Suppression Of iNOS Induction Research Articles

A Nurr1 ligand C-DIM12 attenuates brain inflammation and improves functional recovery after intracerebral hemorrhage in mice

We have previously reported that amodiaquine, a compound that binds to the ligand-binding domain of a nuclear receptor Nurr1, attenuates inflammatory responses and neurological deficits after intracerebral hemorrhage (ICH) in mice. 1,1-Bis(3′-indolyl)-1-(p-chlorophenyl)methane (C-DIM12) is another Nurr1 ligand that recognizes a domain of Nurr1 different from the ligand-binding domain. In the present study, mice were treated daily with C-DIM12 (50 or 100 mg/kg, p.o.) or amodiaquine (40 mg/kg, i.p.), or twice daily with 1400 W (20 mg/kg, i.p.), an inducible nitric oxide synthase (iNOS) inhibitor, from 3 h after ICH induction by microinjection of collagenase into the striatum. C-DIM12 improved the recovery of neurological function and prevented neuron loss in the hematoma, while suppressed activation of microglia/macrophages and expression of inflammatory mediators interleukin-6 and CC chemokine ligand 2. In addition, C-DIM12 as well as amodiaquine preserved axonal structures in the internal capsule and axonal transport function. We also found that C-DIM12 and amodiaquine suppressed the increases of iNOS mRNA expression after ICH. Moreover, 1400 W improved neurological function and prevented neuron loss, activation of microglia/macrophages and axonal transport dysfunction. These results suggest that suppression of iNOS induction contributes to several features of the therapeutic effects of Nurr1 ligands.

Therapeutic effects of s-petasin on disease models of asthma and peritonitis.

To explore the anti-allergic and anti-inflammatory effects of extracts of Petasites genus, we studied the effects of s-petasin, a major sesquiterpene from Petasites formosanus (a butterbur species) on asthma and peritonitis models. In an ovalbumin-induced mouse asthma model, s-petasin significantly inhibited the accumulations of eosinophils, macrophages, and lymphocytes in bronchoalveolar fluids. S-petasin inhibited the antigen-induced degranulation of β-hexosamidase but did not inhibit intracellular Ca2+ increase in RBL-2H3 mast cells. S-petasin inhibited the LPS induction of iNOS at the RNA and protein levels in mouse peritoneal macrophages. Furthermore, s-petasin inhibited the production of NO (the product of iNOS) in a concentration-dependent manner in the macrophages. Furthermore, in an LPS-induced mouse model of peritonitis, s-petasin significantly inhibited the accumulation of polymorpho nuclear and mononuclear leukocytes in peritoneal cavity. This study shows that s-petasin in Petasites genus has therapeutic effects on allergic and inflammatory diseases, such as, asthma and peritonitis through degranulation inhibition in mast cells, suppression of iNOS induction and production of NO in macrophages, and suppression of inflammatory cell accumulation.

Suppressive effects of coumarins from Mammea siamensis on inducible nitric oxide synthase expression in RAW264.7 cells

A methanol extract of the flowers of Mammea siamensis (Calophyllaceae) was found to inhibit nitric oxide (NO) production in lipopolysaccharide-activated RAW264.7 cells. From the extract, two new geranylated coumarins, mammeasins A (1) and B (2), were isolated together with 17 known compounds including 15 coumarins. The structures of 1 and 2 were determined on the basis of their spectroscopic properties as well as of their chemical evidence. Among the isolates, 1 (IC50=1.8μM), 2 (6.4μM), surangins B (3, 5.0μM), C (4, 6.8μM), and D (5, 6.2μM), kayeassamins E (7, 6.1μM), F (8, 6.0μM), and G (9, 0.8μM), mammea A/AD (11, 1.3μM), and mammea E/BB (16, 7.9μM) showed NO production inhibitory activity. Compounds 1, 9, and 11 were found to inhibit induction of inducible nitric oxide synthase (iNOS). With regard to mechanism of action of these active constituents (1, 9, and 11), suppression of STAT1 activation is suggested to be mainly involved in their suppression of iNOS induction.

Japanese Herbal Medicine Hochuekkito Inhibits the Expression of Proinflammatory Biomarker, Inducible Nitric Oxide Synthase, in Hepatocytes

Hochuekkito (TJ-41) is used for the treatment of complaints in patients with general fatigue. However, there is little scientific evidence to demonstrate the liver-protective effects of TJ-41. In the inflamed liver, proinflammatory cytokines stimulate the induction of inducible nitric oxide synthase (iNOS). Over-production of NO by iNOS has been implicated as a factor in liver injury. We examined proinflammatory cytokine-stimulated hepatocytes as a simple in vitro injury model to determine liver-protective effects of TJ-41. The objective was to investigate whether TJ-41 influences iNOS induction and to determine its mechanism. Primary cultured rat hepatocytes were treated with interleukin (IL)-1β in the presence or absence of TJ-41. The induction of iNOS and its signaling pathway were analyzed. IL-1β produced increased levels of NO. This effect was inhibited by TJ-41, which exerted its maximal effects at 6 mg/ml. TJ-41 decreased the levels of iNOS protein and its mRNA expression. Experiments with nuclear extracts revealed that TJ- 41 inhibited the translocation of NF-κB to the nucleus and its DNA binding. TJ-41 also inhibited the activation of Akt, resulting in the reduction of type I IL-1 receptor mRNA and protein expression. Transfection experiments demonstrated that TJ-41 suppressed iNOS induction by the inhibition of promoter transactivation and mRNA stabilization. TJ-41 reduced the expression of an iNOS gene antisense-transcript, which is involved in iNOS mRNA stability. Results indicate that TJ-41 inhibits the induction of iNOS at both transcriptional and post-transcriptional steps, leading to the prevention of NO production. TJ-41 may have therapeutic potential for various liver injuries through the suppression of iNOS induction.

Japanese herbal medicine, inchinkoto, inhibits inducible nitric oxide synthase induction in interleukin‐1β‐stimulated hepatocytes

A herbal medicine, kampo inchinkoto (TJ-135), is used to treat jaundice and liver fibrosis in patients with cirrhosis. In the inflamed liver, proinflammatory cytokines stimulate the induction of inducible nitric oxide synthase (iNOS) gene expression. Over-production of nitric oxide (NO) by iNOS has been implicated as a factor in liver injury. We examined interleukin (IL)-1β-stimulated hepatocytes as a simple in vitro injury model to determine liver-protective effects of TJ-135. The objective was to investigate whether TJ-135 influences iNOS induction and to determine its mechanism. Primary cultured rat hepatocytes were treated with IL-1β in the presence or absence of TJ-135. The induction of iNOS and its signaling pathway were analyzed. IL-1β produced increased levels of NO. This effect was inhibited by TJ-135, which exerted its maximal effects at 3 mg/mL. TJ-135 decreased the levels of iNOS protein and its mRNA expression. Experiments with nuclear extracts revealed that TJ-135 inhibited the translocation of nuclear factor-κB (NF-κB) to the nucleus and its DNA binding. TJ-135 also inhibited the activation of Akt, resulting in the reduction of type I IL-1 receptor mRNA and protein expression. Transfection experiments with iNOS promoter-luciferase constructs demonstrated that TJ-135 suppressed iNOS induction by inhibition of promoter transactivation and mRNA stabilization. TJ-135 reduced the expression of an iNOS gene antisense-transcript. Delayed administration or withdrawal of TJ-135 after IL-1β addition also inhibited iNOS induction. RESULTS indicate that TJ-135 inhibits the induction of iNOS at both transcriptional and post-transcriptional steps, leading to the prevention of NO production. TJ-135 may have therapeutic potential for various liver injuries through the suppression of iNOS induction.

Cancer prevention by antioxidants

Various antioxidants in foods, such as phenolic compounds and carotenoids, were proven to have anticarcinogenic activity. In the case of carotenoids, the mixture of them was found to be very effective. In fact, the development of hepatoma in the high risk group of liver cancer, was significantly suppressed by the treatment with natural carotenoids mixture. The role of nitric oxide (NO) in carcinogenesis has been pointed out, since large quantity of NO has been detected in cancer tissues, and the expression of inducible NO synthase (iNOS) was found to correlate with tumor growth and metastasis. Recently, we found that NO possessed tumor initiating activity in mouse skin carcinogenesis. It has been suggested that some parts of pathological effects induced by NO may depend on peroxynitrite, an active metabolite of NO. Thus, we accessed the tumor initiating activity of peroxynitrite, and found that treatment with peroxynitrite (initiator) plus TPA (promoter) resulted in the formation of skin tumors. Under this experimental condition, it has been proven that natural antioxidants, such as curcumin and nobiletin, showed anti-tumor initiating effect. In the case of nobiletin, suppressive effect on iNOS induction has also been demonstrated. It is of interest that suppression of iNOS induction was also observed in phytoene synthase transgenic mouse. After administration of glycerol (a lung tumor promoter), lower induction of iNOS gene was observed in lung of the phytoene producing mice, comparing with that of control mice. Combinational use of various kinds of antioxidants distributed in foods, e.g., mixture of carotenoids and flavonoids, seems to be effective methods for cancer prevention.

Protein-protein interactions involving inducible nitric oxide synthase.

Nitric oxide (NO) is a signaling and effector molecule that contributes to multiple physiological and pathophysiological processes in the kidney, vasculature, and other tissues. High output NO generation by inducible NO synthase (iNOS) participates in host defense against pathogens and contributes to tissue injury during inflammatory states. Because of its potent reactivity and diffusibility, NO generation by iNOS is subject to multiple levels of regulation, including transcriptional, translational, and post-translational controls, including protein-protein interactions. This review examines the experimental basis for these protein-protein interactions and their known and potential importance for kidney and vascular physiology. Analysis of the biomedical literature in the area. iNOS interacts with the inhibitory molecules Kalirin and NOS-associated protein 1.10 kd (NAP110), which inhibit iNOS homodimerization, as well as activator proteins, the Rac-GTPases. Interactions with caveolin-1 control the intracellular locale and degradation of iNOS in tumor cells. In polarized epithelial cells, associations of iNOS with the scaffolding protein EBP50 position iNOS in the apical membrane near key ion transport proteins that also interact with EPB50. In addition, protein-protein interactions of proteins governing iNOS transcription function to specify activation or suppression of iNOS induction by cytokines. Interactions of iNOS with a diverse group of heterologous proteins provides a selective mechanism to control the activity, spatial distribution, and proximity of iNOS to intended targets, while potentially limiting autotoxicity to the iNOS-expressing cell.

Protein interactions with nitric oxide synthases: controlling the right time, the right place, and the right amount of nitric oxide.

Nitric oxide (NO) is a potent cell-signaling, effector, and vasodilator molecule that plays important roles in diverse biological effects in the kidney, vasculature, and many other tissues. Because of its high biological reactivity and diffusibility, multiple tiers of regulation, ranging from transcriptional to posttranslational controls, tightly control NO biosynthesis. Interactions of each of the major NO synthase (NOS) isoforms with heterologous proteins have emerged as a mechanism by which the activity, spatial distribution, and proximity of the NOS isoforms to regulatory proteins and intended targets are governed. Dimerization of the NOS isozymes, required for their activity, exhibits distinguishing features among these proteins and may serve as a regulated process and target for therapeutic intervention. An increasingly wide array of proteins, ranging from scaffolding proteins to membrane receptors, has been shown to function as NOS-binding partners. Neuronal NOS interacts via its PDZ domain with several PDZ-domain proteins. Several resident and recruited proteins of plasmalemmal caveolae, including caveolins, anchoring proteins, G protein-coupled receptors, kinases, and molecular chaperones, modulate the activity and trafficking of endothelial NOS in the endothelium. Inducible NOS (iNOS) interacts with the inhibitory molecules kalirin and NOS-associated protein 110 kDa, as well as activator proteins, the Rac GTPases. In addition, protein-protein interactions of proteins governing iNOS transcription function to specify activation or suppression of iNOS induction by cytokines. The calpain and ubiquitin-proteasome pathways are the major proteolytic systems responsible for the regulated degradation of NOS isozymes. The experimental basis for these protein-protein interactions, their functional importance, and potential implication for renal and vascular physiology and pathophysiology is reviewed.

Effects of prenylated flavonoids and biflavonoids on lipopolysaccharide-induced nitric oxide production from the mouse macrophage cell line RAW 264.7.

Certain flavonoid derivatives possess anti-inflammatory activity in vitro and in vivo. Besides their antioxidative properties and effects on the arachidonic acid metabolism including cyclooxygenase/lipoxygenase inhibition, some flavones and flavonols were previously found to show inhibitory activity on nitric oxide production by inducible nitric oxide synthase (iNOS; NOS type 2) through suppression of iNOS induction. As part of our continuing investigations, the effects of unique and minor flavonoids (prenylated flavonoids and biflavonoids) on nitric oxide production from lipopolysaccharide-induced macrophage cell line (RAW 264.7) were evaluated in order to establish their inhibitory activity on NO production and correlate this action with their in vivo anti-inflammatory potential. Among the derivatives tested, prenylated compounds including morusin, kuwanon C, and sanggenon D and biflavonoids such as bilobetin and ginkgetin were found to inhibit NO production from lipopolysaccharide (LPS)-induced RAW 264.7 cells at > 10 microM. Inhibition of nitric oxide production was mediated by suppression of iNOS enzyme induction but not by direct inhibition of iNOS enzyme activity. An exception was echinoisoflavanone that inhibited iNOS enzyme activity (IC50 = 83 microM) and suppressed iNOS enzyme induction as well. While most prenylated derivatives showed cytotoxicity to RAW cells at 10-100 microM, all biflavonoids tested were not cytotoxic. Since nitric oxide (NO) produced by inducible NO synthase (iNOS) plays an important role in inflammatory disorders, inhibition of NO production by these flavonoids may contribute, at least in part, to their anti-inflammatory and immunoregulating potential in vivo.

Effects of radiodetoxified endotoxin on nitric oxide production in J774 macrophages and in endotoxin shock

Radiodetoxified lipopolysaccharide (RD-LPS) is a60Co-gamma-irradiated LPS with a modified structure, which decreases its toxic effects. To obtain a better understanding of the mechanism of the reduced toxicity of RD-LPS, here we studied the effect of RD-LPS on the regulation of nitric oxide (NO) production in vitro and in vivo. In control cells, stimulation by native LPS (10 μg/ml) induced the expression of the inducible NO synthase (iNOS) and production of NO, as measured by increase in the concentration of nitrite, breakdown product of NO. Pre-exposure of the cells for 24 h to a subthreshold concentration of RD-LPS (10 ng/ml) induced a complete desensitization to the LPS-induced NO production in comparison to control cells (P < 0.01). On the contrary, pre-exposure of the cells with native LPS (10 ng/ml) did not reduce LPS-induced NO synthesis. RD-LPS induced a smaller production of tumor necrosis factor (TNF) than native LPS, but did not induce a desensitization against subsequent LPS-induced TNF synthesis. ln in vivo studies, pretreatment of rats with repeated doses of sublethal RD-LPS (1 mg/kg/day i.p. for 4 days) inhibited increase of plasma nitrate/nitrite levels, NO production in peritoneal macrophages ex vivo and induction of lung iNOS activity, in response to a high-dose LPS challenge (15 mg/kg i.p.) given at the fifth day. Pretreatment with repeated sublethal doses of the native LPS (1 mg/kg/day i.p.) did not affect NO production in rats subjected to endotoxic shock. The results demonstrate that RD-LPS induces tolerance to the stimulatory effect of LPS on NO synthesis. Suppression of iNOS induction was only observed with RD-LPS, but not with native LPS in the models used herein. It remains to be further investigated whether suppression of iNOS induction by RD-LPS contributes to the protective effects of this compound in shock and inflammation.