Regulation Of Nitric Oxide Production Research Articles

Advancements in the Research of New Modulators of Nitric Oxide Synthases Activity.

Nitric oxide (NO) has been defined as the "miracle molecule" due to its essential pleiotropic role in living systems. Besides its implications in physiologic functions, it is also involved in the development of several disease states, and understanding this ambivalence is crucial for medicinal chemists to develop therapeutic strategies that regulate NO production without compromising its beneficial functions in cell physiology. Although nitric oxide synthase (NOS), i.e., the enzyme deputed to the NO biosynthesis, is a well-recognized druggable target to regulate NO bioavailability, some issues have emerged during the past decades, limiting the progress of NOS modulators in clinical trials. In the present review, we discuss the most promising advancements in the research of small molecules that are able to regulate NOS activity with improved pharmacodynamic and pharmacokinetic profiles, providing an updated framework of this research field that could be useful for the design and development of new NOS modulators.

Comparative Anti-Cancer and Anti-Inflammatory Activities of Essential Oils from the Bark and Flower of Magnolia officinalis Rehd. et Wils.

This study was designed to compare the antioxidant, antitumor and anti-inflammatory effects of essential oils from the bark and flower of Magnolia officinalis Rehd. et Wils. Distillation extraction and steam distillation were used to extract EOs from the bark and flower. The results showed that the contents of EOs of SDE-F and SDE-B were much higher than that of SD-F and SD-B. EOs from the bark were rich in eudesmol (especially α-eudesmol) and exhibited a stronger antioxidant effect than the flower. The anti-tumor effects of SD-B and SD-F on HepG2 and MDA-MB-231 cells were better than that of SDE-B and SDE-F. The inhibitory rates of SD-B and SD-F on MDA-MB-231 cells were 59.21% and 48.27%, exceeding that of positive control 5-fluorouracil (47.04%) at 50 μg/mL. All four EOs exhibited excellent anti-inflammatory activities through the regulation of nitric oxide production and pro-inflammation cytokines in LPS-induced RAW 264.7 cells and they also remarkably suppressed the mRNA expressions of nitric oxide synthase, IL-6 and TNF-α at the concentration higher than that of positive control dexamethasone. These results indicated significant differences in the composition, and anti-inflammatory and anti-tumor activities of EOs extracted by different methods and provided a theoretical basis for their development and utilization.

Metabolites of Culinary-medicinal Mushroom Hericium erinaceus Showed Anti-neuroinflammatory Activity in BV2 Cells and Anti-H2O2-induced Oxidative Stress Activity in SH-SY5Y Cells

Background: Hericium erinaceus is a versatile mushroom with potential therapeutic benefits. A variety of bioactive compounds have been found in this mushroom that have anti-ulcer, antiinflammatory, immunomodulatory, and blood sugar and lipid regulating effects. Objectives: The aim is to isolate and characterize compounds from the rice medium of H. erinaceus and evaluate their potential therapeutic effects on oxidative stress-associated diseases. Methods: Fifteen compounds were isolated and characterized using NMR spectra and ESI-MS. The effects of compounds 3 and 7 on LPS-induced nitric oxide (NO) generation in BV2 cells and H2O2- induced cytotoxicity in SH-SY5Y cells were investigated. The regulation of nitric oxide production and apoptosis-associated proteins Bcl-2 and Bax were also examined. Results: Compounds 3 and 7 exhibited potent inhibitory effects on LPS-induced NO generation in microglial cells and significant resistance to H2O2-induced cytotoxicity in neuronal cells. Molecular docking simulations demonstrated the interaction of compounds 3 and 7 with iNOS and Bax, respectively. Western blot analysis revealed that 7 inhibited NO production and regulated the expression of Bax and Bcl-2, suggesting a protective effect against H2O2-induced apoptosis. Conclusion: Compounds 3 and 7 isolated from H. erinaceus are potential therapeutic candidates for oxidative stress-associated diseases, possibly through their regulation of nitric oxide production and apoptosis-related proteins. Further investigations are warranted to elucidate their mechanisms of action and therapeutic potential.

Hypoxia Dysregulates the Transcription of Myoendothelial Junction Proteins Involved with Nitric Oxide Production in Brain Endothelial Cells.

Myoendothelial junctions (MEJs) are structures that allow chemical signals to be transmitted between endothelial cells (ECs) and vascular smooth muscle cells, which control vascular tone. MEJs contain hemoglobin alpha (Hbα) and endothelial nitric oxide synthase (eNOS) complexes that appear to control the production and scavenging of nitric oxide (NO) along with the activity of cytochrome b5 reductase 3 (CYB5R3). The aim of this study was to examine how hypoxia affected the regulation of proteins involved in the production of NO in brain ECs. In brief, human brain microvascular endothelial cells (HBMEC) were exposed to cobalt chloride (CoCl2), a hypoxia mimetic, and a transcriptional analysis was performed using primers for eNOS, CYB5R3, and Hbα2 with ΔΔCt relative gene expression normalized to GAPDH. NO production was also measured after treatment using 4,5-diaminofluorescein diacetate (DAF-DA), a fluorescent NO indicator. When HBMEC were exposed to CoCl2 for 48 h, eNOS and CYB5R3 messenger RNA significantly decreased (up to -17.8 ± 4.30-fold and -10.4 ± 2.8, respectively) while Hbα2 increased to detectable levels. Furthermore, CoCl2 treatment caused a redistribution of peripheral membrane-generated NO production to a perinuclear region. To the best of our knowledge, this is the first time this axis has been studied in brain ECs and these findings imply that hypoxia may cause dysregulation of proteins that regulate NO production in brain MEJs.

LncRNA SNHG8 regulates the migration and angiogenesis of pHUVECs induced by high glucose via the TRPM7/ERK1/2 signaling axis

This study aimed to evaluate the regulatory effect and molecular mechanism of long noncoding RNA small nucleolus RNA host gene 8 (LncRNA SNHG8) in the migration and angiogenesis of primary human umbilical vein endothelial cells (pHUVECs) under high-glucose (HG) conditions. The HG-induced endothelial injury model was established in vitro.The cell model of silencing SNHG8, overexpressing SNHG8, and silencing TRPM7 was established by transfecting SNHG8-siRNA, SNHG8 plasmid and TRPM7-siRNA into cells with liposomes.The SNHG8 level was determined through reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The expression levels of transient receptor potential melastatin 7 (TRPM7), endothelial nitric oxide synthase (eNOS), p-eNOS, extracellular signal-regulated kinase 1/2(ERK1/2), and p-ERK1/2 were assessed through western blot. Nitric oxide (NO) levels were measured with DAF-FM. pHUVEC migration was examined through wound healing and Transwell assay, and pHUVEC angiogenesis was observed through a tube formation assay. Results showed that HG promoted the expression of lncRNA SNHG8 and TRPM7 and decreased the ratio of p-eNOS/eNOS and p-ERK1/2/ERK1/2 in pHUVECs . NO production, migration , and angiogenesis were inhibited in pHUVECs under HG conditions. Silencing lncRNA SNHG8 and TRPM7 could significantly reverse the HG-induced decrease in eNOS activation, NO production , migration, and angiogenesis . SNHG8 and U0126 (ERK pathway inhibitor) overexpression enhanced the HG effects, whereas using U0126 did not affect the TRPM7 expression. In conclusion, lncRNA SNHG8 participates in HG-induced endothelial cell injury and likely regulates NO production, migration, and angiogenesis of pHUVECs via the TRPM7/ERK1/2 signaling axis.

PLC1 mediated Cycloastragenol-induced stomatal movement by regulating the production of NO in Arabidopsis thaliana

BackgroundAstragalus grows mainly in drought areas. Cycloastragenol (CAG) is a tetracyclic triterpenoid allelochemical extracted from traditional Chinese medicine Astragalus root. Phospholipase C (PLC) and Gα-submit of the heterotrimeric G-protein (GPA1) are involved in many biotic or abiotic stresses. Nitric oxide (NO) is a crucial gas signal molecule in plants.ResultsIn this study, using the seedlings of Arabidopsis thaliana (A. thaliana), the results showed that low concentrations of CAG induced stomatal closure, and high concentrations inhibited stomatal closure. 30 µmol·L−1 CAG significantly increased the relative expression levels of PLC1 and GPA1 and the activities of PLC and GTP hydrolysis. The stomatal aperture of plc1, gpa1, and plc1/gpa1 was higher than that of WT under CAG treatment. CAG increased the fluorescence intensity of NO in guard cells. Exogenous application of c-PTIO to WT significantly induced stomatal aperture under CAG treatment. CAG significantly increased the relative expression levels of NIA1 and NOA1. Mutants of noa1, nia1, and nia2 showed that NO production was mainly from NOA1 and NIA1 by CAG treatment. The fluorescence intensity of NO in guard cells of plc1, gpa1, and plc1/gpa1 was lower than WT, indicating that PLC1 and GPA1 were involved in the NO production in guard cells. There was no significant difference in the gene expression of PLC1 in WT, nia1, and noa1 under CAG treatment. The gene expression levels of NIA1 and NOA1 in plc1, gpa1, and plc1/gpa1 were significantly lower than WT, indicating that PLC1 and GPA1 were positively regulating NO production by regulating the expression of NIA1 and NOA1 under CAG treatment.ConclusionsThese results suggested that the NO accumulation was essential to induce stomatal closure under CAG treatment, and GPA1 and PLC1 acted upstream of NO.

Nitric oxide is required for lung alveolarization revealed by deficiency of argininosuccinate lyase.

Inhaled nitric oxide (NO) therapy has been reported to improve lung growth in premature newborns. However, the underlying mechanisms by which NO regulates lung development remain largely unclear. NO is enzymatically produced by three isoforms of nitric oxide synthase (NOS) enzymes. NOS knockout mice are useful tools to investigate NO function in the lung. Each single NOS knockout mouse does not show obvious lung alveolar phenotype, likely due to compensatory mechanisms. While mice lacking all three NOS isoforms display impaired lung alveolarization, implicating NO plays a pivotal role in lung alveolarization. Argininosuccinate lyase (ASL) is the only mammalian enzyme capable of synthesizing L-arginine, the sole precursor for NOS-dependent NO synthesis. ASL is also required for channeling extracellular L-arginine into a NO-synthetic complex. Thus, ASL deficiency (ASLD) is a non-redundant model for cell-autonomous, NOS-dependent NO deficiency. Here, we assessed lung alveolarization in ASL-deficient mice. Hypomorphic deletion of Asl (AslNeo/Neo) results in decreased lung alveolarization, accompanied with reduced level of S-nitrosylation in the lung. Genetic ablation of one copy of Caveolin-1, which is a negative regulator of NO production, restores total S-nitrosylation as well as lung alveolarization in AslNeo/Neo mice. Importantly, NO supplementation could partially rescue lung alveolarization in AslNeo/Neo mice. Furthermore, endothelial-specific knockout mice (VE-Cadherin Cre; Aslflox/flox) exhibit impaired lung alveolarization at 12weeks old, supporting an essential role of endothelial-derived NO in the enhancement of lung alveolarization. Thus, we propose that ASLD is a model to study NO-mediated lung alveolarization.

Paricalcitol Improves the Angiopoietin/Tie-2 and VEGF/VEGFR2 Signaling Pathways in Adriamycin-Induced Nephropathy.

Renal endothelial cell (EC) injury and microvascular dysfunction contribute to chronic kidney disease (CKD). In recent years, increasing evidence has suggested that EC undergoes an endothelial-to-mesenchymal transition (EndoMT), which might promote fibrosis. Adriamycin (ADR) induces glomerular endothelial dysfunction, which leads to progressive proteinuria in rodents. The activation of the vitamin D receptor (VDR) plays a crucial role in endothelial function modulation, cell differentiation, and suppression of the expression of fibrotic markers by regulating the production of nitric oxide (NO) by activating the endothelial NO synthase (eNOS) in the kidneys. This study aimed to evaluate the effect of paricalcitol treatment on renal endothelial toxicity in a model of CKD induced by ADR in rats and explore mechanisms involved in EC maintenance by eNOS/NO, angiopoietins (Angs)/endothelium cell-specific receptor tyrosine kinase (Tie-2, also known as TEK) and vascular endothelial growth factor (VEGF)-VEGF receptor 2 (VEGFR2) axis. The results show that paricalcitol attenuated the renal damage ADR-induced with antiproteinuric effects, glomerular and tubular structure, and function protection. Furthermore, activation of the VDR promoted the maintenance of the function and structure of glomerular, cortical, and external medullary endothelial cells by regulating NO production. In addition, it suppressed the expression of the mesenchymal markers in renal tissue through attenuation of (transforming growth factor-beta) TGF-β1/Smad2/3-dependent and downregulated of Ang-2/Tie-2 axis. It regulated the VEGF/VEGFR2 pathway, which was ADR-deregulated. These effects were associated with lower AT1 expression and VDR recovery to renal tissue after paricalcitol treatment. Our results showed a protective role of paricalcitol in the renal microvasculature that could be used as a target for treating the beginning of CKD.

PDZK1 upregulates nitric oxide production through the PI3K/ERK2 pathway to inhibit porcine circovirus type 2 replication

Porcine circovirus type 2 (PCV2) is the causative agent of porcine circovirus-associated disease. Changes in host cell gene expression are induced by PCV2 infection. Here, we showed that porcine PDZ Domain-Containing 1 (PDZK1) expression was enhanced during PCV2 infection and that overexpression of PDZK1 inhibited the expression of PCV2 Cap protein. PCV2 genomic DNA copy number and viral titers were decreased in PDZK1-overexpressing PK-15B6 cells. PDZK1 knockdown enhanced the replication of PCV2. Overexpression of PDZK1 activated the phosphoinositide 3-kinase (PI3K)/ERK2 signaling pathway to enhance nitric oxide (NO) levels, while PDZK1 knockdown had the opposite effects. A PI3K inhibitor (LY294002) and a NO synthase inhibitor (L-NAME hydrochloride) decreased the activity of PDZK1 in restricting PCV2 replication. ERK2 knockdown enhanced the proliferation of PCV2 by decreasing levels of NO. Levels of interleukin (IL)− 4 mRNA were reduced in PDZK1 knockdown and ERK2 knockdown PK-15B6 cells. Increased IL-4 mRNA levels were unable to decrease NO production in PDZK1-overexpressing cells. Thus, we conclude that PDZK1 affected PCV2 replication by regulating NO production via PI3K/ERK2 signaling. PDZK1 affected IL-4 expression through the PI3K/ERK2 pathway, but PDZK1 modulation of PCV2 replication occurred independently of IL-4. Our results contribute to understanding the biological functions of PDZK1 and provide a theoretical basis for the pathogenic mechanisms of PCV2.

Effects of arginase inhibition on myocardial Ca2+ and contractile responses.

Nitric oxide (NO) is thought to increase cardiac contractility by increasing cytosolic Ca2+ concentration ([Ca2+]cyt) during excitation. Alternatively, NO could increase the sensitivity of the contractile response to [Ca2+]cyt (Ca2+ sensitivity). Arginase regulates NO production by competing with NO synthase (NOS), and thus, arginase inhibition should increase cardiac contractility by increasing NO production. We hypothesized that arginase inhibition increases cardiac contractility by increasing both [Ca2+]cyt and Ca2+ sensitivity. [Ca2+]cyt and contractile (sarcomere length [SL] shortening) responses to electrical stimulation were measured simultaneously in isolated rat cardiomyocytes using an IonOptix system. In the same cardiomyocytes, measurements were obtained at baseline, following 3‐min exposure to an arginase inhibitor (S‐[2‐boronoethyl]‐l‐cysteine; BEC) and following 3‐min exposure to BEC plus a NOS inhibitor (N G‐nitro‐l‐arginine‐methyl ester; l‐NAME). These responses were compared to time‐matched control cardiomyocytes that were untreated. Compared to baseline, BEC increased the amplitude and the total amount of evoked [Ca2+]cyt, and the extent and velocity of SL shortening in cardiomyocytes, whereas addition of l‐NAME mitigated these effects. The [Ca2+]cyt at 50% contraction and relaxation were not different across treatment groups indicating no effect of BEC on Ca2+ sensitivity. The [Ca2+]cyt and SL shortening responses in time‐matched controls did not vary with time. Arginase inhibition by BEC significantly increased the amplitude and the total amount of evoked [Ca2+]cyt, and the extent and velocity of SL shortening in cardiomyocytes, but did not affect Ca2+ sensitivity. These effects of BEC were mitigated by l‐NAME. Together, these results indicate an effect of NO on [Ca2+]cyt responses that then increase the contractile response of cardiomyocytes.

Inhibitory Effect of FMRFamide on NO Production During Immune Defense in Sepiella japonica.

Neuropeptide Phe-Met-Arg-Phe-NH2 (FMRFamide), specifically existing in invertebrates, plays pivotal roles in various physiological processes. The involvement in neuroendocrine-immune regulation was explored in recent years, and it could modulate nitric oxide (NO) production under immune stress. However, detailed knowledge is still little known. In this study, we identified FMRFamide as an inhibitory factor on NO production in the immune reaction of Sepiella japonica. Firstly, Vibrio harveyi incubation caused significantly upregulated expression of FMRFamide precursor and NO synthase (NOS) in just hatched cuttlefish with quantitative Real-time PCR (qRT-PCR), which indicated that both were likely to be involved in the immune defense. The whole-mount in situ hybridization (ISH) detected FMRFamide precursor and NOS-positive signals appeared colocalization, suggesting that at histological and anatomical levels FMRFamide might interact with NOS. Next, NOS mRNA was highly significantly upregulated at 72 h when FMRFamide precursor mRNA was knocked down effectively with the RNA interference (RNAi) method; the results hinted that FMRFamide was likely to regulate NO production. Continuously, the inflammatory model was constructed in RAW 264.7 cells induced by lipopolysaccharide (LPS), FMRFamide administration resulted in a highly significant reduction of the NO level in dose- and time-response manners. Although the addition of the selected inducible NOS (iNOS) inhibitor had inhibited the NO production induced by LPS, the additional FMRFamide could still furtherly sharpen the process. Collectively, it was concluded that neuropeptide FMRFamide could indeed inhibit NO production to serve as feedback regulation at the late stage of immune response to protect hosts from excessive immune cytotoxicity. The inhibitory effect on NO production could not only be mediated by the NOS pathway but also be implemented through other pathways that needed to be furtherly explored. The results will provide data for comparing the structure and immune function of neuroendocrine-immune system (NEIS) between “advanced” cephalopods and other invertebrates and will provide new information for understanding the NEIS of cephalopods.

HSP90 Modulates T2R Bitter Taste Receptor Nitric Oxide Production and Innate Immune Responses in Human Airway Epithelial Cells and Macrophages.

Bitter taste receptors (T2Rs) are G protein-coupled receptors (GPCRs) expressed in various cell types including ciliated airway epithelial cells and macrophages. T2Rs in these two innate immune cell types are activated by bitter products, including those secreted by Pseudomonas aeruginosa, leading to Ca2+-dependent activation of endothelial nitric oxide (NO) synthase (eNOS). NO enhances mucociliary clearance and has direct antibacterial effects in ciliated epithelial cells. NO also increases phagocytosis by macrophages. Using biochemistry and live-cell imaging, we explored the role of heat shock protein 90 (HSP90) in regulating T2R-dependent NO pathways in primary sinonasal epithelial cells, primary monocyte-derived macrophages, and a human bronchiolar cell line (H441). Immunofluorescence showed that H441 cells express eNOS and T2Rs and that the bitter agonist denatonium benzoate activates NO production in a Ca2+- and HSP90-dependent manner in cells grown either as submerged cultures or at the air–liquid interface. In primary sinonasal epithelial cells, we determined that HSP90 inhibition reduces T2R-stimulated NO production and ciliary beating, which likely limits pathogen clearance. In primary monocyte-derived macrophages, we found that HSP-90 is integral to T2R-stimulated NO production and phagocytosis of FITC-labeled Escherichia coli and pHrodo-Staphylococcus aureus. Our study demonstrates that HSP90 serves as an innate immune modulator by regulating NO production downstream of T2R signaling by augmenting eNOS activation without impairing upstream Ca2+ signaling. These findings suggest that HSP90 plays an important role in airway antibacterial innate immunity and may be an important target in airway diseases such as chronic rhinosinusitis, asthma, or cystic fibrosis.

Deleterious effect of bone marrow-resident macrophages on hematopoietic stem cells in response to total body irradiation

AbstractBone marrow (BM) resident macrophages interact with a population of long-term hematopoietic stem cells (LT-HSCs) but their role on LT-HSC properties after stress is not well defined. Here, we show that a 2 Gy-total body irradiation (TBI)-mediated death of LT-HSCs is associated with increased percentages of LT-HSCs with reactive oxygen species (ROS) and of BM resident macrophages producing nitric oxide (NO), resulting in an increased percentage of LT-HSCs with endogenous cytotoxic peroxynitrites. Pharmacological or genetic depletion of BM resident macrophages impairs the radio-induced increases in the percentage of both ROS+ LT-HSCs and peroxynitrite+ LT-HSCs and results in a complete recovery of a functional pool of LT-HSCs. Finally, we show that after a 2 Gy-TBI, a specific decrease of NO production by BM resident macrophages improves the LT-HSC recovery, whereas an exogenous NO delivery decreases the LT-HSC compartment. Altogether, these results show that BM resident macrophages are involved in the response of LT-HSCs to a 2 Gy-TBI and suggest that regulation of NO production can be used to modulate some deleterious effects of a TBI on LT-HSCs.

Divergent Genetic Regulation of Nitric Oxide Production between C57BL/6J and Wild-Derived PWD/PhJ Mice Controls Postactivation Mitochondrial Metabolism, Cell Survival, and Bacterial Resistance in Dendritic Cells.

Dendritic cell (DC) activation is characterized by sustained commitment to glycolysis that is a requirement for survival in DC subsets that express inducible NO synthase (Nos2) due to NO-mediated inhibition of mitochondrial respiration. This phenomenon primarily has been studied in DCs from the classic laboratory inbred mouse strain C57BL/6J (B6) mice, where DCs experience a loss of mitochondrial function due to NO accumulation. To assess the conservation of NO-driven metabolic regulation in DCs, we compared B6 mice to the wild-derived genetically divergent PWD/PhJ (PWD) strain. We show preserved mitochondrial respiration and enhanced postactivation survival due to attenuated NO production in LPS-stimulated PWD DCs phenocopying human monocyte-derived DCs. To genetically map this phenotype, we used a congenic mouse strain (B6.PWD-Chr11.2) that carries a PWD-derived portion of chromosome 11, including Nos2, on a B6 background. B6.PWD-Chr11.2 DCs show preserved mitochondrial function and produce lower NO levels than B6 DCs. We demonstrate that activated B6.PWD-Chr11.2 DCs maintain mitochondrial respiration and TCA cycle carbon flux, compared with B6 DCs. However, reduced NO production by the PWD Nos2 allele results in impaired cellular control of Listeria monocytogenes replication. These studies establish a natural genetic model for restrained endogenous NO production to investigate the contribution of NO in regulating the interplay between DC metabolism and immune function. These findings suggest that reported differences between human and murine DCs may be an artifact of the limited genetic diversity of the mouse models used, underscoring the need for mouse genetic diversity in immunology research.

Sirtuin 7 Regulates Nitric Oxide Production and Apoptosis to Promote Mycobacterial Clearance in Macrophages.

The host immune system plays a pivotal role in the containment of Mycobacterium tuberculosis (Mtb) infection, and host-directed therapy (HDT) is emerging as an effective strategy to treat tuberculosis (TB), especially drug-resistant TB. Previous studies revealed that expression of sirtuin 7 (SIRT7), a nicotinamide adenine dinucleotide (NAD+)-dependent deacetylase, was downregulated in macrophages after Mycobacterial infection. Inhibition of SIRT7 with the pan-sirtuin family inhibitor nicotinamide (NAM), or by silencing SIRT7 expression, promoted intracellular growth of Mtb and restricted the generation of nitric oxide (NO). Addition of the exogenous NO donor SNAP abrogated the increased bacterial burden in NAM-treated or SIRT7-silenced macrophages. Furthermore, SIRT7-silenced macrophages displayed a lower frequency of early apoptotic cells after Mycobacterial infection, and this could be reversed by providing exogenous NO. Overall, this study clarified a SIRT7-mediated protective mechanism against Mycobacterial infection through regulation of NO production and apoptosis. SIRT7 therefore has potential to be exploited as a novel effective target for HDT of TB.

Glycyrrhizin as a Nitric Oxide Regulator in Cancer Chemotherapy.

Simple SummaryGlycyrrhizin (GL) has anti-cancer, anti-inflammatory, anti-viral, and anti-oxidant activity. In particular, GL reduces multidrug resistance (MDR) in cancer cells, which is a major obstacle to chemotherapy. Nitric oxide (NO) also plays an important role in MDR, and GL affects NO concentration in the tumor microenvironment. However, the effects of GL and NO interaction on MDR have not been reviewed. Here, we review the role of GL as an NO regulator in cancer cells and its subsequent anti-MDR effect and posit that GL is a promising MDR inhibitor for cancer chemotherapy.Chemotherapy is used widely for cancer treatment; however, the evolution of multidrug resistance (MDR) in many patients limits the therapeutic benefits of chemotherapy. It is important to overcome MDR for enhanced chemotherapy. ATP-dependent efflux of drugs out of cells is the main mechanism of MDR. Recent studies have suggested that nitric oxide (NO) can be used to overcome MDR by inhibiting the ATPase function of ATP-dependent pumps. Several attempts have been made to deliver NO to the tumor microenvironment (TME), however there are limitations in delivery. Glycyrrhizin (GL), an active compound of licorice, has been reported to both reduce the MDR effect by inhibiting ATP-dependent pumps and function as a regulator of NO production in the TME. In this review, we describe the potential role of GL as an NO regulator and MDR inhibitor that efficiently reduces the MDR effect in cancer chemotherapy.

Effects of the Extract of Four Herbs (Ginger, Cinnamon, Jujube and Chive) on Regulation of Nitric Oxide Production

Effects of the Extract of Four Herbs (Ginger, Cinnamon, Jujube and Chive) on Regulation of Nitric Oxide Production

Regulation of dimethylarginine dimethylaminohydrolase 2 expression by NF-κB acetylation.

Nitric oxide (NO) serves a crucial role in the kidney and is synthesized by NO synthase (NOS). Asymmetrical dimethylarginine is an endogenous inhibitor of NOS that is metabolized by dimethylarginine dimethylaminohydrolase (DDAH). To investigate the role of acetylation in DDAH2 expression, 293 cells were treated with trichostatin A (TSA), a deacetylase inhibitor and the mRNA and protein levels were assessed using quantitative PCR and western blotting respectively. Its promoter activity was detected using a luciferase assay. The effect of TSA on NF-κB acetylation was tested after immunoprecipitation. The binding of NF-κB to the DDAH2 promoter was analyzed using an electrophoretic mobility shift assay and chromatin immunoprecipitation. TSA upregulated DDAH2 expression and transcriptional activity of the DDAH2 promoter through a NF-κB responsive element, which is located at the -1582 to -1573 position of the DDAH2 promoter. Furthermore, TSA treatment promoted NF-κB acetylation, resulting in enhanced NF-κB binding affinity to its binding site both in vitro and in vivo. Taken together, the present study demonstrated that NF-κB acetylation upregulated DDAH2 expression by enhancing the binding ability of NF-κB to the DDAH2 promoter, resulting in increased promoter activity. The results provided a possible mechanism underlying the regulation of NO production in renal cells and a potential target for treating certain NO-associated renal disorders.

A role for the Sts phosphatases in negatively regulating IFNγ‐mediated production of nitric oxide in monocytes

IntroductionThe atypical Sts phosphatases negatively regulate signaling pathways in diverse immune cell types, with two of their molecular targets being the related kinases Syk and Zap‐70. Mice lacking Sts expression (Sts −/−) are resistant to infection by the live vaccine strain (LVS) of Francisella tularensis. Although the mechanisms underlying the enhanced resistance of Sts −/− mice have not been definitively established, Sts −/− bone marrow‐derived monocytes (BMMs) demonstrate greater clearance of intracellular LVS following ex vivo infection, relative to wild type cells. To determine how the Sts proteins regulate monocyte bactericidal properties, we analyzed responses of infected cells.MethodsMonocyte bacterial clearance was assayed using ex vivo coculture infections followed by colony‐forming unit analysis of intracellular bacteria. Levels of gene expression were quantified by quantitative reverse‐transcription polymerase chain reaction, levels of Nos2 protein levels were quantified by Western blot analysis, and levels of nitric oxide (NO) were quantified directly using the Griess reagent. We characterized monocyte cytokine production via enzyme‐linked immunosorbent assay.ResultsWe demonstrate that Sts −/− monocyte cultures produce elevated levels of interferon‐γ (IFNγ) after infection, relative to wild type cultures. Sts −/− monocytes also demonstrate heightened responsiveness to IFNγ. Specifically, Sts −/− monocytes produce elevated levels of antimicrobial NO following IFNγ stimulation, and this NO plays an important role in LVS restriction. Additional IFNγ‐stimulated genes, including Ip10 and members of the Gbp gene family, also display heightened upregulation in Sts −/− cells. Both Sts‐1 and Sts‐2 contribute to the regulation of NO production, as evidenced by the responses of monocytes lacking each phosphatase individually. Finally, we demonstrate that the elevated production of IFNγ‐induced NO in Sts −/− monocytes is abrogated following chemical inhibition of Syk kinase.ConclusionOur results indicate a novel role for the Sts enzymes in regulating monocyte antibacterial responses downstream of IFNγ.

HDAC6 Mediates Macrophage iNOS Expression and Excessive Nitric Oxide Production in the Blood During Endotoxemia.

Excessive nitric oxide (NO) production and NO-mediated nitrative stress contribute to vascular dysfunction, inflammation, and tissue injury in septic shock. New therapeutic targets are urgently needed to provide better control of NO level during septic shock. In the present study, we investigated the role of HDAC6 in the regulation of NO production and nitrative stress in a mouse model of endotoxin-induced septic shock. HDAC6 deficient mice and a specific HDAC6 inhibitor were utilized in our studies. Our data clearly indicate that HDAC6 is an important mediator of NO production in macrophages. HDAC6 mediates NO production through the regulation of iNOS expression in macrophages. HDAC6 up-regulates iNOS expression in macrophages by modulating STAT1 activation and IRF-1 expression. HDAC6 inhibition potently blocked endotoxin-induced STAT1 activation and iNOS expression in macrophages. Furthermore, HDAC6 contributes to excessive NO production and nitrotyrosine level in the blood and promotes iNOS expression in the lung tissues during septic shock. Our data reveal a novel HDAC6/STAT1/iNOS pathway that mediates excessive NO production and nitrative stress in septic shock.