Nitric Oxide Attenuation Research Articles

Nitric oxide mitigates thalidomide-induced abnormalities during germination and development of fennel seeds

Thalidomide causes teratogenic effects in several animal species and in humans. Accordingly, the World Health Organization banned thalidomide when mothers who took thalidomide during pregnancy delivered abnormal fetuses. After four decades, thalidomide underwent drug "re-purposing" since its antiangiogenic and immunomodulatory effects were therapeutic for multiple myeloma. There are no reports of thalidomide's effects on prokaryotes, but it showed teratogenic effects in Arabidopsis thaliana, an ancestor of the plant kingdom. This proof of concept study clearly shows that thalidomide caused a significant and reproducible decrease in germination rate, nitric oxide (NO) production, and chlorophyll content of fennel plantlets. Thalidomide also induced the formation of abnormal fennel plantlets with stunting, wrinkling, and curling of fennel shoots and leaves. Notably, quantitative analysis showed that thalidomide caused a 50% increase in the formation of abnormal fennel plantlets and that these negative effects of thalidomide showed a 2.50- to 4-fold decrease when fennel seeds were co-incubated with an NO donor (Spermine NoNoate) or a stable cGMP analog 8-bromo Guanosine 3',5'-cyclic monophosphate (8-Bromo-cGMP). This study is important because it confirms that thalidomide's negative effects on fennel seed germination and growth are mediated by attenuation of NO and disruption of NO signaling. This reproducible model of thalidomide-induced, NO-dependent damage in a plant system can be used to further investigate the molecular mechanisms of thalidomide action in plants. Importantly, this study establishes a link between the evolution of development of higher plants and mammals.

Suppressive effects of sulfated polysaccharide ascophyllan isolated from Ascophyllum nodosum on the production of NO and ROS in LPS-stimulated RAW264.7 cells.

In this study, we found that a sulfated polysaccharide isolated from the brown alga Ascophyllum nodosum, ascophyllan, showed suppressive effects on stimulated RAW264.7 cells. Ascophyllan significantly inhibited expression of inducible nitric oxide synthase mRNA and excessive production of nitric oxide (NO) in lipopolysaccharide (LPS)-stimulated RAW264.7 cells in a dose-dependent manner without affecting the viability of RAW264.7 cells. Ascophyllan also reduced the elevated level of intracellular reactive oxygen species (ROS) in LPS-stimulated RAW264.7 cells. Furthermore, preincubation with ascophyllan resulted in concentration-dependent decrease in ROS production in phorbol 12-myristate-13-acetate-stimulated RAW264.7 cells. Our results suggest that ascophyllan can exhibit anti-inflammatory effects on stimulated macrophages mainly through the attenuation of NO and ROS productions.

Neuronal Nitric Oxide Inhibitor 7-Nitroindazole Improved Brain-Derived Neurotrophic Factor and Attenuated Brain Tissues Oxidative Damage and Learning and Memory Impairments of Hypothyroid Juvenile Rats.

Hypothyroidism-associated learning and memory impairment is reported to be connected to oxidative stress and reduced levels of brain-derived neurotrophic factor (BDNF). The effects of neuronal nitric oxide inhibitor 7-nitroindazole (7NI) on brain tissues oxidative damage, nitric oxide (NO), BDNF and memory impairments in hypothyroid juvenile rats were investigated. Male Wistar juvenile rats (20 days old) were divided into five groups, including Martinez et al. (J Neurochem 78 (5):1054-1063, 2001). Control in which vehicle was injected instead of 7NI, (Jackson in Thyroid 8 (10):951-956, 1998) Propylthiouracil (PTU) where 0.05% PTU was added in drinking water and vehicle was injected instead of 7NI, (Gong et al. in BMC Neurosci 11 (1):50, 2010; Alva-Sánchez et al. in Brain Res 1271:27-35, 2009; Anaeigoudari et al. in Pharmacol Rep 68 (2): 243-249, 2016) PTU-7NI 5, PTU-7NI 10 and PTU-7NI 20 in which 5, 10, or 20mg/kg7NI was injected intraperitoneally (i.p.). Following 6 weeks, Morris water maze (MMW) and passive avoidance learning (PAL) tests were used to evaluate the memory. Finally, the hippocampus and the cortex of the rats were removed after anesthesia by urethane to be used for future analysis. The escape latency and traveled path in MWM test was increased in PTU group (P < 0.001). PTU also reduced the latency to enter the dark box of PAL and the time spent and the distance in the target quadrant in MWM test (P < 0.001 and P < 0.01). Treatment with 7NI attenuated all adverse effects of PTU (P < 0.05 to P < 0.001). PTU lowered BDNF and thiol content and superoxide dismutase (SOD) and catalase (CAT) activities in the brain but increased malondialdehyde (MDA) and nitric oxide (NO) metabolites. In addition, 7NI improved thiol, SOD, CAT, thiol, and BDNF but attenuated MDA and NO metabolites. The results of the current study showed that 7NI improvement in the learning and memory of the hypothyroid juvenile rats, which was accompanied with improving of BDNF and attenuation of NO and brain tissues oxidative damage.

Inhibitory role of kinins on microglial nitric oxide and tumor necrosis factor-α production

Brain inflammation is sustained by chronic activation of microglia and the over-production of pro-inflammatory cytokines and nitric oxide (NO), which in turn can be highly neurotoxic. Microglial activation can be regulated by neuropeptides such as bradykinin (BK) and other members of the kinin family. Kinins are well known inflammatory regulators outside the CNS. Although the kinin system is well distributed throughout the brain, the precise role of BK in the CNS is not yet clear. The aim of this study was to examine and characterize the effects of BK and related kinins on the production of NO and TNF-α in microglia. We found that BK and selective agonists for both B1 and B2 receptors, attenuated both NO and TNF-α levels in the media of BV2 microglial cells that had been stimulated with LPS. The effects of BK that were observed in BV2 cells were confirmed in primary neonatal rat microglial cells as well. In addition, all kinin agonists reduced the expression of iNOS and TNF-α protein and mRNA levels in LPS-stimulated BV2 cells. Also, while LPS activated the nuclear factor-κB (NF-κB) pathway, BK inhibited NF-κB activation by preventing degradation of the κB protein (IκB) inhibitor, abolishing translocation of p65 and p50 subunits to the nucleus and inhibiting NF-κB transcription activity. These results suggest a role for bradykinin in modulation of glial inflammation, as evidenced by attenuation of NO and TNF-α synthesis pathways in activated microglial cells.

Attenuation of host NO production by MAMPs potentiates development of the host in the squid-vibrio symbiosis

Bacterial pathogens typically upregulate the host's production of nitric oxide synthase (NOS) and nitric oxide (NO) as antimicrobial agents, a response that is often mediated by microbe-associated molecular patterns (MAMPs) of the pathogen. In contrast, previous studies of the beneficial Euprymna scolopes/Vibrio fischeri symbiosis demonstrated that symbiont colonization results in attenuation of host NOS/NO, which occurs in high levels in hatchling light organs. Here, we sought to determine whether V. fischeri MAMPs, specifically lipopolysaccharide (LPS) and the peptidoglycan derivative tracheal cytotoxin (TCT), attenuate NOS/NO, and whether this activity mediates the MAMPs-induced light organ morphogenesis. Using confocal microscopy, we characterized levels of NOS with immunocytochemistry and NO with a NO-specific fluorochrome. When added exogenously to seawater containing hatchling animals, V. fischeri LPS and TCT together, but not individually, induced normal NOS/NO attenuation. Further, V. fischeri mutants defective in TCT release did not. Experiments with NOS inhibitors and NO donors provided evidence that NO mediates apoptosis and morphogenesis associated with symbiont colonization. Attenuation of NOS/NO by LPS and TCT in the squid-vibrio symbiosis provides another example of how the host's response to MAMPs depends on the context. These data also provide a mechanism by which symbiont MAMPs regulate host development.

Exposure to lanthanum compound diminishes LPS-induced inflammation-associated gene expression: involvements of PKC and NF-κB signaling pathways

Lanthanum chloride, a rare earth compound, possesses antibacterial and cellular immunity regulating properties. However, the underlying molecular mechanisms remain largely unknown. In this study, we examined the effects of lanthanum chloride on the production of nitric oxide (NO) and tumor necrosis factor-alpha (TNF-alpha), the expression of inducible NO synthase (iNOS) and TNF-alpha in RAW 264.7 cells, a mouse macrophage cell line. We found that the LPS-elicited excessive production of NO and TNF-alpha in RAW 264.7 cells was inhibited significantly in the presence of lanthanum chloride, and the attenuation of iNOS and TNF-alpha occurred at mRNA level. Furthermore, the possible signaling components affected by lanthanum chloride in the pathway that lead to LPS-induced iNOS and TNF-alpha expression were explored. The results indicated the involvements of PKC/Ca(2+) and NF-kappaB in the attenuation of NO and pro-inflammatory cytokine production by lanthanum chloride. Our observations suggest a possible therapeutic application of this agent for treating inflammatory diseases.

Responses of Nitric Oxide–cGMP Release in Acupuncture Point to Electroacupuncture in Human Skin In Vivo Using Dermal Microdialysis

The aim of this study was to examine the release of nitric oxide (NO) and cGMP in response to electroacupuncture (EA) stimulation in the acupuncture point (acupoint), compared to the non-meridian control area. Thirty samples of dermal microdialysis data were collected from 24 volunteers at pericardium (PC) 4 and control area. EA was applied to PC 3 by using a 5-V pulse with a duration of 1.0 milliseconds at 10 Hz for 15 minutes. Dialysate samples were continuously collected 20 minutes each before, during, and after EA for two hours. Total nitrite and nitrate (NO(x)(-)) and cGMP in the dialysate were quantified in a blinded fashion. Dialysate NO(x)(-) concentrations were decreased during a 120-minute dialysis in all groups, but reduced NO(x)(-) levels were attenuated predominantly in PC 4 acupoint at 20-40 minutes after EA PC 3. cGMP concentrations were significantly enhanced in acupoint PC 4 by EA PC 3, but not in the non-meridian area. We suggest that the attenuation of NO(x)(-) reduction during dialysis reflects an increase in NO release induced by EA stimulation in acupoint and that cGMP mediates the signaling functions of NO to improve local microcirculation, which, at least in part, contributes to the effects of acupuncture.

Expression of iNOS gene in macrophages stimulated with 17beta-estradiol is regulated by free intracellular Ca2+.

17Beta-estradiol has potent Ca2+ ionophore capability and its signaling in macrophages is mediated through binding to surface and genomic receptors, resulting in transient nitric oxide (NO) elaboration. We decided to examine if the transient release of NO is due to Ca2+ influx pattern or the quenching effect of superoxide (*O2-) through peroxynitrite formation. Differential chelation of intracellular Ca2+ ([Ca2+]i) showed that NO generation was favored by [Ca2+]i concentration of 237 nM. Application of an estrogen receptor antagonist ICI 182 780 resulted in attenuation of estradiol mediated NO release. Studies directed at identifying the possible role of *O2-; in the attenuation of NO showed no supportive evidence. Inhibition of extracellular Ca2+ channel or extracellular and intracellular Ca2+ channels showed data consistent with a case for optimum Ca2+ influx signal favoring iNOS gene expression, accompanied by an elevation in iNOS protein. These data show that Ca2+ influx pattern determines macrophage NO elaboration.

Central integration of muscle reflex and arterial baroreflex in midbrain periaqueductal gray: roles of GABA and NO.

It has been suggested that the midbrain periaqueductal gray (PAG) is a neural integrating site for the interaction between the muscle pressor reflex and the arterial baroreceptor reflex. The underlying mechanisms are poorly understood. The purpose of this study was to examine the roles of GABA and nitric oxide (NO) in modulating the PAG integration of both reflexes. To activate muscle afferents, static contraction of the triceps surae muscle was evoked by electrical stimulation of the L7 and S1 ventral roots of 18 anesthetized cats. In the first group of experiments (n = 6), the pressor response to muscle contraction was attenuated by bilateral microinjection of muscimol (a GABA receptor agonist) into the lateral PAG [change in mean arterial pressure (DeltaMAP) = 24 +/- 5 vs. 46 +/- 8 mmHg in control]. Conversely, the pressor response was significantly augmented by 0.1 mM bicuculline, a GABAA receptor antagonist (DeltaMAP = 65 +/- 10 mmHg). In addition, the effect of GABAA receptor blockade on the reflex response was significantly blunted after sinoaortic denervation and vagotomy (n = 4). In the second group of experiments (n = 8), the pressor response to contraction was significantly attenuated by microinjection of L-arginine into the lateral PAG (DeltaMAP = 26 +/- 4 mmHg after L-arginine injection vs. 45 +/- 7 mmHg in control). The effect of NO attenuation was antagonized by bicuculline and was reduced after denervation. These data demonstrate that GABA and NO within the PAG modulate the pressor response to muscle contraction and that NO attenuation of the muscle pressor reflex is mediated via arterial baroreflex-engaged GABA increase. The results suggest that the PAG plays an important role in modulating cardiovascular responses when muscle afferents are activated.

Effect of Amorphophallus Konjac oligosaccharides on STZ-induced diabetes model of isolated islets

Three oligosaccharide fractions from the root of Amorphophallus Konjac, which was reported with hypoglycemic effects on diabetes subjects, were isolated and studied using the STZ-treated diabetes model. Among them, one fraction named as KOS-A, was found with nitric oxide (NO ) free radical regulation effect, while the other two were not. At concentrations less than 1.5 mM, KOS-A positively decreased STZ-induced NO level of islets, but normal NO release for non-STZ-treated islets was not affected within the range. At 15 mM, KOS-A played a contrary role and increased NO level for islets both with and without STZ-treatment. Islets insulin secretion changed corresponding to NO level in the assay. Increased insulin secretion appeared parallel to the decrease of NO , and normal insulin release was not affected by KOS-A less than 1.5 mM. Structure determination of KOS-A shows that it is a tetrasaccharide with Mw of 666 Da and reductive end of α-D-mannose. These results indicate that low dosage of KOS-A, with its function on attenuating STZ-induced NO level, doesn't alter normal NO and insulin secretion pathways of isolated islets. The NO attenuation function of KOS-A on the diabetes model is mainly resulted from environmental free radical scavenging by the oligosaccharide. Present results also imply the mechanism of clinical Amorphophallus Konjac hypoglycemic function maybe related with free radical attenuation and lower risks of islets damage from NO radical.

Inhibition of nitric oxide synthesis by L-name exacerbates acute lung injury induced by hepatic ischemia-reperfusion.

Hepatic Kupffer cells and pulmonary alveolar macrophages together constitute a macrophage-axis involved in the regulation of regional and systemic inflammatory responses. Systemic inflammatory response syndrome induced by overproduced pro-inflammatory mediators is the major cause of adult respiratory distress syndrome. In the present study, we examined the anti-inflammatory role of nitric oxide (NO) in a rat model of acute lung injury induced by hepatic ischemia-reperfusion (HI/R). The left and median lobes of the liver were subjected to 30 min of ischemia by clamping the relevant branches of hepatic artery and portal vein, followed by a 4-h reperfusion achieved by removal of the vascular clamp. Four groups of animals were studied: sham control + saline; sham control + N(omega)-nitro-L-arginine methyl ester (L-NAME, 10 mg/kg, i.v., 10 min before reperfusion); HI/R + saline; HI/R + L-NAME. Results show that (1) administration of L-NAME to rats subjected to HI/R decreased plasma NO levels; however, the attenuation of NO increased plasma alanine aminotransferase (ALT) activity and superoxide generation in the ischemic lobes of liver, compared to HI/R alone. (2) Inhibition of NO synthesis with L-NAME in rats subjected to HI/R also enhanced systemic inflammatory response as assessed by the increase in the number of circulating leukocytes and levels of plasma tumor necrosis factor-alpha (TNFalpha) and interleukin 1-beta (IL-1beta). (3) The overwhelming systemic inflammatory response induced by administration of L-NAME in rats subjected to HI/R also augmented pulmonary vascular permeability and superoxide generation in the lung tissue. (4) Pulmonary alveolar macrophages isolated from rats subjected to HI/R + L-NAME produced higher levels of TNFalpha and IL-1beta in the supernatant of culture medium than that of rats subjected to HI/R alone. (5) There were no differences between the groups of sham + saline and sham + L-NAME in terms of plasma NO levels and ALT activity, circulating leukocytes, superoxide generation in the liver and lung, lavage protein levels, and TNFalpha and IL-1beta levels in plasma and bronchoalveolar lavage fluid. Our results suggest that inhibition of NO synthesis by L-NAME in rats subjected to HI/R not only augments ischemic liver injury, but also enhances the systemic inflammatory response and exacerbates remote lung injury. The increase in TNFalpha and IL-1beta production by alveolar macrophages may, in part, account for L-NAME-induced enhancement of acute lung injury.