Anti-inflammatory effect of pyroglutamyl-leucine on lipopolysaccharide-stimulated RAW 264.7 macrophages

Aims

Food-derived peptides have been reported to yield a variety of health promoting activities. Pyroglutamyl peptides are contained in the wheat gluten hydrolysate. In the present study, we investigated the effect of pyroglutamyl dipeptides on the lipopolysaccharide (LPS)-induced inflammation in macrophages.

Main methods

RAW 264.7 macrophages were treated with LPS and various concentrations of pyroglutamyl-leucine (pyroGlu-Leu), -valine (pyroGlu-Val), -methionine (pyroGlu-Met), and -phenylalanine (pyroGlu-Phe). Cell viability/proliferation and various inflammatory parameters were measured by the established methods including ELISA and western blotting. The binding of fluorescein isothiocyanate-labeled LPS to RAW 264.7 cells was also measured fluorescently.

Key findings

All the tested dipeptides significantly inhibited the secretion of nitric oxide, tumor necrosis factor (TNF)-α, and interleukin (IL)-6 from LPS-stimulated RAW 264.7 macrophages. Above all, pyroGlu-Leu inhibited the secretion of all these inflammatory mediators even at the lowest dose (200 μg/ml). PyroGlu-Leu dose-dependently suppressed IκBα degradation and MAPK (JNK, ERK, and p38) phosphorylation in LPS-stimulated RAW 264.7 cells. On the other hand, it did not affect the binding of LPS to the cell surface.

Significance

Our results indicated that pyroGlu-Leu inhibits LPS-induced inflammatory response via the blocking of NF-κB and MAPK pathways in RAW 264.7 macrophages.     

Magnolol stimulates lipolysis in lipid-laden RAW 264.7 macrophages

This study investigated the effect of magnolol, a compound isolated from Magnolia officinalis, on lipolysis in lipid-laden RAW 264.7 macrophages. Treatment of macrophages with magnolol led to dissolution of lipid droplets. This phenomenon was accompanied by a dose-dependent release of glycerol and cholesterol and a concomitant reduction in intracellular levels of glycerol and cholesterol. Furthermore, adipose differentiation-related protein (ADRP), a lipid droplet-associated protein, was down-regulated by magnolol in a dose- and time-dependent manner by Western blot analysis. Immunofluorescence studies also showed that ADRP became detached from the surface of lipid droplets after magnolol treatment. The lipolytic effect of magnolol was not mediated through the cAMP-protein kinase A (PKA) system, an authentic lipolytic pathway for macrophages, since magnolol did not induce an increase of intracellular cAMP levels, and pretreatment with either of PKA inhibitors, PKI and KT5720, did not abrogate the lipolytic response to magnolol. We conclude that magnolol induce-lipolysis of lipid-laden macrophages by down-regulation of ADRP expression and detachment of ADRP from the lipid droplet surface by a cAMP-independent mechanism. Lipolysis of lipid-laden macrophages may occur when the amount of ADRP on the surface of lipid droplets is not enough to stabilize the lipid droplets.     

Anti-inflammatory activity of c-phycocyanin in lipopolysaccharide-stimulated RAW 264.7 macrophages

C-phycocyanin (C-PC), found in blue green algae, is often used as a dietary nutritional supplement. C-PC has been found to have an anti-inflammatory activity and exert beneficial effect in various diseases. However, little is known about its mechanism of action. Overproduction of nitric oxide (NO) derived from inducible nitric oxide synthase (iNOS) plays an important role in the pathogenesis of inflammation. The aim of this study was to determine whether C-PC inhibits production of nitrite, an index of NO, and iNOS expression in lipopolysaccharide (LPS)-treated RAW 264.7 macrophages. Our results indicated that C-PC significantly inhibited the LPS-induced nitrite production and iNOS protein expression accompanied by an attenuation of tumor necrosis factor-alpha (TNF-alpha) formation but had no effect on interleukin-10 production in macrophages. Furthermore, C-PC also suppressed the activation of nuclear factor-kappaB (NF-kappaB) through preventing degradation of cytosolic IkappaB-alpha in LPS-stimulated RAW 264.7 macrophages. Thus, the inhibitory activity of C-PC on LPS-induced NO release and iNOS expression is probably associated with suppressing TNF-alpha formation and nuclear NF-kappaB activation, which may provide an additional explanation for its anti-inflammatory activity and therapeutic effect.     

beta-Sitosterol modulates antioxidant enzyme response in RAW 264.7 macrophages

Uncontrolled production of reactive oxygen species contributes to the pathogenesis of diseases such as cancer and cardiovascular disorders. Olive oil exerts remarkable preventive effects on the development of these diseases, which may be due to the action of various components of the olive oil. In fact, several findings suggest that minor components, like phytosterols such as beta-sitosterol, are responsible, at least in part, for these beneficial effects. Our results show that beta-sitosterol reverts the impairment of the glutathione/oxidized glutathione ratio induced by phorbol esters in RAW 264.7 macrophage cultures. These data can be correlated with the increase in manganese superoxide dismutase and glutathione peroxidase activities and the decrease in catalase activity. We also demonstrate that the effects of beta-sitosterol on antioxidant enzymes depend on the estrogen/phosphatidylinositol 3-kinase pathway.     

Oleic acid-induced PKC isozyme translocation in RAW 264.7 macrophages

Fatty acids are important second messengers that mediate various cellular functions, but their role in the formation of macrophage foam cells is not known. High plasma levels of oleic acid (OA) in obese patients are often associated with a high risk for atherosclerosis. In this study, we investigated the protein kinase C (PKC) isozymes involved in OA-induced lipid accumulation in RAW 264.7 macrophages. The results show that OA induces translocation of PKC alpha, beta1, and delta from the cytosol to the cell membrane 5 min after the treatment. After 16 h incubation with OA, PKC delta was found to be colocalized with adipose differentiation-related protein (ADRP) on the surface of lipid droplets, but immunoprecipitation experiments showed that PKC delta was not biochemically associated with ADRP. After 16 h incubation with OA plus phorbol 12-myristate 13-acetate (PMA), PKC delta staining on the lipid droplet surface was not seen, whereas the accumulation of lipid droplets was unaffected. Furthermore, downregulation of PKC delta was confirmed by immunoblotting. These results demonstrate possible involvement of specific PKC isozymes in the early phase of lipid accumulation, possibly during the uptake of OA.     

Natural antioxidants attenuate mycolactone toxicity to RAW 264.7 macrophages

Mycobacterium ulcerans produces a macrolide exotoxin, mycolactone which suppresses immune cells activity, is toxic to most cells and the key virulence factor in the pathogenesis of Buruli ulcer disease. Mycolactone is reported to mediate the production of reactive oxygen species in keratinocytes; cells that play critical role in wound healing. Increased levels of reactive oxygen species have been shown to disrupt the well-ordered process of wound repair; hence, the function of wound-healing cells such as macrophages, keratinocytes, and fibroblast could be impaired in the presence of the reactive oxygen species mediator, mycolactone. To ensure regeneration of tissues in chronic ulcers, with proper and timely healing of the wounds, natural antioxidants that can combat the effects of induced reactive oxygen species in wound-healing cells ought to be investigated. Reactive oxygen species activity was determined in mycolactone-treated RAW 264.7 macrophages and the scavenging ability of the antioxidants (ascorbic acid, gallic acid, and green tea kombucha) against mycolactone-induced reactive oxygen species (superoxide anions) was assessed using fluorescein probe (DCF-DA) and nitroblue tetrazolium dye. Cytotoxicity of the antioxidants, mycolactone, and the protective effect of the antioxidants on the cells upon treatment with mycolactone were determined using the Alamar blue assay. The expression levels of endogenous antioxidant enzyme genes (superoxide dismutase, catalase, and glutathione peroxidase) in response to mycolactone-mediated reactive oxygen species were determined using RT-qPCR. Mycolactone induced the production of reactive oxygen species in RAW 264.7 macrophages, and the resulting superoxide anions were scavenged by some of the antioxidants. The selected endogenous antioxidant enzyme genes in the macrophages were upregulated in the presence of the antioxidants and mycolactone. The exogenously supplied ascorbic acid and green tea kombucha offered moderate protection to the macrophages against the toxicity of mycolactone. We conclude that the results provide insights into alternate and adjunct therapeutic approaches in Buruli ulcer treatment, which could significantly attenuate the toxicity of the pathogenic factor; mycolactone.     

Carnosine modulates nitric oxide in stimulated murine RAW 264.7 macrophages

C14orf28 [alias dopamine receptor-interacting protein (DRIP1)] is belonging to the family of DRIPs. However, the function of C14orf28 in cancer remains unclear. Herein, we found that C14orf28 was upregulated in colorectal cancer tissues compared to the adjacent non-tumor tissues. Overexpression of C14orf28 promoted the cellular proliferation, migration, invasion of colorectal cancer cells. In addition, C14orf28 inhibited apoptosis and promoted the EMT process. To explore the mechanism of dysregulation, C14orf28 was identified to be a target of miR-519d by targeting its 3′UTR. Furthermore, in agreement, C14orf28 overexpression counteracted the inhibitory effect of miR-519d. Together, these results evidenced that C14orf28 downregulated by miR-519d contributes to tumorigenesis and might provide new potential targets for colorectal cancer therapy.     

Lipid hydroperoxides inhibit nitric oxide production in RAW264.7 macrophages

The effects of oxidatively modified low density lipoprotein (oxLDL) on atherogenesis may be partly mediated by alterations in the production of nitric oxide (NO) by vascular cells. Lipid hydroperoxides (LOOH) and lysophosphatidylcholine (lysoPC) are the major primary products of LDL oxidation. The purpose of this study was to characterize the effects of oxLDL, LOOH and lysoPC on NO production and the expression of inducible nitric oxide synthase (iNOS) gene in lipopolysaccharide (LPS) stimulated macrophages. LDL was oxidized using an azo-initiator 2,2'-azobis (2-amidinopropane) HCl (ABAP) and octadecadienoic acid was oxidized by lipoxygenase to generate 13-hydroperoxyl octadecadienoic acid (13-HPODE). Our study showed that oxLDL markedly decreased the production of NO, the levels of iNOS protein and iNOS mRNA in LPS stimulated macrophages. The inhibition potential of oxLDL on NO production and iNOS gene expression depended on the levels of LOOH formed in oxLDL and was not due to oxLDL cytotoxicity. Furthermore, 13-HPODE markedly reduced NO production and iNOS protein levels, whereas lysoPC showed only slight reduction. The effects of 13-HPODE and lysoPC did not require an acetylated LDL carrier. Our results suggest that 13-HPODE is a much more potent inhibitor of NO production and iNOS gene expression than lysoPC in LPS stimulated RAW264.7 macrophages.     

Effects of major dietary antioxidants on inflammatory markers of RAW 264.7 macrophages

Oxidative stresses are involved in the process of chronic inflammatory diseases. The objective of this study was to evaluate and compare the anti-inflammatory activity of major dietary antioxidants. Murine RAW264.7 macrophages (4 x 10(6) cells) were incubated in DMEM containing 10% FBS supplemented with 5 to 20 microM of alpha-tocopherol, beta-carotene or quercetin for 24 hrs, and then treated with LPS for 16 hrs. The medium was collected to measure TNF-alpha, IL-6, PGE2 and nitrite. Expressions of cyclooxygenase-2(COX-2) and inducible nitric oxide synthase(iNOS) were determined using the harvested cells. Results indicate that TNF-alpha and IL-6 accumulations were significantly reduced by 5 to 20 microM quercetin treatment, and 20 microM of alpha-tocopherol treatment. Nitrite release was significantly reduced by 5 microM quercetin treatment. However, PGE2 accumulation was not affected by any of the antioxidants used. Expressions of COX-2 and iNOS were effectively reduced by 5 microM quercetin treatment. These findings indicate that dietary antioxidants possess significant anti-inflammatory activities, and quercetin is the most potent antioxidant.     

Protein oxidation and proteolysis in RAW264.7 macrophages: effects of PMA activation

Macrophages are stimulable cells able to increase the production of reactive oxygen and nitrogen species dramatically for a short period of time. Free radicals and other oxidants are able to oxidize the intracellular protein pool. These oxidized proteins are selectively recognized and degraded by the intracellular proteasomal system. We used the mouse macrophage-like cell line RAW264.7 to test whether macrophagial cells are able to increase their protein turnover after oxidative stress and whether this is accompanied by an increased protein oxidation. Macrophagial cells are particularly susceptible to bolus additions of hydrogen peroxide and peroxynitrite. In further experiments we activated RAW264.7 cells with PMA to test whether the production of endogenous oxidants has analogous effects. A clear dependence of the protein turnover and protein oxidation on the oxidative burst could be measured. In further experiments the role of the proteasomal system in the selective removal of oxidized proteins could be revealed exploring the proteasome specific inhibitor lactacystin. Therefore, although oxidants are able to attack the intracellular protein pool in macrophages, these cells are able to remove oxidized proteins selectively and protect the intracellular protein pool from oxidation.     

Chitinous materials inhibit nitric oxide production by activated RAW 264.7 macrophages

Chitinous materials have been studied in wound healing and artificial skin substitutes for many years. Nitric oxide (NO) has been shown to contribute to cytotoxicity in cell proliferation during inflammation of wound healing. In this study, we examined the effect of chitin and its derivatives on NO production by activated RAW 264.7 macrophages. Chitin and chitosan showed a significantly inhibitory effect on NO production by the activated macrophages. Hexa-N-acetylchitohexaose and penta-N-acetylchitopentaose also inhibited NO production but with less potency. However, N-acetylchitotetraose, -triose, -biose, and monomer of chitin, N-acetylglucosamine and glucosamine had little effect on NO production by the activated cells. These results suggest that the promotive effect of chitinous material on wound healing be related, at least partly, to inhibit NO production by the activated macrophages.     

Echinophora tenuifolia L. inflorescences: phytochemistry and in vitro antioxidant and anti-inflammatory properties in LPS-stimulated RAW 264.7 macrophages

During the last decades, different natural compounds have been demonstrated to modulate inflammatory pathways. In this study, methanolic extract of Echinophora tenuifolia L. inflorescences was investigated for its chemical composition and in vitro antioxidant and anti-inflammatory properties. Phytochemical profile was investigated by means of high-performance thin layer chromatography (HPTLC) and gas chromatography–mass spectrometry analyses. Myristic acid and palmitic acid were found to be the major constituents. Six terpenes were identified: α-phellandrene, o-cymene, dihydroactinidiolide, neophytadiene, phytol and β-amyrin, and two phenolic compounds: carvacrol and ferulic acid. HPTLC analysis of the ethyl acetate fraction highlighted the presence of the flavonoid glycosides rutin and quercitrin. This sample showed the best diphenylpicrylhydrazyl scavenging capacity, with an IC₅₀ value equal to 40.39 μg/ml, and the strongest capacity to protect linoleic acid from peroxidation, as assessed by the β-carotene bleaching test (IC₅₀ = 16.31 μg/ml). All samples inhibited nitric oxide production in cell supernatants in a dose-dependent manner, being the two apolar fractions (n-hexane and dichloromethane) even more active than the positive control indomethacin. A relevant biological activity was observed for dichloromethane fraction (IC₅₀ value equal to 39.97 μg/ml). Obtained results indicate that this sample could be an excellent candidate for further investigations aimed at the development of new antioxidant and anti-inflammatory drugs.     

Identification of S-nitrosylated proteins in endotoxin-stimulated RAW264.7 murine macrophages.

Nitric oxide (NO) is an omnipresent regulator of cell function in a variety of physiologic and pathophysiologic states. In part, NO exerts its actions by S-nitrosylation of target thiols, primarily in cysteine residues. Delineating the functional correlates of S-nitrosylation can begin with identification of the entire population of S-nitrososylated proteins. Recently, the biotin switch technique was developed to allow a proteomic approach to identification of the "universe" of S-nitrsoylated proteins. In this study using endotoxin-stimulated RAW264.7 murine macrophages, we have utilized the biotin-switch technique and protein sequencing to identify S-nitrosylated proteins in this setting. In contrast to other studies utilizing exogenous sources of NO, our approach utilizes endogenous NO synthesis as the basis for S-nitrosylation. Our results indicate multiple unique proteins not previously identified as S-nitrosylation targets: enolase, pyruvate kinase, elongation factor-1 and -2, plastin-2, FRAG-6, CEM-16, and SMC-6. While the ubiquitous nature of NO argues for some degrees of commonality, S-nitrosylation of unique proteins specific to endotoxin stimulated macrophages suggests regulatory mechanisms for which NO is necessary, but not sufficient.     

Mitogen-activated protein kinase pathway mediates DBP-maf-induced apoptosis in RAW 264.7 macrophages

Vitamin D-binding protein-macrophage-activating factor (DBP-maf) is derived from serum vitamin D binding protein (DBP) by selective deglycosylation during inflammation. In the present study, we investigated the effect of DBP-maf on RAW 264.7 macrophages and the underlying intracellular signal transduction pathways. DBP-maf increased proapoptotic caspase-3, -8, and -9 activities and induced apoptosis in RAW 264.7 cells. However, DBP, the precursor to DBP-maf did not induce apoptosis in these cells. Cell cycle analysis of DBP-maf-treated RAW 264.7 cells revealed growth arrest with accumulation of cells in sub-G(0)/G(1) phase. We also investigated the role of mitogen-activated protein kinase (MAPK) pathways in the DBP-maf-induced apoptosis of RAW264.7 cells. DBP-maf increased the phosphorylation of p38 and JNK1/2, while it decreased the ERK1/2 phosphorylation. Treatment with the p38 MAPK inhibitor, SB202190, attenuated DBP-maf-induced apoptosis. PD98059, a MEK specific inhibitor, did not show a significant inhibition of apoptosis induced by DBP-maf. Taken together, these results suggest that the p38 MAPK pathway plays a crucial role in DBP-maf-mediated apoptosis of macrophages. Our studies indicate that, during inflammation DBP-maf may function positively by causing death of the macrophages when activated macrophages are no longer needed at the site of inflammation. In summary, we report for the first time that DBP-maf induces apoptosis in macrophages via p38 and JNK1/2 pathway.     

Anti-inflammatory effect of chrysin on RAW 264.7 mouse macrophages induced with polyinosinic-polycytidylic acid

Chrysin (5,7-Dihydroxyflavone) is an active flavonoid isolated from Scutellariae Radix which has been used to treat pneumonia, laryngopharyngitis, jaundice, shigellosis, and breast mass in Korea, China, and Japan. Chrysin has been already reported to inhibit inducible nitric oxide synthase and cyclooxygenase-2 in lipopolysaccharideinduced macrophages. However, the effect of chrysin on virus-induced macrophages is not fully reported. In this study, the anti-inflammatory effect of chrysin on doublestranded RNA (dsRNA)-induced macrophages was examined. Production of Nitric oxide (NO), various cytokines, as well as calcium release and mRNA expression of CHOP and Fas in dsRNA [polyinosinic-polycytidylic acid]-induced RAW 264.7 mouse macrophages were evaluated. Chrysin restored the cell viability in dsRNA [polyinosinicpolycytidylic acid]-induced RAW 264.7 mouse macrophages at concentrations of up to 50 μM. Chrysin significantly inhibited the production of NO, IL-1α, IL-1β, IL-6, IL-10, IP-10, G-CSF, GM-CSF, LIF, LIX/CXCL5, MCP-1, MCSF, MIP-1α, MIP-1β, MIP-2, RANTES, TNF-α, and VEGF as well as calcium release and mRNA expression of CHOP and Fas in dsRNA [polyinosinic-polycytidylic acid]-induced RAW 264.7 mouse macrophages (P< 0.05). These data suggest that chrysin has anti-inflammatory properties related with its inhibition of nitric oxide, cytokines, chemokines, and growth factors in dsRNA-induced macrophages via the ER stress-CHOP pathway.     

Inhibitory constituents of Nardostachys chinensis on nitric oxide production in RAW 264.7 macrophages

The activity-guided fractionation of the MeOH extract of the rhizomes and roots of Nardostachys chinensis led to the isolation of two new sesquiterpenoids, narchinol B (8) and narchinol C (9), along with 10 known compounds, ursolic acid (1), nardosinone (2), pinoresinol (3), desoxo-narchinol A (4), kanshone B (5), epoxyconiferyl alcohol (6), debilon (7), 4α,5-dimethyl-1,3-dioxo-1,2,3,4,4α,5,6,7-octahydronaphthalene (10), p-coumaric acid (11), and isoferulic acid (12). Their structures were determined using spectroscopic techniques, which included 1D- and 2D-NMR. Among the isolates, compounds 2, 4, 5, 8 and 9 showed inhibitory activity against LPS-induced NO production with IC(50) values of 4.6-21.6 μM.     

In vitro model to estimate gut inflammation using co-cultured Caco-2 and RAW264.7 cells

A system for assessing the anti-inflammatory effect of food factors was developed by establishing a co-culture system with intestinal epithelial Caco-2 cells (apical side) and macrophage RAW264.7 cells (basolateral side). In this system, the stimulation of RAW264.7 cells with lipopolysaccharide was followed by a decrease in transepithelial electrical resistance, which is a marker of the integrity of the Caco-2 monolayer and an increase in TNF-α production from RAW264.7 cells and IL-8 mRNA expression in Caco-2 cells. Treatment with anti-TNF-α antibodies or budesonide suppressed in increase in TNF-α production and IL-8 mRNA expression. These results indicated that this co-culture model could imitate the gut inflammation in vivo. In addition, fucoidan, sulphated polysaccharides from brown algae, was employed as a candidate of evolution and added to the apical side of this model. Fucoidan suppressed IL-8 gene expression through a reduction in TNF-α production from RAW264.7 cells stimulated with lipopolysaccharide.     

Assessing cytotoxicity of boron nitride nanotubes: Interference with the MTT assay

Thanks to a non-covalent wrapping with glycol-chitosan, highly biocompatible and highly concentrated dispersions of boron nitride nanotubes were obtained and tested on human neuroblastoma cells. A systematic investigation of the cytotoxicity of these nanovectors with several complementary qualitative and quantitative assays allowed a strong interference with the MTT metabolic assay to be highlighted, similar to a phenomenon already observed for carbon nanotubes, that would wrongly suggest toxicity of boron nitride nanotubes. These results confirm the high complexity of these new nanomaterials, and the needing of extensive investigations on their exciting potential applications in the biomedical field.