Inhibition Of iNOS Expression Research Articles

Activation of latent transforming growth factor‐β1 by nitric oxide in macrophages: Role of soluble guanylate cyclase and MAP kinases

The inducible nitric oxide (NO) synthase and the cytokine transforming growth factor-beta1 (TGF-beta1), both central modulators of wound healing, interact reciprocally: TGF-beta1 generally suppresses iNOS expression, while NO can induce and activate latent TGF-beta1. We have shown that chemical NO activates recombinant human latent TGF-beta1 by S-nitrosation of the latency-associated peptide (LAP), a cleaved portion of pro-TGF-beta1 that maintains TGF-beta1 in a biologically-inactive state. We hypothesized that cell-associated TGF-beta1 could be activated by NO via known NO-inducible signaling pathways (soluble guanylate cyclase [sGC] and mitogen-activated protein [MAP] kinases). Treatment of mouse RAW 264.7 macrophage-like cells with the NO donor S-nitroso-N-acetyl-D,L-penicillamine (SNAP) led to a dose- and time-dependent increase in cell-associated active and latent TGF-beta1, as assessed by quantitative immunocytochemistry for active TGF-beta1 vs. LAP and partially validated by western blot analysis. Treatment with the sGC inhibitor 1,H-[1,2,4]oxadiazole[4,3-a]quinoxalon-1-one (ODQ) reduced both active and latent TGF-beta1 dose-dependently. SNAP, in the presence or absence of ODQ or the MAP kinase inhibitors, did not affect steady-state TGF-beta1 mRNA levels. Treatment with inhibitors specific for JNK1/2, ERK1/2, and p38 MAP kinases suppressed SNAP-induced active and latent TGF-beta1. Treatment with the cell-permeable cGMP analog 8-Br-cGMP increased both active and latent TGF-beta1. However, TGF-beta1 activation induced by 8-Br-cGMP was not blocked by MAP kinase inhibitors. Our findings suggest that NO activates latent TGF-beta1 via activation of sGC and generation of cGMP and separately via MAP kinase activation, and may shed insight into the mechanisms by which both cGMP production and MAP kinase activation enhance wound healing.

Reply

We agree with the commentary by Drs Chignard and Poupon, particularly with respect to the point that FXR activation suppresses inducible nitric oxide synthase (iNOS) mRNA level induced by lipopolysaccharide (LPS) probably in an nuclear factor (NF)-κB–independent manner. LPS activates not only NF-κB, but also other transcriptional factors, such as AP-1. iNOS is a target gene of both NF-κB and AP-1, so it is possible that FXR activation may suppress iNOS expression induced by LPS in an AP-1 dependent manner. It is known that several nuclear receptors such as GR, PPAR-γ, and LXR can antagonize the activity of NF-κB (Genes Dev 2000;14:2314–2329; Nature 2005;437:759–763; Mol Cell 2007;25:57–70). Our study expands it by adding FXR to this list. In addition, nuclear receptors antagonize NF-κB signaling through different mechanisms (Genes Dev 2000;14:2314–2329; Nature 2005;437:759–763; Mol Cell 2007;25:57–70). Our study (Hepatology 2008;48:1632–1643) indicates that FXR does not affect p65 nuclear translocation but suppresses p65 transactivity by decreasing its DNA binding. It would be interesting to further determine by which mechanism FXR suppresses NF-κB signaling in the future research work. Drs Chignard and Poupon highlight FXR agonists as a potential therapy tools for inflammatory biliary diseases. We would like to point out that they did not mention another critical role of FXR in liver regeneration (Science 2006;312:233–236), which constitutes another level of defense mechanism by which FXR can suppress liver inflammation. Recent studies have indicated that impaired liver regeneration may lead to continuing cell death, which results in chronic inflammation in the liver. Several components released from dead cells are known to activate Kupffer cells (liver resident macrophages) and other cells to induce the expression of proinflammatory cytokines (Cell Res 2008;18:334–342; Science 2009;323:1722–1725). In summary, FXR is a novel cell protector that not only controls the bile acid levels but also directly participates in several cellular defense pathways (Cell Res 2008;18:1087–1095). Finally, our other studies also suggest that FXR agonist ligands offer possible therapies for other liver diseases such as fibrosis and hepatocellular carcinoma (Cancer Res 2007;67:863–867). Hopefully, in the near future, FXR agonists can be developed into drugs for clinical treatment of different liver diseases. Targeting Farnesoid X Receptor in Hepatic and Biliary Inflammatory DiseasesGastroenterologyVol. 137Issue 2PreviewWang YD, Chen WD, Wang M, et al. (Department of Gene Regulation and Drug Discovery, Beckman Research Institute, City of Hope National Medical Center, Duarte, California). Farnesoid X receptor antagonizes nuclear factor kappaB in hepatic inflammatory response. Hepatology 2008;48:1632–1643. Full-Text PDF

Targeting Farnesoid X Receptor in Hepatic and Biliary Inflammatory Diseases

Targeting Farnesoid X Receptor in Hepatic and Biliary Inflammatory Diseases

Anti-cataractogenic effect of curcumin and aminoguanidine against selenium-induced oxidative stress in the eye lens of Wistar rat pups: An in vitro study using isolated lens

The aim of this study was to investigate whether curcumin and aminoguanidine (AG) prevent selenium-induced cataractogenesis in vitro. On postpartum day 8, transparent isolated lens were incubated in 24 well plates containing Dulbecco's Modified Eagle Medium (DMEM). Isolated lens of group I were incubated with DMEM medium alone. Group II: lenses incubated in DMEM containing 100 μM sodium selenite; group III: lenses incubated in DMEM containing 100 μM sodium selenite and 100 μM curcumin; group IV: lenses incubated in DMEM containing 100 μM sodium selenite and 200 μM curcumin; group V: lenses incubated in DMEM containing 100 μM sodium selenite and 100 μM AG; group V: lenses incubated in DMEM containing 100 μM sodium selenite and 200 μM AG. On day 12, cataract development was graded using an inverted microscope and the lenses were analyzed for enzymic as well as non-enzymic antioxidants, lipid peroxidation (LPO), nitric oxide (NO), superoxide anion (O 2 −) and hydroxyl radical generation (OH ) and inducible nitric oxide synthase (iNOS) activity by Western blotting and RT-PCR. All control lenses in group I were clear (0). In groups II and III, all isolated lenses developed cataract with variation in levels (+++ or ++), whereas isolated lenses from groups IV, V and VI were clear (0). In agreement to this, a decrease in antioxidants and increased free radical generation and also iNOS expression were observed in selenium exposed lenses when compared to other groups. AG (100 μM) was found to be more effective in anti-cataractogenic effect than curcumin (200 μM). Curcumin and AG suppressed selenium-induced oxidative stress and cataract formation in isolated lens from Wistar rat pups, possibly by inhibiting depletion of enzymic as well as non-enzymic antioxidants, and preventing uncontrolled generation of free radicals and also by inhibiting iNOS expression. Our results implicate a major role for curcumin and AG in preventing cataractogenesis in selenite-exposed lenses, wherein AG was found to be more potent.

Chloride channel blockers inhibit iNOS expression and NO production in IFNγ-stimulated microglial BV2 cells

Microglial cells play an important role during neuroinflammation in the central nervous system. Among other factors, activated microglia produce nitric oxide (NO), which is toxic to neurons and excessive microglial activation and NO production contribute to the pathology of neurodegenerative diseases. Chloride channels have previously been shown to participate in microglial activation. Here we investigate the effects of established chloride channel blockers with different chemical structures on interferon-γ (IFNγ)-induced activation of the murine microglial cell line, BV2. IFNγ-induced NO production was effectively reduced by NPPB, IAA-94, tamoxifen, NS3728 and NS1652, with NS1652 being the most potent. In contrast, DIDS reduced NO production only at cytotoxic concentrations. Furthermore, NS1652 reduced the protein and mRNA levels of inducible nitric oxide synthase (iNOS), without altering STAT1 phosphorylation. These observations suggest a microglial chloride conductance as a critical permissive factor downstream in the IFNγ-induced iNOS cascade. The nature of the underlying channel is unknown, but the pharmacological profile appears incompatible with the involvement of the volume activated anion conductance (VRAC).

JANEX-1, a JAK3 inhibitor, protects pancreatic islets from cytokine toxicity through downregulation of NF-κB activation and the JAK/STAT pathway

JANEX-1/WHI-P131, a selective Janus kinase 3 (JAK3) inhibitor, has been shown to delay the onset of diabetes in the NOD mouse model. However, the molecular mechanism by which JANEX-1 protects pancreatic β-cells is unknown. In the current study, we investigated the role of JANEX-1 on interleukin (IL)-1β and interferon (IFN)-γ-induced β-cell damage using isolated islets. JANEX-1-pretreated islets showed resistance to cytokine toxicity, namely suppressed nitric oxide (NO) production, reduced inducible form of NO synthase (iNOS) expression, and decreased islet destruction. The molecular mechanism by which JANEX-1 inhibits iNOS expression was mediated through suppression of the nuclear factor κB (NF-κB) and JAK/signal transducer and activator of transcription (STAT) pathways. Islets treated with the cytokines downregulated the protein levels of suppressor of cytokine signaling (SOCS)-1 and SOCS-3, but pretreatment with JANEX-1 attenuated these decreases. Additionally, islets from JAK3 −/− mice were more resistant to cytokine toxicity than islets from control mice. These results demonstrate that JANEX-1 protects β-cells from cytokine toxicity through suppression of the NF-κB and JAK/STAT pathways and upregulation of SOCS proteins, suggesting that JANEX-1 may be used to preserve functional β-cell mass.

Hepatic protection by glycyrrhizin and inhibition of iNOS expression in concanavalin A-induced liver injury in mice

In this study, the possible protective effect of glycyrrhizin (GL), an active compound derived from licorice root, was examined on T cell-mediated liver injury in mice. Mice were subjected to liver injury by intravenous injection of concanavalin A (Con A). They had been treated with GL (i.p.) 30 min before the injection. Liver injury was estimated by measuring serum levels of alanine aminotransaminase (ALT) and aspartate aminotransaminase (AST), and by examining liver sections with hematoxylin-eosin staining. Expression of inducible nitric oxide synthase (iNOS) mRNA and protein in the liver was determined by reverse transcription polymerase chain reaction (RT-PCR) and Western blotting. Serum transaminases and hepatic iNOS levels increased with time after Con A treatment. Expression of iNOS mRNA in the liver was elevated for up to 8 h, and at 8 h, GL (ED(50): 10.5 mg/kg) suppressed the increases in AST and ALT in response to Con A. An increase in iNOS mRNA expression and protein was inhibited by treatment with GL. Furthermore, GL inhibited cell infiltration and the degeneration of hepatocytes in the liver of Con A-treated mice. The present study suggests that the prevention by GL of Con A-induced hepatitis is due partly to the modulation of hepatic iNOS induction and of degeneration of hepatocytes.

Suppression of lipopolysaccharide-stimulated inducible nitric oxide synthase (iNOS) expression by a novel humulene derivative in macrophage cells

In our previous study, we reported the isolation of a novel humulene derivative, 5-hydroxyzerumbone, from Zingiber zerumbet and its inhibitory activity on lipopolysaccharide (LPS)-induced nitric oxide (NO) production in RAW 264.7 mouse macrophage cells. This study was performed to examine its mechanism of action on the regulation of NO production. 5-Hydroxyzerumbone inhibited the expressions of iNOS mRNA and protein in a concentration-dependent manner. Treatment with 5-hydroxyzerumbone also induced the expression of heme oxygenase-1 (HO-1) in macrophage cells. In addition, 5-hydroxyzerumbone inhibited LPS-induced transcriptional activation of NF-κB, indicating that regulation of NF-κB activity might be involved in the inhibition of NO production by 5-hydroxyzerumbone. 5-Hydroxyzerumbone, however, did not affect the degradation of IκB-α and the activation of p38 and ERK in LPS-treated cells. Taken together, these results suggest that down-regulation of LPS-induced NO production by 5-hydroxyzerumbone is mediated by the suppression of iNOS expression through the modulation of NF-κB activation and HO-1 induction in macrophage cells.

Dexamethasone attenuated endotoxin-induced acute lung injury through inhibiting expression of inducible nitric oxide synthase

Application of glucocorticoids in sepsis or severe infection is disputable in clinic. In this experiment, we studied the effect of dexamethasone on nitric oxide synthases and whether dexamethasone could attenuate endotoxin-induced acute lung injury (ALI). SD rats received 5 mg/kg lipopolisaccharide (LPS) injection. Then arterial oxygen partial pressure (PaO2), lung histology, lung tissue nitric oxide (NO) production and expression of nitric oxide synthases (NOS) were detected at 0.5, 1, 2, 3 or 4 h after LPS injection. PaO2 and lung injury deteriorated upon time. Production of NO in lung tissue increased significantly particularly in the first two hours, and this change was mainly due to the over-expression of inducible NOS (iNOS), but not endothelial NOS (eNOS). Furthermore, a tight positive correlation was observed between lung injury score (LIS) and NO production level in lung tissue. Dexamethasone could ameliorate PaO2 and lung damage evidently, which were paralleled by significant decreases in the production of NO and in the expression of iNOS mRNA. In conclusion, dexamethasone could effectively attenuate endotoxin-induced lung injury through inhibiting iNOS expression and activation in the very early stage of ALI.

NF-κB Mediates Mitogen-Activated Protein Kinase Pathway-Dependent iNOS Expression in Human Melanoma

Tumor expression of inducible nitric oxide synthase (iNOS) predicts poor outcomes for melanoma patients. We have reported the regulation of melanoma iNOS by the mitogen-activated protein kinase (MAPK) pathway. In this study, we test the hypothesis that NF-kappaB mediates this regulation. Western blotting of melanoma cell lysates confirmed the constitutive expression of iNOS. Western blot detected baseline levels of activated nuclear extracellular signal-regulated kinase and NF-kappaB. Indirect immunofluorescence confirmed the presence of NF-kappaB p50 and p65 in melanoma cell nuclei, with p50 being more prevalent. Electrophoretic mobility shift assay demonstrated baseline NF-kappaB activity, the findings confirmed by supershift analysis. Treatment of melanoma cells with the MEK inhibitor U0126 decreased NF-kappaB binding to its DNA recognition sequence, implicating the MAPK pathway in NF-kappaB activation. Two specific NF-kappaB inhibitors suppressed iNOS expression, demonstrating regulation of iNOS by NF-kappaB. Several experiments indicated the presence of p50 homodimers, which lack a transactivation domain and rely on the transcriptional coactivator Bcl-3 to carry out this function. Bcl-3 was detected in melanoma cells and co-immunoprecipitated with p50. These data suggest that the constitutively activated melanoma MAPK pathway stimulates activation of NF-kappaB hetero- and homodimers, which, in turn, drive iNOS expression and support melanoma tumorigenesis.

Liquid Perfluorochemical Inhibits Inducible Nitric Oxide Synthase Expression and Nitric Oxide Formation in Lipopolysaccharide-Treated RAW 264.7 Macrophages

Partial liquid ventilation with various types of perfluorocarbon (PFC) has been shown to be beneficial in treating acute lung injury, a clinical outcome that may involve the anti-inflammatory activity of PFC. FC-77 is a type of PFC with relatively higher vapor pressure and evaporative loss than other PFCs during partial liquid ventilation. Overproduction of nitric oxide (NO) by inducible nitric oxide synthase (iNOS) has been proposed to play a crucial role in the pathogenesis of inflammatory diseases. However, whether the iNOS/NO pathway is affected by FC-77 is unknown. Thus, the aim of this study was to investigate whether FC-77 inhibits iNOS expression and NO production in lipopolysaccharide (LPS)–treated RAW 264.7 macrophages. We found that treatment with FC-77 significantly attenuated LPS-induced iNOS expression/activity and production of NO and reactive oxygen species (ROS). FC-77 also attenuated LPS-induced pro-inflammatory cytokine formation, but enhanced interleukin-10 production. Furthermore, the LPS-induced degradation of cytosolic IκB-α and activation of nuclear transcription factor-κB (NF-κB) were also inhibited by FC-77. In conclusion, the present study is the first to demonstrate that FC-77 decreases LPS-induced NO production in macrophages, which may be associated with the suppression of pro-inflammatory cytokines, and ROS production, as well as NF-κB activation. These results also provide a novel explanation for its anti-inflammatory activity.

Overexpression of SIRT1 Protects Pancreatic β-Cells Against Cytokine Toxicity by Suppressing the Nuclear Factor-κB Signaling Pathway

OBJECTIVE—SIRT1, a class III histone/protein deacetylase, is known to interfere with the nuclear factor-κB (NF-κB) signaling pathway and thereby has an anti-inflammatory function. Because of the central role of NF-κB in cytokine-mediated pancreatic β-cell damage, we postulated that SIRT1 might work in pancreatic β-cell damage models.RESEARCH DESIGN AND METHODS—RINm5F (RIN) cells or isolated rat islets were treated with interleukin-1β and interferon-γ. SIRT1 was activated by resveratrol, a pharmacological activator, or ectopic overexpression. The underlying mechanisms of SIRT1 against cytokine toxicity were further explored.RESULTS—Treatment of RIN cells with cytokines induced cell damage, and this damage was well correlated with the expression of the inducible form of nitric oxide (NO) synthase (iNOS) and NO production. However, SIRT1 overexpression completely prevented cytokine-mediated cytotoxicity, NO production, and iNOS expression. The molecular mechanism by which SIRT1 inhibits iNOS expression appeared to involve the inhibition of the NF-κB signaling pathway through deacetylation of p65. In addition, SIRT1 activation by either resveratrol or adenoviral-directed overexpression of SIRT1 could prevent cytokine toxicity and maintain normal insulin-secreting responses to glucose in isolated rat islets.CONCLUSIONS—This study will provide valuable information not only into the mechanisms underlying β-cell destruction but also into the regulation of SIRT1 as a possible target to attenuate cytokine-induced β-cell damage.

Extracts of Magnoliae flos inhibit inducible nitric oxide synthase via ERK in human respiratory epithelial cells

Nitric oxide (NO) is a marker of pulmonary inflammation. In asthma, the levels of exhaled NO are elevated and the source of this increased NO is inducible nitric oxide synthase (iNOS) within airway epithelial cells. Epimagnolin and fargesin are compounds isolated from the ethanol extract of Magnoliae flos, the seed of the Magnolia plant and are used to treat nasal congestion, headache and sinusitis in Asian countries. This study investigated whether epimagnolin and fargesin inhibit extracellular signal-regulated kinase (ERK) activation and decrease iNOS expression and NO production in stimulated human respiratory epithelial cells. An immortal Type II alveolar cell line of human origin (A549) was stimulated by cytomix (CM), composed of IL-1β, TNF-α and IFN-γ, with or without concurrent exposure to M. flos extract (epimagnolin or fargesin). CM-induced levels of NO production, iNOS expression and ERK activation were evaluated. A549 cells stimulated with CM showed increases in iNOS mRNA and protein expression, and NO synthesis. However, treatment with epimagnolin or fargesin decreased levels of iNOS mRNA and protein expression, and NO synthesis. CM stimulated a rapid increase in the activity of ERK, whereas epimagnolin and fargesin inhibited ERK phosphorylation. Epimagnolin and fargesin inhibit iNOS expression and decrease production of NO via ERK pathway in cytokine-stimulated human respiratory epithelial cells.

Apoptosis‐Inducing High .NO Concentrations Are Not Sustained Either in Nascent or in Developed Cancers

Nitric oxide ((.)NO) induces apoptosis at high concentrations by S-nitrosating proteins such as glyceraldehyde-3-phosphate dehydrogenase. This literature analysis revealed that failure to sustain high (.)NO concentrations is common to all cancers. In cervical, gastric, colorectal, breast, and lung cancer, the cause of this failure is the inadequate expression of inducible nitric oxide synthase (iNOS), resulting from the inhibition of iNOS expression by TGF-beta1 at the mRNA level. In bladder, renal, and prostate cancer, the reason for the insufficient (.)NO levels is the depletion of arginine, resulting from arginase overexpression. Arginase competes with iNOS for arginine, catalyzing its hydrolysis to ornithine and urea. In gliomas and ovarian sarcomas, low (.)NO levels are caused by inhibition of iNOS by N-chlorotaurine, produced by infiltrating neutrophils. Stimulated neutrophils express myeloperoxidase, catalyzing H2O2 oxidation of Cl- to HOCl, which N-chlorinates taurine at its concentration of 19 mM in neutrophils. In squamous cell carcinomas of the skin, ovarian cancers, lymphomas, Hodgkin's disease, and breast cancers, low (.)NO concentrations arise from the inhibition of iNOS by N-bromotaurine, produced by eosinophil-peroxidase-expressing infiltrating eosinophils. Eosinophil peroxidase catalyzes the H2O2 oxidation of Br- to HOBr, which N-brominates taurine to N-bromotaurine at its concentration of 15 mM in eosinophils. In microvascularized tumors, the (.)NO concentration is further depleted; (.)NO is rapidly consumed by red blood cells (RBCs) through S-nitrosation of RBC glutathione and hemoglobin, and by oxidation to nitrate by RBC oxyhemoglobin. Angiogenesis-inhibiting antibodies are currently used to treat cancers; their mode of action is not, as previously thought, reduction of the tumor O2 or nutrient supply. They actually decrease the loss of (.)NO to RBCs.

Withaferin A inhibits iNOS expression and nitric oxide production by Akt inactivation and down-regulating LPS-induced activity of NF-κB in RAW 264.7 cells

Induction of inducible nitric oxide synthase (iNOS) expression and nitric oxide (NO) production is thought to have beneficial immunomodulatory effects in acute and chronic inflammatory disorders. In Raw 264.7 cells stimulated with lipopolysaccharide (LPS) to mimic inflammation, withaferin A inhibited LPS-induced expression of both iNOS protein and mRNA in a dose-dependent manner. To investigate the mechanism by which withaferin A inhibits iNOS gene expression, we examined activation of mitogen-activated protein kinases (MAPKs) and Akt in Raw 264.7 cells. We did not observe any significant changes in the phosphorylation of p38 MAPK in cells treated with LPS alone or LPS plus withaferin A. However, LPS-induced Akt phosphorylation was markedly inhibited by withaferin A, while the phosphorylation of p42/p44 extracellular signal-regulated kinases (ERKs) was slightly inhibited by withaferin A treatment. Withaferin A prevented IκB phosphorylation, blocking the subsequent nuclear translocation of nuclear factor-κB (NF-κB) and inhibiting its DNA binding activity. LPS-induced p65 phosphorylation, which is mediated by extracellular signal-regulated kinase (ERK) and Akt pathways, was attenuated by withaferin A treatment. Moreover, LPS-induced NO production and NF-κB activation were inhibited by SH-6, a specific inhibitor of Akt. Taken together, these results suggest that withaferin A inhibits inflammation through inhibition of NO production and iNOS expression, at least in part, by blocking Akt and subsequently down-regulating NF-κB activity.

Effects of Dexmedetomidine on Regulating Endotoxin-Induced Up-Regulation of Inflammatory Molecules in Murine Macrophages

Effects of dexmedetomidine on regulating endotoxin-induced upregulation of inflammatory molecules were elucidated. Murine macrophages (RAW264.7 cells) were treated with lipopolysaccharide (LPS, 100 ng/mL), LPS plus dexmedetomidine (0.01, 0.1, 1, 10, or 100 microM), LPS plus dexmedetomidine plus yohimbine, or LPS plus dexmedetomidine plus idazoxan. The dosages of dexmedetomidine were chosen to correspond to 1, 10, 100, and 1000 times of clinically relevant dosages (i.e., 0.01-0.1 microM). The levels of inducible nitric oxide synthase (iNOS)/nitric oxide (NO), cyclooxygenase-2 (COX-2)/prostaglandin E2 (PGE2), tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), IL-6, and IL-10 were measured. Dexmedetomidine at 0.01 microM did not affect iNOS expression and NO production in activated macrophages. At 1 microM, dexmedetomidine significantly inhibited iNOS expression (up to 20.8% +/- 4.7%) and NO production (up to 26.2% +/- 6.8%). In contrast, dexmedetomidine at 100 microM significantly enhanced iNOS expression (up to 31.5% +/- 7.5%) and NO production (up to 34.9% +/- 5.6%). The effects of dexmedetomidine on COX-2 expression and the production of PGE2, TNF-alpha, IL-1beta, IL-6, and IL-10 paralleled the effects of dexmedetomidine on iNOS. Moreover, these effects were significantly reversed by both of the alpha2-adrenergic receptor antagonists, yohimbine, and idazoxan. Dexmedetomidine at clinically relevant dosages did not significantly affect the expression of inflammatory molecules in activated macrophages. In contrast, dexmedetomidine at dosages higher than clinically relevant ones posted small but significant biphasic effects (inhibiting, then enhancing) on regulating the expression of inflammatory molecules, possibly through the alpha2-adrenergic receptors. However, as the magnitude of changes was relatively small, these effects may not be clinically significant.

Calcineurin inhibitors down-regulate iNOS expression by destabilising mRNA

In inflammation, bacterial products and pro-inflammatory cytokines induce the expression of inducible nitric oxide synthase (iNOS) and formation of high amounts of nitric oxide (NO). In a number of inflammatory diseases NO has pro-inflammatory and cytotoxic effects. The aim of the present study was to investigate the effects of immunosuppressive drugs cyclosporin A (CsA), tacrolimus (FK-506) and pimecrolimus on NO production through iNOS pathway in activated macrophages and fibroblasts. Calcineurin inhibitors (CsA, FK-506 and pimecrolimus) inhibited NO production and iNOS expression in a concentration-dependent manner, CsA being more potent than FK-506 and pimecrolimus. No effect on the activation or activity of the transcription factors nuclear factor-κB (NF-κB) and signal transducer and activator of transcription 1 (STAT-1) was found. CsA, FK-506 and pimecrolimus did not reduce iNOS mRNA levels when measured 6–8 h after the inflammatory stimulus, but significantly lower levels of iNOS mRNA were found after 24 h incubation. Also, in cells transfected with a luciferase gene under the control of 3′ untranslated region (3′UTR) of iNOS, CsA reduced luciferase activity. In conclusion, the results suggest that calcineurin inhibitors cyclosporin A, tacrolimus (FK-506) and pimecrolimus inhibit iNOS expression and NO production in response to inflammatory stimuli by enhancing the decay of iNOS mRNA by a 3′UTR-dependent manner. The findings add our knowledge on the anti-inflammatory effects of CsA, FK-506 and pimecrolimus, and suggest that calcineurin may have a role in the regulation of iNOS expression.

Anti‐inflammatory effects of dimemorfan on inflammatory cells and LPS‐induced endotoxin shock in mice

Dimemorfan (a sigma1 receptor agonist) showed neuroprotective properties in animal models of inflammation-mediated neurodegenerative conditions, but its effects on inflammatory cells and systemic inflammation remain unclear. The effects of dimemorfan on phorbol-12-myristate-13-acetate (PMA)- and N-formyl-methionyl-leucyl-phenylalanine (fMLP)- induced neutrophils and lipopolysaccharide (LPS)-activated microglial cells, as well as LPS-induced endotoxin shock in mice were elucidated. Dimemorfan decreased PMA- and fMLP-induced production of reactive oxygen species (ROS) and CD11b expression in neutrophils, through mechanisms independent of sigma1 receptors, possibly by blocking ROS production and G-protein-mediated intracellular calcium increase. Dimemorfan also inhibited LPS-induced ROS and nitric oxide (NO) production, as well as that of monocyte chemoattractant protein-1 and tumour necrosis factor-alpha (TNF-alpha), by inhibition of NADPH oxidase (NOX) activity and suppression of iNOS up-regulation through interfering with nuclear factor kappa-B (NF-kappaB) signalling in microglial cells. Treatment in vivo with dimemorfan (1 and 5 mg kg(-1), i.p., at three successive times after LPS) decreased plasma TNF-alpha, and neutrophil infiltration and oxidative stress in the lung and liver. Our results suggest that dimemorfan acts via sigma1 receptor-independent mechanisms to modulate intracellular calcium increase, NOX activity, and NF-kappaB signalling, resulting in inhibition of iNOS expression and NO production, and production of pro-inflammatory cytokines. These effects may contribute its anti-inflammatory action and protective effects against endotoxin shock in mice.

Upregulation of estrogen receptor α and mediation of 17β-estradiol vasoprotective effects via estrogen receptor α in basilar arteries in rats after experimental subarachnoid hemorrhage

The authors previously demonstrated that 17beta-estradiol benzoate (E2) treatment prevents subarachnoid hemorrhage (SAH)-induced cerebral vasospasm and preserves endothelial nitric oxide synthase (eNOS) in male rats. Changes in the expression of estrogen receptor (ER) subtypes ERalpha and -beta and their roles in the E2-mediated preservation of eNOS in SAH remain unknown. In the present study the effects of SAH on the expression of ERalpha and -beta in the cerebral arteries were clarified, and the receptor roles in the E2-mediated preservation of eNOS expression in SAH were differentiated. A 2-hemorrhage SAH model was induced by 2 autologous blood injections into the cisterna magna of adult male rats. The effect of SAH on ERalpha and -beta expression was evaluated. Other rats subcutaneously received implanted Silastic tubes containing corn oil with E2 and daily injections of various doses of an ERalpha- (methyl-piperidinopyrazole [MPP]) or ERbeta-selective antagonist (R,R-tetrahydrochrysene) after the first hemorrhage. The protein levels of ERalpha, ERbeta, eNOS, and inducible nitric oxide synthase (iNOS) from basilar arteries were examined using Western blot analysis, and their mRNAs were evaluated by reverse transcription-polymerase chain reaction. The ERalpha but not the ERbeta was upregulated in the basilar artery after SAH. Treatment with MPP eliminated E2-mediated effects in SAH, relieved cerebral vasospasm, preserved eNOS expression, and suppressed iNOS expression. Estrogen receptor alpha is upregulated in the basilar artery after SAH. Note that E2 exerts its protective effects through ERalpha-dependent pathways to relieve cerebral vasospasm and preserve eNOS expression. A selective ERalpha agonist may be the drug of choice for the treatment of patients with SAH.

HO-1 and JAK-2/STAT-1 signals are involved in preferential inhibition of iNOS over COX-2 gene expression by newly synthesized tetrahydroisoquinoline alkaloid, CKD712, in cells activated with lipopolysacchride

We found that CKD712, an S enantiomer of YS49, strongly inhibited inducible nitric oxide synthase (iNOS) and NO induction but showed a weak inhibitory effect on cyclooxygenase-2 (COX-2) and PGE 2 induction in LPS-stimulated RAW 264.7 cells. We, therefore, investigated the molecular mechanism(s) responsible for this by using CKD712 in LPS-activated RAW264.7 cells. Treatment with either SP600125, a specific JNK inhibitor or TPCK, a NF-κB inhibitor, but neither ERK inhibitor PD98059 nor p38 inhibitor SB203580, significantly inhibited LPS-mediated iNOS and COX-2 induction. CKD712 inhibited NF-κB (p65) activity and translocation but failed to prevent JNK activation. However, AG490, a specific JAK-2/STAT-1 inhibitor, efficiently prevented LPS-mediated iNOS induction but not the induction of COX-2, and CKD712 completely blocked STAT-1 phosphorylation by LPS, suggesting that the NF-κB and JAK-2/STAT-1 pathways but not the JNK pathway are important for CKD712 action. Interestingly, CKD712 induced heme oxygenase 1 (HO-1) gene expression in LPS-treated cells. LPS-induced NF-κB and STAT-1 activation was partially prevented by HO-1 overexpression. Furthermore, HO-1 siRNA partly reversed not only the LPS-induced NF-κB activation and STAT-1 phosphorylation but also inhibition of these actions by CKD 712. Additionally, silencing HO-1 by siRNA prevented CKD712 from inhibiting iNOS expression but not COX-2. When examined plasma NO and PGE 2 levels and iNOS and COX-2 protein levels in lung tissues of mice injected with LPS (10 mg/kg), pretreatment with CKD712 greatly prevented NO and iNOS induction in a dose-dependent manner and slightly affected PGE 2 and COX-2 production as expected. Taken together, we conclude that inhibition of JAK-2/STAT-1 pathways by CKD 712 is critical for the differential inhibition of iNOS and COX-2 by LPS in vitro and in vivo where HO-1 induction also contributes to this by partially modulating JAK-2/STAT-1 pathways.