Nitric Oxide Production In Peritoneal Macrophages Research Articles

Curcuma longa L. extract increased immune responses in RAW 264.7 cells and cyclophosphamide-induced BALB/c mice

Curcuma longa L. extract (CLE) exerts various biological functions including antioxidant, anti-inflammation, anticancer, and antiallergenic effects. However, its immune-enhancing capacity remains unclear. Therefore, the immune-enhancing effect of CLE was investigated in RAW 264.7 cells and cyclophosphamide (CPP)-induced immunosuppression model. CLE upregulated nitric oxide (NO) and reactive oxygen species production and increased inducible nitric oxide synthase and cyclooxygenase-2 expression without affecting the RAW 264.7 cells viability. The results of quantitative real-time reverse transcription polymerase chain reaction and sandwich enzyme-linked immunosorbent assay showed that CLE increased the gene expression and protein levels of tumor necrosis factor-α, interleukin-6, and interleukin-1β in RAW 264.7 cells. Moreover, CLE upregulated p65, I kappa B kinase α/β, and I kappa B α (IκBα) phosphorylation and downregulated IκBα expression in RAW 264.7 cells. CLE also increased p65 translocation from the cytoplasmic to the nucleus in RAW 264.7 cells. The oral administration of CLE increased organ indexes (including the spleen and thymus) and NO production in peritoneal macrophages and improved natural killer cell activity in CPP-induced immunosuppression BALB/c mice. Overall, CLE could be a useful health functional food material that can improve innate immunity via macrophage activation.

Anti-inflammatory effects of cavidine in vitro and in vivo, a selective COX-2 inhibitor in LPS-induced peritoneal macrophages of mouse.

Cavidine is an isoquinoline alkaloid which is isolated from Corydalis impatiens. In traditional Tibetan herb, C. impatiens has been widely used for treatment of skin injuries, hepatitis, cholecystitis, and scabies. The present study aimed to evaluate its anti-inflammatory effect and investigate the mechanisms underlying this anti-inflammatory action. We used different inflammation model animals and lipopolysaccharide (LPS)-induced murine peritoneal macrophages to examine the anti-inflammatory function of cavidine. Results indicated pretreatment with cavidine (i.p.) decreased xylene-induced ear edema, formaldehyde-induced paw edema, leukocyte number, and the level of nitric oxide (NO), prostaglandin E2 (PGE2), and tumor necrosis factor-alpha (TNF-α) in acetic acid-induced peritonitis in mice. The data also demonstrated that cavidine significantly inhibited LPS-induced TNF-α, interleukin-6 (IL-6), and NO production in peritoneal macrophages. Moreover, cavidine regulated the expression of cyclooxygenase-2 (COX-2) instead of cyclooxygenase-1 (COX-1) at protein levels. These results suggested that cavidine is a selective COX-2 inhibitor which possesses an anti-inflammatory activity.

Inhibitory effect of TGF-β1 on NO production in peritoneal macrophages from collagen-induced arthritis rats involving the LPS-TLR4 pathway

Transforming growth factor-β1 (TGF-β1) is critical in controlling inflammatory responses and the prevention of autoimmune diseases. Although the effect of TGF-β1 on macrophages from normal mice or rats has been established, little attention has been paid to its effect on disease conditions. In the present study, we investigated the regulatory effect, and possible mechanism, of TGF-β1 exposure on the secretion of nitric oxide (NO) in peritoneal macrophages (PMΦ) obtained from collagen-induced arthritis (CIA) rats. The CIA model was established by immunizing the emulsion of collagen type II and incomplete Freund's adjuvant (IFA) in Wistar rats. PMΦ were incubated with TGF-β1 (5 ng/ml) for 36 h and the supernatant, and cell mRNA and protein were collected. NO concentration was determined using an NO assay kit. The mRNA expression of inducible nitric oxide synthase (iNOS) and Toll-like receptor 4 (TLR4) was determined using reverse transcription-polymerase chain reaction (RT-PCR). The protein expression of iNOS was tested with western blot analysis. The expression of membrane TLR4 was determined by flow cytometry. We discovered that the secretion of NO from the PMΦ of CIA rats increased compared with normal rats. TGF-β1 significantly inhibited the production of NO in the PMΦ from CIA rats. iNOS mRNA and protein expression in the PMΦ from CIA rats may be suppressed by TGF-β1. TLR4 mRNA and protein expression in PMΦ from CIA rats were upregulated with LPS stimulation and treatment with TGF-β1 inhibited their expression. These results demonstrated that TGF-β1 inhibited lipopolysaccharide (LPS)-induced NO production in the PMΦ from CIA rats, which may be due to the inhibition of the LPS-TLR4 signaling pathway.

Abarema cochliacarpos reduces LPS-induced inflammatory response in murine peritoneal macrophages regulating ROS-MAPK signal pathway

Ethnopharmacological relevanceAbarema cochliacarpos (Gomes) Barneby and Grimes (Fabaceae), known by the vulgar name of Babatenã, has been traditionally used in Northeast Brazil, as an anti-inflammatory remedy. Previous studies have demonstrated its anti-inflammatory and antiulcer effects in skin lesion, alcohol gastric ulcer and acute and chronic colitis. AimsThe present study was designed to evaluate the antioxidant and anti-inflammatory effects of the butanolic fraction from A. cochliacarpos (BFAC) and its major flavonoid, (+)-catechin, in LPS-stimulated murine peritoneal macrophages. Moreover, we studied the role of mitogen-activated protein kinase (MAPK)s and NF-kB signaling pathways possibly involved in the beneficial effects. Materials and methodsThe quantification of the extract was carried out by ultra-performance liquid chromatography analysis. Cell viability was determined using SRB assay. Nitric oxide (NO) production was analyzed by Griess method and intracellular reactive oxygen species (ROS) by fluorescence analysis. In addition, cyclooxygenase (COX-2) and inducible nitric oxide synthase (iNOS) expression, MAPK activation and IkappaBalpha (IKBα) degradation, were determined by Western blot. ResultsAfter BFAC characterization, (+)-catechin was revealed as its major constituent. Both BFAC and (+)-catechin, exerted significant anti-oxidant and anti-inflammatory effects inhibiting LPS-induced intracellular ROS and NO production in peritoneal macrophages. Additionally, the extract but also its major component reduced pro-inflammatory proteins expression probably through c-Jun N-terminal kinase and p38 MAPK signaling pathways. ConclusionThese data suggest that the beneficial effects of BFAC might be mediated, at least in part, by the presence of (+)-catechin. Conclusively our findings confirm the potential of A. cochliacarpos as a new therapeutic strategy for the management of inflammatory and oxidative stress-related diseases.

Peningkatan aktivitas fagositosis dan produksi nitrit oksida pada makrofag peritoneum tikus Sprague Dawley yang diberi Lactobacillus plantarum Mut7 dan ekstrak serat ubi jalar

Background: Macrophages play an important role as part of the innate immune response in the gut and they represent one of the first lines of nonspecific defense against bacterial invasion. Previous studies indicated that probiotics and prebiotics may act as an immunomodulator agents. Nevertheless, research on the immunomodulatory effect of local materials has never been performed.Objective: To study the effects of Lactobacillus plantarum Mut7 and sweet potato fiber on the activity and Nitric Oxide (NO) production of peritoneal macrophages of Sprague Dawley rats.Method: Ninety six Sprague Dawley rats aged 8 weeks were divided into two groups; A (not infected with Salmonella typhimurium) and B (infected with S. typhimurium). Each group was divided into 4 subgroups and assigned to standard AIN-93M diet (KON), 109 CFU/ml of L. plantarum Mut7 (PRO), modified AIN-93M diet with sweet potato fiber (PRE), and both component (SIN). After 3 weeks of treatment, 6 rats of each subgroup were sacrificed and the peritoneal macrophages were isolated and analysed for its activity and NO production. The rest of the rats continued the treatments for another 2 weeks. At the end of the experiment, they were sacrificed and the peritoneal macrophage were isolated and analysed for its activity and NO production.Results: Oral administration of L. plantarum Mut7, sweet potato fiber, or both improve phagocytic activity of peritoneal macrophage which was indicated by an increase in the percentage of macrophages that phagocyte latex particles (p<0.05) and an increase in the number of latex particles engulfed by macrophages either after 3 or 5 weeks of treatment (p<0.05). Oral administration of L. plantarum Mut7, sweet potato fiber, or both were unable to increase the nitric oxide production after 3 weeks of treatment (p>0.05), but after 5 weeks of treatment the production of NO was significantly increased (p<0.05).Conclusion: L. plantarum Mut7, sweet potato fiber, or both increase the non specific immune response as they could improve the activity and NO production of peritoneal macrophages.

IL-27 promotes nitric oxide production induced by LPS through STAT1, NF-κB and MAPKs

Interleukin (IL)-27, a member of the IL-6/IL-12 heterodimeric cytokine family, induces pro-inflammatory responses including early T helper (Th)1 differentiation and generation of cytotoxic T lymphocytes, and also anti-inflammatory responses including the differentiation to IL-10-producing regulatory T cells, inhibition of Th2 and Th17 differentiation, and suppression of pro-inflammatory cytokine production. Nitric oxide (NO) is a potent source of reactive nitrogen species that play an important role in killing intracellular pathogens and forms a crucial component of host defense. Inducible NO synthase (iNOS), which catalyzes the production of NO, is induced by a range of stimuli including cytokines and microbes. Recently, IL-27 was reported to play an anti-inflammatory role in microglia by blocking oncostatin M-induced iNOS expression and neuronal toxicity. In the present study, we investigated the effects of IL-27 on NO production in thioglycollate-elicited peritoneal macrophages. IL-27 together with lipopolysaccharide (LPS) induced morphological change into more spread and elongated cells and synergistically enhanced NO production. The combined stimulation also enhanced iNOS mRNA expression and the NO production was abrogated by an iNOS inhibitor, NG-monomethyl l-arginine. The synergistic NO production could be attributed to the augmented Toll-like receptor (TLR)4 mRNA expression by the combination. Signal transducer and activator of transcription (STAT)1 was indispensable for the morphological change and NO production. The combination induced nuclear factor κB (NF-κB) translocation into nuclear and phosphorylation of extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK), and their inhibitors suppressed NO production. These results suggest that in contrast to the anti-proinflammatory role in microglia, IL-27 exerts a pro-inflammatory role by enhancing NO production in peritoneal macrophages stimulated with LPS through activation of STAT1, NF-κB and MAPKs.

Differential regulation of NO availability from macrophages and endothelial cells by the garlic component S-allyl cysteine

Garlic has been used as a traditional medicine for prevention and treatment of cardiovascular diseases. However, the molecular mechanism of garlic’s pharmacological action has not been clearly elucidated. We examined here the effect of garlic extract and its major component, S-allyl cysteine (SAC), on nitric oxide (NO) production by macrophages and endothelial cells. The present study demonstrates that these reagents inhibited NO production through the suppression of iNOS mRNA and protein expression in the murine macrophage cell line RAW264.7, which had been stimulated with LPS and IFNγ. The garlic extract also inhibited NO production in peritoneal macrophages, rat hepatocytes, and rat aortic smooth muscle cells stimulated with LPS plus cytokines, but it did not inhibit NO production in iNOS-transfected AKN-1 cells or iNOS enzyme activity. These reagents suppressed NF-κB activation and murine iNOS promoter activity in LPS and IFNγ-stimulated RAW264.7 cells. In contrast, these reagents significantly increased cGMP production by eNOS in HUVEC without changes in activity, protein levels, and cellular distribution of eNOS. Finally, garlic extract and SAC both suppressed the production of hydroxyl radical, confirming their antioxidant activity. These data demonstrate that garlic extract and SAC, due to their antioxidant activity, differentially regulate NO production by inhibiting iNOS expression in macrophages while increasing NO in endothelial cells. Thus, this selective regulation may contribute to the anti-inflammatory effect and prevention of atherosclerosis by these reagents.

Restoration of macrophage tumoricidal activity by bleomycin correlates with the decreased production of transforming growth factor beta in rats bearing KDH-8 hepatoma cells.

To explore the mechanisms of immuno-modulatory activities of bleomycin, we investigated interferon gamma (IFN gamma) mRNA expression, tumor necrosis factor alpha (TNF alpha) production, nitric oxide (NO) production and macrophage tumoricidal activities in rats bearing KDH-8 hepatoma cells, which secreted a large amount of transforming growth factor beta (TGF beta), and these processes in KDH-8 tumor-bearing rats treated with bleomycin. We found that IFN gamma mRNA expression, TNF alpha production, NO production and macrophage cytotoxic activities were lower in the KDH-8-bearing rats than in normal rats. On the other hand, low-dose bleomycin restored the macrophage cytotoxic activities, NO production, IFN gamma mRNA expression and TNF alpha production in the KDH-8-bearing rats. In vitro experiments showed that KDH-8-derived TGF beta decreased the IFN gamma mRNA expression and TNF alpha production in splenocytes, and NO production in peritoneal macrophages. These results suggest that low-dose bleomycin restored the cytokine production and macrophage tumoricidal activities in the KDH-8-bearing rats by decreasing KDH-8-derived TGF beta.

Methotrexate suppresses nitric oxide production ex vivo in macrophages from rats with adjuvant-induced arthritis.

We examined the effects of methotrexate (MTX) on the level of nitric oxide (NO) produced by peritoneal macrophages from rats with adjuvant-induced arthritis (AA) ex vivo. During the development of AA, paw swelling increased and LPS enhanced the capacity of peritoneal macrophages to produce NO and prostaglandin E2 (PGE2). MTX (0.1 mg/kg, p.o.) treatment for 21 days reduced the paw swelling, and inhibited the increased NO and PGE2 production. However, when MTX (0.1 mg/kg, p.o.) was administered to rats with established AA, these parameters were not significantly influenced. In normal rats, MTX (0.1 mg/kg, p.o.) treatment for 21 days did not change NO and PGE2 production of LPS-stimulated macrophages. On the other hand, macrophages from normal and AA rats cultured in the presence of MTX (1, 10 and 100 microM), were activated by LPS in vitro. MTX did not influence NO or PGE2 production by LPS-stimulated macrophages in normal and AA rats. By contrast, indomethacin (IM) (1.0 mg/kg, p.o.) treatment for 21 days reduced the paw swelling, and inhibited NO and PGE2 production in AA rats. IM inhibited significantly PGE2 production, but did not influence NO production by LPS-stimulated macrophages in vitro. These results suggest that MTX treatment reduces NO production in peritoneal macrophages in AA rats, and these actions of MTX may have an inhibitory effect without the modulation of PGE2.

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

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