Differences in adipose-derived stem cells from rats with various health statuses and their effects in diabetic rats with erectile dysfunction

Abstract

It is essential to use high-quality adipose-derived stem cells (ADSCs) for clinical applications. The functional characteristics of ADSCs differ in the presence of various health conditions. Specifically, recent studies of the therapeutic effects of ADSCs from individuals with diabetes and no erectile dysfunction (NED-ADSCs), ADSCs from those with diabetes and erectile dysfunction (ED-ADSCs), and normal ADSCs have generated conflicting results. Therefore, we aimed to provide evidence for the clinical utility of ADSCs. Diabetes mellitus (DM) was induced in rats by the intraperitoneal injection of streptozotocin (STZ; 40 mg kg⁻¹) following 4 weeks of high-fat diet feeding. After 12 weeks, erectile dysfunction was confirmed using the apomorphine (80 µg kg⁻¹) test, and those with diabetes mellitus-related erectile dysfunction (DMED) were randomly assigned to treatment groups. Each group was administered 1 × 10⁶ allogeneic ADSCs by injection into the corpus cavernosum; then, erectile function and penile histology were evaluated 28 days later. Of the three ADSCs types injected, normal ADSCs exhibited the strongest proliferation and differentiation capabilities. Following ADSCs treatment, there was an increase in smooth muscle cell number and a higher muscle-to-collagen fiber ratio. The expression of neuronal nitric oxide synthase (nNOS) in the dorsal nerve significantly increased. In addition, proteomic analysis revealed that heat shock protein expression was very low in the diabetic rats, but upregulated by stem cell therapy, which may represent a mechanism for the beneficial effects of ADSCs. Therefore, men with diabetes should consider the early preservation of ADSCs to help prevent the onset of erectile dysfunction (ED).

INTRODUCTION

Erectile dysfunction (ED) is the most common form of sexual dysfunction and has a pathophysiology that involves endothelial, neural, hormonal, and psychological factors. Diabetes induces both central and peripheral nerve damage and leads to endothelial dysfunction,1,2,3,4,5 significantly increasing the risk of ED. Previous studies have shown that the prevalence of ED in patients with diabetes ranges from 52.9% to 81.1%.6,7,8,9,10,11,12 Phosphodiesterase type 5 inhibitors (PDE5is) are the first-line treatments for ED and are effective and safe in patients with diabetes mellitus-related erectile dysfunction (DMED).13,14,15 However, their efficacy is lower in people with diabetes. In some patients with DMED, the dysfunction of specific molecular or neural signaling pathways limits the effectiveness of PDE5is, which cannot have their effects through the upstream nitric oxide (NO) pathway.

The emergence of adipose-derived stem cells (ADSCs) offers a promising alternative. ADSCs are easy to isolate and culture, and their differentiation and regenerative ability can lead to improvements in the function of epidermal and smooth muscle cells in the penis. Moreover, through the paracrine secretion of neurotrophic factors, ADSCs can inhibit apoptosis and promote nerve repair.5,16,17 However, the angiogenic potential of mesenchymal stem cells can be compromised by various risk factors and chronic diseases.18 In patients with coronary artery disease and type 2 diabetes mellitus (T2DM), ADSCs exhibit lower angiogenic activity. Although they retain the ability to differentiate into adipocytes and osteoblasts, their proliferative activity is higher, and their shortened telomeres indicate replicative senescence in vitro, which limits the efficacy of autologous ADSCs-based therapies.

The therapeutic efficacy of stem cells also depends on the stage of the disease. Under hyperglycemic conditions, the high blood glucose concentrations impair stem cell function. In the early stages of diabetes, myogenic stem cells (MDSCs) improve the smooth muscle-to-collagen ratio in the corpus cavernosum of rats with long-term T2DM and increase NO production in neurons and endothelial cells. However, MDSCs lose their tissue repair capacity and are ineffective as autologous grafts in the presence of advanced diabetes.19 In vitro study20 has also shown that MDSCs derived from patients with long-term T2DM exhibit cellular damage that renders autologous stem cell therapy ineffective. The effects of diabetes on ADSCs mirror those on MDSCs: diabetes alters the ADSCs,21 by impairing their ability to form vascular networks both in vitro and in vivo.

To determine whether ADSCs derived from individuals with different health conditions differ in their behavior and therapeutic efficacy for DMED, we studied Sprague Dawley (SD) rats with various health statuses. ADSCs were isolated from rats with different stages of DMED to simulate stem cells from patients with distinct health statuses, with the aim of exploring the mechanisms whereby ADSCs have beneficial effects in patients with DMED.

MATERIALS AND METHODS

Animals and grouping

Twenty male SD rats (weight: 200 ± 20 g) were obtained from the Specific Pathogen-Free Animal Center of Dalian Medical University (Dalian, China). The experimental protocol was approved by the Institutional Animal Care and Ethics Committee of Dalian Medical University (Approval No. AEE24011). All procedures were compliant with National Institutes of Health (NIH) guidelines. Following 4 weeks of consumption of a high-fat and high-sucrose diet (Mediscience, Yangzhou, China), the rats were administered either streptozotocin22 (STZ; MedChemExpress, Monmouth Junction, NJ, USA; 40 mg kg⁻¹, n = 14) or citrate buffer solution (Macklin, Shanghai, China; control group, n = 6) by intraperitoneal injection. The fasting blood glucose concentrations of the rats were measured 72 h later, and those with values more than 16.7 mmol l⁻¹ were defined as having DM. After 12 weeks, an erectile response was pharmacologically induced by the subcutaneous injection of apomorphine (APO)23 (Sigma-Aldrich, St. Louis, MO, USA; 80 µg kg⁻¹). Three animals per group in those with erectile dysfunction (ED), those with no erectile dysfunction (NED), and normal controls (Normal) were euthanized. ADSCs were obtained from the inguinal fat pads, and penile tissue was collected for proteomic analysis.

A separate cohort of 50 male SD rats (weight: 200 ± 20 g) were randomly allocated to two groups: an STZ-treated group (n = 40, 40 mg kg⁻¹ STZ in citrate buffer injected intraperitoneally) and a vehicle control group (n = 10, citrate buffer only injected). DMED was induced using an established method as reported.22,23 At 12 weeks of age, the 24 rats with ED were randomly allocated to four groups (n = 6 per group): a group that received phosphate-buffered saline (PBS) injection (DMED + PBS group), a group that received ADSCs isolated from rats with DMED (DMED + ED-ADSCs group), a group that received ADSCs isolated from diabetic rats without ED (DMED + NED-ADSCs group), and a group that received ADSCs isolated from normal rats (DMED + ADSCs group). A Normal + PBS control group (6 of 10) was administered PBS injections.

Extraction, culture and identification of ADSCs

After anesthesia, inguinal fat was harvested from the rats and minced into approximately 1-mm³ pieces. A digestion solution containing 1 ml of type I collagenase17 (10×; Sigma-Aldrich) and 10 ml of Hanks solution (Brunei, Fuzhou, China) was added, and the mixture was incubated in a 37°C water bath for 40 min, with vigorous shaking for 20 s every 10 min. After digestion, the mixture was centrifuged at 4°C and 277g (Sorvall ST1R Plus; Thermo Fisher, Waltham Mass, MA, USA) for 5 min, and the resulting cell pellet was resuspended in a culture bottle. A complete medium (89% Dulbecco’s modified Eagle’s medium/F-12, Sigma-Aldrich; 10% fetal bovine serum, Sigma-Aldrich; and 1% double antibiotics, HyClone, Logan, UT, USA) was added. The cells were cultured at 37°C in a 5% CO₂-containing incubator, and the medium was changed after 2 h.

The ADSCs from individuals with diabetes and no erectile dysfunction (NED-ADSCs) and individuals with diabetes and erectile dysfunction (ED-ADSCs) and normal ADSCs were used after their third passage. The expression of the stem cell surface markers – CD11b, CD29, CD45, and CD9023 (eBioscience, San Diego, CA, USA) were analyzed,15 and a growth curve (Ncmbio, Suzhou, China) was plotted. The cell cycle was assessed using propidium iodide staining (4abio, Suzhou, China). Adipogenic differentiation was induced in vitro for 16 days, and then, Oil Red O (Beyotime, Shanghai, China) staining was performed.24 Osteogenic differentiation was also induced for 20 days, and alizarin red (Cyagen, Santa Clara, CA, USA) staining24 was performed.

Evaluation of erectile function

Erectile function was evaluated 4 weeks after the ADSCs injection. Sodium pentobarbital25 (Sigma-Aldrich; 40 mg kg⁻¹) was administered intraperitoneally. After the onset of anesthesia, a 26-gauge indwelling needle (without a steel core, filled with heparin; Weigao Group Co., Ltd., Weihai, China) was inserted into the left common carotid artery for the measurement of mean arterial pressure (MAP), and a 24-gauge needle was inserted into the corpus cavernosum to measure intracavernous pressure (ICP). The pelvic stellate ganglion was exposed, and the cavernous nerve was isolated. Bipolar electrodes were used for stimulation at 5 V and 25 Hz for 2 ms (Chengdu Taimeng Software Co., Ltd., Chengdu, China) for 1 min at 5-min intervals. The ratio of maximal ICP to the MAP (MaxICP/MAP)25,26 was then calculated to assess erectile function.

Histological evaluation

Hematoxylin and eosin (H&E) staining, Masson’s trichrome staining, and immunochemistry (IHC) were performed according to the manufacturers’ instructions. For H&E staining, rat penile tissue was fixed in 10% formalin (Beyotime) for 24 h, dehydrated, embedded in paraffin, and sectioned at 3-µm thickness. Sections were stained with H&E, and the morphology of the corpus cavernosum and the distribution of smooth muscle cells were evaluated using an upright microscope (Leica, Hessen, Germany).

For Masson’s trichrome staining, paraffin sections were dehydrated with graded concentrations of alcohol and then stained using a Masson’s trichrome staining kit (Coolaber, Beijing, China). The muscle fibers appeared red, and collagen fibers appeared blue. Images were captured using an upright microscope (Leica), and the smooth muscle-to-collagen ratio in the corpus cavernosum was quantitatively analyzed using ImageJ software (Media Cybernetics, Rockville, MD, USA).

The penile corpus cavernosum tissue was fixed, dehydrated, permeabilized, embedded, sectioned, and stained for ultrastructural examination using a transmission electron microscope (JEOL Ltd., Tokyo, Japan).

To evaluate neuronal nitric oxide synthase (nNOS) expression in the dorsal nerve of the penis, paraffin sections were incubated with anti-nNOS antibody (1:100; Proteintech, Wuhan, China), followed by the appropriate secondary antibody (1:5000; Proteintech). The nNOS content, total dorsal nerve area, and area of nNOS-positive fibers were assessed using the upright microscope (Leica).

Western blot

Animal tissue samples were powdered under liquid nitrogen, lysates were prepared, and the constituent proteins were separated by gel electrophoresis (Bio-Rad, Hercules, CA, USA) and transferred to polyvinylidene fluoride membranes. The membranes were blocked in 5% skim milk for 1 h on a shaker and then incubated overnight with primary antibodies against endothelial nitric oxide synthase (eNOS; 1:1000; Proteintech), alpha-smooth muscle actin (α-SMA; 1:1000; Proteintech), ras homolog family member A (RhoA,1:1000, Proteintech), or Rho-associated protein kinase 2 (ROCK2; 1:1000; Proteintech). After incubation with secondary antibodies (1:5000; Proteintech), specific protein bands were visualized using enhanced chemiluminescence reagents. Beta-actin (β-actin) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were used as loading controls, and densitometric analysis was performed using ImageJ software (Media Cybernetics).

Proteomics

Frozen rat penile tissue was powdered under liquid nitrogen and lysed ultrasonically. The lysates were then centrifuged at 12 000g for 10 min, and the supernatants were collected. Their protein concentrations were determined using a BCA kit (Beyotime). The proteins were enzymatically digested according to the manufacturer’s instructions, and the peptides were separated by liquid chromatography, ionized using a capillary ion source, and analyzed using a mass spectrometer (timsTOF Pro; Bruker, Billerica, MA, USA).

Metagenomic DNA extraction and sequencing

Microbial genomic DNA was extracted using an Omega Mag-Bind Soil Kit (Omega Bio-Tek, Norcross, GA, USA), according to the manufacturer’s instructions. The DNA concentration was measured using a Qubit™ 4 fluorometer (Q33238; Invitrogen, Carlsbad, CA, USA), and its integrity was assessed by agarose gel electrophoresis. The DNA was processed using an Illumina TruSeq Nano DNA LT Library Preparation Kit (Illumina, San Diego, CA, USA) to construct a metagenomic shotgun sequencing library with lengths of 400 bp.

Statistical analyses

All experimental data were analyzed using Prism version 9 (GraphPad Software, San Diego, CA, USA) and were expressed as mean ± standard deviation (s.d.). Differences between the two groups were assessed using Student’s t-test, and differences among multiple groups were evaluated using one-way analysis of variance (ANOVA). P < 0.05 was considered to indicate statistical significance.

RESULTS

Characterization of the ADSCs from the different sources

The ADSCs, ED-ADSCs, and NED-ADSCs were all spindle shaped (Figure 1a) and exhibited whorl-like growth patterns. Flow cytometry analysis showed that all the ADSCs types were positive for CD90 and CD29 and negative for CD45 and CD11b (Figure 1b). In vitro differentiation experiments confirmed that all three types of ADSCs showed Oil Red O staining after adipogenesis had been induced (Figure 1c), and Alizarin red staining after osteogenesis had been induced (Figure 1d).

Figure 1.

Phenotypic characterization and proliferative capacity evaluation of ADSCs from rat inguinal fat pads (ADSCs, NED-ADSCs, and ED-ADSCs). (a) Morphology of ADSCs, NED-ADSCs, and ED-ADSCs. (b) Flow cytometry analysis of surface markers (CD90, CD29, CD45, and CD11b) in the third-passage ADSCs, NED-ADSCs, and ED-ADSCs. (c) Oil Red O staining after adipogenic induction for 16 days. (d) Alizarin Red staining after osteogenic induction for 20 days. (e) Cell cycle in the third-passage ADSCs, NED-ADSCs, and ED-ADSCs (n = 3 in each group). (f) Growth curves were generated for ADSCs, NED-ADSCs, and ED-ADSCs at passage 4 (n = 3 in each group). (g) Growth curves were generated for normal ADSCs at passages 3, 5, and 7 (P3, P5, and P7; n = 3 in each group). DMED: diabetic mellitus erectile dysfunction; PBS: phosphate-buffered saline; ADSCs: adipose-derived stem cells; NED-ADSCs: diabetic non-erectile dysfunction ADSCs; ED-ADSCs: diabetic erectile dysfunction ADSCs; PI-A: propidium iodide fluorescence-area; OD: optical density; PE: phycoerythrin; APC: allophycocyanin; PerCP: peridinin-chlorophyll-protein complex; FITC: fluorescein isothiocyanate

The normal ADSCs proliferated faster than the equivalent ED-ADSCs and NED-ADSCs (Figure 1e and 1f). The third-generation ADSCs showed faster growth than the fifth-generation and the seventh-generation ADSCs (Figure 1g).

Effects of ADSCs on erectile function

There were no significant differences in the body masses or blood glucose concentrations of the groups either before or after ADSCs injection (all P > 0.05; Figure 2a and 2b). After 4 weeks of treatment, all three types of ADSCs improved erectile function in DMED rats to varying degrees. The DMED + ADSCs group showed the most substantial improvement, including an increase in the number of erections (Figure 2c). ED-ADSCs did not significantly improve MaxICP/MAP in DMED rats (P > 0.05), whereas the ADSCs and NED-ADSCs did significantly increase MaxICP/MAP (P < 0.001 and P < 0.01, respectively), as shown in Figure 2d and 2e.

Figure 2.

Pre- and post-treatment changes in blood glucose and weight following stem cell therapy and its effect on erectile function. Comparative evaluation of (a) body weight and (b) blood glucose parameters before and after stem cell therapy (n = 6 in each group). (c) Erection count in the APO-induced rat model post-stem cell therapy (n = 6 in each group). (d) Recordings of ICP and MAP response to electrical stimulation of the cavernous nerve. The red cure and green bar represent the ICP values and the duration of cavernous nerve stimulation (60 s), respectively. (e) Comparison of the ratio of the maximum intracavernous pressure to the mean arterial pressure post-stem cell therapy (MaxICP/MAP, n = 3 in each group). ^*P < 0.05; ^**P < 0.01; ^***P < 0.001; ^****P < 0.001. MaxICP: maximum intracavernous pressure; MAP: mean arterial pressure; DMED: diabetic mellitus erectile dysfunction; PBS: phosphate-buffered saline; ADSCs: adipose-derived stem cells; NED-ADSCs: diabetic non-erectile dysfunction ADSCs; ED-ADSCs: diabetic erectile dysfunction ADSCs; ICP: intracavernous pressure; APO: apomorphine.

Effects of the ADSCs from different sources on the penile corpus cavernosum

H&E staining revealed a diabetes-induced reduction in the smooth muscle cell density in the corpus cavernosum of the rats. However, stem cell treatment increased the smooth muscle cell density (Figure 3a). Furthermore, the therapeutic effects of the stem cell treatment were confirmed by Masson’s trichrome staining and immunohistochemistry analysis (Figure 3b and 3c).

Figure 3.

Therapeutic effects of stem cell therapy on corpus cavernosum histomorphology and molecular profile. (a) H&E staining of corpus cavernosum tissue post-stem cell therapy. (b) Masson staining of corpus cavernosum tissue post-stem cell therapy. Smooth muscle and collagen are stained red and blue, respectively. (c) Immunohistochemical evaluation of nNOS expression in the dorsal penile nerve post-stem cell therapy. (d) Ultrastructural analysis of the penile corpus cavernosum by electron microscopy post-stem cell therapy. Green arrows: mitochondria; red arrows: endoplasmic reticulum; yellow arrows: nuclei. (e) Quantitative analysis of nNOS-positive fibers in the dorsal penile nerve (n = 3 in each group). (f) Quantitative comparison of smooth muscle and collagen composition in corpus cavernosum (n = 3 in each group). (g) Expression of representative proteins (a-SMA, ROCK2, RhoA, and ROCK2) in the penile corpus cavernosum. (h) Western blot quantification of α-SMA, eNOS, RhoA, and ROCK2 protein expression levels in rat corpus cavernosum tissues post-stem cell therapy (n = 3 in each group). ^*P < 0.05; ^**P < 001; ^***P < 0.001; ^****P < 0.0001. H&E: hematoxylin and eosin; nNOS: neuronal nitric oxide synthase; α-SMA: alpha-smooth muscle actin; ROCK2: Rho-associated protein kinase 2; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; eNOS: endothelial nitric oxide synthase; RhoA: ras homolog family member A; β-actin: beta-actin; DMED: diabetic mellitus erectile dysfunction; PBS: phosphate-buffered saline; ADSCs: adipose-derived stem cells; NED-ADSCs: diabetic non-erectile dysfunction ADSCs; ED-ADSCs: diabetic erectile dysfunction ADSCs.

Electron microscopy revealed less fibrosis and superior endothelial morphology following ADSCs injection. The mitochondria, endoplasmic reticulum, and nuclei were better defined, implying a restoration of organelle function (Figure 3d). Masson staining showed that DM reduced the ratio of the areas of smooth muscle and collagen fibers in the corpus cavernosum (P < 0.0001; Figure 3e), but that this ratio significantly increased after ADSCs and NED-ADSCs treatment (P < 0.001 and P < 0.01, respectively, Figure 3e). Immunohistochemistry revealed a larger number of nNOS-positive fibers in the dorsal penile nerve after ADSCs treatment (Figure 3f), and the nNOS contents of all the ADSCs-treated groups were higher than those of the DMED + PBS group (all P < 0.05; Figure 3f).

Western blot analysis showed that the α-SMA and eNOS expression in the corpus cavernosum was significantly increased by ADSCs treatment (both P < 0.05; Figure 3g and 3h). DM induced RhoA and ROCK2 expression (both P < 0.01; Figure 3g and 3h), and this was reduced by ADSCs treatments (both P < 0.05; Figure 3g and 3h).

Proteomics

Proteomic analysis of the penile corpus cavernosum of nine rats from the Normal, NED, and ED groups identified a total of 5975 proteins (Figure 4a). Of these, 561 proteins were proteins were differentially expressed in the ED vs NED and NED vs normal groups (Figure 4b and 4c).

Figure 4.

Quantitative proteomic profiling of rat corpus cavernosum following stem cell treatment. (a) Hierarchical clustering heatmap analysis of global protein expression profiles in the rat corpus cavernosum across untreated groups. (b) Comparative analysis of significantly upregulated and downregulated proteins across untreated groups, with Venn diagrams illustrating shared and unique protein expression profiles. (c) Volcano plot analysis identified 317 significantly dysregulated proteins in the rat corpus cavernosum across untreated groups. (d) Hierarchical clustering analysis of differentially expressed proteins in the corpus cavernosum of rats across treatment groups, visualized by heatmap. (e) Volcano plot analysis of all differentially upregulated and downregulated proteins in the corpus cavernosum of rats across treatment groups. (f) Representative differentially expressed proteins (selected from 317 significantly dysregulated proteins, n = 3 in each group). (g) Representative dysregulated proteins identified following stem cell therapy (n = 5 in each group). ^*P < 0.05; ^**P < 0.01; ^***P < 0.001; ^****P < 0.001. Hsp47: heat shock protein 47; Sparc: secreted protein acidic and rich in cysteine; Ppp1r12a: phosphatase protein phosphatase 1 regulatory subunit; MYPT1: myosin phosphatase target subunit 1; DMED: diabetic mellitus erectile dysfunction; PBS: phosphate-buffered saline; ADSCs: adipose-derived stem cells; NED-ADSCs: diabetic non-erectile dysfunction ADSCs; ED-ADSCs: diabetic erectile dysfunction ADSCs; ED: erectile dysfunction group pre-stem cell therapy; NED: non-erectile dysfunction group pre-stem cell therapy; Normal: normal control group pre-stem cell therapy.

A quantitative proteomic analysis of penile tissue from 25 rats in all the treatment groups identified 7262 proteins. Heat shock protein 47 (Hsp47, also known as Serpinh1), protein-lysine 6-oxidase (Lox), and secreted protein acidic and rich in cysteine (Sparc) were significantly downregulated in the rats with DM, and putative protein-lysine deacylase ABHD14B (Abhd14b), prolargin (Prelp), and vitrin (Vit) were significantly upregulated (Figure 4d). The volcano plot showed that 559 proteins were upregulated and 561 downregulated in the presence of DM. Following treatment with ED-ADSCs, NED-ADSCs, or ADSCs, the number of differentially expressed proteins gradually increased compared to the DMED + PBS group (Figure 4e).

Notably, the Hsp47, the Sparc, and the myosin phosphatase protein phosphatase 1 regulatory subunit (Ppp1r12a) showed significant differences in expression between the ED and NED groups (all P < 0.05; Figure 4f). In addition, Serpinh1 expression significantly differed after treatment with NED-ADSCs or ADSCs (P < 0.001 and P < 0.0001, respectively, Figure 4g).

Gut microbiota

ADSCs treatment improved the gut microbial composition of the DM rats. Specifically, treatment caused significant decreases in the Shannon and Simpson indices (P < 0.01 and P < 0.05, respectively, Figure 5a), implying an increase in microbial diversity after the stem cell intervention.

Figure 5.

Fecal microbiota analysis in rats following stem cell therapy. (a) Alpha diversity: Shannon and Simpson index. (b) Beta diversity. (c) Relative abundance analysis of bacterial phyla in gut microbiota. (d) Differential microbial taxa at the phylum level. (e) Genus-level relative abundance of the gut microbiota. (f) Differential microbial taxa at the genus level. (g) Taxonomically distinct bacterial groups identified by LEfSe analysis. ^*P < 0.05; ^**P < 0.01. DMED: diabetic mellitus erectile dysfunction; PBS: phosphate-buffered saline; ADSCs: adipose-derived stem cells; NED-ADSCs: diabetic non-erectile dysfunction ADSCs; ED-ADSCs: diabetic erectile dysfunction ADSCs; p: phylum; c: class; o: order; f: family; g: genus; s: species; LEfSe: linear discriminant analysis effect size; PC1: principal component 1; PC2: principal component 2; LDA: linear discriminant analysis.

Principal coordinates analysis revealed significant differences in the microbial composition of the rats in the various groups (P < 0.01; Figure 5b). At the phylum level, the abundances of the Firmicutes and Bacteroidota were lower in the control group compared to the other groups (P < 0.05 and P < 0.01, respectively; Figure 5c and 5d). At the genus level, the abundance of Vermiculatibacterium and Prevotella were also lower (P < 0.01 and P < 0.05, respectively, Figure 5e and 5f). Linear discriminant analysis effect size (LEfSe) showed that there were significant differences in intestinal flora at different classification levels (P < 0.05, Figure 5g).

DISCUSSION

The pathogenesis of DMED is complex, involving both the central and peripheral nervous systems, and a role of endothelial microorganisms has recently emerged.1 It is widely accepted that diabetic neuropathy, altered neurotransmitter levels, and vascular endothelial dysfunction are central to the development of DMED.1,2,3,4,5 In patients with diabetes, the disordered glucose metabolism leads to sorbitol accumulation, peripheral neuropathy, and abnormalities in penile nerve conduction, resulting in a reduction in NO production, lower cyclic GMP levels in smooth muscle, and impaired sexual sensation and nerve signaling, which all contribute to DMED.27,28 Endothelial dysfunction also plays a critical role, because it leads to decreases in the synthesis and bioactivity of eNOS,28 an increase in oxidative stress,29 increases in the concentrations of advanced glycation end products,30 and impairments in angiogenesis and vascular repair mechanisms,2,31 all of which contribute to an impairment in smooth muscle relaxation and ED.

ADSCs have gained significant attention because of their ready availability, ease of extraction, potential for multidirectional differentiation, and ethical acceptability, especially for DMED treatment. Recent research regarding the mechanisms underpinning the use of ADSCs to treat DMED has primarily focused on the following: (1) the repair of damaged penile vascular endothelium and the increase in vascular endothelial cell numbers;17,32 (2) the recruitment of ADSCs to peripheral nerves and the secretion of neurotrophic factors via paracrine signaling, which promote nerve repair;3,5 and (3) the regulation of smooth muscle and collagen to reduce fibrosis in the penile corpus cavernosum.33,34

Previous studies have shown that diabetes impairs precursor cell function, including that of ADSCs, leading to lower proliferation, growth factor secretion, and angiogenic potential.18,21 Kovanecz et al.19 found that, during the early stages of diabetes, treatment with MDSCs increases the NO levels in nerves and endothelial cells in the corpus cavernosum. However, during the later stages, MDSCs treatment led to inflammation and fibrosis and therefore a failure to repair the damaged tissue. Quaade et al.35 compared ADSCs obtained from non-diabetic rats and those with early diabetes and found that although the latter showed slightly lower levels of proliferation, their proteomic profiles were largely similar to the former, and both types of ADSCs restored the erectile function of diabetic rats. In the present study, we found that the proliferation adipogenic and osteogenic potential, of the NED-ADSCs and ED-ADSCs were lower than those of the ADSCs, consistent with the findings of Quaade et al.35 and Rennert et al.21 These results confirm that diabetes impairs adipose stem cell function and that this impairment progressively worsens as the disease advances.

To characterize the differences in the therapeutic effects of ADSCs derived from donors with different health statuses, we studied an animal model. We found that the erectile function of DMED rats was improved to varying degrees by 4 weeks of treatment with the three types of stem cells. Following treatment with ED-ADSCs, NED-ADSCs, and ADSCs, H&E and Masson staining revealed increases in the density of smooth muscle cells in the corpus cavernosum and a progressive increase in the ratio of muscle to collagen fibers. In addition, electron microscopy showed the presence of fewer collagen fibers, and less endothelial cell proliferation and hypertrophy, with the most pronounced therapeutic effect being present in DMED + ADSCs-treated rats.

Western blot analysis showed an increase in α-SMA expression and a decrease in eNOS expression in the treated rats. In addition, the protein expression of RhoA and ROCK2 was high in the penile corpus cavernosum of DMED rats, consistent with previous findings, whereas there was no significant difference in the expression of ROCK2,36 consistent with the present findings. Therefore, we speculate that the various types of ADSCs may ameliorate DMED by modulating the RhoA/ROCK2 signaling pathway.

The exact mechanisms by which ADSCs treat ED remain unclear. The present proteomic study revealed high Hsp47 expression after ADSCs treatment, which was associated with an improvement in erectile function, suggesting that Hsp47 may be a key mediator of the effects of stem cell therapy on ED. Hsp47 expression was lower after DM was induced and slightly higher after the ADSCs treatment, although the difference was not statistically significant. However, treatment with NED-ADSCs or ADSCs led to a significant increase in Hsp47 expression.

The increase in Hsp47 correlated with the changes in expression of collagen-specific genes, implying a promotion of collagen synthesis, which might aid ED treatment. Although the precise role of Hsp47 in procollagen production remains unclear, several mechanisms have been proposed. These mechanisms include that Hsp47 acts as a molecular chaperone, facilitating the folding and assembly of procollagen molecules, retaining unfolded proteins in the endoplasmic reticulum, and assisting with their correct folding for transport to the Golgi apparatus.37,38 Changes in Hsp47 expression have been shown to be accompanied by alterations in collagen gene expression, suggesting that they might be co-regulated.39 Therefore, we propose that ADSCs may improve erectile function through a mechanism involving Hsp47 and collagen.

DM has also been shown to be associated with lower microbial diversity. However, in the present study, there were no significant differences in the abundance of bacterial phyla or genera, possibly because of the short duration of the study.

In summary, normal ADSCs and NED-ADSCs have therapeutic potential for patients with ED, with normal ADSCs being the most effective, whereas ED-ADSCs are less effective. Because DM has a genetic predisposition40 and T2DM is closely linked to ED, it is important for healthy individuals to preserve ADSCs early, before the onset of DM, for potential subsequent ED treatment. Even those collected during the early stages of DM, before ED, can be used therapeutically, although they may be less effective than those collected from healthy individuals. For patients with established ED, the early extraction and preservation of ADSCs may still be therapeutically useful, albeit to a lesser extent.

AUTHOR CONTRIBUTIOS

TJ and HJ conceived and designed the experiment of this study. GCL and LZ performed experiments and data analysis and wrote the manuscript. LTW assisted with experiments. ZYL participated in the study design. All authors read and approved the final manuscript.

COMPETING INTERESTS

All authors declare no competing interests.