iNOS Expression In RAW Research Articles

Diterpenoids from the Resin of Liquidambar formosana Hance and Their Anti‐inflammatory Activities

AbstractLiquidambarine D (1), a new labdane‐type diterpenoid, and nineteen known compounds including five labdanes (2–6), eight abietanes (7–14), four pimaranes (16–19), and two nor‐diterpenoids (15, 20) were isolated from the resin of Liquidambar formosana Hance. The new compound was characterized by the combination of spectroscopic and computational methods. With the exception of 15‐hydroxydehydroabietic acid (13), all the known compounds were reported for the first time from the resins of Liquidambar species. All these isolates were evaluated for their inhibitory effects in RAW264.7 macrophage cells, revealing that compounds, acetylisocupressic acid (3), (13S)‐15‐acetoxylabd‐8(17)‐en‐19‐oic acid (6), abietic acid (7), dehydroabietic acid (11), abieta‐8,11,13,15‐tetraen‐18‐oic acid (12), and 4‐epi‐isopimaric acid (16) could suppress lipopolysaccharide‐induced iNOS expression in RAW 264.7 cells and disclosing their structure activity relationships.

A mixture of Panax ginseng and Scrophularia buergeriana improves immune function in an immunosuppressed murine model

BackgroundImmunomodulatory drugs are currently used for immunosuppressed individuals, but adverse side effects have been reported. Although Panax ginseng and Scrophularia buergeriana are known to have respective pharmacological properties, the potential of a mixture of Panax ginseng and Scrophularia buergeriana (Isam-Tang, IST) as an immunomodulatory drug has not yet been studied. PurposeThe present study was designed to assess the immunomodulatory activity of IST and p-coumaric acid (pCA), an active compound of IST, in the immune system. MethodsThe levels of immunostimulatory cytokines, nitrite, inducible nitric oxide synthase (iNOS), NF-kB activation, and proliferation were examined in RAW264.7 cells, primary splenocytes and splenic NK cells isolated from normal mouse spleen, and in cyclophosphamide-induced immunosuppressed mice using ELISA, quantitative real-time PCR, Western blotting, and immunofluorescence staining. ResultsIST or pCA treatment increased the production of immunostimulatory cytokines and nitrite and the expression of iNOS in RAW264.7 cells and splenocytes. IST or pCA also induced NF-κB signaling activation and promoted the phagocytic activity of RAW264.7 cells. In addition, the splenocyte proliferation and splenic NK activity were enhanced by IST or pCA. IST or pCA increased the levels of immunostimulatory cytokines in immunosuppressed mice and ameliorated splenic tissue damage. ConclusionThese findings suggest that IST supplementation may be used to enhance immune function.

Anti-inflammatory Constituents from Caulis Trachelospermi.

Caulis Trachelospermi, the stems with leaves of Trachelospermum jasminoides, is a well-known herbal drug of the Apocynaceae family recorded in the Chinese pharmacopeia and used for the treatment of inflammation-related diseases by ethnic minorities of China. The mechanism of anti-inflammatory activity and responsible constituents of T. jasminoides have not been well elucidated in previous studies. Preliminary investigation showed that both the water and the ethyl ester extracts of T. jasminoides exhibited potent inhibitory activity on nitric oxide (NO) production using lipopolysaccharide (LPS)-stimulated murine macrophages. Phytochemical investigation on these extracts afforded 23 compounds, including three new compounds (1: -3: ) identified on the basis of spectroscopic and mass spectrometric data. Anti-inflammatory bioassay showed that compounds 17, 18, 22: , and 23: inhibited significantly the production of NO in a concentration-dependent manner. Further studies indicated that compound 23: inhibited significantly TNF-α and IL-6 produced by LPS-stimulated RAW 264.7 cells with good selectivity, as well as protein expression of iNOS in RAW 264.7 cells. These chemical constituents may contribute to the anti-inflammatory potential of T. jasminoides.

Immune-enhancing effects of postbiotic produced by Bacillus velezensis Kh2-2 isolated from Korea Foods

Postbiotics defined as soluble factors (products or metabolic byproducts) that are released after bacterial lysis or secreted by live bacteria, have attracted considerable attention because of their long shelf life, safety, and beneficial effects. In this study, we investigated the immune-enhancing activities of squid jeotgal (a traditional Korean fermented seafood)-derived Bacillus velezensis Kh2-2 (Kh2-2) postbiotics in vitro, ex vivo, and in vivo. Cell lysates of four Bacillus species were prepared by sonication. In particular, Kh2-2 lysates induced NO production by upregulating iNOS expression in RAW264.7 cells compared with the lysates of B. subtilis Kh2-1, B. vallismortis Kh8-3, and B. amyloliquefaciens Kh3-1. Furthermore, Kh2-2 lysates stimulated immune activation of macrophages by upregulating the NF-κB and MAPK signaling pathways and promoting immune-related cytokine secretion. In the ex vivo study, Kh2-2 lysates stimulated proliferation and polarized Th1 response by inducing the production of IL-2 and IFN-γ and inhibiting IL-10 expression in splenocytes. The in vivo immune-enhancing effects of Kh2-2 lysates and Kh2-2 were further evaluated using a cyclophosphamide (CTX)-induced immunosuppression mouse model. The results showed that oral administration of Kh2-2 lysates improved CTX-induced immunosuppression by enhancing innate and adaptive immunity, stimulating immune-related cytokine secretion, and modulating gut microbiota dysbiosis in mice. Thus, we concluded that Kh2-2 lysates have potential as a functional material for postbiotics with immune-enhancing effects.

Intra-Articular Route for the System of Molecules 14G1862 from Centella Asiatica: Pain Relieving and Protective Effects in a Rat Model of Osteoarthritis.

Current pharmacological therapies for the management of chronic articular diseases are far from being satisfactory, so new strategies need to be investigated. We tested the intra-articular pain relieving properties of a system of molecules from a characterized Centella asiatica extract (14G1862) in a rat model of osteoarthritis induced by monoiodoacetate (MIA). 14G1862 (0.2–2 mg mL−1) was intra-articularly (i.a.) injected 7 days after MIA, behavioural and histological evaluations were performed 14, 30 and 60 days after treatments. Moreover, the effect of 14G1862 on nitrate production and iNOS expression in RAW 264.7 macrophages stimulated with LPS was assessed. In vitro, 14G1862 treatment attenuated LPS-induced NO production and iNOS expression in a comparable manner to celecoxib. In vivo, 14G1862 significantly reduced mechanical allodynia and hyperalgesia, spontaneous pain and motor alterations starting on day 14 up to day 60. The efficacy was higher or comparable to that evoked by triamcinolone acetonide (100 μg i.a.) used as reference drug. Histological evaluation highlighted the improvement of several morphological parameters in MIA + 14G1862-treated animals with particularly benefic effects on joint space and fibrin deposition. In conclusion, i.a. treatment with Centella asiatica is a candidate to be a novel effective approach for osteoarthritis therapy.

Type I Interferons Ameliorate Zinc Intoxication of Candida glabrata by Macrophages and Promote Fungal Immune Evasion.

SummaryHost and fungal pathogens compete for metal ion acquisition during infectious processes, but molecular mechanisms remain largely unknown. Here, we show that type I interferons (IFNs-I) dysregulate zinc homeostasis in macrophages, which employ metallothionein-mediated zinc intoxication of pathogens as fungicidal response. However, Candida glabrata can escape immune surveillance by sequestering zinc into vacuoles. Interestingly, zinc-loading is inhibited by IFNs-I, because a Janus kinase 1 (JAK1)-dependent suppression of zinc homeostasis affects zinc distribution in macrophages as well as generation of reactive oxygen species (ROS). In addition, systemic fungal infections elicit IFN-I responses that suppress splenic zinc homeostasis, thereby altering macrophage zinc pools that otherwise exert fungicidal actions. Thus, IFN-I signaling inadvertently increases fungal fitness both in vitro and in vivo during fungal infections. Our data reveal an as yet unrecognized role for zinc intoxication in antifungal immunity and suggest that interfering with host zinc homeostasis may offer therapeutic options to treat invasive fungal infections.

Inhibition of Inflammatory Responses by Centella asiatica via Suppression of IRAK1-TAK1 in Mouse Macrophages.

Centella asiatica (L.) Urb. (C. asiatica) has been widely treated for inflammation-related diseases in China for thousands of years. While C. asiatica showed relevant effects as traditional medicine, the mechanism of C. asiatica suppressing inflammation has not been thoroughly investigated. Therefore, this study was conducted to reveal the anti-inflammatory mechanism of methanol fraction from C. asiatica (MCA) at the molecular level in murine macrophages. Levels of inflammation-related mediators were observed with treatment of MCA. MCA significantly suppressed nitric oxide production and iNOS expression in RAW 264.7 macrophages. Prostaglandin E2 production was alleviated by MCA via the downregulation of cyclooxygenase-2. MCA treatment also reduced pro-inflammatory tumor necrosis factor-[Formula: see text] and interleukin (IL)-6 levels. LPS/D-GalN-induced acute hepatitis in mouse was alleviated by MCA treatment. In addition, MCA decreased the phosphorylation of inhibitory [Formula: see text]B[Formula: see text] (I[Formula: see text]B[Formula: see text]) at Ser32/36 and thereby blocked I[Formula: see text]B[Formula: see text] degradation. TXY motif phosphorylation in the activation loops of mitogen-activated protein kinases (MAPKs) was also suppressed by MCA treatment. Further investigation revealed that MCA inhibited transforming growth factor-[Formula: see text]-activated kinase 1 (TAK1) phosphorylation and IL-1 receptor-associated kinase (IRAK1) degradation, the upstream kinases activating nuclear factor [Formula: see text]B and MAPKs. Taken together, MCA exhibited anti-inflammatory properties via the downregulation of IRAK1-TAK1 signaling pathways.

Chung Hun Wha Dam Tang attenuates atherosclerosis in apolipoprotein E-deficient mice via the NF-κB pathway

Chung Hun Wha Dam Tang (CHWDT), a traditional Korean herbal formula, has been used for hundreds of years for alleviating dizziness, phlegm, and inflammation. The inhibitory effects of CHWDT on obesity have been reported. However, the effects of CHWDT in atherosclerosis have not yet been explored. Therefore, the aim of the study was to investigate whether CHWDT could confer protection from oxidative stress and inflammation in a high fat diet (HFD)-induced atherosclerosis model. Atherosclerosis was induced by feeding ApoeE−/− mice with HFD for 6 weeks. To examine the in vivo effects of CHWDT on HFD-induced atherosclerosis, mice on HFD for 6 weeks were orally administrated with CHWDT (400 or 800 mg/kg) every other day for an additional 6 weeks and histological features of aorta were determined by Sudan IV and H&E staining. The mRNA levels of TNF-α, SOD1, SOD2, iNOS or eNOS were determined with RT-PCR analysis or western blot analysis for protein levels. ROS generation was measured by CM-2DCFDA or MitoSox staining using FACS analysis or confocal microscopy. CHWDT decreased the mRNA levels of TNF-α and increased the mRNA levels of SOD1, SOD2 and catalase in both aorta and liver tissues of atherosclerotic mice. CHWDT attenuated TNF-α and iNOS expression in RAW 264.7 cells, U937 cells and HUVECs, and restored eNOS expression in HUVECs. CHWDT decreased H2O2-induced cellular ROS generation in RAW 264.7 cells and U937 cells, and also decreased H2O2-induced mitochondrial ROS generation in RAW 264.7 cells. Furthermore, SOD1, SOD2 and catalase mRNA levels were increased by pre-treatment with CHWDT in H2O2 and LPS-stimulated RAW 264.7 cells, as well as in LPS-treated U937 and HUVECs. CHWDT not only decreased LPS-induced NF-κB p65 phosphorylation but also inhibited the translocation of p65 from the cytosol to the nucleus in RAW 264.7 macrophages. These results suggest that CHWDT exerts inhibitory effects on atherosclerosis-induced oxidative stress and inflammation via the NF-κB pathway.

Zinc chelation decreases IFN-β-induced STAT1 upregulation and iNOS expression in RAW 264.7 macrophages

One consequence of lipopolysaccharide (LPS)-induced stimulation of macrophages is the release of Interferon (IFN)-β, and subsequently the activation of the JAK-STAT1 pathway, resulting in the expression of inducible nitric oxide synthase (iNOS). Free intracellular zinc ions (Zn2+) have a profound impact as a second messenger in LPS-dependent gene expression. Previous work had indicated a Zn2+-dependent upregulation of STAT1 mRNA in response to LPS and IFN-β, potentially affecting STAT1-dependent downstream signaling upon pre-incubation with these agents. The aim of the present study was to investigate the long-term influence of Zn2+ chelation on cellular STAT1 levels and their effect on protein levels and activity of iNOS. The LPS- and IFN-β-mediated increase of STAT1 mRNA and protein levels was abrogated by chelation of Zn2+ with the membrane permeable chelator N,N,N′,N′-Tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) in RAW 264.7 macrophages. After 48h pre-incubation together with IFN-β, TPEN also led to reduced nitric monoxide formation in response to a second stimulation with LPS. Nonetheless, the latter was observed regardless of any pre-incubation with IFN-β, suggesting that the effect of treatment with TPEN negatively affects iNOS induction independently from cellular STAT1 levels. In conclusion, long term Zn2+ chelation does affect STAT1 protein expression, but interferes with NO production by a different, yet unknown pathway not involving STAT1. However, as there are many additional STAT1-dependent genes, there might still be effects on targets other than iNOS.

Development and validation of a novel flow cytometric macrophage phenotyping method.

Abstract Macrophages differentiate into pro-inflammatory M1 or anti-inflammatory M2 phenotypes. M2 macrophages suppress anti-tumor responses, and phenotyping tumor-associated macrophages can be of prognostic value. Expression of mRNA of inducible nitric oxide synthase (iNOS) or arginase 1 is a well-accepted marker of murine M1 or M2 macrophages, respectively. However, macrophages need to be isolated and purified in order to evaluate the mRNA expression. There are no reliable and simple methods for detection of macrophage markers which would not require cell purification. Here we report the development and validation of a novel flow cytometric macrophage phenotyping method. Murine macrophage RAW 264.7 cells were treated with endotoxin and IFN-γ to stimulate iNOS expression. Alternatively, RAW 264.7 cells were stimulated with 8-bromo-cAMP to induce arginase 1 expression. Our new technique allowed successful detection of iNOS expression in RAW 264.7 cells by flow cytometry, which correlated well with NO production measured by Griess assay. We also developed a method which allowed us to successfully detect the Arginase I expression by flow cytometry. Arginase I expression also correlated well with arginase activity as measured by an Abnova arginase assay kit. Our results suggest that murine macrophage phenotype can be successfully determined by flowcytometric measurement of iNOS and arginase 1 expression.

Pulmonary Macrophages Attenuate Hypoxic Pulmonary Vasoconstriction via β3AR/iNOS Pathway in Rats Exposed to Chronic Intermittent Hypoxia.

Chronic intermittent hypoxia (IH) induces activation of the sympathoadrenal system, which plays a pivotal role in attenuating hypoxic pulmonary vasoconstriction (HPV) via central β1-adrenergic receptors (AR) (brain) and peripheral β2AR (pulmonary arteries). Prolonged hypercatecholemia has been shown to upregulate β3AR. However, the relationship between IH and β3AR in the modification of HPV is unknown. It has been observed that chronic stimulation of β3AR upregulates inducible nitric oxide synthase (iNOS) in cardiomyocytes and that IH exposure causes expression of iNOS in RAW264.7 macrophages. iNOS has been shown to have the ability to dilate pulmonary vessels. Hence, we hypothesized that chronic IH activates β3AR/iNOS signaling in pulmonary macrophages, leading to the promotion of NO secretion and attenuated HPV. Sprague-Dawley rats were exposed to IH (3-min periods of 4–21% O2) for 8 h/d for 6 weeks. The urinary catecholamine concentrations of IH rats were high compared with those of controls, indicating activation of the sympathoadrenal system following chronic IH. Interestingly, chronic IH induced the migration of circulating monocytes into the lungs and the predominant increase in the number of pro-inflammatory pulmonary macrophages. In these macrophages, both β3AR and iNOS were upregulated and stimulation of the β3AR/iNOS pathway in vitro caused them to promote NO secretion. Furthermore, in vivo synchrotron radiation microangiography showed that HPV was significantly attenuated in IH rats and the attenuated HPV was fully restored by blockade of β3AR/iNOS pathway or depletion of pulmonary macrophages. These results suggest that circulating monocyte-derived pulmonary macrophages attenuate HPV via activation of β3AR/iNOS signaling in chronic IH.

Inhibitory Effects of JEUD-38, a New Sesquiterpene Lactone from Inula japonica Thunb, on LPS-Induced iNOS Expression in RAW264.7 Cells.

We isolated JEUD-38, a new sesquiterpene lactone from Inula japonica Thunb. JEUD-38 dramatically attenuated lipopolysaccharide (LPS)-induced nitric oxide (NO) production. Consistent with this finding, the protein expression of inducible nitric oxide synthase (iNOS) was blocked by JEUD-38 in a concentration-dependent manner. To elucidate the mechanism, we examined the effect of JEUD-38 on LPS-stimulated nuclear factor-κB (NF-κB) nuclear translocation, inhibitory factor-κB (IκB) phosphorylation, and degradation. JEUD-38 reduced the translocation of p65, via abrogating IκB-α phosphorylation and degradation. In addition, JEUD-38 inhibited LPS-stimulated phosphorylation of mitogen-activated protein kinases (MAPKs) including extracellular signal-regulated kinase 1/2 (ERK1/2), c-Jun N-terminal kinase (JNK), and p38. Since iNOS as well as the upstream NF-κB and MAPKs are known to be closely involved in inflammation, these results suggest that JEUD-38 is a promising candidate for prevention and therapy of inflammatory diseases.

SIRT2 ameliorates lipopolysaccharide-induced inflammation in macrophages

IntroductionSIRT2 is a NAD(+)-dependent deacetylases and associated with numerous processes such as infection, carcinogenesis, DNA damage and cell cycle regulation. However, the role of SIRT2 in inflammatory process in macrophage remains unclear. Materials and methodsIn the present study, we have evaluated the regulatory effects of SIRT2 in lipopolysaccharide (LPS)-stimulated macrophages isolated from SIRT2 knockout (KO) and wild type (WT) mice or Raw264.7 macrophage cells. As inflammatory parameters, expression of inducible nitric oxide synthase (iNOS), the productions of nitric oxide, reactive oxygen species (ROS) and M1-macrophage-related factors were evaluated. We also examined the effects of SIRT2 on activation of nuclear factor-kappaB (NFκB) signaling. ResultsSIRT2 deficiency inhibits LPS-induced iNOS mRNA and protein expression in bone marrow derived macrophages. SIRT2-siRNA transfection also suppressed LPS-induced iNOS expression in Raw264.7 macrophage cells. Bone marrow derived macrophages isolated from SIRT2 KO mice produced lower nitric oxide and expressed lower levels of M1-macrophage related markers including iNOS and CD86 in response to LPS than WT mice. Decrease of SIRT2 reduced the LPS-induced reactive oxygen species production. Deficiency of SIRT2 resulted in inhibition of NFκB activation through reducing the phosphorylation and degradation of IκBα. The phosphorylation and nuclear translocation of p65 was significantly decreased in SIRT2-deficient macrophages after LPS stimulation. DiscussionOur data suggested that deficiency of SIRT2 ameliorates iNOS, NO expression and reactive oxygen species production with suppressing LPS-induced activation of NFκB in macrophages.

Anti‐adipogenic and anti‐inflammatory mechanism of nobiletin in co‐culture of adipocyte and macrophage (1045.13)

Adipose tissues are characterized by the enhanced infiltration of macrophages. It is considered that the paracrine loop between adipocytes and macrophages establishes a vicious cycle that aggravates inflammatory changes and insulin resistance in obese adipose tissues. Nobiletin is abundantly present in the fruit peel of Citrus and has several health beneficial effects including antioxidant, anti‐inflammation and anticancer activities. However, not much is known about the anti‐adipogenic and anti‐inflammatory effect of nobiletin in co‐culture system of adipocyte and macrophage. The objective of this study was to evaluate the inhibitory effect of nobiletin on the interaction between adipocytes and macrophages. The results revealed that nobiletin markedly inhibited NO, TNF‐α and MCP‐1 secretion in contact and trans‐well systems of adipocytes and macrophages in a dose‐dependent manner. In addition, nobiletin blocked the expression of adipogenic transcription factors, including PPARγ, C/EBPα and aP2 in 3T3‐L1 cells of transwell system. Also, nobiletin inhibited the expression of iNOS in RAW264.7 cells. These results indicate that nobiletin exhibits anti‐adipogenic and anti‐inflammatory properties in the interaction of adipocytes and macrophages. Nobiletin may be useful for ameliorating the adipogenic and inflammatory changes in obese adipose tissues.

Inhibitory effect of carbazolequinone derivatives on lipopolysaccharide and interferon-γ-induced nitric oxide production in mouse macrophage RAW264.7 cells

The aim of this study was to examine the mechanism underlying the inhibitory effect of our synthesized carbazolequinone derivatives on nitric oxide (NO) production in activated macrophages. Lipopolysaccharide (LPS) and interferon-γ (IFN-γ)-stimulated RAW264.7 macrophages were treated with carbazolequinone derivatives. The NO and prostaglandin E2 (PGE2 ) levels in cell culture supernatants fractions were measured by Greiss and ELISA assay, respectively. The expression of inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2) was assessed by the real-time RT-PCR method. Nuclear factor kappa B (NF-κB) activation was detected by an NF-κB-dependent luciferase reporter assay. Our synthesized carbazolequinone derivatives (7-methoxy-2-methylcarbazole-1,4-quinone, 6-methoxy-2-methylcarbazole-1,4-quinone and 6-chloro-2-methylcarbazole-1,4-quinone) significantly inhibited LPS/IFN-γ-induced NO production and iNOS expression in RAW264.7 cells. They also inhibited the LPS/IFN-γ-mediated induction of COX-2 expression and PGE2 production. In addition, the LPS/IFN-γ-induced transcription activity of NF-κB was attenuated. Using the RAW264.7-tsAM5NE co-culture system, we found that these carbazolequinone derivatives protected neuronally differentiated tsAM5NE cells from NO-induced cell death by inhibiting the production of NO. These results suggest that the three carbazolequinone derivatives inhibit LPS/IFN-γ-induced NO production via iNOS and COX-2 downregulation due to NF-κB inhibition. Therefore, these three carbazolequinone derivatives may be useful for developing a new drug against NO-mediated neurodegenerative diseases.

Exendin‐4 inhibits iNOS expression at the protein level in LPS‐stimulated Raw264.7 macrophage by the activation of cAMP/PKA pathway

Glucagon-like peptide-1 (GLP-1) and its potent agonists have been widely studied in pancreatic islet β-cells. However, GLP-1 receptors are present in many extrapancreatic tissues including macrophages, and thus GLP-1 may have diverse actions in these tissues and cells. Therefore, we examined the mechanism by which exendin-4 (EX-4), a potent GLP-1 receptor agonist, inhibits lipopolysaccharide (LPS)-induced iNOS expression in Raw264.7 macrophage cells. EX-4 significantly inhibited LPS-induced iNOS protein expression and nitrite production. However, Northern blot and promoter analyses demonstrated that EX-4 did not inhibit LPS-induced iNOS mRNA expression and iNOS promoter activity. Electrophoretic mobility shift assay (EMSA) showed that EX-4 did not alter the binding activity of NF-κB to the iNOS promoter. Consistent with the result of EMSA, LPS-induced IκBα phosphorylation and nuclear translocation of p65 were not inhibited by EX-4. Also, actinomycin D chase study and the promoter assay using the construct containing 3'-untranslated region of iNOS showed that EX-4 did not affect iNOS mRNA stability. Meanwhile, cycloheximide chase study demonstrated that EX-4 significantly accelerated iNOS protein degradation. The EX-4 inhibition of LPS-induced iNOS protein was significantly reversed by adenylate cyclase inhibitors (MDL-12330A and SQ 22536), a PKA inhibitor (H-89) and PKAα gene silencing. These findings suggest that EX-4 inhibited LPS-induced iNOS expression at protein level, but not at transcriptional mechanism of iNOS gene and this inhibitory effect of EX-4 was mainly dependent on cAMP/PKA system.

The regulatory effect of veratric acid on NO production in LPS-stimulated RAW264.7 macrophage cells

Veratric acid, a simple benzoic acid derived from plants and fruits, has been reported to have anti-oxidant, anti-inflammation, and blood pressure-lowering effects. This study was designed to evaluate the inhibitory effects of veratric acid on nitric oxide (NO) production in LPS-stimulated RAW264.7 cells. It was found that veratric acid inhibited NO production and inducible nitric oxide synthase (iNOS) expression in LPS-stimulated RAW264.7 cells. The inhibitory effects of veratric acid on the generation of interleukin-6 (IL-6) and interferon-γ (IFN-γ) was determined. Furthermore, veratric acid facilitated the inactivation of glycogen synthase kinase-3β (GSK-3β), STAT-1, and STAT-3 in dose-dependent manner. Notably, NF-κB and members of the mitogen activated protein kinase (MAPK) family including p38, ERK, and JNK were dephosphorylated by veratric acid. These findings suggest that the treatment of veratric acid might be effective in neutralizing the over-expression of NO in inflammatory disorders.

PP026-MON HEPCIDIN STORM AFTER ABDOMINAL SURGERY

Rationale: Glucagon-like peptide-1 (GLP-1) and its potent agonists have been widely studied in pancreatic islet b-cells. However, GLP-1 receptors are present in many extrapancreatic tissues including macrophages, and thus GLP-1 may have diverse actions on these tissues and cells. Therefore, we examined the mechanism by which exendin-4 (EX-4), a potent GLP-1 receptor agonist, inhibits lipopolysaccharide (LPS)-induced iNOS expression in Raw264.7 macrophage cells. Methods: The expression of iNOS, p-IuBa and p65 were detected by Western blot analysis and content of nitrite was measured using Griess reagent. Additionally, iNOS mRNA expression and promoter activity were observed by Northern blot analysis and luciferase assay, respectively. To observe the stability of iNOS mRNA and protein, actinomycin D chase and cycloheximide chase studies were performed, respectively. Also, we examined using adenylate cyclase inhibitor, PKA inhibitor and PKA gene silencing. Results: EX-4 significantly inhibited LPS-induced iNOS protein expression and nitrite production. However, EX-4 did not inhibit LPS-induced iNOS mRNA expression and iNOS promoter activity. Consistent with the result of iNOS promoter, LPS-induced IuBa phosphorylation and nuclear translocation of p65 were not inhibited by EX-4. Also, actinomycin D chase study showed that EX-4 did not affect iNOS mRNA stability. Meanwhile, cycloheximide chase study demonstrated that EX-4 significantly accelerated iNOS protein degradation. The EX-4 inhibition of LPS-induced iNOS protein was significantly reversed by adenylate cyclase inhibitors (MDL-12330A and SQ 22536), a PKA inhibitor (H-89) and PKAa gene silencing. Conclusion: These findings suggest that EX-4 inhibited LPS-induced iNOS protein expression at protein level, but not at transcriptional mechanism of iNOS gene and this inhibitory effect of EX-4 was mainly dependent on cAMP/PKA system.

Small-molecular virulence inhibitors show divergent and immunomodulatory effects in infection models of Salmonella enterica serovar Typhimurium

The virulence-associated Salmonella pathogenicity island 2 (SPI2) type III secretion system supports intracellular replication of Salmonella enterica serovar Typhimurium in macrophage-like RAW264.7 cells. In contrast, the salicylidene acylhydrazide INP0010 and the benzimidazole omeprazole prevent virulence factor-mediated replication of S. Typhimurium in these cells. Here we show that INP0010 enhances expression of inducible nitric oxide synthase (iNOS), nitric oxide (NO) production, the oxidative burst and tumour necrosis factor-alpha (TNFα) release in infected RAW264.7 cells. INP0010 also inhibited SPI2 activity in RAW264.7 cells. The ability of INP0010 to suppress bacterial intracellular replication correlated with NO production. The iNOS inhibitor N-monomethyl-l-arginine restored SPI2 activity and antagonised the bacteriostatic effect of INP0010. Omeprazole, which inhibited iNOS expression in RAW264.7 cells, likewise antagonised INP0010. In infected epithelioid MDCK cells that did not express NO upon infection, INP0010 enhanced bacterial intracellular replication. In Caenorhabditis elegans, INP0010 significantly attenuated the virulence of S. Typhimurium. In this infection model, the attenuating effect of INP0010 was further enhanced by omeprazole. These results demonstrate that chemically unrelated virulence inhibitors may act in an antagonistic or additive manner, that their effect depends on the infection model applied, and that the attenuating effects of INP0010 in part relate to its ability to promote the SPI2 antagonist NO.

Ligustilide prevents LPS-induced iNOS expression in RAW 264.7 macrophages by preventing ROS production and down-regulating the MAPK, NF-κB and AP-1 signaling pathways

Angelica sinensis (AS), an herb used in traditional Chinese medicine, is thought to have anti-inflammatory activities. Ligustilide is its most abundant ingredient. This study sought to determine ligustilide's effects on lipopolysaccharide (LPS)-induced inflammation in RAW 264.7 macrophages. Ligustilide significantly suppressed the production of nitric oxide (NO), prostaglandin E2 (PGE2) and tumor necrosis factor-α (TNF-α). The inhibition of NO was concomitant with a decrease in the protein and mRNA levels of LPS-induced nitric oxide synthase (iNOS). Furthermore, activation of activator protein-1 (AP-1) and nuclear factor κB (NF-κB) in the nucleus and the cytosolic degradation of IκBα were abrogated by ligustilide. Ligustilide also inhibited the phosphorylation of IκB kinase (IKK) and mitogen-activated protein kinases (MAPKs), including p38 MAPK, extracellular signal-regulated kinase (ERK1/2) and c-Jun N-terminal kinase (JNK). The intracellular reactive oxygen species (iROS) level was also significantly decreased. These results suggest that ligustilide exhibits anti-inflammatory activities by blocking the activation of MAPKs/IKK and the downstream transcription factors AP-1 and NF-κB, which may result from ligustilide's down-regulation of iROS production.