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

doi:10.21203/rs.3.rs-5246841/v1

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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

https://doi.org/10.21203/rs.3.rs-5246841/v1

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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

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.

2.1 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% CO₂ 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)×10⁶. 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.

3.1 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. 1A). Both hASCs and melatonin-pretreated hASCs showed typical spindle and fibroblast-like morphologies (Fig. 1B). The levels of growth factors secreted by hASCs were detected by RT-PCR (Fig. 1C). 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. 1D). 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. 1E). There was also no difference in mean particle size (Fig. 1F). Both hASC-EVs and MT-hASC-EVs had a typical cup-like shape under transmission electron microscope (TEM) (Fig. 1G). 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. 2A, 2B). 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. 2C, 2D). 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. 2E, 2F). 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. 2G, 2H). 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. 3A). 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. 3B). 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. 3C, 3D), 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. 3E,3F). 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. 4A) and heatmap (Fig. 4B).

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. 4C). Consistent with that, these target genes were enriched in the "TGF-beta signaling pathway" in KEGG enrichment analysis (Fig. 4D). 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. 5A). 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. 5B,5C). In the ICC experiment, lower TGF-β2 fluorescence signals were detected in CCSMCs after MT-hASC-EVs treatment (Fig. 5D,5E). 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. 6A). 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. 6B). Similarly, MT-hASC- EVs exhibited significantly higher expression levels of these three miRNAs compared to hASC-EVs without melatonin pretreatment (Fig. 6C). 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. 6D).

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. 6E. 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. 6F), 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. 6G). 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.

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.

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.

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.

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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

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Version 1

Abstract

Figures

1 Introduction

2 Materials and Methods

2.1 Cell isolation, culture and identification

2.2 Isolation and characterization of extracellular vesicles.

2.3 In vivo experimental design

2.4 Erectile function evaluation.

2.5 Histological and immunohistochemical analysis

2.6 Fluorescent labeling and in vitro tracing of EVs.

2.7 CCSMC viability and proliferation.

2.8 Cell apoptosis assay

2.9 The miRNA Library Construction and Sequencing.

2.10 Immunocytochemistry (ICC)

2.11 Cell Transfection.

2.12 Dual-luciferase reporter assay.

2.13 Real-Time PCR.

2.14 Western Blots.

2.15 Statistical analysis.

3 Results

3.1 Isolation and characterization of melatonin-pretreated adipose mesenchymal stem cell-derived EVs.

3.3 MT-hASC-EVs could promote the proliferation of CCSMCs and reduce cell apoptosis in vitro.

3.4 High-throughput sequencing revealed that the microRNAs in MT-hASC-EVs can target the TGF-β/Smad axis

3.5 MT-hASC-EVs could reduce the fibrosis of CCSMCs in vitro by inhibiting the TGF-β pathway.

3.6 MT-hASC-EVs inhibit TGF-β2 and Smad3 expression through miR-145-5p.

4 Discussion

5 Conclusions

Declarations

References

Supplementary Files

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