Melatonin-Pretreated Mesenchymal Stem Cell-Derived Exosomes Alleviate Cavernous Fibrosis in a Rat Model of Nerve Injury-induced Erectile Dysfunction via miR-145-5p/TGF-β/Smad Axis

Melatonin-Pretreated Mesenchymal Stem Cell-Derived Exosomes Alleviate Cavernous Fibrosis in a Rat Model of Nerve Injury-induced Erectile Dysfunction via miR-145-5p/TGF-β/Smad Axis | 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 Melatonin-Pretreated Mesenchymal Stem Cell-Derived Exosomes Alleviate Cavernous Fibrosis in a Rat Model of Nerve Injury-induced Erectile Dysfunction via miR-145-5p/TGF-β/Smad Axis Xiaolin Zhang, Mengbo Yang, Xinda Chen, Ming Zhang, Yiliang Peng, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5246841/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 25 Feb, 2025 Read the published version in Stem Cell Research & Therapy → Version 1 posted 5 You are reading this latest preprint version Abstract Backgrounds: Cavernous nerve injury-induced erectile dysfunction (CNI-ED) is a common complication after radical prostatectomy. As a consequence of the concomitant severe fibrosis of the corpus cavernosum, conventional treatment approaches have had little success. Methods: Pre-treatment of adipose-derived stem cells with melatonin allows for the extraction of active exosomes (MT-hASC-EVs) from the conditioned medium. The therapeutic effects of MT-hASC-EVs were assessed in a rat model of CNI-ED, and the anti-fibrotic properties were evaluated. MicroRNA sequencing was used to identify specific microRNAs highly expressed in MT-hASC-EVs, and differential microRNAs were screened for regulatory pathways through target gene enrichment analysis. Finally, the conclusions from bioinformatics analysis were validated through in vitro experiments. Results: Intracavernous injection of MT-hASC-EVs significantly restored erectile function and reduced the extent of corpus cavernosum fibrosis in the CNI-ED rat model. MT-hASC-EVs promoted the proliferation and anti-apoptotic effects of CCSMCs in vitro. Mechanistically, MT-hASC-EVs inhibit fibrosis by delivering miR-145-5p, which targets TGF-β2/Smad3 axis. Conclusions: MT-hASCs-EVs can inhibit cavernous fibrosis and improve erectile function in a rat model of CNI-ED by targeting the miR-145-5p/TGF-β/Smad axis. Melatonin mesenchymal stem cell exosome CNI-ED cavernous fibrosis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1 Introduction Erectile dysfunction (ED) refers to the persistent or recurrent inability to achieve and maintain sufficient penile erection for satisfactory sexual intercourse[ 1 ]. The etiology of ED is complex, and cavernous nerve injury (CNI) is a significant contributing factor, commonly associated with pelvic surgery, pelvic fractures, and post-urethral injury surgeries[ 2 ]. CNI-induced ED following radical prostatectomy has an incidence rate as high as 63%-94%, despite the use of nerve-sparing techniques[ 3 ]. During radical prostatectomy, unavoidable traction, compression, and vascular damage to the cavernous nerves lead to impairment and neurotrophic loss which results in reduced penile blood flow perfusion, leading to sustained hypoxia in the corpus cavernosum[ 3 ]. Subsequent smooth muscle atrophy occurs along with activation of TGF-β/Smad and RhoA/Rock pathways which lead to cavernous fibrosis[ 2 ]. Traditional phosphodiesterase 5 inhibitors (PDE5i, e.g., tadalafil and sildenafil) have limited efficacy in treating CNI- ED due to insensitivity of corpora cavernosa tissue to NO resulting from smooth muscle cell loss and cavernous fibrosis[ 4 , 5 ]. It is worth noting that while regeneration of cavernous nerves could be observed 28 days after CNI in a rat model, subsequent loss of smooth muscle cells and cavernous fibrosis were irreversible[ 6 ]. Therefore, prevention, alleviation, or even reversal of cavernous fibrosis represents an important therapeutic direction for managing CNI-ED. In recent years, stem cells and their derivatives have become new treatment options for ED[ 7 – 9 ], among which extracellular vesicles (EVs) have gained favor among researchers due to their simplicity, convenience, and suitability for industrial production[ 10 ]. EVs are sac-like structures that cells release into the extracellular space[ 11 – 13 ]. Based on diameter and source, they can be divided into exosomes (50–100 nm) and microvesicles (100–1000 nm). As a carrier, EVs transport signal proteins, lipids, and nucleic acids, which can be endocytosed by distant target cells, thereby changing the biological behavior of the target cells and serving as a key factor in cell-to-cell signal communication[ 14 ]. Many reports have suggested that mesenchymal stem cell-derived EVs have a certain therapeutic effect on CNI-ED[ 15 – 18 ]. Melatonin is a hormone secreted by the pineal gland, primarily responsible for regulating biological rhythms and sleep-wake cycles[ 19 ]. In addition, melatonin has been found to have therapeutic effects on several fibrotic diseases including liver fibrosis[ 20 ], pulmonary fibrosis[ 21 ], and renal fibrosis[ 22 ], indicating its potential in counteracting cavernous fibrosis caused by CNI-ED. Furthermore, melatonin exhibits the ability to promote nerve regeneration[ 23 ], thereby improving erectile function through the protection and promotion of repair of cavernous nerves[ 24 , 25 ]. Melatonin can regulate the behavior of MSCs and significantly enhance the therapeutic effects of MSCs-derived EVs in certain diseases. For example, melatonin-pretreated MSCs-derived EVs shows better protective effects against rat renal ischemia-reperfusion injury[ 26 ]; they also show stronger therapeutic effects in diabetic wound healing[ 27 ]. However, it is still unclear whether melatonin-pretreated MSCs-derived EVs can play a therapeutic role in fibrotic diseases. In this study, we found that EVs derived from melatonin-pretreated adipose mesenchymal stem cells (MT-hASC-EVs) exhibit high expression of miR-145-5p through which they can target the TGF-β/Smad axis, inhibit corpus cavernosum fibrosis in the CNI-ED rat model, and subsequently promote the recovery of erectile function. This may represent a potential therapeutic approach for CNI-ED. 2 Materials and Methods 2.1 Cell isolation, culture and identification Human adipose stem cells (hASCs) were isolated from abdominal adipose tissues from healthy female liposuction as previously reported[ 28 ]. hASCs were resuspended in Minimum Essential Medium Alpha (α-MEM, Gibco) supplemented with 10% fetal bovine serum (New Zealand Characterized Fetal Bovine Serum, Hyclone, SH30406.05) and 1% penicillin-streptomycin (Beyotime, CNH) and cultured at 37 ° C in a 5% CO 2 incubator. The medium was changed every 3 days. Cells were passaged when they reached 80% confluence. All cells used in the experiments were at passages 3 to 5. The differentiation properties and stem cell markers of hASCs have been identified in our previous reports[ 28 ]. The isolation of corpus cavernous smooth muscle cells (CCSMCs) was conducted as described previously[ 28 ]. In brief, after anesthesia, the rats were sterilized, and the foreskin and dorsal penile vessels were removed to obtain penile corpus cavernosum tissue. Cavernosal tissue was washed in PBS and cut into small pieces of 1 to 2 mm. Segments were placed on 10-cm cell culture dishes (Corning, USA) containing a minimal volume of DMEM supplemented with 20% FBS and cultured at 37 ° C in a humidified atmosphere of 95% air and 5% CO2. After the explants were attached, more DMEM containing 10% FBS was added and tissue segments that had fallen off from the culture dish were removed. The cells were cultured in high-glucose DMEM (Gibco), supplemented with 10% FBS (Hyclone, SH30406.05), 1% penicillin-streptomycin (Beyotime Biotechnology) at 37°C and 5% CO2. The cells were frozen or passaged once 80–90% confluence was achieved. All cells used in the experiments were at passages 3 to 8. 2.2 Isolation and characterization of extracellular vesicles. An EV-free FBS was prepared by ultracentrifugation at 100 000 × g for 2 h at 4°C and filtered with a 0.22 µm filter. When the ADSC at passage 3 to 5 reached an 80% density, cells were washed with PBS and cultured in culture medium supplemented with EV-free FBS for 48 h, while MT-hASCs were incubated with 10µM melatonin. The media were then collected, and EVs were isolated through a multistep centrifugation. Dead cells, cell debris and microvesicles were removed at 300g for 5min, 3000g for 10min and 10000g for 30min, respectively. The supernatant was then ultracentrifuged at 100 000 × g for 2h (XP-90, Beckman Coulter, USA). The pellets were washed for three times and resuspended with PBS to obtain a suspension of hASC-EVs or MT-hASC-EVs. The total protein concentration of the sEVs was quantified using a micro bicinchoninic acid protein assay kit (Beyotime, CHN). For EV characterization, the EV-positive markers CD81 (Abclonal, A5270, 1:1000), CD9 (Abclonal, A1703, 1:1000), and TSG101 (Abclonal, A2216, 1:1000), and the EV-negative marker, Calnexin (Abclonal, A15631, 1:1000), were identified by western blotting analysis. The size distribution of the EVs was determined using the NanoSight NS300 (Malvern, UK), according to the manufacturer’s instructions. The ultrastructure and morphology of the EVs prepared by the Exosome-TEM-easy kit (101Bio, USA) were observed using transmission electron microscopy (TEM; Hitachi, JP). 2.3 In vivo experimental design Generally, forty 8-week-old Sprague Dawley (SD) rats (males, weighing 250 g each) were used in this study. All rats were maintained on a 12h light/12h dark cycle and were acclimatized for at least 1 week before surgery and allowed free access to standard food and water. Operations and welfare in this study complied with international and Chinese local legislations and National Institutes of Health guide for the care and was guaranteed under the supervision of the Experimental Animal Ethical Committee of Ren Ji Hospital (KY2022-180-B). Bilateral CNI was performed in 30 rats (CNI group), and the other 10 rats were subjected to only laparotomy (Sham group). The construction of the CNI-ED animal model was conducted as previously described[ 29 ]. After anesthesia by intraperitoneal injection of pentobarbital sodium (35 mg/kg), the rats were placed on an isothermal thermal pad. Hair above the abdomen and perineum was shaved with a hair clipper for better visualization. After disinfection, a 2.5-cm midline lower abdominal incision was made to expose the pelvic ganglions (MPG) and cavernous nerves (CNs) on the surface of both sides of prostate. The CNs were isolated bilaterally and crushed 5 mm distal to the MPG of 90s using micro-forceps (Storz, Germany). Then, the CNI group was randomly divided into three groups of 10 rats each, which received intracavernous injection of 1) PBS (0.1mL); 2) hASCs-EVs (100µg in PBS 0.1 ml); 3) MT-hASC-EVs (100µg in PBS 0.1 ml). The intracavernous injection method was performed as previously described[ 8 ]. In brief, the penis was exposed locally and a rubber tourniquet was applied at the base. After injection of 0.1 ml solution into the corpus cavernosum, the tourniquet was removed after 1 min and the penis was restored. The work has been reported in line with the ARRIVE guidelines 2.0. 2.4 Erectile function evaluation. The maximal intracavernous pressure (ICP) and realtime arterial pressure were recorded as previously described[ 28 , 29 ]. 4 weeks after intracavernous injection, SD rats were anesthetized by intraperitoneal injection of pentobarbital sodium (35 mg/kg). A 26-gauge needle connected to a catheter inserted into one side of corpus cavernosum to measure the intracavernous pressure (ICP) while the other end of the catheter was connected to a data collection device (BL-420s, Chengdu Taimeng Software Co.Ltd., China) using a pressure transducer. After exposing the carotid artery of the other side, a 20-gauge cannula filled with heparin saline was punctured in the artery to measure the mean artery pressure (MAP), with the other end of the cannula connected to the BL-420s using a pressure transducer. The CN were dissected and separated in the same way described above, and the CN was stimulated with electrodes with stimulus parameters set at 5 V, 25 Hz and 60 s duration. At the end, the penis was excised for further testing, and then the rats were euthanized with carbon dioxide. 2.5 Histological and immunohistochemical analysis For Masson's trichrome staining, the penile tissues were fixed in 4% paraformaldehyde overnight, which were subsequently dehydrated and embedded in paraffin. Next, the paraffin-embedded tissue was cut into 4-micron sections for staining. After gradient dehydration with xylene, the tissues were stained with a Masson trichrome staining kit (Masson trichrome Staining Kit, Solarbio, USA). Stained sections were observed under microscope and analyzed using Image J software. For immunohistochemical staining, penile tissue sections were rehydrated, and antigen retrieval was performed. The sections were blocked with goat serum for 60 min, then incubated with anti-Desmin antibodies (Proteintech, 16520-1-AP, 1:500) overnight. Then, MaxVision HRP-Polymer immunohistochemistry kit (Maxim, China) was used and the sections weredeveloped in color with diaminobenzidine (DAB). Sections were then counterstained with hematoxylin. Stained sections were observed under microscope and analyzed using Image J software. 2.6 Fluorescent labeling and in vitro tracing of EVs. hASC-EVs and MT-hASC-EVs were labeled with PKH67 dye (Maokang Biotechnology, China) according to the manufacturer's instructions. CCSMCs were incubated with PKH67-labeled EVs for 12h at 37℃. Following fixed with 4% paraformaldehyde and stained with 40, 6-diamidino-2-phenylindole (DAPI, Invitrogen, USA), the cells were observed under the confocal laser scanning microscope (Olympus, Japan). 2.7 CCSMC viability and proliferation. For cell viability evaluation, CCSMCs were seeded in a 96-well plate with a density of 2000 cells per well and incubated with hASC-EVs or MT-hASC-EVs. 24h or 48h later, Cell Counting Kit-8 (Beyotime, China) was added to the medium and incubated at 37℃ for 2h. Cell viability was assessed by OD value of each well using a microplate reader (Biotek, USA). For EdU cell proliferation staining, CCSMCs were seeded in a six-well plate and pre-treated with hASC-EVs or MT-hASC-EVs for 24h. Then, EdU cell proliferation staining was performed using an EdU kit (BeyoClick™ EdU Cell Proliferation Kit with Alexa Fluor 488, Beyotime, China) according to the manufacturer's instructions and nuclei were stained using Hoechst33342 (Beyotime, China). The fluorescence was detected using the fluorescence microscope (Olympus, Japan) and the cells in the proliferative phase were counted using Image J software. 2.8 Cell apoptosis assay Annexin V-FITC/PI cell apoptosis detection kit (Yeasen Biology, China) was used to detect the level of apoptosis under different conditions. CCSMCs seeded in a six-well plate (costar, United States) were pre-treated with hASC-EVs or MT-hASC-EVs for 24 h and then stimulated under hypoxia for 24 h. Subsequently, the cells were collected and stained with Annexin V-FITC and PI probe solution at room temperature for 15 min. The apoptosis rate was detected by Cytoflex (Beckman Coulter, United States). 2.9 The miRNA Library Construction and Sequencing. Total RNA of hASC-EVs and MT-hASC-EVs was extracted by the MagZol (Magen, China) according to the manufacturer’s protocol. The quantity and integrity of RNA yield was assessed by using the Qubit®2.0 (Invitvogen, USA) and Agilent 2200 TapeStation (Agilent Technologies, USA) separately. Briefly, RNAs were ligated with 3’ RNA adapter, and followed by 5’ adapter ligation. Subsequently, the adapter-ligated RNAs were subjected to RT-PCR and amplified with a low-cycle. Then the PCR products were size selected by PAGE gel according to instructions of NEBNext® Multiplex Small RNA Library Prep Set for Illumina® (Illumina, USA). The purified library products were evaluated using the Agilent 2200 TapeStation, The libraries were sequenced by HiSeq 2500(Illumina, USA) with single-end 50bp at Ribobio Co. Ltd (Ribobio, China). The raw reads were processed by filtering out containing adapter, poly ’ N’, low quality, smaller than 17nt reads by FASTQC to get clean reads. Mapping reads were obtained by mapping clean reads to reference genome of by BWA. miRDeep2 was used to identify known mature miRNA based on miRBase21 ( www.miRBase.org ) and predict novel miRNA. The expression levels were normalized by RPM, RPM is equal to (number of reads mapping to miRNA/number of reads in Clean data)×10 6 . Differential expression between two sets of samples was calculated by edgeR algorithm according to the criteria of |log2(Fold Change)|≥1 and p-value < 0.05. TargetScan, miRDB, miRTarBase and miRWalk were used to predict targets gene of selected miRNA. R 4.3.1 and miRPath v.3 ( https://dianalab.e-ce.uth.gr/html/mirpathv3/index.php?r=mirpath )[ 30 ] were used for further Gene Ontology (GO) and KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analysis. 2.10 Immunocytochemistry (ICC) CCSMCs were seeded in 35mm confocal dishes (Bioshark, China) and induced using 10ng/mL recombinant human TGF-β2 (Proeintech, HZ-1092, USA), while hASC-EVs or MT-hASC-EVs were co-incubated. After 48h, CCSMCs were fixed with 4% paraformaldehyde (PFA) and permeabilized with 0.1% Triton X-100 (Sigma, USA). Blocking was performed with goat serum for 1h, followed by incubation with TGF-β2 specific antibody (Ptoteintech, 19999-1-AP, 1:100) overnight. Then, the cells were incubated with green fluorescent secondary antibody (Proteintech, RGAR002, 1:200) for 1h at room temperature. Nuclei were stained using DAPI (Invitrogen, USA). CCSMCs were observed under the confocal laser scanning microscope (Olympus, Japan) and the fluorescent intensity was valued by Image J software. 2.11 Cell Transfection. Cell transfection was performed using Lipofectamine 3000 (Invitrogen, USA), according to the manufacturer’s protocol. The miR-145-5p inhibitor and NC inhibitor, miR-145-5p mimics and NC mimics were obtained from Genomeditech (Shanghai, China), and their sequences were shown in Supplementary Table 2 . 2.12 Dual-luciferase reporter assay. The entire 3ʹ-UTR fragments of Tgfb2 and Smad3 and the mutant form in which the potential miR-145-5p binding sites were mutated were inserted into PGL3-CMV-LUC vector, namely Rat_Tgfb2 WT, Rat_Tgfb2 mut, Rat_Smad3 WT and Rat_Smad3 mut, respectively. Plasmid profiles as well as sequences are shown in Supplementary Fig. 5 and Supplementary Table 5 . These plasmids were respectively, co-transfected with miR-145-5p mimics or mimics NC into HEK293 cells. After 48 hours, cells were collected and luciferase activity was measured using Dual-Luciferase Reporter Assay System (E1910, Promega, Madison, WI, USA). 2.13 Real-Time PCR. The EZ-press RNA Purification Kit (EZB, USA) was used to extract mRNA from the ASCs and CCSMCs. Exosome RNA Purification Kit (EZB, USA) was used to extract total RNA from EVs. A reverse transcription kit (TaKaRa, Japan) was used to synthesize complementary DNA from mRNA. An miRNA 1st Strand cDNA Synthesis Kit (by stem-loop) (Vazyme, China) was used to synthesize complementary DNA from microRNA. The ChamQ Universal SYBR qPCR Master Mix (Vazyme, China) was used to and perform quantitative real-time PCR, according to the manufacturer’s instructions. ACTB and U6 were used as internal controls. The LightCycler 480 real-time PCR system (Roche Diagnostics, Indianapolis, IN, USA) was used for detection. The primers used are listed in Supplementary Table 1 . 2.14 Western Blots. Proteins were extracted using RIPA Lysis Buffer (Beyotime, China) according to the manufacturer’s instructions. The protein concentration was detected using a Bicinchoninic Acid Protein Assay Kit (Beyotime, China). The Protein solution and SDS-PAGE Protein Sample Loading Buffer were mixed and denatured by heating at 95 ° C. 10 µg of proteins were used for electrophoresis per lane. After electrophoresis, the proteins were transferred onto polyvinylidene difluoride membranes. The membranes were blocked with Tris-buffered saline-Tween (with 5% skim milk) and incubated at 4°C overnight with primary antibodies against β-actin (Proeintech, 66009-1-Ig, 1:20000), TGF-β2(Proteinteech, 19999-1-AP, 1:1000), TGF-βRII (Proteintech, 66636-1-Ig, 1:5000), Smad3 (Abclonal, A19115, 1:20000), p-Smad3 (Cell Signaling Technology, #9520, 1:1000), Smad2/Smad3 (Cell Signaling Technology, #3102, 1:1000), p-Smad2/p-Smad3 (Abclonal, AP1343, 1:1000), COL1A1 (Abclonal, A1352, 1:1000), COL3A1 (Abclonal, A3795, 1:2000). After hybridization with the secondary antibody (Proteinteh, 1:10000), bands were observed with enhanced chemiluminescence substrates (Millipore, MA) under a chemiluminescence imaging system (Bio-Rad, USA). The grayscale values of the bands were calculated using Image Lab software. 2.15 Statistical analysis. All quantitative data were expressed as the mean ± standard deviation. Differences between the groups were assessed by oneway analysis of variance (ANOVA), followed by a Student’s t-test using GraphPad Prism v9.0 software. p < 0.05 was considered statistically significant. 3 Results 3.1 Isolation and characterization of melatonin-pretreated adipose mesenchymal stem cell-derived EVs. Primary adipose mesenchymal stem cells were isolated from human adipose tissue. After 48 hours of treatment with 10µM Melatonin, the conditioned medium was collected, from which MT-hASC-EVs were isolated (Fig. 1 A). Both hASCs and melatonin-pretreated hASCs showed typical spindle and fibroblast-like morphologies (Fig. 1 B). The levels of growth factors secreted by hASCs were detected by RT-PCR (Fig. 1 C). After Melatonin treatment, the expression levels of various growth factors in hASCs increased, among which the expression level of SHH (Sonic Hedgehog) increased most significantly as early as 2–4 h of Melatonin treatment. In addition, the expression levels of PDGF, SDF and HGF were also significantly increased after melatonin pretreatment. hASC-EVs and MT-hASC-EVs were isolated from the conditioned medium of hASCs and melatonin-treated hASCs respectively. Western Blot revealed that both hASC-EVs and MT-hASC-EVs both highly expressed CD81, CD9, and the endosomal marker TSG101, but rarely expressed the endoplasmic reticulum marker Calnexin (Fig. 1 D). Nanoparticle Trafficking Analysis (NTA) detection showed that the particle sizes of hASC-EVs and MT-hASC-EVs were mostly distributed in the range of 50-200nm, with a main peak at about 110nm (Fig. 1 E). There was also no difference in mean particle size (Fig. 1 F). Both hASC-EVs and MT-hASC-EVs had a typical cup-like shape under transmission electron microscope (TEM) (Fig. 1 G). These results proved that EVs from hASCs and MT-hASCs were successfully isolated and characterized, while the pretreatment of melatonin had no significant effect on the surface markers, morphology, and particle size distribution of hASC-EVs. 3.2 Intracavernous injection of MT-hASC-EVs could restore erectile function and reduce cavernous fibrosis in a rat model of CNI-ED. In order to evaluate the therapeutic effects of MT-hASC-EVs on CNI-ED, we utilized a rat model of CNI-ED established through bilateral cavernous nerve crush, following which both types of EVs were administered via intracavernous injection. At 4 weeks post-treatment, the erectile function was assessed by measuring intracavernous pressure (ICP) and mean arterial pressure (MAP) (Fig. 2 A, 2 B). There were no significant differences in MAP among all groups. The successful establishment of CNI-ED model was confirmed by a significant decrease of ICP/MAP ratio in the PBS group compared with the sham group. Intracavernous injection of hASC-EVs or MT-hASC-EVs could restore the ICP/MAP ratio to a certain extent. Notably, the therapeutic effects of MT-hASC-EVs were more pronounced compared to that of hASC-EVs. Cavernous fibrosis is one of the most severe pathological changes of CNI-ED, making it a major factor contributing to the refractory of the disease, so we focused on the degree of cavernous fibrosis among all groups. The Masson staining results showed t obvious collagen fiber accumulation and a significant decrease in the ratio of muscle fibers to collagen fibers in PBS group. hASC-EVs and MT-hASC-EVs could alleviate such phenomenon to some extent, with the therapeutic effect of MT-hASC-EVs being more obvious, suggesting the stronger anti-fibrotic effects of MT-hASC-EVs (Fig. 2 C, 2 D). Immunohistochemistry was used to evaluate the content of cavernous smooth muscle, and the results were consistent with the Masson staining results, with significant loss of Desmin + muscle fiber in PBS group and the highest smooth muscle level in MT-hASC-EV group (Fig. 2 E, 2 F). Chronic hypoxia caused by long-term low perfusion after CNI can activate the TGF-β/Smad pathway and induce cavernous nerve fibrosis. Therefore, RT-PCR and Western Blot were used to reveal that MT-hASC-EVs could most significantly reduce the mRNA and protein level of TGF-β2 and Smad3 in cavernous nerve tissue after CNI, thereby reducing the expression of Collagen I (Fig. 2 G, 2 H). These results suggest that MT-hASC-EVs have good effects in relieving CNI-induced cavernous fibrosis by downregulating the TGF-β/Smad pathway. 3.3 MT-hASC-EVs could promote the proliferation of CCSMCs and reduce cell apoptosis in vitro. At the in vivo level, MT-hASC-EVs significantly increased the content of cavernous smooth muscle. To elucidate the mechanism, primary corpus cavernous smooth muscle cells (CCSMCs) were isolated from rat corpus cavernosum for the in vitro experiments. Both PKH67-labeled hASC-EVs and MT-hASC-EVs added to the culture supernatant were significantly up-taken by CCSMCs after 12 hours (Fig. 3 A). After incubation for 24 hours, both MT-hASC-EVs and hASC-EVs significantly enhanced the cell viability of CCSMCs, with a stronger effect observed for MT-hASC-EVs compared to hASC-EVs. The effect became more pronounced after 48 hours (Fig. 3 B). When EdU was added to the culture medium, a higher number of EdU-positive cells was detected in CCSMCs after incubation with MT-hASC-EVs (Fig. 3 C, 3 D), indicating that MT-hASC-EV treatment promoted CCSMC proliferation. Furthermore, given that low oxygen-induced ROS damage is a key factor in CNI-ED progression, we stimulated CCSMCs with low oxygen and measured apoptosis rates under different treatment conditions. After hypoxia treatment, the apoptosis rate of CCSMCs significantly increased, while both MT-ASC-EVs and ASC-EVs had a certain inhibitory effect on CCSMC apoptosis, with the former being more significant (Fig. 3 E, 3 F). Taking all above account, CCSMCs can enhance cell proliferation after uptaking MT-ASC-EVs, while reducing cell apoptosis induced by hypoxia. 3.4 High-throughput sequencing revealed that the microRNAs in MT-hASC-EVs can target the TGF-β/Smad axis EVs contain a large number of microRNAs, as one of the most important effectors in EVs. EVs can target specific mRNAs by delivering microRNAs to target cells and regulate protein expression specifically, thereby achieving the regulation of cell functions. Therefore, microRNAs specifically enriched in MT-hASC-EVs were screened by high-throughput sequencing. Among the co-expressed microRNAs, we selected those with a |Log2(FoldChange)|>1 and p < 0.05 as the screening conditions to screen for differentially expressed microRNAs, ultimately obtaining 12 upregulated microRNAs and 8 downregulated microRNAs, which were displayed in the volcano plot (Fig. 4 A) and heatmap (Fig. 4 B). microRNAs specifically target mRNAs to exert negative regulation, so we focused on the functional enrichment of target genes of 12 up-regulated microRNA. Using the prediction results from miRTarBase or the common prediction results from miRDB, miRWalk, and TargetScan as candidate target genes for the 12 upregulated microRNAs, a total of 4,928 target genes were identified( Supplementary Table 3 ). Gene Ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis were performed on these target genes. In the GO enrichment analysis, several enriched terms were related to "decreased oxygen levels", suggesting significant regulatory roles in cellular responses to hypoxia/anoxia of these microRNAs, and it is worth noting the term "response to transforming growth factor beta" was also enriched (Fig. 4 C). Consistent with that, these target genes were enriched in the "TGF-beta signaling pathway" in KEGG enrichment analysis (Fig. 4 D). In summary, the microRNA sequencing and target gene enrichment analysis suggest that the microRNAs enriched in MT-ASC-EVs may have played a therapeutic role in the treatment of CNI-ED by targeting the TGF-beta pathway, which is consistent with the observation of downregulation of TGF-beta pathway-related proteins in the in vivo experiments. 3.5 MT-hASC-EVs could reduce the fibrosis of CCSMCs in vitro by inhibiting the TGF-β pathway. The results of microRNA sequencing and bioinformatics analysis were validated in vitro. CCSMCs were treated with TGF-β (10ng/mL) for 48h to induce fibrosis, and hASC-EVs or MT-ASC-EVs were simultaneously co-incubated. RT-PCR detection indicated that the mRNA level of Tgfb2 was significantly decreased after MT-ASC-EVs treatment, while the effect of ASC-EVs was not obvious (Fig. 5 A). However, both of them could downregulate the mRNA level of Col1a1 and this may indicate both of the EVs contain microRNAs targeting the mRNA of Col1a1, rather than simply downregulating the expression of Col1a1 through the TGF-β pathway. Western Blot detection revealed that the levels of TGF-β2 and Smad2/3 in CCSMCs were significantly downregulated after MT-ASC-EVs treatment compared with the TGF-β2 group and hASC-EVs group. As for the evaluation index of fibrosis, CollagenI/CollagenIII, MT-hASC-EVs also showed more excellent anti-CCSMCs fibrosis effects (Fig. 5 B, 5 C). In the ICC experiment, lower TGF-β2 fluorescence signals were detected in CCSMCs after MT-hASC-EVs treatment (Fig. 5 D, 5 E). Combining the above results, after MT-hASC-EVs treatment, the activation level of the TGF-β pathway in CCSMCs was significantly downregulated, which may be an important mechanism for the anti-fibrosis effects of MT-hASC-EVs. 3.6 MT-hASC-EVs inhibit TGF-β2 and Smad3 expression through miR-145-5p. To further identify the core microRNAs that play a pivotal role among the differentially expressed miRNAs identified in the microRNA sequencing analysis, KEGG enrichment analysis was conducted on the upregulated miRNAs using the miRPath v3.0 software (Fig. 6 A). The results indicated that, among all upregulated miRNAs, miR-145-5p (targeting 12 genes), as well as miR-23b-3p and miR-23a-3p (both targeting 15 genes), were significantly enriched in the TGF-β signaling pathway ( Supplementary Table 4 ). To validate these results, RT-PCR was first performed to assess the expression of miR-145-5p, miR-23a-3p, and miR-23b-3p in hASCs. Following a 48-hour treatment with 10 µM melatoni, we observed a significant upregulation of these three miRNAs in hASCs (Fig. 6 B). Similarly, MT-hASC- EVs exhibited significantly higher expression levels of these three miRNAs compared to hASC-EVs without melatonin pretreatment (Fig. 6 C). Given that the upregulation of miR-145-5p was particularly pronounced, we chose to focus our subsequent validation efforts on this specific microRNA. After treating CCSMCs with both types of EVs, RT-PCR analysis revealed that MT-hASC-EVs significantly increased the expression level of miR-145–5p (Fig. 6 D). TargetScan version 8.0 ( https://www.targetscan.org/vert_80/ ) predicted binding sites for miR–145–5p within the 3' untranslated region (UTR) of rat Tgfb2 at a 7mer-A1 site and within rat Smad3 at an 8mer site which are illustrated in Fig. 6 E. Dual luciferase reporter assays were performed to determine whether Tgfb2 and Smad3 serve as direct targets for regulation by miR–145–5p. The results demonstrated that co-transfection with a mimic for mir–145–5p led to a significant reduction in luciferase activity from both Tgfb2 WT and Smad3 WT constructs, while no significant changes were observed for Tgfb2 mut or Smad3 mut constructs ( Fig. 6 F), which confirmed the specific targeting. Furthermore, transfection with miR − 145 − 5p mimics into CCSMCs resulted in marked inhibition of TGF − β2-induced activation within TGF − β signaling pathways along with reduced collagen I accumulation; conversely, transfection with miR − 145 − 5p inhibitor blocked this effect significantly (Fig. 6 G). In summary, our analyses indicate that MT-hASC-EVs facilitate fibrosis alleviation by delivering miR − 145 − 5p targeting Tgfb2and Smad3 expressions thereby inhibiting TGF-β signaling pathways within CCSMCs. 4 Discussion Corpus cavernosum nerve injury is one of the important causes of erectile dysfunction, commonly seen after pelvic surgery especially radical prostatectomy, with a postoperative occurrence rate of ED as high as 63%-94% in two years[ 3 ]. Currently, perioperative penile rehabilitation techniques have been adopted in clinical practice, including vacuum negative pressure suction, intracavonous injection of vasoactive agent and oral phosphodiesterase 5 inhibitors, to restore erectile function in CNI-ED patients[ 5 ]. However, about 50%-75% of patients still cannot recover to their preoperative erectile function status, seriously affecting the postoperative quality of life[ 4 ]. The refractoriness of CNI-ED is closely related to its pathogenesis and development mechanism, especially severe corpus cavernosum fibrosis after CNI-ED surgery[ 2 ]. Chronic hypoxia caused by long-term low blood flow perfusion in the penis after CNI can lead to functional impairment of corpus cavernosum smooth muscle cells through ROS accumulation[ 31 ], resulting in severely reduced responsiveness of the corpus cavernosum to nitric oxide (NO)[ 32 ]; on the other hand, hypoxia can also activate TGF-β/Smad[ 33 ] and RhoA/Rock[ 34 ] pathways leading to corporal fibrosis and impaired blood capacity and compliance. Therefore, the response effect of traditional PDE5i drugs on CNI-ED is often unsatisfactory. Thus it is urgent to study new methods for effectively targeting cavernous fibrosis in order to achieve better therapeutic effects for CNI-ED. In recent years, many animal and clinical studies have confirmed that mesenchymal stem cells can promote recovery of erectile function in CNI-ED[ 7 – 9 ]. However clinically stem cell therapy for ED is still confronted with constraints such as cumbersome processes for storage, culture and proliferation; ethical issues; risk of immune rejection; tumorigenicity; pathogen contamination etc[ 35 ]. Besides, a recent study revealed that MSCs could be maintained within the cavernous tissue for only 3 days in CNI-ED rats with no obvious evidence for endothelial and smooth muscle differentiation, indicating that MSC differentiation does not play a major role in treatment efficacy [ 36 ]. The paracrine function, especially extracellular vesicles (EVs), is likely to be one of the main mechanisms for the therapeutic effects of MSCs. MSC-derived EVs exert therapeutic effects while avoiding risks associated with stem cell therapy, making them suitable for industrial preparation, thus having good prospects for clinical translation[ 13 , 37 , 38 ]. Therefore, in this study, we also selected EVs isolated and enriched from the conditioned medium of hASCs for intracavernous injection, in order to achieve better treatment efficiency. Melatonin (MT) is an amine hormone mainly synthesized and secreted by the pineal gland, which has been proved to alleviate CNI-ED by promoting the repair of cavernous nerve[ 24 , 25 ]. However, it should be noted that the cavernous nerve can spontaneously repair after CNI, while downstream cavernous fibrosis is difficult to reverse[ 6 ]. Therefore, neuroprotection and regeneration may not be the only mechanism by which melatonin exerts its therapeutic effect on CNI-ED. Many studies have demonstrated Melatonin's anti-fibrotic effects in various disease models, such as liver fibrosis[ 20 ], pulmonary fibrosis[ 21 ], renal fibrosis[ 22 ], etc. So it is reasonable to infer that the anti-fibrotic effect of Melatonin also plays a rather important role in the remission of CNI-ED, but this has not been elucidated before. In addition, melatonin can also regulate MSCs behavior and significantly enhance the therapeutic effect of EVs derived from MSCs in certain diseases[ 26 , 27 ]. It has also been observed that exosomes isolated from melatonin-stimulated MSCs can to some extent alleviate renal fibrosis in chronic kidney disease[ 39 ]. In order to verify the above conjecture and clarify its intrinsic mechanism, in this study, we enriched EVs produced by hASCs after pretreatment with melatonin. On one hand, MT-hASC-EVs contain melatonin absorbed by hASC during pretreatment which can exert a certain therapeutic effect; on the other hand and more importantly, melatonin can regulate the composition of EVs derived from hASCs, thus enhancing the therapeutic effect. Through in vivo experiments, we found that intracavernous injection of MT-hASC-EVs could significantly ameliorate the erectile function of CNI-ED rats, meanwhile reduce collagen accumulation indicated by Masson staining, and increase Desmin staining distribution in the cavernous tissue. They could also inhibite the expression of TGF-β pathway-related molecules such as TGF-β, phosphorylated-Smad3 and fibrosis marker Col1a1. Similarly, in vitro, MT-hASC-EVs promoted the proliferation of CCSMCs, reduced hypoxia-induced apoptosis, and inhibited TGF-β2-induced fibrosis of CCSMCs by inhibiting the activation of TGF-β pathway. These experiments demonstrated the excellent anti-fibrosis ability of MT-hASC-EVs in the treatment of CNI-ED. EVs exert regulatory effects by delivering their cargo to target cells, among which microRNAs are important components[ 40 ]. microRNAs can recognize the binding sites on the 3'-UTR of target gene mRNAs through their seed sequences (nucleotides 2–8 at the 5' end), causing the translation of mRNAs to be blocked or even degraded[ 41 ]. Therefore, to explore the underlying mechanism of the stronger therapeutic effects of MT-hASC-EVs compared to hASC-EVs, we performed high-throughput microRNA sequencing and comparison of the two types of EVs and identified 20 microRNAs with significant expression differences. Since microRNAs exert regulatory effects by downregulating the expression of target genes when they are highly expressed, the upregulated microRNAs in MT-hASC-EVs should play a more primary regulatory role, but not the down-regulated microRNAs. Thus, enrichment analysis of the target genes of upregulated miRNAs in MT-hASC-EVs revealed significant enrichment in "response to transforming growth factor beta" and "transforming growth factor beta signaling". To identify the most crucial microRNAs, KEGG analysis by miRPath v3 suggested that miR-145-5p, miR-23a-3p, and miR-23b-3p are all related to the TGF-β pathway. Previous studies have reported the regulatory role of miR-145 in various fibrotic diseases, such as myocardial fibrosis (targeting SOX9)[ 42 ], scar hyperplasia (targeting Smad2/3)[ 43 ], liver fibrosis (targeting ADD3)[ 44 ], etc., so we chose miR-145-5p as the most likely effector molecule for further experimental design. The expression of miR-145-5p in both MT-hASCs and MT-hASC-EVs can be significantly upregulated by melatonin, confirming the conclusion of high-throughput sequencing. To clarify the downstream targets of miR-145-5p, we predicted the possible target genes of miR-145-5p using TargetScan v8.0 and sifted potential targets related to the TGF-β pathway. We identified a 7mer-A1 site for miR-145-5p in the 3'UTR of Tgfb2 and an 8mer site in the 3'UTR of Smad3, which were subsequently validated using dual luciferase reporter gene assays. Combining the above results, we confirmed that MT-hASC-EVs can inhibit the occurrence of fibrosis through miR-145-5p/Tgfb2/Smad3 axis. However, our research still has some limitations worth further exploration and improvement. First, MT-hASC-EVs can promote the proliferation of CCSMCs and inhibit apoptosis in vitro, and can also increase the content of Desmin at the in vivo level, but the mechanism behind this is not yet clear. Additionally, we only focused on the changes in the microRNA expression profile of MT-hASC-EVs, while proteins, lipids, and other molecules are also important regulatory molecules that EVs can deliver to target cells, and the potential effector molecules among them are also worth further exploration. 5 Conclusions In conclusion, the present study demonstrated that intracavernosal injection of MT-hASC-EVs could significantly alleviate CNI-ED. MT-hASC-EVs could effectively alleviate the apoptosis and fibrosis of CCSMCs in vitro and in vivo. Mechanistically, microRNA sequencing revealed that miR-145-5p was significantly enriched in MT-hASC-EVs and played a direct and negative regulatory role on Tgfb2 and Smad3 mRNAs by being translocated to CCSMCs. MT-hASCs-EVs can significantly alleviate CNI-ED by inhibiting the occurrence of fibrosis through miR-145-5p/TGF-β/Smad axis (Fig. 7 ). All these suggest that these extracellular vesicles may be potential drugs and materials for CNI-ED treatment. Declarations Ethics approval and consent to participate The study of “Application of mesenchymal stem cells in cavernous nerve injury-induced erectile dysfunction” was approved by the Ethics Committee of Renji Hospital Affiliated to Shanghai Jiao Tong University School of Medicine on Oct.26, 2022, with approval number KY2022-180-B. The patients provided written informed consent for participation in the study and the use of samples. Consent for publication Not applicable. Availability of data and materials The original contributions presented in the study are included in the article/Supplementary Material; further inquiries can be directed to the corresponding author. The matrix of microRNA sequencing is available within the supplementary materials. Competing interests The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Funding This work was supported by the National Natural Science Foundation of China No. 82171611, No. 82371631 and No. 82401887. Authors’ contributions XZ and MY performed most of the experiments, analyzed the data, and drafted the manuscript. XC helped with the isolation of ASCs. MZ and YP helped with the animal experiment. ML designed this study and critically revised the manuscript. All authors have read and approved the final manuscript. Acknowledgement The authors declare that they have not use Artificial intelligence (AI) -generated work in this manuscript. References Yafi FA, Jenkins L, Albersen M, Corona G, Isidori AM, Goldfarb S, et al. Erectile dysfunction . Nat Rev Dis Primers 2016;2:16003. Song G, Hu P, Song J, Liu J, Ruan Y. Molecular pathogenesis and treatment of cavernous nerve injury-induced erectile dysfunction: A narrative review . Front Physiol 2022;13:1029650. Ficarra V, Novara G, Ahlering TE, Costello A, Eastham JA, Graefen M, et al. Systematic review and meta-analysis of studies reporting potency rates after robot-assisted radical prostatectomy . Eur Urol 2012;62:418-30. Philippou YA, Jung JH, Steggall MJ, O'Driscoll ST, Bakker CJ, Bodie JA, et al. Penile rehabilitation for postprostatectomy erectile dysfunction . Cochrane Database Syst Rev 2018;10:CD012414. Liu C, Lopez DS, Chen M, Wang R. Penile Rehabilitation Therapy Following Radical Prostatectomy: A Meta-Analysis . J Sex Med 2017;14:1496-503. Wu YN, Chen KC, Liao CH, Chiang HS. Spontaneous Regeneration of Nerve Fiber and Irreversibility of Corporal Smooth Muscle Fibrosis After Cavernous Nerve Crush Injury: Evidence From Serial Transmission Electron Microscopy and Intracavernous Pressure . Urology 2018;118:98-106. Yan H, Ding Y, Lu M. Current Status and Prospects in the Treatment of Erectile Dysfunction by Adipose-Derived Stem Cells in the Diabetic Animal Model . Sex Med Rev 2020;8:486-91. Yan H, Rong L, Xiao D, Zhang M, Sheikh SP, Sui X, et al. Injectable and self-healing hydrogel as a stem cells carrier for treatment of diabetic erectile dysfunction . Mater Sci Eng C Mater Biol Appl 2020;116:111214. Haahr MK, Harken Jensen C, Toyserkani NM, Andersen DC, Damkier P, Sorensen JA, et al. A 12-Month Follow-up After a Single Intracavernous Injection of Autologous Adipose-Derived Regenerative Cells in Patients with Erectile Dysfunction Following Radical Prostatectomy: An Open-Label Phase I Clinical Trial . Urology 2018;121:203 e6-03 e13. Zhang X, Yang M, Chen X, Lu M. Research progress on the therapeutic application of extracellular vesicles in erectile dysfunction . Sex Med Rev 2024. Cheng L, Hill AF. Therapeutically harnessing extracellular vesicles . Nat Rev Drug Discov 2022;21:379-99. Couch Y, Buzàs EI, Di Vizio D, Gho YS, Harrison P, Hill AF, et al. A brief history of nearly EV‐erything – The rise and rise of extracellular vesicles . Journal of Extracellular Vesicles 2021;10. Pan Z, Sun W, Chen Y, Tang H, Lin W, Chen J, et al. Extracellular Vesicles in Tissue Engineering: Biology and Engineered Strategy . Adv Healthc Mater 2022:e2201384. van Niel G, D'Angelo G, Raposo G. Shedding light on the cell biology of extracellular vesicles . Nat Rev Mol Cell Biol 2018;19:213-28. Li M, Lei H, Xu Y, Li H, Yang B, Yu C, et al. Exosomes derived from mesenchymal stem cells exert therapeutic effect in a rat model of cavernous nerves injury . Andrology 2018;6:927-35. Ouyang X, Han XY, Chen ZH, Fang JF, Huang XN, Wei HB. MSC-derived exosomes ameliorate erectile dysfunction by alleviation of corpus cavernosum smooth muscle apoptosis in a rat model of cavernous nerve injury . Stem Cell Research & Therapy 2018;9:12. Liang L, Shen Y, Dong ZF, Gu X. Photoacoustic image-guided corpus cavernosum intratunical injection of adipose stem cell-derived exosomes loaded polydopamine thermosensitive hydrogel for erectile dysfunction treatment . Bioact Mater 2022;9:147-56. Liu S, Li R, Dou K, Li K, Zhou Q, Fu Q. Injectable thermo-sensitive hydrogel containing ADSC-derived exosomes for the treatment of cavernous nerve injury . Carbohydr Polym 2023;300:120226. Vasey C, McBride J, Penta K. Circadian Rhythm Dysregulation and Restoration: The Role of Melatonin . Nutrients 2021;13. Zhu L, Zhang Q, Hua C, Ci X. Melatonin alleviates particulate matter-induced liver fibrosis by inhibiting ROS-mediated mitophagy and inflammation via Nrf2 activation . Ecotoxicol Environ Saf 2023;268:115717. Hosseinzadeh A, Javad-Moosavi SA, Reiter RJ, Hemati K, Ghaznavi H, Mehrzadi S. Idiopathic pulmonary fibrosis (IPF) signaling pathways and protective roles of melatonin . Life Sci 2018;201:17-29. Repova K, Stanko P, Baka T, Krajcirovicova K, Aziriova S, Hrenak J, et al. Lactacystin-induced kidney fibrosis: Protection by melatonin and captopril . Front Pharmacol 2022;13:978337. Qian Y, Han Q, Zhao X, Song J, Cheng Y, Fang Z, et al. 3D melatonin nerve scaffold reduces oxidative stress and inflammation and increases autophagy in peripheral nerve regeneration . J Pineal Res 2018;65:e12516. Tavukcu HH, Sener TE, Tinay I, Akbal C, Ersahin M, Cevik O, et al. Melatonin and tadalafil treatment improves erectile dysfunction after spinal cord injury in rats . Clin Exp Pharmacol Physiol 2014;41:309-16. Zhang JL, Hui Y, Zhou F, Hou JQ. Neuroprotective effects of melatonin on erectile dysfunction in streptozotocin-induced diabetic rats . Int Urol Nephrol 2018;50:1981-88. Alzahrani FA. Melatonin improves therapeutic potential of mesenchymal stem cells-derived exosomes against renal ischemia-reperfusion injury in rats . Am J Transl Res 2019;11:2887-907. Liu W, Yu M, Xie D, Wang L, Ye C, Zhu Q, et al. Melatonin-stimulated MSC-derived exosomes improve diabetic wound healing through regulating macrophage M1 and M2 polarization by targeting the PTEN/AKT pathway . Stem Cell Res Ther 2020;11:259. Ti Y, Yang M, Chen X, Zhang M, Xia J, Lv X, et al. Comparison of the therapeutic effects of human umbilical cord blood-derived mesenchymal stem cells and adipose-derived stem cells on erectile dysfunction in a rat model of bilateral cavernous nerve injury . Front Bioeng Biotechnol 2022;10:1019063. Li Z, Yin Y, He K, Ye K, Zhou J, Qi H, et al. Intracavernous Pressure Recording in a Cavernous Nerve Injury Rat Model . J Vis Exp 2021. Vlachos IS, Zagganas K, Paraskevopoulou MD, Georgakilas G, Karagkouni D, Vergoulis T, et al. DIANA-miRPath v3.0: deciphering microRNA function with experimental support . Nucleic Acids Res 2015;43:W460-6. Wang HS, Ruan Y, Banie L, Cui K, Kang N, Peng D, et al. Delayed Low-Intensity Extracorporeal Shock Wave Therapy Ameliorates Impaired Penile Hemodynamics in Rats Subjected to Pelvic Neurovascular Injury . J Sex Med 2019;16:17-26. Karakus S, Musicki B, La Favor JD, Burnett AL. cAMP-dependent post-translational modification of neuronal nitric oxide synthase neuroprotects penile erection in rats . BJU Int 2017;120:861-72. Leungwattanakij S, Bivalacqua TJ, Usta MF, Yang DY, Hyun JS, Champion HC, et al. Cavernous neurotomy causes hypoxia and fibrosis in rat corpus cavernosum . J Androl 2003;24:239-45. Cho MC, Park K, Kim SW, Paick JS. Restoration of erectile function by suppression of corporal apoptosis, fibrosis and corporal veno-occlusive dysfunction with rho-kinase inhibitors in a rat model of cavernous nerve injury . J Urol 2015;193:1716-23. Philip S, Bredeson C, Allan DS, Altouri S, Huebsch LB, Atkins H, et al. Complications and Toxicities Associated with Autologous Stem Cell Transplantation for Severe Autoimmune Disease: Single Center Experience . Blood 2018;132. Chen Z, Han X, Ouyang X, Fang J, Huang X, Wei H. Transplantation of induced pluripotent stem cell-derived mesenchymal stem cells improved erectile dysfunction induced by cavernous nerve injury . Theranostics 2019;9:6354-68. Witwer KW, Buzas EI, Bemis LT, Bora A, Lasser C, Lotvall J, et al. Standardization of sample collection, isolation and analysis methods in extracellular vesicle research . J Extracell Vesicles 2013;2. Keshtkar S, Azarpira N, Ghahremani MH. Mesenchymal stem cell-derived extracellular vesicles: novel frontiers in regenerative medicine . Stem Cell Res Ther 2018;9:63. Yea JH, Yoon YM, Lee JH, Yun CW, Lee SH. Exosomes isolated from melatonin-stimulated mesenchymal stem cells improve kidney function by regulating inflammation and fibrosis in a chronic kidney disease mouse model . J Tissue Eng 2021;12. Herrmann IK, Wood MJA, Fuhrmann G. Extracellular vesicles as a next-generation drug delivery platform . Nat Nanotechnol 2021;16:748-59. Miao Y, Fu C, Yu Z, Yu L, Tang Y, Wei M. Current status and trends in small nucleic acid drug development: Leading the future . Acta Pharm Sin B 2024;14:3802-17. Cui S, Liu Z, Tao B, Fan S, Pu Y, Meng X, et al. miR-145 attenuates cardiac fibrosis through the AKT/GSK-3beta/beta-catenin signaling pathway by directly targeting SOX9 in fibroblasts . J Cell Biochem 2021;122:209-21. Shen W, Wang Y, Wang D, Zhou H, Zhang H, Li L. miR-145-5p attenuates hypertrophic scar via reducing Smad2/Smad3 expression . Biochem Biophys Res Commun 2020;521:1042-48. Ye Y, Li Z, Feng Q, Chen Z, Wu Z, Wang J, et al. Downregulation of microRNA-145 may contribute to liver fibrosis in biliary atresia by targeting ADD3 . Plos One 2017;12:e0180896. Supplementary Files AuthorChecklistFull.pdf N1N2N3M1M2M3.all.miRNA.xls SupplementaryMaterials.docx Cite Share Download PDF Status: Published Journal Publication published 25 Feb, 2025 Read the published version in Stem Cell Research & Therapy → Version 1 posted Reviewers agreed at journal 25 Oct, 2024 Reviewers invited by journal 25 Oct, 2024 Editor assigned by journal 17 Oct, 2024 First submitted to journal 17 Oct, 2024 Editorial decision: Major Revision 16 Oct, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-5246841","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":370499545,"identity":"25c7be59-6acd-4071-9dfe-3377766c710f","order_by":0,"name":"Xiaolin Zhang","email":"","orcid":"","institution":"Shanghai Jiao Tong University School of Medicine Affiliated Renji Hospital Department of Urology","correspondingAuthor":false,"prefix":"","firstName":"Xiaolin","middleName":"","lastName":"Zhang","suffix":""},{"id":370499546,"identity":"70d3ed53-2219-4f23-a40a-850fbd897141","order_by":1,"name":"Mengbo Yang","email":"","orcid":"","institution":"Shanghai Jiao Tong University School of Medicine Affiliated Renji Hospital Department of Urology","correspondingAuthor":false,"prefix":"","firstName":"Mengbo","middleName":"","lastName":"Yang","suffix":""},{"id":370499547,"identity":"ed6fa526-61f4-4fb5-8295-c222cf52d29d","order_by":2,"name":"Xinda Chen","email":"","orcid":"","institution":"Shanghai Jiao Tong University School of Medicine Affiliated Renji Hospital Department of Urology","correspondingAuthor":false,"prefix":"","firstName":"Xinda","middleName":"","lastName":"Chen","suffix":""},{"id":370499548,"identity":"f13e1aef-cbfa-4864-b39a-93e53ec743fe","order_by":3,"name":"Ming Zhang","email":"","orcid":"","institution":"Shanghai Jiao Tong University School of Medicine Affiliated Renji Hospital Department of Urology","correspondingAuthor":false,"prefix":"","firstName":"Ming","middleName":"","lastName":"Zhang","suffix":""},{"id":370499549,"identity":"3ddbf443-aedf-425e-9c81-b3bc15fd5631","order_by":4,"name":"Yiliang Peng","email":"","orcid":"","institution":"Shanghai Jiao Tong University School of Medicine Affiliated Renji Hospital Department of Urology","correspondingAuthor":false,"prefix":"","firstName":"Yiliang","middleName":"","lastName":"Peng","suffix":""},{"id":370499550,"identity":"120e9bd2-bbef-44b1-a68e-fa36f4db4299","order_by":5,"name":"Mujun Lu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAz0lEQVRIiWNgGAWjYBACPhAhwcAgx8BwAMRkJqyFDarFmIHhMClagCCxAaKaGC0S2WkSlm130uc3nj8mwVBhndjAfvYAAS252yQk257lbjhwmE2C4Ux6YgNPXgIxWg7nbmAAamFsO5zYIMFjQJSWdPkGkJZ/JGhJYAA5jLGBGC08bzdbSJw7bAj0i7FFwrF04zaeHPxa+NlzN96WKDssLz/j4MMbH2qsZfvZz+DXAgLMEiBS4gADQwIDIqbwAsYPYPsaiFE7CkbBKBgFIxEAAJd+QMnp4l4UAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0002-5946-2061","institution":"Shanghai Jiao Tong University School of Medicine Affiliated Renji Hospital","correspondingAuthor":true,"prefix":"","firstName":"Mujun","middleName":"","lastName":"Lu","suffix":""}],"badges":[],"createdAt":"2024-10-11 14:33:35","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5246841/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5246841/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s13287-025-04173-0","type":"published","date":"2025-02-25T15:57:45+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":67777583,"identity":"623bbd95-5f80-4d8b-8826-b11dda14a721","added_by":"auto","created_at":"2024-10-29 15:13:28","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":13008757,"visible":true,"origin":"","legend":"\u003cp\u003eIsolation and characterization of MT-hASC-EVs. \u003cstrong\u003eA) \u003c/strong\u003eSchematic diagram for the isolation of hASCs and purification of MT-hASC-EVs. \u003cstrong\u003eB) \u003c/strong\u003eFlow cytometry analysis of the surface markers in ASCs. \u003cstrong\u003eC) \u003c/strong\u003eRT-PCR analysis of the mRNA level of several growth factors in hASCs and melatonin-pretreated hASCs at different time (n=3). Error bars: mean ± SD. **p \u0026lt; 0.01, *p\u0026lt;0.05 comparison with MT- group. \u003cstrong\u003eD)\u003c/strong\u003e Western Blot analysis of exosome-positive markers (CD9, CD81, and TSG101) and exosome-negative marker (Calnexin) of hASC-EVs and MT-hASCs-EVs. Original Western Blot bands are presented in Supplementary Figure 1. \u003cstrong\u003eE-F) \u003c/strong\u003eParticle size distribution of the hASC-EVs and MT-hASC-EVs measured by nanoparticle tracking analysis (n=5). \u003cstrong\u003eG) \u003c/strong\u003eTransmission electron microscopy analysis of hASC-EVs and MT-hASC-EVs (scale bar = 100 nm).\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-5246841/v1/03b0070756e5835a8403d3b4.png"},{"id":67777586,"identity":"87d586c0-6660-4ea9-9314-0f57045500f1","added_by":"auto","created_at":"2024-10-29 15:13:29","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":41778481,"visible":true,"origin":"","legend":"\u003cp\u003eMT-hASC-EVs improved erectile function and cavernous fibrosis in CNI-ED rats. \u003cstrong\u003eA)\u003c/strong\u003e ICP and MAP response to electrical stimulation of the cavernous nerve in Sham, PBS, hASC-EVs and MT-hASC-EVs group (n=6). \u003cstrong\u003eB) \u003c/strong\u003eRelative ratio of ICPmax to MAP in four groups. \u003cstrong\u003eC-D)\u003c/strong\u003e Masson’s trichrome staining of corpus cavernous tissue, and the smooth muscle/collagen ratios of the four groups. \u003cstrong\u003eE-F) \u003c/strong\u003eImmunohistochemical staining and the relative density of Desmin in four groups. \u003cstrong\u003eG)\u003c/strong\u003e Western Blot analysis of the relative protein level of Collagen I, TGF-β2 and p-Smad3 in four groups. Original Western Blot bands are presented in Supplementary Figure 2. \u003cstrong\u003eH)\u003c/strong\u003e RT-PCR analysis of the relative mRNA level of Tgfb2 and Col1a1 in four groups (n=3). Error bars: mean ± SD. *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p\u0026lt;0.001, ****p\u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-5246841/v1/e932a25184ebc3e8b85064b2.png"},{"id":67777585,"identity":"79ca83db-4de9-4114-82ae-2c511e96f598","added_by":"auto","created_at":"2024-10-29 15:13:28","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":10551637,"visible":true,"origin":"","legend":"\u003cp\u003eMT-hASC-EVs could promote the proliferation and reduce the hypoxia-induced apoptosis of CCSMCs. \u003cstrong\u003eA)\u003c/strong\u003e Laser scanning confocal microscopy images of PKH67-labeled hASC-EVs and MT-hASC-EVs taken up by CCSMCs. \u003cstrong\u003eB) \u003c/strong\u003eEffects of hASC-EVs and MT-hASC-EVs on cell viability of CCSMCs at different time points indicated by CCK8 assay (n=3). \u003cstrong\u003eC-D)\u003c/strong\u003e EdU staining of CCSMCs treated with hASC-EVs and MT-hASC-EVs as well as counting of EdU-positive cells.(n=3) \u003cstrong\u003eE-F)\u003c/strong\u003e Flow cytometry analysis of hypoxia-induced apoptosis of CCSMCs as indicated by AnnexinV-PI double staining after treatment with hASC-EVs or MT-hASC-EVs (n=3). Error bars: mean ± SD. *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p\u0026lt;0.001, ****p\u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-5246841/v1/dd7744be95743bdd2c7e9423.png"},{"id":67777580,"identity":"d7026e76-1d21-424c-862e-78b60d4fe70c","added_by":"auto","created_at":"2024-10-29 15:13:28","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":3673519,"visible":true,"origin":"","legend":"\u003cp\u003eDifferences in microRNA expression between MT-hASC-EVs and hASC-EVs indicated by high THROUGHPUT Analysis. \u003cstrong\u003eA)\u003c/strong\u003e Volcano plots showing the differentially expressed microRNAs between MT-hASC-EVs and hASC-EVs. \u003cstrong\u003eB)\u003c/strong\u003e Heatmap showing the differential expression levels of miRNAs between MT-hASC-EVs and hASC-EVs. \u003cstrong\u003eC-D)\u003c/strong\u003eSignificant enrichment of target genes of up-regulated miRNAs in GO analysis (D) and KEGG signaling pathways(E). Number of target genes in each pathway (indicated by circle diameters), and significance of enrichment (indicated by blue/red colors).\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-5246841/v1/2f307e6a6e5b27c0a8418547.png"},{"id":67777871,"identity":"64fb80d6-4abc-4f8a-a881-abe8645ba561","added_by":"auto","created_at":"2024-10-29 15:21:28","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":13118109,"visible":true,"origin":"","legend":"\u003cp\u003eMT-hASC-EVs could inhibit the activation of TGF-β pathway and fibrosis of CCSMCs in vitro. \u003cstrong\u003eA)\u003c/strong\u003eRT-PCR analysis showing relative mRNA levels of Tgfb2, Tgfbr2, Smad3 and Col1a1 of CCSMCs after incubation with hASC-EVs and MT-hASC-EVs.(n=3) \u003cstrong\u003eB, C)\u003c/strong\u003eWestern Blot analysis of protein levels of TGF-β2, TGF-βRII, phosphorylated Smad2 and Smad3, Collagen I/Collagen III (n=3). Original Western Blot bands are presented in Supplementary Figure 3. \u003cstrong\u003eD, E)\u003c/strong\u003e Immunofluorescence analysis of TGF-β2 in CCSMCs incubated with hASC-EVs or MT-hASC-EVs and statistical analysis of fluorescence intensity (n=3). Error bars: mean ± SD. *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p\u0026lt;0.001, ****p\u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-5246841/v1/8455220b70b04d78f8c012a2.png"},{"id":67777577,"identity":"32b44267-29b0-446b-b394-449ad554c15d","added_by":"auto","created_at":"2024-10-29 15:13:28","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":4907776,"visible":true,"origin":"","legend":"\u003cp\u003eMT-hASC-EVs could down-regulate TGF-β2/Smad3 pathway through miR-145-5p. \u003cstrong\u003eA)\u003c/strong\u003e KEGG enrichment analysis of up-regulated miRNAs of MT-hASC-EVs by miRPath v3.0. \u003cstrong\u003eB)\u003c/strong\u003e RT-PCR analysis showing the relative expression levels of miR-145-5p, miR-23a-3p and miR-23b-3p in hASCs after treatment of melatonin (n=3). \u003cstrong\u003eC)\u003c/strong\u003e RT-PCR analysis showing the relative expression levels of miR-145-5p, miR-23a-3p and miR-23b-3p in hASC-EVs and MT-hASC-EVs (n=3). \u003cstrong\u003eD)\u003c/strong\u003e RT-PCR analysis showing the relative expression levels of miR-145-5p in CCSMCs after incubation with hASC-EVs and MT-hASC-EVs (n=3). \u003cstrong\u003eE)\u003c/strong\u003e TargetScan v8.0 predicted binding sites for miR–145–5p within the 3' untranslated region (UTR) of rat Tgfb2 and rat Smad3. \u003cstrong\u003eF)\u003c/strong\u003eLuciferase reporter activities could be inhibited by miR-145-5p mimics in 293T which are transfected with wild type Tgfb2 and Smad3 reporter genes, but not mutant Tgfb2 and Smad3 reporter genes (n=3). \u003cstrong\u003eG)\u003c/strong\u003e Western Blot analysis showing the protein levels of TGF-β2, Smad 3, p-Smad2/3 and Collagen I in CCSMCs after transfection with miR-145-5p mimics and inhibitor (n=3). Original Western Blot bands are presented in Supplementary Figure 4. Error bars: mean ± SD. *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p\u0026lt;0.001, ****p\u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-5246841/v1/a45a47dca2c774b2045bc3e7.png"},{"id":67777582,"identity":"7ffbb05c-6691-4ea0-8250-36f4dfe11c6b","added_by":"auto","created_at":"2024-10-29 15:13:28","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":2819078,"visible":true,"origin":"","legend":"\u003cp\u003eMT-hASCs-EVs can significantly alleviate CNI-ED by inhibiting the occurrence of cavernous fibrosis through miR-145-5p/TGF-β/Smad axis.\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-5246841/v1/2dccf6a07dd36f769382b5c1.png"},{"id":77622593,"identity":"cbcd1614-3c13-40cb-b6b5-32a5760d1b0d","added_by":"auto","created_at":"2025-03-03 16:08:39","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":82869023,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5246841/v1/5395dd91-26e0-44ca-906e-6e243f3a7480.pdf"},{"id":67777579,"identity":"6cb4014c-dfee-481e-b9a2-dd45c23bbf4f","added_by":"auto","created_at":"2024-10-29 15:13:28","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":123161,"visible":true,"origin":"","legend":"","description":"","filename":"AuthorChecklistFull.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5246841/v1/05f2d9512f631e77e8cfa6f3.pdf"},{"id":67777581,"identity":"34c8f4ca-fb46-487a-90a8-ec9e0ac4b2b9","added_by":"auto","created_at":"2024-10-29 15:13:28","extension":"xls","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":105177,"visible":true,"origin":"","legend":"","description":"","filename":"N1N2N3M1M2M3.all.miRNA.xls","url":"https://assets-eu.researchsquare.com/files/rs-5246841/v1/26d566cb39d8241311fe2121.xls"},{"id":67777587,"identity":"0f6ddb6d-f21a-4157-8c80-d314e6a36a31","added_by":"auto","created_at":"2024-10-29 15:13:29","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":2780995,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryMaterials.docx","url":"https://assets-eu.researchsquare.com/files/rs-5246841/v1/c7702b1c0e5c4fcb86484b56.docx"}],"financialInterests":"","formattedTitle":"Melatonin-Pretreated Mesenchymal Stem Cell-Derived Exosomes Alleviate Cavernous Fibrosis in a Rat Model of Nerve Injury-induced Erectile Dysfunction via miR-145-5p/TGF-β/Smad Axis","fulltext":[{"header":"1 Introduction","content":"\u003cp\u003eErectile dysfunction (ED) refers to the persistent or recurrent inability to achieve and maintain sufficient penile erection for satisfactory sexual intercourse[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. The etiology of ED is complex, and cavernous nerve injury (CNI) is a significant contributing factor, commonly associated with pelvic surgery, pelvic fractures, and post-urethral injury surgeries[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. CNI-induced ED following radical prostatectomy has an incidence rate as high as 63%-94%, despite the use of nerve-sparing techniques[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. During radical prostatectomy, unavoidable traction, compression, and vascular damage to the cavernous nerves lead to impairment and neurotrophic loss which results in reduced penile blood flow perfusion, leading to sustained hypoxia in the corpus cavernosum[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Subsequent smooth muscle atrophy occurs along with activation of TGF-β/Smad and RhoA/Rock pathways which lead to cavernous fibrosis[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Traditional phosphodiesterase 5 inhibitors (PDE5i, e.g., tadalafil and sildenafil) have limited efficacy in treating CNI- ED due to insensitivity of corpora cavernosa tissue to NO resulting from smooth muscle cell loss and cavernous fibrosis[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. It is worth noting that while regeneration of cavernous nerves could be observed 28 days after CNI in a rat model, subsequent loss of smooth muscle cells and cavernous fibrosis were irreversible[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Therefore, prevention, alleviation, or even reversal of cavernous fibrosis represents an important therapeutic direction for managing CNI-ED.\u003c/p\u003e \u003cp\u003eIn recent years, stem cells and their derivatives have become new treatment options for ED[\u003cspan additionalcitationids=\"CR8\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], among which extracellular vesicles (EVs) have gained favor among researchers due to their simplicity, convenience, and suitability for industrial production[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. EVs are sac-like structures that cells release into the extracellular space[\u003cspan additionalcitationids=\"CR12\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Based on diameter and source, they can be divided into exosomes (50\u0026ndash;100 nm) and microvesicles (100\u0026ndash;1000 nm). As a carrier, EVs transport signal proteins, lipids, and nucleic acids, which can be endocytosed by distant target cells, thereby changing the biological behavior of the target cells and serving as a key factor in cell-to-cell signal communication[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Many reports have suggested that mesenchymal stem cell-derived EVs have a certain therapeutic effect on CNI-ED[\u003cspan additionalcitationids=\"CR16 CR17\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eMelatonin is a hormone secreted by the pineal gland, primarily responsible for regulating biological rhythms and sleep-wake cycles[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. In addition, melatonin has been found to have therapeutic effects on several fibrotic diseases including liver fibrosis[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], pulmonary fibrosis[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], and renal fibrosis[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], indicating its potential in counteracting cavernous fibrosis caused by CNI-ED. Furthermore, melatonin exhibits the ability to promote nerve regeneration[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], thereby improving erectile function through the protection and promotion of repair of cavernous nerves[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Melatonin can regulate the behavior of MSCs and significantly enhance the therapeutic effects of MSCs-derived EVs in certain diseases. For example, melatonin-pretreated MSCs-derived EVs shows better protective effects against rat renal ischemia-reperfusion injury[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]; they also show stronger therapeutic effects in diabetic wound healing[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. However, it is still unclear whether melatonin-pretreated MSCs-derived EVs can play a therapeutic role in fibrotic diseases.\u003c/p\u003e \u003cp\u003eIn this study, we found that EVs derived from melatonin-pretreated adipose mesenchymal stem cells (MT-hASC-EVs) exhibit high expression of miR-145-5p through which they can target the TGF-β/Smad axis, inhibit corpus cavernosum fibrosis in the CNI-ED rat model, and subsequently promote the recovery of erectile function. This may represent a potential therapeutic approach for CNI-ED.\u003c/p\u003e"},{"header":"2 Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n \u003ch2\u003e2.1 Cell isolation, culture and identification\u003c/h2\u003e\n \u003cp\u003eHuman adipose stem cells (hASCs) were isolated from abdominal adipose tissues from healthy female liposuction as previously reported[\u003cspan class=\"CitationRef\"\u003e28\u003c/span\u003e]. hASCs were resuspended in Minimum Essential Medium Alpha (\u0026alpha;-MEM, Gibco) supplemented with 10% fetal bovine serum (New Zealand Characterized Fetal Bovine Serum, Hyclone, SH30406.05) and 1% penicillin-streptomycin (Beyotime, CNH) and cultured at 37 \u0026deg; C in a 5% CO\u003csub\u003e2\u003c/sub\u003e incubator. The medium was changed every 3 days. Cells were passaged when they reached 80% confluence. All cells used in the experiments were at passages 3 to 5. The differentiation properties and stem cell markers of hASCs have been identified in our previous reports[\u003cspan class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e\n \u003cp\u003eThe isolation of corpus cavernous smooth muscle cells (CCSMCs) was conducted as described previously[\u003cspan class=\"CitationRef\"\u003e28\u003c/span\u003e]. In brief, after anesthesia, the rats were sterilized, and the foreskin and dorsal penile vessels were removed to obtain penile corpus cavernosum tissue. Cavernosal tissue was washed in PBS and cut into small pieces of 1 to 2 mm. Segments were placed on 10-cm cell culture dishes (Corning, USA) containing a minimal volume of DMEM supplemented with 20% FBS and cultured at 37 \u0026deg; C in a humidified atmosphere of 95% air and 5% CO2. After the explants were attached, more DMEM containing 10% FBS was added and tissue segments that had fallen off from the culture dish were removed. The cells were cultured in high-glucose DMEM (Gibco), supplemented with 10% FBS (Hyclone, SH30406.05), 1% penicillin-streptomycin (Beyotime Biotechnology) at 37\u0026deg;C and 5% CO2. The cells were frozen or passaged once 80\u0026ndash;90% confluence was achieved. All cells used in the experiments were at passages 3 to 8.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\n \u003ch2\u003e2.2 Isolation and characterization of extracellular vesicles.\u003c/h2\u003e\n \u003cp\u003eAn EV-free FBS was prepared by ultracentrifugation at 100 000 \u0026times; g for 2 h at 4\u0026deg;C and filtered with a 0.22 \u0026micro;m filter. When the ADSC at passage 3 to 5 reached an 80% density, cells were washed with PBS and cultured in culture medium supplemented with EV-free FBS for 48 h, while MT-hASCs were incubated with 10\u0026micro;M melatonin. The media were then collected, and EVs were isolated through a multistep centrifugation. Dead cells, cell debris and microvesicles were removed at 300g for 5min, 3000g for 10min and 10000g for 30min, respectively. The supernatant was then ultracentrifuged at 100 000 \u0026times; g for 2h (XP-90, Beckman Coulter, USA). The pellets were washed for three times and resuspended with PBS to obtain a suspension of hASC-EVs or MT-hASC-EVs. The total protein concentration of the sEVs was quantified using a micro bicinchoninic acid protein assay kit (Beyotime, CHN).\u003c/p\u003e\n \u003cp\u003eFor EV characterization, the EV-positive markers CD81 (Abclonal, A5270, 1:1000), CD9 (Abclonal, A1703, 1:1000), and TSG101 (Abclonal, A2216, 1:1000), and the EV-negative marker, Calnexin (Abclonal, A15631, 1:1000), were identified by western blotting analysis. The size distribution of the EVs was determined using the NanoSight NS300 (Malvern, UK), according to the manufacturer\u0026rsquo;s instructions. The ultrastructure and morphology of the EVs prepared by the Exosome-TEM-easy kit (101Bio, USA) were observed using transmission electron microscopy (TEM; Hitachi, JP).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\n \u003ch2\u003e2.3 In vivo experimental design\u003c/h2\u003e\n \u003cp\u003eGenerally, forty 8-week-old Sprague Dawley (SD) rats (males, weighing 250 g each) were used in this study. All rats were maintained on a 12h light/12h dark cycle and were acclimatized for at least 1 week before surgery and allowed free access to standard food and water. Operations and welfare in this study complied with international and Chinese local legislations and National Institutes of Health guide for the care and was guaranteed under the supervision of the Experimental Animal Ethical Committee of Ren Ji Hospital (KY2022-180-B).\u003c/p\u003e\n \u003cp\u003eBilateral CNI was performed in 30 rats (CNI group), and the other 10 rats were subjected to only laparotomy (Sham group). The construction of the CNI-ED animal model was conducted as previously described[\u003cspan class=\"CitationRef\"\u003e29\u003c/span\u003e]. After anesthesia by intraperitoneal injection of pentobarbital sodium (35 mg/kg), the rats were placed on an isothermal thermal pad. Hair above the abdomen and perineum was shaved with a hair clipper for better visualization. After disinfection, a 2.5-cm midline lower abdominal incision was made to expose the pelvic ganglions (MPG) and cavernous nerves (CNs) on the surface of both sides of prostate. The CNs were isolated bilaterally and crushed 5 mm distal to the MPG of 90s using micro-forceps (Storz, Germany). Then, the CNI group was randomly divided into three groups of 10 rats each, which received intracavernous injection of 1) PBS (0.1mL); 2) hASCs-EVs (100\u0026micro;g in PBS 0.1 ml); 3) MT-hASC-EVs (100\u0026micro;g in PBS 0.1 ml). The intracavernous injection method was performed as previously described[\u003cspan class=\"CitationRef\"\u003e8\u003c/span\u003e]. In brief, the penis was exposed locally and a rubber tourniquet was applied at the base. After injection of 0.1 ml solution into the corpus cavernosum, the tourniquet was removed after 1 min and the penis was restored.\u003c/p\u003e\n \u003cp\u003eThe work has been reported in line with the ARRIVE guidelines 2.0.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\n \u003ch2\u003e2.4 Erectile function evaluation.\u003c/h2\u003e\n \u003cp\u003eThe maximal intracavernous pressure (ICP) and realtime arterial pressure were recorded as previously described[\u003cspan class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e29\u003c/span\u003e]. 4 weeks after intracavernous injection, SD rats were anesthetized by intraperitoneal injection of pentobarbital sodium (35 mg/kg). A 26-gauge needle connected to a catheter inserted into one side of corpus cavernosum to measure the intracavernous pressure (ICP) while the other end of the catheter was connected to a data collection device (BL-420s, Chengdu Taimeng Software Co.Ltd., China) using a pressure transducer. After exposing the carotid artery of the other side, a 20-gauge cannula filled with heparin saline was punctured in the artery to measure the mean artery pressure (MAP), with the other end of the cannula connected to the BL-420s using a pressure transducer. The CN were dissected and separated in the same way described above, and the CN was stimulated with electrodes with stimulus parameters set at 5 V, 25 Hz and 60 s duration. At the end, the penis was excised for further testing, and then the rats were euthanized with carbon dioxide.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\n \u003ch2\u003e2.5 Histological and immunohistochemical analysis\u003c/h2\u003e\n \u003cp\u003eFor Masson\u0026apos;s trichrome staining, the penile tissues were fixed in 4% paraformaldehyde overnight, which were subsequently dehydrated and embedded in paraffin. Next, the paraffin-embedded tissue was cut into 4-micron sections for staining. After gradient dehydration with xylene, the tissues were stained with a Masson trichrome staining kit (Masson trichrome Staining Kit, Solarbio, USA). Stained sections were observed under microscope and analyzed using Image J software.\u003c/p\u003e\n \u003cp\u003eFor immunohistochemical staining, penile tissue sections were rehydrated, and antigen retrieval was performed. The sections were blocked with goat serum for 60 min, then incubated with anti-Desmin antibodies (Proteintech, 16520-1-AP, 1:500) overnight. Then, MaxVision HRP-Polymer immunohistochemistry kit (Maxim, China) was used and the sections weredeveloped in color with diaminobenzidine (DAB). Sections were then counterstained with hematoxylin. Stained sections were observed under microscope and analyzed using Image J software.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\n \u003ch2\u003e2.6 Fluorescent labeling and in vitro tracing of EVs.\u003c/h2\u003e\n \u003cp\u003ehASC-EVs and MT-hASC-EVs were labeled with PKH67 dye (Maokang Biotechnology, China) according to the manufacturer\u0026apos;s instructions. CCSMCs were incubated with PKH67-labeled EVs for 12h at 37℃. Following fixed with 4% paraformaldehyde and stained with 40, 6-diamidino-2-phenylindole (DAPI, Invitrogen, USA), the cells were observed under the confocal laser scanning microscope (Olympus, Japan).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\n \u003ch2\u003e2.7 CCSMC viability and proliferation.\u003c/h2\u003e\n \u003cp\u003eFor cell viability evaluation, CCSMCs were seeded in a 96-well plate with a density of 2000 cells per well and incubated with hASC-EVs or MT-hASC-EVs. 24h or 48h later, Cell Counting Kit-8 (Beyotime, China) was added to the medium and incubated at 37℃ for 2h. Cell viability was assessed by OD value of each well using a microplate reader (Biotek, USA).\u003c/p\u003e\n \u003cp\u003eFor EdU cell proliferation staining, CCSMCs were seeded in a six-well plate and pre-treated with hASC-EVs or MT-hASC-EVs for 24h. Then, EdU cell proliferation staining was performed using an EdU kit (BeyoClick\u0026trade; EdU Cell Proliferation Kit with Alexa Fluor 488, Beyotime, China) according to the manufacturer\u0026apos;s instructions and nuclei were stained using Hoechst33342 (Beyotime, China). The fluorescence was detected using the fluorescence microscope (Olympus, Japan) and the cells in the proliferative phase were counted using Image J software.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\n \u003ch2\u003e2.8 Cell apoptosis assay\u003c/h2\u003e\n \u003cp\u003eAnnexin V-FITC/PI cell apoptosis detection kit (Yeasen Biology, China) was used to detect the level of apoptosis under different conditions. CCSMCs seeded in a six-well plate (costar, United States) were pre-treated with hASC-EVs or MT-hASC-EVs for 24 h and then stimulated under hypoxia for 24 h. Subsequently, the cells were collected and stained with Annexin V-FITC and PI probe solution at room temperature for 15 min. The apoptosis rate was detected by Cytoflex (Beckman Coulter, United States).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n \u003ch2\u003e2.9 The miRNA Library Construction and Sequencing.\u003c/h2\u003e\n \u003cp\u003eTotal RNA of hASC-EVs and MT-hASC-EVs was extracted by the MagZol (Magen, China) according to the manufacturer\u0026rsquo;s protocol. The quantity and integrity of RNA yield was assessed by using the Qubit\u0026reg;2.0 (Invitvogen, USA) and Agilent 2200 TapeStation (Agilent Technologies, USA) separately. Briefly, RNAs were ligated with 3\u0026rsquo; RNA adapter, and followed by 5\u0026rsquo; adapter ligation. Subsequently, the adapter-ligated RNAs were subjected to RT-PCR and amplified with a low-cycle. Then the PCR products were size selected by PAGE gel according to instructions of NEBNext\u0026reg; Multiplex Small RNA Library Prep Set for Illumina\u0026reg; (Illumina, USA). The purified library products were evaluated using the Agilent 2200 TapeStation, The libraries were sequenced by HiSeq 2500(Illumina, USA) with single-end 50bp at Ribobio Co. Ltd (Ribobio, China).\u003c/p\u003e\n \u003cp\u003eThe raw reads were processed by filtering out containing adapter, poly \u0026rsquo; N\u0026rsquo;, low quality, smaller than 17nt reads by FASTQC to get clean reads. Mapping reads were obtained by mapping clean reads to reference genome of by BWA. miRDeep2 was used to identify known mature miRNA based on miRBase21 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ewww.miRBase.org\u003c/span\u003e\u003c/span\u003e) and predict novel miRNA. The expression levels were normalized by RPM, RPM is equal to (number of reads mapping to miRNA/number of reads in Clean data)\u0026times;10\u003csup\u003e6\u003c/sup\u003e. Differential expression between two sets of samples was calculated by edgeR algorithm according to the criteria of |log2(Fold Change)|\u0026ge;1 and p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05. TargetScan, miRDB, miRTarBase and miRWalk were used to predict targets gene of selected miRNA. R 4.3.1 and miRPath v.3 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://dianalab.e-ce.uth.gr/html/mirpathv3/index.php?r=mirpath\u003c/span\u003e\u003c/span\u003e)[\u003cspan class=\"CitationRef\"\u003e30\u003c/span\u003e] were used for further Gene Ontology (GO) and KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analysis.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\n \u003ch2\u003e2.10 Immunocytochemistry (ICC)\u003c/h2\u003e\n \u003cp\u003eCCSMCs were seeded in 35mm confocal dishes (Bioshark, China) and induced using 10ng/mL recombinant human TGF-\u0026beta;2 (Proeintech, HZ-1092, USA), while hASC-EVs or MT-hASC-EVs were co-incubated. After 48h, CCSMCs were fixed with 4% paraformaldehyde (PFA) and permeabilized with 0.1% Triton X-100 (Sigma, USA). Blocking was performed with goat serum for 1h, followed by incubation with TGF-\u0026beta;2 specific antibody (Ptoteintech, 19999-1-AP, 1:100) overnight. Then, the cells were incubated with green fluorescent secondary antibody (Proteintech, RGAR002, 1:200) for 1h at room temperature. Nuclei were stained using DAPI (Invitrogen, USA). CCSMCs were observed under the confocal laser scanning microscope (Olympus, Japan) and the fluorescent intensity was valued by Image J software.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\n \u003ch2\u003e2.11 Cell Transfection.\u003c/h2\u003e\n \u003cp\u003eCell transfection was performed using Lipofectamine 3000 (Invitrogen, USA), according to the manufacturer\u0026rsquo;s protocol. The miR-145-5p inhibitor and NC inhibitor, miR-145-5p mimics and NC mimics were obtained from Genomeditech (Shanghai, China), and their sequences were shown in \u003cstrong\u003eSupplementary Table\u0026nbsp;2\u003c/strong\u003e.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\n \u003ch2\u003e2.12 Dual-luciferase reporter assay.\u003c/h2\u003e\n \u003cp\u003eThe entire 3ʹ-UTR fragments of Tgfb2 and Smad3 and the mutant form in which the potential miR-145-5p binding sites were mutated were inserted into PGL3-CMV-LUC vector, namely Rat_Tgfb2 WT, Rat_Tgfb2 mut, Rat_Smad3 WT and Rat_Smad3 mut, respectively. Plasmid profiles as well as sequences are shown in \u003cstrong\u003eSupplementary Fig.\u0026nbsp;5\u003c/strong\u003e and \u003cstrong\u003eSupplementary Table\u0026nbsp;5\u003c/strong\u003e. These plasmids were respectively, co-transfected with miR-145-5p mimics or mimics NC into HEK293 cells. After 48 hours, cells were collected and luciferase activity was measured using Dual-Luciferase Reporter Assay System (E1910, Promega, Madison, WI, USA).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\n \u003ch2\u003e2.13 Real-Time PCR.\u003c/h2\u003e\n \u003cp\u003eThe EZ-press RNA Purification Kit (EZB, USA) was used to extract mRNA from the ASCs and CCSMCs. Exosome RNA Purification Kit (EZB, USA) was used to extract total RNA from EVs. A reverse transcription kit (TaKaRa, Japan) was used to synthesize complementary DNA from mRNA. An miRNA 1st Strand cDNA Synthesis Kit (by stem-loop) (Vazyme, China) was used to synthesize complementary DNA from microRNA. The ChamQ Universal SYBR qPCR Master Mix (Vazyme, China) was used to and perform quantitative real-time PCR, according to the manufacturer\u0026rsquo;s instructions. ACTB and U6 were used as internal controls. The LightCycler 480 real-time PCR system (Roche Diagnostics, Indianapolis, IN, USA) was used for detection. The primers used are listed in \u003cstrong\u003eSupplementary Table\u0026nbsp;1\u003c/strong\u003e.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\n \u003ch2\u003e2.14 Western Blots.\u003c/h2\u003e\n \u003cp\u003eProteins were extracted using RIPA Lysis Buffer (Beyotime, China) according to the manufacturer\u0026rsquo;s instructions. The protein concentration was detected using a Bicinchoninic Acid Protein Assay Kit (Beyotime, China). The Protein solution and SDS-PAGE Protein Sample Loading Buffer were mixed and denatured by heating at 95 \u0026deg; C. 10 \u0026micro;g of proteins were used for electrophoresis per lane. After electrophoresis, the proteins were transferred onto polyvinylidene difluoride membranes. The membranes were blocked with Tris-buffered saline-Tween (with 5% skim milk) and incubated at 4\u0026deg;C overnight with primary antibodies against \u0026beta;-actin (Proeintech, 66009-1-Ig, 1:20000), TGF-\u0026beta;2(Proteinteech, 19999-1-AP, 1:1000), TGF-\u0026beta;RII (Proteintech, 66636-1-Ig, 1:5000), Smad3 (Abclonal, A19115, 1:20000), p-Smad3 (Cell Signaling Technology, #9520, 1:1000), Smad2/Smad3 (Cell Signaling Technology, #3102, 1:1000), p-Smad2/p-Smad3 (Abclonal, AP1343, 1:1000), COL1A1 (Abclonal, A1352, 1:1000), COL3A1 (Abclonal, A3795, 1:2000). After hybridization with the secondary antibody (Proteinteh, 1:10000), bands were observed with enhanced chemiluminescence substrates (Millipore, MA) under a chemiluminescence imaging system (Bio-Rad, USA). The grayscale values of the bands were calculated using Image Lab software.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\n \u003ch2\u003e2.15 Statistical analysis.\u003c/h2\u003e\n \u003cp\u003eAll quantitative data were expressed as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation. Differences between the groups were assessed by oneway analysis of variance (ANOVA), followed by a Student\u0026rsquo;s t-test using GraphPad Prism v9.0 software. p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"3 Results","content":"\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Isolation and characterization of melatonin-pretreated adipose mesenchymal stem cell-derived EVs.\u003c/h2\u003e \u003cp\u003ePrimary adipose mesenchymal stem cells were isolated from human adipose tissue. After 48 hours of treatment with 10\u0026micro;M Melatonin, the conditioned medium was collected, from which MT-hASC-EVs were isolated (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Both hASCs and melatonin-pretreated hASCs showed typical spindle and fibroblast-like morphologies (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). The levels of growth factors secreted by hASCs were detected by RT-PCR (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). After Melatonin treatment, the expression levels of various growth factors in hASCs increased, among which the expression level of SHH (Sonic Hedgehog) increased most significantly as early as 2\u0026ndash;4 h of Melatonin treatment. In addition, the expression levels of PDGF, SDF and HGF were also significantly increased after melatonin pretreatment.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003ehASC-EVs and MT-hASC-EVs were isolated from the conditioned medium of hASCs and melatonin-treated hASCs respectively. Western Blot revealed that both hASC-EVs and MT-hASC-EVs both highly expressed CD81, CD9, and the endosomal marker TSG101, but rarely expressed the endoplasmic reticulum marker Calnexin (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). Nanoparticle Trafficking Analysis (NTA) detection showed that the particle sizes of hASC-EVs and MT-hASC-EVs were mostly distributed in the range of 50-200nm, with a main peak at about 110nm (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). There was also no difference in mean particle size (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF). Both hASC-EVs and MT-hASC-EVs had a typical cup-like shape under transmission electron microscope (TEM) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG). These results proved that EVs from hASCs and MT-hASCs were successfully isolated and characterized, while the pretreatment of melatonin had no significant effect on the surface markers, morphology, and particle size distribution of hASC-EVs.\u003c/p\u003e \u003cp\u003e \u003cb\u003e3.2 Intracavernous injection of MT-hASC-EVs could restore erectile function and reduce cavernous fibrosis in a rat model of CNI-ED.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eIn order to evaluate the therapeutic effects of MT-hASC-EVs on CNI-ED, we utilized a rat model of CNI-ED established through bilateral cavernous nerve crush, following which both types of EVs were administered via intracavernous injection. At 4 weeks post-treatment, the erectile function was assessed by measuring intracavernous pressure (ICP) and mean arterial pressure (MAP) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). There were no significant differences in MAP among all groups. The successful establishment of CNI-ED model was confirmed by a significant decrease of ICP/MAP ratio in the PBS group compared with the sham group. Intracavernous injection of hASC-EVs or MT-hASC-EVs could restore the ICP/MAP ratio to a certain extent. Notably, the therapeutic effects of MT-hASC-EVs were more pronounced compared to that of hASC-EVs.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eCavernous fibrosis is one of the most severe pathological changes of CNI-ED, making it a major factor contributing to the refractory of the disease, so we focused on the degree of cavernous fibrosis among all groups. The Masson staining results showed t obvious collagen fiber accumulation and a significant decrease in the ratio of muscle fibers to collagen fibers in PBS group. hASC-EVs and MT-hASC-EVs could alleviate such phenomenon to some extent, with the therapeutic effect of MT-hASC-EVs being more obvious, suggesting the stronger anti-fibrotic effects of MT-hASC-EVs (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). Immunohistochemistry was used to evaluate the content of cavernous smooth muscle, and the results were consistent with the Masson staining results, with significant loss of Desmin\u0026thinsp;+\u0026thinsp;muscle fiber in PBS group and the highest smooth muscle level in MT-hASC-EV group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF). Chronic hypoxia caused by long-term low perfusion after CNI can activate the TGF-β/Smad pathway and induce cavernous nerve fibrosis. Therefore, RT-PCR and Western Blot were used to reveal that MT-hASC-EVs could most significantly reduce the mRNA and protein level of TGF-β2 and Smad3 in cavernous nerve tissue after CNI, thereby reducing the expression of Collagen I (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eH). These results suggest that MT-hASC-EVs have good effects in relieving CNI-induced cavernous fibrosis by downregulating the TGF-β/Smad pathway.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e3.3 MT-hASC-EVs could promote the proliferation of CCSMCs and reduce cell apoptosis in vitro.\u003c/h2\u003e \u003cp\u003eAt the in vivo level, MT-hASC-EVs significantly increased the content of cavernous smooth muscle. To elucidate the mechanism, primary corpus cavernous smooth muscle cells (CCSMCs) were isolated from rat corpus cavernosum for the in vitro experiments. Both PKH67-labeled hASC-EVs and MT-hASC-EVs added to the culture supernatant were significantly up-taken by CCSMCs after 12 hours (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). After incubation for 24 hours, both MT-hASC-EVs and hASC-EVs significantly enhanced the cell viability of CCSMCs, with a stronger effect observed for MT-hASC-EVs compared to hASC-EVs. The effect became more pronounced after 48 hours (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). When EdU was added to the culture medium, a higher number of EdU-positive cells was detected in CCSMCs after incubation with MT-hASC-EVs (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD), indicating that MT-hASC-EV treatment promoted CCSMC proliferation. Furthermore, given that low oxygen-induced ROS damage is a key factor in CNI-ED progression, we stimulated CCSMCs with low oxygen and measured apoptosis rates under different treatment conditions. After hypoxia treatment, the apoptosis rate of CCSMCs significantly increased, while both MT-ASC-EVs and ASC-EVs had a certain inhibitory effect on CCSMC apoptosis, with the former being more significant (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE,\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF). Taking all above account, CCSMCs can enhance cell proliferation after uptaking MT-ASC-EVs, while reducing cell apoptosis induced by hypoxia.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e\u003cb\u003e3.4 High-throughput sequencing revealed that the microRNAs in MT-hASC-EVs can target the TGF-β/Smad axis\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eEVs contain a large number of microRNAs, as one of the most important effectors in EVs. EVs can target specific mRNAs by delivering microRNAs to target cells and regulate protein expression specifically, thereby achieving the regulation of cell functions. Therefore, microRNAs specifically enriched in MT-hASC-EVs were screened by high-throughput sequencing. Among the co-expressed microRNAs, we selected those with a |Log2(FoldChange)|\u0026gt;1 and p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 as the screening conditions to screen for differentially expressed microRNAs, ultimately obtaining 12 upregulated microRNAs and 8 downregulated microRNAs, which were displayed in the volcano plot (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA) and heatmap (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003emicroRNAs specifically target mRNAs to exert negative regulation, so we focused on the functional enrichment of target genes of 12 up-regulated microRNA. Using the prediction results from miRTarBase or the common prediction results from miRDB, miRWalk, and TargetScan as candidate target genes for the 12 upregulated microRNAs, a total of 4,928 target genes were identified(\u003cb\u003eSupplementary Table\u0026nbsp;3\u003c/b\u003e). Gene Ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis were performed on these target genes. In the GO enrichment analysis, several enriched terms were related to \"decreased oxygen levels\", suggesting significant regulatory roles in cellular responses to hypoxia/anoxia of these microRNAs, and it is worth noting the term \"response to transforming growth factor beta\" was also enriched (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). Consistent with that, these target genes were enriched in the \"TGF-beta signaling pathway\" in KEGG enrichment analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). In summary, the microRNA sequencing and target gene enrichment analysis suggest that the microRNAs enriched in MT-ASC-EVs may have played a therapeutic role in the treatment of CNI-ED by targeting the TGF-beta pathway, which is consistent with the observation of downregulation of TGF-beta pathway-related proteins in the in vivo experiments.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003e3.5 MT-hASC-EVs could reduce the fibrosis of CCSMCs in vitro by inhibiting the TGF-β pathway.\u003c/h2\u003e \u003cp\u003eThe results of microRNA sequencing and bioinformatics analysis were validated in vitro. CCSMCs were treated with TGF-β (10ng/mL) for 48h to induce fibrosis, and hASC-EVs or MT-ASC-EVs were simultaneously co-incubated. RT-PCR detection indicated that the mRNA level of Tgfb2 was significantly decreased after MT-ASC-EVs treatment, while the effect of ASC-EVs was not obvious (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). However, both of them could downregulate the mRNA level of Col1a1 and this may indicate both of the EVs contain microRNAs targeting the mRNA of Col1a1, rather than simply downregulating the expression of Col1a1 through the TGF-β pathway. Western Blot detection revealed that the levels of TGF-β2 and Smad2/3 in CCSMCs were significantly downregulated after MT-ASC-EVs treatment compared with the TGF-β2 group and hASC-EVs group. As for the evaluation index of fibrosis, CollagenI/CollagenIII, MT-hASC-EVs also showed more excellent anti-CCSMCs fibrosis effects (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB,\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). In the ICC experiment, lower TGF-β2 fluorescence signals were detected in CCSMCs after MT-hASC-EVs treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD,\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE). Combining the above results, after MT-hASC-EVs treatment, the activation level of the TGF-β pathway in CCSMCs was significantly downregulated, which may be an important mechanism for the anti-fibrosis effects of MT-hASC-EVs.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec23\" class=\"Section2\"\u003e \u003ch2\u003e3.6 MT-hASC-EVs inhibit TGF-β2 and Smad3 expression through miR-145-5p.\u003c/h2\u003e \u003cp\u003eTo further identify the core microRNAs that play a pivotal role among the differentially expressed miRNAs identified in the microRNA sequencing analysis, KEGG enrichment analysis was conducted on the upregulated miRNAs using the miRPath v3.0 software (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). The results indicated that, among all upregulated miRNAs, miR-145-5p (targeting 12 genes), as well as miR-23b-3p and miR-23a-3p (both targeting 15 genes), were significantly enriched in the TGF-β signaling pathway (\u003cb\u003eSupplementary Table\u0026nbsp;4\u003c/b\u003e). To validate these results, RT-PCR was first performed to assess the expression of miR-145-5p, miR-23a-3p, and miR-23b-3p in hASCs. Following a 48-hour treatment with 10 \u0026micro;M melatoni, we observed a significant upregulation of these three miRNAs in hASCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). Similarly, MT-hASC- EVs exhibited significantly higher expression levels of these three miRNAs compared to hASC-EVs without melatonin pretreatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). Given that the upregulation of miR-145-5p was particularly pronounced, we chose to focus our subsequent validation efforts on this specific microRNA. After treating CCSMCs with both types of EVs, RT-PCR analysis revealed that MT-hASC-EVs significantly increased the expression level of miR-145\u0026ndash;5p (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTargetScan version 8.0 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.targetscan.org/vert_80/\u003c/span\u003e\u003cspan address=\"https://www.targetscan.org/vert_80/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) predicted binding sites for miR\u0026ndash;145\u0026ndash;5p within the 3' untranslated region (UTR) of rat Tgfb2 at a 7mer-A1 site and within rat Smad3 at an 8mer site which are illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE. Dual luciferase reporter assays were performed to determine whether Tgfb2 and Smad3 serve as direct targets for regulation by miR\u0026ndash;145\u0026ndash;5p. The results demonstrated that co-transfection with a mimic for mir\u0026ndash;145\u0026ndash;5p led to a significant reduction in luciferase activity from both Tgfb2 WT and Smad3 WT constructs, while no significant changes were observed for Tgfb2 mut or Smad3 mut constructs ( Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF), which confirmed the specific targeting. Furthermore, transfection with miR\u0026thinsp;\u0026minus;\u0026thinsp;145\u0026thinsp;\u0026minus;\u0026thinsp;5p mimics into CCSMCs resulted in marked inhibition of TGF\u0026thinsp;\u0026minus;\u0026thinsp;β2-induced activation within TGF\u0026thinsp;\u0026minus;\u0026thinsp;β signaling pathways along with reduced collagen I accumulation; conversely, transfection with miR\u0026thinsp;\u0026minus;\u0026thinsp;145\u0026thinsp;\u0026minus;\u0026thinsp;5p inhibitor blocked this effect significantly (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eG). In summary, our analyses indicate that MT-hASC-EVs facilitate fibrosis alleviation by delivering miR\u0026thinsp;\u0026minus;\u0026thinsp;145\u0026thinsp;\u0026minus;\u0026thinsp;5p targeting Tgfb2and Smad3 expressions thereby inhibiting TGF-β signaling pathways within CCSMCs.\u003c/p\u003e \u003c/div\u003e"},{"header":"4 Discussion","content":"\u003cp\u003eCorpus cavernosum nerve injury is one of the important causes of erectile dysfunction, commonly seen after pelvic surgery especially radical prostatectomy, with a postoperative occurrence rate of ED as high as 63%-94% in two years[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Currently, perioperative penile rehabilitation techniques have been adopted in clinical practice, including vacuum negative pressure suction, intracavonous injection of vasoactive agent and oral phosphodiesterase 5 inhibitors, to restore erectile function in CNI-ED patients[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. However, about 50%-75% of patients still cannot recover to their preoperative erectile function status, seriously affecting the postoperative quality of life[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. The refractoriness of CNI-ED is closely related to its pathogenesis and development mechanism, especially severe corpus cavernosum fibrosis after CNI-ED surgery[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Chronic hypoxia caused by long-term low blood flow perfusion in the penis after CNI can lead to functional impairment of corpus cavernosum smooth muscle cells through ROS accumulation[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e], resulting in severely reduced responsiveness of the corpus cavernosum to nitric oxide (NO)[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]; on the other hand, hypoxia can also activate TGF-β/Smad[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e] and RhoA/Rock[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e] pathways leading to corporal fibrosis and impaired blood capacity and compliance. Therefore, the response effect of traditional PDE5i drugs on CNI-ED is often unsatisfactory. Thus it is urgent to study new methods for effectively targeting cavernous fibrosis in order to achieve better therapeutic effects for CNI-ED.\u003c/p\u003e \u003cp\u003eIn recent years, many animal and clinical studies have confirmed that mesenchymal stem cells can promote recovery of erectile function in CNI-ED[\u003cspan additionalcitationids=\"CR8\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. However clinically stem cell therapy for ED is still confronted with constraints such as cumbersome processes for storage, culture and proliferation; ethical issues; risk of immune rejection; tumorigenicity; pathogen contamination etc[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Besides, a recent study revealed that MSCs could be maintained within the cavernous tissue for only 3 days in CNI-ED rats with no obvious evidence for endothelial and smooth muscle differentiation, indicating that MSC differentiation does not play a major role in treatment efficacy [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. The paracrine function, especially extracellular vesicles (EVs), is likely to be one of the main mechanisms for the therapeutic effects of MSCs. MSC-derived EVs exert therapeutic effects while avoiding risks associated with stem cell therapy, making them suitable for industrial preparation, thus having good prospects for clinical translation[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Therefore, in this study, we also selected EVs isolated and enriched from the conditioned medium of hASCs for intracavernous injection, in order to achieve better treatment efficiency.\u003c/p\u003e \u003cp\u003eMelatonin (MT) is an amine hormone mainly synthesized and secreted by the pineal gland, which has been proved to alleviate CNI-ED by promoting the repair of cavernous nerve[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. However, it should be noted that the cavernous nerve can spontaneously repair after CNI, while downstream cavernous fibrosis is difficult to reverse[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Therefore, neuroprotection and regeneration may not be the only mechanism by which melatonin exerts its therapeutic effect on CNI-ED. Many studies have demonstrated Melatonin's anti-fibrotic effects in various disease models, such as liver fibrosis[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], pulmonary fibrosis[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], renal fibrosis[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], etc. So it is reasonable to infer that the anti-fibrotic effect of Melatonin also plays a rather important role in the remission of CNI-ED, but this has not been elucidated before. In addition, melatonin can also regulate MSCs behavior and significantly enhance the therapeutic effect of EVs derived from MSCs in certain diseases[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. It has also been observed that exosomes isolated from melatonin-stimulated MSCs can to some extent alleviate renal fibrosis in chronic kidney disease[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn order to verify the above conjecture and clarify its intrinsic mechanism, in this study, we enriched EVs produced by hASCs after pretreatment with melatonin. On one hand, MT-hASC-EVs contain melatonin absorbed by hASC during pretreatment which can exert a certain therapeutic effect; on the other hand and more importantly, melatonin can regulate the composition of EVs derived from hASCs, thus enhancing the therapeutic effect. Through in vivo experiments, we found that intracavernous injection of MT-hASC-EVs could significantly ameliorate the erectile function of CNI-ED rats, meanwhile reduce collagen accumulation indicated by Masson staining, and increase Desmin staining distribution in the cavernous tissue. They could also inhibite the expression of TGF-β pathway-related molecules such as TGF-β, phosphorylated-Smad3 and fibrosis marker Col1a1. Similarly, in vitro, MT-hASC-EVs promoted the proliferation of CCSMCs, reduced hypoxia-induced apoptosis, and inhibited TGF-β2-induced fibrosis of CCSMCs by inhibiting the activation of TGF-β pathway. These experiments demonstrated the excellent anti-fibrosis ability of MT-hASC-EVs in the treatment of CNI-ED.\u003c/p\u003e \u003cp\u003eEVs exert regulatory effects by delivering their cargo to target cells, among which microRNAs are important components[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. microRNAs can recognize the binding sites on the 3'-UTR of target gene mRNAs through their seed sequences (nucleotides 2\u0026ndash;8 at the 5' end), causing the translation of mRNAs to be blocked or even degraded[\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Therefore, to explore the underlying mechanism of the stronger therapeutic effects of MT-hASC-EVs compared to hASC-EVs, we performed high-throughput microRNA sequencing and comparison of the two types of EVs and identified 20 microRNAs with significant expression differences. Since microRNAs exert regulatory effects by downregulating the expression of target genes when they are highly expressed, the upregulated microRNAs in MT-hASC-EVs should play a more primary regulatory role, but not the down-regulated microRNAs. Thus, enrichment analysis of the target genes of upregulated miRNAs in MT-hASC-EVs revealed significant enrichment in \"response to transforming growth factor beta\" and \"transforming growth factor beta signaling\". To identify the most crucial microRNAs, KEGG analysis by miRPath v3 suggested that miR-145-5p, miR-23a-3p, and miR-23b-3p are all related to the TGF-β pathway. Previous studies have reported the regulatory role of miR-145 in various fibrotic diseases, such as myocardial fibrosis (targeting SOX9)[\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e], scar hyperplasia (targeting Smad2/3)[\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e], liver fibrosis (targeting ADD3)[\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e], etc., so we chose miR-145-5p as the most likely effector molecule for further experimental design. The expression of miR-145-5p in both MT-hASCs and MT-hASC-EVs can be significantly upregulated by melatonin, confirming the conclusion of high-throughput sequencing. To clarify the downstream targets of miR-145-5p, we predicted the possible target genes of miR-145-5p using TargetScan v8.0 and sifted potential targets related to the TGF-β pathway. We identified a 7mer-A1 site for miR-145-5p in the 3'UTR of Tgfb2 and an 8mer site in the 3'UTR of Smad3, which were subsequently validated using dual luciferase reporter gene assays. Combining the above results, we confirmed that MT-hASC-EVs can inhibit the occurrence of fibrosis through miR-145-5p/Tgfb2/Smad3 axis.\u003c/p\u003e \u003cp\u003eHowever, our research still has some limitations worth further exploration and improvement. First, MT-hASC-EVs can promote the proliferation of CCSMCs and inhibit apoptosis in vitro, and can also increase the content of Desmin at the in vivo level, but the mechanism behind this is not yet clear. Additionally, we only focused on the changes in the microRNA expression profile of MT-hASC-EVs, while proteins, lipids, and other molecules are also important regulatory molecules that EVs can deliver to target cells, and the potential effector molecules among them are also worth further exploration.\u003c/p\u003e"},{"header":"5 Conclusions","content":"\u003cp\u003eIn conclusion, the present study demonstrated that intracavernosal injection of MT-hASC-EVs could significantly alleviate CNI-ED. MT-hASC-EVs could effectively alleviate the apoptosis and fibrosis of CCSMCs in vitro and in vivo. Mechanistically, microRNA sequencing revealed that miR-145-5p was significantly enriched in MT-hASC-EVs and played a direct and negative regulatory role on Tgfb2 and Smad3 mRNAs by being translocated to CCSMCs. MT-hASCs-EVs can significantly alleviate CNI-ED by inhibiting the occurrence of fibrosis through miR-145-5p/TGF-β/Smad axis (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). All these suggest that these extracellular vesicles may be potential drugs and materials for CNI-ED treatment.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003eEthics approval and consent to participate\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study of \u0026ldquo;Application of mesenchymal stem cells in cavernous nerve injury-induced erectile dysfunction\u0026rdquo; was approved by the Ethics Committee of Renji Hospital Affiliated to Shanghai Jiao Tong University School of Medicine on Oct.26, 2022, with approval number KY2022-180-B. The patients provided written informed consent for participation in the study and the use of samples.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eConsent for publication\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAvailability of data and materials\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe original contributions presented in the study are included in the article/Supplementary Material; further inquiries can be directed to the corresponding author. The matrix of microRNA sequencing is available within the supplementary materials.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eCompeting interests \u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eFunding\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Natural Science Foundation of China No. 82171611, No. 82371631 and No. 82401887.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAuthors\u0026rsquo; contributions\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eXZ and MY performed most of the experiments, analyzed the data, and drafted the manuscript. XC helped with the isolation of ASCs. MZ and YP helped with the animal experiment. ML designed this study and critically revised the manuscript. All authors have read and approved the final manuscript.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAcknowledgement\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have not use Artificial intelligence (AI) -generated work in this manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eYafi FA, Jenkins L, Albersen M, Corona G, Isidori AM, Goldfarb S, et al. Erectile dysfunction\u003cem\u003e.\u003c/em\u003e Nat Rev Dis Primers 2016;2:16003.\u003c/li\u003e\n\u003cli\u003eSong G, Hu P, Song J, Liu J, Ruan Y. Molecular pathogenesis and treatment of cavernous nerve injury-induced erectile dysfunction: A narrative review\u003cem\u003e.\u003c/em\u003e Front Physiol 2022;13:1029650.\u003c/li\u003e\n\u003cli\u003eFicarra V, Novara G, Ahlering TE, Costello A, Eastham JA, Graefen M, et al. Systematic review and meta-analysis of studies reporting potency rates after robot-assisted radical prostatectomy\u003cem\u003e.\u003c/em\u003e Eur Urol 2012;62:418-30.\u003c/li\u003e\n\u003cli\u003ePhilippou YA, Jung JH, Steggall MJ, O\u0026apos;Driscoll ST, Bakker CJ, Bodie JA, et al. Penile rehabilitation for postprostatectomy erectile dysfunction\u003cem\u003e.\u003c/em\u003e Cochrane Database Syst Rev 2018;10:CD012414.\u003c/li\u003e\n\u003cli\u003eLiu C, Lopez DS, Chen M, Wang R. Penile Rehabilitation Therapy Following Radical Prostatectomy: A Meta-Analysis\u003cem\u003e.\u003c/em\u003e J Sex Med 2017;14:1496-503.\u003c/li\u003e\n\u003cli\u003eWu YN, Chen KC, Liao CH, Chiang HS. Spontaneous Regeneration of Nerve Fiber and Irreversibility of Corporal Smooth Muscle Fibrosis After Cavernous Nerve Crush Injury: Evidence From Serial Transmission Electron Microscopy and Intracavernous Pressure\u003cem\u003e.\u003c/em\u003e Urology 2018;118:98-106.\u003c/li\u003e\n\u003cli\u003eYan H, Ding Y, Lu M. Current Status and Prospects in the Treatment of Erectile Dysfunction by Adipose-Derived Stem Cells in the Diabetic Animal Model\u003cem\u003e.\u003c/em\u003e Sex Med Rev 2020;8:486-91.\u003c/li\u003e\n\u003cli\u003eYan H, Rong L, Xiao D, Zhang M, Sheikh SP, Sui X, et al. Injectable and self-healing hydrogel as a stem cells carrier for treatment of diabetic erectile dysfunction\u003cem\u003e.\u003c/em\u003e Mater Sci Eng C Mater Biol Appl 2020;116:111214.\u003c/li\u003e\n\u003cli\u003eHaahr MK, Harken Jensen C, Toyserkani NM, Andersen DC, Damkier P, Sorensen JA, et al. A 12-Month Follow-up After a Single Intracavernous Injection of Autologous Adipose-Derived Regenerative Cells in Patients with Erectile Dysfunction Following Radical Prostatectomy: An Open-Label Phase I Clinical Trial\u003cem\u003e.\u003c/em\u003e Urology 2018;121:203 e6-03 e13.\u003c/li\u003e\n\u003cli\u003eZhang X, Yang M, Chen X, Lu M. Research progress on the therapeutic application of extracellular vesicles in erectile dysfunction\u003cem\u003e.\u003c/em\u003e Sex Med Rev 2024.\u003c/li\u003e\n\u003cli\u003eCheng L, Hill AF. Therapeutically harnessing extracellular vesicles\u003cem\u003e.\u003c/em\u003e Nat Rev Drug Discov 2022;21:379-99.\u003c/li\u003e\n\u003cli\u003eCouch Y, Buz\u0026agrave;s EI, Di Vizio D, Gho YS, Harrison P, Hill AF, et al. A brief history of nearly EV‐erything \u0026ndash; The rise and rise of extracellular vesicles\u003cem\u003e.\u003c/em\u003e Journal of Extracellular Vesicles 2021;10.\u003c/li\u003e\n\u003cli\u003ePan Z, Sun W, Chen Y, Tang H, Lin W, Chen J, et al. Extracellular Vesicles in Tissue Engineering: Biology and Engineered Strategy\u003cem\u003e.\u003c/em\u003e Adv Healthc Mater 2022:e2201384.\u003c/li\u003e\n\u003cli\u003evan Niel G, D\u0026apos;Angelo G, Raposo G. Shedding light on the cell biology of extracellular vesicles\u003cem\u003e.\u003c/em\u003e Nat Rev Mol Cell Biol 2018;19:213-28.\u003c/li\u003e\n\u003cli\u003eLi M, Lei H, Xu Y, Li H, Yang B, Yu C, et al. Exosomes derived from mesenchymal stem cells exert therapeutic effect in a rat model of cavernous nerves injury\u003cem\u003e.\u003c/em\u003e Andrology 2018;6:927-35.\u003c/li\u003e\n\u003cli\u003eOuyang X, Han XY, Chen ZH, Fang JF, Huang XN, Wei HB. MSC-derived exosomes ameliorate erectile dysfunction by alleviation of corpus cavernosum smooth muscle apoptosis in a rat model of cavernous nerve injury\u003cem\u003e.\u003c/em\u003e Stem Cell Research \u0026amp; Therapy 2018;9:12.\u003c/li\u003e\n\u003cli\u003eLiang L, Shen Y, Dong ZF, Gu X. Photoacoustic image-guided corpus cavernosum intratunical injection of adipose stem cell-derived exosomes loaded polydopamine thermosensitive hydrogel for erectile dysfunction treatment\u003cem\u003e.\u003c/em\u003e Bioact Mater 2022;9:147-56.\u003c/li\u003e\n\u003cli\u003eLiu S, Li R, Dou K, Li K, Zhou Q, Fu Q. Injectable thermo-sensitive hydrogel containing ADSC-derived exosomes for the treatment of cavernous nerve injury\u003cem\u003e.\u003c/em\u003e Carbohydr Polym 2023;300:120226.\u003c/li\u003e\n\u003cli\u003eVasey C, McBride J, Penta K. Circadian Rhythm Dysregulation and Restoration: The Role of Melatonin\u003cem\u003e.\u003c/em\u003e Nutrients 2021;13.\u003c/li\u003e\n\u003cli\u003eZhu L, Zhang Q, Hua C, Ci X. Melatonin alleviates particulate matter-induced liver fibrosis by inhibiting ROS-mediated mitophagy and inflammation via Nrf2 activation\u003cem\u003e.\u003c/em\u003e Ecotoxicol Environ Saf 2023;268:115717.\u003c/li\u003e\n\u003cli\u003eHosseinzadeh A, Javad-Moosavi SA, Reiter RJ, Hemati K, Ghaznavi H, Mehrzadi S. Idiopathic pulmonary fibrosis (IPF) signaling pathways and protective roles of melatonin\u003cem\u003e.\u003c/em\u003e Life Sci 2018;201:17-29.\u003c/li\u003e\n\u003cli\u003eRepova K, Stanko P, Baka T, Krajcirovicova K, Aziriova S, Hrenak J, et al. Lactacystin-induced kidney fibrosis: Protection by melatonin and captopril\u003cem\u003e.\u003c/em\u003e Front Pharmacol 2022;13:978337.\u003c/li\u003e\n\u003cli\u003eQian Y, Han Q, Zhao X, Song J, Cheng Y, Fang Z, et al. 3D melatonin nerve scaffold reduces oxidative stress and inflammation and increases autophagy in peripheral nerve regeneration\u003cem\u003e.\u003c/em\u003e J Pineal Res 2018;65:e12516.\u003c/li\u003e\n\u003cli\u003eTavukcu HH, Sener TE, Tinay I, Akbal C, Ersahin M, Cevik O, et al. Melatonin and tadalafil treatment improves erectile dysfunction after spinal cord injury in rats\u003cem\u003e.\u003c/em\u003e Clin Exp Pharmacol Physiol 2014;41:309-16.\u003c/li\u003e\n\u003cli\u003eZhang JL, Hui Y, Zhou F, Hou JQ. Neuroprotective effects of melatonin on erectile dysfunction in streptozotocin-induced diabetic rats\u003cem\u003e.\u003c/em\u003e Int Urol Nephrol 2018;50:1981-88.\u003c/li\u003e\n\u003cli\u003eAlzahrani FA. Melatonin improves therapeutic potential of mesenchymal stem cells-derived exosomes against renal ischemia-reperfusion injury in rats\u003cem\u003e.\u003c/em\u003e Am J Transl Res 2019;11:2887-907.\u003c/li\u003e\n\u003cli\u003eLiu W, Yu M, Xie D, Wang L, Ye C, Zhu Q, et al. Melatonin-stimulated MSC-derived exosomes improve diabetic wound healing through regulating macrophage M1 and M2 polarization by targeting the PTEN/AKT pathway\u003cem\u003e.\u003c/em\u003e Stem Cell Res Ther 2020;11:259.\u003c/li\u003e\n\u003cli\u003eTi Y, Yang M, Chen X, Zhang M, Xia J, Lv X, et al. Comparison of the therapeutic effects of human umbilical cord blood-derived mesenchymal stem cells and adipose-derived stem cells on erectile dysfunction in a rat model of bilateral cavernous nerve injury\u003cem\u003e.\u003c/em\u003e Front Bioeng Biotechnol 2022;10:1019063.\u003c/li\u003e\n\u003cli\u003eLi Z, Yin Y, He K, Ye K, Zhou J, Qi H, et al. Intracavernous Pressure Recording in a Cavernous Nerve Injury Rat Model\u003cem\u003e.\u003c/em\u003e J Vis Exp 2021.\u003c/li\u003e\n\u003cli\u003eVlachos IS, Zagganas K, Paraskevopoulou MD, Georgakilas G, Karagkouni D, Vergoulis T, et al. DIANA-miRPath v3.0: deciphering microRNA function with experimental support\u003cem\u003e.\u003c/em\u003e Nucleic Acids Res 2015;43:W460-6.\u003c/li\u003e\n\u003cli\u003eWang HS, Ruan Y, Banie L, Cui K, Kang N, Peng D, et al. Delayed Low-Intensity Extracorporeal Shock Wave Therapy Ameliorates Impaired Penile Hemodynamics in Rats Subjected to Pelvic Neurovascular Injury\u003cem\u003e.\u003c/em\u003e J Sex Med 2019;16:17-26.\u003c/li\u003e\n\u003cli\u003eKarakus S, Musicki B, La Favor JD, Burnett AL. cAMP-dependent post-translational modification of neuronal nitric oxide synthase neuroprotects penile erection in rats\u003cem\u003e.\u003c/em\u003e BJU Int 2017;120:861-72.\u003c/li\u003e\n\u003cli\u003eLeungwattanakij S, Bivalacqua TJ, Usta MF, Yang DY, Hyun JS, Champion HC, et al. Cavernous neurotomy causes hypoxia and fibrosis in rat corpus cavernosum\u003cem\u003e.\u003c/em\u003e J Androl 2003;24:239-45.\u003c/li\u003e\n\u003cli\u003eCho MC, Park K, Kim SW, Paick JS. Restoration of erectile function by suppression of corporal apoptosis, fibrosis and corporal veno-occlusive dysfunction with rho-kinase inhibitors in a rat model of cavernous nerve injury\u003cem\u003e.\u003c/em\u003e J Urol 2015;193:1716-23.\u003c/li\u003e\n\u003cli\u003ePhilip S, Bredeson C, Allan DS, Altouri S, Huebsch LB, Atkins H, et al. Complications and Toxicities Associated with Autologous Stem Cell Transplantation for Severe Autoimmune Disease: Single Center Experience\u003cem\u003e.\u003c/em\u003e Blood 2018;132.\u003c/li\u003e\n\u003cli\u003eChen Z, Han X, Ouyang X, Fang J, Huang X, Wei H. Transplantation of induced pluripotent stem cell-derived mesenchymal stem cells improved erectile dysfunction induced by cavernous nerve injury\u003cem\u003e.\u003c/em\u003e Theranostics 2019;9:6354-68.\u003c/li\u003e\n\u003cli\u003eWitwer KW, Buzas EI, Bemis LT, Bora A, Lasser C, Lotvall J, et al. Standardization of sample collection, isolation and analysis methods in extracellular vesicle research\u003cem\u003e.\u003c/em\u003e J Extracell Vesicles 2013;2.\u003c/li\u003e\n\u003cli\u003eKeshtkar S, Azarpira N, Ghahremani MH. Mesenchymal stem cell-derived extracellular vesicles: novel frontiers in regenerative medicine\u003cem\u003e.\u003c/em\u003e Stem Cell Res Ther 2018;9:63.\u003c/li\u003e\n\u003cli\u003eYea JH, Yoon YM, Lee JH, Yun CW, Lee SH. Exosomes isolated from melatonin-stimulated mesenchymal stem cells improve kidney function by regulating inflammation and fibrosis in a chronic kidney disease mouse model\u003cem\u003e.\u003c/em\u003e J Tissue Eng 2021;12.\u003c/li\u003e\n\u003cli\u003eHerrmann IK, Wood MJA, Fuhrmann G. Extracellular vesicles as a next-generation drug delivery platform\u003cem\u003e.\u003c/em\u003e Nat Nanotechnol 2021;16:748-59.\u003c/li\u003e\n\u003cli\u003eMiao Y, Fu C, Yu Z, Yu L, Tang Y, Wei M. Current status and trends in small nucleic acid drug development: Leading the future\u003cem\u003e.\u003c/em\u003e Acta Pharm Sin B 2024;14:3802-17.\u003c/li\u003e\n\u003cli\u003eCui S, Liu Z, Tao B, Fan S, Pu Y, Meng X, et al. miR-145 attenuates cardiac fibrosis through the AKT/GSK-3beta/beta-catenin signaling pathway by directly targeting SOX9 in fibroblasts\u003cem\u003e.\u003c/em\u003e J Cell Biochem 2021;122:209-21.\u003c/li\u003e\n\u003cli\u003eShen W, Wang Y, Wang D, Zhou H, Zhang H, Li L. miR-145-5p attenuates hypertrophic scar via reducing Smad2/Smad3 expression\u003cem\u003e.\u003c/em\u003e Biochem Biophys Res Commun 2020;521:1042-48.\u003c/li\u003e\n\u003cli\u003eYe Y, Li Z, Feng Q, Chen Z, Wu Z, Wang J, et al. Downregulation of microRNA-145 may contribute to liver fibrosis in biliary atresia by targeting ADD3\u003cem\u003e.\u003c/em\u003e Plos One 2017;12:e0180896.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"stem-cell-research-and-therapy","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scrt","sideBox":"Learn more about [Stem Cell Research \u0026 Therapy](http://stemcellres.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/scrt/default.aspx","title":"Stem Cell Research \u0026 Therapy","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Melatonin, mesenchymal stem cell, exosome, CNI-ED, cavernous fibrosis","lastPublishedDoi":"10.21203/rs.3.rs-5246841/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5246841/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eBackgrounds: Cavernous nerve injury-induced erectile dysfunction (CNI-ED) is a common complication after radical prostatectomy. As a consequence of the concomitant severe fibrosis of the corpus cavernosum, conventional treatment approaches have had little success.\u003c/p\u003e \u003cp\u003eMethods: Pre-treatment of adipose-derived stem cells with melatonin allows for the extraction of active exosomes (MT-hASC-EVs) from the conditioned medium. The therapeutic effects of MT-hASC-EVs were assessed in a rat model of CNI-ED, and the anti-fibrotic properties were evaluated. MicroRNA sequencing was used to identify specific microRNAs highly expressed in MT-hASC-EVs, and differential microRNAs were screened for regulatory pathways through target gene enrichment analysis. Finally, the conclusions from bioinformatics analysis were validated through in vitro experiments.\u003c/p\u003e \u003cp\u003eResults: Intracavernous injection of MT-hASC-EVs significantly restored erectile function and reduced the extent of corpus cavernosum fibrosis in the CNI-ED rat model. MT-hASC-EVs promoted the proliferation and anti-apoptotic effects of CCSMCs in vitro. Mechanistically, MT-hASC-EVs inhibit fibrosis by delivering miR-145-5p, which targets TGF-β2/Smad3 axis.\u003c/p\u003e \u003cp\u003eConclusions: MT-hASCs-EVs can inhibit cavernous fibrosis and improve erectile function in a rat model of CNI-ED by targeting the miR-145-5p/TGF-β/Smad axis.\u003c/p\u003e","manuscriptTitle":"Melatonin-Pretreated Mesenchymal Stem Cell-Derived Exosomes Alleviate Cavernous Fibrosis in a Rat Model of Nerve Injury-induced Erectile Dysfunction via miR-145-5p/TGF-β/Smad Axis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-10-29 15:13:23","doi":"10.21203/rs.3.rs-5246841/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2024-10-26T03:16:46+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-10-25T17:00:51+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-10-18T00:28:49+00:00","index":"","fulltext":""},{"type":"submitted","content":"Stem Cell Research \u0026 Therapy","date":"2024-10-17T04:45:51+00:00","index":"","fulltext":""},{"type":"decision","content":"Major Revision","date":"2024-10-17T02:44:32+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"stem-cell-research-and-therapy","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scrt","sideBox":"Learn more about [Stem Cell Research \u0026 Therapy](http://stemcellres.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/scrt/default.aspx","title":"Stem Cell Research \u0026 Therapy","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"3f362595-ccba-4b72-9f1c-fe1ec35a7284","owner":[],"postedDate":"October 29th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-03-03T16:02:12+00:00","versionOfRecord":{"articleIdentity":"rs-5246841","link":"https://doi.org/10.1186/s13287-025-04173-0","journal":{"identity":"stem-cell-research-and-therapy","isVorOnly":false,"title":"Stem Cell Research \u0026 Therapy"},"publishedOn":"2025-02-25 15:57:45","publishedOnDateReadable":"February 25th, 2025"},"versionCreatedAt":"2024-10-29 15:13:23","video":"","vorDoi":"10.1186/s13287-025-04173-0","vorDoiUrl":"https://doi.org/10.1186/s13287-025-04173-0","workflowStages":[]},"version":"v1","identity":"rs-5246841","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5246841","identity":"rs-5246841","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}