table 1). The Cis group scored significantly lower than the other groups. There is a significant difference between Cis and Cis+Exo (p\u0026lt;0.001 for all comparisons) (Figure 1B).\u003c/p\u003e\n\u003cp\u003eSeminiferous tubule diameters\u0026nbsp;were also evaluated to assess testicular injury. The Cis group had significantly smaller seminiferous tubule diameters than the control group and exo group. Seminiferous tubule diameter increased statistically in the Cis+Exo group compared to the Cis group. (p\u0026lt;0.05) (Figure 1C). There is a significant difference between the control, Exo, Cis (p\u0026lt;0.001), and Cis+Exo groups (p\u0026lt;0.05).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;The Effect of Exo Sperm Quality Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAfter Cis therapy, there was a significant decrease in the number of sperm compared to the control group (p = 0.000) and Exo group (p = 0.007). Between the Cis and Cis+Exo groups, there was a significant increase (p\u0026lt;0.001) (Table 2). Sperm motility analysis showed a significant decrease after Cis therapy when compared to the control group and Exo group (p\u0026lt;0.05). The Cis+Exo group shows an increase in the Cis group, but the difference is not statistically significant. (Table 2).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;Cis-induced autophagy is supressed by Exo\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe contribution of MSC-derived exosomes on the expression levels of autophagy proteins Beclin-1, p62, and LC3-2 following Cis-giving was investigated in this research. In the control groups, immunohistochemical evaluation of autophagy markers indicated a weakly positive cytoplasmic reactivity (Figure 2). The Cis group immunoreactivity intensity for Beclin-1, p62, and LC3-2 was elevated substantially in comparison with the control group (p\u0026lt;0.001 for all comparisons). Following Cis, Exo-treated rats showed a significant decrease in the autophagy markers Beclin-1 (p\u0026lt;0.05), p62, and LC3-2 (p\u0026lt;0.001) (Figure 3).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;SF-1 and StAR İmmunoreactivity\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSF-1 has been shown to modulate the expression of the key protein StAR, which plays a role in the synthesis of testosterone. Leydig cells in the interstitial area, the control groups expressed SF-1 and StAR immunoreactivity (Figure 4A). The Cis group had considerably lower immunoreactive Leydig cell intensity than the control group (p\u0026lt;0.001). SF-1 immunoreactivity in Leydig cells showed a significant increase (p\u0026lt;0.05) in Cis+Exo group rats treated with Exo after Cis (Figure 4B). The StAR expression in the Cis+Exo group did not significantly increase.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;Effect of Exo on testicular oxidative stress and serum hormone levels\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTesticular oxidative stress injury brought on by cisplatin was discovered by measuring antioxidant and oxidant enzyme levels in this study. When compared to the control group, testicular GSH-PX concentration decreased significantly (p\u0026lt;0.05), and oxidative stress was dramatically initiated, as indicated by an elevated MDA level (p\u0026lt;0.01). However, compared to controls, it decreased activity, SOD, and CAT (p\u0026lt;0.001). MDA production was reduced, and MDA imbalance was improved in the Cis+Exo group treated with Exo following Cis (p\u0026lt;0.001). Exo-therapy increased GSH-PX concentration in the Cis+Exo group rats (p\u0026lt;0.05) and significantly decreased oxidative stress by decreasing testicular MDA (p\u0026lt;0.001). When compared to the control group, there was a significant rise in testicular TNF-\u0026alpha; expression in the Cis-treated group (p\u0026lt;0.05). The Exo-treated Cis+Exo group blocked the increase in testicular TNF-\u0026alpha; expression compared to the Cis group, but there was no statistically significant difference (Figure 5).\u003c/p\u003e\n\u003cp\u003eReduced SF-1 and StAR immunoreactivity in Leydig cells resulted in decreased testosterone levels. INHB regulates sperm production by inhibiting the pituitary gland from secreting too much follicle-stimulating hormone (FSH). The presence of androgens in the blood increases inhibin levels, promoting spermatogenesis. Reduced testosterone levels also have a deleterious impact on INHB levels. Cis therapy decreased serum testosterone (p\u0026lt;0.005) and INHB levels (p\u0026lt;0.001) significantly (p\u0026lt;0.001). When compared to the Cis group, Exo therapy increased serum testosterone (p\u0026lt;0.005) and INHB levels (p\u0026lt;0.001) (Figure 6).\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eCis, a chemotherapy drug used to treat several cancers, causes damage to tissue in the testes despite its potent antitumoral activity. Because chemotherapy agents attack dividing cells, the spermatogenesis process is particularly affected. Previous studies reported that exposure to Cis reduced reproductive function. (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e) This study's histological, immunohistochemical, and biochemical results demonstrated Cis's damaging impact on testicular tissue.\u003c/p\u003e \u003cp\u003eAntitumoral drugs that destroy cancer cells, like Cis, damage tissue by causing cells to undergo autophagy and apoptosis. (\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan additionalcitationids=\"CR11 CR12 CR13\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e) Exosomes of BM-derived MSCs have been shown to have therapeutic potential in different articles studying tissue damage and degenerative disorders. (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan additionalcitationids=\"CR34\" citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e) Exosomes function as extracellular organelles with a paracrine/endocrine role, transporting proteins, miRNAs, and enzymes to target cells. (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e) Many studies have reported that MSC exosomes have antiapoptotic, anti-inflammatory, and regenerative effects. (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan additionalcitationids=\"CR34\" citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e)\u003c/p\u003e \u003cp\u003eCis therapy changed the testicular morphology, induced autophagy, and decreased hormone levels, sperm count, and oxidative stress. A single dose of exo-therapy was shown to have considerable regenerative and antioxidant effects, as well as increased autophagic activity. Hormone levels increased significantly, although sperm motility and count were unaffected. The Cis group exhibited degeneration, atrophied tubular epithelium, cavities, and dilatation of seminiferous tubules. A decrease in tubule spermatogenic cells, germ cell infiltration into the lumen, and interstitial edema were reported. These findings were supported by a decrease in the JTBS score and tubule diameter. In studies, it was reported that seminiferous tubule diameters, germinal epithelial thickness, and testosterone synthesis decreased in Cis-treated rats; testicular atrophy and infertility may develop accordingly. (\u003cspan additionalcitationids=\"CR37\" citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e)\u003c/p\u003e \u003cp\u003eExosomes are highly effective active paracrine components that play a vital role in cell communication and have a high potential for tissue repair (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). Exosomes establish cell-cell interactions by delivering proteins, miRNAs, and enzymes to the target cell. Exosomes, as extracellular organelles, thus serve a paracrine/endocrine role. (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e) Exosomal proteins and microRNAs have a wide range of biochemical and physiological functions, including communication, structure and interactions, inflammation, exosome biogenesis, tissue repair and regeneration, and metabolism. (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e) Exo treatment reduced seminiferous tubule degeneration and restored germinal epithelial cell structure. Edema continues in this group. Additionally, the JTBS socket and tubule diameters increased in this group.\u003c/p\u003e \u003cp\u003eCis, an alkalizing chemical, cross-links to the inner and outer chains of DNA, generating 'adduct' forms that can be seen as twisted DNA chains. Sperm are presumably impacted by this mechanism, as is the testicular structure. (\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e) In studies, Cis treatment caused a significant decrease in sperm count and sperm motility in rat testes. (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e) In this study, sperm motility and count were significantly decreased by Cis therapy. A significant increase in sperm count was detected in the Cis\u0026thinsp;+\u0026thinsp;Exo group that received Exo therapy, but sperm motility did not significantly increase. Sperm count and motility increased when Exo was given as only one dose on the third day following Cis therapy, but motility did not significantly increase.\u003c/p\u003e \u003cp\u003eBecause exosome uptake is dependent on intracellular and microenvironmental acidity and tissue injury is frequently characterized by tissue acidosis, exosomes would be preferentially endocytosed by cells in an injured tissue, implying that exosomes could be home to injured tissues. (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e) Furthermore, exosomes are small, nanometer-sized particles that can easily be carried via blood and other bodily fluids, as indicated by the quantity of exosomes seen in most biological fluids, and MSC exosomes can interact with cells in a paracrine and endocrine way. As a result, exosomes have the biophysical capacity to orchestrate MSC interactions with a variety of cell types in both local and distant environments. However, the ability to evoke appropriate cellular responses after tissue injury in order to restore and maintain tissue microenvironment homeostasis would be dependent on the biochemical capability of its protein and RNA cargo. (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e)\u003c/p\u003e \u003cp\u003eAutophagy, which is essential for cellular adaptability or survival, can be triggered by a variety of stressors, including metabolic stress, endoplasmic reticulum (ER) stress, and chemotherapeutic drugs. (\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e) An intense energy is expended in the process of sprematogenesis. In this process, starvation, chemical exposure and radiation negatively affect this process. The connection between apoptotic and autophagic molecular mechanisms in these pathophysiological conditions has been shown by the studies carried out. (\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e) Cis-induced oxidative stress and tissue acidosis in testicular tissue stimulated the apoptotic pathway and autophagy process. Autophagy induced by Cis in testis was detected by increased Beclin-1, LC3-2 and p62 proteins. Exo therapy at a single dose significantly improved in tissue repair via regulating Cis-induced autophagy. Previous studies have showed that exos protected against testicular ischemia/reperfusion injury in rats through antioxidant, anti-inflammatory, and antiapoptotic mechanisms (\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e). It has also been reported that antitumoural drugs trigger the autophagy process. (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e) Exosomes have been shown to have immunoregulatory, anti-inflammatory, regenerative, and anti-apoptotic capacities in therapeutic applications, however it is unclear whether or if such characteristics regulate autophagy. (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e)\u003c/p\u003e \u003cp\u003eOxidative stress, defined as an imbalance between free radicals and the antioxidant defense system, is crucial in disease development. (\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e) Chemotherapy drugs cause mitochondrial damage. This results in an increase in reactive oxygen species and, as a result, an increase in oxidative stress. (\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e) This accumulation of ROS would operate as a cell chemoattractant, attracting MSCs and exosomes to the damage site. (\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e) As a result of the accumulation of ROS at the injury site, MSCs and their exosomes are drawn to the area by this chemoattractant. (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e) The levels of antioxidant enzymes such as GSH-PX, MDA, SOD, and CAT were evaluated in male rats to evaluate the therapeutic effects of Exo on testicular toxicity caused by Cis-induced oxidative stress. The Cis-induced group's GSH-PX tissue concentration, SOD, and CAT activities in testicular homogenate were significantly lower than the control group. Cis increased MDA levels, a marker of testicular lipid peroxidation. TNF-α, an inflammatory marker in the testis, was significantly increased. The results confirmed oxidative stress, which is similar to previous research on gonadal damage caused by cisplatin.(\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e) The exo-treated Cis group's GSH-PX concentration and SOD and CAT activities improved significantly. MDA levels were significantly reduced. Single doz exo caused protection against the up-regulation of testicular TNF-α levels in Cis-inducted rats. However, no significant decrease in TNF-α level was obtained. TNF-α plays an active role as an inflammatory cytokine in Cis-induced testicular damage. (\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e) An increase in TNF-α levels results in impaired spermatogenesis and decreased testosterone levels. Increased ROS production following damage to mitochondria contributes as well to this process by initially apoptosis. (\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e) Studies have shown that exosomes are able to penetrate the body's barrier and reach the damaged area. (\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e) Exosomes with antiapoptotic, anticancer, and antioxidant qualities have been reported to lower oxidative stress in the area of damage. (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e, \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e)\u003c/p\u003e \u003cp\u003eThe primary functions of the testicles are the production of testosterone and sperm. (\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e) Leydig cells in the interstitial tissue of the testes are responsible for the secretion of testosterone. (\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e) In this study, the group that received Cis testosterone had lower testosterone levels. The decrease in SF-1 and StAR protein expression supports the decrease in testosterone levels. NHB is a key hormone that regulates reproductive function. The INHB hormone is a key regulator of FSH, which is released by sertoli cells in the testis. (\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e, \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e) FSH stimulates the seminiferous tubules and the Sertoli cells. It is in charge of starting the spermatogenesis process. Testosterone is required for spermatogenesis to continue. (\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e)\u003c/p\u003e \u003cp\u003eINHB and FSH secretion become negatively correlated during secondary Sertoli cell development and spermatogenesis. Decreased serum INHB indicates sperm damage and infertility. As a result, it is used to measure male spermatogenesis (\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e, \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e). Inhibins also serve as growth factors in Leydig cells via paracrine mechanisms. INHB, in conjunction with testosterone, regulates spermatogenesis indirectly. (\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e) Previous research has shown that CP intake has a direct effect on the Leydig cells, resulting in cellular death and a decrease in secretory activity. (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e) Because of damaged seminiferous tubule epithelium and low testosterone levels, serum INHB levels were considerably lower in the Cis group compared to the control group. INHB levels significantly increased in the exo-treated group after Cis. This recovery process was supported by tissue regeneration in the seminiferous tubule and interstitial areas.\u003c/p\u003e"},{"header":"CONCLUSION","content":"\u003cp\u003eExosomes have several advantages as a cell-free therapy. In recent years, it has shown potential in the treatment of several human diseases. The key advantages of exosomes are that they are natural, can be derived from a person's own cells, and can be targeted to specific cells or tissues.\u003c/p\u003e \u003cp\u003eThis study examined the therapeutic efficacy of bone marrow-derived mesenchymal stem cell exosomes on cisplatin-induced testicular damage in rats. Exosomes, a regeneratif product with anti-cancer efficacy, were found to reduce cisplatin-induced testicular damage in rats. Accordingly, exosomes might be an encouraging agent in treating Cis-induced testiculer damage and degeneration of the germ cell layer. This study shows that a single dose exosomes may treat Cis-induced testicular damage by reducing oxidative stress and regulating enhanced autophagy. In order to see more effective and long-term results, new studies may design to include repeated doses, different dose applications and more localised applications. Studies on the regulation of autophagy at a more molecular level may be more informative to understand its therapeutic effect.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eACKNOWLEDGMENT\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank the staff of Erciyes University ER\u0026Uuml; Experimental Research and Application Centre for the care of the rats in this study. We would like to thank the Gevher Nesibe Genome and Stem Cell Institute of Erciyes University for their support in the generation of BM-MSCs and the exosomes derived from these cells. For this study, no funding were provided.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCONFLİCT OF INTEREST STATEMENT\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe other authors have no conflict of interest to disclose.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eETHICS STATEMENT\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eExperiments were carried out following the Declaration of Helsinki and the National Institutes of Health\u0026apos;s (USA) Guide for the Care and Use of Laboratory Animals. All experimental protocols were approved by the Local Ethic Committee of Animal Experiments of Erciyes University (approval no. 22/185).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDATA AVAİLABİLİTY\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data in this study were obtained from experiments, not from the internet, and therefore are not suitable for data sharing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAUTHORS\u0026rsquo; CONTRİBUTİONS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eH.T.Y.: Conceptualization, data curation, resources, writing-original draft, and writing-review \u0026amp; editing.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eK.T.K: \u0026nbsp;Data curation, formal analysis, methodology, and software.\u003c/p\u003e\n\u003cp\u003eN.B.: Methodology, supervision, and validation.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eZ.B.G.: Conceptualization, methodology, and validation.\u003c/p\u003e\n\u003cp\u003eO.C.M.: \u0026nbsp;Formal analysis, methodology, supervision, and validation.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eE.K.: Formal analysis, methodology, and software.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eG.O.O.: Investigation, project administration, and validation.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eA.Y.: Conceptualization, supervision, visualization, writing \u0026nbsp;and review \u0026amp; editing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFUNDİNG\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThere was no funding source for this work.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eORCID\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHalime Tozak Yıldız: https://orcid.org/0000-0003-4310-6238\u003c/strong\u003e\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eMcSweeney KR, Gadanec LK, Qaradakhi T, Ali BA, Zulli A, Apostolopoulos V. Mechanisms of cisplatin-induced acute kidney injury: Pathological mechanisms, pharmacological ınterventions, and genetic mitigations. Cancers. Basel. 2021 Mar 29;13(7):1572. \u003c/li\u003e\n\u003cli\u003eMoradi M, Goodarzi N, Faramarzi A, Cheraghi H, Hashemian AH, Jalili C. Melatonin protects rats testes against bleomycin, etoposide, and cisplatin-induced toxicity via mitigating nitro-oxidative stress and apoptosis. Biomed Pharmacother. 2021 Jun;138:111481.\u003c/li\u003e\n\u003cli\u003eSallam AO, Rizk HA, Emam MA, Fadl SE, Abdelhiee EY, Khater H. et al. The ameliorative effects of l-carnitine against cisplatin-induced gonadal toxicity in rats. Pak Vet J. 2021;41(1):147-151. Available from: http://dx.doi.org/10.29261/pakvetj/2020.082. \u003c/li\u003e\n\u003cli\u003eMackie EJ, Radford M, Shalet SM. Gonadal function following chemotherapy for childhood Hodgkin\u0026rsquo;sdisease. MedPeidatrOncol. 1996;27:74-8.\u003c/li\u003e\n\u003cli\u003eCherry SM, Hunt PA, Hassold TJ. Cisplatin disrupts mammalian spermatogenesis, but does not affect recombination or chromosome segregation. Mutat Res. 2004;564(2):1115\u0026ndash;1128.\u003c/li\u003e\n\u003cli\u003eDasari S, Njiki S, Mbemi A, Yedjou CG, Tchounwou PB. pharmacological effects of cisplatin combination with natural products in cancer chemotherapy. Int J Mol Sci. 2022 Jan 28;23(3):1532.\u003c/li\u003e\n\u003cli\u003eKelland L. Theresurgence of platinum-based cancer chemotherapy. NatRevCancer. 2007;7(8):573-84.\u003c/li\u003e\n\u003cli\u003eLi X, Kang X, Deng Q, Cai J, Wang Z. Combination of a GnRH agonist with an antagonist prevent sflare-upeffects and protects primordial ovarian follicles in therat ovary from cisplatin-induced toxicity: a controlled experimental animal study. Reprod Biol Endocrino. 2013;11-16.\u003c/li\u003e\n\u003cli\u003eBoekelheide K. Mechanisms of toxic damage to spermatogenesis. J Natl Cancer Inst Monogr. 2005;34:6\u0026ndash;8.\u003c/li\u003e\n\u003cli\u003eAmin A, Hamza A. Effects of ginger and roselle on cisplatin induced reproductive toxicity in rats. Asian J Androl. 2006;8(5):607\u0026ndash;12.\u003c/li\u003e\n\u003cli\u003eAmin A, Hamza AA, Kmbal A, Daoud S. Herbal extracts counteract cisplatin-mediated cell death in rat testis. Asian J Androl. 2008;10(2):291\u0026ndash;7. 39.\u003c/li\u003e\n\u003cli\u003eMesbahzadeh B, Hassanzadeh-Taheri M, Aliparast Ms, Baniasadi P, Hosseini M\u003cem\u003e.\u003c/em\u003e The protective effect of crocin on cisplatin-induced testicular impairment in rats. BMC Urol. 2021;21:117. Available from: https://doi.org/10.1186/s12894-021-00889-2. \u003c/li\u003e\n\u003cli\u003eSueishi K, Mishima K, Makino K, Itoh Y, Tsuruya K, Hirakata H. Protection by a radical scavenger edaravone against cisplatin-induced nephrotoxicity in rats. Eur J Pharmacol. 2002;451:203\u0026ndash;8.\u003c/li\u003e\n\u003cli\u003eWhirledge SD, Garcia JM, Smith RG, Lamb DJ. Ghrelin partially protects against cisplatin-induced male murine gonadal toxicity in a GHSR-1a-dependent manner. Biol Reprod. 2015 Mar;92(3):76.\u003c/li\u003e\n\u003cli\u003eChen B, Li Q, Zhao B, Wang Y. Stem cell-derived extracellular vesicles as a novel potential therapeutic tool for tissue repair. Stem Cells Transl Med. 2017 Sep; 6(9);6(9):1753-1758.\u003c/li\u003e\n\u003cli\u003eMaia J, Caja S, Strano Moraes MC, Couto N, Costa-Silva B. Exosome-based cell-cell communication in the tumor microenvironment. Front Cell Dev Biol. 2018 Feb;6:18.\u003c/li\u003e\n\u003cli\u003eZhang J, Guan J, Niu X, Hu G, Guo S, Li Q, et al. Exosomes released from human induced pluripotent stem cells-derived MSCs facilitate cutaneous wound healing by promoting collagen synthesis and angiogenesis. J Transl Med. 2015 Feb 1;13:49.\u003c/li\u003e\n\u003cli\u003eXie Y, Chen Y, Zhang L, Ge W. The roles of bone-derived exosomes and exosomal micro RNA s in regulating bone remodelling. Journal of Cellular and Molecular Medicine. 2017;21(5):1033-1041.\u003c/li\u003e\n\u003cli\u003eRani S, Ryan AE, Griffin MD, Ritter T. Mesenchymal stem cell derived extracellular vesicles: toward cell-free therapeutic applications. Molecular Therapy. 2015;23:812-823.\u003c/li\u003e\n\u003cli\u003eAnding AL, Baehrecke EH. Autophagy in cell life and cell death. Apoptosis and Development. 2015;114:67\u0026ndash;91.\u003c/li\u003e\n\u003cli\u003eGalluzzi L, Vitale I, Abrams JM, Alnemri ES, Baehrecke EH, Blagosklonny MV. Molecular definitions of cell death subroutines: Recommendations of the nomenclature committee on cell death. Cell Death and Differentiation. 2012;19(1):107\u0026ndash;120.\u003c/li\u003e\n\u003cli\u003eXiao G. Autophagy and Nf-Kb: Fight for fate. Cytokine Growth Factor Rev. 2007 Jun-Aug;18(3-4):233-43.\u003c/li\u003e\n\u003cli\u003eToh WS, Lai RC, Zhang B, Lim SK. MSC exosome works through a protein-based mechanism of action. Biochem Soc Trans. 2018 August 20;46(4):843\u0026ndash;853.\u003c/li\u003e\n\u003cli\u003eToh WS, Zhang BIN, Lai RC, Lim SK. Immune regulatory targets of mesenchymal stromal cell exosomes/small extracellular vesicles in tissue regeneration. Cytotherapy. 2018;20:(12):1419-1426.\u003c/li\u003e\n\u003cli\u003eBang C and Thum T. Exosomes: new players in cell\u0026ndash;cell communication. The International Journal of Biochemistry \u0026amp; Cell Biology. 2012;44:2060-2064.\u003c/li\u003e\n\u003cli\u003eRaposo G and Stoorvogel W. Extracellular vesicles: exosomes, microvesicles, and friends. The Journal of Cell Biology. 2013;200:373-383.\u003c/li\u003e\n\u003cli\u003eAbdel-Latif R, Fathy M, Anwar HA, Naseem M, Dandekar T, Othman EM. Cisplatin-induced reproductive toxicity and oxidative stress: Ameliorative effect of kinetin. Antioxidants. 2022;11(5):863. Available from: https://doi.org/10.3390/antiox11050863. \u003c/li\u003e\n\u003cli\u003eAfsar T, Razak S, Khan MR, Almajwal A. Acacia hydaspica ethyl acetate extract protects against cisplatin-induced DNA damage, oxidative stress and testicular injuries in adult male rats. BMC Cancer. 2017;17(1):883. \u003c/li\u003e\n\u003cli\u003eBahar D, Gonen ZB, Gumusderelioglu M, Onger ME, Tokak EK, Ozturk-Kup F, et al. Repair of rat calvarial bone defect by using exosomes of umbilical cord-derived mesenchymal stromal cells embedded in chitosan/hydroxyapatite scaffolds. Int J Oral Maxillofac Implants. 2022 Sep-Oct;37(5):943-950.\u003c/li\u003e\n\u003cli\u003eBadkoobeh P, Parivar K, Kalantar SM, Hosseini SD, Salabat A. Effect of nano-zinc oxide on doxorubicin- induced oxidative stress and sperm disorders in adult male Wistar rats. Iran J Reprod Med. 2013 May;11(5):355-64.\u003c/li\u003e\n\u003cli\u003eJohnsen SG. Testicular biopsy score count\u0026ndash;a method for registration of spermatogenesis in human testes: normal values and results in 335 hypogonadal males. Hormones. 1970;1:2\u0026ndash;25.\u003c/li\u003e\n\u003cli\u003eShabani R, Ashtari K, Behnam B, Izadyar F, Asgari H, Asghari Jafarabadi M, et al. In vitro toxicity assay of cisplatin on mouse acute lymphoblastic leukaemia and spermatogonial stem cells, Andrologia. 2016;48:(5):584\u0026ndash;94.\u003c/li\u003e\n\u003cli\u003eLai RC, Yeo RW, Lim SK. Mesenchymal stem cell exosomes. Semin Cell Dev Biol. 2015 Apr;40:82-8.\u003c/li\u003e\n\u003cli\u003eLai RC, Tan SS, Teh BJ, Sze SK, Arslan F, de Kleijn DP, Choo A, Lim SK. Proteolytic potential of the msc exosome proteome: ımplications for an exosome-mediated delivery of therapeutic proteasome. Int J Proteomics. 2012;2012:971907.\u003c/li\u003e\n\u003cli\u003eParolini I, Federici C, Raggi C, Lugini L, Palleschi S, De Milito A, et al. Microenvironmental pH is a key factor for exosome traffic in tumor cells. J Biol Chem. 2009 Dec 4; 284:(49):34211-22.\u003c/li\u003e\n\u003cli\u003eNarayana K, Al-Bader M, Mousa A, Khan KM. Molecular effects of chemotherapeutic drugs and their modulation by antioxidants in the testis. Eur J Pharmacol 2012;674:207-216.\u003c/li\u003e\n\u003cli\u003ePrihatno SA, Padeta I, Larasati AD, Sundari B, Hidayati A, Fibrianto, et al. Effects of secretome on cisplatin-induced testicular dysfunction in rats. Vet. World. 2018;11:1349.\u003c/li\u003e\n\u003cli\u003eGholami Jourabi F, Yari S, Amiri P, Heidarianpour A, Hashemi H. The ameliorative effects of methylene blue on testicular damage induced by cisplatin in rats. Andrologia. 2021 Feb;53(1):e13850.\u003c/li\u003e\n\u003cli\u003eGrimshaw MJ, Balkwill FR. Inhibition of monocyte and macrophage chemotaxis by hypoxia and inflammation--a potential mechanism. Eur J Immunol. 2001 Feb;31(2):480-9.\u003c/li\u003e\n\u003cli\u003eYoshida GJ. Therapeutic strategies of drug repositioning targeting autophagy to induce cancer cell death: from pathophysiology to treatment. J. Hematol. Oncol. 2017;10:67.\u003c/li\u003e\n\u003cli\u003eZhang M, Jiang M, Bi Y, Zhu H, Zhou Z, Sha J. Autophagy and apoptosis act as partners to induce germ cell death after heat stress in mice. Plos One. 2012;7:7.\u003c/li\u003e\n\u003cli\u003eGallagher LE, Williamson LE, Chan EY. Advances in autophagy regulatory mechanisms. Cells 2016; 5: 1\u0026ndash;30.\u003c/li\u003e\n\u003cli\u003eZhang W, Yang C, Guo W, Guo X, Bian J, Zhou Q, et al. [Protective effect of bone marrow mesenchymal stem cells-derived exosomes against testicular ischemia-reperfusion injury in rats]. Nan Fang Yi Ke Da Xue Xue Bao. 2018 Jul 30;38(8):910-916.\u003c/li\u003e\n\u003cli\u003eLiu H, Shi M, Li X, Lu W, Zhang M, Zhang T, et al. Adipose mesenchymal stromal cell-derived exosomes Prevent testicular torsion injury via activating PI3K/AKT and MAPK/ERK1/2 pathways. Oxid Med Cell Longev. 2022 Jun 16;2022:8065771.\u003c/li\u003e\n\u003cli\u003eAfsar T, Razak S, Khan MR, Almajwal A. Acacia hydaspica ethyl acetate extract protects against cisplatin-induced DNA damage, oxidative stress and testicular injuries in adult male rats. BMC Cancer. 2017 Dec 21;17(1):883.\u003c/li\u003e\n\u003cli\u003eDasari S, Tchounwou PB. Cisplatin in cancer therapy: molecular mechanisms of action. Eur J Pharmacol. 2014 Oct 5;740:364-78.\u003c/li\u003e\n\u003cli\u003eCocetta V, Ragazzi E, Montopoli M. Mitochondrial Involvement in Cisplatin Resistance. Int J Mol Sci. 2019 Jul 10;20(14):3384.\u003c/li\u003e\n\u003cli\u003eWan M, Li C, Zhen G, Jiao K, He W, Jia X, et al. Injury-activated transforming growth factor \u0026beta; controls mobilization of mesenchymal stem cells for tissue remodeling. Stem Cells. 2012 Nov;30(11):2498-2511. \u003c/li\u003e\n\u003cli\u003eAly HA, Eid BG. Cisplatin induced testicular damage through mitochondria mediated apoptosis, inflammation and oxidative stress in rats: Impact of resveratrol. Endocr. J. 2020;67:969\u0026ndash;980.\u003c/li\u003e\n\u003cli\u003eWang L, He Y, Li Y, Pei C, Olatunji OJ, Tang J, et al. Protective effects of nucleosides-rich extract from cordyceps cicadae against cisplatin ınduced testicular damage. Chem Biodivers. 2020 Nov; 17(11):e2000671.\u003c/li\u003e\n\u003cli\u003eFerreiro ME, Amarilla MS, Glienke L, M\u0026eacute;ndez CS, Gonz\u0026aacute;lez C, Jacobo PV, et al. The inflammatory mediators TNF-\u0026alpha; and nitric oxide arrest spermatogonia GC-1 cell cycle. Reprod. Biol. 2019;19:329\u0026ndash;339. \u003c/li\u003e\n\u003cli\u003eAlvarez-Erviti L, Seow Y, Yin H, Betts C, Lakhal S, Wood MJ. Delivery of siRNA to the mouse brain by systemic injection of targeted exosomes. Nat Biotechnol. 2011 Apr;29(4):341-5.\u003c/li\u003e\n\u003cli\u003eArslan F, Lai RC, Smeets MB, Akeroyd L, Choo A, Aguor EN, et al. Mesenchymal stem cell-derived exosomes increase ATP levels, decrease oxidative stress and activate PI3K/Akt pathway to enhance myocardial viability and prevent adverse remodeling after myocardial ischemia/reperfusion injury. Stem Cell Res. 2013 May;10(3):301-12.\u003c/li\u003e\n\u003cli\u003eGuo XB, Zhai JW, Xia H, Yang JK, Zhou JH, Guo WB, et al. Protective effect of bone marrow mesenchymal stem cell-derived exosomes against the reproductive toxicity of cyclophosphamide is associated with the p38MAPK/ERK and AKT signaling pathways. Asian J Androl. 2021 Jul-Aug;23(4):386-391.\u003c/li\u003e\n\u003cli\u003eHassanzadeh-Taheri M, Hosseini M. Comments on \u0026ldquo;The improvement efects of Gordonia bronchialis on male fertility of rats with diabetes mellitus induced by Streptozotocin\u0026quot;. Pharm Sci. 2020;26:93\u0026ndash;5.\u003c/li\u003e\n\u003cli\u003eHoivik EA, Lewis AE, Aumo L, Bakke M. Molecular aspects of steroidogenic factor 1 (SF-1). Mol Cell Endocrinol. 2010;315:27\u0026ndash;39.\u003c/li\u003e\n\u003cli\u003eYing SY. Inhibins, activins, and follistatins: gonadal proteins modulating the secretion of follicle-stimulating hormone. Endocr Rev 1988:267-93. Available from: https://doi.org/10.1210/edrv-9-2-267. \u003c/li\u003e\n\u003cli\u003eHan, Y., Jiang, T., Liu, A., \u0026amp; Liu, L. Role and regulatory mechanism of inhibin in animal reproductive system. Theriogenology. 2023;202:10-20.\u003c/li\u003e\n\u003cli\u003eYang X. To analyze the relationship between semen quality and male FSH, Inh B level. Modern Med Health Res. 2021:113-5.\u003c/li\u003e\n\u003cli\u003eAndersson AM, Juul A, Petersen JH, M\u0026uuml;ller J, Groome NP, Skakkebaek NE. Serum inhibin B in healthy pubertal and adolescent boys: relation to age, stage of puberty, and follicle-stimulating hormone, luteinizing hormone, testosterone, and estradiol levels. J Clin Endocrinol Metabol. 1997;3976-81. Available from: https://doi.org/10.1210/jcem.82.12.4449. \u003c/li\u003e\n\u003cli\u003ePfaff T, Rhodes J, Bergmann M, Weinbauer GF. Inhibin B as a marker of sertoli cell damage and spermatogenic disturbance in the rat. Birth defects research. Part B, Developmental and reproductive toxicology. 2013; p. 91-103. Available from: https://doi.org/10.1002/bdrb.21046. \u003c/li\u003e\n\u003cli\u003ede Kretser DM, Buzzard JJ, Okuma Y, O\u0026apos;Connor AE, Hayashi T, Lin SY, Morrison JR, Loveland KL, Hedger MP. The role of activin, follistatin and inhibin in testicular physiology. Mol Cell Endocrinol. 2004;225(1-2):57-64.\u003c/li\u003e\n\u003cli\u003eTian M, Liu F, Liu H, Zhang Q, Li L, Hou X, et al. Grape seed procyanidins extract attenuates Cisplatin-induced oxidative stress and testosterone synthase inhibition in rat testes. Syst Biol Reprod Med. 2018;64:246\u0026ndash;59.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Exosome, cisplatin, testicular damage, autophagy","lastPublishedDoi":"10.21203/rs.3.rs-4011219/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4011219/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackround:\u003c/h2\u003e \u003cp\u003eCisplatin is a significant chemotherapy drug that has been used for different cancer types. The infertility and gonadotoxicity caused by metabolites resulting from cisplatin metabolism are two of the most serious side effects of cisplatin. Testicular dysfunction is generally caused by oxidative stress and autophagy that are induced by cisplatin. This study focused on potential exosomes' regenerative, antioxidant, and autophagy-regulating properties might be used to minimize testicular damage resulting from cisplatin.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eFor cisplatin toxicity, one dose of 7.5mg/kg was injected on first day. Exosom (80x10\u003csup\u003e6\u003c/sup\u003e/100\u0026micro;l) was injected single dose on sixth day. To examine potential therapeutic effects of the exosome, tissue samples were taken on eighth day. For examination of testicular morphology, tissues were stained with Hematoxylin\u0026amp;Eosin. Sperm motility and number were examined. Autophagy markers and SF-1,StAR expressions, were evaluated immunohistochemistry. Oxidative stress, inhibin and testosterone levels were analysed.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eCisplatin-induced damage caused to decrease in germinal epithelium, edema in interstitial areas, and degeneration in seminiferous tubules. Sperm motility and quality decreased. When compared to control groups, there was an increase in the expression of Beclin-1, p62, LC3-2, SF-1, and StAR. MDA activity increased,GSH-PX, SOD, and CAT activity decreased. Testosterone and inhibin levels decreased in serum. Tissue morphology was returned to normal, autophagy marker upregulation decreased, and SF-1 and StAR expressions increased in the group that received exosome, following cisplatin. There was a decrease in oxidative stress and increase in hormone levels\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eThese findings suggest that the exosome may offer a promising approach to minimizing Cis-induced reproductive toxicity and tissue damage by reducing autophagy and improving oxidative stress.\u003c/p\u003e","manuscriptTitle":"Exosome Therapy on Cisplatin-Induced Testicular Tissue Toxicity ","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-03-08 06:35:29","doi":"10.21203/rs.3.rs-4011219/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"34c4f8e1-f228-4f77-9645-0f8fc1bc62b4","owner":[],"postedDate":"March 8th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-03-13T21:29:28+00:00","versionOfRecord":[],"versionCreatedAt":"2024-03-08 06:35:29","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4011219","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4011219","identity":"rs-4011219","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}