Inhibition Of iNOS Expression Research Articles

Syntheses of 1,2,3-triazolyl salicylamides with inhibitory activity on lipopolysaccharide-induced nitric oxide production

A 28-membered 1,2,3-triazolyl salicylamide library was synthesized via a Cu(I)-catalyzed azide-alkyne 1,3-dipolar cycloaddition and evaluated for their abilities to inhibit NO production in LPS-activated RAW264.7 macrophage cells. Among 28 analogues, 29 g showed a significant inhibitory activity (IC(50) = 12.8 μM). The inhibitory effects of 29 g on LPS-mediated NO production in macrophage cells appeared to be associated with the suppression of iNOS expression.

Suppressive effects of methoxyflavonoids isolated from Kaempferia parviflora on inducible nitric oxide synthase (iNOS) expression in RAW 264.7 cells

The rhizomes of Kaempferia parviflora Wall. ex Baker have been traditionally used in Thailand to treat abscesses, gout, and peptic ulcers. Previously, we reported that the chloroform fraction of a Kaempferia parviflora extract had an inhibitory effect on rat paw-edema. In the present study, we isolated the constituents of this fraction and investigated the anti-inflammatory mechanism against nitric oxide (NO) production, tumor necrosis factor-α (TNF-α) and the expression of inducible nitric oxide synthase (iNOS) as well as phosphorylated extracellular signal-regulated kinase (p-ERK), and phosphorylated c-Jun N-terminal kinase (p-JNK). In addition, effects of trimethylapigenin (4) on the enzyme activities of protein kinases possibly leading to iNOS expression were examined to clarify the targets. The chloroform fraction was isolated using silica gel column chromatography and HPLC. Isolated compounds were tested against NO and TNF-α using RAW264.7 cells. Cytotoxicity and iNOS, p-ERK and p-JNK expression were also examined. Three active components, 5,7-dimethoxyflavone (2), trimethylapigenin (4), and tetramethylluteolin (5), markedly inhibited the production of NO in lipopolysaccharide (LPS)-activated RAW264.7 cells. Compounds 2, 4, and 5 moderately inhibited production of TNF-α. Compounds 2, 4, and 5 strongly inhibited expression of iNOS mRNA and iNOS protein in a dose-dependent manner, but did not inhibit p-ERK or p-JNK protein expression. The most active compound, 4, did not inhibit the enzyme activity of inhibitor of κB kinases or mitogen-activated protein kinases, but inhibited that of spleen tyrosine kinase (SYK). The mechanism responsible for the anti-inflammatory activity of methoxyflavonoids from the chloroform fraction of the rhizomes of Kaempferia parviflora is mainly the inhibition of iNOS expression, and the inhibition of SYK by 4 may be involved in the suppression of LPS-induced signaling in macrophages.

Antioxidative effects of cinnamomi cortex: A potential role of iNOS and COX-II

Background:Cinnamomi cortex has wide varieties of pharmacological actions such as anti-inflammatory action, anti-platelet aggregation, and improving blood circulation. In this study, we tested to determine whether the Cinnamomi cortex extract has antioxidant activities.Materials and Methods:Antioxidative actions were explored by measuring free radical scavenging activity, NO levels, and reducing power. The mechanism of antioxidative action of Cinnamomi cortex was determined by measuring iNOS and COX-II expression in lipopolysaccharide (LPS) stimulated Raw cells.Results:Seventy percent methanolic extract of Cinnamomi cortex exerted significant 1,1-diphenyl--2--picrylhydrazyl (DPPH) free radicals and NO scavenging activities in a dose-dependent manner. More strikingly, the Cinnamomi cortex extract exerted dramatic reducing power activity (13-fold over control). Production of iNOS induced by LPS was significantly inhibited by the Cinnamomi cortex extract, suggesting that it inhibits NO production by suppressing iNOS expression. Additionally, COX-2 induced by LPS was dramatically inhibited by the Cinnamomi cortex extract.Conclusion:These results suggest that 70% methanolic extract of Cinnamomi cortex exerts significant antioxidant activity via inhibiting iNOS and COX-II induction.

Dual Specificity Phosphatase 1 Regulates Human Inducible Nitric Oxide Synthase Expression by p38 MAP Kinase

The role of dual specificity phosphatase 1 (DUSP1) in inducible nitric oxide synthase (iNOS) expression in A549 human pulmonary epithelial cells, J774 mouse macrophages and primary mouse bone marrow-derived macrophages (BMMs) was investigated. iNOS expression was induced by a cytokine mixture (TNF, IFNγ and IL-1β) in A549 cells and by LPS in J774 cells, and it was inhibited by p38 MAPK inhibitors SB202190 and BIRB 796. Stimulation with cytokine mixture or LPS enhanced also DUSP1 expression. Down-regulation of DUSP1 by siRNA increased p38 MAPK phosphorylation and iNOS expression in A549 and J774 cells. In addition, LPS-induced iNOS expression was enhanced in BMMs from DUSP1(−/−) mice as compared to that in BMMs from wild-type mice. The results indicate that DUSP1 suppresses iNOS expression by limiting p38 MAPK activity in human and mouse cells. Compounds that enhance DUSP1 expression or modulate its function may be beneficial in diseases complicated with increased iNOS-mediated NO production.

Curcumin enhances neuronal survival in N-methyl-d-aspartic acid toxicity by inducing RANTES expression in astrocytes via PI-3K and MAPK signaling pathways

Neuroinflammation, which is characterized by the overproduction of cytokines and chemokines, plays an important role in neurodegenerative diseases, especially in Alzheimer's disease (AD). In the brain, chemokines are predominantly released by astrocytes and microglias. Expression of RANTES, as well as other cytokines, is involved in the inflammatory cascade that contributes to neurodegeneration in AD. Expression of RANTES may also have a neuroprotective effect. We sought to investigate whether curcumin exhibited neuroprotective and antioxidant activity via enhanced RANTES expression by astrocytes in cortical neuron cultures. We evaluated the neuroprotective and anti-neurodegenerative effects of curcumin in NMDA toxicity and in long-term cultures. Pregnant female Sprague-Dawley (SD) rats were used for primary culture of cortical neurons, and neonatal 0- to 2-day-old SD rats were used for primary culture of astrocytes. Cultured astrocytes were conditioned with curcumin to prepare astrocyte-conditioned medium (ACM). Real-time polymerase chain reaction was performed to assess RANTES and iNOS mRNA expression in astrocytes following curcumin treatment. ELISA was used to detect astrocyte-secreted RANTES protein in ACM with curcumin treatment. JAK/STAT, PI-3K, PKC and MAPK inhibitors were used to ascertain whether the effects of curcumin involved these signaling pathways. To evaluate the effects of curcumin-enhanced astrocytes on neuronal survival, cultured cortical neurons treated or untreated with NMDA were incubated in ACM with or without curcumin treatment. Long-term culture (15days in vitro, DIV) was performed to investigate the effects of curcumin-treated astrocytes on the survival of cultured cortical neurons. Neuronal survival rate was assessed by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction activity assay (for cell viability), and the lactate dehydrogenase (LDH) release assay (for cell death). We demonstrated that curcumin enhanced RANTES expression in primary cultured astrocytes, and that this effect was related to activation of PI-3K and MAPK signaling pathways. We found that curcumin inhibited iNOS expression in primary cultured astrocytes in non-stressed condition. We also found that neurons exposed to NMDA and cultured with curcumin treated ACM, which characteristically exhibited elevated RANTES expression showed higher level of cell viability and lower level of cell death. Using a small interfering RNA (siRNA) knockdown model, we found evidence that the basal level of RANTES expression in non-stimulated astrocytes provided neuroprotection. We postulate that the enhanced neuronal survival by curcumin treatment in NMDA toxicity and long-term cultures was in part attributable to elevated astrocyte-derived RANTES expression via activation of PI3K/MAPK signaling pathways.

KAJIAN EFEK ALFA-PINEN TERHADAP i-NOS (INDUCIBLE NITRIC OXIDE SYNTHASE) PADA KULTUR SEL NEURON-GLIA YANG DIPAPAR LIPOPOLISAKARIDA

The aim of this research was to study the effect of a-pinene on number of cell that express iNOS (inducible Nitric Oxide Synthase) on lipopolysaccharide (LPS)-induced neuron-glial culture. Neuron-glial culture were divided into eight groups: (1) normal; (2), (3) and (4) incubated with a-pinene (0,2; 0,4 and 0,8 μg/mL respectively, for 24 hours); (5) incubated with LPS 1 μg/mL; (6), (7) and (8) incubated with a-pinene (0,2; 0,4 and 0,8 μg/mL respectively, for 24 hours) and then induced with LPS (1 μg/mL, on 1 last hour of treatment period). The expression of iNOS in neuron-glial culture was detected by immunocytochemistry using anti-iNOS rabbit polyclonal IgG and goat anti-rabbit IgG Biotin Conjugated. The result shown that iNOS were expressed on normal group. Lipopolysaccharide 1 μg/mL induction causing iNOS expression increased approximately three times higher than normal (from 9,7% became 28,7%). Inducible NOS expression on non LPS-induced neuron-glial culture did not affected by a-pinene. Whereas a-pinene 0,2 and 0,4 μg/mL could suppress iNOS expression on LPSinduced condition. a-pinene 0,2; 0,4 and 0,8 μg/mL could help neuronglial cells survived on LPS-induced condition, but normal neuron-glial culture’s density was decreased when incubated with a-pinene 0,8 μg/mL. Keywords: alpha-pinene, cell culture, glial, iNOS, LPS, neuron

Biochanin-A, an isoflavon, showed anti-proliferative and anti-inflammatory activities through the inhibition of iNOS expression, p38-MAPK and ATF-2 phosphorylation and blocking NFκB nuclear translocation

Biochanin-A, an isoflavone, existing in red clover, cabbage and alfalfa, has an inhibitory and apoptogenic effect on certain cancer cells. However, the actual mechanism by which this compound inhibits proliferation and induces apoptosis in cancer cells and the mechanism of its anti-inflammatory activities have not been well characterized. In this study, we have investigated the anti-inflammatory and anti-proliferative activity of Biochanin-A. The effects of Biochanin-A on RAW 264.7, HT-29 cell lines and mouse peritoneal macrophages have been investigated in vitro. Cell proliferation and anti-inflammatory effects were analyzed by 3-(4-5-dimethylthiozol-2-yl)2-5-diphenyl-tetrazolium bromide (MTT) assay, 3H-thymidine incorporation assay, Western blot, cytokines estimation, Luciferase assay, Electrophoretic mobility shift assay (EMSA) and Kinase assay. Present investigation demonstrated that, Biochanin-A inhibited lipopolysacharide (LPS)-induced nitric oxide(NO) production in macrophage and showed dose dependent inhibition of inducible nitric oxide synthase (iNOS) expression. The induction of NF-κB binding activity by LPS was inhibited markedly by co-incubation with different doses of Biochanin-A. Biochanin-A inhibited the LPS-induced IkB kinase (IKK) activity and nuclear factor kappa beta (NF-κB) activation associated with the inhibition of iNOS expression. LPS-induced phosphorylation of IκBα and p38 MAPK was blocked by Biochanin-A and it inhibited IL-6, IL-1β and TNF-α production in RAW264.7 cells indicating its anti-inflammatory activity in association with anti-proliferation. Biochanin-A is important for the prevention of phosphorylation and degradation of IκBα, thereby blocking NF-κB activation, which in turn leads to decreased expression of the iNOS, thus preventing proliferation and inflammation.

Characterization of anti-oxidative effects of Mori Cortex Radicis

We tested to determine if Mori Cortex Radicis extract has antioxidant activities and its potential mechanism of action was explored. Anti-oxidative effects were tested by measuring free radical and nitric Oxide (NO) scavenging activity, and reducing power. Since iNOS and COX-2 are important enzymes responsible for the production of free radicals in the cell, Mori Cortex Radicis extract was tested as to whether it could inhibit iNOS and COX-2 expression in LPS stimulated Raw cells. 70% methanolic extract of Mori Cortex Radicis exerted significant DPPH free radical and NO scavenging activities. In addition, the Mori Cortex Radicis extract exerted dramatic reducing power with maximal activity observed at 1 mg/ml (11-fold over control). Production of iNOS induced by LPS was significantly inhibited by the Mori Cortex Radicis extract, suggesting it could inhibit NO production by suppressing iNOS expression. COX-2 induced by LPS was also significantly inhibited by the Mori Cortex Radicis extract. The extract contains well known antioxidant components including phenolics, flavonoids and anthocyanin at the concentration of 0.23 mg/g, 42.97 mg/g and 12.08 mg/g, respectively. These results suggest that 70% methanolic extract of Mori Cortex Radicis exerts significant anti-oxidant activity via inhibiting iNOS and COX-2 induction.

Cymbopogon citratus as source of new and safe anti-inflammatory drugs: Bio-guided assay using lipopolysaccharide-stimulated macrophages

Aqueous extracts of Cymbopogon citratus (Cy) leaves are used in traditional medicine for the treatment of inflammatory conditions, however, little is known about their mechanism of action. The aim of this study is to explore the anti-inflammatory properties of Cymbopogon citratus leaves and their polyphenol-rich fractions (PFs), as well its mechanism of action in murine macrophages. A lipid- and essential oil-free infusion of Cy leaves was prepared (Cy extract) and fractionated by column chromatography. Anti-inflammatory properties of Cy extract (1.115 mg/ml) and its PFs, namely phenolic acids (530 μg/ml), flavonoids (97.5 μg/ml) and tannins (78 μg/ml), were investigated using lipopolysaccharide (LPS)-stimulated Raw 264.7 macrophages as in vitro model. As inflammatory parameters, nitric oxide (NO) production was evaluated by Griess reaction, as well as effects on cyclooxygenase-2 (COX-2), inducible NO synthase (iNOS) expression and on intracellular signaling pathways activation, which were analyzed by Western blot using specific antibodies. Cy extract inhibited iNOS expression, NO production and various LPS-induced pathways like p38 mitogen-activated protein kinase (MAPK), c-jun NH(2)-terminal kinase (JNK) 1/2 and the transcription nuclear factor (NF)-κB. The extracellular signal-regulated kinase (ERK) 1/2 and the phosphatidylinositol-3-kinase (PI3K)/Akt activation were not affected by Cy extract. Both phenolic acid- and tannin-rich fractions significantly inhibited NF-κB activation, iNOS expression and NO production but none of the PFs modulated MAPKs or PI3K/Akt activation. Neither Cy extract nor PFs affected LPS-induced COX-2 expression but LPS-induced PGE(2) production is inhibited by Cy extract and by phenolic acid-rich fraction. Our data provide evidence that support the usage of Cymbopogon citratus leaves extract in traditional medicine, and suggest that Cy, in particular its polyphenolic compounds, could constitute a natural source of a new and safe anti-inflammatory drug.

Taraxacum officinale Weber extracts inhibit LPS-induced oxidative stress and nitric oxide production via the NF-κB modulation in RAW 264.7 cells

The common dandelion (Taraxacum officinale G.H. Weber ex Wiggers, Asteraceae) has been widely used in folklore medicine to treat dyspepsia, heartburn, and spleen and liver disorders. To compare the antioxidative and anti-inflammatory activities of Taraxacum officinale methanol extract (TOME) and water extract (TOWE) in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells and assess their constitutional differences, including luteolin, chicoric acid, and total phenol content. Antioxidative enzyme activities, nitric oxide (NO) production, and inducible NO synthase (iNOS) and nuclear factor (NF)-κB expression were estimated by biochemical analysis, the Griess reaction, reverse transcription-polymerase chain reaction, western hybridization, and electrophoretic mobility shift assay. High-performance liquid chromatography and the Folin-Ciocalteau method were used to analyze functional phytochemicals and total phenol content. TOME and TOWE significantly reduced NO production with an IC(50) of 79.9 and 157.5 μg/mL, respectively, without cytotoxicity. Depleted glutathione (GSH) and antioxidative enzyme activities, including superoxide dismutase, catalase, GSH-peroxidase, and GSH-reductase, were restored by dandelion extracts. Both extracts inhibited LPS-stimulated iNOS gene expression and that of its transcription factor, NF-κB, in parallel with nitrite reduction. TOME showed more potent antioxidative and anti-inflammatory capacities than TOWE, which was attributable to its high total phenol, luteolin, and chicoric acid content. These results indicate that TOME and TOWE inhibit oxidative stress and inflammatory responses through elevated de novo synthesis of antioxidative enzymes and suppression of iNOS expression by NF-κB inactivation.

Activation of α7 nicotinic acetylcholine receptors ameliorates indomethacin-induced small intestinal ulceration in mice

Cholinergic anti-inflammatory actions have been shown to result mainly from the activation of α7 nicotinic acetylcholine receptors. Here, we investigated the possible role of α7 nicotinic acetylcholine receptors in the pathogenesis of indomethacin-induced small intestinal ulceration in mice. Male C57BL/6 mice were given indomethacin (10 mg/kg, s.c.), and sacrificed 24 h later. Nicotine (0.3–3 mg/kg) and PNU-282987 (a selective agonist of α7 nicotinic acetylcholine receptors; 1–10 mg/kg) were administered i.p. twice, at 0.5 h before and 8 h after indomethacin treatment, while methyllycaconitine (a selective antagonist of α7 nicotinic acetylcholine receptors; 10 mg/kg was administered twice, at 0.5 h before each nicotine treatment. Indomethacin caused severe hemorrhagic lesions in the small intestine with marked increases in myeloperoxidase (MPO) activity and inducible nitric oxide synthase (iNOS) expression in the mucosa. Pretreatment with nicotine reduced the severity of intestinal lesions in a dose-dependent manner. The protective effect of nicotine was mimicked by PNU-282987 and significantly attenuated by methyllycaconitine. The increases in MPO activity and iNOS expression induced by indomethacin were also significantly suppressed by nicotine and PNU-282987. Immunohistochemical study showed that the expression of α7 nicotinic acetylcholine receptors was clearly enhanced in the submucosa of the damaged area following indomethacin treatment. These results suggest that the activation of α7 nicotinic acetylcholine receptors ameliorates indomethacin-induced small intestinal ulceration, and that this effect may result from the inhibition of iNOS expression and neutrophil migration.

Inhibitors of Nitric Oxide Production fromStemona javanica

In our screening program for bioactive natural products from our library of tropical plants, the extract prepared from the roots of Stemona javanica inhibited NO production in mouse macrophage-like cell line J774.1 stimulated by lipopolysaccharide (LPS). Bioassay-guided fractionation of the extract from S. javanica led to the isolation of two active compounds, stemofoline (1) and stemanthrene C (2). The inhibition mechanism of 1 was proposed to suppress iNOS expression in J774.1 cells stimulated by LPS, whereas that of 2 was due to potent radical scavenging activity resulting in NO inhibitory activity.

Aqueous extract of Lycii fructus suppresses inflammation through the inhibition of nuclear factor kappa B signal pathway in murine raw 264.7 macrophages

Lycii fructus is the fruit of Lycium chinense Miller and is part of the Solanaceae family. Lycii fructus produces various effects such as hypotensive, hypoglycemic, anti-pyretic, and anti-stress activities. Lycii fructus is known to contain betaine, carotene, nicotinic acid, zeaxanthin, and cerebroside. In the present study, the effects of Lycii fructus aqueous extract on lipopolysaccharide (LPS)-induced inflammation in murine raw 264.7 macrophage cells were investigated. In this study we utilized the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, reverse transcriptionpolymerase chain reaction (RT-PCR), Western blotting, and nitric oxide (NO) detection. Lycii fructus aqueous extract suppressed NO production by inhibiting the LPS-induced expressions of inducible nitric oxide synthase (iNOS) and tumor necrosis factor-alpha (TNF-<TEX>$\alpha$</TEX>) mRNA and iNOS protein in murine raw 264.7 macrophage cells. Also, Lycii fructus aqueous extract suppressed the activation of nuclear factor-kappa B (NF-<TEX>${\kappa}B$</TEX>) in the nucleus. These results demonstrated that Lycii fructus aqueous extract causes an anti-inflammatory effect that was likely produced by the suppression of iNOS expression through the down-regulation of NF-<TEX>$\hat{e}B$</TEX> binding activity.

Polyphenols from Cymbopogon citratus inhibit iNOS expression and NO production – a promising source of new anti-inflammatory drugs

Polyphenols from Cymbopogon citratus inhibit iNOS expression and NO production – a promising source of new anti-inflammatory drugs

Nitric oxide synthases and tubal ectopic pregnancies induced by Chlamydia infection: basic and clinical insights

Human ectopic pregnancy (EP) remains a common cause of pregnancy-related first trimester death. Nitric oxide (NO) is synthesized from L-arginine by three NO synthases (NOS) in different tissues, including the Fallopian tube. Studies of knockout mouse models have improved our understanding of the function of NOS isoforms in reproduction, but their roles and specific mechanisms in infection-induced tubal dysfunction have not been fully elucidated. Here, we provide an overview of the expression, regulation and possible function of NOS isoforms in the Fallopian tube, highlighting the effects of infection-induced changes in the tubal cellular microenvironment (imbalance of NO production) on tubal dysfunction and the potential involvement of NOS isoforms in tubal EP after Chlamydia trachomatis genital infection. The non-equivalent regulation of tubal NOS isoforms during the menstrual cycle suggests that endogenous ovarian steroid hormones regulate NOS in an isoform-specific manner. The current literature suggests that infection with C. trachomatis induces an inflammatory response that eventually leads to tubal epithelial destruction and functional impairment, caused by a high NO output mediated by inducible NOS (iNOS). Therefore, tissue-specific therapeutic approaches to suppress iNOS expression may help to prevent ectopic implantation in patients with prior C. trachomatis infection of the Fallopian tube.

Anti-inflammatory and Anticancer Properties of Dichloromethane and Butanol Fractions from the Stem Bark of Broussonetia papyrifera

Broussonetia papyrifera radix, fruits, leaves, and stems exhibit antioxidant, antinociceptive, antityrosinase, anti-inflammatory, and antiplatelet activities. However, studies of the anticancer and anti-inflammatory activities of B. papyrifera stem bark are limited. Yet it is important to evaluate these activities to isolate active compounds for potential medicines and food additive. The dichloromethane fraction of B. papyrifera stem bark was found to induce apoptosis-related DNA fragmentation, increase sub-G1 accumulation, increase morphological changes, and inhibit the proliferation of human colon cancer HT-29 cells. Moreover, it significantly increased p53, caspase-3, and Bax expression in HT-29 cells. In addition, the n-butanol fraction inhibited nitric oxide production in RAW 264.7 macrophages by suppressing iNOS expression. These results suggest that B. papyrifera is a potential natural resource for treating cancer and inflammatory diseases.

Anti-Inflammatory Activity ofCymbopogon citratusLeaf Infusion in Lipopolysaccharide-Stimulated Dendritic Cells: Contribution of the Polyphenols

Cymbopogon citratus, an herb known worldwide as lemongrass, is widely consumed as an aromatic drink, and its fresh and dried leaves are currently used in traditional cuisine. However, little is known about the mechanism of action of C. citratus, namely, the anti-inflammatory effects of its dietary components. Because nitric oxide (NO), produced in large quantities by activated inflammatory cells, has been demonstrated to be involved in the pathogenesis of acute and chronic inflammation, we evaluated the effects of the infusion of dried leaves from C. citratus, as well as its polyphenolic fractions--flavonoid-, tannin-, and phenolic acid-rich fractions (FF, TF, and PAF, respectively)--on the NO production induced by lipopolysaccharide (LPS) in a skin-derived dendritic cell line (FSDC). C. citratus infusion significantly inhibited the LPS-induced NO production and inducible NO synthase (iNOS) protein expression. All the polyphenolic fractions tested also reduced the iNOS protein levels and NO production stimulated by LPS in FSDC cells, without affecting cell viability, with the strongest effects being observed for the fractions with mono- and polymeric flavonoids (FF and TF, respectively). Our results also indicated that the anti-inflammatory properties of FF are mainly due to luteolin glycosides. In conclusion, C. citratus has NO scavenging activity and inhibits iNOS expression and should be explored for the treatment of inflammatory diseases, in particular of the gastrointestinal tract.

INOS-TARGETED 10-23 DNAZYME REDUCES LPS-INDUCED SYSTEMIC INFLAMMATION AND MORTALITY IN MICE

Sepsis and/or systemic inflammatory response syndrome are leading causes of death in intensive care unit patients. NO is a critical player in the pathogenesis of bacterial sepsis. Several studies demonstrate elevation of iNOS in LPS-induced acute inflammatory responses and mortality; however, the effectiveness of its therapeutic suppression in systemic inflammation is largely controversial. Earlier, we have reported that DNAzymes specific to iNOS mRNA efficiently suppress iNOS expression in LPS-stimulated J774 murine macrophages. In the present study, we explored the effects of two of these DNAzymes in BALB/c mice model of LPS-induced lethal systemic inflammation. Experimental animal groups receiving previous injections of iNOS-specific DNAzyme (100 microg, i.p.) showed significantly reduced mortality. Total cell counts of peritoneal lavage and histopathological studies of tissues demonstrated substantial reduction in the leukocytic infiltration and edema in DNAzyme-treated mice. In addition, DNAzyme-injected animals displayed significantly decreased IL-12 serum level, whereas the levels of IL-1[beta], IFN-[gamma], and TNF-[alpha] also declined to a great extent. DNAzyme treatment resulted in significantly reduced NO levels in serum and peritoneal lavage, confirming functional suppression of iNOS gene in LPS-injected mice. These DNAzymes were also able to limit excessive NO production by cytokine and LPS co-challenges in cultured peritoneal macrophages from DNAzyme-treated mice. Estimation of iNOS mRNA and protein expression in the peritoneal macrophages of DNAzyme-administered animals further confirmed the iNOS gene knockdown. All these results indicated that iNOS-specific DNAzymes reduce inflammatory responses and enhance survival in murine model of LPS-induced lethal systemic inflammation.

Abstract 948: Silibinin selectively inhibits growth and progression of urethane-induced lung tumors in wild-type but not inducible nitric oxide synthase (-/-) mice: potential usefulness of micro-computed tomography scanning in lung cancer chemoprevention studies

Abstract Lung cancer is the second most common cancer but accounts for highest mortality in both men and women in the United States and Europe. The overall 5 year survival rate is still 16% underscoring the importance of new approaches to prevention and treatment of this deadly malignancy. Since sustained and excessive nitric oxide (NO) generation is positively correlated with lung cancer development and progression, recent studies have also focused on the involvement of inducible NO synthase (iNOS) and its inhibition by natural and synthetic agents for both prevention and therapy of lung cancer. Here we evaluated the role of iNOS in lung tumor occurrence and progression as well as the angiopreventive efficacy of silibinin employing B6/129J wild-type (WT) and iNOS (−/−) mice. Mice were injected with 1 mg/g body weight (i.p.) urethane once weekly for seven consecutive weeks, and thereafter were divided into two groups and gavaged with vehicle (control) or silibinin (742 mg/kg body weight) 5 days/week for 18 weeks. Using micro-computed tomography (micro-CT), we monitored the growth and progression of lung tumors from 8 through 20 weeks in WT mice. At 25 weeks, mice were sacrificed and their lung tumor multiplicity and diameters recorded. Quantification of micro-CT data showed that silibinin treatment significantly decreases urethane-induced tumor number and size in WT mice. This correlated with the decreases in tumor number and size directly measured at the termination of the study. Genetic ablation of iNOS decreased urethane-induced tumor multiplicity by 89% (P&amp;lt;0.001) compared to WT mice, but unlike its strong chemopreventive efficacy in WT mice, silibinin treatment failed to decrease tumor number or size in iNOS (-/-) mice. Tumors in WT control mice expressed more iNOS than those in silibinin-treated mice. Further analyses of tumors from WT mice showed that compared to controls silibinin treatment significantly decreased newly formed microvessels as measured by nestin immunostaining, activation of STAT3 and NF-κB as determined by pSTAT3 (ser727) and p65NF-κB (ser276) levels, and proliferation as measured by PCNA expression. Whereas a decrease in lung tumor number in iNOS (-/-) mice compared to WT confirms previous studies that iNOS is a critical regulator of lung tumorigenesis; this study identified an essential role of iNOS in lung tumor angiogenesis. The lack of effect of silibinin in iNOS null mice suggests that silibinin exerts its angiopreventive effects at least in part through its inhibition of iNOS expression in lung tumors. Together, our results suggest iNOS as a potential target for treating lung cancer and support the value of micro-CT as a non-invasive imaging modality for evaluating the efficacy of lung cancer chemopreventive agents. (RO1 CA113876) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 948.

Redox modulation as a mechanistic feature of biological effects of cysteine and glutathione mixed disulfide conjugates of Allium thiosulfinates

The objective of this work was to determine if phase II enzyme induction and inhibition of nitric oxide (NO) evolution by cysteine and glutathione mixed disulfide conjugates of Allium thiosulfinates (CySSR/GSSR) were linked to redox homeostasis in cultured cells. The CySSR/GSSR species induced quinone reductase (QR) through the Nrf2 pathway in Hepa 1c1c7 cells, and inhibited iNOS expression and NO evolution in LPS‐activated macrophages. CySSR/GSSR also modulated intracellular redox status by boosting intracellular GSH levels. The effects of the CySSR and GSSR species were progressively attenuated by the addition of exogenous thiol agents that influenced redox status in a dose‐dependent manner in both intracellular and extracellular spaces. Thus, the biological activities of CySSR/GSSR appear to be dependent on both extracellular and intracellular redox status. The effects of CySSR, but not GSSR, treatments resulted in an increase in extracellular thiol levels. Evidence was obtained for a mechanism involving cellular uptake of CySSR by L transporter, disulfide bond reduction by thioredoxin reductase (TRxR) and the export of cysteine and RSH into the medium by multiple drug resistance protein. This study adds to the evidence that Allium organosulfur compounds and metabolic derivatives exert a range of biological effects through a redox‐modulatory mechansism.Supported by USDA‐NRI‐CGP.