This study explored the usefulness of two-dimensional shear wave elastography (2D-SWE) in the early assessment of corpora cavernosa fibrosis (CCF). New Zealand male rabbits were randomly assigned to an experimental group or a control group. Recombinant human transforming growth factor beta 1 (TGF-β1) was injected into the dorsal penis tissue of rabbits in the experimental group. Conventional ultrasound and 2D-SWE examinations were performed before and 20 days after injection. Penile histological analysis was performed by hematoxylin-eosin staining, sirius red staining, and immunohistochemistry. Measurement of 2D-SWE examination results was performed using shear wave elastography quantitative measurement (SWQ). Histological analysis outcomes were the proportion of smooth muscle cells (SMCs), collagen fibers (CFs), collagen type I (Col I), and collagen type III (Col III), as well as the SMCs/CFs ratio, measured by sirius red staining. Other histological analysis outcomes were the positive area proportion (PAP) of TGF-β1 (PAP_T), fibronectin (PAP_F), and Col III (PAP_C), measured by immunohistochemistry. After recombinant human TGF-β1 injection, SWQ was higher in the experimental group than that in the control group (P < 0.001); however, there were no differences in conventional ultrasound results. There were significant differences in histological outcomes between the two groups (all P < 0.05). These results indicated that 2D-SWE was superior for identifying early histological changes in CCF.
Animals ; Elasticity Imaging Techniques/methods* ; Fibrosis ; Male ; Penis/pathology* ; Rabbits ; Transforming Growth Factor beta1/metabolism*

Silent information regulator 2-related enzyme 1 (SIRT1) is an aging-related protein activated with aging. Herein, we evaluated the role of SIRT1 in aging-related erectile dysfunction. The expression of SIRT1 was modulated in aged Sprague-Dawley rats following intragastric administration of resveratrol (Res; 5 mg kg^-1), niacinamide (NAM; 500 mg kg^-1) or Res (5 mg kg^-1) + tadalafil (Tad; phosphodiesterase-5 [PDE5] inhibitor; 5 mg kg^-1) for 8 weeks. Then, we determined erectile function by the ratio of intracavernosal pressure (ICP)/mean systemic arterial pressure (MAP). Cavernosal tissues were extracted to evaluate histological changes, cell apoptosis, nitric oxide (NO)/cyclic guanosine monophosphate (cGMP), the superoxide dismutase (SOD)/3,4-methylenedioxyamphetamine (MDA) level, and the expression of SIRT1, p53, and forkhead box O3 (FOXO3a) using immunohistochemistry, terminal deoxynucleotidyl transferase (TdT)-mediated 2'-deoxyuridine 5'-triphosphate (dUTP) nick-end labeling (TUNEL), enzyme-linked immunosorbent assays, and western blot analysis. Compared with the control, Res treatment significantly improved erectile function, reflected by an increased content of smooth muscle and endothelium, NO/cGMP and SOD activity, and reduced cell apoptosis and MDA levels. The effect of Res was improved by adding Tad. In addition, the protein expression of SIRT1 was increased in the Res group, accompanied by decreased p53 and FOXO3a levels. In addition, inhibition of SIRT1 by NAM treatment resulted in adverse results compared with Res treatment. SIRT1 activation ameliorated aging-related erectile dysfunction, supporting the potential of SIRT1 as a target for erectile dysfunction treatment.
Animals ; Male ; Rats ; Cyclic GMP/metabolism* ; Erectile Dysfunction/metabolism* ; Nitric Oxide/metabolism* ; Penile Erection ; Penis/pathology* ; Phosphodiesterase 5 Inhibitors/pharmacology* ; Rats, Sprague-Dawley ; Sirtuin 1/metabolism* ; Superoxide Dismutase/metabolism* ; Tumor Suppressor Protein p53/metabolism*

We aimed to determine whether combination of LIM-kinase 2 inhibitor (LIMK2i) and phosphodiesterase type-5 inhibitor (PDE5i) could restore erectile function through suppressing cavernous fibrosis and improving cavernous apoptosis in a rat model of cavernous nerve crush injury (CNCI). Seventy 12-week-old Sprague-Dawley rats were equally distributed into five groups as follows: (1) sham surgery (Group S), (2) CNCI (Group I), (3) CNCI treated with daily intraperitoneal administration of 10.0 mg kg^-1 LIMK2i (Group I + L), (4) daily oral administration of 20.0 mg kg^-1 udenafil, PDE5i (Group I + U), and (5) combined administration of 10.0 mg kg^-1 LIMK2i and 20.0 mg kg^-1 udenafil (Group I + L + U). Rats in Groups I + L, I + U, and I + L + U were treated with respective regimens for 2 weeks after CNCI. At 2 weeks after surgery, erectile response was assessed using electrostimulation. Penile tissues were processed for histological studies and western blot. Group I showed lower intracavernous pressure (ICP)/mean arterial pressure (MAP), lower area under the curve (AUC)/MAP, decreased immunohistochemical staining for alpha-smooth muscle (SM) actin, higher apoptotic index, lower SM/collagen ratio, increased phospho-LIMK2-positive fibroblasts, decreased protein kinase B/endothelial nitric oxide synthase (Akt/eNOS) phosphorylation, increased LIMK2/cofilin phosphorylation, and increased protein expression of fibronectin, compared to Group S. In all three treatment groups, erectile responses, protein expression of fibronectin, and SM/collagen ratio were improved. Group I + L + U showed greater improvement in erectile response than Group I + L. SM content and apoptotic index in Groups I + U and I + L + U were improved compared to those in Group I. However, Group I + L did not show a significant improvement in SM content or apoptotic index. The number of phospho-LIMK2-positive fibroblasts was normalized in Groups I + L and I + L + U, but not in Group I + U. Akt/eNOS phosphorylation was improved in Groups I + U and I + L + U, but not in Group I + L. LIMK2/cofilin phosphorylation was improved in Groups I + L and I + L + U, but not in Group I + U. Our data indicate that combined treatment of LIMK2i and PDE5i immediate after CN injury could improve erectile function by improving cavernous apoptosis or eNOS phosphorylation and suppressing cavernous fibrosis. Rectification of Akt/eNOS and LIMK2/cofilin pathways appears to be involved in their improvement.
Animals ; Apoptosis/drug effects* ; Arterial Pressure ; Electric Stimulation ; Erectile Dysfunction/pathology* ; Lim Kinases/antagonists & inhibitors* ; Male ; Nerve Crush ; Nitric Oxide Synthase Type III/metabolism* ; Penis/pathology* ; Peripheral Nerve Injuries/pathology* ; Phosphodiesterase 5 Inhibitors/therapeutic use* ; Phosphorylation ; Pyrimidines/therapeutic use* ; Rats ; Rats, Sprague-Dawley ; Sulfonamides/therapeutic use*

This study aimed to investigate the functional and morphological changes in the corpus cavernosum after cavernous nerve (CN) injury or neurectomy and then reveal whether treatment with the angiotensin II Type 1 receptor antagonist losartan would improve erectile function as well as its potential mechanisms. A total of 48 10-week-old Sprague-Dawley male rats, weighing 300-350 g, were randomly divided into the following four groups (n = 12 per group): sham operation (Sham) group, bilateral cavernous nerve injury (BCNI) group, losartan-treated BCNI (BCNI + Losartan) group, and bilateral cavernous neurectomy (Neurectomy) group. Losartan was administered once daily by oral gavage at a dose of 30 mg kg^-1 day^-1 for 4 weeks starting on the day of surgery. The BCNI and the Neurectomy groups exhibited decreases in erectile response and increases in apoptosis and oxidative stress, compared with the Sham group. Treatment with losartan could have a modest effect on erectile function and significantly prevent corporal apoptosis and oxidative stress. The phospho-B-cell lymphoma 2 (Bcl-2)-associated death promoter (p-Bad)/Bad and phospho-the protein kinase B (p-AKT)/AKT ratios were substantially lower, while the Bcl-2-associated X protein (Bax)/Bcl-2 ratio, nuclear factor erythroid 2-related factor 2 (Nrf2)/Kelch-like ECH-associated protein 1 (Keap-1), transforming growth factor-β 1 (TGF-β 1) and heme oxygenase-1 (HO-1) levels, and caspase-3 activity were higher in the BCNI and Neurectomy groups than in the Sham group. After 4 weeks of daily administration with losartan, these expression levels were remarkably attenuated compared with the BCNI group. Taken together, our results suggested that early administration of losartan after CN injury could slightly improve erectile function and significantly reduce corporal apoptosis and oxidative stress by inhibiting the Akt/Bad/Bax/caspase-3 and Nrf2/Keap-1 pathways.
Angiotensin II Type 1 Receptor Blockers/pharmacology* ; Animals ; Apoptosis/drug effects* ; Denervation ; Disease Models, Animal ; Erectile Dysfunction/metabolism* ; Losartan/pharmacology* ; Male ; Oxidative Stress/drug effects* ; Penile Erection/drug effects* ; Penis/metabolism* ; Rats ; Rats, Sprague-Dawley

Emerging evidence indicates that aldosterone and mineralocorticoid receptors (MRs) are associated with the pathogenesis of erectile dysfunction. However, the molecular mechanisms remain largely unknown. In this study, freshly isolated penile corpus cavernosum tissue from rats was treated with aldosterone, with or without MRs inhibitors. Nuclear factor (NF)-kappa B (NF-κB) activity was evaluated by real-time quantitative PCR, luciferase assay, and immunoblot. The results demonstrated that mRNA levels of the NF-κB target genes, including inhibitor of NF-κB alpha (IκB-α), NF-κB1, tumor necrosis factor-alpha (TNF-α), and interleukin 6 (IL-6), were higher after aldosterone treatment. Accordingly, phosphorylation of p65/RelA, IκB-α, and inhibitor of NF-κB kinase-β was markedly increased by aldosterone. Furthermore, knockdown of MRs prevented activation of the NF-κB canonical pathway by aldosterone. Consistent with this finding, ectopic overexpression of MRs enhanced the transcriptional activation of NF-κB by aldosterone. More importantly, the MRs antagonist, spironolactone blocked aldosterone-mediated activation of the canonical NF-κB pathway. In conclusion, aldosterone has an inflammatory effect in the corpus cavernosum penis, inducing NF-κB activation via an MRs-dependent pathway, which may be prevented by selective MRs antagonists. These data reveal the possible role of aldosterone in erectile dysfunction as well as its potential as a novel pharmacologic target for treatment.
Aldosterone/pharmacology* ; Animals ; Cytokines/biosynthesis* ; Gene Knockdown Techniques ; I-kappa B Kinase/antagonists & inhibitors* ; Interleukin-6/genetics* ; Male ; Mineralocorticoid Receptor Antagonists/pharmacology* ; NF-kappa B/genetics* ; Penis/metabolism* ; Protein Serine-Threonine Kinases/antagonists & inhibitors* ; RNA, Messenger/biosynthesis* ; Rats ; Rats, Inbred WKY ; Receptors, Mineralocorticoid/genetics* ; Signal Transduction/drug effects* ; Spironolactone/pharmacology* ; Transcriptional Activation ; Tumor Necrosis Factor-alpha/biosynthesis* ; NF-kappaB-Inducing Kinase

The present study aimed to identify which mitogen-activated protein kinase (p38 or Jun amino-terminal kinase [JNK]) was involved in cavernosal apoptosis during the acute phase after cavernosal nerve crush injury (CNCI) in rats to ameliorate apoptosis of cavernosal tissue, such as smooth muscle (SM). A total of twenty 10-week-old male Sprague-Dawley rats were divided equally into two groups: sham surgery (S) and CNCI (I). The I group approximated the clinical situation of men undergoing radical prostatectomy using two 60-second compressions of both CNs with a microsurgical vascular clamp. At 2-week postinjury, erectile response was assessed using electrostimulation. Penile tissues were harvested for immunohistochemistry analysis of alpha-SM actin (α-SMA), western blot analysis, and double immunofluorescence analysis of α-SMA and phosphorylated p38 or JNK, as well as double immunofluorescent of TUNEL and phosphorylated p38 or JNK. At 2-week postinjury, the I group had a significantly lower intracavernous pressure (ICP)/mean arterial pressure (MAP) and a lower area under the curve (AUC)/MAP than the S group. The I group also exhibited decreased immunohistochemical staining of α-SMA, an increase in the number of SM cells positive for phosphorylated JNK, an increased number of apoptotic cells positive for phosphorylated JNK, and increased JNK phosphorylation compared with the S group. However, there was no significant difference in p38 phosphorylation expression or the number of SM cells positive for phosphorylated p38 between the two groups. In conclusion, our data suggest that JNK, not p38, is involved in cavernosal apoptosis during the acute phase after partial CN damage.
Animals ; Apoptosis ; Disease Models, Animal ; Electric Stimulation ; MAP Kinase Kinase 4/metabolism* ; Male ; Penile Erection ; Penis/pathology* ; Peripheral Nerve Injuries/pathology* ; Phosphorylation ; Prostatectomy ; Rats ; Rats, Sprague-Dawley ; Signal Transduction ; p38 Mitogen-Activated Protein Kinases/metabolism*

Previous studies have shown that oxidative stress and corporal fibrosis in penile tissues of rats were key pathological factors of erectile dysfunction induced by diabetic mellitus (DMED). Lipoxin A4 (LXA4) was reported to inhibit oxidative stress and fibrosis diseases, while whether it could exert a protective role on erectile function was not clear. Type I diabetic mellitus (DM) was induced in thirty male 10-week-old Sprague-Dawley rats using streptozotocin. Ten weeks later, twenty-two rats with DMED confirmed by an apomorphine test were divided into two groups: the DMED group (n = 11) and the DMED + LXA4 group (n = 11; LXA4 injection daily for 4 weeks). In addition, another ten age-matched rats formed the Control group. We found that erectile function was significantly impaired in the DMED group compared with the Control group, but was improved in the DMED + LXA4 group. Similarly, the over-activated oxidative stress and impaired endothelial function in the DMED group were both improved in the DMED + LXA4 group. Moreover, the DMED group showed serious corporal fibrosis, which was also inhibited by the treatment of LXA4 in the DMED + LXA4 group. Taken together, LXA4 could exert an inhibition role on oxidative stress and fibrosis to improve DMED effectively.
Actins/metabolism* ; Animals ; Anti-Inflammatory Agents, Non-Steroidal/pharmacology* ; Diabetes Mellitus, Experimental/physiopathology* ; Diabetes Mellitus, Type 1/physiopathology* ; Erectile Dysfunction/physiopathology* ; Fibrosis ; Lipoxins/pharmacology* ; Male ; Nitric Oxide/metabolism* ; Nitric Oxide Synthase Type III/metabolism* ; Oxidative Stress/drug effects* ; Penile Erection/drug effects* ; Penis/pathology* ; Rats ; Rats, Sprague-Dawley

Calpain activation contributes to hyperglycemia-induced endothelial dysfunction and apoptosis. This study was designed to investigate the role of calpain inhibition in improving diabetic erectile dysfunction (ED) in mice. Thirty-eight-week-old male C57BL/6J mice were divided into three groups: (1) nondiabetic control group, (2) diabetic mice + vehicle group, and (3) diabetic mice + MDL28170 (an inhibitor of calpain) group. Type 1 diabetes was induced by intraperitoneal injection of streptozotocin at 60 mg kg^-1 body weight for 5 consecutive days. Thirteen weeks later, diabetic mice were treated with MDL28170 or vehicle for 4 weeks. The erectile function was assessed by electrical stimulation of the cavernous nerve. Penile tissues were collected for measurement of calpain activity and the endothelial nitric oxide synthase (eNOS)-nitric oxide (NO)-cyclic guanosine monophosphate (cGMP) pathway. Terminal deoxynucleotidyl transferase 2'-deoxyuridine 5'-triphosphate nick end labeling (TUNEL) staining was used to evaluate apoptosis. Caspase-3 expression and activity were also measured to determine apoptosis. Our results showed that erectile function was enhanced by MDL28170 treatment in diabetic mice compared with the vehicle diabetic group. No differences in calpain-1 and calpain-2 expressions were observed among the three groups. However, calpain activity was increased in the diabetic group and reduced by MDL28170. The eNOS-NO-cGMP pathway was upregulated by MDL28170 treatment in diabetic mice. Additionally, MDL28170 could attenuate apoptosis and increase the endothelium and smooth muscle levels in corpus cavernosum. Inhibition of calpain could improve erectile function, probably by upregulating the eNOS-NO-cGMP pathway and reducing apoptosis.
Animals ; Apoptosis/drug effects* ; Calpain/antagonists & inhibitors* ; Cyclic GMP/biosynthesis* ; Diabetes Complications/drug therapy* ; Diabetes Mellitus, Experimental/complications* ; Dipeptides/therapeutic use* ; Endothelium/metabolism* ; Enzyme Inhibitors/therapeutic use* ; Erectile Dysfunction/etiology* ; In Situ Nick-End Labeling ; Male ; Mice ; Mice, Inbred C57BL ; Muscle, Smooth/metabolism* ; Nitric Oxide Synthase Type III/biosynthesis* ; Penis/enzymology* ; Up-Regulation

We evaluated whether LIM-kinase 2 inhibitor (LIMK2i) could improve erectile function by suppressing corporal fibrosis through the normalization of the Rho-associated coiled-coil protein kinase 1 (ROCK1)/LIMK2/Cofilin pathway in a rat model of cavernous nerve crush injury (CNCI). Sixty 11-week-old male Sprague-Dawley rats were divided equally into five groups: sham surgery (S), CNCI (I), and CNCI treated with low-dose (L), medium-dose (M), and high-dose (H) LIMK2i. The L, M, and H groups were treated with a daily intraperitoneal injection of LIMK2i (2.5, 5.0, and 10.0 mg kg^-1 body weight, respectively) for 1 week after surgery. The erectile response was assessed using electrostimulation at 1 week, postoperatively. Penile tissues were processed for Masson's trichrome staining, double immunofluorescence, and Western blot assay. Erectile responses in the H group improved compared with the I group, while the M group showed only partial improvement. A significantly decreased smooth muscle/collagen ratio and an increased content of fibroblasts positive for phospho-LIMK2 were noted in the I group. The M and H groups revealed significant improvements in histological alterations and the dysregulated LIMK2/Cofilin pathway, except for LIMK2 phosphorylation in the M group. The inhibition of LIMK2 did not affect the ROCK1 protein expression. The content of fibroblasts positive for phospho-LIMK2 in the H group returned to the level found in the S group, whereas it did not in the M group. However, the L group did not exhibit such improvements. Our data suggest that the inhibition of LIMK2, particularly with administration of 10.0 mg kg^-1 body weight LIMK2i, can improve corporal fibrosis and erectile function by normalizing the LIMK2/Cofilin pathway.
Animals ; Cofilin 1/metabolism* ; Electric Stimulation ; Erectile Dysfunction/etiology* ; Fibroblasts/pathology* ; Fibrosis/drug therapy* ; Lim Kinases/antagonists & inhibitors* ; Male ; Penile Diseases/drug therapy* ; Penis/innervation* ; Peripheral Nerve Injuries/pathology* ; Phosphorylation ; Rats ; Rats, Sprague-Dawley ; Signal Transduction/drug effects* ; rho-Associated Kinases/genetics*

Erectile dysfunction (ED) associated with type 2 diabetes is a severe problem that requires effective treatment. Pancreatic kininogenase (PK) has the potential to improve the erectile function of ED patients. This study aims to investigate the effect of PK on erectile function in streptozotocin-induced type 2 diabetic ED rats. To achieve this goal, we divided male Sprague-Dawley rats into five groups. One group was not treated, and the other four groups were treated with saline, sildenafil, PK or sildenafil, and PK, respectively, for 4 weeks after the induction of type 2 diabetic ED. Then, intracavernous pressure under cavernous nerve stimulation was measured, and penile tissue was collected for further study. Endothelial nitric oxide synthase levels, smooth muscle content, endothelium content, cyclic guanosine monophosphate (cGMP) levels in the corpus cavernosum, and neuronal nitric oxide synthase levels in the dorsal penile nerve were measured. Improved erectile function and endothelium and smooth muscle content in the corpus cavernosum were observed in diabetic ED rats. When treating diabetic ED rats with PK and sildenafil at the same time, a better therapeutic effect was achieved. These data demonstrate that intraperitoneal injection of PK can improve erectile function in a rat model of type 2 diabetic ED. With further research on specific mechanisms of erectile function improvement, PK may become a novel treatment for diabetic ED.
Animals ; Cyclic GMP/metabolism* ; Diabetes Mellitus, Experimental/physiopathology* ; Erectile Dysfunction/physiopathology* ; Kallikreins/therapeutic use* ; Male ; Muscle, Smooth, Vascular/physiopathology* ; Nitric Oxide Synthase Type I/metabolism* ; Nitric Oxide Synthase Type III/metabolism* ; Penile Erection/physiology* ; Penis/metabolism* ; Rats ; Rats, Sprague-Dawley ; Sildenafil Citrate/therapeutic use* ; Treatment Outcome ; Urological Agents/therapeutic use*