nNOS Expression Levels Research Articles

(+)-Catechin Alleviates CCI-Induced Neuropathic Pain in Rats by Modulating the IL34/CSFIR Axis and Attenuating the Schwann Cell-Macrophage Cascade Response in the DRG.

The aim of this study was to investigate the potential therapeutic applications of (+)-catechin in the treatment of neuropathic pain. In vivo study, 32 SD rats were randomly divided into four groups: sham group, chronic constriction injury (CCI) group, CCI + ibuprofen group and CCI+ (+)-catechin group. They were subjected to behavioural tests, ELISA, immunohistochemistry and Western blotting. The mechanisms involved were investigated using specific inhibitors in cell experiments. Results of in vivo experiments showed that (+)-catechin could reduce the cold sensitivity pain in a rat model of CCI; ELISA and immunohistochemistry results showed that (+)-catechin could decrease the levels of IL-8, IL-6, TNF-α, CCL2 and CCL5 in serum and the expression levels of nNOS, COX2, IL6, TNF-α, IBA-1 and CSF1R in DRG of CCI rats. Finally, western blot confirmed that (+)-catechin could diminish the levels of IL-34/CSF1R/JAK2/STAT3 signalling pathway in DRG of CCI rats. In vitro studies showed that (+)-catechin reduced IL-34 secretion in LPS-induced RSC96 cells. Meanwhile, (+)-catechin administration in LPS-induced Schwann cell-conditioned medium (L-CM) significantly inhibited the proliferation and migration of RAW264.7 cells; in addition, L-CM+(+)-catechin reduced the activation of the CSF1R/JAK2/STAT3 signalling pathway. (+)-Catechin attenuated the Schwann cell-macrophage cascade response in the DRG by modulating the IL34/CSFIR axis and inhibiting activation of the JAK2/STAT3 pathway, thereby attenuating CCI-induced neuropathic pain in rats.

NMDA receptor activation induces damage of alveolar type II cells and lung fibrogenesis through ferroptosis

Ferroptosis, a newly discovered type of regulated cell death, has been implicated in numerous human diseases. Idiopathic pulmonary fibrosis (IPF) is a progressive and ultimately fatal interstitial lung disease with poor prognosis and limited treatment options. Emerging evidence has linked ferroptosis and glutamate-determined cell fate which is considered a new light on the etiology of pulmonary fibrosis. Here, we observed that N-methyl d-aspartate receptor (NMDAR) activation promoted cell damage and iron deposition in MLE-12 cells in a dose-, time-, and receptor-dependent manner. This mediated substantial Ca2+ influx, upregulated the expression levels of nNOS and IRP1, and affected intracellular iron homeostasis by regulating the expression of iron transport-related proteins (i.e., TFR1, DMT1, and FPN). Excessive iron load promoted the continuous accumulation of total intracellular and mitochondrial reactive oxygen species, which ultimately led to ferroptosis. NMDAR inhibition reduced lung injury and pulmonary fibrosis in bleomycin-induced mice. Bleomycin stimulation upregulated the expression of NMDAR1, nNOS, and IRP1 in mouse lung tissues, which ultimately led to iron deposition via regulation of the expression of various iron metabolism-related genes. NMDAR activation initiated the pulmonary fibrosis process by inducing iron deposition in lung tissues and ferroptosis of alveolar type II cells. Our data suggest that NMDAR activation regulates the expression of iron metabolism-related genes by promoting calcium influx, increasing nNOS and IRP1 expression, and increasing iron deposition by affecting cellular iron homeostasis, ultimately leading to mitochondrial damage, mitochondrial dysfunction, and ferroptosis. NMDAR activation-induced ferroptosis of alveolar type II cells might be a key event to the initiation of pulmonary fibrosis.

Effect of clove on improving running ability in aging mice.

In this study, a D-galactose-induced aging mouse model was established, and Syringa oblata Lindl. extract (SOLE) was administered orally to observe the effect and mechanism of SOLE on the running ability of aging mice. The role of SOLE was evaluated by H&E histopathological observation, detection of serum biochemical indices, and detection of mRNA expression levels by qPCR experiments. The experimental results showed that SOLE could increase the exhaustive running time of aging mice and reduce the oxidative aging of the liver and kidney. At the same time, the levels of BUN, lactic acid, GOT, GPT, MDA, iNOS, and TNF-α in the serum of the mice were decreased, and the relative mRNA expression levels of nNOS, iNOS, TNF-α and syncytin-1 in the liver tissue and skeletal muscle tissue of the mice were downregulated. In addition, it increased the levels of CAT, GSH-Px, and T-SOD in mouse serum and upregulated the relative mRNA expression of Cu/Zn-SOD, Mn-SOD, and CAT in mouse liver tissue and muscle tissue. The analysis results showed that SOLE mainly contained rutin, isoquercitrin, ferulic acid, dihydroquercetin, and quercitrin. In summary, SOLE can enhance the running ability and exercise ability of aging mice and slow down aging. PRACTICAL APPLICATIONS: Clove is used as health tea or traditional Chinese medicine in China, but there is no relevant research and application on the improvement of human exercise ability. This study found this new function of clove, which accumulated a theoretical basis for further popularizing its application.

Inhibition of neuronal nitric oxide synthase protects against hippocampal neuronal injuries by increasing neuropeptide Y expression in temporal lobe epilepsy mice

Neuronal nitric oxide synthase (nNOS) plays a pivotal role in the pathological process of neuronal injury in the development of epilepsy. Our previous study has demonstrated that nitric oxide (NO) derived from nNOS in the epileptic brain is neurotoxic due to its reaction with the superoxide radical with the formation of peroxynitrite. Neuropeptide Y (NPY) is widely expressed in the mammalian brain, which has been implicated in energy homeostasis and neuroprotection. Recent studies suggest that nNOS may act as a mediator of NPY signaling. Here in this study, we sought to determine whether NPY expression is regulated by nNOS, and if so, whether the regulation of NPY by nNOS is associated with the neuronal injuries in the hippocampus of epileptic brain. Our results showed that pilocarpine-induced temporal lobe epilepsy (TLE) mice exhibited an increased level of nNOS expression and a decreased level of NPY expression along with hippocampal neuronal injuries and cognition deficit. Genetic deletion of nNOS gene, however, significantly upregulated hippocampal NPY expression and reduced TLE-induced hippocampal neuronal injuries and cognition decline. Knockdown of NPY abolished nNOS depletion-induced neuroprotection and cognitive improvement in the TLE mice, suggesting that inhibition of nNOS protects against hippocampal neuronal injuries by increasing neuropeptide Y expression in TLE mice. Targeting nNOS-NPY signaling pathway in the epileptic brain might provide clinical benefit by attenuating neuronal injuries and preventing cognitive deficits in epilepsy patients.

Inhibition of interferon-gamma-stimulated melanoma progression by targeting neuronal nitric oxide synthase (nNOS)

Interferon-gamma (IFN-γ) is shown to stimulate melanoma development and progression. However, the underlying mechanism has not been completely defined. Our study aimed to determine the role of neuronal nitric oxide synthase (nNOS)-mediated signaling in IFN-γ-stimulated melanoma progression and the anti-melanoma effects of novel nNOS inhibitors. Our study shows that IFN-γ markedly induced the expression levels of nNOS in melanoma cells associated with increased intracellular nitric oxide (NO) levels. Co-treatment with novel nNOS inhibitors effectively alleviated IFN-γ-activated STAT1/3. Further, reverse phase protein array (RPPA) analysis demonstrated that IFN-γ induced the expression of HIF1α, c-Myc, and programmed death-ligand 1 (PD-L1), in contrast to IFN-α. Blocking the nNOS-mediated signaling pathway using nNOS-selective inhibitors was shown to effectively diminish IFN-γ-induced PD-L1 expression in melanoma cells. Using a human melanoma xenograft mouse model, the in vivo studies revealed that IFN-γ increased tumor growth compared to control, which was inhibited by the co-administration of nNOS inhibitor MAC-3-190. Another nNOS inhibitor, HH044, was shown to effectively inhibit in vivo tumor growth and was associated with reduced PD-L1 expression levels in melanoma xenografts. Our study demonstrates the important role of nNOS-mediated NO signaling in IFN-γ-stimulated melanoma progression. Targeting nNOS using highly selective small molecular inhibitors is a unique and effective strategy to improve melanoma treatment.

Clusterin enhances cell survival by suppressing neuronal nitric-oxide synthase expression in the rhodopsin S334ter-line3 retinitis pigmentosa model

Environmental changes in the retina, including oxidative stress-induced cell death, influence photoreceptor degeneration in Retinitis Pigmentosa (RP). Previously, we tested and discovered that a cytoprotective chaperone protein, clusterin, produced robust preservation of rod photoreceptors of a rat autosomal dominant rhodopsin transgenic model of RP, S334ter-line3. To investigate the biochemical and molecular cytoprotective pathways of clusterin, we examined and compared a known source of cone cell death, nitric oxide (NO), observing nNOS expression using antibody against nNOS in RP retinas with intravitreal injections of saline, clusterin (10 μg/ml), or a non-isoform-selective NOS inhibitor (25 mM), L-NAME, or with an intraperitoneal injection (IP) of L-NAME (100 mg/kg). Rhodopsin-immunoreactive rod photoreceptor cells and nNOS-immunoreactive cells were quantified with immunohistochemistry in the presence or absence of L-NAME or clusterin, and the total nNOS retinal expression was determined by immunoblot analysis. In this study, the level of nNOS expression was significantly up-regulated postnatally (P) at P15 (P < 0.05), P30 (P < 0.001) and P60 (P < 0.0001) in RP retinas compared to normal controls. Clusterin treatment suppressed the up-regulated nNOS expression in RP retinas (P < 0.0001) and was enhanced in Type II amacrine cells. Additionally, IP injection of L-NAME at P15 prolonged rod survival in the later stage of RP retinas (P < 0.001). Conversely, rod survival in L-NAME–treated RP retinas was not equivalent to the rod survival number seen in clusterin-treated retinas, which suggests induction of nNOS expression in RP retinas and its reduction by clusterin is only partly responsible for the rescue observed through the reduction of nNOS expression in S334ter-line3 rat retinas.

Insights into the effects of inducible and neuronal nitric oxide synthase isoenzymes in experimental intestinal heterophyiasis

Background: Heterophyiasis is one of the food-borne trematode infections, caused by the intestinal flukeHeterophyes heterophyes. The exact role of nitric oxide (NO) in the immune response against the majorityof parasites remains controversial. It proved protective against a wide range of protozoan and helminthicparasites. Nevertheless, its role in intestinal heterophyiasis is yet to be explored.Objectives: The study aims to explore the possible roles of inducible nitric oxide synthase (iNOS) andneuronal nitric oxide synthase (nNOS) in experimental intestinal heterophyiasis.Material and Methods: The experimental study design included infection of male puppies with H.heterophyes encysted metacercariae (EMC), followed by treatment with aminoguanidine (AG) and7-nitroindazole (7-NI) drugs, as selective inhibitors of iNOS and nNOS, respectively. Controls includednon-infected and infected untreated puppies. Intestinal tissue sections from all puppies were stained forhistopathological and immunohistochemical (IHC) assessments.Results: Different intensities of iNOS and nNOS isoenzymes were observed in intestinal sections. The studyshowed the highest concentration of iNOS isoenzyme in the infected-7-NI treated group. The control noninfectedpuppies exhibited the highest levels of nNOS expression, with statistical significance (P<0.05).The study also showed that AG significantly reduced the degree of inflammatory cellular infiltrations.Additionally, the over-production of NO worsened the degree of intestinal apoptotic changes.Conclusion: Results obtained in the study suggested that inhibition of iNOS, to some extent, improvedintestinal architecture, while inhibition of nNOS failed to eliminate experimental intestinal heterophyiasis.

PO-01-010 Synergistic effects of extracorporeal shockwave therapy and Korean herbal formulation on erectile dysfunction in diabetic animal model

Studies on low-intensity extracorporeal shockwave therapy (ESWT) for the treatment of refractory erectile dysfunction (ED) have been reported to date, but inconclusive evidence has been obtained. KH-204, a Korean herbal formula has been reported to have anti-oxidative effects many times. We investigated the synergy effect of ESWT with KH-204 in an animal model of diabetes mellitus (DM) - induced ED. Streptozotocin-induced DM rats were divided into 5 groups: group 1, control; group 2, DM; group 3, DM + ESWT; group 4 DM + KH-204; and group 4 DM + ESWT + KH-204. In ESWT groups rats were treated with ESWT 3 times a week for 2 weeks. The KH-204 groups were treated with a daily oral dose of KH-204 for 12 weeks. After all treatments, intracavernous pressure (ICP) was measured, and the cavernous tissues were evaluated. ICP was evaluated as a measurement of erectile function. The DM + ESWT, DM + KH-204, and DM + ESWT + KH-204 groups showed significantly restored erectile function compared with the DM group (p<0.05). Among these groups, the DM+ ESWT + KH-204 group showed the highest ICP. Moreover, ESWT and KH-204 treatment restored smooth muscle contents and many parameters related to potency including vascular endothelial growth factor, eNOS, platelet endothelial cell adhesion molecule, cGMP and nNOS expression levels compared with the DM group (p<0.05).

NNOS in Embryonic Kidney Contributes to Glomerular Maturation

The regulatory role of neuronal nitric oxide synthase (nNOS) in renal and glomerular hemodynamics is well established. In addition, nNOS is involved in cellular migration in other organs, such as the brain. The highest level of nNOS expression occurs on day 6 after birth (P6), when the mouse kidney is still developing. The purpose of this study was to explore a novel, non‐traditional role of nNOS in renal development and glomerular maturation. To determine the timepoints and localization of nNOS expression in embryonic kidney, fluorescent reporter nNos+/−‐Cre+/+/mTmG+/+ mice were induced with tamoxifen at different timepoints during pregnancy. The specific inhibitor of nNOS, 7‐Nitroindazole (7‐NI) was administered daily by ip. injections (20 mg/kg) from E16.5 to P7 in pregnant Pod+/+‐Cre+/+/Confetti+/+ mice. Fetal kidneys were harvested after birth at P7 and further processed for histological analysis. During embryonic kidney development, nNOS expression is detectable in the medullary region as early as E16.5. At E19.5, nNOS is expressed in macula densa cells at the juxtaglomerular apparatus and in a few cells of the medullary collecting duct. At P3, nNOS was localized in the same cell types as in earlier stages. Pharmacological inhibition of nNOS for 10 days by daily 7‐NI treatment did not alter the kidney/body weight ratio compared to vehicle treatment, albeit the 7‐NI treated animal group had growth retardation (n=4 each). The diameter of glomeruli was 20% smaller, and the cumulative podocyte number in glomeruli was decreased by 40% in 7‐NI treated animals (n=4). Albeit 7‐NI treatment resulted in the reduction of body weight likely due to nonrenal effects, the embryonic presence of nNOS in macula densa cells and the negative impact of nNOS inhibition on glomerular diameter and podocyte number suggest a novel, non‐traditional role for nNOS in embryonic kidney development and glomerular maturation.Support or Funding InformationResearch support NIH NIDDK R01 DK64324This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Exercise Training Rescues High Fat Diet-induced Neuronal Nitric Oxide Synthase Expression In Hippocampus And Cortex

Western high-fat diet (HFD) consumption and being overweight induce hippocampal atrophy and deterioration of function. These alterations are associated with mental disorders, such as depression and anxiety. Exercise is an effective therapeutic treatment to combat obesity and enhance brain health. Numerous studies have demonstrated that neuronal nitric oxide synthase (nNOS) is a key regulator of affective behavior. Increased nNOS expression leads to anxiety, while reduced brain nNOS in an enriched environment that includes running exercise has anxiolytic effects. PURPOSE: We investigated whether HFD consumption and exercise training altered nNOS expression in the brain. METHODS: Twenty 4-week-old male C57BL/6J mice were used. After 2 weeks of acclimatization, mice were randomly assigned to a standard diet (SD; n = 5) or HFD group (n = 15). After 6 weeks, HFD-fed mice were further divided into either a non-exercise (HFD; n = 7) or a HFD (12 weeks) with exercise group (HFD+Ex; n = 8). The HFD+Ex group was allowed free access to a running wheel. Western blotting was performed to determine nNOS protein expression levels in the hippocampus (Hp), cortex (Cx) and cerebellum (Ce) from SD, HFD and HFD+Ex mice. RESULTS: Body weights were significantly increased in HFD-fed mice (SD: 26.0 ± 0.4 g; HFD: 36.6 ± 1.5 g; HFD+Ex: 29.1 ± 0.5 g; p < 0.01). Similarly, mesenteric fat weights were increased in the HFD group, while exercise training mitigated this effect (SD: 0.16 ± 0.04 g; HFD: 0.56 ± 0.10; HFD+Ex: 0.25 ± 0.03; p < 0.01). Compared with that of SD mice, Hp and Cx nNOS expression levels increased significantly with HFD feeding (Hp: 1.90 ± 0.28 fold increase, p < 0.05; Cx: 1.89 ± 0.49; p < 0.01). HFD-induced Hp and Cx nNOS expression was reduced in HFD+Ex mice to levels comparable to those of the SD group, though the difference in the Cx was not significant (Hp: 0.86 ± 0.16 fold increase, Cx: 1.48 ± 0.22; p = 0.1003). While Hp and Cx nNOS expression levels were susceptible to HFD consumption and exercise, those in the Ce were unchanged (p > 0.05). CONCLUSION: We conclude that exercise training restores HFD-induced nNOS expression in the Hp and Cx. Our results indicate that HFD-induced brain dysfunction is regulated by nNOS in the Hp and Cx, and exercise has therapeutic potential for mitigating HFD-induced depression and anxiety via the nNOS/NO pathway.

Interaction between interleukin‑6 and angiotensin II receptor 1 in the hypothalamic paraventricular nucleus contributes to progression of heart failure.

The association between interleukin‑6 (IL‑6) and angiotensin II receptor 1 (AT1‑R) in modulating the progression of heart failure (HF) remains to be fully elucidated. The aim of the present study was to investigate the mechanism of IL‑6 and AT1‑R in a model of HF induced by surgery. Male Sprague‑Dawley rats were randomly divided into five groups, including sham surgery and vehicle groups. The animals were treated for 4weeks via paraventricular nucleus infusion with either vehicle, losartan (LOS; 200µg/day), IL‑6 (1µg/day) or LOS and IL‑6 together (LOS+IL‑6). The rats with HF had higher levels of IL‑6, corticotropin‑releasing hormone (CRH) and norepinephrine (NE), and a lower level of neuronal nitric oxide synthase (nNOS), compared with the rats in the sham surgery group. Treatment with LOS attenuated the decrease in nNOS and the increases in IL‑6, CRH and NE; whereas treatment with IL‑6 facilitated the lower expression of nNOS and higher expression levels of IL‑6, CRH and NE. No differences in the expression levels of nNOS, CRH or NE were found between the LOS group and LOS+IL‑6 group. The results of the study demonstrated that IL‑6 contributed to the progression of HF via the AT1‑R pathway.

Exercise training rescues high fat diet-induced neuronal nitric oxide synthase expression in the hippocampus and cerebral cortex of mice

Consumption of a high fat diet (HFD) and being overweight both induce functional deterioration and atrophy of the hippocampus. These alterations are associated with mental disorders such as depression and anxiety. Exercise combats obesity and enhances brain health. There is substantial evidence that neuronal nitric oxide synthase (nNOS) is a key regulator of affective behavior, and that increased brain nNOS leads to anxiety while environmental enrichment (EE), which reduces brain nNOS, has anxiolytic effects. In this study we investigated the effects of HFD with and without exercise on nNOS protein and gene expression levels in the brains of mice. Twelve weeks of HFD consumption increased body and mesenteric fat weight, as well as nNOS protein levels in the hippocampus and cerebral cortex. Six weeks of exercise training reduced body fat and rescued hippocampal and cortical nNOS expression levels in HFD-fed mice. Cerebellar nNOS expression was unaffected by HFD and exercise. Our results suggest that HFD-induced brain dysfunction may be regulated by hippocampal and/or cortical nNOS, and that exercise may have therapeutic potential for the treatment of HFD-induced depression and anxiety via the nNOS/NO pathway. In conclusion, exercise reverses HFD-induced changes in hippocampal and cortical nNOS protein levels in mice.

Effects of environmental enrichment in aged mice on anxiety-like behaviors and neuronal nitric oxide synthase expression in the brain

Previous studies have shown that an enriched environment (EE) has an important effect on brain function via the neuronal nitric oxide synthase/nitric oxide (nNOS/NO) pathway in young and aged animals. However, whether EE induces its effect by altering nNOS expression levels and whether it lowers anxiety-like behaviors in aged mice remains unclear. Here, we show that nNOS expression levels increased with age in the hippocampus and cerebellum in aged mice, but not in the cortex. Moreover, EE reduced anxiety-like behaviors in aged mice and reduced nNOS expression levels in the cerebellum, but not in the cortex. The present study suggests that EE improves anxiety-like behaviors in aged mice by altering nNOS expression levels in the hippocampus or cerebellum.

Role of abnormal nerve conduction in the pathogenesis of congenital choledochal cyst

Objective To observe the expression levels of interstitial cells of Cajal (ICCs) and neurotransmitters in the extrahepatic biliary of congenital choledochal cysts (CCC),and to explore the role of abnormal nerve conduction in the pathogenesis of CCC.Methods A total of 38 child patients with CCC,15 males and 23 females,were randomly enrolled in this study and underwent cyst excision and biliary revascularization.The specimens were obtained from the distal cyst wall,the proximal cyst wall and gallbladder wall respectively and were classified into the corresponding groups.The expression of ICC was detected by immunotluorescence analysis and that of choline acetyltransferas (CHAT) and neuronal nitric oxide synthase (nNOS),the key enzymes for synthesis of acetylcholine and nitric oxide respectively,was analyzed by real-time polymerase chain reaction (RT-PCR).All of data were analyzed by SPSS 13.0 software for Windows.Results There was significant difference in the expression of ICCs between the distal cyst wall and the proximal cyst wall,also between the distal cyst wall and gallbladder wall (P ＜ 0.01).However,there was no significant difference between the the proximal cyst and gallbladder (P ＞ 0.05).The expression level of CHAT in the proximal cyst wall (1.39 ±0.11) and gallbladder wall (1.32 ±0.14) was significantly lower than in the distal cyst wall (1.78 ±0.21,P ＜0.05),but no significant difference existed between the proximal cyst wall and gallbladder wall (P ＞ 0.05).The expression level of nNOS in the proximal cyst wall (1.64 ±0.16) and gallbladder wall (1.60 ±0.13) was higher than in the distal cyst wall (1.14±0.11,P ＜ 0.05),but no significant difference existed between the proximal cyst and gallbladder (P ＞ 0.05).There was no significant difference in the expression levels of CHAT and nNOS in the the distal cyst wall between type Ⅰ CCC (1.95 ±0.27,and 1.14 ±0.12) and type ⅣW CCC (1.81 ±0.08 and 1.11 ± 0.13,P ＞0.05).However,there was significant difference in the expression of them in the proximal cyst wall between type Ⅰ CCC (1.04 ±0.17 and 1.95 ±0.24) and type Ⅳ CCC (1.39 ±0.13 and 1.70 ± 0.20,P ＜ 0.05).Conclusion The differential expression of ICC,and excitatory and inhibitory neurotransmitter in the different parts of extrahepatic biliary of CCC are probably reasons of CCC formation.The abnormal expression and distribution of the two neurotransmitters probably result in the different types of CCC. Key words: Choledochal cyst, congenital; Enteric nerve ; Interstitial cell of Cajal; Neurotransmitter; Choline acetyltransferas; Neuronal nitric oxide synthase

66 Neural Stem Cell Transplantation Restored the Delayed Gastric Emptying in Apolipoprotein E-Knockout Mice

Background. Although the pathogenesis of delayed gastric emptying, namely gastroparesis, is still unclear, diabetes mellitus is major etiology of gastroparesis. Decreased neuronal nitric oxide synthase (nNOS), which induces relaxation of gastric smooth muscle through the production of nitric oxide, is supposed to be one of the mechanisms of gastroparesis (Gastroenterology 113:1535, 1997). However, the effective treatment for gastroparesis has not been developed. While dyslipidemia is a risk factor of diabetic neuropathy, the defect of apolipoprotein E (apoE), a lipid transporter secreted from glial cell, decreased the expression of nNOS in the stomach (Dig Dis Sci. 57:1504, 2012). Furthermore, transplantion of neural stem cell into gastric antral wall was reported to improve delayed gastric emptying in nNOS knockout mouse (Gastroenterology 129:1817, 2005). The aim of the present study is to explore the role of apoE on gastric emptying and evaluate the effect of neural stem cell transplantation against the model of gastroparesis. Methods. The model of type2 diabetes, db/db mice, and apoE knockout mice, which is the model of arteriosclerosis and hyperlipidemia, were used. For measurement of gastric emptying, 0.48 mg of 13C-acetic acid with 0.15 ml liquid meal (Lacol®) was orally administered. The gastric half emptying time (t1/2) measured by 13C breath test was used for evaluation. The expression levels of nNOS, pan-neuronal marker, PGP 9.5, glial fibrillary acidic protein (GFAP), and glia derived neurotrophic factor (GDNF) were examined by immunohistochemistry andWestern blotting. Neural stem cells were isolated from ffLuc-transgenic mouse, which fluorescent protein, venus, and luciferase gene was fused (BBRC 419; 188, 2012). After tertiary neurosphere was formed, it was injected into gastric antral wall by glass needle. Results. Delayed gastric emptying was observed in 27% of db/db mice, while the levels of blood glucose and body weight were not significantly different. The expression of nNOS in gastric antrum and serum level of apoE were significantly decreased in db/db mice which presented delayed gastric emptying. In apoE knockout mice, the gastric emptying was also delayed compared with control. Although, the expression of PGP 9.5 was not changed, the expression of nNOS was significantly decreased in apoE knockout mice. Furthermore, the expression levels of GFAP and GDNF were significantly decreased in apoE knockout mice. Transplantation of neural stem cell significantly improved the delayed gastric emptying in apoE knockout mice. Conclusion. Neural stem cell transplantation restored the delayed gastric emptying in apoE knockout mice, suggesting a promising application of regenerative therapy against gastroparesis.

67 Perturbing NMDA Receptor -PSD-95 Protein Interactions Prevents Selective Loss of Nitric Oxide Synthase (NOS) Containing Neuron and Improves Gastric Emptying in Diabetic Rats

Background. Although the pathogenesis of delayed gastric emptying, namely gastroparesis, is still unclear, diabetes mellitus is major etiology of gastroparesis. Decreased neuronal nitric oxide synthase (nNOS), which induces relaxation of gastric smooth muscle through the production of nitric oxide, is supposed to be one of the mechanisms of gastroparesis (Gastroenterology 113:1535, 1997). However, the effective treatment for gastroparesis has not been developed. While dyslipidemia is a risk factor of diabetic neuropathy, the defect of apolipoprotein E (apoE), a lipid transporter secreted from glial cell, decreased the expression of nNOS in the stomach (Dig Dis Sci. 57:1504, 2012). Furthermore, transplantion of neural stem cell into gastric antral wall was reported to improve delayed gastric emptying in nNOS knockout mouse (Gastroenterology 129:1817, 2005). The aim of the present study is to explore the role of apoE on gastric emptying and evaluate the effect of neural stem cell transplantation against the model of gastroparesis. Methods. The model of type2 diabetes, db/db mice, and apoE knockout mice, which is the model of arteriosclerosis and hyperlipidemia, were used. For measurement of gastric emptying, 0.48 mg of 13C-acetic acid with 0.15 ml liquid meal (Lacol®) was orally administered. The gastric half emptying time (t1/2) measured by 13C breath test was used for evaluation. The expression levels of nNOS, pan-neuronal marker, PGP 9.5, glial fibrillary acidic protein (GFAP), and glia derived neurotrophic factor (GDNF) were examined by immunohistochemistry andWestern blotting. Neural stem cells were isolated from ffLuc-transgenic mouse, which fluorescent protein, venus, and luciferase gene was fused (BBRC 419; 188, 2012). After tertiary neurosphere was formed, it was injected into gastric antral wall by glass needle. Results. Delayed gastric emptying was observed in 27% of db/db mice, while the levels of blood glucose and body weight were not significantly different. The expression of nNOS in gastric antrum and serum level of apoE were significantly decreased in db/db mice which presented delayed gastric emptying. In apoE knockout mice, the gastric emptying was also delayed compared with control. Although, the expression of PGP 9.5 was not changed, the expression of nNOS was significantly decreased in apoE knockout mice. Furthermore, the expression levels of GFAP and GDNF were significantly decreased in apoE knockout mice. Transplantation of neural stem cell significantly improved the delayed gastric emptying in apoE knockout mice. Conclusion. Neural stem cell transplantation restored the delayed gastric emptying in apoE knockout mice, suggesting a promising application of regenerative therapy against gastroparesis.

PD8-11 TIME COURSE OF TREATMENT EFFECT OF AUTOLOGOUS ADIPOSE STROMAL VASCULAR FRACTION IN TYPE 2 DM RATS (ZFDM) WITH ERECTILE DYSFUNCTION

You have accessJournal of UrologyStem Cell Research1 Apr 2014PD8-11 TIME COURSE OF TREATMENT EFFECT OF AUTOLOGOUS ADIPOSE STROMAL VASCULAR FRACTION IN TYPE 2 DM RATS (ZFDM) WITH ERECTILE DYSFUNCTION Geehyun Song, Dalsan You, Bo Hyun Kim, Myoung Jin Jang, Choung-Soo Kim, and Tai Young Ahn Geehyun SongGeehyun Song More articles by this author , Dalsan YouDalsan You More articles by this author , Bo Hyun KimBo Hyun Kim More articles by this author , Myoung Jin JangMyoung Jin Jang More articles by this author , Choung-Soo KimChoung-Soo Kim More articles by this author , and Tai Young AhnTai Young Ahn More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2014.02.805AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Recent evidence suggests that adipose stromal vascular fraction (aSVF) may attenuate cavernosal destruction in diabetic erectile dysfunction (ED). However, so far, no study has investigated the effects of autologous aSVF on the time course of the structural, mechanical, and remodeling properties in diabetes ED. METHODS Thirty two 10-week old male type-II diabetic fa/fa Zucker Fatty Diabetes Mellitus (ZFDM) rats as DM group and ten fa/+ ZFDM lean rats as normal control group underwent weight and blood glucose measurement every one week. At age 22 weeks, in the DM group, intracavernosal (IC) injection of autologous aSVF (n=22) (2 X 106 cells/100μL) as DM treated group and PBS (n=10) (100μL) as DM control group, respectively. We evaluated IC pressure (ICP), mean arterial pressure (MAP), immunohistochemistry (vWF) and western blot (eNOS, nNOS) at 1d (n=3), 3d (n=3), 7d (n=3), 28d (n=10) and 56d (n=3) after IC injection of aSVF. RESULTS Blood glucose (BG) level and body weight (Bwt) steadily increased in DM group between age 10 and 22weeks. Differences in non-fasting BG level between DM and normal control group were evident as early as 11 weeks of age (fa/fa 349.0±123.0mg/dl vs. fa/+ 130.3±6.9mg/dl, p = 0.008), and were maintained until 22 weeks of age (fa/fa 353±189.2 mg/dl vs. fa/+ 124.0±10.9 mg/dl, p = 0.029). However, there were no differences in the Bwt because of same high fat diet. The ratio of maximal ICP and MAP (maxICP/MAP) were significantly increased in DM group treated with aSVF than DM control group treated with PBS after 7 days injecting aSVF and PBS, respectively. At 28 days, maxICP/MAP was shown maximum levels in DM group treated with aSVF. However, at 56 days, maxICP/MAP decreased significantly compared to DM group treated with aSVF at 28 days (0.81±0.13, 0.63±0.04, p=0.022). At days of 1, 3 and 28, vWF were gradually increased in penis of ZFDM rats (18.7 vs. 21.7 vs. 23.7, p=0.06). At 28 days, eNOS on western blot increased significantly from DM-control groups (23.6 vs. 32.8, p<0.001). However, at 28 days after injection of autologous aSVF, the level of nNOS expression in the penile dorsal nerves were similar with DM control and control groups. The rest of the histologic results are pending. CONCLUSIONS Intracavernous injection of aSVF gradually restored erectile function via regeneration of the destructed cavernous endothelium in Type 2 DM. However, in uncontrolled type 2 DM, the regenerative effect of aSVF may decrease after certain period of time functionally. Further investigations are needed on the time course of treatment effect with aSVF. © 2014FiguresReferencesRelatedDetails Volume 191Issue 4SApril 2014Page: e221 Advertisement Copyright & Permissions© 2014MetricsAuthor Information Geehyun Song More articles by this author Dalsan You More articles by this author Bo Hyun Kim More articles by this author Myoung Jin Jang More articles by this author Choung-Soo Kim More articles by this author Tai Young Ahn More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Gastric nNOS reduction accompanied by natriuretic peptides signaling pathway upregulation in diabetic mice.

To investigate the relationship between neuronal nitric oxide synthase (nNOS) expression and the natriuretic peptide signaling pathway in the gastric fundus of streptozotocin (STZ)-induced diabetic mice. Diabetic mice were induced by injection of STZ solution. Immunofluorescence labeling of HuC/D, nNOS and natriuretic peptide receptor-A, B, C (NPRs) in the gastric fundus (GF) was used to observe nNOS expression and whether NPRs exist on enteric neurons. The expression levels of nNOS and NPRs in the diabetic GF were examined by western blotting. An isometric force transducer recorded the electric field stimulation (EFS)-induced relaxation and contraction in the diabetic GF. An intracellular recording method assessed EFS-induced inhibitory junction potentials (IJP) on the GF. GF smooth muscles acquired from normal mice were incubated with different concentrations of the NPRs agonist C-type natriuretic peptide (CNP) for 24 h, after which their nNOS expressions were detected by western blotting. Eight weeks after injection, 43 diabetic mice were obtained from mouse models injected with STZ. Immunofluorescence indicated that the number of NOS neurons was significantly decreased and that nNOS expression was significantly downregulated in the diabetic GF. The results of physiological and electrophysiological assays showed that the EFS-induced relaxation that mainly caused by NO was significantly reduced, while the contraction was enhanced in the diabetic GF. EFS-induced IJP showed that L-NAME sensitive IJP in the diabetic GF was significantly reduced compared with control mice. However, both NPR-A and NPR-B were detected on enteric neurons, and their expression levels were upregulated in the diabetic GF. The nNOS expression level was downregulated dose-dependently in GF smooth muscle tissues exposed to CNP. These findings suggested that upregulation of the NPs signaling pathway may be involved in GF neuropathy caused by diabetes by decreasing nNOS expression.

Protective effect of puerarin on lead-induced mouse cognitive impairment via altering activities of acetyl cholinesterase, monoamine oxidase and nitric oxide synthase

Puerarin (PU), a natural flavonoid, has been reported to have many benefits and medicinal properties. The present study aimed to investigate the protective effects of puerarin on neurotoxicity in mice exposed to lead. ICR mice were exposed to lead acetate in the drinking water (500ppm) with or without puerarin coadministration (100 and 200mgPU/kg intragastrically once daily) for three months. We found puerarin significantly prevented Pb-induced neurotoxicity in a dose-dependent manner, indicated by behavioral indicators. Puerarin also decreased Pb contents in blood and brain. Puerarin increased activities of acetyl cholinesterase (AChE) and monoamine oxidase (MAO) in brain of Pb-treated mice. Moreover, Pb-induced profound elevation of oxidative stress, as evidenced by increasing of lipid peroxidation level and depleting of total antioxidant capacity in brain, were suppressed by treatment with puerarin. Puerarin markedly increased NO production and PKA activity in brain of Pb-treated mice. Western blot analysis showed that puerarin dramatically increased the expression levels of nNOS, eNOS and phosphor-Akt in brains of Pb-treated mice. In conclusion, these results suggested that puerarin can inhibit Pb-induced neurotoxicity, at least in part, by suppressing oxidative stress, reversing the Pb-induced alterations in transmitters and enzymes and modulating the PKA/Akt/NOS signaling pathway.

Electroacupuncture at Zusanli Rescues the Enteric Neuronal Loss in the Stomach of Diabetic Rats

The purpose of this study was to investigate the effects of electroacupuncture at Zusanli acupoint on the enteric neuropathy in diabetic rats. Sprague–Dawley rats were divided into different groups depending on the total electroacupuncture span and frequency. The expression of nitric oxide synthase (nNOS), choline acetyltransferase (CHAT), protein gene product 9.5 (PGP9.5), and doublecortin was significantly decreased in the diabetic group compared with the control group. Long-term electroacupuncture at Zusanli with either high frequency or low frequency could increase the expression levels of nNOS, CHAT, PGP9.5, and doublecortin, and the increase was greater in the high-frequency group. But no obvious changes were seen in the short-term electroacupuncture groups. These results suggest that electroacupuncture at Zusanli can restore the deficiency of enteric neurons in diabetes partly but a comparative long duration of stimuli (6 weeks) is required. The increase of doublecortin may be involved in this positive process.