Implications for Differentiation of Endogenous Stem Cells: Therapeutic Effect from Icariside II on a Rat Model of Postprostatectomy Erectile Dysfunction

Abstract

Self-renewal and differentiation of endogenous stem cells (SCs) are essential for adult tissue homoeostasis and intrinsic healing capacity. In this study, we hypothesize that penis contains a small population of endogenous SCs, which might help rejuvenation of damaged erectile function. In this study, 60 newborn male rats were intraperitoneally injected with 5-ethynyl-2-deoxyuridine (EdU; 50 mg/kg) for the purpose of tracking endogenous SCs. Twelve weeks later, 48 rats underwent bilateral cavernous nerves injury and were randomized into gavage feeding of solvent (vehicle group) or icariside II (0.5, 1.5, and 4.5 mg/kg/day, respectively). Twelve sham-operated rats received vehicle treatment and served as control. The treatments were continued for 4 weeks followed by a washout period of 72 h. Results showed that ICA II treatment significantly restored erectile function and effectively prevented distortion of normal neural anatomy, smooth muscle atrophy, and collagen deposition compared with the vehicle group. The numbers of label-retaining cells (LRCs) coexpressing EdU and differentiated phenotypes (smooth muscle marker α-SMA or Schwann cell marker S100) were significantly higher in the three ICA II-treated groups than those in vehicle group in a dose-dependent manner. In addition, the changing trend of p38 mitogen-activated protein kinase (MAPK) activity in the penis between groups was same as that of the number of differentiated LRCs. Together, these results suggest that the underlying mechanisms of ICA II in ameliorating erectile function and pathological changes appear to involve enhanced endogenous SCs differentiation, which might be regulated by p38 MAPK signaling pathway.

Introduction

Several treatment approaches are currently available for erectile dysfunction (ED) patients, including oral phosphodiesterase type 5 inhibitors (PDE5-Is), intracavernous injection of erectogenic agents, vacuum device, and penile prosthesis implantation. Although the majority of ED patients can be satisfactorily treated with PDE5-Is, the average success rate (43%) of these drugs is significantly lower and varies distinctly in ED patients after radical prostatectomy (RP) [1,2]. In addition, PDE5-Is can only temporarily relieve the symptoms and, therefore, must be taken before sexual intercourse. Thus, several alternative treatment options are still being investigated, including stem cell (SC) therapy, herbs, genetic modifications, and hyperbaric oxygen.

Previous studies have demonstrated that SC had significant benefits for the treatment of ED. However, the traditional SC strategies are restricted by ethical and regulatory issues, limited availability of SC sources, poor cellular retention, and invasive procedures. Stem/progenitor cells, resides in various tissues, play an important role in tissue maintenance and repair. Even without any therapeutic intervention, human body has a robust self-healing capability to repair the damaged organs through endogenous SCs. Thus, SC-based ED therapy should not be limited to a supply-side approach. Mobilization of the body's innate ability to heal cavernous nerves (CN) injury by activating endogenous SCs is likely a meritorious approach in future ED treatment. Recently, we employed the label-retaining cells (LRCs) strategy to identify potential SCs in the penis. Our results showed that the penile LRCs are mainly distributed within the subtunic and perisinusoidal spaces [3].

There are multiple signaling pathways that have been established as SCs regulators, such as p38 mitogen-activated protein kinase (MAPK) and Notch pathways. In particular, p38 pathway is essential for the differentiation of several different stem/progenitor cells. Jones et al. [4] reported that satellite cells are activated from quiescence and undergo myogenic differentiation through the activation of p38 MAPK. Furthermore, adult neural differentiation also can be promoted by the activation of p38 MAPK pathway [5]. However, p38 exhibits contradictory functions in different cell lineages and maturation stages.

Herba Epimedii, a traditional Chinese medicine, has been traditionally used for the treatment of ED for centuries in East Asian countries. Icariin (C₃₃H₄₀O₁₅, 676.67) is one of the most abundant flavonoids in Herba Epimedii [6,7]. Our previous studies [8 –10] have demonstrated that icariin possess notable erectogenic and neurotrophic effects, both in vivo and in vitro models. ICA II (C₂₇H₃₂O₁₀, 514.54), the bioactive form of icariin, lacks a glucose moiety at C-7 and is more bioavailable than icariin [11,12]. Interestingly, a recently published study reported that ICA II can activate p38 MAPK pathway [13].

Endogenous SCs are rare cells, residing in specific SC niches in many organs. Due to the lack of specific SC markers, tissue-resident SCs are not easy to identify. A reliable approach to identify endogenous SCs and their location is based on their asymmetric division, slow cycling, and quiescent nature by using LRCs strategy [14 –16]. Rapidly dividing transit-amplifying cells (TACs) dilute their DNA label with cell divisions. LRCs are the cells that can retain a DNA label after a prolonged period due to infrequent cycling, and have been identified as somatic SCs in some organs [17,18]. 5-Ethynyl-2-deoxyuridine (EdU) is a thymidine analogue that can be incorporated into the newly synthesized DNA of replicating cells, and has been used to identify endogenous SCs [19].

In the present study, EdU were used for tracking the putative endogenous SCs in penis. Furthermore, we investigate the feasibility and underlying mechanisms of ICA II in the treatment of ED in a rat model of bilateral CN injury.

Materials and Methods

Animals

A total of 60 newborn male Sprague Dawley (SD) rats were obtained from the Animal Breeding Center at the Peking University Health Science Center. The experiments were approved by the Institutional Animal Care and Use Committee (IACUC) at our university. For the purpose of tracking putative endogenous SCs, each pup received intraperitoneal injection of EdU (Invitrogen) at a dosage of 50 mg/kg immediately after birth. At the age of 12 weeks, 12 SD rats were randomly selected as sham group (midline abdominal incision with no CN crush) and received vehicle treatment. The other 48 rats underwent bilateral CN crush as described previously [20], were randomly divided into four equal groups, and were treated with solvent (vehicle group) or ICA II (0.5, 1.5, and 4.5 mg/kg/day, respectively) by gavage feeding starting at the day of surgery. Treatment was continued for 4 weeks, followed by a washout period of 72 h before erectile function measurement. After functional testing, the animals were sacrificed and the penises were harvested for histology.

Measurement of intracavernous pressure and mean arterial pressure

Under 5% sodium pentobarbital, the bilateral CN were exposed through midline laparotomy. Two 24-gauge needles connected to PE-50 tubes with heparinized saline (200 IU/mL) were inserted into the corpus cavernosum (CC) and left abdominal aorta, respectively. The other end of the PE-50 tube was connected to a data acquisition system (MP150; Biopac Systems, Inc.). The stimulus parameters were 20 Hz, pulse width of 0.2 ms, 1.5 mA, and duration of 50 s. The intracavernous pressure (ICP) and mean arterial pressure (MAP) were measured simultaneously. The ratio of maximal ICP (mmHg) to MAP (mmHg) was calculated to normalize for variations in systemic blood pressure.

Immunofluorescence and Masson trichrome staining

Freshly dissected penis (mid-shaft portion) was fixed with 2% formaldehyde and 0.002% picric acid in 0.1 M phosphate buffer for 4 h, followed by overnight immersion in 30% sucrose. Tissues were frozen in optimum cutting temperature compound (Sakura Finetek) and stored at −80°C until use. Sections were cut at 5 μm and subjected to immunofluorescence. Primary antibodies were mouse anti-myelin basic protein (MBP) monoclonal antibody (1:400; Abcam), rabbit anti-neurofilament (1:200; Abcam), rabbit anti-S100 (1:100; Sigma), rabbit anti-neuronal nitric oxide synthase (nNOS, 1:400; Abcam), rabbit anti-α-SMA (1:400; Abcam) and mouse anti-endothelial cell antibody (RECA-1, 1:100; Abcam). Smooth muscle was stained with rabbit AlexaFluor-488 conjugated phalloidin (Sigma Aldrich). Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI; Invitrogen).

For evaluation of the number of nNOS-positive nerves, the two largest dorsal nerve branches, the two dorsal arteries, and two fields at the similar parts of the CC were selected. For evaluation of the number of total LRCs or differentiated LRCs, a field at the similar location of the CC and penile dorsal nerve were selected. The cells labeled with EdU (positive for differentiation phenotype or not) were counted as total LRCs. Cells positive for both EdU and differentiation phenotype (α-SMA, a smooth muscle marker; S-100, a Schwann cell marker; or RECA, a endothelial cell marker) were respectively counted as differentiated LRCs. Slides were then stained according to Masson's Trichrome staining protocol for the interstitial collagen deposition. Slides were photographed and recorded using a LEICA DFC 425 C digital microscope camera system (Leica). Computerized histomorphometric analysis was performed using the Image-Pro Plus 6.0 software (Media Cybernetics).

Western blotting

The cellular protein samples were prepared by homogenization of penile tissue in a lysis buffer containing 1% IGEPAL CA-630, 0.5% sodium deoxycholate, 0.1% sodium docecyl sulfate, aprotinin (10 μg/mL), leupeptin (10 μg/mL), and phosphate buffered saline. The cellular lysates from penis containing 20 μg of protein were electrophoresed in sodium dodecyl sulfate–polyacrylamide gel electrophoresis and then transferred to a polyvinylidene fluoride membrane (Millipore Corp.). Primary antibodies were rabbit anti-nNOS (1:1,000; Abcam), rabbit anti-α-SMA (1:1,000; Abcam), rabbit anti-p38 MAPK (1:1,000; CST), rabbit anti-phospho-p38 MAPK (1:1,000; CST), and rabbit anti-GAPDH (1:2,000; Santa Cruz). After the hybridization of secondary antibodies, the resulting images were analyzed with ChemiImager 4000 (Alpha Innotech Corporation).

Statistical analyses

Statistical analyses were performed with the SPSS 17.0 software. Data are expressed as mean±standard deviation. Multiple groups were compared using one-way analysis of variance followed by the Tukey HSD post hoc comparisons. A two-group comparison was done using t-test, and P<0.05 was considered significant.

Results

Assessment of erectile function affected by ICA II

Erectile function was assessed at day 31 postsurgery in all groups. CN crush injury resulted in a significantly decreased ICP/MAP ratio in the vehicle-treated group (0.24±0.01) compared to the sham-treated rats (0.93±0.02). Partial but significant amelioration of erectile function occurred in all ICA II-treated groups relative to vehicle-treated animals, in a dose-dependent fashion. The values of ICP/MAP ratio in the ICA II 1.5 group (0.57±0.02) and ICA II 4.5 (0.71±0.04) were significantly higher than that in the ICA II 0.5 (0.31±0.03) group. But the difference between the ICA II 1.5 group and ICA II 4.5 group did not attain statistical significance (P=0.135). In addition, the ICP/MAP ratios of the sham-treated rats were significantly higher than those in any ICA II-treated group. MAP did not differ significantly among groups (Fig. 1).

FIG. 1.

Evaluating erectile function. (A) ICP recordings after electrostimulation in each group. The upper gray curve represents the ICP values in response to nerve stimulation, and the gray bar below indicates duration of CN stimulation (50 s). (B) Left: ICP change from baseline to peak ICP; Right: ICP normalized over MAP. ICA II 0.5, 1.5, and 4.5, bilateral CN crush treated with ICA II at a dose of 0.5, 1.5, and 4.5 mg/kg/day, respectively. *P<0.05 versus the Vehicle group, ^# P<0.05 versus the ICA II 0.5 group, ^& P<0.05 versus the ICA II 1.5 group, ^@ P<0.05 versus the ICA II 4.5 group. CN, cavernous nerve; ICP, intracavernous pressure; MAP, mean arterial pressure; Sham, the Sham group (midline abdominal incision without CN crush); Vehicle, the Vehicle group (bilateral CN crush with vehicle treatment).

ICA II promote penile nerve regeneration

Transverse distal nerve sections were prepared and neuropathological changes were assessed by double-immunostaining for MBP and neurofilament (NF). In the sham-treated rats, penile dorsal nerves with individual myelin units (MBP-positive) were visible, each forming a 1:1 association with the axon (NF-positive). In the CN injury-induced rats with vehicle treatment, demyelination was clearly seen as fragmented myelin and unmyelinated axons. Axon degeneration was also evident, shown by axonal swelling vacuolization. Image analysis showed that there was a significant decrease in the MBP-positive area in vehicle-treated rats (32.7%±3.8%) after CN injury compared with the sham group (52.8±3.4), indicating Schwann cells loss. ICA II treatment in all groups preserved neural morphology, whereas the difference in the mean MBP% between the vehicle group and ICA II 0.5 group (36.4±3.7) did not attain statistical significance (P=0.222). In addition, the difference in the mean MBP% between the ICA II 1.5 (46.9±2.9) and ICA II 4.5 group (49.8±3.8) also did not attain statistical significance (P=0.345), although all their values were significantly higher than those in the vehicle group and ICA II 0.5 group. Neuropathological changes and comparisons between groups are shown in Fig. 2.

FIG. 2.

Neurodegeneration in the CN distal to the crush lesion. Cross sections of penile dorsal nerve are immunostained for MBP (red) and NF (green). In the sham group, MBP-positive myelin rings are visible and associated with NF-positive axons. Following CN injury, myelin and the axons degenerate. The typical nerve pathological changes included a distortion of normal nerve anatomy, axonal swelling (white arrows), and demyelination in the vehicle-treated group. ICA II treatment in all groups preserved neural morphology, indicating neural regeneration. Bar graph at the lower right corner: Semi-quantitative image analysis of the MBP-positive area, expressed as percentage of the area within the nerve. Original magnification: ×400. Images taken at a higher magnification (×1000) are shown in the insets. *P<0.05 versus the Vehicle group, ^# P<0.05 versus the ICA II 0.5 group. MBP, myelin basic protein; NF, neurofilament. Color images available online at www.liebertpub.com/scd

Regeneration of nNOS-positive nerves and nNOS expression were evaluated by immunofluorescent staining and western blot. CN crush resulted in a significant decrease of the nNOS content in the penis relative to the sham-treated rats. Following treatment, the nNOS expression in the penis was significantly higher in all ICA II-treated groups compared with vehicle-treated CN-injured animals in a dose-dependent manner (Fig. 3).

FIG. 3.

Evaluation of nNOS expression by immunofluorescence staining and western blot. (A) For evaluation of the nNOS-positive nerves, the largest dorsal nerve branches, dorsal arteries, and the fields at the similar part of the corpus cavernosum were selected. Phalloidin staining (green) for smooth muscle and immunostaining for nNOS (red). Original magnification: ×400. (B) Western blot analysis of nNOS; (C) semi-quantitative data of nNOS expression is shown in the bar chart. *P<0.05 versus the Vehicle group, ^# P<0.05 versus the ICA II 0.5 group, ^& P<0.05 versus the ICA II 1.5 group. nNOS, neuronal nitric oxide synthase; Pha, phalloidin. Color images available online at www.liebertpub.com/scd

ICA II improves penile pathological changes

Consistent with the previous observation, CN crush injury resulted in a significant decline of smooth muscle content compared with those sham-operated animals. The content of smooth muscle in the ICA II 1.5 and ICA II 4.5 groups, but not in the ICA II 0.5 group, was significantly higher than that in the vehicle-treated rats. There was no significant difference between the sham-operated group and the ICA II 4.5 group (P=0.064). Comparisons between groups are shown in Fig. 4.

FIG. 4.

Analysis of smooth muscle architecture and content by fluorescent Phalloidin staining and western blot. (A) The left panel shows an overview of the CC, original magnification: ×100. The sinus indicated by the white box is depicted in the corresponding×400 graphs in the right panel. (B) Western blot analysis of α-SMA expression; (C) semi-quantitative data of smooth muscle content is shown in the bar chart. *P<0.05 versus the Vehicle group, ^# P<0.05 versus the ICA II 0.5 group, ^& P<0.05 versus the ICA II 1.5 group. CC, corpus cavernosum. Color images available online at www.liebertpub.com/scd

Penile interstitial collagen deposition was evaluated qualitatively and semi-quantitatively by Masson's Trichrome staining (Fig. 5). Fibrosis was present following CN crush injury, as indicated by the significant increase in collagen staining in vehicle-treated CN-injured animals (0.051±0.006) compared with sham-operated rats (0.106±0.010). ICA II resulted in less interstitial collagen deposition in all treatment groups compared with the vehicle-treated group in a dose-dependent manner. A significant difference (P=0.049) was observed between the ICA II 0.5 (0.061±0.011) and ICA II 4.5 (0.099±0.013) group, whereas ICA II 1.5 (0.086±0.004) was not significantly different from the lowest (P=0.485) and highest doses (P=0.161).

FIG. 5.

Penile interstitial collagen deposition was evaluated by Masson's Trichrome staining. Smooth muscle is stained red and collagen is stained green, original magnification: ×100. Bar graph at the lower right corner depicts semi-quantitative data of the ratio of smooth muscle to collagen content in penile midshaft sections. *P<0.05 versus the Vehicle group, ^# P<0.05 versus the ICA II 0.5 group, ^& P<0.05 versus the ICA II 1.5 group. Color images available online at www.liebertpub.com/scd

ICA II treatment enhances EdU-LRCs to differentiate into smooth muscle cells and Schwann cells

In the sham-operated rats, the LCRs in the CC or penile dorsal nerves were rare, and only a tiny fraction of these cells coexpressed α-SMA (a smooth muscle cell marker), RECA (a endothelial cell marker) or S100 (a Schwann cell marker). There were more LRCs and LRCs coexpressing markers (α-SMA and S100) in the vehicle-treated rats with bilateral CN injury than those in the sham-operated animals; it may be associated with body's self-healing abilities. Although the numbers of LRCs in all CN injury groups (vehicle, ICA II 0.5, ICA II 1.5 and ICA II 4.5 group) were higher than that in the sham group, there were no significant differences among four CN injury groups. It is worth noting that the numbers of LRCs coexpressing α-SMA and S100 were significantly higher in the three ICA II-treated groups than those in the vehicle group in a dose-dependent manner. However, the numbers of LRCs coexpressing RECA among the five groups did not differ significantly (Fig. 6).

FIG. 6.

Evaluation of LRCs. Newborn rats received intraperitoneal injection of 50 mg of EdU per kg body weight immediately after birth. At about 16 weeks post-EdU injection, penile tissues were evaluated by immunofluorescent staining for LRCs and their fate decisions. (A) Note the LRCs in the CC and their coexpression of smooth muscle marker (α-SMA, green) and EdU; the numbers of total LRCs and LRCs coexpressing α-SMA and EdU in these five groups are shown in the bar graph at the bottom. (B) Note the LRCs in the CC and their coexpression of Schwann cell marker (S100, green) and EdU; the numbers of LRCs and LRCs coexpressing S100 and EdU are shown in the bar graph at the bottom. (C) Note the LRCs in the CC and their coexpression of endothelial cell marker (RECA, green) and EdU; the numbers of LRCs and LRCs coexpressing RECA and EdU are shown in the bar graph at the bottom. *P<0.05 versus the Vehicle group, ^# P<0.05 versus the ICA II 0.5 group, ^& P<0.05 versus the ICA II 1.5 group, ^@ P<0.05 versus the ICA II 4.5 group. Edu, 5-ethynyl-2-deoxyuridine; LRCs, label-retaining cells. Color images available online at www.liebertpub.com/scd

The same changing trend as LRCs coexpressing differentiated phenotypes was observed for the expression of phospho-p38 MAPK (p-p38 MAPK), as illustrated in Fig. 7. The expression of p-p38 MAPK was elevated significantly in the vehicle-treated group and all ICA II-treated groups compared with that in the sham-operated rats. However, no significant changes were observed in the total levels of p38 MAPK among groups. These data indicated that p-p38 might play a role in the regulation of LRCs and their fate decisions.

FIG. 7.

Activation of p38 MAPK in the penis. (A) The representative western blot diagrams of p38 and p-p38 after CN crush injury. (B) The mean value of p-p38/p38. (C) Relative expression of p-p38. *P<0.05 versus the Vehicle group, ^# P<0.05 versus the ICA II 0.5 group, ^& P<0.05 versus the ICA II 1.5 group, ^@ P<0.05 versus the ICA II 4.5 group. MAPK, mitogen-activated protein kinase; p38, p38 MAPK; p-p38, phospho-p38 MAPK.

Discussion

Most investigators attribute ED following RP to nerve stretching, thermal damage, ischemia, and local inflammatory [21]. Previous studies have documented the relationship of neuropraxia and some pathophysiological consequences, including CC smooth muscle atrophy, collagen deposition, the decrease in nNOS containing nerve fibers, and Wallerian degeneration [22 –24]. Oral PDE5-Is is one of the most common treatment options after surgery. However, it has had a poor response in post-RP patients [1]. In addition, PDE5-Is can only temporarily relieve symptoms of ED and must be taken before sexual intercourse. In recent years, healing herbs for ED are gaining even more attention [8].

It has been demonstrated that bilateral CN crush injury significantly decreased erectile function [25]. In the present study, we further confirmed this observation, and more importantly, we showed that ICA II treatment significantly improved erectile function in the rat model of cavernosal nerve injury. It has also been reported that CN injury resulted in significant distortion of nerve anatomy and loss of nNOS-positive nerve fibers in rodent model [23]. In this study, we showed that ICA II treatment could preserve neural morphology and improve regeneration of nNOS-positive nerves and overall nNOS expression, when compared with vehicle-treated CN injured rats. We speculated that this preservation of nNOS-positive nerve fibers might be one of the mechanisms underlying the improvement effect of ICA II on erectile function.

It is inevitable that CN injury results in neuropraxia, causing secondary smooth muscle atrophy. In addition, neuropraxia caused loss of nocturnal penile tumescence, which results in persistent penile hypoxia. This vicious spiral appears to result in permanent ED because of veno-occlusive dysfunction induced by progressive cavernosal fibrosis. Losses of smooth muscle and collagen deposition have been consistently identified in the models of CN injury [22]. In this study, we found that CN crush caused significant penile fibrosis and smooth muscle atrophy when compared with those sham-operated animals. More importantly, we observed that ICA II has significant effects on smooth muscle preservation and fibrosis prevention. Thus, the salutary effects of ICA II mentioned above might represent its other therapeutic mechanism for the treatment of ED after CN injury.

In our previous study of STZ-induced ED model, ICA II was also been proved to possess effects on improvement of erectile function, restoration of nNOS-positive nerves, preservation of smooth muscle atrophy, and prevention penile fibrosis [26]. However, the underlying mechanism remains unknown. Stem/progenitor cells, residing in each type of tissue, play a critical role in normal tissue homeostasis and repair. Even without any therapeutic intervention, human body has a robust self-healing capability through endogenous SCs to repair the diseased tissues. We hypothesized that penis contains small populations of endogenous SCs, which are associated with the body's self-healing abilities. Schwann cells, the ensheathing glial cells of the peripheral nerve system, are crucial for normal nerve function and for nerve repair. In the present study, we found that the number of LRCs and LRCs coexpressing α-SMA or S100 was significantly higher in the penis following CN crush compared with that in the sham-operated rats, showing the regenerative capacity of LRCs. More importantly, following ICA II treatment, the numbers of LRCs coexpressing α-SMA and S100 significantly increased in a dose-dependent fashion compared with vehicle-treated rats. These results indicated the therapeutic effects of ICA II on the preservation of nerve anatomy, restoration of nNOS-positive nerves, and smooth muscle content appear to involve enhanced endogenous SCs differentiation. Consistent with the previous research [24], we observed that CN injury did not affect the content of endothelial cells in the CC (Supplementary Fig. S1; Supplementary Data are available online at www.liebertpub.com/scd). In addition, the numbers of LRCs coexpressing RECA among groups did not differ significantly. The above results suggested that the increased LRCs committed to differentiate into damaged cells.

The proper regulation of progenitor/SC self-renewing and differentiation is essential for adult tissue homoeostasis, and disruption of such a balance results in disease. p38 MAPK pathway is essential for the proper differentiation of many hematopoietic, mesenchymal, and epithelial stem/progenitor cells, and disruption of this kinase pathway has pathological consequences in many organs [27]. In addition, accumulating evidence suggests the p38 MAPK pathway is crucially involved in the nerve injury [28]. Although p38 MAPK has been detected to participate in neural degeneration following peripheral nerve injury, it also is abundantly expressed in regenerating nerves, suggesting that the activation of the p38 participates in the normal regeneration process [29]. Interestingly, the effect of ICA II on the activation of p38 MAPK has been observed in a non-ED field [13]. In the current study, the activation of p38 MAPK was observed in vehicle-treated rats. On the other hand, following ICA II treatment, the expression level of p-p38 MAPK was further increased in a dose-dependent fashion when compared with vehicle-treated rats. It is worth noting that the changing trend for p38 MAPK pathway was similar as that for LRCs. These data claimed that p-p38 might play a role in the regulation of LRCs and their fate decisions.

In summary, this study showed that CN injury is associated with ED and some pathophysiological consequences, including loss of smooth muscle, collagen deposition, decrease of nNOS-positive nerves, and Wallerian degeneration. ICA II treatment was able to partially but significantly restore erectile function and these pathological changes. In addition, we also showed that these salutary effects of ICA II appeared to involve enhanced endogenous SCs differentiation, which might be due to p38 MAPK activation. However, it should be cautioned that the limitation within the present study requires further validation. Specifically, the correlation between p38 MAPK pathway and the regulation of endogenous SCs was speculated mainly based on the comparison of different data. In addition, although LRC approach offers a valuable alternative for detecting SCs in laboratory animals, the precise location and characteristics of LRCs in the penis have not been investigated in the past. Therefore, further confirmation of our conclusions with more direct evidences, such as application of a specific inhibitor of p38 MAPK pathway, should be conducted in the future.

Footnotes

Acknowledgments

This work was supported by the National Natural Science Foundation of China (project no. 81272531, 81300478) and the Natural Science Foundation of Jiangsu Province (no. BK20130269).

Author Disclosure Statement

The authors declare no conflict of interest. No competing financial interests exist.

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