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BACKGROUND AND AIM: Nitric oxide (NO) is the intracellular chemical responsible for initiating a penile erection. Despite conflicting clinical data, it continues to be publicized and promoted that orally administered l-arginine, the putative substrate for NO, enhances the erectile response presumably by stimulating NO production by the corporal tissues resulting in an increase in cGMP production. To shed light on this issue, an in vitro study was conducted to explore the effect of direct exogenous administration of l-arginine as well as its precursor and metabolite, l-citrulline, on the NO-cGMP pathway within the cavernosal smooth muscle (CSM) cell. MATERIALS AND METHODS: CSM cells obtained from 8 to 10 week old Sprague-Dawley rats were grown in Dulbecco media with 20% fetal calf serum and then incubated with or without l-arginine (L-ARG) or l-citrulline (L-CIT) in a time course and dose-response manner. Sildenafil (0.4â¯mM), IBMX (1â¯mM), l-NAME (3⯵M), ODQ (5⯵M) and Deta Nonoate (10⯵M) were used as either inhibitors or stimulators of the NO-cGMP pathway. mRNA and protein were extracted and used for the determination of the phosphodiesterase 5 (PDE5). PDE5 activity was determined by luminometry. cGMP content was determined by ELISA. Nitrite formation, an indicator of NO production, was measured in the cell culture media by a colorimetric assay. The cationic (CAT-1) and neutral (SNAT-1) amino acid transporters for L-ARG and L-CIT, respectively, were determined by Western blot. RESULTS: When compared to untreated CSM cells, incubation with 0.25-4.0â¯mM of L-ARG or 0.3-4.8â¯mM of L-CIT anywhere between 3 and 24â¯h did not result in any additional nitrite or cGMP production. The addition of l-NAME, IBMX or ODQ to these L-ARG and L-CIT treated cells did not alter these results. L-CIT but not L-ARG increased PDE5 mRNA and protein content as well as the activity of the PDE5 enzyme. Both CAT-1 and SNAT-1 were expressed in the CSM cells. CONCLUSIONS: This in vitro study demonstrates that exogenous administration of L-ARG or L-CIT failed to stimulate production of either NO or cGMP by the corporal CSM cells. A re-evaluation of the presumptive role of the exogenous administration of L-ARG in improving the synthesis of NO at least at the level of the CSM cells appears warranted.
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Arginina/farmacología , GMP Cíclico/metabolismo , Miocitos del Músculo Liso/metabolismo , Óxido Nítrico/metabolismo , Pene/citología , Animales , Células Cultivadas , Citrulina/farmacología , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 5/metabolismo , Masculino , Músculo Liso/citología , NG-Nitroarginina Metil Éster/farmacología , Nitritos/análisis , Inhibidores de Fosfodiesterasa 5/farmacología , Ratas Sprague-DawleyRESUMEN
INTRODUCTION: COMP-4 is a natural compound-based dietary supplement consisting of the combination of ginger, Paullinia cupana, muira puama and l-citrulline, which when given long-term has been shown in the aged rat to a) upregulate iNOS in the penile smooth muscle cells (SMC), b) reverse the corporal SMC apoptosis and fibrosis associated with corporal veno-occlusive dysfunction (CVOD), and c) improve resulting erectile function. To elucidate the mechanism of how COMP-4 and its individual components modulate the iNOS-cGMP pathway, an in vitro study was conducted using a rat corporal primary SMC culture to determine its effect on NOS, soluble guanylate cyclase (sGC), cGMP and the phosphodiesterase 5 enzyme (PDE5). MATERIALS AND METHODS: Primary SMC cultures using the explant technique were initiated by cutting small pieces of corporal tissue from 8 week old Sprague-Dawley rats. The SMC were grown in Dulbecco media with 20% fetal calf serum. The SMC were then incubated with or without COMP-4 (0.69â¯mg/ml) or its ingredients alone (ginger: 0.225â¯mg/ml; muira puama, Paullinia cupana and l-citrulline each at 0.9â¯mg/ml) for up to 24â¯h mRNA and protein were extracted and used for the determination of NOS, sGC and PDE5 content. cGMP content was determined by ELISA. L-NIL (4⯵M) was used as an inhibitor of iNOS activity. RESULTS: Compared to the control values, COMP-4 upregulated expression of cGMP by 85%, induced a 42 fold increase in sGC as well as a 15 fold increase in both iNOS protein and mRNA content while it decreased both PDE5 mRNA and protein content each by about 50%. L-NIL completely inhibited the effect of COMP-4 on cGMP production. When compared with each of the individual four components of COMP-4, it appears that COMP-4 itself had the most profound effect in modulating each one the specific steps within the iNOS-cGMP pathway. CONCLUSIONS: This in vitro study demonstrates that COMP-4 is capable of activating the endogenous cellular iNOS-cGMP pathway within the CSM cells, which is theorized to be responsible for reducing the fibrosis and apoptosis as well as the CVOD observed in the aging rat penis. Further studies will be necessary in order to determine whether supplementation of COMP-4 on a daily basis may be beneficial in halting or reversing this aging related erectile dysfunction in the clinical setting.
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Citrulina/farmacología , Miocitos del Músculo Liso/efectos de los fármacos , Olacaceae/química , Paullinia/química , Pene/efectos de los fármacos , Zingiber officinale/química , Animales , Apoptosis/efectos de los fármacos , Células Cultivadas , Citrulina/administración & dosificación , Citrulina/química , GMP Cíclico/metabolismo , Masculino , Miocitos del Músculo Liso/metabolismo , Óxido Nítrico/metabolismo , Óxido Nítrico Sintasa de Tipo II/metabolismo , Pene/metabolismo , Ratas , Ratas Sprague-DawleyRESUMEN
In utero exposure to the ubiquitous plasticizer, bisphenol A (BPA) is associated with offspring obesity. As food intake/appetite is one of the critical elements contributing to obesity, we determined the effects of in vivo maternal BPA and in vitro BPA exposure on newborn hypothalamic stem cells which form the arcuate nucleus appetite center. For in vivo studies, female rats received BPA prior to and during pregnancy via drinking water, and newborn offspring primary hypothalamic neuroprogenitor (NPCs) were obtained and cultured. For in vitro BPA exposure, primary hypothalamic NPCs from healthy newborns were utilized. In both cases, we studied the effects of BPA on NPC proliferation and differentiation, including putative signal and appetite factors. Maternal BPA increased hypothalamic NPC proliferation and differentiation in newborns, in conjunction with increased neuroproliferative (Hes1) and proneurogenic (Ngn3) protein expression. With NPC differentiation, BPA exposure increased appetite peptide and reduced satiety peptide expression. In vitro BPA-treated control NPCs showed results that were consistent with in vivo data (increase appetite vs satiety peptide expression) and further showed a shift towards neuronal versus glial fate as well as an increase in the epigenetic regulator lysine-specific histone demethylase1 (LSD1). These findings emphasize the vulnerability of stem-cell populations that are involved in life-long regulation of metabolic homeostasis to epigenetically-mediated endocrine disruption by BPA during early life.
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Apetito , Efectos Tardíos de la Exposición Prenatal , Animales , Apetito/fisiología , Compuestos de Bencidrilo , Femenino , Neurogénesis , Fenoles , Embarazo , RatasRESUMEN
Erectile dysfunction is a common complication for patients undergoing surgeries for prostate, bladder, and colorectal cancers, due to damage of the nerves associated with the major pelvic ganglia (MPG). Functional re-innervation of target organs depends on the capacity of the neurons to survive and switch towards a regenerative phenotype. PDE5 inhibitors (PDE5i) have been successfully used in promoting the recovery of erectile function after cavernosal nerve damage (BCNR) by up-regulating the expression of neurotrophic factors in MPG. However, little is known about the effects of PDE5i on markers of neuronal damage and oxidative stress after BCNR. This study aimed to investigate the changes in gene and protein expression profiles of inflammatory, anti-inflammatory cytokines and oxidative stress related-pathways in MPG neurons after BCNR and subsequent treatment with sildenafil. Our results showed that BCNR in Fisher-344 rats promoted up-regulation of cytokines (interleukin- 1 (IL-1) ß, IL-6, IL-10, transforming growth factor ß 1 (TGFß1), and oxidative stress factors (Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, Myeloperoxidase (MPO), inducible nitric oxide synthase (iNOS), TNF receptor superfamily member 5 (CD40) that were normalized by sildenafil treatment given in the drinking water. In summary, PDE5i can attenuate the production of damaging factors and can up-regulate the expression of beneficial factors in the MPG that may ameliorate neuropathic pain, promote neuroprotection, and favor nerve regeneration.
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Ganglios/metabolismo , Estrés Oxidativo/efectos de los fármacos , Inhibidores de Fosfodiesterasa 5/farmacología , Piperazinas/farmacología , Sulfonamidas/farmacología , Animales , Citocinas/genética , Citocinas/metabolismo , Modelos Animales de Enfermedad , Ganglios/patología , Hemo-Oxigenasa 1/genética , Hemo-Oxigenasa 1/metabolismo , Masculino , NADPH Oxidasas/genética , NADPH Oxidasas/metabolismo , Tejido Nervioso/lesiones , Óxido Nítrico Sintasa de Tipo II/metabolismo , Erección Peniana/efectos de los fármacos , Erección Peniana/fisiología , Pene/inervación , Purinas/farmacología , Ratas , Ratas Endogámicas F344 , Citrato de Sildenafil , Transcriptoma , Regulación hacia Arriba/efectos de los fármacosRESUMEN
OBJECTIVE: To determine the gene expression profile of pelvic ganglia neurones after bilateral cavernosal nerve resection (BCNR) and subsequent treatment with sildenafil in relation to neurotrophic-related pathways. MATERIALS AND METHODS: Fisher rats aged 5 months were subjected to BCNR or sham operation and treated with or without sildenafil (20 mg/kg body-weight in drinking water) for 7 days. Total RNA isolated from pelvic ganglia was subjected to reverse transcription and then to quantitative reverse transcriptase-polymerase chain reaction (PCR) with the RAT-neurotrophic array. Results were corroborated by real-time PCR and western blotting. Another set of animals were injected with a fluorescent tracer at the base of the penis, 7 days before BCNR or sham operation, and were sacrificed 7 days after surgery. Sections of pelvic ganglia were used for immunohistochemistry with antibodies against neurturin, neuronal nitric oxide synthase, tyrosine hydroxylase and glial cell line-derived neurotrophic factor receptor α2. RESULTS: A down-regulation of the expression of neuronal nitric oxide synthase accompanied by changes in the level of cholinergic neurotrophic factors, such as neurturin and its receptor glial cell line-derived neurotrophic factor receptor α2, artemin, neurotrophin-4 and cilliary neurotrophic factor, was observed 7 days after BCNR in pelvic ganglia neurones. Treatment with sildenafil, starting immediately after surgery, reversed all these changes at a level similar to that in sham-operated animals. CONCLUSIONS: Sildenafil treatment promotes changes in the neurotrophic phenotype, leading to a regenerative state of pelvic ganglia neurones. The present study provides a justification for the use of phosphodiesterase 5 inhibitors as a neuroprotective agent after BCNR.
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Ganglios Autónomos/efectos de los fármacos , Receptores del Factor Neurotrófico Derivado de la Línea Celular Glial/genética , Fármacos Neuroprotectores/farmacología , Pene/inervación , Inhibidores de Fosfodiesterasa 5/farmacología , Piperazinas/farmacología , Sulfonas/farmacología , Animales , Ganglios Autónomos/metabolismo , Expresión Génica/efectos de los fármacos , Receptores del Factor Neurotrófico Derivado de la Línea Celular Glial/metabolismo , Masculino , Neuronas/metabolismo , Neurturina/metabolismo , Óxido Nítrico Sintasa de Tipo I/metabolismo , Tratamientos Conservadores del Órgano/métodos , Pelvis/inervación , Pene/efectos de los fármacos , Pene/cirugía , Purinas/farmacología , Ratas , Ratas Endogámicas F344 , Ratas Sprague-Dawley , Transducción de Señal/efectos de los fármacos , Citrato de SildenafilRESUMEN
Developmental programming studies using mouse models have housed the animals at human thermoneutral temperatures (22°C) which imposes constant cold stress. As this impacts energy homeostasis, we investigated the effects of two housing temperatures (22°C and 30°C) on obesity development in male and female offspring of Control and FR dams. Pregnant mice were housed at 22°C (cold-exposed, CE) or 30°C (thermoneutrality, TN) room temperature. At gestational age e10, mice were fed either an ad libitum diet (Control) or were 30% food-restricted (FR) to produce low birth weight newborns. Following delivery, all dams were fed an ad libitum diet and maternal mice continued to nurse their own pups. At 3 weeks of age, offspring were weaned to an ad libitum diet and housed at similar temperatures as their mothers. Body weights and food intake were monitored. At 6 months of age, body composition and glucose tolerance test were determined, after which, brain and adipose tissue were collected for analysis. FR/CE and FR/TN offspring exhibited hyperphagia and were significantly heavier with increased adiposity as compared to their respective Controls. There was sex-specific effects of temperature in both groups. Male offspring at TN were heavier with increased body fat, though the food intake was decreased as compared to CE males. This was reflected by hypertrophic adipocytes and increased arcuate nucleus satiety/appetite ratio. In contrast, female offspring were not impacted by housing temperature. Thus, unlike female offspring, there was a significant interaction of diet and temperature evident in the male offspring with accentuated adverse effects evident in FR/TN males.
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Tejido Adiposo , Obesidad , Embarazo , Humanos , Animales , Masculino , Femenino , Ratones , Obesidad/etiología , Obesidad/metabolismo , Tejido Adiposo/metabolismo , Dieta , Adiposidad , DesteteRESUMEN
Introduction: The COVID-19 pandemic has highlighted the need to identify mechanisms of antiviral host defense against SARS-CoV-2. One such mediator is interferon-g (IFN-γ), which, when administered to infected patients, is reported to result in viral clearance and resolution of pulmonary symptoms. IFN-γ treatment of a human lung epithelial cell line triggered an antiviral activity against SARS-CoV-2, yet the mechanism for this antiviral response was not identified. Methods: Given that IFN-γ has been shown to trigger antiviral activity via the generation of nitric oxide (NO), we investigated whether IFN-γ induction of antiviral activity against SARS-CoV-2 infection is dependent upon the generation of NO in human pulmonary epithelial cells. We treated the simian epithelial cell line Vero E6 and human pulmonary epithelial cell lines, including A549-ACE2, and Calu-3, with IFN-γ and observed the resulting induction of NO and its effects on SARS-CoV-2 replication. Pharmacological inhibition of inducible nitric oxide synthase (iNOS) was employed to assess the dependency on NO production. Additionally, the study examined the effect of interleukin-1b (IL-1ß) on the IFN-g-induced NO production and its antiviral efficacy. Results: Treatment of Vero E6 cells with IFN-γ resulted in a dose-responsive induction of NO and an inhibitory effect on SARS-CoV-2 replication. This antiviral activity was blocked by pharmacologic inhibition of iNOS. IFN-γ also triggered a NO-mediated antiviral activity in SARS-CoV-2 infected human lung epithelial cell lines A549-ACE2 and Calu-3. IL-1ß enhanced IFN-γ induction of NO, but it had little effect on antiviral activity. Discussion: Given that IFN-g has been shown to be produced by CD8+ T cells in the early response to SARS-CoV-2, our findings in human lung epithelial cell lines, of an IFN-γ-triggered, NO-dependent, links the adaptive immune response to an innate antiviral pathway in host defense against SARS-CoV-2. These results underscore the importance of IFN-γ and NO in the antiviral response and provide insights into potential therapeutic strategies for COVID-19.
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COVID-19 , Interferón gamma , Óxido Nítrico , Humanos , Enzima Convertidora de Angiotensina 2 , COVID-19/inmunología , Interferón gamma/inmunología , Óxido Nítrico/inmunología , SARS-CoV-2/fisiología , Replicación ViralRESUMEN
OBJECTIVE: ⢠To investigate whether sustained long-term separate treatments of diabetic inducible nitric oxide synthase knockout (iNOSKo) mice with allopurinol, an antioxidant inhibiting xanthine oxidoreductase, decorin, a transforming growth factor-ß1 (TGFß1) -binding antagonist, and molsidomine, a long-life nitric oxide donor, prevent the processes of diabetes-induced cavernosal fibrosis. MATERIALS AND METHODS: ⢠Eight week old male iNOS knock out (iNOSKo) mice were made diabetic by injecting 150 mg/kg B.W Streptozotocin (1P) with were either left untreated or treated with the oral antioxidant allopurinol (40 mg/kg/day), or decoin (50 mg, 1P, twice), as an anti-TGFß1 agent (n = 8/group). ⢠Glycemia and oxidative stress markers were determined in blood and urine. ⢠Paraffin-embedded tissue sections from the penile shaft were subjected to Masson trichrome staining for the smooth muscle (smc)/collagen ratio, and imunostaining for smc content, profibrotic factors, oxidative stress, cell replication and cell death markers followed by quantitative image analysis. RESULTS: ⢠Eight-week treatment with either allopurinol or decorin counteracted the decrease in smooth muscle cells and the increase in apoptosis and local oxidative stress within the corpora tissue. ⢠Decorin but not allopurinol increased the smooth muscle cell/collagen ratio, whereas allopurinol but not decorin inhibited systemic oxidative stress. ⢠Molsidomine was effective in reducing both local and systemic oxidative stress, but did not prevent corporal fibrosis. CONCLUSION: ⢠Both allopurinol and decorin appear as promising approaches either as a single or a combined pharmacological modality for protecting the diabetic corpora from undergoing apoptosis and fibrosis although their functional effects still need to be defined.
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Antioxidantes/farmacología , Diabetes Mellitus Experimental/complicaciones , Óxido Nítrico Sintasa de Tipo II/deficiencia , Pene/patología , Factor de Crecimiento Transformador beta1/antagonistas & inhibidores , Alopurinol/farmacología , Animales , Apoptosis/efectos de los fármacos , Decorina/farmacología , Inhibidores Enzimáticos/farmacología , Fibrosis/prevención & control , Masculino , Ratones , Ratones Noqueados , Molsidomina/farmacología , Músculo Liso/efectos de los fármacos , Donantes de Óxido Nítrico/farmacología , Estrés OxidativoRESUMEN
INTRODUCTION: It has been shown that phosphodiesterase type 5 (PDE5) inhibitors preserve smooth muscle (SM) content and ameliorate the fibrotic degeneration normally seen in the corpora cavernosa after bilateral cavernosal nerve resection (BCNR). However, the downstream mechanisms by which these drugs protect the corpora cavernosa remain poorly understood. AIM: To provide insight into the mechanism, we aimed to determine the gene expression profile of angiogenesis-related pathways within the penile tissue after BCNR with or without continuous sildenafil (SIL) treatment. METHODS: Five-month-old Fisher rats were subjected to BCNR or sham operation and treated with or without SIL (20 mg/kg/BW drinking water) for 3 days or 45 days (N = 8 rats per group). Total RNAs isolated from the denuded penile shaft and prostate were subjected to reverse transcription and to angiogenesis real-time-polymerase chain reaction arrays (84 genes). Changes in protein expression of selected genes such as epiregulin (EREG) and connective tissue growth factor (CTGF) were corroborated by Western blot and immunohistochemistry. MAIN OUTCOMES MEASURES: Genes modulated by BCNR and SIL treatment. RESULTS: A decreased expression of genes related to SM growth factors such as EREG, platelet-derived growth factor (PDGF), extracellular matrix regulators such as metalloproteinases 3 and 9, endothelial growth factors, together with an upregulation of pro-fibrotic genes such as CTGF and transforming growth factor beta 2 were found at both time points after BCNR. SIL treatment reversed this process by upregulating endothelial and SM growth factors and downregulating pro-fibrotic factors. SIL did not affect the expression of EREG, VEGF, and PDGF in the ventral prostate of BCNR animals. CONCLUSIONS: SIL treatment after BCNR activates genes related to SM preservation and downregulates genes related to fibrosis in the corpora cavernosa. These results provide a mechanistic justification for the use of SIL and other PDE5 inhibitors as protective therapy against corporal SM loss and fibrosis after radical prostatectomy.
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Matriz Extracelular/efectos de los fármacos , Fibrosis/tratamiento farmacológico , Expresión Génica/efectos de los fármacos , Péptidos y Proteínas de Señalización Intercelular/genética , Pene/cirugía , Inhibidores de Fosfodiesterasa 5/farmacología , Piperazinas/farmacología , Sulfonas/farmacología , Animales , Modelos Animales de Enfermedad , Endotelio Vascular/efectos de los fármacos , Factor de Crecimiento Epidérmico/efectos de los fármacos , Epirregulina , Fibrosis/patología , Expresión Génica/genética , Masculino , Metaloproteinasa 1 de la Matriz/efectos de los fármacos , Metaloproteinasa 3 de la Matriz/efectos de los fármacos , Tejido Nervioso/lesiones , Pene/irrigación sanguínea , Pene/inervación , Purinas/farmacología , Ratas , Citrato de Sildenafil , Factor de Crecimiento Transformador beta2/efectos de los fármacos , Factor de Crecimiento Transformador beta2/genéticaRESUMEN
COMP-4, a nutraceutical combination consisting of ginger rhizome, muira puama, Paullinia cupana, and L-citrulline, enhances intracellular nitric oxide (NO) production by the corporal smooth muscle cells (CSMC). This study aims to determine if the previously shown beneficial effect of COMP-4 on the histology and function of the aging penis is associated with an antioxidative effect from endogenously produced NO. Ten-month-old male rats were treated daily for 2 months with COMP-4 or vehicle at which time the corpora and penile dorsal artery (PDA) were evaluated by immunohistochemistry for (a) apoptosis (b) proliferative cell nuclear antigen, (c) heme oxygenase-1 (HO-1), (d) myeloperoxidase (MPO), and (e) nitrotyrosine (NT). CSMC were cultured and incubated with COMP-4 in order to determine intracellular oxidative stress via the GSH/GSSG ratio. In both the corpora and PDA, daily treatment with COMP-4 resulted in an increase in both smooth muscle cell proliferation and HO-1 expression as well as a decrease in MPO. There was no change in either apoptosis or NT expression. In the CSMC cell culture, treatment with COMP-4 increased the intracellular GSH/GSSG ratio. COMP-4 appears to have an antioxidant effect on the aging vascular smooth muscle cells both in the corpora and peripheral vasculature.
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Estrés Oxidativo , Pene , Envejecimiento , Alimentación Animal , Animales , Apoptosis , Arterias , Masculino , Miocitos del Músculo Liso , Pene/metabolismo , RatasRESUMEN
BACKGROUND: The combination of the nutraceuticals, Paullinia cupana, ginger rhizome, muira puama, and the amino acid L-citrulline (COMP-4) has been shown to stimulate the production of inducible nitric oxide synthase (iNOS), nitric oxide (NO), and cGMP in rat corpora cavernosa smooth muscle cells (CSMC). When administered to middle-aged rats, long-term treatment with COMP-4 resulted in both an increase in the number of CSMC and an improvement in erectile function. We, therefore, aimed to determine whether a commercial formulation of COMP-4, Revactin®, could have a similar stimulatory effect on human CSMC. METHODS: Primary human CSMC cultures (HCSMC) were grown and incubated with Revactin® for up to 24 hours. cGMP generation and nitrite formation were determined by ELISA and Griess reaction, respectively. IBMX (1 mM), sildenafil (0.4 mM), and L-NIL (4 µM) were utilized as modulators of the NO-cGMP pathway. iNOS, endothelial NOS (eNOS), and neuronal NOS (nNOS) expressions were determined by Western blot. RESULTS: Revactin® up-regulated both nitrite formation and cGMP expression, achieving the highest expression at 24 hours in the HCSMC. These effects were completely blocked by L-NIL. Revactin® up-regulated iNOS expression, but not that of eNOS or nNOS. CONCLUSIONS: The results presented in this study confirmed that Revactin® activated the iNOS-NO-cGMP pathway intracellularly in HCSMC. It still needs to be determined whether the upregulation of this pathway would be an effective approach for counteracting the fibrosis and apoptosis of the corporal smooth muscle cells associated with aging.
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BACKGROUND: Erectile dysfunction is a disease commonly caused by diabetes mellitus (DMED) and cavernous nerve injury (CNIED). Bioinformatics analyses including differentially expressed genes (DEGs), enriched functions and pathways (EFPs), and protein-protein interaction (PPI) networks were carried out in DMED and CNIED rats in this study. The critical biomarkers that may intervene in nitric oxide synthase (NOS, predominantly nNOS, ancillary eNOS, and iNOS)-cyclic guanosine monophosphate (cGMP)-phosphodiesterase 5 enzyme (PDE5) pathway, an important mechanism in erectile dysfunction treatment, were then explored for potential clinical applications. METHODS: GSE2457 and GSE31247 were downloaded. Their DEGs with a |logFC (fold change)| > 0 were screened out. Database for Annotation, Visualization and Integrated Discovery (DAVID) online database was used to analyze the EFPs in Gene Ontology enrichment and Kyoto Encyclopedia of Genes and Genomes networks based on down-regulated and up-regulated DEGs respectively. PPI analysis of 2 datasets was performed in Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) and Cytoscape. Interactions with an average score greater than 0.9 were chosen as the cutoff for statistical significance. RESULTS: From a total of 1710 DEGs in GSE2457, 772 were down-regulated and 938 were up-regulated, in contrast to the 836 DEGs in GSE31247, from which 508 were down-regulated and 328 were up-regulated. The 25 common EFPs such as aging and response to hormone were identified in both models. PPI results showed that the first 10 hub genes in DMED were all different from those in CNIED. CONCLUSIONS: The intervention of iNOS with the hub gene complement component 3 in DMED and the aging process in both DMED and CNIED deserves attention.
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Biomarcadores/metabolismo , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 5/metabolismo , Disfunción Eréctil/metabolismo , Óxido Nítrico Sintasa/metabolismo , Nucleótidos Cíclicos/metabolismo , Animales , Biología Computacional/métodos , Bases de Datos Genéticas , Complicaciones de la Diabetes/epidemiología , Disfunción Eréctil/epidemiología , Disfunción Eréctil/fisiopatología , Regulación de la Expresión Génica/genética , Ontología de Genes/estadística & datos numéricos , Redes Reguladoras de Genes/genética , Humanos , Masculino , Modelos Animales , Mapas de Interacción de Proteínas/genética , RatasRESUMEN
INTRODUCTION: The link between cannabis use and erectile dysfunction remains unclear. Moreover, the effect of cannabis in tandem with current Western dietary habits is an area in male sexual health that has yet to be explored. This study seeks to investigate the impact of diet and cannabis on penile health in an animal model. AIM: To determine the effects of diet and oral cannabis extract on fibrosis and oxidative stress within the corpora cavernosa of mice. METHODS: This is a pilot animal study in which groups of 2-month old C57BL/6J male mice were fed a normal chow diet (NCD) or high-fat diet (HFD) daily and treated with or without either MJ or THC extract for 2 months. After euthanization, mouse penises were isolated and processed for immunohistochemical studies to determine: (i) smooth muscle cell to collagen content, (ii) myofibroblast proliferation, and (iii) anti-oxidative activity. MAIN OUTCOME MEASURES: Quantitative assessment of immunohistochemical markers of fibrosis and oxidative stress within the corpora cavernosa of mice fed a high-fat diet in combination with either oral marijuana (MJ) or Δ-9-tetrahydrocannabinol extract (THC). RESULTS: The combination of HFD with MJ resulted in: (i) a decrease in the smooth/collagen ratio in the corpora cavernosa, (ii) an increase in alpha-smooth muscle actin expression in the tunica albuginea compatible with myofibroblast proliferation, and (iii) a decrease in heme oxygenase 1 expression indicating an increase in oxidative stress. Significant histological changes were not observed in the HFD + THC group. CONCLUSIONS: HFD combined with oral MJ extract led to structural alterations in erectile tissue that are associated with accelerated corporal fibrosis. However, the addition of THC to the diet did not exacerbate histological changes within the corpora. Further studies are warranted to elucidate the discrepant effects between MJ and THC in order to optimize the therapeutic potential of cannabis and minimize its adverse effects on penile health. S Nguyen, M Mangubat, S Eleswarapu, et al. The Combination of High-Fat Diet and Oral Marijuana Promotes the Development of Fibrosis in the Mouse Corpora Cavernosa. Sex Med 2021;9:100312.
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INTRODUCTION: Endogenously elicited inducible nitric oxide synthase (iNOS) induction counteracts fibrosis and oxidative stress in penile tissues in rat models of Peyronie's disease and erectile dysfunction. AIM: The current study aimed to determine whether the genetic blockade of iNOS expression in the iNOS knock out (iNOS KO) mouse intensifies fibrosis and oxidative stress in the penile corpora cavernosa, and this is exacerbated by streptozotocin (STZ)-induced diabetes and counteracted by insulin. MAIN OUTCOMES MEASURES: Quantitative assessment of histological and biochemical markers in mouse corporal tissue. METHODS: Male iNOS KO and wild type (WT) mice were left untreated or injected with STZ, with or without insulin treatment. At 8 weeks, glycemia, glucosuria, and proteinuria were determined, and corporal tissue sections were obtained and subjected to Masson trichrome staining for smooth muscle (SM)/collagen ratio, and immunostaining for α-smooth muscle actin (ASMA) for, SM content, proliferating cell nuclear antigen (PCNA) for cell replication, TGFß1 as profibrotic factor, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay for apoptosis, and xanthine oxidoreductase (XOR) for oxidative stress. Collagen was estimated by the hydroxyproline reaction. RESULTS: The corporal SM/collagen ratio and SM content were reduced, and collagen content increased in iNOS KO mice as compared with WT mice, but apoptosis was decreased and cell replication increased, whereas TGFß1 and XOR did not vary. Severe hyperglycemia caused in the WT a reduction of the corporal SM/collagen ratio and SM content and an increase in apoptosis without changes in PCNA, TGFß1, or XOR. In the iNOS KO mouse the hyperglycemia-induced alterations were exacerbated, with additional increases in oxidative stress and TGFß1. Insulin normalized glycemia and partially protected the SM in both the WT and the iNOS KO mice. CONCLUSIONS: The antifibrotic, antioxidative, and SM-protective roles of iNOS in the penile corpora cavernosa were confirmed in the iNOS KO/STZ mouse model. These findings support the importance of endogenously-elicited iNOS induction in protecting the penile corpora cavernosa from the pro-fibrotic effects of hyperglycemia.
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Diabetes Mellitus Experimental , Fibrosis , Óxido Nítrico Sintasa/metabolismo , Estrés Oxidativo , Pene/patología , Actinas/metabolismo , Animales , Apoptosis , Proliferación Celular , Colágeno/metabolismo , Hipoglucemiantes/farmacología , Inmunohistoquímica , Etiquetado Corte-Fin in Situ , Insulina/farmacología , Masculino , Ratones , Ratones Noqueados , Óxido Nítrico Sintasa/genética , Pene/metabolismo , Factor de Crecimiento Transformador beta1/metabolismo , Xantina Deshidrogenasa/metabolismoRESUMEN
Maternal high-fat (HF) is associated with offspring hyperphagia and obesity. We hypothesized that maternal HF alters fetal neuroprogenitor cell (NPC) and hypothalamic arcuate nucleus (ARC) development with preferential differentiation of neurons towards orexigenic (NPY/AgRP) versus anorexigenic (POMC) neurons, leading to offspring hyperphagia and obesity. Furthermore, these changes may involve hypothalamic bHLH neuroregulatory factors (Hes1, Mash1, Ngn3) and energy sensor AMPK. Female mice were fed either a control or a high fat (HF) diet prior to mating, and during pregnancy and lactation. HF male newborns were heavier at birth and exhibited decreased protein expression of hypothalamic bHLH factors, pAMPK/AMPK and POMC with increased AgRP. As adults, these changes persisted though with increased ARC pAMPK/AMPK. Importantly, the total NPY neurons were increased, which was consistent with the increased food intake and adult fat mass. Further, NPCs from HF newborn hypothalamic tissue showed similar changes with preferential NPC neuronal differentiation towards NPY. Lastly, the role of AMPK was further confirmed with in vitro treatment of Control NPCs with pharmacologic AMPK modulators. Thus, the altered ARC development of HF offspring results in excess appetite and reduced satiety leading to obesity. The underlying mechanism may involve AMPK/bHLH pathways.
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Animales Recién Nacidos/metabolismo , Dieta Alta en Grasa/efectos adversos , Hiperfagia/etiología , Fenómenos Fisiologicos Nutricionales Maternos , Obesidad/etiología , Efectos Tardíos de la Exposición Prenatal/etiología , Proteínas Quinasas Activadas por AMP/metabolismo , Proteína Relacionada con Agouti/metabolismo , Animales , Apetito/fisiología , Núcleo Arqueado del Hipotálamo/crecimiento & desarrollo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Diferenciación Celular/fisiología , Femenino , Hipotálamo/metabolismo , Masculino , Ratones , Neurogénesis/fisiología , Neuronas/metabolismo , Embarazo , Saciedad/fisiologíaRESUMEN
OBJECTIVES: Grafts are used for vaginal repair after prolapse, but their use to carry stem cells to regenerate vaginal tissue has not been reported. In this study, we investigated whether 1) muscle-derived stem cells (MDSC) grown on small intestinal submucosa (SIS) generate smooth-muscle cells (SMC) in vitro and upon implantation in a rat model of vaginal defects; 2) express markers applicable to the in-vivo detection of vaginal endogenous stem cells; and 3) stimulate the repair of the vagina. METHODS: Mouse MDSC grown on monolayer, SIS, or polymeric mesh, were tested for cell differentiation by immunocytochemistry, Western blot and real-time polymerase chain reaction (PCR). Stem cell markers were screened by DNA microarrays followed by real-time PCR, immunocytochemistry, and Western blot. Rats that underwent hysterectomy and partial vaginectomy were left as such or implanted in the vagina with 4',6-Diamidino-2-Phenylindole (DAPI)-labeled MDSC on SIS, or SIS without MDSC, immunosuppressed, and killed at 2-8 weeks. Immunofluorescence, hematoxylin-eosin, and Masson trichrome were applied to tissue sections. RESULTS: Muscle-derived stem cell cultures on monolayer and on scaffolds differentiate into SMC, as shown by alpha-smooth muscle actin (ASMA), calponin, and smoothelin markers. Muscle-derived stem cells express embryonic stem cell markers Oct-4 and nanog. Dual DAPI/ASMA fluorescence indicated MDSC conversion to SMC. Muscle-derived stem cells/SIS stimulated vaginal tissue repair, including keratin-5 positive epithelium formation and prevented fibrosis at 4 and 8 weeks. Oct-4+ putative endogenous stem cells were identified. CONCLUSION: Muscle-derived stem cells/SIS implants stimulate vaginal tissue repair in the rat, thus autologous MDSC on scaffolds may be a promising approach for the treatment of vaginal repair.
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Trasplante de Células Madre/métodos , Andamios del Tejido , Vagina/cirugía , Animales , Células Cultivadas , Femenino , Mucosa Intestinal , Intestino Delgado , Ratones , Ratones Endogámicos C57BL , Células Musculares/citología , Miocitos del Músculo Liso/citología , Ratas , Ratas Endogámicas F344 , Trasplante Heterólogo , Prolapso Uterino/cirugíaRESUMEN
OBJECTIVES: To determine, in the obese Zucker fa/fa rat (OZR), whether the loss in smooth muscle cells (SMCs) as well as the increase in fibrosis that occurs within the corpora cavernosa accompanying corporal veno-occlusive dysfunction (CVOD), also occurs within the media of the arterial tree. MATERIALS AND METHODS: The penis and aorta from both 7-month-old male diabetic OZR (5 months of diabetes) and aged-matched nondiabetic lean Zucker rats (LZR) rats were harvested (eight per group). The penis and aorta were subjected to histo- or immnohistochemistry, followed by quantitative image analysis (QIA) to determine the contents of SMC, collagen and the pro-fibrotic transforming growth factor (TGF)beta1. The turnover of SMCs was assessed by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labelling (TUNEL) and proliferating cell nuclear antigen (PCNA) assays. Quantitative Western blots determined calponin (SMC marker) and PCNA, and hydroxyproline was used for collagen. In vitro relaxation of corporal strips was measured. RESULTS: In vitro relaxation of corporal tissue from OZR was considerably less than in the LZR. In the media of the penile dorsal artery (PDA) of OZR, there was a considerable reduction in the SMC content and the SMC/collagen ratio, as well as an increase in apoptosis, but there were no changes in PCNA or TGFbeta1 expression, or in the intima-media/lumen ratio. In the aorta of the OZR, in contrast to the PDA, there was a reduction in PCNA as well as a more pronounced decrease in the SMC/collagen ratio, mainly from an increase in collagen, but there were no changes in TGFbeta1 or the wall/lumen morphometry. In the OZR, Western blots of aortic tissue confirmed the decrease in PCNA and a reduction in the SMC marker calponin. CONCLUSIONS: These data show that 5 months after the onset of hyperglycaemia in the OZR, the rats develop both abnormal corporal SMC relaxation and a generalized fibrosis of the arterial media of both the large and small diameter vessels. It is possible that this pan-fibrosis of the media of the arterial system might contribute to the diabetes-related ED that occurs during this period in this rat model.
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Aorta/patología , Diabetes Mellitus Tipo 2/patología , Impotencia Vasculogénica/patología , Pene/irrigación sanguínea , Túnica Media/patología , Animales , Western Blotting , Diabetes Mellitus Tipo 2/complicaciones , Fibroblastos/patología , Fibrosis , Inmunohistoquímica , Impotencia Vasculogénica/etiología , Masculino , Músculo Liso Vascular/patología , Miocitos del Músculo Liso/patología , Pene/patología , Ratas , Ratas ZuckerRESUMEN
BACKGROUND: Recent evidence suggests that treatment of type 2 diabetes with thiazolidinediones [peroxisome proliferator-activated receptor-gamma (PPAR-gamma) agonists], ameliorates glomerulosclerosis and tubulointerstitial fibrosis in the rat kidney. In the current work, we have investigated whether these drugs, and specifically pioglitazone (PGT), act by preventing fibrosis and kidney dysfunction mainly through antioxidant and anti-inflammatory effects, independently of glycaemic control. METHODS: Male 2- to 3-month-old obese Zucker fa/fa (OZR) and ZDF fa/fa rats (ZDFR), and their control the lean Zucker rat (LZR), were used. Diabetic rats were given either a low dose (0.6 mg/kg/day) or a high dose (12 mg/ kg/day) of PGT in the chow for 2 or 4-5 months. Glycaemia, blood pressure, creatinine clearance and proteinuria were determined, and the underlying histopathology was defined with markers of fibrosis, glomerular damage, oxidative stress and inflammation by immunohistochemistry/ quantitative image analysis in tissue sections, and western blots and ad hoc assays in fresh tissue. RESULTS: PGT at low doses given for 4-5 months considerably reduced blood pressure, proteinuria and creatinine clearance. This was associated with amelioration of renal tissue damage and fibrosis, evidenced by the glomerulosclerosis, tubulointerstitial fibrosis, tubular atrophy and podocyte injury indexes, and of oxidative stress and inflammation, as shown by the decrease in the respective markers, although glycaemia remained high and obesity was not affected. CONCLUSIONS: These results indicate that low doses of PGT ameliorate renal fibrosis and preserve renal function in this animal model of metabolic syndrome, independently of glycaemic control or effects on body weight.
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Diabetes Mellitus Tipo 2/tratamiento farmacológico , Glomeruloesclerosis Focal y Segmentaria/tratamiento farmacológico , Hipoglucemiantes/uso terapéutico , Túbulos Renales/patología , Obesidad/tratamiento farmacológico , Tiazolidinedionas/uso terapéutico , Animales , Presión Sanguínea/efectos de los fármacos , Peso Corporal/efectos de los fármacos , Diabetes Mellitus Tipo 2/patología , Fibrosis/prevención & control , Inflamación/prevención & control , Riñón/metabolismo , Riñón/patología , Pruebas de Función Renal , Masculino , Obesidad/patología , Estrés Oxidativo/efectos de los fármacos , Pioglitazona , Ratas , Ratas ZuckerRESUMEN
INTRODUCTION: Corporal veno-occlusive dysfunction (CVOD), which usually is associated with a loss of smooth muscle cells (SMC) and an increase in fibrosis within the corpora cavernosa, can be induced by an injury to the cavernosal nerves. The corporal tissue expresses inducible nitric oxide synthase (iNOS), presumably as an antifibrotic and SMC-protective response. AIMS: We studied the temporal relationship in the corpora between the expression of iNOS, other histological and biochemical changes, and the development of CVOD, after bilateral cavernosal nerve resection (BCNR) in the rat. METHODS: Rats underwent either BCNR or sham operation. Cavernosometry was performed 1, 3, 7, 15, 30, and 45 days (N = 8/groups) after surgery. Penile tissue sections were subjected to Masson trichrome staining for SMC and collagen, and immunodetection for alpha smooth muscle actin, iNOS, neuronal NOS (nNOS), endothelial NOS (eNOS), proliferating cell nuclear antigen (PCNA), and terminal transferase dUTP nick end labeling (TUNEL). Quantitative western blot analysis was done in homogenates. MAIN OUTCOME MEASURES: Time course on the development of fibrosis and CVOD. RESULTS: Following BCNR, CVOD was detectable 30 days later, and it became more pronounced by 45 days. In contrast, the SMC/collagen ratio in the BCNR corpora was reduced at 7 days and bottomed at 30 and 45 days, due in part to the reduction of SMC, presumably caused by an increase in apoptosis peaking at 3 days. PCNA also peaked at 3 days, but then decayed. nNOS was reduced early (3-7 days) and disappeared at 30 days, whereas eNOS was not affected. iNOS was induced at day 3, and steadily increased peaking at 30 days. CONCLUSIONS: CVOD develops in the BCNR rat as a result of the early loss of corporal SMC by the neuropraxia-induced apoptosis, which the initial cell replication response cannot counteract, followed by fibrosis. The time course of iNOS induction supports the antifibrotic role of iNOS.
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Fibrosis/patología , Músculo Liso/patología , Músculo Liso/fisiopatología , Pene , Nervios Periféricos/patología , Nervios Periféricos/fisiopatología , Actinas/metabolismo , Animales , Western Blotting , Endotelio/metabolismo , Etiquetado Corte-Fin in Situ , Masculino , Músculo Liso/metabolismo , Óxido Nítrico Sintasa/metabolismo , Pene/inervación , Pene/patología , Pene/fisiopatología , Antígeno Nuclear de Célula en Proliferación/metabolismo , Distribución Aleatoria , Ratas , Ratas Sprague-DawleyRESUMEN
Osteoporosis represents an imbalance between bone formation and bone resorption. As a result of low estrogen levels, it is markedly prevalent during menopause, thus making such patients susceptible to fractures. Both bone formation and resorption are modulated by nitric oxide (NO). Currently, there are no risk-free pharmaceutical prevention therapies for osteoporosis. COMB-4, a nutraceutical combination of Paullinia cupana, Muira puama, ginger, and L-citrulline, known to activate the NO-cGMP pathway, was reported to accelerate fracture healing in the rat. To determine whether COMB-4 could be effective in preventing menopausal osteoporosis, it was compared to estradiol (E2) in an ovariectomized (OVX) rat osteoporosis model. Nine-month-old female Sprague Dawley rats were divided into SHAM, OVX, OVX+E2, and OVX+COMB-4. After 100 days of treatment, bone mineral density (BMD) and bone mineral content (BMC) were measured by DXA scan. TRAP staining was performed in the femur and lumbar vertebrae. TRACP 5b and osteocalcin levels were assayed in the serum. MC3T3-E1 cells were differentiated into osteoblasts and treated with COMB-4 for one week in order to evaluate calcium deposition by Alizarin staining, cGMP production by ELISA, and upregulation of the nitric oxide synthase (NOS) enzymes by RT-PCR. OVX resulted in a decrease in BMD, BMC, and serum osteocalcin and an increase in serum TRACP 5b. Except for an increase in BMC with COMB-4, both E2 and COMB-4 reverted all bone and serum markers, as well as the number of osteoclasts in the vertebrae, to SHAM levels. Incubation of MC3T3-E1 cells with COMB-4 demonstrated an increase in the three NOS isoforms, cGMP, and calcium deposition. COMB-4 increased BMD in OVX rats by inhibiting bone resorption and increasing calcium deposition presumably via activation of the NO-cGMP pathway. It remains to be determined whether COMB-4 could be a potential nutraceutical therapy for the prevention of premenopausal bone loss.