Exosomes Derived from miR-301a-3p-Modied Adipose-derived Mesenchymal Stem Cells Reverse Hypoxia-induced Erectile Dysfunction in Rat Models

Erectile dysfunction (ED) has often been observed in patients with obstructive sleep apnea (OSA). Research on adipose-derived stem cells (ADSCs)-derived exosomes has shown that they have signicant therapeutic effects in many diseases including ED. In this study, ED was induced in Sprague Dawley (SD) rats using chronic intermittent hypoxia (CIH) exposure. CIH-mediated inuences were then measured in the corpus cavernous smooth muscle cells (CCSMCs). Results showed that miR-301a-3p-enriched exosomes treatment signicantly recovered erectile function in rats and CCSMCs by promoting autophagy and inhibiting apoptosis. The treatment also signicantly recovered the level of alpha Smooth Muscle Actin (α-SMA) in rats and CCSMCs. Bioinformatics predicted that Phosphatase and tensin homolog (PTEN) and Toll-like receptor 4 (TLR4) might be targets of miR-301a-3p. Our results indicate that PTEN-overexpression vectors or TLR4-overexpression vectors reverse the therapeutic effects achieved by miR-301a-3p in CCSMCs indicating that PTEN/HIF-1α and TLR4 signaling pathways play key roles in the progression of ED. The ndings in this study suggest that miR-301a-3p should be considered as a new therapeutic target for treating ED associated with OSA.


Introduction
Erectile dysfunction (ED), also known as inadequate penile erection, is a common clinical entity that mainly affects males older than 40 years [1]. It is de ned as the inability to achieve and maintain an adequate erection to permit satisfactory sexual intercourse [2] resulting in dissatisfaction with sex life in a signi cant proportion of men [3]. Several factors are associated with ED including smoking, hormonal imbalance, general health status of the individual, diabetes mellitus, cardiovascular diseases and psychiatric disorders [4]. In addition, ED is the most common long-term side effect of active therapy for early prostate cancer treatment including robotic-assisted laparoscopic radical prostatectomy (RALP) [5,6]. Despite the use of nerve-sparing techniques, as high as 90% rates of post-RALP ED have been reported mainly due to periprostatic and cavernous nerve (CN) damage [7].
Chronic intermittent hypoxia (CIH) is one of the most important and direct consequences of obstructive sleep apnea (OSA) [8,9] with studies showing a higher ED incidence in male patients with chronic hypoxia [10]. Hypoxia may affect erectile function, neuronal nitric oxide synthase (nNOS) and endothelial nitric oxide synthase (eNOS) expression leading to erectile dysfunction under hypoxic conditions in murine models [11,12]. Constitutively expressed nNOS and eNOS isoforms mediate penile erection by producing nitric oxide (NO), which has been recognized as playing a major role in the physiology of penile erection [1,2]. Catalyzed by NOS and produced from L-Arginine, NO activates guanylate cyclase (GC) which converts guanosine triphosphate (GTP) to cyclic guanosine monophosphate (cGMP) leading to relaxation of vascular smooth muscles and vasodilation [13]. Oral phosphodiesterase type 5 (PDE-5) inhibitors were the rst and most effective form of oral therapies recommended for the treatment of ED [13]. However, potential side effects such as anterior ischemic optic neuropathy and increased risk of stroke have hindered the use of PDE-5 inhibitor [14].
Recent studies have shown that adipose-derived stem cells (ADSCs) therapy has therapeutic effects for ED resulting from cavernous nerve injury [15,16]. Several studies have shown that ADSC-derived exosomes play an important role in ADSC therapy where the use of exosomes derived from microRNAs (miRNAs)-overexpressed ADSCs provides protection from various diseases [17][18][19][20]. Exosomes are membrane vesicles that are secreted by most cells. Exosomes having a diameter of 30-100 nm contain many macromolecular components including proteins, mRNAs, and miRNAs which can regulate intracellular signaling pathways [21]. Studies have found that exosomes derived from ADSCs and mesenchymal stem cells (MSCs) exert therapeutic effect on ED in rat models having diabetes and cavernous nerves injury [22][23][24][25]. Therefore, the study of miRNA-overexpressed ADSC-derived exosomes treatment for ED is vital. Previous studies have shown that miR-301a-3p is a key factor in pancreatic cancer, breast cancer, carcinoma and schizophrenia [26][27][28][29]. One study indicated that the level of miR-301a-3p in the corpus cavernosum of type 2 diabetes mellitus-associated erectile dysfunction (T2DMED) mice was signi cantly decreased compared with normal mice [30]. In addition, our preliminary research found that miR-301a-3p was signi cantly down-regulated in ED patients and rats with CIH-induced ED. This led to our focus on the potential role of miR-301a-3p in the progression of ED.
In this study, we used exosomes derived from miR-301-3p-overexpressed ADSC as the therapeutic medium. We analyzed intracavernous pressure (ICP) and arterial pressure (AP) in rat models after CIH exposure. Expression levels of nNOS and eNOS were measured to investigate the potential effects of miR-301a-3p-enriched exosomes treatment. Analysis of the expression levels of apoptosis, autophagosomes, autolysosomes and other indicators associated with ED was done using CIH exposed murine models.
Results showed that miR-301a-3p-enriched exosomes treatment had signi cant therapeutic effects on rats after CIH exposure. Further study of signaling pathways indicated that phosphatase and tensin homolog (PTEN) and Toll-like receptor 4 (TLR4) might be directly targeted by miR-301a-3p with overexpression of both reversing protection effects induced by miR-301a-3p. Our ndings indicate that miR-301a-3p should be considered as a new therapeutic target in treating ED patients.

Patient samples
A sample size of 30 OSA patients with ED admitted at Shanghai Ninth People's Hospital, Shanghai, China was enrolled. According to hospital records, the patients had been clinically diagnosed with ED. The age of the patients ranged from 30 to 65 years. Patients with hypertension, diabetes, trauma and surgery history were excluded. Thirty healthy people to be used as controls were also recruited from the hospital. Serum samples were taken within 24 hours of symptom onset and frozen in liquid nitrogen. Ethical approval for the study was provided by the Independent Ethics Committee of Shanghai Ninth People's Hospital, Shanghai, China. Guidelines from the Ethics Committee were followed where informed and written consent was obtained from all patients or their advisors before samples were collected.

Culturing ADSCs
Adipose tissues collected from SD rats were washed with pre-cooled phosphate-buffered saline (PBS) and cut into 1mm 2 pieces. Tissues were digested using collagenase followed by centrifugation at 4000 × g for 5 minutes. Obtained cell pellet was then suspended in Dulbecco's modi ed Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS), 1% penicillin-streptomycin and 2 mmol/L L-glutamine. The cells were then cultured in a controlled environment having 5% CO2 and a temperature of 38 °C for 48 hours. Cells were then transferred into fresh culture medium with subsequent subculture every 3 days.
When cells were approximately 90% con uent, they were passaged and used at passage three. Cells were then incubated with conjugated monoclonal antibodies against CD29, CD44, CD90, CD105, CD34 and vWF to con rm the identity of ADSCs. Isotype-identical antibodies (PharMingen) were used as controls.

Isolation of exosomes
Adipose-derived stromal cells collected from miR-301a-3p overexpression, control and NC groups at 80%-90% con uence were washed with PBS and cultured in microvascular endothelial cell growth medium-2 media deprived of FBS. ADSCs were then supplemented using 1 × serum replacement solution (PeproTech) for 24 hours. Dead cells and debris were removed by centrifugation of ADSCs at 300 × g for 10 minutes and 2000 × g for 10 minutes followed by mixing 10 mL of the supernatant with 5 mL of ExoQuick-TC reagent (System Biosciences). The mixture was then centrifuged at 1500 × g for 30 minutes, with the resulting exosome-containing pellet being re-suspended in nuclease-free water. TRIzol-LS (Invitrogen) and Exosomal Protein Extraction (Invitrogen) kits were used for extracting total RNA and protein, respectively. Isolated exosomes were used immediately for experiments or stored at -180 °C. Sizes of puri ed exosomes were determined using a NanoSight LM10 (Malvern Instruments) nanoparticle tracking system.

CIH exposure-induced ED rat model
Thirty-two male SD rats were randomly divided into control, CIH, CIH + exosomes (Exo) and CIH + miR-301a-3p-enriched exosomes (Exo-301a) groups (n = 6). An oxygen sensor was placed at the bottom of the chamber to measure the oxygen content in the CIH exposure chamber over the course of several cycles. Animals were exposed to 2 minutes of 5% O 2 for each 4-minute cycle with each challenge lasting 8 hours.
The challenge was done for eight weeks during the daytime from 8am to 4 pm. Sham group rats were exposed to 21% O 2 . Exosomes (400 µg of protein) were isolated using 200 µL PBS and then administered using intracavernous injection for Exo groups, whereas control rats received an equal volume of PBS. Exosomes were administered to the rats every week for eight weeks.

Erectile function Measurement
After eight weeks of CIH exposure, intracavernous pressure (ICP) and Mean carotid artery pressure (MAP) were measured to evaluate erectile function as described in our previous article [7]. Penile tissues were then harvested, under euthanasia, for further histological studies.

Immuno uorescence
Harvested tissues were immersed in optimal cutting temperature compound and immediately frozen in liquid nitrogen. The tissues were cut into sections having a thickness of 5 mm followed by immuno uorescence staining as described in [31]. Primary antibodies used in this study were eNOS (ab76198), nNOS (ab76067) and Phalloidin (ab176753) all obtained from Abcam. Secondary antibodies included Alexa-488, Texas Red-conjugated antibodies, (1:500; Invitrogen, Carlsbad, CA, USA) and Texas Red goat anti-rabbit IgG (1:200; Life Technologies, Grand Island, NY, USA). Nuclei were stained using DAPI (1:10,000, Invitrogen, Carlsbad, CA, USA). Ratio of positive nNOS counts to DAPI were used to analyze the nerve bers in the dorsal section of the penis. Smooth muscle and endothelial stains were analyzed using the ratio of positively stained areas of phalloidin and eNOS to DAPI in the corpora cavernosa.

CCSMCs culture and CIH exposure
Rats were sacri ced where on a sterile table, the penis was excised and placed in a sterile Petri dish followed by two washes using PBS. The skin around the penis was carefully peeled away, along with the albuginea, urethral sponge, cavernous body, and other vessels. The corpus cavernosum was cut into 1mm 3 tissue blocks that were placed in a cell culture ask containing 0.5% type I collagenase solution (Sigma). Cells were cultured at 37 °C with shaking in a humidi ed atmosphere having 95% air and 5% CO 2 for 3 hours. The cells were then ltered and centrifuged followed by the addition of 3 ml F12 medium (Invitrogen) containing 20% fetal bovine serum (Invitrogen) and incubated at 37 °C and 5% CO 2 . Long, spindle-shaped SMCs were observed at the bottom of the 25 cm 2 culture asks after incubating for 24 hours. For CIH exposure, CCSMCs were exposed to 5 s of 14-15% O 2 during each 60 s cycle for 24 hours.
All cells were cultured for 24 hours followed by co-culturing with miR-301a-3p-enriched exosomes for 48 hours.

Statistical analysis
All the data obtained from this study was analyzed using GraphPad 9.0. One-way ANOVA was used among various groups with p < 0.05 being considered as statistically signi cant.
More detailed materials and methods are in the Supplementary Methods.

Results
3.1 CIH exposure signi cantly down-regulates miR-301a-3p in patients with ED and in rats induced with ED ADSCs obtained from adipose tissues of SD rats displayed a typical cobblestone-like morphology under the microscope (Fig. 1A). Oil Red O staining con rmed that they were undergoing adipogenesis (Fig. 1B). To con rm the identity of ADSCs, they were incubated with conjugated monoclonal antibodies against CD29, CD44, CD90, CD105, CD34 and vWF with isotype-identical antibodies (PharMingen) being used as controls. Results showed that ADSCs were positive for the mesenchymal stem cell (MSC) markers CD29, CD34, CD44, CD90 and CD 105 (Fig. 1C) with the sequence between hsa-miR-301a-3p and rno-miR-301a-3p being similar (Fig. 1D). RT-qPCR analysis of serum samples collected from 30 ED patients showed that expression levels of hsa-miR-301a-3p in ED patients were signi cantly lower than those in healthy patients (p < 0.001) (Fig. 1E). To further determine the expression of rno-miR-301a-3p, CIH exposure was done on SD rats. Results showed that rno-miR-301a-3p expression was inhibited at gene level in CIH exposed rats compared to control group (n = 8, p < 0.01) (Fig. 1F).
ADSC-derived exosomes were isolated and con rmed under a microscope ( Fig. 1G) with results showing that the diameter of most exosomes was approximately 100 mm (Fig. 1H). Examination of ADSCs and exosomes using Western blot resulted in exosomes testing positive against exosome markers TSG101, CD9 and CD63 while ADSCs tested negative (Fig. 1I). After transfection with miR-301a-3p-overexpressed mimic, ADSCs and ADSC-derived exosomes were analyzed using RT-qPCR. Results con rmed the overexpression of miR-301a-3p in both ADSCs and ADSC-derived exosomes, compared to control and negative control groups (Fig. 1I). The results indicated that miR-301a-3p was signi cantly down-regulated in ED patients and CIH exposure rats. In addition, miR-301a-3p mimic had a good transcription e ciency in ADSCs.
3.2 CIH exposure negatively in uences erectile function while miR-301a-3p-enriched exosomes treatment repairs the damage in SD rats Masson trichrome staining of actin and collagen was done for each group where smooth muscle and connective tissue in the corpus cavernosum stained red and blue, respectively. Results indicated a decrease in the proportion of smooth muscle when CIH exposure rats were compared with the sham group after (p < 0.001). When compared with CIH exposure group, miR-301a-3p-enriched exosomes treatment signi cantly promoted the proportion of smooth muscle indicating that exosomes treatment had therapeutic effects on the repair of smooth muscle (p < 0.001) ( Fig. 2A and B). Results obtained after Phalloidin staining indicated that CIH exposure destroyed F-actin. Signi cantly more stained cytoskeleton area was observed after normal exosomes treatment and miR-301a-3p-enriched exosomes treatment with the effects of the latter being more pronounced (Fig. 2C and D).
The ratio of ICP/RT-AP was used to assess erectile function (Fig. 2E) with results showing that CIH exposure signi cantly inhibited erectile function in SD rats (Fig. 2G). Normal exosomes and miR-301a-3penriched exosomes treatments had signi cant effects on recover ICP/RT-AP when compared to CIH exposure group (p < 0.001) with miR-301a-3p-enriched exosomes having more pronounced effects (Fig. 2G). Western blot analysis was done to measure the level of myo broblast formation with results indicating that α-SMA was down-regulated after CIH exposure. However, exosome treatment signi cantly increased the expression of α-SMA when compared with CIH exposure group (Fig. 2F).
To determine the level of nNOS in DNP of the penis, harvested tissues were prepared for immuno uorescence staining and Western blot analysis. Results showed no signi cant changes in the ratio of nNOS-positive nerve counts/DAPI in all areas of CIH exposure groups when compared with Sham groups indicating that CIH exposure did not alter NO release from peripheral nerve endings (Fig. 3A-C). This was con rmed by the results of Western blot analysis which indicated that CIH exposure had no effect on the expression of nNOS. Interestingly, CIH exposure stimulated the expression of iNOS while miR-301a-3p-enriched exosomes reduced its expression (Fig. 3D and E). Immuno uorescence staining of endothelial cells showed that eNOS expression decreased signi cantly after CIH exposure (p < 0.00) when compared to Sham group. Exosomes treatment had positive effects on recovering the expression level of eNOS with miR-301a-3p-enriched exosomes treatment having signi cantly better results (Fig. 3F-H).
Results indicate that CIH exposure negatively affected erectile function while miR-301a-3p-enriched exosomes treatment had signi cant remediation effects on SD rats, including ICP/RT-AP and expression levels of α-SMA and eNOS.

miR-301a-3p directly targets PTEN and TLR4
Serum samples were collected from SD rats in all groups (Sham, CIH, CIH + EXO, CIH + EXO-301a) and analyzed using RT-qPCR to determine the signaling pathway used by miR-301a-3p to in uence erectile function. RT-qPCR results showed that the expression level of miR-301a-3p in serum signi cantly decreased in CIH exposure group when compared to Sham group (p < 0.001). There was no signi cant difference between CIH exposure group and CIH + EXO group, while miR-301a-3p was signi cantly overexpressed in CIH + EXO-301a group (Fig. 4A). Furthermore, the results showed a signi cant increase of PTEN and TLR4 gene levels in CIH and CIH + EXO groups ( Fig. 4B and D). Treatment with miR-301a-3p reversed the expression of PTEN and TLR4 leading to a decrease in PTEN and TLR4 levels ( Fig. 4B and  D). Protein levels of PTEN and TLR4 in each group were con rmed using Western blot analysis (Fig. 4C and E).
Results obtained after Western blot analysis showed that CIH exposure directly induced overexpression of LC3I/II and p65 in the nucleus (Fig. 4C and E) indicating that the level of autophagy was up-regulated by CIH exposure. Up-regulated autophagy was also con rmed by the inhibited expression of p62 (Fig. 4C). Exosomes treatment increased the level of autophagy through overexpressing LC3I/II and p65 while the levels of p62 decreased with miR-301a-3p enriched exosomes having a more pronounced effect (Fig. 4C and E). These results suggest that PTEN and TLR4 can be directly targeted by miR-301a-3p.
Bioinformatics was used to predict the possible targets in the determination of the potential association between miR-301a-3p and PTEN/TLR4 with results showing that both PTEN and TLR4 could be possible targets of miR-301a-3p ( Fig. 6A and C). Dual-luciferase reporter assay results showed that overexpression of miR-301a-3p reduced the intensity of uorescence in CCSMCs transfected with TLR4-WT and PTEN-WT vectors while having no effect on CCSMCs transfected with TLR4-MUT and PTEN-MUT vectors ( Fig. 6B and D). RT-qPCR and Western blot results further con rmed that both TLR4 and PTEN were inhibited at mRNA and protein level after cells were transfected with miR-301a-3p ( Fig. 6E and F).
Combining both sets of results made a clear indication that both PTEN and TLR4 are direct targets of miR-301a-3p.

miR-301a-3p-enriched exosomes inhibits CIH-induced apoptosis and up-regulates CIH-induced overexpression of autophagy in CCSMCs
For CIH exposure, CCSMCs were exposed to 5 s of 14-15% O 2 during every 60 s cycle for 24 h. All cells were cultured for 24 h and Co-culturing with miR-301a-3p-enriched exosomes for 48 h. Results obtained after CIH exposure of CCSMCs indicated that α-SMA was down-regulated at protein level. On the other hand, exosome treatment increased the level of α-SMA after CIH exposure with miR-301a-3p-enriched exosomes treatment having a more signi cant effect than normal exosomes treatment (Fig. 5A). Flow cytometry with Annexin V-FITC staining was used to assess the apoptosis rate with results showing that CIH exposure directly led to a signi cant increase in the apoptosis rate. However, exosomes treatment inhibited apoptosis with miR-301a-3p-enriched exosomes treatment having a signi cantly higher CIHinduced apoptosis rate inhibition than normal exosomes treatment (p < 0.001) ( Fig. 5B and C). Levels of miR-301a-3p, PTEN, and TLR4 were analyzed using RT-qPCR. As we had hypothesized, results indicated that miR-301a-3p levels decreased after CIH exposure (p < 0.01) when compared to control group. There was no signi cant difference between CIH group and CIH + EXO group (Fig. 5D). However, miR-301a-3penriched exosomes treatment led to a signi cant overexpression of miR-301a-3p levels in CCSMCS after CIH exposure (Fig. 5D). Both PTEN and TLR4 levels increased signi cantly after CIH exposure while miR-301a-3p-enriched exosomes treatment signi cantly decreased the mRNA expression level of PTEN and TLR4 (Fig. 5D). Results obtained after Western blot analysis con rmed the expression levels of PTEN and TLR4 ( Fig. 5G and H).
In addition, Western blot results showed that CIH exposure directly induced overexpression of LC3I/II and p65 in the nucleus indicating that the level of autophagy was up-regulated by CIH exposure (Fig. 5G and  H). Increased autophagy was con rmed by the inhibited expression of p62 (Fig. 5G). The level of autophagy was further increased by exosomes treatment, especially treatment with miR-301a-3p-enriched exosomes ( Fig. 5G and H). Autophagic ux analysis was further done where CCSMCs were transfected with mRFP-GFP-LC3 with results showing that the quantity of autophagosomes, autolysosomes, and autophagic vacuoles increased signi cantly after CIH exposure (Fig. 5I-L). There was no signi cant difference between CIH group and CIH + EXO group, while miR-301a-3p led to a signi cant increase of autophagosomes, autolysosomes, and autophagic vacuoles in CCSMCs (( Fig. 5I-L)). Our ndings suggest that miR-301a-3p-enriched exosomes treatment inhibits CIH-induced apoptosis and up-regulates CIH-induced overexpression of autophagy in CCSMCs.
Potential roles of TLR4 were also determined where the effectiveness of TLR4-OE was checked at protein and gene level ( Fig. 7L and M). Flow cytometry results showed that the high CIH-induced apoptosis rate was inhibited by miR-301a-3p. TLR4-OE signi cantly suppressed miR-301a-induced inhibitory effects thereby promoting increased apoptosis ( Fig. 7N and O). Similar to results of CIH + EXO-301a + PTEN-OE group, miR-301a-induced expression of high α-SMA levels was signi cantly suppressed by TLR4-OE vectors (Fig. 7P). In addition, Western blot analysis con rmed that TLR4-OE reversed the miR-301a-3pinhibted expression level of p62 in CCSMCs ( Fig. 7Q and R). When combined, the results suggest that both PTEN-OE and TLR4-OE signi cantly suppressed miR-301a-3p-induced positive effects on autophagy and inhibitory effects on apoptosis.

Discussion
There is an increase in the incidences of prostate cancer in line with increasing male life expectancy. Despite the use of nerve-sparing techniques during the treatment of prostate cancer, rates as high as 90% of post-RALP ED have been reported [7]. Other important risk factors associated with ED are CIH and sleep apnea problems in men [32]. Traditionally, clinical interventions have been limited to managing the chronic form of ED using phosphodiesterase-5 inhibitors, intracavernosal injections, vacuum devices, and penile prostheses. Recently, exosomes have been found to have therapeutic effects on ED in rat models having diabetes and cavernous nerves injury [22,24,25]. In this study, we designed experiments to investigate the effects of CIH exposure in rat models and CCSMCs. The main aim of the study was to determine the role that miR-301a-3p plays in CIH exposure rats and CCSMCs as well as determining its molecular mechanisms.
In the treatment of ED, cavernous smooth muscle plays a key role in recovery of erectile function [33]. Our results indicated that miR-301a-3p-enriched exosomes treatment signi cantly increased the proportion of smooth muscle in CIH exposure rats. Moreover, miR-301a-3p-enriched exosomes led to a signi cantly raised expression of α-SMA. Erectile function recovery was also determined by measuring ICP and RT-AP levels. Generally, both exosome treatments had signi cant effects on recover ICP/RT-AP in CIH exposure rats with miR-301a-3p-enriched exosomes treatment having better therapeutic effects. Production of NO, a key factor in erectile function, was determined by examining the levels of nNOS and eNOS. Results showed that the expression level of eNOS in DNP and sinusoid decreased signi cantly in CIH exposure groups when compared with Sham groups. On the other hand, miR-301a-3p-enriched exosomes treatment led to a signi cantly higher expression of eNOS in CIH exposure rats. Results from this study indicated that miR-301a-3p-enriched exosomes has therapeutic effects on recovering erectile function damaged after CIH exposure.
A previous study recommends using MSC-induced promotion of autophagy to treat ED [34]. In this study, we examined the expression levels of LC3I/II, p62 and p65. Results showed that autophagy was stimulated to a higher level in CIH exposure rats and CCSMCs. miR-301a-3p-enriched exosomes treatment increased the number of autophagosomes, autolysosomes, and autophagic vacuoles in rats and CCSMCs after CIH exposure. The level of apoptosis was assessed using ow cytometry after Annexin V-FITC staining. We found that CIH exposure increased the apoptosis rate in CCSMCs, while miR-301a-3p reversed these effects by decreasing the apoptosis rate. Our ndings supported the prediction by bioinformatics that PTEN and TLR4 could be targets of miR-301-3p. Therapeutic effects of miR-301a-3penriched exosomes treatment on the levels of HIF-1α, α-SMA, autophagy and apoptosis were reversed by both PTEN-OE and TLR4-OE. These ndings suggest that miR-301-3p directly targets PTEN and TLR4 in the regulation of erectile function. However, there is need to further investigate miR-301a-3p, PTEN and TLR4 as possible therapeutic targets for treating ED. In this study, performing CIH-induced injury in cell experiments enabled the measurement of ICP/RT-AP and smooth muscle staining in SD rats. Due to a number of factors, like the function of PTEN-OE and TLR4-OE not being studied in SD rats, this study still needs further development.

Conclusions
In summary, we found that miR-301a-3p might play an important role in the progression of post-RALPrelated or CIH-mediated ED by targeting PTEN and TLR4 thereby affecting the expression levels of α-SMA, eNOS, cell autophagy and apoptosis. Despite miR-301a-3p/PTEN and miR-301a-3p/TLR4 signaling pathway needing further investigation, our ndings suggest that miR-301a-3p should be considered as a new therapeutic target for ED treatment. The study has been examined and certi ed by the Ethics Committee of Shanghai Ninth People's Hospital of Shanghai Jiao Tong University, and informed consent was obtained from all participants included in the study, in agreement with institutional guidelines.

Consent for publication
All authors have agreed to publish this manuscript.

Availability of data and materials
The data generated or analyzed during this study are included in this article, or if absent are available from the corresponding author upon reasonable request.

Figure 6
A and C) Results of rno-miR-301a-3p, TLR4 (wt and mut), and PTEN (wt and mut) sequencing. B and D) Luciferase assay of TLR4 (wt and mut), and PTEN (wt and mut) transfected with miR-301a-3p mimic. E) Expression levels of TLR4 and PTEN in control, miR-NC, miR-301a-3p groups as measured by RT-qPCR. F) Protein levels of TLR4 and PTEN in the three groups as measured by Western blotting. Data are expressed as mean ± SD (n = 6; ***p <0.001).