Stem cell treatment may enhance erectile dysfunction (ED) in individuals with cavernous nerve injury (CNI). Nevertheless, no investigations have directly ascertained the implications of varying amounts of human umbilical cord-derived mesenchymal stem cells (HUC-MSCs) on ED. We compare the efficacy of three various doses of HUC-MSCs as a therapeutic strategy for ED. Sprague-Dawley rats (total = 175) were randomly allocated into five groups. A total of 35 rats underwent sham surgery and 140 rats endured bilateral CNI and were treated with vehicles or doses of HUC-MSCs (1 × 10 6 cells, 5 × 10 6 cells, and 1 × 10 7 cells in 0.1 ml, respectively). Penile tissues were harvested for histological analysis on 1 day, 3 days, 7 days, 14 days, 28 days, 60 days, and 90 days postsurgery. It was found that varying dosages of HUC-MSCs enhanced the erectile function of rats with bilateral CNI and ED. Moreover, there was no significant disparity in the effectiveness of various dosages of HUC-MSCs. However, the expression of endothelial markers (rat endothelial cell antigen-1 [RECA-1] and endothelial nitric oxide synthase [eNOS]), smooth muscle markers (alpha smooth muscle actin [α-SMA] and desmin), and neural markers (neurofilament [RECA-1] and neurogenic nitric oxide synthase [nNOS]) increased significantly with prolonged treatment time. Masson's staining demonstrated an increased in the smooth muscle cell (SMC)/collagen ratio. Significant changes were detected in the microstructures of various types of cells. In vivo imaging system (IVIS) analysis showed that at the 1 st day, the HUC-MSCs implanted moved to the site of damage. Additionally, the oxidative stress levels were dramatically reduced in the penises of rats administered with HUC-MSCs.
Male ; Animals ; Erectile Dysfunction/metabolism* ; Rats, Sprague-Dawley ; Mesenchymal Stem Cell Transplantation/methods* ; Rats ; Penis/pathology* ; Humans ; Disease Models, Animal ; Umbilical Cord/cytology* ; Peripheral Nerve Injuries/complications* ; Mesenchymal Stem Cells ; Nitric Oxide Synthase Type III/metabolism* ; Actins/metabolism* ; Nitric Oxide Synthase Type I/metabolism*

In continuation of research aimed at identifying anti-inflammatory agents from natural sesquiterpenoids, an activity-guided fractionation approach utilizing lipopolysaccharide (LPS)-mediated RAW264.7 cells was employed to investigate chemical constituents from Inula Britannica (I. britannica). Seven novel sesquiterpenoid dimers inulabritanoids A-G (1-7) and two novel sesquiterpenoid monomers inulabritanoids H (8) and I (9) were isolated from I. britannica together with eighteen known compounds (10-27). The structural elucidation was accomplished through comprehensive analysis of 1D and 2D nuclear magnetic resonance (NMR), high-resolution mass spectrometry (HR-MS), and electronic circular dichroism (ECD) spectra, complemented by quantum chemical calculations. Compounds 1, 2, 12, 16, 19, and 26 demonstrated inhibitory effects on NO production, with IC₅₀ values of 3.65, 5.48, 3.29, 6.91, 3.12, and 5.67 μmol·L^-1, respectively. Mechanistic studies revealed that compound 1 inhibited IκB kinase β (IKKβ) phosphorylation, thereby blocking nuclear factor κB (NF-κB) nuclear translocation, and activated the kelch-like ECH-associated protein 1 (Keap1)/nuclear factor erythroid 2-related factor 2 (Nrf2) signal pathway, leading to decreased expression of NADPH oxidase 2 (NOX-2), inducible nitric oxide synthase (iNOS), tumor necrosis factor α (TNF-α), interleukin-6 (IL-6), monocyte chemotactic protein-1 (MCP-1), IL-1β, and IL-1α and increased expression of NAD(P)H: quinone oxidoreductase 1 (NQO-1) and heme oxygenase-1 (HO-1), thus exhibiting anti-inflammatory effects in vitro. These results indicate that dimeric sesquiterpenoids may serve as promising candidates for anti-inflammatory drug development.
Mice ; Animals ; Sesquiterpenes/isolation & purification* ; Anti-Inflammatory Agents/isolation & purification* ; Inula/chemistry* ; RAW 264.7 Cells ; Nitric Oxide ; Molecular Structure ; NF-kappa B/immunology* ; NF-E2-Related Factor 2/immunology* ; Macrophages/immunology* ; Nitric Oxide Synthase Type II/immunology* ; Plant Extracts/pharmacology* ; Lipopolysaccharides ; Tumor Necrosis Factor-alpha/immunology* ; I-kappa B Kinase/genetics*

Cancer pain is one of the most common symptoms in patients with advanced cancer. In this study, we aimed to investigate the effects of the <i>Masi>-related gene C (MrgC) receptors on bone cancer pain. Mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL) were measured after the inoculation of Walker 256 mammary gland carcinoma cells into the tibia of adult Sprague-Dawley rats. The effects of MrgC receptor agonist bovine adrenal medulla 8-22 (BAM8-22) on nociceptive behaviors were investigated after intrathecal injection on days 16 and 17. Glial fibrillary acidic protein (GFAP)-positive cells in the spinal dorsal cord, and calcitonin gene related peptide (CGRP)-, neuronal nitric oxide synthase (nNOS)- and IL-1β-positive neurons in the dorsal root ganglia (DRG) were examined by immunofluorescence staining. The expression of nNOS and IL-1β proteins in the spinal dorsal horn and the DRG was examined by Western blotting after treatment with (Tyr6)-γ2-MSH-6-12 (MSH), which was another MrgC receptor agonist. The results showed that intrathecal injection of BAM8-22 (30 nmol) attenuated mechanical allodynia in a rat model of bone cancer pain and the effects could last for about 60 min, and single administration of BAM8-22 for two consecutive days reduced mechanical allodynia by about half on the third day. Moreover, the number of GFAP-positive cells in the spinal dorsal horn, and the number of CGRP-, nNOS- and IL-1β-positive neurons in the DRG were decreased. Similarly, intrathecal administration of MSH (15 nmol) reduced the expression of nNOS and IL-1β in the spinal dorsal horn and the DRG. In conclusion, activation of MrgC receptors suppresses the activation of astrocytes in the spinal dorsal cord and the expression of CGRP, nNOS, and IL-1β in the spinal dorsal cord and/or DRG, which may underlie the inhibition of bone cancer pain. These findings provide a novel strategy for the treatment of bone cancer pain.
Animals ; Cancer Pain/drug therapy* ; Rats ; Rats, Sprague-Dawley ; Bone Neoplasms/complications* ; Ganglia, Spinal/metabolism* ; Spinal Cord Dorsal Horn/metabolism* ; Receptors, G-Protein-Coupled/genetics* ; Female ; Calcitonin Gene-Related Peptide/genetics* ; Interleukin-1beta/metabolism* ; Peptide Fragments/metabolism* ; Nitric Oxide Synthase Type I/genetics* ; Disease Models, Animal

In the central nervous system, nitric oxide (NO), a free gas with multitudinous bioactivities, is mainly produced from the oxidation of L-arginine by neuronal nitric oxide synthase (nNOS). In the past 20 years, the studies in our group and other laboratories have suggested a significant involvement of nNOS in a variety of neurological and neuropsychiatric disorders. In particular, the interactions between the PDZ domain of nNOS and its adaptor proteins, including post-synaptic density 95, the carboxy-terminal PDZ ligand of nNOS, and the serotonin transporter, significantly influence the subcellular localization and functions of nNOS in the brain. The nNOS-mediated protein-protein interactions provide new attractive targets and guide the discovery of therapeutic drugs for neurological and neuropsychiatric disorders. Here, we summarize the work on the roles of nNOS and its association with multiple adaptor proteins on neurological and neuropsychiatric disorders.
Humans ; Nitric Oxide Synthase Type I/metabolism* ; Adaptor Proteins, Signal Transducing ; Brain/metabolism* ; Nervous System Diseases

Chronic intermittent hypobaric hypoxia (CIHH) is known to have an anti-hypertensive effect, which might be related to modulation of the baroreflex in rats with renal vascular hypertension (RVH). In this study, RVH was induced by the 2-kidney-1-clip method (2K1C) in adult male Sprague-Dawley rats. The rats were then treated with hypobaric hypoxia simulating 5000 m altitude for 6 h/day for 28 days. The arterial blood pressure (ABP), heart rate (HR), and renal sympathetic nerve activity (RSNA) were measured before and after microinjection of L-arginine into the nucleus tractus solitarii (NTS) in anesthetized rats. Evoked excitatory postsynaptic currents (eEPSCs) and spontaneous EPSCs (sEPSCs) were recorded in anterogradely-labeled NTS neurons receiving baroreceptor afferents. We measured the protein expression of neuronal nitric oxide synthase (nNOS) and endothelial NOS (eNOS) in the NTS. The results showed that the ABP in RVH rats was significantly lower after CIHH treatment. The inhibition of ABP, HR, and RSNA induced by L-arginine was less in RVH rats than in sham rats, and greater in the CIHH-treated RVH rats than the untreated RVH rats. The eEPSC amplitude in NTS neurons receiving baroreceptor afferents was lower in the RVH rats than in the sham rats and recovered after CIHH. The protein expression of nNOS and eNOS in the NTS was lower in the RVH rats than in the sham rats and this decrease was reversed by CIHH. In short, CIHH treatment decreases ABP in RVH rats via up-regulating NOS expression in the NTS.
Animals ; Baroreflex ; physiology ; Blood Pressure ; drug effects ; Hypertension ; metabolism ; Hypoxia ; chemically induced ; Kidney ; drug effects ; metabolism ; Male ; Nitric Oxide Synthase Type I ; drug effects ; metabolism ; Rats, Sprague-Dawley ; Solitary Nucleus ; metabolism

BACKGROUND AND OBJECTIVES: Few studies were evaluated the effect of blindness on outcome in animal models, though a potential effect of blinding has been reported in clinical trials. We evaluated the effects of adipose tissue-derived stem cells (ADSCs) on cavernous nerve injury (CNI)-induced erectile dysfunction (ED) in the rat and examined how proper blinding of the outcome assessor affected treatment effect. METHODS AND RESULTS: We searched in Pubmed, EMBASE, Cochrane and Web of Science databases from inception to January 2019. We included CNI animal model, randomized controlled experiments, and ADSC intervention. Erectile function and structural changes were assessed by intracavernous pressure and mean arterial pressure (ICP/MAP) ratios, neuronal nitric oxide synthase (nNOS) levels, cavernous smooth muscle and collagen (CSM/collagen) ratios, and cyclic guanosine monophosphate (cGMP). RESULTS: Nineteen studies were included in the final meta-analysis. The ICP/MAP ratio of the ADSC treatment group increased compared to the control group (SMD=1.33, 95%CI: 1.11~1.56, I²=72%). The nNOS level (SMD=2.29, 95%CI: 1.74~2.84, I²=75%), CSM/collagen (SMD=2.57, 95%CI: 1.62~3.52; I²=85%), and cGMP (SMD=2.96, 95%CI: 1.82~4.10, I²=62%) were also increased in the ADSC treatment group. Preplanned subgroup analysis was conducted to explore the source of heterogeneity. Five studies with blinded outcome assessment were significantly less effective than the unblinded studies (SMD=1.33, 95%CI: 0.86~1.80; SMD=1.81, 95%CI: 1.17~2.46, respectively). CONCLUSIONS: ADSCs might be effective in improving erectile function and structural change in CNI-induced ED. However, non-blinded outcome assessors might cause detection bias and overestimate treatment efficacy. Therefore, the ADSC efficacy must be further evaluated with a rigorous study design to avoid bias.
Animals ; Arterial Pressure ; Bias (Epidemiology) ; Blindness ; Collagen ; Erectile Dysfunction ; Guanosine Monophosphate ; Male ; Models, Animal ; Muscle, Smooth ; Nitric Oxide Synthase Type I ; Population Characteristics ; Rats ; Stem Cells ; Treatment Outcome

ObjectiveTo investigate the protective effect of human urine-derived stem cells (USCs) on erectile function and cavernous structure in rats with cavernous nerve injury (CNI).

METHODSSixty adult male SD rats with normal sexual function were randomly divided into four groups of equal number: sham operation, bilateral CNI (BCNI) model control, phosphate buffered saline (PBS), and USC. The BCNI model was established in the latter three groups of rats by clamping the bilateral cavernous nerves. After modeling, the rats in the PBS and USC groups were treated by intracavernous injection of PBS at 200 μl and USCs at 1×106/200 μl PBS respectively for 28 days. Then, the maximum intracavernous pressure (mICP) and the ratio of mICP to mean arterial pressure (mICP/MAP) of the rats were calculated by electrical stimulation of the major pelvic ganglions, the proportion of nNOS- or NF200-positive nerve fibers in the total area of penile dorsal nerves determined by immunohistochemical staining, the levels of endothelial cell marker eNOS, smooth muscle marker α-SMA and collagen I detected by Western blot, and the smooth muscle to collagen ratio and the cell apoptosis rate in the corpus cavernosum measured by Masson staining and TUNEL, respectively.

RESULTSAfter 28 days of treatment, the rats in the USC group, as compared with those in the PBS and BCNI model control groups, showed significant increases in the mICP (［81 ± 9.9］ vs ［31 ± 8.3］ and ［33 ± 4.2］ mmHg, P <0.05), mICP/MAP ratio (0.72 ± 0.05 vs 0.36 ± 0.03 and 0.35 ± 0.04, P <0.05), the proportions of nNOS-positive nerve fibers (［11.31 ± 4.22］% vs ［6.86 ± 3.08］% and ［7.29 ± 4.84］% , P <0.05) and NF200-positive nerve fibers in the total area of penile dorsal nerves (［27.31 ± 3.12］% vs ［17.38 ± 2.87］% and ［19.49 ± 4.92］%, P <0.05), the eNOS/GAPDH ratio (0.52 ± 0.08 vs 0.31 ± 0.06 and 0.33 ± 0.07, P <0.05), and the α-SMA/GAPDH ratio (1.01 ± 0.09 vs 0.36 ± 0.05 and 0.38 ± 0.04, P <0.05), but a remarkable decrease in the collagen I/GAPDH ratio (0.28 ± 0.06 vs 0.68 ± 0.04 and 0.70 ± 0.10, P <0.05). The ratio of smooth muscle to collagen in the corpus cavernosum was significantly higher in the USC than in the PBS and BCNI model control groups (17.91 ± 2.86 vs 7.70 ± 3.12 and 8.21 ± 3.83, P <0.05) while the rate of cell apoptosis markedly lower in the former than in the latter two (3.31 ± 0.83 vs 9.82 ± 0.76, P <0.01; 3.31 ± 0.83 vs 9.75 ± 0.91, P <0.05).

CONCLUSIONSIntracavernous injection of USCs can protect the erectile function of the rat with cavernous nerve injury by protecting the nerves, improving the endothelial function, alleviating fibrosis and inhibiting cell apoptosis in the cavernous tissue.

Actins ; analysis ; Animals ; Arterial Pressure ; Collagen ; analysis ; Disease Models, Animal ; Erectile Dysfunction ; prevention & control ; Male ; Nitric Oxide Synthase Type I ; analysis ; Nitric Oxide Synthase Type III ; analysis ; Penile Erection ; physiology ; Penis ; innervation ; Pudendal Nerve ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Saline Solution ; administration & dosage ; Stem Cell Transplantation ; methods ; Stem Cells ; Urine ; cytology

The ganglion cardiacum or juxtaductal body is situated along the left recurrent laryngeal nerve in the aortic window and is an extremely large component of the cardiac nerve plexus. This study was performed to describe the morphologies of the ganglion cardiacum or juxtaductal body in human fetuses and to compare characteristics with intracardiac ganglion. Ganglia were immunostained in specimens from five fetuses of gestational age 12–16 weeks and seven fetuses of gestational age 28–34 weeks. Many ganglion cells in the ganglia were positive for tyrosine hydroxylase (TH; sympathetic nerve marker) and chromogranin A, while a few neurons were positive for neuronal nitric oxide synthase (NOS; parasympathetic nerve marker) or calretinin. Another ganglion at the base of the ascending aorta carried almost the same neuronal populations, whereas a ganglion along the left common cardinal vein contained neurons positive for chromogranin A and NOS but no or few TH-positive neurons, suggesting a site-dependent difference in composite neurons. Mixtures of sympathetic and parasympathetic neurons within a single ganglion are consistent with the morphology of the cranial base and pelvic ganglia. Most of the intracardiac neurons are likely to have a non-adrenergic non-cholinergic phenotype, whereas fewer neurons have a dual cholinergic/noradrenergic phenotype. However, there was no evidence showing that chromogranin A- and/or calretinin-positive cardiac neurons corresponded to these specific phenotypes. The present study suggested that the ganglion cardiacum was composed of a mixture of sympathetic and parasympathetic neurons, which were characterized the site-dependent differences in and near the heart.
Aorta ; Calbindin 2 ; Chromogranin A ; Fetus* ; Ganglia ; Ganglion Cysts* ; Gestational Age ; Heart ; Humans* ; Neurons ; Nitric Oxide Synthase Type I ; Phenotype ; Recurrent Laryngeal Nerve ; Skull Base ; Tyrosine 3-Monooxygenase ; Veins

Calcium Channels, L-Type ; genetics ; metabolism ; Esophageal Achalasia ; genetics ; metabolism ; Esophagus ; metabolism ; Humans ; Nitric Oxide Synthase ; metabolism ; Nitric Oxide Synthase Type I ; genetics ; metabolism ; RNA, Messenger ; metabolism ; Real-Time Polymerase Chain Reaction ; Receptors, Calcium-Sensing ; genetics ; metabolism

The traditionally used oriental herbal medicine Moutan Cortex Radicis [MCR; Paeonia Suffruticosa Andrews (Paeoniaceae)] exerts anti-inflammatory, anti-spasmodic, and analgesic effects. In the present study, we investigated the therapeutic effects of differently fractioned MCR extracts in a 6-hydroxydopamine (OHDA)-induced Parkinson's disease model and neuro-blastoma B65 cells. Ethanol-extracted MCR was fractionated by n-hexane, butanol, and distilled water. Adult Sprague-Dawley rats were treated first with 20 μg of 6-OHDA, followed by three MCR extract fractions (100 or 200 mg·kg) for 14 consecutive days. In the behavioral rotation experiment, the MCR extract-treated groups showed significantly decreased number of net turns compared with the 6-OHDA control group. The three fractions also significantly inhibited the reduction in tyrosine hydroxylase-positive cells in the substantia nigra pars compacta following 6-OHDA neurotoxicity. Western blotting analysis revealed significantly reduced tyrosine hydroxylase expression in the substantia nigra pars compacta in the 6-OHDA-treated group, which was significantly inhibited by the n-hexane or distilled water fractions of MCR. B65 cells were exposed to the extract fractions for 24 h prior to addition of 6-OHDA for 30 min; treatment with n-hexane or distilled water fractions of MCR reduced apoptotic cell death induced by 6-OHDA neurotoxicity and inhibited nitric oxide production and neuronal nitric oxide synthase expression. These results showed that n-hexane- and distilled water-fractioned MCR extracts inhibited 6-OHDA-induced neurotoxicity by suppressing nitric oxide production and neuronal nitric oxide synthase activity, suggesting that MCR extracts could serve as a novel candidate treatment for the patients with Parkinson's disease.
Animals ; Anti-Inflammatory Agents ; pharmacology ; therapeutic use ; Antiparkinson Agents ; pharmacology ; therapeutic use ; Cell Death ; drug effects ; Cell Line ; Disease Models, Animal ; Drugs, Chinese Herbal ; chemistry ; Neurons ; pathology ; Nitric Oxide ; analysis ; Nitric Oxide Synthase Type I ; biosynthesis ; Oxidopamine ; toxicity ; Paeonia ; chemistry ; Parkinsonian Disorders ; chemically induced ; drug therapy ; Phytotherapy ; Plant Extracts ; pharmacology ; therapeutic use ; Plants, Medicinal ; Rats ; Rats, Sprague-Dawley ; Substantia Nigra ; drug effects ; enzymology ; Tyrosine 3-Monooxygenase ; genetics ; metabolism