Microenergy acoustic pulse therapy restores urethral wall integrity and continence in a rat model of female stress incontinence.

OBJECTIVE: To determine the outcomes and mechanisms of microenergy acoustic pulse (MAP) therapy in an irreversible rat model of female stress urinary incontinence. MATERIALS AND METHODS: Twenty-four female Sprague-Dawley rats were randomly assigned into four groups: sham control (sham), vaginal balloon dilation and ovariectomy (VBDO), VBDO + ß-aminopropionitrile (BAPN), and VBDO + ß-aminopropionitrile treated with MAP (MAP). MAP therapy was administered twice per week for 4 weeks. After a 1-week washout period, all 24 rats were evaluated with functional and histological studies. The urethral vascular plexus was examined by immunofluorescence staining with antibodies against collagen IV and von Willebrand factor (vWF). The urethral smooth muscle stem/progenitor cells (uSMPCs) were isolated and functionally studied in vivo and in vitro. RESULTS: Functional study with leak point pressure (LPP) measurement showed that the MAP group had significantly higher LPPs compared to VBDO and BAPN groups. MAP ameliorated the decline in urethral wall thickness and increased the amount of extracellular matrix within the urethral wall, especially in the urethral and vaginal elastic fibers. MAP also improved the disruption of the urethral vascular plexus in the treated animals. In addition, MAP enhanced the regeneration of urethral and vaginal smooth muscle, and uSMPCs could be induced by MAP to differentiate into smooth muscle and neuron-like cells in vitro. CONCLUSION: MAP appears to restore urethral wall integrity by increasing muscle content in the urethra and the vagina and by improving the urethral vascular plexus and the extracellular matrix.

[Up-regulated expression of miR-576 inhibits ALK4 expression, regulates JAK/STAT signaling pathway and promotes proliferation and migration of prostatic cancer cells].

OBJECTIVE: To explore the mechanism of the action of the miR-576/ALK4 axis on the progression of prostate cancer (PCa). METHODS: PCa cells were transfected with miR-576 mimics/inhibitor, the proliferation and migration distance of the cells were detected by MTT and scratch wound healing assay, respectively. The targeted regulation effect of miR-576 on ALK4 was verified by dual-luciferase reporter assay. The effects of miR-576 on the mRNA and protein expressions and phosphorylation levels of the ALK4 and JAK/STAT signaling pathway factors JAK2 and STAT3 were determined by qPCR and Western blot, respectively. The C4-2 cells were co-treated with sh-ALK4 and Ruxolitinib for measurement of the proliferation and migration of the PCa cells. RESULTS: Bioinformatics analysis and binding site prediction showed that miR-576 was up-regulated in the PCa cells, and dual-luciferase reporter assay revealed its targeted regulation effect on ALK4 and its impact on the phosphorylation levels of JAK2 and STAT3. Overexpressed miR-576 promoted while knocked-down miR-576 inhibited the proliferation and migration of the PCa cells. sh-ALK4 increased the proliferation and migration of the cells, while Ruxolitinib suppressed the promoting effect of sh-ALK4. CONCLUSION: The expression of miR-576 is up-regulated in PCa, inhibits the expression of ALK4, regulates the activity of the JAK and STAT signaling pathways, and promotes the proliferation and migration of PCa cells.

Knockdown of miR-423-5p simultaneously upgrades the eNOS and VEGFa pathways in ADSCs and improves erectile function in diabetic rats.

This study aimed to explore the possibility of miR-423-5p modified adipose-derived stem cell (ADSCs) therapy on streptozotocin (STZ)-induced diabetes mellitus erectile dysfunction (DMED) rats. MiR-423-5p was knocked down in ADSCs. ADSCs, NC-miR-ADSCs and miR-ADSCs were co-cultured with human umbilical vein endothelial cells (HUVECs). Normal and high glucose media were supplemented. The supernatant and HUVECs were collected for assessment of eNOS and VEGFa expression, cell proliferation, and apoptosis. HUVECs co-cultured with ADSCs or miR-ADSCs exhibited higher eNOS and VEGFa protein expression levels compared to DM groups. MiR-ADSCs enhanced HUVEC proliferation compared to the ADSCs and NC-miR-ADSCs. Lower apoptotic rates were observed when HUVECs were co-cultured with miR-ADSCs, compared to ADSCs and NC-miR-ADSCs. Fifteen male Sprague-Dawley (SD) rats aged 12 weeks were induced to develop diabetes mellitus by intraperitoneal injection with STZ, and five healthy SD rats were used as normal controls. Eight weeks after developing diabetes, the rats received ADSCs and miR-ADSCs via injection into the corpora cavernosa, whereas normal controls and DM controls were injected with saline. Erectile function and histological assessment of penile tissues were performed 8 weeks after injection. The ICP/MAP indicated that erectile function was impaired in the DM rats compared with the normal group. Injection of ADSCs and miR-ADSCs improved erectile function significantly and was associated with the overexpression of eNOS and VEGFa. MiR-423-5p knockdown in ADSCs ameliorated high glucose-mediated damage to HUVECs and improved erectile function in DM rats by inducing eNOS and VEGFa overexpression, indicating that miR-423-5p may be a potential target in the treatment of DMED.

Exosome Released From Schwann Cells May Be Involved in Microenergy Acoustic Pulse-Associated Cavernous Nerve Regeneration.

BACKGROUND: Neurogenic erectile dysfunction (ED) is often refractory to treatment because of insufficient functional nerve recovery after injury or insult. Noninvasive mechano-biological intervention, such as microenergy acoustic pulse (MAP), low-intensity pulsed ultrasound, and low-intensity extracorporeal shockwave treatment, is an optimal approach to stimulate nerve regeneration. AIM: To establish a new model in vitro to simulate nerve injury in neurogenic ED and to explore the mechanisms of MAP in vitro. METHODS: Sprague-Dawley rats were used to isolate Schwann cells (SCs), major pelvic ganglion (MPG), and cavernous nerve with MPG (CN/MPG). SCs were then treated with MAP (0.033 mJ/mm2, 1 Hz, 100 pulses), and SC exosomes were isolated. The MPG and CN/MPG were treated with MAP (0.033 mJ/mm2, 1 Hz) at different dosages (25, 50, 100, 200, or 300 pulses) or exosomes derived from MAP-treated SCs in vitro. OUTCOMES: Neurite growth from the MPG fragments and CN was photographed and measured. Expression of neurotropic factors (brain-derived neurotrophic factor, nerve growth factor, and neurotrophin-3) was checked. RESULTS: Neurite outgrowth from MPG and CN/MPG was enhanced by MAP in a dosage response manner, peaking at 100 pulses. MAP promoted SC proliferation, neurotropic factor (brain-derived neurotrophic factor, nerve growth factor, and neurotrophin-3) expression, and exosome secretion. SC-derived exosomes significantly enhanced neurite outgrowth from MPG in vitro. CLINICAL IMPLICATIONS: MAP may have utility in the treatment of neurogenic ED by SC-derived exosomes. STRENGTH & LIMITATIONS: We confirmed that MAP enhances penile nerve regeneration through exsomes. Limitations of this study include that our study did not explore the exact mechanisms of how MAP increases SC exosome secretion nor whether MAP modulates the content of exosomes. CONCLUSION: This study revealed that neurite outgrowth from MPG was enhanced by MAP and by SC-derived exosomes which were isolated after MAP treatment. Our findings indicate that one mechanism by which MAP induces nerve regeneration is by stimulation of SCs to secrete exosomes. Peng D, Reed-Maldonado AB, Zhou F, et al. Exosome Released From Schwann Cells May Be Involved in Microenergy Acoustic Pulse-Associated Cavernous Nerve Regeneration. J Sex Med 2020;17:1618-1628.

Testis developmental related gene 1 regulates the chemosensitivity of seminoma TCam-2 cells to cisplatin via autophagy.

We previously identified testis developmental related gene 1 (TDRG1), a gene implicated in proliferation of TCam-2 seminoma cells. Recent evidence has revealed that autophagy influences the chemosensitivity of cancer cells to chemotherapy. However, whether TDRG1 protein regulates autophagy in seminoma cells and influences their sensitivity to cis-dichlorodiammine platinum (CDDP) remains unknown. In this study, we used TCam-2 cells and male athymic BALB/c nude mice with xenografts of TCam-2 cells to investigate autophagy, cell viability, apoptosis and the p110ß/Rab5/Vps34 (PI3-kinase Class III) pathway under the conditions of TDRG1 overexpression or knockdown and with or without CDDP treatment. We found that TDRG1 upregulation promoted autophagy in both TCam-2 cells and seminoma xenografts via p110ß/Rab5/Vps34 activation. Inhibition of autophagy reduced cell viability and promoted apoptosis during CDDP treatment of TCam-2 cells. Similarly, TDRG1 knockdown inhibited autophagy, reduced cell viability and promoted apoptosis during CDDP treatment of TCam-2 cells. TDRG1 knockdown inhibited tumour growth and promoted apoptosis in TCam-2 cell xenografts, whereas TDRG1 overexpression had the opposite effect. According to these results, we propose that high expression of TDRG1 promotes autophagy through the p110ß/Rab5/Vps34 pathway in TCam-2 cells. TDRG1 overexpression promotes autophagy and leads to CDDP resistance, whereas TDRG1 knockdown inhibits autophagy and promotes chemosensitivity to CDDP both in vivo and in vitro. This study has uncovered a novel role of TDRG1 in reducing chemoresistance during CDDP treatment and provides potential therapeutic strategies for the treatment of human seminoma.

Smooth Muscle Differentiation of Penile Stem/Progenitor Cells Induced by Microenergy Acoustic Pulses In Vitro.

INTRODUCTION: Modulating tissue-resident stem and progenitor cells with a non-invasive, mechanobiological intervention is an optimal approach for tissue regeneration. Stem cell antigen-1 (Sca-1) has been identified as a stem cell marker within many organs but never within the penis. AIM: To localize and isolate penile stem/progenitor cells (PSPCs) and to evaluate cellular differentiation after exposure to induction medium and microenergy acoustic pulse (MAP) therapy. METHODS: Six male Sprague-Dawley rats were used to isolate PSPCs. Isolation was followed by stem cell characterization and differentiation assays. The PSPCs were then treated with MAP (0.033 mJ/mm2, 1 Hz) at various dosages (25, 50, 100, and 200 pulses) and for different durations (1, 2, 4, 6, or 8 hours) in vitro. MAIN OUTCOME MEASURE: The PSPCs (Sca-1-positive cells) were isolated using the magnetic-activated cell sorting system. PSPC cellular differentiation was assessed after induction with induction medium and with MAP in vitro. Wnt/ß-catenin signaling was also assayed. RESULTS: The PSPCs were successfully localized within the penile subtunic and perisinusoidal spaces, and they were successfully isolated using magnetic-activated cell sorting. The stemness of the cells was confirmed by stem cell marker characterization and by multiple differentiation into smooth muscle cells, endothelial cells, adipocytes, and neurons. MAP-induced PSPCs differentiated into smooth muscle cells by activating the Wnt/ß-catenin signaling pathway in a time- and dosage-dependent manner. CLINICAL IMPLICATIONS: By modulating resident PSPCs, MAP may have utility in the treatment of erectile dysfunction (ED). STRENGTHS & LIMITATIONS: This study provides solid evidence in support of microenergy therapies, including both MAP and low-intensity extracorporeal shock wave therapy, for the treatment of ED. Additional studies are needed and should include additional stem cells markers. Furthermore, studies exploring the underling mechanisms for PSPC activation and differentiation are required. CONCLUSION: PSPCs were successfully identified, localized, and isolated. Additionally, MAP provoked PSPCs to differentiate into smooth muscle cells via the Wnt/ß-catenin signaling pathway. As such, MAP provides a novel method for activating endogenous tissue-resident stem/progenitor cells and might facilitate stem cell regenerative therapy targeting ED. Peng D, Yuan H, Liu T, et al. Smooth Muscle Differentiation of Penile Stem/Progenitor Cells Induced by Microenergy Acoustic Pulses In Vitro. J Sex Med 2019; 16:1874-1884.

Delayed Low-Intensity Extracorporeal Shock Wave Therapy Ameliorates Impaired Penile Hemodynamics in Rats Subjected to Pelvic Neurovascular Injury.

BACKGROUND: Erectile dysfunction (ED) caused by pelvic neurovascular injury (PNVI) is often refractory to treatment. In many cases, erectogenic therapy is administered in a delayed fashion. AIM: To evaluate penile hemodynamic effects and histologic changes associated with delayed low-intensity extracorporeal shock wave therapy (Li-ESWT) after PNVI ED in a rat model. We visualized images using immunofluorescence and 3-dimensional imaging of solvent-cleared organs (3DISCO), a novel imaging technique. METHODS: A total of 32 Sprague-Dawley male rats aged 12 weeks were divided equally into 4 groups: sham surgery as normal controls (NC), PNVI controls (PC), PNVI with very-low-energy Li-ESWT (PVL), and PNVI with low-energy Li-ESWT (PL). Bilateral cavernous nerve crush and internal pudendal bundle ligation were performed in the 3 PNVI groups. Li-ESWT was administered twice a week for 4 weeks in the PL and PVL groups starting at 4 weeks after PNVI. OUTCOMES: Intracavernous pressure (ICP) studies (normalized to mean arterial pressure [MAP]) were conducted in all subject animals. After testing, tissue was harvested for immunofluorescence staining and 3DISCO analysis. RESULTS: Mean ICP/MAP was lower in PC animals compared with NC animals (0.37 ± 0.03 vs 0.91 ± 0.03, respectively; P = .001). The ICP/MAP ratio was significantly higher in PVL and PL animals (0.66 ± 0.07 and 0.82 ± 0.05, respectively) compared with PC animals (P = .002 and .001, respectively). Detailed microstructures and trajectories of nerves and vessels were identified with immunofluorescence and 3DISCO. The PC group had lower density of nerves, axons, neuronal nitric oxide synthase-positive nerves, and Schwann cells in the dorsal penis. Animals in the PL group had significantly higher expression of all of these markers compared with PC animals. CLINICAL IMPLICATIONS: Li-EWST may have utility in the management of severe ED related to PNVI from severe pelvic injury or radical pelvic surgeries, even when administered in a delayed fashion. STRENGTH & LIMITATIONS: This study of a severe ED phenotype involved treatment administered in a delayed fashion, which is more consistent with how therapy likely would be delivered in a real-world clinical context. Moreover, because the treatment commenced at 4 weeks after injury, when nerve and tissue atrophy have already occurred, the results imply that Li-ESWT can be used for regenerative therapy. Additional studies on dose optimization and treatment interval are needed to inform the design of human clinical trials. CONCLUSION: Li-ESWT ameliorates the negative functional and histologic effects of severe pelvic neurovascular injury in a rat model system. 3DISCO provides high-resolution images of neuroanatomy and neural regeneration. Wang HS, Ruan Y, Banie L, et al. Delayed Low-Intensity Extracorporeal Shock Wave Therapy Ameliorates Impaired Penile Hemodynamics in Rats Subjected to Pelvic Neurovascular Injury. J Sex Med 2019;16:17-26.

The effects of microenergy acoustic pulses on animal model of obesity-associated stress urinary incontinence. Part 2: In situ activation of pelvic floor and urethral striated muscle progenitor cells.

AIM: To investigate the possibility and mechanism of microenergy acoustic pulses (MAP) for activating tissue resident stem/progenitor cells within pelvic and urethral muscle and possible mechanism. METHODS: The female Zucker Lean and Zucker Fatty rats were randomly divided into four groups: ZL control, ZLMAP, ZF control, and ZFMAP. MAP was applied at 0.033 mJ/mm2 , 3 Hz for 500 pulses, and the urethra and pelvic floor muscles of each rat was then harvested for cell isolation and flow cytometry assay. Freshly isolated cells were analyzed by flow cytometry for Pax-7, Int-7α, H3P, and EdU expression. Meanwhile, pelvic floor muscle-derived stem cells (MDSCs) were harvested through magnetic-activated cell sorting, MAP was then applied to MDSCs to assess the mechanism of stem cell activation. RESULTS: Obesity reduced EdU-label-retaining cells and satellite cells in both pelvic floor muscle and urethra, while MAP activated those cells and enhanced cell proliferation, which promoted regeneration of striated muscle cells of the pelvic floor and urethral sphincter. Activation of focal adhesion kinase (FAK)/AMP-activated protein kinase (AMPK) /Wnt/ß-catenin signaling pathways by MAP is the potential mechanism. CONCLUSIONS: MAP treatment activated tissue resident stem cells within pelvic floor and urethral muscle in situ via activating FAK-AMPK and Wnt/ß-catenin signaling pathway.

The effects of microenergy acoustic pulses on an animal model of obesity-associated stress urinary incontinence. Part 1: Functional and histologic studies.

AIM: Obesity is a strong independent risk factor for urinary incontinence. Effective therapeutic approaches for obesity-associated stress urinary incontinence (OA-SUI) are lacking as the mechanisms remain unclear. The aim of our study is to explore the impacts of microenergy acoustic pulse (MAP) therapy on urethral and pelvic floor muscle structure and function in female lean and fatty rats. METHODS: A total 24 Zucker fatty (ZF) and 24 Zucker lean (ZL) female 24-week-old rats were grouped into four groups: ZL control, ZLMAP, ZF control, and ZFMAP. For MAP treatment, 500 pulses were delivered at an energy level of 0.033 mJ/mm 2 and a frequency of 3 Hz and were applied twice a week for 4 weeks. After a 1-week washout, all rats underwent conscious cystometry and leak-point pressure (LPP) measurements followed by ex vivo organ-bath assay and histological study. RESULTS: ZF rats had lower LPP as compared to ZL rats, and MAP treatment significantly improved LPP in ZF rats (P < .05). Impaired muscle contractile activity (MCA) in organ-bath study was noted in ZF rats. MAP treatment significantly increased MCA in ZF rats (P < .05) and also increased the thickness of the striated muscle layer and the number of neuromuscular junctions (NMJs). In situ, MAP activated muscle satellite cells significantly (P < .05). CONCLUSIONS: Obesity impairs the function of both the urethral sphincter and the pelvic floor and leads to atrophy and distortion of the striated muscle in obese female rats. These issues contribute to OA-SUI. MAP improves continence by stimulating muscle regeneration and nerve innervation as well as by activating satellite cells.

[Roles of adenosine and cytokines in the prostate tissue of rats with acute bacterial prostatitis].

OBJECTIVE: To investigate the possible roles of adenosine and the cytokines TNF-alpha and IL-10 in the pathogenesis of acute bacterial prostatitis (ABP) in rats. METHODS: Forty-eight male Wistar rats were randomly divided into groups A (ABP), B (ABP + theophylline intervention), C (sham) and D (blank control). ABP models were established by injecting Escherichia coli 0157 into the prostate, and those in group B were treated by intraperitoneal injection of theophylline immediately after modeling. At 4 and 14 days, the prostate tissues of the rats were collected for detection of the expressions of TNF-alpha and IL-10 by immunohistochemistry and the concentration of adenosine by high-performance liquid chromatography. RESULTS: At 4 and 14 days, the concentrations of adenosine were significantly higher in group A ([48.38 +/- 17.27] and [26.54 +/- 11.22] microg/g) than in C ([0.45 +/- 0.25] and [0.46 +/- 0.29] microg/g) and D ([0.41 +/- 0.23] and [0.43 +/- 0.27] microg/g) (P < 0.05), and so were the expressions of TNF-alpha in A (0.23 +/- 0.08 and 0.21 +/- 0.03) than in C (0.07 +/- 0.03 and 0.07 +/- 0.01) and D (0.07 +/- 0.06 and 0.07 +/- 0.06) (P < 0.05), and those of IL-10 in A (0.13 +/- 0.03 and 0.25 +/- 0.01) than in C (0.07 +/- 0.03 and 0.07 +/- 0.03) and D (0.07 +/- 0.01 and 0.07 +/- 0.02) (P < 0.05). Compared with group A, the rats in group B showed significant increases at 4 and 14 days in the severity of inflammation, concentration of adenosine ([86.64 +/- 32.87] and [51.17 +/- 22.96] microg/g, P < 0.05) and expression of TNF-alpha (0.37 +/- 0.08 and 0.32 +/- 0.06, P < 0.05), but exhibited no remarkable difference in the expression of IL-10 (0.12 +/- 0.06 and 0.15 +/- 0.06, P > 0.05). CONCLUSION: Adenosine may affect the progression of inflammation by regulating the expressions of the cytokines TNF-alpha and IL-10 in ABP rats through the adenosine receptor signaling pathway.

Effects of low-intensity pulsed ultrasound on the microorganisms of expressed prostatic secretion in patients with IIIB prostatitis.

To detect and analyze the changes of microorganisms in expressed prostatic secretion (EPS) of patients with IIIB prostatitis before and after low-intensity pulsed ultrasound (LIPUS) treatment, and to explore the mechanism of LIPUS in the treatment of chronic prostatitis (CP). 25 patients (study power was estimated using a Dirichlet-multinomial approach and reached 96.5% at α = 0.05 using a sample size of 25) with IIIB prostatitis who were effective in LIPUS treatment were divided into two groups before and after LIPUS treatment. High throughput second-generation sequencing technique was used to detect and analyze the relative abundance of bacterial 16 s ribosomal variable regions in EPS before and after treatment. The data were analyzed by bioinformatics software and database, and differences with P < 0.05 were considered statistically significant. Beta diversity analysis showed that there was a significant difference between groups (P = 0.046). LEfSe detected four kinds of characteristic microorganisms in the EPS of patients with IIIB prostatitis before and after LIPUS treatment. After multiple comparisons among groups by DESeq2 method, six different microorganisms were found. LIPUS may improve patients' clinical symptoms by changing the flora structure of EPS, stabilizing and affecting resident bacteria or opportunistic pathogens.

Low-intensity pulsed ultrasound ameliorates erectile dysfunction induced by bilateral cavernous nerve injury through enhancing Schwann cell-mediated cavernous nerve regeneration.

BACKGROUND: Cavernous nerve injury-induced erectile dysfunction caused by pelvic surgery or trauma is refractory to conventional medications and required an alternative treatment. Low-intensity pulsed ultrasound is a noninvasive mechanical therapy that promotes nerve regeneration. OBJECTIVES: To investigate the therapeutic effect and potential mechanism of low-intensity pulsed ultrasound in the treatment of neurogenic erectile dysfunction. MATERIALS AND METHODS: Thirty rats were randomly divided into the sham-operated group, bilateral cavernous nerve injury group, and bilateral cavernous nerve injury + low-intensity pulsed ultrasound group. The erectile function was assessed 3 weeks after daily low-intensity pulsed ultrasound treatment. The penile tissues and cavernous nerve tissues were harvested and subjected to histologic analysis. Primary Schwann cells and explants were extracted from adult rats. The effects of low-intensity pulsed ultrasound on proliferation, migration, and nerve growth factor expression of Schwann cells and axonal elongation were examined in vitro. RNA sequencing and western blot assay were applied to predict and verify the molecular mechanism of low-intensity pulsed ultrasound-induced Schwann cell activation. RESULTS: Our study showed that low-intensity pulsed ultrasound promoted Schwann cells proliferation, migration, and neurotrophic factor nerve growth factor expression. Meanwhile, low-intensity pulsed ultrasound exhibits a stronger ability to enhance Schwann cells-mediated neurite outgrowth of major pelvic ganglion neurons and major pelvic ganglion/cavernous nerve explants in vitro. In vivo experiments demonstrated that the erectile function of the rats in the bilateral cavernous nerve injury + low-intensity pulsed ultrasound group was significantly higher than those in the bilateral cavernous nerve injury groups. Moreover, the expression levels of smooth muscle and cavernous endothelium also increased significantly in the bilateral cavernous nerve injury + low-intensity pulsed ultrasound group. In addition, we observed the higher density and number of cavernous nerve regenerating axons in the bilateral cavernous nerve injury + low-intensity pulsed ultrasound group, indicating that low-intensity pulsed ultrasound promotes axonal regeneration following cavernous nerve injury in vivo. RNA sequencing analysis and bioinformatic analysis suggested that low-intensity pulsed ultrasound might trigger the activation of the PI3K/Akt pathway. Western blot assay confirmed that low-intensity pulsed ultrasound activated Schwann cells through TrkB/Akt/CREB signaling. CONCLUSIONS: Low-intensity pulsed ultrasound promoted nerve regeneration and ameliorated erectile function by enhancing Schwann cells proliferation, migration, and neurotrophic factor nerve growth factor expression. The TrkB/Akt/CREB axis is the possible mechanism of low-intensity pulsed ultrasound-mediated Schwann cell activation. Low-intensity pulsed ultrasound-based therapy could be a novel potential treatment strategy for cavernous nerve injury-induced neurogenic erectile dysfunction.

The m6A methylation landscape, molecular characterization and clinical relevance in prostate adenocarcinoma.

Background: Despite the recent progress of therapeutic strategies in treating prostate cancer (PCa), the majority of patients still eventually relapse, experiencing dismal outcomes. Therefore, it is of utmost importance to identify novel viable targets to increase the effectiveness of treatment. The present study aimed to investigate the potential relationship between N6-methyladenosine (m6A) RNA modification and PCa development and determine its clinical relevance. Methods: Through systematic analysis of the TCGA database and other datasets, we analyzed the gene expression correlation and mutation profiles of m6A-related genes between PCa and normal tissues. Patient samples were divided into high- and low-risk groups based on the results of Least Absolute Shrinkage and Selection Operator (LASSO) Cox analysis. Subsequently, differences in biological processes and genomic characteristics of the two risk groups were determined, followed by functional enrichment analysis and gene set enrichment (GSEA) analysis. Next, we constructed the protein-protein interaction (PPI) network of differentially expressed genes between patients in high- and low-risk groups, along with the mRNA-miRNA-lncRNA network. The correlation analysis of tumor-infiltrating immune cells was further conducted to reveal the differences in immune characteristics between the two groups. Results: A variety of m6A-related genes were identified to be differentially expressed in PCa tissues as compared with normal tissues. In addition, the PPI network contained 278 interaction relationships and 34 m6A-related genes, and the mRNA-miRNA-lncRNA network contained 17 relationships, including 91 miRNAs. Finally, the immune characteristics analysis showed that compared with the low-risk group, the levels of M1 and M2 macrophages in the high-risk group significantly increased, while the levels of mast cells resting and T cells CD4 memory resting significantly decreased. Conclusions: This study provides novel findings that can further the understanding of the role of m6A methylation during the progression of PCa, which may facilitate the invention of targeted therapeutic drugs.

LIPUS-SCs-Exo promotes peripheral nerve regeneration in cavernous nerve crush injury-induced ED rats via PI3K/Akt/FoxO signaling pathway.

OBJECTIVE: Clinical treatment of erectile dysfunction (ED) caused by cavernous nerve (CN) injury during pelvic surgery is difficult. Low-intensity pulsed ultrasound (LIPUS) can be a potential strategy for neurogenic ED (NED). However, whether Schwann cells (SCs) can respond to LIPUS stimulation signals is unclear. This study aims to elucidate the signal transmission between SCs paracrine exosome (Exo) and neurons stimulated by LIPUS, as well as to analyze the role and potential mechanisms of exosomes in CN repair after injury. METHODS: The major pelvic ganglion (MPG) neurons and MPG/CN explants were stimulated with LIPUS of different energy intensities to explore the appropriate LIPUS energy intensity. The exosomes were isolated and purified from LIPUS-stimulated SCs (LIPUS-SCs-Exo) and non-stimulated SCs (SCs-Exo). The effects of LIPUS-SCs-Exo on neurite outgrowth, erectile function, and cavernous penis histology were identified in bilateral cavernous nerve crush injury (BCNI)-induced ED rats. RESULTS: LIPUS-SCs-Exo group can enhance the axon elongation of MPG/CN and MPG neurons compared to SCs-Exo group in vitro. Then, the LIPUS-SCs-Exo group showed a stronger ability to promote the injured CN regeneration and SCs proliferation compared to the SCs-Exo group in vivo. Furthermore, the LIPUS-SCs-Exo group increased the Max intracavernous pressure (ICP)/mean arterial pressure (MAP), lumen to parenchyma and smooth muscle to collagen ratios compared to the SCs-Exo group in vivo. Additionally, high-throughput sequencing combined with bioinformatics analysis revealed the differential expression of 1689 miRNAs between the SCs-Exo group and the LIPUS-SCs-Exo group. After LIPUS-SCs-Exo treatment, the phosphorylated levels of Phosphatidylinositol 3-kinase (PI3K), protein kinase B (Akt) and forkhead box O (FoxO) in MPG neurons increased significantly compared to negative control (NC) and SCs-Exo groups. CONCLUSION: Our study revealed that LIPUS stimulation could regulate the gene of MPG neurons by changing miRNAs derived from SCs-Exo, then activating the PI3K-Akt-FoxO signal pathway to enhance nerve regeneration and restore erectile function. This study had important theoretical and practical significance for improving the NED treatment.

Molecular Mechanism of Action of Low-Intensity Extracorporeal Shockwave Therapy for Regenerating Penile and Peripheral Nerves.

Sufficient functional repair of damaged peripheral nerves is a big clinical challenge in terms of long-lasting morbidity, disability, and economic costs. Nerve damage after radical prostatectomy is the most common cause of erectile dysfunction. In recent years, low-intensity extracorporeal shockwave therapy has been explored to improve the outcomes of peripheral nerve repair and regeneration. Research indicated that application of low-intensity extracorporeal shockwave therapy after nerve surgery promoted nerve regeneration and improved the functional outcomes, underlined the mechanisms related to increase of neurotrophic factors, Schwann cells activation, and cellular signaling activation for cell activation and mitosis induced by low-intensity extracorporeal shockwave therapy. We searched PubMed for articles related to research on these topics in both in vitro and in vivo animal models and found numerous studies suggesting that the application low-intensity extracorporeal shockwave therapy could be a novel treatment for erectile dysfunction induced by nerve injury and other disease related to nerve injury.

Tetrathiomolybdate Partially Alleviates Erectile Dysfunction of Type 1 Diabetic Rats Through Affecting Ceruloplasmin/eNOS and Inhibiting Corporal Fibrosis and Systemic Inflammation.

INTRODUCTION: Patients with erectile dysfunction induced by diabetes mellitus (DMED) show a poor effect rate for oral phosphodiesterase type 5 inhibitors (PDE5is). Therefore, the new therapeutic strategy is necessary in patients with DMED. AIM: To investigate whether Tetrathiomolybdate (TM) supplementation could ameliorate DMED by activation of eNOS. METHODS: Twenty-four diabetic rats were induced by intraperitoneal injection of streptozotocin (STZ) and the other 6 normal rats constituted the control group. Eight weeks later, the erectile function of rats was assessed with an apomorphine test. Only some rats with DMED were treated with TM orally every day for 4 weeks; the other rats remained in the same condition for 4 weeks. After 1 week washout, the erectile function of rats in each group was evaluated. Then, the serum concentration of IL-6 and histologic changes of corpus cavernosum were measured. MAIN OUTCOME MEASURE: Erectile function was measured after DMED rats treated with TM. The cavernosum level of Ceruloplasmin (Cp), eNOS, endothelial cell content, corporal fibrosis, apoptosis rate and the serum level of IL-6 were also assayed. RESULTS: Erectile function in the DMED group was significantly impaired compared with the control group and was partly, but significantly, improved in the DMED+TM group. The DMED group showed upregulation of Cp and inhibition of eNOS, but the inhibition was partly reversed in the DMED+TM group. The DMED group showed serious corporal fibrosis. However, TM supplementation partly increased the ratio of smooth muscle to collagen, decreased the ratio of apoptosis. What's more, gavage administration of TM profoundly decreased the serum level of IL-6 in DMED rats. CONCLUSION: TM supplementation inhibits endothelial dysfunction, corporal fibrosis, and systemic inflammation, ultimately leading to partial improvement of DMED in rats. Yin Y, Peng J, Zhou J, et al., Tetrathiomolybdate Partially Alleviates Erectile Dysfunction of Type 1 Diabetic Rats Through Affecting Ceruloplasmin/eNOS and Inhibiting Corporal Fibrosis and Systemic Inflammation. Sex Med 2022;10:100455.

The m6A methyltransferase METTL3 regulates autophagy and sensitivity to cisplatin by targeting ATG5 in seminoma.

BACKGROUND: Our previous work shows Autophagy enhanced resistance to cisplatin in seminoma. The expression of the N6-methyladenosine (m6A) methyltransferases METTL3 was significantly increased in the cisplatin-resistant TCam-2 cell line of seminoma. We aimed to investigate the role of m6A methylation in autophagy and the chemosensitivity of seminoma cells. METHODS: Plasmid and siRNA were used to overexpress and knockdown METTL3. Autophagy was detected by western blot and immunofluorescence, respectively. The expression of downstream targets of METTL3 was detected by quantitative real-time PCR (qRT-PCR) and western blot, and the m6A level of them was detected by MeRIP-qPCR. Chemosensitivity of the TCam-2 cell line was identified through MTT assay. RESULTS: Upon METTL3 overexpression, autophagy of TCam-2 cell line was enhanced and its sensitivity to cisplatin was decreased. The use of autophagy inhibitors 3-methyladenine (3-MA) could reverse the protective effect of METTL3 on TCam-2 cells. We found that the up-regulation of METTL3 could increase the m6A modification level of ATG5 transcript, thus increased expression of ATG5. Moreover, knockdown of ATG5 reduced METTL3-induced autophagy, suggesting that ATG5 was a potential target for METTL3 to promote autophagy. CONCLUSIONS: In summary, our research unveiled the unique mechanism by which m6A methylation regulates autophagy and chemosensitivity of the TCam-2 cell line and METTL3 was a potential target to overcome the cisplatin resistance of seminoma.

Molecular mechanism of action of low-intensity extracorporeal shockwave therapy for regenerating penile and peripheral nerves.

Sufficient functional repair of damaged peripheral nerves is a big clinical challenge in terms of long-lasting morbidity, disability, and economic costs. Nerve damage after radical prostatectomy is the most common cause of erectile dysfunction (ED). In recent years, low-intensity extracorporeal shockwave therapy (Li-ESWT) has been explored to improve the outcomes of peripheral nerve repair and regeneration. Research indicated that application of Li-ESWT after nerve surgery promoted nerve regeneration and improved the functional outcomes, underlined the mechanisms related to increase of neurotrophic factors, Schwann cells activation, and cellular signaling activation for cell activation and mitosis induced by Li-ESWT. We searched PubMed for articles related to research on these topics in both in vitro and in vivo animal models and found numerous studies suggesting that the application Li-ESWT could be a novel treatment for ED induced by nerve injury and other disease related to nerve injury.

Low-intensity pulsed ultrasound for regenerating peripheral nerves: potential for penile nerve.

Peripheral nerve damage, such as that found after surgery or trauma, is a substantial clinical challenge. Much research continues in attempts to improve outcomes after peripheral nerve damage and to promote nerve repair after injury. In recent years, low-intensity pulsed ultrasound (LIPUS) has been studied as a potential method of stimulating peripheral nerve regeneration. In this review, the physiology of peripheral nerve regeneration is reviewed, and the experiments employing LIPUS to improve peripheral nerve regeneration are discussed. Application of LIPUS following nerve surgery may promote nerve regeneration and improve functional outcomes through a variety of proposed mechanisms. These include an increase of neurotrophic factors, Schwann cell (SC) activation, cellular signaling activations, and induction of mitosis. We searched PubMed for articles related to these topics in both in vitro and in vivo animal research models. We found numerous studies, suggesting that LIPUS following nerve surgery promotes nerve regeneration and improves functional outcomes. Based on these findings, LIPUS could be a novel and valuable treatment for nerve injury-induced erectile dysfunction.

Development of Male External Urethral Sphincter and Tissue-Resident Stem/Progenitor Cells in Rats.

Stress urinary incontinence (SUI) after prostate surgery is primarily caused by urethral sphincter damage. There are few effective therapeutic approaches for male SUI due to both insufficient study of the structure of the external urethral sphincter (EUS) and incomplete understanding of the resident EUS stem/progenitor cells. The goals of this study were to localize and to determine the distribution of tissue-resident stem/progenitor cells in the male EUS throughout EUS development and to understand the anatomic temporal patterns of the EUS. Newborn Sprague Dawley rats were intraperitoneally injected with the thymidine analogue, 5-ethynyl-2-deoxyuridine (EdU), and the EUS was harvested at five time points (1, 2, 3, 4, and 8 weeks postinjection). The tissue was then processed for EdU staining and immunofluorescence staining for stem cell markers Ki67 and proliferating cell nuclear antigen. We counted the EdU+ label-retaining cells (LRCs) at each time point and colocalized with each stem cell marker, also we isolated and cultured the cells in vitro. The results revealed that the number of EdU+ LRCs in each EUS cross-section decreased over time and that the LRCs were located immediately under the basal membrane of laminin, densely adherent to the muscle fibers. In addition, the thickness of the striated muscle layer developed much faster than the smooth muscle layer during EUS development. By 4 weeks, the structure of the EUS layers was well differentiated. The EUS resident stem/progenitor cells were isolated with MACS® MicroBeads system, and myogenesis was confirmed. In this study, we defined both the time-course development of the EUS and the distribution of resident stem/progenitor cells. This information is crucial for forthcoming studies regarding male micturition and for development of novel therapeutic approaches for postoperative male SUI.