Induction Of iNOS mRNA Research Articles

Expression of human inducible nitric oxide synthase in response to cytokines is regulated by hypoxia-inducible factor-1

The production of nitric oxide (NO) by inducible NO synthase (iNOS) and the regulation of gene expression by hypoxia-inducible factors (HIFs) are important for many aspects of human cell biology. However, little is known about whether iNOS expression is controlled by HIFs in human cells. Stimulation of A549 human lung epithelial cells with cytokines (TNF, IL-1 and IFNγ) increased the nuclear accumulation of HIF-1 in normoxic conditions. Activation of HIF-1 by hypoxia or CoCl2 was not sufficient to induce iNOS expression. However, pharmacological inhibition of HIF-1 reduced the induction of iNOS expression in A549 cells and primary human astrocytes. Moreover, elimination of HIF-1α expression and activity by CRISPR/Cas9 gene editing significantly reduced the induction of human iNOS gene promoter, mRNA and protein expression by cytokine stimulation. Three putative hypoxia response elements (HRE) are present within the human iNOS gene promoter and elimination of an HRE at −4981 bp reduced the induction of human iNOS promoter activity in response to cytokine stimulation. These findings establish an important role for HIF-1α in the induction of human iNOS gene expression in response to cytokine stimulation.

Regulating Action of in Vitro Hepatitis C Virus Infection on Interferon-Induced Interferon Stimulating Genes in Murine Macrophages

Objectives: The objective of this study was to determine the in vitro regulating effect of Hepatitis C virus (HCV) infection on interferon stimulating genes (ISGs) stimulated by IFN-ү and Imiquimod (TLR7 ligand) on murine macrophages. iNOS and STAT1 were measured by RT-PCR, and immunoblotting (IB). Nitric oxide production was measured by Griess Reagent Assay. Objectives: The objective of this study was to determine the in vitro regulating effect of Hepatitis C virus (HCV) infection on interferon stimulating genes (ISGs) stimulated by IFN-ү and Imiquimod (TLR7 ligand) on murine macrophages. iNOS and STAT1 were measured by RT-PCR, and immunoblotting (IB). Nitric oxide production was measured by Griess Reagent Assay. Results: HCV inhibits IFN induced iNOS mRNA and also protein expression. HCV significantly reduced IFN-ү induced ISGs (iNOS mRNA, iNOS protein, s727-STAT1, tyr701- STAT1). Conclusion: These results indicate that in vitro hepatitis C virus infection is involved in the regulation of IFN-ү induced ISGs in the levels of gene and protein expression of iNOS and STAT1 transcription factors.: HCV inhibits IFN induced iNOS mRNA and also protein expression. HCV significantly reduced IFN-ү induced ISGs (iNOS mRNA, iNOS protein, s727-STAT1, tyr701- STAT1). Conclusion: These results indicate that in vitro hepatitis C virus infection is involved in the regulation of IFN-ү induced ISGs in the levels of gene and protein expression of iNOS and STAT1 transcription factors.

Enzyme-Treated Asparagus Extract Prevents Hydrogen Peroxide-Induced Pro-Inflammatory Responses by Suppressing p65 Nuclear Translocation in Skin L929 Fibroblasts

We recently reported that enzyme-treated asparagus extract (ETAS) attenuates hydrogen peroxide (H(2)0(2))-stimulated matrix metalloproteinase-9 expression in skin fibroblast L929 cells. To further elucidate the anti-aging effects of ETAS on skin, we examined whether ETAS has preventive effects on H202-induced pro-inflammatory responses of skin fibroblasts. H(2)0(2) induced Ser536 phosphorylation and nuclear accumulation of nuclear factor-κB (NF-κB) p65, and increased the mRNA levels .of interleukin-12α (IL-12α)-and inducible nitric oxide synthase (iNOS) in L929 cells. Pretreatment of the cells with JSH-23, an inhibitor of NF-κB nuclear translocation, abolished the H(2)(0(2)-induced expression of IL-12α and iNOS, indicating that the increased transcription is regulated by p65. The H(2)0(2)-stimulated nuclear accumulation of p65 and-induction of IL12a and iNOS mRNA were significantly attenuated after pretreatment with ETAS for 3 h, and these responses were completely abolished when the duration was extended to 24 h. However, ETAS did not affect the H(2)0(2)-stimulated degradation of IκBα and phosphorylation of p65. On the other hand, ETAS treatment for 24 h resulted in decreased protein levels of importin-α. These results suggest that ETAS prevents pro-inflammatory responses by suppressing the p65 nuclear translocation in skin fibroblasts induced by H202.

Influence of Culture Medium on Production of Nitric Oxide and Expression ofInducible Nitric Oxide Synthase by Activated Macrophages In Vitro

Activated macrophage phenotypes were influenced by the culture medium; a murine macrophage-like cell line, J774.1/JA-4, expresses different activated-macrophage phenotypes induced by lipopolysaccharide (LPS) and/or interferon-γ (IFN-γ) when the cells are incubated in either Ham’s F-12 medium (F-12) or Dulbecco’s modified Eagle medium (DMEM). Among these phenotypes, NO production and iNOS expression are the most remarkably influenced by the medium; the induction of iNOS mRNA and iNOS protein is higher in DMEM than in F-12, but NO production by activated macrophages is less in DMEM than in F-12. These results suggest that the interpretation of the experimental results requires consideration of the possibility that the differences obtained by different laboratories were caused by the culture medium used.

Antileishmanial activity of essential oil and 6,7-dehydroroyleanone isolated from Tetradenia riparia

Tetradenia riparia plant is used as a traditional medicine in Africa for the treatment of inflammatory and infectious diseases as like parasitic. Therapy for leishmaniasis caused by Leishmania (Leishmania) amazonensis specie often fails, and the conventional drugs are toxic, expensive, require a long period of treatment, and adverse effects are common. The alternative therapies using natural products are inexpensive and have few or any adverse reaction. These reasons are sufficient to investigate the new natural therapeutic for leishmaniasis. We evaluated the potential of the essential oil (TrEO) and 6,7-dehydroroyleanone (TrROY) isolated from T. riparia on L. (L.) amazonensis promastigote and amastigote forms, cytotoxicity on human erythrocytes and murine macrophages, nitric production and inducible nitric oxide synthase (iNOS) mRNA expression. TrEO was the most effective to promote the Leishmania promastigote death. After 72 h incubation, the lethal dose of TrEO and TrROY that promoted 50% Leishmania death (LD50) were 0.8 μg/mL and 3 μg/mL, respectively. TrEO and TrROY were not cytotoxic to human erythrocytes, but TrROY was toxic to murine macrophages resulting in a low selectivity index. The transmission electronic microscopy showed that TrEO (0.03 μg/mL) was able to modify the promastigote ultrastructures suggesting autophagy as chromatin condensation, blebbing, membranous profiles and nuclear fragmentation. Infected-macrophages treated with TrEO (0.03 μg/mL) or TrROY (10 μg/mL) had an infection index decreased in 65 and 48%. TrEO did not induce iNOS mRNA expression or nitrite production in macrophages infected with Leishmania. TrROY and mainly TrEO promoted the Leishmania death, and TrROY showed loss toxicity to erythrocytes cells. Other compounds derived from T. riparia and the essential oil could be explored to develop a new alternative treatment for leishmaniasis.

Ganglioside GD1a suppresses LPS-induced pro-inflammatory cytokines in RAW264.7 macrophages by reducing MAPKs and NF-κB signaling pathways through TLR4

Gangliosides, sialic acid-containing glycosphingolipids, have been considered to be involved in the development, differentiation, and function of nervous systems in vertebrates. However, the mechanisms for anti-inflammation caused by gangliosides are not clear. In this paper, we investigated the anti-inflammation effects of ganglioside GD1a by using RAW264.7 macrophages. Our data demonstrated that treatment of macrophages with lipopolysaccharide significantly increased the production of NO and pro-inflammatory cytokines. GD1a suppressed the induction of iNOS and COX-2 mRNA and protein expression and secretory pro-inflammatory cytokines in culture medium, such as TNFα, IL-1α and IL-1β. In addition, LPS-induced phosphorylation of mitogen-activating protein kinases and IκBα degradation followed by translocation of the NF-κB from the cytoplasm to the nucleus were attenuated after GD1a treatment. Furthermore, GD1a probably inhibited LPS binding to macrophages and LPS-induced accumulation between TLR4 and MyD88. Taken together, the results demonstrated that ganglioside GD1a inhibited LPS-induced inflammation in RAW 264.7 macrophages by suppressing phosphorylation of mitogen-activating protein kinases and activation of NF-κB through repressing the Toll-like receptor 4 signaling pathway.

Toll like receptor 4 (TLR4) mediates the stimulating activities of chitosan oligosaccharide on macrophages.

The in vivo and in vitro immunostimulating properties of chitosan oligosaccharide (COS) prepared by enzymatic hydrolysis of chitosan and the mechanisms mediating the effects were investigated. Our data showed that the highly active chitosanase isolated could hydrolyze chitosan to the polymerization degree of 3-8. The resulting COS was an efficient immunostimulator. COS markedly enhanced the proliferation and neutral red phagocytosis by RAW 264.7 macrophages. The production of nitric oxide (NO) and tumor necrosis factor alpha (TNF-α) by macrophages was significantly increased after incubation with COS. Oral administration of COS in mice could increase spleen index and serum immunoglobin G (IgG) contents. COS was labeled with FITC to study the pinocytosis by macrophages. Results showed that FITC-COS was phagocyted by macrophages and anti-murine TLR4 antibody completely blocked FITC-COS pinocytosis. RT-PCR indicated that COS treatment of macrophages significantly increased TLR4 and inducible nitric oxide synthase (iNOS) mRNA levels. When cells were pretreated with anti-murine TLR4 antibody, the effect of COS on TLR4 and iNOS mRNA induction was decreased. COS-induced NO secretion by macrophages was also markedly decreased by anti-murine TLR4 antibody pretreatment. In conclusion, the present study revealed that COS possesses potent immune-stimulating properties by activating TLR4 on macrophages.

Tauroursodeoxycholic acid reduces glial cell activation in an animal model of acute neuroinflammation

BackgroundBile acids are steroid acids found predominantly in the bile of mammals. The bile acid conjugate tauroursodeoxycholic acid (TUDCA) is a neuroprotective agent in different animal models of stroke and neurological diseases. However, the anti-inflammatory properties of TUDCA in the central nervous system (CNS) remain unknown.MethodsThe acute neuroinflammation model of intracerebroventricular (icv) injection with bacterial lipopolysaccharide (LPS) in C57BL/6 adult mice was used herein. Immunoreactivity against Iba-1, GFAP, and VCAM-1 was measured in coronal sections in the mice hippocampus. Primary cultures of microglial cells and astrocytes were obtained from neonatal Wistar rats. Glial cells were treated with proinflammatory stimuli to determine the effect of TUDCA on nitrite production and activation of inducible enzyme nitric oxide synthase (iNOS) and NFκB luciferase reporters. We studied the effect of TUDCA on transcriptional induction of iNOS and monocyte chemotactic protein-1 (MCP-1) mRNA as well as induction of protein expression and phosphorylation of different proteins from the NFκB pathway.ResultsTUDCA specifically reduces microglial reactivity in the hippocampus of mice treated by icv injection of LPS. TUDCA treatment reduced the production of nitrites by microglial cells and astrocytes induced by proinflammatory stimuli that led to transcriptional and translational diminution of the iNOS. This effect might be due to inhibition of the NFκB pathway, activated by proinflammatory stimuli. TUDCA decreased in vitro microglial migration induced by both IFN-γ and astrocytes treated with LPS plus IFN-γ. TUDCA inhibition of MCP-1 expression induced by proinflammatory stimuli could be in part responsible for this effect. VCAM-1 inmunoreactivity in the hippocampus of animals treated by icv LPS was reduced by TUDCA treatment, compared to animals treated with LPS alone.ConclusionsWe show a triple anti-inflammatory effect of TUDCA on glial cells: i) reduced glial cell activation, ii) reduced microglial cell migratory capacity, and iii) reduced expression of chemoattractants (e.g., MCP-1) and vascular adhesion proteins (e.g., VCAM-1) required for microglial migration and blood monocyte invasion to the CNS inflammation site. Our results present a novel TUDCA anti-inflammatory mechanism, with therapeutic implications for inflammatory CNS diseases.

Induction of nitric oxide synthase expression by lipopolysaccharide is mediated by calcium-dependent PKCα-β1 in alveolar epithelial cells

Nitric oxide (NO) plays an important role in innate host defense and inflammation. In response to infection, NO is generated by inducible nitric oxide synthase (iNOS), a gene product whose expression is highly modulated by different stimuli, including lipopolysaccharide (LPS) from gram-negative bacteria. We reported recently that LPS from Pseudomonas aeruginosa altered Na⁺ transport in alveolar epithelial cells via a suramin-dependent process, indicating that LPS activated a purinergic response in these cells. To further study this question, in the present work, we tested whether iNOS mRNA and protein expression were modulated in response to LPS in alveolar epithelial cells. We found that LPS induced a 12-fold increase in iNOS mRNA expression via a transcription-dependent process in these cells. iNOS protein, NO, and nitrotyrosine were also significantly elevated in LPS-treated cells. Ca²⁺ chelation and protein kinase C (PKCα-β1) inhibition suppressed iNOS mRNA induction by LPS, implicating Ca²⁺-dependent PKC signaling in this process. LPS evoked a significant increase of extracellular ATP. Because PKC activation is one of the signaling pathways known to mediate purinergic signaling, we evaluated the hypothesis that iNOS induction was ATP dependent. Although high suramin concentration inhibited iNOS mRNA induction, the process was not ATP dependent, since specific purinergic receptor antagonists could not inhibit the process. Altogether, these findings demonstrate that iNOS expression is highly modulated in alveolar epithelial cells by LPS via a Ca²⁺/PKCα-β1 pathway independent of ATP signaling.

EP2and EP4receptors on muscularis resident macrophages mediate LPS-induced intestinal dysmotility via iNOS upregulation through cAMP/ERK signals

Intestinal resident macrophages play an important role in gastrointestinal dysmotility by producing prostaglandins (PGs) and nitric oxide (NO) in inflammatory conditions. The causal correlation between PGs and NO in gastrointestinal inflammation has not been elucidated. In this study, we examined the possible role of PGE2 in the LPS-inducible inducible NO synthase (iNOS) gene expression in murine distal ileal tissue and macrophages. Treatment of ileal tissue with LPS increased the iNOS and cyclooxygenase (COX)-2 gene expression, which lead to intestinal dysmotility. However, LPS did not induce the expression of iNOS and COX-2 in tissue from macrophage colony-stimulating factor-deficient op/op mice, indicating that these genes are expressed in intestinal resident macrophages. iNOS and COX-2 protein were also expressed in dextran-phagocytized macrophages in the muscle layer. CAY10404, a COX-2 inhibitor, diminished LPS-dependent iNOS gene upregulation in wild-type mouse ileal tissue and also in RAW264.7 macrophages, indicating that PGs upregulate iNOS gene expression. EP2 and EP4 agonists upregulated iNOS gene expression in ileal tissue and isolated resident macrophages. iNOS mRNA induction mediated by LPS was decreased in the ileum isolated from EP2 or EP4 knockout mice. In addition, LPS failed to decrease the motility of EP2 and EP4 knockout mice ileum. EP2- or EP4-mediated iNOS expression was attenuated by KT-5720, a PKA inhibitor and PD-98059, an ERK inhibitor. Forskolin or dibutyryl-cAMP mimics upregulation of iNOS gene expression in macrophages. In conclusion, COX-2-derived PGE2 induces iNOS expression through cAMP/ERK pathways by activating EP2 and EP4 receptors in muscularis macrophages. NO produced in muscularis macrophages induces dysmotility during gastrointestinal inflammation.

Cytotoxic and anti-inflammatory cembranoids from the Vietnamese soft coral Lobophytum laevigatum

Four new cembranoids, namely laevigatol A–D ( 1– 4), and six known metabolites ( 5– 10), were isolated from the Vietnamese soft coral Lobophytum laevigatum. The structures of these compounds were elucidated by extensive spectroscopic analyses, and the absolute stereochemistry of 1 was determined using the modified Mosher’s method. Compounds 5, and 7– 10 exhibited cytotoxic activity against selected human cancer cell lines. Compounds 1, 2, 8, and 9 showed dose-dependent inhibitory effects on the TNFα-induced NF-κB transcriptional activity in Hep-G2 cells. Moreover, compounds 1, 2, 8, and 9 significantly inhibited the induction of COX-2 and iNOS mRNA dose-dependently, indicating that these compounds attenuated the synthesis of these transcripts at the transcriptional level.

Anti-inflammatory activity of constituents from Glechoma hederacea var. longituba

Rosmarinic acid, its analogues, and a phenolic compound were obtained from G. hederacea var. longituba. There were two new compounds, methyl isoferuloyl-7-(3,4-dihydroxyphenyl) lactate (1) and benzyl-4'-hydroxy-benzoyl-3'-O-β-D-glucopyranoside (4), and four known compounds (2, 3, 5 and 6). The structures of these compounds were determined on the basis of spectroscopic methods. Each compound was tested by NF-κB luciferase assay and three rosmarinic acid analogues inhibited NF-κB production and the induction of COX-2 and iNOS mRNA in HepG2 cells.

Sivelestat Suppresses iNOS Gene Expression in Proinflammatory Cytokine-Stimulated Hepatocytes

Recent evidence has indicated that sivelestat, a neutrophil elastase inhibitor, has liver-protective effects in a variety of liver injuries. Proinflammatory cytokines including interleukin (IL)-1β stimulate the induction of inducible nitric oxide synthase (iNOS) gene expression, leading to excess production of NO and resulting in liver damage. We hypothesized that inhibition of iNOS induction underlies the protective effects of sivelestat on the liver. The objective of this study was to investigate whether sivelestat directly influences iNOS induction in cultured hepatocytes, which is used as a simple in vitro injury model, and to determine the mechanism involved. Primary cultured rat hepatocytes were treated with IL-1β in the presence or absence of sivelestat. The induction of iNOS and its signaling pathway were analyzed. Sivelestat inhibited the induction of iNOS mRNA and its protein, followed by decreased production of NO. Transfection and iNOS gene antisense-transcript experiments revealed that sivelestat reduced the levels of iNOS mRNA at both the promoter activation and mRNA stabilization steps. However, sivelestat had no effects on the degradation of IκB and nuclear translocation of NF-κB subunit p65, although it moderately blocked the activation of NF-κB. In contrast, sivelestat blocked the upregulation of IL-1 receptor I through the inactivation of phosphatidylinositol 3-kinase/Akt. Delayed sivelestat addition experiments demonstrated that the destabilization of the iNOS mRNA contributed more significantly to the inhibitory effects of sivelestat than the reduction in iNOS mRNA synthesis. Sivelestat may provide useful therapeutic effects through the suppression of iNOS induction involved in liver injury.

A Novel Pathway for Inducible Nitric-oxide Synthase Activation through Inflammasomes

Innate immune recognition of flagellin is shared by transmembrane TLR5 and cytosolic Nlrc4 (NOD-like receptor family CARD (caspase activation recruitment domain) domain containing 4)/Naip5 (neuronal apoptosis inhibitory protein 5). TLR5 activates inflammatory genes through MYD88 pathway, whereas Nlrc4 and Naip5 assemble multiprotein complexes called inflammasomes, culminating in caspase-1 activation, IL-1β/IL-18 secretion, and pyroptosis. Although both TLR5 and Naip5/Nlrc4 pathways cooperate to clear infections, little is known about the relative anti-pathogen effector mechanisms operating through each of them. Here we show that the cytosolic flagellin (FLA-BSDot) was able to activate iNOS, an enzyme previously associated with TLR5 pathway. Using Nlrc4- or Naip5-deficient macrophages, we found that both receptors are involved in iNOS activation by FLA-BSDot. Moreover, distinct from extracellular flagellin (FLA-BS), iNOS activation by intracellular flagellin is completely abrogated in the absence of caspase-1. Interestingly, IL-1β and IL-18 do not seem to be important for FLA-BSDot-mediated iNOS production. Together, our data defined an additional anti-pathogen effector mechanism operated through Naip5 and Nlrc4 inflammasomes and illustrated a novel signaling transduction pathway that activates iNOS.

NF-kappaB dependent and independent mechanisms of quartz-induced proinflammatory activation of lung epithelial cells.

In the initiation and progression of pulmonary inflammation, macrophages have classically been considered as a crucial cell type. However, evidence for the role of epithelial type II cells in pulmonary inflammation has been accumulating. In the current study, a combined in vivo and in vitro approach has been employed to investigate the mechanisms of quartz-induced proinflammatory activation of lung epithelial cells. In vivo, enhanced expression of the inflammation- and oxidative stress-related genes HO-1 and iNOS was found on the mRNA level in rat lungs after instillation with DQ12 respirable quartz. Activation of the classical NF-κB pathway in macrophages and type II pneumocytes was indicated by enhanced immunostaining of phospho-IκBα in these specific lung cell types. In vitro, the direct, particle-mediated effect on proinflammatory signalling in a rat lung epithelial (RLE) cell line was compared to the indirect, macrophage product-mediated effect. Treatment with quartz particles induced HO-1 and COX-2 mRNA expression in RLE cells in an NF-κB independent manner. Supernatant from quartz-treated macrophages rapidly activated the NF-κB signalling pathway in RLE cells and markedly induced iNOS mRNA expression up to 2000-fold compared to non-treated control cells. Neutralisation of TNFα and IL-1β in macrophage supernatant did not reduce its ability to elicit NF-κB activation of RLE cells. In addition the effect was not modified by depletion or supplementation of intracellular glutathione.The results from the current work suggest that although both oxidative stress and NF-κB are likely involved in the inflammatory effects of toxic respirable particles, these phenomena can operate independently on the cellular level. This might have consequences for in vitro particle hazard testing, since by focusing on NF-κB signalling one might neglect alternative inflammatory pathways.

Anti-Inflammatory Role of Cilostazol in Vascular Smooth Muscle Cells In vitro and In vivo

Cilostazol is a selective inhibitor of phosphodiesterase 3, by which it increases intracellular cAMP and activates protein kinase A, thereby inhibiting platelet aggregation and inducing peripheral vasodilation. We investigated whether cilostazol might prevent nuclear factor (NF)-kappaB activation by activating AMP-activated protein kinase (AMPK) in vascular smooth muscle cells (VSMC). Cilostazol was observed to activate AMPK, as well as its downstream target, acetyl-CoA carboxylase, in rat VSMC. Phosphorylation of AMPK with cilostazol was not affected by co-treatment with an adenylate cyclase inhibitor, SQ 22536. Furthermore, a cell-permeable cyclic AMP analog, pCTP-cAMP, did not influence cilostazol-induced AMPK phosphorylation. These findings suggest that cilostazol-induced AMPK activation occurs through a signalling pathway independent of cyclic AMP. Cilostazol dose-dependently inhibited LPS-induced NF-kappaB activation in the present study. It was also observed to inhibit LPS-induced iNOS gene promoter activity and iNOS gene expression, resulting in markedly reduced NO production. An AMPK inhibitor compound C or siRNA for AMPK attenuated the observed cilostazol-induced inhibition of NF-kappaB activation by LPS. Ingestion of cilostazol inhibited NF-kappaB activation, as well as the induction of iNOS mRNA and protein expression, within the aortas of LPS-treated rats. In light of these findings, we suggest that cilostazol might attenuate cytokine-induced expression of the iNOS gene by inhibiting NF-kappaB following AMPK activation in VSMC.

Interleukin-17 Is Required for T Helper 1 Cell Immunity and Host Resistance to the Intracellular Pathogen Francisella tularensis

The importance of T helper type 1 (Th1) cell immunity in host resistance to the intracellular bacterium Francisella tularensis is well established. However, the relative roles of interleukin (IL)-12-Th1 and IL-23-Th17 cell responses in immunity to F. tularensis have not been studied. The IL-23-Th17 cell pathway is critical for protective immunity against extracellular bacterial infections. In contrast, the IL-23-Th17 cell pathway is dispensable for protection against intracellular pathogens such as Mycobacteria. Here we show that the IL-23-Th17 pathway regulates the IL-12-Th1 cell pathway and was required for protective immunity against F.tularensis live vaccine strain. We show that IL-17A, but not IL-17F or IL-22, induced IL-12 production in dendritic cells and mediated Th1 responses. Furthermore, we show that IL-17A also induced IL-12 and interferon-gamma production in macrophages and mediated bacterial killing. Together, these findings illustrate a biological function for IL-17A in regulating IL-12-Th1 cell immunity and host responses to an intracellular pathogen.

Paeonia japonica,Houttuynia cordata, andAster scaberWater Extracts Induce Nitric Oxide and Cytokine Production by Lipopolysaccharide-Activated Macrophages

Natural products are increasingly recognized as potential targets for drug discovery and development. We previously reported that Paeonia japonica, Houttuynia cordata, and Aster scaber enhanced macrophage activation both in vitro and in vivo. In the present study we investigated the immunomodulating effects of these plants on lipopolysacharide (LPS)-stimulated macrophages. An aqueous extract of each plant was administered to female BALB/c mice every other day for 4 weeks. Peritoneal macrophages were then collected and incubated to examine the immunoreactivity of macrophages against LPS at different time points. The expression levels of inducible nitric oxide (NO) synthetase (iNOS), cyclooxygenase (COX)-2, and inhibitory factor kappaB alpha (IkappaBalpha) proteins and the production of NO metabolite (nitrite), prostaglandin (PG) E(2), and the pro-inflammatory cytokines interleukin-1beta, interleukin-6, and tumor necrosis factor-alpha were determined in the activated macrophages treated with extracts from each plant individually or combined. High levels of pro-inflammatory cytokines were produced by A. scaber-, P. japonica-, and H. cordata-treated macrophages following 24 hours of LPS stimulation. P. japonica, H. cordata, and A. scaber treatment also induced the production of nitrate by LPS-treated macrophages. Induction of iNOS mRNA and protein was also different in each group. PGE(2) secretion was up-regulated by all extract-treated macrophages at early time points; however, no significant differences were observed between the groups by 8 hours post-LPS stimulation. Treatment with A. scaber extract resulted in the highest levels of IkappaBalpha degradation. Our findings illustrate that the natural plant products P. japonica, H. cordata, and A. scaber may enhance immune function by modulating ex vivo pro-inflammatory cytokine and NO production as well as the expression of iNOS and COX-2.

Regulation of astrocyte inflammatory responses by the Parkinson's disease‐associated gene DJ–1

The Parkinson's disease (PD)-associated gene DJ-1 mediates direct neuroprotection. The up-regulation of DJ-1 in reactive astrocytes also suggests a role in glia. Here we show that DJ-1 regulates proinflammatory responses in mouse astrocyte-rich primary cultures. When treated with a Toll-like receptor 4 agonist, the bacterial endotoxin lipopolysaccharide (LPS), Dj-1-knockout astrocytes generated >10 times more nitric oxide (NO) than littermate controls. Lentiviral reintroduction of DJ-1 restored the NO response to LPS. The enhanced NO production in Dj-1(-/-) astrocytes was mediated by a signaling pathway involving reactive oxygen species leading to specific hyperinduction of type II NO synthase [inducible NO synthase (iNOS)]. These effects coincided with significantly increased phosphorylation of p38 mitogen-activated protein kinase (MAPK), and p38(MAPK) inhibition suppressed NO production and iNOS mRNA and protein induction. Dj-1(-/-) astrocytes also induced the proinflammatory mediators cyclooxygenase-2 and interleukin-6 significantly more strongly, but not nerve growth factor. Finally, primary neuron cultures grown on Dj-1(-/-) astrocytes became apoptotic in response to LPS in an iNOS-dependent manner, directly demonstrating the neurotoxic potential of astrocytic DJ-1 deficiency. These findings identify DJ-1 as a regulator of proinflammatory responses and suggest that loss of DJ-1 contributes to PD pathogenesis by deregulation of astrocytic neuroinflammatory damage.

Protein Never in Mitosis Gene A Interacting-1 (PIN1) regulates degradation of inducible nitric oxide synthase in endothelial cells

The peptidyl-proline isomerase Protein Never in Mitosis Gene A Interacting-1 (PIN1) increases the level or activity of several transcription factors that can induce the inducible nitric oxide (NO) synthase (iNOS). PIN1 can also regulate mRNA and protein turnover. Here, the effect of depletion of PIN1 on induction of iNOS by Escherichia coli endotoxin (LPS) and interferon-gamma (IFNgamma) in murine aortic endothelial cells (MAEC) was determined. Suppression of PIN1 by 85% with small hairpin RNA enhanced the induction of NO and iNOS protein by LPS-IFNgamma. There was no effect on induction of iNOS mRNA, suggesting a posttranscriptional effect. The enhanced levels of iNOS protein were functionally significant since LPS-IFNgamma was cytotoxic to MAEC lacking PIN1 but not MAEC harboring an inactive control construct, and because cytotoxicity was blocked by the NO synthase inhibitor N(omega)-nitro-L-arginine methyl ester. Consistent with posttranscriptional action, knockdown of PIN1 increased the stability of iNOS protein in cycloheximide-treated cells. Furthermore, loss of iNOS was blocked by the calpain inhibitor carbobenzoxy-valinyl-phenylalaninal but not by the selective proteasome inhibitor epoxomicin. Immunoprecipitation indicated that PIN1 can interact with iNOS. Pull down of iNOS with a wild-type glutathione-S-transferase-PIN1 fusion protein, but not with a mutant of the amino terminal phospho-(serine/threonine)-proline binding WW domain of PIN1, indicated that this domain mediates interaction. The results suggest that PIN1 associates with iNOS and can limit its induction by facilitating calpain-mediated degradation in MAEC.