Corpus Cavernosum Smooth Muscle Research Articles

Caveolin-1 scaffolding domain-derived peptide enhances erectile function by regulating oxidative stress, mitochondrial dysfunction, and apoptosis of corpus cavernosum smooth muscle cells in rats with cavernous nerve injury

AimIncreased corpus cavernosum smooth muscle cells (CCSMCs) apoptosis in the penis due to cavernous nerve injury (CNI) is a crucial contributor to erectile dysfunction (ED). Caveolin-1 scaffolding domain (CSD)-derived peptide has been found to exert potential antiapoptotic properties. However, whether CSD peptide can alleviate CCSMCs apoptosis and ED in CNI rats remains unknown. The study aimed to determine whether CSD peptide can improve bilateral CNI-induced ED (BCNI-ED) by enhancing the antiapoptotic processes of CCSMCs. Main methodsFifteen 10-week-old male Sprague-Dawley (SD) rats were randomly classified into three groups: sham surgery (Sham) group and BCNI groups that underwent saline or CSD peptide treatment respectively. At 3 weeks postoperatively, erectile function was assessed and the penis tissue was histologically examined. Furthermore, an in vitro model of CCSMCs apoptosis was established using transforming growth factor-beta 1 (TGF-β1) to investigate the mechanism of CSD peptide in treating BCNI-ED. Key findingsIn BCNI rats, CSD peptide significantly prevented ED and decreased oxidative stress, the Bax/Bcl-2 ratio, and the levels of caspase3. TGF-β1-treated CCSMCs exhibited severe oxidative stress, mitochondrial dysfunction, and apoptosis. However, CSD peptide partially reversed these alterations. SignificanceExogenous CSD peptide could improve BCNI-ED by inhibiting oxidative stress, the Bax/Bcl-2 ratio, and caspase3 expression in penile tissue. The underlying mechanism might involve the regulatory effects of CSD peptide on oxidative stress, mitochondrial dysfunction, and apoptosis of CCSMCs following CNI. This study highlights CSD peptide as an effective therapy for post-radical prostatectomy ED (pRP-ED).

Cldn4 overexpression promotes penile cavernous smooth muscle cell fibrotic response via the JNK signaling pathway.

Erectile dysfunction (ED), defined as the inability to achieve or maintain a penile erection sufficient to satisfy sexual behavior, is prevalent worldwide. Using previous research, bioinformatics, and experimental confirmation, we aimed to discover genes that contribute to ED through regulating hypoxia in corpus cavernosum smooth muscle cells (CCSMCs). We used the Gene Expression Omnibus to acquire the sequencing data of the corpus cavernosum transcriptome for diabetic ED and nerve injury type ED rats. We intersected the common differentially expressed genes. Further verification was performed using single cell sequencing. Real-time quantitative polymerase chain reaction and immunofluorescence were used to investigate whether the differentially expressed genes are found in the corpus cavernosum. We used induced hypoxia to assess cell viability changes, and we developed a lentivirus overexpressing Cldn4 for in vitro and in vivo experiments to measure changes in JNK signaling, fibrosis, hypoxia, and erectile function. Our results indicate that targeting the JNK pathway and decreasing local hypoxia may be better options for therapeutic intervention to improve erectile function. We identified Cldn4 and found its expression increased in the corpora cavernosa of the 2 datasets. In addition, we found that hypoxia can increase the expression of Cldn4, activate the JNK signaling pathway, and exacerbate fibrosis in CCSMCs. Cldn4 overexpression in CCSMCs activated the JNK signaling pathway and increased fibrotic protein expression. Last, rat corpus cavernosum overexpressing Cldn4 activated the JNK signaling pathway, increased local fibrosis, and impaired erectile function. Through bioinformatics and in vitro and in vivo experiments, we found that Cldn4 has a negative effect on ED, and targeting Cldn4 may provide new ideas for ED treatment. Although we have identified Cldn4 as a potential target for ED treatment, we have only conducted preliminary validation on CCMSCs, and we still need to further validate in other cell lines. CCSMC hypoxia leads to increased Cldn4, in both nerve injury and diabetic ED rat models, and promotes fibrosis by activating the JNK signaling pathway.

Galectin-3 is involved in inflammation and fibrosis in arteriogenic erectile dysfunction via the TLR4/MyD88/NF-κB pathway

Galectin-3 (Gal-3) is a multifunctional protein that has been linked to fibrosis and inflammation in the cardiovascular system. In this study, we examined the impact of Gal-3 on inflammation and fibrosis in patients with arteriogenic erectile dysfunction (A-ED) and the underlying mechanisms involved. To induce arterial injury, we utilized cuffs on the periaqueductal common iliac arteries of Sprague‒Dawley (SD) rats and administered a high-fat diet to co-induce local atherosclerosis. Our results showed that we successfully developed a novel A-ED model that was validated based on histological evidence. In vivo, the vascular lumen of rats subjected to a high-fat diet and cuff placement exhibited significant narrowing, accompanied by the upregulation of Gal-3, Toll-like receptor 4 (TLR4), and myeloid differentiation primary response protein 88 (MyD88) expression in the penile cavernosa. This led to the activation of nuclear factor kappa B 65 (NF-κB-p65), resulting in reduced intracavernosal pressure, endothelial nitric oxide synthase expression, and smooth muscle content, promoting inflammation and fibrosis. However, treatment with Gal-3 inhibitor-modified citrus pectin (MCP) significantly normalized those effects. In vitro, knocking down Gal-3 led to a significant reduction in TLR4, MyD88, and NF-κB-p65 expression in corpus cavernosum smooth muscle cells (CCSMCs), decreasing inflammation levels. In conclusion, inhibiting Gal-3 may improve A-ED by reducing inflammation, endothelial injury, and fibrosis in the penile corpus cavernosum through the TLR4/MyD88/NF-κB pathway. These findings highlight the potential therapeutic target of Gal-3 in A-ED.

Artificial cavernosa-like tissue based on multibubble Matrigel and a human corpus cavernous fibroblast scaffold.

Ex vivo tissue culture of the human corpus cavernosum (CC) can be used to explore the tissue structural changes and complex signaling networks. At present, artificial CC-like tissues based on acellular or three-dimensional (3D)-printed scaffolds are used to solve the scarcity of primary penis tissue samples. However, inconvenience and high costs limit the wide application of such methods. Here, we describe a simple, fast, and economical method of constructing artificial CC-like tissue. Human CC fibroblasts (FBs), endothelial cells (ECs), and smooth muscle cells (SMCs) were expanded in vitro and mixed with Matrigel in specific proportions. A large number of bubbles were formed in the mixture by vortexing combined with pipette blowing, creating a porous, spongy, and spatial structure. The CC FBs produced a variety of signaling factors, showed multidirectional differentiation potential, and grew in a 3D grid in Matrigel, which is necessary for CC-like tissue to maintain a porous structure as a cell scaffold. Within the CC-like tissue, ECs covered the surface of the lumen, and SMCs were located inside the trabeculae, similar to the structure of the primary CC. Various cell components remained stable for 3 days in vitro, but the EC content decreased on the 7th day. Wingless/integrated (WNT) signaling activation led to lumen atrophy and increased tissue fibrosis in CC-like tissue, inducing the same changes in characteristics as in the primary CC. This study describes a preparation method for human artificial CC-like tissue that may provide an improved experimental platform for exploring the function and structure of the CC and conducting drug screening for erectile dysfunction therapy.

Inhibition of YAP1 Rescues Erectile Dysfunction by Inhibiting Phenotypic Modulation through Myocardin in Diabetic Rats

Background. Phenotypic modulation was shown to be related to the pathological mechanism of diabetes mellitus erectile dysfunction (DMED), and excessive expression of YAP1 was measured in the cavernous tissues of rats with DMED. Objectives. This investigation was performed to explore the efficiency and mechanism of gene therapy through the knockdown of YAP1 in the field of ED treatment. Materials and Methods. Corpus cavernosum smooth muscle cells were obtained and cultivated in vitro employing the explant technique. EdU, CCK-8, and collagen gel lattice contraction assays were subsequently performed to assess cell proliferation and contractility, which indicate smooth muscle phenotypes. Results. Initially, overexpression of YAP1 enhanced the proliferation and expression of YAP1 and osteopontin (OPN) while reducing cell contractility and the expression of myocardin, α-SMA, and calpontin, which is consistent with phenotypic modulation. Moreover, knocking down YAP1 resulted in the opposite effects, and the blockade of myocardin attenuated the reversal of phenotypic transformation. Then, 45 screened DMED rats were randomly divided into three equal groups, named streptozotocin (STZ)-induced DMED with intracavernosal injection of Ad-myocardin (DMED + myocardin), DMED with lenti-shYAP1 (DMED + shYAP1) and DMED with PBS (DMED + PBS), and 10 rats were in the negative control group. Staining with H&E and Masson’s trichrome, immunochemistry, and determination of the ratio of the intracavernosal pressure/mean arterial pressure were performed to evaluate penile erection and histological changes. Western blotting was conducted to determine the molecular protein expression levels. In vivo, similar to myocardin overexpression, YAP1 inhibition rescued erectile function, repaired morphology, and maintained the contractile phenotype of smooth muscle tissues 14 days after treatment. Discussion and Conclusion. In conclusion, our study demonstrated the validity of gene therapy by downregulating excessive YAP1 expression to ameliorate ED in diabetic rats, and the ability of YAP1 inhibition to reverse phenotypic modulation was mediated through interaction with myocardin.

Heme-induced corpus cavernosum relaxation and its implications for priapism in sickle cell disease: a mechanistic insight.

Patients with sickle cell disease (SCD) experience intravascular hemolysis, leading to elevated plasma heme levels. This phenomenon has been associated with increased priapism in men with SCD. The heme group can be metabolized by heme oxygenase (HO), generating carbon monoxide (CO), which is known to promote smooth muscle relaxation via soluble guanylyl cyclase (sGC)-cyclic guanosine monophosphate (cGMP). However, the effects of heme on the relaxation responses of corpus cavernosum (CC) have not been investigated. To evaluate the functional and biochemical effects of the heme group on mouse CC smooth muscle in vitro. Male C57BL/6 mice were used. CC tissues were mounted in organ baths. Measurement of cGMP in mice CC was evaluated. The cumulative addition of heme concentrations promoted the relaxation of CC. HO inhibitor (1J, 100μM) or sGC inhibitor (ODQ, 10μM) blocked the relaxing effect of the heme group. Pre-incubation of CC with heme (100μM) enhanced relaxation induced by acetylcholine, sodium nitroprusside, and nitrergic relaxation (electrical field stimulation), which was abolished by 1J or ODQ. The heme group increased the cGMP production in CC, which was abolished by 1J or ODQ. cGMP levels were significantly higher in CC treated with heme, and pre-incubation with compound 1J or ODQ abolished the effect of heme in raising cGMP levels. The HO-CO-sGC-cGMP pathway appears to play a crucial role in promoting CC relaxation. Our study provides novel insight into the role of group heme in CC relaxation and its potential contribution to priapism in SCD. Heme may serve as a pharmacological target for new therapies to prevent priapism.

MicroRNA-145 Gene Modification Enhances the Retention of Bone Marrow-Derived Mesenchymal Stem Cells within Corpus Cavernosum by Targeting Krüppel-Like Factor 4.

The poor retention and ambiguous differentiation of stem cells (SCs) within corpus cavernosum (CC) limit the cell application in erectile dysfunction (ED). Herein, the effects and mechanism of microRNA-145 (miR-145) gene modification on modulating the traits and fate of bone marrow-derived mesenchymal stem cells (BMSCs) were investigated. The effects of miR-145 on cell apoptosis, proliferation, migration, and differentiation were determined by flow cytometry, cell counting kit-8, transwell assays and myogenic induction. Then, the age-related ED rats were recruited to four groups including phosphate buffer saline, BMSC, vector-BMSC, overexpressed-miR-145-BMSC groups. After cell transplantation, the CC were harvested and prepared to demonstrate the retention and differentiation of BMSCs by immunofluorescent staining. Then, the target of miR-145 was verified by quantitative real-time polymerase chain reaction and immunohistochemical. After that, APTO-253, as an inducer of Krüppel-like factor 4 (KLF4), was introduced for rescue experiments in corpus cavernosum smooth muscle cells (CCSMCs) under the co-culture system. In vitro, miR-145 inhibited the migration and apoptosis of BMSCs and promoted the differentiation of BMSCs into smooth muscle-like cells with stronger contractility. In vivo, the amount of 5-ethynyl-2'-deoxyuridine (EdU)+cells within CC was significantly enhanced and maintained in the miR-145 gene modified BMSC group. The EdU/CD31 co-staning was detected, however, no co-staining of EdU/α-actin was observed. Furthermore, miR-145, which secreted from the gene modified BMSCs, dampened the expression of KLF4. However, the effects of miR-145 on CCSMCs could be rescued by APTO-253. Overall, miR-145 modification prolongs the retention of the transplanted BMSCs within the CC, and this effect might be attributed to the modulation of the miR-145/KLF4 axis. Consequently, our findings offer a promising and innovative strategy to enhance the local stem cell-based treatments.

6-Nitrodopamine is an endogenous mediator of the rabbit corpus cavernosum relaxation.

6-Nitrodopamine (6-ND) is a novel endogenous catecholamine that has a potent relaxant action on vascular smooth muscle in vitro. To evaluate the basal release of 6-ND and noradrenaline from rabbit-isolated corpus cavernosum (RbCC) and its relaxing action on this tissue. Rabbit corpus cavernosa were dissected and suspended in a 5-mL organ bath containing oxygenated Krebs-Henseleit's solution. 6-ND and noradrenaline release was quantified by liquid chromatography coupled to tandem mass spectrometry. The relaxant activity of 6-ND was assessed in RbCC strips pre-contracted with endothelin-1 (10nM). Rabbit corpus cavernosum presented basal release of both 6-ND (2.9±0.8ng/mL, n=12) and noradrenaline (1.7±1.3ng/mL, n=12). The 6-ND release was reduced by pre-treatment with Nω -nitro-l-arginine methyl ester (l-NAME) (100 µM), whereas that of noradrenaline was unaffected. Tetrodotoxin (TTX, 1 µM) abolished the noradrenaline release but had no effect on 6-ND release, indicating a non-neurogenic origin for 6-ND. 6-ND and the selective dopamine D2 -agonist L-741,626 caused concentration-dependent RbCC relaxations (pEC50 of 11±0.15 and 11.15±0.28, respectively). Pre-treatment with either l-NAME or the soluble guanylate cyclase inhibitor 1H-[1,2,4] oxadiazolo[4,3-a]quinoxalin-1-on (ODQ) (100 µM) caused a rightward shift of the concentration-response curve to 6-ND, without affecting the L-741,626 responses. In TTX (100nM)-pre-treated preparations, neither l-NAME nor ODQ shifted the 6-ND concentration-response curve. Dopamine, noradrenaline, and adrenaline caused concentration-dependent RbCC contractions. Pre-incubation with 6-ND concentration-dependently inhibited the dopamine-induced contractions, without affecting those induced by either noradrenaline or adrenaline. 6-Nitrodopamine is the most potent endogenous relaxant agent in RbCC ever described and represents a novel mechanism by which NO causes corpus cavernosum smooth muscle relaxation. The finding that 6-ND acts as a truly selective dopamine D2 -receptor antagonist indicates that the balance of dopamine and 6-ND release/synthesis may be the main mechanism that modulates corpus cavernosum smooth muscle tonus in vivo.

D-Galactose induces the senescence and phenotype switch of corpus cavernosum smooth muscle cells.

Studies regarding age-related erectile dysfunction (ED) based on naturally aging models are limited by their high costs, especially for the acquisition of primary cells from the corpus cavernosum. Herein, d-galactose ( d-gal) was employed to accelerate cell senescence, and the underlying mechanism was explored. As predominant functional cells involved in the erectile response, corpus cavernosum smooth muscle cells (CCSMCs) were isolated from 2-month-old rats. Following this, d-gal was introduced to induce cell senescence, which was verified via β-galactosidase staining. The effects of d-gal on CCSMCs were evaluated by terminal deoxynucleoitidyl transferase dUTP nick-end labeling (TUNEL), immunofluorescence staining, flow cytometry, western blot, and quantitative real-time polymerase chain reaction (qRT-PCR). Furthermore, RNA interference (RNAi) was carried out for rescue experiments. Subsequently, the influence of senescence on the corpus cavernosum was determined via scanning electron microscopy, qRT-PCR, immunohistochemistry, TUNEL, and Masson stainings. The results revealed that the accelerated senescence of CCSMCs was promoted by d-gal. Simultaneously, smooth muscle alpha-actin (alpha-SMA) expression was inhibited, while that of osteopontin (OPN) and Krüppel-like factor 4 (KLF4), as well as fibrotic and apoptotic levels, were elevated. After knocking down KLF4 expression in d-gal-induced CCSMCs by RNAi, the expression level of cellular alpha-SMA increased. Contrastingly, the OPN expression, apoptotic and fibrotic levels declined. In addition, cellular senescence acquired partial remission. Accordingly, in the aged corpus cavernosum, the fibrotic and apoptotic rates were increased, followed by downregulation in the expression of alpha-SMA and the concurrent upregulation in the expression of OPN and KLF4. Overall, our results signaled that d-gal-induced accelerated senescence of CCSMCs could trigger fibrosis, apoptosis and phenotypic switch to the synthetic state, potentially attributed to the upregulation of KLF4 expression, which may be a multipotential therapeutic target of age-related ED.

Caveolin-1 scaffolding domain peptide prevents corpus cavernosum fibrosis and erectile dysfunction in bilateral cavernous nerve injury-induced rats.

Corpus cavernosum (CC) fibrosis significantly contributes to post-radical prostatectomy erectile dysfunction (pRP-ED). Caveolin-1 scaffolding domain (CSD)-derived peptide has gained significant concern as a potent antagonist of tissue fibrosis. However, applying CSD peptide on bilateral cavernous nerve injury (BCNI)-induced rats remains uninvestigated. The aim was to explore the therapeutic outcome and underlying mechanism of CSD peptide for preventing ED in BCNI rats according to the hypothesis that CSD peptide may exert beneficial effects on erectile tissue and function following BCNI through limiting collagen synthesis in CC smooth muscle cells (CCSMCs) and CC fibrosis. After completing a random assignment of male Sprague Dawley rats (10 weeks of age), BCNI rats received either saline or CSD peptide treatment, as opposed to sham-operated rats. The evaluations of erectile function (EF) and succedent collection and histological and molecular biological examinations of penile tissue were accomplished 3weeks postoperatively. In addition, the fibrotic model of CCSMCs was used to further explore the mechanism of CSD peptide action in vitro. The assessments of EF, SMC/collagen ratio, α-smooth muscle actin, caveolin-1 (CAV1), and profibrotic indicators expressions were conducted. BCNI rats exhibited significant decreases in EF, SMC/collagen ratio, α-SMA, and CAV1 levels, and increases in collagen content together with transforming growth factor (TGF)-β1/Smad2 activity. However, impaired EF, activated CC fibrosis, and Smad2 signaling were attenuated after 3weeks of CSD peptide treatment in BCNI rats. In vitro, TGF-β1-induced CCSMCs underwent fibrogenetic transformation characterized by lower expression of CAV1, higher collagen composition, and phosphorylation of Smad2; then, the delivery of CSD peptide could significantly block CCSMC fibrosis by inactivating Smad2 signaling. Based on available evidence of CSD peptide in the prevention of ED in BCNI rats, this study can aid in the development and clinical application of CSD peptide targeting pRP-ED. This study provides data to suggest that CSD peptide protects against BCNI-induced deleterious alterations in EF and CC tissues. However, the available evidence still does not fully clarify the detailed mechanism of action of CSD peptide. Administration of CSD peptide significantly retarded collagen synthesis in CCSMCs, limited CC fibrosis, and prevented ED via confrontation of TGF-β1/Smad signaling in BCNI rats.

Therapeutic potential of mesenchymal stem cell-derived exosomal miR-296-5p and miR-337-3p in age-related erectile dysfunction via regulating PTEN/PI3K/AKT pathway

Mesenchymal stem cells (MSCs) are viewed as an increasingly promising treatment for age-related erectile dysfunction (AED). Owing to the limitations of injecting living cells, the injection of exosomes appears to be a more plausible option. However, whether MSC-derived exosomes (MSC-Exos) improve AED and their potential mechanism remains unknown. MSC-Exos were prepared and injected intracavernously into aged rats to determine their effects on AED. Masson's trichrome staining was used to ascertain the changes in the histological structure of the corpus cavernosum. Then miRNA sequencing of MSC-Exos and analysis of the critical exosomal miRNAs were performed, as well as their target pathway enrichment analysis. Real-time quantitative PCR (RT-qPCR) and Western blot assay were performed to reveal the functions of MSC-Exos in regulating the PTEN/PI3K/AKT signaling pathway. Moreover, the effects of MSC-Exos on the corpus cavernosum smooth muscle cells (CCSMCs) apoptosis are explored in vitro. The experimental data validate that intracavernous injection of MSC-Exos ameliorated erectile function in AED rats. Masson's trichrome staining shows MSC-Exos therapy restores the histological structure of the corpus cavernosum by improving the ratios of smooth muscle to collagen. The exosomal miR-296–5p and miR-337–3p target and inhibit PTEN, modulating the PI3K/AKT signaling pathway. Furthermore, exosomes inhibit the apoptosis of CCSMCs. Our findings suggest that MSC-Exos improve AED by delivering miR-296–5p and miR-337–3p to regulate the PTEN/PI3K/AKT signaling pathway. These results bode well for the therapeutic potential of MSC-Exos for AED treatment.

The bitter taste receptor (TAS2R) agonist denatonium promotes a strong relaxation of rat corpus cavernosum

Bitter taste receptors (TAS2R) are found in numerous extra-oral tissues, including smooth muscle (SM) cells in both vascular and visceral tissues. Upon activation, TAS2R stimulate the relaxation of the SM. Nitric oxide (NO)/cyclic guanosine monophosphate (cGMP) signaling pathway is involved in penile erection, and type 5 phosphodiesterase (PDE5) inhibitors, a cGMP-specific hydrolase are used as first-line treatments for erectile dysfunction (ED). Nevertheless, PDE5 inhibitors are ineffective in a considerable number of patients, prompting research into alternative pharmacological targets for ED. Since TAS2R agonists regulate SM contractility, this study investigates the role of TAS2Rs in rat corpus cavernosum (CC). We performed immunohistochemistry to detect TAS2R10, isometric force recordings for TAS2R agonists denatonium and chloroquine, the slow-release H2S donor GYY 4137, the NO donor SNAP, the β-adrenoceptor agonist isoproterenol and electrical field stimulation (EFS), as well as measurement of endogenous hydrogen sulfide (H2S) production. The immunofluorescence staining indicated that TAS2R10 was broadly expressed in the CC SM and to some extent in the nerve fibers. Denatonium, chloroquine, SNAP, and isoproterenol cause potent dose-dependent SM relaxations. H2S production was decreased by NO and H2S synthase inhibitors, while it was enhanced by denatonium. In addition, denatonium increased the relaxations induced by GYY 4137 and SNAP but failed to modify EFS- and isoproterenol-induced responses. These results suggest neuronal and SM TAS2R10 expression in the rat CC, where denatonium induces a strong SM relaxation per se and promotes the H2S- and NO-mediated inhibitory gaseous neurotransmission. Thus, TAS2R10 might represent a valuable therapeutic target in ED.

Effects of endoplasmic reticulum stress on erectile function in rats with cavernous nerve injury.

Erectile dysfunction (ED) occurs in an increasing number of patients after radical prostatectomy and cystectomy, and the phenotypic modulation of corpus cavernosum smooth muscle cells is closely related to ED. To determine whether endoplasmic reticulum stress (ERS) is implicated in the phenotypic modulation of ED induced by bilateral cavernous nerve injury (BCNI). In total, 36 Sprague-Dawley rats were randomly divided into 3 groups: sham, in which rats received sham surgery with bilateral cavernous nerve exposure plus phosphate-buffered saline; control, in which rats received BCNI plus phosphate-buffered saline; and experimental, in which rats received BCNI plus 4-phenylbutyric acid. Analysis of variance and a Bonferroni multiple-comparison test were utilized to evaluate differences among groups. Erectile function, smooth muscle/collagen ratios, and the expression levels of phenotypic modulation and ERS were measured. Two ratios-maximum intracavernosal pressure/mean arterial pressure and smooth muscle/collagen-were decreased in the control group as compared with the sham group. In penile tissue, there was increased expression of GRP78 (78-kDa glucose-regulated protein), p-PERK/PERK (phosphorylated protein kinase R-like endoplasmic reticulum kinase/protein kinase R-like endoplasmic reticulum kinase), caspase 3, CHOP (C/EBP homologous protein), and OPN (osteopontin) but decreased expression of nNOS (neuronal nitric oxide synthase) and α-SMA (α-smooth muscle actin). As compared with the control group, erectile function was improved and pathologic changes were partially recovered in the experimental group. The present study demonstrated that ERS is involved in ED caused by cavernous nerve injury, thereby providing a new target and theoretical basis for clinical treatment. The present study demonstrated for the first time that ERS is related to ED caused by cavernous nerve injury. Inhibition of ERS reverses phenotypic modulation and improves erectile function in rats with BCNI. Additional in vitro studies should be performed to verify these conclusions and explore the specific mechanism of phenotypic modulation. The present study demonstrated that inhibiting ERS reverses phenotypic modulation and enhances erectile function in rats with BCNI.

N-acetylcysteine ameliorates erectile dysfunction in rats with hyperlipidemia by inhibiting oxidative stress and corpus cavernosum smooth muscle cells phenotypic modulation.

Hyperlipidemia is a major risk factor for erectile dysfunction (ED). Oxidative stress and phenotypic modulation of corpus cavernosum smooth muscle cells (CCSMCs) are the key pathological factors of ED. N-acetylcysteine (NAC) can inhibit oxidative stress; however, whether NAC can alleviate pathological variations in the corpus cavernosum and promote erectile function recovery in hyperlipidemic rats remains unclear. A hyperlipidemia model was established using 27 eight-week-old male Sprague-Dawley (SD) rats fed a high-fat and high-cholesterol diet (hyperlipidemic rats, HR). In addition, 9 male SD rats were fed a normal diet to serve as controls (NC). HR rats were divided into three groups: HR, HR+normal saline (NS), and HR+NAC (n = 9 for each group; NS or NAC intraperitoneal injections were administered daily for 16 weeks). Subsequently, the lipid profiles, erectile function, oxidative stress, phenotypic modulation markers of CCSMCs, and tissue histology were analyzed. The experimental results revealed that erectile function was significantly impaired in the HR and HR + NS groups, but enhanced in the HR + NAC group. Abnormal lipid levels, over-activated oxidative stress, and multi-organ lesions observed in the HR and HR + NS groups were improved in the HR + NAC group. Moreover, the HR group showed significant phenotypic modulation of CCSMCs, which was also inhibited by NAC treatment. This report focuses on the therapeutic effect of NAC in restoring erectile function using a hyperlipidemic rat model by preventing CCSMC phenotypic modulation and attenuating oxidative stress.

(091) RNAseq and Pathway Analysis of Rat Penis with Cavernous Nerve Injury and Sonic Hedgehog Treatment by Peptide Amphiphile

Abstract Introduction Erectile dysfunction (ED) treatments are minimally effective in prostatectomy and diabetic patients due to injury to the cavernous nerve (CN). When the CN is injured, loss of innervation initiates penile remodeling, which includes abundant apoptosis in corpora cavernosal smooth muscle, followed by fibrosis. Decreased smooth muscle makes the erectile tissue less able to respond to normal neurotransmitter signaling, and ED results. When developing novel therapies, the goal is to prevent the apoptotic response and penile remodeling, to preserve erectile function. Our previous studies identify Sonic hedgehog (SHH) as a key mediator of penile remodeling. We examine what signaling mechanisms are altered with CN injury, and how SHH impacts changes in signaling to prevent penile remodeling and ED. Objective We performed RNA seq analysis and subsequent pathway analysis, to identify changes in gene expression and signaling pathways that are altered in response to cavernous nerve injury and with SHH treatment by peptide amphiphile (PA), in a rat prostatectomy model of ED. Methods Corpora cavernosa were obtained from Sprague Dawley rats (n=36) that underwent bilateral CN crush, CN crush plus SHH treatment of the penis by PA, or sham surgery (controls). Rats were sacrificed 1, 2 and 4 days after surgery. RNA was extracted using TRIzol, DNase treated, and purified by Qiagen mini kit. RNAseq analysis was performed, and subsequent directional pathway enrichment analysis was examined using Ingenuity software. A two-fold change with a q-value <0.05 was used as a threshold for differential expression. Z value measures were used to predict altered activity for the identified enrichments. Results mRNA representing 523 genes were significantly differentially expressed (>2 fold; Q<0.05) in CN crushed corpora cavernosal tissue relative to sham controls. Ingenuity analysis showed altered activity in canonical signaling and disease-function enrichments. These changes include increased necrosis and netrin, and decreased Integrin linked kinase, integrin, actin cytoskeleton, acute phase response signaling, cell movement, angiogenesis, and myeloid cell movement activity. With SHH PA treatment at the time of CN injury, these changes were reversed or significantly diminished. In addition, SHH PA impacted known mediators of ED by reducing apoptosis, and increasing cell survival, cell viability, muscle cell proliferation, VEGF, p38 MAPK, and PI3K/AKT pathways. Conclusions RNAseq and pathway analysis identified changes in gene expression and pathway signaling that is associated with CN injury in a rat prostatectomy model. Pathway analysis showed increases in processes associated with cell death and a loss of functions necessary for maintenance of corpora cavernosal architecture. Most of the CN injury associated changes were reversed or greatly diminished with SHH treatment of the penis at the time of CN injury by PA. These results support the significant potential of SHH PA to be developed as a preventative treatment for ED in prostatectomy patients. The miriad of altered signaling pathways that were identified with CN injury offer multiple, novel avenues for intervention independently and in combination with SHH PA treatment. Disclosure No

Fermented Gynochthodes officinalis (F.C.How) Razafim. & B.Bremer alleviates diabetic erectile dysfunction by attenuating oxidative stress and regulating PI3K/Akt/eNOS pathway.

As a traditional Chinese medicine, Gynochthodes officinalis (F.C.How) Razafim. & B.Bremer (G. officinalis) has been historically as tonics to treat impotence. Fermentation is an ancient processing method for traditional Chinese medicine. Whether fermentation affects the therapeutic effects of G. officinalis on diabetic erectile dysfunction has so far remained unknown. In this research, we aim to determine the effect of fermented or unfermented G. officinalis root extract on diabetes mellitus-induced erectile dysfunction (DMED) and the potential mechanisms. Candida sp. B5, Lactobacillus sp. Y5 and Lactobacillus sp. R2 are applied for the fermentation of G. officinalis. The optimum fermentation conditions of G. officinalis are investigated. Sprague-Dawley rats were used to establish a diabetic erectile dysfunction model, treated with different concentrations of fermented or unfermented G. officinalis, to compare the effect of fermented or unfermented G. officinalis on DMED and explore underlying mechanisms by assessment of intracavernous pressure, ELISA, Western blot, Masson's trichrome staining, and immunofluorescence. The corpus cavernosum smooth muscle cells (CCSMCs) and Schwann cells were isolated and used to investigate the effect of fermented or unfermented G. officinalis on hydrogen peroxide (H2O2)-induced apoptosis. The results reveal the optimum fermentation conditions of G. officinalis using Lactobacillus sp. Y5 were determined to be 35°C, the ratio of solid to liquid 1:10, and six days of fermentation. The fermentation increases the abundance of major active ingredients within G. officinalis. After fermented or unfermented G. officinalis treatment for eight weeks by oral gavage at a dose of 100mgkg-1 or 300mgkg-1, the results show that the fermentation enhances the effect of G. officinalis on diabetic erectile dysfunction detected by intracavernous pressure. The protein expressions of the PI3K/Akt/eNOS pathway were upregulated in diabetic rats after fermented or unfermented G. officinalis treatment, while the level of oxidative stress was significantly reduced. Meanwhile, Masson's trichrome staining also displayed an improvement in the ratio of smooth muscle to collagen. In vitro experiments confirmed that fermented or unfermented G. officinalis protected CCSMCs and Schwann cells from apoptosis. In contrast, fermented G. officinalis showed a fortified protective effect over unfermented G. officinalis. Our findings suggest that fermentation can increase the composition of main active ingredients in G. officinalis and enhance its role in diabetic erectile dysfunction. It augurs the potential therapeutic application of fermented G. officinalis well for treating diabetic erectile dysfunction.

Overexpression of PRDX2 in Adipose-Derived Mesenchymal Stem Cells Enhances the Therapeutic Effect in a Neurogenic Erectile Dysfunction Rat Model by Inhibiting Ferroptosis.

Neurogenic erectile dysfunction (NED) is a common and serious complication after pelvic surgery. The clinical translation of adipose-derived mesenchymal stem cell (ADSC) therapies in NED remains a major challenge due to their low survival rate and limited therapeutic effect. Peroxiredoxin 2 (PRDX2) is a member of the peroxidase family that exerts its therapeutic effects by inhibiting oxidative stress (OS) and ferroptosis, and PRDX2 is expected to enhance the therapeutic effect of ADSCs in treating NED. The purpose of this study was to investigate whether PRDX2 could improve the survival of ADSCs and determine whether overexpression of PRDX2 in ADSCs (PRDX2-ADSCs) could enhance the therapeutic effect of NED. This study investigated the potential role of PRDX2-ADSCs through a NED model induced by bilateral cavernous nerve injury (BCNI) and three in vitro models established by H2O2-stimulated ADSCs, H2O2-stimulated corpus cavernosum smooth muscle cells (CCSMCs), and RSL3-stimulated CCSMCs. We found that PRDX2 could significantly improve the viability of ADSCs by suppressing apoptosis and OS in H2O2-stimulated ADSCs. We also found that BCNI triggered ferroptosis of the corpus cavernosum, which was manifested by increased reactive oxygen species (ROS), total iron content, and MDA as well as decreased SOD and GSH. Our results further demonstrated changes in the expression of key proteins (GPX4 and ACSL4) in the ferroptosis pathway, whereas PRDX2-ADSCs ameliorated BCNI-induced erectile dysfunction and ferroptosis of the corpus cavernosum in NED rats. Consistently, PRDX2-ADSCs attenuated OS in H2O2-stimulated CCSMCs and inhibited ferroptosis in RSL3-stimulated CCSMCs, as evidenced by the decrease in ROS, total iron content, and MDA and the increase in SOD and GSH together with changes in ferroptosis-related protein (GPX4 and ACSL4) expression. In conclusion, overexpression of PRDX2 in ADSCs enhanced the therapeutic effect in a rat model of neurogenic erectile dysfunction by inhibiting ferroptosis via regulation of the GPX4/ACSL4 axis.

Adipose Derived Mesenchymal Stem Cells-Derived Mitochondria Transplantation Ameliorated Erectile Dysfunction Induced by Cavernous Nerve Injury.

Erectile dysfunction (ED) is a common postoperative complication of pelvic surgery for which there is currently no effective treatment. This study investigated the therapeutic effects and potential mechanisms of adipose derived mesenchymal stem cells-derived mitochondria (ADSCs-mito) transplantation in a rat model of bilateral cavernous nerve injury (CNI) ED. We isolated mitochondria from ADSCs and tested their quality. In vivo, twenty male Sprague Dawley rats were randomly divided into four groups: sham operation group and CNI groups that received intracavernous injection of either phosphate buffer solution, ADSCs-mito or ADSCs. Two weeks after therapy, the erectile function of the rats was evaluated and the penile tissues were harvested for histologic analysis and western blotting. In vitro, the apoptosis rate, reactive oxygen species (ROS), mitochondria derived active oxygen (mtROS) and adenosine triphosphate (ATP) levels were detected in corpus cavernosum smooth muscle cells (CCSMCs) after the incubation with ADSCs-mito. In addition, intercellular mitochondrial transfer was visualized by co-culture of ADSCs and CCSMCs. The ADSCs, ADSCs-mito and CCSMCs were isolated and identified successfully. ADSCs-mito transplantation notably restored the erectile function and smooth muscle content of CNI ED rats. Moreover, the levels of ROS, mtROS and cleaved-caspase 3 were reduced and the levels of superoxide dismutase and ATP were increased after ADSCs-mito transplantation. In CNI ED rats, the mitochondrial structure of cells in penile tissues was destroyed. ADSCs could transfer its own mitochondria to CCSMCs. Pre-treatment with ADSCs-mito could significantly decrease apoptosis rate, ROS levels and mtROS levels as well as restore the ATP level in CCSMCs. ADSCs-mito transplantation significantly ameliorated ED induced by CNI, with similar potency to ADSCs treatment. The ADSCs-mito might exert their effects via anti-oxidative stress, anti-apoptosis and modulating energy metabolism of CCSMCs. Mitochondrial transplantation should be a promising therapeutic method for treating CNI ED in the future.

Isolation, bioassay and 3D-QSAR analysis of 8-isopentenyl flavonoids from Epimedium sagittatum maxim. as PDE5A inhibitors

BackgroundAs known, inhibition of phosphodiesterase 5 (PDE5) has the therapeutic effect on male erectile dysfunction (ED), and the processed folium of Epimedium sagittatum Maxim. (PFES) characterized by 8-isopentenyl flavonoids is a famous herb for treating ED. However, the main flavonoids inhibitory activities, structure–activity relationship (SAR) and signaling pathway have been not systematically studied so that its pharmacodynamic mechanism is unclear.MethodsWe aimed to initially reveal the PFES efficacy mechanism for treating ED. For the first time, 6 main 8-isopentenyl flavonoids (1–6) from PFES were isolated and identified. Then based on HPLC detection, we proposed a novel method to screen inhibitors among them. We further analyze the three-dimensional quantitative structure–activity relationship (3D-QSAR) for those inhibitors.ResultsThe results were verified by cellular effects of the screened flavonoids. Among 6 compounds, Icariin: (1), 2-Oʹʹrhamnosylicaridide II (2) and Baohuoside I (3) were identified with significant activities (IC50 = 8.275, 3.233, 5.473 μM). Then 3D-QSAR studies showed that the replacement of C8 with bulky steric groups as isopentenyl, C3 with positive charge groups and C4' with a hydrogen bond acceptor substituent could increase inhibitory effects. In contrast, the substitution of C7 with bulky steric groups or hydrophilic groups tended to decrease the efficacies. And compounds 1, 2, 3 could increase cGMP level and decrease cytoplasmic Ca2+ of rat corpus cavernosum smooth muscle cells (CCSMCs)by activating PKG.Conclusion8-isopentenyl flavonoids could be the main pharmacodynamic substances of PFES in the treatment for ED, and some had significant PDE5A1 inhibitory activities so as to activate cGMP/PKG/Ca2+ signaling pathway in CCSMCs, that was related to the substituents at the key sites such as C8, C3, C4ʹ and C7 in the characteristic compounds.

Ethnological validation of Ashwagandha (Withania somnifera L. Dunal) ghrita as ‘Vajikarana Rasayana’: In-silico, in-vitro and in-vivoapproach

Ethnopharmacological relevanceProcessing cow ghee (clarified butterfat) with therapeutic herbs, i.e. ghrita, is recognized for augmenting the therapeutic efficacy of plant materials. Ashwagandha ghrita (AG) is an effective Ayurvedic formulation consisting of Indian ginseng, i.e., Withania somnifera (L.) Dunal, the main constituent used to treat infertility, weakness, gynaecological disorders, and general debility. ObjectivesThe present investigation was undertaken to corroborate the ethnopharmacological claim of AG as ‘Vajikarana Rasayana’ for its aphrodisiac potential using bioinformatics (in-silico) and experimental (in-vitro and in-vivo) approaches. MethodsAG was formulated as per the methods reported in Ayurved sarsangraha. AG was further subjected to HPLC, GCMS analysis, and biological (acute toxicity and aphrodisiac) assessment per the standard procedures. Thirty-eight bioactives of Indian ginseng were subjected to computational studies (molecular docking and network pharmacology) to confirm the plausible mechanism. ResultsAG was found to be safe up to 2000 mg/kg body wt., and it showed dose-dependent upsurge (p < 0.01 and p < 0.05, wherever necessary) in mount and intromission frequency, genital grooming, and anogenital sniffing at 150 and 300 mg/kg body weight suggesting aphrodisiac activity. In-vitro studies demonstrated significant relaxation of the Corpus Cavernosal Smooth Muscle at all concentrations in a dose-dependent manner. Furthermore, the results of molecular modelling studies were in agreement with the biological activity and showed interaction with phosphodiesterase-5 as a possible target. ConclusionAG exhibited an aphrodisiac effect and substantiated the traditional claim of Indian ginseng-based ghrita formulation as ‘Vajikarana Rasayana’.